JP2019031470A - Antitumor composition containing nk cell conditioned medium and method for producing the same - Google Patents

Abstract

To provide a novel antitumor composition containing NK cell conditioned medium.SOLUTION: An antitumor composition contains an NK cell conditioned medium obtained by culturing NK cells. There are also provided a method for producing the antitumor composition, and a method for treating tumor.SELECTED DRAWING: None

Description

  The present invention relates to an antitumor composition containing an NK cell culture supernatant and a method for producing the same.

In recent years, immune cell therapy has become widespread and has a certain effect as a fourth treatment after surgery, radiation therapy, and anticancer drug treatment. In particular, natural killer cell (NK cell) therapy has attracted attention over other immune cell therapies because of its high therapeutic effect and many adaptation diseases.
NK cells are one type of cellular immune cell that circulates widely in the body and attacks abnormal cells such as tumor cells and virus-infected cells. Unlike T cells and B cells, NK cells can directly attack abnormal cells without undergoing immunization, and are currently regarded as the most promising cells for tumor immunotherapy.

  The main mechanism by which NK cells kill tumor cells is to release intracellular granules containing proteases having cytotoxic activity such as perforin and granzyme to damage tumor cells and induce apoptosis, and Fas Pathways that induce apoptosis in tumor cells via ligands are known. In any route, NK cells recognize tumor cells via receptors and contact with tumor cells to exert strong cytotoxic activity.

NK cell therapy using NK cells collected from cancer patients using the mechanism by which NK cells damage cancer cells as described above has already been put into practical use (Non-patent Document 1).
Typically, blood is drawn from the patient and lymphocytes in the blood are separated to activate and proliferate NK cells that are returned to the patient. A general method is to repeat this for 6 cools.
However, the conventional NK cell therapy requires multiple autologous blood collections for NK cell collection and culture, which imposes a heavy burden on the patient and is costly because it is a complete custom-made treatment. . Therefore, although there is an effect, there is a reality to be overcome that NK cell therapy remains a special medical treatment that cannot benefit from many patients.
In addition to the above problems, self-NK cell therapy has fateful challenges. This is a problem that a waiting period is required from blood collection until NK cells increase to a necessary amount. This is a serious obstacle for the treatment of time-consuming tumors.

Sutlu et al., Journal of Internal Medicine, 266; 154-181

An object of the present disclosure is to provide a new antitumor composition and a method for producing the same in view of the above situation.
As a result of intensive studies, the inventors have found that the culture supernatant (NKCM) of NK cells has a high tumor suppressing effect.

The present disclosure includes the following aspects.
<1> An antitumor composition comprising a natural killer cell culture supernatant.
<2> The antitumor composition according to <1>, which does not contain natural killer cells.
<3> The antitumor composition according to <1> or <2>, which is cell-free.
<4> The antitumor composition according to any one of <1> to <3>, wherein the natural killer cell is a natural killer cell of another family.
<5> The natural killer cell culture supernatant treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage The antitumor composition according to any one of <1> to <4>.
<6> The antitumor composition according to any one of <1> to <5>, which does not contain serum.
<7> The antitumor composition according to any one of <1> to <6>, wherein the tumor is squamous cell carcinoma.
<8> The antitumor composition according to any one of <1> to <7>, wherein the tumor is tongue cancer.

<9> (1) a step of culturing natural killer cells; and (2) a step of collecting a culture supernatant obtained by culturing the natural killer cells,
A method for producing an antitumor composition, comprising:
<10> The method for producing an antitumor composition according to <9>, wherein the step of culturing the natural killer cell includes a culture in which serum is not contained in the culture solution.
<11> The method for producing an antitumor composition according to <9> or <10>, wherein the natural killer cell is a natural killer cell of another family.
<12> A step of subjecting the collected culture supernatant to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage The method for producing an antitumor composition according to any one of <9> to <11>, further comprising:

<13> A method for treating a tumor, comprising administering an antitumor composition comprising a natural killer cell culture supernatant to a subject in need of treatment.
<14> A method for preventing and / or suppressing progression of a tumor, comprising administering an effective amount of an antitumor composition comprising a natural killer cell culture supernatant to a subject.
<15> Use of a natural killer cell culture supernatant in the production of an antitumor composition.

According to the present invention, an antitumor composition comprising a natural killer cell culture supernatant and a method for producing the same are provided.
NK cells are cells responsible for cellular immunity, and the main mechanism for killing tumor cells is that NK cells recognize tumor cells and release cytotoxic substances such as perforin in the vicinity of the tumor cells, or Fas It is thought to induce apoptosis via a ligand. Therefore, a composition for treating tumors that focuses on the culture supernatant of NK cells has not been studied.
In addition, a high anti-tumor effect that suppresses the growth of solid tumors or reduces the size of solid tumors can be obtained only with a culture supernatant (NKCM) that does not contain NK cells. This is an unexpected result.

  When the culture supernatant of NK cells is used, rejection reaction is suppressed as compared with transplantation of NK cells themselves. Therefore, it is not always necessary to prepare NKCM from own NK cells. Therefore, NKCM can also be prepared from NK cells from other families. By using NK cells from another family, it becomes possible to prepare and store the NK cell culture supernatant in advance, and shorten the waiting period until the NK cells proliferate. In addition, the benefits of using NKCM from other homes, such as the need for blood collection, are immeasurable.

