Genetically modified immune cell expressing chimeric protein of il-2 and il-15 proteins, and chimeric antigen protein, and uses thereof

Abstract

The present invention relates to: a genetically modified immune cell modified to express a chimeric protein of IL-2 and IL-15 proteins, and a chimeric antigen protein; and uses thereof. The present invention, by weakening the binding affinity to IL-2Rβ, inhibits side effects caused by excessive immune cell activity by IL-2, and prevents rapid immune cell exhaustion so as to maintain an adequate level of cell activity and cell survival ability for a long time, and thus exhibits excellent activity for a long period compared to existing CAR-expressing immune cells. Thus, the genetically modified immune cell of the present invention is useful for immune system regulation and the treatment of cancer, infectious diseases, immune-related diseases such as autoimmune diseases and the like.

Description

Genetically engineered immune cells expressing chimeric proteins and chimeric antigen proteins of IL-2 and IL-15 proteins and their uses

[0001] The present invention relates to a chimeric protein of IL-2 protein and IL-15 protein or a variant thereof; and a genetically engineered immune cell modified to express a chimeric antigen protein and the use thereof.

[0002]

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

[0004] IL-2 mediates various immune responses by binding to the IL-2 receptor, which is composed of three individual subunits (IL-2R) of IL-2Rα (CD25), β (CD122), and γ (CD132). The binding affinity for the IL-2 protein varies depending on the different binding of each chain of the IL-2 receptor. Specifically, the trimer IL-2 receptor, composed of α, β, and γ chains, exhibits high affinity for the IL-2 protein; the dimeric IL-2 receptor, composed of β and γ, exhibits intermediate binding affinity (approximately 100 times lower) compared to the trimer; however, the monomer IL-2 receptor, composed solely of the α chain, exhibits low binding affinity for the IL-2 protein. Both IL-2 receptor trimers (α,β,γ) and IL-2 receptor dimers (β,γ) are essential for cellular signaling activity following IL-2 binding (Minami et al., Annu Rev Immunol 11, 245-268 (1993)), but IL-2 receptor monomers (α,CD25) are not essential for cellular signaling (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).

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

[0006] IL-2 receptor trimers (α,β,γ) containing the IL-2 receptor α-chain (CD25) exhibit sustainedly high expression in FoxP3+ CD+4 regulatory T cells. On the other hand, conventional immune effector cells in vivo, such as CD8 T cells and NK cells, are known to express IL-2 receptor dimers (β,γ) during the resting phase and transiently express IL-2 receptor trimers (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 primarily synthesized by activated T cells, particularly CD4+ helper T cells, to stimulate T cell proliferation and differentiation, and to induce the production of cytotoxic T lymphocytes (CTLs). Additionally, it 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 generation, 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] In addition, IL-2 is involved in the maintenance of CD4+CD25+ regulatory T cells (Tregs), known as suppressor T cells, and inhibits the activity of effector functions of T cells, NK cells, etc. through cell-to-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] Since IL-2 or IL-15 can increase the lymphocyte population in vivo and enhance the function of said immune cells, they mediate the increase and activation of anticancer agents or immune cells; however, they possess a dual function in immune responses in that they suppress anti-tumor immunity mediated by CD8+ T cells and NK cells by potently expanding Treg cells expressing high-affinity IL-2 receptors. (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] Serious cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, skin, hematological, and systemic side effects occur in patients receiving immunotherapy with IL-2. Therefore, various IL-2 mutations have been studied to improve the therapeutic efficacy of IL-2 and minimize side effects (US 5,229,109 B).

[0011] One way to address the drawbacks of treatments using IL-2 or IL-15 is to extend the in vivo half-life of IL-2 or IL-15 while simultaneously selectively activating CD8+ T cells and NK cells expressing low-affinity IL-2 receptors. Although many attempts have been made to achieve this, there have been no significant results (Arenas-Ramirez, N., et al., Sci Transl Med, 2016. 8(367): p. 367ra166). It has also been studied that continuous stimulation of lymphocytes, particularly 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 in NK cells in vivo, leading to reduced proliferation capacity and decreased anticancer ability due to an imbalance in the activation mechanism (Frutoso et al, Int J Mol Sci 20(18), 4514(2019)). Therefore, there is a need for continuous improvement of IL-2 variants that can minimize hyporesponsiveness in NK cells.

[0013] Natural killer cells modified to express IL-15 as a membrane protein have been studied to have higher cell proliferation and viability than natural killer cells expressing soluble IL-15 (Masaru Imamura et al, Blood, 2014, 124(7), 1081-1088). However, in the case of natural killer cells modified to express IL-15 as a membrane protein, 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] The inventors have developed a chimeric protein and a variant thereof by substituting a portion of the domain of IL-2 with a domain of IL-15, resulting in a weakened binding affinity to IL-2Rβ and lower STAT5 activity intensity compared to wild-type IL-2. They confirmed that the chimeric protein and the variant exhibited excellent cancer cell proliferation inhibitory activity without causing side effects such as pulmonary edema after administration. Furthermore, they confirmed that when a chimeric protein containing IL-2 and IL-15 sequences is expressed in natural killer cells, it exhibits superior proliferation, survival, and sustained activation capabilities compared to natural killer cells expressing a conventional IL-15 cell membrane protein, and have filed a patent application (Korean Patent Application No. 10-2024-0116364).

[0015] Under the background technology described above, the inventors made diligent efforts to improve the in vivo proliferation and sustained efficacy of immune cells into which chimeric antigen receptors were introduced. As a result, they produced natural killer cells that express both the chimeric proteins of IL-2 and IL-15 and the chimeric antigen receptor. They confirmed that these natural killer cells not only exhibit superior proliferation, survival, and sustained activation capabilities compared to natural killer cells expressing IL-15 cell membrane proteins, but also exhibit superior target cell killing ability compared to existing IL-15 armored CAR-NK cells, 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] (Non-patent literature 18) Ilias Christodoulou et al.,J Immunother Cancer. 2021 Dec 7;9(12):e003894.

[0037]

[0038] The object of the present invention is to provide a genetically modified immune cell that expresses a chimeric protein comprising IL-2 and IL-15 proteins and a chimeric antigen receptor.

[0039] Another objective of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of cancer comprising the genetically modified immune cells.

[0040] Another objective of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of infectious diseases comprising the genetically modified immune cells.

[0041] Another objective of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of autoimmune diseases comprising the genetically modified immune cells.

[0042] Another objective of the present invention is to provide a method for preventing and / or treating cancer using the genetically modified immune cells, an use for preventing and / or treating cancer, and a use for manufacturing a drug for preventing and / or treating cancer.

[0043] Another objective of the present invention is to provide a method for preventing and / or treating infectious diseases using the genetically modified immune cells, an use for preventing and / or treating infectious diseases, and a use for manufacturing a drug for preventing and / or treating infectious diseases.

[0044] Another objective of the present invention is to provide a method for preventing and / or treating autoimmune diseases using the genetically modified immune cells, an use for preventing and / or treating autoimmune diseases, and a use for manufacturing a drug for preventing and / or treating autoimmune diseases.

[0045]

[0046] To achieve the above objective, the present invention provides a chimeric protein comprising IL-2 and IL-15 proteins or a variant thereof; and an immune cell genetically modified to express a chimeric antigen receptor.

[0047] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of cancer comprising the genetically modified immune cells.

[0048] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of infectious diseases comprising the genetically modified immune cells.

[0049] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of autoimmune diseases comprising the genetically modified immune cells.

[0050] The present invention also provides a method for preventing and / or treating cancer comprising the step of administering the genetically modified immune cells, and the use of the genetically modified immune cells for the prevention and / or treatment of cancer and for the manufacture of a drug for the prevention and / or treatment of cancer.

[0051] The present invention also provides a method for preventing and / or treating an infectious disease comprising the step of administering the genetically modified immune cell, and the use of the genetically modified immune cell for the prevention and / or treatment of an infectious disease and for the manufacture of a drug for the prevention and / or treatment of an infectious disease.

[0052] The present invention also provides a method for preventing and / or treating an autoimmune disease comprising the step of administering the genetically modified immune cells, and uses of the genetically modified immune cells for the prevention and / or treatment of an autoimmune disease and for the manufacture of a drug for the prevention and / or treatment of an autoimmune disease.

[0053]

[0054] Figure 1a is a schematic diagram of a chimeric protein containing IL-2 and IL-15 of the present invention.

[0055] Figure 1b is a schematic diagram of a fusion protein containing IL-15Ra (sushi domain) and Fc domain.

[0056] Figures 1c to 1f are SDS-PAGE gel images of prepared and purified fusion proteins GIC-982C1 to GIC-982C15.

[0057] Figure 2 is a graph showing the results of the Octet binding assay for Fc-IL2, GIC-982C1 to GIC-982C6 and GIC-982C12 to GIC-982C15.

[0058] Figure 3 is a graph showing the results of the evaluation of STAT-5 cell signaling mechanism activity of Fc-IL2, GIC-982C1 to GIC-982C6 and GIC-982C13 to GIC-982C15.

[0059] Figure 4a shows the structure of the insertion gene and vector for genetically modifying immune cells to express a chimeric protein containing IL-2 protein and IL-15 protein (control group: mbIL-15, test group mbIL-2 / IL-15). Figure 4b is a schematic diagram of natural killer cells expressing the prepared chimeric protein.

[0060] Figure 5 shows FACS data of genetically modified natural killer cells.

[0061] Figure 6 shows the results of confirming the proliferation ability of each natural killer cell according to the culture period.

[0062] Figure 7 shows the results of confirming the killing ability of genetically modified natural killer cells against tumor cells in vitro.

[0063] Figure 8a shows the structure of an insertion gene and a vector for genetically modifying immune cells to express a chimeric protein and a chimeric antigen receptor containing IL-2 protein and IL-15 protein (left side of figure: Bicsitronic_1 vector, right side of figure: Co-Transduction_2 vector). Figure 8b is a schematic diagram of an immune cell expressing the prepared chimeric protein and chimeric antigen receptor containing IL-2 protein and IL-15 protein.

[0064] Figure 9 shows the results of analyzing the expression efficiency between a control group in which a chimeric antigen receptor and a chimeric protein including IL-2 and IL-15 were introduced, and a control group in which a chimeric antigen receptor was introduced alone or a chimeric antigen receptor and IL-15 were introduced together.

[0065] Figure 10 shows the results confirming the anticancer efficacy of NKG2D single CAR against tumor cells in vitro between the control group and the test group. (Control group: Un-TD NK, NKG2D CAR NK x mbIL-15, Test group: NKG2D CAR NK x mb-IL-2 / IL-15)

[0066] Figure 11 shows the results confirming the anticancer efficacy of anti-HER2 single CARs against tumor cells in vitro between the control group and the test group. (Control group: Un-TD NK, anti-HER2 CAR NK x mbIL-15, Test group: anti-HER2 CAR NK x mb-IL-2 / IL-15)

[0067] Figure 12 shows the results confirming the in vitro cytotoxicity of anti-HER2 X NKG2D dual CAR against tumor cells between the control group and the test group. (Control group: Un-TD NK, anti-HER2 X NKG2D CAR NK x mbIL-15, Test group: anti-HER2 X NKG2D CAR NK x mbIL-2 / IL-15)

[0068] Figure 13 shows the number of cells (a) and viability (b) according to culture time when CAR-NK cells were recultured at a cell density of 0.5 × 10⁶ cells / mL with or without the expression of chimeric proteins including IL-2 protein and IL-15 protein.

[0069] Figure 14 shows the duration of anticancer effects on PC3 cells of CAR-NK cells at 0, 6, and 14 days depending on the presence or absence of chimeric protein expression including IL-2 protein and IL-15 protein.

[0070] Figure 15 shows the duration of the anticancer effect of CAR-NK cells on SKOV3 cells at 0, 6, and 14 days depending on the presence or absence of chimeric protein expression including IL-2 protein and IL-15 protein.

[0071] Figure 16 shows the duration of anticancer effects on HCT116 cells of CAR-NK cells at days 0, 6, and 14 depending on the presence or absence of chimeric protein expression including IL-2 protein and IL-15 protein.

[0072] Figure 17 shows the duration of anticancer effects on A549 CAR-NK cells at 0, 6, and 14 days depending on the presence or absence of chimeric protein expression including IL-2 protein and IL-15 protein.

[0073] Figure 18 shows the duration of anticancer effects on K562 CAR-NK cells at 0, 6, and 14 days depending on the presence or absence of chimeric protein expression including IL-2 protein and IL-15 protein.

[0074]

[0075] Specific details for implementing the invention

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0077] All amino acid sequences and nucleic acid sequences described herein may include not only the sequences themselves but also their biological equivalents. For example, all amino acid sequences and nucleic acid sequences described herein are interpreted to include sequences that exhibit substantial identity with the sequence described in the sequence number. The substantial identity described above refers to a sequence that exhibits at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when any other sequence is aligned to correspond as much as possible with the sequence of the present invention described above and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the internet. BLSAT is available at www.ncbi.nlm.nih.gov / BLAST / . Methods for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0078] Each domain included in the chimeric protein, fusion protein, or chimeric antigen described in this specification may include not only the amino acid sequence described in relation thereto, but also a protein or polypeptide in which a part of the said amino acid sequence is substituted through conservative substitution.

[0079] In this specification, “conservative substitution” means a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of the biological or biochemical function of the polypeptide.

[0080] “Conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are defined in the art and are well known. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In general, conservative substitutions in the sequences of polypeptides and proteins described in this specification do not cause a loss of these functions.

[0081]

[0082] The inventors prepared genetically modified natural killer cells to simultaneously express a chimeric protein and a chimeric antigen receptor based on a novel chimeric protein containing IL-2 and IL-15 with reduced binding affinity to IL-2Rβ and STAT5 activity intensity developed in Korean Patent Application No. 10-2024-0116364, and confirmed that the genetically modified natural killer cells exhibited potent target cell killing ability along with superior proliferation and activity maintenance ability compared to existing CAR-NK cells or IL-15 armored CAR-NK cells.

