Medium composition for culturing t cells and method for culturing t cells using same

Abstract

The present invention relates to a composition for proliferating a T cells, containing a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, and to a method for culturing T cells using same. The T cells cultured according to the present invention increase the proliferation and activity of T cells even without using CD3 / CD28 antibody-bound magnetic beads and proliferate T cells by culturing a patient’s own peripheral blood mononuclear cells and are not likely to cause side effects in the human body, and thus will be widely used as a novel T cell therapeutic agent. Furthermore, in the case of CD8+ T cells cultured as described above, the activity thereof increases, and thus, the CD8+ T cells can be used as a more effective therapeutic agent.

Description

T cell culture medium composition and method for culturing T cells using the same

[0001] The present invention relates to a T cell culture medium composition containing a fusion protein comprising a CD80 protein and an IL-2 wild-type or variant, and a method for culturing T cells using the same.

[0002] Novartis’s Kymriah (active ingredient: tisagenlecleucel, product code: CTL019), which recently received marketing authorization from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), is a gene therapy composed of autologous genetically modified T cells (U.S. Patent No. 9499629). This product is produced from the patient’s own T cells, which are transduced using a lentiviral vector encoding a chimeric antigen receptor (CAR) for human CD19. These T cells act in an antigen-dependent manner and specifically target and destroy CD19-positive B cells in a manner independent of the Major Histocompatibility Complex (MHC).

[0003] The manufacturing process of Kymriah is as follows. T cells are proliferated through two different processes, and the proliferated T cells are stimulated by magnetic beads conjugated with CD3 and CD28 antibodies, after which they are transduced with the CTL019 HIV-1 vector. Subsequently, CAR-expressing autologous T cells are proliferated through culture for a certain period, followed by a washing process to remove impurities such as the CD3 / CD28 conjugated beads. However, recently, Dynabeads, the CD3 / CD28 antibody-conjugated magnetic beads used for T cell proliferation and activation in the Kymriah manufacturing process... ® It has been confirmed that it has the potential to cause acute toxicity.

[0004] Furthermore, replication competent lentiviruses (RCLs) within pharmaceuticals are known to be infectious and can be transmitted to people who come into contact with them. After administering Kymriah to patients who carry the risk of transmitting new lentiviruses through the donation of blood, organs, tissues, or cells for transfusion or transplantation, RCLs may be formed due to vector transfer resulting from the complementarity between the provirus and the host sequence. Therefore, in the case of patients who were previously HIV positive, there is a possibility of generating new HIV viruses. As such, since new replication competent lentiviruses could spread into the human population, it was determined that the theoretical potential adverse effects on the environment are significant.

[0005] Consequently, there is an increasing demand for new autologous T-cell therapies that do not have these side effects.

[0006] The inventors, through diligent efforts to increase the proliferation and activity of T cells without using CD3 / CD28 antibody-conjugated magnetic beads, confirmed that the proliferation and activity of T cells increased by culturing peripheral blood mononuclear cells in a cell culture medium in the presence of a fusion protein containing CD80 protein and IL-2 wild-type or variant, and thus completed the present invention.

[0007] To achieve the above objective, a composition or medium for T cell culture is provided, comprising as an active ingredient a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

[0008] In addition, a method for culturing T cells using a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof is provided.

[0009] In addition, the present invention provides a pharmaceutical composition comprising T cells cultured in a medium containing a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof as an active ingredient.

[0010] T cells cultured using the T cell culture medium composition and the T cell culture method utilizing the present invention exhibit increased proliferation and activity without the use of CD3 / CD28 antibody-bound magnetic beads. Consequently, since impurities such as magnetic beads are not generated, T cells can be produced more safely. Furthermore, because T cells are proliferated by culturing the patient's own peripheral blood mononuclear cells, there is no concern regarding adverse effects on the human body, making it likely to be widely utilized as a novel T cell therapeutic agent.

[0011] Figure 1 is a schematic illustration of one example of the preparation of a fusion protein used in the present invention.

[0012] Figure 2 shows the obtained fusion protein (GI-101) confirmed by SDS-PAGE.

[0013] Figure 3 shows the obtained fusion protein (GI-101) analyzed by size exclusion chromatography (SEC).

[0014] Figure 4 shows the obtained Fc-IL2v2 fusion protein confirmed by SDS-PAGE.

[0015] Figure 5 shows the obtained Fc-IL2v2 fusion protein analyzed by size exclusion chromatography (SEC).

[0016] Figure 6 shows the obtained Fc-IL2wt fusion protein dimer confirmed by SDS-PAGE.

[0017] Figure 7 shows the obtained Fc-IL2wt fusion protein dimer analyzed by size exclusion chromatography (SEC).

[0018] Figure 8 shows the obtained hCD80-Fc-IL2wt fusion protein confirmed by SDS-PAGE.

[0019] Figure 9 shows the obtained hCD80-Fc-IL2wt fusion protein analyzed by size exclusion chromatography (SEC).

[0020] Figure 10 shows the obtained hCD80-Fc fusion protein confirmed by SDS-PAGE.

[0021] Figure 11 shows the obtained hCD80-Fc fusion protein analyzed by size exclusion chromatography (SEC).

[0022] Figure 12 shows the total number of CD4-PBMC cells when cultured in a culture composition containing the basic culture medium of Table 1.

[0023] Figure 13 shows the viability of CD4-PBMC cells when cultured in a culture composition containing the basic culture medium of Table 1.

[0024] Figure 14 shows the total number of CD4-PBMC cells when cultured in a culture composition containing the basic culture medium of Table 2.

[0025] Figure 15 shows the viability of CD4-PBMC cells when cultured in a culture composition containing the basic culture medium of Table 2.

[0026] Figure 16 shows the results of cell surface flow cytometry analysis of CD4-PBMC cells treated with GI-101 or hCD80-Fc and Fc-IL2v additives according to the present invention in a culture composition containing the basic culture medium of Table 1.

[0027] Figure 17 is a graph quantifying the total number of T cells (CD3+CD19- cells) by FACS (Fluorescence-activated cell sorting) analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v additive substances of the present invention.

[0028] Figure 18 is a graph showing the quantification of the number of CD8 T cells by FACS analysis of CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0029] Figure 19 is a graph quantifying the number of CD25+ memory T cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0030] Figure 20 is a graph quantifying the number of central memory T cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0031] Figure 21 shows the results of cell surface flow cytometry analysis of CD4-PBMC cells treated with GI-101 or hCD80-Fc and Fc-IL2v additives according to the present invention in a culture composition containing the basic culture medium of Table 2.

[0032] Figure 22 is a graph quantifying the total number of T cells (CD3+CD19- cells) by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0033] Figure 23 is a graph showing the quantification of the number of CD8 T cells by FACS analysis of CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0034] Figure 24 is a graph quantifying the number of CD25+ memory T cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0035] Figure 25 is a graph quantifying the number of central memory T cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0036] Figure 26 shows the results of intracellular flow cytometry analysis of CD4-PBMC cells treated with GI-101 or hCD80-Fc and Fc-IL2v additives according to the present invention in a culture composition containing the basic culture medium of Table 1.

[0037] Figure 27 is a graph quantifying the number of Granzyme B+ CTL (Cytotoxicity T Lymphocyte) cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v added substances of the present invention.

[0038] Figure 28 is a graph quantifying the number of IFN-γ CTL cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0039] Figure 29 is a graph quantifying the number of perforin+ CTL cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v added substances of the present invention.

[0040] Figure 30 shows the results of intracellular flow cytometry analysis of CD4-PBMC cells treated with GI-101 or hCD80-Fc and Fc-IL2v additives according to the present invention in a culture composition containing the basic culture medium of Table 2.

[0041] Figure 31 is a graph quantifying the number of Granzyme B+ CTL cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101 or hCD80-Fc and Fc-IL2v additives of the present invention.

[0042] Figure 32 is a graph quantifying the number of IFN-γ CTL cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0043] Figure 33 is a graph quantifying the number of perforin+ CTL cells by FACS analysis on CD4- PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 2, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0044] Figure 34 shows the total number of CD8+ PBMC cells when cultured in a culture composition containing the basic culture medium of Table 1.

[0045] Figure 35 shows the viability of CD8+ PBMC cells when cultured in a culture composition containing the basic culture medium of Table 1.

[0046] Figure 36 shows the results of cell surface flow cytometry analysis of CD8+ PBMC cells treated with GI-101 or hCD80-Fc and Fc-IL2v additives according to the present invention in a culture composition containing the basic culture medium of Table 1.

[0047] Figure 37 is a graph quantifying the total number of T cells (CD3+CD19- cells) by FACS analysis on CD8+ PBMC cells cultured for 1 day in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v additive substances of the present invention.

[0048] Figure 38 is a graph showing the quantification of the number of CD8 T cells by FACS analysis of CD8+ PBMC cells cultured for one day in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additive substances of the present invention.

[0049] Figure 39 is a graph quantifying the number of CD25+ memory T cells by FACS analysis on CD8+ PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v additive substances of the present invention.

[0050] Figure 40 is a graph quantifying the number of central memory T cells by FACS analysis on CD8+ PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0051] Figure 41 shows the results of intracellular flow cytometry analysis of CD8+ PBMC cells treated with GI-101 or hCD80-Fc and Fc-IL2v additives according to the present invention in a culture composition containing the basic culture medium of Table 1.

[0052] Figure 42 is a graph quantifying the number of Granzyme B+ CTL cells by FACS analysis on CD8+ PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v added substances of the present invention.

[0053] Figure 43 is a graph quantifying the number of IFN-γ CTL cells by FACS analysis on CD8+ PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1, treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0054] Figure 44 is a graph quantifying the number of perforin+ CTL cells by FACS analysis on CD8+ PBMC cells cultured for 14 days in a culture composition containing the basic culture medium of Table 1 treated with the GI-101, or hCD80-Fc and Fc-IL2v additives of the present invention.

