JP4653465B2 - Method for promoting natural killer cell proliferation and culture composition used therefor - Google Patents

Description

  The present invention relates to a method for promoting the proliferation of natural killer cells (hereinafter sometimes abbreviated as NK cells) present in human leukocytes when cultured in vitro.

  In recent years, cellular immunotherapy that applies the cellular immune function of living bodies has been applied to the treatment and prevention of various diseases including cancer. Especially in the field of cancer treatment, many clinical trials have been conducted all over the world for the past decades, and today it is recognized as the fourth treatment after surgical therapy, chemotherapy and radiation therapy. .

  Taking cancer therapy as an example, cellular immunotherapy is mainly classified into the following three therapies. That is, “LAK therapy” (for example, refer to Non-Patent Document 1) using lymphocytes activated by cytokines such as interleukin-2 (hereinafter abbreviated as IL-2), antigen-specific cytotoxic activity. Applying “activated lymphocyte transfer therapy” (see, for example, Non-Patent Document 2) used for killer T cell therapy and dendritic cells capable of inducing an immune response specific to a cancer antigen “Vaccine therapy” (see, for example, Non-Patent Document 3).

Among these, “LAK therapy” utilizes a phenomenon in which the killing ability of lymphocytes including natural killer cells to cancer cells is remarkably increased by culturing the lymphocyte fraction taken out of the body in the presence of IL-2. It is a therapy to do. That is, LAK therapy refers to a mononuclear cell fraction (lymphocyte + monocyte) or a lymphocyte fraction obtained by removing monocytes from the mononuclear cell fraction from peripheral blood, and the fraction is treated with IL-2. This is a therapy in which the cells are cultured for 1 to 2 weeks to enhance the antitumor activity and then returned to the living body. In the 1980s, Rosenberg et al. Started clinical studies targeting cancer-bearing patients. It has been widely implemented all over the world even now (for example, see Non-Patent Document 4). Thus, a cell cultured in the presence of IL-2 and induced to increase the killing ability against cancer cells is referred to as a LAK cell.
Rosenberg SA, Lotze MT, Muul LM, Leitman S, Chang AE, Ettinghausen SE, Matry YL, Skibber JM, Shiloni E, Vetto JT. N Engl J Med. 1985 Dec 5; 313 (23): 1485-92. Takayama T, Sekine T, Makuchi M, Yamazaki S, Kosuge T, Yamamoto J, Shimada K, Sakamoto M, Hirohashi S, Ohashi Y, Kahiko. Lancet. 2000 Sep 2; 356 (9232): 802-7. Nestle FO, Alijagic S, Gillet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadedorf D. et al. Nat Med. 1998 Mar; 4 (3): 328-32. Rosenberg SA. , Semin Oncol. 1986 Jun; 13 (2): 200-6.

  However, it is known that natural killer cells, which are the main effects of LAK cells, do not always efficiently proliferate in the culture method described above.

  Particularly in patients with cancer, cases in which natural killer cell culture is difficult are often observed. That is, even when cultivated by the above general culture method, almost no proliferation of natural killer cells was observed, and proliferation of other cells, for example, CD56 positive and CD3 positive T cells became dominant, resulting in natural killer cells. LAK cells that are almost free of sucrose are induced.

  As a result of inducing LAK cells from peripheral blood lymphocyte fractions of these patients according to a conventional method mainly for patients with gastrointestinal cancer, in 11 cases out of 20 cases, natural killer cells as well as healthy people LAK cells (natural killer cell ratio: average 20.4%) could be induced, but in 9 out of 20 cases, CD56 positive and CD3 positive T cells were dominant, and natural killer Little cell proliferation was observed (natural killer cell ratio: average 2.2%). Such LAK cells predominantly CD56-positive CD3-positive T cells have significantly lower cytotoxic activity against cancer cells and the ability to produce cytokines such as interferon γ and TNFα than LAK cells predominantly natural killer cells. Therefore, even if LAK cells in which such natural killer cell growth is hardly observed are used, almost no therapeutic effect can be expected.

