CN101580817A - Method for preparing cell group containing cytokine-induced killing cell - Google Patents

Abstract

The present invention provides a method for producing a cell group containing cytokine-induced killer cells, comprising the step of culturing the cell group containing cells capable of differentiating into cytokine-induced killer cells in the presence of fibronectin fragments. The present invention also provides a cell group containing the cytokine-induced killer cells obtained by the above method. The present invention also provides a medicine containing the above-mentioned cell population as an active ingredient. The invention also provides the application of the cell population in medicine preparation.

Description

Method for producing cell population containing cytokine-induced killer cells

technical field

The present invention relates to a highly efficient cell group containing cytokine-induced killer (CIK) cells in adoptive immunotherapy, a production method of the cell group and the like. The proliferation of regulatory T cells (regulatory T cells; Treg cells) in the cell population containing CIK cells involved in the present invention is inhibited, and can be used for early and advanced cancers and various cell proliferation diseases including hematopoietic cells and solid tumors Treatment.

Background technique

In recent years, people have re-examined the treatment methods that cause severe physical pain to patients, such as drug therapy and radiation therapy, and began to pay more and more attention to less painful immunotherapy. The therapy includes adoptive immunotherapy, in which the immune-related cells taken out of the body are cultured to increase the number of cells and/or enhance the activity related to the therapeutic effect, and then transplanted to the patient.

Cells used as effector cells in adoptive immunotherapy include lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TIL) and cytokine-induced killer (CIK) cells. cell.

LAK cells are a cell population obtained by proliferating 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 co-existing anti-CD3 antibody and IL-2 at the time of culture has been developed.

TILs are T cells that infiltrate into tumor tissues, and have more significant tumor antigen specificity than LAK cells. The cells can also be propagated in vitro and used in therapy. However, TILs can only be obtained by removing the patient's tumor tissue, so the collection operation is complicated and the number of cells obtained is very small.

CIK cells can be prepared by culturing peripheral blood mononuclear cells (PBMC) in the presence of interferon (IFN)-γ, anti-CD3 antibody, and IL-2. CIK cells are a rare cell population among PBMCs characterized by being positive for CD3 and CD56, but this cell population can be preferentially proliferated in the absence of target cells. CIK cells exhibit superior cell killing activity against tumor cells in vivo than LAK cells (for example, Non-Patent Document 1).

Fibronectin is a huge glycoprotein with a molecular weight of 250,000 that exists in animal blood, on the surface of cultured cells, and in the extracellular matrix of tissues, and is known to have various functions. It has seven structural domains (see Figure 1). Furthermore, its amino acid sequence contains three similar sequences, and the whole consists of repetitions of the above-mentioned sequences. The three similar sequences are called type I, type II, and type III, respectively. Among them, type III consists of 71-96 amino acid residues, and the identity rate of these amino acid residues is 17-40%.

A variety of fibronectin fragments have been produced by recombinant DNA technology, and some of them are expected to have the effect of inhibiting cancer metastasis (Non-Patent Document 2). In addition, it has also been reported that a fibronectin fragment having a heparin-binding domain has a function of promoting retrovirus infection of cells (Non-Patent Document 3).

In the immunotherapy aimed at transplanting lymphokine-activated cells induced by in vitro cytotoxic T cells (cytotoxic T cells; CTL) or peripheral blood lymphocytes under the action of IL-2 and anti-CD3 antibodies, how to The effects of using fibronectin or its fragments to maintain cytotoxic activity when amplifying and culturing antigen-specific CTLs induced in vitro and how to efficiently amplify and culture lymphocytes in vitro have been discussed (for example, patent documents 1 to 3 ).

Non-Patent Literature 1 J. Immunol., 1994, Vol.153, p1687-1696

Non-Patent Literature 2 J.Biochem., 1991, Vol.110, No.2, p284-291

Non-patent literature 3 Nature Medicine, 1996, Vol.2, No.8, p876-882

Patent Document 1 International Publication No. 03/016511 Pamphlet

Patent Document 2 International Publication No. 03/080817 Pamphlet

Patent Document 3 International Publication No. 2005/019450 Pamphlet

Contents of the invention

During the amplified culture of CIK cells, PBMCs differentiate into multiple cell populations with different properties, so the proportion of CIK cells in the amplified cell population must not be high. On the other hand, the differentiated cell population also contains a cell population that has the function of controlling or suppressing immunity. An object of the present invention is to provide a method for scale-up culture of a cell population for cancer treatment with a small content of unnecessary cell populations for producing CIK cells, which is a preferred cell population for adoptive immunotherapy.

