Il-2 protein and il-15 protein chimeric protein or variant, fusion protein comprising same, and genetically-engineered cell expressing same

Abstract

The present invention relates to an IL-2 protein and IL-15 protein chimeric protein, a variant thereof, a fusion protein comprising some, and / or genetically-engineered cells modified to express any one or more thereof. In the present invention, binding to IL-2Rβ is weakened such that side effects caused by excessive immune cell activity of IL-2 are inhibited, and rapid exhaustion of natural killer cells is prevented such that a suitable level of cell activity and cell survival ability is maintained for a long time. Therefore, the present invention is useful for regulating the immune system and treating immune-associated diseases such as cancer, infectious diseases and autoimmune diseases.

Description

Chimeric proteins or variants of IL-2 protein and IL-15 protein, fusion proteins comprising the same, and genetically engineered cells expressing the same

[0001] The present invention relates to chimeric proteins of IL-2 protein and IL-15 protein, variants thereof, fusion proteins comprising the same, and / or genetically engineered cells modified to express at least one of the same.

[0002]

[0003] Interleukin 2 (IL-2), also known as T-cell growth factor (TCGF), is a globular glycoprotein of 15.5 to 16 kDa that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 is 133 amino acids long and has a quaternary structure composed of four antiparallel, amphipathic α-helices ((Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)).

[0004] IL-2 mediates various immune actions by binding to the IL-2 receptor (IL-2 receptor), which is composed of three individual subunits (IL-2R): IL-2Rα (CD25), β (CD122), and γ (CD132). The binding affinity for IL-2 protein varies depending on the different binding of each chain of the IL-2 receptor. Specifically, the trimeric IL-2 receptor composed of α, β, and γ chains exhibits high affinity for IL-2 protein, the dimeric IL-2 receptor composed of β and γ exhibits an intermediate binding affinity (about 100 times lower) compared to the trimer, but the monomeric IL-2 receptor composed only of the α chain exhibits a low level of IL-2 protein binding affinity. Both the IL-2 receptor trimer (α,β,γ) and the IL-2 receptor dimer (β,γ) are essential for cell signaling activation upon IL-2 binding (Minami et al., Annu Rev Immunol 11, 245-268 (1993)), but the IL-2 receptor monomer (α, CD25) is not essential for cell signaling (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).

[0005] IL-15 is a 14- to 15-kDa glycoprotein that exists in a membrane-bound form by binding to the IL-15 receptor alpha (IL-15Rα) present on the cell surface. The IL-15 / IL-15Rα complex binds to IL-2Rβ (CD122), γ (CD132) and shares a cell signaling mechanism with IL-2 (Marek Jakobisiak et al., Cytokine & Growth Factor Reviews 22, 99-108 (2011)).

[0006] The IL-2 receptor trimer (α, β, γ) containing the IL-2 receptor α-chain (CD25) is consistently expressed at high levels in FoxP3+ CD+4 regulatory T cells. On the other hand, it is known that immune effector cells such as normal in vivo CD8 T cells and NK cells express the IL-2 receptor dimer (β, γ) in the resting state and transiently express the IL-2 receptor trimer (α, β, γ) when the cells are activated (Fontenot et al., Nature Immunol 6, 1142-51 (2005); H.Asao, Encyclopedia of Endocrine Disease, 60-63 (2004)).

[0007] IL-2 is synthesized primarily by activated T cells, particularly CD4+ helper T cells, stimulating the proliferation and differentiation of T cells and inducing the production of cytotoxic T lymphocytes (CTLs). It also induces the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the production, proliferation, and activation of natural killer (NK) cells (reviewed eg in Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).

[0008] Additionally, IL-2 is involved in the maintenance of CD4+CD25+ regulatory T cells (Treg, regulatory T cells), known as suppressive T cells, and suppresses the activity of effector functions such as T cells and natural killer cells through cell-cell contact and the release of immunosuppressive cytokines such as IL-10 or TGF-β (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005)).

[0009] IL-2 or IL-15 can increase the lymphocyte population in vivo and enhance the function of these immune cells, so it has a dual function in the immune response in that it mediates the increase and activation of anticancer agents or immune cells using IL-2 or IL-15, but suppresses antitumor immunity mediated by CD8+ T cells and natural killer cells by strongly expanding regulatory T (Treg) cells expressing the high-affinity IL-2 receptor. (Brandenburg, S., et al., Eur J Immunol, 2008. 38(6): p. 1643-53; Facciabene, A., et al., Cancer Res, 2012. 72(9): p. 2162-71)

[0010] Patients receiving IL-2-based immunotherapy experience serious cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, dermatological, hematological, and systemic adverse effects. Therefore, various IL-2 mutations have been studied to improve the therapeutic efficacy of IL-2 and minimize adverse effects (US 5,229,109 B).

[0011] A way to overcome the shortcomings of treatment using IL-2 or IL-15 is to extend the in vivo half-life of IL-2 or IL-15 while selectively activating CD8 + T cells and natural killer cells expressing low-affinity IL-2 receptors. Many attempts have been made to this end, but no significant results have been achieved (Arenas-Ramirez, N., et al., Sci Transl Med, 2016. 8(367): p. 367ra166). It has been studied that continuous stimulation of lymphocytes, especially natural killer cells, by IL-15 protein actually inhibits immune enhancement and cancer treatment efficacy (Felices et al., JCI Insight, 2018).

[0012] Repeated administration of IL-2 or IL-15 proteins induces hyporesponsiveness of natural killer cells in vivo, resulting in decreased proliferative capacity and reduced anticancer activity due to an imbalance in the activation mechanism (Frutoso et al, Int J Mol Sci 20(18), 4514(2019)). Therefore, continuous improvement of IL-2 variants that can minimize hyporesponsiveness of natural killer cells is necessary.

[0013] Natural killer cells that have been modified to express IL-15 in a membrane protein form have been studied to have higher cell proliferation and survival abilities than natural killer cells that express soluble IL-15 in their native form (Masaru Imamura et al, Blood, 2014, 124(7), 1081-1088). However, in the case of natural killer cells modified to express IL-15 in a membrane protein form, the cell number and proliferation ability were unstable depending on the donor, and in most cases, only maintenance or a slight increase in cell number was observed.

[0014] Under these backgrounds, the present inventors have made great efforts to develop a chimeric protein comprising IL-2 and IL-15 sequences with low affinity and low irritation while extending the half-life of IL-2. As a result, in the case of a chimeric protein in which some domains of IL-2 are replaced with domains of IL-15, or in the case of a variant of a chimeric protein in which some amino acid sequences of the IL-2Rβ binding domain of IL-2 are replaced, the binding affinity to IL-2Rβ is weakened, the intensity of STAT5 activity is lowered, the cancer cell proliferation inhibition activity is excellent, and side effects such as pulmonary edema do not occur after administration.

[0015] Furthermore, when a chimeric protein containing IL-2 and IL-15 sequences is expressed in natural killer cells, it was confirmed that it exhibits superior proliferation, survival, and activation capabilities compared to natural killer cells expressing the conventional IL-15 cell membrane protein, and it was confirmed that it exhibits a strong tumor cell killing ability against various cancer types, thereby completing the present invention.

[0016]

[0017] Prior art literature

[0018] Non-patent literature

[0019] (Non-patent literature 1) Smith, Science 240, 1169-76 (1988)

[0020] (Non-patent literature 2) Bazan, Science 257, 410-413 (1992)

[0021] (Non-patent literature 3) Minami et al., Annu Rev Immunol 11, 245-268 (1993)

[0022] (Non-patent literature 4) Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)

[0023] (Non-patent literature 5) Marek Jakobisiak et al., Cytokine & Growth Factor Reviews 22, 99-108 (2011)

[0024] (Non-patent literature 6) Fontenot et al., Nature Immunol 6, 1142-51 (2005)

[0025] (Non-patent literature 7) H.Asao, Encyclopedia of Endocrine Disease, 60-63 (2004)

[0026] (Non-patent literature 8) reviewed eg in Waldmann, Nat Rev Immunol 6, 595-601 (2009)

[0027] (Non-patent Document 9) Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008)

[0028] (Non-patent literature 10) Malek, Annu Rev Immunol 26, 453-79 (2008)

[0029] (Non-patent literature 11) Fontenot et al., Nature Immunol 6, 1142-51 (2005);

[0030] (Non-patent literature 12) D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005)

[0031] (Non-patent literature 13) Brandenburg, S., et al., Eur J Immunol, 2008. 38(6): p. 1643-53

[0032] (Non-patent literature 14) Arenas-Ramirez, N., et al., Sci Transl Med, 2016. 8(367): p. 367ra166

[0033] (Non-patent literature 15) Felices et al., JCI Insight 3(3):e96219 (2018)

[0034] (Non-patent literature 16) Frutoso et al, Int J Mol Sci 20(18), 4514(2019)

[0035] (Non-patent literature 17) Masaru Imamura et al, Blood, 2014, 124(7), 1081-1088

[0036]

[0037] Summary of the invention

[0038] The purpose of the present invention is to provide a chimeric protein or a variant thereof that can maintain the activity of immune cells for a long time by reducing excessive activity of natural killer cells and delaying cell exhaustion.

[0039] Another object of the present invention is to provide a fusion protein comprising the chimeric protein or a variant thereof.

[0040] Another object of the present invention is to provide a nucleic acid encoding a fusion protein comprising the chimeric protein or a variant thereof.

[0041] Another object of the present invention is to provide a recombinant vector comprising a nucleic acid encoding a fusion protein comprising the chimeric protein or a variant thereof.

[0042] Another object of the present invention is to provide a cell genetically modified to express the chimeric protein or a variant thereof, and / or a fusion protein comprising the same.

[0043] Another object of the present invention is to provide an immune cell genetically modified to express the chimeric protein or a variant thereof, and / or a fusion protein comprising the same.

[0044] Another object of the present invention is to provide a method for producing a chimeric protein, comprising: culturing the genetically modified cell to express the chimeric protein or a variant thereof, and / or a fusion protein comprising the same; and

[0045] The present invention provides a method for producing the chimeric protein or variant thereof, and / or a fusion protein comprising the same, comprising a step of recovering the expressed chimeric protein or variant thereof, and / or a fusion protein comprising the same.

[0046] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell.

