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

Abstract

The present invention provides a pharmaceutical composition for cancer treatment, comprising as active ingredients a fusion protein dimer comprising an IL-2 protein or variant thereof and a CD80 protein or fragment thereof, and an immune checkpoint inhibitor. One embodiment of the present invention, a fusion protein comprising a CD80 fragment, immunoglobulin Fc, and an IL-2 variant, can activate immune cells such as natural killer cells and simultaneously suppress the immune cell-regulating activity of regulatory T cells. Furthermore, when administered in combination with an immune checkpoint inhibitor such as Keytruda, a known PD-1 inhibitor, cancer can be effectively suppressed. Therefore, a pharmaceutical composition comprising as active ingredients a fusion protein comprising an IL-2 protein or variant thereof and a CD80 protein or fragment thereof, and an immune checkpoint inhibitor, can be effectively used in cancer treatment and has high industrial applicability.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for cancer treatment containing, as an active ingredient, a fusion protein dimer containing an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

Background Art

[0002] IL-2 (Interleukin 2) is also called T-cell growth factors (TCGF), and is a globular glycoprotein that plays a central role in lymphocyte production, survival, and homeostasis. The size of the IL-2 protein is 15.5 kDa to 16 kDa and consists of 133 amino acids. IL-2 mediates various immune effects by binding to the IL-2 receptor composed of three individual subunits.

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

[0004] On the one hand, CD80, also known as B7-1, is one of the B7 family of membrane-bound proteins that bind to ligands and transmit costimulatory and coinhibitory responses to participate in immune regulation. CD80 is a transmembrane protein expressed on the surfaces of T cells, B cells, dendritic cells, and monocytes. CD80 is known to bind to CD28, CTLA-4 (CD152), and PD-L1 (programmed cell death ligand 1). CD80, CD86, CTLA-4, and CD28 are involved in the costimulatory-coinhibitory system. For example, they regulate the activity of T cells and are involved in proliferation, differentiation, and survival.

[0005] Also, recently, immune checkpoint inhibitors such as Keytruda (登録商標) ) have attracted attention. Immune checkpoint inhibitors are anti-cancer drugs that assist in activating our body's immune system to attack cancer cells. So far, cancer treatment has focused on killing rapidly dividing cells, which are characteristic of cancer cells, and thus has also affected rapidly dividing cells among normal cells, resulting in side effects. However, immune anti-cancer drugs utilize the immune system of cancer patients to affect cancer cells and are known to have few typical side effects of existing anti-cancer drugs. Anti-PD-1 antibodies such as Keytruda bind to a specific receptor (PD-1) on T cells and block the pathway by which cancer cells avoid the surveillance system of activated T cells, thereby showing an anti-cancer effect through immune reactivation that enables T cells in the human body to attack cancer cells (KR10-2018-0030580A).

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, as a result of research to develop a safe and effective anticancer agent, the present inventors completed the present invention by confirming that a novel fusion protein dimer containing an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof within a single molecule and an immune checkpoint inhibitor exhibit excellent anticancer effects.

Means for Solving the Problems

[0007] In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for treating cancer containing, as an active ingredient, a fusion protein dimer containing an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

Effects of the Invention

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

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

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

[0011] Immune checkpoint inhibitor As used herein, the term "immune checkpoint" refers to an intracellular signaling system that maintains self-tolerance and protects tissues from excessive immune responses that cause damage. Immune checkpoint proteins are cell membrane proteins that regulate immune checkpoints and can suppress the differentiation, proliferation, and activation of immune cells. Specifically, immune checkpoint proteins are expressed in activated T cells and have the function of reducing T cell proliferation, cytokine secretion, and cytotoxicity, and suppressing the excessive activation of T cells. Some immune checkpoints are known as one of the main mechanisms by which tumor cells achieve immune evasion. Therefore, an "immune checkpoint inhibitor" targets immune checkpoint proteins to inhibit or block immune checkpoints, thereby increasing T cell activation, enhancing anti-tumor immunity, and showing an anti-cancer effect. In addition to the advantages of having fewer side effects such as vomiting and hair loss and a greater therapeutic effect than general cytotoxic anti-cancer drugs, immune checkpoint inhibitors utilize an immune response system with excellent memory ability, and it is known that the therapeutic effect persists for a long time even after drug administration is interrupted.

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

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

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

[0015] As used herein, the term "PD-1 (programmed cell death protein 1)" is also known as CD279 and is a protein expressed on the surface of activated T cells. It reacts with PD-L1 (B7-H1) and PD-L2 (B7-DC), which are proteins on the surface of cancer cells, and suppresses the activity of T cells mediated by TCR (T cell receptor) and CD28, as well as the production of growth factors and cytokines, thereby inducing negative signal transduction. PD-1 inhibitors include, for example, pembrolizumab (Keytruda (登録商標) ), MK-3475, nivolumab (Opdivo (登録商標) ), semipramab (Ribtayo (登録商標) ), JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514.

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

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

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

[0019] As used herein, the term "TIM3 (T cell membrane protein 3)" is also referred to as hepatitis A virus cellular receptor 2 (HAVCR2) and is expressed in various immune cells. When activated by binding to the water-soluble protein GAL9 (galectin 9), the influx of intracellular calcium increases, inducing T cell death, which consequently causes immune tolerance. In addition, TIM3, together with GAL9, binds to the cell surface protein CEACAM1 (cell adhesion molecule 1) to suppress the immune activity of T cells, and binds to the water-soluble proteins HMGB1 (high mobility group protein 1) or PTdSer (phospatidyl serine) to suppress immune activity. TIM3 inhibitors may be LY3321367, MBG453, and TSR-022.

[0020] As used herein, the term "LAG3 (lymphocyte activation gene 3)" is referred to as CD223 and binds to MHC (major histocompatibility complex) class II to suppress the proliferation and activity of T cells. LAG3 inhibitors may be IMP321, relatlimab, and GSK2831781.

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

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

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

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

[0025] In the present specification, IL-2 or its variants may also be collectively referred to 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 confirmed by methods known to those skilled in the art.

[0026] One specific example of the IL-2 can have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Also, at this time, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not contain a signal sequence or may have the amino acid sequence of SEQ ID NO: 10. At this time, the IL-2 can be utilized as a concept including a truncated fragment in which a part of the N-terminus or C-terminus of wild-type IL-2 is deleted.

[0027] Also, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are continuously deleted from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Further, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are continuously deleted from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0028] As used herein, the term "IL-2 variant" means a form in which a part of the amino acids of full-length IL-2 or the fragment of IL-2 described above is substituted. That is, the IL-2 variant can have an amino acid sequence different from that of wild-type IL-2 or its fragment. However, the IL-2 variant can have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" can mean, for example, specifically binding to an IL-2 receptor, and this specific binding can be measured by methods known to those skilled in the art.

[0029] Specifically, the IL-2 variant may be one in which a part of the amino acids of wild-type IL-2 is substituted. As a specific example of the IL-2 variant by amino acid substitution, at least one of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10 may be substituted.

[0030] Specifically, the IL-2 variant may be one in which at least any one of the amino acids at positions 38, 42, 45, 61, or 72 in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid. Moreover, in the case where IL-2 is in a form in which a part of the N-terminus of the amino acid sequence of SEQ ID NO: 35 is deleted, the amino acids at positions corresponding complementarily in the amino acid sequence of SEQ ID NO: 10 may be substituted with other amino acids. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 variant may be one in which at least any one of the amino acids at positions 58, 62, 65, 81, or 92 in the amino acid sequence of SEQ ID NO: 35 is substituted with another amino acid. These respectively correspond to the amino acid residues at positions 38, 42, 45, 61, and 72 of the amino acid sequence of SEQ ID NO: 10. According to one specific example, as long as IL-2 activity is maintained, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted. According to another specific example, 1 to 5 amino acids may be substituted.

[0031] As a specific example, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may be one in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Incidentally, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted.

[0032] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may be one in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Note that, as a specific example, the IL-2 variant may be one in which the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted.

[0033] In addition, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may be one in which the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Also, as a specific example, the IL-2 variant may be one in which the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Note that, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted.

[0034] Furthermore, the IL-2 variant may be in a form in which five amino acids are substituted. Specifically, the IL-2 variant may be one in which the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are all substituted with other amino acids. At this time, the "other amino acid" introduced by the above substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, in the amino acid substitution of the IL-2 variant, the 38th amino acid in the amino acid sequence of SEQ ID NO: 10 cannot be substituted with arginine, the 42nd amino acid cannot be substituted with phenylalanine, the 45th amino acid cannot be substituted with tyrosine, the 61st amino acid cannot be substituted with glutamic acid, and the 72nd amino acid cannot be substituted with leucine.

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

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

[0037] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with other amino acids except tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (Y45A).

[0038] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with other amino acids except glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with arginine (E61A).

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

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

[0041] Specifically, the IL-2 variant may have amino acid substitutions at 2, 3, 4, or 5 positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

[0042] In addition, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may be one in which substitutions have occurred at R38A and F42A. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A and Y45A. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A and E61R. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A and L72G. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A and Y45A. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A and E61R. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A and L72G. Note that, as a specific example, the IL-2 variant may be one in which substitutions have occurred at E61R and L72G.

