Pharmaceutical compositions comprising a fusion protein comprising an IL-2 protein and a CD80 protein and an immune checkpoint inhibitor for use in the treatment of cancer

Abstract

The present application provides a pharmaceutical composition for preventing or treating cancer, including, as an active ingredient, a fusion protein dimer including an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor. In one embodiment, the fusion protein including a CD80 fragment, an Fc domain of an immunoglobulin, and an IL-2 variant can activate immune cells such as natural killer cells, and control immune cell regulatory activity of regulatory T cells. Furthermore, the combined administration of the fusion protein and an immune checkpoint inhibitor such as Keytruda known as a PD-1 inhibitor can be effective in inhibiting cancer. Accordingly, the pharmaceutical composition provided by the present application including, as an active ingredient, a fusion protein dimer including an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor can be effectively used for the treatment of cancer, and has a high degree of industrial applicability.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising, as an active ingredient, a fusion protein dimer containing IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor. Background Technology

[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. The IL-2 protein is 15.5 kDa to 16 kDa in size and consists of 133 amino acids. IL-2 mediates various immune responses by binding to the IL-2 receptor, which is composed of three different subunits.

[0003] In addition, IL-2 is mainly synthesized by activated T cells, especially CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells and induces the production of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAK cells).

[0004] Meanwhile, CD80, also known as B7-1, is a member of the B7 family of membrane-bound proteins, which participate in immune regulation by binding to their ligands and transmitting co-stimulatory and co-inhibitory 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, CTLA4 (CD152), and PD-L1. CD80, CD86, CTLA4, and CD28 are involved in the co-stimulatory-co-inhibitory system. For example, they regulate T cell activity and participate in their proliferation, differentiation, and survival.

[0005] In addition, recently, immune checkpoint inhibitors such as Significant attention has been paid to immune checkpoint inhibitors, which are anticancer agents that help attack cancer cells by activating the body's immune system. To date, cancer treatments have focused on killing rapidly dividing cells, a characteristic of cancer cells, thus resulting in side effects that affect not only cancer cells but also rapidly dividing cells in normal cells. However, immunotherapy is known to use the cancer patient's immune system to influence cancer cells, thus minimizing the typical side effects of existing anticancer agents. Anti-PD-1 antibodies such as Keytruda bind to the specific receptor (PD-1) on T cells and block pathways used by cancer cells to evade the active T cell surveillance system, thereby exhibiting anticancer effects through immune reactivation, allowing T cells in the body to attack cancer cells (Korean Patent Publication No. 2018-0030580A). Summary of the Invention

[0006] Technical issues

[0007] Therefore, as a result of research on developing safe and effective anticancer agents, the inventors discovered that a novel fusion protein dimer containing IL-2 protein or a variant thereof and CD80 protein or a fragment thereof in one molecule, as well as an immune checkpoint inhibitor, exhibits excellent anticancer activity, thus completing the present invention.

[0008] Solution to the problem

[0009] To achieve the above objectives, one aspect of the present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising, as active ingredients, a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0010] Invention Effects

[0011] A fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof can activate immune cells via IL-2. Furthermore, a synergistic effect of the fusion protein dimer has been demonstrated when administered in combination with an immune checkpoint inhibitor. Therefore, the pharmaceutical composition for treating cancer provided by this invention, comprising as active ingredients a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor, can be effectively used for cancer prevention and treatment. Attached Figure Description

[0012] Figure 1 A schematic embodiment of the fusion protein dimer is shown.

[0013] Figure 2 A schematic diagram illustrating the mechanism by which fusion protein dimers function in lymph nodes is shown.

[0014] Figure 3 A schematic diagram illustrating the mechanism by which fusion protein dimers function in the tumor microenvironment is shown.

[0015] Figure 4 A schematic diagram of the structure of the fusion protein is shown. Here, GI101 and mGI101 are implementation schemes of the fusion protein; GI101C1, GI101C2, and mGI101C1 are comparative examples comparing the activity of the fusion protein.

[0016] Figure 5 Various implementation schemes of the fusion protein are shown. Human and mouse-derived proteins can be combined to prepare the fusion protein. CD80 and IL-2 proteins can bind to each other through various linkers other than Fc.

[0017] Figure 6 The SDS-PAGE identification results of the obtained fusion protein dimer (GI101) are shown.

[0018] Figure 7 The amount of fusion protein (GI101) was shown to depend on absorbance.

[0019] Figure 8 The results of the fusion protein dimer (GI101) obtained by size exclusion chromatography (SEC) are shown.

[0020] Figure 9 The SDS-PAGE identification results of the obtained mGI101 fusion protein dimer are shown.

[0021] Figure 10 The SDS-PAGE identification results of the obtained GI101C1 fusion protein dimer are shown.

[0022] Figure 11 The SDS-PAGE identification results of the obtained GI101C2 fusion protein dimer are shown.

[0023] Figure 12 The SDS-PAGE identification results of the obtained mGI101C1 fusion protein dimer are shown.

[0024] Figure 13 The SDS-PAGE identification results of the obtained GI102-M45 fusion protein dimer are shown.

[0025] Figure 14 The SDS-PAGE identification results of the obtained GI102-M61 fusion protein dimer are shown.

[0026] Figure 15 The SDS-PAGE identification results of the obtained GI102-M72 fusion protein dimer are shown.

[0027] Figure 16 The binding affinity between hCTLA4 and GI101 is shown.

[0028] Figure 17 The binding affinity between hPD-L1 and GI101 was shown.

[0029] Figure 18 The binding affinity between hPD-L1 and hPD-1 is shown.

[0030] Figure 19 The binding affinity between mCTLA4 and mGI101 is shown.

[0031] Figure 20 The binding affinity between mPD-L1 and mGI101 is shown.

[0032] Figure 21 The results of the identification of the binding affinity between GI-101 (hCD80-Fc-hIL-2v) and CTLA-4 are shown. GI-101 (hCD80-Fc-hIL-2v) was identified as having a high binding affinity to CTLA-4.

[0033] Figure 22 The results of the identification of binding affinity between GI101 and IL-2Rα or IL-2Rβ are shown.

[0034] Figure 23 The results of the identification of the binding affinity between GI101 and IL-2Rα are shown.

[0035] Figure 24 The results of the identification of the binding affinity between GI101 and IL-2Rβ are shown.

[0036] Figure 25 The results of the identification of the binding affinity between IL-2Rα and GI102-M45 are shown.

[0037] Figure 26 The results of the identification of the binding affinity between IL-2Rα and GI102-M61 are shown.

[0038] Figure 27 The results of the identification of the binding affinity between IL-2Rα and GI102-M72 are shown.

[0039] Figure 28 The results of the identification of the binding affinity between IL-2Rβ and GI102-M45 are shown.

[0040] Figure 29 The results of the identification of the binding affinity between IL-2Rβ and GI102-M61 are shown.

[0041] Figure 30 The results of the identification of the binding affinity between IL-2Rβ and GI102-M72 are shown.

[0042] Figure 31 and Figure 32 The results show the amount of IFN-γ secreted by cells when cells were treated and incubated with GI101, GI101C1, GI101C2 or IL-2 at their respective concentrations.

[0043] Figure 33The results show the identification of the effects of GI101, GI101C1, GI101C2 and IL-2 (aldeleukin) on CD8+ T cell proliferation.

[0044] Figure 34 A schematic diagram illustrating the mechanism by which GI101 acts on effector T cells is shown.

[0045] Figure 35 The results of identifying the effects of GI101 and GI102 on the proliferation of CD8+ T cells and CD4+ T cells are shown. Here, (A) shows the ratio of CD8+ T cells to CD4+ T cells, (B) shows the proliferative capacity of CD8+ T cells, and (C) shows the ratio of CD4+ / FoxP3+ Treg cells.

[0046] Figure 36 and Figure 37 The results show the identification of the effects of GI101 and GI101w on the proliferation of CD8+ T cells and NK cells.

[0047] Figure 38 and Figure 39 The results of the identification of the effect of GI101 on effector T cells are shown.

[0048] Figure 40 The results show the identification of the effects of mGI101 and mGI102-M61 on mouse immune cells.

[0049] Figure 41 and Figure 42 The results show the identification of the inhibitory effect of GI101 on T cell activity in cancer cells expressing PD-L1 and CTLA-4.

[0050] Figure 43 The results show the identification of the tumor-suppressive effect of mGI101 (depending on its dose) in mice transplanted with mouse-derived colorectal cancer cells.

[0051] Figure 44 The results of survival assessment for mice that received mGI101-derived colorectal cancer cell transplants are shown.

[0052] Figure 45 The results show the identification of the tumor-suppressive effect of GI101 in mice with mouse-derived colorectal cancer cells.

[0053] Figure 46 The results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in mice with mouse-derived colorectal cancer cells after treatment with hIgG4, anti-PD-1 antibody, or GI101 are shown.

[0054] Figure 47 The results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in mice with mouse-derived colorectal cancer cells after treatment with hIgG4, anti-PD-1 antibody, or GI101 are presented in graphical form.

[0055] Figure 48 The results of FACS analysis of macrophages in cancer tissues from mice with mouse-derived colorectal cancer cells after treatment with hIgG4, anti-PD-1 antibody, or GI101 are shown.

[0056] Figure 49 The results of FACS analysis of macrophages in mouse tissues after mouse-derived colorectal cancer cell transplantation were presented in graphical form.

[0057] Figure 50 The results of FACS analysis of dendritic cells in mouse tissues after mouse-derived colorectal cancer cells were transplanted and treated with hIgG4, anti-PD-1 antibody, or GI101 are shown.

[0058] Figure 51 The results of FACS analysis of dendritic cells in mouse tissues after mouse-derived colorectal cancer cell transplantation were presented in graphical form.

[0059] Figure 52 The results show the identification of the tumor-suppressive effect of GI101 in mice transplanted with mouse-derived lung cancer cells.

[0060] Figure 53 The results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in mice transplanted with mouse-derived lung cancer cells after treatment with hIgG4, anti-PD-1 antibody, or GI101 are presented in graphical form.

[0061] Figure 54 The results of FACS analysis of macrophages in cancer tissues after mice with mouse-derived lung cancer cell transplantation were presented in graphical form.

[0062] Figure 55 The results of FACS analysis of dendritic cells in cancer tissues after mice with mouse-derived lung cancer cell transplantation were presented in graphical form.

[0063] Figure 56The results show the identification of the tumor-suppressive effect of mGI102-M61 in mice with mouse-derived colorectal cancer cells.

[0064] Figure 57 The results of the survival analysis of mice that received mGI102-M61 mouse-derived colorectal cancer cell transplants are shown.

[0065] Figure 58 The results show the identification of the tumor-suppressive effect of mGI101 in mice with mouse-derived colorectal cancer cells.

[0066] Figure 59 The tumor inhibition rate of mGI101 on mice transplanted with mouse-derived colorectal cancer cells is shown.

[0067] Figure 60 The graphs show tumor growth when GI101 and Keytruda were used in combination in mice with human breast cancer cell transplantation. Tumor growth was inhibited in the GI101 or Keytruda treatment groups compared to the control group (hIgG4). Tumor growth was inhibited in the GI101 and Keytruda combination treatment group compared to the control group. Tumor growth was inhibited in the GI101 and Keytruda combination treatment group compared to the GI101 or Keytruda treatment groups.

[0068] Figure 61 The tumor growth inhibition rates when GI-101 and Keytruda were used in combination in mice with human breast cancer cell transplantation are shown. In the IgG4 treatment group, 2 mice showed a tumor growth inhibition rate of 30% or higher, 1 mouse showed a tumor growth inhibition rate of 50% or higher, and 1 mouse showed a tumor growth inhibition rate of 80%. In the GI101 treatment group, 5 mice showed a tumor growth inhibition rate of 30% or higher, 5 mice showed a tumor growth inhibition rate of 50% or higher, and 2 mice showed a tumor growth inhibition rate of 80%. In the Keytruda treatment group, 7 mice showed a tumor growth inhibition rate of 30% or higher, 5 mice showed a tumor growth inhibition rate of 50% or higher, and 3 mice showed a tumor growth inhibition rate of 80%. In the GI101 and Keytruda combination treatment group, 8 mice showed a tumor growth inhibition rate of 30% or higher, 8 mice showed a tumor growth inhibition rate of 50% or higher, and 6 mice showed a tumor growth inhibition rate of 80%.

[0069] Figure 62 The extent of tumor growth in individual experimental animals in each treatment group is shown when GI101 and Keytruda are used in combination in mice with human breast cancer cell transplantation.

[0070] Figure 63The extent of tumor growth in individual experimental animals in the hIgG4 treatment group of human breast cancer cell transplanted mice is shown.

[0071] Figure 64 The extent of tumor growth in individual experimental animals in the GI101 treatment group in mice with human breast cancer cell transplantation is shown.

[0072] Figure 65 The extent of tumor growth in individual experimental animals in the Keytruda treatment group of human breast cancer cell transplanted mice is shown.

[0073] Figure 66 The extent of tumor growth in individual experimental animals in the GI101 and Keytruda combination therapy group in human breast cancer cell transplanted mice is shown.

[0074] Figure 67 The graph shows the tumor growth curves when mGI101 and anti-PD-1 antibody were administered in combination to mice with murine colorectal cancer cells.

[0075] Figure 68 The tumor growth inhibition rate was shown when mGI101 and anti-PD-1 antibody were administered in combination to mice with murine colorectal cancer cells.

[0076] Figure 69 The study showed the extent of tumor growth in individual experimental animals in each treatment group when mGI101 and anti-PD-1 antibody were administered in combination to mice with murine colorectal cancer cells.

[0077] Figure 70 The extent of tumor growth in individual experimental animals in the hIgG4 treatment group of mice with mouse-derived colorectal cancer cells is shown.

[0078] Figure 71 The extent of tumor growth in individual experimental animals in the mGI101 treatment group in mice with mouse-derived colorectal cancer cells is shown.

[0079] Figure 72 The extent of tumor growth in individual experimental animals in the anti-PD-1 antibody treatment group in mice with mouse-derived colorectal cancer cells is shown.

[0080] Figure 73 The extent of tumor growth in individual experimental animals treated with a combination of mGI101 and anti-PD-1 antibody in mouse mice with transplanted murine colorectal cancer cells is shown.

[0081] Figure 74The study showed the extent of tumor growth in individual experimental animals after re-injection of murine colorectal cancer cells. In mice with murine colorectal cancer cell transplantation, the group treated with a combination of mGI101 and anti-PD-1 antibody achieved complete remission of tumor growth.

[0082] Figure 75 The graph shows the tumor growth curves when mGI101 and anti-PD-L1 antibody were administered in combination to mice with mouse-derived colorectal cancer cells.

[0083] Figure 76 The graph shows the tumor growth curves when mGI101 and anti-TIGIT antibody were administered in combination to mice with mouse-derived colorectal cancer cells.

[0084] Best mode for carrying out the invention

[0085] One aspect of the present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising, as an active ingredient, a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0086] Immune checkpoint inhibitors

[0087] As used herein, the term "immune checkpoint" refers to the intracellular signaling system that maintains self-tolerance and protects tissues from excessive immune responses that lead to damage. Immune checkpoint proteins are cell membrane proteins that regulate immune checkpoints and can inhibit the differentiation, proliferation, and activity of immune cells. Specifically, immune checkpoint proteins are expressed in activated T cells to reduce T cell proliferation, cytokine secretion, and cytotoxicity, and to inhibit excessive T cell activity. Some immune checkpoints are known to be among the main mechanisms leading to immune evasion by tumor cells. Therefore, "immune checkpoint inhibitors" target immune checkpoint proteins to inhibit or block immune checkpoints and increase T cell activation, thereby enhancing anti-tumor immunity and thus exhibiting anti-cancer effects. In addition to having fewer side effects such as vomiting and hair loss than conventional cytotoxic anticancer agents and better therapeutic effects, immune checkpoint inhibitors are known to have durable therapeutic effects even after drug administration is discontinued because they utilize the immune response system, which has excellent memory capabilities.

