Interleukin 21 variants, fusion proteins comprising same and uses thereof

Abstract

The present invention relates to an interleukin-21 variant, a fusion protein comprising the interleukin-21 variant and an antibody heavy chain constant region, and uses thereof, and relates to: an interleukin-21 variant in which some amino acids in a wild-type interleukin-21 are substituted and / or deleted; a fusion protein in which an interleukin-21 variant is fused with an antibody (immunoglobulin) heavy chain constant region (Fc) pair comprising a first Fc region and a second Fc region; the use of the interleukin-21 variant or fusion protein for the prevention or treatment of cancer; and methods of culturing immune cells using the interleukin-21 variants or fusion proteins.

Description

Technical Field

[0001] The present invention relates to an interleukin 21 variant, a fusion protein comprising the interleukin 21 variant and an antibody heavy chain constant region, and uses thereof, and more specifically, to: an interleukin 21 variant in which one or more amino acids are replaced and / or deleted from a wild-type interleukin 21, a fusion protein in which an antibody (immunoglobulin) heavy chain constant region (Fc) pair including a first Fc region and a second Fc region is fused to an interleukin 21 variant, uses of the interleukin 21 variant or the fusion protein for preventing or treating cancer, and a method for culturing immune cells using the interleukin 21 variant or the fusion protein. Background Art

[0002] Cytokines are relatively small immune proteins contained in the blood. Cytokines secreted from cells affect other cells or cells that secrete cytokines, thus playing an important role in cell signaling.

[0003] Cytokines are proteins that are essential for biological regulation, inducing cell proliferation and differentiation, and promoting changes in function and activity, and are associated with immune system diseases, allergies, atopy, inflammation, and various types of tumors. In recent years, with the development of immunotherapy and immune cell therapy, the demand for research and medical cytokine proteins used in basic experiments for immune cell culture and cell therapy development has grown exponentially.

[0004] Interleukin-21 (IL21) is a cytokine. IL21 is a member of the IL2 cytokine family, which includes IL4, IL7, IL9, IL13, and IL15. Proteins in this family have been shown to have both anticancer and antiviral effects.

[0005] IL21 is activated by helper T (CD4 +Helper T cells, TH1) cells and natural killer T (NK-T) cells are produced, and are considered to be important regulators of immunity. In particular, IL21 activates the proliferation and cytotoxic effector functions of CD8+ cytotoxic T lymphocytes and natural killer (NK) cells, which can eliminate tumor-infected and virus-infected cells (Spolski et al., Nat Rev Drug Discov. 2014; 13 (5): 379-95). In addition, IL21 stimulates a selected class of B cells. Therefore, IL21 is essential for immunotherapy as a cytokine that induces the proliferation and activation of immune cells (especially NK cells). However, to date, IL-2 is the only cytokine approved for direct clinical administration to patients. IL2 increases the survival rate and activity of NK cells, and is relatively inexpensive, but its disadvantage is that it can inhibit anti-cancer immune function by activating regulatory T cells (which are immunosuppressive cells). Therefore, there is an urgent need to commercialize cytokines that activate NK cells without activating regulatory T cells.

[0006] In this regard, IL21 has recently attracted much attention in anticancer immunotherapy. IL21, like IL2, promotes the proliferation and differentiation of CD4-positive T cells and CD8-positive T cells. On the other hand, it is reported that IL21 does not affect the proliferation of regulatory T cells, so it is expected to be co-administered with anticancer immune cell therapy (Al-Chami et al., Cytokine, 2016; 82: 33-37). In addition, IL21 promotes the differentiation of CD8 T cells into memory cells (Li et al., Journal of Immunology, 2005), and in particular, recent studies have reported that IL21 induces genetic reprogramming of differentiated T cells and acquires stable memory-related transcriptional features (increased Lef1 and Tcf7) when administered with HDAC inhibitors, thereby enabling dedifferentiation into memory cells (Wang et al., Cancer Immunology Research, 2020). It is reported that IL21 has the following effects on NK cells: 1) When FMS-like tyrosine kinase-3 (FLT3-L) is present, IL21 together with IL15 promotes the proliferation of NK cells derived from human CD34-positive hematopoietic progenitor cells (Parrish-Novak et al. Nature, 2000; 408(6808):57-63), 2) When the membrane-bound form of IL21 is expressed in K562 cells used as a nutrient cell line, it promotes the in vitro expansion of NK cells and inhibits senescence (Lee DA et al. Plos One, 2012; 7(1):e30264), 3) When canine NK cells are cultured, proliferation by IL2 and IL15 is promoted (Shin et al., Vet Immunol Immunopathol. 2015; 165(1):1-13), 4) The expression of NKG2D receptor is promoted by activating STAT3 in human NK cells (Zhu et al. al., Blood, 2014; 124(3): 403-411) etc.

[0007] Recombinant human IL21 shows anti-tumor activity in preclinical and clinical trials. However, when systemically administered, IL21 has a short half-life and is consumed by other immune cells expressing IL21 receptors that are difficult to target tumor sites, and is inevitably administered at high concentrations. In addition, the Phase 1 clinical trial of IL21 failed due to the toxicity caused by the simultaneous action of multiple effects on various immune cells (Petrella et al., Journal of Clinical Oncology, 2013). Several attempts have been made to overcome these shortcomings. In order to increase the half-life, a single-IL21-Fc fusion protein is proposed, in which IL21 is combined with the heterodimeric heavy chain constant region (heterodimeric Fc) of the antibody in a monomeric form (Korean Patent No. 10-1928981; U.S. Patent No. US10800825B2). In addition, it is also attempted to develop immune cytokines (antibody-cytokine conjugates) fused with antibodies targeting tumors (Shen et al. Front. Immunol. 2020; 11: 832). However, the protein structure of IL21 has many positively charged amino acids exposed on the surface, and compared with IL2 and IL4, there is a problem of reduced stability. The problem is that when systemically administered, IL21 will be distributed to other cells and tissues in the body and easily decomposed, resulting in a short serum half-life, which ultimately leads to poor pharmacokinetics and is therefore not suitable for use as a therapeutic agent. In order to solve these problems, people have tried to introduce mutations that can reduce positive charges and confer other structural stability in recombinant IL21 (U.S. Patent No. US8,475,784B2; Bondensgaard et al. J. BioI. Chem. 2007; 282 (32): 23326-23336). Nevertheless, there is still a need to develop mutants that can improve the physical properties of IL21 and provide a long half-life.

[0008] Human antibodies (immunoglobulin G (IgG), IgM, IgD, IgE, IgA) existing in nature exist in the form of an assembly of two heavy chains having the same amino acid sequence and two light chains having the same sequence. In this case, the homodimerization between the two identical heavy chains is caused by the non-covalent interaction between the last few domains of the antibody constant region (the CH3 domain of IgG, IgD and IgA, the CH4 domain of IgM, and the CH2 and CH4 domains of IgE) and the disulfide bonds between the hinge regions.

[0009] The antibody-derived heterodimeric heavy chain constant region (heterodimeric Fc) is a technology that forms heterodimeric heavy chain constant regions through engineering, which imparts bonds that favor heterodimerization and are unfavorable or reject homodimerization by forming specific non-covalent bonds on the interaction surface between the last few domains of the constant region, which greatly contributes to the homodimerization of previously naturally occurring antibodies (IgG, IgM, IgA, IgD and IgE). More specifically, the heterodimeric Fc technology induces mutations in the CH3 domains of two different antibody heavy chains through genetic engineering to form a heterodimer with two heavy chains, similar to natural antibodies, with minimal sequence deviation. The EW-RVT heterodimeric Fc mutant used in this article, as a new type of strategic mutant, is a mutant that promotes the formation of heterodimers by: forming selective electrostatic bonds at the CH3 domain interaction surface (K360E CH3A-Q347R CH3B) through sequence and structural analysis of the interactions of conventional mutants, and forming complementary hydrophobic bonds instead of conventional electrostatic bonds (K409W CH3A-D399V CH3B / F405T CH3B) (U.S. Patent No. 9,951,145; Korean Patent No. 1,522,954; Choi HJ et al. Molecular Cancer Therapeutics, 2013; 12(12): 2748-2759). Conventional single-IL21-Fc fusion protein is based on the EW-RVT heterodimeric Fc (γ1 / 2 / 4, EW-RVT) with the sequences of human IgG1, IgG2 and IgG4 introduced into the hinge-CH2-CH3 region (Korean Patent No. 10-1928981; U.S. Patent No. US 10800825B2). The resulting single-IL21-Fc fusion protein can exhibit a more excellent NK cell proliferation promoting function compared to the double-IL21-Fc fused with IL21 and wild-type Fc (γ1 / 2 / 4), and can induce a more excellent anti-tumor effect compared to soluble IL21 or double-IL21-Fc by administering it to the body at a reasonable frequency (Korean Patent No. 10-1928981; U.S. Patent No. US10800825B2).

[0010] The molecular structure analysis of IL21 showed that it affects CD8 +Compared with cytokines (IL-2, IL-12, IL-15, IL-18, IFN) that regulate the proliferation and activation of cytotoxic T lymphocytes and natural killer (NK) cells, amino acids with positively charged side chains are relatively abundant, and finally IL21 has an isoelectric point (pI) of 9.42, so it is positively charged under in vivo pH conditions. In addition, it is predicted that the ionic patches formed by the positively charged amino acids on the surface of the tertiary structure may interact nonspecifically with various receptors and cells in the body, resulting in adverse effects when administered to the body.

[0011] Under these technical backgrounds and limitations, the inventors have developed an interleukin 21 variant, in which one or more amino acids are replaced and / or deleted in wild-type interleukin 21, and a fusion protein, which is a heterodimer containing an interleukin 21 variant, and an antibody (immunoglobulin) heavy chain constant region (Fc) pair including a first Fc region and a second Fc region, wherein the CH3 domain of the first Fc region or the second Fc region has a mutation. Based on this, the present invention is completed. Compared with conventional single-IL21-Fc fusion proteins, the mutants developed according to the present invention are found to have excellent biological activity and anti-tumor activity. Summary of the invention

[0012] Therefore, the present invention is made in view of the above problems, and one object of the present invention is to provide an interleukin-21 variant, wherein one or more amino acid sequences are replaced or deleted from the wild-type interleukin-21.

[0013] Another object of the present invention is to provide a fusion protein comprising the interleukin 21 variant and an antibody (immunoglobulin) heavy chain constant region (Fc) pair.

[0014] Another object of the present invention is to provide a nucleic acid encoding the interleukin-21 variant or fusion protein.

[0015] Another object of the present invention is to provide an expression vector containing the nucleic acid.

[0016] Another object of the present invention is to provide a transformed recombinant cell containing the expression vector.

[0017] Another object of the present invention is to provide a method for producing the interleukin 21 variant or fusion protein.

[0018] Another object of the present invention is to provide a composition for preventing or treating cancer containing the interleukin 21 variant or fusion protein as an active ingredient. Another object of the present invention is to provide a method for preventing or treating cancer containing the interleukin 21 variant or fusion protein as an active ingredient. Another object of the present invention is to provide the use of the interleukin 21 variant or fusion protein in the preparation of a composition for preventing or treating cancer.

[0019] Another object of the present invention is to provide a method for preventing or treating cancer, comprising administering the interleukin 21 variant or fusion protein to a patient.

[0020] Another object of the present invention is to provide a culture method, comprising treating immune cells with the interleukin 21 variant or fusion protein.

[0021] According to one aspect of the present invention, the above-mentioned and other objects can be achieved by providing an interleukin 21 (IL21) variant, wherein one or more of the amino acid sequences selected from the group consisting of positions 50, 54, 75 to 88 and 124 from the N-terminus in the interleukin 21 (IL21) including the amino acid sequence of SEQ ID NO.4 is substituted or deleted.

