WO2017200017A1 - Procédé de préparation cellulaire et récipient de culture cellulaire - Google Patents

Procédé de préparation cellulaire et récipient de culture cellulaire Download PDF

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WO2017200017A1
WO2017200017A1 PCT/JP2017/018575 JP2017018575W WO2017200017A1 WO 2017200017 A1 WO2017200017 A1 WO 2017200017A1 JP 2017018575 W JP2017018575 W JP 2017018575W WO 2017200017 A1 WO2017200017 A1 WO 2017200017A1
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cells
antigen
cell
culture
specific
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PCT/JP2017/018575
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English (en)
Japanese (ja)
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郷史 田中
亮 末永
貴彦 戸谷
棟梁 李
真悟 田路
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東洋製罐グループホールディングス株式会社
株式会社医学生物学研究所
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Publication of WO2017200017A1 publication Critical patent/WO2017200017A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention particularly relates to a cell preparation method capable of efficiently preparing immune cells carrying a specific immune response against viruses, bacteria, cancer, and other immune diseases, and a cell culture container used therefor About.
  • immune cell therapy using autologous or allogeneic allogeneic antigen-specific immune cells and antigen-specific genetically modified immune cells is used for opportunistic infections after hematopoietic cell transplantation, lymphoma, melanoma, nasopharyngeal cancer, blood cancer Effective clinical data for diseases such as autoimmunity is being accumulated.
  • Antigen-specific immune cell therapy has used TIL therapy using tumor infiltrating cells, CTL therapy using cytotoxic T cells, and Perper T cells as the fastest-growing medical technology for the past 20 years.
  • HTL therapy, CAR-T using genetically modified immune cells, CAR-NK therapy, TCR-T therapy, etc. have been developed and are being clinically tested.
  • antigen-specific immune cell therapy is efficient in vitro only for immune cells carrying specific immune responses. Inducing or transforming and growing in sufficient quantities.
  • CTL therapy and HTL therapy antigen-specific cytotoxic T cells (CTL) and helper T cells (HTL) are induced and expanded from peripheral blood mononuclear cells collected from patients. Infused into patients. In many clinical trials using it, the number of cells to be transfused into a patient is ideally 1 ⁇ 10 7 or more. For this reason, for example, when the abundance ratio of antigen-specific immune cells is 10%, the total number of cells at the end of the culture needs to be at least 1 ⁇ 10 8 (several hundred million). In order to reduce the burden on blood collection subjects, it is required to induce and proliferate more antigen-specific immune cells in a single operation.
  • CTL cytotoxic T cells
  • HTL helper T cells
  • CTLs cytotoxic T cells
  • HTLs helper T cells
  • the existence frequency of these antigen-specific immune cells is observed to be biased by individuals, antigen types, and alleles.
  • the viral antigen HCMV which is one of therapeutic targets in opportunistic infections after hematopoietic stem cell transplantation, is exemplified.
  • the number of HLA-A24-restricted HCMV-specific CTLs common in Asians is about one-hundred compared to that of HLA-A2-restricted HCMV-specific CTLs common in Westerners. This is considered to be one of the reasons that induction and proliferation of HLA-A24-restricted HCMV-specific CTLs in vitro are difficult. For this reason, there have been few reports on in vitro preparation methods for clinical applications of specific CTLs other than HLA-A2 restricted.
  • antigen-specific immune cells In order to induce antigen-specific immune cells, for example, antigen-specific CTL requires preparation of antigen-presenting cells. Dendritic cells are often used as antigen-presenting cells, but the preparation of dendritic cells is complicated and expensive. (2) When the induced antigen-specific CTL is further expanded, antigen-presenting cells are used (see Non-Patent Document 1). Therefore, the same problem as the above (1) occurs. In addition, in order to prepare antigen-presenting cells for use in proliferation, it is necessary to collect blood from the donor again.
  • the burden on the donor is large.
  • nonspecific stimulation with OKT3 or lectin may be used.
  • this method lacks efficiency because cells other than the antigen-specific CTL are relatively likely to proliferate.
  • the REM method is known as a relatively efficient growth method. Since the REM method requires a large amount of peripheral blood mononuclear cells and EBV-LCL, there is a serious problem such as virus contamination, so it is not a practical method for commercialization.
  • peripheral blood mononuclear without preparing antigen-presenting cells in order to realize practical use of cell therapy using antigen-specific immune cells, particularly antigen-specific CTLs.
  • a preparation method has been proposed in which antigen-specific immune cells can be induced from peripheral blood mononuclear cells quickly and easily by co-culturing spheres with antigen-specific peptides and stimulating substances.
