Method for preparing dendritic cells using platelet lysates

Abstract

The purpose of the present invention is to provide a method for preparing a dendritic cell from a monocyte using a platelet lysate.　Provided is a method for preparing a dendritic cell having cytotoxicity from a monocyte, the method comprising culturing a monocyte separated from peripheral blood by non-adhesive culture using a serum-free culture medium containing a human platelet lysate (HPL), GM-CSF and PEG conjugated interferon-α, adding prostaglandin E2 and OK432 to the resultant culture, and further culturing the resultant mixture by non-adhesive culture.

Description

Technical Field

[0001] The present invention relates to a method for preparing dendritic cells from monocytes.

Background Art

[0002] Dendritic cells (DC) are powerful antigen-presenting cells in vivo and are known to induce an immune response by presenting an antigen to T cells. In addition, it is known that DCs not only act directly on T cells but also on B cells, NK cells, NKT cells, etc., and play a central role in the immune reaction. Immature DCs acquire high T cell-stimulating ability with an increase in the expression of CD40, CD80, CD86, etc. by receiving antigen stimulation, migrate to peripheral lymphoid tissues, and induce an immune response by activating T cells specific to the antigen taken up.

[0003] Generally, several types of cytokines are known as substances that are recognized to be able to induce the differentiation of dendritic cells from hematopoietic progenitor cells. For example, there are many reports on the induction of DC differentiation by the combined use of GM-CSF and IL-4 (Non-Patent Document 1). In addition, substances that can induce DC differentiation alone or in combination with other cytokines have also been reported (Non-Patent Document 2). For example, TNF-α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-13, IL-15, HGF (Hepatocyte growth factor), CD40 ligand, M-CSF, Flt3 ligand, c-kit ligand, TGF-β, etc. have been reported.

[0004] In the method of inducing DC differentiation by combining GM-CSF and IL-4, it is carried out by an adherent culture method. Mononuclear cells (monocytes and lymphocytes) are seeded on a culture dish, the lymphocytes are washed, and the adherent monocytes are used for culture. Culturing is performed for 5 to 7 days in the presence of GM-CSF / IL-4, the cells are collected by washing with the medium and scraping (physically peeling off), and the medium is replaced with a medium (Fresh medium) containing an adjuvant (immunostimulant) OK432 to produce mature DCs.

[0005] In addition, a method for preparing dendritic cells using G-CSF (Patent Document 1) and a method for preparing dendritic cells by non-adherent culture using IFN (Patent Document 2) have been reported.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for preparing dendritic cells from monocytes using platelet lysate.

Means for Solving the Problems

[0009] Conventionally, methods for preparing dendritic cells (DCs) from monocytes in peripheral blood have been reported, but in the conventional methods, the yield of DCs has not been sufficient. Furthermore, for using DCs in cancer treatment, DCs having activities such as cytotoxicity in addition to strong antigen-presenting ability and phagocytic ability have been required.

[0010] The inventors diligently conducted research to increase the yield of dendritic cells (DCs) and produce highly functional DCs. As a result, they discovered that by using platelet lysate (HPL), GM-CSF, and PEGylated interferon-α (PEG-IFN-α), and further isolating monocytes from peripheral blood, and then producing DCs through non-adherent culture, i.e., suspension culture, they were able to produce optimized DCs in a short period of time, increasing the yield of DC production, and the resulting DCs also possessed strong cytotoxicity, thus completing the present invention.

[0011] In other words, the present invention is as follows. [1] A method for preparing cytotoxic dendritic cells from monocytes, comprising culturing monocytes isolated from peripheral blood in a serum-free medium containing human platelet lysate (HPL), GM-CSF, and PEGylated interferon α by non-adherent culture, and then adding prostaglandin E2 and OK432 and culturing further by non-adherent culture. [2] A method for preparing dendritic cells from monocytes according to [1], comprising culturing for 2 to 5 days in non-adherent culture using serum-free medium containing human platelet lysate (HPL), GM-CSF, and PEGylated interferon α, followed by the addition of prostaglandin E2 and OK432 and further culturing for 1 to 2 days. [3] A method for preparing dendritic cells from monocytes according to [1] or [2], wherein monocytes are cultured in serum-free medium containing 1-10 (v / v)% human platelet lysate (HPL), 100 U / mL-10,000 U / mL GM-CSF, 500 ng / mL-5 μg / mL PEGylated interferon α, 5 ng / mL-50 ng / mL prostaglandin E2, and 5 μg / mL-50 μg / mL OK432. [4] A method for preparing dendritic cells from any of the monocytes described in [1] to [3], wherein the serum-free medium is DCO-K. [5] A method for preparing dendritic cells from any of the monocytes described in [1] to [4], wherein the viability of the obtained dendritic cells is 90% or more and the yield, which is the ratio of the number of obtained dendritic cells to the number of monocytes in culture, is 15% or more. [6] A method for preparing dendritic cells from any of the monocytes [1] to [5], wherein the resulting dendritic cells are positive for CD14, CD16, CD56, CD83, CD86, CCR7 (CD197), HLA-ABC, and HLA-DR. [7] Dendritic cells obtained by a method for preparing dendritic cells from any of the monocytes described in [1] to [6]. A pharmaceutical composition comprising the dendritic cells of [8] [7]. [9] A pharmaceutical composition of [8] having anti-cancer immune activity and usable for cancer treatment.

[10] A method for separating monocytes, comprising culturing peripheral blood mononuclear cells in an adherent culture vessel using serum-free medium containing human platelet lysate (HPL) for 15 minutes to 3 hours, removing non-adherent cells, and collecting adherent cells.

[11] A method for isolating monocytes according to

[10] , using serum-free medium containing 1-10 (v / v)% human platelet lysate (HPL).

[12] A method for isolating monocytes in serum-free medium, DCO-K, as in

[10] or

[11] .

[13] A differentiation and induction agent for cytotoxic dendritic cells from monocytes, comprising human platelet lysate (HPL), GM-CSF, PEGylated interferon α, prostaglandin E2, and OK432.

[14] A cytotoxic dendritic cell differentiation and induction agent from monocytes of

[13] , comprising human platelet lysate (HPL), GM-CSF and PEGylated interferon α, and a dendritic cell maturation agent comprising prostaglandin E2 and OK432.

[15] Furthermore, any of the methods [1] to [6], wherein a cancer-specific antigen is added to prepare dendritic cells that have cancer antigen-specific dendritic cell-toxicity. Dendritic cells with cancer antigen-specific dendritic cell-toxicity obtained by the method of

[16]

[15] .

[17] A pharmaceutical composition comprising dendritic cells of

[16] having anti-cancer immune activity and usable for cancer treatment. This specification includes the disclosures of Japanese Patent Application No. 2020-184317, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0012] The present invention provides a method for preparing dendritic cells (DCs) that includes culturing isolated monocytes in non-adherent culture in the presence of HPL, GM-CSF, PEGylated interferon (IFN)-α (PEG-IFN-α), prostaglandin E2 (PGE2), and OK432. This method allows for the rapid and high yield production of highly cytotoxic DCs. The resulting DCs can be suitably used in cancer immunotherapy. [Brief explanation of the drawing]

