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

Abstract

There is provided a pharmaceutical composition for preventing or treating cancer 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. The fusion protein containing an CD80 fragment, an Fe domain of an immunoglobulin, and an IL-2 variant in an embodiment can activate immune cells such as natural killer cells as well as control the immune cell regulatory activity of regulatory T cells. In addition, the combined administration of the fusion protein and an immune checkpoint inhibitor such as Keytruda known as a PD-1 inhibitor can effectively inhibit cancer. Therefore, a pharmaceutical composition containing, 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 can be effectively applied to the treatment of cancer and has high industrial applicability.

Description

A pharmaceutical composition for treating cancer comprising a fusion protein comprising IL-2 protein and CD80 protein and an immune checkpoint inhibitor

[0001] The present invention relates to a pharmaceutical composition 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 as active ingredients.

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

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

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

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

[0006] Accordingly, the inventors of the present invention completed the present invention by confirming that a novel fusion protein dimer and immune checkpoint inhibitor comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof in one molecule exhibits excellent anticancer effects as a result of research to develop a safe and effective anticancer agent.

[0007] To achieve the above purpose, one aspect of the present invention provides a pharmaceutical composition 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 as active ingredients.

[0008] A fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof can activate immune cells by IL-2. Furthermore, it was confirmed that a synergistic effect is exhibited when administered in combination with an immune checkpoint inhibitor. Therefore, a pharmaceutical composition for treating cancer comprising the fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof and an immune checkpoint inhibitor as active ingredients can be usefully used for the prevention and treatment of cancer.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0024] Figure 16 shows the binding affinity between hCTLA4 and GI101.

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

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

[0027] Figure 19 shows the binding affinity between mCTLA4 and mGI101.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0081] One aspect of the present invention provides a pharmaceutical composition 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 as active ingredients.

[0082] immune checkpoint inhibitors

[0083] The term "immune checkpoint," as used herein, refers to an intracellular signaling system that maintains self-tolerance and protects tissues from excessive immune responses that cause damage. Immune checkpoint proteins are membrane proteins that regulate immune checkpoints and can suppress the differentiation, proliferation, and activity of immune cells. Specifically, immune checkpoint proteins are expressed in activated T cells, reducing T cell proliferation, cytokine secretion, and cytotoxicity, and suppressing excessive T cell activity. Some immune checkpoints are known to be a key mechanism by which tumor cells evade immune responses. Therefore, "immune checkpoint inhibitors" target immune checkpoint proteins to inhibit or block immune checkpoints, thereby increasing T cell activation and enhancing anti-tumor immunity, resulting in anticancer effects. Immune checkpoint inhibitors have the advantage of having fewer side effects, such as vomiting and hair loss, than conventional cytotoxic anticancer agents, and offer greater therapeutic efficacy. Furthermore, they utilize the immune response system's excellent memory, which is known to provide long-lasting therapeutic effects even after drug administration is discontinued.

[0084] 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.

[0085] 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.

[0086] The term "CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4)" used in this specification refers to CD152, which is expressed on the membrane surface of activated T cells. It binds to CD80 (B7-1) and CD86 (B7-2) of antigen-presenting cells and inhibits T cell activity. CTLA-4 inhibitors include ipilimumab (Yervoy). ® ) and tremelimumab.

[0087] As used herein, the term "PD-1 (programmed cell death protein 1)" refers to CD279, a protein 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 inhibits T-cell activation, growth factor and cytokine production mediated by TCR (T cell receptor) and CD28, thereby inducing negative signal transduction. PD-1 inhibitors include, for example, pembrolizumab (Keytruda). ® ), MK-3475, nivolumab (Opdivo ® ), cemiplimab (Liptayo ® ), JTX-4014, spartalizumab, camrelizumab, scintillimab, tislelizumab, toripalimab, dostalimab, INCMGA00012, AMP-224, and AMP-514.

[0088] The term "PD-L1 (Programmed death-ligand 1)" used in this specification refers to CD274 and B7-H1, and is a protein on the surface of cancer cells or hematopoietic cells. PD-L1 on the surface of cancer cells can bind to PD-1 on the surface of T cells. The PD-L1 inhibitors include, for example, atezolizumab and avelumab (Bavencio). ® ), durvalumab (Imfinzi) ® ), KN035, CK-301, AUNP12, CA-170 and BMS-986189.

[0089] 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 of T cells and inhibits T cell activity, growth, and cytokine production, thereby negatively regulating T cell-mediated immune responses.

[0090] As used herein, the term "HVEM (herpesvirus entry mediator)" 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 binds to various partner proteins to regulate inflammation and immune responses. When combined with BTLA (B and T lymphocyte attenuator, CD272) or CD160 on T cells, it inhibits T cell immune activity. On the other hand, when combined with TNFSF14 (LIGHT), it induces dendritic cell maturation, T cell proliferation, and cytokine production, thereby activating inflammation and immune responses.

[0091] As used herein, the term "TIM3 (T cell membrane protein 3)" is also referred to as hepatitis A virus cellular receptor 2 (HAVCR2) and is expressed in various immune cells. When activated by binding to the soluble protein GAL9 (galectin 9), intracellular calcium influx increases, inducing T cell death, which ultimately leads to immune tolerance. In addition, TIM3 inhibits T cell immune activity by binding to the cell surface protein CEACAM1 (cell adhesion molecule 1) together with GAL9, and also inhibits immune activity by binding to the soluble proteins HMGB1 (high mobility group protein 1) or PTdSer (phospatidyl serine). TIM3 inhibitors may be LY3321367, MBG453, and TSR-022.

[0092] As used herein, the term "LAG3 (lymphocyte activation gene 3)" refers to CD223, which binds to MHC (major histocompatibility complex) class II and inhibits T cell proliferation and activity. LAG3 inhibitors may be IMP321, lilatrimab, and GSK2831781.

[0093] As used herein, the term "VISTA (V-domain Ig suppressor of T cell activation)" belongs to the B7 family (B7-H5) and is expressed in various immune cells to suppress T cell proliferation, activity, and cytokine production. The VISTA inhibitor may be JNJ-63723283.

[0094] As used herein, the term "KIR (killer cell immunoglobulin-like receptor)" refers to a membrane protein expressed on NK cells and T cells, and is a family of proteins with genetic diversity and homology. Among them, KIR2DL1, KIR2DL2 / L3, KIR3DL1, and KIR3DL2 can bind to MHC class I and suppress the cellular immune activity of NK cells.

[0095] The term "TIGIT (T cell immunoglobulin and ITIM domain)" used in this specification is a membrane protein expressed on the surface of NK cells and T cells, which binds to CD155, CD112, and CD113 to suppress immune activity.

[0096] Fusion protein comprising IL-2 protein and CD80 protein and dimer thereof

[0097] As used herein, the term "IL-2" or "interleukin-2" means any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Furthermore, the IL-2 may be wild-type IL-2 or a variant thereof.

[0098] In this specification, IL-2 or its variants are collectively referred to as "IL-2 protein" or "IL-2 polypeptide." IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind to, for example, the IL-2 receptor. This specific binding can be confirmed by methods known to those skilled in the art.

[0099] A specific example of the IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not include a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. In this case, the IL-2 may be used as a concept that includes a fragment in which a portion of the N-terminus or C-terminus of the wild-type IL-2 is deleted (truncated).

[0100] In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0101] As used herein, the term "IL-2 variant" refers to a form in which a portion of an amino acid sequence of full-length IL-2 or a fragment of the above-mentioned IL-2 is substituted. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may mean, for example, specific binding to an IL-2 receptor, and this specific binding can be measured by a method known to those skilled in the art.

[0102] Specifically, the IL-2 variant may be one in which some of the amino acids of wild-type IL-2 are substituted. As a specific example of the IL-2 variant by amino acid substitution, at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 may be substituted.

[0103] Specifically, the IL-2 variant may be one in which at least one of the 38th, 42nd, 45th, 61st, or 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid. In addition, when IL-2 is in a form in which a part of the N-terminus of the amino acid sequence of SEQ ID NO: 35 is deleted, the amino acid at the complementarily corresponding position in the amino acid sequence of SEQ ID NO: 10 may be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 variant may be one in which at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in the amino acid sequence of SEQ ID NO: 35 is substituted with another amino acid. These correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues of the amino acid sequence of SEQ ID NO: 10, respectively. In one embodiment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids may be substituted, as long as IL-2 activity is maintained. In another embodiment, from one to five amino acids may be substituted.

[0104] In one specific example, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10.

[0105] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted.

[0106] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Additionally, as a specific example, the IL-2 variant may have amino acids substituted at positions 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0107] Furthermore, the IL-2 variant may be a form in which five amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are all substituted with different amino acids.

[0108] At this time, the "other amino acid" introduced by the above substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, in the amino acid substitution of the IL-2 variant, the 38th position in the amino acid sequence of SEQ ID NO: 10 cannot be substituted with arginine, the 42nd position cannot be substituted with phenylalanine, the 45th position cannot be substituted with tyrosine, the 61st position cannot be substituted with glutamic acid, and the 72nd position cannot be substituted with leucine.