[0043] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may be one in which substitutions have occurred at R38A, F42A, and Y45A. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, F42A, and E61R. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, F42A, and L72G. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A, E61R, and L72G. Note that, as a specific example, the IL-2 variant may be one in which substitutions have occurred at Y45A, E61R, and L72G.

[0044] In addition, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may be one in which substitutions have occurred at R38A, F42A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, F42A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, F42A, E61R, and L72G. Also, as a specific example, the IL-2 variant may be one in which substitutions have occurred at R38A, Y45A, E61R, and L72G. Note that, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A, Y45A, E61R, and L72G.

[0045] Furthermore, the IL-2 variant may have substitutions at R38A, F42A, Y45A, E61R, and L72G.

[0046] Preferably, one specific example of the IL-2 variant may be one in which any one combination selected from the following combinations (a) to (d) has a substitution in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R (d) R38A / F42A / L72G

[0047] At this time, when IL-2 has the amino acid sequence of SEQ ID NO: 35, it can have an amino acid substitution at a position corresponding complementarily to SEQ ID NO: 10. Also, even when IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, the amino acid at the position corresponding complementarily to SEQ ID NO: 10 may be substituted.

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

[0049] In addition, the IL-2 variant may be characterized by having low toxicity in vivo. At this time, the low toxicity in vivo may be a side effect induced by the binding of IL-2 to the alpha chain (IL-2Rα) of the IL-2 receptor. A variety of IL-2 variants have been developed to improve the side effects caused by the binding of IL-2 and IL-2Rα, and such IL-2 variants can use those disclosed in US Patent 5,229,109 and Korean Patent 1667096. In particular, the IL-2 variant described in the present application has a low binding force to the alpha chain (IL-2Rα) of the IL-2 receptor and has lower in vivo toxicity than wild-type IL-2.

[0050] As used herein, the term "CD80" is also referred to as "B7-1" and is a membrane protein present in dendritic cells, activated B cells, and monocytes. CD80 provides a co-stimulatory signal essential for the activation and survival of T cells. CD80 is known as a ligand for two different proteins, CD28 and CTLA-4, present on the surface of T cells. CD80 is composed of 288 amino acids and may specifically have the amino acid sequence of SEQ ID NO: 11. Also, as used herein, the "CD80 protein" means the full-length CD80 or a CD80 fragment.

[0051] As used herein, the term "CD80 fragment" means a cleaved form of CD80. Further, the CD80 fragment may be the extracellular domain of CD80. As a specific example of the CD80 fragment, it may be one from which the 1st to 34th amino acids from the N-terminus, which is the signal sequence of CD80, are removed. Specifically, a specific example of the CD80 fragment may be a protein composed of the 35th to 288th amino acids of SEQ ID NO: 11. Also, a specific example of the CD80 fragment may be a protein composed of the 35th to 242nd amino acids of SEQ ID NO: 11. Also, a specific example of the CD80 fragment may be a protein composed of the 35th to 232nd amino acids of SEQ ID NO: 11. Also, a specific example of the CD80 fragment may be a protein composed of the 35th to 139th amino acids of SEQ ID NO: 11. Note that a specific example of the CD80 fragment may be a protein composed of the 142nd to 242nd amino acids of SEQ ID NO: 11. As an example, the CD80 fragment may have the amino acid sequence of SEQ ID NO: 2.

[0052] Also, the IL-2 protein and the CD80 protein may be bound by a linker or a carrier. Specifically, the IL-2 or its variant and the CD80 (B7-1) or its fragment may be bound by a linker or a carrier. As used herein, linker and carrier may be used interchangeably.

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

[0054] In addition, the Fc domain of the immunoglobulin may be not only a wild-type Fc domain but also an Fc domain variant. Also, the term "Fc domain variant" as used herein may be different from the glycosylation pattern of the wild-type Fc domain, have an increased sugar chain compared to the wild-type Fc domain, have a decreased sugar chain compared to the wild-type Fc domain, or be in a form in which the sugar chain is deglycosylated. Note that an aglycosylated Fc domain is also included. The Fc domain or variant may be adjusted to have a certain number of sialic acid, fucosylation, and glycosylation by culture conditions or host gene recombination.

[0055] In addition, the sugar chains of the Fc domain of immunoglobulins can be modified by ordinary methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Further, the Fc domain variant may be in a form in which the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, or IgM are mixed. Note that the Fc domain variant may be in a form in which some amino acids of the Fc domain are substituted with other amino acids. As a specific example of the Fc domain variant, it may have the amino acid sequence of SEQ ID NO: 12.

[0056] The fusion protein can have a structure in which CD80 and IL-2 proteins are respectively linked to the N-terminus and C-terminus thereof using the Fc domain as a linker (or carrier), or a structure in which IL-2 and CD80 are linked. The linkage between the N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 is optionally carried out by a linker peptide.

[0057] Specifically, the fusion protein may be composed of the following structural formula (I) or (II): N’-X-[Linker(1)]n-Fc domain-[Linker(2)]m-Y-C’ (I) N’-Y-[Linker(1)]n-Fc domain-[Linker(2)]m-X-C’ (II) At this time, in the structural formulas (I) and (II), the N’ is the N-terminus of the fusion protein, the C’ is the C-terminus of the fusion protein, the X is the CD80 protein, the Y is the IL-2 protein, the Linker(1) and Linker(2) are peptide linkers, the n and m are each independently 0 or 1.

[0058] Preferably, the fusion protein may consist of the structural formula (I). The IL-2 protein is as described above. Also, the CD80 protein is as described above. According to one specific example, the IL-2 protein may be an IL-2 variant in which 1 to 5 amino acids are substituted as compared with wild-type IL-2. The CD80 protein may be a truncated fragment in which about 34 amino acid residues are continuously deleted from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having an activity of binding to T cell surface receptors CTLA-4 and CD28.

[0059] Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. According to another specific example, the fusion protein includes a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. At this time, the identity can be determined by, for example, percent homology or homology comparison software such as NCBI's BlastN software.

[0060] A peptide linker (1) is included between the CD80 protein and the Fc domain. The peptide linker (1) may consist of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. As a specific example, the peptide linker (1) may consist of 30 amino acids. Also, the peptide linker (1) can contain at least one cysteine. Specifically, it can contain 1, 2, or 3 cysteines. Note that the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one specific example, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3.

[0061] The peptide linker (2) may consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 amino acids. As a specific example, the peptide linker (2) may be (G4S)n (where n is an integer from 1 to 10). At this time, in (G4S)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. As one embodiment, the peptide linker (2) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5.

[0062] Another aspect of the present invention provides a dimer in which two fusion proteins containing the IL-2 protein and the CD80 protein are bound. The fusion protein containing the IL-2 or its variant and CD80 or its fragment is as described above.

[0063] At this time, the binding between the fusion proteins constituting the dimer may be carried out by a disulfide bond formed by cysteine present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same or may be different fusion proteins from each other. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

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

[0065] The cancer may 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.

[0066] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and body weight, the degree of the disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for preventing or treating cancer of the present invention, as long as the active ingredient can exhibit anti-cancer activity, it is contained in an arbitrary amount (effective amount) depending on the use, dosage form, compounding purpose, etc., but the normal effective amount is determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient that can induce an anti-cancer effect. Such an effective amount can be determined experimentally within the normal ability range of those skilled in the art.

[0067] As used herein, the term "treatment" is used in the sense of including all therapeutic and prophylactic processes. At this time, prophylaxis is used in the sense of alleviating or reducing an individual's pathological condition or disease. In one specific example, the term "treatment" includes all applications and any form of dosing for treating diseases in mammals including humans. Further, the term includes suppressing or delaying a disease or the progression of a disease; restoring or treating a damaged or defective function to partially or completely alleviate the disease; or stimulating an inefficient process; including the meaning of alleviating a serious disease.

[0068] As used herein, the term "efficacy" can be determined by one or more parameters such as survival or disease-free survival over a certain period of time such as one year, five years, or ten years. Moreover, the parameter can include that the size of at least one tumor is suppressed in an individual.

[0069] Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate can also affect efficacy. Therefore, "improved efficacy" (for example, improvement in efficacy) can result from improved pharmacokinetic parameters and improved efficacy, and is measured by comparing the clearance rate and tumor growth in test animals or human subjects, or comparing parameters such as survival, recurrence rate, or disease-free survival.

[0070] As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition effective to prevent or treat a target disease, which is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and causes no side effects. The level of the effective amount can be determined by factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the excretion ratio, the treatment period, the ingredients including the formulated or co-administered drugs, and other factors well known in the medical field. In one specific example, the therapeutically effective amount means the amount of a drug effective to treat cancer.

[0071] At this time, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. Any carrier can be used as long as it is a non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes and inert solids are included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) are also included in the pharmaceutical composition.

