WO2023048518A1 - Fusion protein dimer comprising pd-1 and il-15 and use thereof - Google Patents

Abstract

The present invention relates to a fusion protein comprising the PD-1 protein and IL-15 protein, and a use thereof. The fusion protein including PD-1, IL-15, and prolonged serum persistent Fc according to a specific embodiment can activate immune cells such as NK cells and effectively increase the half-life of the immune cells in the body. Therefore, a pharmaceutical composition containing the fusion protein as an active ingredient increases immune activity in the body and thus can be utilized as an anticancer drug for patients who do not respond to immune checkpoint inhibitor treatment and relapsed patients.

Description

Fusion protein dimers including PD-1 and IL-15 and uses thereof

The present invention relates to a fusion protein comprising a PD-1 protein and an IL-15 protein and uses thereof. Specifically, the present invention relates to a novel fusion protein dimer comprising PD-1, IL-15, and long-acting Fc in blood, which has an increased half-life in blood and has an immune-enhancing effect, and uses thereof for cancer treatment.

Programmed death receptor-1 (PD-1) is an immune checkpoint that has recently been in the limelight. It is mainly involved in the control of T cell activation and can regulate the strength and duration of an immune response. Under normal circumstances, PD-1 mediates and maintains body tissue autoimmune tolerance, prevents the immune system from over-activating and damaging autologous tissue during the inflammatory response, thus preventing autoimmune diseases from occurring. It works. However, in pathological situations, PD-1 is known to be involved in the development and development of tumor immunity and various autoimmune diseases (Sara Pilotto et.al. , Anticancer Agents Med Chem. , 15(3):307-313 , 2015).

PD-1 is mainly expressed on the surface of activated T cells, and is also expressed on B cells, NK cells, monocytes, and dendritic cells (DCs). The ligands of PD-1, PD-L1 and PD-L2, are expressed on tumor cells, activated B and T cells, dendritic cells, and macrophages. PD-1 binds to these ligands and induces T-cell apoptosis, thereby weakening the cellular immune response. Thus, blocking the PD-1/PD-L1 or PD-L2 pathway is a promising therapeutic approach being explored in many types of cancer research (Miguel F. Sanmamed et. al. , Cancer J. , 20(4): 256-261, 2014).

Meanwhile, IL-15 (Interleukin-15) is a cytokine structurally similar to IL-2 and is expressed in macrophages, monocytes, dendritic cells, fibroblasts, and the like. IL-15 exhibits biological activity by binding to IL-15 receptors composed of IL-15Rα, IL-2Rβ, and γc subunits. Specifically, IL-15 and IL-15Rα are co-expressed in activated dendritic cells and function in the form of an IL-15/IL-15Rα complex. The IL-15/IL-15Rα complex binds to IL-2Rβ/γc on NK cells and T cells and consequently induces differentiation and proliferation of T cells and NK cells. In other words, it is possible to efficiently kill tumor cells by overcoming immune suppression through continuous cytolytic activity of T cells and NK cells (Ying Yang et.al. , Cancers , 12(12):3586, 2020). .

Meanwhile, in the CH2-CH3 portion of immunoglobulin Fc, there is an FcRn (protection receptor) binding site that lengthens the half-life of the antibody (Korean Patent Registration No. 10-1957431). FcRn is an MHC class I-related protein expressed in vascular endothelial cells and binds to IgG and albumin. Antibody fragments without Fc, i.e., without an FcRn-binding site, have a half-life in the body of around 2 to 3 hours, but IgG1, IgG2, and IgG4 with an FcRn-binding site have an average half-life of 3 weeks in the body, which is longer than other proteins. .

Accordingly, the present inventors studied to develop an anti-cancer drug having an increased half-life in the body and excellent immune enhancing efficacy. As a result, a novel fusion protein including PD-1, IL-15 and long-acting Fc in blood activates immune cells, The present invention was completed by confirming that the half-life was increased without affecting the anticancer activity.

To achieve the above object, one aspect of the present invention provides a fusion protein comprising a PD-1 protein and an IL-15 protein.

Another aspect of the present invention provides a fusion protein dimer in which the two fusion proteins are combined.

Another aspect of the present invention provides a polynucleotide encoding the fusion protein.

Another aspect of the present invention provides a vector containing the polynucleotide.

Another aspect of the present invention provides a host cell transformed with the vector.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer disease comprising the fusion protein or fusion protein dimer as an active ingredient.

Another aspect of the present invention provides the use of the fusion protein for treating cancer disease.

A fusion protein containing PD-1, IL-15, and long-acting Fc in the blood can activate immune cells by PD-1 and IL-15, and maximize half-life in vivo by long-acting Fc in the blood. . Therefore, since the fusion protein can efficiently attack cancer cells, it can be usefully used in the treatment of cancer diseases.

1 shows the obtained fusion proteins (BNS002; PD-1 D-Fc(29)-IL-15, PD-1 D-Fc(41)-IL-15, and PD-1 D-Fc(wt)-IL -15) was confirmed by SDS-PAGE.

Figure 2 shows the results of analyzing the purity of the fusion protein (BNS002) obtained by performing SEC-HPLC analysis.

Figure 3 confirms the concentration of the fusion protein (BNS002) obtained using a UV spectrophotometer.

Figure 4 confirms the thermodynamic stability of the fusion protein (BNS002) obtained using fluorescence and SLS (Static Light Scattering) detectors.

5 confirms the structural stability of the fusion protein (BNS002) obtained using a Fourier-transform infrared spectroscopy (FT-IR) spectrophotometer.

6 confirms the molecular weight of the fusion protein (BNS002) obtained through SEC-MALS (Size Exclusion Chomatography - Multi-Angle Laser Light Scattering) analysis.

7 is a result of confirming the binding affinity between the obtained fusion protein (BNS002) and hPD-L1 (human programmed cell death ligand-1).

8 is a result of confirming the binding affinity between hPD-1 protein (human programmed cell death protein-1) and hPD-L1.

9 is a result of confirming the binding affinity between the obtained fusion protein (BNS002) and hPD-L2 (human programmed cell death ligand-2).

10 is a result of confirming the binding affinity between hPD-1 protein and hPD-L2.

11 is a result of confirming the binding affinity between the obtained fusion protein (BNS002) and hIL-15Rα (human Interleukin-15 receptor alpha).

12 is a result of confirming the binding affinity between hIL-15 protein and hIL-15Rα (human Interleukin-15 receptor alpha).

13 is a result of confirming the binding affinity between the obtained fusion protein (BNS002) and hIL-2Rβ (human Interleukin-15 receptor beta).

14 is a result of confirming the binding affinity between hIL-15 protein and hIL-2Rβ (human Interleukin-15 receptor beta).

15 is a result of confirming the binding affinity between the obtained fusion protein (BNS002) and the hFcRn receptor (human neonatal Fc receptor).

16 is a result of confirming the binding affinity between the obtained fusion protein (BNS002) and PD-L1 through ELISA analysis.

17 shows the result of confirming the binding affinity between the obtained fusion protein (BNS002) and PD-L2 through ELISA analysis.

18 shows the result of confirming the binding affinity between the obtained fusion protein (BNS002) and IL-15Rα through ELISA analysis.

19 shows the result of confirming the binding affinity between the obtained fusion protein (BNS002) and IL-2Rβ (Interleukin-2 receptor beta) through ELISA analysis.

20 shows the result of confirming the binding affinity between the obtained fusion protein (BNS002) and the hFcRn receptor through Lumit ^™ FcRn binding immunoassay analysis.

21 shows the result of confirming the binding affinity between the obtained fusion protein (BNS002) and the IL-15 receptor through PathHunter ^® eXpress Dimerization Assay analysis.

22 shows the obtained fusion protein (BNS002) and IL-15 respectively treated with peripheral blood mononuclear cells (PBMC), and the amount of IFN-γ secreted from PBMC containing immune cells during culture was measured through ELISA analysis. This is the result.

23 confirms the effect of the obtained fusion protein (BNS002) and IL-15 protein on the proliferation of CD8+ T cells through FACS analysis.

24 confirms the effect of the obtained fusion protein (BNS002) and IL-15 protein on the proliferation of CD4+ T cells through FACS analysis.

Figure 25a confirms the effect of the obtained fusion protein (BNS002) and IL-15 protein on NK cell proliferation through FACS analysis.

Figure 25b is a graph of the results of confirming the effect of the obtained fusion protein (BNS002) and IL-15 protein on the proliferation of NK cells through FACS analysis.

26 is a result of confirming the effect of the obtained fusion protein (BNS002) on effector T cells through PD-L1/PD-1 blocking assay.

27 shows the results of confirming the effect of the obtained fusion protein (BNS002) on effector T cells through PD-L2/PD-1 blocking assay.

FIG. 28 schematically shows a lentivirus vector map used to construct a cell line expressing Firefly luciferase and green fluorescent protein (GFP). Here, CMV is a promoter derived from Cytomegalovirus, and RSV is a promoter derived from respiratory syncytial virus.

29 shows the results of measuring the luminescence signal (left) and fluorescence signal (right) of the human melanoma cell line (A375-Luc-GFP) expressing firefly luciferase and GFP established in the present invention.

30 shows PD-L1 and PD-L2 genes (left) specifically expressed in human melanoma cell line (A375-Luc-GFP) and PBMC containing immune cells according to an embodiment of the present invention. The expression pattern of the PD-1 gene (right), which is expressed as , was confirmed through reverse transcription PCR.

31 shows the IFN-secreted from the cells upon treatment with the obtained fusion protein (BNS002) and IL-15 in co-cultured human melanoma cell line (A375-Luc-GFP) and peripheral blood mononuclear cells (PBMC), respectively. This is the result of measuring the amount of γ through ELISA analysis.

32 shows the treatment of co-cultured human melanoma cell line (A375-Luc-GFP) and peripheral blood mononuclear cells (PBMC) with the obtained fusion protein (BNS002), and the cancer cell killing effect by treatment time was measured by dehydrogenase, 　succi It was analyzed by measuring succinate-tetrazolium reductase activity.

Figure 33 shows the cancer cell killing effect after treatment of the obtained fusion protein (BNS002) on co-cultured human melanoma cell line (A375-Luc-GFP) and peripheral blood mononuclear cells (PBMC) for 48 hours. It was confirmed by measuring the activity of tetrazolium reductase.

34 shows the co-cultured human melanoma cell line (A375-Luc-GFP) and peripheral blood mononuclear cells (PBMC) treated with the obtained fusion protein (BNS002) at different concentrations, and the cancer cell killing effect at each treatment concentration was measured by the dehydrogenase 　succi It was analyzed by measuring nate-tetrazolium reductase activity.

35 confirms the cancer cell line (A375-Luc-GFP)-specific antibody dependent cellular cytotoxicity (ADCC) activity of the obtained fusion protein (BNS002).

36 schematically shows administration and experimental schedules for confirming the anticancer effect of the fusion protein (BNS002) in mice with mouse-derived colon cancer cells implanted therein.

37 is a result confirming the tumor suppression effect by the fusion protein (BNS002) in mice with mouse-derived colorectal cancer cells implanted therein.

38 is a result confirming the statistical significance of the tumor suppression effect by the fusion protein (BNS002) in mice with mouse-derived colorectal cancer cells implanted therein.

Figure 39 confirms the degree of immune cell activity in cancer tissue by the obtained fusion protein (BNS002), and in detail, mice with mouse-derived colorectal cancer cells were treated with the fusion protein (BNS002) and excised on day 35 in the cancer tissue. The results of FACS analysis of the activity levels of phagocytes, dendritic cells (DC), CD4+ cells, CD8+ cells and NK cells are shown in graphs.

40 schematically shows administration and experimental schedules for confirming the anticancer effect of the fusion protein (BNS002) in humanized mice implanted with human-derived lung cancer cells (H460).

41 is a result confirming the tumor suppression effect by administration of the fusion protein (BNS002) in humanized mice implanted with human-derived lung cancer cells (H460).

42 is a result confirming the statistical significance of the tumor suppression effect by administration of the fusion protein (BNS002) in humanized mice implanted with human-derived lung cancer cells (H460).

43 schematically shows administration and experimental schedules for confirming the anticancer effect of the fusion protein (BNS002) in humanized mice implanted with human-derived colorectal cancer cells (HCT116).

44 is a result confirming the concentration-dependent tumor suppression effect by administration of the fusion protein (BNS002) in humanized mice implanted with human-derived colorectal cancer cells (HCT116).

45 is a result of confirming the degree of body weight change according to the administration of each concentration of fusion protein (BNS002) in humanized mice implanted with human-derived colorectal cancer cells (HCT116).

46 schematically shows administration and experimental schedules for confirming the anticancer effect between a fusion protein (BNS002 or BNS002S) and a control drug in humanized mice implanted with human-derived colorectal cancer cells (HCT116).

47 compares the tumor suppression effect by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized mice implanted with human-derived colorectal cancer cells (HCT116) compared to control BNS001D (PD-1 D-Fc) and BNS002I (Fc-IL-15) fusion protein alone or combined administration.

48 compares the degree of body weight change by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized mice implanted with human-derived colorectal cancer cells (HCT116) compared to control BNS001D (PD-1 D-Fc) and BNS002I (Fc-IL-15) fusion protein alone or combined administration.

49 shows the tumor suppression effect by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized colorectal cancer cell (HCT116) implanted humanized mice in positive control groups atezolizumab, pembrolizumab, and Abel This is a result compared to Lumab administration.

50 shows the degree of weight change by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized mice implanted with human-derived colorectal cancer cells (HCT116) in positive control groups atezolizumab, pembrolizumab, and Abel This is a result compared to Lumab administration.

51 compares the tumor suppression effect by administration of a fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived colorectal cancer cells (HCT116), as a control, BNS001D (PD-1 D -Fc) and BNS002I (Fc-IL-15) fusion protein alone or combined administration.

52 compares the degree of body weight change by administration of fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived colon cancer cells (HCT116), BNS001D (PD-1 D -Fc) and BNS002I (Fc-IL-15) fusion protein alone or combined administration.

53 shows the tumor suppression effect by administration of a fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized colon cancer cell (HCT116) implanted humanized mice as positive control groups, atezolizumab and pembroli. This is a result compared to administration of Zumab and Avelumab.

54 shows the degree of weight change by administration of fusion proteins (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived colorectal cancer cells (HCT116) in positive control groups, atezolizumab and pembroli This is a result compared to administration of Zumab and Avelumab.

55 schematically shows administration and experimental schedules for confirming the anticancer effect between a fusion protein (BNS002 or BNS002S) and a control drug in humanized mice implanted with human-derived lung cancer cells (A549).

56 shows the tumor suppression effect by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized mice implanted with human-derived lung cancer cells (A549) as positive controls: atezolizumab, pembrolizumab, and avelumab. This is the result of comparison with administration.

57 shows the degree of body weight change by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized mice implanted with human-derived lung cancer cells (A549) as positive controls: atezolizumab, pembrolizumab, and avelumab. This is the result of comparison with administration.

58 is a result confirming the concentration-dependent tumor suppression effect by administration of a fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice harboring human-derived lung cancer cells (A549).

59 is a result of confirming the degree of body weight change according to administration of fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) at different concentrations in humanized mice implanted with human-derived lung cancer cells (A549).

60 shows the tumor suppression effect by administration of a fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived lung cancer cells (A549) in positive control groups, atezolizumab and pembrolizumab. and the results compared with administration of Avelumab.

61 shows the degree of weight change by administration of fusion proteins (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived lung cancer cells (A549) in positive control groups, atezolizumab and pembrolizumab. and the results compared with administration of Avelumab.

62 schematically shows administration and experimental schedules for confirming the anticancer effect between a fusion protein (BNS002 or BNS002S) and a control drug in humanized mice implanted with human-derived breast cancer cells (MDA-MB-231).

63 shows the tumor suppression effect by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized breast cancer cells (MDA-MB-231) implanted humanized mice in positive control groups, atezolizumab and pembroli. This is a result compared to administration of Zumab and Avelumab.