[0088] Specifically, immune checkpoint inhibitors can target CTLA-4, PD-1, PD-L1, PD-L2, B7-H4, HVEM (herpesvirus invasion mediator), BTLA, TIM3, GAL9, LAG3, VISTA, KIR, or TIGIT.

[0089] In particular, immune checkpoint inhibitors may include, but are not limited to, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-TIM3 antibodies, anti-GAL9 antibodies, anti-LAG3 antibodies, anti-VISTA antibodies, anti-KIR antibodies, and anti-TIGIT antibodies.

[0090] As used herein, the term "cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)" is referred to as CD152 and is expressed on the membrane surface of activated T cells. CTLA-4 binds to antigen-presenting cells CD80 (B7-1) and CD86 (B7-2) and inhibits T cell activity. CTLA-4 inhibitors may include ipilimumab. And trimelimumab.

[0091] As used herein, the term "programmed cell death protein 1 (PD-1)" is referred to as CD279 and is expressed on the surface of activated T cells. PD-1 reacts with the proteins PD-L1 (B7-H1) and PD-L2 (B7-DC) on the surface of cancer cells and inhibits TCR (T cell receptor) and CD28-mediated T cell activity, growth factor and cytokine production to induce negative signaling. PD-1 inhibitors can be, for example, pembrolizumab. MK-3475, Nivolumab Cimipril JTX-4014, Spartazumab, Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, Dotalimab, INCMGA00012, AMP-224, and AMP-514.

[0092] As used herein, the term "programmed death-ligand 1 (PD-L1)" is referred to as CD274 or B7-H1 and is a protein present on the surface of cancer cells or hematopoietic cells. PD-L1 present on the cancer surface can bind to PD-1 present on the surface of T cells. PD-L1 inhibitors can be, for example, atezolizumab or avelumumab. Dvalumab KN035, CK-301, AUNP12, CA-170, and BMS-986189.

[0093] As used herein, the term "B7-H4" refers to VTCN1, a T-cell activation inhibitor containing the V-set domain, expressed on the membrane surface of antigen-presenting cells. B7-H4 binds to the CD28 protein of T cells and inhibits T-cell activity, growth, and cytokine production, thereby negatively regulating the T-cell-mediated immune response.

[0094] As used herein, the term "herpesvirus invasion mediator (HVEM)" is referred to as CD270 and also as tumor necrosis factor receptor superfamily member 14 (TNFRSF14). HVEM is expressed on the membrane surfaces of various immune cells, including T cells, and binds to various chaperone proteins to modulate inflammatory and immune responses. When HVEM binds to B lymphocyte attenuators and T lymphocyte attenuators (BTLA, CD272) or CD160, T cell immune activity is suppressed. On the other hand, when HVEM binds to TNFSF14 (LIGHT), HVEM induces dendritic cell maturation, T cell proliferation, and cytokine production to activate inflammatory and immune responses.

[0095] As used herein, the term "T cell membrane protein 3 (TIM3)" is also known as hepatitis A virus cell receptor 2 (HAVCR2) and is expressed in various immune cells. When TIM3 is activated by binding to the water-soluble protein GAL9 (galactagogue 9), intracellular calcium influx increases to induce T cell apoptosis, which in turn leads to immune tolerance. Additionally, along with GAL9, TIM3 binds to cell adhesion molecule 1 (CEACAM1) (a cell surface protein) to suppress T cell immune activity and binds to high-mobility group box 1 (HMGB1) or phosphatidylserine (PTdSer) (a water-soluble protein) to suppress immune activity. TIM3 inhibitors include LY3321367, MBG453, and TSR-022.

[0096] As used herein, the term "lymphocyte activation gene 3 (LAG3)" is referred to as CD223, and it binds to major histocompatibility complex (MHC) class II to inhibit T cell proliferation and activity. LAG3 inhibitors may include IMP321, piracetamab, and GSK2831781.

[0097] As used herein, the term "V-domain Ig inhibitor of T cell activation (VISTA)" belongs to the B7 family (B7-H5) and is expressed in various immune cells to inhibit T cell proliferation, activity, and cytokine production. A VISTA inhibitor may be JNJ-63723283.

[0098] As used herein, the term "killer cell immunoglobulin-like receptor (KIR)" refers to membrane-bound proteins 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 to inhibit the cellular immune activity of NK cells.

[0099] As used herein, the term “T cell immunoglobulin and ITIM domain (TIGIT)” refers to membrane-bound proteins expressed on the surface of NK cells and T cells that can bind to CD155, CD112, and CD113 to suppress immune activity.

[0100] Fusion proteins and their dimers containing IL-2 and CD80 proteins

[0101] As used herein, unless otherwise stated, the term "IL-2" or "interleukin-2" refers to any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (such as humans) and rodents (such as mice and rats). IL-2 can be obtained from animal cells, including IL-2 obtained from recombinant cells capable of producing IL-2. Additionally, IL-2 can be wild-type IL-2 or a variant thereof.

[0102] In this specification, IL-2 or its variants may be expressed collectively by the terms "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 identified by methods known to those skilled in the art.

[0103] One embodiment of IL-2 may have an amino acid sequence as shown in SEQ ID NO:35 or SEQ ID NO:36. Here, IL-2 may also be a mature form. Specifically, mature IL-2 may not contain a signal sequence and may have an amino acid sequence as shown in SEQ ID NO:10. Here, IL-2 may be used under the concept of containing a fragment of wild-type IL-2, wherein a portion of the N-terminus or C-terminus of wild-type IL-2 is truncated.

[0104] Additionally, the IL-2 fragment can be in the form of a truncated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids from the N-terminus of a protein having an amino acid sequence as shown in SEQ ID NO:35 or SEQ ID NO:36. Additionally, the IL-2 fragment can be in the form of a truncated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive amino acids from the C-terminus of a protein having an amino acid sequence as shown in SEQ ID NO:35 or SEQ ID NO:36.

[0105] As used herein, the term "IL-2 variant" refers to a form in which a portion of the amino acids in full-length IL-2 or a fragment of IL-2 described above are substituted. That is, an IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, an IL-2 variant may have activity equivalent to or similar to wild-type IL-2. Here, "IL-2 activity" may, for example, refer to specific binding to the IL-2 receptor, which can be measured by methods known to those skilled in the art.

[0106] Specifically, IL-2 variants can be obtained by substituting a portion of the amino acids in wild-type IL-2. One embodiment of the IL-2 variant obtained by amino acid substitution can be obtained by substituting at least one of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence shown in SEQ ID NO:10.

[0107] Specifically, IL-2 variants can be obtained by replacing at least one of the amino acids at positions 38, 42, 45, 61, or 72 in the amino acid sequence shown in SEQ ID NO:10 with another amino acid. Additionally, when IL-2 is in a form where a portion of the N-terminus of the amino acid sequence shown in SEQ ID NO:35 is truncated, the amino acid at the position complementary to the amino acid in the amino acid sequence shown in SEQ ID NO:10 can be replaced by another amino acid. For example, when IL-2 has the amino acid sequence shown in SEQ ID NO:35, its IL-2 variants can be obtained by replacing at least one of the amino acids at positions 58, 62, 65, 81, or 92 in the amino acid sequence shown in SEQ ID NO:35 with another amino acid. These amino acid residues correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues in the amino acid sequence shown in SEQ ID NO:10, respectively. According to one embodiment, as long as this IL-2 variant retains IL-2 activity, it can replace 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. According to another embodiment, it can replace 1 to 5 amino acids.

[0108] In one embodiment, the IL-2 variant can be a form in which two amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting amino acids at positions 38 and 42 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38 and 45 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38 and 61 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38 and 72 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 42 and 45 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 42 and 61 of the amino acid sequence shown in SEQ ID NO:10. In one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 42 and 72 of the amino acid sequence shown in SEQ ID NO:10. In another embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 45 and 61 of the amino acid sequence shown in SEQ ID NO:10. In yet another embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 45 and 72 of the amino acid sequence shown in SEQ ID NO:10. In yet another embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 61 and 72 of the amino acid sequence shown in SEQ ID NO:10.

[0109] Furthermore, the IL-2 variant can be a form in which three amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting amino acids at positions 38, 42, and 45 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 42, and 61 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 42, and 72 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 45, and 61 of the amino acid sequence shown in SEQ ID NO:10. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 45, and 72 of the amino acid sequence shown in SEQ ID NO:10. Additionally, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 61, and 72 of the amino acid sequence shown in SEQ ID NO:10. Additionally, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 42, 45, and 61 of the amino acid sequence shown in SEQ ID NO:10. Additionally, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 42, 45, and 72 of the amino acid sequence shown in SEQ ID NO:10. Additionally, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 45, 61, and 72 of the amino acid sequence shown in SEQ ID NO:10.

[0110] Additionally, the IL-2 variant can be a form in which four amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting amino acids at positions 38, 42, 45, and 61 of the amino acid sequence shown in SEQ ID NO:10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 42, 45, and 72 of the amino acid sequence shown in SEQ ID NO:10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 45, 61, and 72 of the amino acid sequence shown in SEQ ID NO:10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 38, 42, 61, and 72 of the amino acid sequence shown in SEQ ID NO:10. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting amino acids at positions 42, 45, 61, and 72 of the amino acid sequence shown in SEQ ID NO:10.

[0111] Furthermore, IL-2 variants can be forms in which five amino acids are substituted. Specifically, IL-2 variants can be obtained by substituting each of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence shown in SEQ ID NO:10 with another amino acid.

[0112] Here, the "other amino acid" introduced by substitution can 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, regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 38 cannot be substituted with arginine, amino acid position 42 cannot be substituted with phenylalanine, amino acid position 45 cannot be substituted with tyrosine, amino acid position 61 cannot be substituted with glutamic acid, and amino acid position 72 cannot be substituted with leucine.

[0113] Regarding the amino acid substitutions for the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 38 (arginine) may be substituted with an amino acid other than arginine. Preferably, regarding the amino acid substitutions for the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 38 (arginine) may be substituted with alanine (R38A).

[0114] Regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 42 (phenylalanine) may be substituted with an amino acid other than phenylalanine. Preferably, regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 42 (phenylalanine) may be substituted with alanine (F42A).

[0115] Regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 45 (tyrosine) may be substituted with an amino acid other than tyrosine. Preferably, regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 45 (tyrosine) may be substituted with alanine (Y45A).

[0116] Regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 61 (glutamic acid) may be substituted with an amino acid other than glutamic acid. Preferably, regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 61 (glutamic acid) may be substituted with arginine (E61R).

[0117] Regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 72 (leucine) may be substituted with an amino acid other than leucine. Preferably, regarding the amino acid substitutions of the IL-2 variant, in the amino acid sequence shown in SEQ ID NO:10, amino acid position 72 (leucine) may be substituted with glycine (L72G).

[0118] Specifically, the IL-2 variant can be obtained by substitution of at least one of the amino acid sequences selected from the group consisting of R38A, F42A, Y45A, E61R and L72G, as shown in SEQ ID NO:10.

[0119] Specifically, IL-2 variants can be obtained by substituting two, three, four, or five amino acids at positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

[0120] Additionally, the IL-2 variant can be in a form where two amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting R38A and F42A. In one embodiment, the IL-2 variant can be obtained by substituting R38A and Y45A. In another embodiment, the IL-2 variant can be obtained by substituting R38A and E61R. In another embodiment, the IL-2 variant can be obtained by substituting R38A and L72G. In another embodiment, the IL-2 variant can be obtained by substituting F42A and Y45A. In another embodiment, the IL-2 variant can be obtained by substituting F42A and E61R. In another embodiment, the IL-2 variant can be obtained by substituting F42A and L72G. In another embodiment, the IL-2 variant can be obtained by substituting E61R and L72G.

[0121] Furthermore, the IL-2 variant can be a form in which three amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting R38A, F42A, and Y45A. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting R38A, F42A, and E61R. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting R38A, F42A, and L72G. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting R38A, Y45A, and E61R. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting R38A, Y45A, and L72G. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting F42A, Y45A, and E61R. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting F42A, Y45A, and L72G. Alternatively, in one embodiment, the IL-2 variant can be obtained by substituting F42A, E61R, and L72G. Alternatively, in one implementation, the IL-2 variant can be obtained by replacing Y45A, E61R, and L72G.

[0122] Additionally, the IL-2 variant can be a form in which four amino acids are substituted. Specifically, the IL-2 variant can be obtained by substituting R38A, F42A, Y45A, and E61R. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting R38A, F42A, Y45A, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting R38A, F42A, E61R, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting F42A, Y45A, E61R, and L72G. Furthermore, in one embodiment, the IL-2 variant can be obtained by substituting F42A, Y45A, E61R, and L72G.

[0123] In addition, IL-2 variants can be obtained by replacing R38A, F42A, Y45A, E61R, and L72G.

[0124] Preferably, one embodiment of the IL-2 variant may comprise any of the following substitution combinations (a) to (d) selected from the amino acid sequence shown in SEQ ID NO:10:

[0125] (a)R38A / F42A

[0126] (b)R38A / F42A / Y45A

[0127] (c)R38A / F42A / E61R

[0128] (d)R38A / F42A / L72G

[0129] Here, when IL-2 has the amino acid sequence shown in SEQ ID NO:35, amino acid substitutions may be present at positions complementary to the amino acid sequence shown in SEQ ID NO:10. Furthermore, even when IL-2 is a fragment of the amino acid sequence of SEQ ID NO:35, amino acid substitutions may be present at positions complementary to the amino acid sequence shown in SEQ ID NO:10.

[0130] Specifically, IL-2 variants may have amino acid sequences as shown in SEQ ID NO:6, 22, 23 or 24.

[0131] In addition, IL-2 variants are characterized by low in vivo toxicity. Here, low in vivo toxicity may refer to the side effects caused by IL-2 binding to the IL-2 receptor α chain (IL-2Rα). Various IL-2 variants have been developed to mitigate the side effects caused by IL-2 binding to IL-2Rα, and such IL-2 variants can be those disclosed in U.S. Patent No. 5,229,109 and Korean Patent No. 1,667,096. In particular, the IL-2 variants described in this application have low binding affinity to the IL-2 receptor α chain (IL-2Rα) and therefore exhibit lower in vivo toxicity than wild-type IL-2.

[0132] As used herein, the term "CD80," also known as "B7-1," is a membrane protein found in dendritic cells, activated B cells, and monocytes. CD80 provides a co-stimulatory signal essential for T cell activation and survival. CD80 is known to be a ligand for two distinct proteins, CD28 and CTLA-4, which are present on the surface of T cells. CD80 consists of 288 amino acids and may specifically have the amino acid sequence shown in SEQ ID NO:11. Furthermore, as used herein, the term "CD80 protein" refers to the full-length CD80 or a fragment of CD80.

[0133] As used herein, the term "CD80 fragment" refers to a cleaved form of CD80. Additionally, the CD80 fragment can be an extracellular domain of CD80. One embodiment of the CD80 fragment can be obtained by eliminating amino acids 1 through 34 from the N-terminus of the signal sequence that is CD80. Specifically, one embodiment of the CD80 fragment can be a protein composed of amino acids 35 through 288 of SEQ ID NO:11. Another embodiment of the CD80 fragment can be a protein composed of amino acids 35 through 242 of SEQ ID NO:11. Another embodiment of the CD80 fragment can be a protein composed of amino acids 35 through 232 of SEQ ID NO:11. Another embodiment of the CD80 fragment can be a protein composed of amino acids 35 through 139 of SEQ ID NO:11. Another embodiment of the CD80 fragment can be a protein composed of amino acids 142 through 242 of SEQ ID NO:11. In one embodiment, the CD80 fragment may have the amino acid sequence of SEQ ID NO:2.

[0134] In addition, IL-2 and CD80 proteins can be linked to each other via adapters or vectors. Specifically, IL-2 or its variants and CD80 (B7-1) or fragments thereof can be linked to each other via adapters or vectors. In this specification, adapters and vectors are used interchangeably.