[0072] Specifically, the pharmaceutical composition contains, in addition to the active ingredient, a pharmaceutically acceptable carrier and is manufactured into a parenteral dosage form by a route of administration in a conventional manner known in the art. Here, the meaning of "pharmaceutically acceptable" is that it does not suppress the activity of the active ingredient and has no more toxicity than the applicable (formulated) subject can tolerate.

[0073] When the pharmaceutical composition is manufactured into a parenteral dosage form, together with a suitable carrier, it is formulated into the form of an injection, a transdermal administration agent, a nasal inhalant, and a suppository by a method known in the art. When formulating into an injection, as a suitable carrier, sterile water, ethanol, polyols such as glycerol or propylene glycol, or a mixture thereof can be used. Preferably, an isotonic solution such as a drip solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, 5% dextrose, etc. can be used. The formulation of pharmaceutical compositions is known in the art. Specifically, references such as [Remington’s Pharmaceutical Sciences (19th ed., 1995)] can be referred to. The said reference is regarded as a part of this specification.

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

[0075] The subjects to which the said pharmaceutical composition can be applied (prescribed) are mammals and humans, and particularly preferably humans. The pharmaceutical composition of the present application can additionally contain, in addition to the active ingredient, any compound or natural extract that has already been verified for safety and is known to have a therapeutic effect on anti-cancer activity for the purpose of increasing and enhancing anti-cancer activity.

[0076] Still another aspect of the present invention provides a cancer treatment kit comprising a fusion protein dimer containing an IL-2 protein or its variant and a CD80 protein or its fragment, and an immune checkpoint inhibitor.

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

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

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

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

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

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

[0083] In a fusion protein of a specific example of the present invention, immune cells such as natural killer cells can be activated by the activity of IL-2. Therefore, it can be effectively utilized for cancer. In particular, compared with the wild type, an IL-2 variant in which 2 to 5 amino acids are substituted at positions, particularly an IL-2 variant containing 2, 3, 4, or 5 amino acid substitutions at positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, has been confirmed to show characteristics in which the binding force to the alpha chain of the IL-2 receptor is reduced and the pharmacological side effects conventionally associated with IL-2 are improved. Therefore, such an IL-2 variant, when used alone or in the form of a fusion protein, can reduce the occurrence of vascular (or capillary) leak syndrome (VLS), which is a problem of conventionally known IL-2.

Example

[0084] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely for exemplifying the present invention, and the scope of the present invention is not limited only to these.

[0085] I. Production of fusion protein Production Example 1. Production of hCD80-Fc-IL-2 variant (2M): GI101 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 8) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) in which 2 amino acids are substituted, in this order from the N-terminus, was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Further, the vector was transfected into CHO cells (Expi-CHO TM) was introduced to express the fusion protein of SEQ ID NO: 9. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and in an environment with a CO2 concentration of 8%. Then the culture broth was collected and the fusion protein was purified. The purified fusion protein was named "GI101".

[0086] Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was bound under the conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Then, it was eluted with 100 mM acetic acid at pH 3 containing 100 mM NaCl. After putting 20% 1 M Tris-HCl at pH 9 into the collection tube, the fusion protein was collected. The collected fusion protein was dialyzed against PBS buffer for 16 hours for replacement.

[0087] Thereafter, size exclusion chromatography was performed using a TSKgel G3000SWXL column (TOSOH Bioscience), and the absorbance at a wavelength of 280 nm was measured over time to ensure a high-concentration fusion protein. At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with coomassie blue to confirm its purity (Figure 6). When detected using NanoDrop, it was confirmed that the fusion protein was contained at a concentration of 2.78 mg / ml (Figure 7). Also, the results of analysis using size exclusion chromatography are as shown in Figure 8.

[0088] Production Example 2. Production of mCD80-Fc-IL-2 variant (2M): mGI101 To produce a fusion protein containing mouse CD80, Fc domain, and IL-2 variant, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 14) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), mCD80 (SEQ ID NO: 13), Ig hinge (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted, in this order from the N-terminus, was synthesized by Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein of SEQ ID NO: 15. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "mGI101".

[0089] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 9). When detected at an absorbance of 280 nm using NanoDrop, it was confirmed that the fusion protein was contained at a concentration of 1.95 mg / ml.

[0090] Production Example 3. Production of hCD80-Fc: GI101C1 To produce a fusion protein containing a human CD80 fragment and Fc domain, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 16) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), CD80 fragment (SEQ ID NO: 2), Ig hinge (SEQ ID NO: 3), and Fc domain (SEQ ID NO: 4) was synthesized by Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHOTM ) was introduced into [the cells] to express the fusion protein of SEQ ID NO: 17. After the vector was introduced, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO₂ concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI101C1".

[0091] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 10). When detected at an absorbance of 280 nm using NanoDrop, it was confirmed that the fusion protein was contained at a concentration of 3.61 mg / ml.

[0092] Production Example 4. Production of Fc-IL-2 variant (2M): GI101C2 To produce a fusion protein containing an Fc domain and an IL-2 variant, a polynucleotide containing, in this order from the N-terminus, 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) (SEQ ID NO: 6) in which two amino acids are substituted, was synthesized by ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded onto a pcDNA3_4 vector. Also, the said vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein of SEQ ID NO: 19. After the vector was introduced, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO₂ concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI101C2".

[0093] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 11). When detected at an absorbance of 280 nm using a NanoDrop, it was confirmed that the fusion protein was contained at a concentration of 4.79 mg / ml.

[0094] Production Example 5. Production of mCD80-Fc: mGI101C1 To produce a fusion protein containing mouse CD80 and the Fc domain, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 20) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), mouse CD80 (SEQ ID NO: 13), Ig hinge (SEQ ID NO: 3), and Fc domain (SEQ ID NO: 4) in this order from the N-terminus was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein of SEQ ID NO: 21. After introducing the vector, the culture was incubated for 7 days at 37 °C, 125 rpm, and a CO2 concentration of 8%, and then the culture solution was collected to purify the fusion protein. The purified fusion protein was named "mGI101C1".

[0095] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 12). When detected at an absorbance of 280 nm using a NanoDrop, it was confirmed that the fusion protein was contained at a concentration of 2.49 mg / ml.

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

Table 1

[0097] Production Example 6. Production of CD80-Fc-IL-2: GI101w To produce a fusion protein containing a human CD80 fragment, an Fc domain, and human IL-2, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 31) encoding a fusion protein containing, in this order from the N-terminus, a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and mature human IL-2 (SEQ ID NO: 10) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHO TM ) to express the fusion protein of SEQ ID NO: 32. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI101w". The purification and collection of the fusion protein were performed in the same manner as in Production Example 1.

[0098] Production Example 7. Production of hCD80-Fc-IL-2 Variant (3M): GI102-M45 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) in which three amino acids are substituted (R38A, F42A, Y45A) (GI102-M45), a polynucleotide containing a nucleotide sequence (SEQ ID NO: 25) encoding a fusion protein containing, in this order from the N-terminus, a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 22) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHO TM) was introduced to express the fusion protein of SEQ ID NO: 26. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI102-M45".

[0099] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 13).

[0100] Production Example 8. Production of hCD80-Fc-IL-2 mutant (3M): GI102-M61 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 mutant (3M) (R38A, F42A, E61R) (GI101-M61) with three amino acids substituted, a polynucleotide containing the nucleotide sequence (SEQ ID NO: 27) encoding the fusion protein containing a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 mutant (SEQ ID NO: 23) in this order from the N-terminus was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHO (商標) ) to express the fusion protein of SEQ ID NO: 28. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI102-M61".

[0101] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 14).

[0102] Production Example 9. Production of hCD80-Fc-IL-3M: GI102-M72 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) in which three amino acids were substituted (R38A, F42A, L72G) (GI102-M72), a polynucleotide containing the nucleotide sequence (SEQ ID NO: 29) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 24) in this order from the N-terminus was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded onto the pcDNA3_4 vector. Further, the vector was introduced into CHO cells (Expi-CHO (商標) ) to express the fusion protein of SEQ ID NO: 30. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI102-M72".

[0103] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 15).

[0104] Production Example 10. Production of mCD80-Fc-IL-3M: mGI102-M61 To produce a fusion protein containing a mouse CD80 fragment, an Fc domain, and an IL-2 variant (3M) in which three amino acids are substituted (R38A, F42A, E61R) (GI102-M61), a polynucleotide containing the nucleotide sequence (SEQ ID NO: 33) encoding a fusion protein containing 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) in this order from the N-terminus was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. Also, the vector was introduced into CHO cells (Expi-CHO (商標) ) to express the fusion protein of SEQ ID NO: 34. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected and the fusion protein was purified. The purified fusion protein was named "mGI102-M61".

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

[0106] II. Confirmation of Binding Affinity between Fusion Protein and Ligand To confirm the binding affinity between the fusion protein and the ligand, the binding affinity was measured using Octet RED 384.