[0022] According to another aspect of the present invention, a fusion protein is provided, which is a heterodimer and contains the interleukin 21 variant and an antibody (immunoglobulin) heavy chain constant region (Fc) pair comprising a first Fc region and a second Fc region, wherein the Fc pair has a mutation in the CH3 domain of the first Fc region or the second Fc region.

[0023] According to another aspect of the present invention, a nucleic acid encoding the interleukin 21 variant or fusion protein is provided.

[0024] According to another aspect of the present invention, an expression vector containing the nucleic acid is provided.

[0025] According to another aspect of the present invention, a transformed recombinant cell containing the expression vector is provided.

[0026] According to another aspect of the present invention, there is provided a method for producing an interleukin-21 variant or fusion protein, comprising:

[0027] (a) culturing cells; and (b) harvesting the interleukin 21 variant or fusion protein from the cultured cells.

[0028] According to another aspect of the present invention, a composition for preventing or treating cancer containing the interleukin 21 variant or fusion protein as an active ingredient is provided. According to another aspect of the present invention, a method for preventing or treating cancer is provided, comprising administering the interleukin 21 variant or fusion protein to a patient. According to another aspect of the present invention, use of the interleukin 21 variant or fusion protein in the preparation of a medicament for preventing or treating cancer is provided.

[0029] According to another aspect of the present invention, a culture method is provided, comprising treating immune cells with the interleukin 21 variant or fusion protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Fig. 1A is a schematic diagram showing a single-IL21-Fc fusion protein (IL21 monomer-Fc fusion protein) constructed by introducing human IL21 cytokine into EW-RVT heterodimeric Fc in the form of a monovalent monomer. The EW-RVT heterodimeric Fc used herein is an Fc (γ1 / 4) having a hinge region sequence of human IgG1 with EW-RVT mutations introduced and a CH2-CH3 region of human IgG4. "Single" means monomer.

[0031] Fig. 1B is a schematic diagram showing a double-IL21-Fc fusion protein (IL21 dimer-Fc fusion protein) constructed by introducing human IL21 cytokine into wild-type Fc in the form of a divalent dimer, as a comparative example of Fig. 1A. The wild-type Fc used herein is an Fc (γ1 / 4) having a hinge region sequence of human IgG1 and a CH2-CH3 region of human IgG4. "Dual" means a divalent dimer.

[0032] Figure 2 Schematic diagram showing CH3A (EW) and CH3B (RVT) of the Fc (γ1 / 4) expression vector used for expressing and purifying the fusion protein of FIG. 1A in animal cells.

[0033] Figure 3 The results of transiently expressing and purifying the specified wild-type Fc (γ1 / 4, WT), heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), and dual-IL21-Fc and single-IL21-Fc (in the single-IL21-Fc fusion protein, the ratio of expression vector CH3A (EW) to CH3B (RVT) for animal cell transformation = 1: 1, 1.5: 1, 2: 1) fusion proteins in HEK293F cells by co-transfection, separating 5 μg of protein on 10% SDS-PAGE under non-reducing and reducing conditions, and analyzing its size and binding form by Coomassie Brilliant Blue staining are shown. "Non-reducing" means non-reducing conditions, "reducing" means reducing conditions, and "M" means protein marker.

[0034] Figure 4 The results of size exclusion chromatography (SEC) analysis of constructed single-IL21-Fc fusion protein (ratio of expression vector CH3A (EW) and CH3B (RVT) for animal cell transformation = 1:1, 1.5:1, 2:1) and wild-type Fc (γ1 / 4, WT) and heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT) are shown. In the SEC analysis, the running buffer was 1X PBS (pH 7.4, 12mM phosphate, 137mM NaCl, 2.7mM KCl), the flow rate was 0.75ml / min, and the protein injection amount was 30μg (30μl was injected at 1mg / ml).

[0035] Figure 5 The structure of human IL21 wild type (PDB ID: 3tgx) visualized using the Chimera X program is shown, in which each amino acid side chain is highlighted with a designated color according to its characteristics. Blue represents amino acids with positively charged side chains, red represents amino acids with negatively charged side chains, gray represents amino acids with hydrophobic side chains, and green represents other amino acids.

[0036] Figure 6A shows the results of size exclusion chromatography analysis of the constructed single-IL21-Fc fusion protein. In size exclusion chromatography, the running buffer was 1X PBS (pH 7.4, 137 mM NaCl), the flow rate was 0.75 ml / min, and the protein injection amount was 30 μg (30 μl injected at 1 mg / ml).

[0037] Fig. 6B shows the results of analyzing the constructed single-IL21-Fc fusion protein using size exclusion chromatography. In size exclusion chromatography, the running buffer was high salt PBS (pH 7.4, 12mM phosphate, 2.7mM KCl, 500mM NaCl), the flow rate was 0.75ml / min, and the protein injection amount was 30μg (injected 30μl at 1mg / ml).

[0038] Figure 7 The diagram shows the sequence changes of the amino acid substitution variants of IL21 constructed in the present invention compared with the human wild-type IL21, as well as the related nomenclature and basis.

[0039] Figure 8The results of transiently expressing and purifying single-IL21(NKQ)-Fc and single-IL21(DDD)-Fc as amino acid substitution variants of single-IL21-Fc fusion protein and single-IL21-Fc fusion protein as control by co-transfection in HEK293F cells, separating 5 μg of protein on 10% SDS-PAGE under non-reducing and reducing conditions, and analyzing its size and binding form by Coomassie Brilliant Blue staining are shown. "Non-reducing" means non-reducing conditions, "reducing" means reducing conditions, and "M" means protein marker.

[0040] Fig. 9 The results of size exclusion chromatography (SEC) analysis of single-IL21 (DDD) -Fc, single-IL21 (NKQ) -Fc and single-IL21 (NQNT) -Fc as amino acid substitution variants of single-IL21-Fc fusion protein and single-IL21-Fc fusion protein as control group are shown. In SEC analysis, the running buffer was 1X PBS (pH 7.4, 12mM phosphate, 137mM NaCl, 2.7mM KCl), the flow rate was 0.75ml / min, and the protein injection amount was 30μg (30μl was injected at 1mg / ml).

[0041] Fig.10 The diagram shows the sequence changes of the IL21 orthologous sequence grafted variant constructed in the present invention compared with the human wild-type IL21, and the image related to the nomenclature and basis thereof.

[0042] Fig.11 Shown is the transient expression and purification of single-IL21 (helix 1)-Fc and single-IL21 (helix 2)-Fc as single-IL21-Fc fusion protein orthologous sequence grafted variants and single-IL21-Fc fusion protein as a control in HEK293F cells by co-transfection, and then 5 μg of protein was separated on 10% SDS-PAGE under non-reducing and reducing conditions, and its size and structure of the bound form were analyzed by Coomassie Brilliant Blue staining. "Non-reducing" means non-reducing conditions, "reducing" means reducing conditions, and "M" means protein marker.

[0043] Fig.12The results of size exclusion chromatography analysis of single-IL21 (helix 1)-Fc and single-IL21 (helix 2)-Fc as orthologous sequence grafted variants of single-IL21-Fc fusion protein, and single-IL21-Fc fusion protein, single-IL21-Fc fusion protein, single-IL21 (DDD)-Fc, single-IL21 (NQNT)-Fc and single-IL21 (DDD NQNT)-Fc as controls are shown. In the SEC analysis, the running buffer was 1X PBS, the flow rate of the running buffer was 0.75 ml / min, and the protein injection amount was 30 μg (30 μl was injected at 1 mg / ml).

[0044] Fig.13 The binding affinity of IL21 to IL21 receptor on the cell surface of single-IL21-Fc fusion protein and single-IL21-Fc fusion protein variants analyzed by FACS is shown. Jurkat cell lines (human CD4+T-cell lines) expressing human IL21 receptors were treated with single-IL21-Fc fusion proteins, single-IL21(NKQ)-Fc, single-IL21(DDD)-Fc and wild-type Fc at 40nM and 200nM concentrations, and fluorescently labeled with anti-human IgG4 antibodies and antibodies fused to fluorescent material FITC, and then fluorescence intensity was measured using FACScalibur.

[0045] Figure 14 shows the results of the identification of the effects of single-IL21-Fc fusion proteins and single-IL21-Fc fusion protein variants on NK cell proliferation. In the presence of 100 gray gamma-irradiated Jurkat (KL-1) cells and EBV-transformed LCL cells, NK cells were cultured by adding IL-2 (30 ng / ml of monomeric IL-2Fc) to PBMCs isolated from normal blood.

[0046] FIG. 14A : Timeline of NK cell culture method and schedule.

[0047] FIG. 14B : Table showing the yield and percentage of NK cells obtained on days 0, 6 and 14.

[0048] FIG. 14C : Line graph showing the NK cell yields obtained on days 0, 6 and 14.

[0049] FIG. 14C : Bar graph showing NK cell yields obtained on days 0, 6 and 14.

[0050] Figure 15 shows the results of the identification of the effects of single-IL21-Fc fusion proteins and single-IL21-Fc fusion protein variants on NK cell proliferation. NK cells were cultured by adding IL-2 (30 ng / ml of monomeric IL-2Fc) to PBMCs from which CD4 / CD8 T cells were removed in the presence of 100 gray gamma-irradiated Jurkat (KL-1) cells and EBV-transformed LCL cells.

[0051] FIG. 15A : Timeline of NK cell culture method and schedule.

[0052] FIG. 15B : Table showing the yield and percentage of NK cells obtained on days 0, 6 and 14.

[0053] FIG. 15C : Line graph showing NK cell yields obtained on days 0, 6 and 14.

[0054] FIG. 15C : Bar graph showing NK cell yields obtained on days 0, 6 and 14. DETAILED DESCRIPTION

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which the invention belongs. Generally speaking, the nomenclature used herein is well known and commonly used in the art.

[0056] In one aspect, the present invention relates to an interleukin 21 (IL21) variant, wherein one or more selected from the group consisting of the amino acid sequences at positions 50, 54, 75 to 88 and 124 from the N-terminus in interleukin 21 (IL21) including the amino acid sequence of SEQ ID NO.4 is substituted or deleted.

[0057] Interleukin 21 (IL21) variants may include one or more amino acid substitutions or deletions in human IL21 of SEQ ID NO: 4, including amino acid residues 1 to 131 (denoted herein as "positions 1-131", excluding the signal peptide and propeptide) corresponding to the sequence of the mature form of human IL21 cytokine.

[0058] This is achieved by a method comprising: (1) selecting amino acid sequences and regions that may lead to deteriorated properties through IL21 structural analysis; (2) designing amino acids to be replaced to improve the properties in the selected amino acid positions and regions; and (3) fusing the designed IL21 variants with the heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), followed by expression / purification and then analyzing the biological properties.

[0059] The identification and improvement of the properties of therapeutic protein candidates are very important for the production of therapeutic proteins. The properties of therapeutic proteins may affect the therapeutic efficacy, pharmacodynamics and side effects of the protein. The properties of therapeutic proteins may be reduced due to the net charge, hydrophobicity and improper folding of the protein in the body. The properties may be degraded when the net charge in the body is strongly positive or negative or when it is highly hydrophobic in the body and cannot be dissolved in an aqueous environment, or when the protein cannot maintain a proper folding state in the body. This problem can be overcome by selecting and alternating amino acid mutations that cause the properties to deteriorate while maintaining the structure and function of the protein.

