WO2011052638A1

WO2011052638A1 - Process for production of cytokine-induced killer cells

Info

Publication number: WO2011052638A1
Application number: PCT/JP2010/069072
Authority: WO
Inventors: 芽衣子神; 美津子出野; 竜嗣榎
Original assignee: タカラバイオ株式会社
Priority date: 2009-10-28
Filing date: 2010-10-27
Publication date: 2011-05-05
Also published as: JPWO2011052638A1; JP5097856B2; CN102575231A; CN102575231B; HK1170533A1

Abstract

A process for producing cytokine-induced killer cells, comprising the steps of (a) culturing a cell mass that contains cells capable of being differentiated into CD3-positive CD56-positive cells and/or CD3-positive CD56-positive cells in the presence of a CD3 ligand, (b) culturing the cell mass produced in step (a) in the absence of a CD3 ligand, and (c) culturing the cell mass produced in step (b) in the presence of a CD3 ligand; and others.

Description

Method for producing cytokine-induced killer cells

The present invention relates to a method for producing a cytokine-induced killer cell (CIK cell) having high efficacy in adoptive immunotherapy, a CIK cell obtained by the production method, a medicine containing the CIK cell, and the like.
This application claims priority to Japanese Patent Application No. 2009-247347 filed on Oct. 28, 2009, and the entire contents of Japanese Patent Application No. 2009-247347 are incorporated in the present application. Is.

In recent years, therapies with heavy physical burdens on patients such as drug therapy and radiation therapy have been reviewed, and interest in immunotherapy methods with less burden has increased. The 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, tumor infiltrating lymphocytes (TIL), and cytotoxic T lymphocytes (cytotoxic T lymphocytes) are used as effector cells in adoptive immunotherapy. CTL) and cytokine-induced killer (CIK) cells are known.

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. Regarding the preparation of LAK cells, 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. However, since TIL needs to be obtained by extracting the tumor tissue of the patient, it has a problem that the sampling operation is complicated and the number of cells obtained is small.

CTL induces and proliferates lymphocytes specifically for tumor-associated antigens in the presence of HLA (human leukocyte antigen) -restricted tumor-associated antigens (peptides, proteins, etc.), antigen-presenting cells, and IL-2. Cell populations that can be obtained in a highly functional manner. However, CTL is difficult to cultivate in large quantities and has the property of recognizing only the cells that express the tumor-related antigen used at the time of induction. It has the problem of being significantly different.

CIK cells are a cell population prepared from peripheral blood mononuclear cells (PBMC) by culturing in the presence of interferon (IFN) -γ, anti-CD3 antibody, and IL-2. Yes, and characterized as a cell population containing CD3-positive and CD56-positive cells. CD3-positive CD56-positive cells are rare cells in PBMC, but 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). However, the ratio of CD3-positive CD56-positive cells contained in CIK cells produced by the conventional method is generally about 20%, and the proliferation of CIK cells is about 10 times after 3 weeks of culture. Very low. For this reason, in order to culture a large amount of CIK cells, blood is often collected from a patient, and there is a problem that physical burden is large (for example, Non-Patent Documents 2 and 3).

In the process of expansion culture of CIK cells, PBMC differentiate into many different cell populations, and thus the proportion of CD3-positive CD56-positive cells in the expanded cell population is not high. An object of the present invention is to provide a method for producing CIK cells, which can efficiently obtain large amounts of CD3-positive CD56-positive cells.

To summarize the present invention, the first aspect of the present invention is that (a) a cell population capable of differentiating into CD3 positive CD56 positive cells and / or a cell population containing CD3 positive CD56 positive cells is cultured in the presence of a CD3 ligand. (B) culturing the cell population obtained in step (a) in the absence of CD3 ligand, and (c) culturing the cell population obtained in step (b) in the presence of CD3 ligand. The present invention relates to a method for producing cytokine-induced killer cells. In the first embodiment, the culture of the cell population in at least one of steps (a) to (c) may be performed in the presence of interferon-γ and / or interleukin-2. In the first embodiment, the CD3 ligand is exemplified by an anti-CD3 antibody. In the first embodiment, the culture in the step (a) may be performed in the presence of a fibronectin fragment and a CD3 ligand. Examples of the fibronectin fragment include those containing a region selected from the group consisting of a VLA-4 binding region, a VLA-5 binding region, and a heparin binding region.

The second aspect of the present invention relates to a cytokine-induced killer cell that can be obtained by the production method of the first aspect of the present invention.

The third aspect of the present invention relates to a medicine containing the cytokine-induced killer cell of the second aspect of the present invention as an active ingredient.

The fourth aspect of the present invention relates to a method for treating or preventing a disease, which comprises the step of administering to a subject an effective amount of the cytokine-induced killer cells of the second aspect of the present invention.

A fifth aspect of the present invention relates to the use of the cytokine-induced killer cells of the second aspect of the present invention for the manufacture of a medicament.
The sixth aspect of the present invention also relates to a cytokine-induced killer cell according to the second aspect of the present invention for use in the treatment of diseases.

In a seventh aspect of the present invention, (A) a step of culturing a cell population capable of differentiating into a CD3-positive CD56-positive cell and / or a CD3-positive CD56-positive cell in a medium containing a CD3 ligand; B) reducing the CD3 ligand concentration in the medium, (C) culturing the cell population in a medium having a decreased CD3 ligand concentration, and (D) adding the CD3 ligand to the medium and further culturing the cell population. The present invention relates to a method for producing cytokine-induced killer cells, comprising a step.

According to the present invention, there is provided a method for producing CIK cells capable of expanding and culturing a cell population containing a high amount of CD3-positive CD56-positive cells having high cytotoxic activity in large quantities. High-quality CIK cells obtained in large quantities by the production method exhibit a high therapeutic effect in vivo, and thus are extremely useful for treatment of diseases by cell therapy.

The present inventors surprisingly cultivated a cell population cultured in the presence of CD3 ligand in the absence of CD3 ligand, and further cultured in the presence of CD3 ligand, and surprisingly both CD3 and CD56 on the cell surface. The inventors have found that a large number of cell populations containing a high ratio of cells expressing molecules can be obtained, thereby completing the present invention.

Hereinafter, the present invention will be specifically described.
(1) Production method of CIK cell of the present invention The production method of the present invention is a method for producing a CIK cell, that is, a cell population highly containing a sub-cell population characterized as CD3 positive and CD56 positive. The method for producing CIK cells of the present invention comprises (a) culturing a cell population that can be differentiated into CD3 positive CD56 positive cells and / or a cell population containing CD3 positive CD56 positive cells in the presence of a CD3 ligand, (b) step Culturing the cell population obtained by (a) in the absence of CD3 ligand, and (c) culturing the cell population obtained by step (b) in the presence of CD3 ligand.

In the conventional CIK cell production method, cells that can differentiate into CD3 positive CD56 positive cells and / or a cell population containing CD3 positive CD56 positive cells are stimulated with CD3 ligand, and then in the absence of CD3 ligand. It was completed by culturing. The reason why culture is performed in the absence of CD3 ligand after stimulation with CD3 ligand is that excessive stimulation (high concentration, long-term stimulation, etc.) with CD3 ligand suppresses cell proliferation. On the other hand, the CIK cell production method of the present invention is a cell population to which stimulation by CD3 ligand [the above step (a); hereinafter, CD3 ligand stimulation in this step is referred to as “initial stimulation”]. Is cultured in the absence of CD3 ligand [the above step (b)] and then stimulated again with CD3 ligand [the above step (c); hereinafter, the CD3 ligand stimulation in this step is referred to as “re-stimulation”] It is characterized by adding.