[0107] Experimental Example 1. Confirmation of Binding Affinity between hCTLA-4 and GI101 AR2G biosensor (Amine Reactive 2 ndGen, ForteBio, Cat: 18-5092) was pre-hydrated by adding 200 μl of distilled water to a Microplate-96-well (Greiner Bio-one, Cat: 655209). The ligand for the AR2G biosensor (CTLA-4, Human CTLA-4 / CD152, His tag, Sino Biological, Cat: 11159-H08H) was diluted to a concentration of 5 μg / ml with an acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095) at a concentration of 10 mM. Also, GI101 for the ligand was diluted with 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095) to concentrations of 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM. The activation buffer was prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095) in distilled water. 80 μl of each reagent was placed in a Microplate-384-well (Greiner Bio-one, Cat: 781209) and the program was set.

[0108] As a result, the binding affinity between hCTLA-4 and GI101 was measured as shown in FIG. 16.

[0109] Experimental Example 2. Confirmation of the binding affinity between hPD-L1 / GI101 and hPD-L1 / PD-1 Ni-NTA (Nickel charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101) was pre-hydrated by adding 200 μl of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) to each well of a Microplate-96-well. The ligand to be attached to the Ni-NTA biosensor (Human PD-L1 / B7-H1 protein, His-tag, Sino biological, Cat: 10084-H08H) was diluted to a concentration of 5 μg / ml with 1X Ni-NTA kinetic buffer. GI101 for the ligand was diluted with 1X Ni-NTA kinetic buffer to 1,000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM. Also, human PD-1 / PDCD1 (Human PD-1 / PDCD1, Fc Tag, Sino Biological, Cat: 10377-H02H) for the ligand was diluted with 1X Ni-NTA kinetic buffer to concentrations of 2,000 nM, 1,000 nM, 500 nM, 250 nM, or 125 nM. Then, 80 μl of each reagent was added to each well of a Microplate-384-well and the program was set.

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

[0111] Experimental Example 3. Confirmation of the binding affinity between mCTLA-4 and mGI101 The binding affinity between mCTLA-4 and mGI101 was confirmed in the same manner as in Experimental Example 1. At this time, the equipment used was as follows: Biosensor: AR2G, Ligand: mCTLA-4 (recombinant mouse CTLA-4 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0112] As a result, the binding affinity between mCTLA-4 and mGI101 was measured as shown in Fig. 19.

[0113] Experimental Example 4. Confirmation of the binding affinity between mPD-L1 and mGI101 The binding affinity between mPD-L1 and mGI101 was confirmed in the same manner as in Experimental Example 1. At this time, the equipment used was as follows. Biosensor: AR2G, Ligand: mPD-L1 (recombinant mouse mGI101 B7-H1 / PD-L1 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

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

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

[0116] Subsequently, the binding of various concentrations of GI-101 (hCD80-Fc-hIL-2v), GI-101C1 (hCD80-Fc), Ipilimumab (Bristol-Myers Squibb), and GI-101C2 (Fc-hIL-2v) was measured for 300 seconds, and the dissociation was also measured for 300 seconds. The binding kinetics analysis was performed using Octet Data analysis HT software ver10 provided by Pall. The results are shown in Figure 21.

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

[0118] The ligand for the AR2G biosensor (IL-2Rα-His Tag, Acro, Cat: ILA-H52H9) was diluted to a concentration of 5 μg / ml with an acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095) at a concentration of 10 mM. After activating the AR2G biosensor with a buffer prepared by mixing EDC at a concentration of 400 mM and sulfo-NHS at a concentration of 100 mM, the diluted ligand was loaded onto the AR2G biosensor for 300 seconds to be immobilized.

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

[0120] Subsequently, various concentrations of GI101, GI101w, or Proleukin (Novartis, hIL-2) were loaded onto the ligand for 300 seconds, after which binding was measured, and dissociation was also measured for 300 seconds. Binding kinetics analysis was performed using Octet Data analysis HT software ver.10 provided by Pall. The results are shown in Figures 22 to 24.

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

[0122] Experimental Example 7. Measurement of Binding Affinity between Fusion Protein and Ligand To confirm the binding affinity between the fusion protein and the ligand, the binding affinity was measured using Octet RED 384.

[0123] Experimental Example 7.1. Confirmation of Binding Affinity between IL-2 alpha receptor and GI101-M45, GI101-M61, and GI101-M72 AR2G biosensor (Amine Reactive 2 ndGen, ForteBio, Cat: 18 - 5092) was pre - hydrated by adding 200 μl of distilled water (DW) to each well of a Microplate - 96 - well (Greiner Bio - one, Cat: 655209). The ligand (Human IL - 2R alpha protein, His Tag, Acro, ILA - H52H9) for the biosensor was diluted to a concentration of 5 μg / ml with 10 mM acetate pH 5 buffer (AR2G reagent Kit, ForteBio, Cat: 18 - 5095). The analytes (GI101 - M45, GI101 - M61, GI101 - M72) for the ligand were diluted to 500 nM, 250 nM, 125 nM, and 62.5 nM respectively with 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18 - 5095). The activation buffer was prepared by adding 20 mM EDC and 10 mM s - NHS (AR2G reagent Kit, ForteBio, Cat: 18 - 5095) to DW. 80 μl of each reagent was added to a Microplate - 384 - well (Greiner Bio - one, Cat: 781209) and the program was set up.

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

[0125] Experimental Example 7.2. Confirmation of the binding affinity of GI102 - M45, GI102 - M61, and GI102 - M72 for IL - 2Rβ The Ni-NTA biosensor was pre-hydrated by adding 200 μl of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) to each well of a Microplate-96-well. The ligand to be attached to the biosensor (Human IL-2R beta protein, His-Tag, Acro, CD2-H5221) was diluted with 1X Ni-NTA kinetic buffer to a concentration of 2 μg / ml. GI102-M45, GI102-M61 or GI102-M72 attached to the ligand was diluted with 1X Ni-NTA kinetic buffer to concentrations of 500 nM, 250 nM, 125 nM or 62.5 nM. 80 μl of each reagent was added to each well of a Microplate-384-well and the program was set up.

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

[0127] III. Confirmation of the immunological activity of the fusion protein Experimental Example 8. Confirmation of the amount of IFN-γ produced by the fusion protein Experimental Example 8.1. Culturing of CFSE-labeled PBMC PBMC (Peripheral blood mononuclear cells) isolated from humans were reacted with CellTrace CFSE dye at a concentration of 1 μM at 37 °C for 20 minutes to be labeled with CFSE (carboxyfluorescein succinimidyl ester). CFSE that did not bind to the cells was reacted with culture medium, which was 5 times the staining reaction solution, for 5 minutes and then removed by centrifugation at 1,300 rpm for 5 minutes. The PBMC labeled with CFSE was resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acids, and 2 mM L-glutamine), and then 1×10 5 cells per well were placed in a Microplate-96-well, and treated with 5 μg / ml of PHA (Lectin from Phaseolus Vulgaris, red kidney bean, Sigma-Aldrich, St. Louis, MO, USA, cat No. L1668-5MG) and GI101, GI101C1, GI101C2, or IL-2 (Aldesleukin; human recombinant IL-2, Novartis), and cultured in an incubator at 37 °C and 5% CO2 for 6 days.

[0128] At this time, GI101, GI101C1, GI101C2, and IL-2 were treated at concentrations of 1 nM, 10 nM, or 100 nM. The cells were analyzed by FACS, and human IFN-γ present in the culture medium was measured using an ELISA kit (Biolegend, San Diego, CA, USA, cat No. 430103).

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

[0130] Experimental Example 8.3. Human IFN-γ ELISA The amount of human IFN-γ secreted into the supernatant of each sample in which cells had been cultured was measured using a human IFN-γ ELISA kit (Biolegend, cat No.430103). Briefly, the anti-human-IFN-γ antibody was placed in an ELISA plate and reacted overnight at 4 °C for coating. Subsequently, it was blocked with a PBS solution containing 1% BSA at room temperature for 1 hour. After washing with a washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample were appropriately diluted and added, and then reacted at room temperature for 2 hours. After the reaction was completed, the plate was washed and a secondary antibody (detection antibody) was added and reacted at room temperature for 1 hour. After washing with the washing buffer, an Avidin-HRP solution was added and reacted at room temperature for 30 minutes, and then a substrate solution was added to induce a color reaction in the dark at room temperature for 20 minutes. Finally, H2SO4 was added to stop the color reaction, and the absorbance at 450 nm was measured using an Epoch Microplate Spectrophotometer (BioTek instruments, Winooski, VT, USA) for concentration calculation.

[0131] As a result, it was confirmed that the amount of IFN-γ secretion in the cells treated with GI101 was significantly increased compared to the cells treated with GI101C1, GI101C2 or IL-2 (Figs. 31 and 32).

[0132] Experimental Example 9. Confirmation of the effect of GI101 on the proliferation of CD8+ T cells PBMC (Peripheral blood mononuclear cells) isolated from humans were reacted with CellTrace CFSE dye at a concentration of 1 μM at 37°C for 20 minutes to be labeled with CFSE. The CFSE that did not bind to the cells was reacted with a culture medium that was 5 times the staining reaction solution for 5 minutes, and then removed by centrifugation at 1,300 rpm for 5 minutes. The PBMC labeled with CFSE were resuspended in a culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acids and 2 mM L-glutamine), and then 1×10 5 cells per well were placed in a Microplate-96-well.