[0047] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating infectious diseases comprising the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell.

[0048] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an autoimmune disease comprising the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell.

[0049] Another object of the present invention is to provide a method for preventing or treating cancer using the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell, a use for preventing or treating cancer, and a use for manufacturing a drug for preventing or treating cancer.

[0050] Another object of the present invention is to provide a method for preventing or treating an infectious disease using the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell, a use for preventing or treating an infectious disease, and a use for manufacturing a medicament for preventing or treating an infectious disease.

[0051] Another object of the present invention is to provide a method for preventing or treating an autoimmune disease using the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell, a use for preventing or treating an autoimmune disease, and a use for manufacturing a drug for preventing or treating an autoimmune disease.

[0052]

[0053] To achieve the above purpose, the present invention provides a chimeric protein or a variant thereof comprising IL-2 and IL-15 proteins.

[0054] The present invention also provides a fusion protein comprising the chimeric protein or a variant thereof.

[0055] The present invention also provides a genetically modified cell expressing the chimeric protein, a variant thereof and / or a fusion protein comprising the same.

[0056] The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the modified natural killer cells.

[0057] The present invention also provides a pharmaceutical composition for preventing or treating an infectious disease comprising the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell.

[0058] The present invention also provides a pharmaceutical composition for preventing or treating an autoimmune disease comprising the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell.

[0059] The present invention also provides a method for preventing or treating cancer, a use for preventing or treating cancer, and a use for manufacturing a medicament for preventing or treating cancer, comprising administering the chimeric protein or a variant thereof, a fusion protein, and / or the genetically modified cell.

[0060] The present invention also provides a method for preventing or treating an infectious disease, comprising administering the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell, a use for preventing or treating an infectious disease, and a use for manufacturing a medicament for preventing or treating an infectious disease.

[0061] The present invention also provides a method for preventing or treating an autoimmune disease, comprising administering the chimeric protein or a variant thereof, a fusion protein and / or the genetically modified cell, a use for preventing or treating an autoimmune disease, and a use for manufacturing a medicament for preventing or treating an autoimmune disease.

[0062]

[0063] Figure 1a is a schematic diagram of the IL-2 and / or IL-15 chimeric protein of the present invention.

[0064] Figure 1b is a schematic diagram of a fusion protein comprising IL-15Ra (sushi domain) and an Fc domain.

[0065] Figures 1c to 1f are SDS-PAGE gel photographs of the manufactured and purified fusion proteins GIC-982C1 to GIC-982C15.

[0066] Figure 2 is a diagram showing the results of an Octet binding assay experiment for Fc-IL2, GIC-982C1 to GIC-982C6, and GIC-982C12 to GIC-982C15.

[0067] Figure 3 is a diagram showing the results of evaluating the STAT-5 cell signaling pathway activity of Fc-IL2, GIC-982C1 to GIC-982C6, and GIC-982C13 to GIC-982C15.

[0068] Figure 4a illustrates the structure of an insert gene and vector for genetically modifying natural killer cells to express a chimeric protein comprising IL-2 and IL-15 proteins (control group: mbIL-15, experimental group: mbIL-2 / IL-15). Figure 4b is a schematic diagram of natural killer cells expressing the manufactured chimeric protein.

[0069] Figure 5 shows FACS data of genetically modified natural killer cells.

[0070] Figure 6 shows the results of confirming the proliferation ability of each natural killer cell according to the culture period.

[0071] Figure 7 shows the results of confirming the killing ability against tumor cells in vitro when genetically modified natural killer cells are treated.

[0072]

[0073] The invention is described in detail and has specific implementation examples.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0075]

[0076] The present inventors have attempted to develop a low-affinity, low-irritant recombinant cytokine that can maintain the killing ability against cancer cells while suppressing the side effects caused by the strong cell killing ability of IL-2. By adjusting the non-specific and strong toxicity of IL-2 such that a chimeric protein in which some domains of IL-2 are replaced with the domain of IL-15 has a weakened binding affinity for IL-2Rβ and a lower intensity of STAT5 activity compared to wild-type IL-2, a novel chimeric protein has been developed that exhibits excellent cancer cell proliferation inhibition activity without the side effects of existing IL-2, such as pulmonary edema.

[0077]

[0078] Accordingly, the present invention relates, in one aspect, to a chimeric protein comprising an IL-2 protein and an IL-15 protein.

[0079]

[0080] Chimeric protein containing IL-2 and IL-15 proteins

[0081] Interleukin 2 (IL-2) is a protein encoded by the IL-2 gene in humans. It is also called T-cell growth factor (TCGF). It is a globular glycoprotein, weighing 15.5 to 16 kDa, that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 is 133 amino acids long and has a quaternary structure composed of four antiparallel, amphipathic α-helices.

[0082] In the case of human IL-2, IL-2 can be composed of a structure in which a “Helix A” domain, an “AB loop” domain, a “Helix B” domain, a “BC loop” domain, a “Helix C” domain, a “CD loop” domain, and a “Helix D” domain are sequentially connected, and it has been reported that the “AB loop” domain, the “Helix B” domain, and the “CD loop” domain can be involved in IL-2Rα binding, and the “Helix C” domain can be involved in IL-2Rβ binding (Cassell, Current Pharmaceutical Design, 2002, 8, 2171-2183).

[0083] The amino acid or nucleic acid sequence of human IL-2 is as described in Table 1 below:

[0084]

[0085] IL-15 (Interleukin 15) is a protein encoded by the IL-15 gene in humans. It is a 14 to 15 kDa glycoprotein that exists in a membrane-bound form by binding to the IL-15 receptor alpha (IL-15Rα) present on the cell surface. The IL-15 / IL-15Rα complex binds to IL-2Rβ (CD122), γ (CD132) and shares a cell signaling mechanism with IL-2.

[0086] In the case of human IL-15, IL-15 can be composed of a structure in which the “Helix A” domain, “AB loop” domain, “Helix B” domain, “BC loop” domain, “Helix C” domain, “CD loop” domain, and “Helix D” domain are sequentially connected, and it has been reported that the “AB loop” domain, “Helix B” domain, and “CD loop” domain can be involved in binding to IL-15Rα (Lowe, Journal of Molecular Biology, 2011, 406, 160-175).

[0087] The sequence of human IL-15 is as shown in Table 2 below:

[0088]

[0089] IL-2 or IL-15 proteins can increase the lymphocyte population and enhance the function of these immune cells in vivo, so IL-2 or IL-15 can be used to mediate the increase and activation of anticancer agents or immune cells. However, they have a dual function in the immune response in that they suppress antitumor immunity mediated by CD8+ T cells and natural killer cells by strongly expanding regulatory T cells expressing the high-affinity IL-2 receptor. In particular, repeated administration of IL-2 or IL-15 proteins induces hyporesponsiveness of natural killer cells in vivo, resulting in decreased proliferative capacity, rapid exhaustion, and decreased anticancer ability due to an imbalance in their activation mechanisms.

[0090] In one embodiment of the present invention, the chimeric protein may be characterized by comprising a portion of an IL-2 amino acid sequence and a portion of an IL-15 amino acid sequence.

[0091] In one embodiment of the present invention, the chimeric protein may include a portion of the IL-2 amino acid sequence and a portion of the IL-15 amino acid sequence, directly or indirectly linked via a linker or the like.

[0092] In one embodiment of the present invention, the chimeric protein may be characterized in that a portion of the IL-2 amino acid sequence is substituted with the IL-15 amino acid sequence. In the present specification, a protein comprising an amino acid sequence in which a specific amino acid sequence is substituted is used interchangeably with the term “hybrid protein.”

[0093] When a part of the IL-2 amino acid sequence is replaced with an IL-15 amino acid sequence, as in the chimeric protein of the present invention, the intensity of the signaling mechanism of the IL-2Rβ (CD122), γ (CD132) dimeric receptor is controlled, thereby preventing excessive activity of natural killer cells and delaying cell exhaustion, thereby maintaining the activity of immune cells for a long time.

[0094] In one embodiment of the present invention, at least one domain selected from the group consisting of the “Helix A” domain, the “AB loop” domain, the “Helix B” domain, the “BC loop” domain, the “Helix C” domain, the “CD loop” domain, and the “Helix D” domain of IL-2 may be substituted with the “Helix A” domain, the “AB loop” domain, the “Helix B” domain, the “BC loop” domain, the “Helix C” domain, the “CD loop” domain, or the “Helix D” domain of IL-15.

[0095] In a preferred embodiment of the present invention, at least one domain selected from the group consisting of the “AB loop” domain, the “Helix B” domain and the “CD loop” domain of IL-2 may be substituted with the “AB loop” domain, the “Helix B” domain or the “CD loop” domain of IL-15.

[0096] In a preferred embodiment of the present invention, the “AB loop” domain, “Helix B” domain and “CD loop” domain of IL-2 may be replaced with the “AB loop” domain, “Helix B” domain or “CD loop” domain of IL-15, respectively, and for example, the amino acid sequence of the chimeric protein or the nucleic acid sequence encoding it may include or consist of the amino acid sequence of SEQ ID NO: 33 described in Table 3 below or the nucleic acid sequence encoding it of SEQ ID NO: 34.

[0097]

[0098] A variant of a chimeric protein comprising IL-2 and IL-15 proteins

[0099] From another perspective, the present invention relates to a variant of a chimeric protein comprising the IL-2 and IL-15 proteins.

[0100] In the case of a variant of a chimeric protein in which a part of the amino acid sequence of the IL-2Rβ binding domain of IL-2 is mutated, as in the present invention, the binding affinity for IL-2Rβ is further weakened and the intensity of STAT5 activity is lowered, thereby preventing excessive activity of natural killer cells and delaying cell exhaustion, thereby maintaining the activity of immune cells for a long time.

[0101] In this specification, “variant” is used as a concept that includes not only mutations of some amino acid residues in the wild-type amino acid sequence, preferably substitutions, deletions, insertions, etc. of amino acid residues, but also truncation of some amino acid residues at the N-terminus or C-terminus, etc., and therefore, in the present invention, a variant of a chimeric protein is used as a broad concept that includes “a fragment of a chimeric protein variant.”

[0102] As used herein, “substitution” means a modification that involves replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function.