When a medium containing CD3 ligand is used for initial stimulation, after the initial stimulation, the medium is diluted to reduce the concentration of CD3 ligand to a level where no cell growth inhibitory effect is observed, and further culturing of the cell population is continued. By doing so, an effect equivalent to that of the above step (b) can be obtained. That is, (A) a step of culturing cells that can differentiate into CD3 positive CD56 positive cells and / or a cell population containing CD3 positive CD56 positive cells in a medium containing CD3 ligand, (B) CD3 ligand of the medium CIK cell production comprising the steps of reducing the concentration, (C) culturing the cell population in a medium having a reduced CD3 ligand concentration, and (D) adding the CD3 ligand to the medium and further culturing the cell population. The method is also an embodiment of the production method of the present invention. The step (A) of this embodiment can be regarded as the initial stimulation, and the step (D) can be regarded as the restimulation. The concentration of CD3 ligand in the “medium with reduced CD3 ligand concentration” in the above step (C) is not particularly limited as long as the cell growth inhibitory action by CD3 ligand is not observed. For example, step (A) A concentration of about 1/50 to 1/2 times the concentration of CD3 ligand in the medium is exemplified.

The “cell population that can be differentiated into CD3 positive CD56 positive cells and / or CD3 positive CD56 positive cells” used in the production method of the present invention can be obtained from peripheral blood, bone marrow, umbilical cord blood and the like. Examples are cell populations or populations of hematopoietic cells separated from these materials. Preferably, PBMC is used in the present invention. Moreover, you may use for this invention the cell population which becomes substantially from the CD3 positive CD56 positive cell isolate | separated from these cell populations. Separation can be performed by a known method using, for example, a cell sorter, magnetic beads, an affinity column, or the like. Any of these cell populations collected directly from a living body or cryopreserved can be used in the present invention. In addition, the cell population capable of differentiating into CD3-positive CD56-positive cells and / or the cell population containing CD3-positive CD56-positive cells is pre-cultured in the presence of interferon-γ before being used in the present invention. Also good.

The CD3 ligand used in the present invention is not particularly limited as long as it is a substance having binding activity to CD3. For example, anti-CD3 antibody, particularly preferably anti-CD3 monoclonal antibody such as OKT3 [J. Immunol. 124, No. 6, 2708-2713 (1980)].

The concentration of the CD3 ligand in the medium is not particularly limited. For example, when an anti-CD3 monoclonal antibody is used, for example, 0.001 to 500 μg / mL, particularly 0.01 to 100 μg / mL is suitable for initial stimulation. For re-stimulation, for example, 0.001 to 100 μg / mL, particularly 0.005 to 50 μg / mL is preferable. In addition to dissolving CD3 ligand in the medium and coexisting with it, an appropriate solid phase, for example, cell culture equipment such as a petri dish, flask, bag, etc. (culture vessel; open type or closed type) Or may be immobilized 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. If the CD3 ligand is immobilized on the solid phase, the above components and the obtained cell population can be easily separated by simply separating the cells and the solid phase after the completion of the culture. Mixing of components can be prevented. The amount of the immobilized CD3 ligand may be selected so as to have a ratio similar to a desired concentration when the component or carrier is used for culturing and the component is dissolved in a medium. If a desired effect is acquired, it will not specifically limit.

There is no particular limitation on the culture conditions of the above-described step (a), that is, “the step of culturing cells that can differentiate into CD3 positive CD56 positive cells and / or cell populations containing CD3 positive CD56 positive cells in the presence of CD3 ligand”. 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. The culture period is not particularly limited, and examples thereof include 1 to 10 days, preferably 1 to 7 days. The number of cells at the start of the culture is not particularly limited, but preferably 1 × 10 ⁴ to 1 × 10 ⁸ cells / mL, more preferably 1 × 10 ⁵ to 5 × 10 ⁷ cells / mL. Illustrated.

The above-mentioned step (b), that is, “the step of culturing the cell population obtained in step (a) in the absence of CD3 ligand” includes, for example, cell population and CD3 from the culture obtained in step (a). It is carried out by separating the ligand and further culturing the cell population in the absence of CD3 ligand. Separation of the cell population and the CD3 ligand is not particularly limited to the present invention. For example, when using a cell culture device in which the CD3 ligand is immobilized in the step (a), the cell population is separated from the CD3 ligand. It is carried out by transferring to other cell culture equipment that is not immobilized. In addition, for example, when a medium containing free CD3 ligand is used in step (a), the medium can be replaced with a medium not containing CD3 ligand. There are no particular limitations on the culture conditions in step (b), and 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. The culture period in the step (b) is not particularly limited, and examples thereof include 1 to 20 days, preferably 1 to 10 days.

The above step (c), ie, “the step of culturing the cell population obtained in step (b) in the presence of CD3 ligand” is not particularly limited to the present invention, but was obtained by step (b). You may implement by adding a CD3 ligand to a culture, and you may implement by moving the culture obtained by the process (b) to the cell culture equipment by which CD3 ligand was fix | immobilized. There are no particular limitations on the culture conditions of step (c), and 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. The culture period is not particularly limited, and examples thereof include 1 to 20 days, preferably 1 to 15 days.

Further, after the step (c) is performed, a step of culturing the cell population obtained by the step (c) in the absence of CD3 ligand may be further performed as the step (d). The culture conditions in the step (d) are not particularly limited, and 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. The culture period when step (d) is carried out is not particularly limited, and examples thereof include 1 to 20 days, preferably 1 to 15 days.

In the production method of the present invention, cell culture equipment (containers) such as petri dishes, flasks, bags, large culture tanks, bioreactors and the like can be used as culture equipment. As the bag, a CO ₂ gas permeable bag for cell culture is suitable. When a large amount of cells are required, a large culture tank may be used. The culture can be carried out in either an open system or a closed system, but is preferably carried out in a closed system.

In the production method of the present invention, the initial stimulation with CD3 ligand for cells that can differentiate into CD3 positive CD56 positive cells and / or cell populations containing CD3 positive CD56 positive cells is not particularly limited, but preferably Is performed in the presence of a fibronectin fragment. In addition, restimulation with CD3 ligand can also be performed in the presence of fibronectin.

The concentration of the fibronectin fragment used in the present invention is not particularly limited. For example, 0.001 to 500 μg / mL, particularly 0.01 to 500 μg / mL is preferable. The fibronectin fragment can be used by being immobilized on a cell culture device or a cell culture carrier, in addition to being dissolved in the medium and coexisting. The immobilization of the fibronectin fragment on the solid phase can be carried out, for example, by bringing a fragment dissolved in an appropriate buffer into contact with the solid phase, such as WO 97/18318 pamphlet or WO 00/09168 pamphlet. It can implement also by the method as described in (1). 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.

Fibronectin fragments may be those obtained from nature (such as those obtained by fragmenting natural fibronectin by enzymatic digestion) or those produced by recombinant DNA technology. Fibronectin fragments are, for example, Ruoslati E. [J. Biol. Chem. 256, No. 14, pp. 7277-7281 (1981)], can be produced in substantially pure form from naturally occurring substances. As used herein, “substantially pure form of fibronectin fragments” means that they are essentially free of other proteins that are naturally present with fibronectin. In the present invention, as the fibronectin fragment, a single molecular species may be used, or a plurality of molecular species may be mixed and used.

