WO2011129348A1

WO2011129348A1 - Composition for treatment of cancer which is produced from cancer-tissue-derived cell mass or cancer cell aggregate, and process for production of immunotherapeutic agent and method for evaluation of efficacy of immunotherapy both using the composition

Info

Publication number: WO2011129348A1
Application number: PCT/JP2011/059132
Authority: WO
Inventors: 正宏井上
Original assignee: 株式会社Ｒｅｉメディカル; 地方独立行政法人大阪府立病院機構
Priority date: 2010-04-13
Filing date: 2011-04-13
Publication date: 2011-10-20
Also published as: KR20130055591A

Abstract

Disclosed are an immunotherapy and a method for evaluating the efficacy of an immunotherapy, both of which utilize a novel cancer-tissue-derived cell mass (a cell mass derived from a cancer tissue) or a novel cancer cell aggregate which can reflect the in vivo behavior of a cancer cell accurately. A composition for treating cancer is prepared by chemically or photodynamically treating a cancer-tissue-derived cell mass or a cancer cell aggregate. The composition for treating cancer can be used as a cancer vaccine which is intended to be directly administered to a patient from which the cell mass or the cell aggregate is derived, or as a medicinal agent which is intended to be contacted in vitro with cells derived from blood collected from the patient. Further, the efficacy of an immunotherapy can be evaluated by measuring the activity of a killer T-cell in blood over time using a cancer-tissue-derived cell mass or a cancer cell aggregate.

Description

Composition for cancer treatment obtained from cancer tissue-derived cell mass or cancer cell aggregate, method for producing immunotherapeutic agent using the same, and method for evaluating immunotherapeutic effect

The present invention relates to a composition for cancer treatment obtained from a cancer tissue-derived cell mass or a cancer cell aggregate, a method for producing an immunotherapeutic agent using the same, and a method for evaluating an immunotherapeutic effect. More specifically, the present invention relates to a composition for treating cancer obtained by treating cancerous tissue-derived cell masses or cancer cell aggregates capable of reconstructing cancer in vitro and retaining proliferation ability, and immunity using the same The present invention relates to a method for producing a therapeutic agent and a method for evaluating an immunotherapeutic effect.

In recent years, various researches have been accumulated to overcome cancer, and as a result, treatment results for early cancer are dramatically improved. However, treatment of advanced cancer is still difficult, and cancer continues to be the leading cause of Japanese death. According to the Ministry of Health, Labor and Welfare's 2007 demographic data, more than 340,000 people die annually from cancer.

At present, chemotherapy, surgery and radiation therapy are the mainstream in cancer treatment, but immunotherapy is also used as another option.

For immunotherapy, active immunotherapy, which directly administers a vaccine to the living body to enhance the immune activity of the living body, and immune-related cells such as cytotoxic T lymphocytes and dendritic cells, are once activated outside the body, and then the body is restarted. There is a passive immunotherapy with the technique of returning to. In any of these methods, as a so-called immunogen, a tumor antigen peptide that has been found to be universally expressed in various types of cancer (for example, WT1 peptide (Patent Document 1), HLA-A2 restriction An antigen peptide (patent document 2) etc. can be used. On the other hand, it is also possible to use a cancer tissue containing an unidentified antigen instead of the thus-defined antigenic peptide. Such antigenic peptides or conjugates or fusions of treated products of cancer tissue with dendritic cells are also known.

Furthermore, there is also disclosed a vaccine in which cancer tissues and cells are immobilized on microparticles and mixed with cytokines (Patent Document 3).

WO 2007/097358 JP, 2009-65835, A JP, 2001-10961, A

An object of the present invention is to provide a composition for treating cancer obtained from a cancer tissue-derived cell mass or a cancer cell aggregate.

Another object of the present invention is to provide a method for producing an immunotherapeutic agent from a cancer tissue-derived cell mass or a cancer cell aggregate.

The present inventors have intensively studied to provide a cancer vaccine or immunotherapy having a very high therapeutic effect by using an antigen with high specificity and little extraneous contamination for individual cancer patients. The present inventors have found a novel composition for treating cancer and a method for preparing an immunotherapeutic agent using a novel cancer tissue-derived cell mass or cancer cell aggregate, and have completed the present invention.

The present invention relates to a composition for treating cancer obtained by treating a cancer tissue-derived cell mass or a cancer cell aggregate.

The process may be a chemical process.

The chemical treatment may be enzyme treatment or formalin treatment.

The process may be a photodynamic process.

The treatment may be radiation treatment.

The composition for treating cancer may be a cancer vaccine for oral or parenteral administration to patients derived therefrom.

The composition for treating cancer may be for contacting blood-derived cells obtained from blood from a patient in vitro.

The blood may be peripheral blood.

The blood-derived cells can be dendritic cells derived from monocytes.