  • Non-Patent Document 2 since the antigen-specific immune cell culture methods that have been performed so far involve complicated steps, a 24-well flat plate (see Non-Patent Document 2), a flat flask (see Non-Patent Document 3), agitation culture There is no choice but to rely on open culture vessels such as tanks (see Non-Patent Document 4).
  • EBV-LCL EBV-LCL cells are pulsed with an antigen peptide and then inactivated by X-ray (40 Gy) irradiation. Thereafter, the collected 1 ⁇ 10 6 peripheral blood mononuclear cells are mixed at a ratio of 40: 1 and seeded on a 24-well flat plate. At that time, a maximum of 2 mL of medium is added per well.
  • a culture method using EBV-LCL is given as an example of a relatively efficient preparation method. Specifically, in this culture method, EBV-LCL cells are pulsed with an antigen peptide and then inactivated by X-ray (40 Gy) irradiation. Thereafter, the collected 1 ⁇ 10 6 peripheral blood mononuclear cells are mixed at a ratio of 40: 1 and seeded on a 24-well flat plate. At that time, a maximum of 2 mL of medium is added per well.
  • the cells need to be centrifuged many times, resuspended in fresh medium and seede
  • the proportion of antigen-specific immune cells in the total cells changes dynamically compared to the early phase of induction, so essential nutrients and cytokines are timed with appropriate amounts. You have to control Lee.
  • culture in an open system requires frequent medium exchange work and must be performed in a cell processing facility to prevent contamination by bacteria and viruses from the outside. Cost.
  • the number of cells finally obtained with a 24-well flat plate is 1 ⁇ 10 7 or less. Without scaling up with multiple plates, a sufficient number of cells for treatment cannot be obtained. Furthermore, culture vessels such as flat plates and flat flasks have a limited volume and are very unsuitable for large-scale cell preparation.
  • the film material of the flat culture bag is very soft, and it can be stored in the culture container when it is placed at an angle, or when the film is left standing, the film is distorted, and the surrounding environment and slight vibration derived from the incubator
  • the cells inside are concentrated near a part of the container or the periphery of the container, causing damage due to physical collision between the cells and uneven cell density in the liquid phase, which causes cell growth to decrease. ing.
  • the present invention has been made in view of the above circumstances, and in order to realize the practical application of antigen-specific immune cell therapy, it has both convenience and safety, and at low cost and in large quantities efficiently and efficiently. It is an object of the present invention to provide a cell preparation method capable of suitably preparing a cell culture vessel and a cell culture vessel used therefor.
  • the cell preparation method according to the present invention is a cell preparation method for preparing antigen-specific immune cells by co-culture of peripheral blood mononuclear cells and an antigen-specific peptide or a transforming virus, comprising:
  • a target cell is prepared by precipitating cells in the concave portion using a plurality of concave portions provided on one surface of the inner wall of the container.
  • the cell culture container according to the present invention is a cell culture container used in the above-described cell preparation method, and includes a container main body made of a gas permeable plastic film and an injection port 3, and the container main body Has a bulging shape with a sealed periphery, and a plurality of bowl-shaped recesses having an opening diameter of 1.5 mm or more serving as a cell culture part are provided on the bottom surface of the container body.
  • a target cell can be prepared by a simple operation without requiring a complicated operation.
  • the time required for preparation becomes very short.
  • all the culture steps can be performed in a closed system. This provides an improvement in safety. By ensuring a high level of safety, the level of necessary equipment can be reduced and the risk of contamination can be reduced.
  • a cell culture container 1 shown in FIG. 1 includes a container body 2 made of a gas permeable plastic film and an injection port 3 made of a tubular member through which a medium, cells, and the like can flow.
  • the container body 2 has a bulging shape in which the peripheral part is sealed and swelled in a pillow shape, and an opening diameter r serving as a cell culture part is 1.5 mm or more on the bottom surface 2b of the container body 2.
  • a plurality of bowl-shaped recesses 4 are provided.
  • the top surface 2a of the container body 2 is a flat surface.
  • the bowl-shaped recess 4 formed on the bottom surface 2b of the container body 2 is preferably formed in a U-shaped cross section so that cells can easily gather at the bottom, but is not limited thereto.
  • a diameter-reduced structure in which the area of the bottom is smaller than the opening area so that cells can easily gather at the bottom may be used.
  • a semicircular section, a V-shaped section It may be in the shape of an inverted cone.
  • the depth d of the bowl-shaped recess 4 is The ratio d / r of the depth d to the diameter r is preferably 0.05 to 1.