For useful information regarding the preparation of fibronectin fragments that can be used in the present invention and the fragments, see, for example, 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.

In the present invention, a fibronectin fragment having cell adhesion activity and / or heparin binding activity can be preferably used. In fibronectin, there is a region having an activity of binding to integrin on the cell surface. Examples of the region include a VLA (very rate antigen) -4 or VLA-5 binding region. A region called IIICS exists at a site near the C-terminal side of fibronectin. Here, a region consisting of 25 amino acids called CS-1 is included, and 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.

Also, 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 containing the amino acid sequence of SEQ ID NO: 2 and is a recombinant fibronectin fragment having strong cell adhesion activity.

Furthermore, 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.

In the present invention, in addition to a fragment containing each region alone, 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. As a fibronectin fragment having a plurality of binding regions in the molecule, H-296 (amino acid sequence represented by SEQ ID NO: 6 in the sequence listing) containing VLA-4 binding region and heparin binding region, VLA-5 binding region and CH-271 containing a heparin binding region (amino acid sequence represented by SEQ ID NO: 7 in the sequence listing), VLA-4 binding region, VLA-5 binding region, and CH-296 containing a heparin binding region (sequence listing) A polypeptide such as C-CS1 (amino acid sequence represented by SEQ ID NO: 9 in the Sequence Listing) containing VLA-4 binding region and VLA-5 binding region is exemplified. These various polypeptides are described in Non-Patent Document 2, and can be prepared according to the disclosure. CH-296 is sold by Takara Bio Inc. under the name of Retronectin (registered trademark).

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 as long as the desired effect of the fibronectin fragment is obtained. 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. In addition, for example, C-274 or H-271 may have a deletion of 1 or 2 type III repeat sequence.

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. For example, in such a 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. For example, the above Williams D.C. A. In these methods, by using heparin (eg, 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 viewpoint of safety that the quality is uniform and the risk of contamination with viruses is low, in addition to ease of acquisition and handling. 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 medium used in the method for producing CIK cells of the present invention is not particularly limited in any of steps (a) to (c), and a known medium that can be used for lymphocyte expansion culture or the like may be used. For example, a commercially available medium can be appropriately selected and used. These media may contain cytokines, appropriate proteins, or other components in addition to the original components. Usually, a medium containing at least one selected from the group consisting of IFN-γ and IL-2 is used in the present invention. The concentration of IFN-γ 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. Furthermore, cytokines such as IL-1α, IL-7, and 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.

These components can be used in any of the steps (a) to (c), and the present invention is not particularly limited. For example, IFN-γ is used in the step (a). IL-2 is preferably used, and IL-2 is preferably used in all the steps (a) to (c).

In addition, serum or plasma may be added to the medium. 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 viewpoint of the above, those derived from the self are preferred. An isolated serum component such as human serum albumin may also be added.

In addition, cells that have been treated to deprive growth ability may be added to the medium. In the present specification, the “treatment for depriving the proliferation ability” is not particularly limited as long as it is a treatment for losing or reducing the proliferation ability of the cell. For example, the treatment is performed by chemical treatment and / or physical treatment. Can do. Examples of the chemical treatment include chemical agents (formalin and the like), anticancer agents (mitotic inhibitors such as mitomycin C and the like), heating / heating, freeze-thawing, and ultrasonic treatment. Moreover, as said physical process, when implementing the said process by irradiation of a radiation, for example, a gamma ray and a X-ray are used, for example. Irradiation is not particularly limited as long as the proliferation ability of irradiated cells is lost. For example, 100 to 20000 R (0.88 to 175.40 Gy), preferably 1000 to 8000 R (8.77 to 70). .16 Gy). The number of CIK cells obtained can be increased by adding cells that have been treated to deprive the growth ability of the medium in the production method of the present invention. In addition, a cell population exhibiting high cytotoxic activity against lung cancer cell lines and the like can be obtained.

Cells that have been treated to deprive them of proliferative ability are cells that have lost or lost their ability to proliferate such as cell division or DNA synthesis. For example, cells that have been subjected to radiation treatment have a reduced proliferative ability, but immediately after the treatment, they exhibit the same morphology and traits as living cells, and maintain the metabolic ability to secrete proteins such as cytokines. It is desirable that the “cell that has been treated to deprive proliferative ability” is derived from the patient itself. The “cells that have been treated to deprive growth ability” used in the present invention are not particularly limited, but are derived from the patient who has been treated to deprive proliferation ability by irradiation of radiation. A cell (Autologous-Irradiated cell; AIC) is preferably exemplified.

The cell concentration of the “cells that have been treated to deprive the growth ability” used in the method of the present invention is not particularly limited. For example, 1 to 1 × 10 ⁸ cells / mL, preferably 10 to 5 × 10 ⁷ cells / mL, more preferably 1 × 10 ² to 2 × 10 ⁷ cells / mL are exemplified.

In addition, the medium used in the method for producing CIK cells of the present invention may contain a biological response modifier. “Biological response modifier” means a group of substances called non-biological response modifier (BRM) that improve non-specific immune response ability in the living body.

Examples of biological response modifiers include bacterial-derived preparations [OK-432, BCG (Bacillus Calmette Guerin), Streptococcus pyogenes, Corynebacterium parvum and their cell wall skeletons], Basidiomycete-derived polysaccharides (lentinan, schizophyllan, PSK, etc.) Piran copolymers, levamisole, etc.) or cytokines are known. “OK-432” means a general name of a bacterial-derived preparation obtained by treating a weakly toxic natural mutant strain (Su strain) of group A type 3 hemolytic streptococci (Streptococcus pyogenes) with penicillin. This formulation is marketed under the trade name Pisibanil®. In a preferred embodiment, the present invention uses a biological response modifier, which is a microorganism-derived preparation, and particularly preferably OK-432.

The concentration of the biological response modifier in the medium is not particularly limited, but for example, when OK-432 is used, 0.001 to 1 KE / mL, preferably 0.005 to 0.5 KE. / ML, more preferably 0.01 to 0.2 KE / mL. Usually, the biological response modifier can be added to the medium at the beginning of the culture.

The method for producing CIK cells of the present invention can further include a step of introducing a foreign gene into the cell population. The term “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. Sendai virus vectors and the like are used. A retroviral vector or a lentiviral vector that can stably incorporate a foreign gene contained in the vector into the chromosomal DNA of the introduced cell is particularly preferred. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells. In addition, a substance that improves the efficiency of gene transfer, such as RetroNectin (registered trademark), can be used during gene transfer.

As a gene introduction method that does not use a viral vector, 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. In this case, 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 antisense nucleic acid, siRNA (in addition to those encoding proteins such as enzymes, cytokines, receptors, etc.) Small interfering RNA), ribozyme encoding, 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 enhancer sequences or terminator sequences may be present in the vector.

According to the method of the present invention, a gene encoding an enzyme related to resistance to a drug used for treatment of a patient such as cancer (for example, a multidrug resistance gene) is introduced to impart drug resistance to CIK cells. Can do. By using such a cell population, it is possible to combine immunotherapy and drug therapy, and thus it is possible to obtain a higher therapeutic effect. On the other hand, conversely to the above-described embodiment, a gene that confers sensitivity to a specific drug (for example, thymidine kinase gene) can be introduced to give CIK cells 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.