[0060] Human IL21 has a four-helix (helix A, B, C to D) structure, which is arranged in a typical way of class 1 cytokines, up-up-down-down, and a ring structure with a connecting helical structure. From the structural characteristics, in the mature form sequence of human IL21, amino acids 4 to 23 form helix A, amino acids 41 to 56 form helix B, amino acids 62 to 74 form helix C, and amino acids 102 to 120 form helix D. When binding to the IL21 receptor, 14 IL21 residues form van der Waals bonds with 16 IL21 receptor residues, among which arginine 3, arginine 7 and glutamine 10 in the IL21 helix A sequence, and arginine 74 and lysine 71 in the helix C sequence are the most important amino acids known. In the present invention, in order to improve the biological properties in vivo without affecting the structure and function of wild-type IL21, an amino acid replacement strategy and an orthologous sequence transplantation strategy are used.

[0061] Specifically, the interleukin-21 variant may include one or more amino acid substitutions or deletions selected from the group consisting of the following amino acid substitutions and deletions in the amino acid sequence of SEQ ID NO: 4:

[0062] (1) K50N,

[0063] (2) K54Q,

[0064] (3) K75L,

[0065] (4) P76D,

[0066] (5) P77R,

[0067] (6)S78N,

[0068] (7)T79L,

[0069] (8) R83N, R83D, R83L,

[0070] (9) R84K, R84D, R84W,

[0071] (10) Q85G, Q85S,

[0072] (11) K86N, K86L,

[0073] (12)H87A,

[0074] (13) R88T, R88G, R88N,

[0075] (14) R124Q, R124D,

[0076] (15) a deletion of amino acids at positions 79 to 82, and

[0077] (16) The amino acids at positions 81 to 84 are missing.

[0078] Specifically, the interleukin 21 variant may include one or more amino acid substitutions and / or deletions selected from the group consisting of the following amino acid substitutions and deletions in the amino acid sequence of SEQ ID NO: 4:

[0079] (1) R83N, R84K, R124Q;

[0080] (2) R83D, R84D, R124D;

[0081] (3) K50N, K54Q, K86N, R88T; and

[0082] (4)K50N, K54Q, R83D, R84D, K86N, R88T, R124D.

[0083] According to the present invention, a single-IL21-Fc fusion protein orthologous sequence graft variant is constructed by replacing the K75 to R88 sequence including the atypical region of IL21 with a complementary human IL4 sequence and an orthologous IL4 sequence. Based on this, the interleukin 21 variant may include one or more amino acid substitutions and / or deletions selected from the group consisting of the following amino acid substitutions and / or deletions in the amino acid sequence of SEQ ID NO: 4:

[0084] (1) K75L, P76D, P77R, S78N and deletions of amino acids at positions 79 to 82, R83L, R84W, Q85G, K86L, H87A, R88G; and

[0085] (2) K75L, P76D, P77R, S78N, T79L, deletion of amino acids at positions 81 to 84, and Q85S, K86L, H87A, R88N.

[0086] In a specific embodiment, the interleukin 21 variant may include a sequence selected from the group consisting of SEQ ID NOs: 5 to 10.

[0087] As used herein, the term "mutation (or variation)" refers to a modification of a protein that includes replacing an amino acid with an amino acid having similar biochemical properties, but does not result in loss of structure and biological or biochemical function of the protein. The term "amino acid substitution" refers to a substitution of an amino acid with an amino acid having a side chain that structurally retains the amino acid but has different chemical properties. Naturally occurring amino acids can be divided into amino acids with side chains that exhibit similar properties, which is well known in the art. These categories include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). The present invention provides IL21 variants constructed by amino acid substitutions of human IL21, and shows that the IL21 variants retain activity.

[0088] The term "orthologous sequence transplantation" used herein means replacing amino acids at the same position of IL21 with reference to a specific sequence region of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15) belonging to the same family as IL21. The results of the structural analysis of IL21 show that the CD loop structure from helix C to helix D and the C-terminal region are atypical regions with uncertain structures. Although there are many reasons for the formation of atypical regions, the biggest reason is the formation of an unstable structure. Therefore, it is speculated that the sequence of the CD loop (with reference to the cytokine sequence belonging to the same family as IL21) expected to form the most stable structure is transplanted into this region and the constructed IL21 mutant will have improved properties over the wild type.

[0089] The human IL21 variant of the present invention can include not only the sequence of the IL21 variant disclosed herein, but also its biological equivalent, as long as it can maintain the expected function. For example, the amino acid sequence of the antibody can be additionally varied to further improve the physical properties or other biological properties of IL21. This variation includes, for example, the deletion, insertion and / or replacement of the amino acid sequence residues of the antibody. Such amino acid mutations are based on the relative similarity of the amino acid sequence residue substitution bases, such as their hydrophobicity, hydrophilicity, charge and size. By analyzing the size, shape and type of the substitution base, it can be seen that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are considered to be biological functional equivalents.

[0090] In another aspect, the present invention relates to a fusion protein which is a heterodimer comprising an interleukin 21 variant and an antibody (immunoglobulin) heavy chain constant region (Fc) pair comprising a first Fc region and a second Fc region, wherein the Fc pair has a mutation in the CH3 domain of the first Fc region or the second Fc region.

[0091] The present invention relates to a fusion protein in which the interleukin-21 (IL21) cytokine is fused in monomeric form to the heterodimeric heavy chain constant region Fc of an antibody (hereinafter referred to as "IL21 monomer-Fc fusion protein", "heterodimeric Fc fused IL21 monomer protein" or "single-IL21-Fc fusion protein"), or a fusion protein in which an interleukin-21 variant is fused in monomeric form to the heterodimeric heavy chain constant region Fc of an antibody (hereinafter referred to as "IL21 monomer-Fc fusion protein variant", "heterodimeric Fc fused IL21 variant monomer protein" or "single-IL21-Fc fusion protein variant").

[0092] The present invention also relates to a fusion protein in which the interleukin-21 (IL21) cytokine is fused in a dimer form to the heterodimeric heavy chain constant region Fc (heterodimeric Fc) of an antibody (referred to as "IL21 dimer-Fc fusion protein variant", "heterodimeric Fc fused IL21 variant dimer protein" or "dimer-IL21-Fc fusion protein variant").

[0093] As an interleukin-21 variant, the mutant is designed by performing amino acid mutations on wild-type IL21 to reduce the non-specific reactivity of IL21 and increase structural stability, so that when the mutant is expressed in the form of a single-IL21-Fc fusion protein variant, the expression level of the fusion protein is increased, the formation of undesirable oligomers is reduced under physiological conditions, and the biological properties are improved so that it can exist in a monomeric form.

[0094] Specifically, the present invention provides a technology for developing IL21 variants with improved biological properties compared to a single-IL21-Fc fusion protein, which is a fusion protein of wild-type IL21 but has improved properties to retain the ability to bind to an IL21 receptor expressed on the cell surface and the ability to proliferate immune cells.

[0095] The present invention provides a technology for developing a single-IL21-Fc fusion protein variant, which includes an IL21 variant, which can: 1) reduce side effects that may occur in vivo, 2) improve pharmacokinetics, 3) replace frequent biological administration required due to the short half-life of the recombinant protein IL21 cytokine, and 4) solve the disadvantage of high cost by reducing the non-specific interaction of the single-IL21-Fc fusion protein under physiological conditions, which single-IL21-Fc fusion protein is a fusion protein containing wild-type IL21.

[0096] First, a single-IL21-Fc fusion protein monomer fusion protein was constructed, in which wild-type IL21 was fused with the heterodimeric heavy chain constant region EW-RVT heterodimeric Fc (γ1 / 4, EW-RVT) based on the hinge region sequence of human IgG1 and the CH2-CH3 region of human IgG4, which introduced the EW-RVT mutation. Then, the structure of wild-type IL21 was analyzed, sequences that may affect the physical properties and specificity of the protein were selected, and mutants were designed in which the amino acid units were replaced or inserted with sequences of the same series of cytokines. The mutant was expressed in the form of a single-IL21-Fc fusion protein to develop a single-IL21-Fc fusion protein variant that can increase the expression amount, reduce the formation of undesirable oligomers under physiological conditions, and exist in a monomeric form.

[0097] This article uses EW-RVT heterodimeric Fc (γ1 / 4, EW-RVT), which introduces EW-RVT mutations based on Fc (γ1 / 4) with an IgG1 hinge region sequence and a CH2-CH3 region of human IgG4. In addition, a wild-type Fc (γ1 / 4, WT) with a hinge region sequence of human IgG1 and a CH2-CH3 region of human IgG4 was used to construct a dual-IL21-Fc fusion protein (IL21 dimer-Fc fusion protein) as a comparative example and compared.

[0098] Based on this, the present invention provides a single-IL21-Fc fusion protein variant, wherein the wild-type IL21 amino acid sequence of the single-IL21-Fc fusion protein is replaced or transplanted with a cytokine sequence of a direct homolog. More specifically, the mechanism of constructing IL21 to develop a single-IL21-Fc fusion protein variant is: 1) by reducing the surface positive charge of the IL21 protein, a fusion protein with a longer half-life in vivo is produced, which improves the stability of the substance, reduces nonspecific interactions in vivo, and has a longer half-life in vivo; 2) by transplanting sequences that may cause IL21 structural instability into the sequences of cytokines IL-2 and IL-4 belonging to the same family as IL21, and comparing the sequences of IL-2 and IL-4, it is confirmed that the fusion protein simulates the better stability of IL-2 and IL-4 in vivo; and 3) it is confirmed that the receptor binding ability is maintained in a cell line expressing the human IL21 receptor when compared with the single-IL21-Fc fusion protein. Based on this mechanism, the present invention is completed.

[0099] The heterodimeric Fc fusion protein variant containing an interleukin 21 variant according to the present invention provides a protein capable of maintaining the function and activity of a conventional single-IL21-Fc fusion protein by maintaining its structure.

[0100] As used herein, "Fc region" refers to a region including a CH2 domain, a CH3 domain, and a hinge domain derived from an antibody. As used herein, "the first Fc region and the second Fc region mutate to promote the formation of a heterodimeric heavy chain constant region (heterodimeric Fc)" means that antibodies present in the natural system have a homodimeric heavy chain constant region (homodimeric Fc) form, wherein the two Fc regions have the same sequence, and by inducing a mutation in a portion of the sequence of the Fc region, the formation of a heterodimeric heavy chain constant region (heterodimeric Fc) is promoted by a specific non-covalent bond between the first Fc region and the second Fc region, and homodimers are reduced or preferably almost not formed.

[0101] Preferably, the mutation that promotes the formation of the heterodimeric heavy chain constant region (heterodimeric Fc) of the first Fc region and the second Fc region according to the present invention may include a mutation that promotes the formation of the heterodimeric heavy chain constant region (heterodimeric Fc) derived from each of the CH3 domains included in the first Fc region and the second Fc region of the antibody.

[0102] In the present invention, the heterodimeric heavy chain constant region includes a first Fc region and a second Fc region, and the first Fc region and the second Fc region are characterized in that the CH3 domain is mutated to promote the formation of the heterodimeric heavy chain constant region.

[0103] The CH3 domain mutation of the first Fc region or the second Fc region includes at least one mutation selected from the following mutations, and the mutation position is determined according to the EU index:

[0104] (1) the substitution at position K370 in the CH3 domain of the first Fc region is K370E, K370R, K370M, K370D or K370H;

[0105] (2) position K409 in the CH3 domain of the first Fc region is replaced by K409W;

[0106] (3) position E357 in the CH3 domain of the second Fc region is substituted with E357N, E357D, E357A, E357I, E357G or E357M, and position S364 is substituted with S364T or S364W; and

[0107] (4) In the CH3 domain of the second Fc region, position F405 is substituted with F405T, and position D399 is substituted with D399V.

[0108] The CH3 domain of the first Fc region or the second Fc region may further include the following substituted amino acids, and the mutation positions are determined according to the EU index:

[0109] (i) a cysteine ​​(C) substituted at position Y349 in the CH3 domain of the first Fc region; or

[0110] (ii) Cysteine ​​substituted at position S354 in the CH3 domain of the second Fc region (C).