[0103] In one embodiment of the present invention, the variant of the chimeric protein may include a mutation, preferably an amino acid substitution, at at least one or more positions of aspartic acid (Asp(D), Aspartic acid) at position 73, leucine (Leu(L), Leucine) at position 74, serine (Ser(S), Serine) at position 76, asparagine (Asn(N), Asparagine) at position 77, and valine (Val(V), Valine) at position 80 relative to the amino acid sequence of SEQ ID NO: 33.

[0104] In one embodiment of the present invention, the variant of the chimeric protein may comprise at least one amino acid substitution of D73E, L74F, S76D, S76E, S76N, S76K, S76L, N77D and V80L with respect to the amino acid sequence of SEQ ID NO: 33.

[0105] 본 발명의 일 구현예에서, 키메라 단백질의 변이체는 D73E / L74F, D73E / S76D, D73E / S76E, D73E / S76N, D73E / S76K, D73E / S76L, D73E / N77D, D73E / V80L, L74F / S76D, L74F / S76E, L74F / S76N, L74F / S76K, L74F / S76L, L74F / N77D, L74F / V80L, S76D / N77D, S76D / V80L, S76E / N77D, S76E / V80L, S76N / N77D, S76N / V80L, S76K / N77D, S76K / V80L, S76L / N77D, S76L / V80L, D73E / L74F / S76D, D73E / L74F / S76E, D73E / L74F / S76N, D73E / L74F / S76K, D73E / L74F / S76L, D73E / L74F / N77D, D73E / L74F / V80L, D73E / S76D / N77D, D73E / S76D / V80L, D73E / S76E / N77D, D73E / S76E / V80L, D73E / S76N / N77D, D73E / S76N / V80L, D73E / S76K / N77D, D73E / S76K / V80L, D73E / S76L / N77D, D73E / S76L / V80L, D73E / N77D / V80L, S76D / N77D / V80L, S76E / N77D / V80L, S76N / N77D / V80L, S76K / N77D / V80L, S76L / N77D / V80L, D73E / L74F / S76D / N77D, D73E / L74F / S76E / N77D, D73E / L74F / S76N / N77D, D73E / L74F / S76K / N77D, D73E / L74F / S76L / N77D, D73E / L74F / S76D / V80L, D73E / L74F / S76E / V80L, D73E / L74F / S76N / V80L, D73E / L74F / S76K / V80L, D73E / L74F / S76L / V80L, L74F / S76D / N77D / V80L, L74F / S76E / N77D / V80L, L74F / S76N / N77D / V80L, L74F / S76K / N77D / V80L, L74F / S76L / N77D / V80L,It may include a combination of one selected from among D73E / L74F / S76D / N77D / V80L, D73E / L74F / S76E / N77D / V80L, D73E / L74F / S76N / N77D / V80L, D73E / L74F / S76K / N77D / V80L, D73E / L74F / S76L / N77D / V80L.

[0106] In a preferred embodiment of the present invention, the variant of the chimeric protein may comprise at least one amino acid substitution of D73E, L74F, S76D, N77D and V80L with respect to the amino acid sequence of SEQ ID NO: 33.

[0107] In a preferred embodiment of the present invention, the amino acid sequence or nucleic acid sequence of the variant of the chimeric protein may comprise or consist of an amino acid sequence of SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75 as set forth in Table 4 below, or a nucleic acid sequence of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72 or 74 encoding the same.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] A fusion protein comprising a chimeric protein comprising IL-2 and IL-15 proteins or a variant thereof

[0115] In another aspect, the present invention relates to a fusion protein comprising the chimeric protein or a variant thereof.

[0116] In this specification, the fusion protein refers to a protein comprising a fusion domain or amino acid sequence other than a chimeric protein comprising IL-2 and IL-15 proteins or a variant thereof.

[0117] In the present invention, the fusion protein may be characterized by an additional peptide sequence (fusion domain) fused to a chimeric protein or a variant thereof.

[0118] The term “fusion domain” of the present invention means an additional domain or moiety that can be directly or indirectly bound to and included in the chimeric protein of the present invention or a variant thereof, and the fusion domain is used to mean both a polypeptide having a function and a polypeptide having no function.

[0119] In the present invention, the fusion domain may be fused to, but is not limited to, impart expression characteristics such as secretion from a cell, cell surface expression or anchoring to a cell membrane, or intracellular localization; add a substrate or other recognition sequence for post-translational modification; or alter tissue localization, tissue exclusion, or other ADME characteristics; or add a protein or peptide having another function.

[0120] In the present invention, the fusion domain may be, for example, a transmembrane domain, a hinge domain, an intracellular signaling domain, an immune regulatory domain, a localization domain, an immune costimulatory factor / receptor, a cytokine, a growth factor, an albumin binding domain, an Fc domain, a transferrin fusion domain, albumin, PEG, hyaluronic acid, or other therapeutic peptides, but is not limited thereto, and may include any domain (or moiety) that is not fatal to the expression or activity of the chimeric protein comprising the IL-2 and IL-15 proteins of the present invention or a variant thereof.

[0121] In the present invention, when it is desired to fix a fusion protein expressed in a modified natural killer cell to the cell membrane or to express it on the cell surface, the fusion domain may be characterized by including a membrane protein or a membrane-penetrating domain.

[0122] In the present invention, “membrane protein” means a protein inserted into or attached to the surface of a membrane formed by a lipid bilayer, and includes “intrinsic membrane protein” which is a protein inserted into a lipid bilayer, and “extrinsic membrane protein” which is a protein attached to the surface of a lipid bilayer. “Intrinsic membrane protein” belongs to one type of “membrane protein” that crosses the membrane.

[0123] In the present invention, an "intrinsic membrane protein" is a protein that penetrates the cell membrane, and includes a "transmembrane domain" that penetrates the membrane, an "intracellular domain" located inside the cell, and an "extracellular domain" exposed to the outside of the cell. The number of transmembrane domains varies depending on the membrane protein. Since the lipid bilayer is composed of highly hydrophobic lipids, the transmembrane domains that pass through this region are mostly composed of hydrophobic amino acids. Almost all transmembrane domains are structured in an α-helix structure. In membrane proteins with multiple transmembrane domains, the transmembrane domains are arranged in a circle to form a cylindrical structure. This cylindrical structure opens and closes in response to specific signals, thereby serving as a conduit for the transport of specific ions or biological substances. The transmembrane domains of a very limited number of membrane proteins are structured in a β-sheet structure. A cylindrical structure formed by the circular arrangement of multiple domains in a β-sheet structure is called a "β-barrel."

[0124] In the present invention, in the case of an “extrinsic membrane protein,” a lipoprotein is formed by covalent bonding between a protein molecule and a lipid molecule, and as a result of hydrophobic bonding between the lipid molecule of the lipoprotein and the lipid monolayer of the cell membrane, the protein is fixed to the surface of the cell membrane, and an example is Glycosylphosphatidylinositol (GPI)-anchored protein. GPI fixes the protein to the surface of the lipid bilayer by inserting the fatty acid of GPI into the lipid monolayer and covalently bonding the phosphate group on the other side with the protein.

[0125] In the present invention, examples of the membrane protein include a receptor, a ligand, an immunoglobulin, a glycophorin, or a combination thereof. The membrane protein may be characterized by being selected from the group consisting of, but not limited to, specific examples of IL-15 receptor alpha (IL-15Rα), CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, FcεRIγ, T-cell receptors (TCRs, for example, TCRα and / or TCRβ), nicotinic acetylcholine receptors, GABA receptors, and fragments thereof. Specific examples of immunoglobulins include IgG, IgA, IgM, IgE, IgD, or a combination thereof. Specific examples of glycophorins include, but are not limited to, glycophorin A, glycophorin D, or a combination thereof.

[0126] In the present invention, the fusion protein may additionally include a hinge domain or a sushi domain in addition to the transmembrane domain. The term "hinge domain" as used herein refers to a series of amino acid sequences present between the transmembrane domain and the extracellular domain of a membrane-anchored protein. In the present invention, the domains included in the fusion protein may be in the form of a combination of domains derived from the same protein, or in the form of a chimeric protein in which domains derived from different proteins are combined.

[0127] In one embodiment of the present invention, the fusion protein may include an IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof in addition to the IL-2 and IL-15 chimeric protein of the present invention or a variant thereof.

[0128] In the present invention, the IL-15 receptor alpha (IL-15Rα) protein fragment may include a “sushi domain”, which is the shortest region of the receptor possessing IL-15 binding activity.

[0129] In the present invention, the IL-15 receptor alpha (IL-15Rα) protein fragment may include a “sushi domain,” which is the shortest region of the receptor possessing IL-15 binding activity, and a “transmembrane domain,” which is a cell membrane-penetrating region of the IL-15 receptor alpha. In the present invention, the fusion protein may be characterized as a fusion protein in which the IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof is fused to a chimeric protein or a variant thereof.

[0130] In the present invention, the human IL-15 receptor alpha (IL-15Rα) and fragment sequences thereof are, for example, as described in Table 5 below, but are not limited thereto.

[0131]

[0132] In the present invention, the chimeric protein or its variant; and the IL-15 receptor alpha (IL-15Rα) protein may be characterized by being directly or indirectly linked, for example, by being linked by a linker. In one embodiment of the present invention, the fusion protein may be characterized by comprising a structure represented by the following structural formula (I) or structural formula (II).

[0133] N'-X-[L1]nY-C'(structural formula (I))

[0134] N'-Y-[L1]nX-C'(structural formula (II))

[0135] Here,

[0136] N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein,

[0137] X is a chimeric protein comprising IL-2 and IL-15 proteins or a variant thereof,

[0138] Y is IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof,

[0139] L1 is the linker,

[0140] n is an integer greater than or equal to 0 or 1, preferably 0 or 1.

[0141] In the present invention, the fragment of the IL-15Rα protein may include at least one of the IL15Rα membrane protein domain and the IL15Rα sushi domain. For example, the IL15Rα membrane protein domain may include or consist of the amino acid sequence represented by SEQ ID NO: 79, and the IL15Rα sushi domain may include or consist of the amino acid sequence represented by SEQ ID NO: 81, but is not limited thereto.

[0142] In the present invention, the linker may be any of various linkers known in the art. For example, the linker may include, but is not limited to, a hinge domain between the transmembrane domain and the extracellular domain, a flexible linker, a rigid linker, etc.