[0013] [Figure 1] This is a diagram showing the protocol for Preliminary Examination 1. [Figure 2-1] This figure shows the observed morphology of cells on day 1 in preliminary test 1. [Figure 2-2] This figure shows the observed morphology of cells on day 2 in preliminary test 1. [Figure 3] This figure shows the results of detecting the cell surface antigen of IFN-DCs prepared in DCO-K medium alone using flow cytometry with a labeled antibody in preliminary test 1. [Figure 4] This figure shows the results of detecting the cell surface antigen of IFN-DCs prepared in DCO-K+ABS medium using labeled antibody by flow cytometry in preliminary test 1. [Figure 5] This figure shows the results of detecting the cell surface antigen of IFN-DCs prepared in DCO-K+HPL medium using flow cytometry with a labeled antibody in preliminary test 1. [Figure 6] This figure shows the results of detecting the cell surface antigen of IFN-DCs prepared in AIM-V medium using flow cytometry in preliminary test 1. [Figure 7] This figure shows the results of evaluating the purity and lymphocyte contamination rate of IFN-DCs during IFN-DC recovery using flow cytometry in preliminary study 1. [Figure 8] This figure shows the summary results of the viable cell rate and yield in preliminary experiment 1. [Figure 9] This is a diagram showing the protocol for Preliminary Examination 2. [Figure 10] This figure shows the observed morphology of cells in preliminary test 2. [Figure 11] This figure shows the results of detecting the cell surface antigen of IFN-DCs cultured with DCO-K alone using flow cytometry with a labeled antibody in preliminary test 2. [Figure 12] This figure shows the results of detecting the cell surface antigen of IFN-DCs cultured in DCO-K+ABS using labeled antibody flow cytometry in preliminary test 2. [Figure 13] This figure shows the results of detecting the cell surface antigen of IFN-DCs cultured in DCO-K+HPL using labeled antibody flow cytometry in preliminary test 2. [Figure 14] This figure shows the results of evaluating the purity and lymphocyte contamination rate of IFN-DCs during IFN-DC recovery using flow cytometry in preliminary study 2. [Figure 15] This figure shows the summary results of the viable cell rate and yield in preliminary experiment 2. [Figure 16] This is a diagram showing the protocol for Preliminary Examination 3. [Figure 17] This figure shows the observed morphology of cells in preliminary examination 3. [Figure 18] This figure shows the results of detecting the cell surface antigen of IFN-DCs cultured in HPL 5(v / v)% using labeled antibody flow cytometry in preliminary test 3. [Figure 19] This figure shows the results of detecting the cell surface antigen of IFN-DCs cultured in HPL 2.5(v / v)% using flow cytometry with a labeled antibody in preliminary test 3. [Figure 20] This figure shows the results of evaluating the purity and lymphocyte contamination rate of IFN-DCs during IFN-DC recovery using flow cytometry in preliminary study 3. [Figure 21] This figure shows the summary results of the viable cell rate and yield in preliminary experiment 3. [Figure 22] This figure shows the protocol for Preliminary Examination 4. [Figure 23] This figure shows the viability rate, yield, and lymphocyte fraction contamination rate when IFN-DCs were prepared using DCO-K medium supplemented with HPL at various concentrations (0(v / v)%, 1(v / v)%, 5(v / v)%, 10(v / v)%) in preliminary test 4. [Figure 24] This figure shows the results of flow cytometry evaluation of the phenotypes of IFN-DCs prepared with HPL (0(v / v)%, 1(v / v)%, 5(v / v)%, 10(v / v)%) at each concentration in Preliminary Test 4. [Figure 25] This figure shows the results of detecting the cell surface antigen of IFN-DCs cultured in HPL 10(v / v)% using flow cytometry with a labeled antibody in preliminary test 4. [Figure 26] This is a diagram showing the protocol for Preliminary Examination 5. [Figure 27-1] This figure shows the observed cell morphology and the composition of the maturation cocktail in preliminary experiment 5. [Figure 27-2] This figure shows the results of evaluating the lymphocyte contamination rate during IFN-DC retrieval in preliminary study 5 using flow cytometry. [Figure 28] This figure shows the viability rate, yield, and lymphocyte fraction contamination rate when IFN-DCs were produced using each matured cocktail in preliminary test 5. [Figure 29] This figure shows the results of the phenotypic analysis of IFN-DCs prepared using each matured cocktail in preliminary test 5. [Figure 30] This is a diagram showing the protocol for Preliminary Examination 6. [Figure 31] This figure shows the results of the cytotoxic activity measurement of HPL-IFN-DCs prepared using fresh or cryopreserved PBMCs in preliminary test 6 (Case 1). [Figure 32] This figure shows the results of the cytotoxic activity measurement of HPL-IFN-DCs prepared using fresh or cryopreserved PBMCs in preliminary test 6 (Case 2). [Figure 33] This is a diagram showing the protocol for Preliminary Examination 7. [Figure 34] This figure shows the results of flow cytometry analysis of the cytotoxic T cell induction ability of HPL-IFN-DCs prepared in serum-free medium (AIM-V) in preliminary test 7. [Figure 35] This figure shows the protocol for Test 1. [Figure 36-1] This figure shows the observed morphology of cells in Experiment 1. [Figure 36-2] This figure shows the viability, yield, and purity of IFN-DCs and HPL-IFN-DCs recovered after maturation in Experiment 1. [Figure 37] This figure shows the results of flow cytometry analysis of the effect of HPL on the phenotype of IFN-DCs in Study 2. [Figure 38] This diagram shows the protocol for Test 3. [Figure 39] This figure shows the antigen phagocytic activity and antigen resolution ability of IFN-DC and HPL-IFN-DC in Study 3. [Figure 40] This figure shows the protocol for Test 4. [Figure 41] This figure shows the results of measuring cytokines (IL-10, TGF-β, IFN-γ, TNF-α, IL-12(p70), IL-6) involved in the induction of cytotoxic T cells secreted from HPL-IFN-DCs in this study 4. [Figure 42] This figure shows the protocol for Test 5. [Figure 43-1] This figure shows the results of co-culturing CD8-positive T cells with IFN-DCs and HPL-IFN-DCs pre-pulsed with MART1 peptide in Study 5, and detecting MART1-specific cytotoxic T cells by flow cytometry at days 14 and 21. [Figure 43-2] This figure shows the number of MART1-specific CD8+ T cells when CD8-positive T cells and IFN-DCs and HPL-IFN-DCs pre-pulsed with MART1 peptide were co-cultured in this study 5. [Figure 43-3]This figure shows the percentage of MART1-specific CD8+ T cells when CD8-positive T cells and IFN-DCs and HPL-IFN-DCs pre-pulsed with MART1 peptide were co-cultured in this study 5. [Figure 44] This figure (Part 1) compares the cytotoxic T cell induction ability of IFN-DCs and HPL-IFN-DCs in this study 5. [Figure 45] This figure (part 2) compares the cytotoxic T cell induction ability of IFN-DCs and HPL-IFN-DCs in this study 5. [Figure 46] This figure (part 3) compares the cytotoxic T cell induction ability of IFN-DCs and HPL-IFN-DCs in this study 5. [Figure 47] This figure shows the protocol for Test 6. [Figure 48-1] This figure shows spot images illustrating the antigen-specific IFN-γ production ability of cytotoxic T cells induced by IFN-DCs and HPL-IFN-DCs. [Figure 48-2] This figure shows the antigen-specific IFN-γ production ability of cytotoxic T cells induced by IFN-DCs and HPL-IFN-DCs, as indicated by the amount of IFN-γ produced. [Figure 49] This figure summarizes the excellent viability, recovery rate, and purity of HPL-IFN-DC. [Figure 50] This figure summarizes the traits of HPL-IFN-DC. [Figure 51] This figure summarizes the results of the functional evaluation of HPL-IFN-DC. [Figure 52] This figure shows a method for producing IFN-DC using HPL. [Figure 53] This figure shows the state of monocytes that underwent selective adhesion culture during the production of IFN dendritic cells using HPL. [Figure 54] This figure shows flow cytometry of monocytes cultured under selective adhesion culture during the generation of IFN dendritic cells using HPL. [Figure 55]This figure shows the results of the phenotypic analysis of HPL-IFN-DC. [Figure 56] This figure shows the induction of MART-1 antigen-specific cytotoxic T cells by IFN-DC or HPL-IFN-DC. [Figure 57] This diagram shows the protocol for the WT1-CTL induction test. [Figure 58] This figure shows the methods for preparing IL-4-DC (Figure 58A) and HPL-IFN-DC (Figure 58B) used in the WT1-CTL induction test. [Figure 59] This figure shows a comparison of WT1-induced CTLs using IL-4-DC or HPL-IFN-DC with added WT1. [Figure 60] This figure shows the total number of WT1-CTLs induced by IL-4-DC (WT1 post-pulse) or HPL-IFN-DC (WT1 pre-pulse). [Modes for carrying out the invention]

[0014] The present invention will be described in detail below. In this specification, "A~B" (where A and B are numerical values) means "A or greater and B or less" unless otherwise specified. In this specification, "%" means "v / v%" unless otherwise specified.

[0015] The present invention relates to a method for separating monocytes from mononuclear cells, and a method for preparing dendritic cells (DCs) from monocytes.

[0016] Mononuclear cells are white blood cells, and they are divided into monocytes and lymphocytes. Mononuclear cells include peripheral blood mononuclear cells (PBMCs), bone marrow mononuclear cells, spleen cell mononuclear cells, and umbilical cord blood mononuclear cells. Among these, peripheral blood mononuclear cells are preferred. Mononuclear cells can also be separated using apheresis. Mononuclear cells may be fresh, unfrozen, or frozen. Even when frozen mononuclear cells are used, the cytotoxic activity of the dendritic cells obtained in the end does not decrease.

[0017] In the method for preparing dendritic cells from monocytes according to the present invention, the monocytes may be those isolated by the method for separating monocytes from mononuclear cells according to the present invention, or monocytes isolated by other methods. The monocytes include peripheral blood-derived monocytes, bone marrow-derived monocytes, spleen cell-derived monocytes, and umbilical cord blood-derived monocytes, among which peripheral blood-derived monocytes are preferred. The monocytes are characterized by being CD14-positive, and when collecting monocytes from a living organism, they can be separated using FACS (Fluorescent-activated cell sorter), flow cytometer, magnetic separator, etc., with the presence of CD14 as an indicator. They can also be separated using apheresis. Furthermore, they can be separated by density gradient centrifugation using Ficoll®, etc. The animal species from which the monocytes are derived is not limited, and mammals such as mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, monkeys, and humans can be used. For FACS and flow cytometers, for example, FACS Vantage (Becton Dickinson Corporation) and FACS Calibur (Becton Dickinson Corporation) can be used. For magnetic separation devices, for example, autoMACS® (Miltenyi Biotec) can be used. For instance, peripheral mononuclear cells (PBMCs) can be isolated using CD14-bound CD14 microbeads, with CD14 expression as the indicator, utilizing AutoMACS® and CliniMACS® technologies.

[0018] 1. Isolation of monocytes from mononuclear cells In the method for separating monocytes from mononuclear cells of the present invention, mononuclear cells are seeded in an adherent culture dish, cultured, and the monocytes are separated from lymphocytes by adhering them to the culture dish.

[0019] In this process, a serum-free medium (a medium without serum) supplemented with platelet lysate (PL) is used as the culture medium. Preferably, human platelet lysate (HPL) derived from human platelets is used. HPL is purified human platelet lysate and can be purified from platelets in plasma. HPL contains platelet-derived growth factors such as PDGF, TGF-β, IGF-1, and EGF.

[0020] The method for preparing HPL is not limited, but for example, it can be obtained by freezing and thawing platelets. Specifically, to lyse platelets, 1.5 × 10⁶ of plasma is added. 9 Platelets can be frozen at -80°C and then thawed at a rate of / mL. Furthermore, platelets pooled from multiple donors are preferable. Commercially available HPL can be used. For example, UltrGRO(trademark)-PURE, UltrGRO(trademark)-PURE GI (AventaCell BioMedical), etc., can be used. HPL exhibits little lot-to-lot variation within the same manufacturer, and also little variation between manufacturers.

[0021] In vitro culture of mononuclear cells can be performed using well-known human lymphoid cell culture techniques.

[0022] The serum-free medium to which HPL is added is not limited; any medium that can be used for culturing human lymphoid cells may be used. For example, DCO-K (Nissui Pharmaceutical Co., Ltd.), AIM-V (registered trademark, Thermo Fisher Scientific), X-VIVO5 (registered trademark), HL-1 (trademark, Lonza Co., Ltd.), BIOTARGET (trademark)-1 SFM (Cosmo Bio Co., Ltd.), DMEM, MEM, RPMI1640, IMDM, etc. can be used. Among these, DCO-K (Nissui Pharmaceutical Co., Ltd.) is preferred.

[0023] These serum-free media can be used by adding 1-10% (v / v), preferably 2-7.5% (v / v), more preferably 2.2-5.3% (v / v), and especially preferably 2.5-5% (v / v) of the above-mentioned HPL. As described above, HPL shows little variation between lots within the same manufacturer and also little variation between manufacturers, so the same effect can be obtained by using HPL at the above concentrations, regardless of the manufacturer or lot.

[0024] Because monocytes have the characteristic of strongly adhering to containers, mononuclear cells can be cultured by adherent culture. The monocytes adhere to culture containers such as culture dishes, petri dishes, plates, and flasks, and then separated and recovered by removing cells that do not adhere. Any adherent cell culture container capable of cell adhesion should be used. A wide range of commercially available adherent cell culture containers can be used. Either low-adhesion or high-adhesion culture containers may be used.

[0025] The pH during culture is preferably around 6 to 8. Culture is usually carried out at approximately 30 to 40°C for 15 minutes to 12 hours, more preferably 15 minutes to 6 hours, more preferably 15 minutes to 3 hours, more preferably 15 minutes to 1 hour, more preferably 20 minutes to 45 minutes, and most preferably 25 minutes to 35 minutes. If the culture time exceeds one day, the cells will detach and become suspended. During culture, the culture medium may be changed, aerated, and stirred as needed. For example, carbon dioxide may be added, preferably at a concentration of 2.5 to 10%, more preferably 2.5 to 7.5%, and more preferably 5%.

[0026] After adherent culture, non-adherent cells can be removed by washing, and monocytes can be isolated as adherent culture cells. In this case, washing is performed 1 to 5 times, preferably 2 times.

[0027] 2. Method for preparing dendritic cells (DCs) from monocytes Dendritic cells can be prepared using monocytes isolated by the above method of separating monocytes from mononuclear cells. The isolated monocytes are cultured in non-adherent culture, i.e., suspension culture. To perform non-adherent culture, a non-adherent incubator such as a plate, dish, or flask can be used. A non-adherent incubator is an incubator in which the surface of the culture dish is coated with a compound such as a superhydrophilic polymer, phospholipid polymer, or MPC polymer, or is treated hydrophilically without using a coating agent, so that cells do not adhere to it. For example, low-adherence culture dishes such as HydroCell® (CellSeed Co.), EZ-BindShut® II (Iwaki), Nunclon® Vita, and Lipidure® Coat (NOF Co., Ltd.) can be used.