[0055] Figure 45 shows the results of analyzing the apoptotic effect of Her2 protein-recognizing T cells cultured in a culture composition containing the basic culture medium of Table 1 treated with the GI-101 additive of the present invention on BT-474 (ATCC® HTB-20™) cancer cells.

[0056] Figure 46 shows the results of analyzing the apoptotic effect of Her2 protein-recognizing T cells cultured in a culture composition containing the basic culture medium of Table 1 treated with the GI-101 additive of the present invention on CAMA-1 (ATCC® HTB-21™) cancer cells.

[0057] Composition and medium for T cell proliferation

[0058] One aspect of the present invention provides a composition for T cell proliferation comprising a fusion protein comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof. Additionally, the invention provides a medium for T cell proliferation or culture comprising said fusion protein dimer as an active ingredient.

[0059] The above T cell proliferation medium may be a medium to which a fusion protein dimer comprising the IL-2 protein or a variant thereof and the CD80 protein or a fragment thereof is added to a T cell culture medium. In this case, the T cell culture medium may include any one selected from the group consisting of amino acids, sugars, inorganic salts, and vitamins. Preferably, the T cell culture medium may include all of amino acids, sugars, inorganic salts, and vitamins. Additionally, the medium may further include FBS (Fetal Bovine Serum), HEPES (Hydroxyethyl piperazine ethyl sulfonic acid), proteins, carbohydrates, mercaptoethanol, and growth factors.

[0060] As used herein, the term "medium for cell culture" refers to a medium used to culture cells, specifically a medium for culturing T cells, more specifically CD8+ cells. It contains components required by cells for cell growth and survival in vitro, or components that aid in cell growth and survival. Specifically, said components may be vitamins, essential or non-essential amino acids, and trace elements.

[0061] The cell culture medium according to the present invention is composed of an amino acid component, a vitamin component, an inorganic salt component, other components, and purified water, and

[0062] a) The above amino acid component is at least one amino acid selected from the group consisting of glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-threonine, L-serine, L-cysteine, L-methionine, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, L-lysine, L-arginine, L-histidine, L-phenylalanine, L-tyrosine, L-tryptophan, L-proline, β-alanine, β-aminobutyric acid, ornithine, citrulline, homoserine, triiodotyrosine, thyroxine, and deoxyphenylalanine, or a combination thereof, preferably glycine, L-alanine, L-arginine, L-cysteine, L-glutamine, L-histidine, L-lysine, L-methionine, At least one amino acid selected from the group consisting of L-proline, L-serine, L-threonine, and L-valine, or a combination thereof,

[0063] b) The above vitamin component is at least one vitamin selected from the group consisting of biotin, calcium D-pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, choline chloride, i-inositol, and ascorbic acid, or a combination thereof, and preferably, at least one vitamin selected from the group consisting of i-inositol, thiamine hydrochloride, niacinamide, and pyridoxine hydrochloride, or a combination thereof.

[0064] c) The above inorganic salt component is at least one inorganic salt or a combination thereof selected from the group consisting of calcium chloride (CaCl2) (anhydrous), copper sulfate pentahydrate (CuSO4-5H2O), ferric sulfate heptahydrate (FeSO4-7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4) (anhydrous), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), sodium hydrogen phosphate monohydrate (NaH2PO4-H2O), zinc sulfate heptahydrate (ZnSO4-7H2O), ferric nitrate nonahydrate (Fe(NO3)3·9H2O), and sodium bicarbonate (NaHCO3), and preferably, sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl2) (anhydrous), and sodium hydrogen phosphate. At least one inorganic salt selected from the group consisting of monohydrates (NaH2PO4-H2O) or a combination thereof, and

[0065] d) The above other component is at least one other component selected from the group consisting of D-glucose (dextrose), sodium pyruvate, hypoxanthine Na, thymidine, linoleic acid, lipoic acid, adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine HCl, and 2'-deoxyguanosine, or a combination thereof, and preferably may be sodium pyruvate.

[0066] e) Purified water is used to dissolve the above amino acids, vitamins, inorganic salts and other components, and may be water obtained through one or more distillations or purified through a filter.

[0067] In addition, the cell culture medium according to the present invention may further include growth factors or cytokines. The growth factors may include IGF, bFGF, TGF, HGF, EGF, VEGF, or PDGF, either alone or in combination of two or more types, but are not specifically limited thereto. The cytokines may include IL-1, IL-4, IL-6, IFN-γ, IL-10, or IL-17, either alone or in combination of two or more types, but are not specifically limited thereto.

[0068] As used herein, the term "T cell" refers to a type of lymphocyte responsible for antigen-specific adaptive immunity. T cells are classified into naive T cells, which have not yet encountered an antigen; mature T cells, which have encountered an antigen; and memory T cells. In this case, the mature effector T cells include helper T cells, cytotoxic T cells, and natural killer T cells. Preferably, the T cells may be CD8+ T cells.

[0069] As used herein, the term "helper T cell" (or "Th cell") refers to a cell that promotes humoral immunity by regulating the differentiation and activation of other leukocytes. Because it possesses the CD4 protein on its surface, it is also called a CD4+ T cell. Helper T cells can be further classified into Th1, Th2, Th17, and Treg depending on their specific functions. Th1 cells secrete interferon gamma (IFN-γ) and tumor necrosis factor beta (TNF-β), thereby inducing the fusion of endosomes and lysosomes within macrophages to form endolysosomes. Meanwhile, Th2 cells secrete various types of interleukin (IL) to induce B cells to differentiate into plasma cells. Th17 cells secrete interleukin-17 (IL-17) to cause neutrophils to assemble.

[0070] As used herein, the term "regulatory T cell (Treg)" includes natural regulatory T cells (nTreg) or induced regulatory T cells (iTreg). In this specification, regulatory T cells include CD4+CD25+ T cells, CD4+CD25+CD127low / - T cells, or CD4+CD25+Foxp3+ T cells. These regulatory T cells maintain immune homeostasis by suppressing immune responses and block autoimmune responses, etc.

[0071] As used herein, the term "cytotoxic T cell" refers to a cell that kills virus-infected cells or tumor cells by secreting cytotoxic substances such as granzymes or perforins. It is also called a CD8 T cell because it possesses the CD8 protein on its surface. Unlike helper T cells, it eliminates viruses and cancer cells by mediating cellular immunity.

[0072] As used herein, the term "natural killer T cell" refers to one of the effector T cells distributed in smaller proportions compared to helper T cells and cytotoxic T cells. Natural killer T cells have T cell antigen receptors (TCRs) on their cell surface similar to those of T cells, but also possess natural killer cell-specific molecules such as NK1.1. Natural killer T cells play a role in regulating immune responses by secreting gamma interferon, interleukin-4, etc.

[0073] As used herein, the term "memory T cell" refers to a T cell that has the potential to function as an effector T cell by rapidly becoming activated when the antigen invades again, after recognizing the antigen and undergoing differentiation and selection processes to survive for a long period. Naive T cells become activated upon encountering an antigen, or effector T cells differentiate into long-surviving memory T cells under the influence of interleukin-7 and interleukin-15.

[0074] At this time, the fusion protein dimer comprising the IL-2 protein or its variant and the CD80 protein or its fragment may be contained in the culture medium at an amount of 1 nM to 2,000 nM. Additionally, the dimer may be contained at an amount of 1 nM to 1,000 nM, or 1 nM to 500 nM. Furthermore, the dimer may be contained at an amount of 2 nM to 300 nM, 5 nM to 100 nM, 10 nM to 80 nM, 20 nM to 70 nM, or 40 nM to 50 nM. Specifically, the fusion protein dimer may be contained in the medium at an amount of 1 nM, 3.2 nM, 10 nM, or 50 nM.

[0075] A fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof

[0076] As used herein, the terms “IL-2” or “Interleukin-2” mean any wild-type IL-2 obtained from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise noted. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Additionally, the IL-2 may be wild-type IL-2 or a variant thereof.

[0077] In this specification, IL-2 or variants thereof are collectively referred to as "IL-2 protein" or "IL-2 polypeptide." IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind to, for example, an IL-2 receptor. This specific binding can be verified through methods known to those skilled in the art.

[0078] One embodiment of the above IL-2 may have the amino acid sequence of SEQ ID NO. 35 or SEQ ID NO. 36. In addition, the above IL-2 may be in a mature form. Specifically, the above mature IL-2 may not contain a signal sequence and may have the amino acid sequence of SEQ ID NO. 10. In this case, the above IL-2 may be used as a concept comprising a fragment in which a part of the N-terminus or C-terminus of wild-type IL-2 is truncated.

[0079] In addition, the above IL-2 fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the N-terminus of a protein having the amino acid sequence of SEQ ID NO. 35 or SEQ ID NO. 36. In addition, the above IL-2 fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO. 35 or SEQ ID NO. 36.

[0080] As used herein, the term "IL-2 variant" refers to a form in which some of the amino acids of full-length IL-2 or the fragment of IL-2 described above are substituted. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or its fragment. However, the IL-2 variant may have activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may mean, for example, specific binding to an IL-2 receptor, and this specific binding may be measured by methods known to those skilled in the art.

[0081] Specifically, the IL-2 variant may be one in which some of the amino acids of wild-type IL-2 are substituted. One specific example of an IL-2 variant by amino acid substitution may be one in which at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 is substituted.

[0082] Specifically, the IL-2 variant may be one in which at least one of the 38th, 42nd, 45th, 61st, or 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 is substituted with another amino acid. In addition, if IL-2 is in a form in which a portion of the N-terminus of the amino acid sequence of SEQ ID NO. 35 is deleted, the amino acid at the complementary position in the amino acid sequence of SEQ ID NO. 10 may be substituted with another amino acid. For example, if IL-2 has the amino acid sequence of SEQ ID NO. 35, the IL-2 variant may be one in which at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in the amino acid sequence of SEQ ID NO. 35 is substituted with another amino acid. These correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues of the amino acid sequence of SEQ ID NO. 10, respectively. According to one embodiment, as long as IL-2 activity is maintained, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted. According to another embodiment, 1 to 5 amino acids may be substituted.