  • the arrangement of the bowl-shaped recesses 4 is preferably staggered as shown in the figure so that the occupied area of the bowl-shaped recesses 4 occupying the bottom surface 2b is as large as possible. May be.
  • the size of the container body 2 is not particularly limited, but is preferably, for example, 50 to 500 mm in length and 50 to 500 mm in width.
  • Such a cell culture container 1 can be manufactured as follows, for example. First, a top surface side plastic film to be the top surface 2 a of the container body 2 and a bottom surface side plastic film to be the bottom surface 2 b of the container body 2 are prepared. The bottom side plastic film is molded so as to swell while leaving its peripheral portion, and the bowl-shaped recess 4 is formed in the swelled portion. These can be formed by general vacuum forming, pressure forming, or the like. By appropriately adjusting the mold or the like, the bulging shape and the shape of the bowl-shaped concave portion 4 are formed to a desired shape. can do.
  • the bottom side plastic film and the top side plastic film molded as described above are overlapped, and the peripheral part is heat-fused with a tubular member that forms the injection port 3 at a predetermined position. Seal by wearing and trim the periphery if necessary. Thereby, the cell culture container 1 as shown in FIG. 1 can be manufactured.
  • the gas permeability of the plastic film forming the container body 2 is determined according to the gas permeability test method of JIS K 7126.
  • the oxygen permeability measured at a test temperature of 37 ° C. is 3000 ml / (m 2 ⁇ day ⁇ atm. ) Or more, more preferably 6000 ml / (m 2 ⁇ day ⁇ atm) or more.
  • it is preferable that a part or all of the plastic film has transparency.
  • the material used for the plastic film forming the container body 2 is not particularly limited as long as it has a desired gas permeability.
  • examples thereof include thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomer, polystyrene elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP). Silicone can also be used. These may be used as a single layer, or may be used by laminating the same or different materials, but has a layer that functions as a sealant layer in consideration of heat fusion when sealing the periphery. Is preferred.
  • the thickness of the plastic film used to form the container body 2 is 30 to 200 ⁇ m so that the container body 2 has an appropriate shape retaining property that does not crush the bowl-shaped recess 4 while having flexibility. Is preferred.
  • the injection port 3 is composed of a tubular member through which a culture medium, cells, and the like can circulate.
  • the tubular member forming the injection port 3 is molded into a predetermined shape by injection molding, extrusion molding, or the like using, for example, a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, or FEP.
  • a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, or FEP.
  • the specific form of the injection port 3 is not particularly limited.
  • a port having an injection port made of an elastic body such as rubber may be used, or a luer lock type port may be used so that injection with an injection needle is possible.
  • the injection port 3 is used for injecting cells such as peripheral blood mononuclear cells, and also for extracting target cells such as prepared antigen-specific immune cells.
  • one injection port 3 is provided, but a plurality of ports may be provided to cope with an operation error.
  • contamination can be prevented and identification can be made by differentiating the color, shape, etc. of the cap so that an operation error can be prevented more reliably. .
  • the cell culture vessel 1 In order to prepare a target cell by inducing or transforming antigen-specific immune cells from peripheral blood mononuclear cells with such a cell culture vessel 1, for example, the cell culture vessel 1 is connected to the injection port 3.
  • a peripheral blood mononuclear cell or other cell isolated from the patient's peripheral blood while maintaining a closed system via a liquid delivery tube, an antigen-specific peptide prepared according to the patient's HLA type, a stimulant, and A predetermined medium or the like is injected into the container body 2 at a predetermined mixing ratio.
  • Peripheral blood mononuclear cells and antigen-specific peptides injected into the container body 2 settle in the medium and are divided into the respective saddle-shaped recesses 4, gather at the bottom of each, and are inhibited from moving in the container. .
  • peripheral blood mononuclear cells and antigen-specific peptides can be co-cultured in a state in which the cells are in close contact with each other, and effective antigen presentation can be performed against a very low frequency of antigen-specific immune cells. It is possible to achieve and efficiently induce antigen-specific immune cells.
  • each bowl-shaped recess 4 substantially the same shape, it is possible to obtain a substantially uniform cell density for each bowl-shaped recess 4. And when the cell culture vessel 1 is placed at an inclination or when it is left still, the cells gathered in the bowl-shaped recess 4 due to the distortion of the film, the slight vibration derived from the surrounding environment or the incubator, etc. Can form a certain agglomeration within each bowl-shaped recess 4 without moving to the other bowl-shaped recess 4. As a result, the cell density can be maintained in an efficiently inducible state, and the cells can be induced and proliferated without causing a decrease in proliferation rate due to crowding or a decrease in induction efficiency due to dispersion. As a result, homogeneous cell growth can be realized, which is effective in reducing dead cells and improving induction efficiency.
