WO2009139413A1 - Procédé de production de masse cellulaire contenant une cellule tueuse induite par les cytokines - Google Patents

Procédé de production de masse cellulaire contenant une cellule tueuse induite par les cytokines Download PDF

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WO2009139413A1
WO2009139413A1 PCT/JP2009/058917 JP2009058917W WO2009139413A1 WO 2009139413 A1 WO2009139413 A1 WO 2009139413A1 JP 2009058917 W JP2009058917 W JP 2009058917W WO 2009139413 A1 WO2009139413 A1 WO 2009139413A1
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cell
cells
cell population
culture
present
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Japanese (ja)
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希山 ▲赤▼
秀宝 任
津浦 于
慧 李
水 曹
秀梅 安
郁之進 加藤
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タカラバイオ株式会社
天津医科大学附属▲中▼瘤医院
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Priority to CN2009801129125A priority Critical patent/CN102027104A/zh
Publication of WO2009139413A1 publication Critical patent/WO2009139413A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/58—Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates to a cytokine-inducing killer (CIK) cell-containing cell population having high efficacy in adoptive immunotherapy, a method for producing the cell population, and the like.
  • CIK cytokine-inducing killer
  • the proliferation of regulatory T cells is suppressed, and various cell proliferative properties including hematopoietic cells and solid tissue tumors in addition to early and late cancers. Can be used to treat disease.
  • Such therapy includes, for example, adoptive immunotherapy in which immune-related cells taken out of the body are cultured to increase the number of cells, and / or the activity related to the therapeutic effect is enhanced and transplanted to a patient.
  • lymphokine-activated killer (LAK) cells As cells used as effector cells in adoptive immunotherapy, lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TIL), and cytokine-induced killer (CIK) ) Cells are known.
  • LAK lymphokine-activated killer
  • TIL tumor-infiltrating lymphocytes
  • CIK cytokine-induced killer
  • LAK cells are a cell population that can be obtained by growing lymphocytes in the presence of interleukin (IL) -2, and have the property of lysing tumor cells but not normal cells.
  • IL interleukin
  • a method of coexisting an anti-CD3 antibody together with IL-2 during culture has been developed.
  • TIL is a T cell infiltrating a tumor tissue, and has a tumor antigen specificity remarkably higher than that of a LAK cell.
  • the cells can also be grown outside the body and used for therapy.
  • TIL needs to be obtained by extracting the tumor tissue of the patient, it has a problem that the collection operation is complicated and the number of cells obtained is small.
  • CIK cells are prepared from peripheral blood mononuclear cells (PBMC) by culturing in the presence of interferon (IFN) - ⁇ , anti-CD3 antibody, and IL-2.
  • CIK cells are characterized as a cell population containing CD3-positive and CD56-positive cells, a rare cell population in PBMC that can be preferentially grown in the absence of target cells .
  • CIK cells exhibit cytotoxic activity against tumor cells superior to LAK cells in vivo (for example, Non-Patent Document 1).
  • Fibronectin is a huge glycoprotein with a molecular weight of 250,000 present in animal blood, on the surface of cultured cells, and in the extracellular matrix of tissues, and is known to have a variety of functions.
  • the domain structure is divided into seven (see FIG. 1).
  • the amino acid sequence includes three types of similar sequences, and the whole is constituted by repetition of these sequences. Three similar sequences are called type I, type II and type III, respectively.
  • type III is composed of 71 to 96 amino acid residues, and the concordance rate of these amino acid residues is 17 to 40%.
  • Non-patent Document 2 Several types of fibronectin fragments have been produced by recombinant DNA technology, and some of them are expected to have cancer metastasis inhibitory activity. Furthermore, it has been reported that a fibronectin fragment having a heparin-binding domain has a function of promoting infection of retrovirus cells (Non-patent Document 3).
  • lymphokine-activated killer cells obtained by in vitro culture from cytotoxic T cells (CTL) or peripheral blood lymphocytes, etc. induced by the action of IL-2 and anti-CD3 antibodies.
  • CTL cytotoxic T cells
  • IL-2 and anti-CD3 antibodies Use fibronectin and its fragments for questions such as how to maintain cytotoxic activity when antigen-specific CTLs induced outside the body are expanded and how efficiently lymphocytes can be expanded outside the body.