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

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

[0135] Experimental Example 10. Confirmation of the effect of GI101 and GI102 on the proliferation of CD8+ T cells Human PBMCs were purchased from Allcells (Lot#3014928, USA). The CellTrace CFSE dye at a concentration of 1M was used, and this was reacted with human PBMCs at room temperature for 20 minutes under light-blocking conditions. It was reacted with the CellTrace CFSE dye at a concentration of 1μM at 37°C for 20 minutes to label with CFSE. The CFSE that did not bind to the cells was reacted with the culture medium, which was 5 times the staining reaction solution, for 5 minutes, and then removed by centrifugation at 1,300 rpm for 5 minutes. The PBMCs labeled with CFSE were resuspended in the culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM non-essential amino acids, and 2 mM L-glutamine), and then 1×10 5 cells per well were placed in a Microplate-96-well.

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

[0137] The cultured cells were examined for the degree of proliferation of these cells by measuring the ratio of the cells not labeled with CFSE among CD8+ T cells by the FACS analysis method using anti-human CD4-PE antibody (BioLegend, U.S.A.), anti-human CD8-PE / Cy7 antibody (BioLegend, USA), and anti-human FoxP3-APC antibody (BioLegend, U.S.A.).

[0138] As a result, in the groups treated with GI101, GI102_M61, GI101C2, and Proleukin, the ratio of CD8+ T cells was significantly increased compared to the control group (No stimulus), the group treated with anti-CD3 antibody alone, and the GI101C1-treated group. Also, compared to the negative control group (No stimulation) and the treatment with anti-CD3 antibody alone, GI101, GI101C2, and Proleukin significantly increased the proliferation of CD4+ / FoxP3+ Treg cells, while GI102 and GI101C1 did not significantly increase the proliferation of CD4+ / FoxP3+ Treg cells (Figure 35).

[0139] Experimental Example 11. Confirmation of the effect of GI101 or GI101w on the proliferation of CD8+ T cells and NK cells Seven-week-old C57BL / 6 mice purchased from Orient Bio (Korea) were divided into three groups of three mice each, and PBS, GI101, or GI101w was intraperitoneally injected. At this time, GI101 and GI101w were each prepared to be 40.5 μg in 200 μl of PBS and intraperitoneally injected. Five days after the injection, the spleens were removed from the mice in each group to isolate cells, and the total number of cells was measured using a hematocytometer. The ratio of CD8+ T cells and NK cells in the spleen cells was examined by FACS analysis in which the spleen cells were stained with APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific blue-CD8α antibody (BD; 53-6.7). Thus, the numbers of CD8+ T cells and NK cells present in the spleen were calculated.

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

[0141] Experimental Example 12. Confirmation of the effect of GI101 on the function of T cells The experiment was conducted using the CTLA-4 blockade bioassay kit (Promega cat No. JA4005). Briefly, CTLA-4 Effector cells stored in liquid nitrogen were thawed in a 37°C water bath for 3 minutes. Then, 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). After that, 25 μl of the mixture was added to each well of a 96-well white cell culture plate (SPL, cat No. 30196). Next, 25 μl of GI101 at various concentrations was added. In the case of the negative control group, 25 μl of assay buffer was added. Then, until the aAPC / Raji cells were prepared, the 96-well white cell culture plate was covered and left at room temperature. aAPC / Raji cells stored in liquid nitrogen were thawed in a 37°C water bath for 3 minutes. Then, 0.8 ml of aAPC / Raji cells were thoroughly mixed with 3.2 ml of pre-warmed assay buffer. After that, 25 μl of the mixture was added to each well of the plate, and the plate was incubated in a 37°C, 5% CO2 incubator for 16 hours. After the reaction, the plate was left at room temperature for 15 minutes. Then, Bio-Glo reagent was added carefully to avoid generating bubbles. Bio-Glo reagent was also added to three locations in the peripheral outer wells to be used as blanks to correct the background signal. After reacting at room temperature for 10 minutes, luminescence was measured using Cytation3 (BioTek instruments, Winooski, VT, USA). The final data analysis was calculated as RLU (GI101 - background) / RLU (No treatment - background).

[0142] As a result, it was confirmed that GI101 binds to CTLA-4 expressed on effector T cells and rather activates them without suppressing T cell function (Figures 38 and 39).

[0143] Experimental Example 13: Confirmation of the Effects of mGI101 and mGI102 on Immune Cells Seven-week-old C57BL / 6 mice purchased from Orient (Korea) were divided into three groups of three mice each, and PBS, 3 mg / kg, 6 mg / kg, 12 mg / kg of GI101, or 3 mg / kg, 6 mg / kg, 12 mg / kg of mGI102 (mGI102-M61) were administered intravenously. Spleen tissues were removed from the mice in each group on days 1, 3, 5, 7, and 14 after injection. Subsequently, the numbers of effector CD8+ T cells, NK cells, and Treg cells in the spleen tissues were calculated using the FACS analysis method with their respective antibodies, and the ratios of effector CD8+ T cells and NK cells to Treg cells were calculated respectively. Information on the antibodies used for each cell analysis is as follows:

[0144] Effector CD8+ T cell: PB anti-mouse CD3ε antibody (Biolegend, #155612; KT3.1.1), FITC anti-mouse CD8α antibody (BD, #553031, 53-6.7), PE / Cy7 anti-mouse CD44 antibody (Biolegend, #103030; IM7), APC anti-mouse CD122 antibody (Biolegend, #123214; TM-β1) NK cell: PB anti-mouse CD3ε antibody (Biolegend, #155612; KT3.1.1), PE anti-mouse NK-1.1 (Biolegend, #108708; PK136) Treg cell: FITC anti-mouse CD3 antibody (Biolegend, #100204; 17A2), PB anti-mouse CD4 antibody (Biolegend, #100531; RM4-5), PE anti-mouse CD25 antibody (Biolegend, #102008; PC61), APC anti-mouse Foxp3 antibody (Invitrogen, #FJK-16s, 17-5773-82).

[0145] As a result, in the groups administered with mGI101 or mGI102 (mGI102-M61), CD8+ T cells and NK cells were significantly increased compared to the PBS-administered group at the time points from 3 days to 14 days after administration. Also, it was confirmed that in the group administered with mGI102, the ratios of activated CD8+ T cells / Treg cells and NK cells / Treg cells were significantly increased compared to the PBS-administered group at the time points from 3 days to 7 days after administration (Figure 40).

[0146] IV. Confirmation of the anti-cancer effect of the fusion protein Experimental Example 14. Confirmation of the effect of GI101 on the suppression of T cell activity by cancer cells expressing PD-L1 and CTLA-4 The NCl-H292 cancer cell line expressing PD-L1 and CTLA-4 was cultured in a culture medium containing 10 μg / ml Mitomycin C (Sigma) for 3 hours, and then Mitomycin C was washed and removed from the culture medium. Then, the NCl-H292 cancer cell line treated with Mitomycin C at a cell number of 5×10 4 was cultured with human PBMC at a cell number of 1×10 5 in a Microplate-96-well. At this time, 5 μg / ml of PHA (Sigma) was treated for the activity of T cells. Also, GI101C1 and GI101 at a concentration of 50 nM were reacted with IgG1-Fc (Biolegend) or abatacept (=Orencia; Bristol-Myers Squibb) at a concentration of 50 nM at 4°C for 30 minutes, and then treated with NCl-H292 cancer cells. After 3 days, the supernatant of the cell culture was collected, and the amount of IFN-γ was quantified using an ELISA kit (Biolegend).

[0147] As a positive control group, human PBMC stimulated with PHA in the absence of the NCl-H292 cancer cell line treated with Mitomycin C was used, and as a negative control group, human PBMC stimulated with PHA in the presence of the NCl-H292 cancer cell line treated with Mitomycin C was used. The experimental method using the IFN-γ ELISA kit was carried out in the same manner as in Experimental Example 9.3.

[0148] As a result, GI101 effectively activated the immune response suppressed by cancer cell lines overexpressing PD-L1. In addition, it was confirmed that GI101 suppressed the signal transduction of CTLA-4 expressed on effector T cells (Figs. 41 and 42).

[0149] Experimental Example 15. Confirmation of the anti-cancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells After a 7-day adaptation period, BALB / c mice (female, 7 weeks old) purchased from Orient Bio were mixed with 5×10 6 cells of CT-26 cancer cell line (ATCC, U.S.A.) in 0.05 ml of phenol red-free Matrigel matrix (BD), and 0.1 ml of the mixture was subcutaneously administered to the right dorsal part of the mice for allograft. After the cancer cells were transplanted and a certain period of time had passed, the tumor volume was measured, and individuals with a tumor volume reaching approximately 28 mm 3 were selected. Then, based on the tumor size and body weight of the selected mice, 10 mice per group were classified to be equal. Thereafter, using a disposable syringe (31G, 1 ml), hIgG4 was administered to the negative control group at a dose of 6 mg / kg. mGI101 was intravenously administered to the experimental groups at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. After the first administration, a total of 3 administrations were carried out once every 3 days. The tumor size was measured daily.