In addition to the above, foreign genes to be introduced include, for example, nucleic acids encoding tumor antigen-specific T cell receptors (TCR), or antibodies specific for molecules expressed on tumor cells (Such as anti-CD19 antibodies), receptor molecules (such as TCR), ligands, or extracellular regions containing a portion thereof, transmembrane domains, and one or more other signal-related molecules that transduce signals into the cell Nucleic acids encoding chimeric receptors composed of regions can also be used. Useful information regarding the chimeric receptor can be found, for example, in Marc-Marina V. Et al. [Expert Opin. Biol. Ther. No. 9, No. 5, 579-591 (2009)]. By introducing a nucleic acid encoding the above-mentioned TCR or chimeric receptor into CIK cells, cytotoxic activity specific to the target tumor cells can be imparted to CIK cells. Examples of the extracellular region of the chimeric receptor include, for example, the extracellular region of an antibody or receptor molecule, or an antigen recognition site of an antibody or a ligand recognition site of a receptor molecule, and a spacer / derived from another antibody or receptor molecule (such as CD28). The extracellular region formed by fusing with the hinge region is exemplified. Examples of the intracellular region of the chimeric receptor include an intracellular region of a signal-related molecule present on lymphocytes. Examples of the intracellular region of the signal-related molecule include the intracellular region of CD3ζ chain, CD28, 4-1BB, CD134, FcR-γ, Syk-PTK, or these signal transduction domains.

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, for example, using an anti-CD3 antibody or an anti-CD56 antibody by a known method using a cell sorter, magnetic beads, an affinity column, or the like. The cell population thus separated is enriched with cells having high cytotoxic activity, and is expected to exhibit a higher therapeutic effect.

(2) CIK cell of the present invention The present invention provides a CIK cell obtainable by the above-described method for producing a CIK cell of the present invention. The CIK cell of the present invention contains a higher proportion of CD3-positive CD56-positive cells than CIK cells produced by the conventional method, and thus exhibits higher cytotoxic activity in vivo. Has a therapeutic effect.

The ratio of the CD3-positive CD56-positive cells in the CIK cells of the present invention is not particularly limited, but for example, 20% or more of the total number of cells, preferably 25% or more, more preferably 30% or more. Is exemplified.

(3) Medicament of the present invention The present invention provides a medicament (therapeutic agent or prophylactic agent) containing CIK cells as an active ingredient. The therapeutic agent containing the CIK cells is suitable for use in immunotherapy. In immunotherapy, CIK cells suitable for treatment of a patient are administered to the patient by routes such as intravenous, transarterial, subcutaneous, intraperitoneal, etc., for example, by administration by injection or infusion. The therapeutic agent of the present invention does not particularly limit the present invention, but for example, cancer, leukemia, malignant tumor, hepatitis, infectious diseases (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. In addition, the therapeutic agents include donor lymphocyte infusion, anticancer drug treatment, radiation therapy, antibody therapy, thermal therapy for the purpose of preventing infection in immune deficient conditions such as bone marrow transplantation, radiation irradiation, or remission of relapsed leukemia. It can be used in combination with conventional therapies such as therapy and other immunotherapy.

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, a stabilizer and the like, and prepared as an instillation or an injection. The content of the CIK cell 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 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 to 1 × 10 ^10. cells / mL, and more preferably 1 × 10 ⁵ to 2 × 10 ⁹ cells / mL. 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. Furthermore, 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.

(4) Therapeutic or preventive method of the present invention The present invention also provides a method for treating or preventing a disease, comprising administering to a subject an effective amount of CIK cells obtainable by the above-described method. The term “subject” as used herein refers to, for example, a patient suffering from a disease as described above.

In the present specification, the effective amount is the amount of the cell population that can exert a therapeutic or prophylactic effect when the CIK cells are administered. The specific effective amount is appropriately set according to the administration form, administration method, purpose of use, age, weight, symptom, etc. of the subject and is not constant. There is no limitation on the administration method, and for example, it may be administered by drip, injection or the like, similar to the above-mentioned medicine.

The present invention also provides the use of CIK cells obtainable according to the present invention for the manufacture of a medicament. Furthermore, the present invention also provides CIK cells obtainable by the present invention for use in the treatment of diseases. The manufacturing method of the said pharmaceutical is performed like the above-mentioned pharmaceutical. Further, the disease to which the drug is administered is not particularly limited, but is the same as the aforementioned drug.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these descriptions.

Example 1 CIK cell culture using anti-human CD3 antibody and retronectin (1) Separation and storage of PBMC Ingredients were collected from human healthy donor A, donor B and donor C with informed consent. (The component blood collection referred to here is blood collection for the purpose of collecting mononuclear cells). Dulbecco's PBS (manufactured by Invitrogen or Nissui Pharmaceutical; hereinafter referred to as DPBS) or 1% human serum albumin (formulation name Buminate: manufactured by Baxter, hereinafter referred to as HSA) After approximately 2-fold dilution with a physiological saline containing 1% HSA / physiological saline, Ficoll-Paque PREMIUM or Ficoll-Paque PLUS (both manufactured by GE Healthcare Biosciences) The diluted component blood samples were layered 30 mL each and centrifuged at 700 × g for 20 minutes at room temperature. After centrifugation, among the separated layers, the PBMC layer was collected with a pipette and filled up to 45 mL using RPMI 1640 (manufactured by Invitrogen, Sigma, or Wako Pure Chemical Industries) or 1% HSA / saline. And centrifuged at 650 × g and 4 ° C. for 10 minutes, and the supernatant was removed. The same washing operation was performed three times in total while decreasing the centrifugal acceleration in steps of 600 × g and 500 × g.

Thus, PBMCs collected from each donor were suspended in a stock solution consisting of an equal volume mixture of CP-1 (manufactured by Kyokuto Pharmaceutical) containing 8% HSA and RPMI 1640 and stored in liquid nitrogen. These stored PBMCs were rapidly thawed in a 37 ° C. water bath, washed with GT-T503 (Takara Bio) containing 10 μg / mL DNase (Calbiochem), and the number of viable cells was calculated by trypan blue staining. Later, it used for each following experiment.

(2) Preparation of anti-human CD3 antibody and retronectin-immobilized plate Anti-human CD3 antibody (formulation name: orthoclone OKT3 injection, Janssen Pharma) at a final concentration of 5 μg / mL or 1 μg / mL on a 12-well cell culture plate (Corning) And ACD-A solution (Terumo) containing Retronectin (registered trademark, manufactured by Takara Bio Inc.) at a final concentration of 5 μg / mL was added in an amount of 0.45 mL / well. Next, after 5 hours of incubation at 37 ° C. in the presence of 5% CO _{2, the} ACD-A solution was removed from each well to obtain a cell culture plate on which the anti-CD3 antibody and retronectin were immobilized. In addition, cells with anti-CD3 antibody immobilized by performing the same operation as above except that an anti-human CD3 antibody having a final concentration of 5 μg / mL or 1 μg / mL and using no ACD-A solution without retronectin is used. A culture plate (a plate on which Retronectin is not immobilized) was also prepared. Each plate prepared here was washed twice with DPBS and once with RPMI 1640 and then used for each of the following experiments.