[0028] The isolated monocytes are first differentiated into dendritic cells (DCs). This differentiation inducement yields immature DCs. Subsequently, the immature DCs are cultured in the presence of specific cytokines to mature them, thereby obtaining mature DCs with cytotoxic activity.

[0029] Differentiation into DCs can be induced by culturing in a serum-free medium containing cytokines and HPL that have DC differentiation-inducing activity. As the serum-free medium, the serum-free medium described in the method for separating monocytes from mononuclear cells described above can be used, and among these, DCO-K (Nissui Pharmaceutical Co., Ltd.) is preferred. Furthermore, the HPL described in the method for separating monocytes from mononuclear cells described above can be used, and the concentration added should also be as described in the method for separating monocytes from mononuclear cells described above.

[0030] Cytokines with dendritic cell differentiation-inducing activity can be GM-CSF (granulocyte-monocyte colony-stimulating factor) and IFN-α. PEGylated interferon (IFN)-α (PEG-IFN-α) is preferred as the IFN-α.

[0031] PEG-IFN-α is formed by bonding polyethylene glycol (PEG) to IFN-α. PEG-IFN-α-2b is preferred as the PEG-IFN-α. Commercially available PEG-IFN preparations can be used as the PEG-IFN-α. An example of a commercially available PEG-IFN-α preparation is PEGINTRON (registered trademark) (generic name: Peginterferon Alfa-2b (Genetic Recombination)), which is a PEG-IFN-α-2b preparation.

[0032] PegIntron® is represented by the structural formula H3C-(O-CH2CH2)n-OCO-Interferon alfa-2b, and consists of one molecule of methoxypolyethylene glycol (average molecular weight: approximately 12,000) covalently bonded via a carbonyl group to one amino acid residue (Cys1, His7, Lys31, His34, Lys49, Lys83, Lys112, Lys121, Tyr129, Lys131, Lys133, Lys134, Ser163, and Lys164) of interferon alfa-2b (recombinant) (molecular weight: 19268.91). Its molecular weight is approximately 32,000, and its molecular formula is C 86 0H 1353 N 229 O 255 It is represented as S9. Its CAS registry number is 215647-85-1.

[0033] The concentration of GM-CSF used in culture is, for example, 10 times the amount of monocytes. 4 ~10 7When used at a concentration of cells / mL, it is 100 U / mL to 10,000 U / mL, preferably 500 U / mL to 2,000 U / mL, more preferably 800 U / mL to 1,200 U / mL, and particularly preferably 1,000 U / mL. Alternatively, it is 10 ng / mL to 1,000 ng / mL, preferably 20 ng / mL to 200 ng / mL, more preferably 20 ng / mL to 100 ng / mL. The concentration of PEG-IFN-α is 100 ng / mL to 10 μg / mL, preferably 500 ng / mL to 5 μg / mL, more preferably 500 ng / mL to 2 μg / mL.

[0034] Culturing in the presence of HPL, GM-CSF and PEG-IFN-α is carried out for 2 to 5 days, preferably 3 to 4 days, more preferably 3 days. Immature DCs can be obtained by culturing in the presence of HPL, GM-CSF and PEG-IFN-α.

[0035] Maturation of immature DCs is carried out by culturing the immature DCs in a maturation medium. The maturation medium uses a serum-free medium containing HPL, GM-CSF, PEG-IFN-α, prostaglandin E2 (PGE2) and OK-432. GM-CSF, PEG-IFN-α and prostaglandin E2 are cytokines. As the serum-free medium, the serum-free medium described in the method for separating monocytes from the above-mentioned monocytes can be used, and among them, DCO-K (Nissui Pharmaceutical Co., Ltd.) is preferable. Also, for HPL, the HPL described in the method for separating monocytes from the above-mentioned monocytes can be used, and the addition concentration is also as described in the method for separating monocytes from the above-mentioned monocytes.

[0036] The concentration of GM-CSF used for culturing is, for example, when monocytes are 10 4 ~10 7When used at a concentration of cells / mL, the concentration is 100 U / mL to 10,000 U / mL, preferably 500 U / mL to 2,000 U / mL, more preferably 800 U / mL to 1,200 U / mL, and especially preferably 1,000 U / mL. Alternatively, it is 10 ng / mL to 1,000 ng / mL, preferably 20 ng / mL to 200 ng / mL, and more preferably 20 ng / mL to 100 ng / mL. The concentration of PEG-IFN-α is 100 ng / mL to 10 μg / mL, preferably 500 ng / mL to 5 μg / mL, and more preferably 500 ng / mL to 2 μg / mL. The concentration of PGE2 is 1 ng / mL to 100 ng / mL, preferably 5 ng / mL to 50 ng / mL, and more preferably 5 ng / mL to 20 ng / mL. The concentration of OK432 is 1 μg / mL to 100 μg / mL, preferably 5 μg / mL to 50 μg / mL, and more preferably 5 μg / mL to 20 μg / mL.

[0037] By examining the expression of surface antigens in monocytes or DCs using FACS or similar methods, the appropriate concentration for obtaining cells of the desired degree of differentiation can be determined.

[0038] By culturing in a mature medium for 10 to 48 hours, preferably 10 to 36 hours, more preferably 10 to 24 hours, and especially preferably 18 to 24 hours, cytotoxic dendritic cells (DCs) can be obtained.

[0039] The total culture period for separating monocytes from mononuclear cells and further maturing them is 3 to 7 days, preferably 4 to 6 days, more preferably 4 to 5 days, and most preferably 4 days.

[0040] DCs prepared by the present invention, which involve culturing in a serum-free medium containing cytokines such as HPL and IFN, are called HPL-IFN-DCs. In contrast, DCs prepared by culturing in a serum-free medium that differs from the serum-free medium used to prepare HPL-IFN-DCs, except that it does not contain HPL, are called IFN-DCs.

[0041] 3. Characteristics of the obtained HPL-IFN-DC (1) Viability and yield In the method of the present invention, since dendritic cells (DCs) are produced from monocytes by non-adherent culture, the viability rate of DCs is high and the yield is also high. The viability rate of the obtained DCs is 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher, and even more preferably 97% or higher, which are the standards of the NIH (National Institutes of Health). Furthermore, the recovery rate of DCs (the ratio of the number of obtained viable DCs to the number of seeded monocytes) is 5% or higher, preferably 10% or higher, more preferably 15% or higher, and particularly preferably 20% or higher. In addition, the purity of the DCs is 90% or higher, preferably 95% or higher. HPL-IFN-DCs have higher viability rates, yields, and purity than IFN-DCs.

[0042] (2) Surface antigen HPL-IFN-DCs are characterized by the morphological presence of dendrites, and further analysis by FACS and other methods reveals that they are positive for the surface antigens CD14, CD16, CD56, CD83, CD86, CCR7 (CD197), HLA-ABC, and HLA-DR. CD14 is a monocyte marker, CD56 is a cell adhesion molecule, CD197 (CCR7) is a molecule that promotes migration to lymph nodes, and CD11c is a dendritic cell marker. In addition, CD80 and CD40 are costimulatory molecules involved in antigen presentation to T cells, CD83 is a dendritic cell maturation marker, and HLA-DR is a molecule involved in antigen presentation.

[0043] Whether these surface antigens are positive or negative can be determined by microscopic observation, for example, by checking whether cells are stained with antibodies labeled with chromogenic enzymes, fluorescent compounds, etc., that are antibodies against these antigens. For example, the presence or absence of surface antigens can be determined by immunostaining cells with these antibodies. It can also be determined using magnetic beads to which the antibodies are bound. Furthermore, the presence or absence of surface antigens can be determined using FACS or flow cytometry. A negative result for surface antigens means that, as described above, the cells are not sorted as positive when analyzed using FACS, and no expression is detected when expression is examined by immunostaining. Even if the expression is too low to be detected by these methods, it is still considered negative.

[0044] Comparing the expression of surface antigens in HPL-IFN-DC and IFN-DC, the expression of CD14, CD56, CCR7 (CD197), and CD11c was increased in HPL-IFN-DC compared to IFN-CD. When the percentage of cells expressing each surface antigen (positive cells (%)) in the cell population was calculated by flow cytometry, CD14 was less than 60% (median 35.8%) in IFN-DC compared to more than 50% (median 83.6%) in HPL-IFN-DC; CD56 was less than 60% (median 37.6%) in IFN-DC compared to more than 50% (median 68.4%) in HPL-IFN-DC; and CCR7 (CD197) was less than 30% (median 10.3%) in IFN-DC compared to more than 20% (median 37.8%) in HPL-IFN-DC.

[0045] Specifically, the percentage of CD14-positive cells in HPL-IFN-DCs is 1.5 to 2.5 times that of IFN-DCs, the percentage of CD56-positive cells in HPL-IFN-DCs is 1.5 to 2.5 times that of IFN-DCs, and the percentage of CCR7 (CD197)-positive cells in HPL-IFN-DCs is 2.5 to 5 times, preferably 3 to 5 times, that of IFN-DCs.

[0046] On the other hand, in HPL-IFN-DCs, the expression of CD80, CD83, CD40, and HLA-DR is reduced compared to IFN-CDs. When the percentage of cells expressing each surface antigen (positive cells (%)) in a cell population was calculated by flow cytometry, CD80 was expressed in over 15% of IFN-DCs (median 84.0%) compared to less than 60% of HPL-IFN-DCs (median 33.1%), CD83 was expressed in over 60% of IFN-DCs (median 86.8%) compared to less than 80% of HPL-IFN-DCs (median 64.2%), CD40 was expressed in over 55% of IFN-DCs (median 98.6%) compared to less than 95% of HPL-IFN-DCs (median 66.9%), and HLA-DR was expressed in over 95% of IFN-DCs (median 99.8%) compared to less than 100% of HPL-IFN-DCs (median 92.7%).

[0047] Specifically, the percentage of CD80-positive cells in HPL-IFN-DCs is 0.3 to 0.5 times that of IFN-DCs, the percentage of CD83-positive cells in HPL-IFN-DCs is 0.6 to 0.9 times that of IFN-DCs, the percentage of CD40-positive cells in HPL-IFN-DCs is 0.5 to 0.8 times that of IFN-DCs, and the percentage of HLA-DR-positive cells in HPL-IFN-DCs is 0.8 to 0.95 times that of IFN-DCs.

[0048] (3) Antigen phagocytic ability and decomposition ability HPL-IFN-DCs exhibit improved antigen phagocytosis and antigen degradation capabilities compared to IFN-DCs. For example, when 100 μg / mL FITC-dextran (Molecular Probes, Eugene, OR, USA) and 10 μg / mL DQ-ovalbumin (Molecular Probes) were added to maturation medium and incubated for 24 hours, the recovered IFN-DCs or HPL-IFN-DCs were washed twice with PBS and then resuspended in 1(v / v)% FBS-PBS. The following results were obtained when phagocytosis and antigen degradation capabilities were evaluated by flow cytometry: FITC-dextranΔMFI (antigen phagocytosis) was less than 30 (average 17.1) for IFN-DCs, while it was greater than 50 (average 68) for HPL-IFN-DCs. Furthermore, the DQ-Ovalbumin ΔMFI (antigen resolution index) was 450 or less (average 270.9) in IFN-DCs, while it was 350 or more (average 589.7) in HPL-IFN-DCs.