[0109] In the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than arginine. Preferably, in the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (R38A).

[0110] In the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than phenylalanine. Preferably, in the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (F42A).

[0111] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (Y45A).

[0112] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with arginine (E61A).

[0113] In the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with glycine (L72G).

[0114] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10.

[0115] Specifically, the IL-2 variant may have amino acid substitutions at two, three, four or five positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0116] In addition, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A and F42A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and L72G are substituted. Additionally, as a specific example, the IL-2 variant may have substitutions of E61R and L72G.

[0117] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A, F42A, and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at Y45A, E61R, and L72G.

[0118] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and E61R. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, Y45A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at F42A, Y45A, E61R, and L72G.

[0119] Furthermore, the IL-2 variant may have substitutions such as R38A, F42A, Y45A, E61R, and L72G.

[0120] Preferably, one specific example of the IL-2 variant may be one in which a substitution of any one of the following combinations (a) to (d) has occurred in the amino acid sequence of SEQ ID NO: 10:

[0121] (a) R38A / F42A

[0122] (b) R38A / F42A / Y45A

[0123] (c) R38A / F42A / E61R

[0124] (d) R38A / F42A / L72G

[0125] At this time, if IL-2 has the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementarily corresponding to SEQ ID NO: 10. In addition, even if IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, the amino acid at a position complementarily corresponding to SEQ ID NO: 10 may be substituted.

[0126] Specifically, the variant of IL-2 may have an amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

[0127] In addition, the IL-2 variant may be characterized by low toxicity in vivo. In this case, the low toxicity in vivo may be a side effect caused by IL-2 binding to the alpha chain (IL-2Rα) of the IL-2 receptor. In order to improve the side effects caused by the binding of IL-2 and IL-2Rα, various IL-2 variants have been developed, and such IL-2 variants may be those disclosed in U.S. Patent No. 5,229,109 and Republic of Korea Patent No. 1,667,096. In particular, the IL-2 variant described in the present application has a low binding affinity to the alpha chain (IL-2Rα) of the IL-2 receptor, and thus has lower in vivo toxicity than wild-type IL-2.

[0128] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides costimulatory signals essential for the activation and survival of T cells. CD80 is known to be a ligand for two different proteins present on the surface of T cells, CD28 and CTLA-4. CD80 consists of 288 amino acids, and may specifically have the amino acid sequence of SEQ ID NO: 11. Furthermore, as used herein, "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0129] As used herein, the term "CD80 fragment" refers to a truncated form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. A specific example of the CD80 fragment may be one in which the 1st to 34th amino acids from the N-terminus, which is the signal sequence of CD80, are removed. Specifically, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 288th of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 242nd of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 232nd of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 139th of SEQ ID NO: 11. Additionally, one specific example of the CD80 fragment may be a protein consisting of amino acids 142 to 242 of SEQ ID NO: 11. In one embodiment, the CD80 fragment may have an amino acid sequence of SEQ ID NO: 2.

[0130] Additionally, the IL-2 protein and the CD80 protein may be linked by a linker or carrier. Specifically, the IL-2 or a variant thereof and the CD80 (B7-1) or a fragment thereof may be linked by a linker or carrier. In the present specification, the terms linker and carrier are used interchangeably.

[0131] The linker connects two proteins. A specific example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, or an Fc domain of an immunoglobulin. In this case, the Fc domain of the immunoglobulin refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of the immunoglobulin, and does not include the variable region of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. In this case, the Fc domain of the wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4.

[0132] In addition, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. In addition, the term "Fc domain variant" as used herein may have a different glycosylation pattern from that of the wild-type Fc domain, or may have increased glycosylation compared to the wild-type Fc domain, decreased glycosylation compared to the wild-type Fc domain, or may have deglycosylated glycosylation. An aglycosylated Fc domain is also included. The Fc domain or variant may have a controlled number of sialic acid, fucosylation, or glycosylation through culture conditions or genetic manipulation of the host.

[0133] In addition, the sugar chain of the Fc domain of the immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a form in which the Fc region of the immunoglobulin is mixed with that of IgG, IgA, IgE, IgD, or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. A specific example of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12.

[0134] The fusion protein may have a structure in which CD80 and IL-2 proteins are linked to the N-terminus and C-terminus, respectively, of the Fc domain as a linker (or carrier), or IL-2 and CD80 are linked to each other. The linkage of the N-terminus or C-terminus of the Fc domain to CD-80 or IL-2 may be arbitrarily achieved by a linker peptide.

[0135] Specifically, the fusion protein may be composed of the following structural formula (I) or (II):

[0136] N'-X-[linker (1)]n-Fc domain-[linker (2)]mYC' (I)

[0137] N'-Y-[linker (1)]n-Fc domain-[linker (2)]mXC' (II)

[0138] At this time, in the structural formulas (I) and (II),

[0139] The above N' is the N-terminus of the fusion protein,

[0140] The above C' is the C-terminus of the fusion protein,

[0141] The above X is CD80 protein,

[0142] The above Y is IL-2 protein,

[0143] The above linker (1) and linker (2) are peptide linkers,

[0144] The above n and m are each independently O or 1.

[0145] Preferably, the fusion protein may be composed of structural formula (I). The IL-2 protein is as described above. In addition, the CD80 protein is as described above. According to one specific example, the IL-2 protein may be an IL-2 variant in which one to five amino acids are substituted compared to wild-type IL-2. The CD80 protein may be a fragment in which up to about 34 amino acid residues are consecutively deleted (truncated) from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having binding activity to T cell surface receptors CTLA-4 and CD28.

[0146] Specifically, the fusion protein may have an amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. In another specific embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. Here, the identity can be determined, for example, by percent homology, homology comparison software such as BlastN software of the National Center of Biotechnology Information (NCBI).

[0147] A peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may be comprised of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one specific example, the peptide linker (1) may be comprised of 30 amino acids. In addition, the peptide linker (1) may include at least one cysteine. Specifically, it may include one, two, or three cysteines. In addition, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one specific example, the peptide linker (1) may be a peptide linker comprised of the amino acid sequence of SEQ ID NO: 3.

[0148] The above peptide linker (2) may be composed of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one specific example, the peptide linker (2) may be (G4S)n (wherein n is an integer from 1 to 10). In this case, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may be a peptide linker composed of an amino acid sequence of SEQ ID NO: 5.

[0149] Another aspect of the present invention provides a dimer comprising two fusion proteins comprising the IL-2 protein and the CD80 protein. The fusion protein comprising the IL-2 or a variant thereof and the CD80 or a fragment thereof is as described above.

[0150] At this time, the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond via a cysteine ​​present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same, or may be different fusion proteins. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

[0151] A pharmaceutical composition comprising a fusion protein dimer comprising the 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.

[0152] The above cancer may be selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0153] The preferred dosage of the pharmaceutical composition above varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for preventing or treating cancer of the present invention, the active ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit anticancer activity. A typical effective amount will be determined within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. Here, "effective amount" refers to the amount of the active ingredient capable of inducing an anticancer effect. This effective amount can be experimentally determined within the normal ability range of those skilled in the art.

[0154] As used herein, the term "treatment" can be used to encompass both therapeutic treatment and preventative treatment. In this context, prevention can be used to mean alleviating or reducing a pathological condition or disease in an individual. In one embodiment, the term "treatment" encompasses all applications or any form of medication for treating a disease in mammals, including humans. Furthermore, the term encompasses inhibiting or slowing the progression of a disease or condition; restoring or repairing damaged or defective functions, thereby partially or completely alleviating a disease; or stimulating an inefficient process; or alleviating a serious condition.

[0155] As used herein, the term "efficacy" may be determined by one or more parameters, such as survival or disease-free survival over a period of time, such as one year, five years, or ten years. Furthermore, the parameters may include suppression of the size of at least one tumor in the subject.

[0156] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and enhanced potency, and can be measured by comparing clearance rates and tumor growth in test animals or human subjects, or by comparing parameters such as survival, relapse rate, or disease-free survival.

[0157] Here, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. In one specific embodiment, the therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

[0158] At this time, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0159] Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the tolerable level of the intended subject.

[0160] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. Methods for formulating pharmaceutical compositions are known in the art, and specific references can be made to literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc. The above literature is considered to be a part of the present specification.

[0161] The preferred dosage of the above pharmaceutical composition may range from 0.01 ug / kg to 10 g / kg per day, or from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once a day or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0162] The above pharmaceutical composition can be applied (prescribed) to mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of this application may additionally include any compound or natural extract whose safety has already been verified and known to have a therapeutic effect on anticancer activity.

[0163] 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.

[0164] Another aspect of the present invention provides the use of a composition for combination 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.

[0165] Another aspect of the present invention provides the use of a composition for combination administration comprising a fusion protein comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof and an immune checkpoint inhibitor for enhancing the therapeutic effect of cancer.