(3) CIK cell culture GT-T503 (hereinafter referred to as 0.5% HAB / 0.2% HSA / GT-T503) containing 0.5% HumanAB serum (manufactured by Lonza) and 0.2% HSA The PBMC derived from donor A prepared in Example 1- (1) was suspended at 0.53 × 10 ⁶ cells / mL. To the various immobilized culture plates prepared in Example 1- (2), 1 mL / well of the above PBMC suspension was added, and 0.5% HAB / 0.2% HSA / GT-T503 was further added to 0.75 mL. Per well to give a total of 1.75 mL / well. Next, IL-2 (preparation name Proleukin: manufactured by Chiron or Novartis) and IFN-γ (formulation name Immunomax-γ Note: manufactured by Shionogi & Co., Ltd.) were respectively added to each well at a final concentration of 1000 U / mL. Then, these plates were cultured at 37 ° C. in the presence of 5% CO ₂ (culture day 0). On day 4 of culture, a portion of the cell solution in each well was diluted 12.5 times with 0.5% HAB / 0.2% HSA / GT-T503, and 10 mL of the diluted solution was added to a T-25 cell culture flask. (culture area 10 cm ^2, Corning) was poured to that upright, wherein after addition of IL-2 to a final concentration of 500 U / mL, the culture was continued at this flask. On day 8 of culture, a portion of the cell solution in each flask was diluted 4-fold with 0.5% HAB / 0.2% HSA / GT-T503, and 10 mL of the diluted solution was added to a new T-25 cell culture. Pour into a flask (standing), add IL-2 to a final concentration of 500 U / mL, and then add free anti-human CD3 antibody to a final concentration of 50 ng / mL (anti-human CD3 Antibody restimulation group). At this time, a group to which no anti-human CD3 antibody was added was set as a control group. The culture was continued for the anti-human CD3 antibody restimulation group and the control group, and on day 11 of the culture, a part of the cell fluid in each flask of each group was 0.5% HAB / 0.2% HSA / GT-T503. After diluting 4 times with, pour 10 mL of the diluted solution into a new T-25 cell culture flask (standing), add IL-2 to a final concentration of 500 U / mL, and then add to this flask. The culture was continued.

The culture was continued until the 14th day from the start of the culture, and the number of viable cells was measured by the trypan blue staining method on the 14th day. . The results are shown in Table 1. In the table below,-means no use and + means use.

(4) Analysis of CD3-positive CD56-positive (CD3 ⁺ CD56 ⁺ ) cell and CD8-positive (CD8 ⁺ ) cell content ratio Regarding the cells on day 14 after the start of culture prepared in Example 1- (3), CD3 ⁺ CD56 ⁺ The cell content ratio and the CD8 ⁺ cell content ratio were analyzed with a flow cytometer (Cytomics FC500: manufactured by Beckman Coulter). That is, the cells on the 14th day after the start of culture were washed with DPBS, and then the cells were described as DPBS containing 1% bovine serum albumin (Sigma, hereinafter referred to as BSA) (hereinafter referred to as 1% BSA / DPBS). PC5-labeled mouse anti-human CD3 antibody, FITC-labeled mouse anti-human CD8 and RD1-labeled mouse anti-human CD56 antibody (all manufactured by Beckman Coulter, Inc.) were added. Similarly, FITC-labeled mouse IgG1 / RD1-labeled mouse IgG1 / PC5-labeled mouse IgG1 (all manufactured by Beckman Coulter) was added to a part of each cell population. Each antibody was added and incubated at 4 ° C. for 30 minutes. After incubation, the cells were washed with DPBS containing 0.1% BSA (hereinafter referred to as 0.1% BSA / DPBS) and suspended again in DPBS. These cells were subjected to flow cytometry, and the CD3 ⁺ CD56 ⁺ cell content ratio and CD8 ⁺ cell content ratio were calculated. The results are shown in Table 2.

As shown in Table 2, a high ratio of CD3 ⁺ CD56 ⁺ cells was obtained by restimulation with anti-human CD3 antibody. In addition, CD8 ⁺ cells called active bodies of CIK cells were obtained at a high ratio. Moreover, these effects were further enhanced by combining the anti-human CD3 antibody and retronectin to give initial stimulation to the cell population. Furthermore, these effects were remarkable regardless of the immobilized concentration of the anti-human CD3 antibody used as the initial stimulus. As shown in Table 1, CIK cells containing CD3 ⁺ CD56 ⁺ cells at a very high ratio were obtained with a high expansion culture rate.

From this, it was shown that the production method of the present invention is a method for efficiently obtaining CD3 ⁺ CD56 ⁺ CD8 ⁺ cells that play a large role in the antitumor activity of CIK cells. In addition, since CIK cells having a high content of CD3 ⁺ CD56 ⁺ cells can be cultured in large quantities, it was revealed that this is a method for producing a cell population exhibiting a high therapeutic effect.

(5) Evaluation of expansion culture rate of CD3 ⁺ CD56 ⁺ cells PBMC derived from donor A prepared in Example 1- (1) and cells 14 days after the start of culture prepared in Example 1- (3) For CD1, the CD3 ⁺ CD56 ⁺ cell content ratio was measured in the same manner as in Example 1- (4). However, in consideration of contamination of erythrocytes in PBMC, FITC-labeled mouse anti-human CD3 antibody, RD1-labeled mouse anti-human CD56 antibody and PC5-labeled mouse anti-human CD45 antibody (all manufactured by Beckman Coulter) were used, and CD45-positive CD3 ⁺ CD56 ⁺ cells were defined as CD3 ⁺ CD56 ⁺ cells at the start of culture.

Using the CD3 ⁺ CD56 ⁺ cell content ratio thus calculated and the expansion culture rate (converted to the total number of cells) calculated in Example 1- (3), CD3 ⁺ CD56 ⁺ cells were cultured during the culture period (14 days). The multiplication ratio of CD3, that is, the CD3 ⁺ CD56 ⁺ cell expansion culture ratio was calculated [the following formula (1)]. The content ratio of CD3 ⁺ CD56 ⁺ cells (CD3 ⁺ CD56 ⁺ cell content ratio at the start of culture) in PBMC thawed and used this time was 1.54%. The results are shown in Table 3.

As shown in Table 3, by combining the anti-human CD3 antibody and retronectin to give initial stimulation to the cell population, and further restimulating with the anti-human CD3 antibody during the culture, CD3 for 14 days in culture. ⁺ Proliferation of CD56 ⁺ cells was markedly increased. In addition, these effects were exhibited in a wide concentration range regardless of the immobilized concentration of the anti-human CD3 antibody used as the initial stimulus.

Therefore, according to the production method of the present invention, CD3 ⁺ CD56 ⁺ cells, which are considered to be active components of CIK cells, can be proliferated preferentially, and CD3 plays a major role in the antitumor activity of CIK cells. It was revealed that ⁺ CD56 ⁺ cells can be obtained efficiently.

Example 2 CIK cell culture using anti-human CD3 antibody and retronectin (comparison of IFN-γ added and use of an autologous-irradiated cell)
(1) Preparation of Autologous-irradiated cell (hereinafter referred to as AIC) PBMC derived from donor A prepared in Example 1- (1) was suspended in 0.5% HAB / 0.2% HSA / GT-T503. After turbidity, 3400R (29.8 Gy) X-rays were irradiated using an X-ray irradiation apparatus (hereinafter, the cells after X-ray irradiation are described as PBMC AIC). The prepared PBMC AIC was suspended again in 0.5% HAB / 0.2% HSA / GT-T503 so as to be 1.06 × 10 ⁶ cells / mL.

(2) CIK cell culture derived from donor A prepared in Example 1- (1) so as to be 1.06 × 10 ⁶ cells / mL in 0.5% HAB / 0.2% HSA / GT-T503 PBMCs were suspended (described as IFN-γ the day before untreated group). However, a group was also set in which PBMCs prepared in the same manner on the day before the start of culture were cultured overnight in the presence of IFN-γ (final concentration 1000 U / mL) and then suspended at the cell concentration at the start of the culture (hereinafter referred to as the following). , Described as IFN-γ the day before treatment group).