[0143] More specifically, the linker means an amino acid sequence of sufficient length to allow the protein to form appropriate secondary and tertiary structures. In some embodiments, the linker is a peptide linker comprising at least one, but less than 100 amino acids, for example a peptide linker of 2 to 60 amino acids, preferably 10 to 40 amino acids, more preferably 15 to 40 amino acids, even more preferably 19 to 30 amino acids, and most preferably 20 to 26 amino acids. In some embodiments, the linker has 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30 amino acid residues.

[0144] In the present invention, the linker is CD4-derived Hinge, CD8α-derived Hinge, CD28-derived Hinge, CD34-derived Hinge, IgG1-derived Hinge, IgG2-derived Hinge, IgG3-derived Hinge, IgG4-derived Hinge, PLrigid, 2aa GS linker, 6aa [GS]x linker, 10aa [GS]x linker, 10 aa flexible protein domain linker, 8 aa protein domain linker, Flexible linker 2x (GGGS), flexible linker 2x (GGGGS), 13 amino acids linker [GGGS GGGGS GGGS], Split fluorophore linker; Freiburg standard, 15 aa flexible glycine-serine protein domain linker; Freiburg standard, Short Linker (Gly-Gly-Ser-Gly), Middle Linker ( Gly-Gly-Ser-Gly)x2, Long Linker (Gly-Gly-Ser-Gly)x3, Glycine linker, (HL5)2 peptide helical linker, rigid;separate the domains of fusion proteins, Glycine-Serine linker (GSGGS), Glycine-Serine linker (GSSGS), (G2S)3 linker, SEG-Linker, GSAT-Linker, Z-EGFR-1907_Short-Linker, Z-EGFR-1907_Middle-Linker, Z-EGFR-1907_SEG-Linker, (Gly4Ser)3 Flexible Peptide Linker or (SSSSG)x2 serine glycine linker, Whitlow 218 linker(GSTGSGSKPGSGEGSTKG) etc. However, it is not limited to this.

[0145] In the present invention, the linker may perform additional functions, such as 1) improving biological activity, increasing expression yield, and improving pharmacokinetic profile, in addition to fusion of the chimeric protein of the present invention or a variant thereof with a functional domain, 2) not having a negative effect on the expression, secretion, or functional activity of each domain of the fusion protein, and 3) not being limited thereto, as long as it does not exhibit immunogenicity.

[0146] In the present invention, preferably, the linker is QSFGLLDPK (CD3-derived hinge amino acid sequence, SEQ ID NO: 83) or a variant thereof, LSEGDKVKMDSRIQVLSRGVNQT (CD4-derived hinge amino acid sequence, SEQ ID NO: 84) or a variant thereof, KPTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (CD8α-derived hinge amino acid sequence, SEQ ID NO: 85) or a variant thereof, IEVMYPPPYLDNERSNGTIIHVKGKHLCPSPLFPGPSKP (CD28-derived hinge amino acid sequence, SEQ ID NO: 86) or a variant thereof, ELPTQGTFSNVSTNVS (CD34-derived hinge amino acid sequence, SEQ ID NO: 87) or a variant thereof, (GGGGS)n (n is an integer greater than or equal to 1, SEQ ID NO: 88), or It may be, but is not limited to, GSTGSGSKPGSGEGSTKG (Whitlow 218 linker, SEQ ID NO: 89).

[0147] In the present invention, preferably, the linker may include a sequence represented by SEQ ID NO: 87, 88 or 89, but is not limited thereto.

[0148] In the present invention, the fusion protein may include an Fc domain of an immunoglobulin.

[0149] The Fc domain of the immunoglobulin may comprise the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of the immunoglobulin. The Fc domain of the immunoglobulin may not comprise the variable regions of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably may be IgG4.

[0150] In addition, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. In addition, the term "Fc domain variant" as used herein may have a different glycosylation pattern from that of the wild-type Fc domain, or may have increased glycosylation compared to the wild-type Fc domain, decreased glycosylation compared to the wild-type Fc domain, or may have a deglycosylated form. The Fc domain of the immunoglobulin may include an aglycosylated Fc domain. The Fc domain or variant may have sialic acid, fucosylation, or glycosylation content controlled through culture conditions or genetic manipulation of the host.

[0151] Additionally, the sugar chains of the Fc domain of an immunoglobulin can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Furthermore, the Fc domain variant may be a mixed form of the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, or IgM.

[0152] In the present invention, when the fusion protein includes an Fc domain, the chimeric protein of the present invention and / or its variant may be linked to the N'-terminus and / or C'-terminus of the Fc domain, and another fusion domain may be linked to a terminus other than the terminus to which the chimeric protein or its variant is linked.

[0153]

[0154] In the present invention, for example, the fusion protein may include an IL-2 / IL-15 chimeric protein or a variant thereof, or a sushi domain of IL-15 receptor alpha.

[0155] For example, the fusion protein may have the following structural formula (III) or structural formula (IV).

[0156] As a specific example, the fusion protein may include the following structural formula (III) or structural formula (IV):

[0157] N'-X-[L1]lY-[L2]n-[H]m-Fc domain- C' (structural formula (Ⅲ))

[0158] N'-[H]m-Fc domain-[L1]lY-[L2]nX- C' (structural formula (Ⅳ))

[0159] Here,

[0160] N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein,

[0161] X is a chimeric protein comprising IL-2 and IL-15 proteins or a variant thereof,

[0162] Y is IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof,

[0163] L1 and L2 are linkers,

[0164] H is the Fc hinge domain of immunoglobulin,

[0165] l, n, and m are each independently integers greater than or equal to 0 or 1, preferably 0 or 1.

[0166] In the present invention, the Fc hinge domain may be a hinge domain derived from IgG, IgA, IgE, IGD or IgM, and preferably may be a hinge domain of IgG4, but is not limited thereto.

[0167] In the present invention, for example, when the fusion protein is to be presented on the cell membrane surface, a membrane protein or a membrane penetrating domain may be additionally included.

[0168] In the present invention, the fragment of the IL-15Rα protein may include at least one of the IL-15Rα membrane protein domain and the IL-15Rα sushi domain.

[0169] In the present invention, the fusion protein may additionally include an immune regulatory domain or an intracellular signaling domain.

[0170] In the present invention, the immune regulatory domain or intracellular signaling domain is a domain located in the cytoplasmic direction of a membrane-anchored protein, and refers to a site that activates or suppresses an immune response when a target antigen binds to the extracellular domain.

[0171] In the present invention, the immune regulatory domain or intracellular signal transduction domain may be an intracellular signal transduction domain capable of activating natural killer cells to an appropriate level, and may be characterized by being an intracellular signal transduction domain derived from, for example, IL-15Rα, CD3, CD28, CD40L, ICOS, OX40, 4-1BB, TNFR2 DAP10, 2B4, CD3ζ, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3-derived domains, but is not limited thereto.

[0172]

[0173] In the present invention, the fusion protein may be multimerized into dimers, trimers, or more depending on the linker, fusion domain, etc. included. In one embodiment of the present invention, the fusion protein is used in a dimer form through the hinge region including the Fc domain, but is not limited thereto.

[0174]

[0175] As in the embodiments of the present invention, the fusion protein may include or consist of an amino acid sequence selected from, for example, SEQ ID NOs: 90 to 133, but is not limited thereto.

[0176] As in the embodiment of the present invention, the fusion protein may include or consist of, for example, the amino acid sequence of SEQ ID NO: 185, but is not limited thereto.

[0177]

[0178] Nucleic acids and recombinant vectors

[0179] In another aspect, the present invention relates to a nucleic acid encoding the chimeric protein or a variant thereof, or the fusion protein.

[0180] As used herein, “nucleic acid” may be present in cells, a cell lysate, or may be present in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. A nucleic acid of the present invention may be, for example, DNA or RNA.

[0181] In the present invention, the nucleic acid encoding the chimeric protein may include or consist of the nucleic acid sequence of SEQ ID NO: 34.

[0182] In the present invention, the nucleic acid encoding the variant of the chimeric protein may comprise or consist of a nucleic acid sequence of SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or 74.

[0183] In the present invention, the sequence of the nucleic acid encoding the fusion protein can be easily designed based on the amino acid sequence of the chimeric protein or its variant protein described in the present invention or the nucleic acid sequence encoding it, and the amino acid sequence of the linker or fusion domain additionally included.

[0184] In the present invention, the nucleic acid encoding the fusion protein may include or consist of a nucleic acid sequence selected from SEQ ID NOs: 135 to 178, but is not limited thereto.

[0185] In the present invention, the nucleic acid encoding the fusion protein may include or consist of the nucleic acid sequence of SEQ ID NO: 186, but is not limited thereto.

[0186]

[0187] In another aspect, the present invention relates to a recombinant vector containing the nucleic acid of the present invention.

[0188] In the present invention, the recombinant vector can be used without limitation by appropriately selecting a vector known in the art as long as it is a vector capable of inducing protein expression of a nucleic acid encoding the peptide. For example, when Escherichia coli is used as a host, a vector containing a T7 series (T7A1, T7A2, T7A3, etc.), lac, lacUV5, temperature-dependent (λphoA, phoB, rmB, tac, trc, trp or 1PL promoter) can be used, and when yeast is used as a host, a vector containing an ADH1, AOX1, GAL1, GAL10, PGK or TDH3 promoter can be used, and in the case of Bacillus, a vector containing a P2 promoter can be used. However, this is only a listing of some embodiments, and in addition to the vector containing the promoter, as long as it is suitable for the host as a vector containing a promoter for inducing expression of the peptide according to the present invention, a person skilled in the art can appropriately select and use various vectors known in the art without limitation.

[0189] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein. However, the present invention encompasses other forms of vectors known or becoming known in the art that have equivalent functions. Protein expression vectors used in E. coli include the pET series from Novagen (USA); the pBAD series from Invitrogen (USA); the pHCE or pCOLD series from Takara (Japan); and the pACE series from Xenofocus (South Korea). In Bacillus subtilis, protein expression can be achieved by inserting the target gene into a specific region of the genome, or by using vectors such as the pHT series from MoBiTech (Germany). Protein expression is also possible in molds and yeast using genome insertion or self-replicating vectors. Protein expression vectors for plants can be used using the T-DNA system of Agrobacterium tumefaciens or Agrobacterium rhizogenes. Typical expression vectors for expression in mammalian cell cultures are based on, for example, pRK5 (EP 307,247), pSV16B (WO 91 / 08291), and pVL1392 (Pharmingen).