  • the effects of have been studied (for example, Patent Documents 1 to 3).
  • An object of the present invention is to provide a method for expanding a cell population for cancer treatment with a low content of unnecessary cell population for the production of CIK cells, which is said to be a suitable cell population for adoptive immunotherapy. It is in.
  • the first invention of the present invention includes a step of culturing a cell population containing cells that can differentiate into cytokine-induced killer cells in the presence of a fragment of fibronectin
  • the present invention relates to a method for producing a cell population containing CIK cells.
  • a method for producing a cell population containing CIK cells that is cultured in the presence of a fragment of fibronectin containing a region selected from: A VLA-4 binding region, VLA-5 binding region and heparin binding region.
  • a preferred example of the fibronectin fragment used in the first invention is a fibronectin fragment comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, and 5.
  • the first invention there is provided a method for producing a cell population containing CIK cells, wherein the culture in the presence of a fibronectin fragment is carried out in the presence of CD3 ligand, interferon- ⁇ and interleukin-2.
  • the CD3 ligand is not limited as long as it can be used in the present invention, but is preferably carried out using an anti-CD3 antibody.
  • the second invention of the present invention relates to a cell population containing CIK cells obtainable by the method of the first invention of the present invention.
  • the third invention of the present invention relates to a medicament containing the cell population of the second invention of the present invention as an active ingredient.
  • the fourth invention of the present invention relates to a method for treating or preventing a disease comprising the step of administering an effective amount of the cell population of the second invention of the present invention to a subject.
  • the disease may be any disease that is sensitive to the cell population of the second invention of the present invention.
  • the cell population of the second invention may be a cell population derived from a subject (patient) who has a disease requiring treatment or prevention, and may be a cell population derived from a donor different from the subject in some cases.
  • the fifth invention of the present invention relates to the use of the cell population of the second invention for the manufacture of a medicament.
  • the production method of the present invention provides a production method for expanding and culturing a cell population containing a high amount of CIK cells having high cytotoxic activity in large quantities.
  • a high-quality cell population obtained in a large amount by the production method exhibits a high therapeutic effect in vivo, and thus is extremely useful for treatment of diseases by cell medicine.
  • the present invention has found that by culturing PBMC in the presence of a fibronectin fragment, the cell population obtained contains cells expressing both CD3 and CD56 molecules at a high ratio on the cell surface. It has come to be completed.
  • the present invention will be specifically described below.
  • Fragment of fibronectin used in the present invention The fibronectin fragment described in the present specification is obtained from nature (such as those obtained by fragmenting natural fibronectin by enzymatic digestion), or recombinant DNA. Any of those manufactured by technology may be used. Fibronectin fragments can be obtained, for example, from Luoslati E. [J. Biol. Chem. 256, No. 14, pp. 7277-7281 (1981)], can be produced in substantially pure form from naturally occurring substances.
  • the substantially pure fibronectin fragments described herein mean that they are essentially free of other proteins that are naturally present with fibronectin.
  • a single molecular species may be used, or a plurality of molecular species may be mixed and used.
  • the useful information regarding the preparation of the fibronectin fragment that can be used in the present invention and the fragment includes corn bullet A. R. [EMBO J. et al. Vol. 4, No. 7, 1755-1759 (1985)], and Sekiguchi K .; [Biochemistry, Vol. 25, No. 17, 4936-4941 (1986)] and the like. Further, the nucleic acid sequence encoding fibronectin or the amino acid sequence of fibronectin is Genbank Accession No. NM_002026 and NP_002017.
  • a fibronectin fragment having cell adhesion activity and / or heparin binding activity can be preferably used.
  • fibronectin there is a region having an activity of binding to integrin on the cell surface. Examples of the region include a VLA (very late antigen) -4 or VLA-5 binding region.
  • a region called IIICS exists at a site near the C-terminal side of fibronectin.
  • CS-1 a region consisting of 25 amino acids
  • This region shows binding activity to VLA-4.
  • the amino acid sequence of the CS-1 region is shown in SEQ ID NO: 1 in the sequence listing.
  • fibronectin has a repetitive sequence called type III, and the 10th type repetitive sequence from the N-terminal side has a cell binding region.
  • the amino acid sequence of the 10th type III repeat sequence is shown in SEQ ID NO: 2 in the sequence listing.