Next, the present invention is outlined. The first invention of the present invention relates to a method for producing a cell population containing CIK cells, comprising the step of culturing the cell population containing cells capable of differentiating into cytokine-induced killer cells in the presence of fibronectin fragments . The aspect of the first invention can provide a method for producing a cell population containing CIK cells cultured in the presence of a fibronectin fragment containing a region selected from the VLA-4 binding domain, the VLA-5 binding domain, and the heparin binding domain .

In addition, as a preferable example of the fibronectin fragment used in the first invention, a fibronectin fragment containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 5 can be mentioned. Furthermore, as a preferred embodiment of the first invention, there is also provided a method for producing a cell population containing CIK cells cultured in the presence of fibronectin fragments in the presence of CD3 ligand, interferon-γ, and interleukin-2. The CD3 ligand is not limited as long as it can be used in the present invention, and it is preferably carried out using an anti-CD3 antibody.

The second invention of the present invention relates to a cell population containing CIK cells obtained by the method of the first invention of the present invention.

The third invention of the present invention relates to a medicine 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 to a subject an effective amount of the cell population of the second invention of the present invention. In addition, the disease may be any disease as long as it is susceptible to the cell population of the second invention of the present invention. In addition, the cell population of the second invention may be a cell population derived from a subject (patient) suffering from a disease requiring treatment or prevention and receiving the administration of the cell population, or may be derived from a donor other than the subject in some cases. cell population.

The fifth invention of the present invention relates to the use of the cell group of the second invention for the production of pharmaceuticals.

The production method of the present invention provides a production method capable of mass-scale-up culturing a cell population rich in CIK cells having high cytotoxic activity. The high-quality cell population obtained in large quantities by this production method exerts a highly effective therapeutic effect in the living body, and therefore is extremely useful for the treatment of diseases using cell medicine.

Description of drawings

Fig. 1 is a schematic diagram showing domains of fibronectin.

Figure 2 is a graph showing the phenotype of cell populations obtained by scale-up culture.

Detailed ways

The present inventors have completed the present invention by finding that by culturing PBMCs in the presence of fibronectin fragments, cells expressing both molecules CD3 and CD56 on the cell surface contain a high proportion of the resulting cell population.

Next, the present invention will be specifically described.

(1) Fragment of fibronectin used in the present invention

The fragments of fibronectin described in this specification may be any of fragments obtained naturally (fragments obtained by enzymatic fragmentation of natural fibronectin, etc.) or fragments obtained by DNA recombination technology. The fibronectin fragment can be obtained, for example, according to the content disclosed in Rust (Luosura-Tei) E. et al. [J.Biol.Chem., Vol. Substances derived from nature are manufactured in a substantially pure form. Here, the substantially pure fibronectin fragment referred to in this specification means that it does not substantially contain other proteins that naturally exist together with fibronectin. In the present invention, a single molecular species may be used for the fibronectin fragment, or a mixture of multiple molecular species may be used.

Fibronectin fragments that can be used in the present invention and useful information on the preparation of the fragments can be found in Non-Patent Document 2 and Koarnblihtt A.R. et al. [EMBO J., Vol. 4, No. 7, 1755-1759 (1985)], Sekiguti K. et al [Biochemistry, Vol. 25, No. 17, 4936-4941 (1986)] et al. In addition, the nucleic acid sequence encoding fibronectin or the amino acid sequence of fibronectin has been disclosed in Genbank Accession No. NM_002026, NP_002017.

In the present invention, fibronectin fragments having cell adhesion activity and/or heparin binding activity can be preferably used.

In fibronectin, there is a region that has the activity of binding to integrin on the cell surface. Examples of the aforementioned domains include VLA (very late antigen)-4 and VLA-5 binding domains.

A region called IIICS exists near the C-terminal side of fibronectin. It contains a region consisting of 25 amino acid sequences called CS-1, which exhibits 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.

In addition, fibronectin has a repeat sequence called type III, and there is a cell-binding region in the tenth type III repeat sequence from the N-terminal side. The amino acid sequence of the tenth type III repeat sequence is shown in SEQ ID NO: 2 in the sequence listing. Among the above sequences, the sequence that plays a major role in binding to VLA-5 is Arg-Gly-Asp-Ser (RGDS) four amino acids shown in SEQ ID NO: 3 in the Sequence Listing. C-274, which consists of the amino acid sequence shown in SEQ ID NO: 4, is a polypeptide containing the amino acid sequence of SEQ ID NO: 2, and is a recombinant fibronectin fragment with strong cell adhesion activity.

In addition, fibronectin has heparin-binding activity. The heparin-binding domain of fibronectin corresponds to the 12th to 14th from the N-terminal side of the above-mentioned 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 composed of this heparin-binding domain.