[0190] The term "expression control sequence" refers to a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism. Such control sequences include a promoter for initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. For example, a control sequence suitable for prokaryotes includes a promoter, optionally an operator sequence, and a ribosome binding site. For eukaryotes, this includes a promoter, a polyadenylation signal, and an enhancer. The factor most influencing the level of gene expression in a plasmid is the promoter. Promoters suitable for high expression include the SRα promoter and a cytomegalovirus-derived promoter.

[0191] Any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention. Examples of useful expression control sequences include, in addition to the promoters described above, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd-encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the phosphatases, e.g., Pho5, the promoter of the yeast alpha-mating system, and any other sequence known to regulate the expression of genes in prokaryotes or eukaryotes or their viruses, and any combination thereof. The T7 RNA polymerase promoter Φ can be usefully used to express proteins in E. coli.

[0192] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. This can be a gene and regulatory sequence(s) that are linked in such a way that an appropriate molecule (e.g., a transcriptional activating protein) enables gene expression when bound to the regulatory sequence(s). For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and are in reading frame. However, an enhancer need not be in contact. These sequences are joined by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional methods.

[0193] The term "expression vector" as used herein typically refers to a recombinant carrier into which a fragment of heterologous DNA has been inserted, typically a fragment of double-stranded DNA. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in the host cell. Once within the host cell, the expression vector can replicate independently of the host chromosomal DNA, producing multiple copies of the vector and its inserted (heterologous) DNA.

[0194] As is well known in the art, to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function within the selected expression host. Preferably, the expression control sequences and the gene are contained within a single expression vector that also includes a bacterial selection marker and an origin of replication. If the expression host is a eukaryotic cell, the expression vector may further include expression markers useful in the eukaryotic expression host.

[0195]

[0196] Genetically modified cells expressing chimeric proteins comprising IL-2 and IL-15 proteins, variants thereof and / or fusion proteins comprising the same

[0197] In another aspect, the present invention relates to a cell genetically modified to express a chimeric protein of the present invention, a variant thereof and / or a fusion protein comprising the same.

[0198] In the present invention, the genetically modified cell can be produced by any method without limitation as long as it is modified to express the chimeric protein, its variant, and / or fusion protein.

[0199] In the present invention, preferably, the modified natural killer cell may be characterized in that a nucleic acid encoding the chimeric protein, a variant thereof, and / or a fusion protein, or a recombinant vector including the same, is introduced into a host cell.

[0200] In the present invention, the host cell may refer to an expression cell into which a gene or recombinant vector has been introduced to produce a protein, etc. The host cell may be used without limitation as long as it is a cell capable of expressing the chimeric protein or a variant thereof, or the fusion protein, and is preferably a eukaryotic cell, more preferably a yeast cell, an insect cell, an animal cell, and most preferably an animal cell. For example, a CHO cell line or HEK cell line, which are mainly used for the expression of fusion proteins, may be used, but is not limited thereto.

[0201] A wide variety of expression host / vector combinations can be used to express the chimeric protein or variant thereof, or the fusion protein of the present invention. Suitable expression vectors for eukaryotic hosts include, for example, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors usable in bacterial hosts include bacterial plasmids, exemplified by those obtained from E. coli, such as pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9, and derivatives thereof, plasmids with a wider host range, such as RP4, phage DNA, exemplified by a wide variety of phage lambda derivatives, such as λ and λNM989, and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells include the 2μ plasmid and its derivatives. A useful vector for insect cells is pVL 941.

[0202] The recombinant vector can be introduced into a host cell by methods such as transformation or transfection. The term "transformation" as used herein refers to the introduction of DNA into a host cell, thereby rendering the DNA replicable as an extrachromosomal element or through chromosomal integration. The term "transfection" as used herein refers to the acceptance of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0203] It should be understood that not all vectors and expression control sequences are equally effective in expressing the DNA sequences of the present invention. Similarly, not all hosts will function equally well in the same expression system. However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without undue experimental burden or deviation from the scope of the present invention. For example, when selecting a vector, consideration should be given to the host, as the vector must replicate within it. The vector's copy number, its ability to control copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered. When selecting an expression control sequence, various factors should be considered, such as the relative strength of the sequence, its modulation potential, and its compatibility with the DNA sequences of the present invention, particularly with respect to potential secondary structures. The unicellular host should be selected by considering factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the scope of these variables, those skilled in the art can select various vector / expression control sequence / host combinations that can express the DNA sequence of the present invention in fermentation or large-scale animal culture. When attempting to clone cDNA by expression cloning, the binding method, the panning method, the film emulsion method, etc. can be applied as screening methods.

[0204] The above genes and recombinant vectors can be introduced into host cells using various conventionally known methods. The genes encoding the chimeric protein or its variants, or the fusion protein, can be directly introduced into the genome of the host cell and exist as chromosomal elements. It will be apparent to those skilled in the art that inserting the genes into the host cell's genomic chromosome will have the same effect as introducing a recombinant vector into the host cell.

[0205]

[0206] Method for producing a chimeric protein comprising IL-2 and IL-15 proteins or a variant thereof, and / or a fusion protein comprising the same

[0207] In one aspect, the present invention relates to a method for producing the chimeric protein or a variant thereof, and / or the fusion protein.

[0208] The chimeric protein of the present invention or a variant thereof, and / or the fusion protein can be produced by various protein production methods known in the art.

[0209] In the present invention, the chimeric protein or its variant, and / or the fusion protein can be produced using the genetically modified cell.

[0210] For example, the manufacturing method comprises a step of culturing the genetically modified cell to express the chimeric protein or a variant thereof, and / or the fusion protein;

[0211] It may be characterized by including a step of recovering the expressed chimeric protein or variant thereof, and / or the fusion protein.

[0212] In the present invention, the chimeric protein or its variant, the protein complex, or the fusion protein can also be produced in a cell-free protein synthesis system.

[0213] Cell-free protein synthesis systems are methods for synthesizing proteins in vitro by adding substrates or enzymes to cell lysates or extracts and utilizing the key elements (adenosine triphosphate (ATP), amino acids, etc.) required for protein synthesis. This cell-free protein synthesis system can overcome the shortcomings of existing protein production methods using cells. Cell-free protein synthesis extracts only the intracellular machinery and its factors related to protein production from cells, and artificially repeats only the protein synthesis process outside the cell while excluding the physiological regulatory mechanisms of the cell, thereby mass-producing the target protein in a short period of time. This allows for high-speed protein synthesis without going through a cell culture process. In addition, unlike the cell culture process where protein expression occurs within the space of the cell membrane and cell wall, it is a completely open system with no physical barriers, and has the advantage of being able to freely modify the conditions of protein synthesis for application to various studies. In addition, intracellular protein synthesis systems have significant problems with production and yield if the produced proteins exhibit cytotoxicity, but cell-free protein synthesis systems can produce peptides or proteins without these problems.

[0214] In the present invention, in addition to the examples of host cells for the purpose of protein expression and production, immune cells and the like may be used as host cells and modified to express the chimeric protein of the present invention, variants thereof, and / or fusion proteins comprising the same. In such cases, the function or characteristics of the host cell (e.g., immune cell) may be modified or improved by the expression of the chimeric protein of the present invention, variants thereof, and / or fusion proteins comprising the same.

[0215] In particular, in one embodiment of the present invention, in order to develop genetically modified natural killer cells that maintain an appropriate level of cell activity and cell survival ability for a longer period of time than natural or previously reported modified natural killer cells, when a chimeric protein including IL-2 and IL-15 proteins is expressed in natural killer cells, not only is the expression yield superior to that of other proteins, but also side effects due to excessive immune cell activity of IL-2 are suppressed, and rapid exhaustion of natural killer cells is prevented, thereby maintaining a significantly superior level of cell activity and cell survival ability for a longer period of time. In addition, it was confirmed that the genetically modified natural killer cells exhibit excellent cancer cell killing ability without side effects such as pulmonary edema.

[0216] Accordingly, the present invention relates, in one aspect, to a modified immune cell expressing a chimeric protein comprising IL-2 and IL-15 proteins, a variant thereof and / or a fusion protein comprising the same.

[0217]

[0218] Genetically modified immune cells expressing chimeric proteins comprising IL-2 and IL-15 proteins, variants thereof and / or fusion proteins comprising the same

[0219] The term “modified immune cell expressing a chimeric protein comprising IL-2 and IL-15 proteins, a variant thereof and / or a fusion protein comprising the same” of the present invention means an immune cell genetically modified to express a chimeric protein of the present invention, a variant thereof or a fusion protein comprising the same.

[0220] The immune cells of the present invention may be characterized by being genetically modified to express the chimeric protein, a variant thereof, and / or the fusion protein.

[0221] In the present invention, the immune cells are preferably animal-derived, more preferably human-derived immune cells, and may include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, effector T cells, helper T cells, etc.), natural killer cells (NK cells), NKT cells, dendritic cells, as well as cells capable of differentiating into immune cells, such as hematopoietic stem cells, iPSCs, adult stem cells, etc.

[0222] In the present invention, it is most preferably characterized in that the immune cells are natural killer cells.

[0223] In the present invention, the "natural killer cell" ("NK cell") refers to a type of cytotoxic lymphocyte of the immune system. NK cells provide a rapid response to virus-infected cells and respond to transformed cells. Typically, immune cells detect peptides from pathogens presented on the surface of infected cells by major histocompatibility complex (MHC) molecules, triggering cytokine release and resulting in cell lysis or apoptosis. However, NK cells are unique in that they have the ability to recognize stressed cells regardless of whether peptides from pathogens are present on MHC molecules. These cells were originally named "natural killers" because they did not require prior activation to kill their targets. NK cells are known to be large granular lymphocytes (LGLs) that differentiate and mature in the bone marrow, from which they then enter the circulation.

[0224] In some embodiments, the immune cell is a mammalian immune cell. Examples of "mammalian" or "mammal" include primates (e.g., humans), canines, felines, rodents, porcines, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats, and mice. In certain embodiments, the mammalian immune cell is a human-derived immune cell.