The appearance of the tumor of the tumor-bearing mouse on the 15th day after the SCCVII cell injection is shown. The appearance of the tumor-bearing mice of the experimental group treated with NKCM and the control group not treated on the 35th day after SCCVII cell injection is shown. The time-dependent change of the tumor diameter of the experimental group which was treated with NKCM and the control group which was not treated is shown in the tumor-bearing mice. The time-dependent change of the NK cell activity of the experimental group which was treated with NKCM and the control group which was not treated in cancer-bearing mice is shown. 2 shows HE-stained images of tumor tissues of an experimental group treated with NKCM and a control group not treated with cancer-bearing mice. The appearance of the tongue cancer after arterial chemotherapy and NKCM treatment in Case 1 is shown. It is a figure which shows the external appearance of the tongue cancer after NKCM treatment of case 2, and the metastatic focus of the left lower jaw. FIG. 6 is a diagram showing a PET image of a metastasis in the lower left jaw after NKCM treatment in Case 2. The HE-stained image of the tissue obtained by puncturing the primary lesion and lymph node of Case 2 is shown.

<Anti-tumor composition containing natural killer (NK) cell culture supernatant>
The antitumor composition according to the present disclosure includes an NK cell culture supernatant obtained by culturing NK cells.
In the present disclosure, the “antitumor effect” is used in a commonly used meaning and refers to having an effect of suppressing cancer growth or invasion in vitro or in vivo.

In the antitumor composition containing the NK cell culture supernatant according to the present disclosure, the NK cell culture supernatant obtained by culturing NK cells is used as an active ingredient of the antitumor composition. Although the mechanism by which the NK cell culture supernatant shows an antitumor effect is not clear, it is considered as one of the mechanisms that the NK cell culture supernatant contains a physiologically active substance such as cytokine.
However, the main mechanism of conventional NK cell therapy is based on the cytotoxic activity of NK cells, and the NK cell culture supernatant alone can prevent the reduction or increase of solid tumors. Obtaining a tumor effect is an unexpected phenomenon.
Note that the present invention is not limited to the estimation.

  The NK cell culture supernatant in the present disclosure is obtained by culturing NK cells. In the following description, the manufacturing method is also described.

Natural killer cells (NK cells) are one type of lymphocytes that work in innate immunity. Unlike T cells, it exhibits cytotoxic activity against cancer cells or cells infected with viruses without requiring sensitization.
In the present disclosure, NK cells refer to lymphocytes that are positive for the surface antigen CD56. CD3 is negative in most NK cells. However, there is a subset of T cells that are positive for CD3 and positive for CD56. Such cells have the characteristics of natural killer cells and T cells, and are called natural killer T cells. In the present disclosure, natural killer T cells are also included in natural killer cells.

The method for collecting NK cells is well known and may be followed by a conventional method, and the method is not particularly limited.
NK cells can be conveniently collected from human or non-human animal blood. The blood is not particularly limited, and peripheral blood, arterial blood, umbilical cord blood or the like can be used. Generally, whole blood is collected from a subject, and a peripheral blood mononuclear cell fraction containing NK cells may be used as a starting material by density gradient centrifugation using Ficoll (registered trademark). Alternatively, peripheral blood mononuclear cells (PBMC) may be separated and collected using a blood component separation device or the like.

A CD56-positive fraction may be separated from PBMC using an apparatus such as flow cytometry. It is also possible to specifically grow NK cells by culturing PBMC. For example, the cells may be cultured under various culture conditions as described in Sutlu et al., Journal of Internal Medicine, 266; 154-181. Alternatively, a commercially available kit for specifically growing NK cells such as BINKIT (registered trademark, manufactured by Japan Biotherapy Research Institute Co., Ltd.) can also be used.
Alternatively, NK cells may be prepared by inducing differentiation of stem cells such as iPS cells, embryonic stem cells or adult stem cells, or NK cells are prepared by inducing differentiation of hematopoietic progenitor cells obtained from peripheral blood, umbilical cord blood, etc. May be.

In the conventional NK cell therapy, NK cells are collected from a patient to be administered, processed into NK cells, and then administered to the person. As the anti-tumor composition containing the NK cell culture supernatant of the present disclosure, a culture supernatant obtained by culturing autologous NK cells may be used, or NK cells of other homes not to be administered are cultured. The culture supernatant obtained in this way may be used.
That is, the antitumor composition containing the NK cell culture supernatant of the present disclosure can be administered to anyone regardless of whether the NK cell is derived from the home or the home.

The use of a culture supernatant obtained by culturing NK cells of another family has the following advantages.
1) There is no need for blood collection.
2) It can be produced and stored in large quantities in advance.
3) Significant cost reduction can be expected and many patients can receive NK immunotherapy.
4) Since NKCM can be stored, there is no waiting period until the start of treatment.
As described above, the culture supernatant derived from the allogeneic NK cell can be considered as a kind of biologic, and its industrial value as a new tumor therapeutic agent is extremely high. Moreover, the NK cell culture supernatant with a higher antitumor effect can be obtained by using the NK cell derived from a healthy person as an allogeneic NK cell.