The present invention also provides a process of contacting blood-derived cells recovered from blood derived from a patient with the composition for treating cancer described in any of the above, and recovering the blood-derived cells activated by contact. A method of producing an immunotherapeutic agent for inoculating said patient, comprising the steps of

The blood may be peripheral blood.

In the above method for producing an immunotherapeutic agent, the blood-derived cells may be monocytes, and may further include the step of inducing monocytes to dendritic cells.

The present invention is also a method of evaluating the effects of immunotherapy, comprising contacting blood-derived cells derived from a patient who has been subjected to immunotherapy with a cancer tissue-derived cell mass or a cancer cell aggregate. About.

The method of evaluating the effect of the above immunotherapy may further include the step of measuring the cytotoxicity of the blood-derived cells.

The blood-derived cells can be isolated cytotoxic T cells.

In the method of evaluating the effect of the immunotherapy, the blood-derived cells are isolated natural killer cells, and the step of contacting may further cause an antibody to coexist.

In the method of evaluating the effect of the above immunotherapy, the patient who has been subjected to the immunotherapy is a patient who has been subjected to an immunotherapy using a cancer tissue-derived cell mass or a cancer cell aggregate, and blood-derived cells derived from the patient However, it may be provided as a plurality of samples collected while changing the elapsed time since administration of immunotherapy.

The composition for treating cancer of the present invention is obtained from an individual patient and does not contain unnecessary impurities other than cells. Therefore, it is possible to elicit an immune response which is highly specific to the cancerous tissue of the individual patient and which reliably attacks the cancer. In addition, since they can be stored in vitro, and can be grown as needed, they can be prepared at any time according to the condition of the patient. Such a composition for treating cancer can also be used as a directly administered cancer vaccine, and can be conveniently used to specifically activate blood cells in vitro. Furthermore, in the present invention, the effect of immunotherapy can also be simply examined using such a composition for cancer treatment, a cancer tissue-derived cell mass or a cancer cell aggregate itself.

It is a figure which shows the cancer tissue origin cell mass used for this invention. It is a figure showing the change in shape and proliferation ability of the cancer tissue origin cell mass used for the present invention in the culture process in vitro. Tumor tissue (two on the right side of the figure) obtained by implanting a cancer tissue-derived cell mass used in the present invention into a mouse and tumor tissue excised from the inside of the body which is derived from the cancer tissue-derived cell mass (left side 2 of the figure) 1) and FIG. It is a figure which shows the cancer tissue origin cell mass used for this invention obtained from various cancer tissue. It is the figure which compared the state before freezing (left) and 24 hours after thawing | decompression (right) of the cancer tissue origin cell mass used by this invention. It is a figure which shows a cancer cell aggregate used by this invention. It is a figure which shows the cancer cell aggregate obtained from the sample used by this invention. It is a figure which shows the change by the time progress after freezing and thawing the cancer cell aggregate used by this invention. It is a figure which shows the flow cytometry analysis result of the untreated cancer tissue origin cell mass used by this invention, and the cancer tissue origin cell mass processed. It is a graph which shows the flow cytometry analysis result of the untreated cancer tissue origin cell mass used by this invention, and the cancer tissue origin cell mass processed. It is the figure which carried out the western blot analysis of the protein relevant to the apoptosis induction of the untreated cancer tissue origin cell mass used by this invention, and the cancer tissue origin cell mass processed.

The cancer tissue-derived cell mass of the present invention is an isolated product separated from or treated as a mass containing three or more cancer cells from a cancer tissue obtained from an individual, or a culture thereof, and retains proliferation ability in vitro. It can be something that

Here, “the separated material separated and treated as a mass containing three or more cancer cells from cancer tissue obtained from an individual” is obtained by treating cancer tissue obtained from cancer generated in vivo. It refers to an isolate containing three or more, preferably eight or more cancer cells. Such isolates do not include those that have been separated into single cells, and do not include constructs that have been separated into single cells and then reassembled. However, this separated material includes not only those immediately after being separated from the living body, but also those which have been kept in physiological saline for a certain period of time and those which have been frozen or refrigerated.

“Cancer tissue obtained from an individual” refers to cancer tissue obtained by excision by surgery etc., as well as cancer tissue obtained so that it can be handled in vitro for histological examination with an injection needle or an endoscope. Point to.

“Culture of the isolate obtained as a mass separated from a cancer tissue obtained from an individual as a mass containing three or more cancer cells” is obtained by treating a cancer tissue obtained from a cancer generated in vivo It refers to what is obtained by culturing in vitro the isolate separated as a mass containing three or more cancer cells. The culture time is not particularly limited as long as it is present in the medium even for a short time. Such a culture often exhibits a substantially spherical or elliptical spherical shape by culturing for a certain period of time, preferably 3 hours or more. The culture here includes a substantially spherical or spheroidal culture after such a given period of time, and an atypical culture up to that. Furthermore, an irregular shape obtained by further dividing such a substantially spherical or spheroidal culture, and a substantially spherical or spheroid obtained by further culture are also referred to as the culture herein. Preferably, the cells contained in the cancer tissue-derived cell mass are a population of pure cancer cells only.