  Therefore, the benefits of LAK therapy can be obtained even in patients who have difficulty in growing natural killer cells as described above, for example, patients in whom CD56-positive CD3-positive T cells with low antitumor activity are dominant even if cultured as usual. The development of a new cell culture method that can be enjoyed, that is, a new culture method of LAK cells that enables the natural killer cells with strong antitumor activity to grow well, is awaited. Further, even in the case of a patient who has a good natural killer cell proliferation by the conventional methods, it is expected that the effect of LAK therapy will be further enhanced by further promoting the proliferation.

  On the other hand, in recent years, antibody drugs mainly composed of monoclonal antibodies specific to tumor antigens such as Rituxan and Herceptin have been developed and used for treatment. One of the mechanisms of antitumor action of these drugs is natural killer cells. (Carson WE et al., Eur J Immunol. 2001 Oct; 31 (10): 3016-25. And Maloney DG et al., Semin Oncol. 2002 Feb; 29 (). 1 Suppl 2): 2-9.). Therefore, if more natural killer cells can be proliferated, not only the effectiveness of LAK therapy is expected to be increased, but also by using the antibody drug and LAK therapy in combination as described above, the effect is improved. It is expected to increase synergistically, and as a result, it will be possible to provide more useful treatments for cancers that exhibit various aspects.

  Therefore, an object of the present invention is to provide a culture method in which the growth of natural killer cells is promoted and a culture composition suitable for the culture method as compared with conventional LAK cell culture methods.

  As a result of diligent studies to solve such problems, the inventors have cultivated a group of cells containing natural killer cells in a medium containing a ligand of CD56 molecule expressed by natural killer cells, thereby It has been found that growth can be selectively promoted.

  CD56 molecule is one of the adhesion forms of neural cell adhesion molecule (NCAM), which is one of adhesion molecules, and is known to be expressed in natural killer cells and a subset of some T cells in peripheral blood. However, it has never been known so far that the addition of the ligand promotes the growth of these cells. According to the method according to the present invention, natural killer cell proliferation is hardly observed by induction with IL-2 alone, and even in a cell group in which CD56-positive CD3-positive T cells and the like are dominant, proliferation of natural killer cells. Is fully promoted. Moreover, even in the case of a cell group in which natural killer cells proliferate originally, a cell group containing more natural killer cells can be induced.

Examples of cell groups containing natural killer cells include mononuclear cells and lymphocyte fractions obtained from blood, and those cell groups can be easily prepared according to known methods by those skilled in the art. is there.
As one aspect of the invention, when culturing a cell group containing natural killer cells, the present invention increases the growth of natural killer cells by adding glycosaminoglycans, which are one of the ligands of the CD56 molecule, to the medium. It provides a culture method that significantly promotes.

  As the glycosaminoglycan used in the invention, for example, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and the like can be used. These are polysaccharides in which sugars having sulfate groups are regularly arranged, and all have a function as a ligand that binds to the CD56 molecule.

  These glycosaminoglycans are readily available as reagents, including some pharmaceutical grades. These are mainly purified from animal skin, cartilage, etc., but their origin is not specified. It is also possible to use artificial glycosaminoglycans that are chemically synthesized using the constituent sugars of each glycosaminoglycan as well as those naturally derived from the above.

  The concentration of glycosaminoglycan added at the time of culture is not particularly limited, but if the concentration is low, a sufficient effect cannot be expected, and if the concentration is too high, it is not preferable for cell growth. For example, in the case of heparin, the final concentration is in the range of 0.1 unit / mL to 1000 unit / mL, preferably 0.5 unit / mL to 500 unit / mL, most preferably 1 Unit / mL to 100 units / mL. In the case of chondroitin sulfate, heparan sulfate, and dermatan sulfate, the final concentration is in the range of 0.1 to 1000 μg / mL, preferably 0.5 μg / mL to 500 μg / mL, and most preferably 1 μg / mL to 300 μg / mL. Range.

  These glycosaminoglycans may be used alone or as a mixture of two or more.

  Furthermore, as a ligand for the CD56 molecule, not only the glycosaminoglycan which is a polysaccharide itself but also a form in which glycosaminoglycan is bound to a core protein, that is, a proteoglycan having glycosaminoglycan as a constituent component of a sugar chain is used. Is also possible.