[0111] Specifically, the mutation of the CH3 domain of the first Fc region or the second Fc region may include one or more mutations selected from the following mutations (provided that the mutation position is determined according to the EU index):

[0112] (1) replacing the amino acid residue at position K360 in the CH3 domain of the first Fc region with K360E;

[0113] (2) replacing the amino acid residue at position E347 in the CH3 domain of the second Fc region with E347R;

[0114] (3) replacing the amino acid residue at position K409 in the CH3 domain of the first Fc region with K409W; and

[0115] (4) The amino acid residue at position F405 in the CH3 domain of the second Fc region is substituted with F405T, and the amino acid residue at position D399 is substituted with D399V.

[0116] The CH3 domain of the first Fc region or the second Fc region may further include the following substituted amino acids, and the mutation positions may be determined according to the EU index:

[0117] (i) a cysteine ​​(C) substituted at position Y349 in the CH3 domain of the first Fc region; or

[0118] (ii) Cysteine ​​substituted at position S354 in the CH3 domain of the second Fc region (C).

[0119] In a specific embodiment according to the present invention, the CH3 domain mutations of the first Fc region and the second Fc region include: (1) position K360 in the CH3 domain of the first Fc region is replaced by K360E, and position K409 in the CH3 domain of the first Fc region is replaced by K409W; and (2) position E347 in the CH3 domain of the second Fc region is replaced by E347R, position F405 is replaced by F405T, and position D399 is replaced by D399V, and the mutation positions are determined according to the EU index (hereinafter referred to as "EW-RVT").

[0120] The term "EW-RVT heterodimeric Fc" used herein means EW-RVT heterodimeric Fc (γ1 / 4, EW-RVT) into which EW-RVT mutations are introduced based on Fc (γ1 / 4) having the hinge region sequence of human IgG1 and the CH2-CH3 region of human IgG4.

[0121] Preferably, the CH3 domain included in the antibody-derived first Fc region and the second Fc region according to the present invention may have a sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.

[0122] The first Fc region and the second Fc region may be derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE, preferably, the first Fc region and the second Fc region may be derived from IgG1, IgG2, IgG3 or IgG4, and the first Fc region and the second Fc region may be derived from isotype antibodies.

[0123] In particular, the antibody-derived first Fc region and second Fc region according to the present invention preferably have the sequence of the IgG4-derived CH3 domain described in Table 1.

[0124] The term “γ1 / 4” as used herein means an antibody heavy chain constant region domain in which the hinge region uses the IgG1 isotype which is widely adopted in commercial therapeutic antibodies due to its high stability, the CH2 and CH3 regions use the IgG4 isotype which prevents the occurrence of antibody-specific functions such as unwanted ADCC (antibody-dependent cellular cytotoxicity) / CDC (complement-dependent cytotoxicity), and the Fc fused thereto is referred to as “Fc (γ1 / 4)”.

[0125] [Table 1]

[0126]

[0127] Specifically, the fusion protein according to the present invention may include an interleukin 21 variant, which includes a sequence selected from the group consisting of sequences of SEQ ID NOS: 5 to 10, and an antibody (immunoglobulin) heavy chain constant region (Fc) pair comprising a first Fc region and a second Fc region, the first Fc region including the sequence of SEQ ID NO: 1, and the second Fc region including the sequence of SEQ ID NO: 2.

[0128] In another aspect, the present invention relates to nucleic acids encoding interleukin 21 variants or fusion proteins. Nucleic acids encoding interleukin 21 variants or fusion proteins of the present invention can be isolated to recombinantly produce interleukin 21 variants or fusion proteins.

[0129] The term "nucleic acid" as used herein is intended to encompass both DNA (gDNA and cDNA) and RNA molecules, and nucleotides as basic building blocks of nucleic acids include nucleotides of natural origin and analogs with modified sugar or base moieties. The sequence of the nucleic acid encoding the IL21 variant according to the present invention may vary. Such variations include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0130] DNA encoding interleukin 21 variants or fusion proteins can be easily isolated or synthesized using conventional molecular biological techniques (e.g., by using oligonucleotide probes that can specifically bind to CH3A (EW) and CH3B (RVT) DNA), and the nucleic acid is isolated, inserted into a replicable vector, further cloned (amplified with DNA) or further expressed. Based on this, in another aspect, the present invention relates to a recombinant expression vector containing the nucleic acid.

[0131] The term "expression vector" as used herein refers to a tool for expressing a target gene in a host cell, and includes plasmid vectors, cosmid vectors, and viral vectors such as phage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. Vector components generally include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. The interleukin 21 variant or nucleic acid encoding the antibody is operably linked to a promoter, a transcription termination sequence, and the like.

[0132] The term "operably linked" means a functional connection between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of binding sites for a transcriptional regulator) and another nucleic acid sequence, and enables the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0133] When prokaryotic cells are used as hosts, it usually includes an effective promoter capable of transcription (such as tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, it includes a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter, a β-actin promoter, a human hemoglobin promoter, or a human muscle creatine promoter), or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter), and generally has a polyadenylation sequence as a transcription termination sequence.

[0134] In another aspect, the present invention relates to a host cell transformed with a recombinant expression vector. The host cell used to produce the antibody of the present invention can be a prokaryote, yeast or a higher eukaryotic cell, but is not limited thereto.

[0135] Prokaryotic host cells such as Escherichia coli, strains of Bacillus (such as Bacillus subtilis and Bacillus thuringiensis), Streptomyces spp., Pseudomonas spp. (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus spp. (e.g., Staphylococcus carnosus) can be used.

[0136] Of greatest interest are animal cells, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, and HT1080.

[0137] The term "fusion" as used herein means the integration of two molecules with different or identical functions or structures, which can be performed by any physical, chemical or biological method, which can combine an antibody (immunoglobulin) heavy chain constant region (Fc) pair including a first Fc region and a second Fc region with an interleukin 21 variant. The fusion is preferably performed through a linker peptide, and the linker peptide can mediate the fusion of the biologically active molecule at each position of the interleukin 21 variant of the present invention with an antibody (immunoglobulin) heavy chain constant region (Fc) pair including a first Fc region and a second Fc region.

[0138] The interleukin-21 variant can bind to the N-terminus of the first Fc region or the second Fc region. The interleukin-21 variant can be fused to the N-terminus of the first Fc region or the second Fc region in a monomeric form. By performing amino acid mutations on wild-type IL21, a mutant that reduces the nonspecific reactivity of IL21 and increases structural stability is designed, and when the mutant is expressed in the form of a monomeric IL21-Fc fusion protein variant, the expression amount of the fusion protein is increased, the formation of undesirable oligomers under physiological conditions is reduced, and the biological properties are improved, so that it can exist in a monomeric form.

[0139] IL-21 or its variant can be connected to the first Fc region or the second Fc region through a linker. The linker can be a peptide linker and can have a length of about 10-25aa. For example, the linker can include hydrophilic amino acids, such as glycine and / or serine, but is not limited thereto.

[0140] Specifically, the linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are 1 to 10, respectively), but the linker may be, for example, (GnS)m (wherein n and m are 1 to 10, respectively).

[0141] In another aspect, the present invention relates to a composition for preventing or treating cancer, which contains an interleukin 21 variant or fusion protein as an active ingredient.

[0142] In another aspect, the present invention relates to a method for preventing or treating cancer, comprising administering an interleukin 21 variant or fusion protein to a patient.

[0143] In particular, the single-IL21-Fc fusion protein variant according to the present invention activates the proliferation of T cells and / or NK cells and increases toxicity, thereby exhibiting anti-tumor or anti-cancer effects. The single-IL21-Fc fusion protein variant according to the present invention is used to treat cancer-related diseases.

[0144] For example, the cancer can be selected from the group consisting of colon cancer, melanoma, breast cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma, and blood cancer.

[0145] In another aspect, the present invention relates to a composition for culturing immune cells, which contains the interleukin 21 variant or fusion protein according to the present invention. In another aspect, the present invention relates to a culturing method, comprising treating immune cells with the interleukin 21 variant or fusion protein according to the present invention.

[0146] In some cases, the composition may further contain one selected from the group consisting of a culture medium, serum, a supplement, and a combination thereof. The culture medium may include one selected from the group consisting of amino acids, sugars, inorganic salts, and vitamins. Preferably, the cell culture medium may include all of amino acids, sugars, inorganic salts, and vitamins.

[0147] The composition contains a culture medium for culturing cells, and specifically includes a culture medium for culturing NK cells, more specifically CD3-CD56+ cells. The composition contains components required for cell growth and survival in vitro, or components that promote cell growth and survival. Specifically, these components may include vitamins, essential or non-essential amino acids and trace elements.

[0148] The culture medium contains amino acid components, vitamin components, inorganic salt components, other components and purified water.

[0149] a) the amino acid component comprises at least one amino acid or a combination thereof selected from the group consisting of glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-threonine, L-serine, L-cysteine, L-methionine, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, L-lysine, L-arginine, L-histidine, L-phenylalanine, L-tyrosine, L-tryptophan, L-proline, β-alanine, γ-aminobutyric acid, ornithine, citrulline, homoserine, triiodotyrosine, thyroxine and deoxyphenylalanine, preferably comprises at least one amino acid or a combination thereof selected from the group consisting of glycine, L-alanine, L-arginine, L-cysteine, L-glutamine, L-histidine, L-lysine, L-methionine, L-proline, L-serine, L-threonine and L-valine, and

[0150] b) the vitamin component may include at least one vitamin or a combination thereof selected from the group consisting of biotin, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, choline chloride, i-inositol and ascorbic acid, and preferably includes at least one vitamin or a combination thereof selected from the group consisting of i-inositol, thiamine hydrochloride, niacinamide and pyridoxine hydrochloride,

[0151] c) the inorganic salt component includes at least one inorganic salt or a combination thereof selected from the group consisting of calcium chloride (CaCl2) (anhydrous), copper sulfate pentahydrate (CuSO4-5H2O), iron sulfate heptahydrate (FeSO4·7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4) (anhydrous), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O), zinc sulfate heptahydrate (ZnSO4·7H2O), iron nitrate nonahydrate (Fe(NO3)3·9H2O) and sodium bicarbonate (NaHCO3), preferably includes at least one inorganic salt or a combination thereof selected from the group consisting of sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl2) (anhydrous) and sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O), and

[0152] d) Other ingredients include at least one selected from the group consisting of D-glucose (dextrose), sodium pyruvate, hypoxanthine Na, thymidine, linoleic acid, lipoic acid, adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine HCl and 2'-deoxyguanosine, preferably sodium pyruvate.

[0153] e) Purified water is used to dissolve ingredients such as amino acids, vitamins, inorganic salts, etc. It can be obtained by one or more distillations, or purified by filters.

[0154] In addition, the composition may further contain growth factors or cytokines. The growth factor may be IGF, bFGF, TGF, HGF, EGF, VEGF or PDGF alone or in combination, but is not particularly limited thereto. The cytokine may be IL-1, IL-4, IL-6, IFN-γ, IL-10, IL-15, IL-17 or IL-21 alone or in combination, but is not particularly limited thereto.

[0155] In addition, the composition may further contain an antibody that activates natural killer cells. The antibody that activates natural killer cells may be an anti-CD3 antibody, an anti-CD2 antibody, an anti-CD335 antibody, etc., alone or in combination, but is not particularly limited thereto. In addition, the composition may contain beads to which the antibody that activates natural killer cells is bound.

[0156] In particular, the medium composition for culturing NK may further contain one selected from the group consisting of IL-15, IL-21, and a combination thereof.