[0166] Another aspect of the present invention provides the use of a combination 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.

[0167] Another aspect of the present invention provides a method for preventing or treating cancer, and / or a method for improving the therapeutic effect, comprising administering to a subject a combination composition comprising 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 combined, and an immune checkpoint inhibitor.

[0168] The subject may be a subject suffering from cancer. In addition, the subject 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 a fusion protein dimer comprising two of the fusion proteins, is as described above.

[0169] The route of administration, dosage, and frequency of administration of the above fusion protein or fusion protein dimer can be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and an ordinary person skilled in the art can select the optimal administration method, dosage, and frequency of administration within an appropriate range.

[0170] The fusion protein of one specific example of the present invention can activate immune cells such as natural killer cells due to the activity of IL-2. Therefore, it can be effectively utilized for cancer. In particular, it was confirmed that an IL-2 variant having amino acid substitutions at two to five positions compared to the wild type, particularly an IL-2 variant having amino acid substitutions at two, three, four, or five positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, exhibits characteristics that improve the pharmacological side effects of conventional IL-2 by lowering the binding affinity for the alpha chain of the IL-2 receptor. Therefore, such an IL-2 variant, when used alone or in the form of a fusion protein, can reduce the occurrence of vascular (or capillary) leak syndrome (VLS), which is a problem of conventional IL-2.

[0171] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0172] I. Fusion protein manufacturing

[0173] Manufacturing Example 1. Manufacturing of hCD80-Fc-IL-2 variant (2M): GI101

[0174] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide comprising a base sequence (SEQ ID NO: 8) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 9. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101".

[0175] The fusion protein was purified using chromatography using MabSelect SuRe protein A resin. The fusion protein was bound in 25 mM Tris, 25 mM NaCl, pH 7.4. The solution was eluted with 100 mM NaCl, 100 mM acetic acid (pH 3). The fusion protein was collected by adding 20% ​​1 M Tris-HCl (pH 9) to the collection tube. The collected fusion protein was dialyzed against PBS buffer for 16 h.

[0176] After that, the absorbance at a wavelength of 280 nm was measured over time using size exclusion chromatography using a TSKgel G3000SWXL column (TOSOH Bioscience) to secure a high concentration of fusion protein. At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 6). When detected using NanoDrop, it was confirmed that the fusion protein was included at a concentration of 2.78 mg / ml (Fig. 7). In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 8.

[0177] Manufacturing Example 2. Manufacturing of mCD80-Fc-IL-2 variant (2M): mGI101

[0178] To produce a fusion protein comprising mouse CD80, Fc domain and IL-2 variants, a polynucleotide including a base sequence (SEQ ID NO: 14) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), mCD80 (SEQ ID NO: 13), Ig hinge (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 15. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "mGI101".

[0179] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 9). The fusion protein was confirmed to be present at a concentration of 1.95 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0180] Manufacturing Example 3. Manufacturing of hCD80-Fc: GI101C1

[0181] To produce a fusion protein comprising a human CD80 fragment and an Fc domain, a polynucleotide comprising a base sequence (SEQ ID NO: 16) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4) was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 17. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO2 concentration of 8%, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101C1".

[0182] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 10). The fusion protein was confirmed to be present at a concentration of 3.61 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0183] Manufacturing Example 4. Manufacturing of Fc-IL-2 variant (2M): GI101C2

[0184] To produce a fusion protein comprising an Fc domain and an IL-2 variant, a polynucleotide comprising a signal peptide (SEQ ID NO: 1), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a base sequence (SEQ ID NO: 18) encoding a fusion protein comprising an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 19. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101C2".

[0185] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 11). The fusion protein was confirmed to be present at a concentration of 4.79 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0186] Manufacturing Example 5. Manufacturing of mCD80-Fc: mGI101C1

[0187] To produce a fusion protein comprising mouse CD80 and Fc domains, a polynucleotide comprising a base sequence (SEQ ID NO: 20) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), mouse CD80 (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 21. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "mGI101C1".

[0188] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 12). The fusion protein was confirmed to be present at a concentration of 2.49 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0189] The fusion proteins produced in the above manufacturing examples 1 to 5 are summarized and shown in Table 1 below.

[0190] Classification N-terminal linker C-terminal Manufacturing example 1 (GI101) hCD80 fragment Fc domain hIL-2m Manufacturing example 2 (mGI101) mCD80 fragment Fc domain hIL-2m Manufacturing example 3 (GI101C1) CD80 fragment Fc domain - Manufacturing example 4 (GI101C2) - Fc domain IL-2m Manufacturing example 5 (mGI101C1) mCD80 fragment Fc domain -

[0191] Manufacturing Example 6. Manufacturing of CD80-Fc-IL-2: GI101w

[0192] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and human IL-2, a polynucleotide comprising a base sequence (SEQ ID NO: 31) encoding a fusion protein in this order from the N-terminus, including a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and mature human IL-2 (SEQ ID NO: 10), was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 32. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO2 concentration of 8%, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101w". The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1.

[0193] Manufacturing Example 7. Manufacturing of hCD80-Fc-IL-2 variant (3M): GI102-M45

[0194] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, Y45A) (GI102-M45), a polynucleotide comprising a base sequence (SEQ ID NO: 25) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 22) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 26. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M45".

[0195] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 13).

[0196] Manufacturing Example 8. Manufacturing of hCD80-Fc-IL-2 variant (3M): GI102-M61

[0197] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, E61R) (GI101-M61), a polynucleotide comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a fusion protein comprising an IL-2 variant (SEQ ID NO: 23) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 28. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M61".

[0198] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 14).

[0199] Manufacturing Example 9. Manufacturing of hCD80-Fc-IL-3M: GI102-M72

[0200] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, L72G) (GI102-M72), a polynucleotide comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a fusion protein comprising an IL-2 variant (SEQ ID NO: 24) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 30. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO2 concentration of 8%, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M72".

[0201] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 15).

[0202] Manufacturing Example 10. Manufacturing of mCD80-Fc-IL-3M: mGI102-M61

[0203] To produce a fusion protein comprising a mouse CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, E61R) (GI102-M61) with three amino acid substitutions, a polynucleotide comprising a signal peptide (SEQ ID NO: 1), an mCD80 fragment (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a fusion protein comprising an IL-2 variant (SEQ ID NO: 23) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 34. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "mGI102-M61".

[0204] The above purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1.

[0205] II. Confirmation of binding affinity between fusion protein and ligand

[0206] To confirm the binding affinity of the fusion protein and ligand, the binding affinity was measured using Octet RED 384.

[0207] Experimental Example 1. Confirmation of binding affinity between hCTLA-4 and GI101.

[0208] AR2G biosensor (Amine Reactive 2 ndgen, ForteBio, Cat: 18-5092) were pre-hydrated in 200 ㎕ of distilled water in a Microplate-96-well (GreinerBio-one, Cat: 655209). The ligand to be attached to the AR2G biosensor (CTLA-4, Human CTLA-4 / CD152, His tag, Sino Biological, Cat: 11159-H08H) was diluted to a concentration of 5 ㎍ / ㎖ in 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095). Additionally, GI101 to be bound to the ligand was diluted in 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095) to a concentration of 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM. Activation buffer was prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095) in distilled water. 80 μl of each reagent was added to a 384-well microplate (GreinerBio-one, Cat: 781209), and the program was set.

[0209] As a result, the binding affinity between hCTLA-4 and GI101 was measured as shown in Figure 16.

[0210] Experimental Example 2. Confirmation of binding affinity between hPD-L1 / GI101 and hPD-L1 / PD-1.

[0211] Ni-NTA (Nickel charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101) was prehydrated in 200 ㎕ of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. The ligand to be attached to the Ni-NTA biosensor (Human PD-L1 / B7-H1 protein, His-tag, Sino biological, Cat: 10084-H08H) was diluted in 1X Ni-NTA kinetic buffer to a concentration of 5 ㎍ / ㎖. GI101 to be attached to the ligand was diluted in 1X Ni-NTA kinetic buffer to 1,000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM. Additionally, human PD-1 / PDCD1 (Human PD-1 / PDCD1, Fc Tag, Sino Biological, Cat: 10377-H02H) to be bound to the ligand was diluted in 1X Ni-NTA kinetic buffer to a concentration of 2,000 nM, 1,000 nM, 500 nM, 250 nM, or 125 nM. Then, 80 μl of each reagent was added to a 384-well Microplate, and the program was set.

[0212] As a result, the binding affinity between hPD-L1 and GI101 was measured as shown in Figure 17. In addition, the binding affinity between hPD-L1 and hPD-1 was measured as shown in Figure 18.

[0213] Experimental Example 3. Confirmation of binding affinity between mCTLA-4 and mGI101.

[0214] The binding affinity between mCTLA-4 and mGI101 was confirmed using the same method as in Experimental Example 1. The equipment used here was as follows: Biosensor: AR2G, Ligand: mCTLA-4 (recombinant mouse CTLA-4 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0215] As a result, the binding affinity between mCTLA-4 and mGI101 was measured as shown in Figure 19.