[0083] In one embodiment, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may be in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be one in which the 42nd and 61st amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be one in which the 42nd and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be one in which the 45th and 61st amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be one in which the 45th and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be one in which the 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted.

[0084] Furthermore, the above IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the above IL-2 variant may be in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Also, in one embodiment, the above IL-2 variant may be in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Also, in one embodiment, the above IL-2 variant may be in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Also, in one embodiment, the above IL-2 variant may be in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Also, in one embodiment, the above IL-2 variant may be in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, in one embodiment, the IL-2 variant may be substituted with the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10. In addition, in one embodiment, the IL-2 variant may be substituted with the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO. 10. In addition, in one embodiment, the IL-2 variant may be substituted with the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10. In addition, in one embodiment, the IL-2 variant may be substituted with the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10.

[0085] Additionally, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may be in which the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Additionally, in one embodiment, the IL-2 variant may be in which the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Additionally, in one embodiment, the IL-2 variant may be in which the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. Additionally, in one embodiment, the IL-2 variant may be in which the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted. In addition, as one specific example, the IL-2 variant may be one in which the 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are substituted.

[0086] Furthermore, the above IL-2 variant may be in a form in which five amino acids are substituted. Specifically, the above IL-2 variant may be in which the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO. 10 are all substituted with other amino acids.

[0087] At this time, the "other amino acid" introduced by the above substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, regarding the amino acid substitution of the above IL-2 variant, the 38th amino acid in the amino acid sequence of SEQ ID NO. 10 cannot be substituted with arginine, the 42nd cannot be substituted with phenylalanine, the 45th cannot be substituted with tyrosine, the 61st cannot be substituted with glutamic acid, and the 72nd cannot be substituted with leucine.

[0088] In the amino acid substitution of the above IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with other amino acids excluding arginine. Preferably, in the amino acid substitution of the above IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with alanine (R38A).

[0089] In the amino acid substitution of the above IL-2 variant, phenylalanine, the 42nd amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with other amino acids excluding phenylalanine. Preferably, in the amino acid substitution of the above IL-2 variant, phenylalanine, the 42nd amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with alanine (F42A).

[0090] In the amino acid substitution of the above IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with other amino acids excluding tyrosine. Preferably, in the amino acid substitution of the above IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with alanine (Y45A).

[0091] In the amino acid substitution of the above IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with other amino acids excluding glutamic acid. Preferably, in the amino acid substitution of the above IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with arginine (E61A).

[0092] In the amino acid substitution of the above IL-2 variant, leucine, the 72nd amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with other amino acids excluding leucine. Preferably, in the amino acid substitution of the above IL-2 variant, leucine, the 72nd amino acid in the amino acid sequence of SEQ ID NO. 10, may be substituted with glycine (L72G).

[0093] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO. 10.

[0094] Specifically, the IL-2 variant may undergo amino acid substitutions at two, three, four, or five positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

[0095] Additionally, the above IL-2 variant may be a form in which two amino acids are substituted. Specifically, the above IL-2 variant may be substituted with R38A and F42A. Additionally, in one embodiment, the above IL-2 variant may be substituted with R38A and Y45A. Additionally, in one embodiment, the above IL-2 variant may be substituted with R38A and E61R. Additionally, in one embodiment, the above IL-2 variant may be substituted with R38A and L72G. Additionally, in one embodiment, the above IL-2 variant may be substituted with F42A and Y45A. Additionally, in one embodiment, the above IL-2 variant may be substituted with F42A and E61R. Additionally, in one embodiment, the above IL-2 variant may be substituted with F42A and L72G. In addition, as one specific example, the IL-2 variant may be substituted with E61R and L72G.

[0096] Furthermore, the above IL-2 variant may be a form in which three amino acids are substituted. Specifically, the above IL-2 variant may be substituted with R38A, F42A, and Y45A. Also, in one embodiment, the above IL-2 variant may be substituted with R38A, F42A, and E61R. Also, in one embodiment, the above IL-2 variant may be substituted with R38A, F42A, and L72G. Also, in one embodiment, the above IL-2 variant may be substituted with R38A, Y45A, and E61R. Also, in one embodiment, the above IL-2 variant may be substituted with R38A, Y45A, and L72G. Also, in one embodiment, the above IL-2 variant may be substituted with F42A, Y45A, and E61R. Additionally, in one embodiment, the IL-2 variant may be substituted with F42A, Y45A, and L72G. Additionally, in one embodiment, the IL-2 variant may be substituted with F42A, E61R, and L72G. Additionally, in one embodiment, the IL-2 variant may be substituted with Y45A, E61R, and L72G.

[0097] Additionally, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be substituted with R38A, F42A, Y45A, and E61R. Additionally, in one embodiment, the IL-2 variant may be substituted with R38A, F42A, Y45A, and L72G. Additionally, in one embodiment, the IL-2 variant may be substituted with R38A, F42A, E61R, and L72G. Additionally, in one embodiment, the IL-2 variant may be substituted with R38A, Y45A, E61R, and L72G. Additionally, in one embodiment, the IL-2 variant may be substituted with F42A, Y45A, E61R, and L72G.

[0098] Furthermore, the above IL-2 variant may have undergone substitution with R38A, F42A, Y45A, E61R, and L72G.

[0099] Preferably, one embodiment of the IL-2 variant may have a substitution of any one of the following combinations (a) to (d) in the amino acid sequence of SEQ ID NO. 10:

[0100] (a) R38A / F42A

[0101] (b) R38A / F42A / Y45A

[0102] (c) R38A / F42A / E61R

[0103] (d) R38A / F42A / L72G

[0104] In this case, if IL-2 has the amino acid sequence of SEQ ID NO. 35, it may have an amino acid substitution at a position complementary to SEQ ID NO. 10. Also, even if IL-2 is a fragment of the amino acid sequence of SEQ ID NO. 35, an amino acid at a position complementary to SEQ ID NO. 10 may be substituted.

[0105] Specifically, the variant of IL-2 may have the amino acid sequence of SEQ ID NO. 6, 22, 23, or 24.

[0106] In addition, the above IL-2 variant may be characterized by having low toxicity in vivo. In this case, the low toxicity in vivo may refer to side effects caused by the binding of IL-2 to the alpha chain (IL-2Rα) of the IL-2 receptor. Various IL-2 variants have been developed to mitigate side effects caused by the binding of IL-2 to IL-2Rα, and such IL-2 variants may be those disclosed in U.S. Patent No. 5,229,109 and Korean Patent No. 10-1667096. In particular, the IL-2 variant described in this application has a low binding affinity to the alpha chain (IL-2Rα) of the IL-2 receptor, resulting in lower in vivo toxicity compared to wild-type IL-2.

[0107] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present in dendritic cells, activated B cells, and monocytes. CD80 provides co-stimulatory signals essential for the activation and survival of T cells. CD80 is known as a ligand for two different proteins present on the surface of T cells, CD28 and CTLA-4. CD80 consists of 288 amino acids and, specifically, may have the amino acid sequence of SEQ ID NO. 11. Additionally, as used herein, "CD80 protein" refers to the full-length CD80 or the CD80 fragment.

[0108] The term "CD80 fragment" as used in this specification refers to a truncated form of CD80. Additionally, the CD80 fragment may be an extracellular domain of CD80. One embodiment of the CD80 fragment may be a CD80 signal sequence in which the 1st to 34th amino acids from the N-terminus are excluded. Specifically, one embodiment of the CD80 fragment may be a protein composed of the 35th to 288th amino acids of SEQ ID NO. 11. Additionally, one embodiment of the CD80 fragment may be a protein composed of the 35th to 242nd amino acids of SEQ ID NO. 11. Additionally, one embodiment of the CD80 fragment may be a protein composed of the 35th to 232nd amino acids of SEQ ID NO. 11. Additionally, one embodiment of the CD80 fragment may be a protein composed of the 35th to 139th amino acids of SEQ ID NO. 11. In addition, one embodiment of the CD80 fragment may be a protein composed of the 142nd to 242nd amino acids of SEQ ID NO. 11. In one embodiment, the CD80 fragment may have the amino acid sequence of SEQ ID NO. 2.

[0109] Additionally, the IL-2 protein and the CD80 protein may be bound by a linker or a carrier. Specifically, the IL-2 or a variant thereof and the CD80 (B7-1) or a fragment thereof may be bound by a linker or a carrier. In this specification, the linker and the carrier may be used interchangeably.

[0110] The above linker connects two proteins. An example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, or the Fc domain of an immunoglobulin. In this case, the Fc domain of the immunoglobulin refers to a protein that includes the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of the immunoglobulin, but does not include the variable regions of the heavy and light chains and the light chain constant region 1 (CH1) of the immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. In this case, the Fc domain of wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO. 4.

[0111] In addition, the Fc domain of the above 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. Additionally, aglycosylated Fc domains are also included. 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.

[0112] In addition, the glycosylation of the Fc domain of the immunoglobulin can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a form in which the Fc region of the immunoglobulin IgG, IgA, IgE, IgD, or IgM is mixed. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. One specific example of the Fc domain variant may be having the amino acid sequence of SEQ ID NO. 12.

[0113] The fusion protein may have a structure in which CD80 and IL-2 proteins are connected to the N-terminus and C-terminus, respectively, using the Fc domain as a linker (or carrier) (Fig. 1). The connection of CD-80 or IL-2 to the N-terminus or C-terminus of the Fc domain may be arbitrarily formed by a linker peptide.

[0114] Specifically, the fusion protein may be composed of the following structural formula (I) or (II):

[0115] N'-X-[linker(1)]n-Fc domain-[linker(2)]mYC' (I)

[0116] N'-Y-[linker(1)]n-Fc domain-[linker(2)]mXC' (II)

[0117] At this time, in the above structural formulas (I) and (II),

[0118] The above N' is the N-terminus of the fusion protein, and

[0119] The above C' is the C-terminus of the fusion protein, and

[0120] The above X is the CD80 protein, and

[0121] The above Y is an IL-2 protein, and

[0122] The above linker (1) and linker (2) are peptide linkers, and

[0123] The above n and m are each independently O or 1.