An anti-human CD3 antibody and retronectin-immobilized plate prepared in the same manner as in Example 1- (2) (where the anti-human CD3 antibody-immobilized concentration was 0.1 μg / mL) was added to the PBMC (IFN -Γ-day-untreated group) or PBMC (IFN-γ-day-treated group) was added at a rate of 0.875 mL / well, and the PBMC AIC prepared in Example 2- (2) was added to the group using AIC by 0. 875 mL / well was added to give a total of 1.75 mL / well. For the group not using AIC, 0.575% HAB / 0.2% HSA / GT-T503 was added at 0.875 mL / well instead of AIC. IL-2 and IFN-γ were added to each well to a final concentration of 1000 U / mL, and the culture was started at 37 ° C. in the presence of 5% CO ₂ (culture day 0). The culture method was carried out in the same manner as in Example 1- (3), and dilutions on the 4th, 8th and 11th days of culture, and the anti-human CD3 antibody restimulation group and anti-human CD3 antibody were not added. Group setting and the like were performed in the same manner as in Example 1- (3). The culture was continued until the 14th day, and the number of viable cells was measured by the trypan blue staining method on the 11th and 14th days after the start of the culture, and the expanded culture rate (total number of cells) was compared with the number of cells at the start of the culture. Conversion) was calculated. The results are shown in Table 4.

⁽³⁾ CD3 + in CD56 ⁺ cells and CD8 ⁺ cells Analysis embodiment of the content ratio 1- (4) and the same method, for example 2- (2) culture the fourteenth day after the initiation of the cells prepared CD3 ⁺ CD56 ⁺ cell content ratio and CD8 ⁺ cell content ratio were analyzed. The results are shown in Table 5.

As shown in Table 5, by combining the anti-human CD3 antibody and retronectin to give initial stimulation to the cell population, and further restimulating with the anti-human CD3 antibody during the culture, the CD3 ⁺ CD56 ⁺ cells were A high ratio was obtained. These effects were widely exhibited regardless of the difference in the IFN-γ treatment method for PBMC or the use of AIC. This reveals that the production method of the present invention can be widely applied to existing CIK cell culture methods and can efficiently obtain CD3 ⁺ CD56 ⁺ cells, which are important components in CIK cells. It was.

(4) Evaluation of expansion culture rate of CD3 ⁺ CD56 ⁺ cells Each of PBMC derived from donor A prepared in Example 1- (1) and cells on day 14 after the start of culture prepared in cell Example 2- (2) For CD1, the CD3 ⁺ CD56 ⁺ cell content ratio was measured in the same manner as in Example 1- (4).

CD3 ⁺ CD56 ⁺ cells proliferated during the culture period (14 days) using the calculated CD3 ⁺ CD56 ⁺ cell content ratio and the expanded culture rate (converted to the total number of cells) calculated in Example 2- (2) The magnification (ie, CD3 ⁺ CD56 ⁺ cell expansion culture rate) was calculated in the same manner as in Example 1- (5). Note that the content ratio of CD3 ⁺ CD56 ⁺ cells at the start of the culture was 0.6%. The results are shown in Table 6.

As shown in Table 6, the combination of anti-human CD3 antibody and retronectin gives initial stimulation to the cell population, and restimulation with anti-human CD3 antibody in the middle of the culturing leads to CD3 ⁺ CD56 ⁺ cell Proliferation increased markedly, and the CD3 ⁺ CD56 ⁺ cell expansion culture rate during 14 days of culture increased markedly. In addition, the expansion culture rate further increased by combining AIC. These effects were exhibited regardless of the difference in the IFN-γ treatment method for PBMC.

From this, it became clear that the production method of the present invention is a method for efficiently obtaining CD3 ⁺ CD56 ⁺ cells, which are important constituents in CIK cells.

Example 3 CIK cell culture using anti-human CD3 antibody and retronectin (comparison of anti-human CD3 antibody concentration at restimulation)
(1) Preparation of PBMC AIC PBMC AIC was prepared in the same manner as in Example 2- (1) except that donor B-derived PBMC or donor C-derived PBMC were used instead of donor A-derived PBMC.

(2) CIK cell culture CIK cells were cultured in the same manner as in Example 2- (2). However, as PBMC, PBMC derived from donor B or PBMC derived from donor C (using the same donor-derived PBMC as the donor used in Example 3- (1)) was used, and IFN-γ the day before treatment was not performed. In addition, the concentration of the anti-human CD3 antibody at the time of restimulation with the anti-human CD3 antibody on the 8th day of the culture was set to 50 ng / mL or 200 ng / mL.

The culture is continued until the first 14 days of culture, and the number of viable cells is measured by trypan blue staining on the 14th day after the start of the culture. Was calculated. The results are shown in Table 7.

⁽³⁾ CD3 + by CD56 ⁺ cells and CD8 ⁺ cells Analysis embodiment of the content ratio 1- (4) and the same method, for example 3- (2) culture the fourteenth day after the initiation of the cells prepared CD3 ⁺ CD56 ⁺ cell content ratio was analyzed. The results are shown in Table 8.

As shown in Table 8, CD3 ⁺ CD56 ⁺ cells can be obtained by combining the anti-human CD3 antibody and retronectin to give initial stimulation to the cell population, and by re-stimulating with the anti-human CD3 antibody during the culture. Was obtained at a high ratio. These effects were exhibited regardless of the concentration of the anti-human CD3 antibody at the time of donor or restimulation.

From this, it became clear that the production method of the present invention is a method by which CD3 ⁺ CD56 ⁺ cells, which are important components in CIK cells, can be obtained efficiently.

(4) Evaluation of expansion culture rate of CD3 ⁺ CD56 ⁺ cells Example 1- (1) PBMC derived from donor B, donor C-derived PBMC and cells After the start of culture prepared in Example 3- (2) For each of the cells on day 14, the content ratio of CD3 ⁺ CD56 ⁺ cells was measured in the same manner as in Example 1- (4).

Using the calculated CD3 ⁺ CD56 ⁺ cell content ratio and the expanded culture rate (converted to the total number of cells) calculated in Example 3- (2), CD3 ⁺ CD56 ⁺ cells were observed during the culture period (14 days). The multiplication factor (ie, CD3 ⁺ CD56 ⁺ cell expansion culture rate) was calculated in the same manner as in Example 1- (5). The content ratio of CD3 ⁺ CD56 ⁺ cells at the start of culture was 1.62% for both donors. The results are shown in Table 9.

As shown in Table 9, by combining the anti-human CD3 antibody and retronectin to give initial stimulation to the cell population, and further stimulating again with the anti-human CD3 antibody during the culture, ⁺ Proliferation of CD56 ⁺ cells was markedly increased. These effects were exhibited regardless of the concentration of the anti-human CD3 antibody at the time of donor and restimulation.

From this, it became clear that the production method of the present invention is a method for efficiently obtaining CD3 ⁺ CD56 ⁺ cells, which are important components in CIK cells.

Example 4 CIK Cell Culture Using Gas-Permeable Culture Bag (1) Preparation of Anti-Human CD3 Antibody and RetroNectin Immobilized MultiLife 215 Gas-permeable Culture Bag MultiLife (Registered Trademark) Sealed to a Culture Area of 86 cm ² 10.4 mL of ACD-A solution containing anti-human CD3 antibody with a final concentration of 0.1 or 0.3 μg / mL and RetroNectin (registered trademark) with a final concentration of 5 μg / mL was added to 215 (manufactured by Takara Bio Inc.) Incubated for 5 hours at 37 ° C. in the presence of 5% CO ₂ . The thus prepared anti-CD3 antibody / retronectin-immobilized MultiLife 215 was washed 3 times with 1% HSA / physiological saline immediately before use.