[0225] In the present invention, the chimeric protein, its variant, and / or the fusion protein expressed in the immune cell may be characterized by being secreted extracellularly, expressed on the cell membrane, and / or expressed on the cell surface.

[0226] In the present invention, a signal sequence may be included for transport of the chimeric protein, its variant, and / or the fusion protein.

[0227] In the present invention, the signal sequence (signal peptide or leader sequence) may be located at the N-terminus of the fusion protein. The signal sequence functions to enable the fusion protein to move to a desired location. For example, the signal sequence may be cleaved after being transported to a transport channel such as an endoplasmic reticulum or a translocon, and may be characterized by being cleaved at a residue within the signal sequence, but is not limited thereto.

[0228] In the present invention, the signal sequence (signal peptide or leader sequence) may be located at the N-terminus of the fusion protein. The signal sequence functions to enable the fusion protein to move to a desired location. For example, the signal sequence may be cleaved after being transported to a transport channel such as an endoplasmic reticulum or a translocon, and may be characterized by being cleaved at a residue within the signal sequence, but is not limited thereto.

[0229] In the present invention, the chimeric protein, its variant, and / or the fusion protein may be more preferably characterized by being fixed to the cell membrane of an immune cell or expressed on the cell surface.

[0230] In the present invention, the chimeric protein, its variants and / or fusion proteins may be directly or indirectly (e.g., ionic, non-ionic, covalent) bound (conjugated; fused) to the surface of the immune cell (e.g., on the surface of the immune cell or within the membrane of the cell) using any of various linkers known in the art (Hermanson, G., Bioconjugate Techniques, Academic Press 1996).

[0231] In the present invention, the fusion protein may preferably be characterized as a fusion protein comprising a structure of structural formula (I) or structural formula (II), as in the embodiment, but is not limited thereto.

[0232] In the present invention, the fusion protein may be characterized by being presented on the cell membrane of an immune cell, but is not limited thereto.

[0233] The genetically modified immune cells of the present invention can maintain an appropriate level of immune cell activity and cell survival ability for a long period of time, and thus can be usefully used as a cell immunotherapy agent using the modified immune cells for the prevention or treatment of various diseases.

[0234] Therefore, from another aspect, the present invention relates to the use of the genetically modified immune cells for the prevention or treatment of diseases.

[0235] The present invention relates to a pharmaceutical composition for preventing or treating a disease comprising the genetically modified immune cell.

[0236] The present invention relates to a method for preventing or treating a disease, comprising a step of administering the genetically modified immune cells.

[0237] The present invention relates to the use of the genetically modified immune cells for the manufacture of a pharmaceutical agent for the prevention or treatment of diseases.

[0238] In the present invention, any disease reported to be preventable or treatable using immune cells, preferably natural killer cells, can be used without limitation, and examples thereof include, but are not limited to, cancer, infectious diseases, and autoimmune diseases.

[0239] The above cancer is used with the same meaning as 'tumor', and the preventive or therapeutic composition according to the present invention can be applied to all types of cancer, including solid cancer and blood cancer. Unlike blood cancer, solid cancer refers to cancer that is formed in a mass in an organ, and cancer that occurs in most organs falls under this category. In the present invention, cancer can be selected from the group consisting of colon cancer, melanoma, stomach cancer, liver cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, bladder cancer, kidney cancer, gallbladder cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0240] The preventive or therapeutic pharmaceutical composition according to the present invention may contain a pharmaceutically effective amount of the genetically modified immune cells alone, or may include one or more pharmaceutically acceptable carriers, excipients, or diluents. The "pharmaceutically effective amount" herein refers to an amount sufficient to prevent, improve, and treat disease symptoms.

[0241] The above "pharmaceutically acceptable" means physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal upset or dizziness or similar reactions when administered to humans. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included.

[0242] The composition of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. The formulation can be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.

[0243] The composition of the present invention can be administered through various routes including intravenous, transdermal, subcutaneous, intramuscular or oral, and the dosage of the active ingredient can be appropriately selected depending on various factors such as the route of administration, the patient's age, sex, weight and severity of the condition, and the preventive or therapeutic composition according to the present invention can be administered in combination with a known therapeutic agent or compound having an effect of preventing, improving or treating the target disease.

[0244]

[0245] Although specific amino acid sequences and nucleic acid sequences are described in the present invention, it will be apparent to those skilled in the art that amino acid sequences and nucleic acid sequences encoding the same that are substantially identical to the enzymes to be implemented in the present invention fall within the scope of the present invention. Substantially identical includes cases where the homology of the amino acid or nucleic acid sequences is very high, and also means proteins that share structural characteristics or have the same function as those used in the present invention, regardless of the homology of the sequences. A protein in which a portion of a sequence other than the sequence constituting the core of the present invention is deleted, or a fragment of a nucleic acid sequence encoding the same, may also be included in the present invention, and therefore the present invention includes all amino acid or nucleic acid sequences that have the same function as those used in the present invention, regardless of the length of the fragment.

[0246]

[0247] Example

[0248] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0249]

[0250] Example 1. Selection of IL-2 / IL-15 fusion protein candidates with down-regulated binding affinity to IL-2R (IL-2Rβ) compared to IL-2 and Fc-IL-2.

[0251] Example 1-1: Preparation of a fusion protein comprising an IL-2 / IL-15 chimeric protein, IL-15Rα, and Fc domain

[0252] To produce a fusion protein comprising a chimeric protein comprising an Fc domain, IL-15Rα (sushi domain) and IL-2 / IL-15 or a variant thereof, a nucleic acid of SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 or 74 encoding an amino acid sequence of SEQ ID NO: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75 is synthesized through the gBlock gene fragments service of Integrated DNA Technologies. It was loaded into the pcDNA3.4 vector through cloning (Fig. 1b).

[0253] The structure of the manufactured fusion protein is as follows:

[0254] N'-[hinge domain]-[Fc domain]-[linker]-[IL15Rα / sh]-[linker]-[IL-2 / IL-15 chimeric protein or variant thereof]-C'; or

[0255] N'-[IL-2 / IL-15 chimeric protein or variant thereof]-[linker]-[IL15Rα / sh]-[linker]-[hinge domain]-[Fc domain]-C'

[0256] The vectors constructed above were introduced into CHO cells (Expi-CHO™) to express each fusion protein. After vector introduction, the cells were cultured for 5 days at 37°C, 125 RPM, and CO28%, and the culture medium was collected and the fusion proteins were purified. The names of the purified fusion proteins, the IL-2 / IL-15 chimeric proteins or variants thereof they each contain, and their amino acid and nucleic acid sequence numbers are as follows: Tables 6 and 7

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] GIC-982C1 to GIC-982C15 were purified using an open column containing Protein A resin. The Repligen Protein A resin was washed and equilibrated using PBS and Protein A binding buffer from Thermo. The supernatant, filtered through a 0.22 μm filter, was then reacted with the Protein A resin. The Protein A resin reacted on the PBS-washed open column was collected, and the column was washed with Protein A binding buffer equivalent to 10 times the resin volume. Afterwards, Binding buffer (1 / 10 times the volume of Elution buffer) was added to a collection tube, and then Thermo's IgG elution buffer was added to elute and collect. The collected fusion protein was replaced with PBS buffer.

[0301] At this time, the purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and confirmed by staining with Coomassie blue (Fig. 1c to Fig. 1f).

[0302]

[0303] Example 1-2: Measurement of IL-2Rβ binding affinity using the Octet binding assay

[0304] To confirm the binding affinity of GIC-982C1 to GIC-982C6 and GIC-982C12 to GIC-982C15 to IL-2Rβ, an Octet binding assay was performed using a Ni-NTA sensor chip (ForteBio 18-5101). Fc-IL2 was selected as a control group for analysis. First, the sensor chip was activated by soaking it in distilled water and incubating it for 5 minutes. The sensor was baselined in PBS for 3 minutes, and then immersed in a solution containing 6 μg / ㎖ of IL-2Rβ-his (Acro CD2-H5221) to activate the sensor. After baselined in PBS for 3 minutes, the sensor was immersed in a solution containing 1 / 2 of the test substance serially diluted from 2000 nM to 31.25 nM for 5 minutes to allow association to the IL-2Rβ. At this time, a PBS buffer without the test substance was included as a blank value for the analysis. Afterwards, the cells were transferred back to PBS and dissociated for 10 minutes. All steps were performed at room temperature, and the plates were shaken at 1,000 rpm. The experimental results derived through ForteBio Data Analysis software are shown in Table 8 and Figure 2 below.

[0305] As a result of confirming the binding affinity through Octet, GIC-982C1 to GIC-982C6 and GIC-982C12 to GIC-982C15 showed relatively higher dissociation constants (Kd) than Fc-IL2, confirming that the binding affinity to IL-2Rβ was weakened.

[0306]

[0307] Example 1-3: Confirmation of IL-2 receptor (IL-2R)-mediated STAT5 mechanism activation effect using STAT5 signaling assay

[0308] To evaluate the STAT5 activity of GIC-982C1 to GIC-982C6 and GIC-982C12 to GIC-982C15, HEK-Blue CD122 / 132 cells (Invivogen hkb-il2bg) expressing IL-2Rβ and common γ chain were cultured and the STAT5 activity was analyzed. IL2-Fc was selected as a comparison group for the analysis.

[0309] The HEK-Blue CD122 / 132 cells were cultured using a medium composed of “DMEM (Gibco, Cat No. 10569010) + 10% FBS (Gibco, Cat No. 26140079) + penicillin / streptomycin (Gibco, Cat No. 15140122) + Normocin (Invivogen, Ant-Nr-2) + 1X HEK-Blue™ Selection (Invivogen, Hb-Sel) + 1 ㎍ / ㎖ Puromycin (Invivogen, Ant-Pr-1)”, and when conducting an activity test, “DMEM (Gibco, Cat No. 10569010) + 10% FBS (Gibco, Cat No. 26140079) + penicillin / streptomycin (Gibco, Cat No. 15140122)” medium without the selection antibiotics was used. were used. The cultured cells were harvested by treatment with Trypsin-EDTA, washed with added medium. The harvested HEK-Blue CD122 / 132 cells were centrifuged at 300 xg for 5 minutes, the supernatant was removed, and resuspension was performed by adding medium. After that, the HEK-Blue CD122 / 132 cells were counted and diluted with medium to prepare 2.5 x 10^5 cells / ㎖. The protein of the control group was diluted to the highest concentration to be used in the test (100 ng / ㎖ based on IL-2), loaded onto the plate, and serially diluted by 1 / 10. 20 ㎕ of the diluted protein was loaded onto a flat-bottom plate, and 180 ㎕ (45,000 cells) of the previously prepared cells were added. The plate was cultured for 24 hours at 37℃ and CO₂ 5% conditions.