The culture medium of NK cell can use what is usually used. For example, DMEM, DMEM / F12 or DME culture solution containing 10-15% fetal bovine serum (FBS) (in addition to these culture solutions, LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol and the like can be appropriately contained.) or a commercially available culture solution [for example, NK cell initial medium and NK cell passage medium contained in BINKIT (registered trademark) (manufactured by Biotherapy Laboratories)] included.
The culture conditions are not particularly limited. For example, the culture may be performed with shaking at 37 ° C. in the presence of 5% CO ₂ .

From the viewpoint that the invasiveness to the collection target is low and a sufficient number of NK cells can be obtained, it is preferable to culture the collected peripheral blood mononuclear cells to proliferate the NK cells. Preferably, the culture is performed until the number of cells reaches about 1.0 × 10 ⁹ .
By culturing the collected peripheral blood mononuclear cells or isolated NK cells as starting materials under the conditions described in, for example, Sutlu et al., Journal of Internal Medicine, 266; 154-181, NK Cells can be grown.
It is also possible to selectively proliferate NK cells among peripheral blood mononuclear cells by culturing using a commercially available kit such as BINKIT (registered trademark, manufactured by Nippon Biotherapy Laboratories, Inc.). In order to promote proliferation, the cells may be cultured with various feeder cells.
Alternatively, NK cells can also be obtained by removing CD3 + positive blood cells from the collected peripheral blood mononuclear cells or cultured peripheral blood mononuclear cells by the bead method or the like.

From the viewpoint of enhancing the antitumor effect of the antitumor composition of the present disclosure, it is preferable that the NK cell used for NK cell culture is activated. Moreover, proliferation can also be promoted by activating NK cells.
The method for activating NK cells is not particularly limited. For example, the method of adding interleukin-2 (IL-2) or interleukin-15 to the culture solution of NK cells can be mentioned.
Further, NK cells can be activated by culturing mixed with cancer cell lines such as K-562 and HFWT, or by adding a protein prepared from these cells.
Various methods for activating NK cells are known, see, eg, Granzin et al., Front Immunol. 2017, 26; 8: p458 etc. can be used.

The NK cell culture supernatant contained in the antitumor composition of the present disclosure is preferably serum-free because unnecessary reactions such as allergic reactions may be suppressed.
For example, a culture supernatant without serum can be prepared by culturing NK cells in a serum-free medium (serum-free medium). A serum-free culture supernatant can also be obtained by performing subculture once or a plurality of times and culturing the last or last several subcultures in a serum-free medium. On the other hand, serum-free culture supernatant can also be obtained from the collected culture supernatant by removing the serum using dialysis or solvent replacement using a column.

  In order to prepare a culture supernatant of NK cells that does not contain serum, a serum-free medium may be used throughout the whole process or for the last or several subcultures. In addition to DMEM, Iskov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI 1640 medium, etc. can be used as the basic medium. Two or more basic media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (GIBCO)) can be mentioned. Examples of ingredients that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.

The culture time for obtaining the culture supernatant is, for example, 5 hours to 20 days, and may be 1 day to 14 days, or 6 days to 14 days. The culture temperature is, for example, 36 ° C to 38 ° C, for example, 37 ° C, and the CO ₂ concentration is 4 to 6%, for example, 5%. The culture may be performed, for example, by three-dimensional culture under non-adhesive conditions, for example, suspension culture (for example, dispersion culture, aggregation suspension culture, etc.)

  After culturing, NK cell culture supernatant can be obtained by separating and removing cell components. In the present disclosure, the culture supernatant is not only the supernatant itself from which the cell components are separated and removed from the culture solution, but also various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution) , Desalted, preserved, etc.) are also included in the range. Details of the culture supernatant treatment method will be described later. In the present disclosure, the culture supernatant does not contain cell components. For this reason, the culture supernatant in this indication does not contain the NK cell used for culture.

  The antitumor composition according to the present disclosure includes a culture supernatant of NK cells as an active ingredient. In one embodiment, the antitumor composition does not contain NK cells as a whole composition. In another embodiment, the antitumor composition does not contain cells (regardless of cell type) as a whole composition. That is, it is cell-free. The anti-tumor composition of this embodiment is clearly distinguished from various compositions containing NK cells as a matter of course by this characteristic. A typical example of this embodiment is an antitumor composition that does not contain NK cells and is composed only of the culture supernatant of NK cells.

  The anti-tumor composition according to the present disclosure may additionally contain other components. Additional ingredients that may be included include other pharmaceutically acceptable ingredients (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives). Agents, physiological saline, etc.). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tiger tumor and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used. Antibiotics, pH adjusting agents, cytokines (eg, tumor necrosis factor (eg, TNF-α), interferon (eg, IFN-α, β, or γ)) and the like may be contained.

  The final form of the antitumor composition according to the present disclosure is not particularly limited. Examples of forms are liquid (liquid, gel, etc.) and solid (powder, fine granules, granules, etc.). The anti-tumor composition according to the present disclosure may have a form suitable for inhalation, for example, a liquid form that can be sprayed in a nebulizer or a diffuser.