  • the top surface 2a of the container body 2 has a flat shape, and sinks into each bowl-shaped recess 4 by pressing down the top surface 2a and contacting the bottom of the bowl-shaped recess 4 using flexibility. Cells can be easily resuspended. Thereby, even if the cell density for each hook-shaped recess 4 is biased, the cells can be dispersed almost uniformly in each hook-shaped recess 4.
  • the contents can be easily discharged from the injection port 3 by inverting and tilting the container, so that a plastic container with a 24-hole flat plate is used.
  • a pipette By using a pipette, it is possible to omit the time-consuming work of injecting and collecting cells per well.
  • the cells can be filled and the proliferated cells can be recovered while maintaining the closed system, contamination such as microorganisms such as bacteria and mycoplasmas and viruses can be reduced. Therefore, it is possible to more suitably culture cells that are infused or embedded in the body, or are attached to the damaged part.
  • the container body 2 is made of a plastic film having gas permeability, a sufficient amount of oxygen can be supplied to cells in culture even when a closed system is constructed.
  • the cell culture container 1 of the present embodiment all the culture steps can be performed in a closed system. This provides an improvement in safety. By ensuring a high level of safety, the level of necessary equipment can be reduced and the risk of contamination can be reduced. Furthermore, since the container body 2 is formed using a plastic film, it is lightweight even if the capacity is increased and is not bulky. Therefore, the container body 2 is suitable for large-scale cell culture. A sufficient amount of medium can be injected.
  • the cells targeted by the present invention include cells that can induce antigen-specific immune cells by co-culture with antigen-specific peptides and stimulating substances, and cells that can be transformed into antigen-specific genetically modified immune cells by co-culture with a virus solution. It is. It is particularly suitable for peripheral blood mononuclear cells.
  • the culture medium and cells, and the antigen-specific peptide or the virus solution for transformation are injected into the cell culture container 1 from the injection port 3, and the cells that settle in the culture medium gather at the bottom of each bowl-shaped recess 4. Like that. At this time, the culture medium is injected to such an extent as to cover not only the bowl-shaped recess 4 but also the bottom surface 2 b of the container body 2.
  • a liquid medium is used, and it is appropriately selected according to the cell type.
  • Medium suitable for culturing antigen-specific immune cells for example, RPMI1640, AIM-V, Dulbecco's modified Eagle medium (DULBECCO'sdimodified eagle medium), X-VIVO (manufactured by Bio Whittaker), ALYS505N (Cell Science Laboratory)
  • cytokine-containing medium can be prepared by adding IL-2 or the like to KBM series lymphocyte culture medium (manufactured by Kojin Bio). Serum, plasma, serum albumin, antibiotics, L-glutamine and the like may also be added.
  • IL-2 it is preferable to use recombinant human IL-2 (for example, PROLEUKIN provided by Chiron and Teseleukin provided by Shionogi & Co.).
  • the content of IL-2 is, for example, 10 to 1,000 IU / mL.
  • Antigen-specific peptides include viruses (cytomegalovirus, Epstein-Barr virus, adenovirus, etc.), tumor antigens (WT1, hTERT, MN / CA9, gp100, MART1, TRP1, TRP2, tyrosinase, MAGE1, MAGE2, MAGE3, MAGE6 NY-ESO-1, MUM1, BAGE, GAGE1, GAGE2, CEA, PSA, etc.).
  • viruses cytomegalovirus, Epstein-Barr virus, adenovirus, etc.
  • tumor antigens WT1, hTERT, MN / CA9, gp100, MART1, TRP1, TRP2, tyrosinase, MAGE1, MAGE2, MAGE3, MAGE6 NY-ESO-1, MUM1, BAGE, GAGE1, GAGE2, CEA, PSA, etc.
  • WT1, hTERT tumor antigens
  • MN / CA9 gp100
  • retrovirus vectors including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai Viral vectors such as viral vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors can be used.
  • retrovirus vectors including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors
  • AAV adeno-associated virus vectors
  • simian virus vectors vaccinia virus vectors
  • Sendai Viral vectors such as viral vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors
  • ESV vectors Epstein-Barr virus
  • HSV vectors Sendai Viral vectors
  • those lacking replication ability are preferable so that they cannot self-replicate in infected cells.