64 shows the degree of body weight change by administration of a fusion protein (BNS002; PD-1 D-Fc-IL-15) in humanized mice implanted with human-derived breast cancer cells (MDA-MB-231) in positive control groups, atezolizumab and pembroli This is a result compared to administration of Zumab and Avelumab.

65 shows the tumor suppression effect by administration of a fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived breast cancer cells (MDA-MB-231) as a positive control group, atezolizumab. , This is a result of comparison with administration of pembrolizumab and avelumab.

66 shows the degree of body weight change by administration of a fusion protein (BNS002S; PD-1D-Fc-IL-15/IL-15Ra) in humanized mice implanted with human-derived breast cancer cells (MDA-MB-231) as a positive control group, atezolizumab. , This is a result of comparison with administration of pembrolizumab and avelumab.

67 is a schematic diagram of an embodiment of a fusion protein (BNS002) dimer.

68 is a schematic diagram of an embodiment of a fusion protein (BNS002S) dimer.

Fusion protein comprising PD-1 protein and IL-15 protein

One aspect of the present invention provides a fusion protein comprising PD-1 protein and IL-15 protein.

In addition, the fusion protein may further include an IL-15 binding protein.

In addition, the fusion protein may further include an Fc region.

PD-1 protein or fragment thereof

As used herein, the term "PD-1 (programmed cell death protein 1 or programmed death receptor-1)" is a type of reciprocal inhibitory receptor belonging to the B7-CD28 family, an immune checkpoint also known as CD279. (immune checkpoint) protein. PD-1 is widely expressed on the surface of activated T cells, natural killer T cells, B cells and macrophages. When PD-1 binds to its ligand, programmed cell death ligand-1 (PD-L1) or programmed cell death ligand-2 (PD-L2), intracellular signaling inhibits CD3- and CD28-mediated T cell activation. induce Downregulation of the T cell activation results in a decrease in T cell proliferation, IFN-γ secretion, IL-2 secretion, and the like. PD-1 and its ligand known to be expressed in various types of cancer cells (e.g., melanoma, liver cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, breast cancer, lymphoma, glioma, etc.) Interaction with PD-L1 or PD-L2 inactivates T cells and acts as a mechanism for cancer cells to evade the immune system's attack. Therefore, blocking the interaction between PD-1 and PD-L1 or PD-L2 activates T cells in the tumor microenvironment to eliminate tumor cells.

As used herein, the term "PD-1", "programmed cell death receptor-1" or "programmed death protein-1" refers to a mammal, e.g., a primate (e.g., a human), unless otherwise indicated. ) and any wild-type PD-1 obtained from any vertebrate source, including rodents (eg, mice and rats). The PD-1 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing PD-1. Also, the PD-1 may be wild-type PD-1 or a fragment thereof.

PD-1 is a type I membrane protein consisting of 288 amino acids, and its structure consists of an extracellular IgV-like domain, a transmembrane domain, and a cytoplasmic domain. The amino acid sequence of PD-1 is GenBank accession NO. NP_005009.2 or UniProtkB No. Q15116 (for the amino acid sequence of mouse PD-1, see Genbank accession No. NP_032824.1). In addition, PDCD1 (programmed cell death 1) gene as a sequence encoding the PD-1 is GenBank accession NO. It may be a nucleotide sequence corresponding to a coding sequence (CDS) among the sequences described in NM_005018.3 (see GenBank accession No. NM_008798.3 for mouse sequence).

As used herein, “PD-1 protein” refers to full-length PD-1 or PD-1 fragments. In the present specification, PD-1 or fragments thereof are collectively referred to as "PD-1 protein". PD-1, PD-1 proteins and PD-1 fragments specifically bind to, for example, PD-1's ligand, PD-L1 or PD-L2. This specific binding can be confirmed by methods known to those skilled in the art.

The ligands of PD-1, PD-L1 and PD-L2, are proteins expressed on the surface of cancer cells. PD-L1 is also known as B7-H1 or CD274, and PD-L2 is also known as B7-DC or CD273. When PD-L1 or PD-L2 binds to PD-1 present in T cells, it suppresses the function of T cells by changing the immune checkpoint function. That is, T cell function is suppressed by the interaction of PD-1 and PD-L1 or PD-L2, and ultimately cancer cells evade the attack of immune cells.

As used herein, the term "PD-1 fragment" means a truncated form of PD-1. In addition, the PD-1 fragment may be an extracellular domain of PD-1. One specific example of the PD-1 fragment may be one in which the 1st to 24th amino acids from the N-terminus, which is the signal sequence of PD-1, are excluded. Specifically, one specific example of the PD-1 fragment may be a protein composed of the 25th to 288th amino acids of SEQ ID NO: 18. In addition, one specific example of the PD-1 fragment may be a protein composed of 25th to 170th amino acids of SEQ ID NO: 18. In addition, one specific example of the PD-1 fragment may be a protein composed of the 25th to 150th amino acids of SEQ ID NO: 18. In addition, one specific example of the PD-1 fragment may be a protein composed of 25th to 144th amino acids of SEQ ID NO: 18. In one embodiment, the PD-1 fragment may have the amino acid sequence of SEQ ID NO: 2 or 39.

In addition, one specific example of the PD-1 fragment may be a protein composed of amino acids 25th to 169th of SEQ ID NO: 31. In one embodiment, the PD-1 fragment may have the amino acid sequence of SEQ ID NO: 23.

IL-15 or a variant thereof

As used herein, the term "IL-15" or "interleukin-15" is a cytokine structurally similar to IL-2, and belongs to a family having a four α-helix structure. IL-15 is expressed in macrophages, monocytes, dendritic cells, fibroblasts, and the like. IL-15 binds to the receptor for IL-15, which consists of IL-15Rα (IL-15 receptor alpha), IL-2Rβ (IL-2 receptor beta; also known as IL-15Rβ or CD122), and γc (also known as CD132) subunits. binding to exhibit biological activity. Specifically, IL-15 and IL-15Rα are co-expressed in activated dendritic cells and function in the form of an IL-15/IL-15Rα complex. The IL-15/IL-15Rα complex binds to IL-2Rβ/γc on NK cells and T cells and consequently induces differentiation and proliferation of T cells and NK cells. That is, it is possible to efficiently kill tumor cells by overcoming immunosuppression by continuous cytolytic activity of T cells and NK cells. In addition, IL-15 promotes the induction of immunoglobulin synthesis by B cells and the regulation of lymphoid homeostasis. Moreover, IL-15 shares the IL-2Rβ and IL-2Rγ receptors with IL-2, but does not bind to the IL-2Rα (CD25) receptor and thus does not activate regulatory T cells (Treg) that suppress immunity. There are advantages to not

As used herein, the term "IL-15" or "interleukin-15", unless stated otherwise, includes mammals such as primates (eg humans) and rodents (eg mice and rats). Any wild-type IL-15 obtained from any vertebrate source. The IL-15 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-15. In addition, the IL-15 may be wild-type IL-15, an isoform, or a variant thereof.

IL-15 has two IL-15 isoforms with different signal peptide lengths, but these two isoforms produce the same mature IL-15. IL-15 isoform has IL-15 LSP (Isoform IL-15-S48AA; Interlekin-15 isoform 1 preproprotein, Homo sapiens , GenBank accession No. NP_000576.1) with a long signal peptide of 48 amino acids, 21 There is an IL-15 SSP (Isoform IL-15-S21AA; Interleukin-15 isofrom 2 preproprotein, Homo sapiens , GenBank accession No. NP_751915.1) in which a short signal peptide of four amino acids is present. The two IL-15 isoforms share 11 amino acids (CFSAGLPKTEA) between the N-terminal signal sequences, and produce the same mature IL-15, although there are differences in intracellular trafficking. The IL-15 LSP isoform is secreted from the Golgi apparatus, early endosomes, and endoplasmic reticulum (ER), whereas the IL-15 SSP isoform is not secreted and exists in the cytoplasm and nucleus.

IL-15 consists of a signal peptide and a mature polypeptide, the amino acid sequence of which is GenBank accession No. NP_000576.1, NP_751915.1, CAA71044.1 or UniProtkB No. P40933 (see Genbank accession No. NP_001241676.1 for the amino acid sequence of mouse IL-15). In addition, as a sequence encoding the IL-15, the IL-15 gene is GenBank accession NO. It may be a nucleotide sequence corresponding to a coding sequence (CDS) among the sequences described in NM_000585.5, NM_172175.3 or Y09908.1 (for mouse sequences, see GenBank accession No. NM_001254747.1).

Specifically, IL-15 consists of 136 amino acids, and may consist of a secretion signal sequence of amino acid residues 1 (Met) to 22 (Ala) and a mature polypeptide of amino acid residues 23 (Asn) to 136 (Ser). In addition, IL-15 consists of 162 amino acids, and may consist of a secretion signal sequence of amino acid residues 1 (Met) to 48 (Ala) and a mature polypeptide of amino acid residues 49 (Asn) to 162 (Ser). In addition, IL-15 consists of 135 amino acids, and may consist of a secretion signal sequence of amino acid residues 1 (Met) to 21 (Ala) and a mature polypeptide of amino acid residues 22 (Asn) to 135 (Ser).

As used herein, “IL-15 protein” refers to full-length IL-15, IL-15 fragments, or IL-15 variants. In the present specification, IL-15, IL-15 fragments, or variants thereof are collectively referred to as "IL-15 protein" or "IL-15 polypeptide". IL-15, IL-15 proteins, IL-15 polypeptides, and IL-15 variants specifically bind to, for example, IL-15Rα or IL-2Rβ. This specific binding can be confirmed by methods known to those skilled in the art.

As used herein, one specific example of the IL-15 may have an amino acid sequence of SEQ ID NO: 19, 20, 21 or 32. Also, at this time, the IL-15 may be in a mature form. Specifically, the matured IL-15 may not contain a signal sequence and may have the amino acid sequence of SEQ ID NO: 6 or 24. In this case, the IL-15 may be used as a concept including a fragment in which a part of the N-terminus or C-terminus of wild-type IL-15 is truncated.

In addition, the fragment of IL-15 is 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 19. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids may be deleted. In addition, the fragments of IL-15 are 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 19. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids may be deleted.

In addition, the fragments of IL-15 are 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 20. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids may be deleted. In addition, the fragments of IL-15 are 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 20. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids may be deleted.

In addition, the fragments of IL-15 are 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 21. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids may be deleted. In addition, the fragment of IL-15 is continuously 1, 2, 3, 4, 5, 6, 7, 8, 9 from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 21. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids may be deleted.

In addition, the fragments of IL-15 are 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 32. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids may be deleted. In addition, the fragments of IL-15 are 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 32. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids may be deleted.

As used herein, the term "IL-15 variant" refers to a full-length IL-15 or a form in which a part of the amino acid of the aforementioned IL-15 fragment is substituted. That is, the IL-15 mutant may have an amino acid sequence different from wild-type IL-15 or a fragment thereof. However, the IL-15 variant may have an activity equivalent to or similar to that of wild-type IL-15. Here, “IL-15 activity” may mean, for example, binding specifically to IL-15Rα or IL-2Rβ, and this specific binding can be measured by a method known to those skilled in the art.

Specifically, the IL-15 mutant may be one in which some amino acids of wild-type IL-15 are deleted and/or substituted. One specific example of an IL-15 mutant by amino acid deletion and/or substitution is the secretion signal sequence of 1 (Met) to 22 (Ala) in the amino acid sequence of SEQ ID NO: 19, as disclosed in US Patent No. 10,450,359. deletion, and amino acid substitution of Asn(N) with Asp(D) or Asn(N) with Ala(A) at position 72 of the mature 114 amino acid peptide. In addition, the secretion signal sequence of 1 (Met) to 48 (Ala) is deleted from the amino acid sequence of SEQ ID NO: 20, and Asn (N) is converted to Asp (D) or Asn (N) at position 72 of the mature 114 amino acid peptide. ) may be amino acid substitution with Ala (A). In addition, the secretion signal sequence of 1 (Met) to 21 (Ala) was deleted from the amino acid sequence of SEQ ID NO: 21, and Asn (N) was converted to Asp (D) or Asn (N) at position 72 of the mature 114 amino acid peptide. ) may be amino acid substitution with Ala (A). The IL-15 variant may be characterized in that the activity of IL-15 is maintained or improved compared to wild-type IL-15.

In addition, the PD-1 protein and the IL-15 protein may be linked by a linker or a carrier. Specifically, the PD-1 or a fragment thereof and the IL-15 or a variant thereof may be linked by a linker or a carrier. In this specification, a linker and a carrier are also used interchangeably.

linker

The linker connects the two proteins. One specific example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, or an Fc domain of an immunoglobulin. At this time, the Fc domain of the immunoglobulin includes heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin, and includes variable regions and light chain constant region 1 (CH1) of heavy and light chains of immunoglobulin. protein that does not The immunoglobulin may be IgG, IgA, IgE, IgD or IgM, preferably IgG1. In this case, the Fc domain of wild-type immunoglobulin G1 may have the amino acid sequence of SEQ ID NO: 14.

In addition, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. In addition, the term "Fc domain variant" as used herein refers to a glycosylation pattern that is different from that of the wild-type Fc domain, increased sugar chains compared to the wild-type Fc domain, reduced sugar chains compared to the wild-type Fc domain, or sugar chains removed. (deglycosylated) form. Also included are aglycosylated Fc domains. The Fc domain or variant may have sialic acid, fucosylation, and glycosylation of numbers adjusted through culture conditions or genetic manipulation of the host.

In addition, the sugar chain of the Fc domain of immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a mixed form of an immunoglobulin Fc region of IgG, IgA, IgE, IgD or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. One specific example of the Fc domain variant is Q81R, M198L, L79G, T136S, L138A, Y177V, Q81R/M198L, L79G/M198L, T136S/L138A/Y177V or Q81R/T136S/L138A/Y177V in the amino acid sequence of SEQ ID NO: 14. Substitution of /M198L may have occurred. Specifically, one specific example of the Fc domain variant may have an amino acid sequence of SEQ ID NO: 4, 10, 55, 88 or 97.

As disclosed in Korean Patent Registration No. 10-1957431, one specific example of the Fc domain variant by amino acid substitution is an Fc domain variant comprising an amino acid substitution that regulates binding and dissociation to FcRn (neonatal Fc receptor). This may include, in particular, Fc variants that exhibit increased binding affinity to FcRn at low pH and show no substantially altered binding at high pH, or functional variants thereof.

The Fc domain variant has a binding affinity to FcRn of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more at pH 5.6 to 6.2 (preferably pH 5.8 to 6.0) compared to the wild-type Fc domain. 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more, or more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or 7-fold greater than the wild-type Fc domain more than 8 times, more than 9 times, more than 10 times, more than 20 times, more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, or more than 100 times It can be.

The degree of dissociation of the Fc domain variant from the neonatal Fc receptor (FcRn) at pH 7.0 to 7.8 (preferably pH 7.2 to 7.6) may be the same as or substantially unchanged compared to the wild-type Fc domain.

In addition, the Fc domain variant may be characterized in that half-life is increased compared to the wild type. Specifically, the half-life of the Fc domain variant is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more compared to the wild-type Fc domain. Increased by at least 100% or more, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold more than the wild-type Fc domain there is.

As used herein, the term "neonatal Fc receptor (FcRn)" or "neonatal Fc receptor" is an MHC class I related protein expressed in vascular endothelial cells and binds to IgG and albumin. The characteristic point is that the binding between IgG and FcRn is strong when the pH is slightly acidic, and there is no binding force at neutral pH. Therefore, IgG entering cells by pinocytosis or endocytosis binds strongly to FcRn, a type of Fc gamma receptor (FcγR), in endosomes under pH 6.0 conditions, and forms degradative lysosomes. ) pathway, and once circulated back to the cell membrane, IgG rapidly dissociates from FcRn in the bloodstream at pH 7.4. By this receptor-mediated recycling mechanism (receptor mediated recycling mechanism), FcRn is known to effectively block the degradation of IgG in the lysosome to extend the half-life of IgG.