Conventionally, immune cell therapy using NK cells has been put into practical use. When cells are used, the cells collected from the living body are cultured and then collected and administered. In conventional NK cell immunotherapy, the culture supernatant is usually discarded and replaced with a physiological buffer or the like in the course of a series of operations. Therefore, the culture supernatant is not actively included in the end. In view of this, in terms of including the culture supernatant of NK cells, the antitumor composition according to the present disclosure is substantially different from the wording of the composition using NK cells as an active ingredient. Are also distinguished.
However, in some embodiments, the antitumor composition according to the present disclosure may include NK cells in addition to the culture supernatant of NK cells.

The antitumor composition according to the present disclosure exhibits an antitumor effect by containing a culture supernatant of NK cells. The antitumor composition according to the present disclosure has a tumor suppressing effect even in vivo, and has an effect comparable to that of conventional NK cell therapy.
In addition, the antitumor composition of the present invention may be administered before the onset of a tumor and used prophylactically for a subject whose tumor is not diagnosed. For example, nasal spray dosage forms can be conveniently administered and are suitable for prophylactic use. By using the antitumor composition according to the present disclosure habitually, a wide variety of tumors can be suppressed at an extremely early stage of development.

In this disclosure,
There is also provided a method for producing an antitumor composition, comprising: (1) culturing NK cells; and (2) collecting the culture supernatant obtained by culturing the NK cells.
The description of the antitumor composition containing the NK cell culture supernatant can also be applied to the production method of the present disclosure.
As a preferred example of the method for preparing the NK cell culture supernatant, the following example can be given.
(I) Collecting whole blood from a healthy person (for example, about 50 mL).
(Ii) A fraction containing peripheral blood mononuclear cells (PBMC) is separated by density gradient centrifugation using Ficoll (registered trademark) or the like.
(Iii) The separated PBMCs are subjected to conditions of 37 ° C. and 5% CO ₂ in a DMEM medium containing 10% FBS using BINKIT for NK Cells Expansion from PBMCs (trade name, manufactured by Nippon Biotherapy Co., Ltd.). And NK cells are grown according to the method of use of the kit.
(Iv) Interleukin 2 (IL-2) 6000 IU / mL is added to the culture solution, and cultured for 14 days under conditions of 7 ° C. and 5% CO ₂ , and activated while growing NK cells.
(V) After the number of cells grows to about 1.0 × 10 ⁹ , the cells are collected by centrifugation and suspended in PBS. You may wash about twice with PBS.
(Vi) Replace with 400 mL of serum-free DMEM medium not containing phenol red and not containing IL-2, and culturing for 48 hours under conditions of 37 ° C. and 5% CO ₂ .
(Vii) Collect the culture supernatant by centrifugation.

  The production method of the present disclosure includes at least one selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, storage, and desalting of the collected culture supernatant. You may further include the process of giving a process. By including such a process, handling, storage, and transportation of the antitumor composition become easier. The production method may further include a step of adding an additional component to the collected culture supernatant. By adding such additional ingredients, it is possible to improve the overall properties of the anti-tumor composition. About each process and an additional component, the matter described in description of the antitumor composition which concerns on this indication is applied as it is. Subjecting the collected culture supernatant to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, storage, and desalting; When both steps of adding additional components to the collected culture supernatant are included, both of the steps may be performed first, and may be performed in parallel when possible.

  In the step (2), the culture supernatant of NK cells is collected. For example, the culture solution can be collected by suction with a dropper or pipette. The collected culture supernatant is used as it is or after one or more treatments as an active ingredient of the antitumor composition according to the present disclosure. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (eg, 4 ° C., −10 ° C. to −80 ° C.). . Note that the culture supernatant of NK cells shows the desired action without complicated and sophisticated purification. For this reason, the anti-tumor composition according to the present disclosure can be produced by a simple process. The fact that a complicated purification step is not required is also advantageous in that a decrease in activity associated with purification can be avoided.

<Concentration method of NK cell culture supernatant>
The anti-tumor composition according to the present disclosure may be concentrated. The preferred concentration factor is 1.1 to 50 times, more preferably 2 to 20 times, and particularly preferably 4 to 10 times. As a method for concentrating the NK cell culture supernatant, a method generally used for concentrating the culture supernatant can be applied. Examples of the concentration method include the following two methods.
1. Spin column concentration method NK cell culture supernatant is concentrated using Amicon (registered trademark) Ultra-15 (Merck). The specific operation procedure is as follows. After dispensing 15 ml, centrifuge at 3500 rpm for 15 minutes. The NK cell culture supernatant can be concentrated about 1.2 to 20 times by the spin column concentration method.

2. Ethanol precipitation concentration method The culture supernatant is concentrated using the ethanol precipitation method. The specific operation procedure is as follows.
(i) 45 mL of 100% ethanol is added to 5 ml of the culture supernatant, mixed, and left at −20 ° C. for 60 minutes.
(ii) Centrifuge at 4 ° C. and 15000 g for 15 minutes.
(iii) Remove the supernatant, add 10 mL of 90% ethanol and stir well.
(iv) Centrifuge at 4 ° C. and 15000 g for 5 minutes.
(v) The supernatant is removed, and the resulting pellet is dissolved in 500 μL of sterilized water, collected in a micro test tube, and used as a concentrated NK cell culture supernatant.

<Method of freeze-drying NK cell culture supernatant>
In addition, the NK cell culture supernatant contained in the antitumor composition according to the present disclosure may be frozen or lyophilized, or may be obtained by dissolving the lyophilized culture supernatant in an appropriate solvent. . Thereby, good storage stability is obtained. As a lyophilization method of the NK cell culture supernatant, a method usually used for lyophilization of the culture supernatant can be applied.