The cancer tissue-derived cell mass of the present invention can maintain its growth ability in vitro at a temperature of 37 ° C. under cell culture conditions of a 5% CO ₂ incubator for at least 10 days or more, preferably 13 It means that the growth ability can be maintained for a period of a day or more, more preferably 30 days or more.

Such a cancer tissue-derived cell mass can retain its proliferative ability for a period of 10 days or more, preferably 13 days or more, more preferably 30 days or more, by continuing the culture as it is. By performing mechanical division, the proliferative capacity can be maintained virtually indefinitely.

The mechanical division can be performed using a scalpel, a knife, scissors, an ophthalmologic sharp blade or the like. Alternatively, it can be performed by attaching an injection needle to a syringe and repeating aspiration and discharge of the cancer tissue-derived cell mass together with the culture solution. For example, a 1 ml syringe and a 27G injection needle are preferably used in the present invention, but the invention is not limited thereto.

Here, the medium for culturing the cancer tissue-derived cell mass of the present invention is not particularly limited, but preferably, a medium for animal cell culture is used. Particularly preferably, a serum-free medium for stem cell culture is used. Such serum-free medium is not particularly limited as long as it can be used to culture stem cells. A serum-free medium refers to a medium free of unprepared or unpurified serum, and can be used by adding a purified blood-derived component or animal tissue-derived component (eg, growth factor).

The serum-free medium of the present invention can be prepared using a medium used for culturing animal cells as a basal medium. As a basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, DMEM medium, RPMI 1640 medium, Fischer's medium And combinations thereof.

A serum substitute can be added to such serum-free medium to culture the cancer tissue-derived cell mass of the present invention. The serum substitute suitably contains, for example, albumin, amino acid (eg, non-essential amino acid), transferrin, fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol or 3 'thiol glycerol, or equivalents thereof, etc. It can be

Commercially available serum substitutes can also be used in the culture method of the present invention. Such commercially available serum substitutes include, for example, knockout serum replacement (KSR), Chemically-defined Lipid concentrated fatty acid concentrate (Gibco), and Glutamax (Gibco).

The medium for culturing the cancer tissue-derived cell mass of the present invention may also contain vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, inorganic salts and the like.

In particular, any serum-free medium such as serum-free medium containing EGF and bFGF, serum-free medium containing bFGF and serum substitute such as knockout serum replacement (KSR, manufactured by Invitrogen) can be preferably used. . The content of serum substitute or EGF or the like is preferably 10 to 30% w / v of the whole medium.

Such a medium is not limited, but commercially available products include STEMPRO human ES cell serum-free medium (Gibco).

The incubator used to culture the cancer tissue-derived cell mass is not particularly limited as long as it can generally culture animal cells, and for example, flasks, tissue culture flasks, dishes, petri dishes, for tissue culture Dishes, multi dishes, microplates, micro well plates, multi plates, multi well plates, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc. may be mentioned.

The culture vessel is non-cell-adherent, and is preferably three-dimensionally cultured in the presence of a cell supporting substrate such as extracellular matrix (ECM) in the medium. The cell support matrix may be for adhesion of cancer tissue-derived cell mass. Examples of such a cell support substrate include matrigel using an extracellular matrix, for example, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin and fibronectin. Such conditions are suitably used particularly when the cancer tissue-derived cell mass of the present invention is desired to be expanded.

Other culture conditions can be set appropriately, for example, the culture temperature is preferably, but not limited to, about 30 to 40 ° C. Most preferably, it is 37 ° C. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 2 to 5%.

The cancer tissue-derived cell mass of the present invention can be cultured in such a medium and culture conditions. Furthermore, the culture of the cancer tissue-derived cell mass may, depending on its individual nature, require co-culture with other cells, or may require the presence of additional specialized supplements such as hormones.

Specifically, co-culture may be performed with feeder cells. As feeder cells, stroma cells such as fetal fibroblasts can be used. Specifically, although not limited thereto, NIH3T3 and the like are preferable.

Alternatively, for certain types of breast cancer, uterine cancer and prostate cancer, it is preferable to culture in the presence of a hormone. Specifically, estrogens for breast cancer, progesterone for uterine cancer, testosterone for prostate cancer and the like are not limited thereto, and various hormones can be added to advantageously adjust culture conditions. Furthermore, the presence of such hormones indicates the hormonal dependence of the cancer of the originating patient by examining the behavior after culture of the cell mass derived from cancer tissue, for example, the state of life or death or the state of proliferation. There is a possibility that the efficacy of antihormonal drug treatment can be predicted.