[0135] The linker connects two proteins. One embodiment of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, the Fc domain of an immunoglobulin, etc. Here, the Fc domain of an immunoglobulin refers to a protein containing the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin but not the heavy chain and light chain variable regions and the light chain constant region 1 (CH1) of an immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and may preferably be IgG4. Here, the Fc domain of wild-type immunoglobulin G4 may have an amino acid sequence as shown in SEQ ID NO:4.

[0136] Furthermore, the Fc domain of an immunoglobulin can be an Fc domain variant or a wild-type Fc domain. Additionally, as used herein, the term "Fc domain variant" can refer to a form that differs from the wild-type Fc domain in its glycosylation pattern, exhibiting high glycosylation, low glycosylation, or deglycosylation compared to the wild-type Fc domain. This also includes non-glycosylated Fc domains. Through culture conditions or host genetic manipulation, the Fc domain or its variants can be adapted to have regulated amounts of sialic acid, fucosylation, or glycosylation.

[0137] Furthermore, the glycosylation of the Fc domain of immunoglobulins can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Additionally, Fc domain variants can be mixtures of the corresponding Fc regions of immunoglobulins IgG, IgA, IgE, IgD, and IgM. Furthermore, Fc domain variants can be forms in which some amino acids in the Fc domain are substituted with other amino acids. One embodiment of the Fc domain variant may have the amino acid sequence shown in SEQ ID NO:12.

[0138] Fusion proteins may have a structure in which an Fc domain is used as a linker (or vector), and CD80 and IL-2 proteins, or IL-2 and CD80 proteins, are respectively linked to the N-terminus and C-terminus of the linker or vector. The link between the N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 may optionally be achieved via a linker peptide.

[0139] Specifically, the fusion protein may be composed of the following structural formulas (I) or (II):

[0140] N'-X-[Connector(1)] n -Fc structural domain-[joint(2)] m -Y-C'(I)

[0141] N'-Y-[Connector(1)] n -Fc structural domain-[joint(2)] m -X-C'(II)

[0142] Here, in structural formulas (I) and (II),

[0143] N' is the N-terminus of the fusion protein.

[0144] C' is the C-terminus of the fusion protein.

[0145] X is CD80 protein.

[0146] Y represents the IL-2 protein.

[0147] Linkers (1) and (2) are peptide linkers, and

[0148] n and m are each independently 0 or 1.

[0149] Preferably, the fusion protein may be composed of structure (I). The IL-2 protein is as described above. Additionally, the CD80 protein is as described above. According to one embodiment, the IL-2 protein may be an IL-2 variant having one to five amino acid substitutions compared to wild-type IL-2. The CD80 protein may be a fragment obtained by truncating up to about 34 consecutive amino acid residues from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having activity of binding T cell surface receptors CTLA-4 and CD28.

[0150] Specifically, the fusion protein may have an amino acid sequence as shown in SEQ ID NO:9, 26, 28, or 30. According to another 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 with the amino acid sequence of SEQ ID NO:9, 26, 28, or 30. Here, identity is, for example, a percentage of homology, and can be determined using homology comparison software such as BlastN software from the National Center for Biotechnology Information (NCBI).

[0151] The peptide linker (1) may be located between the CD80 protein and the Fc domain. The peptide linker (1) may consist of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 consecutive amino acids. In one embodiment, the peptide linker (1) may consist of 30 amino acids. Additionally, the peptide linker (1) may contain at least one cysteine ​​residue. Specifically, the peptide linker (1) may contain one, two, or three cysteine ​​residues. Furthermore, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one embodiment, the peptide linker (1) may be a peptide linker consisting of an amino acid sequence as shown in SEQ ID NO:3.

[0152] The peptide linker (2) may consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 consecutive amino acids. In one embodiment, the peptide linker (2) may be (G4S). n (Where n is an integer from 1 to 10). Here, in (G4S) n In this context, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) can be a peptide linker composed of an amino acid sequence as shown in SEQ ID NO:5.

[0153] In another aspect of the invention, a dimer obtained by combining two fusion proteins is provided, each of which comprises an IL-2 protein and a CD80 protein. The fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof is as described above.

[0154] Here, the binding between the fusion proteins constituting the dimer can be achieved through, but is not limited to, disulfide bonds formed by cysteine ​​residues present in the linker. The fusion proteins constituting the dimer can be identical fusion proteins or different fusion proteins. Preferably, the dimer can be a homodimer. One embodiment of the fusion protein constituting the dimer can be a protein having the amino acid sequence shown in SEQ ID NO:9.

[0155] The pharmaceutical compositions of the present invention comprise, as active ingredients, a fusion protein dimer containing IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor, exhibiting efficacy in preventing or treating cancer.

[0156] The cancer can be selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0157] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration of administration, and can be appropriately selected by those skilled in the art. In the pharmaceutical compositions of the present invention for treating or preventing cancer, the active ingredient can be contained in any amount (effective amount), depending on the application, dosage form, admixture purpose, etc., as long as the active ingredient exhibits anticancer activity. Its conventional effective amount will be determined in the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the term "effective amount" refers to the amount of active ingredient capable of inducing an anticancer effect. This effective amount can be determined experimentally within the scope of common knowledge of those skilled in the art.

[0158] As used herein, the term "treatment" may be used to mean both therapeutic and preventative treatment. Here, prevention may be used to mean the reduction or alleviation of an individual's pathological condition or disease. In one embodiment, the term "treatment" includes the application or any form of application for the treatment of a disease in mammals, including humans. Additionally, the term includes the suppression or slowing of disease or disease progression; and includes the meaning of restoring or repairing impaired or lost function resulting in partial or complete remission of the disease; stimulating inefficient processes; or alleviating severe disease.

[0159] As used herein, the term "efficacy" refers to the ability to be determined by one or more parameters, such as survival or disease-free survival over a time period such as one year, five years, or ten years. Additionally, parameters may include the suppression of the size of at least one tumor in an individual.

[0160] Pharmacokinetic parameters such as bioavailability and baseline parameters such as clearance can also affect efficacy. Therefore, “enhanced efficacy” (e.g., improved efficacy) may be due to enhanced pharmacokinetic parameters and improved efficacy, which can be measured by comparing clearance and tumor growth in test animals or human subjects, or by comparing parameters such as survival, recurrence, or disease-free survival.

[0161] As used herein, the term "therapeutic effective amount" or "pharmaceutical effective amount" refers to an amount of compound or composition that is effective in preventing or treating the disease, sufficient to provide a reasonable medical benefit / risk ratio for treating the disease, and without causing side effects. The level of an effective amount may be determined based on factors including the patient's health status, the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, the formulation or concomitant drugs, and other factors known in the medical field. In one embodiment, a therapeutically effective amount means an amount of drug that is effective in treating cancer.

[0162] Here, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be any carrier, provided that it is a non-toxic substance suitable for delivery to a patient. Distilled water, alcohols, fats, waxes, and inert solids may be included as carriers. The pharmaceutical composition may also contain pharmaceutically acceptable adjuvants (buffers, dispersants).

[0163] Specifically, a pharmaceutical composition can be prepared as a parenteral formulation using conventional methods known in the art, depending on its route of administration, by including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the term "pharmaceuticalally acceptable" means that the toxicity of the carrier does not exceed a level that the subject receiving the application (prescription) can tolerate, while not inhibiting the activity of the active ingredient.

[0164] When a pharmaceutical composition is prepared as a parenteral formulation, it can be formulated with a suitable carrier in the form of an injection, transdermal patch, nasal inhaler, or suppository, according to methods known in the art. In the case of an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, can be used as suitable carriers; and isotonic solutions, such as Ringer's solution, phosphate-buffered saline (PBS) containing triethanolamine or sterile water for injection, and 5% glucose, are preferred. Formulations of pharmaceutical compositions are known in the art, and specific references can be made to Remington's Pharmaceutical Sciences (19th edition, 1995), etc. This document is considered part of this specification.

[0165] The preferred dosage of the pharmaceutical composition may be from 0.01 μg / kg to 10 g / kg daily, or from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the illness, and route of administration. This dosage may be administered once daily or divided into several doses per day. This dosage should not be construed as limiting the scope of the invention in any way.

[0166] Subjects to whom the (prescription) pharmaceutical composition may be administered are mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of this application may further comprise any compound or natural extract that has been safely verified and is known to have anticancer activity, in order to enhance or strengthen the anticancer activity.

[0167] In another aspect of the invention, a kit for treating cancer is provided, the kit comprising a fusion protein dimer containing IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0168] In another aspect of the invention, there is provided the use of a composition for combined administration for the prevention or treatment of cancer, the composition 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.

[0169] In another aspect of the invention, there is provided the use of a composition for combined administration to enhance the therapeutic effect against cancer, the composition comprising a fusion protein containing IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0170] In another aspect of the invention, there is provided the use of a composition for combined administration in the preparation of a medicament for treating cancer, the composition comprising a fusion protein containing IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0171] In another aspect of the invention, a method for preventing or treating cancer and / or enhancing the therapeutic effect on cancer is provided, the method comprising administering to a subject a composition for combined administration, the composition comprising a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, or a fusion protein dimer wherein two of the fusion proteins are linked together, and an immune checkpoint inhibitor.

[0172] Subjects may be individuals with cancer. Alternatively, subjects may be mammals, preferably humans. The fusion protein comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, or a fusion protein dimer comprising two of these fusion proteins linked together, is as described above.

[0173] The route, dosage, and frequency of administration of fusion proteins or fusion protein dimers and NK cells can vary depending on the patient's condition and the presence or absence of side effects; therefore, fusion proteins or fusion protein dimers can be administered to subjects in various ways and amounts. Those skilled in the art can select the optimal method, dosage, and frequency of administration within appropriate limits.

[0174] Due to the activity of IL-2, the fusion protein in embodiments of the present invention can activate immune cells such as natural killer cells. Therefore, this fusion protein can be effectively used for cancer. In particular, IL-2 variants with 2 to 5 amino acid substitutions, especially those containing amino acid substitutions at 2, 3, 4, or 5 positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G, have been identified as having low binding capacity to the α chain of the IL-2 receptor and thus exhibit improved properties associated with the pharmacological side effects of conventional IL-2. Therefore, when used alone or as a fusion protein, this IL-2 variant can reduce the incidence of vascular (or capillary) leakage syndrome (VLS), a known problem with conventional IL-2.

[0175] Mode of implementing the present invention

[0176] The present invention will be described in more detail below through the following embodiments. However, the following embodiments are for illustrative purposes only, and the scope of the invention is not limited thereto.

[0177] I. Preparation of fusion proteins

[0178] Preparation Example 1. Preparation of hCD80-Fc-IL-2 variant (2M): GI101

[0179] To generate a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide was synthesized using ThermoFisherScientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:8), which, starting from the N-terminus, comprises 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) with two amino acid substitutions (SEQ ID NO:6). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The vector was introduced to express the fusion protein of SEQ ID NO:9. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI101”.

[0180] Purification was performed using chromatography with MabSelect SuRe protein A resin. The fusion protein was bound to the precipitate under conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Elution was then performed with 100 mM NaCl, 100 mM acetic acid, and pH 3. The fusion protein was collected by placing 20% ​​1 M Tris-HCl (pH 9) in a collection tube. The collected fusion protein was then dialyzed with PBS buffer for 16 hours.

[0181] Then, absorbance at 280 nm was measured using size exclusion chromatography on a TSKgel G3000SWXL column (TOSOH Bioscience) to obtain highly concentrated fusion proteins. Here, the isolated and purified fusion proteins were subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie blue to check their purity. Figure 6 The concentration of the fusion protein was determined to be 2.78 mg / ml when measured using NanoDrop. Figure 7 ).in addition, Figure 8 Results obtained using size exclusion chromatography are provided.

[0182] Preparation Example 2. Preparation of mCD80-Fc-IL-2 variant (2M): mGI101

[0183] To generate a fusion protein containing mouse CD80, an Fc domain, and an IL-2 variant, a polynucleotide was synthesized using ThermoFisherScientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:14), which, starting from the N-terminus, contains a signal peptide (SEQ ID NO:1), mCD80 (SEQ ID NO:13), an Ig hinge (SEQ ID NO:3), an Fc domain (SEQ ID NO:4), a linker (SEQ ID NO:5), and an IL-2 variant (2M) (R38A, F42A) with two amino acid substitutions (SEQ ID NO:6). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The vector was introduced to express the fusion protein of SEQ ID NO:15. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “mGI101”.

[0184] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 9 The concentration of the fusion protein was found to be 1.95 mg / ml when detected by absorbance at 280 nm using NanoDrop.

[0185] Preparation Example 3. Preparation of hCD80-Fc: GI101C1

[0186] To generate a fusion protein containing a human CD80 fragment and an Fc domain, a polynucleotide was synthesized using ThermoFisherScientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide contains a nucleotide sequence encoding the fusion protein (SEQ ID NO:16), which includes 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). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TMThe vector was introduced to express the fusion protein of SEQ ID NO:17. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI101C1”.

[0187] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 10 When the absorbance was detected at 280 nm using NanoDrop, the concentration of the fusion protein was observed to be 3.61 mg / ml.

[0188] Preparation Example 4. Preparation of Fc-IL-2 variant (2M): GI101C2

[0189] To generate a fusion protein containing an Fc domain and an IL-2 variant, a polynucleotide was synthesized using ThermoFisher Scientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide contained the nucleotide sequence encoding the fusion protein (SEQ ID NO:18), which, starting from the N-terminus, contained a signal peptide (SEQ ID NO:1), an Fc domain (SEQ ID NO:4), a linker (SEQ ID NO:5), and an IL-2 variant (2M) (R38A, F42A) with two amino acid substitutions (SEQ ID NO:6). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The fusion protein of SEQ ID NO:19 was expressed. After introduction of the vector, the culture was incubated for 7 days at 37°C, 125 rpm and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI101C2”.

[0190] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 11 The concentration of the fusion protein was found to be 4.79 mg / ml when detected by absorbance at 280 nm using NanoDrop.

[0191] Preparation Example 5. Preparation of mCD80-Fc: mGI101C1

[0192] To generate a fusion protein containing mouse CD80 and Fc domains, a polynucleotide was synthesized using ThermoFisher Scientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:20), which, starting from the N-terminus, contains a signal peptide (SEQ ID NO:1), mCD80 (SEQ ID NO:13), an Ig hinge (SEQ ID NO:3), and an Fc domain (SEQ ID NO:4). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The vector was introduced to express the fusion protein of SEQ ID NO:21. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “mGI101C1”.

[0193] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 12 When the absorbance was detected at 280 nm using NanoDrop, the concentration of the fusion protein was observed to be 2.49 mg / ml.

[0194] The fusion proteins prepared in Examples 1 to 5 are summarized in Table 1 below.

[0195] [Table 1]

[0196]

[0197] Preparation Example 6. Preparation of CD80-Fc-IL-2: GI101w

[0198] To generate a fusion protein containing a human CD80 fragment, an Fc domain, and human IL-2, a polynucleotide was synthesized using ThermoFisherScientific's Invitrogen GeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:31), which, starting from the N-terminus, contains 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). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The fusion protein of SEQ ID NO:32 was expressed. After introduction of the vector, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI101w”. The purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1.

[0199] Preparation Example 7. Preparation of hCD80-Fc-IL-2 variant (3M): GI102-M45

[0200] To generate 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 was synthesized using ThermoFisher Scientific's InvitrogenGeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:25), which, starting from the N-terminus, comprises 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). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The vector was introduced to express the fusion protein of SEQ ID NO:26. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI102-M45”.

[0201] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 13 ).

[0202] Preparation Example 8. Preparation of hCD80-Fc-IL-2 variant (3M): GI102-M61

[0203] To generate 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) (GI102-M61), a polynucleotide was synthesized using ThermoFisher Scientific's InvitrogenGeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:27), which, starting from the N-terminus, comprises a signal peptide (SEQ ID NO:1), a CD80 fragment (SEQ ID NO:2), an Ig hinge (SEQ ID NO:3), an Fc domain (SEQ ID NO:4), a linker (SEQ ID NO:5), and an IL-2 variant (SEQ ID NO:23). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The vector was introduced to express the fusion protein of SEQ ID NO:28. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI102-M61”.