(2) Expansion of CIK cell population 1.2 × 10 ⁷ cells of PBMC prepared in Example 1- (1) were added to the anti-CD3 antibody / retronectin-immobilized CultureLife 215 prepared in Example 4- (1). IL-2 was added to a final concentration of 1000 U / mL, and IFN-γ was added to a final concentration of 1000 U / mL. 0.5% autologous plasma (PBMC donor-derived inactivated plasma) ) And GT-T503 (hereinafter referred to as 0.5% plasma / 0.2% HSA / GT-T503) containing 0.2% HSA, and finally the total liquid volume was 40 mL. The culture bag to which the cell solution was added was cultured at 37 ° C. in the presence of 5% CO ² (culture day 0). On the fourth day of the culture, 23.4 mL of the cell solution was transferred to a gas permeable culture bag, MultiLife (registered trademark) Eva (manufactured by Takara Bio Inc.) sealed so that the culture area was 300 cm ² , and a final concentration of 500 U / mL. After adding IL-2 so that the total amount was 0.5 mL plasma / 0.2% HSA / GT-T503, the total liquid volume was finally adjusted to 300 mL. On the 8th day, 75 mL of the cell solution in the MultiLife (registered trademark) Eva was transferred to a gas permeable culture bag, MultiLife (registered trademark) Eva, which was newly sealed to have a culture area of 300 cm ^2. % Plasma / 0.2% HSA / GT-T503 was diluted 4-fold to a total volume of 300 mL, and IL-2 was added to a final concentration of 500 U / mL. Furthermore, restimulation was performed by adding an anti-human CD3 antibody to a final concentration of 50 ng / mL. On the 11th day, after diluting twice with 0.5% plasma / 0.2% HSA / GT-T503 to make the total volume 600 mL, IL-2 was added so that the final concentration was 500 U / mL. Added. Incubation continued until day 15. The number of viable cells was counted by trypan blue staining for the cells sampled on the 15th day after the start of the culture, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. Further, the CD3 + CD56 + cell content ratio was measured by the same method as in Example 1- (4). The results are shown in Table 10.

As shown in Table 10, even when the production method of the present invention was performed using a gas-permeable culture bag, a cell population containing CD3 ⁺ CD56 ⁺ cells at a high ratio was obtained at a high expansion culture rate. These effects were exhibited regardless of the anti-human CD3 antibody concentration at the time of initial stimulation.

Example 5 CIK cell culture using OK-432 (comparison with or without AIC)
(1) Preparation of anti-human CD3 antibody and retronectin-immobilized plate Anti-human CD3 antibody and retronectin-immobilized plate were prepared in the same manner as in Example 1- (2). However, the anti-human CD3 antibody was adjusted to a final concentration of 0.1 μg / mL.

(2) Expansion culture of CIK cell population Using the anti-human CD3 antibody and retronectin-immobilized plate prepared in Example 5- (1), culture was performed in the same manner as in Example 2- (2). However, the IFN-γ previous day treatment group was not set, and OK-432 (formulation name: Pishibanil: manufactured by Chugai Pharmaceutical Co., Ltd.) was added to all groups so that the final concentration was 0.05 KE / mL. Moreover, the influence of AIC addition was also examined. Incubation continued until day 15. The number of viable cells was counted by trypan blue staining for the cells sampled on the 15th day after the start of the culture, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. Further, the CD3 ⁺ CD56 ⁺ cell content ratio was measured by the same method as in Example 1- (4). The results are shown in Table 11.

As shown in Table 11, even when OK-432 was added, a cell population containing CD3 ⁺ CD56 ⁺ cells at a high ratio was obtained at a high expansion culture ratio. Moreover, the expansion culture rate increased significantly by adding AIC.

(3) Measurement of cytotoxic activity The cytotoxic activity of the cells prepared in Example 5- (2) on the 15th day after the start of the culture was measured. Cytotoxic activity is determined by measuring cytotoxic activity using Calcein-AM [Lichtenfels R.C. (Lichtenfelds R. et al.), J. Immunol. Methods, Vol. 172, No. 2, pp. 227-239 (1994)]. That is, the chronic myeloid leukemia cell line K562 cell and the lung cancer cell line A549 cell were suspended in RPMI 1640 containing 5% fetal calf serum (manufactured by HyClone) so as to be (1-2) × 10 ⁶ cells / mL, respectively. Thereafter, Calcein-AM (manufactured by Dojindo Laboratories) was added to a final concentration of 25 μM and cultured at 37 ° C. for 1 hour. The cells thus obtained were washed with a medium not containing Calcein-AM and used as Calcein-labeled target cells.

15% after the start of culture prepared in Example 5- (2) as effector cells, 5% Human AB type serum, 0.1 mM so as to be 3 × 10 ⁶ cells / mL, 1 × 10 ⁵ cells / mL After dilution with RPMI 1640 containing NEAA mixture, 1 mM sodium pyruvate, 2 mM L-glutamine (all manufactured by Lonza), and 1% penicillin-streptomycin (manufactured by Gibco BRL) or 100 μg / mL streptomycin sulfate (manufactured by Meiji Seika) 100 μL / well was dispensed into each well of a well cell culture plate (Corning). Subsequently, 100 μL of Calcein-labeled target cells prepared to 1 × 10 ⁵ cells / mL were added to each well. At this time, the ratio of the effector cell (E) to the Calcein-labeled target cell (T), that is, the E / T ratio, was set to two values of 30 and 10. The plate containing the cell suspension was centrifuged at 400 × g for 1 minute and then incubated at 37 ° C. in the presence of 5% CO ₂ for 4 hours. Thereafter, 100 μL of the culture supernatant was collected from each well, and the amount of calcein released into the culture supernatant was measured with a fluorescent plate reader (Mithras LB 940: manufactured by Bertrud) (excitation 485 nm / measurement 535 nm). “Cytotoxic activity (%)” was calculated according to the following formula (2).

In the above formula, the minimum release amount is the calcein release amount of a well containing only calcein labeled target cells, and indicates the calcein spontaneous release amount from the calcein labeled target cells. The maximum release amount indicates the release amount of Calcein when the cell is completely destroyed by adding 0.1% surfactant Triton X-100 (manufactured by Nacalai Tesque) to Calcein-labeled target cells. The results of the cytotoxic activity measurement are shown in Table 12.

As shown in Table 12, the cell population obtained by the production method of the present invention exhibited high cytotoxic activity. Moreover, it became clear that the cell population which shows high cytotoxic activity with respect to a lung cancer cell line can be obtained by adding AIC.

Example 6 Gene introduction into CIK cells (1) Preparation of anti-human CD3 antibody and retronectin-immobilized plate An anti-human CD3 antibody and retronectin-immobilized plate were prepared in the same manner as in Example 1- (2). However, the anti-human CD3 antibody was adjusted to a final concentration of 0.3 μg / mL.

(2) Construction of AcGFP Expression Retroviral Vector Plasmid An AcGFP expression vector MT-AcGFP was prepared as follows. Using pIRES2-AcGFP1 (manufactured by Clontech) as a template, PCR was performed using an AcGFP5 primer consisting of the nucleic acid sequence shown in SEQ ID NO: 10 of the sequence listing and an AcGFP3 primer consisting of the nucleic acid sequence shown in SEQ ID NO: 11 of the sequence listing, The amplified fragment was inserted into a pMT vector [pM vector described in Gene Therapy Vol. 7, Item 797-804 (2000)] to prepare an MT-AcGFP plasmid.