[0124] Preferably, the fusion protein may be composed of structural formula (I). The IL-2 protein is as described above. Also, the CD80 protein is as described above. According to one embodiment, the IL-2 protein may be an IL-2 variant in which one to five amino acids are substituted compared to wild-type IL-2. The CD80 protein may be a fragment in which up to about 34 amino acid residues are truncated consecutively from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD80 protein may be an extracellular immunoglobulin-like domain having activity of binding to T-cell surface receptors CTLA-4 and CD28.

[0125] Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO. 9, 26, 28, or 30. According to another embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO. 9, 26, 28, or 30. In this case, identity may be determined, for example, through percentage homology, or through homology comparison software such as the BlastN software of NCBI (National Center of Biotechnology Information).

[0126] A peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may consist of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one embodiment, the peptide linker (1) may consist of 30 amino acids. Additionally, the peptide linker (1) may include at least one cysteine. Specifically, it may include one, two, or three cystes. Additionally, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one embodiment, the peptide linker (1) may be a peptide linker composed of the amino acid sequence of SEQ ID NO. 3.

[0127] The peptide linker (2) may consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one embodiment, the peptide linker (2) may be (G4S)n (wherein n is an integer from 1 to 10). In this case, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may be a peptide linker composed of the amino acid sequence of SEQ ID NO. 5.

[0128] Another aspect of the present invention provides a dimer comprising two fusion proteins combined, the IL-2 protein and the CD80 protein. The fusion protein comprising the IL-2 or a variant thereof and the CD80 or a fragment thereof is as described above.

[0129] At this time, the binding between the fusion proteins constituting the dimer may be formed by disulfide bonds through cysteine ​​present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be identical, but may be different fusion proteins. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO. 9.

[0130] T cell culture method

[0131] In another aspect, the present invention relates to a method for culturing T cells comprising the step of culturing cells in the T cell proliferation medium composition. In the present invention, the cells may be characterized as being peripheral blood mononuclear cells (PBMCs). Additionally, the cells may be CD8+ T cells or CD8+ T cells derived from PBMCs.

[0132] In the present invention, the T cell culture medium composition may be characterized by containing 0.1 nM to 1,000,000 nM of fusion protein dimer, and more specifically, may contain 1 nM to 1,000 nM or 1.6 nM to 100 nM of fusion protein. Specifically, the fusion protein dimer may be contained in the T cell medium at 1 nM, 1.6 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, or 50 nM.

[0133] In a specific embodiment of the present invention, PBMCs were cultured using a cell culture medium composition containing 1.6 nM of the above-mentioned IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, or 50 nM of the above-mentioned IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof. Additionally, when culturing CD8+ T cells, a cell culture medium composition containing 1.6 nM of the above-mentioned IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof was used.

[0134] In the present invention, the step of culturing the cells may be characterized by being performed for 7 to 21 days. More specifically, the step of culturing the cells may be performed for 9 to 15 days.

[0135] In a specific embodiment of the present invention, the step of culturing PBMCs was performed for 14 days. As used herein, the term "PBMC" refers to peripheral blood mononuclear cells (PBMCs) and includes T cells, B cells, NK cells, and monocytes.

[0136] The term "CD4-PBMC" as used in this specification refers to peripheral blood mononuclear cells that do not contain immune cells expressing CD4 (cluster of differentiation 4) glycoprotein, and preferably do not contain CD4+ helper T cells.

[0137] As used in this specification, the term "T cell" includes CD4+ or CD8+ T cells.

[0138] As used herein, the term "CD8+ T cell" includes cytotoxic T cells (CD8+) or memory T cells.

[0139] As used herein, the term “Memory T cell” includes effector memory T cell, central memory T cell, tissue-resident memory T cell, peripheral memory T cell, or CD25+ memory T cell.

[0140] In one embodiment of the present invention, it was confirmed that T cells cultured by a culture method comprising the step of culturing in a medium composition containing a fusion protein dimer comprising the IL-2 protein or a variant thereof and the CD80 protein or a fragment thereof showed a significantly increased number of cells and an increased amount of IFN-γ secreted by CD4+ or CD8+ T cells compared to T cells cultured in a medium composition containing Fc-IL2v2, Fc-IL2v3, or rhIL-2.

[0141] Obtained T cells and their uses

[0142] In another aspect of the present invention, T cells obtained by the above-described culture method are provided. In this case, it was confirmed that the T cells obtained by the above-described culture method, preferably CD8+ T cells, showed increased secretion of Granzyme B, IFN-γ, and Perforin compared to CD8+ T cells obtained by another culture method. Accordingly, it was confirmed that the cancer-killing ability of the CD8+ T cells was increased (Figs. 45 and 46).

[0143] In another aspect of the present invention, a pharmaceutical composition for treating cancer is provided, comprising T cells obtained by the method described above as an active ingredient.

[0144] The dosage of the above pharmaceutical composition may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients included in the composition, the type of formulation, the patient's age, weight, general health condition, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and drugs used concurrently.

[0145] In addition, the above pharmaceutical composition may be administered to an individual by various methods known in the art. The route of administration can be appropriately selected by a person skilled in the art, taking into account the method of administration, the volume of body fluid, viscosity, etc.

[0146] The above cancer may be any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0147] The composition of the present invention may include pharmaceutically acceptable carriers and / or additives, etc. For example, it may include sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants, surfactants, suspending agents, isotonic agents, or preservatives, etc. In addition, organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrix, polylactic acid polymer or copolymer, polyethylene glycol polymer or copolymer and chemical derivatives thereof, and mixtures thereof may be used, but are not limited thereto. As the stabilizer, for example, dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc. may be used. For example, the antioxidant may be chelating agents such as erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallic acid, propyl gallic acid, or sodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate, etc. For example, the suspension agent may be methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragant malt, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate, etc. For example, the isotonic agent may be D-mannitol, sorbitol, etc. Examples of the above preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, etc.

[0148] Treatment method using obtained T cells

[0149] In another aspect of the present invention, a method for treating cancer is provided, comprising the step of administering the T cells to an individual having cancer. At this time, the cancer is as described above.

[0150] In another aspect of the present invention, the use of the T cells for cancer treatment is provided.

[0151] T cell activation composition

[0152] In another aspect of the present invention, a composition for enhancing the antigen recognition efficiency of T cells is provided, comprising as an active ingredient a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

[0153] In another aspect of the present invention, a composition for enhancing T cell cancer killing is provided, comprising as an active ingredient a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

[0154] The fusion protein dimer comprising the above-mentioned IL-2 protein or a variant thereof and the CD80 protein or a fragment thereof is as described above. The activation of T cells is synergistically induced by the composition comprising the above-mentioned fusion protein dimer, thereby enhancing the recognition efficiency of T cells for target antigens and ultimately effectively enhancing the cytotoxic ability against target cells, namely cancer cells, so that it can be applied to anticancer immunotherapy.

[0155] T cell activation method using cancer antigens

[0156] In another aspect of the present invention, a method for in vitro activation and proliferation of CD8+ T cells is provided, comprising the step of simultaneously culturing peripheral blood mononuclear cells (PBMCs) or T cells and cancer antigens in a medium containing a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof. In this case, the T cells are CD8+ T cells, as described above.