[0204] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 14 ).

[0205] Preparation Example 9. Preparation of hCD80-Fc-IL-3M: GI102-M72

[0206] To generate 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 was synthesized using ThermoFisher Scientific's InvitrogenGeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:29), which, starting from the N-terminus, comprises 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:24). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO). TM The fusion protein of SEQ ID NO:30 was expressed. After introduction of the vector, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “GI102-M72”.

[0207] The fusion protein was purified and recovered in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was checked by Coomassie blue staining. Figure 15 ).

[0208] Preparation Example 10. Preparation of mCD80-Fc-IL-3M: mGI102-M61

[0209] To generate a fusion protein comprising a mouse CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, E61R) (GI102-M61), a polynucleotide was synthesized using ThermoFisher Scientific's InvitrogenGeneArt gene synthesis service. Specifically, the polynucleotide contains the nucleotide sequence encoding the fusion protein (SEQ ID NO:33), which, starting from the N-terminus, comprises a signal peptide (SEQ ID NO:1), an mCD80 fragment (SEQ ID NO:13), an Ig hinge (SEQ ID NO:3), an Fc domain (SEQ ID NO:4), a linker (SEQ ID NO:5), and an IL-2 variant (SEQ ID NO:23). This polynucleotide was inserted into the pcDNA3_4 vector. Furthermore, this vector was introduced into CHO cells (Expi-CHO).TM The vector was introduced to express the fusion protein of SEQ ID NO:34. After introduction, the culture was incubated for 7 days at 37°C, 125 rpm, and 8% CO2. The culture was then harvested and the fusion protein was purified. The purified fusion protein was named “mGI102-M61”.

[0210] The fusion protein was purified and recovered in the same manner as in Preparation Example 1.

[0211] II. Identification of binding affinity between fusion proteins and their ligands

[0212] To determine the binding affinity between the fusion protein and its ligand, the binding affinity was measured using an Octet RED 384.

[0213] Example 1. Identification of binding affinity between hCTLA-4 and GI101

[0214] AR2G biosensor (Amine Reactive 2) nd The ligand (CTLA-4, human CTLA-4 / CD152, His tag, Sino Biological, catalog number: 11159-H08H) to be liganded to the AR2G biosensor was pre-hydrated with 200 μl of distilled water in a 96-well microplate (GreinerBio-One, catalog number: 655209). The ligand (CTLA-4, human CTLA-4 / CD152, His tag, Sino Biological, catalog number: 11159-H08H) to be liganded to the AR2G biosensor was diluted to a concentration of 5 μg / mL with 10 mM acetate buffer (pH 5, AR2G kit, ForteBio, catalog number: 18-5095). Additionally, the GI101 to be liganded was diluted to concentrations of 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM with 1X AR2G kinetic buffer (AR2G kit, ForteBio, catalog number: 18-5095). To prepare the activation buffer, mix 20 mM EDC and 10 mM s-NHS (AR2G kit, ForteBio, catalog number: 18-5095) in distilled water. Place 80 μl of each reagent into a 384-well microplate (Greiner Bio-One, catalog number: 781209) and set the program.

[0215] Result: As Figure 16 The binding affinity between hCTLA-4 and GI101 was measured.

[0216] Example 2. Identification of binding affinity between hPD-L1 / GI101 and hPD-L1 / PD-1

[0217] In 96-well microplates (GreinerBio-One, catalog number: 655209), Ni-NTA (nickel-loaded Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101) was pre-hydrated with 200 μl of 1X Ni-NTA kinetic buffer (10× kinetic buffer, ForteBio, 18-1042). The ligand to be liganded to the Ni-NTA biosensor (human PD-L1 / B7-H1 protein, His tag, Sino Biological, catalog number: 10084-H08H) was diluted to a concentration of 5 μg / mL with 1X Ni-NTA kinetic buffer. GI101 to be liganded was diluted to 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM with 1× Ni-NTA kinetic buffer. Additionally, the human PD-1 / PDCD1 (human PD-1 / PDCD1, Fc tag, Sino Biological, catalog number: 10377-H02H) to be ligand-liganded was diluted to concentrations of 2,000 nM, 1,000 nM, 500 nM, 250 nM, or 125 nM using 1×Ni-NTA kinetic buffer. Then, 80 μl of each reagent was placed in a 384-well microplate and the program was set.

[0218] Result: As Figure 17 The binding affinity between hPD-L1 and GI101 was measured, as shown. Additionally, as... Figure 18 The binding affinity between hPD-L1 and hPD-1 was measured.

[0219] Example 3. Identification of binding affinity between mCTLA-4 and mGI101

[0220] The binding affinity between mCTLA-4 and mGI101 was detected in the same manner as in Example 1. The equipment used here is as follows: biosensor: AR2G, ligand: mCTLA-4 (recombinant mouse CTLA-4Fc chimera, R&D Systems, catalog number: 434-CT-200), analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0221] Result: As Figure 19 The binding affinity between mCTLA-4 and mGI101 was measured.

[0222] Example 4. Identification of binding affinity between mPD-L1 and mGI101

[0223] The binding affinity between mPD-L1 and mGI101 was identified in the same manner as in Example 1. The equipment used here is as follows: Biosensor: AR2G; Ligand: mPD-L1 (recombinant mouse mGI101 B7-H1 / PD-L1 Fc chimera, R&DSystems, catalog number: 434-CT-200); Analytes: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0224] Result: As Figure 20 The binding affinity between mPD-L1 and mGI101 was measured.

[0225] Example 5. Identification of the binding affinity between GI-101 (hCD80-Fc-hIL-2v) and CTLA-4.

[0226] Binding kinetics were measured using an Octet RED 384 instrument (ForteBio, Pall Life Science) at 30°C and 1,000 rpm with stirring. CTLA-4 binding capacity was measured using an Amine Reactive 2-generation (AR2G) biosensor chip, and PD-L1 binding capacity was measured using a nickel-loaded Tris-NTA (Ni-NTA) biosensor chip. The AR2G biosensor chip was activated with a combination of 400 mM EDC and 100 mM Sulfo-NHS. The human CTLA-4-His tag (Sino Biological, catalog number: 11159-H08H) was then diluted to 5 μg / ml with 10 mM acetate buffer (pH 5), loaded onto the AR2G biosensor chip, and fixed for 300 seconds.

[0227] Then, the binding time of various concentrations of CTLA-4 to 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 their dissociation was also measured for 300 seconds. Binding kinetics were analyzed using Octet data analysis HT software version 10 provided by Pall. The results are shown in... Figure 21 middle.

[0228] Example 6. Identification of binding affinity between IL-2Rα or IL-2Rβ and GI101

[0229] The binding capacity of IL-2Rα was measured using an AR2G biosensor, and the binding capacity of IL-2Rβ was measured using a Ni-NTA biosensor (Ni-loaded Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101).

[0230] The ligand (IL-2Rα-His tag, Acro, catalog number: ILA-H52H9) to be linked to the AR2G biosensor was diluted to a concentration of 5 μg / mL using 10 mM acetate buffer (pH 5, AR2G kit, ForteBio, catalog number: 18-5095). The AR2G biosensor was activated with a buffer prepared by mixing 400 mM EDC and 100 mM Sulfo-NHS, and then the diluted ligand was loaded onto the AR2G biosensor for 300 seconds and immobilized.

[0231] Simultaneously, the ligand (IL-2Rβ-His tag, Acro, catalog number: CD2-H5221) to be linked to the Ni-NTA biosensor was diluted to a concentration of 5 μg / ml using 1×Ni-NTA kinetic buffer. The diluted ligand was then loaded onto the Ni-NTA biosensor for 600 seconds and fixed.

[0232] Then, various concentrations of GI101, GI101w, or Novartis (hIL-2) to be ligand-ligand-bound were loaded onto the ligands for 300 seconds. Binding and dissociation were then measured for 300 seconds. Binding kinetics were analyzed using Octet data analysis HT software version 10 provided by Pall. The results are shown below. Figures 22 to 24 middle.

[0233] Results: Compared with GI101w and interleukin, GI101 showed low binding capacity to the IL-2 receptor α chain IL-2Rα and high binding capacity to IL-2Rβ.

[0234] Example 7. Measuring the binding affinity between the fusion protein and the ligand.

[0235] To determine the binding affinity between the fusion protein and its ligand, the binding affinity was measured using an Octet RED 384.

[0236] Example 7.1. Identification of binding affinity of IL-2α receptor to GI101-M45, GI101-M61 or GI101-M72

[0237] The AR2G biosensor (Amine Reactive 2nd gen, ForteBio, catalog number: 18-5092) was pre-hydrated in a 96-well microplate (GreinerBio-One, catalog number: 655209) with 200 μl of distilled water (DW). The ligands to be linked to the biosensor (human IL-2Rα protein, His tag, Acro, ILA-H52H9) were diluted to a concentration of 5 μg / mL with 10 mM acetate buffer (pH 5) (AR2G kit, ForteBio, catalog number: 18-5095). The analytes to be linked to the ligands (GI101-M45, GI101-M61, GI101-M72) were diluted to 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively, with 1×AR2G kinetic buffer (AR2G kit, ForteBio, catalog number: 18-5095). Activation buffers were prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G kit, ForteBio, catalog number: 18-5095) in a DW. 80 μl of each reagent was placed in a 384-well microplate (Greiner Bio-One, catalog number: 781209) and the program was set.

[0238] Results: The binding affinity between the IL-2α receptor and GI101-M45 was as follows: Figure 25 As shown. Additionally, the binding affinity between the IL-2α receptor and GI101-M61 is as follows: Figure 26 As shown, and the binding affinity between the IL-2α receptor and GI101-M72 is as follows: Figure 27 As shown.

[0239] Example 7.2. Identification of the binding affinity of GI102-M45, GI102-M61, and GI102-M72 to IL-2Rβ

[0240] In 96-well microplates, the Ni-NTA biosensor was pre-hydrated with 200 μl of 1×Ni-NTA kinetic buffer (10× kinetic buffer, ForteBio, 18-1042). The ligand to be liganded to the biosensor (human IL-2Rβ protein, His tag, Acro, CD2-H5221) was diluted to a concentration of 2 μg / mL with 1×Ni-NTA kinetic buffer. GI102-M45, GI102-M61, or GI102-M72 to be liganded were diluted to concentrations of 500 nM, 250 nM, 125 nM, or 62.5 nM with 1×Ni-NTA kinetic buffer. 80 μl of each reagent was placed in a 384-well microplate, and the program was set.

[0241] Result: As Figure 28 The binding affinity between IL-2Rβ and GI102-M45 was measured, as shown in the figure. Figure 29 The binding affinity between IL-2Rβ and GI102-M61 was measured, as shown. Additionally, as... Figure 30 The binding affinity between IL-2Rβ and GI102-M72 was measured.

[0242] III. Identifying the immunological activity of the fusion protein

[0243] Example 8. Identification of IFN-γ production induced by the fusion protein

[0244] Example 8.1. Cultivation of CFSE-labeled PBMCs

[0245] Peripheral blood mononuclear cells (PBMCs) isolated from humans were labeled with carboxyfluorescein succinimide (CFSE) by reacting with 1 μM CellTrace CFSE dye at 37 °C for 20 min. Unbound CFSE was removed by reacting with medium containing 5 times the volume of the staining reaction solution for 5 min, followed by centrifugation at 1,300 rpm for 5 min. The CFB-labeled PBMCs were resuspended in medium (RPMI 1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acids, and 2 mM L-glutamine) and then sputtered at 1 × 10⁻⁶ ppm. 5 Cells / well were added to 96-well microplates. Treatment was performed with 5 μg / ml PHA (lectin derived from common bean, red kidney bean, Sigma-Aldrich, St. Louis, MO, USA, catalog number: L1668-5MG) and GI101, GI101C1, GI101C2, or IL-2 (aldeleukin; recombinant human IL-2, Novartis), and incubated at 37°C in a 5% CO2 incubator for 6 days.

[0246] Here, the therapeutic concentrations of GI101, GI101C1, GI101C2, and IL-2 were 1 nM, 10 nM, or 100 nM. Cells were analyzed by FACS, and the presence of human IFN-γ in the culture medium was measured using an ELISA kit (Biolegend, San Diego, CA, USA, catalog number: 430103).

[0247] Example 8.2. FACS Analysis

[0248] The cell pellet obtained by removing the supernatant was washed with FACS buffer (3% fetal bovine serum (FBS), 10 mM EDTA, 1 M HEPES, 100 U / ml penicillin / streptomycin, 10 μg / ml 1 mM sodium pyruvate), and then reacted with an Fc blocker (Biolegend, catalog number: 422302) at 4°C for 5 min. Afterwards, the pellet was treated with APC anti-CD3 antibody (Biolegend, catalog number: 300412) and PE anti-CD8a antibody (Biolegend, catalog number: 300908) and the reaction was incubated at 4°C for 20 min. The resulting product was then washed with FACS buffer. The cell pellet was resuspended in FACS buffer and analyzed using BD LSRFortessa (BD Biosciences, San Diego, CA, USA) and FlowJo software.

[0249] Example 8.3. ELISA detection of human IFN-γ

[0250] The amount of human IFN-γ secreted into the supernatant of each sample was measured using a Human IFN-γ ELISA kit (Biolegend, catalog number: 430103). In short, anti-human IFN-γ antibodies were added to the ELISA plate and the reaction was incubated overnight at 4°C to allow the antibodies to coat the plate. The plate was then blocked with PBS solution supplemented with 1% BSA at room temperature for 1 hour. The plate was washed with wash buffer (PBS solution of 0.05% Tween-20), and then the standard solutions and individual samples were appropriately diluted and added. The reaction was then allowed to proceed at room temperature for 2 hours.

[0251] After the reaction was complete, the plate was washed and a second antibody (detection antibody) was added. The reaction was allowed to proceed at room temperature for 1 hour. The plate was washed with washing buffer, and then an avidin-HRP solution was added. The reaction was allowed to proceed at room temperature for 30 minutes. The substrate solution was added, and the colorimetric reaction was induced in the dark at room temperature for 20 minutes. Finally, H₂SO₄ was added to terminate the colorimetric reaction, and the absorbance at 450 nm was measured using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

[0252] Therefore, it was found that cells treated with GI101 showed a significant increase in IFN-γ secretion compared to cells treated with GI101C1, GI101C2, or IL-2. Figure 31 and Figure 32 ).

[0253] Example 9. Identification of the effect of GI101 on CD8+ T cell proliferation

[0254] 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 min. Unbound CFSE was removed by reacting with medium containing 5 times the volume of staining reaction solution for 5 min, followed by centrifugation at 1,300 rpm for 5 min. The CFB-labeled PBMCs were resuspended in medium (RPMI 1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acids, and 2 mM L-glutamine), and then thawed at 1 × 10⁻⁶ ppm. 5 The amount of cells / well added to the 96-well microplate.

[0255] Cells were then treated with 1 μg / ml anti-CD3ε antibody (Biolegend, catalog number: L1668-5MG) and GI101, GI101C1, GI101C2, or adeleukin (Novartis), and incubated for 6 days at 37°C in a 5% CO2 incubator. Cells were also treated with 100 nM GI101, GI101C1, GI101C2, and IL-2. Cell proliferation was assessed by measuring the proportion of unlabeled CFSE-positive CD8+ T cells using FACS analysis with APC-TCRαβ and PE-CD8α antibodies.

[0256] The results showed that GI101 activated CD8+ T cell proliferation to a similar degree as wild-type IL-2, adefovir. Figure 33 and Figure 34 ).

[0257] Example 10. Identification of the effects of GI101 and GI102 on CD8+ T cell proliferation

[0258] Human PBMCs were purchased from Allcells (Lot #3014928, USA). Human PBMCs were reacted with 1M CellTrace CFSE dye at room temperature for 20 minutes in the dark. Cells were labeled with CFSE by reacting with 1μM CellTrace CFSE dye at 37°C for 20 minutes. Unbound CFSE was removed by reacting with medium containing 5 times the volume of the staining reaction solution for 5 minutes, followed by centrifugation at 1,300 rpm for 5 minutes. CFB-labeled PBMCs were resuspended in medium (RPMI 1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acids, and 2 mM L-glutamine), and then thawed at 1×10⁻⁶ ppm. 5 The amount of cells / well added to the 96-well microplate.