[0150] As a result, it was confirmed that in the experimental groups administered with mGI101 at doses of 6 mg / kg and 12 mg / kg, there was significant suppression compared to the negative control group at some measurement time points and at the end of the test (Fig. 43). In addition, as a result of measuring the survival rate, it was confirmed that in the experimental group administered with mGI101 at a dose of 6 mg / kg, there was significant improvement compared to the negative control group at some measurement time points and at the end of the test (Fig. 44).

[0151] Experimental Example 16. Confirmation of the anti-cancer effect of GI101 in mice implanted with mouse-derived colon cancer cells Experimental Example 16.1. Confirmation of tumor suppression effect After a 7-day adaptation period, BALB / c mice (female, 7 weeks old) obtained by assignment from Orient Bio were subcutaneously administered with 5 × 10 6 cells of CT-26 cancer cell line (ATCC, U.S.A.) suspended in 0.1 ml of PBS into the right dorsal part of the mice for allogeneic transplantation. After transplantation of cancer cells and after a certain period of time, the tumor volume was measured, and individuals that reached about 50 mm 3 ~200 mm 3 were selected. Then, based on the tumor size and body weight of the selected mice, they were classified into 10 mice per group so as to be equal. Thereafter, using a disposable syringe (31G, 1 ml), no drug was administered to the negative control group, and 5 mg / kg dose of anti-PD-1 antibody or 5 mg / kg dose of anti-PD-1 antibody and 5 mg / kg dose of anti-CTLA-4 antibody were intravenously administered to the positive control group. 0.1 mg / kg or 1 mg / kg dose of GI101 was intravenously administered to the experimental group. After the first administration, a total of 3 administrations were carried out once every 3 days. The tumor size was measured every day.

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

[0153] Experimental Example 16.2. Analysis of immune cells in cancer tissue The mice in each group of the above Experimental Example 16.1 were sacrificed when the tumor volume was on average 200 mm 3Upon reaching [the specified point], the cancer tissue was sacrificed and collected. Subsequently, to analyze the immune cells within the cancer tissue, after separating the cancer tissue to the single-cell level, FACS analysis of the immune cells in the cancer tissue was performed using the following antibodies. Specifically, the antibodies used were Anti-mouse-CD3 (Biolegend, Cat.No.100320), Anti-mouse-CD4 (Biolegend, Cat.No.100526), Anti-mouse-CD8 (Biolegend, Cat.No.100750), Anti-mouse-FoxP3 (eBioscience, Cat.No.12-5773-82), Anti-mouse-CD25 (Biolegend, Cat.No.102049), Anti-mouse-CD44 (eBioscience, Cat.No.61-0441-82), Anti-mouse-PD-1 (Biolegend, Cat.No.135218), Anti-mouse-IFN-gamma (Biolegend, Cat.No.505832), Anti-mouse-CD49b (Biolegend, Cat.No.108906), Anti-mouse-H2 (Invitrogen, Cat.No.A15443), Anti-mouse-CD11c (Biolegend, Cat.No.117343), Anti-mouse-CD80 (eBioscience, Cat.No.47-4801-82), Anti-mouse-CD86 (Biolegend, Cat.No.104729), Anti-mouse-F4 / 80 (eBioscience, Cat.No.47-4801-82), and Anti-mouse-CD206 (eBioscience, Cat.No.17-2061-80).

[0154] As a result, compared with the positive control group that received a single administration of the anti-PD-1 antibody at a dose of 5 mg / kg, the CD8+ T cells were significantly increased in the experimental group administered with 0.1 mg / kg of GI101 (*p<0.05, Figures 46 and 47). Furthermore, in both experimental groups administered with GI101, the expression of IFN-γ in T cells was significantly increased compared with the negative control group (*p<0.05, Figures 46 and 47). Also, in the experimental group administered with 0.1 mg / kg of GI101, M1 macrophages were increased compared with the negative control group and the positive control group that received a single administration of the anti-PD-1 antibody (Figures 48 and 49). In addition, in both experimental groups administered with GI101, the expression of CD86 in macrophages and dendritic cells was increased (*p<0.05, Figures 48 to 51).

[0155] Experimental Example 17. Confirmation of the anti-cancer effect of GI101 in mice implanted with mouse-derived lung cancer cells Experimental Example 17.1. Confirmation of tumor suppression effect After a 7-day adaptation period, C57BL / 6 mice (female, 7 weeks) obtained by sublicense from Orient Bio (Korea) were subcutaneously administered with 0.1 ml of PBS containing 5×10 6 cells of the LLC2 cancer cell line (ATCC, U.S.A.) at the right dorsal part of the mouse for allograft. After the cancer cells were transplanted and a certain period of time had passed, the tumor volume was measured, and individuals that reached about 50 mm 3 ~200 mm 3 were selected. Then, based on the tumor size and body weight of the selected mice, 10 mice per group were classified to be equal. Thereafter, using a disposable syringe (31G, 1 ml), no drug was administered to the negative control group, and the anti-PD-1 antibody at a dose of 5 mg / kg or the anti-PD-1 antibody at a dose of 5 mg / kg and the anti-CTLA-4 antibody at a dose of 5 mg / kg were intravenously administered to the positive control group. The experimental groups were intravenously administered with 0.1 mg / kg or 1 mg / kg of GI101. After the first administration, a total of 3 administrations were carried out once every 3 days. The tumor size was measured every day.

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

[0157] Experimental Example 17.2. Analysis of Immune Cells in Cancer Tissues When the tumor volume of the mice in each group of the above Experimental Example 17.1 reached an average of 200 mm 3 they were sacrificed and the cancer tissues were collected. Then, in order to analyze the immune cells in the cancer tissues, FACS analysis was performed in the same manner as in Experimental Example 16.2.

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

[0159] Experimental Example 18. Confirmation of Anticancer Effect of mGI102-M61 in Mice Implanted with Mouse-derived Colorectal Cancer Cells After a 7-day adaptation period, BALB / c mice (female, 7 weeks old) obtained from OriGene Technologies were mixed with 5×10 6 cells of CT-26 cancer cell line (ATCC, U.S.A.) and 0.05 ml of phenol red-free Matrigel matrix (BD), and 0.1 ml of the mixture was administered subcutaneously to the right dorsal part of the mice for allograft. After the cancer cells were transplanted and a certain period of time had passed, the tumor volume was measured, and individuals with a tumor volume reaching approximately 28 mm 3 were selected. Then, based on the tumor size and body weight of the selected mice, 10 mice per group were classified to be equal. Thereafter, using a disposable syringe (31G, 1 ml), hIgG4 was administered to the negative control group at a dose of 6 mg / kg. mGI102-M61 was intravenously administered to the experimental groups at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. After the first administration, a total of 3 administrations were carried out once every 3 days. The tumor size was measured every day.

[0160] As a result, it was confirmed that in the experimental group administered with mGI102-M61 at a dose of 12 mg / kg, it was significantly suppressed at some measurement time points and at the end of the test compared to the negative control group (Figure 56). In addition, as a result of measuring the survival rate, it was confirmed that in the experimental group administered with mGI102-M61 at a dose of 12 mg / kg, it was significantly improved at some measurement time points and at the end of the test compared to the negative control group (Figure 57).

[0161] Experimental Example 19. Confirmation of the anti-cancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells After a 7-day adaptation period, BALB / c mice (female, 7 weeks) obtained from Orient Bio (Korea) were mixed with CT-26 cancer cell line (ATCC, USA) at a cell count of 5×10 6 in 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously at 0.1 ml each to the right dorsal part of the mice for orthotopic transplantation. After a certain period had elapsed after transplanting the cancer cells, the tumor volume was measured, and individuals that reached about 200 mm 3 ~250 mm 3 were selected. Then, based on the tumor size and body weight of the selected mice, 10 mice were classified into each group to make them equal.

[0162] Thereafter, using a disposable syringe (31G, 1 ml), hIgG4 was administered to the negative control group at a dose of 4 mg / kg. mGI101 was intravenously administered to the experimental group at doses of 1 mg / kg, 4 mg / kg, or 6 mg / kg. Additionally, groups administered with 4.9 mg / kg of mCD80 or 2.8 mg / kg of Fc-IL-2v (GI101C2) were also set as control groups. Moreover, a group administered with 4.9 mg / kg of mCD80 and 2.8 mg / kg of Fc-IL-2v (GI101C2) simultaneously was also set as a control group.

[0163] In the measurement of tumor volume, it was confirmed that in the mGI101 administration group at a dose of 6 mg / kg, it was significantly suppressed compared to the negative control group at some measurement time points and at the end of the test. It was revealed that the tumor growth inhibition rate was superior compared to the combined administration group of mCD80 and Fc-IL-2v (GI101C2) (Figures 58 and 59).