  As another aspect of the invention, when culturing a cell group, the present invention comprises a leukocyte mononuclear cell fraction or lymphocyte fraction, and heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, etc. The present invention provides a culture method that significantly promotes the growth of natural killer cells by co-culturing with another cell that expresses proteoglycan.

  Cells that can be used for co-culture can be searched by a flow cytometer or immunostaining by an ordinary method using antibodies against each glycosaminoglycan. In addition, cells whose cell surface glycosaminoglycan is decomposed with an enzyme such as heparitinase, chondroitinase, etc., and the growth promoting action of natural killer cells disappears or attenuates can be used for co-culture. Examples of cells that satisfy such conditions include the liver cancer cell line HuH2 (specific examples are shown below), the myeloid leukemia cell line K562, the breast cancer cell line MCF7, and the gallbladder cancer cell line AG. Examples of cells that can be used in the present invention are not limited to these cell lines.

  Although the mixing ratio of the cell line and lymphocyte used for co-culture is not specified, it is preferably mixed in the range of 1: 1 to 1: 100, more preferably in the range of 1: 5 to 1:20. be able to.

  In addition, in order to prevent an unfavorable situation in which the cell line itself used for co-culture is proliferated at the time of culture, it is preferable to perform a treatment for suppressing its division and proliferation ability in advance. This treatment can be performed by techniques commonly used by those skilled in the art, such as culturing with mitomycin C or irradiating with radiation before using the cell line for co-culture.

  Furthermore, the present invention also provides a culture composition for carrying out the present invention.

  This culture composition is based on a medium generally used for culturing lymphocytes, and glycosaminoglycan as an additive for promoting the growth of natural killer cells, proteoglycan containing glycosaminoglycan as a constituent, And / or a cell to which proteoglycan expressing glycosaminoglycan as a constituent is added.

Standard media used to culture lymphocytes include RPMI-1640 media, AIM-V media, X-VIVO media, and the like. Therefore, these basic media can be used as they are as the basic medium, but they are not limited to these media, and any medium composition can be used as long as it is capable of lymphocyte culture. It does not matter. And the culture composition of the said invention is comprised by adding heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate etc. to such a basic composition as glycosaminoglycan. Glycosaminoglycan is 0.1 units / mL to 1000 units / mL, preferably 0.5 units / mL to 500 units / mL, most preferably 1 units / mL to 100 units / mL in heparin at final concentrations. In the concentration range, and in chondroitin sulfate, heparan sulfate, and dermatan sulfate, in the concentration range of 0.1 μg / mL to 1000 μg / mL, preferably 0.5 μg / mL to 500 μg / mL, most preferably 1 μg / mL to 300 μg / mL. Provided in blended form.

  According to the method of the present invention, the growth of natural killer cells can be selectively promoted by culturing a cell group containing natural killer cells in a medium containing a ligand for CD56 molecule. According to this method, it becomes possible to efficiently proliferate natural killer cells even in a cell group in which natural killer cell proliferation was not induced by IL-2. Even in a group of cells that are easily induced, a culture containing more natural killer cells can be obtained. If the cell composition containing the natural killer cells thus obtained in a high ratio is used, the effect of LAK therapy can be enhanced, and a useful therapeutic method can be obtained by combining LAK therapy with antibody drugs such as Rituxan and Herceptin. It will be possible to provide

The effects of the invention will be described below with specific examples.

  Promotion of natural killer cell (= NK cell) proliferation by heparan sulfate, chondroitin sulfate A, and dermatan sulfate in samples from healthy individuals.