[0049] In other words, the FITC-dextran ΔMFI (antigen phagocytic activity) in HPL-IFN-DC is 2 to 6 times, preferably 3 to 5 times, the FITC-dextran ΔMFI (antigen phagocytic activity) in IFN-DC is 1.5 to 3 times the DQ-Ovalbumin ΔMFI (antigen resolution) in HPL-IFN-DC is 1.5 to 3 times the DQ-Ovalbumin ΔMFI (antigen resolution) in IFN-DC.

[0050] (4) Cytokine production capacity The production of the following cytokines is 1 × 10⁻⁶ of mature HPL-IFN-DCs. 6 The cytokine levels were measured using a Bio-plex assay kit (Bio-Rad Labs) after suspending the cells in DCO-K medium to a cell density of cells / mL, seeding in a culture dish, culturing at 37°C and 5% CO2 for 24 hours, and then collecting the culture supernatant. The production amount is the average of multiple measurements, for example, n=6 measurements.

[0051] In HPL-IFN-DCs, the production of IL-12(p70), a Th1 cytokine that enhances the induction of cytotoxic T cells, is significantly lower than in IFN-DCs. The average production level in IFN-DCs is 1.1 pg / mL, while the average production level in HPL-IFN-DCs is 0.18 pg / mL.

[0052] On the other hand, the production of Th2 cytokines IL-10 and TGF-β, which suppress the induction of cytotoxic T cells, is increased in HPL-IFN-DC compared to IFN-DC. For IL-10, the average production in IFN-DC was 11.47 pg / mL, while the average production in HPL-IFN-DC was 132.7 pg / mL. Similarly, for TGF-β, the average production in IFN-DC was 8.02 pg / mL, while the average production in HPL-IFN-DC was 9.38 pg / mL.

[0053] In other words, the amount of IL-10 produced in HPL-IFN-DC is 8 to 15 times, preferably 9 to 13 times, the amount of IFN-DC produced, and the amount of TGF-β produced in HPL-IFN-DC is 1.1 to 1.5 times the amount of IFN-DC produced.

[0054] Furthermore, the production of TNF-α and IL-6, which induce inflammatory responses and are involved in T cell activation and differentiation, is increased in HPL-IFN-DCs compared to IFN-DCs. For TNF-α, the average production in IFN-DCs is 412.5 pg / mL, while the average production in HPL-IFN-DCs is 1144.4 pg / mL. Similarly, for IL-6, the average production in IFN-DCs is 302.3 pg / mL, while the average production in HPL-IFN-DCs is 2883 pg / mL.

[0055] In other words, the amount of TNF-α produced in HPL-IFN-DC is 2 to 4 times the amount produced in IFN-DC, and the amount of IL-6 produced in HPL-IFN-DC is 8 to 15 times, preferably 8 to 13 times, the amount produced in IFN-DC.

[0056] Therefore, the presence of HPL in the culture medium during DC differentiation and maturation reduces Th1 / Th2 cytokines.

[0057] (5) Cytotoxic T cell inducing ability HPL-IFN-DCs exhibit increased cytotoxic T cell induction compared to IFN-DCs.

[0058] (6) Antigen-specific IFN-γ production capacity by induced cytotoxic T cells In HPL-IFN-DCs, antigen-specific IFN-γ production by induced cytotoxic T cells is increased compared to IFN-DCs.

[0059] 4. Dendritic cell therapy Dendritic cells (DCs) prepared by the method of the present invention can be used in dendritic cell therapy. Examples of dendritic cell therapy include cancer immunotherapy, also known as dendritic cell vaccine therapy. For example, dendritic cells can be prepared from monocytes of a subject by the method of the present invention, and the obtained dendritic cells can be returned to the subject to be used for cancer treatment or prevention. In this case, the prepared dendritic cells can act non-specifically to cancer types and exert cancer therapeutic effects. Furthermore, by adding cancer-specific antigens specific to a particular cancer when preparing dendritic cells and culturing them, the cancer-specific antigens can be taken up by the dendritic cells, making it possible to obtain dendritic cells with cancer-type-specific anti-cancer immune activity. Adding cancer-specific antigens specific to a particular cancer when preparing dendritic cells and culturing them is called pulsing dendritic cells with cancer-specific antigens. Pulsing may be performed by adding cancer-specific antigens when preparing cytotoxic dendritic cells from monocytes, or by culturing dendritic cells with cancer-specific antigens after preparing cytotoxic dendritic cells from monocytes. The former is called prepulsing, and the latter is called postpulsing. Furthermore, obtaining dendritic cells with cancer-specific anti-cancer immune activity is referred to as inducing cancer antigen-cytotoxic dendritic cells. Examples of cancer-specific antigens include WT1 peptide in leukemia and other various cancers, HER2 / neu in breast cancer, CEA (carcinoembryonic antigen) in colorectal cancer, MART-1 (melan-a protein) and MEGA (melanoma antigen) in melanoma, GPC3 (glypican 3) in hepatocellular carcinoma, and PAP (prostate acid phosphatase) and PSMA (prostate-specific membrane antigen) in prostate cancer. The present invention states that these dendritic cells can induce cancer-specific cytotoxic T cells (CTLs). Dendritic cells with cancer-specific anti-cancer immune activity can be used to treat lung cancer, gastric cancer, pancreatic cancer, liver cancer, rectal cancer, colon cancer, breast cancer, esophageal cancer, uterine cancer, kidney cancer, bladder cancer, lymphoma / leukemia, brain tumors, urethral cancer, renal pelvis / ureteral cancer, mesothelioma, and other cancers.

[0060] Furthermore, the proliferation of cancer antigen-specific CTLs in the test subjects can be confirmed by the tetramer assay or the Elispot assay.

[0061] The present invention provides a method for preparing cytotoxic dendritic cells from monocytes, comprising culturing monocytes isolated from peripheral blood by non-adherent culture in a serum-free medium containing human platelet lysate (HPL), GM-CSF, and PEGylated interferon α, and then adding prostaglandin E2 and OK432 and culturing further by non-adherent culture, wherein the method further includes adding a cancer-specific antigen when adding prostaglandin E2 and OK432, thereby preparing dendritic cells that exhibit cancer antigen-specific cytotoxicity from monocytes. In this method, for example, the cells may be cultured for 2 to 5 days by non-adherent culture in a serum-free medium containing human platelet lysate (HPL), GM-CSF, and PEGylated interferon α, and then prostaglandin E2, OK432, and a cancer-specific antigen may be added and the cells cultured for a further 1 to 2 days. The concentration of the cancer-specific antigen is not limited, but is 0.1 to 1000 μg / mL, preferably 1 to 500 μg / mL, and more preferably 5 to 300 g / mL.

[0062] Furthermore, the present invention includes dendritic cells having cancer antigen-specific cytotoxicity obtained by the method for preparing dendritic cells having cancer antigen-specific cytotoxicity from monocytes as described above.

[0063] Furthermore, it can also be used to treat bacterial and viral infections. In the treatment of infections, dendritic cells (DCs) prepared by culturing monocytes in the presence of HPL, GM-CSF, PEG-IFN-α, PGE2, and OK432 using the method of the present invention are useful. The prepared dendritic cells can be administered to the subject by intradermal, subcutaneous, intravenous, or intra-lymph node administration. The dosage and timing of administration can be appropriately determined according to the type of disease, the severity of the disease, and the subject's condition.

[0064] 5. DC Differentiation and Induction Agents The present invention encompasses dendritic cell (DC) differentiation and induction agents from monocytes, comprising HPL, GM-CSF, and PEG-IFN-α. These DC differentiation and induction agents may also be referred to as DC preparations. The DC differentiation and induction agents may further comprise PGE2 and OK432. The DC differentiation and induction agent may consist of a first reagent comprising HPL, GM-CSF, and PEG-IFN-α and a second reagent comprising PGE2 and OK432, and the present invention also encompasses DC differentiation and induction kits comprising these first and second reagents. The first reagent comprising HPL, GM-CSF, and PEG-IFN-α is used to differentiate and induce immature DCs, and the second reagent comprising PGE2 and OK432 is used to mature immature DCs.

[0065] By the method of the present invention, DCs are induced as mature DCs. Furthermore, the present invention also encompasses DCs obtained by the method of the present invention and cell populations containing said DCs. The cell population contains 10% or more, 30% or more, 50% or more, 70% or more, 90% or more, or 95% or more DCs. [Examples]

[0066] The present invention will be specifically described by the following embodiments, but the present invention is not limited to these embodiments. In this embodiment, DCs prepared using a culture medium supplemented with HPL and IFN are called HLP-IFN-DCs, and DCs prepared using a culture medium supplemented with IFN but without HPL are called IFN-DCs.

[0067] [Example 1] Establishment of a monocyte isolation method and IFN-DC production method using serum-free medium (DCO-K) with optimized additives (ABS or HPL). This method was conducted as a preliminary test.

[0068] The objective of this example was to establish a method for separating monocytes from peripheral blood mononuclear cells and a method for producing IFN-DCs using serum-free medium (DCO-K) (manufactured by Nissui Pharmaceutical Co., Ltd.) with optimized concentrations of additives (Human serum type AB (ABS) (biowest) and Human platelet lysate (HPL) (AnentaCell Biomedical)). In this example, we show an example using DCO-K as the serum-free medium, but similar results can be obtained with other serum-free media.

[0069] The evaluation criteria are listed below. (1) IFN-DCs were prepared using DCO-K medium optimized for ABS or HPL supplementation, and cell morphology was observed using a phase-contrast microscope (EVOS® FL Cell Imaging System). (2) The viability of IFN-DCs was measured by staining dead cells with trypan blue, and the yield and purity were evaluated using flow cytometry (FCM). (3) Cells were stained with antibodies against DC markers to which the fluorescent dyes FITC, PE, and APC were added, and the phenotype of IFN-DCs was examined by flow cytometry.

[0070] In the preparation of dendritic cell (DC) vaccines, adherent culture is commonly used to isolate monocytes, the raw material for the vaccine, from peripheral blood mononuclear cells (PBMCs: including monocytes and lymphocytes). Monocytes have the characteristic of strongly adhering to the culture dish. Patient-derived PBMCs collected by apheresis were suspended in serum-free medium (DCO-K) or AIM medium alone (conventional method, AIM-V medium) prepared with additives (final concentration 5(v / v)% ABS or 5(v / v)% HPL), and seeded in adherent culture dishes. By culturing at 37°C and 5% CO2 for 24 hours or 30 minutes, the cells adhered to the bottom of the culture dish, and monocytes (raw material for IFN-DC vaccine) and lymphocytes were separated. Next, adherent cells were differentiated into IFN-DCs using DCO-K medium or AIM medium supplemented with 1 μg / mL PEG-Intron, 100 ng / mL GM-CSF, and a final concentration of 5(v / v)% HPL. Three days after the start of differentiation, the cells were harvested and matured into IFN-DCs by culturing them for 18-24 hours in a low-adhesion culture dish (Sumitomo Bakelite, Prime surface) using a maturation medium mixed with various reagents (1 μg / mL PEG-Intron, 100 ng / mL GM-CSF, 10 μg / mL OK432, 10 ng / mL PGE2) and 20 μg / mL tumor antigen peptide (WT-1: Wilms tumor 1). Preliminary tests 1-7 were performed using IFN-DCs prepared under these various conditions.