  The present disclosure also provides a method for treating tumors, comprising administering an antitumor composition according to the present disclosure to a subject in need of treatment in an effective amount. The subjects include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc. ).

The present disclosure also provides a method for preventing and / or suppressing progression of a tumor, comprising administering an effective amount of the antitumor composition according to the present disclosure to a subject.
In general, the more advanced a tumor, the more difficult it is to treat. For this reason, early detection of such tumors is important, but early detection is not always reliable. Considering this point, it is desirable that treatment can be started in advance before tumor development or at the stage when a minute tumor has occurred, but as long as the drugs used for such treatment are used continuously, the subject of administration It is desirable that the burden on the person is small. Meeting these requirements has been difficult with conventional knowledge. However, the antitumor composition according to the present disclosure can be administered nasally and has wide efficacy in general tumors. Therefore, the antitumor composition has high effectiveness in the early treatment (progression suppression) of a minute tumor before onset or very early as described above.

The dose of the antitumor composition is, for example, 0.1 mg / kg / day to 1000 mg / kg / day, and 1 mg / kg / day to 100 mg / kg in terms of the amount of untreated culture supernatant. / Day may be sufficient. Moreover, the administration method is not particularly limited. For example, administration of the anti-composition is preferably parenteral administration, and the parenteral administration may be systemic administration or local administration. Examples of topical administration include injection into the target tissue, application or spraying. Examples of methods for administering the anti-tumor composition include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration (transpulmonary absorption) and Nasal administration and the like can be mentioned. Of these, nasal administration, transpulmonary administration and the like are preferable because they are minimally invasive. In addition, nasal administration is particularly effective for tumors related to the brain because there is no need to worry about the possibility of passage through the blood-brain barrier (BBB). As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.
The administration method can be selected by those skilled in the art based on the type of tumor to be treated, the organ, and the like.

In some embodiments, in addition to the antitumor composition according to the present disclosure, other substances having antitumor activity may be administered.
For example, an anti-tumor agent, immune cells such as NK cells, T cells, NKT cells, B cells, and dendritic cells are administered to a subject simultaneously with the anti-tumor composition of the present disclosure or at separate timings. be able to. When using immune cells, it is preferable to perform a treatment for activating immune functions after collection. When the antitumor composition and an antitumor agent or immune cell are used in combination, a first component containing the antitumor composition and an antitumor agent or immune cell containing the second component Administration may be performed using a kit comprising the components. Each component may be a separate capsule, for example, or a separate ampoule or vial. In this case, you may administer a 2nd component with respect to the treatment target which administered the 1st component simultaneously with the administration of a 1st component, or after administration. Here, “simultaneous” does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without a substantial time difference is also included in the concept of “simultaneous” herein.

Preferable examples of the drug having an antitumor action include the following. For example, ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, gemtuzumab ozogamicin, sunitinib, cetuximab, sorafenib, dasatinib, tamivaroten, trastuzumab, tetizumab, bartuximab, pantizumab And various cytokines such as IL-2, IL-15, interferon-α, interferon-β or interferon-γ.
In addition to the antitumor composition according to the present disclosure, by administering the drug having the antitumor action, a synergistic strong antitumor action can be exerted.

The antitumor compositions of the present disclosure are effective against a wide variety of cancers in mammals, including humans. Examples of cancer include oral cancer such as pharyngeal cancer, laryngeal cancer, and tongue cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, uterine cancer, vulvar cancer, skin cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer. Gallbladder cancer, kidney cancer, prostate cancer, malignant melanoma, thyroid cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, hemangiosarcoma, fibrosarcoma, leukemia, malignant lymphoma, myeloma It is done.
The tissue classification of cancer may be any of squamous cell carcinoma, adenocarcinoma or other cancer, but is preferably squamous cell carcinoma. Examples of squamous cell carcinoma include oral cancer such as pharyngeal cancer, laryngeal cancer and tongue cancer, lung cancer, esophageal cancer, uterine cancer, vulvar cancer, skin cancer, kidney cancer and the like.
The antitumor composition according to the present disclosure has a high antitumor effect even for solid cancer.

  The anti-tumor effect may be achieved based on the ability of endogenous immune cells. The antitumor composition according to the present disclosure contains various components such as cytokines, and these components are considered to stimulate the ability of endogenous immune cells such as NK cells to exert an antitumor effect. It is done.

  The administration target of the antitumor composition of the present disclosure is not particularly limited. For example, in humans or non-human mammals (including pet animals, farm animals, laboratory animals, specifically mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc.) There may be.

  According to the present disclosure, use of an NK cell culture supernatant obtained by culturing NK cells in the production of an antitumor composition is also provided. For the details of the NK cell culture supernatant, the antitumor composition and the method of use, see the above explanation (the description of the antitumor composition according to the present disclosure and the production method thereof, and various treatment methods according to the present disclosure) ).

  Examples of the present invention will be described below, but the present invention is not limited thereto. Further,% in the examples is based on weight (mass) unless otherwise specified.