As used herein, the term "Fc gamma receptor (FcγR)" is an Fc receptor for IgG, and there are three types of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). An Fc receptor is a molecule that binds to immunoglobulin and allows the bound antibody to perform various biological functions independently of an antigen binding site, and is distributed on the surface of various cells and tissues. Fc receptors are involved in cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), secretion of mediators such as cytokines, phagocytosis, initiation of oxidation, and regulation of antibody production.

The fusion protein may have a structure in which PD-1 and IL-15 proteins are connected to the N-terminus and C-terminus of the Fc domain as a linker (or carrier), or IL-15 and PD-1 are connected, respectively. Connection between the N-terminus or C-terminus of the Fc domain and PD-1 or IL-15 may be optionally achieved by a linker peptide.

IL-15 binding protein

The fusion protein including the PD-1 protein or a fragment thereof and the IL-15 protein or a variant thereof may additionally include an IL-15 binding protein.

As used herein, the term "IL-15 binding protein" may be a protein or peptide that specifically binds to IL-15. In this case, one specific example of the IL-15 binding protein may be “the “sushi” domain of IL-15Rα. The "sushi" domain, which corresponds to a portion of the extracellular domain of the IL-15Rα, is required for binding to IL-15. The sushi domain of IL-15Rα is the sushi domain of mammalian IL-15Rα, preferably the sushi domain of primate IL-15Rα, more preferably the sushi domain of human IL-15Rα. One specific example of the “sushi” domain of the IL-15Rα may have the amino acid sequence of SEQ ID NO: 95. In addition, the “sushi” domain of IL-15Rα may be a fragment of SEQ ID NO: 95 (ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 95)).

Fusion protein structure

Specifically, the fusion protein may be any one of the groups consisting of structural formulas I to IV:

N'-A-[L ₁ ]n-Fc domain-[L ₂ ]mB-[L ₃ ] _l -SC' I

N'-A-[L ₁ ]n-Fc domain-[L ₂ ]mS-[L ₃ ] _l -BC' II

N'-S-[L ₃ ] _l -B-[L ₁ ]n-Fc domain-[L ₂ ]mAC' III

N'-B-[L ₃ ] _l -S-[L ₁ ]n-Fc domain-[L ₂ ]mAC' IV

At this time, in the structural formulas I to IV,

N' is the N-terminus of the fusion protein,

The C' is the C-terminus of the fusion protein,

A is PD-1 protein or a fragment thereof;

B is an IL-15 protein or a variant thereof;

S is an IL-15 binding protein,

Wherein L ₁ , L ₂ and L ₃ are each a peptide linker,

The above n, m and l are each independently 0 or 1.

In this case, PD-1 protein or fragment thereof, IL-15 protein or variant thereof, IL-15 binding protein, peptide linker, and Fc domain are as described above.

Specifically, the form in which the IL-15 binding protein is removed from the structure of the fusion protein may have structural formula V or VI:

N′-A-[L ₁ ]n-Fc domain-[L ₂ ]mBC′ V

N'-B-[L ₁ ]n-Fc domain-[L ₂ ]mAC' VI

At this time, in the structural formulas V and VI,

N' is the N-terminus of the fusion protein,

The C' is the C-terminus of the fusion protein,

A is PD-1 protein or a fragment thereof;

B is an IL-15 protein or a variant thereof;

Wherein L ₁ and L ₂ are each a peptide linker,

The n and m are each independently 0 or 1. In this case, the PD-1 protein or a fragment thereof, the IL-15 protein or a variant thereof, the peptide linker, and the Fc domain are as described above.

Preferably, the fusion protein may have structural formula V. The PD-1 protein and the IL-15 protein are each as described above. According to one embodiment, the PD-1 protein may be a truncated fragment of up to about 24 amino acid residues consecutively from the N-terminus or C-terminus of wild-type PD-1. The IL-15 protein may be a fragment in which about 21 to 48 amino acid residues are consecutively deleted from the N-terminus or C-terminus of wild-type IL-15. Alternatively, the IL-15 protein may be an IL-15 mutant in which some amino acids of an IL-15 fragment are deleted and/or substituted.

Specifically, the fusion protein may have an amino acid sequence of SEQ ID NO: 8, 12, 16, 26, 29, 33, 37, 41, 46, 49, 57, 60, 77, 80, 83, 90 or 93 . According to another embodiment, the fusion protein is for the amino acid sequence of SEQ ID NO: 8, 12, 16, 26, 29, 33, 37, 41, 46, 49, 57, 60, 77, 80, 83, 90 or 93 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity It includes a polypeptide having. At this time, the identity may be determined through, for example, percent homology, homology comparison software such as NCBI (National Center of Biotechnology Information) BlastN software.

A peptide linker L ₁ may be included between the PD-1 protein and the Fc domain. The peptide linker L ₁ may consist of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, or 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one embodiment, the peptide linker L ₁ may consist of 30 amino acids. In addition, the peptide linker L ₁ may include at least one cysteine. Specifically, it may contain 1, 2 or 3 cysteines. In addition, the peptide linker L ₁ may be derived from the hinge of an immunoglobulin. In one embodiment, the peptide linker L ₁ may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3 or 43.

The peptide linker L ₂ or L ₃ may consist of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one embodiment, the peptide linker L ₂ or L ₃ may be (G ₄ S)n (where n is an integer from 1 to 10) or include this. In this case, in (G4S)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker L ₂ may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5, 44 or 62. In one embodiment, the peptide linker L ₃ may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 96.

Another aspect of the present invention provides a fusion protein dimer in which two fusion proteins including the PD-1 protein or a fragment thereof and the IL-15 protein or a variant thereof are linked.

The fusion protein comprising the PD-1 or a fragment thereof and the IL-15 protein or a variant thereof is as described above.

Another aspect of the present invention provides a fusion protein dimer in which two fusion proteins including the PD-1 protein or a fragment thereof, the IL-15 protein or a variant thereof, and an IL-15 binding protein are linked.

The fusion protein comprising the PD-1 or a fragment thereof, the IL-15 protein or a variant thereof, and the IL-15 binding protein is as described above.

At this time, the binding between the fusion proteins constituting the dimer may be made by a disulfide bond by a cysteine present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same or may be different fusion proteins. Preferably, the dimer may be a homodimer. One embodiment of the fusion protein constituting the dimer is SEQ ID NO: 8, 12, 16, 26, 29, 33, 37, 41, 46, 49, 57, 60, 77, 80, 83, 90 or 93 amino acids It may be a protein having a sequence.

Polynucleotides encoding fusion proteins

Another aspect of the present invention provides a polynucleotide encoding a fusion protein comprising PD-1 protein and IL-15 protein. Specifically, the polynucleotide may include the nucleotide sequence of SEQ ID NO: 9, 13, 17, 27, 30, 35, 38, 42, 47, 50, 58, 61, 78, 81, 84, 91 or 94 there is. The fusion protein comprising the PD-1 protein and the IL-15 protein is as described above. The polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases. When preparing a nucleotide sequence by chemical synthesis, a synthesis method well known in the art, for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis methods on solid supports, and the like.

According to one embodiment, the polynucleotide is at least about 70% SEQ ID NO: 9, 13, 17, 27, 30, 35, 38, 42, 47, 50, 58, 61, 78, 81, 84, 91 or 94 , at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92% , at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity. can

The polynucleotide may additionally include a nucleic acid encoding a signal sequence or a leader sequence. As used herein, the term "signal sequence" refers to a signal peptide that directs the secretion of a target protein. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence is an amino acid sequence that initiates protein movement through an endoplasmic reticulum (ER) membrane. In one embodiment, the signal sequence may have the amino acid sequence of SEQ ID NO: 1 or 22.

The signal sequence is well known in the art and usually includes 16 to 30 amino acid residues, but may include more or less amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence throughout the membrane lipid bilayer during migration of the immature polypeptide.

After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. In this case, the signal sequence may be tPa (tissue plasminogen activation), HSV gDs (signal sequence of herpes simplex virus glycoprotein D), or growth hormone secretion signal sequence. Preferably, secretion signal sequences used in higher eukaryotic cells including mammals and the like can be used. In addition, as the signal sequence, a signal sequence included in wild-type PD-1 and/or IL-15 may be used, or a codon frequently expressed in the host cell may be substituted for use.

A vector loaded with a polynucleotide encoding a fusion protein

Another aspect of the present invention provides a vector containing the polynucleotide.

The vector can be introduced into a host cell and then recombinated and inserted into the genome of the host cell. Alternatively, the vector is understood to be a nucleic acid vehicle comprising a polynucleotide sequence capable of autonomous replication as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogues thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

Specifically, the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids derived from spp. (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal viral vectors (retrovirus ), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the expression level and modification of the protein of the vector appear differently depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.

As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean transcription of DNA sequences, translation of mRNA transcripts and secretion of fusion protein products or fragments thereof. A useful expression vector can be RcCMV (Invitrogen, Carlsbad) or variants thereof. The expression vector includes a human cytomegalovirus (CMV) promoter for promoting continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence for increasing the steady-state level of RNA after transcription. can do.

Transformed cells expressing the fusion protein

Another aspect of the present invention provides a transformed host cell into which the vector is introduced.

Host cells of the transformed cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, mammalian cells, plants, insects, fungi, or cells of cellular origin. As an example of the prokaryotic cell, Escherichia coli may be used. In addition, yeast may be used as an example of a eukaryotic cell. In addition, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells, or HEK293T cells may be used as the mammalian cells. , It is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art can be used.

In addition, when the expression vector is introduced into the host cell, the CaCl ₂ precipitation method, the Hanahan method with increased efficiency by using a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ precipitation method, the electroporation method, and the calcium phosphate precipitation method , protoplast fusion method, agitation method using silicon carbide fibers, agrobacteria-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine, and drying/inhibition-mediated transformation methods, etc. may be used.

As described above, the sugar chain pattern of the fusion protein (e.g., sialic acid, fucosylation, glycosylation).

Fusion protein production method

Another aspect of the present invention provides a method for producing a fusion protein comprising PD-1 protein and IL-15 protein, comprising culturing the transformed cell. Specifically, the production method includes i) culturing the transformed cells to obtain a culture product; and ii) recovering the fusion protein from the culture.

The method of culturing the transformed cells may be performed using a method widely known in the art. Specifically, the culture may be cultured continuously in a batch process or an injection batch or a repeated injection batch process (fed batch or repeated fed batch process).

Use of fusion protein or dimer thereof

Another aspect of the present invention is for the treatment or prevention of cancer, comprising as an active ingredient a fusion protein comprising PD-1 protein and IL-15 protein or a fusion protein dimer in which the two fusion proteins are combined, and/or It provides a pharmaceutical composition capable of increasing the therapeutic effect (efficacy).

The fusion protein including the PD-1 protein and the IL-15 protein or the fusion protein dimer in which the two fusion proteins are combined are as described above.

The cancer is colorectal cancer, melanoma, stomach cancer, liver cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, gallbladder cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma , head and neck cancer, salivary gland cancer and lymphoma.

A preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing cancer disease of the present invention, the active ingredient may be included in any amount (effective amount) according to the purpose of use, formulation, combination, etc., as long as it can exhibit anticancer activity. A typical effective amount is the entire composition. On a weight basis it will be determined within the range of 0.001% to 20.0% by weight. Here, "effective amount" refers to the amount of an active ingredient capable of inducing an anticancer effect. Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

As used herein, the term "treatment" may be used to include both therapeutic treatment and prophylactic treatment. At this time, prevention may be used in the sense of alleviating or reducing the pathological condition or disease of the subject. In one embodiment, the term “treatment” includes any form of administration or application to treat a disease in a mammal, including humans. The term also includes inhibiting or slowing the disease or progression of the disease; restoring or repairing a damaged or missing function, thereby partially or completely alleviating the disease; or stimulate inefficient processes; It includes the meaning of alleviating serious illness.

As used herein, the term "efficacy" refers to survival or disease-free survival over a period of time, such as 1 year, 5 years, or 10 years, by one or more parameters, such as disease-free survival. can be determined Additionally, the parameter may include inhibition of the size of at least one tumor in the subject.

Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate may also affect efficacy. Thus, "enhanced efficacy" (eg, improvement in efficacy) can be attributed to improved pharmacokinetic parameters and improved efficacy, comparing clearance rates and tumor growth in test animals or human subjects, or reducing survival, recurrence rates, or disease. It can be measured by comparing parameters such as survival in the absence.

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

In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. A pharmacologically acceptable adjuvant (buffer, dispersant) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity more than is adaptable to the subject of application (prescription).

When the pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterilization for injection water, isotonic solutions such as 5% dextrose, and the like can be used. Formulation of pharmaceutical compositions is known in the art, and may be specifically referred to in Remington's Pharmaceutical Sciences (19th ed., 1995). These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition is in the range of 0.01 μg/kg to 10 g/kg, or 0.01 mg/kg to 1 g/kg per day depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. can be Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application may further include any compound or natural extract known to have a therapeutic effect on anticancer activity and whose safety has already been verified for enhancement or enhancement of anticancer activity.

Another aspect of the present invention provides the use of a fusion protein comprising PD-1 protein and IL-15 protein for treating cancer diseases.

Another aspect of the present invention provides the use of a fusion protein comprising a PD-1 protein and an IL-15 protein to enhance the therapeutic effect of cancer disease.

Another aspect of the present invention provides the use of a fusion protein comprising a PD-1 protein and an IL-15 protein for preparing a drug for treating cancer.

Another aspect of the present invention is a method for treating cancer disease comprising administering to a subject a fusion protein comprising PD-1 protein and IL-15 protein or a fusion protein dimer in which the two fusion proteins are combined; and/or a method for enhancing the therapeutic effect.

The subject may be an individual suffering from a cancer disease. Also, the subject may be a mammal, preferably a human. The fusion protein including the PD-1 protein and the IL-15 protein or the fusion protein dimer in which the two fusion proteins are combined are as described above.

The administration route, dosage and frequency of administration of the fusion protein or fusion protein dimer can be 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 the optimal administration method, dosage and frequency of administration are A person skilled in the art can select an appropriate range. In addition, the fusion protein or fusion protein dimer may be administered in combination with other drugs or physiologically active substances known to have therapeutic effects on the disease to be treated, or formulated in combination with other drugs.

Cell culture medium containing fusion protein or dimer thereof

Another aspect of the present invention provides a cell culture medium containing a fusion protein comprising PD-1 protein and IL-15 protein or a fusion protein dimer in which the two fusion proteins are linked.

In this case, the cells may be T cells or natural killer cells. The cell culture medium may be a medium in which a fusion protein including PD-1 protein and IL-15 protein or a fusion protein dimer in which the two fusion proteins are combined is added to a cell culture medium. The cell culture medium may include any one selected from the group consisting of amino acids, sugars, inorganic salts, and vitamins. Preferably, the medium for cell culture may contain all amino acids, sugars, inorganic salts and vitamins.

As used herein, the term "cell culture medium" refers to a medium used to culture cells, specifically immune cells, more specifically CD4+ or CD8+ T cells; or a medium for culturing NK cells. Contains components required by cells for cell growth and survival in vitro, or contains components that help cell growth and survival. Specifically, the components may be vitamins, essential or non-essential amino acids, and trace elements. The medium may be a medium used for culturing cells, preferably eukaryotic cells, more preferably T cells or NK cells.

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

I. Preparation of fusion protein: BNS002

Preparation Example 1. Preparation of hPD-1 D-Fc(29)-hIL-15

Signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), Ig hinge to which a linker is attached to produce a fusion protein including human PD-1 fragment, long-acting Fc (29) domain and IL-15 (SEQ ID NO: 3), Fc (29) domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in order from the N-terminus to a fusion protein (SEQ ID NO: 7) A polynucleotide containing the coding nucleotide sequence (SEQ ID NO: 9) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfecting the CHO cells with the expression vector, cultured for 9 days according to the manufacturer's Max Protocol, the culture medium was recovered, and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 8) is referred to as "hPD-1 D-Fc(29)-hIL-15" or "BNS002(29)", and thus a fusion protein containing PD-1 and IL-15 The dimer was collectively referred to as "BNS002".