In the present invention, in addition to fragments containing each region alone, fragments in which two or more of the above-mentioned regions are directly or through a suitable linker (leader) can also be used. The regions derived from fibronectin contained in the above fragments may be the same or different. As a fragment of fibronectin having multiple binding domains in the molecule, H-296 (amino acid sequence represented by SEQ ID NO: 6 in the Sequence Listing) containing a VLA-4 binding domain and a heparin binding domain, a fragment containing a VLA-5 binding CH-271 (the amino acid sequence represented by the sequence number 7 of the sequence listing) of the VLA domain and the heparin binding domain, CH-296 (represented by the sequence number 8 of the sequence listing) containing the VLA-4 binding domain, the VLA-5 binding domain and the heparin binding domain amino acid sequence), polypeptides such as C-CS1 (amino acid sequence represented by SEQ ID NO: 9 in the Sequence Listing) containing a VLA-4 binding domain and a VLA-5 binding domain. The above-mentioned various polypeptides are described in Non-Patent Document 2, and can be produced based on the contents disclosed therein. CH-296 is marketed by Takara Biotechnology Co., Ltd. under the name Retronectin (Retronectin: registered trademark).

As the fragment used in the present invention, as long as the desired effect of the present invention can be obtained, it may have the same function as the fragment containing at least part of the amino acid sequence of natural fibronectin exemplified above. A fragment of a polypeptide whose amino acid sequence has undergone one or more amino acid substitutions, deletions, insertions or additions. For example, a fragment obtained by deleting one or two type III repeat sequences from C-274 or H-271.

The cell-adhesive activity can be obtained by measuring the binding of the fragment (the cell-binding domain thereof) used in the present invention to cells by a known method. For example, the above method includes the method of Williams D.A. et al. [Nature, Vol. 352, pp. 438-441 (1991)]. This method is a method of measuring the binding of cells to immobilized fragments on culture plates. The heparin-binding activity can be determined by measuring the heparin-binding activity of the fragment (the heparin-binding domain) used in the present invention by a known method. For example, the binding of fragments to heparin can be evaluated by the same method by replacing the cells in the method of Williams D.A. et al. with heparin, for example, using labeled heparin.

In view of the ease of acquisition and handling, the uniformity of quality, and the low risk of virus contamination, it is preferable to use recombinant fibronectin fragments in the present invention. The molecular weight of the fibronectin fragment used in the present invention is not particularly limited, but is preferably 1 to 200 kD, more preferably 5 to 190 kD, more preferably 10 to 180 kD. This molecular weight can be measured, for example, by SDS-polyacrylamide gel electrophoresis.

(2) Method for producing a cell population containing CIK cells

Next, a method for producing a CIK cell-containing cell population according to the present invention will be specifically described. The present invention relates to a method for producing a cell population enriched for CIK cells, a subpopulation of cells characterized by being CD3 positive and CD56 positive. The method of the present invention is characterized by comprising the step of culturing a cell population containing cells capable of differentiating into CIK cells in the presence of the above-mentioned fibronectin fragment.

The cell population containing cells capable of differentiating into CIK cells used in the production method of the present invention may, for example, be a cell population obtained from peripheral blood, bone marrow, or umbilical cord blood, or a blood cell-like cell population isolated from these materials. The present invention preferably uses PBMCs. The above-mentioned cell populations can be fresh cell populations collected from organisms or cryopreserved cell populations, both of which can be used in the present invention.

The concentration of fibronectin fragments in the production method of the present invention is not particularly limited, but is preferably, for example, 0.001 to 500 μg/mL, particularly 0.01 to 500 μg/mL. In addition to dissolving and co-existing in the culture medium, fibronectin fragments can also be used on suitable solid phases such as Petri dishes, flasks, bags and other cell culture equipment (vessels for culture; including open systems and closed systems) or beads, Used after being immobilized on a carrier for cell culture such as a membrane or glass slide. Materials for these solid phases are not particularly limited as long as they can be used in cell culture. The amount of immobilized fibronectin is selected according to the same ratio as the concentration required when the above-mentioned equipment or carrier is used for culture, as long as the desired effect can be obtained. limit.

The immobilization method of the fibronectin fragment on the solid phase is not particularly limited, and for example, the immobilization can be carried out by bringing the fragment dissolved in an appropriate buffer into contact with the solid phase. In addition, the immobilization of fibronectin fragments can also be carried out by the method described in International Publication No. 97/18318 pamphlet and International Publication No. 00/09168 pamphlet.