Example 1: Preparation of human natural killer cell culture supernatant (NKCM) <Preparation of NKCM stock solution and NKCM 5-fold concentrated solution>
<Preparation of natural killer cell culture supernatant (NKCM)>
Peripheral blood was collected from healthy volunteers, centrifuged, and the lymphocyte fraction was collected. Peripheral mononuclear cells (PBMC) were separated by Ficoll-Hypaque specific gravity method (GE Health Care).
Next, the PBMC is suspended in the NK cell initial medium contained in the BIKIT for NK Cells Expansion from PBMCs (trade name, manufactured by Biotherapy Laboratories), and the NKCM cell initial cocktel contained in the BINKIT is added. The cells were cultured in a DMEM medium supplemented with% FBS for 3 days in a suspension culture method using magnetic beads under conditions of 5% CO ₂ and 37 ° C. to specifically proliferate NK cells. Subsequently, the obtained NK cells were used in 10% FBS-added DMEM medium supplemented with interleukin 2 (IL-2) 6000 IU / mL, using magnetic beads under conditions of 5% CO ₂ and 37 ° C. The culture was grown for 14 days using the conventional suspension culture method and grown to 100 million cells.

The culture solution was centrifuged to collect NK cells (number of cells: 1.0 × 10 ⁹ ). The collected cells were suspended in PBS and washed twice.
Next, the medium was replaced with 400 ml of serum-free DMEM medium containing no phenol red and IL-2 and cultured for 48 hours. The culture supernatant was collected by centrifugation, and the collected culture supernatant was further centrifuged at 2000 rpm for 3 minutes to remove the precipitate, followed by filtration to obtain an NK cell culture supernatant (NKCM). This is called NKCM stock solution. Later, the culture supernatant was collected. NK cells were discarded.
In the above method, NKCM obtained by culturing using a medium to which IL-2 is not added when cultured for 14 days is referred to as “fresh NKCM”.
<Preparation of NKCM 5-fold concentrated solution>
The NKCM stock solution was passed through a centrifugal filter unit (Amicon Ultra 15 (trade name) (manufactured by Merck)), dispensed 15 ml each into a test tube, and centrifuged at 3500 rpm for 15 minutes. The supernatant was collected and used as an NKCM 5-fold concentrated solution.
Unless otherwise specified, the NKCM stock solution and the NKCM 5-fold concentrated solution refer to those obtained from activated NK cells activated by adding IL-2 and culturing.
<Virus inspection>
HBs antigen (precision), HBs antibody (precision), HBc antibody, HIV antigen / antibody RPR qualitative, HTLV-I third generation, HCV antibody, human parvo B19 IgG, endotoxin, mycoplasma culture, sterility test (direct method) were performed. All results were negative.

Example 2: Verification of antitumor effect in mice <Creation and treatment of cancer-bearing animals>
SCCVII cells derived from mouse skin squamous cell carcinoma (supplied by Dr. Masamasa Nishimura, Kinki University) under the trunk of a 7-week-old female C3H / HeJ mouse (purchased from Chubu Scientific Materials) (5th passage, 5X10 ⁵ / 0.2 mL) was injected, and administration of NKCM was started on the 15th day when the tumor cells had engrafted and the tumor diameter had grown to 15 mm (the injection date was defined as day 0).
In the experimental group, C3H / HeJ mice (n = 4) were nasally administered with the NKCM 5-fold concentrated solution prepared above at a dose of 10 μmL once a day for 14 days. In the control group (n = 4), physiological saline was similarly administered instead of the NKCM 5-fold concentrated solution.

<Evaluation method>
(1) Measurement of tumor diameter The major and minor diameters of tumors of the tumor-bearing mice in the experimental group and the control group are represented as 0, 5, 15, 20, 25, 30 when the SCCVII cell injection date is 0 days. On day 35, measurements were made using calipers. One half of the sum of the major axis and the minor axis was taken as the tumor diameter.

When the day of CCVII cell injection was defined as day 0, there was no significant difference in the tumor diameter between the experimental group and the control group on day 15, and the average value was 15 ± 3 mm (FIG. 1A). On day 35, tumor diameters increased in control mice not receiving NKCM, with a maximum of 35 mm. On the other hand, the tumor growth was stopped in the mice administered with NKCM, and the tumor was reduced in one case (FIG. 1B, FIG. 2, Table 1).
From the above, it can be seen that NKCM has an antitumor effect.

(2) Measurement of NK cell activity NK cell activity was measured by the ⁵¹ Cr release method on the 0th day, the 15th day, and the 35th day when the injection date of the SCCVII cells was 0 day. Blood was collected from the tumor-bearing mice of the experimental group and the control group, and NK cells were separated from the blood as described above. ⁵¹ Cr-target cells (human chronic myeloid leukemia-derived K562 cell line) are added to NK cells at three ratios of NK cells: ⁵¹ Cr-target cells of 1: 1, 3: 1, and 10: 1. In addition, the cells were cultured in a U-bottom 96-well microplate in 200 μL of complete RPMI medium at 5% CO ₂ and 37 ° C. for 4 hours. ⁵¹ Cr-target cells were prepared by culturing with ⁵⁰ μCi of ⁵¹ Cr for 1 hour and washing twice with a culture solution not containing ⁵¹ Cr.
Subsequently, the culture supernatant was recovered, a gamma counter (CRC-55tW (trade name), calibration pin Tech Co., Ltd.) using ^a radiation dose of free ⁵¹ Cr 51 Cr-sensitivity energy window (300-400KeV) It was measured. This was taken as the experimental free value.
In addition, ⁵¹ Cr-target cells were treated in the same manner as when experimental free values were determined except that NK cells were not added, and the radiation dose of ⁵¹ Cr obtained as natural free values. The radiation dose of ⁵¹ Cr obtained by adding 4% SDS to ⁵¹ Cr-target cells and incubating was defined as the maximum free value.
NK cell activity was calculated as follows.