The fusion protein in the recovered culture medium was first purified using Affinity Chromatography with Protein A Resin (MabSelect Sure ^TM ; Cytiva). After binding the fusion protein with Buffer A (20 mM Sodium Phosphate, 150 mM NaCl, pH 7.1) and washing the resin, only the bound fusion protein was eluted with Buffer B (100 mM Citrate, pH 3.0). The eluted fusion protein was neutralized (pH 7.0) with 1 M Tris-HCl (pH 9.0) buffer, concentrated using a centrifugal filter (Amicon Ultra-15, Merck), and buffer exchanged with PBS. The buffer-exchanged fusion protein was secondarily purified by performing size exclusion chromatography using a HiLoad ^® 26/600 Superdex ^® 200 pg column (Cytiva). The separated and purified fusion protein was quantified by IGGHB analysis of Cedex Bio HT Analyzer (Roche), and the purity was analyzed by performing SDS-PAGE and SEC-HPLC analysis (FIG. 1 and FIG. 2).

Preparation Example 2. Preparation of hPD-1 D-Fc(41)-hIL-15

Signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), Ig hinge to which a linker is attached to produce a fusion protein including human PD-1 fragment, long-acting Fc (41) domain and IL-15 (SEQ ID NO: 3), Fc (41) domain (SEQ ID NO: 10), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in order from the N-terminus to a fusion protein (SEQ ID NO: 11) A polynucleotide containing the coding nucleotide sequence (SEQ ID NO: 13) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfecting the CHO cells with the expression vector, cultured for 9 days according to the manufacturer's Max Protocol, the culture medium was recovered, and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 12) is referred to as "hPD-1 D-Fc(41)-hIL-15" or "BNS002(41)", and thus a fusion protein containing PD-1 and IL-15 The dimer was collectively referred to as "BNS002".

Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above. The separated and purified fusion protein was quantified by IGGHB analysis of Cedex Bio HT Analyzer (Roche), and the purity was analyzed by performing SDS-PAGE and SEC-HPLC analysis (FIG. 1 and FIG. 2).

Preparation Example 3. Preparation of hPD-1 D-Fc(wt)-hIL-15

Signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), linker-linked Ig hinge to produce a fusion protein including human PD-1 fragment, long-acting Fc (wt) domain and IL-15 (SEQ ID NO: 3), Fc (wt) domain (SEQ ID NO: 14), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in order from the N-terminus to a fusion protein (SEQ ID NO: 15) A polynucleotide containing the coding base sequence (SEQ ID NO: 17) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfecting the CHO cells with the expression vector, cultured for 9 days according to the manufacturer's Max Protocol, the culture medium was recovered, and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 16) is referred to as "hPD-1 D-Fc(wt)-hIL-15" or "BNS002(wt)", and thus a fusion protein containing PD-1 and IL-15 The dimer was collectively referred to as "BNS002".

Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above. The separated and purified fusion protein was quantified by IGGHB analysis of Cedex Bio HT Analyzer (Roche), and the purity was analyzed by performing SDS-PAGE and SEC-HPLC analysis (FIG. 1 and FIG. 2).

Preparation Example 4. Preparation of mPD-1 D-Fc(29)-mIL-15

Signal peptide (SEQ ID NO: 22), PD-1 fragment (SEQ ID NO: 23), linker-linked Ig hinge to produce a fusion protein including mouse PD-1 fragment, long-acting Fc (29) domain and IL-15 (SEQ ID NO: 3), Fc (29) domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 24) in order from the N-terminus to a fusion protein (SEQ ID NO: 25) A polynucleotide containing the coding base sequence (SEQ ID NO: 27) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 26) is referred to as "mPD-1 D-Fc (29) -mIL-15" or "mBNS002 (29)", and thus a fusion protein containing mPD-1 and mIL-15 The dimer was collectively referred to as "BNS002". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 5. Preparation of mPD-1 D-Fc(wt)-mIL-15

Signal peptide (SEQ ID NO: 22), PD-1 fragment (SEQ ID NO: 23), linker-linked Ig hinge to produce a fusion protein including mouse PD-1 fragment, persistent Fc (wt) domain and IL-15 (SEQ ID NO: 3), Fc (wt) domain (SEQ ID NO: 14), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 24) in order from the N-terminus to a fusion protein (SEQ ID NO: 28) A polynucleotide containing the coding nucleotide sequence (SEQ ID NO: 30) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 29) is referred to as "mPD-1 D-Fc (wt) -mIL-15" or "mBNS002 (wt)", and thus a fusion protein containing PD-1 and IL-15 The dimer was collectively referred to as "BNS002". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 6. Preparation of hPD-1 D-Fc(29)-mIL-15

Signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), linker-linked Ig Fusion protein (SEQ ID NO: 36 ) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 37) was called "hPD-1 D-Fc(29)-mIL-15", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 7. Preparation of hPD-1 ECD-Fc(29)-hIL-15

To produce a fusion protein including human PD-1 fragment, long-acting Fc (29) domain and IL-15, signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 39), Ig hinge to which linker is attached (SEQ ID NO: 3), Fc (29) domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in order from the N-terminus to a fusion protein (SEQ ID NO: 40) A polynucleotide containing the coding nucleotide sequence (SEQ ID NO: 42) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 41) was called "hPD-1 ECD-Fc(29)-hIL-15", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 8. Preparation of hIL-15-Fc(29)-hPD-1 D

Signal peptide (SEQ ID NO: 22), IL-15 (SEQ ID NO: 6), and an Ig hinge coupled with a linker to produce a fusion protein including a human IL-15 fragment, a persistent Fc (29) domain, and a PD-1 fragment (SEQ ID NO: 43), an Fc (29) domain (SEQ ID NO: 4), a linker (SEQ ID NO: 44), and a PD-1 fragment (SEQ ID NO: 2) in the above order from the N-terminus (SEQ ID NO: 45) A polynucleotide containing the nucleotide sequence encoding (SEQ ID NO: 47) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 46) was called "hIL-15-Fc(29)-hPD-1 D", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 9. Preparation of hIL-15-Fc(29)-hPD-1 ECD

Signal peptide (SEQ ID NO: 22), IL-15 (SEQ ID NO: 6), and an Ig hinge coupled with a linker to produce a fusion protein including a human IL-15 fragment, a persistent Fc (29) domain, and a PD-1 fragment (SEQ ID NO: 43), an Fc (29) domain (SEQ ID NO: 4), a linker (SEQ ID NO: 44), and a PD-1 fragment (SEQ ID NO: 39) in the above order from the N-terminus (SEQ ID NO: 48) A polynucleotide containing the nucleotide sequence encoding (SEQ ID NO: 50) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 49) was called "hIL-15-Fc(29)-hPD-1 ECD", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 10. Preparation of hPD-1 D-Fc(29, H)-hIL-15

To produce a fusion protein including human PD-1 fragment, long-acting Fc (29, H) domain and IL-15, a signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), and a linker are combined. Fusion protein comprising Ig hinge (SEQ ID NO: 3), Fc (29, H) domain (SEQ ID NO: 55), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in the above order from the N-terminus (SEQ ID NO: 55) 56) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 57) is called "hPD-1 D-Fc (29, H) -hIL-15", and the fusion protein dimer containing PD-1 and IL-15 is called "BNS002". referred to as ". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 11. Preparation of hPD-1 D-Fc(29, H)-mIL-15

To produce a fusion protein containing a human PD-1 fragment, a persistent Fc (29, H) domain, and a mouse IL-15 fragment, a signal peptide (SEQ ID NO: 1), a PD-1 fragment (SEQ ID NO: 2), and a linker are required. A fusion protein comprising a combined Ig hinge (SEQ ID NO: 3), an Fc (29, H) domain (SEQ ID NO: 55), a linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 24) in the above order from the N-terminus. A polynucleotide containing the nucleotide sequence (SEQ ID NO: 61) encoding (SEQ ID NO: 59) was synthesized through GenScript's Gene Synthesis service and introduced into pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 60) is called "hPD-1 D-Fc (29, H) -mIL-15", and the fusion protein dimer containing PD-1 and IL-15 is called "BNS002". referred to as ". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 12. Preparation of hPD-1 ECD-Fc(wt)-hIL-15

Signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 39), Ig hinge to which a linker is attached to produce a fusion protein including human PD-1 fragment, long-acting Fc (wt) domain and IL-15 (SEQ ID NO: 3), Fc (wt) domain (SEQ ID NO: 14), linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in order from the N-terminus to a fusion protein (SEQ ID NO: 76) A polynucleotide containing the coding base sequence (SEQ ID NO: 78) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 77) was called "hPD-1 ECD-Fc(wt)-hIL-15", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 13. Preparation of hIL-15-Fc(wt)-hPD-1 D

Signal peptide (SEQ ID NO: 22), IL-15 (SEQ ID NO: 6), Ig hinge coupled with a linker to produce a fusion protein including a human IL-15 fragment, a persistent Fc (wt) domain, and a PD-1 fragment (SEQ ID NO: 43), Fc (wt) domain (SEQ ID NO: 14), linker (SEQ ID NO: 44) and PD-1 fragment (SEQ ID NO: 2) in the above order from the N-terminus (SEQ ID NO: 79) A polynucleotide containing the nucleotide sequence encoding (SEQ ID NO: 81) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 80) was called "hIL-15-Fc(wt)-hPD-1 D", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 14. Preparation of hIL-15-Fc(wt)-hPD-1 ECD

Signal peptide (SEQ ID NO: 22), IL-15 (SEQ ID NO: 6), Ig hinge coupled with a linker to produce a fusion protein including a human IL-15 fragment, a persistent Fc (wt) domain, and a PD-1 fragment (SEQ ID NO: 43), Fc (wt) domain (SEQ ID NO: 14), linker (SEQ ID NO: 44) and PD-1 fragment (SEQ ID NO: 39) in the above order from the N-terminus (SEQ ID NO: 82) A polynucleotide containing the nucleotide sequence encoding (SEQ ID NO: 84) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 83) was called "hIL-15-Fc(wt)-hPD-1 ECD", and the fusion protein dimer containing PD-1 and IL-15 was called "BNS002". collectively. Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 15. Preparation of hPD-1 D-Fc(wt, H)-hIL-15

To produce a fusion protein including human PD-1 fragment, long-acting Fc (wt, H) domain and IL-15, a signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), and a linker are combined. A fusion protein comprising an Ig hinge (SEQ ID NO: 3), an Fc (wt, H) domain (SEQ ID NO: 88), a linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in the above order from the N-terminus (SEQ ID NO: 88) 89) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 90) was referred to as "hPD-1 D-Fc (wt, H)-hIL-15", and the fusion protein dimer containing PD-1 and IL-15 was designated as "BNS002". referred to as ". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 16. Preparation of hPD-1 D-Fc (wt, H) -mIL-15

To produce a fusion protein containing a human PD-1 fragment, a persistent Fc (wt, H) domain and a mouse IL-15 fragment, a signal peptide (SEQ ID NO: 1), a PD-1 fragment (SEQ ID NO: 2), and a linker are Containing the combined Ig hinge (SEQ ID NO: 3), Fc (wt, H) domain (SEQ ID NO: 88), linker (SEQ ID NO: 5) and mouse IL-15 fragment (SEQ ID NO: 24) in the above order from the N-terminus A polynucleotide containing the nucleotide sequence (SEQ ID NO: 94) encoding the fusion protein (SEQ ID NO: 92) was synthesized through GenScript's Gene Synthesis service and introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 93) is called "hPD-1 D-Fc (wt, H) -mIL-15", and the fusion protein dimer containing PD-1 and IL-15 is called "BNS002". referred to as ". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

Preparation Example 17. Preparation of PD-1D-Fc-IL-15Rα/IL-15 (BNS002S)

To produce a fusion protein including human PD-1 fragment, long-acting Fc (29) domain and IL-15Rα/IL-15, signal peptide (SEQ ID NO: 1), PD-1 fragment (SEQ ID NO: 2), and linker Combined Ig hinge (SEQ ID NO: 3), Fc (29) domain (SEQ ID NO: 4), linker 1 (SEQ ID NO: 5), IL-15Rα (SEQ ID NO: 95), linker 4 (SEQ ID NO: 96) and IL-15 ( A polynucleotide containing the nucleotide sequence (SEQ ID NO: 35) encoding the fusion protein (SEQ ID NO: 34) containing SEQ ID NO: 6) in the above order from the N-terminus was synthesized through Cosmogenetech's Gene Synthesis service, and pcDNA3. 4 TOPO vector (Thermo Fisher Scientific).

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfecting the CHO cells with the expression vector, cultured for 8 days according to the manufacturer's Max Protocol, the culture medium was recovered, and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 33) is referred to as "hPD-1 D-Fc-hIL-15Rα/hIL-15", and thus a fusion protein dimer containing PD-1 and IL-15Rα/IL-15 was collectively referred to as "BNS002S".

The fusion protein in the recovered culture medium was first purified using Affinity Chromatography with Protein A Resin (KANEKA). After binding the fusion protein with DPBS buffer and washing the resin, only the bound fusion protein was eluted with 0.1 M glycine buffer (pH 3.3). The eluted fusion protein was subjected to buffer exchange by dialysis with DPBS buffer for one day, and secondary purification was performed by size exclusion chromatography using a HiLoad ^® 16/600 Superdex ^® 200 pg column (Cytiva). The separated and purified fusion protein was quantified using NanoDrop, and the purity was analyzed by performing SDS-PAGE and SEC-HPLC analysis.

Preparation Example 18. Preparation of BNS001D (PD-1 D-Fc)

To produce a fusion protein containing a human PD-1 fragment and a persistent Fc (29) domain, a signal peptide (SEQ ID NO: 1), a PD-1 fragment (SEQ ID NO: 2), and an Ig hinge (SEQ ID NO: 3) to which a linker is coupled ), a polynucleotide containing the nucleotide sequence (SEQ ID NO: 100) encoding the fusion protein (SEQ ID NO: 98) containing the Fc (29) K domain (SEQ ID NO: 97) in the above order from the N-terminus of Cosmogenetech Co., Ltd. It was introduced into pcDNA3.4 TOPO vector (Thermo Fisher Scientific) through Gene Cloning service.

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfection into CHO cells with the above expression vector, the cells were cultured for 11 days according to the manufacturer's Max Protocol, and then the culture medium was recovered and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 99) was referred to as "hPD-1 D-Fc", and the fusion protein dimer including PD-1 and Fc was collectively referred to as "BNS001D". Fusion protein purification and recovery were performed in the same manner as in Preparation Example 17.

Preparation Example 19. Preparation of BNS002I (Fc-IL-15)

To produce a fusion protein containing a persistent Fc (29) domain and a human IL-15 fragment, a signal peptide (SEQ ID NO: 22), an Ig hinge (SEQ ID NO: 57), an Fc (29) domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5) and IL-15 (SEQ ID NO: 6) in this order from the N-terminus, and a polynucleotide comprising a nucleotide sequence (SEQ ID NO: 54) encoding a fusion protein (SEQ ID NO: 52) was prepared by CosmoGenetech. It was introduced into the pcDNA3.4 TOPO vector (Thermo Fisher Scientific) through Gene Cloning.

The expression vector was introduced into CHO cells (ExpiCHO-S ^™ , Thermo Fisher Scientific) to express the fusion protein. After transfecting the CHO cells with the expression vector, cultured for 10 days according to the manufacturer's Max Protocol, the culture medium was recovered, and the fusion protein was purified. The purified fusion protein (SEQ ID NO: 53) was referred to as "Fc-IL-15", and the fusion protein dimer comprising Fc and IL-15 was collectively referred to as "BNS002I". Purification and recovery of the fusion protein were performed in the same manner as in Preparation Example 1 above.

II. Purity analysis of BNS002 fusion protein

Experimental Example 1. SEC-HPLC analysis

To confirm the purity of the separated and purified fusion protein, SEC-HPLC experiments were performed. Size exclusion high performance liquid chromatography (HPLC) was performed on a Waters Alliance HPLC system with a UV detector. The column (TSK G3000 SW _XL , 5 μm SEC column) was purchased from Tosoh Bioscience. 100 mM NaPi, 150 mM NaCl (pH 7.0) was used as a mobile phase. The purity of the fusion protein was analyzed by measuring absorbance at a wavelength of 280 nm over time at a flow rate of 0.5 mL/min under 100% isocratic transfer conditions (FIG. 2).