[0259] Then, CFB-labeled PBMCs were treated with 1 μg / ml anti-CD3ε antibody (OKT3, eBioscience, USA) and GI101, GI101C1, GI101C2 or interleukin (Novartis) and incubated for 7 days at 37°C in a 5% CO2 incubator. Here, cells were treated with 10 μM of GI101, GI101C1, GI101C2 and IL-2.

[0260] The proliferation of incubated cells was examined by measuring the proportion of unlabeled CFSE-positive CD8+ T cells using FACS analysis with anti-human CD4-PE antibody (Biolegend, USA), anti-human CD8-PE / Cy7 antibody (Biolegend, USA), and anti-human FoxP3-APC antibody (Biolegend, USA).

[0261] Therefore, compared with the control group (no stimulation), the anti-CD3 antibody monotherapy group, and the GI101C1 treatment group, the GI101, GI102_M61, GI101C2, and adeleukin treatment groups showed a significant increase in the proportion of CD8+ T cells. Furthermore, compared with the negative control group (no stimulation) and the anti-CD3 monotherapy group, the GI101, GI101C2, and adeleukin treatment groups showed a significant increase in the proliferation of CD4+ / FoxP3+ Treg cells, while the GI102 and GI101C1 treatment groups did not show a significant increase in CD4+ / FoxP3+ Treg cell proliferation. Figure 35 ).

[0262] Example 11. Identification of the effects of GI101 or GI101w on the proliferation of CD8+ T cells and NK cells.

[0263] Seven-week-old C57BL / 6 mice purchased from Orient Bio (Korea) were randomly divided into three groups of three mice each and intraperitoneally injected with PBS, GI101, or GI101w. GI101 and GI101w were prepared as 40.5 μg solutions in 200 μl PBS and administered intraperitoneally to the mice. Five days after injection, spleens were harvested from each group. Cells were isolated, and the total cell count was measured using a hematology counter. The proportions of CD8+ T cells and NK cells in the spleen cells were determined by FACS analysis using APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific Blue-CD8α antibody (BD; 53-6.7). The number of CD8+ T cells and NK cells present in the spleen was then calculated.

[0264] Results: Compared with GI101w, GI101 activated the proliferation of CD8+ T cells and NK cells in vivo. Figure 36 and Figure 37 ).

[0265] Example 12. Identifying the effect of GI101 on T cell function

[0266] The experiment was performed using the CTLA-4 Blockade Bioassay Kit (Promega, catalog number: JA4005). The experiment is briefly described as follows: CTLA-4 effector cells held in liquid nitrogen were thawed in a 37°C water bath for 3 minutes, and 0.8 ml of CTLA-4 effector cells were thoroughly mixed with 3.2 ml of pre-warmed assay buffer (90% RPMI + 10% fetal bovine serum). The mixture was then added at 25 μl / well to a 96-well leukocyte culture plate (SPL, catalog number: 30196). Next, 25 μl of GI101 at various concentrations was added. For the negative control, 25 μl of assay buffer was added. The 96-well leukocyte culture plate was then covered and placed at room temperature until the aAPC / Raji cells were prepared.

[0267] aAPC / Raji cells held in liquid nitrogen were thawed in a 37°C water bath for 3 minutes, and 0.8 mL of aAPC / Raji cells was thoroughly mixed with 3.2 mL of preheated detection buffer. Then, 25 μL of the mixture was added to each well of the plate, and the reaction was incubated at 37°C in a 5% CO2 incubator for 16 hours. After the reaction was complete, the product was allowed to stand at room temperature for 15 minutes, and then Bio-Glo reagent was added, carefully avoiding the formation of air bubbles. Bio-Glo reagent was then added to the three outermost wells as blanks to correct for background signal. The reaction was allowed to proceed at room temperature for 10 minutes, and then fluorescence measurements were performed using a Cytation 3 (BioTek Instruments, Inc., Winooski, VT, USA). Final data analysis was performed by calculating RLU (GI101 - background) / RLU (no treatment background).

[0268] The results showed that GI101, which is linked to CTLA-4, is expressed on effector T cells and activates T cell function rather than suppresses it. Figure 38 and Figure 39 ).

[0269] Example 13. Identification of the effects of mGI101 and mGI102 on immune cells

[0270] Seven-week-old C57BL / 6 mice purchased from Orient Bio (Korea) were randomly divided into three groups of three mice each and administered intravenously 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. Spleens were harvested from each group on days 1, 3, 5, 7, and 14 post-injection. The spleen tissue was then analyzed using the corresponding antibodies via FACS to calculate the number of effector CD8+ T cells, NK cells, and Treg cells, and the ratio of effector CD8+ T cells and NK cells to Treg cells was calculated. Information regarding the antibodies used in each cell assay is as follows:

[0271] Effector CD8+ T cells: 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)

[0272] NK cells: PB anti-mouse CD3ε antibody (Biolegend, #155612; KT3.1.1), PE anti-mouse NK-1.1 (Biolegend, #108708; PK136)

[0273] Treg cells: 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).

[0274] Results: Compared with the PBS administration group, the groups receiving mGI101 or mGI102 (mGI102-M61) showed a significant increase in the number of CD8+ T cells and NK cells at time points 3 to 14 days after administration. Furthermore, compared with the PBS administration group, the groups receiving mGI102 showed a significant increase in the ratios of activated CD8+ T cells / Treg cells and NK cells / Treg cells at time points 3 to 14 days after administration. Figure 40 ).

[0275] IV. Identifying the anticancer effects of fusion proteins

[0276] Example 14. Identifying the effect of GI101 on inhibiting T cell activity in cancer cells expressing PD-L1 and CTLA-4.

[0277] The NCl-H292 cancer cell line expressing PD-L1 and CTLA-4 was cultured in medium containing 10 μg / ml mitomycin C (Sigma) for 3 hours, followed by washing with medium to remove mitomycin C. Then, 5 × 10⁻⁶ cells were cultured... 4 NCl-H292 cancer cell line treated with mitomycin C and 1×10 5 Human PBMCs were incubated in 96-well microplates. T cell activity was enhanced with 5 μg / ml PHA (Sigma). Additionally, 50 nM GI101C1 and GI101 were reacted with 50 nM IgG1-Fc (Biolegend) or abatacept (=Orencia; Bristol-Myers Squibb) at 4°C for 30 min, and the resulting mixture was used to treat NCl-H292 cancer cells. After 3 days, the supernatant of the cell culture was collected, and the amount of IFN-γ was determined using an ELISA kit (Biolegend).

[0278] As a positive control, human PBMCs stimulated with PHA in the absence of mitomycin C treatment of the NCI-H292 cancer cell line were used; as a negative control, human PBMCs stimulated with PHA in the presence of mitomycin C treatment of the NCI-H292 cancer cell line were used. Detection was performed using the IFN-γ ELISA kit in the same manner as in Example 9.3.

[0279] Results: GI101 effectively activated the immune response, which was suppressed by cancer cell lines overexpressing PD-L1. Furthermore, GI101 was found to inhibit CTLA-4 signaling on effector T cells. Figure 41 and Figure 42 ).

[0280] Example 15. Identification of the anticancer effect of mGI101 in mice transplanted with mouse-derived colorectal cancer cells.

[0281] Female BALB / c mice (7 weeks old) obtained from Orient Bio were given a 7-day acclimatization period. Then, 5 × 10⁸ mice were... 6 CT-26 cancer cell line (ATCC, USA) cells were mixed with 0.05 ml of phenol red-free matrix gel (BD) and allogeneic transplantation of the mixture was performed by subcutaneous injection of 0.1 ml into the right dorsal region of mice. Tumor volume was measured at a certain time after transplantation, and tumors reaching approximately 28 mm were selected. 3 The subjects were then selected, and the mice were uniformly grouped into groups of 10 mice each, based on tumor size and body weight. The negative control group was then administered hIgG4 at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were given mGI101 intravenously at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. Injections were given three times, every three days after the first injection. Tumor size was measured daily.

[0282] Results: In the groups receiving 6 mg / kg and 12 mg / kg doses of mGI101, tumor size was significantly suppressed compared with the negative control group at some measurement time points and at the end of the retest. Figure 43 Furthermore, as a result of measuring survival, significant improvements were observed in the group that received a 6 mg / kg dose of mGI101 compared to the negative control group at several measurement time points and at the end of the trial. Figure 44 ).

[0283] Example 16. Identification of the anticancer effect of GI101 in mice transplanted with mouse-derived colorectal cancer cells.

[0284] Example 16.1. Identification of tumor-suppressive effect

[0285] Female BALB / c mice (7 weeks old) obtained from Orient Bio were given a 7-day acclimatization period. Then, 5 × 10⁸ mice were... 6 CT-26 cancer cell line (ATCC, USA) cells were suspended in 0.1 ml of PBS and allogeneic transplantation of the suspension was performed via subcutaneous injection of 0.1 ml into the right dorsal region of mice. Tumor volume was measured at a certain time post-transplantation, and tumors reaching approximately 50 mm in size were selected. 3 Up to 200mm 3 The subjects were then selected, and mice were uniformly grouped into groups of 10 mice each, based on tumor size and body weight. Subsequently, using a disposable syringe (31G, 1ml), the positive control group was intravenously injected with either 5 mg / kg of anti-PD-1 antibody or 5 mg / kg of both anti-PD-1 antibody and anti-CTLA-4 antibody, without administration of the drug to the negative control group. The experimental group received an intravenous injection of 0.1 mg / kg or 1 mg / kg of GI101. Injections were administered three times, every three days after the first injection. Tumor size was measured daily.

[0286] Results: In mice transplanted with the CT-26 cancer cell line, all groups receiving 0.1 mg / kg or 1 mg / kg doses of anti-PD-1 antibody, anti-PD-1 antibody, and anti-CTLA-4 antibody or GI101 showed significant inhibition of tumor growth compared to the negative control group. In particular, the experimental group receiving GI101 at a dose of 0.1 mg / kg showed significant tumor inhibition compared to the anti-PD-1 antibody treatment group (*p < 0.05). Figure 45 ).

[0287] Example 16.2. Analysis of immune cells in cancer tissue

[0288] When the tumor volume reaches an average of 200mm 3At that time, mice in each group in Example 16.1 were sacrificed and cancer tissue was collected. The cancer tissue was then isolated to the single-cell level for analysis of immune cells. The following antibodies were used for FACS analysis of the immune cells in the cancer tissue: anti-mouse CD3 (Biolegend, catalog number: 100320), anti-mouse CD4 (Biolegend, catalog number: 100526), ​​anti-mouse CD8 (Biolegend, catalog number: 100750), anti-mouse FoxP3 (eBioscience, catalog number: 12-5773-82), anti-mouse CD25 (Biolegend, catalog number: 102049), anti-mouse CD44 (eBioscience, catalog number: 61-0441-82), anti-mouse PD-1 (Biolegend, catalog number: 135218), and anti-mouse IFN-γ (Biolegend, catalog number: 100320). Anti-mouse CD49b (Biolegend, catalog number: 108906), anti-mouse H2 (Invitrogen, catalog number: A15443), anti-mouse CD11c (Biolegend, catalog number: 117343), anti-mouse CD80 (eBioscience, catalog number: 47-4801-82), anti-mouse CD86 (Biolegend, catalog number: 104729), anti-mouse F4 / 80 (eBioscience, catalog number: 47-4801-82), and anti-mouse CD206 (eBioscience, catalog number: 17-2061-80) were used as antibodies.

[0289] Results: Compared with the positive control group receiving only 5 mg / kg of anti-PD-1 antibody, the experimental group receiving 0.1 mg / kg of GI101 showed a significant increase in CD8+ T cells (*p < 0.05). Figure 46 and Figure 47 Furthermore, compared with the negative control group, all experimental groups receiving GI101 showed a significant increase in IFN-γ expression levels in T cells (*p<0.05). Figure 46 and Figure 47 In addition, compared with the negative control group and the positive control group that received only anti-PD-1 antibody, the experimental group receiving 0.1 mg / kg of GI101 showed an increase in M1 macrophages. Figure 48 and Figure 49 Additionally, all experimental groups receiving GI101 showed increased CD86 expression levels in macrophages and dendritic cells (*p < 0.05). Figures 48 to 51 ).

[0290] Example 17. Identification of the anticancer effect of GI101 in mice transplanted with mouse-derived lung cancer cells.

[0291] 17.1. Identification of tumor-suppressive effects

[0292] Female, 7-week-old C57BL / 6 mice obtained from Orient Bio were given a 7-day acclimatization period. Then, 5 × 10⁸ mice were... 6 A single-cell LLC2 cancer cell line (ATCC, USA) was suspended in 0.1 ml of PBS, and allogeneic transplantation of the suspension was performed by subcutaneous injection of 0.1 ml into the right dorsal region of mice. Tumor volume was measured at a certain time after transplantation, and tumors reaching approximately 50 mm in size were selected. 3 Up to 200mm 3 The subjects were then selected, and mice were uniformly grouped into groups of 10 mice each, based on tumor size and body weight. Using a disposable syringe (31G, 1ml), the positive control group was administered either 5 mg / kg of anti-PD-1 antibody or 5 mg / kg of both anti-PD-1 antibody and anti-CTLA-4 antibody intravenously, without drug administration to the negative control group. The experimental group received either 0.1 mg / kg or 1 mg / kg of GI101 intravenously. Injections were administered three times, every three days after the first injection. Tumor size was measured daily.

[0293] Results: Compared with the negative control group, all experimental groups showed significant tumor inhibition (*p<0.05). Figure 52 ).

[0294] Example 17.2. Analysis of immune cells in cancer tissue

[0295] When the tumor volume reaches an average of 200mm 3 At that time, mice from each group in Example 17.1 were sacrificed and cancer tissue was collected. Then, FACS analysis was performed in the same manner as in Example 16.2 to analyze the immune cells in the cancer tissue.

[0296] As a result, compared with the positive control group that received only anti-PD-1 antibody, the experimental group that received GI101 at a dose of 0.1 mg / kg showed a significant increase in CD8+ T cells (*p<0.05). Figure 59 Furthermore, compared with the negative control group, all experimental groups receiving GI101 showed a significant increase in IFN-γ expression levels (*p<0.05). Figure 59 Additionally, all experimental groups receiving GI101 showed increased CD86 expression levels in macrophages and dendritic cells (*p < 0.05). Figures 53 to 55 ).

[0297] Example 18. Identification of the anticancer effect of mGI102-M61 in mice transplanted with mouse-derived colorectal cancer cells.

[0298] Female BALB / c mice (7 weeks old) obtained from Orient Bio were given a 7-day acclimatization period. Then, 5 × 10⁸ mice were... 6 CT-26 cancer cell line (ATCC, USA) cells were mixed with 0.05 ml of phenol red-free matrix gel (BD) and allogeneic transplantation of the mixture was performed by subcutaneous injection of 0.1 ml into the right dorsal region of mice. Tumor volume was measured at a certain time after transplantation, and tumors reaching approximately 28 mm were selected. 3 The subjects were then selected, and the mice were uniformly grouped into groups of 10 mice each, based on tumor size and body weight. The negative control group was then injected with hIgG4 at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were given intravenous injections of mGI102-M61 at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. Injections were given every three days after the first injection, for a total of three injections. Tumor size was measured daily.

[0299] Results: Compared with the negative control group, the experimental group receiving 12 mg / kg of mGI102-M61 showed significant inhibition of tumor growth at some measurement time points and at the end of the trial. Figure 56 Furthermore, as a result of measuring survival, it was determined that the experimental group receiving a dose of 12 mg / kg mGI102-M61 showed significant improvement at some measurement time points and at the end of the trial compared with the negative control group. Figure 57 ).