(3) Production of Producer Cells Eco-friendly from 293T cells transformed with plasmids pGP, pE-eco and MT-AcGFP prepared in Example 6- (1) included in Retrovirus Packaging Kit (manufactured by Takara Bio Inc.) A retrovirus with a tropic envelope was made. The obtained retrovirus was infected with producer cell PG13 (ATCC #: CRL-10686) three times, and a plurality of clones were obtained by limiting dilution. A clone of one strain was selected based on the measurement result of the amount of viral RNA in the culture supernatant by real-time PCR and the result of the positive cell rate of AcGFP measured by flow cytometry for the cultured cells in contact with the supernatant. Was used as a retroviral vector producer cell in the following experiments.

(4) Preparation of virus solution The culture supernatant of the producer cells produced in Example 6- (3) was removed, washed once with DPBS, treated with Trypsin / EDTA, and the cells were detached, and 10% fetal bovine After suspending in serum (manufactured by Invitrogen) and D-MEM (manufactured by Sigma Aldrich) containing 50 U / mL penicillin / 50 mg / mL streptomycin (manufactured by Nacalai Tesque), the cell density was 25000 in 100 mm dish (manufactured by Iwaki). It seed | inoculated so that it might become a cell / cm < ² >. After culturing for 24 hours, the culture supernatant was removed, and 8 mL of fresh medium was added. After further culturing for 24 hours, the supernatant was collected, filtered through a 0.45 μm filter, and stored at −80 ° C.

(5) Preparation of retronectin-immobilized plate (at gene introduction)
A DPBS solution containing anti-human CD3 antibody having a final concentration of 5 μg / mL and Retronectin (registered trademark) having a final concentration of 25 μg / mL was added to a 12-well non-treatment plate (manufactured by Corning) at a rate of 0.95 mL / well. Next, after incubation at 37 ° C. for 5 hours in the presence of 5% CO _{2, the} DPBS solution was removed from each well to obtain a cell culture plate on which the anti-CD3 antibody and retronectin were immobilized. The plate prepared here was washed 3 times with DPBS and then used for each of the following experiments.

(6) Preparation of gene transfer plate (immobilization of virus)
1 mL / well of the virus solution prepared in Example 6- (4) was added to the plate prepared in Example 6- (5), and centrifuged at 32 ° C. and 1000 × g for 2 hours. However, wells to which no virus solution was added were also prepared. At this time, virus diluted solution containing no virus (D-MEM containing 5% fetal bovine serum) was added at 1 mL / well and centrifuged in the same manner. After centrifugation, the virus solution was removed by suction and washed by adding 1 mL / well of DPBS containing 1.5% HSA.

(7) Gene transfer to CIK cell population and expansion culture Culture using the anti-human CD3 antibody and retronectin immobilized plate prepared in Example 6- (1) in the same manner as in Example 2- (2) Went. However, the IFN-γ previous day treatment group was not set. Furthermore, gene transfer (hereinafter referred to as a single infection group) on the fourth day of culture, gene transfer (hereinafter referred to as a double infection group) on days 4 and 5 after the start of culture, A non-gene transfer group (hereinafter referred to as a control group) that does not infect the virus was set to the infection group. That is, in the single infection group, 0.4 mL of the cell solution was transferred to the plate prepared in Example 6- (6) on the 4th day from the start of culture, and 0.5% HAB / 0.2% HSA / GT-T503 was transferred. Of IL-2 was added to a final concentration of 500 U / mL. The culture was continued on this plate until the 8th day. In the twice infected group, 1.5 mL of the cell solution was transferred to the plate prepared in Example 6- (6) on the 4th day from the start of the culture, and 0.5% HAB / 0.2% HSA / GT-T503 was added as 1. After adding 5 mL and diluting 2-fold, IL-2 was added to a final concentration of 500 U / mL. Furthermore, on the fifth day of the culture, 0.8 mL of the cell solution was transferred to the plate prepared in Example 6- (6), and 4.2 mL of 0.5% HAB / 0.2% HSA / GT-T503 was added. IL-2 was added to a final concentration of 500 U / mL after doubling dilution. The culture was continued on this plate until the 8th day. In the control group, the same operation was performed using a plate to which no virus was fixed in both the once-infected group and the twice-infected group. The culture was continued until day 14. The number of viable cells was counted by trypan blue staining for the cells sampled on the 14th day after the start of the culture, and the expansion culture rate was calculated by comparison with the number of cells at the start of the culture. Further, the CD3 ⁺ CD56 ⁺ cell content ratio was measured by the same method as in Example 1- (4). Furthermore, the expression rate of the protein AcGFP expressed by gene introduction was analyzed with a flow cytometer as gene introduction efficiency. The results are shown in Table 13.

As shown in Table 13, even when gene transfer was performed during CIK cell expansion culture, CIK cells expressing the target gene were obtained while maintaining a high CD3 ⁺ CD56 ⁺ cell ratio. It was also found that the higher the number of infections, the higher the gene transfer efficiency.

The present invention makes it possible to provide a large amount of CIK cells containing a high proportion of CD3-positive CD56-positive cells, which is said to be a sub-cell population suitable for adoptive immunotherapy. The cell population obtainable by the present invention is useful in adoptive immunotherapy.

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.
SEQ ID NO: 10; Designed oligonucleotide primer to amplify DNA fragment encodeing AcGFP.
SEQ ID NO: 11; Designed oligonucleotide primer to amplify DNA fragment encodeing AcGFP.

Claims

A method for producing cytokine-induced killer cells, comprising the following steps (a) to (c):
(A) culturing a cell population containing cells capable of differentiating into CD3-positive CD56-positive cells and / or CD3-positive CD56-positive cells in the presence of a CD3 ligand;
(B) culturing the cell population obtained in step (a) in the absence of CD3 ligand; and (c) culturing the cell population obtained in step (b) in the presence of CD3 ligand.
The production method according to claim 1, wherein the culture of the cell population in at least one of steps (a) to (c) is performed in the presence of interferon-γ and / or interleukin-2.
The production method according to claim 1, wherein the CD3 ligand is an anti-CD3 antibody.
The production method according to claim 1, wherein the culture in the step (a) is performed in the presence of a fibronectin fragment and a CD3 ligand.
The production method according to claim 4, wherein the fibronectin fragment contains a region selected from the group consisting of:
A VLA-4 binding region;
A VLA-5 binding region; and a heparin binding region.
A cytokine-induced killer cell obtainable by the method according to any one of claims 1 to 5.
A medicament comprising the cytokine-induced killer cell according to claim 6 as an active ingredient.
A method for treating or preventing a disease, comprising a step of administering an effective amount of the cytokine-induced killer cell according to claim 6 to a subject.
The cytokine-induced killer cell according to claim 6, for use in the treatment of a disease.
A method for producing cytokine-induced killer cells, comprising the following steps (A) to (D):
(A) a step of culturing a cell population capable of differentiating into a CD3 positive CD56 positive cell and / or a cell population containing a CD3 positive CD56 positive cell in a medium containing a CD3 ligand;
(B) reducing the CD3 ligand concentration in the medium;
(C) culturing the cell population in a medium having a reduced CD3 ligand concentration; and (D) adding CD3 ligand to the medium and further culturing the cell population.