[0310] To prepare QUANTI-Blue™ Solution (Invivogen, rep-qbs) for detection, 750 ㎕ of QB reagent and QB buffer were added to 73.5 ㎖ of distilled water, vortexed to mix well, and incubated at room temperature for 10 minutes. 20 ㎕ of the supernatant from the plate cultured for 24 hours was loaded onto a new plate, and 180 ㎕ of the previously prepared QUANTI-Blue™ Solution was added to each plate, and incubated for 1 hour in a light-shielded state at 37℃. The absorbance measured at 630 nm using a microplate reader is as shown in Figure 3, and the EC50 results derived from this are as shown in Table 9.

[0311] As a result, GIC-982C1 to GIC-982C5 and GIC-982C13 to GIC-982C15 were confirmed to have relatively lower STAT5 activity intensity than Fc-IL2, and were confirmed to be substances capable of preventing cell hyperactivity while maintaining an appropriate level of cell activity.

[0312]

[0313] Example 1-4: Confirmation of anticancer efficacy and improvement of side effects of IL-2 / IL-15 fusion protein in an in vivo mouse anticancer model.

[0314] To confirm the anticancer effect of IL-2 / IL-15 chimeric protein and improvement of pulmonary edema side effect due to immune hyperactivity, Balb / c mice were injected with 5x10 colon cancer cell line CT26. 5A mouse model was created by subcutaneously administering cells / head. The volume of the generated tumors was measured using a Vernier caliper, and group separation was performed when the volume reached approximately 70 mm3. Afterwards, IL2-Fc, GIC-982C1, GIC-982C4, and GIC-982C15 were administered intraperitoneally to the mice twice a week for two weeks, for a total of four times, at a dose of 10 mg / kg each. The tumor volumes were measured on days 1, 4, 7, 11, 14, 16, and 18 after administration to confirm the anticancer efficacy. The results are shown in Table 10.

[0315] Additionally, the condition of the mice was observed to determine whether there were any adverse effects from the administration. Dead mice were visually inspected for adverse reactions and necropsied to determine the condition of their internal organs. To assess the occurrence of adverse drug reactions, the weight of the excised lungs was measured, dried for two days, and then reweighed to calculate the wet / dry ratio. The results are shown in Table 11.

[0316] As a result, Fc-IL2 caused death due to pulmonary edema caused by immune hyperactivation under conditions of a high dose of 10 mpk, whereas GIC-982C1, GIC-982C4, and GIC-982C15 exhibited excellent cancer cell proliferation inhibitory activity. In addition, Fc-IL2 increased wet lung weight, whereas GIC-982C1, GIC-982C4, and GIC-982C15 showed wet / dry ratios at the same level as the vehicle, confirming that pulmonary edema did not occur.

[0317]

[0318]

[0319] Example 2. Production and efficacy confirmation of immune cells modified to express IL-2 / IL-15 fusion protein.

[0320] Example 2-1: Plasmid construction

[0321] Using a retro-viral vector (Biovec pharma, SinVec(K)-GFP-BSD, Vec-033), two clone vectors containing the amino acid sequences of SEQ ID NOs: 183 and 185 and the nucleic acid sequences of SEQ ID NOs: 184 and 186 as shown in Table 12 below were constructed, and the schematic diagram is as shown in Figure 4A.

[0322] A schematic diagram of an immune cell expressing the IL-2 / IL-15 fusion protein of the above vector is as shown in 4B, and the form of the expressed fusion protein is as follows:

[0323] N'-[IL-2 / IL-15 chimeric protein]-[linker]-[IL15Rα]-C'

[0324]

[0325]

[0326]

[0327] Example 2-2: Retrovirus production and concentration

[0328] To produce BaEV-pseudotyped retrovirus, 6.0 x 10 6293Vec-BaEV cells (Biovec pharma, Vec-006) were cultured in T75 flasks at 37°C in a CO25% incubator for 24 h. When 80% confluent, 15 μg of the plasmid encoding the chimeric protein was transfected using lipofectamine 2000 (Invitrogen, 11668500). 48 h after transfection, the culture medium containing the retrovirus was harvested, centrifuged at 500 × g at 4°C for 10 min, and passed through a 0.45 μm pore PVDF membrane filter (MILLIPORE, SE1M003M00) to remove cell debris. The culture medium containing retrovirus from which cell debris had been removed was mixed with Retro-X concentrator (Clontech, 631456) reagent at a ratio of 3:1 (virus culture medium: Retro-X concentrator reagent) for retrovirus concentration and incubated under 4℃ refrigerated conditions. After 18 hours of incubation, the retrovirus pellet obtained by centrifugation at 1500 xg at 4℃ for 45 minutes was resuspended in Opti-MEM medium (Gibco, 11058021) and concentrated. The concentrated retrovirus was stored at -80℃.

[0329]

[0330] Example 2-3: Preparation of natural killer cells derived from human peripheral blood mononuclear cells (PBMCs)

[0331] Blood collected from different healthy donors was centrifuged at 400 x g for 30 min using a Ficoll-Hypaque gradient, and PBMCs were isolated by collecting the buffy coat. The isolated PBMCs were counted using an ADAM-MC2 automated cell counter (NanoEnTek), and the cells were transferred to a new tube and centrifuged at 350 x g at 4°C for 10 min to obtain CD3(-) cells. After centrifugation, the supernatant was removed and 1 x 10 7 80 ㎕ CliniMACS buffer (20% human serum albumin, EDTA 2 mM) and 22 ㎕ CD3 magnetic beads (Miltenyi biotech, 130-050-101) were dispensed per cell number, the cell pellet was suspended, and the reaction was incubated for 15 minutes at 4℃ under light protection. After the reaction, 10 ㎖ CliniMACS buffer was dispensed for washing, centrifuged at 350 xg, 4℃ for 10 minutes, and 1x10 8 The cell pellet was suspended by dispensing 0.5 ml of CliniMACS buffer per cell.

[0332] After wetting the LD column (Miltenyi Biotec, 130-042-901) with 3 ml of CliniMACS buffer, the cell suspension was passed through the column to obtain CD3(-) cells. For washing, 10 ml of CliniMACS buffer was added, and the cell pellet was centrifuged at 350 xg and 4°C for 10 minutes. The cell pellet was resuspended in CliniMACS buffer and the cell number was measured using an automatic cell counter. After that, 1 x 10 7The cell pellet was suspended by dispensing 80 ㎕ of CliniMACS buffer and 22 ㎕ of CD56 magnetic beads (Miltenyi biotech, 130-050-401) per cell number, and the reaction was allowed to proceed for 15 minutes at 4℃ under light protection. For washing, 10 ㎖ of CliniMACS buffer was dispensed, centrifuged at 350 xg, 4℃ for 10 minutes, and 1x10 8 The cell pellet was suspended by dispensing 0.5 ml of CliniMACS buffer per cell.

[0333] After wetting the LS column (Miltenyi Biotec, 130-042-401) with 3 ml of CliniMACS buffer, the cell suspension was poured. After that, the column was separated from the magnet stand, and 5 ml of CliniMACS buffer was added, and CD3(-) CD56(+) natural killer cells were obtained in a new tube by applying pressure with a piston. The obtained cells were centrifuged at 350 x g at 4°C for 10 minutes. After centrifugation, the cell pellet was resuspended in CliniMACS buffer, and the cell number was measured using an automated cell counter. 10–20 x 10 per ml was used for CryoStor CS10 cryopreservative (Biolife solution, 210102). 6 The cells were suspended to ensure a constant cell count. The suspended cells were dispensed into cryogenic vials (1 ml each), frozen once using a cell freezing container (Corning, CLS432001), and then transferred to an LN2 tank for secondary freezing.

[0334]

[0335] Example 2-4: Production of natural killer cells expressing a chimeric protein containing IL-2 and IL-15

[0336] For the culture of the isolated natural killer cells of Example 2-3, 100 μl of CD335 (NKp46)-biotin and 100 μl of CD2-biotin included in the NK cell activation / expansion kit (Miltenyi Biotec, 130-094-483) were dispensed into a new tube, mixed, and mixed with 500 μl of Anti-biotin MACSIBead. 300 μl of CliniMACS buffer was added to the same tube, and the mixture was prepared by reacting for 2 hours at 4.0 rpm and 4°C using a microtube rotator (Thermo scientific, Hulamixer, 15920D). The frozen natural killer cells of Example 2-3 were thawed, the cell number was measured, and the previously prepared beads were washed with 1 ml of CliniMACS buffer and 1 x 10 6 5 μl of beads per cell were suspended in CN5-101 medium (CTS™ NK-Xpander™ Medium, 1X supplement (Gibco, A5019001), 5% Human AB serum (Milan Analytica AG, #000084) and 50 nM GI-101 (GI-inovation, 8.2 mg / ml)) and inoculated into 6-well plates, and cultured for 4 days at 37°C and 5% CO2.

[0337] To infect the retrovirus prepared in Example 2-2, 10 μg / ml retronectin (Takara, T100B) was coated on a surface-untreated 12-well plate according to the protocol provided by the manufacturer, 300 μl of retrovirus corresponding to an MOI of 1 to 10 was added to each well, and the retrovirus was adsorbed to the retronectin-coated plate by centrifugation at 2,000 xg, 32°C, for 2 hours. After removing the supernatant, the plate was washed once with 1 ml of DPBS (WELGENE, LB001-02). On the 4th day from the start of the culture, the cell number of natural killer cells was measured and 0.8 x 10 6 cells / ㎖ cell density. 1 ㎖ was dispensed into each well plate coated with the prepared natural killer cells, and an additional 0.6 ㎖ of CN5-101 medium was dispensed. Centrifuged at 1,000 x g, 32 ℃, 15 minutes to combine the retrovirus and natural killer cells, and then cultured at 37 ℃, 5% CO2 conditions for 3 days to inoculate 0.5 x 10 6 Cells / ㎖ were inoculated and subcultured.