[0300] Example 19. Identification of the anticancer effect of mGI101 in mice transplanted with mouse-derived colorectal cancer cells.

[0301] Female BALB / c mice (7 weeks old) obtained from Orient Bio were given a 7-day acclimatization period. Then, 5 × 10⁸ mice were... 6 CT-26 cancer cell line (ATCC, USA) cells were mixed with 0.05 ml of phenol red-free matrix gel (BD) and allogeneic transplantation of the mixture was performed by subcutaneous injection of 0.1 ml into the right dorsal region of mice. Tumor volume was measured at a certain time after transplantation, and tumors reaching approximately 200 mm² were selected. 3 Up to 250mm 3 The subjects were then selected, and the mice were then evenly grouped into groups of 10 mice each, based on tumor size and weight.

[0302] Then, using a disposable syringe (31G, 1ml), hIgG4 was injected at a dose of 4 mg / kg into the negative control group. For the experimental groups, mGI101 was administered intravenously at doses of 1 mg / kg, 4 mg / kg, or 6 mg / kg. Additionally, groups receiving 4.9 mg / kg mCD80 or 2.8 mg / kg Fc-IL-2v (GI101C2) were designated as control groups. Furthermore, groups receiving both 4.9 mg / kg mCD80 and 2.8 mg / kg Fc-IL-2v (GI101C2) were designated as control groups.

[0303] In tumor volume measurements, the group receiving 6 mg / kg mGI101 showed significant inhibitory effects at several measurement time points and at the end of the trial, compared to the negative control. Superior tumor growth inhibition rates were observed compared to the group receiving the combination of mCD80 and Fc-IL-2v (GI101C2). Figure 58 and Figure 59 ).

[0304] In summary, in the tumor growth inhibition efficacy test of BALB / c mice allogeneically transplanted with CT-26 (a BALB / c mouse-derived colorectal cancer cell line), the test substance mGI101 demonstrated tumor inhibitory efficacy under the test conditions compared with mCD80 and IL-2v alone; and it was identified that mGI101 showed superior anticancer efficacy compared with the group receiving the combination of mCD80 and IL-2v. Figure 58 and Figure 59 In particular, compared with the negative control group and the group receiving the combination of mCD80 and Fc-IL2v (GI101C2), the group receiving a dose of 6 mg / kg of mGI101 showed a significant inhibitory effect on tumor size.

[0305] V. Determination of the anticancer effect of combined administration of fusion protein dimers and immune checkpoint inhibitors

[0306] Example 20. Determination of the anticancer effect of combined administration of GI101 and anti-PD-1 antibody in mice with human breast cancer cell transplantation.

[0307] This test used a humanized mouse model, created by xenografting human PBMCs into NSGb2m mice, to evaluate the tumor growth inhibition effect following intraperitoneal injection, alone or in combination, of GI101 (as the test material) and Keytruda (Pembrolizumab, MSD) (an anti-PD-1 antibody) (as the positive control material), in a tumor model transplanted with xenograft MDA-MB-231 cells (as human breast cancer cells).

[0308] Dilute the stock solutions of the test materials, negative control materials, and positive control materials described in Table 2 with excipients according to each dose.

[0309] [Table 2]

[0310]

[0311] Human breast cancer cells MDA-MB-231 (Homo sapiens, human mammary gland / breast; derived from metastatic site: pleural effusion) were purchased from the Korean Cell Bank (Korea) and used in this experiment. Fetal bovine serum (FBS, 16000-044, Thermofishers Scientific, USA), penicillin-streptomycin (10,000 U / ml penicillin and 10,000 μg / ml streptomycin) (15140122, Thermofishers Scientific, USA) and RPMI 1640 (A1049101, Thermofishers Scientific, USA) were mixed, with the components per 100 ml as shown in the table below.

[0312] [Table 3]

[0313] name Composition (ml) FBS 10 Penicillin-Streptomycin 1 RPMI1640 89 Total volume 100

[0314] The cells used in the experiment were thawed, placed in flasks for cell culture, and cultured in a 5% CO2 incubator at 37°C (MCO-170M, Panasonic, Japan). Cells were suspended in trypsin-EDTA (catalog number: 25200-072, Thermofisher Scientific, USA). Cell suspension was collected by centrifugation (125xg, 5 min), transferred to fresh culture medium and fresh flasks, and passaged. On the day of cell line transfer, the cultured cells were placed in centrifuge tubes, recovered, and then centrifuged (125xg, 5 min) to discard the supernatant. Cell suspension (5 × 10⁶ cells / mL) was then prepared using PBS (catalog number: LB001-04, Welgene Inc., KOREA). 6 Cells / 0.05ml) were stored on ice until inoculated. For this experiment, 8-week-old female NSGb2m (NOD.Cg-B2m) were used. tm1Unc Prkdc scid Il2rg tm1Wjl / SzJ) mice were purchased from JoongABio (Korea) and used. Body weight was measured the day after the end of quarantine and acclimatization. Then, a suspension of human PBMC cells (5 × 10⁶) prepared for healthy animals was used. 6Cells (0.2 ml / cell) were loaded into a disposable syringe and injected into the tail vein of the animal. General symptoms were observed once a day after cell transplantation.

[0315] The MDA-MB-231 cell suspension (5 × 10⁻⁶) will be prepared by... 6 A solution prepared using phenol red-free matrix gel (0.05 ml, 356237, BD, USA) was added to each cell line (0.05 ml) and filled into a disposable syringe. The solution was then injected subcutaneously (0.1 ml / animal) into the right back of animals that had received human PBMC transplantation. General symptoms were observed daily during the engraftment and growth phases after cell line transplantation.

[0316] After a certain period following cell transplantation, tumor volume was measured in mice that did not show abnormal health conditions, and 32 individuals were selected so that the average tumor volume of each group reached 40 mm. 3 Up to 80mm 3 Based on tumor volume and body weight, the selected animals were divided into 4 groups (8 animals in each group) as evenly as possible.

[0317] The test groups are shown in Table 4. The test material was injected into the animals using a disposable syringe (31G, 1ml) twice a week for a total of 4 injections.

[0318] [Table 4]

[0319]

[0320] During the observation period, observe general symptoms such as appearance, behavior, and excretion daily, and identify dead animals. Measure body weight on the day of cell line transplantation, twice a week, and on the day of animal euthanasia.

[0321] During the observation period, the maximum length (L) and vertical width (W) of the tumor were measured three times a week using a digital caliper (mitutoyo, Japan), and the tumor volume (TV) was calculated using the following equation.

[0322] Equation 1

[0323] TV(mm 3 ) = (W 2 ×L) / 2

[0324] Equation 2

[0325] %TGI (tumor growth inhibition) = (1 - (Ti - T0) / (Vi - V0)) × 100

[0326] The tumor volume of each individual was set to the measurement value at the time of classification before application.

[0327] On days 21, 25, 28, and 31 post-tumor transplantation, the drugs listed in Table 4 were administered. Results showed that tumor growth was inhibited in the GI101 or Keytruda monotherapy groups compared to the control group (hIgG4). Tumor growth was also inhibited in the GI101 and Keytruda combination therapy groups compared to the control group. Furthermore, tumor growth was inhibited in the GI101 and Keytruda combination therapy groups compared to the GI101 or Keytruda monotherapy groups. Figure 60 ).

[0328] Tumor growth inhibition rates were calculated at the end of the experiment (day 42 post-tumor transplantation) compared to day 1 of drug treatment (day 17 post-tumor transplantation). Results: In the hIgG4 treatment group, 2 mice had tumor growth inhibition rates of 30% or higher, 1 mouse had a tumor growth inhibition rate of 50% or higher, and 1 mouse had a tumor growth inhibition rate of 80% or higher; in the GI101 treatment group, 5 mice had tumor growth inhibition rates of 30% or higher, 5 mice had tumor growth inhibition rates of 50% or higher, and 2 mice had tumor growth inhibition rates of 80% or higher; in the Keytruda treatment group, 7 mice had tumor growth inhibition rates of 30% or higher, 5 mice had tumor growth inhibition rates of 50% or higher, and 3 mice had tumor growth inhibition rates of 80% or higher; in the GI101 and Keytruda combination treatment group, 8 mice had tumor growth inhibition rates of 30% or higher, 8 mice had tumor growth inhibition rates of 50% or higher, and 6 mice had tumor growth inhibition rates of 80% or higher. Figure 61 ).

[0329] Additionally, when GI101 and Keytruda were used in combination in mice transplanted with human breast cancer cells, the extent of tumor growth in individual experimental animals within each treatment group was shown as follows: Figures 62 to 66 middle.

[0330] Example 21. Detection of the anticancer effect of combined administration of mGI101 and anti-PD-1 antibody in mice with transplanted mouse-derived colorectal cancer cells.

[0331] This study evaluated the tumor growth inhibition effect of mGI101 (as the test material) and anti-PD-1 antibody (as the positive control material) after transplanting allogeneic mouse colon adenocarcinoma cells (MC38) into a C57BL / 6 mouse tumor model.

[0332] Mouse colon adenocarcinoma cells (MC38), a mouse-derived colorectal cancer cell line, were purchased from Kerafast (USA) and used for testing. MC38 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). Cultured cells were harvested using trypsin and resuspended in PBS. 1 × 10⁶ cells were then cultured. 6 MC38 cells were subcutaneously injected into the right abdomen of 7-week-old female C57BL / 6 mice to establish an allogeneic tumor transplantation model.

[0333] Based on tumor volume (30mm) 3 Mice were randomly assigned (n=5 per group). Tumor grafts were identified approximately 2 days after cell inoculation. The test group assignments and test materials administered are shown in Table 5.

[0334] [Table 5]

[0335]

[0336]

[0337] During the trial period, clinical symptoms such as disease and behavioral changes were observed daily, and dead animals were identified. Animals were euthanized after the trial period. The size of MC38 solid tumors was measured using a tumor 3D scanner (TM900, Peria Belgium). Mean weight loss and percentage change, as well as mean tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared to the solvent control group.

[0338] All statistical calculations were performed using Prism 8.0 (GraphPad Software Inc., USA). Tumor volume measurements were compared by one-way ANOVA (end time) followed by a Bonferroni multiple comparison test. A p-value < 0.05 was considered statistically significant.

[0339] Following administration of mGI101 and co-administration of mGI101 and anti-PD-1 antibody, all experimental animals remained healthy and showed no signs of pathological abnormalities. Figures 67 to 73The results of combination therapy with mGI101 and / or an anti-PD-1 antibody targeting MC38 tumors are presented. Anti-cancer effects were observed in the drug treatment groups compared to the control group, and tumor size was significantly different during the 16-day test period. MC38 tumors are known in previous literature as a model for anti-PD-1 antibody responses, and their anti-cancer effects were also observed in the anti-PD-1 antibody administration group in this trial (p > 0.01). As much anti-cancer effect was also found in the mGI101 (6 mpk) monotherapy group as in the anti-PD-1 antibody administration group (p > 0.01). The mGI101 (0.6 mpk) + anti-PD-1 (5 mpk) combination therapy group showed significant anti-cancer effects (p > 0.0001).

[0340] Individual tumor sizes in each experimental group are shown below. Figures 69 to 73 Based on individual tumor size results, slight tumor regression was observed in some animals in the anti-PD-1 antibody administration group. The mGI101 (6 mpk) monotherapy group showed better tumor growth inhibition than the anti-PD-1 antibody administration group. Tumor size remained the same until day 5-7, but regrowth occurred after day 7. The combination administration group (GI101 (0.6 mpk) + anti-PD-1 antibody (5 mpk)) showed significant tumor growth inhibition. In particular, two mice in the combination administration group showed a complete response (tumor-free).

[0341] MC38 cells were re-injected into the left ventral region of two mice in the combination therapy group that showed complete remission (opposite to the initial injection site of cancer cells). These mice were continuously administered anti-PD-1 antibody (5mpk, BIW) until day 32. Figure 74 A small tumor (>30 mm) was found in one of the two mice. 3 However, the tumor size stopped growing until day 35. Figure 69 After the tumor was re-injected, no tumor was found in another mouse. Figure 69 and Figure 74 ).

[0342] In summary, the antitumor efficacy of mGI101 alone and in combination with an anti-PD-1 antibody was tested in an MC38 allogeneic tumor model. The results showed that the combination administration group (GI101 (0.6 mpk) + anti-PD-1 (5 mpk)) exhibited the best antitumor efficacy. Both experimental animals in the combination administration group showed complete responses, and the mice that showed complete responses exhibited anticancer effects after re-injection of MC38 (Table 6).

[0343] [Table 6]

[0344]

[0345]

[0346] Example 22. Detection of the anticancer effect of combined administration of mGI101 and anti-PD-L1 antibody in mice with transplanted mouse-derived colorectal cancer cells.

[0347] This study evaluated the tumor growth inhibition effect of intraperitoneal administration of mGI101 (as test material) and anti-PD-L1 antibody (BioXcell, Cat#BE0101) (as positive control material) in a BALB / c mouse tumor model after transplantation of allogeneic mouse colon cancer cells (CT26) into the mouse.

[0348] CT26 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). Cultured cells were harvested using trypsin and resuspended in PBS. 5 × 10⁶ cells were then cultured in PBS. 5 CT26 cells were subcutaneously injected into the right abdomen of 7-week-old female BALB / c mice to establish an allogeneic tumor transplantation model.

[0349] Based on tumor volume (50mm) 3 Up to 120mm 3 Mice were randomly assigned (n=4 per group). Tumor grafts were identified approximately 2 days after cell inoculation. The test group assignments and test materials administered are shown in Table 7.

[0350] [Table 7]

[0351]

[0352] During the trial period, clinical symptoms such as disease and behavioral changes were observed daily, and dead animals were identified. Animals were euthanized after the trial period. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria Belgium). Mean weight loss and percentage change, as well as mean tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared to the solvent control group.

[0353] All statistical calculations were performed using Prism 8.0 (GraphPad Software Inc., USA). Tumor volume measurements were compared by one-way ANOVA (end time) followed by a Bonferroni multiple comparison test. A p-value < 0.05 was considered statistically significant.

[0354] The antitumor efficacy of mGI101 alone and in combination with an anti-PD-L1 antibody was tested in the CT26 allogeneic tumor model. Results: The combination administration group (mGI101 (3mpk) + anti-PD-L1 (10mpk)) showed the best antitumor efficacy. Figure 75 ).

[0355] Example 23. Detection of the anticancer effect of combined administration of mGI101 and anti-TIGIT antibody in mice with transplanted mouse-derived colorectal cancer cells.

[0356] This study evaluated the tumor growth inhibition effect of mGI101 (as the test material) and an anti-TIGIT antibody (as the positive control material) specifically binding to the extracellular domain (ECD) of anti-TIGIT with the amino acid sequence shown in SEQ ID NO:39) after transplantation of allogeneic mouse colon cancer cells (CT26) into a BALB / c mouse tumor model.

[0357] CT26 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). Cultured cells were harvested using trypsin and resuspended in PBS. 5 × 10⁶ cells were then cultured in PBS. 5 CT26 cells were subcutaneously injected into the right abdomen of 7-week-old female BALB / c mice to establish an allogeneic tumor transplantation model.

[0358] Based on tumor volume (50mm) 3 Up to 120mm 3 Mice were randomly assigned (n=5 per group). Tumor grafts were identified approximately 2 days after cell inoculation. The test group assignments and test materials administered are shown in Table 8.

[0359] [Table 8]

[0360]

[0361] During the trial period, clinical symptoms such as disease and behavioral changes were observed daily, and dead animals were identified. Animals were euthanized after the trial period. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria Belgium). Mean weight loss and percentage change, as well as mean tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared to the solvent control group.

[0362] All statistical calculations were performed using Prism 8.0 (GraphPad Software Inc., USA). Tumor volume measurements were compared by one-way ANOVA (end time) followed by a Bonferroni multiple comparison test. A p-value < 0.05 was considered statistically significant.