[0157] As used herein, the term "cancer antigen" refers to a cancer cell-specific protein presented on the surface of cancer cells or secreted into the bloodstream by cancer cells. Such cancer antigens are utilized in the diagnosis of specific cancers or in anticancer vaccines for anticancer treatment. Cancer antigens include PSCA (prostate stem cell antigen), HER-2 (human epidermal growth factor receptor 2), MUC1 (mucin 1), CA15-3 (cancer antigen 15-3), CA19-9 (cancer antigen 19-9), CA27-29 (cancer antigen 27-29), CA125 (cancer antigen 125), and CA195 (cancer antigen). 195), PSA (prostate-specific antigen), CA549 (cancer antigen 549), CEA (carcinoembryonic antigen), ACTH (adrenocorticotropic hormone), AFP (alpha-fetoprotein), bcl-2 (B-cell lymphoma 2), β-2 microglobulin, calcitonin, Cathepsin D, chromogranin-A, EFGR (epidermal growth factor receptor), Gastrin, human chorionic gonadotropin (hCG), α-hCG (alpha subunit of hCG), β-hCG (beta subunit of hCG), LDH (lactic dehydrogenase), NSE (neuron-specific enolase), pancreatic polypeptide, proinsulin C-peptide, thyroglobulin, TDT (terminal deoxynucleotidal transferase),TPA(tissue polypeptide antigen), 케라틴19(KRT19, keratin19), ETA(epithelial tumor antigen), 타이로시네이즈(tyrosinase), MAGEA1(melanoma-associated antigen family member A1), MAGEA2(melanoma-associated antigen family member A2), MAGEA3(melanoma-associated antigen family member A3), MAGEA4(melanoma-associated antigen family member A4), MAGEA6(melanoma-associated antigen family member A6), MAGEA9(melanoma-associated antigen family member A9), MAGEA10(melanoma-associated antigen family member A10), MAGEA11(melanoma-associated antigen family member A11), MAGEA12(melanoma-associated antigen family member A12), MAGEC1(melanoma-associated antigen family member C1), MAGEC2(melanoma-associated antigen family member C2), TRP-2(tyrosinase related protein 2), EpCAM(epithelial cell adhesion molecule), GPC3(glypican 3), MSLN(mesothelin), BTA(bladder tumor antigen), ROR1(Receptor Tyrosine Kinase Like Orphan Receptor 1), 사이토케라틴단편21-1(CYFRA21-1, cytokeratin fragment 21-1), CTAG2(cancer / testis antigen 2),BAGE(B melanoma antigen), LRPAP1(LDL receptor related protein associated protein 1), LY6K(lymphocyte antigen 6 family member K), SAGE1(sarcoma antigen 1), SPA17(sperm surface protein 17), SSX-2(SSX family member 2), SSX-4(SSX family member 4), ALDH1A1(aldehyde dehydrogenase 1 family member A1), CSAG2(chondrosarcoma-associated gene family member 2), XAGE1B(X antigen family member 1B), CALCA(Calcitonin gene-related peptide 1), CD274(Programmed cell death 1 ligand 1), CD45(Receptor-type tyrosine-protein phosphatase C), CPSF1(Cleavage and polyadenylation specificity factor subunit 1), DKK1(Dickkopf-related protein 1), ENAH(Protein enabled homolog), EPHA3(Ephrin type-A receptor 3), EZH2(Histone-lysine N-methyltransferase EZH2), FGF(fibroblast growth factor), HEPACAM(Hepatocyte cell adhesion molecule), HPN(Serine protease hepsin), IDO1(indoleamine 2), IMP3(U3 small nucleolar ribonucleoprotein 3), IL13RA2(interleukin 13 receptor subunit alpha 2), CES2(carboxylesterase 2), KLK4(Kallikrein-4),KIF20A(Kinesin-like protein KIF20A), LGSN(lengsin), CSF1(Macrophage colony-stimulating factor 1), CSPG4(chondroitin sulfate proteoglycan 4), MDK(midkine), MMP-2(matrix metallopeptidase 2), MMP-7(matrix metallopeptidase 7), MUC5AC(mucin 5AC), MART1(melanoma antigen recognized by T-cells 1), BCL2L1(Bcl-2-like protein 1), Nectin-4, PLIN2(perilipin 2), PAX5(paired box 5), PLAC1(Placenta-specific protein 1), ZNF395(zinc finger protein 395), PRAME(Melanoma antigen preferentially expressed in tumors), FOLH1(folate hydrolase 1), RGS5(Regulator of G protein signaling 5), RNF43(RING finger protein 43), DCDC2(Doublecortin domain-containing protein 2), SCRN1(Secernin-1), SOX10(Transcription factor SOX-10), SCGB2A2(secretoglobin family 2A member 2), Mammaglobin-A, BIRC5(Survivin; baculoviral IAP repeat containing 5), Surivin, NYESO1(New York Esophageal Squamous Cell Carcinoma-1), TROP2(Trophoblast cell surface antigen 2), TERT(telomerase reverse transcriptase), TPBG(trophoblast glycoprotein),VEGF (vascular endothelia growth factor), WT1 (Wilms tumor protein 1), WDR46 (WD repeat-containing protein 46), PMEL (premelanosome protein), ANKRD30A (ankyrin repeat domain 30A), GPR143 (G protein-coupled receptor 143), ACP3 (prostatic acid phosphatase), RAB38 (Ras-related protein Rab-38), α-TSH (alpha subunit thyroid stimulating hormone), c-Met (tyrosine-protein kinase Met), CD133, KK-LC-1 (Kita-Kyushu lung cancer antigen-1), CD70, GPNMB (Glycoprotein Nmb), MUC16 (Mucin 16), etc. In addition to the above, various mutant proteins, including specific mutants of tumor suppressor genes such as p53 (phosphoprotein 53), can also be used as cancer antigens.

[0158] The present invention will be described in more detail below through examples. These examples are solely for illustrating 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.

[0159] Preparation Example 1. Preparation of hCD80-Fc-IL-2 variant (2M): GI-101

[0160] To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide containing a nucleotide sequence (SEQN 8) encoding a fusion protein, comprising a signal peptide (SEQN 1), a CD80 fragment (SEQN 2), a linker-attached Ig hinge (SEQN 3), an Fc domain (SEQN 4), a linker (SEQN 5), and a two-amino acid substituted IL-2 variant (2M) (R38A, F42A) (SEQN 6) in that order from the N-terminus, was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into the pcDNA3_4 vector. Additionally, the above vector was loaded into CHO cells (Expi-CHO TM The fusion protein of sequence number 9 was expressed by introducing it into the vector. After introducing the vector, the culture medium was collected and the fusion protein was purified after 7 days of incubation in an environment at 37°C, 125 RPM, and a CO2 concentration of 28%. The purified fusion protein dimer was named "GI-101".

[0161] Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was conjugated under conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Subsequently, it was eluted with 100 mM acetic acid at 100 mM NaCl and pH 3. After adding 20% ​​1 M Tris-HCl at pH 9 to the collection tube, the fusion protein was collected. The collected fusion protein was dialyzed with PBS buffer for 16 hours.

[0162] Subsequently, high concentrations of fusion protein were obtained by measuring the absorbance at a wavelength of 280 nm over time using size exclusion chromatography with a TSKgel G3000SWXL column (TOSOH Bioscience). At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 2). When detected using NanoDrop, it was confirmed that the fusion protein was present at a concentration of 2.78 mg / mL. In addition, the results of the analysis using size exclusion chromatography are shown in Fig. 3.

[0163] Preparation Example 2. Preparation of Fc-IL-2 variant (2M) dimer: Fc-IL-2v2

[0164] To produce a fusion protein containing an Fc domain and an IL-2 variant, a polynucleotide containing a nucleotide sequence (Sequence No. 45) encoding a fusion protein, comprising a signal peptide (Sequence No. 1), an Ig hinge (Sequence No. 38), an Fc domain (Sequence No. 4), a linker (Sequence No. 5), and a two-amino acid substituted IL-2 variant (2M) (R38A, F42A) (Sequence No. 6), was synthesized via ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into the pcDNA3_4 vector. Additionally, the vector was introduced into CHO cells (Expi-CHO™) to express the fusion protein of Sequence No. 44. After introducing the vector, the cells were cultured for 7 days in an environment at 37°C, 125 RPM, and a CO2 concentration of 28%, after which the culture medium was collected and the fusion protein dimer was purified. The above-described purified fusion protein dimer was named "Fc-IL2v2".

[0165] The purification and collection of the fusion protein were performed in the same manner as in Preparation Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Comash Blue (Fig. 4). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of analysis using size exclusion chromatography are shown in Fig. 5.

[0166] Preparation Example 3. Preparation of Fc-IL-2 dimer: Fc-IL-2wt

[0167] To produce a fusion protein containing an Fc domain and wild-type IL-2, a polynucleotide containing a nucleotide sequence encoding a fusion protein (SEQ No. 43) including a signal peptide (SEQ No. 1), an Ig hinge (SEQ No. 38), an Fc domain (SEQ No. 4), a linker (SEQ No. 5), and wild-type IL-2 (SEQ No. 10) in this order from the N-terminus was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into the pcDNA3_4 vector. In addition, the above vector was introduced into CHO cells (Expi-CHO™) to express the fusion protein of SEQ No. 42. After introducing the vector, the cells were cultured for 7 days in an environment at 37°C, 125 RPM, and a CO2 concentration of 8%, after which the culture medium was collected and the fusion protein dimer was purified. The purified fusion protein dimer was named "Fc-IL2wt".

[0168] The purification and collection of the fusion protein were performed in the same manner as in Preparation Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Comash Blue (Fig. 6). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of analysis using size exclusion chromatography are shown in Fig. 7.

[0169] Preparation Example 4. Preparation of hCD80-Fc-IL-2 wild-type dimer: hCD80-Fc-IL-2wt

[0170] To produce a fusion protein containing a human CD80 fragment, an Fc domain, and IL-2 wild-type protein, a polynucleotide containing a nucleotide sequence (SEQN 41) encoding a fusion protein comprising a signal peptide (SEQN 1), a CD80 fragment (SEQN 2), a linker-bound Ig hinge (SEQN 3), an Fc domain (SEQN 4), a linker (SEQN 5), and IL-2 wild-type (SEQN 10) in that order from the N-terminus was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into the pcDNA3_4 vector. Additionally, the above vector was loaded into CHO cells (Expi-CHO TM The fusion protein of sequence number 46 was expressed by introducing it into the vector. After introducing the vector, the culture medium was collected and the fusion protein dimer was purified after 7 days of incubation in an environment at 37°C, 125 RPM, and a CO2 concentration of 28%. The purified fusion protein dimer was named "hCD80-Fc-IL2wt".

[0171] Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was conjugated under conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Subsequently, it was eluted with 100 mM acetic acid at 100 mM NaCl and pH 3. After adding 20% ​​1M Tris-HCl at pH 9 to the collection tube, the fusion protein was collected. The collected fusion protein was dialyzed with PBS buffer for 16 hours.

[0172] Subsequently, high concentrations of fusion protein were obtained by measuring the absorbance at a wavelength of 280 nm over time using size exclusion chromatography with a TSKgel G3000SWXL column (TOSOH Bioscience). At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 8). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of the analysis using size exclusion chromatography are shown in Fig. 9.

[0173] Preparation Example 5. Preparation of hCD80-Fc dimer: hCD80-Fc

[0174] To produce a fusion protein containing a human CD80 fragment and an Fc domain, a polynucleotide (SEQN 39) containing a nucleotide sequence encoding a fusion protein, comprising a signal peptide (SEQN 1), a CD80 fragment (SEQN 2), a linker-bound Ig hinge (SEQN 3), and an Fc domain (SEQN 4) in that order from the N-terminus, was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into the pcDNA3_4 vector. Additionally, the above vector was loaded into CHO cells (Expi-CHO TM The fusion protein of sequence number 40 was expressed by introducing it into the vector. After introducing the vector, the culture medium was collected and the fusion protein dimer was purified after 7 days of incubation in an environment at 37°C, 125 RPM, and a CO2 concentration of 28%. The purified fusion protein dimer was named "hCD80-Fc".

[0175] Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was conjugated under conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Subsequently, it was eluted with 100 mM acetic acid at 100 mM NaCl and pH 3. After adding 20% ​​1 M Tris-HCl at pH 9 to the collection tube, the fusion protein was collected. The collected fusion protein was dialyzed with PBS buffer for 16 hours.