[0164] As a conclusion, in the tumor growth inhibition efficacy test of CT-26, a colon cancer cell line derived from BALB / c mice, transplanted into BALB / c mice, the test substance mGI101 demonstrated tumor suppression efficacy against mCD80 and IL-2v single agents under the test conditions, and an anti-cancer efficacy superior to that of the combined administration group of mCD80 and IL-2v was confirmed (Figures 58 and 59). In particular, in the mGI101 administration group at a dose of 6 mg / kg, the tumor size was significantly suppressed compared to the negative control group and the combined administration group of mCD80 and Fc-IL2v (GI101C2).

[0165] V. Confirmation of the anti-cancer effect by combined administration of fusion protein dimer and immune checkpoint inhibitor Experimental Example 20. Confirmation of the anti-cancer effect by combined administration of GI101 and anti-PD-1 antibody in mice implanted with human-derived breast cancer cells This test used a humanized mouse model prepared by xenotransplanting human PBMC into NSGb2m mice. In a tumor model xenotransplanted with human-derived breast cancer cells, MDA-MB-231 cells, the test substance GI101 and the anti-PD-1 antibody Keytruda (Pmembrolizumab, MSD) as a positive control substance were intraperitoneally administered alone and in combination, and then the tumor growth inhibitory effect was evaluated.

[0166] The stock solutions of the test substance, negative control substance, and positive control substance described in Table 2 were diluted by adding excipients according to each dose.

[0167]

Table 2

[0168] MDA-MB-231, which is a human-derived breast cancer cell (Homo sapiens, human mammary gland / breast; derived from the metastatic site: pleural effusion), was purchased from the Korea cell line bank (Korea) and used in the test. The cell culture medium had the composition shown in the following table. Per 100 ml, fetal bovine serum (FBS, 16000-044, Thermofisher scientific, U.S.A.), penicillin-streptomycin; 10,000 units / ml penicillin and 10,000 μg / ml streptomycin (15140122, Thermofisher scientific, U.S.A.), and RPMI1640 (A1049101, Thermofisher scientific, U.S.A.) were mixed and used.

[0169]

Table 3

[0170] The cells used in the test were thawed and placed in a cell culture flask, and cultured in an incubator (MCO-170M, Panasonic, Japan) at 37°C and 5% CO2. They were suspended using Trypsin-EDTA (Cat.25200-072, Thermofisher scientific, U.S.A.). The suspended cells were collected by centrifugation (125xg, 5 minutes) using a centrifuge, transferred to a new medium and a new flask, and subcultured. After the cells cultured on the cell line transplantation day were put into a centrifuge tube, after collection, they were centrifuged (125xg, 5 minutes), the supernatant was discarded, and a cell suspension (5×10 6 cells / 0.05 ml) was made with PBS (Cat.LB 001-04, Welgene, KOREA) and stored on ice until inoculation.

[0171] For the test, 8-week-old female NSGb2m (NOD.Cg-B2m tm1Unc Prkdc scid Il2rg tm1WjlThe / SzJ) mice were purchased from ChoongAng Bio (Korea) and used. After the end of the quarantine and acclimation period, the body weight was measured the next day, and then a human-derived PBMC cell suspension (5×10 6 cells / 0.2 ml) prepared for healthy animals was filled into a disposable syringe and administered to the tail vein of the animals. After cell transplantation, the general symptoms were observed once a day.

[0172] The prepared MDA-MB-231 cell suspension (5×10 6 cells / 0.05 ml) was added with a phenol red-free Matrigel matrix (0.05 ml, 356237, BD, U.S.A.), and the prepared solution was filled into a disposable syringe and administered subcutaneously at 0.1 ml / head to the right dorsal part of the animals transplanted with human PBMC. After transplantation of the cell line, the general symptoms were observed once a day during the engraftment and growth period.

[0173] After a certain period of time after cell transplantation, the tumor volume was measured for animals without abnormalities in their health status, and 32 individuals were selected so that the average of each group reached 40 - 80 mm 3 . The selected animals were grouped into 4 groups of 8 animals per group as evenly as possible based on the tumor volume and body weight.

[0174] The test groups were constituted as shown in Table 4. The test substance was administered to the animals using a disposable syringe (31G, 1 ml), and the administration frequency was 2 times / week for a total of 4 administrations.

[0175]

Table 4

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

[0177] Three times a week during the observation period, the long axis (maximum length, L) and short axis (perpendicular width, W) of the tumor were measured using a digital caliper (Mitutoyo, Japan), and the tumor volume (TV) was calculated by substituting into the following formula. <Equation 1> TV (mm 3 ) = (W 2 ×L) / 2 <Equation 2> %TGI (Tumor Growth Inhibition) = (1 - (Ti - T0) / (Vi - V0)) × 100

[0178] The tumor volume before administration of each individual was set to the value measured at the time of group separation. After tumor transplantation, the drugs listed in Table 4 were administered on days 21, 25, 28, and 31, respectively. As a result, compared with the control group (hIgG4), tumor growth in the GI101 and Keytruda single-treatment groups was inhibited. Compared with the control group, tumor growth in the GI101 and Keytruda combination-treatment group was inhibited. Compared with the GI101 and Keytruda single-treatment groups, tumor growth in the GI101 and Keytruda combination-treatment group was inhibited (Figure 60).

[0179] When calculating the tumor growth inhibition rate at the end of the experiment (day 42 after tumor transplantation) compared with day 1 of drug treatment (day 21 after tumor transplantation), in the hIgG4 treatment group, there were 2 animals with a tumor growth inhibition rate of 30% or more, 1 animal with 50% or more, and 1 animal with 80% or more. In the GI101 treatment group, there were 5 animals with a tumor growth inhibition rate of 30% or more, 5 animals with 50% or more, and 2 animals with 80% or more. In the Keytruda treatment group, there were 7 animals with a tumor growth inhibition rate of 30% or more, 5 animals with 50% or more, and 3 animals with 80% or more. In the GI101 and Keytruda combination-treatment group, there were 8 animals with a tumor growth inhibition rate of 30% or more, 8 animals with 50% or more, and 6 animals with 80% or more (Figure 61).

[0180] In addition, the degree of tumor growth of individual experimental animals in each treatment group when GI101 and Keytruda were combined in human-derived breast cancer cell-implanted mice is shown in Figures 62 to 66.

[0181] Experimental Example 21. Confirmation of Anticancer Effect by Combined Administration of mGI101 and Anti-PD-1 Antibody in Mice Implanted with Mouse-Derived Colorectal Cancer Cells In this study, in a tumor model in which C57BL / 6 mice were syngrafted with MC38 (murine colon adenocarcinoma cells) cells, the test substance mGI101 and the anti-PD-1 antibody as a positive control substance were intraperitoneally administered alone and in combination, and then the tumor growth inhibitory effect was evaluated.

[0182] MC38 (murine colon adenocarcinoma cells), a rodent-derived colorectal cancer cell, was purchased from Kerafast (USA) and used in the test. MC38 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin and then suspended in PBS. To establish a syngeneic transplantation tumor model, 1 × 10 6 MC38 cells were injected s.c. into the right flank of C57BL / 6 female mice (7 weeks old).

[0183] The mice were randomly assigned 5 per group based on the tumor volume (30 mm 3 ). Tumor grafts were confirmed approximately 2 days after cell inoculation. The test groups were constituted as shown in Table 5, and the test substances were administered.

[0184]

Table 5

[0185] Clinical symptoms such as diseases and behavioral changes were observed once a day during the test period, and dead animals were confirmed. At the end of the test period, the animals were sacrificed. The size of the MC38 solid cancer was measured using a tumor 3D scanner (TM900, Peria, belgium). The average body weight loss and percentage change and the average tumor growth inhibition were calculated for each experimental group. The antitumor efficacy was evaluated compared with the vehicle control group.

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

[0187] All test animals maintained a healthy state without pathological abnormalities after administration of mGI101 and combination administration with anti-PD-1 antibody. The results of combination therapy using mGI101 and / or anti-PD-1 antibody against MC38 tumors are as shown in Figures 67 to 73. Compared with the control group, an anti-cancer effect was observed in the drug-treated group, and the difference in tumor size became prominent during the 16-day test period. MC38 tumors are known as a reaction model to anti-PD-1 antibody in previous literature, and an anti-cancer effect was also observed in the anti-PD-1 antibody administration group of this test (p > 0.01). An anti-cancer effect appeared in both the anti-PD-1 antibody administration group alone and the mGI101 (6mpk) single administration group (p > 0.01). The combination administration group of mGI101 (0.6mpk) + anti-PD-1 (5mpk) showed a significantly excellent anti-cancer effect (p > 0.0001).

[0188] The size of individual tumors by test group is as shown in Figures 69 to 73. According to the results of the size of individual tumors, mild tumor regression was observed in some animals in the anti-PD-1 antibody administration group. The mGI101 (6mpk) single administration group showed a more excellent tumor growth inhibitory effect compared with the anti-PD-1 antibody administration group. The tumor size was maintained at the same size until 5 - 7 days, but regrew after 7 days. The tumor size was maintained at the same size until 5 - 7 days, but regrew after 7 days. The combination administration group (GI101 (0.6mpk) + anti-PD-1 antibody (5mpk)) showed significantly excellent tumor growth inhibition. In particular, two of the combination administration group showed complete response (no tumor).