60,000,000 peripheral blood mononuclear cells were obtained from 30 mL of peripheral blood of healthy persons using Lymphosepar (manufactured by Immunobiological Laboratories) according to a conventional method. Thereafter, the cells are suspended in AIM-V medium (manufactured by Invitrogen), seeded in a 150 cm ² culture flask, cultured in a CO ₂ incubator at 37 ° C. for 60 minutes, and then the floating cells are collected. 33,000,000 lymphocyte fractions were obtained. This lymphocyte fraction was suspended in AIM-V medium containing 10% fetal bovine serum and IL-2 (1,000 international units / mL), and 1,000,000 cells / 1 mL / mL were cultured in a 24-well culture plate. Heparan sulfate (manufactured by Seikagaku Corporation), chondroitin sulfate A (manufactured by Seikagaku Corporation) or dermatan sulfate was added to the medium to a final concentration of 100 μg / mL. Cultivate in a CO ₂ incubator at 37 ° C., and on day 7 of culture, add 1 mL of AIM-V medium containing 10% fetal calf serum and IL-2 (1,000 international units / mL) to each well to suspend the cells. After that, the cell suspension was seeded in 2 wells and cultured in the same manner on day 11 of culture, with 2 wells in place of 4 wells, and on day 14 of culture, cells were collected and subjected to hemocytometer. Board As well as measuring the number of cells used, NK cells (CD3-negative, CD56-positive, CD16-positive) the ratio of measured using the flow cytometer was calculated number NK cells.

The results are shown in Table 1. NK cells that had been 8.5% in the peripheral blood lymphocyte fraction at the time prior to the start of culture were added 100 μg / mL heparan sulfate, chondroitin sulfate A or dermatan sulfate in the medium for 2 weeks. By culturing, the numbers were 48.2%, 24.2%, and 49.2%, respectively, and the NK cell numbers were 72.5 times, 23.5 times, and 112.7 times, respectively, at the start of the culture.
On the other hand, as a comparative example, when cultured for 2 weeks under the condition where glycosaminoglycan was not added, the ratio of NK cells increased only to 17.0%, and the number of cells remained at 16.5 times.

Promotion of proliferation of NK cells by heparin in samples from healthy individuals.

Blood mononuclear cells were prepared from 20 mL of normal human peripheral blood using Lymphosepar according to a conventional method, and the cells were then suspended in AIM-V medium and seeded in a 75 cm ² culture flask. _The cells were cultured in a ₂ incubator for 60 minutes, and then the floating cells were collected to obtain 13,000,000 lymphocyte fractions. This lymphocyte fraction was suspended in AIM-V medium containing 10% fetal bovine serum and IL-2 (1,000 international units / mL), and 1,000,000 cells / 1 mL / mL were cultured in a 24-well culture plate. It seed | inoculated so that it might become a well. Heparin (Mochida Pharmaceutical) was added to a final concentration of 5 units / mL. Thereafter, the cells were cultured in a CO ₂ incubator at 37 ° C., and on the seventh day of the culture, 1 mL of AIM-V medium containing 10% fetal calf serum and IL-2 (1,000 international units / mL) was added to each well, and the cells were suspended. After turbidity, the cell suspension was seeded in 2 wells, and on the 11th day of culture, 2 wells were seeded in 4 wells and cultured. On the 14th day of culture, the cells were collected and the number of cells was counted using a hemocytometer, and the ratio of NK cells (CD3 negative, CD56 positive, CD16 positive) was measured using a flow cytometer. Cell number was calculated.

The results are shown in Table 2. NK cells that were 4.1% in the time before the start of culture, that is, peripheral blood lymphocyte fraction, were cultured for 2 weeks by adding 5 units / mL heparin at a final concentration in the medium. The number of NK cells was 21.6 times that at the start of culture.
On the other hand, as a comparative example, when cultured for 2 weeks without adding heparin, the ratio of NK cells increased only to 5.5%, and the number of cells remained 10.2 times.

Promotion of selective proliferation of NK cells by heparin in samples from patients with cancer.

The lymphocyte fraction prepared from the peripheral blood of a patient with liver cancer was suspended in an AIM-V medium containing fetal bovine serum 10%, IL-2 (1,000 international units / mL) in a 24-well culture plate, It seed | inoculated so that it might become 1,000,000 piece / 1mL / well. Heparin (manufactured by Mochida Pharmaceutical Co., Ltd.) was added to a final concentration of 5 units mL (5 units / mL). Thereafter, the cells were cultured in a CO ₂ incubator at 37 ° C., and on the seventh day of the culture, 1 mL of AIM-V medium containing 10% fetal calf serum and IL-2 (1,000 international units / mL) was added to each well, and the cells were suspended. After turbidity, the cell suspension was seeded in 2 wells, and on the 11th day of culture, 2 wells were seeded in 4 wells and cultured. On the 14th day of culture, the cells were collected and counted using a hemocytometer, and NK cells (CD3 negative, CD56 positive, CD16 positive) and CD56 positive T cells (CD3) were measured using a flow cytometer. (Positive, CD56 positive, CD16 negative) were measured, and the number of each cell was calculated.