[0216] Experimental Example 4. Confirmation of binding affinity between mPD-L1 and mGI101.

[0217] The binding affinity between mPD-L1 and mGI101 was confirmed using the same method as Experimental Example 1. The equipment used here is as follows. Biosensor: AR2G, Ligand: mPD-L1 (recombinant mouse mGI101 B7-H1 / PD-L1 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0218] As a result, the binding affinity between mPD-L1 and mGI101 was measured as shown in Figure 20.

[0219] Experimental Example 5. Confirmation of the binding affinity of GI-101 (hCD80-Fc-hIL-2v) to CTLA-4.

[0220] Binding kinetics measurements were performed using an Octet RED 384 instrument (ForteBio, Pall Life Science) at 30°C and 1,000 rpm with agitation. The binding to CTLA-4 was measured using an Amine Reactive 2 generation (AR2G) biosensor chip, and the binding to PD-L1 was measured using a nickel-charged Tris-NTA (Ni-NTA) biosensor chip. Human CTLA-4-His Tag (Sino Biological, Cat: 11159-H08H) was activated on the AR2G biosensor chip with a combination of 400 mM EDC and 100 mM sulfo-NHS, diluted to 5 μg / mL with 10 mM acetate buffer (pH 5), loaded onto the AR2G biosensor for 300 s, and immobilized.

[0221] Then, the binding to various concentrations of GI-101 (hCD80-Fc-hIL-2v), GI-101C1 (hCD80-Fc), Ipilimumab (Bristol-Myers Squibb), and GI-101C2 (Fc-hIL-2v) was measured for 300 seconds, and the dissociation was also measured for 300 seconds. The binding kinetics were analyzed using Octet Data analysis HT software ver. 10 provided by Pall. The results are shown in Fig. 21.

[0222] Experimental Example 6. Confirmation of binding affinity between IL-2Rα or IL-2Rβ and GI101.

[0223] Binding to IL-2Rα was measured using an AR2G biosensor, and binding to IL-2Rβ was measured using a Ni-NTA biosensor (Nickel charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101).

[0224] The ligand (IL-2Rα-His Tag, Acro, Cat: ILA-H52H9) to be attached to the AR2G biosensor was diluted to a concentration of 5 μg / mL in 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095). After activating the AR2G biosensor with a buffer prepared by mixing 400 mM EDC and 100 mM sulfo-NHS, the diluted ligand was loaded onto the AR2G biosensor for 300 s and immobilized.

[0225] Meanwhile, the ligand (IL-2Rβ-His Tag, Acro, Cat: CD2-H5221) to be attached to the Ni-NTA biosensor was diluted to a concentration of 5 μg / mL in 1X Ni-NTA kinetic buffer. The diluted ligand was loaded onto the Ni-NTA biosensor for 600 seconds and immobilized.

[0226] Afterwards, various concentrations of GI101, GI101w, or Proleukin (Novartis, hIL-2) were loaded onto the ligand for 300 seconds, and the binding and dissociation were measured for 300 seconds. The binding kinetics were analyzed using Octet Data analysis HT software ver. 10 provided by Pall. The results are shown in Figs. 22 to 24.

[0227] As a result, it was confirmed that GI101 had a lower binding affinity for IL-2Rα of the IL-2 receptor and a higher binding affinity for IL-2Rβ compared to GI101w and Proleukin.

[0228] Experimental Example 7. Measurement of binding affinity between fusion protein and ligand

[0229] To confirm the binding affinity of the fusion protein and ligand, the binding affinity was measured using Octet RED 384.

[0230] Experimental Example 7.1. Confirmation of the binding affinity of GI101-M45, GI101-M61, and GI101-M72 with the IL-2 alpha receptor.

[0231] AR2G biosensor (Amine Reactive 2 nd gen, ForteBio, Cat: 18-5092) were pre-hydrated by adding 200 ㎕ of distilled water (DW) to a 96-well Microplate (GreinerBio-one, Cat: 655209). The ligand (Human IL-2 R alpha protein, His Tag, Acro, ILA-H52H9) to be attached to the biosensor was diluted to a concentration of 5 ㎍ / ㎖ in 10 mM acetate pH 5 buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095). The analytes to be bound to the ligand (GI101-M45, GI101-M61, GI101-M72) were diluted to 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively, in 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095). Activation buffer was prepared in DW with a concentration of 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095). 80 μl of each reagent was added to a 384-well microplate (GreinerBio-one, Cat: 781209) and the program was set.

[0232] As a result, the binding affinity between the IL-2 alpha receptor and GI101-M45 is as shown in Figure 25. In addition, the binding affinity between the IL-2 alpha receptor and GI101-M61 is as shown in Figure 26, and the binding affinity between the IL-2 alpha receptor and GI101-M72 is as shown in Figure 27.

[0233] Experimental Example 7.2. Confirmation of the binding affinity of GI102-M45, GI102-M61, and GI102-M72 for IL-2Rβ.

[0234] Ni-NTA biosensors were prehydrated in 200 ㎕ of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. The ligands (Human IL-2 R beta protein, His-Tag, Acro, CD2-H5221) to be attached to the biosensor were diluted in 1X Ni-NTA kinetic buffer to a concentration of 2 ㎍ / ㎖. GI102-M45, GI102-M61, or GI102-M72 to be attached to the ligands were diluted in 1X Ni-NTA kinetic buffer to a concentration of 500 nM, 250 nM, 125 nM, or 62.5 nM. 80 ㎕ of each reagent was added to a 384-well microplate, and the program was set.

[0235] As a result, the binding affinity between IL-2Rβ and GI102-M45 was measured as shown in Figure 28, and the binding affinity between IL-2Rβ and GI102-M61 was measured as shown in Figure 29. In addition, the binding affinity between IL-2Rβ and GI102-M72 was measured as shown in Figure 30.

[0236] III. Confirmation of immune activity of fusion proteins

[0237] Experimental Example 8. Confirmation of IFN-γ production by fusion protein

[0238] Experimental Example 8.1. Culturing CFSE-labeled PBMCs

[0239] Peripheral blood mononuclear cells (PBMCs) isolated from humans were labeled with carboxyfluorescein succinimidyl ester (CFSE) by reacting with 1 μM CellTrace CFSE dye at 37°C for 20 minutes. CFSE not bound to cells was removed by reacting with culture medium five times the volume of the dye reaction solution for 5 minutes and centrifuging at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U / mL penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM nonessential amino acids, and 2 mM L-glutamine) and then seeded in a 96-well microplate at a density of 1 × 10 per well. 5 The number of cells was added, and 5 ㎍ / ㎖ of PHA (Lectin from Phaseolus Vulgaris, red kidney bean, Sigma-Aldrich, St. Louis, MO, USA, cat No. L1668-5MG) and GI101, GI101C1, GI101C2, or IL-2 (Aldesleukin; human recombinant IL-2, Novartis) were treated and cultured for 6 days in a 37°C, 5% CO2 incubator.

[0240] At this time, GI101, GI101C1, GI101C2, and IL-2 were treated at concentrations of 1 nM, 10 nM, or 100 nM. Cells were analyzed by FACS, and human IFN-γ present in the culture medium was measured using an ELISA kit (Biolegend, San Diego, CA, USA, cat No. 430103).

[0241] Experimental Example 8.2. FACS Analysis

[0242] The cell pellet from which the supernatant was removed was washed with FACS buffer (3% fetal calf serum, 10 mM EDTA, 1 M HEPES, 100 unit / mL penicillin, streptomycin, 1 mM sodium pyruvate) and then incubated with Fc blocker (Biolegend, cat NO. 422302) at 4°C for 5 minutes. Subsequently, the pellet was treated with APC anti-CD3 Ab (Biolegend, cat NO. 300412) and PE anti-CD8a Ab (Biolegend, cat NO. 300908), incubated at 4°C for 20 minutes, and then washed with FACS buffer. The cell pellet was resuspended in FACS buffer and analyzed using BD LSR Fortessa (BD biosciences, San Diego, CA, USA) and FlowJo Software.

[0243] Experimental Example 8.3. Human IFN-γ ELISA

[0244] The amount of human IFN-γ secreted into the supernatant of each cell culture sample was measured using a human IFN-γ ELISA kit (Biolegend, cat. No. 430103). Briefly, anti-human IFN-γ antibodies were added to ELISA plates and coated overnight at 4°C. The plates were then blocked with 1% BSA in PBS for 1 hour at room temperature. After washing with washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample were appropriately diluted and added, followed by incubation at room temperature for 2 hours.