The cancer tissue-derived cell mass of the present invention can also be cultured in suspension culture. In suspension culture, a cancer tissue-derived cell mass is cultured in a non-adhesive condition to a culture vessel in a medium. As such suspension culture, for example, embryoid body culture method (Keller et al., Curr. Opin. Cell Biol. 7, 862-869 (1995)), SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288- 296 (2005); see WO 2005/123902). There is no particular limitation, but it may be used, for example, when forming or maintaining a stable cancer tissue-derived cell mass having a substantially spherical shape, and sometimes having a basement membrane-like substance.

The cancer tissue-derived cell mass of the present invention also includes those immediately after separation from the cancer tissue-derived cell mass of an individual, those after refrigeration and cryopreservation, and also their cultures. The culture may be performed for a period of preferably 3 hours or more, more preferably at least 10 hours and up to 36 hours, more preferably at least 24 hours to 36 hours.

The number of cancer cells constituting the cancer tissue-derived cell mass is at least 3 or more, preferably 8 or more, more preferably 10 or more, still more preferably 20 or more, and most preferably 50 or more. When the cancer tissue-derived cell mass of the present invention is an isolate, it is preferably 1000 or less, more preferably about 500 or less. It is possible to increase the number of cultures after culturing the isolates by culturing. However, even if it is a culture, it is preferably 10,000 or less, more preferably 5,000 or less.

In the present invention, the term "cancer cell" is used in a commonly used meaning, and refers to a cell in which the order seen in normal cells, that is, unlimited division / proliferation and departure from apoptosis, is disrupted in vivo. More specifically, it refers to a cell that has lost or extremely attenuated cell growth control function, and typically acquires infinite proliferation ability at a high frequency of 80% or more, many of which also have invasive transfer ability. This means that the cells are often provided, and as a result, humans and other mammals, in particular, mammals, are cells that are regarded as malignant neoplasms leading to death.

In the present invention, the type of cancer tissue from which the present invention is derived is not particularly limited, and lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwannoma and meningioma occur in mammals and other animals. It may be adenoma, melanoma, leukemia, lymphoid malignancies, etc., but in particular, it is preferable that it is a carcinoma arising in epithelial cells of a mammal. Examples of carcinomas arising in such epithelial cells include non-small cell lung cancer, hepatocellular carcinoma, biliary tract cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, endometrial cancer, bladder cancer, Pharyngeal cancer, breast cancer, salivary adenocarcinoma, renal cancer, prostate cancer, labia cancer, anal cancer, penile cancer, testicular cancer, thyroid cancer, head and neck cancer and the like. There is no particular limitation on mammals and other animals, but animals belonging to primates including monkeys and humans, animals belonging to rodents such as mice, squirrels and rats, animals belonging to rabbits, cats such as dogs and cats Animals belonging to the eye are exemplified.

Among them, in the present invention, especially derived from colon cancer tissue, from ovarian cancer tissue, from breast cancer tissue, from lung cancer tissue, from prostate cancer tissue, from renal cancer tissue, from bladder cancer tissue, from pharyngeal cancer tissue, or from pancreatic cancer It is particularly preferable to derive from, but not limited to.

In the case of a cancer tissue-derived cell mass derived from colon cancer tissue, the included cancer cells are not particularly limited, but may express CD133.

Separation treatment of cancerous tissue obtained from cancer generated in vivo includes, but is not limited to, enzymatic treatment of cancerous tissue obtained from an individual.

The enzyme treatment may be treatment with one of collagenase, trypsin, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. The enzyme treatment conditions may be in an isotonic salt solution buffered to a physiologically acceptable pH, for example about 6 to 8, preferably about 7.2 to 7.6, such as PBS or Hanks balanced salt solution For example, at about 20 to 40 ° C., preferably about 25 to 39 ° C., for a sufficient time to degrade connective tissue, eg, about 1 to 180 minutes, preferably 30 to 150 minutes, for which sufficient concentration It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

Without limitation, conditions for this enzyme treatment include treatment with mixed enzymes including collagenase. For example, a mixture comprising one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin, and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV Treatment with enzymes is included.

Such mixed enzymes include, but are not limited to, Liberase Blendzyme 1 (registered trademark) and the like.

Alternatively, the cancer tissue-derived cell mass of the present invention may contain three or more cancer cell aggregates and exhibit a substantially spherical shape or an elliptical spherical shape.

Although not limited thereto, it may include basement membrane-like substances present on the outer peripheral surface of the cancer cell aggregate.

Here, although not particularly limited, the cancer cells forming the aggregate may have one or more surface antigens selected from the group consisting of CD133, CD44, CD166, CD117, CD24, and ESA on the cell surface. There are many. CD133, CD44, CD166, CD117, CD24, and ESA are surface antigens generally expressed on leukocytes such as lymphocytes, fibroblasts, epithelial cells, cells such as tumor cells. These surface antigens not only function as cell-cell and cell-matrix adhesion but also are involved in various signal transductions, but are also surface markers of various stem cells.

In the present invention, when the cell group "expresses" a surface antigen such as CD133, 80% or more, preferably 90% or more, more preferably substantially all of the surface antigen is present in the cell group. Points to the state shown.