  • the sequence that plays a central role in binding to VLA-5 in the above sequence is 4 amino acids of Arg-Gly-Asp-Ser (RGDS) shown in SEQ ID NO: 3 in the sequence listing.
  • C-274 consisting of the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, and is a recombinant fibronectin fragment having strong cell adhesion activity.
  • fibronectin has an activity of binding to heparin.
  • the heparin-binding region of fibronectin corresponds to the 12th to 14th positions from the N-terminal side of the type III repeat sequence.
  • H-271 consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing is a recombinant fibronectin fragment consisting of this heparin binding region.
  • a fragment in which two or more of these regions are linked directly or via an appropriate linker can be used.
  • the regions derived from fibronectin contained in the fragment may be the same or different.
  • H-296 amino acid sequence represented by SEQ ID NO: 6 in the sequence listing
  • VLA-4 binding region and heparin binding region VLA-5 binding region
  • CH-271 containing the heparin binding region
  • VLA-4 binding region CH-296 containing the VLA-5 binding region and heparin binding region (of the sequence listing)
  • the fragment used in the present invention is a fragment having a function equivalent to that of the fragment containing at least a part of the amino acid sequence of natural fibronectin exemplified above. It may consist of a polypeptide having an amino acid sequence having substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of the constituent polypeptide. Further, for example, one obtained by deleting one or two type III repeat sequences from C-274 or H-271 may be used.
  • the cell adhesion activity can be examined by assaying the binding between a fragment (its cell binding domain) used in the present invention and a cell by a known method.
  • Williams D.C. A. [Nature, Vol. 352, pp. 438-441 (1991)].
  • This method is a method for measuring the binding of cells to a fragment immobilized on a culture plate.
  • the heparin binding activity can be examined by assaying the binding of the fragment (its heparin binding domain) used in the present invention to heparin using a known method.
  • the above Williams D.C. A. In these methods, by using heparin, for example, labeled heparin, instead of cells, the binding between the fragment and heparin can be evaluated in the same manner.
  • the use of the recombinant fibronectin fragment in the present invention is preferable from the standpoint of safety that it is easy to obtain and handle, the uniformity of its quality, and the low risk of contamination with viruses and the like.
  • the molecular weight of the fibronectin fragment used in the present invention is not particularly limited, but is preferably 1 to 200 kDa, more preferably 5 to 190 kDa, and further preferably 10 to 180 kDa.
  • the molecular weight can be measured, for example, by SDS-polyacrylamide gel electrophoresis.
  • the present invention is a method for producing CIK cells, ie, cell populations that are highly enriched in subcell populations characterized as CD3 positive and CD56 positive.
  • the method of the present invention is characterized by comprising culturing a cell population containing cells capable of differentiating into CIK cells in the presence of the fibronectin fragment described above.
  • the cell population containing cells that can differentiate into CIK cells used in the production method of the present invention is a cell population that can be obtained from peripheral blood, bone marrow, umbilical cord blood, or the like, or separated from these materials.
  • a population of blood cells is exemplified.
  • PBMC is used in the present invention. Any of these cell populations collected directly from a living body or stored frozen can be used in the present invention.
  • the concentration of the fibronectin fragment is not particularly limited. For example, 0.001 to 500 ⁇ g / mL, particularly 0.01 to 500 ⁇ g / mL is preferable.
  • an appropriate solid phase for example, cell culture equipment such as a petri dish, flask or bag (including culture vessels; open and closed) Alternatively, it may be used by immobilizing it on a cell culture carrier such as beads, a membrane or a slide glass.
  • the material of the solid phase is not particularly limited as long as it can be used for cell culture.
  • the amount of fibronectin fragment to be immobilized is selected so that when the above equipment or carrier is subjected to culture, the ratio is the same as the desired concentration when the component is dissolved in the medium. If an effect is acquired, it will not specifically limit.
  • the method for immobilizing the fibronectin fragment on the solid phase is not particularly limited.
  • the fibronectin fragment can be immobilized by bringing a fragment dissolved in an appropriate buffer into contact with the solid phase.
  • the fibronectin fragment can also be immobilized by the methods described in WO 97/18318 pamphlet and WO 00/09168 pamphlet.