[0071] Preliminary Examination 1 Preliminary Study 1: IFN-DCs were prepared using DCO-K medium or AIM-V medium alone, supplemented with a final concentration of 5(v / v)% HPL or 5(v / v)% ABS, during 24-hour adherent culture, differentiation, and maturation of peripheral blood mononuclear cells. Cell morphology, viability, purity (DC fraction defined from FSC / SSC using a flow cytometer, and purity calculated by the percentage of the DC fraction), lymphocyte contamination rate, and phenotype were compared (n=1). Figure 1 shows the protocol for Preliminary Study 1.

[0072] PBMCs were suspended in serum-free medium (DCO-K) or AIM medium alone (conventional method) prepared with additives (final concentration 5(v / v)% ABS or 5(v / v)% HPL), seeded in adherent culture dishes (using low-adhesion dishes), and after 30 minutes, non-adhented cells were washed and their morphology was observed using a phase-contrast microscope (Day 1). The observed cell images are shown in Figure 2. (a) shows the results of culturing with DCO-K alone, (b) with DCO-K + ABS, (c) with DCO-K + HPL, and (d) with AIM-V.

[0073] Typically, in the process of producing dendritic cells, when separating monocytes from patient-derived PBMCs (Primary Blood Cells) collected by apheresis, peripheral blood mononuclear cells are seeded in serum-free medium (AIM-V), washed after 30 minutes, and then subjected to 24 hours of adherent culture before being washed again for non-adherent cells. Therefore, patient-derived PBMCs collected by apheresis were cultured for 24 hours in serum-free medium (DCO-K) supplemented with ABS or HPL, and the cells were observed using a phase-contrast microscope (n=1).

[0074] PBMCs were suspended in serum-free medium (DCO-K) or AIM medium alone (conventional method) prepared with additives (final concentration 5(v / v)% ABS or 5(v / v)% HPL), seeded in adherent culture dishes, and after 30 minutes, non-adherent cells were washed (Day 1). Cell morphology was then observed after further washing with the medium 24 hours later (Day 2). The results are shown in Figure 2-2. Compared to (a), in DCO-K medium with added ABS or HPL ((b) and (c)), many cells became suspended and detached during washing. After adhesion, cells became suspended after standing for 1 day. In the conventional method (d), adherent and suspended cells could be clearly distinguished after washing.

[0075] Cell surface antigens expressed in IFN-DCs produced under each condition were detected by flow cytometry using labeled antibodies (n=1).

[0076] Figure 3 shows the results for IFN-DCs prepared using only DCO-K medium. In IFN-DCs prepared using only DCO-K medium, the expression of costimulatory molecules CD40, CD86, and CD80, which are involved in antigen presentation to T cells, as well as CD83, an indicator of dendritic cell maturation, and HLA-DR and HLA-ABC, which are involved in antigen presentation, were detected. Furthermore, immature-like dendritic cells (CD80) - / CD83 - / CD86 - Although HLADR / HLA-ABC subfractions were detected, this suggests that the cell maturation reaction was impaired due to the cell's state.

[0077] Figure 4 shows the results of IFN-DCs prepared in DCO-K+ABS medium. In (b), when IFN-DCs were prepared in DCO-K medium supplemented with serum (ABS), it was observed that they exhibited a phenotypic expression similar to that in (a). (a) showed even more heterogeneous subfractions (CD80 - / CD83 - / CD86 - ) was recognized.

[0078] Figure 5 shows the results of IFN-DCs (HPL-IFN-DCs) prepared in DCO-K+HPL medium. In (c), compared with (a) and (b) and (d) using conventional medium, decreased expression of CD80, CD86, and CD83 and increased expression of CD14, CD16, CCR7, HLA-DR, and HLA-ABC were observed. In particular, the expression of CD14, CD16, and CD56 was significantly increased (CD14 ++ CD16 + CD56 + CCR7+HLA-ABC + DR + (A homogeneous cell population), (d) showed a phenotype completely different from conventional IFN-DCs.

[0079] Figure 6 shows the results of IFN-DCs prepared using conventional AIM-V medium. The conventional method showed weak positivity for CD14 and expression of CD80, CD86, CD83, HLA-DR, HLA-ABC, and CD40, which was similar to the phenotype reported in related literature (Terutsugu Koya et.al. Scientific reports 7, Article number 42145: 2017).

[0080] IFN-DCs produced under each condition were recovered, and the purity and lymphocyte contamination rate of the IFN-DCs at the time of recovery, which are indicators of quality for the production of the DC vaccine, were evaluated by flow cytometry. Differentiation induction was started immediately after 30 minutes of adhesion. The results are shown in Figure 7. While a large amount of lymphocyte contamination was observed in (a) and (d), a decrease in lymphocyte contamination rate was observed in IFN-DCs produced with the addition of ABS (a) or HPL (c), and a particularly significant decrease was observed when HPL was added. Purity was highest in (c). This suggests that when HPL is added, lymphocyte-like floating cells may be removed by detachment during the process of sorting monocytes and lymphocytes from PBMCs on Day 2.

[0081] Figure 8 shows a summary of the viability rate and yield. The viability rate in serum-free medium DCO-K (a) with HPL was very high. The yields of (c) and (d) were similar, (a) was slightly lower, and (b) was significantly lower. Yield % = number of viable cells at Day 6 harvest / number of viable cells at Day 1 seeding. In terms of viability at harvest, IFN-DC (c) prepared using DCO-K medium with HPL showed a very high value.

[0082] A summary of the first preliminary exam is provided below. Compared to the conventional method (d), it was confirmed that IFN-DCs could be produced (a) using DCO-K (Nissui Pharmaceutical Co., Ltd.), a serum-free medium. IFN-DCs produced using HPL-supplemented DCO-K medium (c) showed improved cell viability and purity compared to the other groups ((a), (b), (d)). Furthermore, in terms of phenotype, CD14 ++ CD16 +CD56 + These exhibit phenotypes different from conventional IFN-DCs, such as CD40. + CD86 + HLA-ABC + HLA-DR + They formed an extremely uniform population of cells.

[0083] Based on the above, HPL and DCO-K are suitable for IFN-DC production in terms of viability and purity. However, a decrease in yield is predicted because cells float and detach after 1 day of standing after adhesion during the monocyte separation process from PBMCs. Therefore, the process involves washing twice with each medium after the adhesion reaction 30 minutes after seeding, followed by differentiation induction.

[0084] Preliminary Examination 2 In a preliminary study of peripheral blood mononuclear cells over 30 minutes of adhesion culture, differentiation, and maturation, IFN-DCs were prepared using DCO-K medium supplemented with 5(v / v)% HPL or 5(v / v)% ABS, or AIM-V medium alone. Cell morphology, viability, purity, lymphocyte contamination rate, and phenotype were compared (n=1).

[0085] Figure 9 shows the protocol for preliminary test 2. In the process of separating monocytes and lymphocytes from PBMCs collected from apheresis, adherent culture was performed for 30 minutes using DCO-K medium supplemented with (b) a final concentration of 5(v / v)% ABS or (c) a final concentration of 5(v / v)% HPL. Subsequently, the cells were washed twice with the medium and then examined by phase-contrast microscopy (n=1). The observed images of the cells are shown in Figure 10. (a) shows the results of culturing with DCO-K alone, (b) shows the results of culturing with DCO-K + ABS, and (c) shows the results of culturing with DCO-K + HPL.

[0086] In (a), many lymphocyte-like cells are mixed in with adherent cells, suggesting that they may not have been removed by washing. In the DCO-K medium with additives ((b) and (c)), compared to (a), cells adhered to the bottom can be observed, suggesting that many lymphocyte-like cells were removed by washing. After washing, GM-CSF / IFN-α was added to initiate differentiation induction.

[0087] The expression of cell surface antigens in IFN-DCs prepared under each condition was evaluated by flow cytometry (n=1).

[0088] Figure 11 shows the results when cultured with DCO-K alone (a). Compared with the results of preliminary test 1 (a), a large number of weakly positive CD14, CD80, CD86, CD83, HLA-ABC, and HLA-DR positive cells were detected, showing a phenotype similar to the conventional method (preliminary test 1 (d)).

[0089] Figure 12 shows the results when cultured with DCO-K+ABS(b). Compared to (a), increased CD14 expression and decreased CD80 / CD83 expression were observed, showing characteristics similar to the phenotype of immature dendritic cells.

[0090] Figure 13 shows the results when cultured with DCO-K+HPL(c). Compared with the other groups ((a) and (b)), there was a decrease in CD80 / CD83 expression and CD14 ++ CD16 + CD56 + HLA-DR / HLA-ABC + A homogeneous cell population was observed, showing a similar trend to that of preliminary study 1.

[0091] IFN-DCs produced under each condition were recovered, and the purity and lymphocyte contamination rate of the IFN-DCs at the time of recovery, which are indicators of quality for DC vaccine production, were evaluated by flow cytometry. The results are shown in Figure 14. Compared with the other groups ((a) and (b)), the lymphocyte contamination rate was significantly lower when DCO-K medium (c) supplemented with HPL was used in the monocyte isolation step 30 minutes after PBMC seeding. The lymphocyte contamination rate was less than 1%. Even when differentiation induction was started immediately after adhesion at 30 minutes, a large amount of lymphocyte contamination was observed in (a).

[0092] Figure 15 shows a summary of the viability rate and yield. Yield % = number of viable cells at Day 5 harvest / number of viable cells at Day 1 seeding. The inoculation time was shortened by one day to Day 5. In terms of viability, the HPL-supplemented DCO-K medium (c) showed a significantly higher value compared to the other groups ((a) and (b)) (n=1).

[0093] A summary of the preliminary examination 2 is provided below. In the monocyte isolation process from patient-derived PBMCs collected by apheresis, differentiation into IFN-DCs was possible from the adhesion reaction 30 minutes after seeding. Using DCO-K medium (c) supplemented with HPL resulted in significantly higher viability, purity, and recovery rates compared to other groups ((a) and (b)). Regarding cell surface antigen expression, under conditions with HPL supplementation, CD14 was expressed similarly to preliminary study 1. ++ CD16 + CD56 + CD86 + , CCR7 + HLA-ABC + HLA-DR + The cells showed a homogeneous population, but the CD80 and CD83 fractions tended to be lower. Using DCO-K medium supplemented with ABS resulted in low cell viability and yield, and further decreased the CD80 fraction, so it was excluded from subsequent preliminary studies.