In the present specification, the “basement membrane-like substance” preferably includes, but is not limited to, collagen, laminin, nidogen, proteoglycans such as heparan sulfate proteoglycan, and / or glycoproteins such as fibronectin. It refers to a substance. In the present invention, a laminin-containing basement membrane-like substance is preferred.

Laminin is a macromolecular glycoprotein that constitutes the basement membrane. The functions of laminin are diverse and are involved in cellular functions such as, for example, cell adhesion, intercellular signaling, proliferation of normal cells and cancer cells. Laminin has a structure in which each of three different subunits is linked by a disulfide bond, and eleven types are found according to different types of each subunit.

Among these, laminin 5 is usually produced only from epithelial cells and is known as a component having an activity to promote adhesion of epithelial cells to basement membrane and motor function. This laminin 5 has a structure in which one each of α3 chain, β3 chain, and γ2 chain form a complex, and in particular, γ2 chain is considered to be unique to LN5, and is contained in other LN molecular species. Absent.

The cancer tissue-derived cell mass of the present invention may have a configuration in which the outer periphery of a collection of cancer cells is totally enclosed in a membrane formed by such a basement membrane-like substance. Such forms can be analyzed by electron microscopic observation of cancer tissue-derived cell masses or immunostaining of basement membrane components, or a combination of both. Here, the aggregate of cancer cells is preferably an aggregate that does not purely contain cells other than cancer cells.

The presence of laminin can be detected, for example, by contacting an antibody that recognizes laminin, for example, a mouse laminin-derived rabbit antibody from Sigma-Aldrich, with a cancer tissue-derived cell mass and measuring an antibody-antigen reaction.

In addition, it is also possible to use a specific antibody that identifies up to the type of laminin. For example, the presence of laminin 5 can be detected, for example, by contacting an antibody having reactivity with the above-mentioned unique γ2 chain or a fragment thereof with a cancer tissue-derived cell mass and measuring the reaction of the antibody. it can.

In the cancer tissue-derived cell mass of the present invention, it is preferable that the thin membranous basement-like substance is formed to about several μm, preferably about 40 to 120 nm, depending on the size of the mass, but is not limited thereto.

The size of the cancer tissue-derived cell mass of the present invention is not limited, and includes irregularly shaped particles having a particle diameter or volume average particle diameter of about 8 μm to 10 μm. Things are also included. Preferably, the diameter is 40 μm to 1000 μm, more preferably 40 μm to 250 μm, and still more preferably 80 μm to 200 μm.

The cancer tissue-derived cell mass of the present invention often has one or more sequences particularly selected from the group consisting of a shelf array, a sheet array, an overlay array and a syncytial array, but is not particularly limited.

In the cancer tissue-derived cell mass of the present invention, typically, a step of subjecting a fragment of cancer tissue excised from a living body to an enzyme treatment; and among the enzyme-treated products, a mass containing three or more cancer cells is selected and recovered. And a method comprising the steps of

Furthermore, without limitation, the cancer tissue-derived cell mass of the present invention can be prepared by a method comprising the step of culturing the component thus recovered for 3 hours or more.

First, a cancer tissue removed from a living body can be minced as it is, or can be first maintained in animal cell culture medium before mincing. Such animal cell culture media include, but are not limited to, Dulbecco's MEM (such as DMEM F12), Eagle's MEM, RPMI, Ham's F12, alpha MEM, Iscove's modified Dulbecco, and the like. At this time, it is preferable to perform suspension culture in a cell non-adhesive culture vessel.

It is also preferred that the cancerous tissue be washed prior to mincing. Such washings include, but are not limited to, acetate buffer (acetate + sodium acetate), phosphate buffer (phosphate + sodium phosphate), citrate buffer (citric acid + sodium citrate), boric acid A buffer solution such as a buffer solution, a tartaric acid buffer solution, a Tris buffer solution, or a phosphate buffered saline can be used. In the present invention, particularly preferably, tissue washing can be performed in HBSS. The number of times of washing is appropriate once to three times.

The fragmentation can be performed by dividing the tissue after washing with a knife, scissors, a cutter (manually, automatically) or the like. The size and shape after fragmentation are not particularly limited, and may be random, but preferably have a uniform size of 1 mm to 5 mm square, more preferably 1 mm to 2 mm square.

The debris obtained in this way is then subjected to an enzyme treatment. Such enzyme treatment may be treatment with one of collagenase, trypsin, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. The enzyme treatment conditions may be in an isotonic salt solution buffered to a physiologically acceptable pH, for example about 6 to 8, preferably about 7.2 to 7.6, such as PBS or Hanks balanced salt solution For example, at about 20 to 40 ° C., preferably about 25 to 39 ° C., for a sufficient time to degrade connective tissue, eg, about 1 to 180 minutes, preferably 30 to 150 minutes, for which sufficient concentration It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

Without limitation, the conditions for this enzyme treatment may be, for example, treatment with a mixed enzyme comprising collagenase. More preferably, one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin, and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV Treatment with mixed enzymes is included.