  • culturing in the presence of a fibronectin fragment is performed in the presence of a CD3 ligand.
  • the CD3 ligand is not particularly limited as long as it is a substance having a binding activity to CD3.
  • an anti-CD3 antibody is exemplified, and an anti-CD3 monoclonal antibody is particularly preferably exemplified.
  • OKT3 J. Immunol. 124, No. 6, 2708-2713 (1980)].
  • concentration of the CD3 ligand in the medium is not particularly limited.
  • CD3 ligand can also be used by immobilizing it on cell culture equipment or cell culture carrier by the same operation as fibronectin fragment. If the fibronectin fragment and the CD3 ligand are immobilized on a solid phase, the above components and the obtained cell population can be easily separated by simply separating the cell and the solid phase after the end of the culture. It is possible to prevent the components from being mixed in.
  • the medium used in the method for producing a CIK cell-containing cell population of the present invention is not particularly limited, and a known medium that can be used for expansion culture of lymphocytes can be used.
  • a commercially available medium is appropriately selected. Can be used.
  • These media may contain cytokines, appropriate proteins, or other components in addition to the original components.
  • a medium containing IFN- ⁇ and IL-2 is used in the present invention.
  • the concentration of IFN- ⁇ in the medium is not particularly limited, but is 50 to 10,000 U / mL, and more preferably 100 to 5000 U / mL.
  • the concentration of IL-2 in the medium is not limited, but is 10 to 5000 U / mL, preferably 50 to 2000 U / mL.
  • cytokines such as IL-1 ⁇ , IL-7 or IL-12 may be added to the medium.
  • the concentration of the component in the medium is not particularly limited as long as a desired effect is obtained.
  • the amount added to these media is not particularly limited, but is exemplified by more than 0% to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be decreased in stages and used.
  • the origin of serum or plasma may be either self (meaning that the origin is the same as the cell to be cultured) or non-self (meaning that the origin is different from the cell to be cultured). From the standpoint of the above, self-derived ones are preferable.
  • An isolated serum component such as human serum albumin may also be added.
  • the production of the CIK cell-containing cell population of the present invention is carried out using the above-mentioned various components or medium in addition to the fibronectin fragment.
  • the number of cells at the start of the culture used in the present invention is not particularly limited, but is preferably 1 ⁇ 10 4 cells / mL to 1 ⁇ 10 8 cells / mL, more preferably 1 ⁇ 10 5 cells / mL. mL to 5 ⁇ 10 7 cells / mL are exemplified.
  • culture conditions The conditions used for normal cell culture can be used. For example, it is possible to 37 ° C., and cultured under conditions, such as 5% CO 2.
  • operations such as diluting a cell culture solution by adding a fresh medium at an appropriate time interval, exchanging the medium, or exchanging equipment for cell culture can be performed.
  • cell culture equipment such as a petri dish, a flask, a bag, a large culture tank, or a bioreactor can be used.
  • bag a CO 2 gas permeable bag for cell culture is suitable.
  • a large culture tank may be used.
  • the culture can be carried out in either an open system or a closed system, but the culture is preferably carried out in a closed system.
  • the culture period in the method for producing a CIK cell-containing cell population of the present invention is not particularly limited.
  • the total number of days is 4 to 30 days, preferably 6 to 25 days.
  • the culture in the presence of the fibronectin fragment may be the entire period during the culture period, or may be an arbitrary partial period. That is, any cell culture step including a culture step in the presence of a fibronectin fragment is included in the present invention. It is particularly preferable to carry out the culture in the presence of the fibronectin fragment at least at an initial stage during the entire culture period, and it is more preferable to start the culture in the presence of the fibronectin fragment.
  • the culture in the presence of the active ingredient of the present invention is preferably carried out for at least 1 day, more preferably 2 days or more from the beginning of the culture.
  • CD3 ligand is used at the time of culturing, there is no particular limitation on the use period, but for example, CD3 ligand is allowed to coexist when fibronectin fragment is used.
  • the fibronectin fragment and the CD3 ligand are used only in the initial stage during the entire culture period.
  • the culture is carried out for the coexistence of the fibronectin fragment and the CD3 ligand from the start of the culture to the 7th day, preferably from the start of the culture to the 5th day.
  • CIK cell-containing cell population is produced by continuing culture in the absence of both components.