[0083] It will be obvious to a person skilled in the art that the significant effect of the simultaneous introduction of the chimeric protein and CAR prepared in the embodiments of the present invention will also show equivalent effects in other types of cells capable of introducing CAR, such as T cells and Treg cells.

[0084]

[0085] Accordingly, in one aspect, the present invention relates to a chimeric protein or a variant thereof comprising IL-2 protein and IL-15 protein; and an immune cell genetically modified to express a chimeric antigen receptor.

[0086]

[0087] Chimeric proteins including IL-2 and IL-15 proteins

[0088] The term “chimeric protein comprising IL-2 and IL-15 proteins” of the present invention means a protein comprising all or part of the IL-2 amino acid sequence and all or part of the IL-15 amino acid sequence, and may be used interchangeably with “IL-2 / IL-15 chimeric protein” herein.

[0089] The term “IL-2 (Interleukin 2)” in the present invention refers to a protein encoded by the IL-2 gene in humans, also known as T-cell growth factors (TCGF), and is a globular glycoprotein of 15.5 kDa to 16 kDa that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 has a length of 133 amino acids and forms a quaternary structure consisting of four antiparallel, amphipathic α-helices.

[0090] In the case of human IL-2, IL-2 can be configured to include 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 linked, and it has been reported that the "AB loop" domain, "Helix B" domain, and "CD loop" domain may be involved in IL-2Rα binding, and the "Helix C" domain may be involved in IL-2Rβ binding (Cassell, Current Pharmaceutical Design, 2002, 8, 2171-2183).

[0091] The amino acid or nucleic acid sequences of human IL-2 are as listed in Table 1 below:

[0092]

[0093] The term “IL-15 (Interleukin 15)” in the present invention refers to a protein encoded by the IL-15 gene in humans, which is a glycoprotein of 14 kDa to 15 kDa that exists in a membrane-bound form attached to the cell surface through binding with IL-15 receptor alpha (IL-15Rα) present on the cell surface, and the IL-15 / IL-15Rα complex binds to IL-2Rβ (CD122) and γ (CD132) to share a cell signaling mechanism with IL-2.

[0094] 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 may be involved in IL-15Rα binding (Lowe, Journal of Molecular Biology, 2011, 406, 160-175).

[0095] The sequence of human IL-15 is as listed in Table 2 below:

[0096]

[0097] Since full-length IL-2 and IL-15 proteins can increase lymphocyte populations and enhance the function of immune cells in vivo, IL-2 or IL-15 can be used to mediate the increase and activation of anticancer agents or immune cells. However, they possess a dual function in immune response in that they suppress antitumor immunity mediated by immune cells such as CD8+ T cells and natural killer cells by strongly expanding regulatory T cells expressing high-affinity IL-2 receptors. In particular, repeated administration of IL-2 or IL-15 proteins induces hyporesponsiveness in immune cells (e.g., natural killer cells), leading to reduced proliferative capacity, rapid exhaustion, and decreased anticancer ability due to an imbalance in activation mechanisms.

[0098] In one embodiment of the present invention, the chimeric protein may be characterized by comprising a portion of the IL-2 amino acid sequence and a portion of the IL-15 amino acid sequence.

[0099] 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 connected directly or indirectly through a linker, etc.

[0100] In one embodiment of the present invention, the chimeric protein may be characterized by being prepared in which a portion of the IL-2 amino acid sequence is substituted with an IL-15 amino acid sequence. In this specification, a protein comprising an amino acid sequence in which a specific amino acid sequence is substituted is used interchangeably with "hybrid protein."

[0101] When a portion of the IL-2 amino acid sequence is substituted with the IL-15 amino acid sequence, as in the chimeric protein of the present invention, the intensity of the signaling mechanism of IL-2Rβ (CD122) and IL-2Rγ (CD132) dimer receptors is regulated to prevent excessive activation of immune cells (e.g., natural killer cells), and at the same time, cell exhaustion is delayed, thereby maintaining the activity of immune cells for a long time.

[0102] In one embodiment of the present invention, at least one domain selected from the group consisting of the “Helix A” domain, “AB loop” domain, “Helix B” domain, “BC loop” domain, “Helix C” domain, “CD loop” domain and “Helix D” domain of IL-2 may be substituted with the “Helix A” domain, “AB loop” domain, “Helix B” domain, “BC loop” domain, “Helix C” domain, “CD loop” domain or “Helix D” domain of IL-15.

[0103] 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.

[0104] In a preferred embodiment of the present invention, the “AB loop” domain, “Helix B” domain and “CD loop” domain of IL-2 may be substituted 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 be composed of the amino acid sequence of SEQ ID NO. 33 or the nucleic acid sequence of SEQ ID NO. 34 encoding it as listed in Table 3 below.

[0105]

[0106] Variants of chimeric proteins including IL-2 and IL-15 proteins

[0107] In another aspect, the present invention relates to a variant of a chimeric protein comprising the IL-2 and IL-15 proteins.

[0108] In the case of a variant of a chimeric protein in which a portion of the amino acid sequence of the IL-2Rβ binding domain of IL-2 is mutated as in the present invention, the binding affinity to IL-2Rβ is further weakened and the STAT5 activity intensity is lowered, thereby preventing excessive activation of immune cells and delaying cell exhaustion, which can maintain the activity of immune cells for a long time.

[0109] In this specification, the term "variant" is used as a concept that includes not only variations of some amino acid residues in the wild-type amino acid sequence, preferably substitutions, deletions, insertions, etc. of amino acid residues, but also cleavages of some amino acid residues at the N-terminus and / or C-terminus; therefore, in the present invention, the term "variant of a chimeric protein" is used in a broad sense that includes "fragments of a chimeric protein variant."

[0110] In this specification, "substitution" means a modification comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of biological and / or biochemical function.

[0111] In one embodiment of the present invention, a variant of the chimeric protein may include a mutation, preferably a substitution of an amino acid, at at least one of the following positions with respect to the amino acid sequence of SEQ ID NO. 33: aspartic acid at position 73 (Asp(D), Aspartic acid), leucine at position 74 (Leu(L), Leucine), serine at position 76 (Ser(S), Serine), asparagine at position 77 (Asn(N), Asparagine), and valine at position 80 (Val(V), Valine).

[0112] In one embodiment of the present invention, a variant of the chimeric protein may include at least one amino acid substitution among D73E, L74F, S76D, S76E, S76N, S76K, S76L, N77D, and V80L with respect to the amino acid sequence of SEQ ID NO. 33.

[0113] 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 variant selected from D73E / L74F / S76D / N77D / V80L, D73E / L74F / S76E / N77D / V80L, D73E / L74F / S76N / N77D / V80L, D73E / L74F / S76K / N77D / V80L, and D73E / L74F / S76L / N77D / V80L.

[0114] In a preferred embodiment of the present invention, a variant of the chimeric protein may include at least one amino acid substitution among D73E, L74F, S76D, N77D, and V80L with respect to the amino acid sequence of SEQ ID NO. 33, and most preferably may include an amino acid substitution of S76D.

[0115] In a preferred embodiment of the present invention, the amino acid sequence or nucleic acid sequence of the variant of the chimeric protein may include or be composed of the amino acid sequence of SEQ ID NOs 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75 listed in Table 4 below, or the nucleic acid sequence of SEQ ID NOs 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, or 74 encoding the same.

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] * Underlines in the amino acid sequence indicate mutation locations

[0122]

[0123] Expression forms of chimeric proteins or variants thereof, including IL-2 and IL-15 proteins

[0124] In the present invention, the chimeric protein or its variant can be expressed in various forms without limitation in genetically modified immune cells.

[0125] In the present invention, the chimeric protein or a variant thereof in the genetically modified immune cell may be characterized by being expressed and secreted extracellularly in the genetically modified immune cell, expressed on the cell membrane, and / or expressed on the cell surface.

[0126] In the present invention, a signal sequence may be included for the transport of the chimeric protein or a variant thereof.

[0127] In the present invention, the signal sequence (signal peptide or leader sequence) may be located at the N-terminus of the chimeric protein or a variant thereof. The signal sequence functions to enable the expressed protein to move to a desired location. For example, the signal sequence may be cleaved after being moved to a transport channel such as an endoplasmic reticulum or a translocone, and may be characterized by cleaving at a residue within the signal sequence, but is not limited thereto.

[0128] In the present invention, more preferably, the chimeric protein or its variant may be characterized by being fixed to the cell membrane of a genetically modified immune cell or presented on the cell surface.

[0129] In the present invention, most preferably, the chimeric protein or its variant may be characterized by being immobilized on the cell membrane of a genetically modified immune cell and presented on the cell surface.

[0130] In the present invention, various domains may be fused to allow the chimeric protein or its variant to be expressed and anchored to a cell membrane. The expression forms of the chimeric protein or its variant according to the present invention into various fusion proteins are described in detail in the section concerning fusion proteins containing chimeric proteins or variants thereof including IL-2 and IL-15 proteins.

[0131] In the present invention, the chimeric protein or a variant thereof may be directly or indirectly (e.g., ionic, non-ionic, covalent) conjugated (conjugated; fused) to the surface of the immune cell (e.g., on the surface of the immune cell and / or within the membrane of the cell) using any various linker known in the art (see reference [Hermanson, G., Bioconjugate Techniques, Academic Press 1996]).

[0132]

[0133] In the present invention, the chimeric protein or its variant may be expressed alone without fusion with another domain, or may be expressed in the form of a fusion protein comprising another fusion domain or amino acid.

[0134] In the present invention, more preferably, the chimeric protein or a variant thereof may be expressed in the form of a fusion protein comprising other fusion domains and / or amino acids.

[0135] In the present invention, the fusion protein means that an additional peptide sequence (fusion domain) is fused to a chimeric protein or a variant thereof.

[0136] The term "fusion domain" in the present invention refers to an additional domain or moiety that may be directly or indirectly bound to and included in the chimeric protein or a variant thereof of the present invention, and the term "fusion domain" is used to include both polypeptides that possess function and polypeptides that do not possess function.

[0137] In the present invention, the fusion domain may be fused to confer expression characteristics such as secretion from a cell, cell surface expression or anchoring to a cell membrane, or intracellular localization; to add a substrate or other recognition sequence for post-translational modification; or to modify tissue localization, tissue exclusion or other ADME characteristics, or to add a protein or peptide having another function, but is not limited thereto.

[0138] 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 co-stimulatory 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 or its variant comprising the IL-2 and IL-15 proteins of the present invention.

[0139] In the present invention, when the chimeric protein or a variant thereof is to be fixed to a cell membrane or expressed on a cell surface, the chimeric protein or a variant thereof may be expressed as a fusion protein including a membrane protein or a transmembrane domain.

[0140] In the present invention, "membrane protein" refers to a protein inserted into or attached to the surface of a membrane composed of a lipid bilayer, and includes "intrinsic membrane proteins" which are proteins inserted into the lipid bilayer, and "extrinsic membrane proteins" which are proteins attached to the surface of the lipid bilayer. "Intrinsic membrane proteins" belong to one of the types of "membrane proteins" that cross the membrane.

[0141] In the present invention, "intrinsic membrane protein" is a protein that penetrates the cell membrane and comprises a "transmembrane domain" that penetrates the membrane, an "intracellular domain" located inside the cell, and an "extracellular domain" exposed outside the cell. The number of transmembrane domains varies depending on the membrane protein. Since the lipid bilayer is composed of highly hydrophobic lipids, transmembrane domains passing through this region are mostly composed of hydrophobic amino acids. Almost all transmembrane domains are composed of an α-helical structure. In membrane proteins having multiple transmembrane domains, the transmembrane domains are arranged in a circular pattern to form a cylindrical structure. This cylindrical structure is used as a channel to transport specific ions or biological substances by opening and closing in response to specific signals. The transmembrane domains of an extremely limited number of membrane proteins are composed of a β-sheet structure. A cylindrical structure formed by the circular arrangement of multiple domains composed of a β-sheet structure is called a "β-barrel."

[0142] In the present invention, in the case of "external membrane proteins," a protein molecule and a lipid molecule are connected by a covalent bond to form a lipoprotein, and as a result of hydrophobic bonding occurring 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; an example is the GPI (Glycosylphosphatidylinositol)-fixation protein. By inserting the fatty acid of GPI into the lipid monolayer and connecting the phosphate group on the other side to the protein by a covalent bond, GPI fixes the protein to the surface of the lipid bilayer.

[0143] In the present invention, examples of the membrane protein include receptors, ligands, immunoglobulins, glycophorins, or combinations thereof. The membrane protein may be characterized as being selected from the group consisting of, but is not limited to, IL-15 receptor alpha (IL-15Rα), CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, FcεRIγ, T-cell receptors (TCR, e.g., TCRα and / or TCRβ), nicotinic acetylcholine receptors, GABA receptors, and fragments thereof. Specific examples of immunoglobulins include IgG, IgA, IgM, IgE, IgD, or combinations thereof. Specific examples of glycophorins include, but are not limited to, glycophorin A, glycophorin D, or combinations thereof.

[0144] In the present invention, the chimeric protein or a variant thereof may be fused with a hinge domain and / or a sushi domain in addition to a transmembrane domain. The term “hinge domain” in the present invention refers to a series of amino acid sequences existing between the transmembrane domain and the extracellular domain of a membrane-fixed 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, as well as a chimeric protein form in which domains derived from different proteins are combined.

[0145] In one embodiment of the present invention, the chimeric protein or a variant thereof may be expressed as a fusion protein comprising an IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof.

[0146] 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.

[0147] 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 the cell membrane permeability 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.

[0148] In the present invention, human IL-15 receptor alpha (IL-15Rα) and the sequence of its fragments are, for example, as described in Table 5 below, but are not limited thereto.

[0149]

[0150] In the present invention, the chimeric protein or its variant; and the IL-15 receptor alpha (IL-15Rα) protein may be characterized as being directly or indirectly connected, for example, by being connected by a linker.

[0151] In one embodiment of the present invention, the chimeric protein or a variant thereof may be characterized as being expressed as a fusion protein comprising the structure of structural formula (I) or structural formula (II) below.