[0157] IL-15 and IL-21 are types of interleukins (IL), which refer to proteinaceous bioactive substances produced by immune cells such as lymphocytes, monocytes and macrophages. IL-15 and IL-21 promote the proliferation of natural killer cells and can be used when using monocytes as raw material cells to culture natural killer cells. However, when IL-15 and IL-21 are used alone or in combination, there is a problem of low proliferation rate and purity (Biossel L.et al.,Biology of Blood and Marrow Transplantation, 14, 1031-1038, 2008).

[0158] Specifically, the culture medium can be an animal cell culture medium such as DMEM (Dulbecco's modified Eagle's medium), EDM (endothelial differentiation medium), MEM (bare essential medium), BME (basal medium of Eagle), RPMI 1640, F-10, F-12, a-MEM (a-basic essential medium), G-MEM (Glasgow's minimal essential medium), Iscove's modified Dulbecco's medium, AIM-V medium, X-vivo TM 15 medium or NK MACS medium. In one embodiment of the present invention, the medium can be AIM-V medium, X-vivo medium, TM 15 medium or NK MACS medium.

[0159] The term "serum" as used herein refers to the transparent supernatant separated from the blood after the blood has completely coagulated. In addition, serum should be added to the synthetic culture medium used to culture animal cells, usually serum from cattle, horses or humans. Bovine serum can be selected from fetal bovine serum (FBS), newborn calf serum, calf serum or cow serum depending on the time of blood collection. Human serum can be human serum from a donor with AB type blood, and can be human AB serum that can minimize the immune response caused by the lack of antibodies to type A blood and type B blood antigens. In addition, CTS immune cell SR and the like can be used as a substitute for "serum". In one embodiment of the present invention, human AB serum or CTS immune cell SR is used.

[0160] Supplements can be GlutaMAX (Gibco TM ), an L-glutamine substitute, is used to improve stability and cell activity during cell culture. In addition, the supplement can be NK MACS supplement (Miltenyi Biotec, 130-113-102).

[0161] The immune cells may include at least one selected from the group consisting of T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor infiltrating lymphocytes (TIL), and dendritic cells.

[0162] Immune cells, particularly NK cells, produced by the above method are provided. Pharmaceutical compositions containing NK cells for preventing or treating cancer are provided. The dosage of the pharmaceutical composition can be adjusted according to a variety of factors, including: the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients included in the composition, the type of preparation, and the patient's age, weight, general health, sex and diet, administration time, administration route and secretion rate of the composition, treatment cycle and concurrently used drugs.

[0163] Based on the active ingredient NK cells, the dosage of the pharmaceutical composition can be 1x10 2 cells / kg to 1.0x10 13 cells / kg, or 1x10 7 cells / kg to 1.5x10 11 In this case, the drug can be administered once a day or divided into several doses.

[0164] Hereinafter, the present invention will be described in more detail with reference to Examples. However, it is obvious to those skilled in the art that these Examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0165] Example 1. Construction of single-IL21-Fc fusion protein

[0166] The Fc variant used in this article for constructing fusion proteins is based on IgG4, in which the EW-RVT mutation is introduced to form a heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT) of a heterodimer. According to previous reports, in the construction of immunocytokines as a fusion form of antibodies and cytokines, the antibody-specific functions of IgG1, such as ADCC / CDC, promote in vivo clearance instead. Compared with IgG1, IgG4 isomers with almost no ADCC / CDC function are used to construct all fusion proteins specified in this article (Gillies SD et al., 1999). In particular, human IL21 uses a hinge region to connect to the heavy chain constant region without an additional peptide linker to promote interaction with the IL21 receptor. The hinge region used here is a widely used IgG1-derived hinge, and the cysteine ​​residues in the upper hinge region (except for the cysteine ​​residues used to form a duplex in the core hinge region) are replaced by serine residues to prevent the formation of unwanted disulfide bonds during protein fusion (mutated hinge, Figure 2 ).

[0167] In an isotype-specific variant that is expected to maintain a similar heterodimer-forming ability as the previously reported IgG1-based EW-RVT variant, a human IL21 (interleukin 21, Table 2, SEQ ID NO: 5) fusion protein was constructed using an IgG4-based variant (IgG4-EW-RVT, Choi et al, 2013; Korean Patent No. 10-1522954). IL21 exists in nature and is a cytokine that functions as a monomer, and is active only when one IL21 binds to one IL21 receptor (IL21R) and one γc chain. Therefore, a heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT) was used to connect only one IL21 to one of the different Fc variants (CH3A or CH3B) to maintain the IL21 monomer form that exists in nature.

[0168] Fig. 1A is a schematic diagram showing a single-IL21-Fc fusion protein having a CH3 mutation pair introduced. The DNA frame of the coding IL21-Fc (γ1 / 4, EW) having a CH3 mutation pair introduced was cloned in-frame into the animal cell expression vector pcDNA3.1 (+) (Invitrogen, the U.S.) with a CMV promoter to form a signal sequence-IL21 mature form-mutation hinge-CH2-CH3A. The IL21 used here is human IL21 (Uniprot entry name, Q9HBE4; sequence number 4), and only the DNA sequence encoding the mature form (excluding the signal sequence) is used.

[0169] As a comparative example of a single-IL21-Fc fusion protein, a double-IL21-Fc fused to a wild-type Fc was constructed. In a similar manner as described above, the DNA sequence encoding the mature form of human IL21 was cloned into the animal cell expression vector pcDNA3.1 (+) (Invitrogen, the United States) containing wild-type IgG4 CH3 using NotI / HindIII restriction enzymes to form a signal sequence-IL21 mature form-mutant hinge-CH2-CH3 structure. Table 2 shows the amino acid sequence of human IL21 used to construct a single-IL21-Fc fusion protein and a double-IL21-Fc fusion protein. The amino acid sequence was constructed as a mature form of IL21 of 131 amino acid peptides (amino acid number 32 to amino acid number 162 of human IL21) (Parrish-Novak et al. Nature, 2000; 408 (6808): 57-63), which is numbered the same as the previously studied single-IL21-Fc (Korean Patent No. 10-1928981; U.S. Patent Registration No. US10800825B2). That is, residues 32 to 1 of the mature form of IL21 are referred to herein as residues 1-131 (see Tables 2 and 4).

[0170] [Table 2]

[0171]

[0172] Example 2. Construction and expression / purification of Fc and mono-IL21-Fc fusion proteins

[0173] The expression of wild-type Fc (γ1 / 4, WT) and heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), dual-IL21-Fc fusion protein and single-IL21-Fc fusion protein (the ratio of CH3A (EW) and CH3B (RVT) expression vectors fused with human IL21 of single-IL21-Fc fusion protein was 1:1, 1.5:1, 2:1) was transiently transfected into HEK293-F (Invitrogen) cells with a mixture of plasmid encoding each protein and polyethyleneimine (PEI) (Polyscinece) and cultured in serum-free FreeStyle medium. TM The incubation was performed in a shake flask with 293 expression medium (Invitrogen). The details of the method are as follows:

[0174] 100mL For transient transfection, HEK293-F cells were plated at 1.0x10 6 The cells were seeded at a density of 10 cells / ml in 90 ml of medium in a shake flask (Corning) and incubated at 135 rpm under 8% CO2. After 24 hours, the corresponding CH3A and CH3B plasmids were added to 4 ml of medium to produce fusion proteins including the respective heterodimeric heavy chain constant regions. 293 expression medium (Invitrogen), a total of 125 μg (1.25 μg / ml), more specifically, 125 μg for Fc (γ1 / 4, WT), 62.5 μg CH3A and 62.5 μg CH3B for the heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), 125 μg for bi-IL21-Fc, 62.5 μg CH3A and 62.5 μg CH3B for the single-IL21-Fc fusion protein CH3A (EW): CH3B (RVT) = 1:1, 75 μg CH3A and 50 μg CH3B for CH3A (EW): CH3B (RVT) = 1.5:1, and 83.3 μg CH3A and 41.7 μg CH3B for CH3A (EW): CH3B (RVT) = 2:1. CH3B, after filtration, is mixed with 6ml culture medium (3.75μg / ml) of PEI diluted with 375μg, and reacts at room temperature for 10 minutes.Then the mixed culture medium of reaction is added to the cells in the 90ml previously inoculated, and the shortest culture is 5 days and the longest culture is 7 days.As a result, the protein produced by the cell is secreted outside the cell and accumulated in the culture medium.Therefore, protein A sepharose column (GE healthcare) is used to purify protein from the cell culture supernatant collected by centrifugation at 3800rpm for 25 minutes after cell culture.At this time, according to the standard scheme provided by protein A column company, purification is carried out, and after buffer is replaced with 1X PBS, Vivaspin 30,000MWCO (Satorius) centrifugal concentrator is used to concentrate the eluted protein.The solution in BCA protein assay kit (Thermo) is used to measure the absorbance of purified protein at 562nm wavelength, and is measured according to the standard curve drawn. Table 3 shows the purified yield of the expressed wild-type Fc (γ1 / 4, WT), heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), single-IL21-Fc fusion protein (ratio of CH3A (EW): CH3B (RVT) expression vector used during transfection = 1: 1, 1.5: 1, 2: 1) and double-IL21-Fc. The experiment was performed three times independently, and the results are expressed as the standard error of the mean (mean ± SD). The yield verification results show that the single-IL21-Fc fusion protein has a significantly higher yield than the double-IL21-Fc fusion protein.

[0175] [Table 3]

[0176]

[0177] Purified wild-type Fc (γ1 / 4, WT) and heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), single-IL21-Fc fusion protein (the ratio of expression vectors of CH3A (EW) and CH3B (RVT) during transfection = 1:1, 1.5:1, 2:1) and dual-IL21-Fc fusion protein (5 μg) were analyzed by SDS-PAGE under 10% non-reducing and reducing conditions ( Figure 3 ). Under non-reducing conditions, single-IL21-Fc fusion protein was observed at 71kDa, and double-IL21-Fc fusion protein was observed at 86kDa. Under reducing conditions, double-IL21-Fc has a band at 43kDa, including the single-IL21-Fc fusion protein of two plasmids including the heterodimeric heavy chain constant region CH3A (EW) and heterodimeric heavy chain constant region CH3B (RVT) of IL21 fusions, and has two bands at 43 and 28kDa. Heterodimeric heavy chain constant region CH3A and CH3B each predict protein size to be about 25kDa, and human IL21 predicts protein size to be 15kDa, but due to the N-glycosylation of human IL21 and Fc, bands greater than expected protein size are observed under reducing and non-reducing conditions. The SDS-PAGE results of single-IL21-Fc fusion protein under non-reducing conditions vary according to the plasmid ratio. At EW:RVT = 1:1 and 1.5:1, a single-IL21-Fc fusion protein in which the heterodimeric heavy chain constant regions CH3A (EW) and CH3B (RVT) were mixed at a ratio of 1:1 was not formed, and a band that appeared to be a dimer formed by two monomers of the heterodimeric heavy chain constant region CH3B (RVT) was observed, whereas such a band was not observed at EW:RVT = 2:1. Therefore, the single-IL21-Fc fusion protein and the single-IL21-Fc fusion protein variants described below were all constructed with a plasmid ratio of EW:RVT = 2:1.

[0178] Example 3. Evaluation of properties of single-IL21-Fc fusion protein

[0179] Figure 4 It is a chromatogram of the change of mAU at 280nm over time (minutes) after SEC (size exclusion chromatography) to analyze the physical properties of the expressed wild-type Fc (γ1 / 4, WT), heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT) and single-IL21-Fc fusion proteins (expression vector ratio of heterodimeric heavy chain constant region CH3A (EW): CH3B (RVT) after conversion = 1:1, 1.5:1, 2:1). According to the following specific protocol, the measurement was performed using an HPLC (Agilent Technologies, USA) instrument and a Superdex200 10 / 300GL column (Cytiva, USA).