  • the production method of the present invention may further include a step of separating a CD3 positive and CD56 positive cell population from the cell population obtained by the above method. Separation can be performed by a known method using, for example, a cell sorter, a magnetic bead, or an affinity column. The cell population thus separated is enriched with cells having high cytotoxic activity, and is expected to exhibit a higher therapeutic effect.
  • the present invention provides a CIK cell-containing cell population obtainable by the above-described method for producing a CIK cell-containing cell population of the present invention. Since the CIK cell-containing cell population of the present invention contains a higher proportion of CD3-positive CD56-positive cells than the CIK cell-containing cell population produced by the conventional method, it exhibits stronger cytotoxic activity in vivo. And has a high therapeutic effect. Furthermore, the cell population has a very excellent feature in therapeutic use, such as a low content of regulatory T cells (regulatory T cells; Treg cells) having an action of suppressing cellular immunity.
  • regulatory T cells regulatory T cells
  • the present invention also provides a medicament (therapeutic agent or prophylactic agent) containing a CIK cell-containing cell population as an active ingredient.
  • the therapeutic agent containing the cell population is suitable for use in immunotherapy.
  • immunotherapy a CIK cell-containing cell population suitable for treating a patient is administered to a patient by an administration method such as intravenous, arterial, subcutaneous, or intraperitoneal injection.
  • the therapeutic agent of the present invention does not particularly limit the present invention, but for example, cancer, leukemia, malignant tumor, hepatitis, or infectious disease (for example, influenza, tuberculosis, AIDS, MRSA infection, VRE infection or deep It is very useful in the treatment of diseases sensitive to CIK cells, such as superficial mycosis.
  • the therapeutic agent can be used for bone marrow transplantation, infectious disease prevention after irradiation, or donor lymphocyte infusion for remission of relapsed leukemia.
  • the therapeutic agent and prophylactic agent of the present invention follow known methods in the pharmaceutical field, for example, using a cell population produced by the method of the present invention as an active ingredient, known organic or inorganic carriers suitable for parenteral administration, shaping It can be mixed with an agent or a stabilizer and prepared as an infusion or injection.
  • the content of the CIK cell-containing cell population of the present invention in the therapeutic agent, the dosage of the therapeutic agent, and various conditions relating to the therapeutic agent can be determined as appropriate according to known immunotherapy.
  • the content of the CIK cell-containing cell population of the present invention in medicine is not particularly limited. For example, it is preferably 1 ⁇ 10 3 to 1 ⁇ 10 11 cells / mL, more preferably 1 ⁇ 10 4.
  • the dose of the medicament of the present invention is not particularly limited. For example, it is preferably 1 ⁇ 10 6 to 1 ⁇ 10 12 cells / day, more preferably 1 ⁇ 10 7 to per adult day. Examples include 5 ⁇ 10 11 cells / day, more preferably 1 ⁇ 10 8 to 2 ⁇ 10 11 cells / day.
  • immunotherapy with the therapeutic agent may be used in combination with drug treatment by administration of a known drug, radiation therapy, or treatment by surgical operation.
  • the method for producing a CIK cell-containing cell population of the present invention can further include a step of introducing a foreign gene into the cell population.
  • foreign gene means a gene that is artificially introduced into a CIK cell to be transfected, and includes a gene derived from the same species as the cell to be transfected.
  • the means for introducing a foreign gene is not particularly limited, and an appropriate one can be selected and used by a known gene introduction method.
  • the gene transfer step can be performed at any point in the production method of the present invention. For example, it is preferable from the viewpoint of work efficiency to carry out at the same time, during or after the production of the cell population. Gene transfer can be performed with or without a viral vector. A lot of literature has already been published on details of these methods.
  • the viral vector is not particularly limited, and is usually a known viral vector used in gene transfer methods, for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a simian virus vector, a vaccinia virus vector. Alternatively, a Sendai virus vector or the like is used.
  • a retroviral vector or a lentiviral vector that can stably incorporate a foreign gene into the vector into the chromosomal DNA of the introduced cell is particularly preferred.
  • those lacking replication ability are preferable so that they cannot self-replicate in infected cells.
  • a substance that improves gene transfer efficiency such as RetroNectin (registered trademark, manufactured by Takara Bio Inc.), can also be used during gene transfer.