[0152] N'-X-[L1]nY-C'(structural formula(I))

[0153] N'-Y-[L1]nX-C'(structural formula(II))

[0154] Here,

[0155] N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, and

[0156] X is a chimeric protein containing IL-2 and IL-15 proteins or a variant thereof, and

[0157] Y is the IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof, and

[0158] L1 is the linker, and

[0159] n is an integer greater than or equal to 0 or 1, preferably 0 or 1.

[0160] In the present invention, the fragment of the IL-15Rα protein may include one or more of the IL-15Rα membrane protein domain and the IL-15Rα sushi domain. For example, the IL-15Rα membrane protein domain may include or be composed of the amino acid sequence represented by SEQ ID NO. 79, and the IL-15Rα sushi domain may include or be composed of the amino acid sequence represented by SEQ ID NO. 81, but is not limited thereto.

[0161] In the present invention, the linker may be any of the various linkers known in the art. For example, the linker may include a hinge domain between a transmembrane domain and an extracellular domain, a flexible linker, a rigid linker, etc., but is not limited thereto.

[0162] More specifically, the linker refers to 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 fewer than 100, amino acids, e.g., 2 to 60 amino acids, preferably 10 to 40 amino acids, more preferably 15 to 40 amino acids, much more preferably 19 to 30 amino acids, 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.

[0163] In the present invention, the linker comprises a CD4-derived Hinge, a CD8α-derived Hinge, a CD28-derived Hinge, a CD34-derived Hinge, an IgG1-derived Hinge, an IgG2-derived Hinge, an IgG3-derived Hinge, an IgG4-derived Hinge, PLrigid, a 2aa GS linker, a 6aa [GS]x linker, a 10aa [GS]x linker, a 10 aa flexible protein domain linker, an 8 aa protein domain linker, a Flexible linker 2x (GGGS), a flexible linker 2x (GGGGS), a 13 amino acids linker [GGGS GGGGS GGGS], a Split fluorophore linker; a Freiburg standard, and a 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 It may be linker(GSTGSGSKPGSGEGSTKG), etc., but It is not limited.

[0164] In the present invention, the linker may perform additional functions in addition to the fusion of the chimeric protein or a variant thereof with the functional domain, such as 1) improving biological activity, increasing expression yield and improving pharmacokinetic profile, 2) not negatively affecting the expression, secretion and / or functional activity of each domain of the fusion protein, and 3) not exhibiting immunogenicity, but is not limited thereto.

[0165] In the present invention, preferably, the linker is QSFGLLDPK (CD3-derived hinge amino acid sequence, SEQ NO. 83) or a variant thereof, LSEGDKVKMDSRIQVLSRGVNQT (CD4-derived hinge amino acid sequence, SEQ NO. 84) or a variant thereof, KPTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (CD8α-derived hinge amino acid sequence, SEQ NO. 85) or a variant thereof, IEVMYPPPYLDNERSNGTIIHVKGKHLCPSPLFPGPSKP (CD28-derived hinge amino acid sequence, SEQ NO. 86) or a variant thereof, ELPTQGTFSNVSTNVS (CD34-derived hinge amino acid sequence, SEQ NO. 87) or a variant thereof, (GGGGS)n (n is an integer greater than or equal to 1, SEQ NO. 88), or It may be GSTGSGSKPGSGEGSTKG (Whitlow 218 linker, sequence number 89), but is not limited thereto.

[0166] In the present invention, preferably, the linker may include the sequence represented by SEQ ID NO. 87, 88, or 89, but is not limited thereto.

[0167] In the present invention, the chimeric protein or a variant thereof may be expressed as a fusion protein comprising the Fc domain of immunoglobulin.

[0168] The Fc domain of the immunoglobulin may include a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) of the immunoglobulin. The Fc domain of the immunoglobulin may not include a variable region of the heavy and light chains and a light chain constant region 1 (CH1) of the immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4.

[0169] Additionally, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. Furthermore, the term "Fc domain variant" as used herein may differ from the glycosylation pattern of the wild-type Fc domain, or may have an increased glycosylation pattern compared to the wild-type Fc domain, a decreased glycosylation pattern compared to the wild-type Fc domain, or 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, and glycosylation whose content is controlled through culture conditions or genetic manipulation of the host.

[0170] In addition, the glycosylation of the Fc domain of 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 in a mixed form of the Fc regions of immunoglobulin IgG, IgA, IgE, IgD, or IgM.

[0171] In the present invention, when the fusion protein comprises an Fc domain, the chimeric protein of the present invention or a variant thereof may be connected to the N' end and / or C' end of the Fc domain, and another fusion domain may be connected to a end other than the end to which the chimeric protein or a variant thereof is connected.

[0172] 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.

[0173] For example, the fusion protein may be like the following structural formula (III) or structural formula (IV).

[0174] In one embodiment, the fusion protein may include the following structural formula (III) or structural formula (IV):

[0175] N'-X-[L1]lY-[L2]n-[H]m-Fc domain- C' (structural formula(III))

[0176] N'-[H]m-Fc domain-[L1]lY-[L2]nX- C' (structural formula(IV))

[0177] Here,

[0178] N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, and

[0179] X is a chimeric protein containing IL-2 and IL-15 proteins or a variant thereof, and

[0180] Y is the IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof, and

[0181] L1 and L2 are linkers,

[0182] H is the Fc hinge domain of immunoglobulin, and

[0183] l, n, and m are each independently integers of 0 or 1 or greater, preferably 0 or 1.

[0184] 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.

[0185] In the present invention, for example, when presenting the chimeric protein or a variant thereof on the surface of a cell membrane, it may be expressed as a fusion protein further comprising a membrane protein or a membrane-penetrating domain.

[0186] In the present invention, the fragment of the IL-15Rα protein may include one or more of the IL-15Rα membrane protein domain and the IL-15Rα sushi domain.

[0187] In the present invention, the chimeric protein or a variant thereof may be expressed as a fusion protein further comprising an immune regulatory domain or an intracellular signal transduction domain.

[0188] In the present invention, the immune regulatory domain or intracellular signaling domain refers to a domain located in the cytoplasmic direction of a membrane-fixed protein, which activates or inhibits an immune response.

[0189] In the present invention, the immune regulatory domain or intracellular signal transduction domain may be an intracellular signal transduction domain capable of activating immune cells to an appropriate level, and may be characterized as 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 a domain derived from B7-H3, but is not limited thereto.

[0190] In the present invention, the fusion protein may be polymeric into a dimer, a trimer, or more depending on the linker, fusion domain, etc. In one embodiment of the present invention, the fusion protein containing the Fc domain was used in a dimer form through the hinge region, but is not limited thereto.

[0191] As in the embodiments of the present invention, the fusion protein may include or be composed of an amino acid sequence selected from SEQ ID NOs 91 to 134, in a preferred example.

[0192] As in the embodiments of the present invention, the fusion protein may include or be composed of the amino acid sequence of SEQ ID NO. 94, most preferably.

[0193]

[0194] Chimeric Antigen Receptor (CAR)

[0195] The genetically modified immune cell of the present invention is characterized by expressing a chimeric antigen receptor together with the chimeric protein or a variant thereof.

[0196] The present invention was completed by confirming that co-expression through the introduction of a chimeric protein containing IL-2 and IL-15 or a variant thereof into CAR-NK cells can improve the proliferative capacity of CAR-NK cells and improve the sustainability of efficacy.

[0197] Therefore, it is readily understood that the examples of chimeric antigen receptors described below and the chimeric antigen receptors used in the embodiments of the present invention are described and used exemplarily to represent CAR-NK, and are not limited thereto.

[0198] Accordingly, in the present invention, the described chimeric antigen receptor is used in the sense that it includes, without limitation, conventionally known chimeric antigen receptor technologies or future known chimeric antigen receptor technologies that can be introduced into NK cells. The term “Chimeric antigen receptor (CAR)” of the present invention is a synthetic structure designed to induce an immune response against a target antigen and a cell expressing the corresponding antigen.

[0199] In the present invention, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0200] First-generation CARs included an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and used only CD3ζ as the signaling domain, but had problems such as minimal therapeutic effect on cancer and short duration.

[0201] To enhance responsiveness to immune cells, a second-generation CAR was manufactured by combining a co-stimulatory domain (CD28 or CD137 / 4-1BB) with CD3ζ, and compared to the first-generation CAR, the number of CAR-containing immune cells remaining in the body increased significantly. While the second-generation CAR utilized a single co-stimulatory domain, the third-generation CAR utilized two or more co-stimulatory domains. To achieve the expansion and persistence of CAR-containing immune cells in vivo, the co-stimulatory domain can be combined with 4-1BB, CD28, or OX40, etc.

[0202] 4th generation CARs include additional genes encoding cytokines such as IL-12 or IL-15 to allow for additional expression of CAR-based immune proteins of the cytokines, and 5th generation CARs additionally include an interleukin receptor chain, e.g., IL-2Rβ, for immune cell enhancement.

[0203] In the present invention, the extracellular domain may include one or more extracellular binding domains.

[0204] In the present invention, the extracellular binding domain may be characterized by binding to two or more target antigens.

[0205] The term "extracellular binding domain" refers to a portion of the CAR comprising an antigen binding domain having the ability to specifically bind to a target antigen. The extracellular binding domain may comprise any protein, polypeptide, oligopeptide, or peptide having the ability to specifically recognize and bind to a biological molecule (e.g., cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or cytokine, etc.). The binding domain comprises any naturally occurring, synthetic, semi-synthetic, or recombinated binding partner for the target biological molecule.

[0206] The term "specifically binding" in the present invention means the binding of a molecule to another molecule with a binding affinity greater than that of background binding. For example, an extracellular binding domain specifically binds to a target molecule when it binds to or associates with the target molecule with an affinity of about 10⁻⁵ M or greater or Ka (i.e., the equilibrium dissociation constant of a specific binding interaction having units of 1 / M). Alternatively, the affinity may be defined as the equilibrium dissociation constant of a specific binding interaction (Kd) having units of M (e.g., 10⁻⁵ M to 10⁻¹³ M or less).

[0207] The affinity of the extracellular binding domain and CAR according to the present invention can be easily measured by binding association or substitution analysis using conventional techniques, for example, competitive ELISA (enzyme-linked immunosorbent assay) or labeled ligands, or surface-plasmon resonance devices such as the Biacore T100 (available from Biacore Incorporated, Piscataway, NJ), or optical biosensor technologies such as EPIC systems or EnSpire available from Corning and Perkin Elmer, respectively.

[0208] In the present invention, the target antigen of the chimeric antigen receptor may include, without limitation, any molecule intended to induce immune activity of the genetically modified immune cells of the present invention. For example, the target antigen of the chimeric antigen receptor may be an antigen expressed in a specific organ, tissue, or cell in vivo, a biomarker of a specific disease, a molecule related to the pathology of a specific disease, or an immunomodulatory molecule, but is not limited thereto.

[0209] In the present invention, the target antigen of the chimeric antigen receptor may be characterized as, for example, an antigen present in an individual, organ, tissue, cell, or microenvironment having a tumor, autoimmune disease, or infectious disease, but is not limited thereto.

[0210] In the present invention, the target antigen of the chimeric antigen receptor may be characterized as being a tumor-associated antigen. In the present invention, tumor-associated antigens may include all molecules expressed in a tumor or in the surrounding environment of a tumor, for example, tumor 4-1BB, 5T4, integrin, activin, amyloid beta, angiopoetin (angiopoetin 1 or 2), angiopoetin analog 3, B cell maturation antigen (BCMA), B-cell activating factor (BAFF), B7-H3, complement 5, CCR4, CCR5, CCL11, CD2, CD3, CD4, CD6, CD11a, CD16A, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD32B, CD33, CD38, CD40, CD45, CD46, CD47, CD52, CD56, CD62, CD70, CD73, CD74, CD79b, CD80, CD105, CD123, CD154, CD166, CD262, CD278, CD319, CD326, Carcinoembryonic antigen (CEA), CGRP, Claudin-18, c-Met, CSF-1, CSF-1 receptor, CTLA4, DLL3, EGF receptor, Hemophilia factor, Fc receptor, FGF23, Folate receptor, GD2, Glucocorticoid-induced TNF receptor (GITR), Glypican 3, GM-CSF, HER2, HER3, TROP2, Hepatocyte Growth Factor (HGF), Interferon receptor, Interferon gamma, IgE, IGF-1 receptor, Interleukin 1, Interleukin 2 receptor, Interleukin 4, Interleukin 4 receptor, Interleukin 5,Interleukin-5 receptor, Interleukin-6, Interleukin-6 receptor, Interleukin-8, Interleukin-12 / 23, Interleukin-13, Interleukin-17A, Interleukin-17 receptor A, Interleukin-23, Interleukin-31 receptor, Interleukin-36 receptor, Lymphocyte-activation gene 3 (LAG3), Lysyl oxidase homolog 2 (LOXL2), Mesothelin, Mucin-1, Mucin-16, Nectin-4, Nerve Growth Factor (NGF), OX40, Proprotein Convertase Subtilisin / Kexin type 9 (PCSK9), PD-1, PD-L1, Phospholipase C, RANKL (Receptor activator of nuclear factors kappa B ligand), Tyrosine-protein kinase transmembrane receptor (ROR1), Sialic acid binding Ig-like lectin 15 (Siglec-15), Transforming growth factor beta (TGFβ), TIGIT (T-cell innunoreceptor with immunoglobulin and ITIM domain), T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), Tissue factor, Tissue factor pathway inhibitor (TFPI), TORP-2, Tumor necrosis factor (TNF),It may be selected from the group consisting of thymic stromal lymphopoietin (TSLB), colony-stimulating factor 1 receptor (CSF1R), vascular endothelial growth factor (VEGF), VEGF receptor, vWF (von Willebrand Factor), and NKG2DL; in the embodiments of the present invention, a chimeric antigen receptor targeting HER-2 and NKG2DL was used as a representative example, but is not limited thereto.