It was confirmed that the purified BNS002 fusion protein had a purity of 98% or higher (FIG. 2).

III. Concentration analysis of BNS002 fusion protein

Experimental Example 2. Concentration analysis

The fusion protein was diluted to a concentration of 0.2, 0.4, 0.6, 0.8, or 1.0 mg/mL. UV absorbance was measured in the wavelength range of 230 nm to 400 nm using a UV spectrophotometer (Little lunatic, Unchained Labs). After diluting each concentration of fusion protein to a concentration of 0.2 mg/mL, acid hydrolysis was performed. The hydrolyzed amino acid was derivatized and UPLC (Ultra Performance Liquid Chromatography) was performed using a Waters ACQUITY UPLC system. As a column, an AccQ-Tag Ultra RP column (2.1 mm×100 mm, 1.7 μm, 130 Å) was used. Mobile phase A was started with 5% AccQ-tag A solvent and mobile phase B was AccQ-tag B solvent, starting with an A:B initial ratio of 99.9:0.1, and a gradient system with a ratio of 40.4:59.6 by 7.5 minutes. performed. The sample injection amount was 1 μl, the flow rate was 0.7 mL/min, and the column temperature was 55°C. The data system used Enpower 3 Software, and the data were fit with the standard solution (I.S.) added in the sample pretreatment step.

The extinction coefficient (Extinction coefficinet) was determined by checking the content of the separated and purified BNS002 fusion protein according to the absorbance. The concentration of the fusion protein was confirmed by the extinction coefficient, and it was confirmed that the fusion protein was included at a concentration of 1.99 mg/mL (FIG. 3).

IV. Physical property analysis of BNS002 fusion protein

Experimental Example 3. Thermodynamic stability analysis

To confirm the thermodynamic stability of the separated and purified fusion protein, the T _m , T _Agg & T _onset analysis was performed. The buffer was exchanged with ultrapure water for the fusion protein, and the sample was prepared to have 1.25 times the protein concentration required for analysis using UNCHAINED LABS Little Lunatic equipment. 1.25× of the prepared sample and 5× of the formulation buffer were diluted with ultrapure water. The prepared sample was loaded into Uni (UNCHAINED LABS), and T _m , T _agg & T _onset were measured with a Fluorescence & SLS detector at a wavelength of 266 nm from 15 to 95 ° C. Data were analyzed using UNCHAINED LABS software Uncle Analysis V4.01.

The thermodynamic stability of the isolated and purified BNS002 fusion protein was measured as shown in FIG. 4 .

Experimental Example 4. Structural stability analysis

To confirm the structural stability of the separated and purified fusion protein, secondary structure analysis was performed using an FT-IR spectrophotometer (Thermo Nicolet IS50, Single-Bounce ATR Diamond Crystal attached). A sample was prepared so that the concentration of the fusion protein was 10 mg/mL. As for the analysis conditions of the FT-IR equipment, the phase resolution was set to 4 cm ^-1 and the data spacing was set to 1 cm ^-1 , and measurements were repeated 128 times in the range of 500 to 4000 cm ^-1 . In order to analyze the secondary structure content of the fusion protein, zero order FT-IR spectral data in the range of 1700 to 1600 cm ^-1 was used. At this time, as the ATR sampling accessory is used for analysis, ATR correction is performed on the initially acquired FT-IR spectrum data, and 2300 cm in the subtracted spectrum obtained after correction The water vapor band of the ^-1 site was removed. In addition, a final zero-order FT-IR spectrum was obtained by fitting with a baseline correction and an 11-point Savitzky-Golay function. Among the zero-order FT-IR spectra obtained through FT-IR analysis, data in the 1700 to 1600 cm ^-1 interval were calculated with a linear-fit function to obtain a second derivative spectrum. Peaks were extracted from the second differential spectrum, and the extracted peaks were fitted with a Gaussian function. The size and position of the peak were adjusted with a tolerance of 3% to secure the final extracted spectrum. The secondary structure was assigned according to the wavenumber for the peaks extracted from the second differential spectrum, and the secondary structure content in each sample was calculated by calculating the peak area for the assigned peak.

Secondary structure analysis of the separated and purified BNS002 fusion protein was measured as shown in FIG. 5 .

Experimental Example 5. Molecular Weight Analysis

To confirm the molecular weight of the separated and purified fusion protein, TSK G3000 SW _XL , 5 μm SEC column (Tosoh Bioscience, LLC) and UFLC Shimadzu LC-20AD pump (Shimadzu co.) were used for 40 minutes at a flow rate of 0.5 mL/min. 100 μl of each sample was separated. 100 mM NaPi, 150 mM NaCl (pH 6.8) was used as a sample running buffer. After HPLC separation, analysis was performed using a Dawn ^® Heleos II MALS detector at 662 nm wavelength and an Optilab T-rEx ^® refractive index detector (Wyatt) at 658 nm wavelength. Data were analyzed using Astra software (Wyatt) ver 6.1.5.

SEC-MALS (Size Exclusion Chomatography - Multi-Angle Laser Light Scattering) analysis of the separated and purified BNS002 fusion protein was measured as shown in FIG. 6 .

V. Confirmation of kinetic binding affinity between BNS002 fusion protein and ligand or receptor

In order to confirm the dynamic binding affinity between the BNS002 fusion protein and the ligand, the binding affinity was measured by surface plasmon resonance (SPR) analysis using a Biacore T200 (GE Healthcare).

Experimental Example 6. Confirmation of binding affinity between hPD-L1 ligand and BNS002 fusion protein

PD-L1 ligand was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and amine coupled on a CM5 sensor chip (GE Healthcare) pre-activated with HBS-EP, pH 7.4. A ring was used to secure it to about 1000 RU. Sensorgrams were recorded by flowing dilutions of BNS002 fusion protein prepared with HBS-EP, pH 7.4, in a range of concentrations from 0.78 nM to 400 nM. The binding between the BNS002 fusion protein and the PD-L1 ligand was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between the hPD-L1 ligand and the BNS002 fusion protein was measured as shown in FIG. 7 .

Experimental Example 7. Confirmation of binding affinity between hPD-L1 ligand and hPD-1 protein

To confirm the strength of the binding affinity between the BNS002 fusion protein and the hPD-L1 ligand, the binding affinity between the hPD-1 protein and the hPD-L1 ligand was compared. PD-L1 ligand was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and amine coupled on a CM5 sensor chip (GE Healthcare) pre-activated with HBS-EP, pH 7.4. A ring was used to secure it to about 1000 RU. Sensorgrams were recorded by flowing dilutions of hPD-1 protein prepared with HBS-EP, pH 7.4, ranging in concentration from 0.78 nM to 400 nM. The association between hPD-L1 ligand and hPD-1 protein was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between the hPD-L1 ligand and the hPD-1 protein was measured as shown in FIG. 8 . It was confirmed that the binding affinity between the BNS002 fusion protein and the hPD-L1 ligand was similar to that between the hPD-1 protein and the hPD-L1 ligand.

Experimental Example 8. Confirmation of binding affinity between hPD-L2 ligand and BNS002 fusion protein

PD-L2 ligand was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and amine coupled on a CM5 sensor chip (GE Healthcare) pre-activated with HBS-EP, pH 7.4. A ring was used to secure it to about 1000 RU. Sensorgrams were recorded by flowing dilutions of BNS002 fusion protein prepared with HBS-EP, pH 7.4, in a range of concentrations from 0.78 nM to 400 nM. The binding between the BNS002 fusion protein and the PD-L2 ligand was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between the hPD-L2 ligand and the BNS002 fusion protein was measured as shown in FIG. 9 .

Experimental Example 9. Confirmation of binding affinity between hPD-L2 ligand and hPD-1 protein

To confirm the strength of the binding affinity between the BNS002 fusion protein and the hPD-L2 ligand, the binding affinity between the hPD-1 protein and the hPD-L2 ligand was compared. PD-L2 ligand was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and amine coupled on a CM5 sensor chip (GE Healthcare) pre-activated with HBS-EP, pH 7.4. A ring was used to secure it to about 1000 RU. Sensorgrams were recorded by flowing dilutions of hPD-1 protein prepared with HBS-EP, pH 7.4, ranging in concentration from 0.78 nM to 400 nM. The association between the hPD-L2 ligand and the hPD-1 protein was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between the hPD-L2 ligand and the hPD-1 protein was measured as shown in FIG. 10 . It was confirmed that the binding affinity between the BNS002 fusion protein and the hPD-L2 ligand was similar to that between the hPD-1 protein and the hPD-L2 ligand.

Experimental Example 10. Confirmation of binding affinity between hIL-15Rα and BNS002 fusion protein

hIL-15Rα (human Interleukin-15 receptor alpha) was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and pre-activated with HBS-EP, pH 7.4. GE Healthcare) using amine coupling to about 1000 RU. Sensorgrams were recorded by flowing dilutions of BNS002 fusion protein prepared with HBS-EP, pH 7.4 in a range of concentrations from 50 nM to 1600 nM. The binding between the BNS002 fusion protein and hIL-15Rα was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between the hIL-15 receptor and the BNS002 fusion protein was measured as shown in FIG. 11 .

Experimental Example 11. Confirmation of binding affinity between hIL-15Rα and hIL-15 protein

To confirm the strength of the binding affinity between the BNS002 fusion protein and hIL-15Rα, the binding affinity between the hIL-15 protein and hIL-15Rα was compared. hIL-15Rα was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and amine-coupled onto a CM5 sensor chip (GE Healthcare) pre-activated with HBS-EP, pH 7.4. was fixed up to about 1000 RU. Sensorgrams were recorded by flowing hIL-15 protein dilutions prepared with HBS-EP, pH 7.4 in a range of concentrations from 12.5 nM to 200 nM. Association between hIL-15Rα and hIL-15 protein was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between hIL-15Rα and hIL-15 protein was measured as shown in FIG. 12 . The binding affinity between the BNS002 fusion protein and hIL-15Rα was confirmed to be 169 to 296 nM, but the binding affinity between the hIL-15 protein and hIL-15Rα was not confirmed.

Experimental Example 12. Confirmation of binding affinity between hIL-2Rβ and BNS002 fusion protein

hIL-2Rβ (human Interleukin-15 receptor beta) was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and pre-activated with HBS-EP, pH 7.4. GE Healthcare) using amine coupling to about 1000 RU. Sensorgrams were recorded by flowing dilutions of BNS002 fusion protein prepared with HBS-EP, pH 7.4 in a range of concentrations from 50 nM to 1600 nM. The binding between the BNS002 fusion protein and hIL-2Rβ was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between hIL-2Rβ and the BNS002 fusion protein was measured as shown in FIG. 13 .

Experimental Example 13. Confirmation of binding affinity between hIL-2Rβ and hIL-15 protein

To confirm the strength of the binding affinity between the BNS002 fusion protein and hIL-2Rβ, the binding affinity between the hIL-15 protein and hIL-2Rβ was compared. hIL-2Rβ was diluted to 3-4 μg/mL in C ₂ H ₃ NaO ₂ (pH 4.0, pH 5.5) and amine-coupled onto a CM5 sensor chip (GE Healthcare) pre-activated with HBS-EP, pH 7.4. was fixed up to about 1000 RU. Sensorgrams were recorded by flowing hIL-15 protein dilutions prepared with HBS-EP, pH 7.4, ranging in concentration from 0.39 nM to 200 nM. Association between hIL-2Rβ and hIL-15 protein was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation period of 10 minutes. The sensor chip surface was regenerated by injecting 10 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between hIL-2Rβ and hIL-15 proteins was measured as shown in FIG. 14 . The binding affinity between the BNS002 fusion protein and hIL-2Rβ was confirmed to be about 200 times lower than that between the hIL-15 protein and hIL-2Rβ.

Experimental Example 14. Confirmation of binding affinity between hFcRn receptor and BNS002 fusion protein

The hFcRn receptor was diluted to 5 μg/mL in C ₂ H ₃ NaO ₂ (pH 5.0) and pre-activated with 20 mM phosphate, 150 mM NaCl, pH 6.0 and pH 7.4 on a CM5 sensor chip (GE Healthcare). was fixed up to about 1000 RU using amine coupling. Sensorgrams were recorded by flowing diluted fusion protein solutions prepared with 20 mM phosphate, 150 mM NaCl, 0.05% Tween20, pH 6.0 in various concentrations ranging from 3.125 nM to 400 nM. Fusion protein association was measured at a flow rate of 30 μl/min with an association period of 4 minutes and a dissociation phase of 10 minutes. The sensor chip surface was regenerated by injecting 50 mM NaOH between each run. Binding kinetics were analyzed using data analysis software (Ver3.2) and data were fit using a 1:1 binding model.

As a result, the binding affinity between the hFcRn receptor and the BNS002 fusion protein was measured as shown in FIG. 15 . Among the BNS002 fusion proteins, the PD-1 D-Fc(29)-IL-15 and PD-1 D-Fc(41)-IL-15 proteins have a higher affinity for the hFcRn receptor than the PD-1 D-Fc(wt)-IL-15 protein. The binding affinity for was confirmed to be strong.

VI. Confirmation of immunological binding affinity between BNS002 fusion protein and ligand or receptor

To confirm the binding affinity between the BNS002 fusion protein and the ligand, the binding affinity was measured by immunological analysis.

Experimental Example 15. Confirmation of binding affinity between BNS002 fusion protein and PD-L1 ligand through ELISA

0.1 mL of PD-L1-Fc ligand at a concentration of 100 ng/mL was immobilized in a 96-well plate (High binding plate, corning, #CT9018) at 4°C for 16 hours, followed by 250 μl of blocking buffer. blocked for 2 hours at room temperature. After blocking, the washing process was performed 4 times with 300 μl of wash buffer. Then, after subdividing the serially diluted BNS002 fusion protein into 50 μl portions, 50 μl of the sample diluent was added thereto. 25 ng/mL diluted detection antibody (Biotinylated Goat anti-human IL-15 Ab) was added at 50 μl/well and reacted at room temperature for 2 hours made it The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. 100 μl of streptavidin-HRP diluted at a ratio of 1:200 using 1× assay buffer was added to the well, and reacted at room temperature for 1 hour. The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. After treating each well with 100 μl of TMB solution, it was reacted for 30 minutes at room temperature with light blocked. After the reaction, 100 μl of stop solution was added to each well, and absorbance was measured using a plate reader (wavelength: 450 nm). At this time, each experiment was conducted in duplicate.

As a result of ELISA analysis, the binding affinity between the BNS002 fusion protein and the PD-L1 ligand was measured as shown in FIG. 16 .

Experimental Example 16. Confirmation of binding affinity between BNS002 fusion protein and PD-L2 ligand through ELISA

0.1 mL of PD-L2-Fc ligand at a concentration of 100 ng/mL was immobilized in a 96-well plate (High binding plate, corning, #CT9018) at 4°C for 16 hours, and then incubated at room temperature with 250 μL of blocking buffer. Blocked for an hour. After blocking, the washing process was performed 4 times with 300 μl of washing buffer. Then, 50 μl of the serially diluted BNS002 fusion protein was subdivided, and then 50 μl of the sample dilution was added. 25 ng/mL diluted detection antibody (Biotinylated Goat anti-human IL-15 Ab) was added at 50 μl/well and reacted at room temperature for 2 hours made it The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. 100 μl of streptavidin-HRP diluted at a ratio of 1:200 using 1× assay buffer was added to the well and reacted at room temperature for 1 hour. The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. After treating each well with 100 μl of TMB solution, it was reacted for 30 minutes at room temperature with light blocked. After the reaction, 100 μl of the stop solution was added to each well, and absorbance was measured using a plate reader (wavelength: 450 nm). At this time, each experiment was conducted in duplicate.

As a result of ELISA analysis, the binding affinity between the BNS002 fusion protein and the PD-L2 ligand was measured as shown in FIG. 17 .