In a preferred embodiment of the present invention, the culture 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 has CD3-binding activity, and examples thereof include anti-CD3 antibodies, particularly preferably anti-CD3 monoclonal antibodies, such as OKT3 [J. Immunol., Vol. 124, No. 6 No. 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, it is preferably, for example, 0.001 to 500 μg/mL, especially 0.01 to 100 μg/mL. The CD3 antibody can also be used after being immobilized on a cell culture device or a cell culture carrier by the same procedure as the fibronectin fragment.

If fibronectin fragments and CD3 ligands are immobilized on a solid phase, the above-mentioned components can be easily separated from the obtained cell population by simply separating the cells from the solid phase after the culture, and the above-mentioned components can be prevented from mixing into the cell population. .

The medium used in the production method of the CIK cell-containing cell population of the present invention is not particularly limited, and known mediums that can be used in lymphocyte expansion culture and the like can be used, for example, commercially available mediums can be appropriately selected and used. The above-mentioned medium may contain cytokines, appropriate proteins and other components in addition to its original constituents. Generally, the present invention uses a medium containing IFN-γ and IL-2. The concentration of IFN-γ in the medium is not particularly limited, and may be 50-10000 U/mL, more preferably 100-5000 U/mL. The concentration of IL-2 in the medium is also not particularly limited, and may be 10-5000 U/mL, preferably 50-2000 U/mL. In addition, cytokines such as IL-1α, IL-7, and IL-12 may be added to the medium. The concentration of this component in the medium is not particularly limited as long as the desired effect can be obtained.

Serum or plasma can be added to the medium for culturing. The amount of them added in the culture medium is not particularly limited, such as greater than 0% to 20% by volume, and the amount of serum or plasma can be changed according to different culture stages. For example, it can be used by reducing the serum or plasma concentration in stages. In addition, the source of serum or plasma may be self (meaning the same source as the cultured cells) or non-self (meaning different source from the cultured cells), from the viewpoint of safety , preferably self-sourced serum or plasma. In addition, isolated and purified serum components such as human serum albumin may also be added.

Production of the CIK cell-containing cell population of the present invention is carried out using the above-mentioned various components and culture medium in addition to the fibronectin fragment. The number of cells at the beginning of the culture used in the present invention is not particularly limited, for example, preferably 1×10 ⁴ cells/mL to 1×10 ⁸ cells/mL, more preferably 1×10 ⁵ cell/mL to 5×10 ⁷ cells/mL . In addition, the culture conditions are not particularly limited, and conditions commonly used for cell culture can be used. For example, it can be cultured under conditions such as 37°C and 5% CO ₂ . Operations such as adding fresh medium at appropriate intervals to dilute the cell culture solution, replacing the medium, or replacing equipment for cell culture can also be performed.

Cell culture equipment (containers) such as petri dishes, flasks, bags, large culture tanks, and bioreactors can be used in the production method of the present invention. As the bag, a CO ₂ gas-permeable bag for cell culture is preferable. In cases where large numbers of cells are required, large culture tanks can be used. Culturing can be carried out in either an open system or a closed system, but it is preferable to carry out cultivation in a closed system.

In the method for producing a CIK cell-containing cell population of the present invention, the culture period is not particularly limited, and for example, culture can be performed for a total of 4 to 30 days, preferably 6 to 25 days. In the production method of the present invention, the culture in the presence of the fibronectin fragment may be the entire period of the culture period, or may be an arbitrary part of the period. That is, as long as part of the cell culture process includes the culture in the culture process in the presence of fibronectin fragments, it belongs to the present invention. It is particularly preferred to carry out the culture in the presence of the fibronectin fragment at least in the initial stage of the entire culture period, more preferably to start the culture in the presence of the fibronectin fragment. The cultivation in the presence of the active ingredient of the present invention is preferably carried out at least 1 day or more, more preferably 2 days or more after the initiation of the culture. When a CD3 ligand is used for culture, the period of use is not particularly limited. For example, when a fibronectin fragment is used, the CD3 ligand is allowed to coexist.

In a preferred embodiment of the present invention, fibronectin fragments and CD3 ligands are used only in the initial stages of the entire culture period. For example, from the beginning of the culture to the seventh day, preferably from the beginning of the culture to the fifth day, the culture is carried out in the presence of the fibronectin fragment and the CD3 ligand, and then under the condition that neither of these two components is present. The culture is continued under the above conditions to produce a cell population containing CIK cells, but the present invention is not limited thereto.

In addition, the production method of the present invention may further include the step of further isolating CD3-positive and CD56-positive cell populations from the cell populations obtained by the above method. Separation can be performed by known means using, for example, a cell sorter, magnetic beads, an affinity column, and the like. The cell population thus separated is rich in cells with high cytotoxic activity, and it can be expected to exert a better therapeutic effect.