[0176] Subsequently, high concentrations of fusion protein were obtained by measuring the absorbance at a wavelength of 280 nm over time using size exclusion chromatography with a TSKgel G3000SWXL column (TOSOH Bioscience). At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 10). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of the analysis using size exclusion chromatography are shown in Fig. 11.

[0177] Preparation Example 1. Culture composition for T cell culture

[0178] T cell culture media were prepared with the following composition. At this time, after preparing the basic culture media of Tables 1 and 2 below, GI-101 or hCD80-Fc + Fc-IL-2v were added before use according to each addition condition of Table 3.

[0179] Basic Culture Medium 1 Ingredient Name Manufacturer Cat.# Volume Final Concentration CTS™ AIM V™ SF Mt Hermo 0870 112D Kto 1L - Human Serum Sigma-Aldrich H4522-100ML 5 mL 5% Gluta MAX Gibco A12860-0110 mL 10% (1×)

[0180] Basic Culture Medium 2. Ingredient Name | Manufacturer | Cat. # | Volume | Final Concentration LM-Xuri T Cell Expansion Medium | Cytiva 2918523 | 1.00 | 1L | Human Serum Sigma-Aldrich H4522 | 100ML | 5 mL | 5% Penicillin-Streptomycin Welgene LS-202-02 | 10 mL | Penicillin 100 U / mL and Streptomycin 100 µg / mL

[0181] Additive Classification Ingredient Name Manufacturer Amount Final Concentration Addition Condition 1 GI-10 1GI-Innovation Add immediately before use 1.6nM Addition Condition 2 CD80-Fc+Fc-IL2v2 GI-Cell Add immediately before use CD80-Fc(1.6nM)+ Fc-IL2v2(1.6nM)

[0182] Example 1. Confirmation of T cell proliferation and activity following CD4-PBMC cell culture

[0183] Example 1.1. CD4-PBMC Cell Isolation and Culture

[0184] Human peripheral blood mononuclear cells (PBMC) (Zen-Bio. Inc, Research Triangle Park, NC, USA, Cat# SER-PBMC-200-F) were thawing and dissolved in 5 mL of the basic culture medium of Tables 1 and 2, and the cells were washed by centrifugation at 300xg for 5 minutes. Subsequently, CD4+ cells were removed from the PBMC using human CD4 micro beads (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat# 130-045-101) and a magnetic cell separation system.

[0185] 1×10 PBMCs with CD4+ cells removed (CD4- PBMCs) 6The cells were suspended in culture medium to a concentration of cells / mL and dispensed 1 mL into each well of a 24-well plate. Subsequently, an anti-human CD3 antibody (clone: ​​OKT3, Biolegend, Cat# 317326) was added to each well containing cells to a concentration of 1 μg / mL. At the same time, additional substances (Table 3) were added to the basic culture medium (Tables 1 and 2) to a concentration of 1.6 nM each, and the cells were cultured at 37°C under 5% CO2 conditions. After 4 days, 1 mL of a cell culture composition containing the anti-human CD3 antibody and additional substances (Table 3) to the basic medium components (Tables 1 and 2) to a concentration of 1.6 nM each was added to each well, and the cells were collected on the 14th day of culture to calculate the cell count and cell viability.

[0186] Specifically, the proliferation results of CD4-PBMC cells in a T cell culture medium composition including the basic culture medium of Table 1 are shown in Table 4 and Fig. 12, and the cell viability is as shown in Table 5 and Fig. 13. In addition, the proliferation results of CD4-PBMC cells in a T cell culture medium composition including the basic culture medium of Table 2 are shown in Table 6 and Fig. 14, and the cell viability is as shown in Table 7 and Fig. 15.

[0187] Classification Treatment Material Culture Date 0 Day 7 Day 9 Day 11 Day 14 CD4- PBMC Total Cell Number GI-101 (1.6 nM) 1,000,000 1,646,000 3,519,000 6,791,670 14,058,757 hCD80-Fc + Fc-IL2v (1.6 nM) 1,000,000 1,826,000 4,194,000 7,968,600 19,921,500

[0188] Classification Treatment Material Culture Day 0 Day 7 Day 9 Day 11 Day 14 Day CD4- PBMC Cell Viability (%) GI-101 (1.6 nM) 92.4 292.2 893.2 795.9 897.2 1 hCD80-Fc + Fc-IL2v (1.6 nM) 92.4 292.5 195.5 496.8 97.6 9

[0189] Classification Treatment Material Culture Day 0 Day 7 Day 9 Day 11 Day 14 CD4- PBMC Total Cell Number GI-101 (1.6nM) 1,000,000 996,000 1,948,000 2,820,000 7,078,200 hCD80-Fc + Fc-IL2v (1.6nM) 1,000,000 1,132,000 2,300,000 1,912,000 6,260,000

[0190] Classification Treatment Material Culture Day 0 Day 7 Day 9 Day 11 Day 14 Day CD4- PBMC Cell Viability (%) GI-101 (1.6nM) 91.868 3.78 4.728 7.248 9.09h CD80-Fc + Fc-IL2v (1.6nM) 91.868 4.718 6.77 9.878 6.81

[0191] Example 1.2. Flow cytometry method using cell surface staining

[0192] FACS analysis was performed to confirm the phenotype of CD4-(CD4 depletion) cells cultured for 14 days in the basic medium components (Tables 1 and 2) and additives (Table 3) according to Example 1.1 above. For each cell group, 2×10 5 ~ 3×10 5 Cells were dispensed into U-bottomed 96-well plates, and 100 µl of FACS buffer (PBS, 3% FBS, 10 mM EDTA, 20 mM HEPES, 10 µg / mL polymyxin B, 1× antibiotics, 1 mM sodium pyruvate) was added to each well, and the cells were washed by centrifuging at 300xg for 5 minutes.

[0193] Human TruStain FcX™ (BioLegend, Cat# 422302) was diluted 1:200 in FACS buffer, 50 µl was added to each well of the cell pellet, and the cells were incubated at 4°C for 10 minutes. For cell surface analysis, 2 µl of the antibody listed in Table 8 was mixed per 50 µl of FACS buffer, 50 µl was dispensed into each well, and the cells were incubated at 4°C for 20 minutes. Subsequently, 100 µl of FACS buffer was added, and the cells were washed by centrifuging at 300xg for 5 minutes. After washing, the cells were resuspended in FACS buffer and analyzed using a BD FACS Celesta flow cytometer (BD Science, San Jose, CA, USA) and Flowjo TM The cell phenotype was confirmed using software.

[0194] As a result, the cell surface flow cytometry results (FACS Plot) of CD4- PBMC cells cultured for 14 days with each additive in the T cell culture medium composition including the basic culture medium of Table 1 are shown in Figure 16. In addition, the results of confirming the number of T cells, specifically the total number of T cells, CD8 T cells, CD25+ T cells, and central memory T cells, are as shown in Table 9 and Figures 17 to 20.

[0195] In addition, the results of cell surface flow cytometry analysis (FACS Plot) of CD4- PBMC cells cultured for 14 days with each additive in the T cell culture medium composition including the basic culture medium of Table 2 are shown in Figure 21. Furthermore, the results of confirming the number of T cells, specifically the total number of T cells, CD8 T cells, CD25+ T cells, and central memory T cells, are as shown in Table 10 and Figures 22 to 25.

[0196] As a result of flow cytometry analysis through cell surface staining in Example 1.2, it was confirmed that when GI-101 was included as an additive, the total number of T cells, CD8 T cells, CD25+ T cells, and central memory T cells all proliferated significantly regardless of the basic culture medium.

[0197] Component: Surface staining antibody Products: Color, Clone Manufacturer: Cat.#anti-human CD3PEUCHT1BioLegend300428anti-human CD4PerCP-Cy5.5OKT4BioLegend317428anti-human CD8APCSK1BioLegend344722anti-human CD8FITCRPA-T8BioLegend301006anti-human CD14FITCM5E2BioLegend301804anti-human CD19BV480SJ25C1BD Biosciences566103anti-human CD25APC / Cy7M-A251BD Biosciences557753anti-human CD45RABV711HI100BioLegend304138anti-human CD45ROPE-CF594UCHL1BD Biosciences562299anti-human CD56PE / Cy7HCD56BioLegend318318anti-human CD62LBV510DREG-56BioLegend304844anti-human CD69AF700FN50BioLegend310922anti-human CD127BV650A019D5BioLegend351326anti-human CD197 (CCR7)BV605G043H7BioLegend353224anti-human HLA-DR, DP, DQBV785Tu39BD Biosciences555557Intracellular staining antibodyanti-human PerforinAPCdG9Biolegend308112anti-human Granyzme BPE / Cy7QA16A02Biolegend372214anti-human IFN-γBV4214S.B3Biolegend502532

[0198] Total number of T cells treated with the substance 1) (CD3+CD19- cells)CD8 T cell count 2)(CD3+CD19-CD8+ cells)CD25+ memory T cell count 3) (CD25+ Memory CTL) Central Memory T cell count 4) (Central Memory CTL)GI-101 (1.6nM)18,487,15216,601,46213,347,57610,807,552hCD80-Fc + Fc-IL2v (1.6nM)12,765,35111,259,0408,669,4616,901,791

[0199] 1) Total T cell count : Total CD4- PBMC count × CD3+CD19- T cell ratio

[0200] 2) CD8 T cell count : Total T cell count × CD8 T cell ratio

[0201] 3) CD25+ memory cell count : CD8 T cell count × CD25+ memory cell ratio

[0202] 4) Central memory T cell count: CD8 T cell count × Central memory cell ratio

[0203] Total number of T cells treated with the substance 1) (CD3+CD19- cells)CD8 T cell count 2) (CD3+CD19-CD8+ cells)CD25+ memory T cell count 3) (CD25+ Memory CTL) Central Memory T cell count 4) (Central Memory CTL)GI-101 (1.6nM)6,568,5705,708,0874,771,9611,746,675hCD80-Fc + Fc-IL2v (1.6nM)5,790,5004,840,8583,906,5721,287,668