[0211] In the present invention, the target antigen of the chimeric antigen receptor may be characterized as being an autoimmune disease-related antigen. In the present invention, the autoimmune disease-related antigen may include all molecules related to autoimmunity, and the autoimmune disease-related antigen may be an immune cell target antigen, an autoantibody or a fragment thereof, or a cytokine, but is not limited thereto. The autoimmune disease-related antigen may be selected from the group consisting of, for example, CD19, CD20, BCMA, TACI (Transmembrane Activator and Calcium-Modulator and Cyclophilin Ligand Interactor), CD5, CD4, PD-1, IL-17RA, BAFF (B-Cell Activating Factor), APRIL (A Proliferation-Inducing Ligand), TNF-α, IL-2, IL-6R, CD95, CTLA-4, CCR7, and CXCR4, but is not limited thereto.

[0212] In the present invention, the target antigen of the chimeric antigen receptor may be characterized as being an antigen associated with an infectious disease. In the present invention, the antigen associated with an infectious disease may be characterized as being a specific antigen expressed by an infectious agent (e.g., virus, bacteria, fungi, parasites, etc.) or an immunomodulatory molecule that pathologically occurs in an infectious disease (e.g., inflammatory cytokine), but is not limited thereto. In the present invention, the extracellular binding domain may include an antibody that specifically binds to the target antigen or an antigen-binding fragment thereof.

[0213] The "antibody" comprises an antibody and an antibody fragment containing at least one binding site that specifically binds to a specific target of interest, wherein the target may be an antigen or a receptor capable of interacting with a specific antibody. The "antibody" comprises, but is not limited to, an immunoglobulin molecule or an antigen-binding or receptor-binding portion thereof. The antigen or receptor to which the antibody binds, or a specific fragment or portion of the target, is generally known as an epitope or an epitope. The "antibody" also comprises, but is not limited to, natural antibodies and their variants, fragments of natural antibodies and their variants, peptibodias and their variants, and antibody mimics that mimic the structure and / or function of an antibody or a specific fragment or portion thereof, including single-strand antibodies and their fragments. The antibody may be a mouse antibody, a human antibody, a humanized antibody, a camel IgG, a single variable neoantigen receptor (VNAR), a shark heavy chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single-domain antibody (dAb), an anti-individual-specific antibody, a bispecific, multispecific, or multimeric antibody, or a fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single-chain fragment variable (scFv), tandem scFv(scFv)2, single-chain Fab(scFab), disulfide-stabilized Fv(dsFv), minibody, diabody, DuoBody®, triabody, tetrabody, single-domain antigen-binding fragment (sdAb), camelid heavy-chain IgG and Nanobody® fragments, recombinant heavy-chain-only antibodies (VHH), and other antibody fragments that retain the binding specificity of the antibody.

[0214] The above "antibody" refers to a conjugate that specifically binds to an immunomodulatory molecule. The scope of the present invention includes not only the complete antibody form that specifically binds to an immunomodulatory molecule, but also the antigen-binding fragment of said antibody molecule.

[0215] As used herein, "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3) and the backbone region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0216] "Complementary determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refer to amino acid residues of the antibody variable domain that are present for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3.

[0217] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0218] In the present invention, the term "fragment" of an antibody refers to a fragment possessing antigen-binding function and is used to include scFv, Fab, F(ab')2, and Fv fragments. Among the antibody fragments, Fab has a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and possesses one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment possessing only a heavy chain variable region and a light chain variable region. Two-chain Fvs have a variable region of the heavy chain and a variable region of the light chain connected by non-covalent bonds, while single-chain Fvs (scFvs) generally have a variable region of the heavy chain and a variable region of the light chain connected by covalent bonds through a peptide linker or directly connected at the C-terminus, so they can form a dimer-like structure similar to two-chain Fvs. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction cleavage of the whole antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), and can also be produced through recombinant DNA technology.

[0219] A “single-strand Fv” or “scFv” antibody fragment contains the VH and VL domains of the antibody, which are present within a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain to enable scFv to form a structure intended for antigen binding.

[0220] In the present invention, in a scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, VH and VL can be connected through a linker.

[0221] The above linker may be a peptide linker and may have a length of about 10-25 aa. For example, it may include hydrophilic amino acids such as glycine and / or serine.

[0222] The above linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), but the above linker may be, for example, (GnS)m (where n and m are each 1 to 10).

[0223] In the embodiments of the present invention, a single specific chimeric antigen receptor comprising SEQ ID NO. 188 or 190, or a dual specific chimeric antigen receptor having both 188 and 190, was used as an extracellular binding domain that specifically binds to NKG2DL or HER-2, but is not limited thereto.

[0224] In the present invention, the chimeric antigen receptor provides CARs comprising an extracellular binding domain comprising one or more antibodies that specifically bind to NKG2DL or HER-2. The CARs may be monospecific or multispecific (e.g., bispecific or higher number of specificities), and the CARs may be monovalent or multivalent (e.g., divalent, trivalent, or higher number of valencies). Exemplary monospecific or bispecific CAR antigen receptors and their exemplary sequences are shown in Table 13, and a list of structures and vectors is shown in FIG. 8. The chimeric antigen receptor may be characterized by comprising a transmembrane domain.

[0225] The "transmembrane domain" mentioned above is a part of the CAR that fuses an extracellular binding portion and an intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The transmembrane domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. In the present invention, the transmembrane domain is the alpha (α), beta (β), or zeta (ζ) chain of a T-cell receptor (TCR), CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD86, CD134, CD137, CD154, CD166, 4-1BB, OX40, FcεRIγ, transferrin receptor (TfR), IL-2R, IL-15R, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, IL-7, IL-12, IL-15, It may be characterized as being derived from a protein selected from the group consisting of KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, and CS1, but is not limited thereto.

[0226] The above-mentioned transmembrane domain can be attached to the extracellular binding domain of the CAR through a linker. For example, the linker may be a short oligo- or polypeptide linker of 2 to 10 amino acids in length, preferably a glycine (G)-serine (S) doublet, but is not limited thereto.

[0227] The binding domain of a CAR is generally followed by one or more "hinge domains." Accordingly, in the present invention, the extracellular domain may be characterized by additionally including a signal peptide (SP) and / or a hinge together with the binding domain. The extracellular domain is a site where the main signal is transmitted, located outside the cell membrane, and is a domain for specifically recognizing a target.

[0228] The term "hinge domain" in the present invention refers to a part of a CAR that plays a crucial role in localizing an extracellular binding domain containing an antigen binding site away from the surface of the working cell to enable appropriate cell / cell contact, antigen binding, and activation. A CAR generally comprises one or more hinge domains between an extracellular binding domain and a transmembrane domain. The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise amino acid sequences of naturally occurring immunoglobulin hinge domains or modified immunoglobulin hinge domains, such as, for example, IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, and IgM. "A modified hinge region" is (a) a naturally occurring hinge region having up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), (b) a portion of a naturally occurring hinge region of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) length having up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), or (c) a core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids It refers to a portion of a naturally occurring hinge region that includes (which may be of length). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (e.g., a serine residue).The hinge domain includes a hinge domain derived from the extracellular region of type 1 membrane proteins such as CD8, CD4, CD28, and CD7, which may be a wild-type hinge domain from these molecules or may be modified.

[0229] In the present invention, any transmembrane domain including the hinge domain can be used as long as it can connect the extracellular domain and the intracellular signaling domain across the cell membrane.

[0230] In some cases, the coupling domain and the membrane-penetrating domain may be connected by a spacer domain. Preferably, the spacer domain may be a hinge domain.

[0231] In the present invention, the chimeric antigen receptor may be characterized by including an intracellular signaling domain.

[0232] In the present invention, the intracellular signal transduction domain is a portion located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and refers to a region that activates the immune response of the immune cell by transducing a signal into the cell when a binding domain included in the extracellular domain binds to a target antigen.

[0233] In the present invention, the intracellular signaling domain may be characterized by including an intracellular signaling domain and / or a co-stimulatory signaling domain.

[0234] The above signaling domain can induce activation of normal effector functions in immune cells where the CAR is located. For example, it can induce lysis activation and / or helper activation through the secretion of cytokines. The above signaling domain may include a truncated fragment of an intracellular signaling domain sufficient to transduce effector function signals.

[0235] The intracellular signaling domain is a primary signaling domain selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d; It may be characterized by comprising a co-stimulatory signaling domain selected from the group consisting of ligands that specifically bind to CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB(CD137), OX40(CD134), CDS, ICAM-1, ICOS(CD278), LFA-1(CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3, and CD83.

[0236] The term "intracellular signaling domain" in the present invention refers to a portion of a CAR involved in transmitting a message of effective CAR binding to a target antigen into an immune effector cell to induce an effector cell function, for example, activation including the release of cytotoxic factors to CAR-bound target cells, cytokine production, proliferation, and cytotoxic activity, and / or other cellular responses induced by antigen binding to the extracellular binding domain of the CAR. An effector function refers to a specific function of a cell, for example, the effector function of an immune cell may be cytolytic activity, or may support or activate activities including the secretion of cytokines. Accordingly, an intracellular signaling domain refers to a portion of a protein that transmits effector function signals and directs the cell to perform a specific function.

[0237] Immune cell activation is mediated by two different classes of intracellular signaling domains. For example, immune cell activation is mediated by a primary signaling domain that initiates antigen-dependent primary activation and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Accordingly, the intracellular signaling domain may be characterized by including a "primary signaling domain" and a "co-stimulatory signaling domain."

[0238] The term "primary signaling domain" in the present invention refers to a signaling domain that regulates immune cell activation in a stimulating or inhibitory manner. A primary signaling domain acting in a stimulating manner may contain a signaling motif known as an immune receptor tyrosine-based activation motif and / or an ITAM. An ITAM containing a primary signaling domain may be selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d, but is not limited thereto.

[0239] The term "co-stimulatory signaling domain" in the present invention refers to an intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory signaling domain refers to a part of a CAR that includes an intracellular signaling domain of a co-stimulatory molecule. It may include, but is not limited to, a co-stimulatory signaling domain selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), HVEM (Herpesvirus Entry Mediator), IL-2Rβ, IL-15Rα, SLAMF7, CTLA-4 GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, and NKG2C.

[0240] The chimeric antigen receptor may be characterized by including two or more intracellular signaling domains, and when including two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other. They may be connected via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acids, and a glycine-serine sequence may be cited as an example of such a linker sequence. The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (where n and m are each 1 to 10), and may be, for example, (GnS)m (where n and m are each 1 to 10), but is not limited thereto.

[0241] In the present invention, the intracellular signaling domain may be characterized by comprising one or more selected from the group consisting of the co-stimulation signaling domain of CD28 or DAP10 and the intracellular signaling domain of CD3 zeta, but is not limited thereto.

[0242] In the present invention, the chimeric antigen receptor may be characterized by comprising a sequence selected from the group consisting of SEQ ID NOs 200, 202, and 204, most preferably, but is not limited thereto.

[0243] In the present invention, the chimeric antigen receptor may further include an immune function-promoting factor of an immune cell, and the immune function-promoting factor of the immune cell may be characterized as being an interleukin signal sequence. The interleukin signal sequence may be characterized as inducing the expression of IL-12, IL-8, or IL-2, but is not limited thereto. Additionally, IL-7 or CCL19 may be examples of immune function-promoting factors for T cells among the immune cells.

[0244] In the present invention, the chimeric antigen receptor may additionally include a signal peptide in front of the extracellular domain. The nucleic acid encoding the chimeric antigen receptor may be characterized by further including a nucleic acid encoding the signal peptide. The signal peptide may be, for example, IgE, GM-CSF, CD8α signal peptide, etc., but is not limited thereto.

[0245]

[0246] genetically modified immune cells expressing chimeric proteins or variants thereof, including IL-2 and IL-15 proteins, and chimeric antigen receptors

[0247] The immune cells of the present invention are genetically modified to express the chimeric protein or a variant thereof and a chimeric antigen receptor.

[0248] In the present invention, the immune cell of the present invention may be manufactured through a method that is not limited to the genetically modified chimeric protein or a variant thereof; and a modification that expresses a chimeric antigen receptor.

[0249] In the present invention, preferably, the genetically modified immune cell may be characterized by the introduction into the host cell a nucleic acid encoding the chimeric protein or a variant thereof, and a nucleic acid encoding the chimeric antigen receptor.

[0250] As used herein, “nucleic acid” may be present in cells or cell lysates, or may be present in a partially purified or substantially pure form. Nucleic acid is “isolated” or “substantially purified” when purified from other cellular components or other contaminants, e.g., nucleic acids or proteins of other cells, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acid of the present invention may be, for example, DNA and / or RNA.

[0251] In the present invention, the nucleic acid encoding the chimeric protein may include or be composed of the nucleic acid sequence of SEQ ID NO. 34.

[0252] In the present invention, the nucleic acid encoding the variant of the chimeric protein may include or be composed of the nucleic acid sequence of SEQ ID NOs 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, or 74.

[0253] In the present invention, when the chimeric protein or its variant is fused with an additional domain, 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 or the nucleic acid sequence encoding it, in addition to the amino acid sequence of the linker and the fusion domain additionally included in addition to the amino acid sequence of the chimeric protein or its variant protein described in the present invention.

[0254] The above nucleic acid (polynucleotide) can be modified by codon optimization, which is due to the degeneracy of codons, and it will be well understood by a person skilled in the art that there exist many nucleotide sequences encoding polypeptides or fragments of their variants. Some of these polynucleotides (nucleic acids) possess minimal homology with the nucleotide sequences of any naturally occurring gene. In particular, polynucleotides (nucleic acids) that are variable due to differences in codon utilization, e.g., polynucleotides (nucleic acids) optimized for codon selection in humans, primates, and / or mammals, are preferred.

[0255] In the present invention, the nucleic acid encoding the chimeric protein or a variant thereof, and the nucleic acid encoding the chimeric antigen receptor can be recombined into a vector and introduced into an immune cell.

[0256] In the present invention, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. The vector may be a plasmid, a phage particle, and / or simply a potential genomic insert. Upon transformation into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention.

[0257] In the present invention, the vector may be a linear molecule or a circular molecule. The vector may be integrated or non-integrated. Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes.