[0245] After the reaction was completed, the plate was washed, the secondary antibody (detection antibody) was added, and the plate was incubated at room temperature for 1 hour. After washing with washing buffer, the Avidin-HRP solution was added, and the reaction was incubated at room temperature for 30 minutes. The substrate solution was added, and the color reaction was induced for 20 minutes in the dark at room temperature. Finally, the color reaction was stopped by adding H2SO4, and the absorbance was measured at 450 nm using an Epoch Microplate Spectrophotometer (BioTek instruments, Winooski, VT, USA), and the concentration was calculated.

[0246] As a result, it was confirmed that the amount of IFN-γ secreted by cells treated with GI101 was significantly increased compared to cells treated with GI101C1, GI101C2, or IL-2 (Fig. 31 and Fig. 32).

[0247] Experimental Example 9. Confirmation of the effect of GI101 on the proliferation of CD8+ T cells.

[0248] Peripheral blood mononuclear cells (PBMCs) isolated from humans were labeled with CFSE by reacting with 1 μM CellTrace CFSE dye at 37°C for 20 minutes. CFSE not bound to cells was removed by reacting with culture medium five times the volume of the dye reaction solution for 5 minutes and centrifuging at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U / mL penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM nonessential amino acids, and 2 mM L-glutamine) and then seeded at 1 × 10 per well in a 96-well microplate. 5 I put in as many cells as I could.

[0249] Afterwards, 1 μg / ml of anti-CD3ε antibody (Biolegend cat. No. L1668-5MG) and GI101, GI101C1, GI101C2, or Proleukin (Novartis) were treated and cultured in a 37°C, 5% CO2 incubator for 6 days. At this time, GI101, GI101C1, GI101C2, and IL-2 were treated to the cells at a concentration of 100 nM. The proliferation of the cultured cells was investigated by measuring the percentage of cells not labeled with CFSE among CD8+ T cells by FACS analysis using APC-TCRαβ antibody and PE-CD8α antibody.

[0250] As a result, it was confirmed that GI101 activates the proliferation of CD8+ T cells to a similar degree to wild-type IL-2, Proleukin, in vitro (Figs. 33 and 34).

[0251] Experimental Example 10. Confirmation of the effects of GI101 and GI102 on the proliferation of CD8+ T cells.

[0252] Human PBMCs were purchased from Allcells (Lot#3014928, USA). A 1 M CellTrace CFSE dye was used, and the cells were incubated with human PBMCs at room temperature for 20 minutes under light-protected conditions. CFSE labeling was achieved by incubating the cells with a 1 μM CellTrace CFSE dye at 37°C for 20 minutes. Unbound CFSE was removed by centrifugation at 1,300 rpm for 5 minutes after incubation with culture medium five times the volume of the staining solution for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM nonessential amino acids, and 2 mM L-glutamine) and seeded at 1x10 per well in a 96-well Microplate.5 I put in as many cells as I could.

[0253] Afterwards, CFSE-labeled PBMCs were treated with 1 μg / ml of anti-CD3ε antibody (OKT3, eBioscience, USA) and GI101, GI101C1, GI101C2, or Proleukin (Novartis) and cultured for 7 days in a 37°C, 5% CO2 incubator. At this time, GI101, GI101C1, GI101C2, and IL-2 were treated to the cells at a concentration of 10 μM.

[0254] The proliferation of cultured cells was investigated by measuring the percentage of cells not labeled with CFSE among CD8+ T cells using FACS analysis using anti-human CD4-PE antibody (BioLegend, USA), anti-human CD8-PE / Cy7 antibody (BioLegend, USA), and anti-human FoxP3-APC antibody (BioLegend, USA).

[0255] As a result, the GI101, GI102_M61, GI101C2, and Proleukin-treated groups significantly increased the proportion of CD8+ T cells compared to the control group (No stimulus), anti-CD3 antibody alone, and GI101C1-treated groups. In addition, compared to the negative control group (No stimulus) 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).

[0256] Experimental Example 11. Confirmation of the effect of GI101 or GI101w on the proliferation of CD8+ T cells and NK cells.

[0257] Seven-week-old C57BL / 6 mice purchased from Orient Bio (Korea) were divided into three groups of three each and injected intraperitoneally with PBS, GI101, or GI101w. GI101 and GI101w were each prepared at 40.5 μg in 200 μl of PBS and injected intraperitoneally. Five days after injection, spleens were removed from each group, and cells were isolated and the total cell number was measured using a hematocytometer. The splenocytes were stained with APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific blue-CD8α antibody (BD; 53-6.7) and analyzed by FACS to determine the proportion of CD8+ T cells and NK cells in the spleen cells. Therefore, the number of CD8+ T cells and NK cells present in the spleen was calculated.

[0258] As a result, it was confirmed that GI101 activated the proliferation of CD8+ T cells and NK cells in vivo more than GI101w (Figs. 36 and 37).

[0259] Experimental Example 12. Confirming the Effect of GI101 on T Cell Function

[0260] The experiment was conducted using the CTLA-4 blockade bioassay kit (Promega cat No. JA4005), and the experiment is briefly described as follows. CTLA-4 Effector cells stored in liquid nitrogen were thawed in a 37℃ water bath for 3 minutes, 0.8 ml of CTLA-4 Effector cells were mixed well with prewarmed 3.2 ml assay buffer (90% RPMI + 10% fetal bovine serum), and 25 μl was added to each well of a 96-well white cell culture plate (SPL, cat No. 30196). Then, 25 μl of GI101 at various concentrations was added. For the negative control, 25 μl of assay buffer was added. Then, the 96-well white cell culture plate was covered and left at room temperature until preparing aAPC / Raji cells.

[0261] aAPC / Raji cells stored in liquid nitrogen were thawed in a 37°C water bath for 3 minutes, 0.8 ml of aAPC / Raji cells were mixed well with preheated 3.2 ml assay buffer, and 25 μl of the cells were added to each well of the plate and incubated for 16 hours in a 37°C, 5% CO2 incubator. After the reaction was complete, the plate was left at room temperature for 15 minutes and then Bio-Glo reagent was added, taking care not to create bubbles. Bio-Glo reagent was also added to three of the outermost wells as blanks to compensate for the background signal. After incubation for 10 minutes at room temperature, luminescence was measured using Cytation 3 (BioTek instruments, Winooski, VT, USA). The final data were calculated as RLU (GI101-background) / RLU (No treatment-background).

[0262] As a result, it was confirmed that GI101 binds to CTLA-4 expressed on effector T cells and activates the function of T cells rather than inhibiting it (Figs. 38 and 39).

[0263] Experimental Example 13. Confirming the Effects of mGI101 and mGI102 on Immune Cells

[0264] Seven-week-old C57BL / 6 mice purchased from Orient (Korea) were divided into three groups of three each and intravenously administered PBS, GI101 at 3 mg / kg, 6 mg / kg, or 12 mg / kg, or mGI102 (mGI102-M61) at 3 mg / kg, 6 mg / kg, or 12 mg / kg. Spleen tissues were removed from each group on days 1, 3, 5, 7, and 14 after injection. The numbers of effector CD8+ T cells, NK cells, and Treg cells were counted by FACS analysis using each antibody, and the ratio of effector CD8+ T cells and NK cells to Treg cells was calculated, respectively. The information on the antibodies used in each cell analysis is as follows:

[0265] 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)

[0266] NK cell: PB anti-mouse CD3ε antibody (Biolegend, # 155612; KT3.1.1), PE anti-mouse NK-1.1 (Biolegend, # 108708; PK136)

[0267] 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).

[0268] As a result, in the group administered mGI101 or mGI102 (mGI102-M61), CD8+ T cells and NK cells significantly increased compared to the PBS administration group from day 3 to day 14 after administration. In addition, it was confirmed that the ratio of activated CD8+ T cells / Treg cells and NK cells / Treg cells in the group administered mGI102 significantly increased compared to the PBS administration group from day 3 to day 7 after administration (Fig. 40).

[0269] IV. Confirmation of the anticancer effect of the fusion protein

[0270] Experimental Example 14. Confirmation of the effect of GI101 on suppressing T cell activation by cancer cells expressing PD-L1 and CTLA-4.

[0271] NCl-H292 cancer cell line expressing PD-L1 and CTLA-4 was cultured in culture medium containing 10 μg / ml Mitomycin C (Sigma) for 3 hours, and then Mitomycin C was removed by washing with culture medium. After that, 5x10 4 NCl-H292 cancer cell line treated with Mitomycin C at a density of 1x10 5Human PBMCs were cultured in 96-well microplates. At this time, 5 μg / ml of PHA (Sigma) was treated to activate T cells. In addition, GI101C1 and GI101 at a concentration of 50 nM were reacted with 50 nM of IgG1-Fc (Biolegend) or abatacept (=Orencia; Bristol-Myers Squibb) at a concentration of 50 nM at 4°C for 30 minutes, and then treated to NCl-H292 cancer cells. After 3 days, the supernatant of the cell culture was collected, and the amount of IFN-γ was quantified using an ELISA kit (Biolegend).