  • the number of cells to be seeded in each hook-shaped recess 4 is preferably about 1 ⁇ 10 4 to 1 ⁇ 10 6 cells / mL although it depends on the size of the hook-shaped recess 4. This is because, when the cell density is less than 1 ⁇ 10 4 cells / mL, the probability that the cells meet each other is low, and the induction efficiency is low. On the other hand, if it exceeds 1 ⁇ 10 6 cells / mL, the rate of cell death increases, so the cell growth efficiency decreases.
  • the cell culture container 1 After elapse of a predetermined time (for example, 1 to 4 days), the cell culture container 1 is taken out from the incubator, and a growth medium is injected into the cell culture container 1 from the injection port 3. After returning to the incubator and culturing for a predetermined time (for example, 2 to 7 days), the growth medium is injected again from the injection port 3. Thereafter, the culture in the incubator is continued. After this, the cell recovery operation is started.
  • the interval from the injection of the growth medium to the next injection of the growth medium is, for example, 1 to 6 days.
  • the interval between the first injection operation and the second injection operation may be 3 days, and the interval between the second injection operation and the third injection operation may be 2 days. The intervals here are not necessarily unified.
  • the growth medium contains cytokines such as IL-2, IL-15, or both.
  • cytokines such as IL-2, IL-15, or both.
  • the content of IL-2 is, for example, 10 to 1,000 IU / mL.
  • the content of IL-15 is, for example, 10 to 100 ng / mL.
  • An antigen-specific peptide is added. By further adding an antigen-specific peptide, expression of the cell surface stimulating factor is promoted. Thereby, in the subsequent proliferation step, the proliferation rate of the induced antigen-specific immune cells is increased and can be proliferated with high efficiency.
  • the amount of the antigen-specific peptide in the additional medium is, for example, 0.01-100 ⁇ g / mL.
  • the anti-CD3 antibody to be used can be prepared by an immunological technique using a CD3 molecule or a part thereof.
  • a commercially available anti-CD3 antibody can also be used.
  • An example of a commercially available anti-CD3 antibody is OKT-3 antibody (manufactured by Janssen Pharma). From the viewpoint of safety, it is preferable to use an OKT-3 antibody that has received regulatory approval.
  • the anti-CD3 antibody may be a monoclonal antibody or a polyclonal antibody.
  • an anti-CD3 antibody that is a monoclonal antibody.
  • Two or more types of anti-CD3 antibodies can be used in combination.
  • the amount of anti-CD3 antibody in the medium is, for example, 0.01 to 10 ⁇ g / mL.
  • the anti-PD-1 antibody By adding anti-PD-1 antibody, immune checkpoint PD-1 molecule that inhibits the proliferation of antigen-specific immune cells can be blocked, and induced antigen-specific immune cells can be proliferated with high efficiency. it can.
  • the anti-PD-1 antibody to be used can be prepared by an immunological technique using the PD-1 molecule or a part thereof.
  • a commercially available anti-PD-1 antibody can also be used.
  • An example of a commercially available anti-PD-1 antibody is nivolumab (manufactured by Ono Pharmaceutical Co., Ltd.). It is preferable to use an anti-PD-1 antibody that has received regulatory approval from the viewpoint of safety.
  • the anti-PD-1 antibody may be a monoclonal antibody or a polyclonal antibody.
  • an anti-PD-1 antibody that is a monoclonal antibody.
  • Two or more types of anti-PD-1 antibodies can be used in combination.
  • the amount of anti-PD-1 antibody in the medium is, for example, 0.01 to 10 ⁇ g / mL.
  • immune checkpoint molecules that inhibit the proliferation of antigen-specific immune cells include PD-L1, CTLA-4, TIGHT, TIM-3, LAG-3, B7-H3, B7-H4, There are many BTLA, VISTA, and the like. Antibodies to these molecules can be added to the medium as well. Two or more types of anti-immune checkpoint molecule antibodies can be used in combination.
  • a small molecule inhibitor that inhibits the expression / metabolic pathway of immune checkpoint molecules and regulatory T cells (Treg) or improves the expression of HLA molecules is added.
  • substances that inhibit the expression of immune checkpoint molecules include low molecular weight compounds such as Nafamostat Methylate, which is a serine protease inhibitor that suppresses the expression of PD-L1.
  • the substance that suppresses the expression of Treg include, for example, a low molecular weight compound that inhibits only effect Tregs (CD4 + FOXP3highCD45RAlow) cells, or a low molecular compound that is an indoleamine 2,3-dioxygenase inhibitor that inhibits Treg-like cells.
  • Examples of substances that improve the expression of HLA molecules include Lapatinib Ditosylate, which is a MAPK inhibitor. It is preferable to add one or more of the low molecular weight compounds to the growth medium.
  • the amount of the low molecular compound in the medium is, for example, 1 pM to 1 ⁇ M.