(3) Cell populations containing CIK cells

The present invention provides a CIK cell-containing cell population obtained by the above method for producing a CIK cell-containing cell population of the present invention. The CIK cell-containing cell population of the present invention exhibits stronger cytotoxicity in vivo because it contains a higher ratio of CIK cells, that is, CD3-positive CD56-positive cells, than the CIK-cell-containing cell population produced by conventional methods activity, to achieve a good therapeutic effect. In addition, the above-mentioned cell population also has the following advantages, that is, the content of regulatory T cells (regulatory T cells; Treg cells) capable of suppressing cellular immunity is low, so it is very beneficial for therapeutic application.

The present invention also provides a drug (therapeutic or preventive) containing the CIK cell-containing cell population as an active ingredient. The above-mentioned therapeutic agents containing this cell population are suitable for use in immunotherapy. In immunotherapy, for example, by intravenous, arterial, subcutaneous, intraperitoneal administration methods such as injection or drip, a CIK cell-containing cell population suitable for patient treatment is administered to a patient. Therapeutic agents of the present invention are very useful for the treatment of diseases that are sensitive to CIK cells such as cancer, leukemia, malignant tumors, hepatitis or infectious diseases (such as influenza, tuberculosis, AIDS, MRSA infection, VRE infection, deep mycoses) and the like. Useful, but the present invention is not limited thereto. In addition, this therapeutic agent can also be used in donor lymphocyte transfusion for the purpose of preventing and treating infectious diseases after bone marrow transplantation or radiation exposure, and alleviating relapsed leukemia symptoms.

The therapeutic agent and preventive agent of the present invention can be prepared according to well-known methods in the field of pharmacy, for example, the cell population prepared by the method of the present invention is used as an active ingredient, and a known organic or inorganic carrier suitable for parenteral administration, Excipients, stabilizers, etc. are mixed to make drips and injections. In addition, 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 related to the therapeutic agent can be appropriately determined according to known immunotherapy. For example, the content of the CIK cell-containing cell population of the present invention in the drug is not particularly limited, for example, preferably 1×10 ³ to 1×10 ¹¹ cells/mL, more preferably 1×10 ⁴ to 1×10 ¹⁰ cells/mL, More preferably 1×10 ⁵ -2×10 ⁹ cells/mL. In addition, the dosage of the drug of the present invention is not particularly limited. For example, for adults, it is preferably 1×10 ⁶ to 1×10 ¹² cells/day, more preferably 1×10 ⁷ to 5×10 ¹¹ cells/day, More preferably, it is 1×10 ⁸ to 2×10 ¹¹ cells/day. In addition, immunotherapy using this therapeutic agent can be used in combination with known drug therapy by administration of drugs, radiation therapy, and surgical treatment.

The method for producing a cell population containing CIK cells of the present invention may further include a step of introducing a foreign gene into the cell population. In addition, "exogenous gene" refers to a gene artificially introduced into a CIK cell to be introduced into a gene, and also includes a gene derived from the same kind of cell as the cell to be introduced into.

The means for introducing the foreign gene is not particularly limited, and an appropriate method can be selected and used from known gene introduction methods. The gene introduction step can be carried out at any time in the production method of the present invention. From the viewpoint of work efficiency, for example, it is preferably performed simultaneously with, during, or after the production of the above-mentioned cell population. Gene introduction may or may not be performed using viral vectors. The specific content of the method has been disclosed in many documents.

The aforementioned viral vectors are not particularly limited, and generally known viral vectors used in gene transfer methods, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, simian virus vectors, vaccinia virus vectors, or Sendai virus vectors are used wait. Retroviral vectors and lentiviral vectors capable of stably integrating foreign genes into the chromosomal DNA of cells to be introduced are particularly preferred. As the above-mentioned viral vector, a replication-deficient vector that cannot self-replicate in infected cells is preferable. In addition, at the time of gene transfer, a substance capable of improving gene transfer efficiency, such as Lunacuzin (registered trademark, manufactured by Takara Biotechnology Co., Ltd.), can also be used.

As a gene transfer method not using a viral vector, a method using a carrier such as ribosome, ligand-polylysine, or a calcium phosphate method, electroporation method, gene gun method, or the like can be used. At this time, a foreign gene integrated in 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 above-mentioned cells (for example, genes encoding proteins such as enzymes, cytokines, receptors, and genes encoding antisense nucleic acid or siRNA (small interfering RNA), ribozymes can be used. Gene). These foreign genes are used, for example, by being inserted into vectors or plasmids to be expressed under the control of an appropriate promoter. In addition, control sequences such as enhancer sequences or termination sequences may also exist in the vector.