[0272] As a positive control group, human PBMCs stimulated with PHA in the absence of NCl-H292 cancer cell lines treated with Mitomycin C were used, and as a negative control group, human PBMCs stimulated with PHA in the presence of NCl-H292 cancer cell lines treated with Mitomycin C were used. The experimental method using the IFN-γELISA kit was carried out in the same manner as Experimental Example 9.3.

[0273] As a result, GI101 effectively activated the immune response suppressed by PD-L1-overexpressing cancer cell lines. In addition, GI101 was confirmed to inhibit the signaling of CTLA-4 expressed on effector T cells (Figs. 41 and 42).

[0274] Experimental Example 15. Confirmation of the anticancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells.

[0275] BALB / c mice (female, 7 weeks old) received from Orient Bio were placed in a 5x10 group after a 7-day acclimation period. 6 CT-26 cancer cell line (ATCC, USA) was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously (0.1 ml) to the right dorsal region of mice for allografting. After a certain period of time after transplanting the cancer cells, the tumor volume was measured to be approximately 28 mm3 After selecting the individuals that reached the target, they were divided into groups of 10 mice each based on the tumor size and body weight of the selected mice. After that, hIgG4 was administered to the negative control group at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental group was administered mGI101 intravenously at a dose of 3 mg / kg, 6 mg / kg, or 12 mg / kg. After the first administration, administration was performed once every three days for a total of three times. The tumor size was measured every day.

[0276] As a result, it was confirmed that the experimental groups administered 6 mg / kg and 12 mg / kg doses of mGI101 showed significant inhibition compared to the negative control group at some measurement time points and the end of the test (Fig. 43). In addition, as a result of measuring the survival rate, it was confirmed that the experimental group administered 6 mg / kg dose of mGI101 showed significant improvement compared to the negative control group at some measurement time points and the end of the test (Fig. 44).

[0277] Experimental Example 16. Confirmation of the anticancer effect of GI101 in mice implanted with mouse-derived colon cancer cells.

[0278] Experimental Example 16.1. Confirmation of tumor suppression effect

[0279] BALB / c mice (female, 7 weeks old) received from Orient Bio were placed in a 5x10 group after a 7-day acclimation period. 6 CT-26 cancer cell line (ATCC, USA) was suspended in 0.1 ml of PBS and administered subcutaneously to the right dorsal region of mice for allografting. After transplanting the cancer cells, the tumor volume was measured after a certain period of time and was approximately 50 mm 3 200 mm in diameter 3After selecting the individuals that reached the target, they were divided into groups of 10 mice each based on the tumor size and body weight of the selected mice. After that, no drug was administered to the negative control group using a disposable syringe (31G, 1 ml), and the positive control group was intravenously administered 5 mg / kg of anti-PD-1 antibody or 5 mg / kg of anti-PD-1 antibody and 5 mg / kg of anti-CTLA-4 antibody. The experimental group was intravenously administered 0.1 mg / kg or 1 mg / kg of GI101. After the first administration, a total of 3 administrations were performed once every 3 days. The tumor size was measured every day.

[0280] As a result, in CT-26 cancer cell line implanted mice, compared to the negative control group, all groups administered anti-PD-1 antibody, anti-PD-1 antibody and anti-CTLA-4 antibody, and 0.1 mg / kg or 1 mg / kg GI101 significantly inhibited tumor growth. In particular, the experimental group administered 0.1 mg / kg GI101 showed a significant tumor inhibition effect compared to the anti-PD-1 antibody treatment group (* p < 0.05) (Fig. 45).

[0281] Experimental Example 16.2. Analysis of Immune Cells in Cancer Tissue

[0282] The tumor volume of each group of mice in the above experimental example 16.1 was on average 200 mm 3When the tumor was reached, the tumor was sacrificed and the tumor tissue was collected. After that, the tumor tissue was separated into single cells to analyze the immune cells in the tumor tissue, and FACS analysis of the immune cells in the cancer tissue was performed using the following antibodies. Antibodies specifically include Anti-mouse-CD3 (Biolegend, Cat. No. 100320), Anti-mouse-CD4 (Biolegend, Cat. No. 100526), ​​Anti-mouse-CD8 (Biolegend, Cat. No. 100750), Anti-mouse-FoxP3 (eBioscience, Cat. No. 12-5773-82), Anti-mouse-CD25 (Biolegend, Cat. No. 102049), Anti-mouse-CD44 (eBioscience, Cat. No. 61-0441-82), Anti-mouse-PD-1 (Biolegend, Cat. No. 135218), Anti-mouse-IFN-gamma (Biolegend, Cat. No. 505832), Anti-mouse-CD49b (Biolegend, Cat. No. 108906), Anti-mouse-H2 (Invitrogen, Cat. No. A15443), Anti-mouse-CD11c (Biolegend, Cat. No. 117343), Anti-mouse-CD80 (eBioscience, Cat. No. 47-4801-82), Anti-mouse-CD86 (Biolegend, Cat. No. 117343). 104729), Anti-mouse-F4 / 80 (eBioscience, Cat. No. 47-4801-82), and Anti-mouse-CD206 (eBioscience, Cat. No. 17-2061-80) were used.

[0283] As a result, in the experimental group administered 0.1 mg / kg of GI101, CD8+ T cells significantly increased compared to the positive control group administered 5 mg / kg of anti-PD-1 antibody alone (* p < 0.05, Figs. 46 and 47). Furthermore, in all experimental groups administered GI101, the expression of IFN-γ in T cells significantly increased compared to the negative control group (* p < 0.05, Figs. 46 and 47). In addition, in the experimental group administered 0.1 mg / kg of GI101, M1 macrophages increased compared to the negative control group and the positive control group administered anti-PD-1 antibody alone (Figs. 48 and 49). Additionally, CD86 expression in macrophages and dendritic cells increased in all experimental groups administered GI101 (*p < 0.05, Figs. 48 to 51).

[0284] Experimental Example 17. Confirmation of the anticancer effect of GI101 in mice implanted with mouse-derived lung cancer cells.

[0285] Experimental Example 17.1. Confirmation of tumor suppression effect

[0286] C57BL / 6 mice (female, 7 weeks old) obtained from Orient Bio (Korea) were given 5x10 after a 7-day acclimation period. 6 LLC2 cancer cell line (ATCC, USA) was suspended in 0.1 ml of PBS and administered subcutaneously to the right dorsal region of mice for allografting. After transplanting the cancer cells, the tumor volume was measured after a certain period of time and was approximately 50 mm 3 200 mm in diameter 3After selecting the individuals that reached the target, they were divided into groups of 10 mice each based on the tumor size and body weight of the selected mice. After that, no drug was administered to the negative control group using a disposable syringe (31G, 1 ml), and the positive control group was intravenously administered 5 mg / kg of anti-PD-1 antibody or 5 mg / kg of anti-PD-1 antibody and 5 mg / kg of anti-CTLA-4 antibody. The experimental group was intravenously administered 0.1 mg / kg or 1 mg / kg of GI101. After the first administration, administration was performed once every three days for a total of three times. The tumor size was measured every day.

[0287] As a result, a significant tumor suppression effect was observed in all experimental groups compared to the negative control group (*p <0.05) (Fig. 52).

[0288] Experimental Example 17.2. Analysis of Immune Cells in Cancer Tissue

[0289] Mice from each group in Experimental Example 17.1 were sacrificed when the tumor volume reached an average of 200㎣, and cancer tissue was collected. Thereafter, FACS analysis was performed using the same method as Experimental Example 16.2 to analyze immune cells within the cancer tissue.

[0290] As a result, in the experimental group administered 0.1 mg / kg of GI101, CD8+ T cells significantly increased compared to the positive control group administered anti-PD-1 antibody alone (* p < 0.05, Fig. 59). Furthermore, in all experimental groups administered GI101, the expression of IFN-γ significantly increased compared to the negative control group (* p < 0.05, Fig. 59). In addition, in all experimental groups administered GI101, the expression of CD86 in macrophages and dendritic cells increased (* p < 0.05, Figs. 53 to 55).

[0291] Experimental Example 18. Confirmation of the anticancer effect of mGI102-M61 in mice implanted with mouse-derived colon cancer cells.

[0292] BALB / c mice (female, 7 weeks old) received from Orient Bio were placed in a 5x10 group after a 7-day acclimation period. 6 CT-26 cancer cell line (ATCC, USA) was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and injected subcutaneously into the right dorsal area of ​​the mice (0.1 ml each) for allografting. After a certain period of time after cancer cell transplantation, the tumor volume was measured, and mice that reached approximately 28㎣ were selected. Then, the selected mice were divided into groups of 10 mice each based on the tumor size and body weight. After that, hIgG4 was administered to the negative control group at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were administered mGI102-M61 at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg intravenously. After the first administration, administration was performed once every three days for a total of three times. The tumor size was measured daily.

[0293] As a result, it was confirmed that the experimental group administered 12 mg / kg of mGI102-M61 showed significant inhibition compared to the negative control group at some measurement time points and the end of the test (Fig. 56). In addition, as a result of measuring the survival rate, it was confirmed that the experimental group administered 12 mg / kg of mGI102-M61 showed significant improvement compared to the negative control group at some measurement time points and the end of the test (Fig. 57).