[0258] In addition, the recombinant vector may be used without limitation by appropriately selecting any vector known in the art, provided that it is a vector capable of inducing protein expression of the nucleic acid encoding the peptide.

[0259] In the present invention, the vector may be characterized as being selected from the group consisting of DNA, RNA, plasmid, lentivirus vector, adenovirus vector, sendaivirus vector, adeno-associated virus vector (AVV), retrovirus vector and non-viral vector, but is not limited thereto.

[0260] In the present invention, the nucleic acid encoding the chimeric protein or its variant and the nucleic acid encoding the chimeric antigen receptor may be characterized by being recombined into a single vector and introduced into an immune cell. When the nucleic acid encoding the chimeric protein or its variant and the nucleic acid encoding the chimeric antigen receptor are recombined into a single vector and transformed, for example, bicistronic vectors such as pIRES vector, pBI vector, p2A vector, pCMV-IRES vector, and pDual-Glo vector may be used, but are not limited thereto.

[0261] In the present invention, the nucleic acid encoding the chimeric protein or a variant thereof, and the nucleic acid encoding the chimeric antigen receptor may be characterized by being recombined into respective vectors and introduced into immune cells. The two vectors may be introduced into immune cells simultaneously or sequentially through co-transformation.

[0262] Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0263] In the above vector, the nucleic acid encoding the antibody is "operably linked" when positioned in a functional relationship with other nucleic acid sequences in the vector. This may be a gene and regulatory sequence(s) linked in such a way that an appropriate molecule (e.g., a transcription-activating protein) enables gene expression when it binds to the regulatory sequence(s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a pre-sequence protein participating in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretion leader, are in contact and exist within the reading frame. However, the enhancer does not require contact. The linkage of these sequences is performed by ligation at a convenient restriction enzyme site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional methods.

[0264] In the present invention, the vector may include an expression-regulating sequence.

[0265] The term "expression control sequence" refers to a DNA sequence essential for the expression of a coding sequence operably linked in a specific host organism. Such control sequences include a promoter for carrying out transcription, an optional operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating 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, it includes a promoter, a polyadenylation signal, and an enhancer. The factor that most influences the expression level of a gene in a plasmid is the promoter. For high expression, SRα promoters and cytomegalovirus-derived promoters are preferably used.

[0266] To express the DNA sequence of the present invention, any of the wide variety of expression regulatory sequences may be used in the vector. Examples of useful expression regulatory sequences include, in addition to the promoters described above, early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, T3 and T7 promoters, the major operator and promoter regions of phage lambda, the regulatory regions of fd code proteins, promoters for 3-phosphoglycerate kinase or other glycolases, promoters of said phosphatase, e.g., Pho5, promoters of the yeast alpha-mating system, and other sequences of configuration and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The T7 RNA polymerase promoter Φ can be usefully used to express proteins in E. coli.

[0267] As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression regulatory sequences that are functional within the selected expression host. Preferably, the expression regulatory sequences and the gene are contained within a single expression vector that includes both a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector may further include expression markers useful within the eukaryotic expression host.

[0268] The above-mentioned recombinant vector may be introduced into host cells, such as immune cells, by methods such as transformation or transfection. As used herein, the term "transformation" means the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. As used herein, the term "transfection" means the acceptance of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0269] Of course, it must be understood that not all vectors and expression regulatory sequences function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, a person skilled in the art can make an appropriate selection among various vectors, expression regulatory sequences, and hosts without exceeding the scope of the present invention. For example, when selecting a vector, the host must be considered, as the vector must be replicated within it. The copy number of the vector, the ability to control the copy number, and the expression of other proteins encoded by said vector, such as antibiotic markers, must also be considered. When selecting an expression regulatory sequence, various factors must also be considered. For example, the relative strength of the sequence, its modulation capabilities, and compatibility with the DNA sequence of the present invention, particularly in relation to potential secondary structures, must be taken into account. A unicellular host must be selected by considering factors such as the selected vector, the toxicity and secretion characteristics of the product encoded by the DNA sequence of the present invention, 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 range of these variables, a person skilled in the art may select various vector / expression control sequence / host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture. Binding methods, panning methods, film emulsion methods, etc., may be applied as screening methods when attempting to clone cDNA by expression cloning.

[0270] In the present invention, the nucleic acid encoding the chimeric protein or a variant thereof, and the nucleic acid encoding the chimeric antigen receptor, may be directly introduced into the genome of a host cell and exist as chromosomal factors. It will be obvious to those skilled in the art to which the present invention pertains that inserting the gene into the genomic chromosome of a host cell will produce the same effect as introducing a recombinant vector into the host cell.

[0271] The immune cell of the present invention is characterized by being genetically modified to express the chimeric protein or a variant thereof; and a chimeric antigen receptor.

[0272] In the present invention, as an exemplary method for producing the genetically modified immune cells, the recombinant vector can be introduced into immune cells as host cells.

[0273] In the present invention, the genetically modified immune cell may be prepared by introducing both the nucleic acid encoding the chimeric protein or a variant thereof and the nucleic acid encoding the chimeric antigen receptor, or by introducing the remaining nucleic acid into an immune cell in which either of the two encoding nucleic acids has already been introduced. For example, it may be prepared by introducing the nucleic acid encoding the chimeric protein or a variant thereof into a CAR-expressing immune cell known in the art, but is not limited thereto.

[0274] The term “immune cell” in this invention refers to any cell involved in the initiation and / or promotion of an immune response. More specifically, it refers to an immune effector cell.

[0275] In the present invention, the immune cells may include, without limitation, any immune cells capable of introducing a chimeric antigen protein, such as 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, mast cells, as well as cells capable of differentiating into immune cells, such as hematopoietic stem cells, iPSCs, adult stem cells, etc., but are not limited thereto.

[0276] In the present invention, most preferably, the immune cell may be characterized as being a natural killer cell.

[0277] The term "natural killer cell" ("NK cell") in the present invention 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 provided by major histocompatibility complex (MHC) molecules on the surface of infected cells, triggering cytokine release and causing 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 named "natural killers" due to the initial concept that they do not require prior activation to kill targets. NK cells are known to be large granular lymphocytes (LGLs) that differentiate and mature in the bone marrow, subsequently entering the circulatory system from the bone marrow.

[0278] In the present invention, the immune cell may preferably be an immune cell of mammalian origin. Examples of "of mammals" or "mammals" include primates (e.g., humans), canids, felines, rodents, pigs, ruminants, etc., and more specifically include, but are not limited to, humans, monkeys, dogs, cats, horses, cattle, sheep, goats, rabbits, guinea pigs, rats, and mice. Most preferably, the immune cell may be an immune cell of human origin.

[0279] In the present invention, the natural killer cells may be used without limitation and, for example, may be selected from the group consisting of distributable natural killer cells known in the art, or NK cell sources including peripheral blood, peripheral blood lymphocytes (PBL), peripheral blood mononuclear cells (PBMC), bone marrow, umbilical cord blood (or cord blood), isolated NK cells, NK cells derived from induced pluripotent stem cells (iPSC), NK cells derived from embryonic stem cells, and combinations thereof, but are not limited thereto.

[0280] When the genetically modified immune cells of the present invention are used for therapeutic purposes, the nucleic acid encoding the chimeric protein or a variant thereof, and the nucleic acid encoding the chimeric antigen receptor may be an autologous immune cell or an allogeneic immune cell isolated from the target to be treated.

[0281] The genetically modified immune cell of the present invention may be characterized by expressing the chimeric protein or a variant thereof; and a chimeric antigen receptor.

[0282] The genetically modified immune cell of the present invention may be characterized by expressing the chimeric protein or a variant thereof; and a chimeric antigen receptor, thereby fixing to the cell membrane and / or presenting it on the cell surface.

[0283] In the present invention, the chimeric protein or its variant; and the extracellularly presented domain when the chimeric antigen receptor is presented on the cell surface can be easily understood by a person skilled in the art by the sequence of the expressed fusion protein or chimeric antigen receptor.

[0284] In the present invention, the chimeric protein or a variant thereof; and the chimeric antigen receptor may be expressed separately.

[0285] For example, in the present invention, when the nucleic acid encoding the chimeric protein or a variant thereof and the nucleic acid encoding the chimeric antigen receptor are recombined into a bicistron vector, the recombinant vector may include a ribosomal skipping sequence for the separate expression of each protein.

[0286] In the present invention, the ribosomal skip sequence may be included between the nucleic acid encoding the chimeric protein or a variant thereof and the nucleic acid encoding the chimeric antigen receptor.

[0287] The term “ribosomal skipping” in the present invention refers to a translation cleavage sequence used to separate and express two proteins within mRNA, and may be used interchangeably with “self-cleaving.” In the present invention, the ribosomal skip sequence may include, but is not limited to, the sequence of SEQ ID NO. 90 below:

[0288] Sequence No. 90: DX1EX2NPGP, where X1 is V or I, and X2 is any amino acid.

[0289] In the present invention, the ribosome skip sequence may be a sequence encoding a 2A peptide or a CHYSEL (cis-acting hydrolase element) sequence, and preferably, it may be a 2A peptide selected from the group consisting of T2A, P2A, E2A, and F2A, but is not limited thereto.

[0290] In the present invention, the chimeric protein or its variant; and the chimeric antigen receptor may be characterized by being expressed as a single protein and then cleaved and separated.

[0291] For example, if the chimeric protein or its variant; and the chimeric antigen receptor are expressed as a single protein and then cleaved to separate, a cleavable linker may be included between the chimeric protein or its variant; and the chimeric antigen receptor. The cleavable linker may be readily selected from those well known in the art.

[0292] 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 time, and furthermore, since the activity of immune cells against target cells can be enhanced and maintained for a long period, they can be usefully used as a cell therapy agent for the prevention and / or treatment of various diseases.

[0293] Accordingly, in another aspect, the present invention relates to the use of the genetically modified immune cells for the prevention and / or treatment of disease.

[0294] The present invention relates to a pharmaceutical composition for the prevention and / or treatment of diseases comprising the genetically modified immune cells.

[0295] The present invention relates to a method for preventing and / or treating a disease, comprising the step of administering the genetically modified immune cells.

[0296] The present invention relates to the use of the genetically modified immune cells for the manufacture of a drug for the prevention and / or treatment of disease.

[0297] In the present invention, the disease may be used without limitation as long as it is a disease reported to be preventable and / or treatable using immune cells, and diseases treatable with immune cells, in particular immune cells to which chimeric antigen receptors have been introduced, are well known in the art. Examples include, but are not limited to, cancer, infectious diseases, and autoimmune diseases.

[0298] The term "cancer" is used interchangeably with "tumor," and the composition for prevention or treatment according to the present invention is applicable to all types of cancer, including solid cancer and blood cancer. Unlike blood cancer, a solid tumor refers to cancer formed as a mass within an organ, and most cancers occurring in most organs fall under this category.

[0299] In the present invention, cancer may be selected from the group consisting of colorectal cancer, melanoma, gastric 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, chronic myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, and multiple myeloma.

[0300] The pharmaceutical composition for prevention and / or treatment according to the present invention may comprise the genetically modified immune cells alone in a pharmaceutically effective amount, or may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, a pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat the symptoms of a disease.

[0301] The term "pharmaceuticalally acceptable" means physiologically acceptable and, when administered to humans, does not typically cause allergic reactions such as gastrointestinal disorders or dizziness, or similar reactions. Examples of the excipients may include, but are not limited to: surfactants, preferably polysorbate-based nonionic surfactants; buffers such as neutral buffered saline, phosphate buffered saline; sugars or sugar alcohols such as glucose, mannose, sucrose, dextran, or mannitol; amino acids, proteins, or polypeptides such as glycine or histidine; antioxidants; chelating agents such as EDTA or glutathione, e.g.; penetrating agents; adjuvants; and preservatives.

[0302] The composition of the present invention may 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 may be, for example, in the form of a sterile injectable solution available for immediate use, but is not limited thereto.

[0303] The composition of the present invention may be administered via various routes including intravenous, transdermal, subcutaneous, intramuscular, or oral, and the dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, the patient's age, gender, weight, and severity of the patient, and the composition for prevention and / or treatment according to the present invention may be administered in combination with known therapeutic agents or compounds having the effect of preventing, improving, or treating a target disease.

[0304] Although specific amino acid sequences and nucleic acid sequences have been described in this invention, it will be obvious to those skilled in the art that amino acid sequences substantially identical to the enzyme intended for implementation in this invention and nucleic acid sequences encoding them fall within the scope of this invention. "Substantially identical" includes cases where the homology of the amino acid or nucleic acid sequences is very high, and also refers to proteins that share structural features or have the same function as those used in this invention, regardless of sequence homology. Proteins in which sequences other than the core sequence of this invention are partially deleted, or fragments of nucleic acid sequences encoding them, may also be included in this invention; therefore, this invention includes all amino acid or nucleic acid sequences having the same function as those used in this invention, regardless of the length of the fragment.

[0305]

[0306] Examples

[0307] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0308]

[0309] Example 1. Selection of IL-2 / IL-15 fusion protein candidates with down-modulated binding affinity to IL-2R (IL-2Rβ) relative to IL-2 and Fc-IL-2

[0310] Example 1-1: Preparation of a fusion protein containing IL-2 / IL-15 chimeric protein, IL-15Rα, and Fc domains

[0311] As an IL-2 / IL-15 containing chimeric protein or a variant thereof, a fusion protein was designed as shown in FIG. 1b by additionally fusing an Fc domain and IL-15Rα (sushi domain) based on nucleic acids SEQ ID NOs. 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, or 74 encoding the amino acid sequence of SEQ ID NOs. 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75, and the gBlock gene of Integrated DNA Technologies After synthesis using the fragments service, it was loaded into the pcDNA3.4 vector via cloning (Fig. 1b).