  • a method using a carrier such as liposome or ligand-polylysine, a calcium phosphate method, an electroporation method, a particle gun method, or the like can be used.
  • a foreign gene incorporated into plasmid DNA, linear DNA or RNA is introduced.
  • the foreign gene to be introduced is not particularly limited, and any gene desired to be introduced into the cell (for example, an enzyme, cytokine, or receptor-encoding protein, antisense nucleic acid or siRNA ( small interfering RNA), ribozyme-encoding genes, etc.
  • These foreign genes can be used, for example, inserted into a vector or a plasmid so as to be expressed under the control of an appropriate promoter. Control sequences such as sequences or terminator sequences may be present in the vector.
  • a gene encoding an enzyme associated with resistance to a drug used for treatment of a patient such as cancer is introduced to impart drug resistance to a CIK cell-containing cell population.
  • a gene that confers sensitivity to a specific drug for example, thymidine kinase gene
  • thymidine kinase gene can be introduced to give the CIK cell-containing cell population susceptibility to the drug. In such a case, it becomes possible to remove the cells after transplanted into the living body by administering the drug.
  • the present invention also provides a method for treating or preventing a disease comprising administering to a subject an effective amount of a CIK cell-containing cell population obtainable by the aforementioned method.
  • subject refers to, for example, a patient suffering from a disease as described above.
  • an effective amount is an amount of the cell population that can exert a therapeutic or prophylactic effect when the CIK cell-containing cell population is administered.
  • the specific effective amount is appropriately set according to the administration form, administration method, purpose of use and age, weight, symptom, etc. of the subject and is not constant.
  • the administration method There is no limitation also on the administration method, and for example, it may be administered by drip or injection or the like, similar to the above-mentioned medicine.
  • the present invention also provides the use of a CIK cell-containing cell population for the manufacture of a medicament.
  • the manufacturing method of the said pharmaceutical is performed like the above-mentioned pharmaceutical.
  • the disease to which the drug is administered is not particularly limited, but is the same as the aforementioned drug.
  • Example 1 CIK cell expansion culture using fibronectin fragment CH-296
  • Isolation of PBMC Five cancer patients with informed consent (2 renal cancer, lung cancer, liver cancer and thyroid gland) After collecting blood components from one cancer each), the cell fraction containing PBMC was layered on Ficoll and centrifuged, and peripheral blood mononuclear cells (PBMC) in the intermediate layer were collected.
  • PBMC peripheral blood mononuclear cells
  • (2) Immobilization of anti-human CD3 antibody and CH-296 A T225 flask was loaded with anti-human CD3 antibody having a final concentration of 5 ⁇ g / mL and CH-296 having a final concentration of 15 ⁇ g / mL (RetroNectin (registered trademark), manufactured by Takara Bio Inc.).
  • PBS phosphate buffered saline
  • CIK cells amplified by stimulation with anti-CD3 antibody and CH-296 were designated as R-CIKs, or CIK cells amplified by stimulation with anti-CD3 antibody alone were designated as C-CIKs.
  • Example 1- (3) The C-CIKs and R-CIKs phenotypes prepared in Example 1- (3) were subjected to flow cytometry after staining the cells with fluorescently labeled antibodies.
  • CD3 positive cells, CD4 positive cells, or CD8 positive cells were stained with anti-CD3 antibody, anti-CD4 antibody, or anti-CD8 antibody, respectively.
  • CD3-positive CD56-positive cells were stained with anti-CD3 antibody and anti-CD56 antibody and evaluated as both positive cells.
  • Treg cells were stained with anti-CD4 antibody, anti-CD25 antibody and anti-CD127 antibody, and evaluated as CD4 positive CD25 positive CD127 negative cells.
  • the horizontal axis represents the detected surface antigen
  • the vertical axis represents the positive rate (%) of the surface antigen.
  • Example 2 Antitumor effect of CIK cells (1) Transfer of CIK cells to cancer patients For each cancer patient, the autologous R-CIKs prepared in Example 1 was transferred twice in 4 days. Was one cycle, and an average of 3.9 ⁇ 10 10 doses were administered. All patients received a minimum of two cycles, one of which received three cycles.
  • PBMC are differentiated into many cell populations by expansion culture, and the differentiated ones include cell populations that control or suppress immunity.