[0294] Experimental Example 19. Confirmation of the anticancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells.

[0295] BALB / c mice (female, 7 weeks old) obtained from Orient Bio (Korea) were placed in a 5x10 group after a 7-day acclimation period. 6CT-26 cancer cell line (ATCC, USA) was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously to the right dorsal region of mice at a dose of 0.1 ml for allografting. After a certain period of time after cancer cell transplantation, the tumor volume was measured and found to be approximately 200 mm After selecting the individuals that reached 250㎣, the selected mice were divided into groups of 10 each based on the tumor size and body weight.

[0296] Afterwards, hIgG4 was administered to the negative control group at a dose of 4 mg / kg using a disposable syringe (31G, 1 ml). The experimental group was administered mGI101 intravenously at a dose of 1 mg / kg, 4 mg / kg, or 6 mg / kg. Additionally, the group administered 4.9 mg / kg of mCD80 or 2.8 mg / kg of Fc-IL-2v (GI101C2) was also set as the control group. In addition, the group administered 4.9 mg / kg of mCD80 and 2.8 mg / kg of Fc-IL-2v (GI101C2) simultaneously was also set as the control group.

[0297] In tumor volume measurements, it was confirmed that the mGI101 administration group at a dose of 6 mg / kg showed significant inhibition compared to the negative control group at some measurement time points and the end of the test. Compared to the group administered with the combination of mCD80 and Fc-IL-2v (GI101C2), the tumor growth inhibition rate was superior to that of the combination administration group (Figs. 58 and 59).

[0298] In conclusion, in the tumor growth inhibition efficacy test of CT-26, a BALB / c mouse-derived colon cancer cell line allografted into BALB / c mice, the test substance mGI101 demonstrated tumor inhibition efficacy over mCD80 and IL-2v single agents under the test conditions, and confirmed superior anticancer efficacy compared to the combination administration group of mCD80 and IL-2v (Fig. 58 and Fig. 59). In particular, the tumor size was significantly suppressed in the mGI101 administration group at a dose of 6 mg / kg compared to the negative control group and the combination administration group of mCD80 and Fc-IL2v (GI101C2).

[0299] V. Confirmation of the anticancer effect of combined administration of fusion protein dimers and immune checkpoint inhibitors.

[0300] Experimental Example 20. Confirmation of the anticancer effect of combined administration of GI101 and anti-PD-1 antibody in mice implanted with human breast cancer cells.

[0301] This study evaluated the tumor growth inhibitory effect after intraperitoneal administration of the test substance GI101 and the anti-PD-1 antibody Keytruda (Pmembrolizumab, MSD) as a positive control substance in a tumor model xenografted with human PBMCs into NSGb2m mice, using a humanized mouse model.

[0302] The test substance, negative control substance, and positive control substance stock solutions listed in Table 2 were diluted by adding excipients according to each capacity.

[0303] -Test substance Positive control substance Negative control substance Excipient substance name GI101 Keytruda hIgG4PBS Appearance Transparent liquid Transparent liquid Transparent liquid Transparent liquid Ingredient Fc fusion protein Anti-PD-1 antibody --pH7.5---Storage conditions Refrigerated storage (4℃) Refrigerated storage (4℃) Refrigerated storage (4℃) Refrigerated storage (4℃) Handling precautions Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use-

[0304] Human breast cancer cells, MDA-MB-231 (Homo sapiens, human mammary gland / breast; derived from the pleural effusion), were purchased from the Korea Cell Line Bank (Korea) and used for the test. The cell culture medium was used with the composition shown in the table below, containing fetal bovine serum (FBS, 16000-044, Thermofisher scientific, USA), penicillin-streptomycin; 10,000 units / mL penicillin and 10,000 μg / mL streptomycin (15140122, Thermofisher scientific, USA), and RPMI1640 (A1049101, Thermofisher scientific, USA) per 100 mL.

[0305] Name Composition (㎖)FBS10Penicillin-Streptomycin1RPMI164089Total volume100

[0306] The cells to be used in the test were thawed, placed in cell culture flasks, and cultured in a 37°C, 5% CO2 incubator (MCO-170M, Panasonic, Japan). Trypsin-EDTA (Cat. 25200-072, Thermofisher scientific, USA) was used to suspend the cells. The suspended cells were collected by centrifugation (125 × g, 5 min) using a centrifuge, transferred to new medium and a new flask, and subcultured. On the day of cell line transplantation, the cultured cells were placed in a centrifuge tube, collected, centrifuged (125 × g, 5 min), the supernatant was discarded, and the cell suspension (5 × 10 ) was resuspended in PBS (Cat. LB 001-04, Welgene, KOREA). 6 Cell count / 0.05 ml) was prepared and stored on ice until inoculation. The test was performed on 8-week-old female NSGb2m (NOD.Cg-B2m tm1Unc Prkdc scid Il2rg tm1Wjl / SzJ) mice were purchased from Jung-A Bio (Korea) and used. After the quarantine and acclimatization period, body weights were measured the next day, and human-derived PBMC cell suspension (5x10) prepared for healthy animals was used. 6 (Cell number / 0.2 ml) was filled into a disposable syringe and administered into the animal's caudal vein. General symptoms were observed once daily after cell transplantation.

[0307] Prepared MDA-MB-231 cell suspension (5x10 6 A solution was prepared by adding phenol red-free Matrigel matrix (0.05 ml, 356237, BD, USA) to 0.05 ml of cell number (cell number / 0.05 ml), filled into a disposable syringe, and administered subcutaneously to the right dorsal area of ​​the animals transplanted with human PBMC at 0.1 ml / head. General symptoms were observed once daily during the engraftment and growth period after transplantation of the cell line.

[0308] After transplanting the cells, tumor volume was measured for animals with no abnormalities in health for a certain period of time, and 32 animals were selected so that the average for each group reached 40 to 80㎣. The selected animals were divided into four groups of eight animals each, with tumor volume and body weight being as even as possible.

[0309] The test group was composed as shown in Table 4. The test substance was administered to the animals using a disposable syringe (31G, 1 ml), and the administration frequency was 2 times / week, for a total of 4 administrations.

[0310] Group Dose (mg / kg) Amount of administered liquid (㎖ / kg) Number of animals G1hIgG46108G2GI1016108G3Keytruda5108G7GI101 + Keytruda6 + 5108

[0311] During the observation period, general symptoms such as appearance, behavior, and excrement were observed daily, and dead animals were identified. Body weights were measured on the day of cell line transplantation, twice a week, and on the day of animal sacrifice.

[0312] During the observation period, the maximum length (L) and perpendicular width (W) of the tumor were measured three times a week using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) was calculated by substituting the values ​​into the following formula.

[0313] <Mathematical Formula 1>

[0314] TV (㎣) = (W 2 ХL) / 2

[0315] <Mathematical Formula 2>

[0316] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0317] The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0318] After tumor transplantation, the drugs listed in Table 4 were administered on days 21, 25, 28, and 31, respectively. As a result, tumor growth in the GI101 and Keytruda monotherapy groups was inhibited compared to the control group (hIgG4). Tumor growth in the GI101 and Keytruda combination treatment group was inhibited compared to the control group. Tumor growth in the GI101 and Keytruda combination treatment group was inhibited compared to the GI101 and Keytruda monotherapy group (Figure 60).

[0319] As a result of calculating the tumor growth inhibition rate at the end of the experiment (day 42 after tumor transplantation) compared to the first day of drug treatment (day 21 after tumor transplantation), the hIgG4 treatment group had tumor growth inhibition rates of 30% or more in 2 mice, 50% or more in 1 mouse, and 80% or more in 1 mouse; the GI101 treatment group had tumor growth inhibition rates of 30% or more in 5 mice, 50% or more in 5 mice, and 80% or more in 2 mice; the Keytruda treatment group had tumor growth inhibition rates of 30% or more in 7 mice, 50% or more in 5 mice, and 80% or more in 3 mice; and the GI101 and Keytruda combination treatment group had tumor growth inhibition rates of 30% or more in 8 mice, 50% or more in 8 mice, and 80% or more in 6 mice (Fig. 61).

[0320] In addition, the degree of tumor growth of individual experimental animals in each treatment group when GI101 and Keytruda were administered together in human-derived breast cancer cell-implanted mice is shown in Figures 62 to 66.

[0321] Experimental Example 21. Confirmation of the anticancer effect of combined administration of mGI101 and anti-PD-1 antibody in mice implanted with mouse-derived colon cancer cells.

[0322] This study evaluated the tumor growth inhibitory effect after intraperitoneal administration of the test substance mGI101 and the positive control substance anti-PD-1 antibody, either alone or in combination, in a tumor model in which MC38 (murine colon adenocarcinoma cells) cells were transplanted into C57BL / 6 mice.