Next, among the enzyme-treated products thus obtained, it is preferable to sort and recover a mass containing three or more cancer cells. The method of sorting and recovering is not particularly limited, and any method known to those skilled in the art of sorting sizes can be used.

As a method of distributing the size, a simple method is by visual observation, separation by phase contrast microscope, or sieve, but it is not particularly limited as long as it is a separation method by particle diameter available to those skilled in the art. In the case of using a sieve, it is preferable to recover a component which passes a sieve mesh size of 20 μm and does not pass 500 μm. More preferably, components that pass a sieve mesh size of 40 μm and not pass 250 μm are recovered.

Here, a mass containing three or more cancer cells to be sorted is a cancer tissue-derived cell mass of the present invention, and has a range of sizes. The size within a certain range includes small particles having a volume average particle diameter of about 8 μm to 10 μm, but in the case of a spherical shape, the diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, and more preferably 40 μm to 250 μm. In the case of an elliptical shape, the major diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, more preferably 40 μm to 250 μm, and in the case of indeterminate shape, the volume average particle diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm. And more preferably 40 μm or more and 250 μm or less. The volume average particle diameter can be measured by evaluating the particle size distribution and the particle shape using a phase contrast microscope (IX70; manufactured by Olympus Corporation) with a CCD camera attached.

The separated processed product as a sorted and recovered component thus obtained or the culture thereof is the cancer tissue-derived cell mass of the present invention. The culture may be one in which the separation and recovery component is present in the culture medium for a short time, for example, at least 3 hours or more, preferably 10 hours to 36 hours, more preferably 24 hours. By culturing for a period of ̃36 hours, it may be in the shape of a substantially spherical shape or a substantially elliptical shape. The culture time may be more than 36 hours, several days, 10 days or more, 13 days or more, or 30 days or more.

The culture can be carried out as it is in the medium for a long period of time, but preferably, by carrying out mechanical division periodically during the culture, the proliferative capacity can be maintained virtually infinitely.

The cancer tissue-derived cell mass of the present invention, for example, has a high degree of establishment in transplantation into xenogeneic animals even with 10 or less cancer tissue-derived cell masses having a diameter of 100 micrometers (corresponding to 1000 cells or less). Therefore, the cancer tissue-derived cell mass of the present invention is useful for simple preparation of mouse and other cancer model animals, and more rigorous examination of cancer tissue, evaluation of drug sensitivity, or treatment such as radiation therapy. Evaluation of aspects is possible.

The cancer tissue-derived cell mass of the present invention can be cryopreserved and can retain its proliferative ability under normal storage conditions.

The cancer cell aggregate of the present invention is a cancer tissue-derived cell mass or a cancer tissue obtained from an individual, which is single-cellified and then individual cells in the single cell complex or completely up to individual cells. A group of several cells that were not separated or formed by aggregation of the individual cells and some cells that were not completely separated into individual cells into a total of three or more cells Aggregates or cultures thereof, which are capable of retaining their ability to grow in vitro.

Here, "a single cell of a cancer tissue-derived cell mass or a cancer tissue obtained from an individual" means that at least a portion of the cancer tissue-derived cell mass or the obtained cancer tissue is separated into single cells in vitro. To perform the process of Thus, typically, after such treatment, some cells may co-exist without being separated individually, even in the presence of cells separated into individual single cells. Even in the case, it corresponds to "to unicellularize" as referred to herein. At this time, in a state where it is not separated up to the individual, there are aggregates of up to 10 cells, preferably those of 2 to 3 cells.

The term “aggregation to 3 or more cells” means individual cancer tissues obtained from cancers generated in vivo or individual cancer clusters obtained from the cancer tissue-derived cell masses found by the present inventors. It refers to a state in which several cell aggregates or combinations thereof that were not separated from one another or individually are included so as to include at least three or more cells.

When subjecting a cancer tissue-derived cell mass or a cancer tissue obtained from a cancer generated in vivo to a single cellification treatment, there is no limitation, but it is included to enzymatically treat the cancer tissue obtained from an individual .

The enzyme treatment is typically treated with trypsin, dispase, and optionally, collagenase, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. possible. The enzyme treatment conditions may be in an isotonic salt solution buffered to a physiologically acceptable pH, for example about 6 to 8, preferably about 7.2 to 7.6, such as PBS or Hanks balanced salt solution For example, at about 20 to 40 ° C., preferably about 25 to 39 ° C., for a sufficient time to degrade connective tissue, eg, about 1 to 180 minutes, preferably 30 to 150 minutes, for which sufficient concentration It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

Although not limited, this enzyme treatment may typically be trypsin or dispase treatment alone.

After the single-cellification treatment, cells that are usually separated individually are obtained. However, it also includes cells which are not completely separated individually.

Such cells may be allowed to aggregate as such, but preferably, for example, the ROCK inhibitor is allowed to aggregate immediately after single cell treatment.