According to the method of the present invention, drug resistance can be imparted to a cell population including CIK cells by introducing genes encoding enzymes related to resistance to drugs used in the treatment of cancer and other patients (for example, multidrug resistance genes). Using such a cell population can combine immunotherapy and drug therapy, thereby achieving better therapeutic effects. On the other hand, in contrast to the above-mentioned method, by introducing a gene (for example, a thymidine kinase gene) that confers sensitivity to a specific drug, it is also possible to impart sensitivity to the drug to a cell population including CIK cells. In this case, the cells transplanted into the living body can be removed 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 the CIK cell-containing cell population obtained by the above method. The subject in this specification is, for example, a patient suffering from the above-mentioned diseases.

The effective amount in this specification refers to the amount of the above-mentioned cell group containing CIK cells that can achieve therapeutic or preventive effects after administration of the cell group. The specific effective amount is appropriately set depending on the form of administration, the method of administration, the purpose of use, and the age, body weight, and symptoms of the subject, and is not a fixed value. The method of administration is not limited, and for example, it can be administered by drip or injection similarly to the above-mentioned medicines.

In addition, the present invention also provides the use of CIK cell-containing cell populations for the manufacture of pharmaceuticals. The manufacturing method of this drug is the same as that of the above-mentioned drug. In addition, there are no special restrictions on the diseases for which this drug is administered, and it is the same as the above-mentioned drugs.

Example

Below, the present invention will be described more specifically in conjunction with examples, but the present invention is not limited thereto.

Example 1 Scaled-up culture of CIK cells using fibronectin fragment CH-296

(1) Isolation of PBMC

After the blood components were collected from 5 patients who gave informed consent (2 for kidney cancer, 1 for lung cancer, 1 for liver cancer, and 1 for thyroid cancer), the cell fraction containing PBMC was superimposed and centrifuged on Ficoll, and the peripheral blood mononuclear cells in the middle layer were recovered (PBMC).

(2) Immobilization of anti-human CD3 antibody and CH-296

Into a T225 flask was added a phosphate-buffered physiological solution containing an anti-human CD3 antibody at a final concentration of 5 μg/mL and CH-296 (Lacronac (registered trademark), manufactured by Takara Biotechnology Co., Ltd.) at a final concentration of 15 μg/mL. 40 mL of saline (PBS) or 40 mL of PBS containing only anti-human CD3 antibody at a final concentration of 5 μg/mL, and then incubated overnight at 4°C. PBS containing the antibody and CH-296 was removed by suction from the culture equipment before use, and washed twice with PBS for each test.

(3) Scale-up culture of CIK cells

The PBMC 9×10 ⁸ cells isolated in Example 1-(2) were suspended in 60 ml of GT-T503 medium containing 0.2% human serum albumin, 1000 U/mL IFN-γ, and 300 U/mL IL-2 ( Takara Bioengineering Co., Ltd., hereinafter referred to as "complete GT-T503 medium"), were added to the four anti-CD3 antibodies prepared in Example 1-(2) and the CH-296-immobilized T225 flasks or four anti-CD3 CD3 antibody was immobilized in a T225 flask, and culture was started at 37° C. in 5% CO ₂ (cultivation day 0).

On the first day (the day after the start of the culture), 150 mL of complete GT-T503 medium was added to each flask, and the culture was continued.

On the fourth day, all the cells suspended in the culture medium in each flask were collected, and transferred to an unimmobilized gas-permeable culture bag CultiLife Eva (manufactured by Takara Bioengineering Co., Ltd.). At this point, add complete GT-T503 medium to make each bag 700 mL, and culture at 37°C in 5% CO ₂ .

On day 7 and day 9, 400 mL of complete GT-T503 medium was added to each bag for subculture, and the culture was continued until day 14. The final culture solution in each bag is 1500mL.

Calculate the number of viable cells on the last day of culture, compare it with the number of cells at the beginning of culture, and calculate the scale-up culture rate. The results are shown in Table 1. In addition, the increased CIK cells stimulated with anti-CD3 antibody and CH-296 were designated as R-CIKs, and the increased CIK cells stimulated with only anti-CD3 antibody were designated as C-CIKs.

Table 1

Amplified culture rate (fold) C-CIKs 9.2 R-CIKs 35.7

As can be seen from Table 1, the increase rate of CIK cells can be significantly improved by allowing fibronectin fragments to coexist at the initial stage of culture.

(4) CIK cell phenotype analysis

After the cells were stained with fluorescently labeled antibodies, they were provided to a flow cytometer to analyze the phenotypes of the C-CIKs and R-CIKs prepared in Example 1-(3). CD3+ cells, CD4+ cells, and CD8+ cells were stained with anti-CD3 antibody, anti-CD4 antibody, and anti-CD8 antibody, respectively. CD3+CD56+ cells were stained with anti-CD3 antibody and anti-CD56 antibody and evaluated as double positive cells. Treg cells were stained with anti-CD4 antibody, anti-CD25 antibody, and anti-CD127 antibody, and evaluated as CD4+CD25+CD127- cells.