[0204] 1) Total T cell count : Total CD4- PBMC count × CD3+CD19- T cell ratio

[0205] 2) CD8 T cell count : Total T cell count × CD8 T cell ratio

[0206] 3) CD25+ memory cell count : CD8 T cell count × CD25+ memory cell ratio

[0207] 4) Central memory T cell count: CD8 T cell count × Central memory cell ratio

[0208] Example 1.3. Flow cytometry method using intracellular staining

[0209] 2×10 for each cell group 5 ~ 3×10 5 Cells were dispensed into separate round-bottom 96-well plates and treated with the media of Tables 1 and 2 supplemented with 1× Cell Stimulation Cocktail (eBioscience, Cat# 00-4970-93). After incubation at 37°C for 4 hours, the cells were washed with FACS buffer containing 1× monensin (Biolegend, Cat# 420701). FACS buffer supplemented with Human TruStain FcX™ (BioLegend, Cat# 422302) and 1× monensin was diluted to a ratio of 1:200, 50 µl was dispensed into each cell group, and the cells were incubated at 4°C for 10 minutes. Cell surface staining antibodies (CD3, CD4, CD8) from the antibodies in Table 8 were mixed at a rate of 2 µl per 50 µl in FACS buffer mixed with 1×monensin, 50 µl was dispensed into each well, and incubated at 4°C for 20 minutes. Afterward, the body was washed twice with FACS buffer mixed with 1×monensin.

[0210] After washing, the cells were fixed and made permeable using BD Cytofix / Cytoperm™ (BD Biosciences, Cat# 554714) according to the manufacturer's protocol. The intracellular staining antibodies (Perforin, Granzyme B, IFN-γ) listed in Table 8 were mixed at a rate of 2 µl per 50 µl of 1× BD Perm / Wash™ (BD Biosciences, Cat# 554723), dispensed into each well at 50 µl, and incubated at 4°C for 20 minutes. Subsequently, the cells were washed once with 1× BD Perm / Wash™ buffer and two more times with FACS buffer. The washed cells were resuspended in FACS buffer, then aligned using a BD FACS Celesta flow cytometer, and Flowjo TM The results were analyzed using software.

[0211] As a result, the intracellular flow cytometry (FACS Plot) results of CD4-PBMC cells treated with each additive in the T cell culture medium composition including the basic culture medium of Table 1 are shown in Fig. 26. In addition, the results of confirming the number of CTL cells expressing Granzyme B, IFN-γ, and Perforin treated with each additive in the T cell culture medium composition including the basic culture medium of Table 1 are as shown in Table 11 and Figs. 27 to 29.

[0212] In addition, the results of intracellular flow cytometry analysis (FACS Plot) of CD4-PBMC cells treated with each additive in the T cell culture medium composition including the basic culture medium of Table 2 are shown in Fig. 30. Furthermore, the results of confirming the number of CTL cells expressing Granzyme B, IFN-γ, and Perforin treated with each additive in the T cell culture medium composition including the basic culture medium of Table 2 are as shown in Table 12 and Figs. 31 to 33.

[0213] Treatment substance Granzyme B+ CTL cell count 5) (CD8+ Granzyme B+ cells) IFN-γ+ CTL cell count 6) (CD8+IFNγ+ cells) Perforin CTL cell count 7) (CD8+ Perforin+ cells)GI-101 (1.6nM)7,190,8653,335,3371,774,767hCD80-Fc + Fc-IL2v (1.6nM)6,022,4202,744,1291,696,751

[0214] 2) CD8 T cell count : Total T cell count × CD8 T cell ratio

[0215] 5) Granzyme B+ CTL cell count: CD8 T cell count 2) × CD8+ Granzyme B+ cell ratio

[0216] 6) IFNg+ CTL cell count: CD8 T cell count 2) × CD8+ IFNg+ cell ratio

[0217] 7) Perforin CTL cell count: CD8 T cell count 2) × CD8+ Perforin+ Cell Ratio

[0218] Treatment substance Granzyme B+ CTL cell count 5) (CD8+ Granzyme B+ cells) IFN-γ+ CTL cell count 6) (CD8+ IFNγ+ cells) Perforin CTL cell count 7) (CD8+ Perforin+ cells)GI-101 (1.6nM)6,398,976 4,334,152 4,083,470hCD80-Fc + Fc-IL2v (1.6nM)5,700,406 3,708,768 3,837,261

[0219] 2) CD8 T cell count : Total T cell count × CD8 T cell ratio

[0220] 5) Granzyme B+ CTL cell count: CD8 T cell count 2)× CD8+ Granzyme B+ cell ratio

[0221] 6) IFNg+ CTL cell count: CD8 T cell count 2) × CD8+ IFNg+ cell ratio

[0222] 7) Perforin CTL cell count: CD8 T cell count 2) × CD8+ Perforin+ Cell Ratio

[0223] Example 2. Confirmation of T cell proliferation and activity following CD8+ T cell culture

[0224] Example 2.1. CD8+ T cell isolation and culture

[0225] Human peripheral blood mononuclear cells (PBMC) (Zen-Bio. Inc, Research Triangle Park, NC, USA, Cat# SER-PBMC-200-F) were thawed and loosened with 5 mL of basic medium components (Tables 1 and 2), and then the cells were washed by centrifuging at 300xg for 5 minutes. Subsequently, CD8+ cells were obtained from the PBMC using human CD8 micro beads (MiltenyiBiotec, Bergisch-Gladbach, Germany, Cat# 130-045-201) and a magnetic cell separation system.

[0226] 1×10 isolated CD8+ T cells 6The cells were suspended in culture medium to a concentration of cells / mL and dispensed into 24-well plates at a concentration of 1 mL. Subsequently, an anti-human CD3 antibody (clone: ​​OKT3, Biolegend, Cat# 317326) was added to each well containing cells to a concentration of 1 μg / mL. At the same time, in addition to the basic components of the medium (Tables 1 and 2), 1.6 nM of additives (Table 3, simultaneous treatment with GI-101 and hCD80-Fc and Fc-IL2 variants) were added, and the cells were cultured at 37°C under 5% CO2 conditions. On the 4th day of culture, 1 mL of a cell culture composition containing 1.6 nM of an additive (Table 3, simultaneous treatment with GI-101 and hCD80-Fc and Fc-IL2 variants) in addition to the anti-human CD3 antibody and the basic components of the medium (Tables 1 and 2) was added to each well, and the cell number and cell viability were calculated after obtaining samples on the 14th day of culture.

[0227] Specifically, the proliferation results of CD8+ PBMC cells in a T cell culture medium composition including the basic culture medium of Table 1 are shown in Table 13 and Figure 34, and the cell viability is as shown in Table 14 and Figure 35.

[0228] Classification Treatment Material Culture Day 0 Day 6 Day 8 Day 12 CD8+ PBMC Total Cell Number (CD8+ PBMC Total Cell Number) GI-101 (1.6nM) 1,000,000 2,440,000 2,394,240 11,065,600 h CD80-Fc + Fc-IL2v (1.6nM) 1,000,000 1,412,000 1,119,000 8,775,000

[0229] Classification Treatment Material Culture Day 0 Day 7 Day 9 Day 11 Day CD8+ PBMC Cell Viability (%) GI-101 (1.6 nM) 95.1 396.2 793.9 196.2 4h CD80-Fc + Fc-IL2v (1.6 nM) 95.1 394.7 89.0 795.3 4

[0230] Example 2.2. Flow cytometry method using cell surface staining

[0231] FACS analysis was performed to confirm the phenotype of CD8+ (CD8 isolation) cells cultured for 12 days in a composition containing the basic medium components of Table 1 and the additives of Table 3 according to Example 2.1 above. For each cell group, 2-3 × 10⁶ 5 Cells were dispensed into U-bottomed 96-well plates, and 100 µl of FACS buffer (PBS, 3% FBS, 10 mM EDTA, 20 mM HEPES, 10 µg / mL polymyxin B, 1× antibiotic, 1 mM sodium pyruvate) was added to each well and the cells were washed by centrifuging at 300xg for 5 minutes.

[0232] Human TruStain FcX™ (BioLegend, Cat# 422302) was diluted 1:200 in FACS buffer, 50 µl was added to each well of the cell pellet, and the cells were incubated at 4°C for 10 minutes. For cell surface analysis, 2 µl of the antibody listed in Table 8 was mixed per 50 µl of FACS buffer, 50 µl was dispensed into each well, and the cells were incubated at 4°C for 20 minutes. Subsequently, 100 µl of FACS buffer was added, and the cells were washed by centrifuging at 300xg for 5 minutes. After washing, the cells were resuspended in FACS buffer and analyzed using a BD FACS Celesta flow cytometer (BD Science, San Jose, CA, USA) and Flowjo TM The cell phenotype was confirmed using software.

[0233] As a result, the cell surface flow cytometry results (FACS Plot) of CD8+ PBMC cells cultured for 12 days with each additive in the T cell culture medium composition including the basic culture medium of Table 1 are shown in Fig. 36. In addition, the results of confirming the number of T cells, CD8 T cells, CD25+ T cells, and central memory T cells are as shown in Table 15 and Figs. 37 to 40.