[0363] The antitumor efficacy of mGI101 alone and in combination with anti-TIGIT antibody was tested in the CT26 allogeneic tumor model. Results: The combination administration group (mGI101 (3mpk) + anti-TIGIT (20mpk)) showed the best antitumor efficacy. Figure 76 No antitumor effect was observed in the anti-TIGIT antibody alone group compared to the control group. However, the combined administration of anti-TIGIT antibody and mGI101 showed significantly better antitumor effect compared to the mGI101 alone group. SEQUENCE LISTING <110> GI Medical New <120> Pharmaceutical compositions for treating cancer include fusion proteins comprising IL-2 and CD80 proteins and immune checkpoint inhibitors. <130> P22112955WP <150> KR 10-2019-0154632 <151> 2019-11-27 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide (TPA) <400> 1 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala 20 25 <210> 2 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> CD80 fragment (hB7-1:35-242) <400> 2 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 4​​​​​​​​​​​​​​​​​​​​​​ 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 <210> 3 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Hinge area <400> 3 Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 20 25 30 <210> 4 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Immunoglobulin Fc domain <400> 4 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly 210 215 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> connector <400> 5 Gly Gly Gly Gly Ser 1 5 <210> 6 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> IL-2 variant with two amino acid substitutions: hIL-2M <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 7 <211> 617 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein containing IL-2 variant and CD80 fragment <400> 7 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Val Ile His Val Thr Lys Glu 20 25 30 Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu 35 40 45 Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val 50 55 60 Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn 65 70 75 80 Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala 85 90 95 Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr 100 105 110 Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser 115 120 125 Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro 130 135 140 Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro 145 150 155 160 Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile 165 170 175 Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser 180 185 190 Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu 195 200 205 Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr 210 215 220 Thr Lys Gln Glu His Phe Pro Asp Asn Gly Ser Gly Gly Gly Gly Ser 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly 245 250 255 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 260 265 270 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg 275 280 285 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 290 295 300 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 305 310 315 320 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 325 330 335 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 340 345 350 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 355 360 365 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 370 375 380 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 385 390 395 400 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 405 410 415 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 420 425 430 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 435 440 445 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 450 455 460 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly 465 470 475 480 Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 485 490 495 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 500 505 510 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe 515 520 525 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 530 535 540 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 545 550 555 560 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 565 570 575 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 580 585 590 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 595 600 605 Cys Gln Ser Ile Ile Ser Thr Leu Thr 610 615 <210> 8 <211> 1857 <212> DNA <213> Artificial Sequence(人工序列) <220> <223> Nucleotides encoding the fusion protein (GI101) <400> 8 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc 120 tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa 180 aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac 240 cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct 300 gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag 360 cacctggctg aagtgacact gtccgtgaag gccgactttc ccacaccttc catctccgac 420 ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct 480 gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg 540 tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc 600 accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc 660 aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct 720 ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct 780 ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct 840 areaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct 900 caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 960 aagaccaagc ctagagag acagttcaac tccacctaca gagtggtgtc cgtgctgacc 1020 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1080 ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag 1140 gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc 1200 ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct 1260 gagaacact aaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac 1320 tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg 1380 ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt 1440 ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat 1500 ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg 1560 accgccatgc tgaccgctaa gttctacatg cccaagaagg ccaccgagct gaagcacctc 1620 cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag 1680 aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg 1740 aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa 1800 tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac ctgatga 1857 <210> 9 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> GI101 <400> 9 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 325 330 335 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser 450 455 460 Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu 465 470 475 480 Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 485 490 495 Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 500 505 510 Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 515 520 525 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 530 535 540 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 545 550 555 560 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 565 570 575 Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585 590 <210> 10 <211> 133 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> Mature human IL-2 (hIL-2) <400> 10 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 11 <211> 288 <212> PRT <213> Artificial Sequence(人工序列)<​​​​​​​​​​​​​​​​​ Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> 12 <211> 215 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> Fc domain variant <400> 12 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 13 <211> 306 <212> PRT <213> Artificial Sequence <220> <223> mCD80 protein <400> 13 Met Ala Cys Asn Cys Gln Leu Met Gln Asp Thr Pro Leu Leu Lys Phe 1 5 10 15 Pro Cys Pro Arg Leu Ile Leu Leu Phe Val Leu Leu Ile Arg Leu Ser 20 25 30 Gln Val Ser Ser Asp Val Asp Glu Gln Leu Ser Lys Ser Val Lys Asp 35 40 45 Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Glu Asp Glu Ser 50 55 60 Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val Val Leu Ser Val 65 70 75 80 Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys Asn Arg Thr Leu 85 90 95 Tyr Asp Asn Thr Thr Tyr Tyr Ser Leu Ile Ile Leu Gly Leu Val Leu Ser 100 105 110 Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys Glu Arg Gly Thr 115 120 125 Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser Ile Lys Ala Asp 130 135 140 Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp Thr 145 150 155 160 Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe 165 170 175 Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile Asn Thr Thr Ile 180 185 190 Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser Ser Gln Leu Asp 195 200 205 Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu Ile Lys Tyr Gly 210 215 220 Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro Glu Asp 225 230 235 240 Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Phe Gly Ala Gly Phe Gly 245 250 255 Ala Val Ile Thr Val Val Val Ile Val Val Ile Ile Lys Cys Phe Cys 260 265 270 Lys His Arg Ser Cys Phe Arg Arg Asn Glu Ala Ser Arg Glu Thr Asn 275 280 285 Asn Ser Leu Thr Phe Gly Pro Glu Glu Ala Leu Ala Glu Gln Thr Val 290 295 300 Phe Leu 305 <210> 14 <211> 1848 <212> DNA <213> Artificial Sequence(人工序列) <220> <223> Nucleotides encoding the fusion protein (mGI101) <400> 14 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgga cgagcagctc tccaagtccg tgaaggataa ggtcctgctg 120 ccttgccggt acaactctcc tcacgaggac gagtctgagg accggatcta ctggcagaaa 180 cacgacaagg tggtgctgtc cgtgatcgcc ggaaagctga aagtgtggcc tgagtacaag 240 aacaggaccc tgtacgacaa caccacctac agcctgatca tcctgggcct cgtgctgagc 300 gatagaggca cctattcttg cgtggtgcag aagaaagagc ggggcaccta cgaagtgaag 360 cacctggctc tggtcaagct gtccatcaag gccgacttca gcacccctaa catcaccgag 420 tctggcaacc cttccgccga caccaagaga atcacctgtt tcgcctctgg cggcttccct 480 aagcctcggt tctcttggct ggaaaacggc agagagctgc ccggcatca taccaccatt 540 tctcaggacc cagagtccga gctgtacacc atctccagcc agctcgactt taacaccacc 600 agaaaccaca ccatcaagtg cctgattaag tacggcgacg cccacgtgtc cgaggacttt 660 acttgggaga aacctcctga ggaccctcct gactctgat ctggcggcgg aggttctggc 720 ggaggtggaa gcggaggcgg aggatctgct gagtctaagt atggccctcc ttgtcctcca 780 tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag 840 gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa 900 gaggaccctg aggtgcagtt caatggtac gtggacggcg tggagtgca caacgccaag 960 accaagccta gagaggaaca gttcaactcc acctatagag tggtgtccgt gctgaccgtg 1020 ctgcaccagg attggctgaa cggcaagg tacaagtgca aggtgtccaa caagggcctg 1080 ccttccagca tcgaaagac catcagcaag gctaagggcc agcctaggga acccaggtt 1140 tacaccctgc ctccaagcca agaggaatg accagaacc aggtccct gacctgcctg 1200 gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgag 1260 aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttctt tctgtactct 1320 cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg 1380 cacgaggctc tgcacaacca ctacacccag aagtccctgt ctctgtctct tggaggtggt 1440 ggcggttctg cccctacctc cagctctacc aagaaaaccc agctccagtt ggagcatctg 1500 ctgctggacc tccagatgat cctgaatggc atcaacaatt acaagaaccc caagctgacc 1560 gccatgctga ccgctaagtt ctacatgccc aagaaggcca ccgagctgaa gcacttgcag 1620 tgcctggaag aggaactgaa gcccctggaa gaagtgctga atctggccca gtccaagaac 1680 ttccacctga ggcctaggga cctgatctcc aacatcaacg tgatcgtgct ggaactgaaa 1740 ggctccgaga caaccttcat gtgcgagtac gccgacgaga cagccaccat cgtggaattt 1800 ; ctgaaccggt ggatcacctt ctgccagagc atcatctcca cactgacc 1848 <210> 15 <211> 616 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> fusion protein(mGI101) <400> 15 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Val Asp Glu Gln Leu Ser Lys 20 25 30 Ser Val Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His 35 40 45 Glu Asp Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val 50 55 60 Val Leu Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys 65 70 75 80 Asn Arg Thr Leu Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly 85 90 95 Leu Val Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys 100 105 110 Glu Arg Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser 115 120 125 Ile Lys Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro 130 135 140 Ser Ala Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro 145 150 155 160 Lys Pro Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile 165 170 175 Asn Thr Thr Ile Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser 180 185 190 Ser Gln Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu 195 200 205 Ile Lys Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys 210 215 220 Pro Pro Glu Asp Pro Pro Asp Ser Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro 245 250 255 Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 325 330 335 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340 345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 355 360 365 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 370 375 380 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 420 425 430 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 450 455 460 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly 465 470 475 480 Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln 485 490 495 Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn 500 505 510 Asn Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr 515 520 525 Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu 530 535 540 Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn 545 550 555 560 Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val 565 570 575 Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp 580 585 590 Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys 595 600 605 Gln Ser Ile Ile Ser Thr Leu Thr 610 615 <210> 16 <211> 1437 <212> DNA <213> Artificial Sequence (Artificial Sequence) <220> <223> Nucleotides encoding the fusion protein (GI101C1) <400> 16 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc 120 tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa 180 aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac 240 cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct 300 gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag 360 cacctggctg aagtgacact gtccgtgaag gccgactttc ccacaccttc catctccgac 420 ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct 480 gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg 540 tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc 600 accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc 660 aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct 720 ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct 780 ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct 840 areaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct 900 caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 960 aagaccaagc ctagagag acagttcaac tccacctaca gagtggtgtc cgtgctgacc 1020 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1080 ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag 1140 gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc 1200 ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct 1260 gagaacact aaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac 1320 tctcgcctga ccgtggacaa gtctaggtgg caaggggca acgtgttctc ctgctctgtg 1380 ctgcacgagg cctgcacaa tcactacacc cagaagtcc tgtctctgtc cctgggc 1437 <210> 17 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> GI101C1 (GI101C1) <400> 17 Will Ile His Will Thr Lys Glu Will Lys Glu Will Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Will Be Valid Glu Glu Leu Only Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 The Trp Pro Glu Tyr Lys Asn was formed by the Phe Asp 50 55 60 Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 325 330 335 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Leu Gly 450 <210> 18 <211> 1176 <212> DNA <213> Artificial Sequence <220> <223> GI101C2 (GI101C2) <400> 18 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccatctc acgccgctga gtctaagtac ggccctcctt gtcctccatg tcctgctcca 120 gaagctgctg gcggaccctc tgtgttcctg tttcctccaa agcctaagga ccagctcatg 180 atctctcgga cccctgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccctgag 240 gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga 300 gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat 360 tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc ttccagcatc 420 gaaaagacca tctccaaggc taagggccag cctagggaac cccaggttta caccctgcct 480 ccaagccaag aggaaatgac caagaaccag gtgtccctga cctgcctggt caagggcttc 540 tacccttccg acattgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag 600 accacacctc ctgtgctgga ctccgacggc tccttctttc tgtactctcg cctgaccgtg 660 gacaagtcta ggtggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg 720 cacaatcact acacccagaa gtccctgtct ctgtctcttg gcggaggcgg aggatctgct 780 cctacctcca gctccaccaa gaaaacccag ctccagttgg agcatctgct gctggacctc 840 cagatgatcc tgaatggcat caacaattac aagaacccca agctgaccgc catgctgacc 900 gctaagttct acatgcccaa gaaggccacc gagctgaagc acctccagtg cctggaagag 960 gaactgaagc ccctggaaga agtgctgaat ctggcccagt ccaagaactt ccacctgagg 1020 cctagggacc tgatctccaa catcaacgtg atcgtgctgg aactgaaagg ctccgagaca 1080 accttcatgt gcgagtacgc cgacgagaca gccaccatcg tggaatttct gaaccggtgg 1140 atcaccttct gccagtccat catctccaca ctgacc 1176 <210> 19 <211> 367 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> Fusion protein (GI101C2) <400> 19 Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 1 5 10 15 Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 25 30 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 35 40 45 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 50 55 60 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 65 70 75 80 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 85 90 95 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 100 105 110 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 115 120 125 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 130 135 140 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 145 150 155 160 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 165 170 175 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 180 185 190 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 195 200 205 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 210 215 220 Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 225 230 235 240 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 245 250 255 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala 260 265 270 Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 275 280 285 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 290 295 300 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 305 310 315 320 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 325 330 335 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 340 345 350 Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 355 360 365 <210> 20 <211> 1434 <212> DNA <213> Artificial Sequence <220> <223> Nucleotides encoding the fusion protein (mGI101C1) <400> 20 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgga cgagcagctc tccaagtccg tgaaggataa ggtcctgctg 120 ccttgccggt acaactctcc tcacgaggac gagtctgagg accggatcta ctggcagaaa 180 cacgacaagg tggtgctgtc cgtgatcgcc ggaaagctga aagtgtggcc tgagtacaag 240 aacaggaccc tgtacgacaa caccacctac agcctgatca tcctgggcct cgtgctgagc 300 gatagaggca cctattcttg cgtggtgcag aagaaagagc ggggcaccta cgaagtgaag 360 cacctggctc tggtcaagct gtccatcaag gccgacttca gcacccctaa catcaccgag 420 tctggcaacc cttccgccga caccaagaga atcacctgtt tcgcctctgg cggcttccct 480 aagcctcggt tctcttggct ggaaaacggc agagagctgc ccggcatcaa taccaccatt 540 tctcaggacc cagagtccga gctgtacacc atctccagcc agctcgactt taacaccacc 600 agaaaccaca ccatcaagtg cctgattaag tacggcgacg cccacgtgtc cgaggacttt 660 acttgggaga aacctcctga ggaccctcct gactctggat ctggcggcgg aggttctggc 720 ggaggtggaa gcggaggcgg aggatctgct gagtctaagt atggccctcc ttgtcctcca 780 tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag 840 gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa 900 gaggaccctg aggtgcagtt caattggtac gtggacggcg tggaagtgca caacgccaag 960 accaagccta gagaggaaca gttcaactcc acctatagag tggtgtccgt gctgaccgtg 1020 ctgcaccagg attggctgaa cggcaaagag tacaagtgca aggtgtccaa caagggcctg 1080 ccttccagca tcgaaaagac catcagcaag gctaagggcc agcctaggga accccaggtt 1140 tacaccctgc ctccaagcca agaggaaatg accaagaacc aggtgtccct gacctgcctg 1200 gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgag 1260 aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttctt tctgtactct 1320 cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg 1380 cacgaggctc tgcacaacca ctacacccag aagtccctgt ctctgtccct gggc 1434 <210> 21 <211> 478 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> Fusion protein (mGI101C1) <400> 21 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Val Asp Glu Gln Leu Ser Lys 20 25 30 Ser Val Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His 35 40 45 Glu Asp Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val 50 55 60 Val Leu Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys 65 70 75 80 Asn Arg Thr Leu Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly 85 90 95 Leu Val Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys 100 105 110 Glu Arg Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser 115 120 125 Ile Lys Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro 130 135 140 Ser Ala Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro 145 150 155 160 Lys Pro Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile 165 170 175 Asn Thr Thr Ile Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser 180 185 190 Ser Gln Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu 195 200 205 Ile Lys Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys 210 215 220 Pro Pro Glu Asp Pro Pro Asp Ser Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro 245 250 255 Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 325 330 335 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340 345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 355 360 365 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 370 375 380 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 420 425 430 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 450 455 460 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 465 470 475 <210> 22 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> IL‑2 change body:IL‑2(3M, M45) <400> 22 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Ala Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 23 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> IL‑2 change body:IL‑2(3M, M61) <400> 23 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 24 <211> 133 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> IL-variant: IL-2 (3M, M72) <400> 24 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 25 <211> 1851 <212> DNA <213> Artificial Sequence(人工序列) <220> <223> Nucleotides encoding the fusion protein (GI102-M45) <400> 25 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc 120 tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa 180 aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac 240 cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct 300 gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag 360 cacctggctg aagtgacact gtccgtgaag gccgactttc ccacaccttc catctccgac 420 ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct 480 gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg 540 tctcaggacc ccgaaaccga gctctacgct gtgtcctcca agctggactt caacatgacc 600 accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc 660 aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct 720 ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct 780 ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct 840 areaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct 900 caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 960 aagaccaagc ctagagag acagttcaac tccacctaca gagtggtgtc cgtgctgacc 1020 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1080 ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag 1140 gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc 1200 ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct 1260 gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt cttctgtac 1320 tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg 1380 ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt 1440 ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat 1500 ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg 1560 accgccatgc tgaccgctaa gttcgccatg cccaagaagg ccaccgagct gaagcacctc 1620 cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag 1680 aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg 1740 aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa 1800 tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c 1851 <210> 26 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein (GI102-M45) <400> 26 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125​​​​​Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 325 330 335 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser 450 455 460 Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu 465 470 475 480 Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 485 490 495 Ala Met Leu Thr Ala Lys Phe Ala Met Pro Lys Lys Ala Thr Glu Leu<00\02025>500 505 510 Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 515 520 525 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 530 535 540 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 545 550 555 560 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 565 570 575 Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585 590<\ <210> 27 <211> 1851 <212> DNA <213> Artificial Sequence(人工序列) <220> <223> Nucleotides encoding the fusion protein (GI102 - M61) <400> 27 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc 120 tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa 180 aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac 240 cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct 300 gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag 360 cacctggctg aagtgacact gtccgtgaag gccgactttc ccacaccttc catctccgac 420 ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct 480 gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg 540 tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc 600 accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc 660 aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct 720 ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct 780 ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct 840 areaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct 900 caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 960 aagaccaagc ctagagag acagttcaac tccacctaca gagtggtgtc cgtgctgacc 1020 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1080 ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag 1140 gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc 1200 ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct 1260 gagaacact aaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac 1320 tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg 1380 ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt 1440 ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat 1500 ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg 1560 accgccatgc tgaccgctaa gttctacatg cccaagaagg ccaccgagct gaagcacctc 1620 cagtgcctgg aaagggaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag 1680 aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg 1740 aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa 1800 tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c 1851 <210> 28 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> GI102‑M61 <400> 28 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 325 330 335 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser 450 455 460 Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu 465 470 475 480 Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 485 490 495 Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 500 505 510 Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val 515 520 525 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 530 535 540 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 545 550 555 560 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 565 570 575 Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585 590 <210> 29 <211> 1857 <212> DNA <213> Artificial Sequence <220> <223> Nucleotides encoding the fusion protein (GI102-M72) <400> 29 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc 120 tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa 180 aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac 240 cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct 300 gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag 360 cacctggctg aagtgacact gtccgtgaag gccgactttc ccacaccttc catctccgac 420 ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct 480 gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg 540 tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc 600 accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc 660 aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct 720 ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct 780 ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct 840 aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct 900 caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 960 aagaccaagc ctagagag acagttcaac tccacctaca gagtggtgtc cgtgctgacc 1020 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1080 ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag 1140 gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc 1200 ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct 1260 gagaacact aaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac 1320 tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg 1380 ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt 1440 ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat 1500 ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg 1560 accgccatgc tgaccgctaa gttctacatg cccaagaagg ccaccgagct gaagcacctc 1620 cagtgcctgg aagagact gaagcccctg gaagaggtgc tgaatggggc ccagtccaag 1680 aacttccacc tgaggccacg ggacctgatc agcacatca acgtcgt gctggactg 1740 aagggctccg agacacctt tgtgcgag tacgccgacg agacagccac catcgtggaa 1800 tttctgaacc gtgatcac cttctgaccag agcatcatct cacactgac ctgatga 1857 <210> 30 <211> 592 <212> PRT <213> Artificial Sequence <220> <223> Section (GI102-M72) <400> 30 Will Ile His Will Thr Lys Glu Will Lys Glu Will Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Will Be Valid Glu Glu Leu Only Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 The Trp Pro Glu Tyr Lys Asn was formed by the Phe Asp 50 55 60 Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 325 330 335 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser 450 455 460 Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu 465 470 475 480 Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 485 490 495 Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 500 505 510 Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 515 520 525 Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 530 535 540 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 545,550,555,560 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 565,570,575 Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 580,585,590 <210> 31 <211> 1851 <212> DNA <213> Artificial Sequence <220> <223> GI101w (GI101w) <400> 31 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc 120 tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa 180 aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac 240 cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct 300 gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag 360 cacctggctg aagtgacact gtccgtgaag gccgactttc ccacaccttc catctccgac 420 ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct 480 gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg 540 tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc 600 accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc 660 aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct 720 ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct 780 ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct 840 aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct 900 caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 960 aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc 1020 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1080 ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag 1140 gttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc 1200 ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct 1260 gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt cttctgtac 1320 tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg 1380 ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt 1440 ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat 1500 ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg 1560 acccgcatgc tgacctttaa gttctacatg cccaagaagg ccaccgagct gaagcacctc 1620 cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag 1680 aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg 1740 aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa 1800 tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c 1851 <210> 32 <211> 592 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Fusion protein (GI101w) <400> 32 Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1 5 10 15 Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20 25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn 35 40 45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn 50 55 60 Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65 70 75 80 Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85 90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser 100 105 110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys 115 120 125 Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130 135 140 Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150 155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr 165 170 175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn 180 185 190 Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195 200 205 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 325 330 335 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser 450 455 460 Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu 465 470 475 480 Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr 485 490 495 Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu 500 505 510 Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 515 520 525 Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 530 535 540 Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 545 550 555 560 Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 565 570 575 Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585 590 <210> 33 <211> 1848 <212> Ms <213> Artificial Sequence <220> <223> Nucleotides encoding the fusion protein (mGI102-M61) <400> 33 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg 60 tctccttctc acgctgtgga cgagcagctc tccaagtccg tgaaggataa ggtcctgctg 120 ccttgccggt acaactctcc tcacgaggac gagtctgagg accggatcta ctggcagaaa 180 cacgacaagg tggtgctgtc cgtgatcgcc ggaaagctga aagtgtggcc tgagtacaag 240 aacaggaccc tgtacgacaa caccacctac agcctgatca tcctgggcct cgtgctgagc 300 gatagaggca cctattcttg cgtggtgcag aagaaagagc ggggcaccta cgaagtgaag 360 cacctggctc tggtcaagct gtccatcaag gccgacttca gcacccctaa catcaccgag 420 tctggcaacc cttccgccga caccaagaga atcacctgtt tcgcctctgg cggcttccct 480 aagcctcggt tctcttggct ggaaaacggc agagagctgc ccggcatcaa taccaccatt 540 tctcaggacc cagagtccga gctgtacacc atctccagcc agctcgactt taacaccacc 600 agaaaccaca ccatcaagtg cctgattaag tacggcgacg cccacgtgtc cgaggacttt 660 acttgggaga aacctcctga ggaccctcct gactctgat ctggcggcgg aggttctggc 720 ggaggtggaa gcggaggcgg aggatctgct gagtctaagt atggccctcc ttgtcctcca 780 tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag 840 gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa 900 gaggaccctg aggtgcagtt caatggtac gtggacggcg tggagtgca caacgccaag 960 accaagccta gagaggaaca gttcaactcc acctatagag tggtgtccgt gctgaccgtg 1020 ctgcaccagg attggctgaa cggcaagg tacaagtgca aggtgtccaa caagggcctg 1080 ccttccagca tcgaaagac catcagcaag gctaagggcc agcctaggga acccaggtt 1140 tacaccctgc ctccaagcca agaggaatg accagaacc aggtccct gacctgcctg 1200 gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgg 1260 aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttct tctgtactct 1320 cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg 1380 cacgaggctc tgcacaacca ctacacccag aagtccctgt ctctgtctct tggaggtggt 1440 ggcggttctg cccctacctc cagctctacc aagaaaaccc agctccagtt ggagcatctg 1500 ctgctggacc tccagatgat cctgaatggc atcaacaatt acaagaaccc caagctgacc 1560 gccatgctga ccgctaagtt ctacatgccc aagaaggcca ccgagctgaa gcacttgcag 1620 tgcctggaaa gggaactgaa gcccctggaa gaagtgctga atctggccca gtccaagaac 1680 ttccacctga ggcctaggga cctgatctcc aacatcaacg tgatcgtgct ggaactgaaa 1740 ggctccgaga caaccttcat gtgcgagtac gccgacgaga cagccaccat cgtggaattt 1800 ctgaaccggt ggatcacctt ctgccagagc atcatctcca cactgacc 1848 <210> 34 <211> 616 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> Fusion protein (mGI102-M61) <400> 34 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Val Asp Glu Gln Leu Ser Lys 20 25 30 Ser Val Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His 35 40 45 Glu Asp Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val 50 55 60 Val Leu Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys 65 70 75 80 Asn Arg Thr Leu Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly 85 90 95 Leu Val Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys 100 105 110 Glu Arg Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser 115 120 125 Ile Lys Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro 130 135 140 Ser Ala Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro 145 150 155 160 Lys Pro Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile 165 170 175 Asn Thr Thr Ile Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser 180 185 190 Ser Gln Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu 195 200 205 Ile Lys Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys 210 215 220 Pro Pro Glu Asp Pro Pro Asp Ser Gly Ser Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro 245 250 255 Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 325 330 335 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340 345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 355 360 365 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 370 375 380 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 420 425 430 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 450 455 460 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly 465 470 475 480 Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln 485 490 495 Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn 500 505 510 Asn Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr 515 520 525 Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg 530 535 540 Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn 545 550 555 560 Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val 565 570 575 Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp 580 585 590 Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys 595 600 605 Gln Ser Ile Ile Ser Thr Leu Thr 610 615 <210> 35 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> wild-type hIL‑2 <400> 35 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 36 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> IL-2 signal chain <400> 36 Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Ala Pro Thr Ser Ser Ser Thr 20 25 30 Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met 35 40 45 Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met 50 55 60 Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His 65 70 75 80 Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn 85 90 95 Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser 100 105 110 Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe 115 120 125 Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn 130 135 140 Arg Trp Ile Thr Phe Cys Gln Ile Ile Ser Thr Leu Thr 145 150 155 <210> 37 <211> 474 <212> DNA <213> Artificial Sequence(Artificial Sequence) <220> <223> Thanks for watching IL‑2 snowflakes <400> 37 atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg tctccttctc acgctgcccc taccagctcc tctaccaaga aaacccagct ccagttggag catctgctgc tggacctcca gatgattctg aacgggatca acaactata gaaccccaag ctgacccgca tgctgacctt tagttctac atgcccaaga aggccaccga gctgaagcac ctccagtgcc tggagaga actgaagccc ctggagagg tgctgaatct ggcccagtcc aagaacttcc acctgaggcc acgggacctg atcagcaaca tcaacgtgat cgtgctggaa ctgaagggct ccgagacaac ctttatgtgc gagtacgccg acgagacagc caccatcgtg gaatttctga accggtggat caccttctgc cagagcatca tctccacact gacc <210> 38 <211> 591 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein(mGI‑101) <400> 38 Val Asp Glu Gln Leu Ser Lys Ser Val Lys Asp Lys Val Leu Leu Pro 1 5 10 15 Cys Arg Tyr Asn Ser Pro His Glu Asp Glu Ser Glu Asp Arg Ile Tyr 20 25 30 Trp Gln Lys His Asp Lys Val Val Leu Ser Val Ile Ala Gly Lys Leu 35 40 45 Lys Val Trp Pro Glu Tyr Lys Asn Arg Thr Leu Tyr Asp Asn Thr Thr 50 55 60 Tyr Ser Leu Ile Ile Leu Gly Leu Val Leu Ser Asp Arg Gly Thr Tyr 65 70 75 80 Ser Cys Val Val Gln Lys Lys Glu Arg Gly Thr Tyr Glu Val Lys His 85 90 95 Leu Ala Leu Val Lys Leu Ser Ile Lys Ala Asp Phe Ser Thr Pro Asn 100 105 110 Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp Thr Lys Arg Ile Thr Cys 115 120 125 Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe Ser Trp Leu Glu Asn 130 135 140 Gly Arg Glu Leu Pro Gly Ile Asn Thr Thr Ile Ser Gln Asp Pro Glu 145 150 155 160 Ser Glu Leu Tyr Thr Ile Ser Ser Gln Leu Asp Phe Asn Thr Thr Arg 165 170 175 Asn His Thr Ile Lys Cys Leu Ile Lys Tyr Gly Asp Ala His Val Ser 180 185 190 Glu Asp Phe Thr Trp Glu Lys Pro Pro Glu Asp Pro Pro Asp Ser Gly 195 200 205 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 325 330 335 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser 450 455 460 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln 465 470 475 480 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala 485 490 495 Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys 500 505 510 His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu 515 520 525 Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile 530 535 540 Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr 545 550 555 560 Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu 565 570 575 Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr 580 585 590 <210> 39 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Extracellular domain of anti-TIGIT <400> 39 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 10​​​​