  • TLR Toll-like receptor
  • RIG-I-like receptor A substance that activates immunity is added through an immune receptor such as Toll-like receptor (TLR) or RIG-I-like receptor.
  • TLR Toll-like receptor
  • RIG-I-like receptor examples of the immunostimulatory component include mushroom components having an immunostimulatory activity (extract and bulk powder), seaweed (mechab and mozuku) components (extract and bulk powder), and the like. These may use only 1 type and may use 2 or more types together.
  • Immuno Potentators immuno Potentators
  • lipoproteins TLR1 ligands
  • peptidoglycans TLR2 ligands
  • Poly (I: C) Polyinosinic-acidic acid sodium
  • examples include dsRNA (salt) typified by dsRNA (ligand of TLR3), lipopolysaccharide (Lipopolysaccharide; LPS) (ligand of TLR4), ssRNA (ligand of TLR7), Cp GDNA (ligand of TLR9), and the like.
  • TLR is expressed in the T cell itself and has the ability to directly control the function of the T cell.
  • CD8 positive T cells express TLR2 and directly enhance cell proliferation and cell viability by costimulation of antigen and TLR2 ligand, reducing the need for indirect costimulation derived from dendritic cells To help.
  • stimulation with TLR ligands (especially TLR3, TLR7, and TLR9) increases the ability of foreign antigens to “cross-present” by MHC class I, and allows antigen-specific CD8-positive T cells to be cytotoxic T cells ( CTL) can be effectively differentiated.
  • Step of collecting antigen-specific immune cells A small amount of cells are collected from the cell culture vessel 1 during cell culture, and the frequency and amount of antigen-specific immune cells are determined using the antigen-specific immune cell quantification method described below. Measure. Then, it moves to a collection process.
  • the injection port 3 of the cell culture vessel 1 is used. That is, the proliferated antigen-specific immune cells are transferred from the injection port 3. After transferring the cells, a cell washing step by centrifugation may be incorporated. Such a cleaning process may be repeated (for example, 2 to 4 times).
  • MHC-multimer method Using MHC-monomer and / or MHC-multimer (hereinafter also collectively referred to as MHC-multimer, etc.) produced using the same peptide as the antigen-specific peptide used for induction of antigen-specific immune cells,
  • the antigen-specific immune cells being prepared or prepared can be quantified.
  • the quantification can be performed, for example, as follows. A sample collected from the cell culture solution is reacted with an appropriate concentration of MHC-multimer or the like. Antigen-specific immune cells bound to the MHC-multimer and the like are stained with a labeled dye, and are counted using a flow cytometer, a microscope or the like.
  • Quantitative method 2 A method for quantifying cytokines and / or chemokines such as IFN ⁇ (interferon gamma), TNF ⁇ (tumor necrosis factor alpha), and interleukin produced by antigen-specific immune cells by stimulating the collected cells with an antigen-specific peptide. is there.
  • IFN ⁇ interferon gamma
  • TNF ⁇ tumor necrosis factor alpha
  • interleukin interleukin produced by antigen-specific immune cells by stimulating the collected cells with an antigen-specific peptide.
  • Method 1 by cytokine quantification (quantitative method for intracellular IFN ⁇ producing cells)
  • the collected cells are suspended in an appropriate medium at a cell concentration of about 2 ⁇ 10 6 / mL, and an antigen-specific peptide is added. Further, an intracellular protein transport inhibitor (for example, Brefeldin A, Monensin, etc.) is added, and the cells are cultured at 37 ° C. for 5 to 16 hours in a 5% CO 2 constant temperature bath.
  • an intracellular protein transport inhibitor for example, Brefeldin A, Monensin, etc.
  • T cell marker antibody anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody
  • MHC-multimer fix cells, perform membrane permeabilization, and react with dye-labeled anti-IFN ⁇ antibody
  • Method 3 by cytokine quantification (method of quantifying IFN ⁇ secreted into the culture supernatant)
  • the prepared cell sample is suspended in a suitable medium at a cell concentration of about 2 ⁇ 10 6 / mL, and an antigen-specific peptide is added. Incubate for 24-48 hours at 37 ° C. in a 5% CO 2 thermostat. After the culture, the IFN ⁇ concentration contained in the culture supernatant is quantified using a commercially available ELISA kit (for example, Quantikine ELISA Human IFN ⁇ Immunoassay manufactured by R & D Systems).
  • ⁇ Quantitative method 3 Quantification is performed using cell surface protein specific antibodies.