ROCK refers to Rho-associated coiled-coil kinase (ROCK: GenBank accession number: NM_005406), which is one of the main effector molecules of Rho GTPase, and is known to control diverse physiological phenomena. (Also called Rho-linked kinase). As a ROCK inhibitor, Y27632 etc. are illustrated, for example. In addition, Fasudil (HA1077), H-1152, Wf-536 (all available from Wako Pure Chemical Industries, Ltd.), and derivatives thereof, antisense nucleic acid against ROCK, RNA interference-inducing nucleic acid, and the like And vectors that contain it.

Treatments separated to single cells or aggregates of 10 or less cells by trypsinization (for example, but not limited to, 0.25% trypsin-EDTA, treatment at 37 ° C. for 5 minutes) are subjected to 96-well culture plate prior to aggregation. At a low density (eg 500 cells / 0.32 cm ² , medium volume about 0.15 ml). The ROCK inhibitor can be added to the maintenance culture solution immediately or after culturing for several days, at a concentration of about 1 to 100 μM, preferably about 10 μM.

Such aggregates can be cultured in vitro. The culture time is not particularly limited as long as it is present in the medium even for a short time. Such a culture often exhibits a substantially spherical or elliptical spherical shape by culturing for a certain period of time, preferably 3 hours or more. The culture here includes a substantially spherical or spheroidal culture after such a given period of time, and an atypical culture up to that. Furthermore, an irregular shape obtained by further dividing such a substantially spherical or spheroidal culture, and a substantially spherical or spheroid obtained by further culture are also referred to as the culture herein.

The cancer cell aggregate of the present invention can maintain its growth ability in vitro, at least 10 days or more, preferably 13 days, under cell culture conditions at a temperature of 37 ° C. in a 5% CO ₂ incubator. This means that the growth ability can be maintained for a period of 30 days or more, more preferably.

Such cancer cell aggregates can retain their growth ability for a period of 10 days or more, preferably 13 days or more, more preferably 30 days or more, by continuing the culture as they are, but furthermore, they can By carrying out selective division, or by further performing unicellularization treatment and aggregation, the proliferative ability can be maintained virtually indefinitely.

Here, the medium for culturing the cancer cell aggregate of the present invention is the same as the medium for culturing a cancer tissue-derived cell mass.

The cancer cell aggregates of the present invention can be cultured in such media and culture conditions. Furthermore, the culture of cancer cell aggregates may, depending on its individual nature, be preferred if co-culture with other cells is preferred or the presence of additional specialized supplements such as hormones.

Alternatively, for certain types of breast cancer, uterine cancer and prostate cancer, it is preferable to culture in the presence of a hormone as in the case of the cancer tissue-derived cell mass. Specifically, estrogens for breast cancer, progesterone for uterine cancer, testosterone for prostate cancer and the like are not limited thereto, and various hormones can be added to advantageously adjust culture conditions. Furthermore, the presence of such hormones reveals the hormonal dependence of the originating patient's cancer by examining the behavior of cancer cell aggregates after culture, such as how life or death or growth changes. It may be possible to predict the efficacy of antihormonal drug treatment.

The cancer cell aggregate of the present invention can also be cultured in suspension culture, like the cancer tissue-derived cell mass.

The number of cancer cells constituting the cancer cell aggregate is at least 3 or more, preferably 8 or more, more preferably 10 or more, still more preferably 20 or more, and the number is not particularly limited. When the cancer cell aggregate of the present invention is an isolated substance, it is preferably 1000 or less, more preferably about 500 or less. It is possible to increase the number of cultures after culturing the isolates by culturing. However, even if it is a culture, it is preferably 10,000 or less, more preferably 5,000 or less.

The size of the cancer cell aggregate of the present invention is not limited, and includes irregularly shaped particles having a particle size or volume average particle size of about 8 μm to 10 μm, and those grown 1 mm or larger in particle size after culture Also included. Preferably, the diameter is 40 μm to 1000 μm, more preferably 40 μm to 250 μm, and still more preferably 80 μm to 200 μm.

The cancer cell aggregate of the present invention often has one or more sequences particularly selected from the group consisting of a shelf array, a sheet array, an interlayer array and a syncytial array, but is not particularly limited.

The cancer cell aggregate of the present invention typically comprises the steps of: converting the cancerous tissue removed from the living body into single cells; and aggregating the cells in the single cellification into three or more cells. It can be prepared by the method.

Furthermore, without limitation, the cancer cell aggregate of the present invention can be prepared by a method comprising the step of culturing the aggregated component for 3 hours or more.