Experimental Example 17. Confirmation of binding affinity between BNS002 fusion protein and IL-15Rα by ELISA

IL-15Rα at a concentration of 100 ng/mL was immobilized at 0.1 mL each in a 96-well plate (High binding plate, corning, #CT9018) at 4°C for 16 hours, then blocked with 250 μl of blocking buffer at room temperature for 2 hours did After blocking, the washing process was performed 4 times with 300 μl of washing buffer. Then, 50 μl of the serially diluted BNS002 fusion protein was subdivided, and then 50 μl of the sample dilution was added. 25 ng/mL diluted detection antibody (Biotinylated Goat anti-human PD-1 Ab) was added at 50 μl/well and reacted at room temperature for 2 hours made it The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. 100 μl of streptavidin-HRP diluted at a ratio of 1:200 using 1× assay buffer was added to the well, and reacted at room temperature for 1 hour. The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. After treating each well with 100 μl of TMB solution, it was reacted for 30 minutes at room temperature with light blocked. After the reaction, 100 μl of the stop solution was added to each well, and absorbance was measured using a plate reader (wavelength: 450 nm). At this time, each experiment was conducted in duplicate.

As a result of ELISA analysis, the binding affinity between the BNS002 fusion protein and IL-15Rα was measured as shown in FIG. 18 .

Experimental Example 18. Confirmation of binding affinity between BNS002 fusion protein and IL-2Rβ by ELISA

IL-2Rβ at a concentration of 100 ng/mL was immobilized at 0.1 mL each in a 96-well plate (High binding plate, corning, #CT9018) at 4°C for 16 hours, then blocked with 250 μl of blocking buffer at room temperature for 2 hours did After blocking, the washing process was performed 4 times with 300 μl of washing buffer. Then, 50 μl of the serially diluted BNS002 fusion protein was subdivided, and then 50 μl of the sample dilution was added. 25 ng/mL diluted detection antibody (Biotinylated Goat anti-human PD-1 Ab) was added at 50 μl/well and reacted at room temperature for 2 hours made it The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. 100 μl of streptavidin-HRP diluted at a ratio of 1:200 using 1× assay buffer was added to the well, and reacted at room temperature for 1 hour. The reaction-completed plate was washed 4 times with 400 μl of washing buffer, and the remaining solution was completely removed. After treating each well with 100 μl of TMB solution, it was reacted for 30 minutes at room temperature with light blocked. After the reaction, 100 μl of the stop solution was added to each well, and absorbance was measured using a plate reader (wavelength: 450 nm). At this time, each experiment was conducted in duplicate.

As a result of ELISA analysis, the binding affinity between the BNS002 fusion protein and IL-2Rβ was measured as shown in FIG. 19 .

Experimental Example 19. Confirmation of binding affinity between BNS002 fusion protein and hFcRn receptor through FcRn binding immunoassay

In order to measure the binding ability of the BNS002 fusion protein to hFcRn, which can exhibit an effect function for hFcRn (human neonatal Fc receptor), analysis was performed using Promega's Lumit ^TM FcRn binding immunoassay kit. Since hFcRn is known to have pH-dependent binding ability, the BNS002 fusion proteins were prepared by correcting the pH to 6.0 using a pH adjustment buffer and diluting each concentration with an FcRn assay buffer. 25 μl of the Tracer-LgBit solution in the kit was dispensed into each well, and 25 μl of BNS002 fusion proteins diluted by concentration were dispensed into each well. Thereafter, 50 μl of the hFcRn-SmBiT solution in the kit was dispensed into each well and reacted at room temperature for 1 hour, and 25 μl of Lumit ^TM FcRn Detection reagent was added to each well and reacted for 5 minutes. After reacting, it was analyzed using Synergy Neo2 (BioTek).

As a result of the Lumit ^™ FcRn binding immunoassay analysis, the binding affinity between the BNS002 fusion protein and the FcRn receptor was measured as shown in FIG. 20 .

VII. Confirmation of binding affinity between BNS002 fusion protein and IL-15 receptor through cell-based assay

Experimental Example 20. Confirmation of cell-based binding affinity between BNS002 fusion protein and IL-15 receptor

To measure the binding affinity of the BNS002 fusion protein to the IL-15 receptor, PathHunter ^® eXpress Dimerization Assay Kit (Eurofins) was used, and PathHunter ^® eXpress Dimerization Cells stored in liquid nitrogen were warmed with Cell Plating Reagent. Thawed using The thawed cells were dispensed in 96-well plates (SPL) by 100 μl, treated with BNS002 fusion protein at each concentration, and then treated with 110 μl of PathHunter ^® Flash Detection reagent in each well. After treatment, it was reacted for 1 hour at room temperature and under dark conditions. After the reaction, chemiluminescence was measured using Synergy Neo2 (BioTek). Based on the measured values, the EC ₅₀ (Half maximal effective concentration; concentration of a drug that can show about half of the maximum effect that the drug can exhibit when the drug is administered) for the fusion protein was confirmed.

As a result of PathHunter ^® eXpress Dimerization Assay measurement, the binding affinity between the BNS002 fusion protein and the IL-15 receptor was measured as shown in FIG. 21 .

VIII. Confirmation of immune activity of BNS002 fusion protein

Experimental Example 21. Confirmation of IFN-γ production by BNS002 fusion protein

Experimental Example 21.1. Culturing of PBMCs

To actively culture peripheral blood mononuclear cells (PBMC) isolated from humans, anti-CD3 antibody (OKT3, 1 μg/ml, invitrogen) and anti-CD28 antibody (CD28 .2, 1 μg/ml, Invitrogen) was treated with culture medium (RPMI1640 medium: FBS 10%, penicillin/streptomycin containing 200 μl) and coated overnight at 4°C. The next day, the PBMCs were thawed slowly at 37°C, centrifuged at 1,500 rpm at 4°C for 5 minutes, and then supplemented with RPMI1640 culture medium (FBS 10%, penicillin/streptomycin 200 μl, anti-CD3/anti-CD28 antibody (1 μg/each)). ㎖)) and cultured in a 37°C, 5% CO ₂ incubator. At this time, the BNS002 fusion protein and IL-15 were treated with or without anti-CD3/anti-CD28 antibody (each 1 μg/ml), and cultured in an incubator for 3 days.

Experimental Example 21.2. Confirmation of human IFN-γ secretion through ELISA analysis

In Experimental Example 21.1, the amount of human IFN-γ secreted in the culture supernatant of each sample was measured using a human IFN-γ ELISA kit (Biolegend, cat No.430103). An anti-human-IFN-γ antibody was added to the ELISA plate and reacted overnight at 4° C. to coat the plate. Thereafter, blocking was performed for 1 hour at room temperature with a PBS solution to which 1% BSA was added. After washing with washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample were diluted 2-fold and reacted at room temperature for 2 hours. After the reaction was completed, the plate was washed and a secondary detection antibody was added thereto and reacted at room temperature for 1 hour. After washing with a washing buffer, an avidin-HRP solution was added and reacted at room temperature for 30 minutes, and a substrate solution was added to induce a color reaction for 20 minutes at room temperature and under dark conditions. Finally, H ₂ S0 ₄ was added to stop the color reaction, and the concentration was calculated by measuring the absorbance at 450 nm using Synergy Neo2 (BioTek).

As a result, it was confirmed that the amount of IFN-γ secretion increased statistically significantly in cells treated with BNS002 fusion protein compared to cells treated with IL-15 protein (FIG. 22).

Experimental Example 22. Confirmation of the effect of the BNS002 fusion protein on the proliferation of CD8+ T cells

PBMCs isolated from humans were thawed at 37°C and centrifuged at 1,500 rpm at 4°C for 5 minutes. After centrifugation, the suspension was removed, and resuspended in RPMI1640 culture medium (FBS 10%, penicillin/streptomycin 200 μl, BNS002 fusion protein, IL-15 protein (each 10 μg/ml)) at 37°C, 5% CO ₂ cultured in an incubator. The cultured PBMCs were washed twice with cold PBS (Sigma, pH 7.4 ^{), centrifuged, and then 1.5 cells per 100 μl (10 6 cells} /100 μl) with PBS (containing 1% FBS). Resuspended in a microtube for ml. Thereafter, the cells were stained with anti-CD8-PE-Texas Red (1:100, invitrogen, cat No. MHCD0817) for 30 minutes at room temperature. After dispensing 30 μl per well in a V-bottom 96-well plate, the degree of proliferation of these cells was confirmed by measuring the ratio of unlabeled cells among CD8+ T cells by flow cytometry.

As a result, it was confirmed that the BNS002 fusion protein activates the proliferation of CD8+ T cells to a degree similar to that of the IL-15 protein (FIG. 23).

Experimental Example 23. Confirmation of the effect of the BNS002 fusion protein on the proliferation of CD4+ T cells

PBMCs isolated from humans were thawed at 37°C and centrifuged at 1,500 rpm at 4°C for 5 minutes. After centrifugation, the suspension was removed, and resuspended in RPMI1640 culture medium (FBS 10%, penicillin/streptomycin 200 μl, BNS002 fusion protein, IL-15 protein (each 10 μg/ml)) at 37°C, 5% CO ₂ cultured in an incubator. The cultured PBMCs were washed twice with cold PBS (Sigma, pH 7.4 ^{), centrifuged, and then 1.5 cells per 100 μl (10 6 cells} /100 μl) with PBS (containing 1% FBS). Resuspended in a microtube for ml. Thereafter, the cells were stained with anti-CD4-PE-Pacific Blue (2 μg/10 ⁶ cells, BioLegend, cat No. 344620) for 30 minutes at room temperature. After dispensing 30 μl per well in a V-bottom 96-well plate, the degree of proliferation of these cells was confirmed by measuring the ratio of unlabeled cells among CD4+ T cells by flow cytometry.

As a result, it was confirmed that the BNS002 fusion protein activated the proliferation of CD4+ T cells to a degree similar to that of the IL-15 protein, but did not increase the proliferation of CD4+/FoxP3+ Treg cells (FIG. 24).

Experimental Example 24. Confirmation of the effect of the BNS002 fusion protein on the proliferation of NK cells

PBMCs isolated from humans were slowly thawed at 37°C and centrifuged at 1,500 rpm at 4°C for 5 minutes. After centrifugation, the suspension was removed, and resuspended in RPMI1640 culture medium (FBS 10%, penicillin/streptomycin 200 μl, BNS002 fusion protein, IL-15 protein (each 10 μg/ml)) at 37°C, 5% CO ₂ cultured in an incubator. The cultured PBMCs were washed twice with cold PBS (Sigma, pH 7.4 ^{), centrifuged, and then 1.5 cells per 100 μl (10 6 cells} /100 μl) with PBS (containing 1% FBS). Resuspended in a microtube for ml. Then, anti-CD56-PE (0.4 μg/10 ⁶ cells, BioLegend, cat No. 362508) and anti-CD16-APC (5 μl/10 ⁶ cells, BioLegend, cat No. 302012) for 30 minutes at room temperature. dyed. After dispensing 30 μl per well in a V-bottom 96-well plate, the degree of proliferation of these cells was confirmed by measuring the ratio of unlabeled cells among CD56+/CD16+ NK cells by flow cytometry.

As a result, it was confirmed that the BNS002 fusion protein activates the proliferation of NK cells to a degree similar to that of the IL-15 protein (FIGS. 25a and 25b).

Experimental Example 25. Confirmation of the effect of BNS002 fusion protein on T cell function

Experimental Example 25.1. PD-L1/PD-1 blocking assay

Experiments were conducted using a PD-1/PD-L1 blockade bioassay kit (Promega cat No. J1252).

PD-L1 aAPC/CHO-K1 cells (target cells) stored in liquid nitrogen were thawed in a constant temperature water bath at 37° C. for 1 minute, and then suspended in a pre-warmed culture medium (Ham's F12: FBS 10%). 100 μl per well was dispensed into a 96-well white plate (SPL, cat No. 30196) and cultured in a 37°C, 5% CO ₂ incubator. The next day, take out the plate, remove 95 μl of the culture medium, add 40 μl of 2-fold serially diluted BNS002 fusion protein, Atezolizumab, and PD-1-Fc to each well, and Jurkat NFAT-Luc/PD- During the time to prepare 1 cell (Effector cell), the plate was covered and stored at room temperature. After thawing the effector cells stored in liquid nitrogen for 1 minute in a constant temperature water bath at 37 ° C and suspending them in pre-warmed culture medium (Ham's F12: FBS 10%), the target cells and samples were prepared. 40 μl was dispensed per each well of the plate and cultured for 6 hours in a 37° C., 5% CO ₂ incubator.

After the reaction was complete, the plate was taken out and Bio-Glo reagent was added, being careful not to create bubbles. Bio-Glo reagent was also added to the three edge wells and used as a blank to correct the background signal. After reacting at room temperature for 30 minutes, luminescence was measured with Synergy Neo2 (BioTek).

As a result, it was confirmed that the BNS002 fusion protein binds to PD-1 expressed in effector T cells and activates the function of T cells rather than suppressing them (FIG. 26).

Experimental Example 25.2. PD-L2/PD-1 blocking assay

Experiments were conducted using a PD-1/PD-L2 blockade bioassay kit (Promega cat No. CS187131-1).

PD-L2 aAPC/CHO-K1 cells (target cells) stored in liquid nitrogen were thawed in a constant temperature water bath at 37° C. for 1 minute, and then suspended in a pre-warmed culture medium (Ham's F12: FBS 10%). 100 μl per well was dispensed into a 96-well white plate (SPL, cat No. 30196) and cultured in a 37°C, 5% CO ₂ incubator. The next day, take out the plate, remove 95 μl of the culture medium, add 40 μl of BNS002 fusion protein and PD-L2-Fc serially diluted 2 times per well, and Jurkat NFAT-Luc/PD-1 cells (Effector cell). During the preparation time, the plate was covered and stored at room temperature. After thawing the effector cells stored in liquid nitrogen for 1 minute in a constant temperature water bath at 37 ° C and suspending them in pre-warmed culture medium (Ham's F12: FBS 10%), the target cells and samples were prepared. 40 μl was dispensed per each well of the plate and cultured for 6 hours in a 37° C., 5% CO ₂ incubator.

After the reaction was complete, the plate was taken out and Bio-Glo reagent was added, being careful not to create bubbles. Bio-Glo reagent was also added to the three edge wells and used as a blank to correct the background signal. After reacting at room temperature for 30 minutes, luminescence was measured with Synergy Neo2 (BioTek).

As a result, it was confirmed that the BNS002 fusion protein binds to PD-1 expressed in effector T cells and activates the T cells instead of suppressing them (FIG. 27).

IX. Confirmation of anticancer effect of BNS002 fusion protein

Experimental Example 26. Establishment of cancer cell line for confirmation of anticancer effect on BNS002 fusion protein

Human melanoma cell line A375 (ATCC, CRL-1619) cells were seeded in 1×10 ⁵ cells in a 6-well plate and cultured in a 37° C., 5% CO ₂ incubator. The next day, after removing the culture medium, lentivirus transfected with firefly luciferase (fLuc) and GFP genes (GenTarget, cat No. LVP914-G) (FIG. 28) 25 MOI (Multiplicity of infection, 25 μl) was added and incubated for 4 hours. After culturing for 4 hours, the cells were recovered with 0.05% Trypsin-EDTA (Gibco) solution, and then transferred to a 150 mm culture dish (TPP) and cultured. During cultivation, only GFP-expressing cells were selectively cultured while observing under a fluorescence microscope, and Firefly luciferase-expressing human melanoma cell line A375-Luc-GFP cell line was established.

The A375-Luc-GFP cell line was dispensed with 5×10 ⁵ cells (n=3) in a 96-well white or black plate with 100 μl of cell culture medium, and then serially diluted in half with 100 μl of cell culture medium. (8 points). After adding 100 μl of Bio-Glo solution per well, a luciferase or fluorescence (GFP) image was acquired for 1 second using Neo2 equipment. As the number of cells in the A375-Luc-GFP cell line increased, the intensity of the luminescence or fluorescence signal also increased (FIG. 29).