  • the present invention has made it possible to provide a large amount of high-quality cell populations with a low content of unnecessary cell populations in expanded culture to CIK cells, which is said to be a suitable cell population for adoptive immunotherapy. .
  • the cell population obtainable by the present invention is extremely useful as a cell population having remarkable efficacy in adoptive immunotherapy using the cell population.
  • SEQ ID NO: 1 Partial region of fibronectin named CS-1.
  • SEQ ID NO: 2 Partial region of fibronectin named III-10.
  • SEQ ID NO: 3 Partial region of fibronectin in III-10.
  • SEQ ID NO: 4 Fibronectin fragment named C-274.
  • SEQ ID NO: 5 Fibronectin fragment named H-271.
  • SEQ ID NO: 6 Fibronectin fragment named H-296.
  • SEQ ID NO: 7 Fibronectin fragment named CH-271.
  • SEQ ID NO: 8 Fibronectin fragment named CH-296.
  • SEQ ID NO: 9 Fibronectin fragment named C-CS1.

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Abstract

La présente invention concerne un procédé de production de masse cellulaire contenant une cellule tueuse induite par les cytokines (CIK), caractérisé en ce qu'il comprend une étape de culture d'une masse cellulaire contenant une cellule pouvant être différenciée en une cellule CIK en présence d'un fragment d'une fibronectine, et analogues.
PCT/JP2009/058917 2008-05-16 2009-05-13 Procédé de production de masse cellulaire contenant une cellule tueuse induite par les cytokines WO2009139413A1 (fr)

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WO2012096376A1 (fr) * 2011-01-14 2012-07-19 タカラバイオ株式会社 Procédé d'obtention de lymphocytes t régulateurs
EP2543719A1 (fr) 2011-07-08 2013-01-09 Zellwerk GmbH Bioréacteur Mäander et procédé d'expansion, de différenciation et de récolte dynamiques de cellules hématopoïétiques
CN105154398A (zh) * 2015-07-17 2015-12-16 深圳爱生再生医学科技有限公司 Cik及其制备方法
CN114717187A (zh) * 2022-03-09 2022-07-08 中科东方细胞科技有限公司 一种cik细胞及其制备方法与应用

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CN107812013B (zh) * 2017-10-20 2020-07-28 胥萍 一种治疗耐药结核的生物制剂及其制备方法
CN109762785B (zh) * 2018-12-10 2022-06-24 苏州近岸蛋白质科技股份有限公司 一种高效应用于人nk细胞非滋养层体外培养的方法

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Cited By (10)

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Publication number Priority date Publication date Assignee Title
WO2011052638A1 (fr) * 2009-10-28 2011-05-05 タカラバイオ株式会社 Procédé de production de cellules tueuses induites par la cytokine
JP5097856B2 (ja) * 2009-10-28 2012-12-12 タカラバイオ株式会社 サイトカイン誘導キラー細胞の製造方法
WO2012096376A1 (fr) * 2011-01-14 2012-07-19 タカラバイオ株式会社 Procédé d'obtention de lymphocytes t régulateurs
JPWO2012096376A1 (ja) * 2011-01-14 2014-06-09 タカラバイオ株式会社 制御性ｔ細胞の製造方法
JP5911810B2 (ja) * 2011-01-14 2016-04-27 タカラバイオ株式会社 制御性ｔ細胞の製造方法
EP2543719A1 (fr) 2011-07-08 2013-01-09 Zellwerk GmbH Bioréacteur Mäander et procédé d'expansion, de différenciation et de récolte dynamiques de cellules hématopoïétiques
DE102011106914A1 (de) 2011-07-08 2013-01-10 Zellwerk Gmbh Mäander- Bioreaktor und Verfahren zu dynamischen Expansion, Differenzierung und Ernte von hämatopoetischen Zellen
CN105154398A (zh) * 2015-07-17 2015-12-16 深圳爱生再生医学科技有限公司 Cik及其制备方法
CN114717187A (zh) * 2022-03-09 2022-07-08 中科东方细胞科技有限公司 一种cik细胞及其制备方法与应用
CN114717187B (zh) * 2022-03-09 2023-11-07 中科东方细胞科技有限公司 一种cik细胞及其制备方法与应用

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