[0234] Total number of T cells treated with the substance 8) (CD3+CD19- cells)CD8 T cell count 9) (CD3+CD19-CD8+ cells)CD25+ memory T cells 10) (CD25+ Memory CTL) Central Memory T cell count 11) (Central Memory CTL)GI-101 (1.6nM)10,988,14110,702,4498,262,2914,773,292hCD80-Fc + Fc-IL2v (1.6nM)8,722,3508,547,9035,325,3443,017,410

[0235] 8) Total T cell count : Total CD8+ PBMC count × CD3+CD19- T cell ratio

[0236] 9) CD8 T cell count : Total T cell count × CD8 T cell ratio

[0237] 10) CD25+ memory cell count : CD8 T cell count × CD25+ memory cell ratio

[0238] 11) Central memory T cell count: CD8 T cell count × Central memory cell ratio

[0239] Example 2.3. Flow cytometry method using intracellular staining

[0240] 2×10 for each cell group 5 ~ 3×10 5Cells were dispensed into separate round-bottom 96-well plates and treated with the media of Tables 1 and 2 supplemented with 1× Cell Stimulation Cocktail (eBioscience, Cat# 00-4970-93). After incubation at 37°C for 4 hours, the cells were washed with FACS buffer containing 1× monensin (Biolegend, Cat# 420701). FACS buffer supplemented with Human TruStain FcX™ (BioLegend, Cat# 422302) and 1× monensin was diluted to a ratio of 1:200, 50 µl was dispensed into each cell group, and the cells were incubated at 4°C for 10 minutes. Cell surface staining antibodies (CD3, CD4, CD8) from the antibodies in Table 8 were mixed at a rate of 2 µl per 50 µl in FACS buffer mixed with 1×monensin, 50 µl was dispensed into each well, and incubated at 4°C for 20 minutes. Afterward, the body was washed twice with FACS buffer mixed with 1×monensin.

[0241] After washing, the cells were fixed and made permeable using BD Cytofix / Cytoperm™ (BD Biosciences, Cat# 554714) according to the manufacturer's protocol. The intracellular staining antibodies (Perforin, Granzyme B, IFN-γ) listed in Table 8 were mixed at a rate of 2 µl per 50 µl of 1× BD Perm / Wash™ (BD Biosciences, Cat# 554723), dispensed into each well at 50 µl, and incubated at 4°C for 20 minutes. Subsequently, the cells were washed once with 1× BD Perm / Wash™ buffer and two more times with FACS buffer. The washed cells were resuspended in FACS buffer, then aligned using a BD FACS Celesta flow cytometer, and Flowjo TM The results were analyzed using software.

[0242] As a result, the intracellular flow cytometry (FACS Plot) results of CD8+ PBMC cells cultured for 12 days according to each additive treatment in the T cell culture medium composition including the basic culture medium of Table 1 are shown in Fig. 41. In addition, the results of confirming the number of CTL cells expressing Granzyme B, IFN-γ, and Perforin according to each additive treatment in the T cell culture medium composition including the basic culture medium of Table 1 are as shown in Table 16 and Figs. 42 to 44.

[0243] Treatment substance Granzyme B+ CTL cell count 12) (CD8+ Granzyme B+ cells) IFN-γ+ CTL cell count 13) (CD8+IFNγ+ cells) Perforin CTL cell count 14) (CD8+ Perforin+ cells)GI-101 (1.6nM)10,578,714 10,134,408 5,437,459 hCD80-Fc + Fc-IL2v (1.6nM)8,345,025 8,153,089 4,765,009

[0244] 9) CD8 T cell count : Total T cell count × CD8 T cell ratio

[0245] 12) Granzyme B+ CTL cell count: CD8 T cell count 9) × CD8+ Granzyme B+ cell ratio

[0246] 13) IFNg+ CTL cell count: CD8 T cell count 9) × CD8+ IFNg+ cell ratio

[0247] 14) Perforin CTL cell count: CD8 T cell count 9) × CD8+ Perforin+ Cell Ratio

[0248] Example 3. Confirmation of the cancer cell death effect of Her2-recognizing T cells treated with GI101

[0249] Example 3.1. T cell activation

[0250] 1×10⁶ CD4-deficient (CD4-) PBMC cells obtained in Example 1 above were counted using a cell counter 6 After suspending the cells in the basic components of T cell medium (Table 1) to a concentration of cells / mL, 10 mL aliquots were seeded into T75 flasks. Subsequently, 1 µg / mL to 10 µg / mL of Her2 protein (Acro, Cat# HE2-H5225- 1 mg) was inoculated into the medium containing the seeded cells and cultured at 37°C under 5% CO2 conditions for 2 hours.

[0251] Example 3.2. Restimulation of activated T cells

[0252] Cells cultured with Her2 protein (Acro, Cat# HE2-H5225-1 mg) for 2 hours in Example 3.1 above were simultaneously cultured for 7 days with 50 nM of additives (GI-101 and Proleukin) (Proleukin, Novartis, USA) (Proleukin, Novartis, USA) in addition to the basic medium components of Table 1 and 0.1 μg / mL to 1 μg / mL of OKT3 (Biolegend).

[0253] The cells cultured for the above 7 days were re-stimulated on the 4th day of culture by co-culturing with T cells inoculated with activated Her2 protein (Acro, Cat# HE2-H5225-1 mg) in the same manner as described in Example 3.1, so that the cell-to-cell ratio was 1:1. At this time, the cell saturation was 1×10⁶ 6 It was ensured not to exceed cells / mL.

[0254] Example 3.3. Analysis of the cancer cell death effect of activated T cells

[0255] To confirm the cancer cell killing effect of cultured activated T cells, the cells obtained from Example 3.2 were co-cultured with two types of cancer cells, CAMA-1 (ATCC® HTB-21™; breast cancer cell line); BT-474 (ATCC® HTB-20™; breast cancer cell line)), at various ratios (1:1, 5:1, 10:1) for 24 hours. Afterward, the cells were stained with Annexin-V and 7-AAD, and the cancer cell killing effect was evaluated using a flow cytometer.

[0256] Specifically, 1×10 of the above two types of cancer cells each 6 The activated T cells obtained from Example 3.2 were suspended in the basic components of T cell medium (Table 1) to a concentration of cells / mL and seeded into a round-bottom 96-well plate. The activated T cells obtained from Example 3.2 were suspended in the basic components of T cell medium (Table 1) and seeded into 96-well plates with cancer cell ratios (E:T) of 1:1, 5:1, and 10:1, and co-cultured for 24 hours at 37°C under 5% CO2 conditions. After 24 hours of co-culture, the cells were centrifuged at 1,300 rpm for 5 minutes, and the supernatant was removed.

[0257] 100 µl of preheated 0.25% trypsin was added to each well containing the co-cultured cells and incubated at 37°C for 5 minutes. The isolated cells were washed with FACS buffer and stained with an anti-human CD45 antibody (Clone HI30, eBioscience, Cat# 25-0459-42). Subsequently, the cells were stained with Annexin-V and 7-AAD (BioLegend, USA, Cat# 640922) using the FITC Annexin-V Apoptosis Detector Kit containing 7-AAD, according to the manufacturer's protocol. After staining, the cells were incubated at 4°C for 20 minutes, followed by the addition of 100 µl of FACS buffer and centrifugation at 300xg for 5 minutes. Afterward, the cells were resuspended in FACS buffer and analyzed using a BD FACS Celesta flow cytometer (BDscience, San Jose, Ca, USA) and Flowjo TM The cancer cell death effect was confirmed using software.

[0258] The cancer cell killing effect of T cells confirmed through the above experiment is shown in Figures 45 and 46.

Claims

1. A composition for in vitro proliferating a CD8+CD45RO+ memory T cell comprising as an active ingredient a fusion protein dimer,wherein the fusion protein comprises the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]m-Y-C' - formula (I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]m-X-C' - formula (II), wherein, N′ is the N-terminus of the fusion protein, C′ is the C-terminus of the fusion protein, X is the CD80 protein or the fragment thereof, Y is the IL-2 protein variant, the linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1, wherein the IL-2 protein variant comprises any one of the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R, (d) R38A / F42A / L72G,wherein the CD80 protein fragment comprises an extracellular domain of CD80. 2. The composition according to claim 1, wherein the CD8+CD45RO+ memory T cell is a CD8+CD25+CD45RO+ memory T cell, a CD8+CD62L+CD45RO+ memory T cell, or a CD8+CD25+CD62L+CD45RO+ memory T cell. 3. The composition according to claim 1, wherein the IL-2 variant comprises the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24. 4. The composition according to claim 1, wherein the fragment of CD80 comprises the 35th to 242nd amino acids in the amino acid sequence of SEQ ID NO: 11. 5. The composition according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. 6. A medium for in vitro proliferating a CD8+CD45RO+ memory T cell comprising a fusion protein dimer, wherein the fusion protein comprises the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]m-Y-C' - formula (I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]m-X-C' - formula (II), wherein, N′ is the N-terminus of the fusion protein, C′ is the C-terminus of the fusion protein, X is the CD80 protein or the fragment thereof, Y is the IL-2 protein variant, the linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1, wherein the IL-2 protein variant comprises any one of the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R, (d) R38A / F42A / L72G,wherein the CD80 protein fragment comprises an extracellular domain of CD80. 7. The medium according to claim 6, further comprising a medium for culturing a T cell. 8. The medium according to claim 7, wherein the medium for culturing a T cell includes an amino acid, a sugar, an inorganic salt, and a vitamin.

9. A method for in vitro culturing a CD8+CD45RO+ memory T cell comprising:culturing CD8+ T cells in a presence of a fusion protein dimer,wherein the fusion protein comprises the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]m-Y-C' - formula (I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]m-X-C' - formula (II), wherein, N′ is the N-terminus of the fusion protein, C′ is the C-terminus of the fusion protein, X is the CD80 protein or the fragment thereof, Y is the IL-2 protein variant, the linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1, wherein the IL-2 protein variant comprises any one of the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R, (d) R38A / F42A / L72G,wherein the CD80 protein fragment comprises an extracellular domain of CD80. 10. The method according to claim 9, wherein the CD8+ T cell is obtained from peripheral blood mononuclear cells (PBMCs).

11. The method according to claim 9, wherein the culturing is performed for 7 to 21 days.

12. The method according to claim 9, wherein the CD8+CD45RO+ memory T cell is a CD8+CD25+CD45RO+ memory T cell, a CD8+CD62L+CD45RO+ memory T cell, or a CD8+CD25+CD62L+CD45RO+ memory T cell.

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