The results are shown in Table 3. NK cells, which were 5.0% in the peripheral blood lymphocyte fraction before the start of culture, were cultured for 2 weeks by adding 5 units / mL heparin at a final concentration in the medium. The number of NK cells was 30.0 times that at the start of culture.
As a comparative example, when cultured for 2 weeks under the condition where heparin was not added, the ratio of NK cells increased only to 16.5%, and the number of cells remained at 16.6 times.
On the other hand, CD56-positive T cells account for 11.0% in the peripheral blood lymphocyte fraction, and 32.5% (15.7 times the number of cells) when cultured with heparin, without adding heparin. When cultured, it was about 44.7% (20.5 times the number of cells).
From the above results, it was clarified that heparin has an action of promoting the proliferation of NK cells, but has an action of functioning suppressively on the proliferation of CD56-positive T cells.

Promotion of selective proliferation of NK cells by chondroitin sulfate A and dermatan sulfate in a sample derived from a cancer-bearing patient in which CD56-positive T cells are predominant by IL-2.

Lymphocyte fractions prepared from peripheral blood of gastric cancer patients are suspended in AIM-V medium containing fetal bovine serum 10%, IL-2 (1,000 international units / mL) in a 24-well culture plate. , 000,000 cells / 1 mL / well. In addition, chondroitin sulfate A or dermatan sulfate was added to the medium so as to have a final concentration of 100 μg / mL. Thereafter, the cells were cultured in a CO ₂ incubator at 37 ° C., and on the seventh day of the culture, 1 mL of AIM-V medium containing 10% fetal calf serum and IL-2 (1,000 international units / mL) was added to each well, and the cells were suspended. After turbidity, the cell suspension was seeded in 2 wells, and on the 11th day of culture, 2 wells were seeded in 4 wells and cultured. On the 14th day of culture, the cells were collected and the number of cells was counted using a hemocytometer, and the ratio of NK cells (CD3 negative, CD56 positive, CD16 positive) and CD3 positive, CD56 were measured using a flow cytometer. The ratio of positive cells was measured, and the number of NK cells and the number of CD56 positive T cells were calculated.

The results are shown in Table 4. NK cells, which were 1.3% in the peripheral blood lymphocyte fraction at the time before the start of culture, were cultured for 2 weeks under the condition where glycosaminoglycan was not added, the number of cells increased 4.5 times. However, the ratio fell to 0.5%. However, when 100 μg / mL of chondroitin sulfate A or dermatan sulfate was added to the medium at the final concentration and cultured for 2 weeks, the ratio of NK cells was 2.4% and 2.3%, respectively, and the number of NK cells was also started. It was revealed that the proliferation of NK cells was significantly promoted by 26.1 times and 26.3 times the time.
On the other hand, the number of CD56-positive T cells, which was 1.2% in the peripheral blood lymphocyte fraction, increased to 420.8 times by culturing for 2 weeks under the condition where glycosaminoglycan was not added. The ratio was about 42.2%. On the other hand, when cultured for 2 weeks in the presence of 100 μg / mL of chondroitin sulfate A or dermatan sulfate, the ratios of CD56-positive T cells were 36.9% and 39.8%, respectively, and the number of cells was also the same as at the beginning of the culture. 458.3 times and 521.8 times, and the proliferation of CD56 positive T cells was almost the same as when no glycosaminoglycan was added.
From the above results, it was confirmed that the addition of chondroitin sulfate A or dermatan sulfate selectively promotes the growth of NK cells.

Promotion of selective proliferation of NK cells by co-culture with glycosaminoglycan-expressing cells in samples from healthy individuals.