Claims

1. A pharmaceutical composition for the prevention or treatment of cancer, said pharmaceutical composition comprising, as an active ingredient, a fusion protein dimer comprising an IL-2 variant and a CD80 fragment, and an immune checkpoint inhibitor; The fusion protein described herein is composed of the following structural formula (I): N'-X-[Connector(1)] n -Fc structural domain-[joint(2)] m -YC' (I) in, N' is the N-terminus of the fusion protein. C' is the C-terminus of the fusion protein. X represents the CD80 fragment, wherein the CD80 fragment is the extracellular domain of the CD80 protein, consisting of amino acids 35 to 242 of SEQ ID NO:

11. Y represents an IL-2 variant consisting of substitutions of R38A and F42A in the amino acid sequence shown in SEQ ID NO:

10. The linker (1) is a peptide linker composed of 30 to 40 amino acids. The linker (2) is a peptide linker composed of 5 to 15 amino acids. n and m are each independently 0 or 1; And the immune checkpoint inhibitor mentioned therein is at least one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody and anti-TIGIT antibody.

2. The pharmaceutical composition for the prevention or treatment of cancer according to claim 1, wherein, The fusion protein has the amino acid sequence shown in SEQ ID NO:

9.

3. The pharmaceutical composition for the prevention or treatment of cancer according to claim 1, wherein, The anti-PD-1 antibody is selected from any one of the following groups: pembrolizumab, nivolumab, cimiprimab, JTX-4014, spartazumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dotalimab, INCMGA00012, AMP-224, and AMP-514; or, The anti-PD-L1 antibody is selected from any one of the following groups: atezolizumab, avelumumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189.

4. The pharmaceutical composition for the prevention or treatment of cancer according to claim 1, wherein, The cancer is selected from any one of the following groups: stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

5. The use of a fusion protein dimer comprising an IL-2 variant and a CD80 fragment, and an immune checkpoint inhibitor, in the prevention or treatment of cancer; The fusion protein described herein is composed of the following structural formula (I): N'-X-[Connector(1)] n -Fc structural domain-[joint(2)] m -YC' (I) in, N' is the N-terminus of the fusion protein. C' is the C-terminus of the fusion protein. X represents the CD80 fragment, wherein the CD80 fragment is the extracellular domain of the CD80 protein, consisting of amino acids 35 to 242 of SEQ ID NO:

11. Y represents an IL-2 variant consisting of substitutions of R38A and F42A in the amino acid sequence shown in SEQ ID NO:

10. The linker (1) is a peptide linker composed of 30 to 40 amino acids. The linker (2) is a peptide linker composed of 5 to 15 amino acids. n and m are each independently 0 or 1; The immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-TIGIT antibody. And the cancer mentioned therein is breast cancer or colorectal cancer.

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