Experimental Example 27. Confirmation of the effect of BNS002 fusion protein on cancer cells expressing PD-L1 and PD-L2

Experimental Example 27.1. Co-culture of cancer cells and PBMCs

PBMC isolated from humans were stained by reacting with membrane-dye (Red) (Sigma, cat No. PKH26) dye at a concentration of 1.25 μM at room temperature for 1 minute, and then the staining reaction was stopped by adding the same volume of FBS. The dye not bound to the cells was centrifuged at 400×g for 10 minutes to remove and wash the suspension, and then resuspended in RPMI1640 culture medium (FBS 10%, penicillin/streptomycin 200 μl). At this time, a total of three washing operations were performed. After resuspending in RPMI1640 culture medium (FBS 10%, penicillin/streptomycin 200 μl), fluorescence was observed. For co-culture of fluorescently stained PBMC and established cancer cell line (A375-fLuc-GFP), A375-fLuc-GFP cells (1×10 ⁵ ) were divided and fluorescently stained PBMC (1×10 ⁶ ) were placed in 6 wells. Divided into plates and co-cultured. At this time, the anti-CD3 antibody (OKT3, 1 μg/ml, Invitrogen) and the anti-CD28 antibody (CD28.2, 1 μg/ml, Invitrogen) were co-treated or not treated, and the BNS002 fusion protein (10 μg/ml) ㎖), and cultured for 3 days in a 37°C, 5% CO ₂ incubator.

Experimental Example 27.2. Confirmation of PD-L1, PD-L2, and PD-1 gene expression in co-cultured cancer cells and PBMCs

Malignant melanoma cells A375 (ATCC, # CRL-1619) and PBMC (ATCC, # To confirm the transcription and expression of PD-L1, PD-L2 and PD1 genes in PCS-800-011), reverse transcription PCR was performed. PBMCs were co-stimulated with an anti-CD3 antibody (1 μg/ml, Invitrogen) and an anti-CD28 antibody (1 μg/ml, Invitrogen), followed by active culture. Total RNA was extracted from each cell line, cDNA was synthesized (TAKARA, RR036A), PCR was performed using a primer set (Table 1), and PD-L1/L2 and PD-1 specific replication was performed in A375 and PBMC. The products (120 bp, 173 bp, 289 bp) were identified.

Primer name order sequence number PD-L1 F: 5'-TGGCATTTGCTGAACGCATTT-3' 101 PD-L1 R: 5'-TGCAGCCAGGTCTAATTGTTTT-3' 102 PD-L2 F: 5'-CAGCAATGTGACCCTGGAAT-3' 103 PD-L2 R: 5'-GGACTTGAGGTATTGTGGAACG-3' 104 PD-1 F: 5'-TGCAGCTTCTCCAACACATC-3' 105 PD-1 R: 5'-CTGCCCTTCTCTCTGTCACC-3' 106 GAPDH-2 F: 5'-AGCCGCATCTTCTTTTGCGT-3' 107 GAPDH-2 R: 5'-TGACGAACATGGGGGCATCA-3' 108

As a result, PD-1-specific replication products (120 bp, 173 bp, 289 bp) were confirmed in PBMCs in which PD-L1 and PD-L2 genes and immune cells were present in malignant melanoma cells (A375) (FIG. 30). ).

Experimental Example 27.3. Confirmation of human IFN-γ secretion through ELISA analysis

In Experimental Example 27.1, the amount of human IFN-γ secreted in the culture supernatant of each sample was measured using a human IFN-γ ELISA kit (Biolegend, cat No.430103). An anti-human-IFN-γ antibody was added to the ELISA plate and reacted overnight at 4° C. to coat the plate. Thereafter, blocking was performed for 1 hour at room temperature with a PBS solution to which 1% BSA was added. After washing with washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample were diluted to a concentration of 7.5 nM and reacted at room temperature for 2 hours. After the reaction was completed, the plate was washed and a secondary detection antibody was added thereto and reacted at room temperature for 1 hour. After washing with washing buffer, an avidin-HRP solution was added and reacted at room temperature for 30 minutes. A color reaction was induced for 20 minutes under room temperature and dark conditions by adding a substrate solution. Finally, H ₂ S0 ₄ was added to stop the color reaction, and the concentration was calculated by measuring the absorbance at 450 nm using Synergy Neo2 (BioTek).

As a result, it was confirmed that the amount of IFN-γ secretion increased statistically significantly in cells treated with BNS002 fusion protein compared to cells treated with IL-15 protein (FIG. 31).

Experimental Example 27.4. Confirmation of cancer cell killing effect

To confirm the immune cell activity and tumor cell killing effect, 5 μg/mL of anti-CD3 antibody (OKT3, 1 μg/mL, invitrogen) and anti-CD28 antibody (CD28.2, 1 μg/mL) were applied to the co-cultured cells. , Invitrogen) was treated with or without BNS002 fusion protein at a concentration of 50 nM, and cultured in a 37°C, 5% CO ₂ incubator for 72 hours. After co-culture for 24 hours, 48 hours and 72 hours to quantify the degree of cancer cell killing, the wells were washed twice with PBS, and 10 μl of EZ-Cytox (Daeillab, Cat. No., EZ-1000) was added. and reacted at room temperature for 30 minutes. After the reaction, absorbance at 450 nm was measured using Synergy Neo2 (BioTek).

After BNS002 fusion protein treatment, the degree of cancer cell killing measured on day 1 (24 hours), day 2 (48 hours), and day 3 (72 hours) was measured as shown in FIG. 32 .

In addition, as a result of comparing the degree of cancer cell killing measured 2 days after treatment with BNS002 fusion protein for statistical analysis of the cancer cell killing effect, the cancer cell killing effect was statistically more significant in cells treated with BNS002 fusion protein than in untreated cells. It was confirmed that the increase was confirmed (FIG. 33).

In addition, as a result of comparing the degree of cancer cell killing measured 2 days after treatment with various concentrations of BNS002 fusion protein for EC ₅₀ analysis on the cancer cell killing effect, cells treated with BNS002 fusion protein had a higher cancer cell killing effect than untreated cells. It was confirmed that increased (FIG. 34).

Experimental Example 28. Confirmation of cancer cell line-specific antibody-dependent cytotoxicity (ADCC) activity of BNS002 fusion protein

The A375 cell line was suspended in a culture medium (RPMI1640: FBS 10%, Antibiotic-Antimycotic 1%), dispensed into a 96-well white plate at a cell concentration of 1×10 ⁴ cells/100 μl, and cultured overnight in an incubator. The next day, after removing 95 μl of the culture medium from the plate in which the A375 cells were seeded, 25 μl of ADCC Assay Buffer (ADCC Assay Buffer: RPMI1640 culture medium, low IgG serum 0.25%) prepared in advance was added to effector cells ( NFAT-luc/FcγRIIIa) was incubated at room temperature while preparing. After suspending 630 μl of ADCC Bioassay Effector Cell (Jurkat/NFAT-luc/FcγRIIIa) in 3.6 mL of pre-warmed ADCC assay buffer, 25 μl of each was dispensed into each well. 25 μl of 2.5-fold serially diluted BNS002 fusion protein samples were treated in each well. Close the lid of the well plate and incubate for 6 hours in a 37°C, 5% CO ₂ incubator. After incubation, the cells were kept at room temperature for about 15 minutes, and then each well was treated with 75 μl of Bio-Glo™ Luciferase Assay Reagent. After 30 minutes, the luciferase activity was measured using Synergy Neo2 (BioTek).

The luminescent activity of the ADCC Assay is a test method that can obtain results only when target cells (A375) with surface antigens (PD-L1/L2), specific antibodies (BNS002 fusion protein), and effector cells expressing FcγRIIIa are present. ADCC activity for the fusion protein was measured as shown in FIG. 35 .

Experimental Example 29. Confirmation of anticancer effect of BNS002 fusion protein in mouse-derived colon cancer cell allograft mice

Experimental Example 29.1. Confirmation of tumor suppression effect

A frozen mouse tumor cell line, specifically colorectal carcinoma cell line CT26 cell line 1 vial, was heat inactivated using RPMI1640 medium containing 10% FBS (Gibco, 10082-147). After thawing, the cells were placed in a flask for cell culture and cultured in an incubator at 37°C and 5% CO ₂ . After culturing, the cells were washed with PBS, diluted 10-fold with 2.5% Trypsin-EDTA (Gibco, 15090), and added thereto to separate the cells. After cell separation, the supernatant was removed by centrifugation (1,000 rpm, 5 minutes), and a cell suspension was obtained with a fresh medium. After confirming viability using a microscope, cell lines were prepared by diluting in a medium at a concentration of 5.0×10 ⁶ cells/mL. As mice, 5-week-old male BALB/c mice (Coretech) were used. When the cell line was transplanted, it was subcutaneously administered at a dose of 5.0×10 ⁵ cells/0.1 mL/head. Cancer cells were transplanted, and after a certain period of time, the volume of the tumor was measured, and objects reaching about 60-90 mm ³ were isolated, and BNS002 fusion protein was intravenously administered at a dose of 5 mg/kg. A total of 4 administrations were performed once every 3 days after the first administration. At this time, PBS was administered as a negative control group, and atezolizumab, an anti-PD-L1 antibody at 5 mg/kg, was administered as a positive control group. To confirm the anticancer effect, the size of the tumor was measured twice a week (FIG. 36).

As a result, it was confirmed that the size of the tumor in the CT26 cancer cell line implanted mice treated with the BNS002 fusion protein was significantly reduced compared to the negative control group and the positive control group (FIG. 37).

As a result of confirming the statistical significance of tumor suppression in the BNS002 fusion protein administration group and the negative control group, it was observed that the tumor suppression was statistically significant at some measurement time points (FIG. 38).

Experimental Example 29.2. Immune cell analysis in cancer tissue

Among the mice of Experimental Example 29.1, the tumors extracted from only 3 mice per group were placed in RPMI medium containing 10% FBS and then used for FACS analysis. In order to analyze immune cells in cancer tissues, cancer tissues are separated into single cells, and then analyzed for T cells, NK cells, DC (Dendritic Cell), and macrophages using the antibodies listed in Table 2 below was carried out.

effector T cells NK cells, DCs, macrophages CD3-efluor ^® 450 (Thermo, 48-0031-82)
CD45-PerCP-cyanine 5.5 (Thermo, 45-0451-82)
CD4-PE-cy7 (Thermo, 25-0041-82)
CD8-APC (Thermo, 17-0081-82) CD3-efluor ^® 450 (Thermo, 48-0031-82)
CD45-Super bright 600 (Thermo, 63-0451-82)
CD335-PE (Thermo, 12-3351-82)
CD11c-Percp-cy5 (Thermo, 45-0114-82)
F4/80-PE-cy7 (Thermo, 25-4801-82)
CD49b-APC (Thermo, 17-5971-82)
CD11b-APC-eFluor 770 (Thermo, 47-0112-82)

As a result, macrophages, dendritic cells, CD4+ T cells, and CD8+ T cells increased in the experimental group administered with the BNS002 fusion protein compared to the positive and negative control groups (FIG. 39). In addition, in the experimental group administered with the BNS002 fusion protein, NK cells were significantly increased compared to the positive control group and the negative control group (FIG. 39).

Experimental Example 30. Confirmation of the anticancer effect of the BNS002 fusion protein in humanized mice transplanted with human-derived lung cancer cells (H460) and human peripheral blood mononuclear cells

Thaw 1 vial of frozen human tumor cell line, specifically lung carcinoma cell line H460 cell line, using RPMI1640 medium containing heat inactivated 10% FBS (Gibco, 10082-147) And, it was put into a flask for cell culture and cultured in a 37°C, 5% CO ₂ incubator. After culturing, the cells were washed with PBS, diluted 10-fold with 2.5% Trypsin-EDTA (Gibco, 15090), and added thereto to separate the cells. After cell separation, the supernatant was removed by centrifugation at 1,000 rpm for 5 minutes, and a cell suspension was obtained with a fresh medium. After confirming viability using a microscope, a cell line was prepared by diluting in a medium at a concentration of 5.0×10 ⁷ cells/mL. In addition, peripheral blood mononuclear cells (PBMC) isolated from humans were actively cultured for one week as in Experimental Example 21.1. After confirming viability using a microscope, a cell line was prepared by diluting in a medium at a concentration of 2.5×10 ⁸ cells/mL. A 6-week-old male NXG mouse (Janvier-labs) was used as the mouse, and after a one-week acclimatization process, body weight was measured to match the average weight of each group and regrouped. When cell lines were transplanted, 5.0×10 ⁶ cells (H460) and 2.5×10 ⁷ cells (hPBMC)/0.1 mL/head were subcutaneously administered. The next day after transplantation of cancer cells, after anesthesia with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical), BNS002 fusion protein was administered through the tail vein at a dose of 10 mg/kg. A total of six doses were administered once every 3 to 4 days after the first dose. At this time, saline (JW Pharmaceutical Co., Ltd.) was administered as a negative control group, and atezolizumab (Evidentic), an anti-PD-L1 antibody at 10 mg/kg, or PD-1 D-Fc was administered as a positive control group. administered. To confirm the anticancer effect, the size of the tumor was measured twice a week (FIG. 40).

As a result, the tumor size of the H460 cancer cell line-transplanted mice treated with the BNS002 fusion protein was reduced compared to the negative control group and the positive control group (FIG. 41), and it was confirmed that this decrease was statistically significant (FIG. 42).

Experimental Example 31. Confirmation of anticancer effect of BNS002 or BNS002S fusion protein in humanized mice transplanted with human-derived colorectal cancer cells (HCT116) and human peripheral blood mononuclear cells

Experimental Example 31.1. Confirmation of anticancer effect by dose of BNS002 fusion protein

A cell line was prepared in the same manner as in Experimental Example 30, except that the HCT116 cell line, a colon carcinoma cell line, was used as a frozen human tumor cell line. In addition, peripheral blood mononuclear cells (PBMC) isolated from humans were actively cultured for one week as in Experimental Example 21.1. After confirming viability using a microscope, a cell line was prepared by diluting in a medium at a concentration of 2.5×10 ⁸ cells/mL. As mice, 6-week-old male NSG mice (NOD.Cg-B2mtm1UncPrkdcscidIl2rgtm1Wjl/SzJ) (Orient Bio) were used, and administration of human peripheral blood mononuclear cells 1×10 ⁷ cells (hPBMC)/0.1 mL/head was used to prepare humanized mice. A liquid dose was administered subcutaneously into the tail vein. Humanized mice were identified by measuring hCD45+ expression in the blood by FACS analysis 14 days after administration of human peripheral blood mononuclear cells. After confirming the humanized mouse, when the cell line HCT116 was transplanted, 3×10 ⁶ cells were administered subcutaneously. Two days after transplanting cancer cells, anesthetize with inhalational anesthetic (2% isoflurane, Hana Pharmaceutical), and then administer BNS002 fusion protein (PD-1 D-Fc-IL-15) at doses of 5, 10, and 20 mg/kg. was administered intraperitoneally. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, saline (JW Choongwa Pharmaceutical) was administered as a negative control group, and the size of the tumor was measured twice a week to confirm the anticancer effect (FIG. 43).

As a result, it was confirmed that the size of the tumor was significantly reduced in a concentration-dependent manner, statistically significant, compared to the negative control group (Vehicle treatment) in the animal experiment of humanized mice transplanted with HCT116 cancer cell line treated with the BNS002 fusion protein. In particular, the anticancer effect was the best when administered at a dose of 10 mg/kg (FIG. 44).

On the other hand, it was confirmed that there was no effect on body weight change according to BNS002 fusion protein treatment (FIG. 45).