NK cell activity (%) = (experimental free value−natural free value) / (maximum free value−natural free value) × 100

NK cells: ⁵¹ Cr—Calculate NK cell activity (%) for each of the cases where the number of target cells is 1: 1, 3: 1, 10: 1, and evaluate the NK cell activity using the average value thereof. It was.

The measurement results of NK cell activity are shown in Table 2 and FIG. In the experimental group, NK cell activity gradually increased after NKCM administration, whereas in the control group, NK cell activity decreased or remained unchanged.
The NK cell activity on the 0th day when the SCCVII cell infusion day was 0 days was 6.7% in the control group and 6.5% in the experimental group. The NK cell activity on the 15th day was slightly decreased in both the experimental group and the control group as compared with the 0th day, 5.7% in the control group and 5.8% in the experimental group. On day 35, NK cell activity was significantly increased to 7.6% in the treatment group compared to 5.0% in the control group (p <0.05).

From the above, it can be seen that NKCM has an effect of increasing NK cell activity.

<Histological observation of tumor>
On day 35, when the injection date of SCCVII cells was 0 day, tumors were excised from each mouse in the experimental group and the control group, fixed in formalin, and embedded in paraffin to prepare tissue sections. . Tissue sections were stained with hematoxylin and eosin (HE) by a conventional method.
In the control mice not administered with NKCM, proliferatively active tumor cells were observed in the tumor. In contrast, in mice administered NKCM. Many necrotic tumor cells were observed (FIG. 4).
It is thought that NKCM killed tumor cells and led to cell death.

Example 3 Comparison of Healthy Person-derived NKCM and Cancer Patient-derived NKCM SCCVII strain was prepared from squamous cell carcinoma naturally occurring in C3H mice (provided by Dr. Masamasa Nishimura, Kinki University). The cells were grown in DMEM medium containing 10% FBS under conditions of 5% CO ₂ and 37 ° C.
SCCVII cells were collected, 5000 cells / well were seeded on a 6-well flat multiplate, and cultured in DMEM medium containing 10% FBS under conditions of 5% CO ₂ and 37 ° C. At this time, (1) NKCM stock solution (with IL-2 activation) obtained from the peripheral blood mononuclear cells of 5 cancer patients by the method described in Example 1, and (2) peripheral blood of 5 healthy individuals A fresh NKCM stock solution (without IL-2 activation) obtained from mononuclear cells without activation with IL-2 during the 14-day culture in the method described in Example 1, or (3) 5 healthy subjects 20% by volume of the NKCM stock solution (with IL-2 activation) obtained from the peripheral blood mononuclear cells by the method described in Example 1 was added, and the MTT method (Mosmann T. et al., 3 days later) was added. Cell proliferation ability was evaluated by Journal of immunological methods 65 (1-2): pp55-63).

  The evaluation results are shown in Table 3. In Table 3, the standard proliferative capacity indicates the number of cells when cultured under the above conditions without adding anything to SCCVII. In all groups, the proliferation of SCCVII cells was suppressed. The inhibitory effect is highest in the NKCM stock solution derived from healthy individuals activated by IL-2 in (3), and then in the NKCM stock solution derived from healthy individuals not activated by IL-2 in (2). (1) NKCM stock solution derived from cancer patients was the lowest. NK cells collected from healthy individuals showed a higher antitumor effect than NK cells derived from cancer patients.

  Conventionally, in NK cell therapy, NK cells collected from the self are used. In cancer patients, the activity of NK cells is often reduced. Therefore, in the conventional NK cell therapy, a method in which NK cells are activated with IL-2 and then returned to the body is often performed. NKCM derived from healthy human NK cells showed a higher antitumor effect than NKCM derived from cancer patients without NK cell activation, and showed a higher antitumor effect by NK cell activation. As described above, an antitumor composition having a remarkably high antitumor effect was obtained by preparing the antitumor composition of the present disclosure using NK cells of a healthy person.

Example 4: Clinical study Case 1
<Patient background>
A 58-year-old man with squamous cell carcinoma on the right lingual margin. The TNM classification according to the criteria of the International Union for Cancer (UICC) was T3N0M0.
The patient received arterial infusion chemotherapy with bleomycin but the tumor continued to grow. Before the start of NKCM administration, an induration of 25 mm × 30 mm was observed from the right lingual side to the center, and an ulcer of 20 mm × 25 mm was accompanied at the center. Moreover, the measured value of the SCC antigen was 3.5 ng / mL, and the NK cell activity was 15%.
<Administration method>
50 mL of NKCM (stock solution) was administered intravenously once a week for a total of 4 times. In addition, NKCM 5-fold concentrated solution 5 mL was administered once a week for a total of 28 nasal administrations in parallel.
<Progress>
Tumor growth stopped at 4 weeks after the start of NKCM administration, and ulcer reduction and pain relief were observed. At the end of administration, the tumor was shrinking (FIG. 5). As a blood finding, the SCC antigen decreased to 0.5 ng / mL, and the NK cell activity improved to 29%. Currently, there is no recurrence of the tumor and the course is good.
There were no complications such as nausea, respiratory failure, fever, general malaise, and allergic reactions that seemed to be related to other NKCM administration.