[0323] Murine colon adenocarcinoma cells (MC38), derived from rodents, were purchased from Kerafast (USA) and used for the test. MC38 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in phosphate-buffered saline (PBS). To establish an allograft tumor model, 1x10 cells were seeded subcutaneously into the right flank of C57BL / 6 female mice (7 weeks old). 6 MC38 cells from the dog were injected.

[0324] Mice had tumor volumes (30 mm 3 ) were randomly assigned to each group. Tumor grafts were identified approximately 2 days after cell inoculation. The test groups were organized and the test substances were administered as shown in Table 5.

[0325] Experimental group Administration route, administration cycle Administration dose Number of animals G1 Vehicle control (hIgG4) ipBIW x 16 days 10 mg / kg 5 G2 mGI 10 1 i.p. day 1, 5, 9 6 mg / kg 6 G3 Anti-PD-1 antibody (clone RMP1-14, InVivoMab) ipBIW x 16 days 5 mg / kg 5 G4 mGI 101 + anti-PD-1 antibody i.p. day 1, 5, 9 (mGI 101) 0.6 mg / kg 5 i.p. BIW x 16 days (anti-PD-1 antibody) 5 mg / kg

[0326] During the experimental period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. At the end of the experimental period, animals were sacrificed. The size of MC38 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated by comparison with the vehicle control group.

[0327] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Comparisons of tumor volume measurements were performed using one-way ANOVA (end time) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant.

[0328] All test animals remained healthy without any pathological abnormalities after mGI101 administration and combination therapy with anti-PD-1 antibodies. The results of combination therapy using mGI101 and / or anti-PD-1 antibodies on MC38 tumors are shown in Figures 67 to 73. Compared to the control group, an anticancer effect was observed in the drug-treated group, and the difference in tumor size was noticeable during the 16-day test period. MC38 tumors are known as a model for response to anti-PD-1 antibodies in the previous literature, and an anticancer effect was also observed in the anti-PD-1 antibody-treated group in this study (p> 0.01). The anticancer effect was observed in the mGI101 (6 mpk) monotherapy group as well as in the anti-PD-1 antibody-treated group (p> 0.01). The mGI101 (0.6 mpk) + anti-PD-1 (5 mpk) combination treatment group showed a significantly superior anticancer effect (p> 0.0001).

[0329] The individual tumor sizes by test group are shown in Figures 69 to 73. According to the individual tumor size results, mild tumor regression was observed in some animals in the anti-PD-1 antibody administration group. The mGI101 (6 mpk) monotherapy group showed a superior tumor growth inhibition effect compared to the anti-PD-1 antibody administration group. The tumor size remained the same size until days 5-7, but regrew after day 7. The tumor size remained the same size until days 5-7, but regrew after day 7. The combination administration group (GI101 (0.6 mpk) + anti-PD-1 antibody (5 mpk)) showed significantly superior tumor growth inhibition. In particular, two animals in the combination administration group showed complete response (no tumor).

[0330] Two mice in the combination treatment group that achieved complete remission were reinjected with MC38 cells into the left flank (contralateral to the initial tumor cell injection site). These mice were maintained on anti-PD-1 antibody (5 mpk, BIW) for up to 32 days (Fig. 74). A small tumor (>30 ㎣) was observed in one of the two mice, but the tumor size did not grow further until day 35 (Fig. 69). The other mouse showed no tumor after reinjection (Figs. 69 and 74).

[0331] In conclusion, the antitumor efficacy of mGI101 alone and in combination with anti-PD-1 antibody was tested in the MC38 allogeneic tumor model. The combination treatment group (GI101 (0.6 mpk) + anti-PD-1 (5 mpk)) showed the best antitumor efficacy. Complete response (CR) was observed in two animals in the combination treatment group, and the MC38-reinjected CR mice showed tumor-resistant effects (Table 6).

[0332] DaysAftertreatmentCR mouseNo.1Tumor Vol.(mm 3 )No.2Tumor Vol.(mm3 )D178.223.6D284.513.5D336.30D400D500D600D700D800D900D1000D1100D1200D1300D1400D1500D16 00D1700D1800D1900D2000D2100D2200D2300D2400*D2500D2600D2700D2800D2900D3000D3100D3212.80

[0333] Experimental Example 22. Confirmation of the anticancer effect of combined administration of mGI101 and anti-PD-L1 antibody in mice implanted with mouse-derived colon cancer cells.

[0334] This study evaluated the tumor growth inhibitory effect after administering the test substance mGI101 and the positive control substance anti-PD-L1 antibody (BioXcell, Cat# BE0101) alone or in combination in a tumor model in which CT26 (murine colon carcinoma cells) cells were transplanted allogeneically into BALB / c mice.

[0335] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish an allograft tumor model, 5x10 5 CT26 cells from dogs were injected.

[0336] Mice were treated with different tumor volumes (50–120 mm 3 ) were randomly assigned to four groups. Tumor grafts were confirmed approximately two days after cell inoculation. The test groups were organized and the test substances were administered as shown in Table 7.

[0337] Experimental group Administration route, administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ipBIW x 9 days - 4 G2 mGI 101 i.v. QW x 9 days 3 mg / kg 4 G3 Anti-PD-L1 antibody (BioXcell, Cat# BE0101) ipBIW x 9 days 10 mg / kg 4 G4 mGI 101 + anti-PD-L1 antibody i.v. QW x 9 days (mGI 101) 3 mg / kg 4 i.p. BIW x 9 days (anti-PD-L1 antibody) 10 mg / kg

[0338] During the experimental period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. At the end of the experimental period, animals were sacrificed. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated by comparison with the vehicle control group.

[0339] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Comparisons of tumor volume measurements were performed using one-way ANOVA (end time) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant.

[0340] In the CT26 allogeneic tumor model, the antitumor efficacy of mGI101 alone and in combination with anti-PD-L1 antibody was tested, and the combination group (mGI101 (3 mpk) + anti-PD-L1 (10 mpk)) showed the best antitumor efficacy (Fig. 75).

[0341] Experimental Example 23. Confirmation of the anticancer effect of combined administration of mGI101 and anti-TIGIT antibodies in mice implanted with mouse-derived colon cancer cells.

[0342] This study evaluated the tumor growth inhibitory effect after administering the test substance mGI101 and the positive control substance, an anti-TIGIT antibody that specifically binds to the extracellular domain (ECD) of TIGIT having the amino acid sequence of SEQ ID NO: 39, alone or in combination in a tumor model in which CT26 (murine colon carcinoma cells) cells were transplanted into BALB / c mice.

[0343] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish an allograft tumor model, 5x10 5 CT26 cells from dogs were injected.

[0344] Mice were treated with different tumor volumes (50–120 mm 3 ) were randomly assigned to each group. Tumor grafts were identified approximately 2 days after cell inoculation. The test groups were organized and the test substances were administered as shown in Table 8.

[0345] Experimental group Administration route, administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ipBIW x 9 days - 5 G2 mGI 101 i.v. QW x 9 days 3 mg / kg 5 G3 Anti-TIGIT antibody (Merck, MK-7684) ipBIW x 9 days 20 mg / kg 5 G4 mGI 101 + anti-TIGIT antibody i.v. QW x 9 days (mGI 101) 3 mg / kg 5 i.p. BIW x 9 days (anti-TIGIT antibody) 20 mg / kg

[0346] During the experimental period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. At the end of the experimental period, animals were sacrificed. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated by comparison with the vehicle control group.

[0347] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Comparisons of tumor volume measurements were performed using one-way ANOVA (end time) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant.

[0348] In the CT26 allogeneic tumor model, the antitumor efficacy of mGI101 alone and in combination with anti-TIGIT antibodies was tested. The combination group (mGI101 (3 mpk) + anti-TIGIT (20 mpk)) showed the greatest antitumor efficacy (Fig. 76). While the anti-TIGIT antibody monotherapy group showed no antitumor effect compared to the control group, combination therapy with mGI101 showed significantly superior antitumor efficacy compared to the mGI101 monotherapy group.

Claims

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

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

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

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

5. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the fusion protein has an amino acid sequence of sequence number 9.

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

7. In paragraph 6, The above anti-CTLA-4 antibody is any one selected from the group consisting of ipilimumab and tremelimumab, The above anti-PD-1 antibody is any one selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, scintillimab, tislelizumab, toripalimab, dostalimab, INCMGA00012, AMP-224, and AMP-514, The above 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 above anti-TIM3 antibody is any one selected from the group consisting of LY3321367, MBG453, and TSR-022, The above anti-LAG3 antibody is any one selected from the group consisting of IMP321, relatimab and GSK2831781, or A pharmaceutical composition for preventing or treating cancer, wherein the anti-VISTA antibody is composed of JNJ-63723283.

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

9. A method for preventing or treating cancer, comprising administering to a subject 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 IL-2 protein or a variant thereof and CD80 protein or a fragment thereof and an immune checkpoint inhibitor for the prevention or treatment of cancer.