  • Antigen-specific immune cells that specifically recognize an antigen-specific peptide are stimulated by binding to the peptide, and expression of cell surface proteins (eg, CD137, CD107a, CD107b, CD63, CD69, etc.) may be enhanced. It has been reported. Therefore, by mixing a cell stimulated with an antigen-specific peptide or the like and a labeled antibody that specifically recognizes a cell surface protein, the antigen-specific immune cell binds to the labeled antibody and is stained with a labeled dye. Stained antigen-specific immune cells can be counted and quantified using a flow cytometer, a microscope or the like. Furthermore, by adding an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody or the like labeled with a dye different from the labeled antibody, a cell subset of antigen-specific immune cells can be determined simultaneously.
  • the cell culture container of the present invention is used for inducing and proliferating antigen-specific immune cells from peripheral blood mononuclear cells by co-culturing peripheral blood mononuclear cells and antigen-specific peptides. It can be suitably used as a cell culture container.
  • isolated peripheral blood mononuclear cells The amount of antigen-specific immune cells necessary for treatment can be prepared in about 14 days to 1 month.
  • the cell culture container of the present invention can also be used as a culture container used for co-culturing peripheral blood mononuclear cells and specific viruses to produce and proliferate genetically modified immune cells from peripheral blood mononuclear cells.
  • Example 1 Using a gas permeable plastic film (manufactured by Toyo Seikan Group Holdings Co., Ltd .: the same material as the polyethylene-based special multilayer film used for the cell culture bag “CCB-st”), the cell culture container shown in FIG. A culture vessel similar to 1 was produced.
  • the bowl-shaped recess 4 was formed.
  • Example 1 A culture vessel was produced in the same manner as in Example 1 except that the bottom portion 2b of the vessel body 2 was not formed with the bowl-shaped recess 4 and the bottom portion 2b was made to be a flat surface similar to the top surface 2a.
  • Antigen-specific CTL induction step (a) Isolation of peripheral blood mononuclear cells 20 mL of peripheral blood is collected from an HLA-A24 positive healthy person using a syringe to which a small amount of heparin is added, and Ficoll specific gravity centrifugation is used. Peripheral blood mononuclear cells were isolated by (3 ⁇ 10 7 cells).
  • Example 1 and Example 2 After standing in the incubator for a while, an image of the cells was obtained with a microscope system equipped with a digital camera, and the sedimentation state of the cells in the culture containers of Example 1 and Example 2 was confirmed.
  • a microscope system equipped with a digital camera
  • As the microscope an inverted fluorescence phase contrast microscope BZ-X700 (manufactured by Keyence Corporation) was used. The magnification used 4 times and 40 times lenses. The result is shown in FIG.
  • FIG. 2 it was found that the injected cells settled at the bottom center of the U-shaped cross section of the bowl-shaped recess serving as the cell culture part. While relatively high density cells are gathered in the center of the bottom of the U-shaped cross section, almost no cells can be confirmed in other areas. In this way, the cells in the container body are divided into a plurality of bowl-shaped recesses, and the movement of the container wall in the extending direction is suppressed, and the cells settle to the center of the bottom having a U-shaped cross section. It was possible to cultivate the cells while bringing them into close contact with each other, and prepared an environment in which antigen-specific immune cells can be efficiently induced against a very low frequency of antigen-specific immune cells.
  • the culture vessel was taken out 2 days after the start of culture. Next, 1 mL of IL-2 containing induction medium (IL-2 content: 300 IU / mL) was injected into the culture vessel from the injection port. After culturing at 37 ° C. in a 5% CO 2 incubator for 5 days, 1 mL of IL-2 containing induction medium (IL-2 content: 50 IU / mL) was injected into the culture vessel from the injection port.
  • IL-2 content IL-2 content: 50 IU / mL
  • the cell suspension and trypan blue staining solution were mixed at 1: 4, and the number of cells was quickly measured with a hemocytometer.
  • the number of viable cells obtained in the culture container of Comparative Example 1 was 1.04 ⁇ 10 7
  • the number of viable cells obtained in the culture containers of Example 1 and Example 2 was The numbers were 1.182 ⁇ 10 7 and 1.20 ⁇ 10 7 , respectively.
  • the culture containers of Example 1 and Example 2 provided with the bowl-shaped recess, more living cells were obtained than when cultured in the culture container of Comparative Example 1 of a flat bottom type.
  • the result of quantifying the antigen-specific CTL is shown in FIG.
  • the numbers in the dot plot development diagram are expressed as UL (upper left), UR (upper right), LL (lower left), and LR (lower right) when the development diagram is divided into four parts.
  • the percentage of UR for (UR + LR) is shown.