[0189] MC38 cells were reinjected into the left flanks (opposite the site of initial injection of cancer cells) of two mice in the combination treatment group that showed complete remission. These mice maintained anti-PD-1 antibody administration (5 mpk, BIW) until day 32 (Figure 74). A small-sized tumor (>30 mm 3 ) was observed in one of the two mice, but the tumor size did not grow further until day 35 (Figure 69). No tumor was observed in the other mouse after tumor reinjection (Figures 69 and 74). In conclusion, as a result of testing the antitumor efficacy of mGI101 alone and in combination with an anti-PD-1 antibody in the MC38 syngeneic tumor model, the most excellent antitumor efficacy was exhibited in the combination treatment group (GI101 (0.6 mpk) + anti-PD-1 (5 mpk)). Complete response occurred in two of the experimental animals in the combination treatment group, and the complete remission mice reinjected with MC38 showed a cancer resistance effect (Table 6).

[0190]

Table 6

[0191] Experimental Example 22. Confirmation of the Anticancer Effect by the Combined Administration of mGI101 and an Anti-PD-L1 Antibody in Mice Implanted with Mouse-Derived Colorectal Cancer Cells In this study, in a tumor model in which CT26 (murine colon carcinoma cells) cells were syngrafted into BALB / c mice, the test substance mGI101 and the anti-PD-L1 antibody (BioXcell, Cat#BE0101) as a positive control substance were administered alone and in combination, and then the tumor growth inhibitory effect was evaluated.

[0192] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin and then suspended in PBS. To establish a syngrafted tumor model, 5 × 10 5 CT26 cells were injected subcutaneously into the right flanks of BALB / c female mice (7 weeks old).

[0193] Mice were randomly assigned four per group based on the tumor volume (50 - 120 mm 3 ). Tumor grafts were confirmed approximately 2 days after cell inoculation. The test groups were constituted as shown in Table 7, and the test substances were administered.

[0194]

Table 7

[0195] Clinical symptoms such as disease and behavioral changes were observed once daily during the test period, and dead animals were confirmed. At the end of the test period, the animals were sacrificed. The size of CT26 solid cancer was measured using a tumor 3D scanner (TM900, Peria, belgium). Mean body weight loss and percentage change and mean tumor growth inhibition were calculated for each experimental group. The anti-tumor efficacy was evaluated compared to the vehicle control group.

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

[0197] As a result of testing the anti-tumor efficacy of mGI101 alone and in combination with an anti-PD-L1 antibody in a CT26 syngeneic tumor model, the most excellent anti-tumor efficacy was observed in the combination administration group (mGI101 (3 mpk) + anti-PD-L1 (10 mpk)) (Figure 75).

[0198] Experimental Example 23. Confirmation of the anti-cancer effect by combined administration of mGI101 and an anti-TIGIT antibody in mice implanted with mouse-derived colon cancer cells This study evaluated the tumor growth inhibitory effect of the test substance mGI101 and an anti-TIGIT antibody that specifically binds to the extracellular domain (ECD) of TIGIT with the amino acid sequence of SEQ ID NO: 39 as a positive control substance, either alone or in combination, in a tumor model established by syngrafting CT26 (murine colon carcinoma cells) cells into BALB / c mice.

[0199] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). The cultured cells were harvested using trypsin and then suspended in PBS. To establish a syngraft tumor model, 5 × 10 5 CT26 cells were injected subcutaneously into the right flank of BALB / c female mice (7 weeks old).

[0200] Mice were randomly assigned 5 per group based on tumor volume (50 - 120 mm 3 ). Tumor grafts were confirmed approximately 2 days after cell inoculation. The test groups were constituted as shown in Table 8, and the test substances were administered.

[0201]

Table 8

[0202] Clinical symptoms such as disease and behavioral changes were observed once daily during the test period, and dead animals were identified. At the end of the test period, the animals were sacrificed. The size of the CT26 solid cancer was measured using a tumor 3D scanner (TM900, Peria, belgium). Mean body weight loss and percentage change and mean tumor growth inhibition were calculated for each experimental group. The anti-tumor efficacy was evaluated compared to the vehicle control group.

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

[0204] In the CT26 syngeneic tumor model, the anti-tumor efficacy against mGI101 alone and in combination with an anti-TIGIT antibody was tested. As a result, the most excellent anti-tumor efficacy was observed in the combination administration group (mGI101 (3mpk) + anti-TIGIT (20mpk)) (Figure 76). No anti-tumor effect was observed in the anti-TIGIT antibody alone administration group compared to the control group. However, when administered in combination with mGI101, it showed a significantly superior anti-tumor effect compared to the mGI101 alone administration group. A further aspect of the present invention is described below: [Item 1] A pharmaceutical composition for the prevention or treatment of cancer, comprising a fusion protein dimer containing an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor as active ingredients. [Item 2] The pharmaceutical composition for the prevention or treatment of cancer according to Item 1, wherein the IL-2 protein or a variant thereof and the CD80 protein or a fragment thereof are bound by a linker. [Item 3] The pharmaceutical composition for the prevention or treatment of cancer according to Item 1, wherein the IL-2 protein has the amino acid sequence of SEQ ID NO: 10. [Item 4] The pharmaceutical composition for the prevention or treatment of cancer according to Item 1, wherein the CD80 has the amino acid sequence of SEQ ID NO: 11. [Item 5] The pharmaceutical composition for the prevention or treatment of cancer according to Item 1, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 9. [Item 6] The immune checkpoint inhibitor is any one selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-TIM3 antibody, an anti-GAL9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-BTLA antibody, and an anti-TIGIT antibody, and is a pharmaceutical composition for preventing or treating cancer according to item 1. [Item 7] The anti-CTLA-4 antibody is any one selected from the group consisting of ipilimumab and tremelimumab, The anti-PD-1 antibody is any one selected from the group consisting of pembrolizumab, nivolumab, semipilimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514, The anti-PD-L1 antibody is any one selected from the group consisting of atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189, The anti-TIM3 antibody is any one selected from the group consisting of LY3321367, MBG453, and TSR-022, The anti-LAG3 antibody is any one selected from the group consisting of IMP321, relatlimab, and GSK2831781, or The anti-VISTA antibody is JNJ-63723283. A pharmaceutical composition for preventing or treating cancer according to item 6. [Item 8] The cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, and is a pharmaceutical composition for preventing or treating cancer according to item 1. [Item 9] A method for preventing or treating cancer, comprising the step of administering to an individual suffering from cancer a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor. [Item 10] Use of a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor for preventing or treating cancer.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a fusion protein dimer containing an IL-2 variant and a CD80 fragment and an immune checkpoint inhibitor as an active ingredient: Here, the fusion protein has the following structural formula (I): N'-X-[Linker(1)]n-Fc domain-[Linker(2)]m-Y-C' (I) In the formula, N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, X is a CD80 fragment, and the CD80 fragment is the extracellular domain of the CD80 protein, Y is an IL-2 variant containing substitutions of R38A and F42A in the amino acid sequence of SEQ ID NO: 10, Linker(1) is a peptide linker consisting of 30 to 40 amino acids, Linker(2) is a peptide linker consisting of 5 to 15 amino acids, n and m are each independently 0 or 1 as shown, The immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-TIGIT antibody.

2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the extracellular domain of CD80 consists of the amino acid sequence from position 35 to position 242 of SEQ ID NO:

11.

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

9.

4. The immune checkpoint inhibitor is an anti-PD-1 antibody selected from the group consisting of pembrolizumab, nivolumab, semipilumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514, or an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189, the pharmaceutical composition for preventing or treating cancer according to claim 1.

5. The cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, the pharmaceutical composition for preventing or treating cancer according to claim 1. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a fusion protein dimer containing an IL-2 variant and a CD80 fragment, and administered in combination with an immune checkpoint inhibitor: Here, the fusion protein has the following structural formula (I): N'-X-[Linker(1)]n-Fc domain-[Linker(2)]m-Y-C'(I) In the formula, N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, X is a CD80 fragment, and the CD80 fragment is the extracellular domain of the CD80 protein, Y is an IL-2 variant containing substitutions of R38A and F42A in the amino acid sequence of SEQ ID NO: 10, Linker(1) is a peptide linker consisting of 30 to 40 amino acids, Linker(2) is a peptide linker consisting of 5 to 15 amino acids, n and m are each independently 0 or 1 as shown, The immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-TIGIT antibody. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an immune checkpoint inhibitor, and administered in combination with a fusion protein dimer containing an IL-2 variant and a CD80 fragment: Here, the fusion protein has the following structural formula (I): N'-X-[Linker(1)]n-Fc domain-[Linker(2)]m-Y-C'(I) In the formula, N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, X is a CD80 fragment, and the CD80 fragment is the extracellular domain of the CD80 protein, Y is an IL-2 variant containing substitutions of R38A and F42A in the amino acid sequence of SEQ ID NO: 10, Linker(1) is a peptide linker consisting of 30 to 40 amino acids, Linker(2) is a peptide linker consisting of 5 to 15 amino acids, n and m are each independently 0 or 1 as shown, The immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-TIGIT antibody.

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