The lymphocyte fraction prepared from the peripheral blood of a healthy person is suspended in an AIM-V medium containing fetal bovine serum 10% and IL-2 (1,000 international units / mL) in a 24-well culture plate. , 000,000 pieces / 2 mL / well. In addition, a HuH2 cell line (liver cancer cell line) that has been cultured for 30 minutes in the presence of mitomycin C at a final concentration of 30 μm in advance to stop its division and proliferation ability to become 200,000 cells / 2 mL / well. (Number of lymphocyte fractional cells: number of HuH2 cells = 10: 1). In addition, some HuH2 cells were previously subjected to enzyme treatment at 37 ° C. for 4 hours in phosphate buffered saline with 10 mU / mL heparitinase I (manufactured by Seikagaku Corporation) at a final concentration of glycoproteins on the cell surface. It was compared with the case of using cells that had been digested with saminoglycan and not subjected to enzyme treatment.

The results are shown in Table 5. NK cells that had been 6.8% in the peripheral blood lymphocyte fraction before the start of culture were cultured in the presence of IL-2 for 7 days. The NK cell ratio was 10.4%, the number of cells. Increased by 1.9 times, but by co-culture with HuH2 cells, the ratio of NK cells was 14.5%, the number of cells was 3.7 times that at the start of culture, and the proliferation of NK cells was significantly promoted. It became clear. On the other hand, when co-cultured with HuH2 cells pretreated with heparitinase I in advance, the ratio of NK cells was 11.9%, and the number of NK cells was 2.5 times that at the start of the culture. In the HuH2 cells digested with the glycosaminoglycan, a significant decrease in the effect of promoting the proliferation of NK cells was observed as compared with the untreated HuH2 cells.
From the above results, the proliferation of NK cells is selectively promoted by co-culture with HuH2 cells, and the proliferation promoting action of NK cells is an action via glycosaminoglycan expressed by HuH2 cells. Was confirmed.

Industrial use of the invention

  The present invention relates to a method for culturing and expanding natural killer cells present in human leukocytes in vitro. Natural killer cells cultured by the method described in the present invention can be used for cell immunotherapy for the treatment and / or prevention of cancer, viral diseases and the like.

Claims (10)

A method of promoting the growth of natural killer cells,
A method comprising culturing a cell group containing natural killer cells in a medium containing at least one of the following (a) to (c) :
(A) A glycosaminoglycan containing one or more substances selected from the group consisting of heparin, chondroitin sulfate, heparan sulfate, and dermatan sulfate.
(B) A proteoglycan comprising the glycosaminoglycan of (a) as a constituent component.
(C) A cell that expresses at least one of the glycosaminoglycan of (a) and the proteoglycan having the glycosaminoglycan of (a) as a constituent component.   The method according to claim 1, wherein the cell group is a mononuclear cell or lymphocyte fraction obtained from blood.   The method according to claim 1 or 2, wherein the medium further contains interleukin-2. The method according to any one of claims 1 to 3, wherein the cell of (c) is a cell whose proliferation promoting ability of natural killer cells is reduced by heparitinase treatment or chondroitinase treatment. The method according to any one of claims 1 to 4, wherein the cell group is derived from a cancer patient. A culture composition for promoting the growth of natural killer cells comprising at least one of the following (a) to (c):
(A) A glycosaminoglycan containing one or more substances selected from the group consisting of heparin, chondroitin sulfate, heparan sulfate, and dermatan sulfate .
(B) A proteoglycan comprising the glycosaminoglycan of (a) as a constituent component.
(C) A cell that expresses at least one of the glycosaminoglycan of (a) and the proteoglycan having the glycosaminoglycan of (a) as a constituent component . The culture composition according to claim 6, wherein the natural killer cell is contained in a cell group that is a mononuclear cell or lymphocyte fraction obtained from blood. The culture composition according to claim 6 or 7, further comprising interleukin-2. The culture composition according to any one of claims 6 to 8, wherein the cell (c) is a cell whose natural killer cell proliferation promoting ability is reduced by heparitinase treatment or chondroitinase treatment. The culture composition according to any one of claims 6 to 9, wherein the natural killer cell is derived from a cancer patient.