  • the X-axis is CD8, and the Y-axis is a dot plot development view showing the fluorescence intensity for the MHC-tetramer mixed reagent on a log scale.
  • the HCMV-specific CTL induced in the culture container of Comparative Example 1 had a positive rate of 0.13%, but the HCMV-specific CTL induced in the culture container of Example 1 was a positive rate of 0.54% and in Example 2 a positive rate of 1.13% was detected.
  • the number of CTL cells was calculated on the right side of FIG.
  • the number of CTL cells induced up to 7 days after the start of culture was 4,160 (Comparative Example 1), 17,730 (Example 1), and 36,000 (Example 2) in three types of culture vessels, respectively. )Met.
  • 4.26 times of CTL cells were obtained in the culture container of Example 1, and 8.65 times in the culture container of Example 2.
  • the frequency of tetramer positive cells and the number of CTL cells in the example shown in FIG. From this, it is clear that the saddle-shaped concave portion having a U-shaped cross section is effective in inducing antigen-specific CTL.
  • Antigen-specific CTL proliferation step In the above-described antigen-specific immune cell induction step, a sufficient positive rate of antigen-specific immune cells was obtained, and the conditions suitable for culturing immune cells as they were (typically Is cultured in an incubator set at 37 ° C. and 5% CO 2 (start of growth culture). Thereafter, the culture (2 days) was carried out in the order of injection of the growth medium from the injection port. Thereafter, the culture in the incubator is continued. By the above operation, an antigen-specific CTL was prepared in a short time of 14 days from the start of culture in steps (1) and (2). The resulting cells were subjected to cell counting and flow cytometry analysis. The results are shown in FIGS.
  • the total number of viable cells on the 14th day from the start of culture was 2.99 ⁇ 10 7 (Comparative Example 1), 3.17 ⁇ 10 7 (Example 1), and 3. The number was 32 ⁇ 10 7 (Example 2).
  • the number of CTL cells was 0.27 ⁇ 10 5 (Comparative Example 1) and 3.90 ⁇ 10 5 (Example 1), respectively. It was 5.22 ⁇ 10 5 (Example 2), which was found to be greatly different.
  • About 14.4 times as many CTL cells as the culture container of Example 1 and about 19.3 times as much as the culture container of Example 2 were obtained with respect to the culture container of Comparative Example 1.
  • the antigen-specific CTL was successfully prepared in high purity and in large quantities in spite of a short period of 14 days from the start of culture in steps (1) and (2) (excluding the time required for preparation of PBMC and plasma). did.
  • Antigen-specific CTL recovery step An example of the recovery operation after preparing the antigen-specific CTL is shown. First, the contents (that is, antigen-specific CTL) are dispensed into an appropriate container using the infusion port of the culture container. Next, after centrifugation, the supernatant (culture solution) is discarded, and a cryopreservation solution (CP-1 supplemented with 8% human serum albumin: manufactured by Kyokuto Pharmaceutical Co., Ltd.) is suspended to suspend the cells. . Subsequently, the cell suspension is transferred to an appropriate cryopreservation container and stored frozen (eg, ⁇ 130 to ⁇ 150 ° C.). In use, the cells are thawed and the cells are washed, and then the cells are infused into the patient using an infusion bag.
  • a cryopreservation solution CP-1 supplemented with 8% human serum albumin: manufactured by Kyokuto Pharmaceutical Co., Ltd.
  • the container main body 2 has a rectangular shape and includes the injecting port 3 on one side of the short side, but is not limited thereto.
  • the shape of the container body 2 may be a square shape, an oval shape, a circular shape, or the like, and can be various shapes as necessary.
  • the position where the injection port 3 is provided can be changed as appropriate.
  • the present invention can be used particularly in fields that require a large amount of cells in a short period of time, such as cell culture for regenerative medicine, etc., by increasing cell induction efficiency and cell growth rate.

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Abstract

Afin d'obtenir l'utilisation pratique d'une thérapie cellulaire immunologique spécifique à un antigène, un récipient de culture cellulaire 1 dans lequel de multiples parties en creux 4, qui servent de sections de culture cellulaire, sont disposées sur une surface des parois du récipient est utilisé, de sorte que les cellules sont déposées dans les parties en creux 4 pour préparer les cellules souhaitées. De cette manière, il devient possible de préparer de manière appropriée des immunocytes spécifiques à un antigène en une grande quantité, simplement et en toute sécurité, à faible coût et avec une efficacité élevée.
PCT/JP2017/018575 2016-05-19 2017-05-17 Procédé de préparation cellulaire et récipient de culture cellulaire WO2017200017A1 (fr)

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