[0312] The structure of the designed fusion protein is as follows:

[0313] N'-[hinge domain]-[Fc domain]-[linker]-[IL-15Rα / sh]-[linker]-[IL-2 / IL-15 chimeric protein or variant thereof]-C'; or

[0314] N'-[IL-2 / IL-15 chimeric protein or its variant]-[linker]-[IL-15Rα / sh]-[linker]-[hinge domain]-[Fc domain]-C'

[0315] The vectors constructed above were introduced into CHO cells (Expi-CHO™) to express each fusion protein. After introducing the vectors, the cells were cultured for 5 days at 37°C, 125 RPM, and a CO2 concentration of 8%, after which 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 their variants contained therein, and their amino acid and nucleic acid sequence numbers are as shown in Tables 6 and 7 below:

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] GIC-982C1 to GIC-982C15 were purified using an open column containing Protein A resin. Repligen's Protein A resin was washed and equilibrated using PBS and Thermo's Protein A binding buffer. Subsequently, the supernatant filtered through a 0.22 µm filter was reacted with the Protein A resin. The Protein A resin reacted in the open column, which had been washed with PBS, was collected, and the column was washed using Protein A binding buffer equivalent to 10 times the volume of the resin. Then, binding buffer was placed in a collection tube at 1 / 10 the volume of the elution buffer, followed by the addition of Thermo's IgG elution buffer to elute and collect the proteins. The collected fusion proteins were then rehydrated with PBS buffer.

[0355] At this time, the isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and confirmed by staining with Comash Blue (Figs. 1c to 1f).

[0356]

[0357] Example 1-2: Measurement of IL-2Rβ binding affinity using Octet binding assay

[0358] An octet binding assay was performed using a Ni-NTA sensor chip (ForteBio 18-5101) to confirm the binding affinity of GIC-982C1 to GIC-982C6 and GIC-982C12 to GIC-982C15 to IL-2Rβ. Fc-IL2 was selected as the control group for the analysis. First, the sensor chip was immersed in distilled water and incubated for 5 minutes to activate it. The sensor was established as a baseline in PBS for 3 minutes and then immersed in a solution containing 6 μg / ml of IL-2Rβ-his (Acro CD2-H5221). Subsequently, the sensor was established as a baseline in PBS for 3 minutes and then immersed for 5 minutes in test substances serially diluted by half from 2000 nM to 31.25 nM to associate with IL-2Rβ. During this process, PBS buffer containing no test substance was included as a blank value for the analysis. Afterward, the plates were transferred back to PBS and dissociated for 10 minutes. All steps were performed at room temperature, and the plates were shaken at 1000 RPM. The test results obtained using ForteBio Data Analysis software are shown in Table 8 and Figure 2 below.

[0359] As a result of confirming the binding affinity through octet analysis, GIC-982C1 to GIC-982C6 and GIC-982C12 to GIC-982C15 exhibited relatively higher dissociation constants (Kd) than Fc-IL2, confirming that the binding affinity to IL-2Rβ was weakened.

[0360]

[0361] Examples 1-3: Confirmation of the activating effect of the IL-2 receptor (IL-2R)-mediated STAT5 mechanism using a STAT5 signaling assay

[0362] 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 a common γ chain were cultured, and STAT5 activity was analyzed. IL2-Fc was selected as a control group for the analysis.

[0363] The above 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 μg / ml Puromycin (Invivogen, Ant-Pr-1)", and for the activity test, a medium without added screening antibiotics was used, which was "DMEM (Gibco, Cat No. 10569010) + 10% FBS (Gibco, Cat No. 26140079) + penicillin / streptomycin (Gibco, Cat No. 15140122)". The cultured cells were treated with Trypsin-EDTA, collected, and washed with added medium. The collected HEK-Blue CD122 / 132 cells were centrifuged at 300 xg for 5 minutes, the supernatant was removed, and medium was added for resuspension. Subsequently, the HEK-Blue CD122 / 132 cells were counted to 2.5 x 10⁶ 5 The protein was prepared by diluting it with medium to a concentration of cells / mL. The control group protein was diluted to the highest concentration to be used in the test (100 ng / mL based on IL-2) and loaded onto plates, followed by serial dilution in 1 / 10 increments. 20 µl of the diluted protein was loaded onto flat-bottom plates, and 180 µl (45,000 cells) of the previously prepared cells were added. The plates were incubated for 24 hours at 37°C under 5% CO₂ conditions.

[0364] To prepare the QUANTI-Blue™ Solution (Invivogen, rep-qbs) for detection, 750 µl each of QB reagent and QB buffer were added to 73.5 ml of distilled water, mixed thoroughly by vortexing, and incubated at room temperature for 10 minutes. 20 µl of the supernatant from the plate incubated for 24 hours was taken and loaded onto a new plate, 180 µl of the previously prepared QUANTI-Blue™ Solution was added, and the plates were incubated for 1 hour under light-blocking conditions at 37°C. The absorbance measured at 630 nm using a microplate reader is shown in Figure 3, and the resulting EC50 results are shown in Table 9.

[0365] As a result, it was confirmed that GIC-982C1 to GIC-982C5 and GIC-982C13 to GIC-982C15 had a relatively lower STAT5 activity intensity than Fc-IL2, and were identified as substances capable of preventing cell overactivity while maintaining an appropriate level of cell activity.

[0366]

[0367] Examples 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

[0368] To confirm the anticancer efficacy of IL-2 / IL-15 chimeric proteins and the improvement of pulmonary edema side effects caused by immune hyperactivation, the colon cancer cell line CT26 was introduced into Balb / c mice at a rate of 5x10 5A mouse model was prepared by administering cells / head subcutaneously. The volume of the generated tumors was measured using a vernier caliper, and group separation was performed when the volume reached approximately 70 mm³. Subsequently, IL2-Fc, GIC-982C1, GIC-982C4, and GIC-982C15 were each administered intraperitoneally to mice at a dose of 10 mg / kg twice a week for a total of four times over two weeks. Tumor volumes were measured on days 1, 4, 7, 11, 14, 16, and 18 after administration to confirm the anticancer efficacy, and the results are shown in Table 10.

[0369] In addition, the condition of the mice was observed to check for any adverse effects from the administration. For individuals that died, adverse reactions were visually examined, and the condition of internal organs was checked through necropsy. To evaluate whether adverse effects occurred due to drug administration, the weight of the excised lungs was measured, dried for 2 days, and then weighed again to calculate the Wet / Dry Ratio. The results are shown in Table 11.

[0370] As a result, Fc-IL2 caused death due to pulmonary edema resulting from immune hyperactivation under high dose conditions of 10 mpk, whereas GIC-982C1, GIC-982C4, and GIC-982C15 exhibited excellent cancer cell proliferation inhibitory activity. In addition, Fc-IL2 showed an increase in wet lung weight, whereas GIC-982C1, GIC-982C4, and GIC-982C15 showed a wet / dry ratio at the same level as Vehicle, confirming that no pulmonary edema occurred.

[0371]

[0372]

[0373] Example 2. Preparation of immune cells modified to express IL-2 / IL-15 fusion protein and verification of their effects

[0374] Example 2-1: Plasmid Construction

[0375] Using a retrovirus vector (Biovec pharma, SinVec(K)-GFP-BSD, Vec-033), two clone vectors containing nucleic acid sequences of sequence numbers 185 and 187 encoding a fusion protein containing amino acids of sequence numbers 184 and 186 listed in Table 12 below were constructed, and the schematic diagram is as shown in Fig. 4A.

[0376] A schematic diagram of an immune cell expressing the IL-2 / IL-15 fusion protein of the above vector is shown in Fig. 4B, and the form of the expressed fusion protein is as follows:

[0377] N'-[IL-2 / IL-15 chimeric protein]-[linker]-[IL-15Rα]-C'

[0378]

[0379]

[0380]

[0381] Example 2-2: Retrovirus Production and Concentration

[0382] To produce the BaEV-pseudotype retrovirus, 6.0 x 10 on the day before transduction 6293Vec-BaEV cell line (Biovec pharma, Vec-006) was cultured in T75 flasks at 37°C in a CO2 5% incubator for 24 hours. When the cell density reached 80%, 15 µg of a plasmid encoding a chimeric protein was transfected using lipofectamine 2000 (Invitrogen, 11668500). 48 hours after transfection, the culture containing the retrovirus was harvested, and cell debris was removed by centrifugation at 500 xg for 10 minutes at 4°C and passing through a 0.45 µm pore PVDF membrane filter (MILLIPORE, SE1M003M00). The culture medium containing retroviruses 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 reacted under refrigerated conditions at 4°C. After reacting for 18 hours, the retrovirus pellet obtained by centrifugation at 1500 xg for 45 minutes at 4°C was concentrated by resuspension in Opti-MEM medium (Gibco, 11058021). The concentrated retroviruses were stored at -80°C.

[0383]

[0384] Example 2-3: Preparation of Human Peripheral Blood Mononuclear Cell (PBMC) Derived Natural Killer Cells

[0385] Blood samples collected from different healthy donors were centrifuged at 400 xg for 30 minutes using a Ficoll-Hypaque concentration gradient, and the buffy coat was collected to isolate PBMCs. The isolated PBMCs were counted using an ADAM-MC2 automated cell counter (NanoEnTek), and to obtain CD3(-) cells, they were transferred to a new tube and centrifuged at 350 xg for 10 minutes at 4°C. After centrifugation, the supernatant was removed to obtain 1 x 10 7 80 µl of CliniMACS buffer (20% human serum albumin, 2mM EDTA) and 22 µl of CD3 magnetic beads (Miltenyi Biotech, 130-050-101) were dispensed per cell count to suspend the cell pellet, and the mixture was incubated for 15 minutes at 4°C under light blockage. After the reaction, 10 ml of CliniMACS buffer was dispensed for washing, followed by centrifugation at 350 xg at 4°C for 10 minutes, and then 1 x 10 8 0.5 ml of CliniMACS buffer was dispensed per cell to suspend the cell pellet.

[0386] 3 mL of CliniMACS buffer was run over an LD column (Miltenyi Biotec, 130-042-901), and the cell suspension was run through it to collect CD3(-) cells that passed through the column. For washing, 10 mL of CliniMACS buffer was added and centrifuged at 350 xg at 4°C for 10 minutes. The cell pellet was then resuspended in CliniMACS buffer, and the cell count was measured using an automated cell counter. Subsequently, 1 x 10⁶ 780 µl of CliniMACS buffer and 22 µl of CD56 magnetic beads (Miltenyi biotech, 130-050-401) were dispensed per cell count to suspend the cell pellet, and the reaction was carried out for 15 minutes at 4°C under light blockage conditions. For washing, 10 ml of CliniMACS buffer was dispensed and centrifuged at 350 xg at 4°C for 10 minutes, after which 0.5 ml of CliniMACS buffer was dispensed per 1 x 10⁸ cells to suspend the cell pellet.

[0387] 3 mL of CliniMACS buffer was flowed onto an LS column (Miltenyi Biotec, 130-042-401) to moisten it, and then the cell suspension was flowed through it. Subsequently, the column was removed from the magnet stand, and 5 mL of CliniMACS buffer was added. CD3(-) CD56(+) Natural Killer cells were collected in a new tube by applying pressure with a piston. The collected cells were centrifuged at 350 xg for 10 minutes at 4°C. After centrifugation, the cell pellet was resuspended in CliniMACS buffer, and the cell count was measured using an automated cell counter. The cells were resuspended in CryoStor CS10 cryopreservation agent (Biolife solution, 210102) to a cell count of 10–20 x 10⁶ cells per 1 mL. Suspended cells were dispensed in 1 ml aliquots into cryogenic vials, frozen once using a cell freezing container (Corning, CLS432001), and then transferred to an LN2 tank for secondary freezing and storage.

[0388]

[0389] Examples 2-4: Preparation of natural killer cells expressing chimeric proteins containing IL-2 and IL-15

[0390] For the culture of the isolated natural killer cells of Examples 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 and mixed, then 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 it for 2 hours at 4.0 rpm and 4°C using a microtube rotator (Thermo Scientific, Hulamixer, 15920D). After thawing the frozen natural killer cells from Examples 2-3 and counting the cells, the previously prepared beads were washed with 1 ml of CliniMACS buffer to obtain 1 x 10⁶ 6 Based on 5 µl of beads per cell, the cells were suspended in CN5-101 medium (CTS™ NK-Xpander™ Medium, containing 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.

[0391] To infect with the retrovirus prepared in Example 2-2, 10 µg / ml retronectin (Takara, T100B) was coated onto an untreated 12-well plate according to the manufacturer's protocol. 300 µl of retrovirus corresponding to an MOI of 1–10 was added to each well, and the retrovirus was adsorbed onto 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). The number of natural killer cells was measured on day 4 from the start of culture, and 0.8 x 10⁶ cells were added to CN5-101 medium. 6 Suspended at a cell density of cells / mL. 1 mL of the prepared natural killer (NK) cell-coated well was dispensed into each well, followed by an additional 0.6 mL of CN5-101 medium. After centrifuging at 1,000 xg, 32°C, for 15 minutes to combine the retrovirus with NK cells, the cells were cultured at 37°C, 5% CO2 conditions at 3-day intervals to yield 0.5 x 10⁶ 6 Subcultured by inoculation at a cell density of cells / mL.

[0392] 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 Equal cell counts at a cell density of cells / mL were cultured in suspension 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)). Subsequently, cell counts were measured separately under CN5-101 and CN5 medium conditions, and 0.5 x 10⁶ 6 5 ml of cells were inoculated at a cell density of cells / ml and subcultured.

[0393]

[0394] Examples 2-5: Expression analysis of chimeric proteins including IL-2 and IL-15

[0395] Natural killer cells were obtained on the 3rd day after inducing retroviral transduction, and 0.2 x 10⁶ cells were placed in a 96-well plate using an automated cell counter. The plate was filled to 250 µl with FACS buffer (DPBS containing 2% FBS) and centrifuged at 350 xg 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 stained sample (Invitrogen, L34955A), and reacted at 4°C for 30 minutes. Subsequently, 150 µl of FACS buffer was added, and the plate was centrifuged at 350 xg for 5 minutes at 4°C. The supernatant was removed, and the cell pellet was resuspended in 200–250 µl of FACS buffer and dispensed into FACS tubes. The expression of IL-15Rα, which constitutes the chimeric protein, was measured using a flow cytometer (FACSymphony™ A3 Cell analyzer, BD).