[0180] Prepare filtered and degassed tertiary water and 1X PBS (pH 7.4, 12mM phosphate, 137mM NaCl, 2.7mM KCl). Prepare wild-type Fc (γ1 / 4, WT), heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT), double-IL21-Fc and single-IL21-Fc fusion proteins at a concentration of 1 μg / ml by dilution (plasmid ratio CH3A (EW): CH3B (RVT) = 1: 1, 1.5: 1, 2: 1), and add 45 μl of each of the obtained dilutions to 250 μl of inserts with Polyemer Feet 100 / PK (Agilent Technologies, USA), and inject into an autosampler vial to prepare the final sample to be analyzed. In addition, markers of unknown protein sample sizes that can be distinguished were prepared under the same concentration conditions. The marker corresponding to 150 kDa is alcohol dehydrogenase, and the marker corresponding to 66 kDa is BSA.

[0181] First, a tertiary water equivalent to 2 to 3 times the volume of the column is passed at 0.5 ml / min to remove the initial storage buffer (20% ethanol) from the column and standardize at the same time. After the process is completed, a physiological running buffer equivalent to 2 to 3 times the volume of 1X PBS (pH7.4, 137 mM NaCl) is passed at 0.75 ml / min to complete the standardization process, and then the analysis is started. The prepared analysis sample is placed in the device and analyzed at a flow rate set to 0.75 ml / min, with an injection volume of 30 μg (only 30 μl of the prepared 45 μl sample is injected), and the analysis is performed for 40 minutes, and the post-time is set to 10 minutes. When the sample of the determined injection volume is injected with the sample injector, it flows into the tube through which the buffer flows and is injected into the column. When the buffer containing the sample passes through the column compressed with a porous resin, the time required for the protein in the sample to elute from the column varies according to the molecular weight (i.e., size) of the protein, and its physical properties are analyzed by measuring the chromatogram obtained by the absorbance change at 280 nm. At this point, the chromatogram can be interpreted based on the following principles: as the size of the protein decreases, the amount of protein passing through the inside of the porous resin increases, and the protein elutes later; as the size of the protein increases, the protein passes through the column from the outside of the porous resin, so the protein elutes faster.

[0182] The results of SEC analysis of wild-type Fc (γ1 / 4, WT), heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT) and single-IL21-Fc fusion protein (CH3A (EW): CH3B (RVT) = 1: 1, 1.5: 1, 2: 1 at the time of conversion) showed that the peak of the single-IL21-Fc fusion protein did not match the expected peak compared to the marker, but a peak corresponding to a smaller protein was observed. This means that the biological properties of the single-IL21-Fc fusion protein in the storage buffer 1X PBS under physiological conditions are poor, and the reasons will be described later. Unstable biological properties mean that the single-IL21-Fc fusion protein cannot perform the desired function in vivo and may have side effects and immunogenicity, which indicates that the biological properties of the single-IL21-Fc fusion protein need to be improved.

[0183] Example 4. Analysis of SEC results of mono-IL21-Fc fusion protein depending on the composition of the assay buffer

[0184] The results of SEC analysis performed in Example 3 showed that the properties of the single-IL21-Fc fusion protein were poor. To determine the reason, the structure of human IL21 was analyzed, and the results were as follows: Figure 5 As shown. There are 22 amino acids with positive side chains on the IL21 surface. Among the amino acids with positive side chains, 10 arginines have the greatest impact. The isoelectric point of IL21 is 9.42, and IL21 is positively charged under pH 7.4 conditions. Based on this, it is speculated that the positive side chains on the IL21 surface have non-specific interactions with the column for SEC analysis. Therefore, using high salt PBS (12mM phosphate, 500mM NaCl, 2.7mM KCl, pH 7.4), i.e., a running buffer containing high concentrations of salt, it produces an environment that can offset the positive surface charge of IL21, and SEC analysis is performed in the same manner as the SEC analytical method described in Example 4. The results show that a peak with a level corresponding to a single-IL21-Fc fusion protein is observed. The results are shown in Figure 6 B. As a control, 1X PBS (which provides a biological environment as a running buffer) is used to analyze a single-IL21-Fc fusion protein, and the results are shown in Figure 6 A. The results showed that a peak corresponding to a protein of a single-IL21-Fc fusion protein was observed, indicating that human IL21 was strongly positively charged in a physiological condition buffer, and therefore interacted nonspecifically with the column during SEC analysis, and no peak was observed under 1X PBS conditions. In addition to SEC analysis, the positive surface charge of IL21 causes side effects when injected into a living body due to nonspecific binding to cells and tissues other than IL21 receptors, and leads to poor pharmacokinetics, making it difficult to act as a therapeutic agent. Therefore, in the following examples, an IL21 variant with a reduced positive surface charge was constructed.

[0185] Example 5. Construction and expression / purification of IL21 variants of single-IL21-Fc fusion proteins

[0186] In order to reduce the positive surface charge of IL21, single-IL21 (NKQ) -Fc, single-IL21 (DDD) -Fc, single L21 (NQNT) -Fc and single-IL21 (DDD NQNT) -Fc were constructed as variants of substituted amino acids with positive side chains (sequence information is provided in Table 4). Specifically, first, in order to obtain information about the amino acids exposed on the surface of IL21, the ChimeraX program was used to analyze the structure with the previously elucidated protein structure (PDB ID: 3TGX) as a sample. The results of the structural analysis showed that 22 amino acids had a positive surface charge, 10 of which were arginine, which had a high aqueous solution pKa (~ 13.8). In addition, when the structure was examined by the Chimera X program, atypical regions in which the structure could not be accurately identified were observed. By replacing amino acids in atypical regions or replacing amino acids in identified structural regions, single-IL21-Fc fusion protein amino acid substitution variants were constructed.

[0187] The atypical region greatly promotes the formation of unstable structures. The nonspecificity of IL21 increases, the possibility of oligomer formation increases, and the physical properties are deteriorated due to the presence of three arginines (83R, 84R, 124R) in the atypical region. In particular, IL21 has nonspecific interactions with the SEC column, inducing results such as Figure 4 Therefore, with reference to the central (rabbit, pig) sequence (R83N, R84K, R124Q), the arginine in the atypical region was replaced with an uncharged polar amino acid or a lysine with a weaker positive charge than arginine, and a new clone was constructed, which was called "single-IL21 (NKQ) -Fc" (SEQ ID NO: 5).

[0188] A new clone was constructed by replacing arginine (R83, R84, R124) in the atypical region with aspartic acid (R83D, R84D, R124D), which has the strongest negative side chain among amino acids, and was named "mono-IL21(DDD)-Fc" (SEQ ID NO: 6).

[0189] In addition, when the IL21 structure was analyzed using the Chimera X program (PDB ID: 3TGX), amino acids located in the L21 receptor binding region or that did not form significant bonds with surrounding residues and whose side chains were facing the surface (K50, K54, K86, R88) were selected, and a new clone was constructed by replacing uncharged polar amino acids (K50N, K54Q, K86N, R88T) with reference to the orthologous sequence, which was called "Single-IL21 (NQNT) -Fc" (SEQ ID NO: 7).

[0190] A new clone was constructed by replacing amino acids (K50N, K54Q, R83D, R84D, K86N, R88T, R124D), including the replacement strategy of mono-IL21(DDD)-Fc and mono-IL21(NQNT)-Fc, which was called "mono-IL21(DDD NQNT)-Fc" (SEQ ID NO: 8).

[0191] The basis and replacement strategy for constructing clones in this example are shown in Figure 7 middle.

[0192] To construct single-IL21(NKQ)-Fc, single-IL21(DDD)-Fc, single-IL21(NQNT)-Fc and single-IL21(DDDNQNT)-Fc clones, NotI / HindIII were used for in-frame cloning into the animal cell expression vector pcDNA3.1(+) (Invitrogen, USA) to form signal sequence-IL21 (variant) mature form-mutated hinge-CH2-CH3A (IgG4, EW).

[0193] HEK293-F (Invitrogen) cells were transiently transfected with a mixture of polyethyleneimine (PEI) (Polyscinece) and expression vectors of IgG4 CH3A (EW) and IgG4 CH3B (RVT) fused with wild-type and mutant human IL21 (NKQ or DDD or NQNT or DDD NQNT) at a ratio of 2:1 and cultured in serum-free FreeStyle TM The cells were cultured in shake flasks with 293 expression medium (Invitrogen) and single-IL21-Fc fusion proteins, single-IL21(NKQ)-Fc, single-IL21(DDD)-Fc, single-IL21(NQNT)-Fc and single-IL21(DDD NQNT)-Fc were constructed. The details of this method are as follows.

[0194] For transient transfection, HEK293-F cells were cultured at 1.0 × 10 6The cells were seeded at a density of 10 cells / ml in 90 ml of medium in a shake flask (Corning) and incubated at 135 rpm under 8% CO2. After 24 hours, the corresponding CH3A and CH3B plasmids were diluted in 4 ml 293 expression medium (Invitrogen), the total amount is 125μg (1.25μg / ml), more specifically, the dilution ratio is IgG4 CH3A (EW): IgG4CH3B (RVT) = 2: 1 = 83.3μg: 41.7μg, filtered, mixed with 6ml medium (3.75μg / ml) diluted with 375μg of PEI, and reacted at room temperature for 10 minutes. The reacted mixed medium is then added to the cells in the previously inoculated 90ml, and the shortest culture is 5 days and the longest culture is 7 days. As a result, the protein produced by the cells, that is, the fusion protein containing the constant region of the heterodimeric heavy chain, is secreted by the cells to the outside of the cells and accumulated in the culture medium. Therefore, a protein A sepharose column (GE healthcare) was used to purify the protein from the cell culture supernatant collected by centrifugation at 3,800rpm for 25 minutes after cell culture. At this time, the standard protocol provided by the protein A column company was used for purification. After replacing the storage buffer (PBS, pH 7.4), the eluted protein was concentrated using a Vivaspin 30,000MWCO (Satorius) centrifugal concentrator. The absorbance of the purified protein was measured at a wavelength of 562nm using the solution in the BCA protein assay kit (Thermo), and the standard curve was drawn for determination (Table 5). The experiment was performed twice independently, and the results were expressed as the standard error of the mean (mean ± SD). The yield verification results showed that the single-IL21-Fc fusion protein variant had an overall higher yield compared to the single-IL21-Fc fusion protein. This shows that there is no problem with the expression of the single-IL21-Fc fusion protein amino acid substitution mutant constructed by reason.

[0195] [Table 4]

[0196]

[0197] [Table 5]

[0198]

[0199] Purified mono-IL21-Fc fusion proteins, mono-IL21(NKQ)-Fc and mono-IL21(DDD)-Fc (5 μg) were analyzed by SDS-PAGE under 10% non-reducing and reducing conditions ( Figure 8). Under non-reducing conditions, single-IL21-Fc fusion protein, single-IL21 (NKQ)-Fc and single-IL21 (DDD)-Fc were observed at 71kDa. Under reducing conditions, single-IL21-Fc fusion protein, single-IL21 (NKQ)-Fc and single-IL21 (DDD)-Fc of two plasmids including heterodimeric heavy chain constant region CH3A (EW) fused to human wild-type IL21 and variant (NKQ or DDD) and heterodimeric heavy chain constant region CH3B (RVT) had two bands at 43 and 28kDa. Heterodimeric heavy chain constant region CH3A and CH3B each predicted protein size of about 25kDa, and human IL21 predicted protein size of 15kDa, but due to the N-glycosylation of human IL21 and Fc, bands larger than the expected protein size were observed under reducing and non-reducing conditions. The results of SDS-PAGE showed that there were no problems in the expression of the different dimeric heavy chain constant regions of the amino acid substitution variants of the single-IL21-Fc fusion protein and in the use thereof to form fusion proteins.