The result is shown in Figure 1. In the figure, the abscissa indicates the detected surface antigen, and the ordinate indicates the positive rate of the surface antigen.

As can be seen from FIG. 1 , the ratio of Treg cells was significantly decreased in the cell population prepared using fibronectin fragments during stimulation. It is well known that Treg cells have the effect of reducing anti-tumor immune activity, so the low ratio of Treg cells in the CIK cells to be transplanted means that a good anti-tumor effect can be obtained.

Example 2 Anti-tumor effect of CIK cells

(1) Transplantation of CIK cells into cancer patients

To each cancer patient, the self-derived R-CIKs prepared in Example 1 were implanted twice in 4 days as a round of administration, with an average of 3.9×10 ¹⁰ administered. All patients received at least 2 rounds of dosing, and one patient received 3 rounds of dosing.

(2) Anti-tumor effect

No serious adverse signs associated with cell administration were found in all R-CIKs-administered cancer patients.

By administration of R-CIKs, improvement of clinical symptoms and recovery of physiological functions were found in all patients. In addition, a significant decrease in calcitonin, a tumor marker, was found in patients with thyroid cancer. In addition, the tumor size of lung cancer patients was measured by PET-CT, and it was found that the tumor shrunk by about 50%.

In summary, R-CIKs is a very safe cell preparation with good anti-tumor effect.

Possibility of industrial use

PBMCs are differentiated into multiple cell populations through amplified culture, and the differentiated cell populations contain cell populations that control or suppress immunity. According to the present invention, by expanding and culturing CIK cells, which are known as the preferred cell population in conventional adoptive immunotherapy, it is possible to provide a high-quality cell population with a low content of a large number of unnecessary cell populations. The cell population obtained by the present invention is extremely useful in adoptive immunotherapy using the cell population and has a remarkable effect.

Sequence Listing Free Content

SEQ ID NO: 1; Local domain CS-1 of fibronectin.

SEQ ID NO: 2; Local domain III-10 of fibronectin.

Local region of fibronectin in SEQ ID NO: 3; III-10.

SEQ ID NO: 4; Fibronectin fragment C-274.

SEQ ID NO: 5; Fibronectin fragment H-271.

SEQ ID NO: 6; Fibronectin fragment H-296.

SEQ ID NO: 7; Fibronectin fragment CH-271.

SEQ ID NO: 8; Fibronectin fragment CH-296.

SEQ ID NO: 9; Fibronectin fragment C-CS1.

sequence listing

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<223> Local domain of fibronectin CS-1