[0094] Preliminary Examination 3 In a preliminary study of 3:30 minutes of adherent culture of peripheral blood mononuclear cells, monocytes and lymphocytes were sorted using DCO-K medium supplemented with HPL at various concentrations (2.5(v / v)%). Subsequently, cell morphology, viability, purity, lymphocyte contamination rate, and phenotype of IFN-DCs prepared in DCO-K medium without HPL supplementation during differentiation and maturation were compared (n=1).

[0095] Figure 16 shows the protocol for preliminary test 3. In preliminary experiment 3, during the IFN-DC preparation process, no significant difference was observed in the initial cell appearance when DCO-K medium supplemented with HPL at various concentrations (2.5(v / v)%) was used only in the monocyte isolation step using a low-adhesion culture dish with PBMCs (n=1). The observed cell images are shown in Figure 17. (a) shows the results of culturing at 2.5(v / v)%, and (b) shows the results of culturing at 5(v / v)%.

[0096] The expression of cell surface antigens in IFN-DCs was evaluated by flow cytometry (n=1). Figure 18 shows the results when cultured with HPL 5(v / v)%, and Figure 19 shows the results when cultured with HPL 2.5(v / v)%.

[0097] IFN-DCs prepared under each condition were recovered, and the purity and lymphocyte contamination rate of the IFN-DCs at the time of recovery, which are indicators of quality for the production of DC vaccines, were evaluated by flow cytometry. The results are shown in Figure 20. In the monocyte isolation step using low-adhesion culture dishes with PBMCs, IFN-DCs prepared using DCO-K medium supplemented with HPL at each concentration (2.5(v / v)%) showed a low lymphocyte contamination rate (n=1).

[0098] Figure 21 shows a summary of the viability rate and yield. Yield % = number of viable cells at Day 5 harvest / number of viable cells at Day 1 seeding. The inoculation time was shortened by one day to Day 5. Both groups showed a viability rate of 76-77%, and no significant difference was observed (n=1).

[0099] A summary of the preliminary exam 3 is provided below. In the IFN-DC fabrication process, when HPL was used only in the monocyte separation step, no significant difference in monocyte adhesion performance was observed. Furthermore, a significant decrease in the lymphocyte contamination rate of IFN-DCs was observed, but the viability rate and yield were lower than those under conditions with HPL added (preliminary studies 1-2) during differentiation and maturation. The phenotype was similar to that of IFN-DCs produced using only DCO-K (from preliminary studies 1 and 2:(a)). Therefore, improvements in viability, yield, and lymphocyte contamination can be expected when producing IFN-DCs using HPL.

[0100] Preliminary Examination 4 Preliminary study 4 investigated the optimal concentration of HPL for preparing IFN-DCs.

[0101] In a preliminary study of peripheral blood mononuclear cells under adhesion culture, differentiation, and maturation processes for 4:30 minutes, the cell morphology, viability, purity, lymphocyte contamination rate, and phenotype of IFN-DCs prepared using DCO-K medium supplemented with various HPL concentrations (0-10 (v / v)%) were compared (n=3).

[0102] Figure 22 shows the protocol for preliminary test 4. In preliminary study 4, the viability, yield, lymphocyte fraction contamination rate, and phenotypic changes were evaluated when IFN-DCs were prepared using DCO-K medium supplemented with HPL at various concentrations (0(v / v)%, 1(v / v)%, 5(v / v)%, 10(v / v)%) from the monocyte isolation process to differentiation and maturation. The results are shown in Figure 23. A represents the viability, B represents the yield, and C represents the lymphocyte fraction contamination rate. Compared to IFN-DCs prepared with DCO-K alone (a), the viability and yield were highest when using 5(v / v)% HPL (c) (n=3).

[0103] The phenotypes of IFN-DCs prepared at various concentrations (0(v / v)%, 1(v / v)%, 5(v / v)%, and 10(v / v)%) were evaluated by flow cytometry (n=3). The results are shown in Figure 24.

[0104] Compared to DCO-K medium alone (a), the expression of CD14 and CD56 increased in an HPL concentration-dependent manner, while the expression of CD80 and CD83 decreased but recovered in an HPL concentration-dependent manner. Furthermore, when evaluated with a dot plot, the expression of CD86 increased in an HPL concentration-dependent manner. + HLA - ABC + DR+ A convergence of a uniform cell population was observed.

[0105] The expression of cell surface antigens in IFN-DCs cultured at HPL 1-10 (v / v)% was evaluated by flow cytometry (n=1). The results for culture at HPL 10 (v / v)% are shown in Figure 25. A convergence of CD80 / CD86 and HLA-ABC / HLA-DR cell populations was observed in an HPL concentration-dependent manner.

[0106] A summary of the preliminary examination 4 is provided below. IFN-DCs were prepared using DCO-K medium supplemented with HPL at various concentrations (1(v / v)%, 5(v / v)%, and 10(v / v)%) from monocyte isolation to differentiation and maturation. The viability, yield, purity, and phenotype were evaluated by flow cytometry. In IFN-DCs prepared with HPL concentrations of 1-10(v / v)%, a concentration-dependent recovery of CD80 / CD86 expression levels and convergence of HLA-ABC / HLA-DR cell populations were observed. Furthermore, IFN-DCs prepared with HPL at a concentration of 5(v / v)% showed the highest viability and yield. Based on the results of preliminary study 4, it is suggested that a concentration of 5(v / v)% HPL is optimal for the preparation of HPL-IFN-DCs, considering manufacturing cost, viability, yield, and purity.

[0107] Preliminary Examination 5 Preliminary study 5: We compared the changes in phenotype, viability, yield, and purity when reagents (HPL, OK432, Cytokines) were added during the maturation process when IFN-DCs were prepared using HPL (n=1).

[0108] Figure 26 shows the protocol for preliminary test 5. In preliminary study 5, the necessity of each reagent in the maturation medium during the HPL-IFN-DC synthesis process was evaluated (n=1). During the maturation process of HPL-IFN-DCs, maturation cocktails (maturation mediums) ((a)-(d)) of each composition were used. Figure 27-1 shows the composition (B) of the maturation cocktails ((a)-(d)) used and microscopic images of the IFN-DCs (A). As shown in Figure 27-1, GM-CSF, IFN-α2b, and PGE2 were used as cytokines added to the maturation cocktails.

[0109] In the maturation process of HPL-IFN-DCs, dendrites were observed in all cases when using maturation cocktails of each composition ((a) to (d)), and no clear changes in cell morphology were observed.

[0110] The lymphocyte contamination rate during IFN-DC recovery was evaluated by flow cytometry. The results are shown in Figure 27-2. In preliminary study 5, HPL (5(v / v)%) was used in the monocyte adhesion separation step, so the lymphocyte contamination rate was less than 1% in all cases (n=1).

[0111] Figure 28 shows the viability rate (A), yield (B), and lymphocyte contamination rate (C) of IFN-DCs produced under each condition. Removing HPL, OK432, or cytokines during the maturation process resulted in lower viability rates and yields (n=1).

[0112] The phenotypic analysis of HPL-IFN-DCs prepared using mature culture medium was evaluated by flow cytometry (n=1).

[0113] Figure 29 shows the results of phenotypic analysis of IFN-DCs prepared under each condition. Compared to (a), the mature medium (b), which excluded HPL, tended to show lower expression of CD80, CCR7, CD40, and CD11c. Comparing (a) and (c), removing cytokines and OK432 from the mature medium resulted in a decrease in the expression of CD83, CD40, and CCR7, which are indicators of the antigen-presenting ability of DCs.

[0114] The results of preliminary study 5 showed that the presence or absence of HPL during maturation in the HPL-IFN-DC synthesis process affected the viability and yield of HPL-IFN-DCs. Furthermore, when OK432 and cytokines were removed from the maturation medium, a decrease in the expression of CD83 and CD40, which are involved in antigen presentation, and a decrease in the expression of CCR7, which is involved in lymphocyte induction, were observed, suggesting a decline in the functional aspects of HPL-IFN-DCs. Therefore, the addition of HPL, cytokines, and OK432 is essential in the HPL-IFN-DC synthesis process.

[0115] Preliminary Examination 6 One of the characteristics of IFN-DCs is their cytotoxic activity, which kills cancer cells. We investigated the cytotoxicity of IFN-DCs prepared using HPL-supplemented DCO-K medium (HPL-IFN-DCs). In addition, to evaluate whether the storage conditions of the starting material (PBMCs) of HPL-IFN-DCs affect cytotoxicity, we compared the cytotoxicity of HPL-IFN-DCs prepared from fresh PBMCs and those prepared from cryopreserved PBMCs.

[0116] Preliminary test 6: The chronic myeloid leukemia cell line K562 (ATCC, Mianassas, VA, USA) was added to PBS containing 0.1(v / v)% FBS with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE; 5 μM; Molecular Probes) in a 1 × 10⁻¹⁶ solution. 6 The cells were suspended in cells / mL and reacted at 37°C for 10 minutes, after which they were washed with AIM-V medium. Using AIM-V medium containing 10% FBS, 5 × 10 5HPL-IFN-DC (Effector, unstained) cells and CFSE-stained cancer cells (K562:Target) were mixed in an E:T ratio of 50:1, and the mixture was incubated at 37°C for 18 hours. After washing twice with FACS flow buffer, the cells were stained with 2 μg / mL propidium iodide (PI; Sigma-Aldrich Co. LLC., Tokyo, Japan) for 10 minutes to determine dead cells, and the results were analyzed using a flow cytometer. The percentage of PI-positive cells among CFSE-positive K562 cells, excluding naturally occurring dead cells, was evaluated as cytotoxicity (% cytotoxicity) (n=2).

[0117] Figure 30 shows the protocol for preliminary test 6. Previously, Koya et al. reported that IFN-DCs prepared using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) to purify monocytes from patient-derived PBMCs and then using serum-free medium (AIM-V) possessed cytotoxic activity (Koya et al. Scientific Report 7, Article number: 42145: 2017).

[0118] Therefore, we measured the cytotoxic activity of IFN-DCs prepared using serum-free medium supplemented with HPL (DCO-K). Furthermore, since IFN-DC cytotoxic activity may be lost through cryopreservation, we also evaluated cytotoxic activity with and without freezing (n=2).

[0119] Figures 31 and 32 show the results of cytotoxic activity measurements of HPL-IFN-DCs prepared using fresh or cryopreserved PBMCs. Figure 31 shows the results using sample #10, and Figure 32 shows the results using IFNDC-KMU-000 as the sample. A represents the control (k562), B represents the results using fresh PBMCs, and C represents the results using frozen PBMCs. In Figure 31, the percentage of fresh HPL-IFN-DCs was 4.2% and the percentage of frozen HPL-IFN-DCs was 3.8%, while in Figure 32, the percentage of fresh HPL-IFN-DCs was 1.6% and the percentage of frozen HPL-IFN-DCs was 1.8%.

[0120] HPL-IFN-DCs prepared using DCO-K medium supplemented with HPL exhibited equivalent cytotoxic activity regardless of whether they were frozen or not; no differences were observed.