[0396] As a result, as shown in Figure 5, 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 showed a superior expression efficiency of 88% compared to 25.1% for the membrane-bound IL-15 protein (mbIL-15).

[0397]

[0398] Examples 2-6: In-vitro cell count measurement and comparison of cell viability

[0399] The cultured natural killer cells were divided into groups of the same cell density and number on the third day after transduction and cultured for 19 days in CN5 medium and for 25 days in CN5-101 medium supplemented with GI-101. On days 0, 4, 7, 10, 13, 16, 19, 22, and 25, cells attached to the bottom of the flask were detached using a scraper (SPL, 90020), and clumped cells were separated by pipetting to obtain samples necessary for cell count measurement. The obtained cells were analyzed using an automated cell counter to determine the cell density of viable cells (cell density, x 10⁻¹⁰). 6 cells / mL), 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 the viable cells by the total volume of the cell culture medium (mL), and thereafter by 0.5 x 10⁻⁶ 6 Natural killer cells were suspended in 5 ml at a cell density of cells / ml and subcultured. Thus, the total number of cells on day 7 was calculated as follows.

[0400] Predicted total number of cells = Live cell density (live cell density, x 10 6 cells / ㎖) / Cell density inoculated on the previous culture day (0.5 x 10 6 cells / ㎖) x Total number of cells on the previous culture day (x 10 6 cells)

[0401] The fold expansion value was calculated using the calculated total number of cells and the predicted total number of cells. The fold expansion was calculated by dividing the total number of cells or the predicted total number of cells for each culture day by the total number of cells on day 0 of culture to determine the multiple, and the results under culture conditions that stimulated cell proliferation by including GI-101 (cell proliferation-promoting protein) or without GI-101 are shown in Figure 6.

[0402] As a result, as shown in Figure 6, it was confirmed that natural killer cells (mbIL-2 / IL-15) expressing membrane-bound chimeric proteins including IL-2 and IL-15 exhibited superior proliferation and survival abilities compared to normal natural killer cells (UTD) without transformation and natural killer cells (mbIL-15) expressing membrane-bound IL-15 proteins, with or without stimulation by GI-101 (cell proliferation-promoting protein).

[0403]

[0404] Example 3. Confirmation of 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

[0405] Example 3-1: Confirmation of in vitro cytotoxic efficacy using various cancer cell lines

[0406] Target tumor cell lines (SKOV3, HCT116, A549) for in vitro cytotoxicity evaluation were infected with a lentivirus (Satorious, 4475) containing GFP and a puromycin resistance gene, and cultured in R10 medium (10% fetal bovine serum, 1% penicillin-streptomycin) containing 0.5 µg / ml puromycin; only the transduced tumor cells were selected and cultured. The target tumor cell lines were cultured at 3 x 10⁶ saturates one day prior to the in vitro cytotoxicity evaluation. 4 Cell count / mL (A549 and HCT116 cell lines) or 5 x 10 4 Target tumor cell lines suspended in R10 medium at a density of cell count / mL (SKOV3 cell line) were dispensed at a density of 100 µL per well of a 96-well plate (Corining, 3799) and cultured for 16 hours at 37°C under 5% CO2 conditions.

[0407] The mbIL-2 / IL-15 expressing natural killer cells prepared according to Examples 2-4 were thawed and washed, then dispensed into 96-well plates attached with target tumor cells prepared the previous day at an E / T (effector-to-target) ratio of 10:1 or 3:1. The plates were imaged for 96 hours at 4-hour intervals per well using an Incucyte®S3 instrument (Sartorius) installed inside a cell culture incubator while the cells were in a live state. Subsequently, GFP-fluorescent positive target tumor cells were counted and analyzed from the stored images.

[0408] As a result, as shown in Figure 7, mbIL-2 / IL-15 expressing natural killer cells were found to have high cytotoxic activity in three types of tumor cells compared to UTD and mbIL-15.

[0409]

[0410] Example 4. Preparation of a carrier fusion protein comprising an IL-2 / IL-15 fusion protein and a chimeric antigen receptor

[0411] Example 4-1: Plasmid Construction

[0412] To introduce a fusion protein containing the amino acid sequence of SEQ ID NO. 184 or 186 listed in Table 13 below and a chimeric antigen receptor containing the amino acid sequence of SEQ ID NO. 200, 202, or 204, a clonal vector containing the nucleic acid sequence of SEQ ID NO. 185 or 187 encoding a fusion protein containing IL-15 wild-type or chimeric protein and the nucleic acid sequence of SEQ ID NO. 201, 203, or 205 encoding a chimeric antigen receptor was constructed using a retrovirus vector (Biovec pharma, SinVec(K)-GFP-BSD, Vec-033), and the schematic diagram is as shown in FIG. 8a.

[0413] A schematic diagram of an immune cell expressing the fusion protein and chimeric antigen receptor of the above vector is shown in FIG. 8b, and the morphology of the expressed fusion protein and chimeric antigen receptor is as follows, respectively:

[0414] N'-[IL-2 / IL-15 chimeric protein]-[linker]-[IL-15Rα]-C'

[0415] N'-[antigen recognition domain]-[hinge]-[transmembrane domain]-[intracellular domain]-C'

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423] Example 4-2: Retrovirus Production and Concentration

[0424] To produce the retrovirus BaEV-pseudotype, 6.0 x 10 days before transfection 6293Vec-BaEV cell lines (Biovec pharma, Vec-006) were cultured in T175 flasks at 37°C in a CO25% incubator for 24 hours. When the cell density reached 70–80%, a fusion protein containing 15 µg of IL-2 / IL-15 chimeric protein and a plasmid containing a gene encoding a chimeric antigen receptor were transfected using lipofectamine® 2000 (Invitrogen, 11668500) according to the manufacturer's test method. 48 hours after transfection, the supernatant containing retroviruses was harvested, and cell residues were removed by passing it through a 0.45 µm pore PVDF membrane filter (MILLIPORE, SE1M003M00). The supernatant containing retroviruses from which cellular residues had been removed was mixed with Retro-X™ concentrator (Clontech, 631456) reagent at a ratio of 3:1 (virus culture: Retro-X concentrator reagent) for retrovirus concentration and reacted for 18 hours at 4°C. After the reaction was complete, the retrovirus pellet obtained by centrifugation at 1500 xg at 4°C for 45 minutes was re-suspended in Opti-MEM™ medium (Gibco, 11058021) to concentrate the retroviruses. The concentrated retroviruses were aliquoted into frozen vials and stored at -80°C until use.

[0425]

[0426] Example 4-3: Preparation of Human Peripheral Blood Mononuclear Cell (PBMC) Derived Natural Killer Cells

[0427] Leukocytes obtained from healthy donors via leukocyte apheresis were centrifuged at 400 xg for 30 minutes at room temperature using a Ficoll-Hypaque concentration gradient, and PBMCs were isolated from the buffy coat. Cell counts were measured using an ADAM™-MC2 automated cell counter (NanoEnTek), and to obtain CD3(-) cells, the cells were transferred to a new tube and centrifuged at 350 xg for 10 minutes at 4°C. After centrifugation, the supernatant was removed to obtain 1 x 10 7 80 µl of CliniMACS® buffer (20% human serum albumin, 2mM EDTA) and 22 µl of CD3 magnetic beads (Miltenyi Biotech, 130-050-101) were dispensed per cell count to suspend the cell pellet, and the mixture was incubated in the dark at 4°C for 15 minutes. After the reaction, 10 ml of CliniMACS® buffer was dispensed for washing, and the mixture was centrifuged at 350 xg at 4°C for 10 minutes, followed by 1 x 10 8 The cell pellet was suspended by dispensing 0.5 ml of CliniMACS® buffer per cell count.

[0428] 3 mL of CliniMACS® buffer was run over an LD column (Miltenyi Biotec, 130-042-901) to moisten it, and the cell suspension was run through it to collect CD3(-) cells that passed through the column. For washing, 10 mL of CliniMACS® buffer was added, and the mixture was centrifuged at 350 xg at 4°C for 10 minutes. The cell pellet was then resuspended in CliniMACS® buffer, and the cell count was measured using an automated cell counter. Subsequently, 1 x 10⁶ 7The cell pellet was suspended by dispensing 80 µl of CliniMACS® buffer and 22 µl of CD56 magnetic beads (Miltenyi Biotech, 130-050-401) per cell count, and incubated for 15 minutes under light-shielded, refrigerated conditions at 4°C. For washing, 10 ml of CliniMACS® buffer was added and centrifuged at 350 xg at 4°C for 10 minutes, followed by 1 x 10 8 The cell pellet was suspended by dispensing 0.5 ml of CliniMACS® buffer per cell count.

[0429] 3 mL of CliniMACS® buffer was flowed onto an LS column (Miltenyi Biotec, 130-042-401) to moisten it, and then the cell suspension was flowed through it. Subsequently, the column was detached from the magnet stand, and 5 mL of CliniMACS® buffer was added. Pressurization was applied using a piston to obtain CD3(-) CD56(+) natural killer cells in a new tube. The obtained cells were centrifuged at 350 x g for 10 minutes at 4°C. After centrifugation, the cell pellet was resuspended in CliniMACS® buffer, and the cell count was measured using an automated cell counter. 10–20 x 10⁶ cells per 1 mL of CryoStor® CS10 cryopreservation agent (Biolife solution, 210102) 6 The cells were suspended to the required number. 1 ml of the suspended cells were dispensed into cryogenic vials, frozen at -80°C for about 16 hours using Coolcell® (Corning, CLS432001), and then transferred to a liquid nitrogen tank for storage until use.

[0430]

[0431] Example 4-4: Preparation of natural killer cells expressing chimeric proteins containing IL-2 and IL-15 and chimeric antigen receptors

[0432] For the culture of the isolated natural killer cells of Example 4-3, the NK cell activation / expansion kit (Miltenyi Biotec, 130-094-483) was used. 100 µl of CD335(NKp46)-biotin and 100 µl of CD2-biotin included in the kit were dispensed into a new tube 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 it using a microtube rotator (Thermo scientific, Hulamixer, 15920D) at 4.0 rpm under 4°C refrigerated conditions for 2 hours. After thawing the frozen natural killer cells from Example 4-3 and counting the cells, the previously prepared natural killer cell activation beads were washed with 1 ml of CliniMACS® buffer to obtain 1 x 10⁶ 6 Based on 5 µl of beads per cell, the cells were suspended in CN5-101 medium (containing 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 under 5% CO2 conditions.

[0433] On the 4th day from the start of natural killer cell culture, to transfect the retrovirus prepared in Example 4-2, 10 μg / ml retronectin (Takara, T100B) was coated onto an untreated surface 12-well plate at a concentration of 10 μg / ml, 300 μl of retrovirus corresponding to 1 to 10 MoI was added to each well, and the retrovirus was adsorbed onto the retronectin-coated plate by centrifuging at 2,000 xg at 32°C for 2 hours. After removing the supernatant, the plate was washed once with 1 ml of DPBS (WELGENE, LB001-02).

[0434] Natural killer cell survival, cell viability (%), cell density (x 10) 6 cells / mL) and the dead cell count were measured, and 0.8 x 10⁶ were added to CN5-101 medium. 6 The cells were suspended to an adjusted cell density of cells / mL. After first dispensing 0.6 mL of CN5-101 medium, 1 mL was dispensed into well plates coated with the prepared natural killer cells. Additionally, after centrifuging at 1,000 xg at 32°C for 15 minutes to combine the retrovirus and natural killer cells, the cells were incubated at 37°C under 5% CO2 conditions at 3-day intervals to obtain 0.5 x 10 6 Subcultured by inoculation at a cell density of cells / mL.

[0435] On the third day of culture after transduction, the expression of chimeric proteins and chimeric antigen receptors was confirmed using a flow cytometer, and 0.5 x 10 6 The same number of cells were cultured in suspension in CN5-101 containing GI-101 at a cell density of cells / mL. Subsequently, cell counts and viability were checked at 3-day intervals until the end of culture, and 0.5 x 10 6 Subcultured at a cell density of cells / mL.

[0436]

[0437] Examples 4-5: Analysis of expression of chimeric proteins including IL-2 and IL-15 and chimeric antigen receptors

[0438] Natural killer cells were obtained on days 3 and 9 after retrovirus transduction, and 0.2 x 10⁶ were counted using an automated cell counter. 6Cells were placed in a 96-well plate, and FACS buffer (DPBS containing 2% FBS) was filled to 250 µl and centrifuged at 350 xg for 5 minutes. After centrifugation, the supernatant was removed, and the cell pellet was suspended in a 200 ng / mL solution of His-Tag-labeled human HER2 / ErbB2 (498-648) protein (ACROBiosystems, HE2-H52H4) and reacted at 4°C for 20 minutes. Subsequently, 150 µl of FACS buffer was added, and the mixture was centrifuged at 350 xg for 5 minutes at 4°C. The supernatant was removed, and the sample was suspended in FITC-labeled anti-CD56 antibody (Biolegend, 362546), BV421-labeled anti-IL-15Rα antibody (BD, 747704), anti-NKG2D antibody (Biolegend, 320808), and LIVE / DEAD Fixable violet stained sample (Invitrogen, L34955A). The sample was then reacted for 30 minutes in the dark at 4°C. Afterward, 150 µl of FACS buffer was added, and the sample was centrifuged at 350 xg for 5 minutes at 4°C. The supernatant was removed, the cell pellet was resuspended in 200–250 µl of FACS buffer, and centrifuged once more at 350 xg for 5 minutes at 4°C. After centrifugation, the samples were dispensed into FACS tubes, and the expression of the chimeric protein and chimeric antigen receptor was measured using a flow cytometer (FACSymphony™ A3 Cell analyzer, BD). As a result of the measurement, as shown in Figure 9, it was confirmed that the group in which the chimeric protein containing IL-2 and IL-15 was introduced showed superior expression efficiency compared to the group in which the chimeric antigen receptor was introduced alone or the group in which the chimeric antigen receptor and IL-15 were introduced together.