[0200] Example 6. Evaluation of properties of amino acid substitution variants of single-IL21-Fc fusion protein

[0201] Figure 8 It is a chromatogram of mAU at 280 nm after SEC (size exclusion chromatography) over time (minutes) to analyze the physical properties of expressed single-IL21(NKQ)-Fc, single-IL21(DDD)-Fc and single-IL21(NQNT)-Fc. The measurement was performed using an HPLC (Agilent Technologies, USA) instrument and a Superdex200 10 / 300GL column (Cytiva, USA) according to the following specific protocol.

[0202] Prepare filtered and degassed tertiary water and 1X PBS (pH7.4, 137mM NaCl) (12mM phosphate, 137mM NaCl, 2.7mM KCl, pH 7.4). Prepare single-IL21-Fc fusion protein, single-IL21 (NKQ)-Fc, single-IL21 (DDD)-Fc and single-IL21 (NQNT)-Fc at a concentration of 1 μg / ml by dilution, and add 45 μl of each of the obtained dilutions to 250 μl with Polyemer Feet 100 / PK (Agilent Technologies, the U.S.) inserts, and inject into an automatic sampler vial to prepare the final sample to be analyzed. In addition, markers of unknown protein sample sizes that can be distinguished were prepared under the same concentration conditions. The marker corresponding to 150kDa is alcohol dehydrogenase, and the marker corresponding to 66kDa is BSA.

[0203] First, tertiary water equivalent to 2 to 3 times the volume of the column was passed at 0.5 ml / min to remove the initial storage buffer (20% ethanol) from the column and standardize at the same time. After the process is completed, a running buffer equivalent to 2 to 3 times the volume of 1X PBS (pH7.4, 137 mM NaCl) was passed at 0.75 ml / min to complete the standardization process, and then the analysis was started. The prepared analysis sample was placed in the device and analyzed at a flow rate of 0.75 ml / min, with an injection volume of 30 μg (only 30 μl of the prepared 45 μl sample was injected), and the analysis was performed for 40 minutes, with the post-time set to 10 minutes.

[0204] The results of SEC analysis of single-IL21-Fc fusion protein, single-IL21(NKQ)-Fc, single-IL21(DDD)-Fc and single-IL21(NQNT)-Fc showed that the peak corresponding to the expressed fusion protein of the single-IL21(NQNT)-Fc fusion protein variant was significantly increased compared with the single-IL21-Fc fusion protein. This supports the hypothesis that the positive surface charge of human IL21 causes nonspecific interactions with the column during SEC analysis, which leads to unexpected peaks during the analysis. In addition, the single-IL21-Fc fusion protein variant that alleviates this problem has improved biological properties, reduced nonspecific interactions when injected into the body, reduced drug side effects, and improved pharmacodynamics.

[0205] Example 7. Construction and expression / purification of IL21 orthologous sequence transplant mutants of single-IL21-Fc fusion protein

[0206] Unlike the single-IL21-Fc fusion protein variant constructed by amino acid substitution in Example 6 to further improve the biological properties of the single-IL21-Fc fusion protein, a single-IL21-Fc fusion protein variant was developed using a new strategy. Specifically, first, the secondary structure formation patterns of three cytokines, IL-2 (PDB ID: 1IAR) and IL-4 (PDB ID: 2B5I), which belong to the same family as IL21, were compared with IL21 using PSIPRED, a secondary structure prediction platform, and multiple sequence alignment. The sequence analysis results showed that IL21 had a shorter helix C and a significantly longer loop structure connecting helix C and helix D compared to IL2 and IL4, and also had an atypical region between the loop structures. It is speculated that these structural differences may make the structure of IL21 unstable. Therefore, the complementary sequences of the corresponding regions of IL2 and IL4 were compared with those of IL21, and the sequence of IL4 with more hydrophilic residues than human IL2 was used to construct a single-IL21-Fc fusion protein orthologous sequence transplant mutant. During sequence transplantation, the structural instability may be caused due to the large number of hydrophobic amino acid residues. Therefore, the K77 to R90 sequence including the atypical region of IL21 was changed to a complementary human IL4 sequence and an orthologous IL4 sequence to construct a single-IL21-Fc fusion protein orthologous sequence transplant variant (sequence information is provided in Table 6). The variant using the human IL4 sequence is called "single-IL21 (helix 1)-Fc (SEQ ID NO: 9)", and the variant using the orthologous IL4 sequence is called "single-IL21 (helix 2)-Fc" (SEQ ID NO: 10).

[0207] The basis and replacement strategy of the clones constructed in this example are shown in Fig. 9 middle.

[0208] To construct mono-IL21 (helix 1)-Fc and mono-IL21 (helix 2)-Fc, NotI / HindIII were used for in-frame cloning into the animal cell expression vector pcDNA3.1(+) (Invitrogen, USA) to form signal sequence-IL21 (helix 1) mature form-mutated hinge-CH2-CH3A (IgG4, EW) and signal sequence-IL21 (helix 2) mature form-mutated hinge-CH2-CH3A (IgG4, EW).

[0209] HEK293-F (Invitrogen) cells were transiently transfected with polyethyleneimine (PEI) (Polyscinece) and a mixture of IgG4 CH3A (EW) and IgG4 CH3B (RVT) expression vectors fused with wild-type human IL21 or IL21 (helix 1) or IL21 (helix 2) at a ratio of 2:1 and cultured in serum-free FreeStyle medium. TM The resulting products were cultured in shake flasks in 293 expression medium (Invitrogen) to construct single-IL21-Fc fusion proteins, single-IL21 (helix 1)-Fc and single-IL21 (helix 2)-Fc. The details of this method are as follows.

[0210] For transient transfection, HEK293-F cells were plated at 1.0x10 6 The cells were seeded at a density of 10 cells / ml in 90 ml of medium in a shake flask (Corning) and incubated at 135 rpm under 8% CO2. After 24 hours, the corresponding CH3A and CH3B plasmids were diluted in 4 ml 293 expression medium (Invitrogen), the total amount is 125μg (1.25μg / ml), more specifically, the dilution ratio is CH3A (EW): CH3B (RVT) = 2: 1 = 8 3.3μg: 41.7μg, filtered, mixed with 6ml medium (3.75μg / ml) diluted with 375μg of PEI, and reacted at room temperature for 10 minutes. The reacted mixed culture medium is then added to the previously inoculated 90ml cells, and the shortest culture is 5 days and the longest culture is 7 days. As a result, the protein produced by the cells, that is, the fusion protein containing the heterodimeric heavy chain constant region, is secreted outside the cells by the cells and accumulated in the culture medium. Therefore, a protein A sepharose column (GE healthcare) was used to purify the protein from the cell culture supernatant collected by centrifugation at 3800rpm for 25 minutes after cell culture. At this time, the protein A column was purified according to the standard protocol provided by the company, and after replacing the storage buffer (PBS, pH 7.4), the eluted protein was concentrated using a Vivaspin 30,000MWCO (Satorius) centrifugal concentrator. The absorbance of the purified protein was measured at a wavelength of 562nm using the solution in the BCA protein assay kit (Thermo), and the standard curve was drawn for determination (Table 7). The experiment was performed three times independently, and the results were expressed as the standard error of the mean (mean ± SD). The yield verification results showed that the yield of single-IL21 (helix 1)-Fc and single-IL21 (helix 2)-Fc was similar to that of single-IL21-Fc fusion protein.

[0211] [Table 6]

[0212]

[0213] [Table 7]

[0214]

[0215] Purified mono-IL21-Fc fusion protein, mono-IL21(helix 1)-Fc and mono-IL21(helix 2)-Fc (5 μg) were analyzed by SDS-PAGE under 10% non-reducing and reducing conditions ( Fig.11 ). Under non-reducing conditions, single-IL21-Fc fusion protein, single-IL21 (helix 1)-Fc, and single-IL21 (helix 2)-Fc were observed at 71 kDa. Under reducing conditions, a single-IL21-Fc fusion protein comprising two plasmids containing a heterodimeric heavy chain constant region CH3A (EW) fused to human wild-type IL21 and IL21 (helix 1) and a heterodimeric heavy chain constant region CH3B (RVT) had two bands at 43 and 28 kDa. The heterodimeric heavy chain constant region CH3A (EW) fused to IL21 (helix 2) was observed to have a band larger than 43 kDa. This is believed to be due to the fact that during the construction of IL21 (helix 2), another N-glycosylation region (N90-T92) was generated when the centromere IL4 sequence was transplanted. The results of SDS-PAGE analysis showed that there were no problems with the expression of each heterodimeric heavy chain constant region and the formation of fusion proteins therethrough.

[0216] Example 8. Evaluation of properties of IL21 orthologous amino acid substitution variants of single-IL21-Fc fusion proteins

[0217] Fig.12 It is a chromatogram of mAU at 280 nm after SEC (size exclusion chromatography) to compare the physical properties between the IL21 orthologous amino acid substitution variants of the expressed single-IL21-Fc fusion protein and the previously constructed single-IL21-Fc fusion protein, single-IL21 (DDD)-Fc, single-IL21 (NQNT)-Fc and single-IL21 (DDD NQNT)-Fc. According to the following specific protocol, HPLC (Agilent Technologies, USA) instrument and Superdex200 10 / 300GL column (Cytiva, USA) were used for measurement.

[0218] Prepare tertiary water and 1X PBS (pH7.4, 137mM NaCl) that have been filtered and degassed. Prepare the single-IL21-Fc fusion protein, single-IL21 (DDD)-Fc, single-IL21 (NQNT)-Fc, single-IL21 (DDD NQNT)-Fc, single-IL21 (helix 1)-Fc and single-IL21 (helix 2)-Fc of construction by dilution with 1 μ g / ml concentration, and add 45 μ l of each of the obtained dilutions to 250 μ l with Polyemer Feet 100 / PK (Agilent Technologies, the U.S.) insert, and inject into the autosampler vial to prepare the final sample to be analyzed. In addition, standard samples of unknown protein sample sizes that can be distinguished were prepared under the same concentration conditions. The standard sample corresponding to 150 kDa is alcohol dehydrogenase, and the standard sample corresponding to 66 kDa is BSA.

[0219] First, tertiary water equivalent to 2 to 3 times the volume of the column was passed at 0.5 ml / min to remove the initial storage buffer (20% ethanol) from the column and standardize at the same time. After the process is completed, a running buffer equivalent to 2 to 3 times the volume of 1X PBS (pH7.4, 137 mM NaCl) was passed at 0.75 ml / min to complete the standardization process, and then the analysis was started. The prepared analysis sample was placed in the device and analyzed at a flow rate of 0.75 ml / min, with an injection volume of 30 μg (only 30 μl of the prepared 45 μl sample was injected), and the analysis was performed for 40 minutes, with the post-time set to 10 minutes.

[0220] The properties of the orthologous sequence grafted variants of the mono-IL21-Fc fusion protein were analyzed by size exclusion chromatography. Fig.10 It can be seen that mono-IL21 (helix 1) -Fc and mono-IL21 (helix 2) -Fc have a higher main peak than the mono-IL21-Fc fusion protein. In particular, compared with the mono-IL21-Fc fusion protein, the mono-IL21 (helix 2) -Fc has a lower oligomer ratio and a higher main peak. It is speculated that this is the reason why the structural stability is further improved by reducing the number of arginines with positive side chain charges in the sequence change portion from 3 to 1 and replacing the atypical region with a helical structure.