Example 5: Clinical study Case 2
<Patient background>
An 80-year-old woman with squamous cell carcinoma on the left lingual margin. The TNM classification according to the criteria of the International Union for Cancer (UICC) was T4aN3M0. At the first visit, a superficial so-called cauliflower-like large 35 mm × 20 mm mass was observed on the left tongue. Biopsy revealed moderately differentiated squamous cell carcinoma. At that time, the SCC antigen was 6.5 ng / mL and the NK cell activity was 10%.
Arterial injection chemotherapy (bleomycin) and radiation treatment almost eliminated the tongue tumor, SCC antigen decreased to 1.2 ng / mL, NK cell activity improved to 12%, and the tumor almost disappeared.
However, one year later, a metastatic lesion was found in the lower left jaw, so partial neck dissection was performed.
<Administration method>
From the day after partial neck dissection, 50 mL of NKCM (stock solution) was administered intravenously once a week for a total of 4 times. In addition, NKCM 5-fold concentrated solution 5 mL was administered once a week for a total of 28 nasal administrations in parallel.
<Progress>
At the end of NKCM administration, cervical metastases remained, but the SCC antigen maintained 1.5 ng / mL and NK cell activity of 20%. From the end of administration of NKCM to the present, no tumor growth was observed in the cervical metastases and in the oral cavity, no other metastases were found, and the general condition was good (FIG. 6A). The absence of an increase in metastasis in the left lower jaw was also confirmed by positron emission tomography (PET) images (FIG. 6B). Furthermore, the HE-stained images of the tissues obtained by puncturing the primary lesion and the submandibular lymph node were both grade 3 (only cancer cells considered to be non-viable were recognized) in the classification of the great stars and Shimozato. (FIG. 6C).
There were no complications such as nausea, respiratory failure, fever, general malaise, and allergic reactions that seemed to be related to other NKCM administration.

As described above, it has been clarified that the NK cell culture supernatant (NKCM) of another family has a tumor control effect equivalent to that of the own NK cell therapy. As shown in cases 1 and 2, the antitumor composition of the present disclosure also had an antitumor effect on solid cancers such as tongue cancer and metastases thereof.
Thus, the culture supernatant of NK cells that does not contain NK cells shows a high tumor control effect and is useful as a new cancer therapeutic agent. A strong rejection is expected when living cells from other families are administered intravascularly, but the culture supernatant has no allergic reactions even if they are derived from other cells. Many experiments by the inventors regarding Qing proved that no allergic reaction was observed in this other family NKCM.

NKCM is an epoch-making tumor therapeutic agent that can be expected to have the same effect while overcoming the weaknesses of conventional NK cell therapy.
Although the mechanism by which NKCM exhibits an antitumor effect as high as that of NK cell therapy is unknown, it is considered that cytokines and the like contained in NKCM activated NK cells in patients. However, it was an unexpected result that administration of cytokines such as IFNα / β contained in NKCM with a single dose of about 50 mL can provide an antitumor effect comparable to NK cell therapy.

<Storage stability of NK cell culture supernatant>
Three specimens of NK cell culture supernatant prepared from three different persons were stored for 3 months under the temperature conditions shown in Table 4, and the TNF-α concentration in the culture solution was consigned to SRL and measured by the ELIZA method. The TNF-α concentration immediately after preparation of each culture supernatant was also measured in the same manner. The results are shown in Table 4. In any specimen, at −20 ° C. or −80 ° C., the decrease in TNF-α was slight after storage for 3 months.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually described to be incorporated by reference, Incorporated herein by reference.

Claims (12)

An antitumor composition comprising a natural killer cell culture supernatant. The antitumor composition according to claim 1, which does not contain natural killer cells. The antitumor composition according to claim 1 or 2, which is cell-free. The antitumor composition according to any one of claims 1 to 3, wherein the natural killer cell is a natural killer cell derived from a healthy person. The natural killer cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage. The antitumor composition as described in any one of Claims 1-4. The antitumor composition according to any one of claims 1 to 5, which does not contain serum. The antitumor composition according to any one of claims 1 to 6, wherein the tumor is squamous cell carcinoma. The antitumor composition according to any one of claims 1 to 7, wherein the tumor is tongue cancer. (1) a step of culturing natural killer cells; and (2) a step of collecting a culture supernatant obtained by culturing the natural killer cells,
A method for producing an antitumor composition, comprising: The method for producing an antitumor composition according to claim 9, wherein the step of culturing the natural killer cells includes a culture in which serum is not contained in a culture solution. The method for producing an antitumor composition according to claim 9 or 10, wherein the natural killer cell is a natural killer cell derived from a healthy person. The method further includes the step of subjecting the collected culture supernatant to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. The manufacturing method of the anti-tumor composition as described in any one of Claims 9-11.