[0338] Specifically, the expression of the chimeric protein was confirmed using a flow cytometer on the third day of culture after transduction, and 0.5 x 10 6 The same number of cells were cultured at a cell density of 10 cells / ㎖ in CN5-101 medium containing GI-101 and CN5 medium without GI-101 (CTS™ NK-Xpander™ Medium, 1X supplement (Gibco, A5019001), 5% Human AB serum (Milan Analytica AG, #000084)). Afterwards, the cell number was measured under each condition of CN5-101 medium and CN5 medium, and 0.5 x 10 6 Subculture was performed by inoculating 5 ml of cells at a cell density of 10 cells / ml.

[0339]

[0340] Example 2-5: Expression analysis of chimeric proteins containing IL-2 and IL-15

[0341] Natural killer cells were obtained on the third day after retroviral transduction and counted at 0.2 x 10 using an automated cell counter. 6 Cells were placed in a 96-well plate, filled with 250 ㎕ of FACS buffer (DPBS containing 2% FBS), and centrifuged at 350 × g for 5 minutes. After centrifugation, the supernatant was removed, and the cell pellet was suspended in FITC-labeled anti-CD56 antibody (Biolegend, 362546), BV650-labeled anti-IL-15Rα antibody (BD, 747701), and LIVE / DEAD Fixable violet staining sample (Invitrogen, L34955A) and reacted at 4 ° C for 30 minutes. After that, 150 ㎕ of FACS buffer was added, and centrifuged at 350 × g, 4 ° C for 5 minutes. The supernatant was removed, the cell pellet was resuspended in 200-250 μl of FACS buffer, dispensed into a FACS tube, and the expression of IL-15Rα, which constitutes the chimeric protein, was measured using a flow cytometer (FACSymphonyTM A3 Cell analyzer, BD).

[0342] As a result, it was confirmed that natural killer cells expressing a membrane-bound chimeric protein (mbIL-2 / IL-15) containing IL-2 and IL-15 on the cell membrane surface, as shown in Fig. 5, showed an expression efficiency of 88%, which was superior to that of the membrane-bound IL-15 protein (mbIL-15) at 25.1%.

[0343]

[0344] Example 2-6: In-vitro cell counting and cell viability comparison

[0345] The above cultured natural killer cells were divided into the same cell density and cell number on the 3rd day after transduction and cultured for 19 days in CN5 medium conditions and for 25 days in CN5-101 medium supplemented with GI-101. The cells attached to the bottom of the flask were removed with a scraper (SPL, 90020) on days 0, 4, 7, 10, 13, 16, 19, 22, and 25, and the clumped cells were dissociated by pipetting to obtain samples necessary for cell counting. The obtained cells were used to determine the cell density (cell density, x 10) of viable cells using an automatic cell counter. 6 cells / ㎖), cell viability (%), and dead cell count were measured, respectively. The total cell count on days 0 and 4 of culture was calculated by multiplying the cell density of viable cells by the total volume (㎖) of cell culture medium, and thereafter, 0.5 x 10 6 Natural killer cells were suspended and subcultured at a cell density of 5 cells / ㎖. The total cell number after 7 days was calculated as follows.

[0346] Predicted total cell count = live cell density (x 10) 6 cells / ㎖) / inoculated cell density on the previous culture day (0.5 x 10 6 cells / ㎖) x total number of cells on previous culture days (x 10 6 cells)

[0347] The fold expansion value was calculated using the calculated total cell number and the predicted total cell number. The fold expansion rate was calculated by dividing the total cell number or predicted total cell number on each culture day based on the total cell number on day 0 of culture, and the results under culture conditions that stimulated cell proliferation by including GI-101 (a cell proliferation-promoting protein) or excluding GI-101 are shown in Figure 6.

[0348] As a result, it was confirmed that natural killer cells (mbIL-2 / IL-15) expressing a membrane-bound chimeric protein containing IL-2 and IL-15, as shown in Fig. 6, exhibited superior proliferation and survival abilities under both GI-101 (cell proliferation-promoting protein) stimulation and non-stimulation conditions compared to untransformed normal natural killer cells (UTD) and natural killer cells expressing membrane-bound IL-15 protein (mbIL-15).

[0349]

[0350] Example 3. Confirmation of the anticancer efficacy and improvement in side effects of IL-2 / IL-15 fusion protein and natural killer cells expressing it compared to the control group.

[0351] Example 3-1: In vitro cell killing efficacy using various cancer cell lines

[0352] Target tumor cell lines (SKOV3, HCT116, A549) for in vitro cytotoxicity evaluation were infected with lentivirus (Satorious, 4475) containing GFP and puromycin resistance genes, and cultured in R10 medium (10% fetal bovine serum, 1% penicillin-streptomycin) containing 0.5 ㎍ / ㎖ of puromycin. Only transduced tumor cells were selected and cultured. The target tumor cell lines were seeded at 3 x 10 one day before the in vitro cytotoxicity evaluation. 4 Number of cells / mL (A549 and HCT116 cell lines) or 5 x 10 4 The target tumor cell line was suspended in R10 medium at a density of 10 cells / ml (SKOV3 cell line) and dispensed 100 μl per well of a 96-well plate (Corining, 3799) and cultured for 16 hours at 37°C and 5% CO2 conditions.

[0353] The mbIL-2 / IL-15 expressing natural killer cells manufactured by Example 2-4 were thawed, washed, and then dispensed into 96-well plates with target tumor cells prepared the previous day attached at an E / T (effector-to-target) ratio of 10:1 or 3:1. The plates were imaged for each well every 4 hours for 96 hours in a live cell state using an Incucyte®S3 device (Sartorius) installed inside a cell culture incubator. Afterwards, the positive target tumor cells showing GFP fluorescence were counted in the stored images and analyzed.

[0354] As a result, as shown in Fig. 7, mbIL-2 / IL-15 expressing natural killer cells were able to confirm high cell killing ability in three tumor cells compared to UTD and mbIL-15.

[0355]

[0356] Natural killer cells modified to express a chimeric protein comprising IL-2 and IL-15 proteins according to the present invention, a variant thereof, or a fusion protein comprising the same suppress side effects caused by excessive immune cell activity of IL-2, and prevent rapid exhaustion of natural killer cells, thereby maintaining an appropriate level of cell activity and cell survival ability for a long time. Accordingly, the present invention provides a protein complex comprising the chimeric protein comprising IL-2 and IL-15 proteins with excellent innate immune function, a fusion protein comprising the same, and natural killer cells modified to express the same.

[0357]

[0358] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0359]

[0360] Electronic file attached.

Claims

1. A chimeric protein or variant thereof in which the AB loop domain, helix B domain, and CD loop domain of IL-2 are replaced with the AB loop domain, helix B domain, and CD loop domain of IL-15, respectively.

2. A chimeric protein or variant thereof, characterized in that it comprises the amino acid sequence of SEQ ID NO: 33 in the first paragraph.

3. A chimeric protein or variant thereof, characterized in that in the first paragraph, the variant comprises a substitution at at least one amino acid position selected from the group consisting of amino acid positions 73, 74, 76, 77, and 80 in the sequence of SEQ ID NO:

33.

4. A chimeric protein or variant thereof, characterized in that in the first paragraph, the variant comprises at least one amino acid substitution selected from the group consisting of D73E, L74F, S76D, S76E, S76N, S76K, S76L, N77D, and V80L in the sequence of SEQ ID NO:

33.

5. A chimeric protein or variant thereof, characterized in that in the first paragraph, the variant comprises at least one amino acid substitution selected from the group consisting of D73E, L74F, S76D, N77D, and V80L in the sequence of SEQ ID NO:

33.

6. A chimeric protein or variant thereof, characterized in that the variant comprises an amino acid sequence of SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75 in the first paragraph.

7. A fusion protein comprising the chimeric protein of paragraph 1 or a variant thereof.

8. A fusion protein according to claim 7, characterized in that it further comprises an IL-15 receptor alpha protein or a fragment thereof.

9. A fusion protein according to claim 8, characterized in that the fragment of the IL-15 receptor alpha protein comprises a sushi domain of the IL-15 receptor alpha protein.

10. A fusion protein according to claim 7, characterized in that it additionally comprises an Fc domain.

11. A nucleic acid encoding a chimeric protein of paragraph 1 or a variant thereof, or a fusion protein containing the same.

12. A vector containing the nucleic acid of Article 11.

13. A host cell into which the nucleic acid of paragraph 11 or the vector of paragraph 12 has been introduced.

14. A genetically modified immune cell expressing the chimeric protein of paragraph 1 or a variant thereof, or a fusion protein containing the same.

15. A genetically modified immune cell according to claim 14, characterized in that the genetically modified immune cell is a natural killer cell.

16. A genetically modified immune cell according to claim 14, wherein the fusion protein comprises a membrane protein.

17. A genetically modified immune cell according to claim 16, wherein the membrane protein is selected from the group consisting of IL-15 receptor alpha (IL-15Rα), CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, FcεRIγ, T-cell receptor, nicotinic acetylcholine receptor, GABA receptor, and fragments thereof.

18. In claim 14, the genetically modified immune cell characterized in that the fusion protein comprises a structure represented by the following structural formula (I) or structural formula (II): N'-X-[L1]nY-C'(structural formula (I)) N'-Y-[L1]nX-C'(structural formula (II)) Here, X is the chimeric protein or chimeric protein variant, Y is IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof, L1 is the linker, n is an integer greater than or equal to 0 or 1.

19. The chimeric protein of paragraph 1 or a variant thereof; The fusion protein of Article 7; and A pharmaceutical composition for preventing or treating cancer, infectious disease, or autoimmune disease, comprising at least one of the genetically modified immune cells of Article 14.

20. A method for producing a chimeric protein or a variant thereof, or a fusion protein comprising the chimeric protein of claim 1, or a variant thereof, or a fusion protein comprising the same, comprising a step of culturing the host cell of claim 13 to produce the chimeric protein or a variant thereof, or a fusion protein comprising the same.

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