[0221] Example 9. Evaluation of receptor binding ability of single-IL21-Fc fusion protein

[0222] In order to evaluate the binding ability of the constructed single-IL21-Fc fusion protein variants to the IL21 receptor, the receptor binding ability of single-IL21-Fc fusion protein, single-IL21(NKQ)-Fc and single-IL21(DDD)-Fc was evaluated using Jurkat cell line (a CD4-positive human T cell line expressing IL21 receptor). 6 Cells were prepared from each sample, washed with washing buffer (PBS + 1% FBS), and then treated with single-IL21-Fc fusion protein, single-IL21 (NKQ) -Fc and single-IL21 (DDD) -Fc at 200nM and 50nM for 1 hour at 4°C. The product was washed again with washing buffer and treated with secondary antibody anti-human IgG4 FITC at 4°C in the dark for 30 minutes. The product was then washed with washing buffer and analyzed using a flow cytometer FACScalibur (BD bioscience, USA).

[0223] The binding ability to the IL21 receptor was evaluated by labeling the experimental group subjected to the treatment process of the single-IL21-Fc fusion protein and the single-IL21 (variant)-Fc and the control group not subjected to the process with a fluorescent substance and measuring the intensity of the fluorescent signal. The results are shown in Fig.13 The results showed that single-IL21 (variant)-Fc also had a fluorescence signal similar to that of single-IL21-Fc fusion protein, and the signal intensity also increased with the increase of the treated protein concentration. This indicates that the receptor binding ability of single-IL21 (variant)-Fc is retained, so there is no problem with biological activity.

[0224] Example 10. Evaluation of the biological activity of single-IL21-Fc fusion protein variants

[0225] According to due process, consent was obtained to collect human blood, and Ficoll (Ficoll-paqueTM PLUS, GE healthcare) was used to carefully place the blood on the Ficoll layer at a ratio of 1: 2, and centrifuged at 2,000rpm for 30 minutes to separate peripheral blood mononuclear cells (PBMC) from the buffy coat separated by density (specific gravity) difference (Figure 14). In addition, magnetic beads (Myltenyi Biotech) were used to remove CD4 T cells and CD8T cells and then group culture was performed to enrich the NK cells in the PBMC separated by Ficoll (Figure 15). Then, Jurkat cells and EBV-LCL cells irradiated with 100Gy of gamma were cultured with peripheral blood mononuclear cells in a ratio of 1: 1 in RPMI 1640 (Corning) supplemented with 10% FBS (fetal bovine serum, HyClone), 1% penicillin-streptomycin (Gibco) and IL-2-Fc (monomer, 30ng / ml) culture medium. At this time, cells were treated once with 30 ng / ml of 7 types of single Fc-IL21 fusion protein variants (mono-IL21-Fc, mono-IL21(NKQ)-Fc, mono-IL21(DDD)-Fc, mono-IL21(NQNT)-Fc, mono-IL21(DDD NQNT)-Fc, mono-IL21(helix 1)-Fc, mono-IL21(helix 2)-Fc) on day 0. RPMI medium containing 30 ng / ml of IL-2-Fc (monomer) was added every 3 to 4 days, and the cell number was adjusted to 2.5×10 on day 6. 5 Cells / ml were transferred to flasks for culture. On the 6th and 10th days, the cells were fluorescently stained with CD3- (FITC) and CD56+ (APC), the percentage of NK cells was measured by flow cytometry, and the NK cell expansion rate was finally calculated. The results showed that when PBMC was used, NQNT and DDD-NQNT had significantly higher yields than WT-IL21 (Figure 14), while when PBMC with CD4 and CD8 T cells removed were used, NQNT and spiral-1 had high yields (Figure 15). It can be seen that the NK cell proliferation and expansion capacity in the group treated with a single Fc-IL21 fusion protein variant is improved. It can be seen that when the group is further treated with a single Fc-IL21 fusion protein variant using a conventional treatment method during the in vitro expansion culture of NK cell therapeutic agents, a large number of NK cells can be expanded compared with conventional methods.

[0226] Industrial Applicability

[0227] The expected limitation of the existing single-IL21-Fc fusion protein disclosed herein is that due to the high isoelectric point of wild-type IL21, the net charge is positive in the body pH environment, and cationic patches are formed on the surface. In addition to the IL21 receptor, it may interact nonspecifically with various tissues and cells. Therefore, when the single-IL21-Fc fusion protein is injected into the body, its biological properties in the body may be poor, thereby causing side effects such as excessive immune response through nonspecific interactions with other cells and tissues, and resulting in poor pharmacodynamics. As a strategy to overcome this problem, a single-IL21-Fc fusion protein or a variant thereof fused to a heterodimeric heavy chain constant region Fc (γ1 / 4, EW-RVT) retains the structure of a conventional single-IL21-Fc fusion protein, the ability to bind to the IL21 receptor, and the biological activity including the proliferation ability and activation ability of T cells and NK cells, thereby improving the physical properties in the body, reducing side effects, and inducing an effect of increasing the half-life while maintaining anti-tumor function.

[0228] The single-IL21-Fc fusion protein or its variant according to the present invention is easy to develop into a therapeutic drug based on high production yield, and can maintain the binding ability of the conventional single-IL21-Fc fusion protein to the IL21 receptor and the original biological function (enhanced T cell proliferation and toxicity, and enhanced NK cell proliferation and toxicity), thereby exhibiting effective anti-cancer activity.

[0229] The single-IL21-Fc fusion protein or variants thereof according to the present invention can be used as a pharmaceutical composition, which can increase the proliferation and activation capacity of T cells and NK cells for immune cell therapy in vivo and in vitro.

[0230] The single-IL21-Fc fusion protein or variant thereof according to the present invention is used for basic experiments on immune cell culture to increase the proliferation and activation capacity of T cells and NK cells for immune cell therapy and for research on the development of cell therapy.

[0231] The construction strategy of the single-IL21-Fc fusion protein or its variants according to the present invention provides a strategy for constructing variants suitable for substances used as pharmaceutical compositions for the treatment and prevention of various diseases, which, except for IL21, are expected to have poor biological properties, side effects and poor pharmacodynamics due to high positive charges in vivo.

[0232] Although the specific configuration of the present invention has been described in detail, those skilled in the art will appreciate that the description is provided for the purpose of illustration to set forth the preferred embodiment and should not be interpreted as limiting the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0233] A separate electronic copy of the sequence listing is attached.

Claims

1. An interleukin 21 (IL21) variant, wherein One or more selected from the group consisting of amino acid sequences at positions 50, 54, 75 to 88 and 124 from the N terminus in interleukin 21 (IL21) including the amino acid sequence of SEQ ID NO. 4 is substituted or deleted.

2. The interleukin 21 (IL21) variant according to claim 1, wherein The interleukin 21 variant comprises one or more selected from the group consisting of the following amino acid substitutions and deletions in the amino acid sequence of SEQ ID NO: 4: (1) K50N; (2) K54Q; (3) K75L; (4) P76D; (5) P77R; (6)S78N; (7) T79L; (8) R83N, R83D, R83L; (9) R84K, R84D, R84W; (10) Q85G, Q85S; (11) K86N, K86L; (12)H87A; (13) R88T, R88G, R88N; (14) R124Q, R124D; (15) deletion of amino acids at positions 79 to 82; and (16) The amino acids at positions 81 to 84 are missing.

3. The interleukin 21 (IL21) variant according to claim 1, wherein The interleukin-21 variant comprises one or more selected from the group consisting of the following amino acid substitutions and deletions in the amino acid sequence of SEQ ID NO: 4: (1) R83N, R84K, R124Q; (2) R83D, R84D, R124D; (3)K50N, K54Q, K86N, R88T; (4)K50N, K54Q, R83D, R84D, K86N, R88T, R124D; (5) K75L, P76D, P77R, S78N and deletions of amino acids at positions 79 to 82, R83L, R84W, Q85G, K86L, H87A, R88G; and (6) K75L, P76D, P77R, S78N, T79L, deletion of amino acids at positions 81 to 84, and Q85S, K86L, H87A, R88N.

4. The interleukin 21 (IL21) variant according to claim 1, wherein The interleukin 21 variant comprises a sequence selected from the group consisting of SEQ ID NOs: 5 to 10. 5 . A fusion protein comprising the interleukin 21 variant according to any one of claims 1 to 4 .

6. The fusion protein according to claim 5, further comprising an antibody (immunoglobulin) heavy chain constant region (Fc) pair, wherein the antibody (immunoglobulin) heavy chain constant region (Fc) pair comprises a first Fc region and a second Fc region, in, The antibody (immunoglobulin) heavy chain constant region (Fc) pair has a mutation in the CH3 domain of the first Fc region or the second Fc region.

7. The fusion protein according to claim 6, wherein The interleukin 21 variant is bound to the N-terminus of the first Fc region or the second Fc region.

8. The fusion protein according to claim 6, wherein The IL-21 or variant thereof is bound to the first Fc region or the second Fc region via a linker.

9. The fusion protein according to claim 8, wherein The linker includes (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are each 1 to 10).

10. The fusion protein according to claim 6, wherein Each of the first Fc region and the second Fc region is derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE.

11. The fusion protein according to claim 6, wherein The mutation of the CH3 domain of the first Fc region or the second Fc region comprises at least one selected from the group consisting of the following mutations, wherein the mutation position is determined according to the EU index: (1) the amino acid at position K360 in the CH3 domain of the first Fc region is substituted with K360E; (2) the amino acid at position K409 in the CH3 domain of the first Fc region is replaced by K409W; (3) the amino acid at position E347 in the CH3 domain of the second Fc region is substituted with E347R; and (4) The amino acid at position F405 in the CH3 domain of the second Fc region is substituted with F405T, and the amino acid at position D399 in the CH3 domain of the second Fc region is substituted with D399V.

12. The fusion protein according to claim 11, wherein The CH3 domain of the first Fc region or the second Fc region further comprises the following substituted amino acids, wherein the mutation positions are determined according to the EU index: (i) a cysteine ​​(C) substituted at position Y349 in the CH3 domain of said first Fc region; or (ii) Cysteine ​​(C) substituted at position S354 in the CH3 domain of the second Fc region.

13. The fusion protein according to claim 6, wherein The CH3 domain mutations of the first Fc region and the second Fc region comprise the following mutations, wherein the mutation positions are determined according to the EU index: (1) the amino acid at position K360 in the CH3 domain of the first Fc region is substituted with K360E, and the amino acid at position K409 is substituted with K409W; and (2) In the CH3 domain of the second Fc region, the amino acid at position E347 is substituted with E347R, the amino acid at position F405 is substituted with F405T, and the amino acid at position D399 is substituted with D399V.

14. The fusion protein according to claim 6, wherein The fusion protein comprises: The interleukin 21 variant comprises a sequence selected from the group consisting of sequences of SEQ ID NO: 5 to 10; and The antibody (immunoglobulin) heavy chain constant region (Fc) pair comprises a first Fc region and a second Fc region, wherein the first Fc region comprises the sequence of SEQ ID NO: 1, and the second Fc region comprises the sequence of SEQ ID NO:

2.

15. A nucleic acid encoding the interleukin 21 variant according to any one of claims 1 to 4.

16. An expression vector comprising the nucleic acid according to claim 14.

17. A transformed recombinant cell comprising the expression vector according to claim 15.

18. A method for producing an interleukin 21 variant, comprising: (a) culturing the cell according to claim 16; as well as (b) harvesting the interleukin 21 variant from the cultured cells.

19. A nucleic acid encoding the fusion protein according to claim 5.

20. An expression vector comprising the nucleic acid according to claim 19. A transformed recombinant cell comprising the expression vector according to claim 20.

22. A method for producing a fusion protein, comprising: (a) culturing the cell according to claim 21; as well as (b) harvesting the fusion protein from the cultured cells.

23. A composition for preventing or treating cancer, comprising the interleukin 21 variant according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 13 as an active ingredient.

24. A culture method comprising treating immune cells with the interleukin 21 variant according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 13.

25. The culture method according to claim 24, wherein: The immune cells include at least one selected from the group consisting of T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor infiltrating lymphocytes (TIL) and dendritic cells.

Images