Experimental Example 31.2. Confirmation of anticancer effect between BNS002 fusion protein (PD-1 D-Fc-IL-15) and control drug

Humanized mice were prepared by preparing the HCT116 cell line and PBMC in the same manner as in Experimental Example 31.1. Two days after transplanting HCT116 cancer cells into humanized mice, they were anesthetized with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical). Then, BNS002 fusion protein (PD-1 D-Fc-IL-15) was intraperitoneally administered at a dose of 10 mg/kg. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, saline was administered as a negative control group, and BNS001D (PD-1 D-Fc) and BNS002I (Fc-IL-15) fusion proteins were administered alone or in combination as a comparative control group. In addition, commercially available antibodies atezolizumab (Anti-PD-L1 (Tecentriq, Roche)), Avelumab (Anti-PD-L1 (Bavencio, Merck)) and pembrolizumab (Pembrolizumab; Anti-PD-L1 (Bavencio, Merck)) were used as positive controls. PD-1 (Keytruda, MSD)) was administered. To confirm the anticancer effect of the administered substance, tumor size and body weight were measured twice a week (FIG. 46).

As a result, it was confirmed that the size of the tumor was significantly reduced statistically compared to the negative control group in the animal experiment of humanized mice transplanted with HCT116 cancer cell line treated with the BNS002 fusion protein (PD-1 D-Fc-IL-15) (FIG. 47). and Figure 49).

It was confirmed that the size of the tumor in the BNS002 fusion protein-administered group was smaller than that of the comparative control groups, BNS001D (PD-1 D-Fc) and BNS002I (Fc-IL-15)-administered groups. In particular, it was confirmed that the BNS002 fusion protein administration group had a smaller tumor size than the BNS001D (PD-1 D-Fc) + BNS002I (Fc-IL-15) combination administration group (FIG. 47).

In addition, the BNS002 fusion protein administration group was statistically significant compared to the commercial antibodies Pembrolizumab (Anti-PD-1 (Keytruda, MSD)) and Avelumab (Anti-PD-L1 (Bavencio, Merck)) administration groups used as positive control groups. It was confirmed that the size of the tumor was significantly reduced. Compared to the atezolizumab (Anti-PD-L1 (Tecentriq, Roche)) administration group, it was confirmed that the BNS002 fusion protein showed an equivalent level of tumor size reduction (FIG. 49).

On the other hand, it was confirmed that there was no effect on body weight change according to BNS002 fusion protein treatment (FIGS. 48 and 50).

Experimental Example 31.3. Confirmation of anticancer effect between BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra) and control drug

Humanized mice were prepared by preparing the HCT116 cell line and PBMC in the same manner as in Experimental Example 31.1. Two days after transplanting HCT116 cancer cells into humanized mice, they were anesthetized with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical). Then, the BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra) was intraperitoneally administered at a dose of 5 mg/kg. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, the negative control group, comparative control group, and positive control group were administered under the same conditions as Experimental Example 31.2. Thereafter, tumor size and weight were measured twice a week to confirm the anticancer effect of the administered material (FIG. 46).

As a result, it was confirmed that the size of the tumor was significantly reduced statistically compared to the negative control group in the animal experiment of humanized mice transplanted with HCT116 cancer cell line treated with the BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra). (FIGS. 51 and 53).

It was confirmed that the size of the tumor in the BNS002S fusion protein-administered group was reduced compared to the comparison control group, BNS001D (PD-1 D-Fc) and BNS002I (Fc-IL-15)-administered groups. In particular, it was confirmed that the BNS002S fusion protein administration group had a smaller tumor size than the BNS001D (PD-1 D-Fc) + BNS002I (Fc-IL-15) combination administration group (FIG. 51).

In addition, the BNS002S fusion protein-administered group had a tumor size compared to the commercially available antibodies Pembrolizumab (Anti-PD-1 (Keytruda, MSD)) and Avelumab (Anti-PD-L1 (Bavencio, Merck))-administered groups used as positive control groups. It was confirmed that it was significantly reduced. Compared to the atezolizumab (Anti-PD-L1 (Tecentriq, Roche)) administration group, it was confirmed that the BNS002S fusion protein showed an equivalent level of tumor size reduction (FIG. 53).

On the other hand, it was confirmed that there was no effect on body weight change according to BNS002 fusion protein treatment (FIGS. 52 and 54).

Experimental Example 32. Confirmation of anticancer effect of BNS002 or BNS002S fusion protein in humanized mice transplanted with human-derived lung cancer cells (A549) and human peripheral blood mononuclear cells

Experimental Example 32.1. Confirmation of anticancer effect between BNS002 fusion protein (PD-1 D-Fc-IL-15) and control drug

A humanized mouse was prepared by preparing the A549 cell line and PBMC in the same manner as in Experimental Example 31.1, except that the A549 cell line, which is a lung cancer (NSCLC) cell line, was used as a frozen human tumor cell line. Two days after transplanting A549 cancer cells into humanized mice, they were anesthetized with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical). Then, BNS002 fusion protein (PD-1 D-Fc-IL-15) was intraperitoneally administered at a dose of 10 mg/kg. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, saline (JW Pharmaceutical) was administered as a negative control group, and commercially available antibodies atezolizumab (Anti-PD-L1 (Tecentriq, Roche)) and avelumab (Avelumab; Anti-PD-L1 (Tecentriq, Roche)) were administered as positive controls. L1 (Bavencio, Merck)) and pembrolizumab (Anti-PD-1 (Keytruda, MSD)) were administered. To confirm the anticancer effect of the administered substance, tumor size and body weight were measured twice a week (FIG. 55).

As a result, it was confirmed that the size of the tumor was significantly reduced statistically significantly compared to the negative control group in the animal experiment of humanized mice transplanted with the A549 cancer cell line treated with the BNS002 fusion protein (PD-1 D-Fc-IL-15). In addition, the BNS002 fusion protein administration group was commercially available antibodies atezolizumab (Anti-PD-L1 (Tecentriq, Roche)), pembrolizumab (Anti-PD-1 (Keytruda, MSD)) and avelumab (Anti-PD-L1 (Keytruda, MSD)) used as a positive control group. PD-L1 (Bavencio, Merck)) showed an equivalent level of tumor size reduction compared to the administration group (FIG. 56).

On the other hand, it was confirmed that there was no effect on body weight change according to BNS002 fusion protein treatment (FIG. 57).

Experimental Example 32.2. BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra) and anticancer effect confirmed by dose

Humanized mice were prepared by preparing the A549 cell line and PBMC in the same manner as in Experimental Example 32.1. Two days after transplanting A549 cancer cells into humanized mice, after anesthesia with inhalation anesthetic (2% isoflurane, Hana Pharmaceutical), BNS002S (PD-1D-Fc-IL-15/IL) was administered at doses of 1 and 5 mg/kg. -15Ra) fusion protein was intraperitoneally administered. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, saline was administered as a negative control group, and tumor size and body weight were measured twice a week to confirm the anticancer effect of the administered material.

As a result, in the animal experiment of A549 cancer cell line transplanted with humanized mice treated with the BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra), the tumor size was statistically significant compared to the negative control group (Vehicle treatment) in a concentration-dependent manner. It was confirmed that it was significantly reduced. In particular, the anticancer effect was the best when administered at a dose of 5 mg/kg (FIG. 58).

On the other hand, it was confirmed that there was no effect on body weight change according to the BNS002S fusion protein treatment (FIG. 59).

Experimental Example 32.3. Confirmation of anticancer effect between BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra) and control drug

Humanized mice were prepared by preparing the A549 cell line and PBMC in the same manner as in Experimental Example 32.1. Two days after transplanting A549 cancer cells into humanized mice, they were anesthetized with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical). Then, the BNS002S fusion protein was intraperitoneally administered at a dose of 5 mg/kg. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, the negative control group and the positive control group were administered under the same conditions as Experimental Example 32.1. Tumor size and body weight were measured twice a week to confirm the anticancer effect of the administered material.

As a result, it was confirmed that the size of the tumor was significantly reduced statistically compared to the negative control group in the animal experiment of humanized mice transplanted with the A549 cancer cell line treated with the BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra). . In addition, the BNS002S fusion protein administration group was commercially available antibodies atezolizumab (Anti-PD-L1 (Tecentriq, Roche)), pembrolizumab (Anti-PD-1 (Keytruda, MSD)) and avelumab (Anti-PD-L1 (Keytruda, MSD)) used as a positive control group. PD-L1 (Bavencio, Merck)) showed an equivalent level of tumor size reduction compared to the administration group (FIG. 60). On the other hand, it was confirmed that there was no effect on body weight change according to the BNS002S fusion protein treatment (FIG. 61).

Experimental Example 33. Confirmation of anticancer effect of BNS002 or BNS002S fusion protein in humanized mice transplanted with human breast cancer cells (MDA-MB-231) and human peripheral blood mononuclear cells

Experimental Example 33.1. Confirmation of anticancer effect between BNS002 fusion protein (PD-1 D-Fc-IL-15) and control drug

As a frozen human tumor cell line, except for using the breast carcinoma (TNBC) cell line MDA-MB-231 cell line, the MDA-MB-231 cell line and PBMC were prepared in the same manner as in Experimental Example 31.1 to prepare a humanized mouse. . Two days after transplanting MDA-MB-231 cancer cells into humanized mice, they were anesthetized with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical). Then, BNS002 fusion protein (PD-1 D-Fc-IL-15) was intraperitoneally administered at a dose of 10 mg/kg. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, saline (JW Pharmaceutical) was administered as a negative control group, and commercially available antibodies atezolizumab (Anti-PD-L1 (Tecentriq, Roche)) and avelumab (Avelumab; Anti-PD-L1 (Tecentriq, Roche)) were administered as positive controls. L1 (Bavencio, Merck)) and pembrolizumab (Anti-PD-1 (Keytruda, MSD)) were administered. To confirm the anticancer effect of the administered substance, tumor size and body weight were measured twice a week (FIG. 62).

As a result, it was confirmed that the BNS002 fusion protein (PD-1 D-Fc-IL-15) treated MDA-MB-231 cancer cell line transplant humanized mouse animal experiment did not show the effect of reducing tumor size compared to the negative control group. (FIG. 63). In addition, there was no effect on body weight change according to BNS002 fusion protein treatment (FIG. 64).

Experimental Example 33.2. Confirmation of anticancer effect between BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra) and control drug

Humanized mice were prepared by preparing the MDA-MB-231 cell line and PBMC in the same manner as in Experimental Example 33.1. Two days after transplanting MDA-MB-231 cancer cells into humanized mice, they were anesthetized with an inhalational anesthetic (2% isoflurane, Hana Pharmaceutical). Then, the BNS002S fusion protein was intraperitoneally administered at a dose of 5 mg/kg. A total of 4 doses were administered once every 3 to 4 days after the first dose. At this time, the negative control group and the positive control group were administered under the same conditions as Experimental Example 33.1. Tumor size and body weight were measured twice a week to confirm the anticancer effect of the administered material.

As a result, in the animal experiment of humanized mice transplanted with MDA-MB-231 cancer cell line treated with the BNS002S fusion protein (PD-1D-Fc-IL-15/IL-15Ra), the size of the tumor was statistically significant compared to the negative control group. confirmed a decrease. In addition, the BNS002S fusion protein administration group was commercially available antibodies atezolizumab (Anti-PD-L1 (Tecentriq, Roche)), pembrolizumab (Anti-PD-1 (Keytruda, MSD)) and avelumab (Anti-PD-L1 (Keytruda, MSD)) used as positive control groups. Compared to the PD-L1 (Bavencio, Merck)) administration group, it was confirmed that the size of the tumor was significantly reduced (FIG. 65). On the other hand, it was confirmed that there was no effect on body weight change according to the BNS002S fusion protein treatment (FIG. 66).

Claims (28)

A fusion protein comprising PD-1 protein and IL-15 protein. According to claim 1, The PD-1 protein and the IL-15 protein are linked by a linker, a fusion protein. According to claim 1, The PD-1 protein has the amino acid sequence of SEQ ID NO: 2, 23 or 39, the fusion protein. According to claim 1, The IL-15 protein has the amino acid sequence of SEQ ID NO: 6 or 24, the fusion protein. According to claim 2, The linker is an immunoglobulin Fc domain, the fusion protein. According to claim 5, The Fc domain is a wild-type or mutant, fusion protein. According to claim 6, The wild type of the Fc domain has the amino acid sequence of SEQ ID NO: 14, the fusion protein. According to claim 6, The variant of the Fc domain is a substitution of Q81R, M198L, L79G, T136S, L138A, Y177V, Q81R/M198L, L79G/M198L, T136S/L138A/Y177V or Q81R/T136S/L138A/Y177V/M198L in the amino acid sequence of SEQ ID NO: 14. This is what happened, the fusion protein. According to claim 8, The variant of the Fc domain will have an amino acid sequence of SEQ ID NO: 4, 10, 55, 88 or 97, the fusion protein. According to claim 6, The mutant of the Fc domain has an increased half-life compared to the wild type, the fusion protein. According to claim 1, The fusion protein is any one selected from the group consisting of the following structural formulas I to IV: N'-A-[L ₁ ]n-Fc domain-[L ₂ ]mB-[L ₃ ] _l -SC' I N'-A-[L ₁ ]n-Fc domain-[L ₂ ]mS-[L ₃ ] _l -BC' II N'-S-[L ₃ ] _l -B-[L ₁ ]n-Fc domain-[L ₂ ]mAC' III N'-B-[L ₃ ] _l -S-[L ₁ ]n-Fc domain-[L ₂ ]mAC' IV At this time, in the structural formulas I to IV, N' is the N-terminus of the fusion protein, The C' is the C-terminus of the fusion protein, A is PD-1 protein or a fragment thereof; B is an IL-15 protein or a variant thereof; S is an IL-15 binding protein, Wherein L ₁ , L ₂ and L ₃ are each a peptide linker, The above n, m and l are each independently 0 or 1. According to claim 11, Wherein L ₁ is a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3 or 43, the fusion protein. According to claim 11, Wherein L ₂ is a peptide linker consisting of the amino acid sequence of SEQ ID NO: 5, 44 or 62, the fusion protein. According to claim 11, Wherein L ₃ is a peptide linker consisting of the amino acid sequence of SEQ ID NO: 96, the fusion protein. According to claim 11, The fusion protein is a fusion protein consisting of the following structural formula V or VI: N′-A-[L ₁ ]n-Fc domain-[L ₂ ]mBC′ V N'-B-[L ₁ ]n-Fc domain-[L ₂ ]mAC' VI At this time, in the structural formulas V and VI, N' is the N-terminus of the fusion protein, The C' is the C-terminus of the fusion protein, A is PD-1 protein or a fragment thereof; B is an IL-15 protein or a variant thereof; Wherein L ₁ and L ₂ are each a peptide linker, The n and m are each independently 0 or 1. According to claim 1, The fusion protein is a sequence of 85% or more of the amino acid sequence of SEQ ID NO: 8, 12, 16, 26, 29, 33, 37, 41, 46, 49, 57, 60, 77, 80, 83, 90 or 93 That which has the identity, fusion protein. A fusion protein dimer in which two fusion proteins according to any one of claims 1 to 16 are combined. According to claim 17, The fusion protein dimer is a homodimer, the fusion protein dimer. A polynucleotide encoding the fusion protein of any one of claims 1 to 16. According to claim 19, The polynucleotide is 85% or more of the nucleotide sequence of SEQ ID NO: 9, 13, 17, 27, 30, 35, 38, 42, 47, 50, 58, 61, 78, 81, 84, 91 or 94 A polynucleotide having identity. A vector comprising the polynucleotide of claim 19. A host cell transformed with the vector of claim 21. The fusion protein of any one of claims 1 to 16; Or a fusion protein dimer of claim 17 or claim 18; cancer disease prevention or treatment pharmaceutical composition comprising as an active ingredient. According to claim 23, The pharmaceutical composition, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. According to claim 23, The cancer is colorectal cancer, melanoma, stomach cancer, liver cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, gallbladder cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma , Head and neck cancer, any one selected from the group consisting of salivary gland cancer and lymphoma, the pharmaceutical composition. Use of the fusion protein dimer of claim 17 or claim 18 for preparing a drug for preventing or treating cancer. Use of the fusion protein dimer of claim 17 or 18 for preventing or treating cancer disease. A method for preventing or treating cancer disease comprising administering the fusion protein dimer of claim 17 or claim 18 to a subject.

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