[0439]

[0440] Example 5: Evaluation of in vitro anticancer efficacy of natural killer cells expressing chimeric proteins including IL-2 and IL-15 and chimeric antigen receptors

[0441] Cytotoxicity tests were conducted using the incucyte® instrument to evaluate the in vitro cytotoxicity against target tumor cell lines. GFP-introduced puromycin-resistant tumor cell lines (HCT116-GFP, human HER2 KO HCT116-GFP, SKOV3-GFP) used as target tumor cell lines were subcultured in R10 medium (RPMI 1640, 10% fetal bovine serum, 1% penicillin-streptomycin) containing 0.5 µg / ml puromycin until use in the test. For the cytotoxicity test, the target tumor cell lines were washed in puromycin-free R10 medium, resuspended, and placed in 96-well plates (Corining, 3596) at a rate of 3 x 10⁶ cells per well. 3 cells / 100 µl (HCT116 cell line) or 5 x 10 3 Cells were dispensed at a rate of 100 µl per 100 cells (SKOV3 cell line). Subsequently, the cells were cultured at 37°C under 5% CO2 conditions for 16 hours to allow for cell attachment. Effective cells (Un-TD NK, CAR NK x mbIL-15, CAR NK x mb-IL-2 / IL-15) prepared according to Example 4-4 and frozen were thawed. Cell suspensions were prepared with an effector cell to tumor cell ratio of 1:1 or 0.3:1 and cultured together with target cells prepared the previous day at 37°C under 5% CO2 conditions. Cell morphology and GFP fluorescence intensity expressed in the cells were imaged at 4-hour intervals for 84 hours using an Incucyte®S3 instrument (Sartorius). Cytocytic efficacy was analyzed by counting the number of viable target cells based on the GFP fluorescence intensity and cell morphology of the captured images, thereby analyzing the anticancer effect in the groups treated with natural killer cells compared to the untreated group.

[0442]

[0443] Example 5-1: Evaluation of NKG2D CAR-NK Monotherapy Efficacy

[0444] The anticancer efficacy was evaluated by treating HCT-116 WT and SKOV3 cell lines with Un-TD NK, NKG2D CAR-NK, NKG2D CAR x mbIL-15 NK, and NKG2D CAR x mbIL-2 / IL-15 NK at an effector cell:tumor cell ratio of 1:1 or 0.3:1. As a result, as shown in Figure 10, excellent anticancer efficacy was confirmed in the group treated with NKG2D CAR x mbIL-2 / IL-15 NK in SKOV3 cell lines with low NKG2D expression.

[0445]

[0446] Example 5-2: Evaluation of anti-HER2 single CAR-NK anticancer efficacy

[0447] The anticancer efficacy was evaluated by treating 371HCT-116 WT, HCT116 HER2 KO, and SKOV3 cell lines with Un-TD NK, anti-HER2 CAR-NK, anti-HER2 CAR x mbIL-15 NK, and anti-HER2 CAR x mbIL-2 / IL-15 NK at an effector cell:tumor cell ratio of 1:1 or 0.3:1. As a result, as shown in Figure 11, no significant results were obtained between the test groups in the HCT-116 cell line, but the anticancer efficacy of the anti-HER2 CAR x mbIL-2 / IL-15 NK treatment group was confirmed to be excellent in the SKOV3 cell line.

[0448]

[0449] Example 5-3: Evaluation of anti-HER2 X NKG2D dual CAR-NK anticancer efficacy

[0450] The anticancer efficacy of Un-TD NK, Anti-HER2xNKG2D CAR x mbIL-15 NK, and Anti-HER2xNKG2D CAR x mbIL-2 / IL-15 NK was evaluated by treating HCT-116 WT, HCT116 HER2 KO, and SKOV3 cell lines with an effector cell:tumor cell ratio of 1:1 or 0.3:1. As a result, as shown in Figure 12, the superior anticancer efficacy of anti-HER2 X NKG2D CAR x mbIL-2 / IL-15 CAR-NK compared to anti-HER2 X NKG2D CAR x mbIL-15 CAR-NK was confirmed in HER2-deficient HCT-116 and SKOV3 cell lines.

[0451]

[0452] Example 6: Evaluation of sustained anticancer efficacy based on the presence or absence of chimeric protein expression including IL-2 and IL-15

[0453] Example 6-1: Confirmation of cell number and viability based on chimeric protein expression

[0454] To evaluate the persistence of cytokine expression and the type of expression on the surface of CAR-NK cells at an in vitro level, Un-TD NK, NKG2D CAR x mbIL-15 NK, and NKG2D CAR x mbIL-2 / IL-15 NK prepared and frozen according to Example 4-4 were thawed and recultured in CN5 medium that does not contain auxiliary proteins.

[0455] In the case of Un-TD NK and NKG2D CAR-NK x mbIL-15 NK, the cell count was temporarily maintained until 3 days after the start of culture, but thereafter, as the culture period progressed, the cell count decreased and showed a tendency to decrease below the initial level after 6 days of culture (Fig. 13a).

[0456] In contrast, NKG2D CAR x mbIL-2 / IL-15 NK increased in cell number during the initial stages of culture and maintained a cell number above the initial level until day 14 of culture. Additionally, when comparing viability measured on the same culture day, NKG2D CAR x mbIL-2 / IL-15 NK showed a tendency to maintain a relatively high viability throughout the entire culture period (Fig. 13b).

[0457] These results indicate that NKG2D CAR x mbIL-2 / IL-15 NK is significantly superior to CAR-NKs that do not express cytokines or express IL-15 in their ability to maintain cell number and viability over the long term under reculture conditions where no helper protein is supplied after thawing.

[0458]

[0459] Example 6-2: Confirmation of sustained anticancer efficacy depending on the presence of chimeric protein expression

[0460] To evaluate whether differences in persistence depending on the presence and type of cytokine expression on the surface of CAR-NK cells affect the anticancer effect, Un-TD NK, NKG2D CAR x mbIL-15 NK, and NKG2D CAR x mbIL-2 / IL-15 NK, which were recultured in a medium without auxiliary proteins immediately after thawing and for 6 and 14 days, were treated to confirm the cell killing effect.

[0461] Immediately after thawing, NKG2D CAR x mbIL-15 NK and NKG2D CAR x mbIL-2 / IL-15 NK exhibited similar cytotoxic effects against each tumor cell line, whereas Un-TD NK showed relatively lower anticancer efficacy from the moment of thawing. Furthermore, as the culture period progressed, the anticancer efficacy of NKG2D CAR x mbIL-15 NK showed a tendency to gradually decrease, whereas NKG2D CAR x mbIL-2 / IL-15 NK maintained a higher cytotoxic effect compared to NKG2D CAR x mbIL-15 NK and Un-TD NK on the same day of culture and against the same tumor cell line. Therefore, it was confirmed that NKG2D CAR x mbIL-2 / IL-15 NK demonstrated excellent persistence of anticancer efficacy against various tumor cell lines even under conditions of long-term re-culture after thawing (Figs. 14 to 18).

[0462]

[0463] The present invention relates to a chimeric protein of IL-2 protein and IL-15 protein or a variant thereof; and genetically modified immune cells modified to express a chimeric antigen protein (CAR). The genetically modified immune cells of the present invention suppress side effects caused by excessive immune cell activation by IL-2 and prevent rapid exhaustion of immune cells, thereby maintaining an appropriate level of cell activity and cell survival ability for a long time, and thus exhibit superior activity over a long period compared to existing CAR-expressing immune cells. Therefore, the genetically modified immune cells of the present invention are useful as an immune cell therapeutic agent for regulating the immune system and treating immune-related diseases such as cancer, infectious diseases, and autoimmune diseases.

[0464]

[0465] Foregoing, specific parts of the content of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0466]

[0467] I have attached the electronic file.

Claims

1. Chimeric proteins including IL-2 and IL-15 or variants thereof; and genetically modified immune cells expressing chimeric antigen receptors.

2. The genetically modified immune cell according to claim 1, wherein the chimeric protein is characterized in that the AB loop domain, helix B domain, and CD loop domain of IL-2 are substituted with the AB loop domain, helix B domain, and CD loop domain of IL-15, respectively.

3. A genetically modified immune cell according to claim 1, characterized in that the chimeric protein comprises the amino acid sequence of SEQ ID NO.

33.

4. A genetically modified immune cell according to claim 1, wherein the variant of the chimeric protein comprises a substitution at at least one amino acid position selected from the group consisting of the 73rd, 74th, 76th, 77th, and 80th amino acid positions in the sequence of SEQ ID NO.

33.

5. A genetically modified immune cell according to claim 1, wherein the variant of the chimeric protein 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.

6. A genetically modified immune cell according to claim 1, wherein the variant of the chimeric protein 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.

7. A genetically modified immune cell according to claim 1, wherein the variant of the chimeric protein comprises the amino acid sequence of SEQ ID NOs 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

8. A genetically modified immune cell according to claim 1, wherein the chimeric protein or a variant thereof is fused with a membrane protein or a transmembrane domain.

9. A genetically modified immune cell according to claim 8, 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.

10. A genetically modified immune cell according to claim 8, wherein the chimeric protein or a variant thereof is fused with an IL-15 receptor alpha protein or a fragment thereof.

11. A genetically modified immune cell according to claim 8, characterized in that the fragment of the IL-15 receptor alpha protein comprises the sushi domain of the IL-15 receptor alpha protein.

12. A genetically modified immune cell according to claim 1, characterized by expressing a fusion protein comprising a structure represented by the following structural formula (I) or structural formula (II). N'-X-[L1] n -Y-C'(Structural Formula(I)) N'-Y-[L1] n -X-C'(Structural Formula(II)) Here, X is the above chimeric protein or chimeric protein variant, and Y is the IL-15 receptor alpha (IL-15Rα) protein or a fragment thereof, and L1 is the linker, and n is an integer greater than or equal to 0 or 1.

13. A genetically modified immune cell according to claim 1, wherein the chimeric antigen receptor comprises an antibody or an antigen-binding fragment thereof that specifically binds to a target antigen.

14. In paragraph 13, the above target antigen is 4-1BB, 5T4, integrin, activin, amyloid beta, angiopoetin (angiopoetin 1 or 2), angiopoetin analog 3, B cell maturation antigen (BCMA), B-cell activating factor (BAFF), B7-H3, complement 5, CCR4, CCR5, CCL11, CD2, CD3, CD4, CD6, CD11a, CD16A, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD32B, CD33, CD38, CD40, CD45, CD46, CD47, CD52, CD56, CD62, CD70, CD73, CD74, CD79b, CD80, CD105, CD123, CD154, CD166, CD262, CD278, CD319, CD326, Carcinoembryonic antigen (CEA), CGRP, Claudin-18, c-Met, CSF-1, CSF-1 receptor, CTLA4, DLL3, EGF receptor, Hemophilia factor, Fc receptor, FGF23, Folate receptor, GD2, Glucocorticoid-induced TNF receptor (GITR), Glypican 3, GM-CSF, HER2, HER3, TROP2, Hepatocyte Growth Factor (HGF), Interferon receptor, Interferon gamma, IgE, IGF-1 receptor, Interleukin 1, Interleukin 2 receptor, Interleukin 4, Interleukin 4 receptor, Interleukin 5, Interleukin 5 receptor, Interleukin 6, Interleukin 6 receptor, Interleukin 8, Interleukin 12 / 23, Interleukin 13, Interleukin 17A, Interleukin 17 receptor A, Interleukin 23, Interleukin 31 receptor, Interleukin 36 receptor,Lymphocyte-activation gene 3 (LAG3), Lysyl oxidase homolog 2 (LOXL2), Mesothelin, Mucin-1, Mucin-16, Nectin-4, Nerve Growth Factor (NGF), OX40, Proprotein Convertase Subtilisin / Kexin type 9 (PCSK9), PD-1, PD-L1, Phospholipase C, RANKL (Receptor activator of nuclear factors kappa B ligand), Tyrosine-protein kinase transmembrane receptor (ROR1), Sialic acid binding Ig-like lectin 15 Ig-like lectin 15 (Siglec-15), Transforming growth factor beta (TGFβ), TIGIT (T-cell innunoreceptor with immunoglobulin and ITIM domain), T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), Tissue factor, Tissue factor pathway inhibitor (TFPI), TORP-2, Tumor necrosis factor (TNF), Thymic stromal lymphopoietin (TSLB), Colony stimulating factor 1 receptor (CSF1R),A genetically modified immune cell characterized by having one or more tumor-associated antigens selected from the group consisting of vascular endothelial growth factor (VEGF), VEGF receptor, vWF (von Willebrand Factor), and NKG2D ligand.

15. In claim 1, the chimeric antigen receptor comprises the alpha (α), beta (β), or zeta (ζ) chain of a T-cell receptor (TCR), CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8, CD8a, CD8b, CD9, CD16, CD22, CD27, CD28, CD28H, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD86, CD134, CD137, CD154, CD166, 4-1BB, OX40, FcεRIγ, transferrin receptor (TfR), IL-2R, IL-15R, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, IL-7, IL-12, A genetically modified immune cell characterized by comprising a transmembrane domain derived from a protein selected from the group consisting of IL-15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, and CS1.

16. A genetically modified immune cell according to claim 1, wherein the chimeric antigen receptor comprises one or more signaling domains selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d.

17. A genetically modified immune cell according to claim 1, wherein the chimeric antigen receptor comprises one or more co-stimulating signaling domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), HVEM (Herpesvirus Entry Mediator), IL-2Rβ, IL-15Rα, SLAMF7, CTLA-4 GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, and NKG2C.

18. A genetically modified immune cell according to claim 1, wherein the chimeric antigen receptor comprises the sequence of SEQ ID NO. 200, 202, or 204.

19. Genetically modified immune cell according to claim 1, characterized in that the immune cell is a natural killer cell.

20. A pharmaceutical composition for the prevention or treatment of cancer, infectious diseases, or autoimmune diseases comprising genetically modified immune cells according to any one of claims 1 to 19.

21. A method for treating a disease of cancer, an infectious disease, or an autoimmune disease, comprising the step of administering a genetically modified immune cell of any one of claims 1 to 19 to a subject who requires it.

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