[0121] Preliminary study 6 showed that HPL-IFN-DC exhibited lower cytotoxic activity, one of the characteristics of IFN-DCs. Furthermore, there was no difference in cytotoxic activity between HPL-IFN-DCs prepared from fresh and frozen PBMCs, indicating no effect from freezing of the raw materials.

[0122] Preliminary Examination 7 In preliminary test 7, CD8 in HPL-IFN-DC + We evaluated the T cell induction ability. IFN-DCs or HPL-IFN-DCs (both using AIM medium as the base medium) prepulsed with the cancer antigen MART-1 (Melanoma Antigen Recognized by T cell-1) produced from patients with HLA-A*02:01, and 1 × 10⁻¹⁴ 6Peripheral blood lymphocytes (PBLs) were mixed in a 1:10 ratio and cultured for 3 days in AIM-V medium supplemented with IL-2 (5 ng / mL), IL-7 (5 ng / mL), and IL-15 (10 ng / mL). Subsequently, AIM-V medium containing 10(v / v)% ABS was replenished according to cell proliferation, and IFN-DC or HPL-IFN-DC was added again on days 7 and 14 from the start of culture. Cells were harvested on day 21, and antigen presentation ability was evaluated from the induction of MART1-specific CD8 T cells. The harvested cells were stained with CD8-FITC, CD3-APC, T-select HLA-A*0201 MART-1 tetramer-ELAGIGILTV-PE, and MART1-specific CD8 T cells were evaluated using a flow cytometer. + T cells were detected (n=1).

[0123] Figure 33 shows the protocol for preliminary test 7. The cytotoxic T cell induction ability of HPL-IFN-DCs prepared using serum-free medium (AIM-V) was analyzed by flow cytometry (n=1). The results are shown in Figure 34. A shows the analysis results for CD8+ T cells, B for IFN-DCs, and C for HPL-IFN-DCs. Compared to IFN-DCs prepared using serum-free medium (AIM-V), HPL-IFN-DCs showed lower antigen-presenting ability when HPL was added (IFN-DC: 3.28%, HPL-IFN-DC: 1.55%). The percentages in the dot plot represent CD8+ T cells. + This shows the rate of MART-1-specific CTL induction in T cells.

[0124] In IFN-DCs prepared by adding 5% (v / v) HPL to AIM-V, MART1-specific CD8 + The T cell induction ability was low. This suggests that the difference in composition between AIM-V and DCO-K mediums affected the antigen-presenting ability of IFN-DCs.

[0125] Conclusion in the preliminary examination The validity of a novel IFN-DC synthesis method using monocytes was evaluated, and the process was finalized. The preparation method using serum-free medium (DCO-K) with added HPL was estimated to be an inventive and novel process based on the results regarding monocyte adhesion performance (purification of raw materials) and viability, yield, and purity (lymphocyte contamination rate) in the IFN-DC differentiation induction and maturation process.

[0126] The phenotype of the processed product, mature HPL-IFN-DC, is CD86. + HLA - ABC + DR + It exhibits a homogeneous cell population, and the CD14 and CD56 positivity rates increase upon addition of HPL. + , CD80 + , CD83 + Cellular expression showed a concentration-dependent pattern.

[0127] HPL was successfully used in DCO-K with 1-10 (v / v)% additive for monocyte-derived IFN-DC synthesis. In HPL-IFN-DCs, no increase in killer activity was observed despite CD56 expression. When IFN-DCs were prepared using HPL with previously evaluated AIM-V, their antigen-presenting ability was lower compared to AIM-V alone.

[0128] Based on the above, we determined that the optimal method for producing IFN-DCs for clinical application is a combination of serum-free medium (DCO-K) and 5(v / v)% HPL, which involves monocyte adhesion, differentiation induction, and maturation. In Example 2 (the main test) below, this manufacturing process was investigated.

[0129] [Example 2] Establishment of a monocyte isolation method and IFN-DC production method using serum-free medium (DCO-K) supplemented with HPL (5(v / v)%). This example was conducted as a main test. The preliminary test in Example 1 established a protocol for separating monocytes from patient-derived PBMCs (30 minutes) and producing IFN-DCs using serum-free medium (DCO-K) supplemented with HPL (5(v / v)%) during the differentiation and maturation process. Confirmed protocol: Peripheral blood mononuclear cells (PBMCs) derived from patients and collected by apheresis were seeded in adherent culture dishes using serum-free medium (DCO-K) prepared with a final concentration of 5(v / v)% HPL. Cells were cultured at 37°C and 5% CO2 for 30 minutes to adhere to the bottom of the culture dish, and monocytes and lymphocytes were separated. Subsequently, the adherent cells were induced to differentiate into IFN-DCs using DCO-K medium supplemented with 1 μg / mL PEG-Intron, 100 ng / mL GM-CSF, and HPL. Three days after the start of differentiation, the cells were harvested and matured into IFN-DCs by culturing them in low-adhesion culture dishes for 18-24 hours using maturation medium mixed with various reagents (10 μg / mL OK432, 10 ng / mL PGE2) and 20 μg / mL tumor antigen peptide (WT-1: Wilms tumor 1). The protocol is shown in Figure 35. This study was conducted using HPL-IFN-DC prepared using this established protocol (n=6).

[0130] Main Exam 1 In this study, we compared the cell viability, recovery rate, and purity of HPL-IFN-DC and IFN-DC (n=6). Peripheral blood mononuclear cells collected from patients by apheresis were suspended in DCO-K medium supplemented with HPL (5(v / v)%) and seeded in adherent culture dishes. The cells were cultured at 37°C and 5% CO2 for 30 minutes, and monocytes were separated by washing non-adherent cells. Adherent cells were induced to differentiate into IFN-DCs by adding differentiation induction medium supplemented with PEG-Intron and GM-CSF. Three days after differentiation, the cells were harvested and matured by suspending them in maturation medium supplemented with various reagents (PEG-Intron, GM-CSF, PGE2, OK432) and simultaneously seeding them in low-adherence culture dishes. After 24 hours, the cells were harvested and their morphology was observed using a phase-contrast microscope. Figure 36-1 shows the observed images. A shows the image of IFN-DCs, and B shows the image of HPL-IFN-DCs. The presence of dendrites suggests differentiation into DCs. No changes in cell morphology were observed with or without HPL.

[0131] Furthermore, the viability, yield, and purity of IFN-DCs recovered after maturation were compared. The results are shown in Figure 36-2. A represents viability, B represents yield, and C represents purity. A significant increase was observed in IFN-DCs prepared with HPL (HPL-IFN-DC) (viability: IFN-DC, 84.2%; HPL-IFN-DC, 95.5%; yield: IFN-DC, 14.1%; HPL-IFN-DC, 25.4%; purity: IFN-DC, 83.1%; HPL-IFN-DC, 99.1%). From the results of this study 1, it became clear that IFN-DCs prepared with HPL (5(v / v)%) showed high values ​​for viability, yield, and purity.

[0132] Main Exam 2 In Study 2, the effect of HPL on the phenotype of IFN-DCs was analyzed by flow cytometry (n=6). The results are shown in Figure 37. Compared with IFN-DCs, HPL-IFN-DCs prepared with HPL showed a significant increase in the expression of CD14 (a monocyte marker), CD56 (a cell adhesion molecule), CCR7 (CD197), which promotes migration to lymph nodes, and CD11c, one of the dendritic cell markers. In addition, a significant decrease in the expression of CD80 and CD40, costimulatory molecules involved in antigen presentation to T cells, CD83 (a dendritic cell maturation marker), and HLA-DR, which is involved in antigen presentation, was observed.

[0133] Main Exam 3 In Study 3, the antigen phagocytic activity and decomposition ability of HPL-IFN-DCs and IFN-DCs were evaluated by flow cytometry using FITC-dextran and DQ-ovalbumin. During the maturation process, 100 μg / mL FITC-Dextran (Molecular Probes, Eugene, OR, USA) and 10 μg / mL DQ ovalbumin (Molecular Probes) were added to the maturation medium, and the cells were incubated for 24 hours. Subsequently, the recovered IFN-DCs or HPL-IFN-DCs were washed twice with PBS and resuspended in 1(v / v)% FBS-PBS, and their phagocytic activity and decomposition ability were evaluated by flow cytometry (n=6). The protocol is shown in Figure 38.

[0134] The antigen phagocytic activity and antigen resolution of IFN-DCs and HPL-IFN-DCs were investigated by flow cytometry using FITC-dextran and DQ-Ovalbumin (n=6). The results are shown in Figure 39. The uptake of FITC-dextran and the resolution of DQ-OVA were examined, and the antigen phagocytic activity and antigen resolution are shown in the ΔMFI dot plot. A shows the results for FITC-dextran, and B shows the results for DQ-Ovalbumin. Compared to IFN-DC, IFN-DC with added HPL showed higher antigen phagocytosis and decomposition capabilities (FITC-dextran ΔMFI: IFN-DC, 17.1; HPL-IFN-DC, 68.0; DQ-Ovalbumin ΔMFI: IFN-DC, 270.9; HPL-IFN-DC, 589.7).

[0135] Main Exam 4 In this study (Study 4), we evaluated the ability of IFN-DCs and HPL-IFN-DCs to produce various cytokines. Mature HPL-IFN-DCs prepared using the confirmed IFN-DC protocol were processed in 1 × 10⁻¹⁶ units. 6 Cells were suspended in DCO-K medium to a cell density of cells / mL and seeded in a culture dish. After 24 hours of incubation at 37°C and 5% CO2, the culture supernatant was collected. Various cytokines (IL-6, IL-10, IL-12 (p70), IFN-γ, TNF-α) were measured in the collected culture supernatant using the Bio-plex assay kit (Bio-Rad Labs). TGF-β was also measured using the Human TGF-beta 1 Quantikine ELISA Kit (R & D systems) (n=6). The protocol is shown in Figure 40.

[0136] Next, cytokines involved in the induction of cytotoxic T cells secreted from HPL-IFN-DCs (IL-10, TGF-β, IFN-γ, TNF-α, IL-12(p70), IL-6) were measured using the Bio-plex assay kit (Bio-Rad Labs) (n=6). The results are shown in Figure 41. Compared to IFN-DCs, IL-12 (p70), a Th1 cytokine that enhances the induction of cytotoxic T cells, was significantly lower in HPL-IFN-DCs (IL-12 production: IFN-DC, 1.1 pg / mL; HPL-IFN-DC, 0.18 pg / mL), while no change was observed in IFN-γ, which has a similar effect (IFN-γ production: IFN-DC, 0.59 pg / mL; HPL-IFN-DC, 0.38 pg / mL). Conversely, Th2 cytokines IL-10 and TGF-β, which suppress the induction of cytotoxic T cells, showed an increasing trend in HPL-IFN-DC (IL-10 production: IFN-DC, 11.47 pg / mL; HPL-IFN-DC, 132.7 pg / mL; TGF-β production: IFN-DC, 8.02 pg / mL, HPL-IFN-DC, 9.38 pg / mL). The secretion of TNF-α and IL-6, which induce inflammatory responses and are involved in T cell activation and differentiation, was significantly increased in HPL-IFN-DC (IL-6 production: IFN-DC, 302.3 pg / mL; HPL-IFN-DC, 2883 pg / mL; TNF-α: IFN-DC, 412.5 pg / mL; HPL-IFN-DC, 1144.4 pg / mL). It has been revealed that HPL alters the Th1 / Th2 cytokines produced from IFN-DCs.