<400>1

Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly

1 5 10 15

Pro Glu Ile Leu Asp Val Pro Ser Thr

20 25

<210>2

<211>94

<212>PRT

<213> Artificial

<220>

<223> Local domain III-10 of fibronectin

<400>2

Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr

1 5 10 15

Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr

20 25 30

Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe

35 40 45

Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro

50 55 60

Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp

65 70 75 80

Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr

85 90

<210>3

<211>4

<212>PRT

<213> Artificial

<220>

Local region of fibronectin in <223>III-10

<400>3

Arg Gly Asp Ser

<210>4

<211>274

<212>PRT

<213> Artificial

<220>

<223> Fibronectin Fragment C-274

<400>4

Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val

1 5 10 15

Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg

20 25 30

Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser

35 40 45

Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu

50 55 60

Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro

65 70 75 80

Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp

85 90 95

Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro

100 105 110

Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe

115 120 125

Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile

130 135 140

Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val

145 150 155 160

Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser

165 170 175

Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro

180 185 190

Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr

195 200 205

Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu

210 215 220

Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys

225 230 235 240

Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly

245 250 255

Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu

260 265 270

Ile Asp

<210>5

<211>271

<212>PRT

<213> Artificial

<220>

<223> Fibronectin Fragment H-271

<400>5

Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr

1 5 10 15

Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr

20 25 30

Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile

35 40 45

Asn Leu Ala Pro Asp Ser Ser Ser Ser Val Val Val Ser Gly Leu Met Val

50 55 60

Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr

65 70 75 80

Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro

85 90 95

Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile

100 105 110

Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala

115 120 125

Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp

130 135 140

Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys

145 150 155 160

Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val

165 170 175

Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu

180 185 190

Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala

195 200 205

Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro

210 215 220

Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile

225 230 235 240

Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu

245 250 255

Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr

260 265 270

<210>6

<211>296

<212>PRT

<213> Artificial

<220>

<223> Fibronectin Fragment H-296

<400>6

Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr

1 5 10 15

Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr

20 25 30

Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile

35 40 45

Asn Leu Ala Pro Asp Ser Ser Ser Ser Val Val Val Ser Gly Leu Met Val

50 55 60

Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr

65 70 75 80

Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro

85 90 95

Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile

100 105 110

Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala

115 120 125

Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp

130 135 140

Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys

145 150 155 160

Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val

165 170 175

Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu

180 185 190

Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala

195 200 205

Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro

210 215 220

Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile

225 230 235 240

Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu

245 250 255

Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr Asp

260 265 270

Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro

275 280 285

Glu Ile Leu Asp Val Pro Ser Thr

290 295

<210>7

<211>549

<212>PRT

<213> Artificial

<220>

<223> Fibronectin Fragment CH-271

<400>7

Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val

1 5 10 15

Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg

20 25 30

Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser

35 40 45

Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu

50 55 60

Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro

65 70 75 80

Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp

85 90 95

Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro

100 105 110

Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe

115 120 125

Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile

130 135 140

Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val

145 150 155 160

Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser

165 170 175

Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro

180 185 190

Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr

195 200 205

Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu

210 215 220

Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys

225 230 235 240

Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly

245 250 255

Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu

260 265 270

Ile Asp Lys Pro Ser Met Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe

275 280 285

Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn

290 295 300

Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr

305 310 315 320

Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val

325 330 335

Val Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala

340 345 350

Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr

355 360 365

Leu Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr

370 375 380

Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr

385 390 395 400

Gly Phe Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln

405 410 415

Arg Thr Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln

420 425 430

Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala

435 440 445

Arg Ser Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro

450 455 460

Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser

465 70 475 480

Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu

485 490 495

Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly

500 505 510

Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr

515 520 525

Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile

530 535 540

Gly Arg Lys Lys Thr

545

<210>8

<211>574

<212>PRT

<213> Artificial

<220>

<223> Fibronectin Fragment CH-296

<400>8

Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val

1 5 10 15

Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg

20 25 30

Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser

35 40 45

Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu

50 55 60

Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro

65 70 75 80

Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp

85 90 95

Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro

100 105 110

Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe

115 120 125

Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile

130 135 140

Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val

145 150 155 160

Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser

165 170 175

Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro

180 185 190

Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr

195 200 205

Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu

210 215 220

Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys

225 230 235 240

Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly

245 250 255

Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu

260 265 270

Ile Asp Lys Pro Ser Met Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe

275 280 285

Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn

290 295 300

Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr

305 310 315 320

Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val

325 330 335

Val Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala

340 345 350

Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr

355 360 365

Leu Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr

370 375 380

Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr

385 390 395 400

Gly Phe Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln

405 410 415

Arg Thr Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln

420 425 430

Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala

435 440 445

Arg Ser Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro

450 455 460

Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser

465 470 475 480

Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu

485 490 495

Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly

500 505 510

Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr

515 520 525

Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile

530 535 540

Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His

545 550 555 560

Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

565 570

<210>9

<211>302

<212>PRT

<213> Artificial

<220>

<223> Fibronectin Fragment C-CS1

<400>9

Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val

1 5 10 15

Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg

20 25 30

Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser

35 40 45

Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu

50 55 60

Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro

65 70 75 80

Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp

85 90 95

Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro

100 105 110

Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe

115 120 125

Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile

130 135 140

Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val

145 150 155 160

Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser

165 170 175

Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro

180 185 190

Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr

195 200 205

Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu

210 215 220

Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys

225 230 235 240

Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly

245 250 255

Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu

260 265 270

Ile Asp Lys Pro Ser Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His

275 280 285

Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr

290 295 300

Claims (8)

1. A method for producing a cell population containing cytokine-induced killer cells, comprising the step of culturing the cell population containing cells capable of differentiating into cytokine-induced killer cells in the presence of fibronectin fragments. 2. The production method according to claim 1, wherein culturing is carried out in the presence of a fibronectin fragment containing a region selected from the group consisting of VLA-4 binding domain, VLA-5 binding domain and heparin binding domain. 3. The production method according to claim 2, wherein culturing is performed in the presence of a fibronectin fragment containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 5. 4 . The production method according to claim 1 , wherein the culture is carried out in the presence of fibronectin fragments in the co-existence of CD3 ligand, interferon-γ, and interleukin-2. 5. A cell population comprising cytokine-induced killer cells obtained by the method according to any one of claims 1 to 4. 6. A drug comprising the cell population according to claim 5 as an active ingredient. A method for treating or preventing a disease, comprising the step of administering an effective amount of the cell population according to claim 5 to a subject. 8. The use of the cell group according to claim 5, characterized in that it is used for the preparation of medicines.

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