[0137] Main Exam 5 In this study 5, MART1-specific CD8 was observed in IFN-DC and HPL-IFN-DC. + The T cell induction ability was evaluated. The protocol is shown in Figure 42. The ability of HPL-IFN-DCs prepared using DCO-K medium supplemented with HPL to induce cytotoxic T cells was evaluated (n=6).

[0138] CD8-positive T cells were co-cultured with IFN-DCs and HPL-IFN-DCs pre-pulsed with MART1 (Melanoma Antigen Recognized by T cell-1) peptide, and MART1-specific cytotoxic T cells were detected by flow cytometry at days 14 and 21. Figure 43-1 shows the flow cytometry analysis results, and Figure 43-2 shows the MART1-specific CD8 cells in each treatment group. + The number of T cells is shown in Figure 43-3, and MART1-specific CD8 + This shows the percentage of T cells (MART-CTLs, MART1-specific CTL-positive cells). A significant increase in MART1-specific cytotoxic T cell induction was observed in HPL-IFN-DCs compared to IFN-DCs at days 14 and 21 (The median of the positive cell number of MART-1 tetramer+ CTLs at Day 14: CD8). + T cells, 1.37 × 10 3 cells; CD8 + T cells + IFN-DC, 2.45 × 10⁻⁶ 4 cells; CD8 + T cells + HPL IFN-DC, 2.25×10 5 cells; The median of the positive cell number of MART-1 tetramer+ CTLs at Day21: CD8 + T cells, 3.64 × 10 3 cells; CD8 + T cells + IFN-DC, 2.54 × 10⁻⁶ 5 cells; CD8 + T cells + HPL IFN-DC, 1.45×10 6 cells; n = 6).

[0139] We compared the cytotoxic T cell induction ability of IFN-DCs and HPL-IFN-DCs using single-group significance tests (comparison was performed only on Day 14 and Day 21).

[0140] Here, we present dot plot graphs for five cases (Case 2, Case 3, Case 4, Case 5, and Case 6) as Figure 44 (A: Case 2, B: Case 3), Figure 45 (A: Case 4, B: Case 5), and Figure 46 (Case 6) (refer to the above for graph format).

[0141] Main Exam 6 The antigen-specific IFN-γ production ability of cytotoxic T cells induced by IFN-DCs and HPL-IFN-DCs was evaluated using the Elispot assay (n=6). The protocol is shown in Figure 47. Figure 48-1 shows the spot image, and Figure 48-2 shows the amount of IFN-γ secreted (produced). Compared with IFN-DCs, HPL-IFN-DCs showed a significant increase in antigen-specific IFN-γ secretion from cytotoxic T cells.

[0142] Summary of the test results Figures 49-51 summarize the detailed numerical results of tests 1-6. As shown in Figure 49, HPL-IFN-DCs exhibited superior viability, recovery rate, and purity. Furthermore, as shown in Figure 50, HPL-IFN-DCs possess characteristics not found in conventional dendritic cells (DCs). Figure 51 shows the results of the functional evaluation of DCs. In the functional evaluation of HPL-IFN-DCs, it was found that they had higher antigen phagocytic ability, cytokine production ability, and cytotoxic T cell induction ability compared to IFN-DCs.

[0143] The results of this study showed that IFN-DCs prepared using serum-free medium (DCO-K) supplemented with 5(v / v)% HPL exhibited improved monocyte separation during the manufacturing process, as well as improved viability, yield, and purity of the final product. Furthermore, evaluation of the functional aspects of dendritic cells, including antigen presentation ability, phagocytic ability, and decomposition ability, led to the conclusion that this is an inventive and novel method for preparing IFN-DCs.

[0144] From the phenotypic results of HPL-IFN-DC, CD14 + CD56 + CD86 +, CCR7 + HLA-ABC / DR + It shows a homogeneous cell population, CD56 + , CD80 + , CD83 + The proportion of cells showed HPL concentration-dependent increases in expression and possessed novel traits that do not fit into the currently reported DC fractions.

[0145] Furthermore, the proportions of CD80 and CD40, which are costimulatory molecules involved in antigen presentation ability to T cells, and CD83, a maturation marker for dendritic cells, were decreased in HPL-IFN-DCs. Despite decreased secretion of IL-12 (p70), one of the Th1 cytokines that induce cytotoxic T cells, and increased secretion of IL-10, an inhibitory Th2 cytokine, the antigen presentation ability was high, indicating a remarkable ability to induce cytotoxic T cells.

[0146] The method for producing IFN-DCs using serum-free medium supplemented with HPL (DCO-K) showed improved viability, recovery rate, and purity compared to the non-supplemented method. Because it exhibited superior antigen-presenting, decomposition, and phagocytic abilities, it is expected to be a novel DC vaccine beneficial for cancer immunity and infection control. [Example 3] WT1 peptide pulsed IFN dendritic cell vaccine Manufacturing of IFN-DOC using HPL Peripheral blood mononuclear cells (PBMCs) were suspended in culture medium and seeded in dishes. After 30 minutes, non-adherent cells were removed by washing, and differentiation was induced from adherent monocytes using GM-CSF and IFN-α. On Day 4, OK-432, PGE2, and peptides were added, and cells were harvested after 18-24 hours. The protocol is shown in Figure 52. On Day 5, significant cluster formation, characteristic of dendritic cells, was observed. Selective adhesion culture of monocytes in the generation of IFN dendritic cells using HPL. The AIM-V medium used in the conventional method was a research-grade reagent and was not manufactured under controlled conditions for clinical use. Therefore, culture was performed using GMP-grade DCO-K medium (serum-free medium, known components). When seeding peripheral blood mononuclear cells, it is possible to selectively adhere monocytes by using HPL compared to adhering monocytes to DCO-K medium alone. The culture state of dendritic cells is shown in Figure 53. Figure 53A shows IFN-DCs prepared without HPL, and Figure 53B shows IFN-DCs prepared with HPL (HPL-IFN-DCs). Flow cytometry is shown in Figure 54. From the flow cytometry images, the contamination of lymphocyte fractions was significantly reduced in IFN-DCs prepared with HPL (HPL-IFN-DCs) (Figure 54A) compared to IFN-DCs prepared without HPL (Figure 54B) (IFN-DCs, 22.1%; HPL-IFN-DCs, 0.88%). Phenotypic analysis of HPL-IFN-DC Monocytes were selectively adhered using HPL, differentiated using GM-CSF and IFN-α, and matured with picibanil or PGE2. The phenotype was then observed using a flow cytometer. The results are shown in Figure 55. Expression of the cell surface markers CD11c, CD40, CD56, CD80, CD83, CD86, HLA-ABC, and HLA-DR, which have been reported in IFN-dendritic cells, was confirmed. Induction of MART-1 antigen-specific cytotoxic T cells by IFN-DCs or HPL-IFN-DCs In vitro CTL induction studies were conducted by co-culturing IFN-DCs or HPL-IFN-DCs incorporating the MART-1 26-35 A27L peptide with CD8+ T cells. MART-1-specific cytotoxicity T lymphocytes (CTLs) were detected 21 days after the start of culture. The results are shown in Figure 56. Compared with IFN-DCs (Figure 56A), HPL-IFN-DCs (Figure 56B) showed a higher induction of MART-1-specific CTLs (IFN-DC, 0.69%; HPL-IFN-DC, 5.47%). Comparison of WT1-induced CTLs using IL-4-DC or HPL-IFN-DC with added WT1. In vitro CTL induction studies were performed on dendritic cells (DCs) and CD8+ T cells incorporating the WT1 antigen. Cells were harvested 21 days after the start of culture, and the percentage of WT1-CTL induction induced by WT1-tetramer analysis was evaluated. The protocol for the WT1-CTL induction study is shown in Figure 57. The methods for preparing IL-4-DCs and HPL-IFN-DCs used in the WT1-CTL induction study are shown in Figure 58. IL-4-DCs harvested on Day 7 were treated with WT1-235 killer peptide at 100 μg / ml at 4°C for 30 minutes before being used in the study (WT1 peptide post-pulse). HPL-IFN-DCs were prepared by adding WT1-235 killer peptide to the maturation cocktail on Day 4, and HPL-IFN-DCs harvested on Day 5 were used in the study (WT peptide pre-pulse). The results of the evaluation of the percentage of WT1-CTL induction induced by WT1-tetramer analysis are shown in Figure 59. Compared to existing IL-4-DCs, HPL-IFN-DCs showed superior WT-CTL induction ability. Figure 60 shows the total number of WT1-CTLs induced by IL-4-DC (WT1 post-pulse) or HPL-IFN-DC (WT1 pre-pulse). After stimulating CD8+ T cells three times with each DC (up to Day 21), an increase in WT1-CTLs was observed. Higher induction was confirmed with HPL-IFN-DC compared to IL-4-DC. CD8+ T cells only were used as a negative control without stimulation by each DC. [Industrial applicability]

[0147] Dendritic cells (DCs) prepared by the method of the present invention can be used in dendritic cell therapy. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A method for preparing cytotoxic dendritic cells from monocytes, comprising culturing monocytes isolated from peripheral blood by adherent culture in serum-free medium containing human platelet lysate (HPL), GM-CSF, and PEGylated interferon α, and then adding prostaglandin E2 and OK432 and further culturing in a low-adhesion culture dish.

2. A method for preparing dendritic cells from monocytes according to claim 1, comprising culturing for 2 to 5 days by adherent culture in a serum-free medium containing human platelet lysate (HPL), GM-CSF, and PEGylated interferon α, followed by the addition of prostaglandin E2 and OK432 and further culturing for 1 to 2 days in a low-adhesion culture dish.

3. A method for preparing dendritic cells from monocytes according to claim 1 or 2, comprising culturing monocytes in a serum-free medium containing 1-10 (v / v)% human platelet lysate (HPL), 100 U / mL to 10,000 U / mL of GM-CSF, 500 ng / mL to 5 μg / mL of PEGylated interferon α, 5 ng / mL to 50 ng / mL of prostaglandin E2, and 5 μg / mL to 50 μg / mL of OK432.

4. A method for preparing dendritic cells from monocytes according to any one of claims 1 to 3, wherein the viability of the obtained dendritic cells is 90% or more, and the yield, which is the ratio of the number of obtained dendritic cells to the number of monocytes during culture, is 15% or more.

5. A method for preparing dendritic cells from monocytes according to any one of claims 1 to 4, wherein the dendritic cells obtained are positive for CD14, CD16, CD56, CD83, CD86, CCR7 (CD197), HLA-ABC, and HLA-DR.

6. Furthermore, the method according to any one of claims 1 to 5, wherein a cancer-specific antigen is added to prepare dendritic cells having cancer antigen-specific dendritic cell-toxicity.

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