First, when the cancer cell aggregate of the present invention is obtained from a cancer tissue-derived cell mass, it is directly subjected to the enzyme treatment, but the cancer tissue removed from the living body is converted into a single cell by being directly subjected to the enzyme treatment. While it is also possible, it is preferable to minify prior to enzyme treatment. Prior to fragmentation, it can be maintained in animal cell culture medium. Such animal cell culture media include, but are not limited to, Dulbecco's MEM (such as DMEM F12), Eagle's MEM, RPMI, Ham's F12, alpha MEM, Iscove's modified Dulbecco, and the like. At this time, it is preferable to perform suspension culture in a cell non-adhesive culture vessel.

The debris obtained in this way is then subjected to an enzyme treatment. Such enzyme treatment may be mainly trypsin treatment as described above. Enzyme treatment conditions may be from 20 ° C. to 45 ° C., minutes to hours.

Next, the cells in the single-cell material thus obtained are allowed to aggregate to three or more cells. Prior to aggregation, preferably, the ROCK inhibitor can be added rapidly to a single cell.

Here, an aggregate containing three or more cancer cells obtained by aggregation is a cancer cell aggregate of the present invention, and has a range of sizes. The size within a certain range includes small particles having a volume average particle diameter of about 8 μm to 10 μm, but in the case of a spherical shape, the diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, and more preferably 40 μm to 250 μm. In the case of an elliptical shape, the major diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, more preferably 40 μm to 250 μm, and in the case of indeterminate shape, the volume average particle diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm. And more preferably 40 μm or more and 250 μm or less. The volume average particle diameter can be measured by evaluating the particle size distribution and the particle shape using a phase contrast microscope (IX70; manufactured by Olympus Corporation) with a CCD camera attached.

The aggregate thus obtained or the culture thereof is the cancer cell aggregate of the present invention. The culture may be one in which the separation / collection component isolate is present in the culture medium for a short time, for example, at least 3 hours or more, preferably 10 hours to 36 hours, more preferably 24 hours. By culturing for a period of time to 36 hours, it may be in the shape of a substantially spherical or substantially elliptical sphere. The culture time may be more than 36 hours, several days, 10 days or more, 13 days or more, or 30 days or more.

Furthermore, the cancer cell aggregate of the present invention has a high degree of establishment in transplantation into xenogeneic animals even if, for example, 10 or less cancer cell aggregates having a diameter of 100 micrometers (corresponding to 1000 cells or less). Therefore, the cancer cell aggregate of the present invention is useful for the simple preparation of mouse and other cancer model animals, and more rigorous examination of cancer tissues, evaluation of drug sensitivity, and treatment modes including radiation therapy. Can be evaluated.

The cancer cell aggregate of the present invention can be cryopreserved and can retain its proliferative ability under normal storage conditions.

The cancer tissue-derived cell mass or cancer cell aggregate of the present invention thus obtained exhibits the same behavior as cancer tissue in vivo in vitro, can be stably cultured, and retains the proliferation ability. Do.

The cancer tissue-derived cell mass or cancer cell aggregate of the present invention thus obtained exhibits the same behavior as cancer tissue in vivo in vitro, can be stably cultured, and retains the proliferation ability. Do. In addition, the proportion of unnecessary contaminants other than cancer cells, such as cells other than cancer cells derived from living bodies and connective tissues, is very small or almost nonexistent. Therefore, it is very conveniently used for immunotherapy.

The cancer tissue-derived cell mass or cancer cell aggregate of the present invention can be cryopreserved, and can retain its proliferative ability under normal storage conditions. Therefore, it can also be conveniently used to measure the effect of immunotherapy over time.

As a specific embodiment of using a cancer tissue-derived cell mass or a cancer cell aggregate for immunotherapy, for example, they may be chemically treated to prepare a composition for cancer treatment. Here, the chemical treatment includes formalin treatment, enzyme treatment and the like. Alternatively, a composition for cancer treatment can be prepared by photodynamic treatment. Such photodynamic processing includes radiation irradiation processing and ultraviolet irradiation processing.

For example, in the case of enzyme treatment, once formed, cancer tissue-derived cell masses or cancer cell aggregates are trypsinized (eg, 37 ° C., 60 minutes) to completely separate into single cells, Leave as it is. Thus, cells separated into single cells undergo apoptosis and die, but peptides contained in these cells can be targets for antigen-presenting cells.

For example, in the case of radiation treatment, radiation is irradiated to such an amount that can promote apoptosis of the cancer tissue-derived cell mass or cancer cell aggregate of the present invention to promote cell apoptosis.

The composition for cancer treatment thus obtained is directly administered to a patient. The method of administration may be oral but preferably is parenteral administration such as injection. Administration with an immune adjuvant is also preferred to enhance immunity. Included is any form of administration in which the composition is absorbed into the subject without absorption through the intestine. Exemplary parenteral administrations include, but are not limited to, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, or intraarticular administration.

When administering the preparation, a certain amount of cancer tissue-derived cell masses or cancer cell aggregates or a degradation product thereof may be dispersed (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate etc.) Aqueous solution (eg, for injection) together with preservatives (eg, methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, phenol etc.), tonicity agents ( It is produced by dissolving, suspending or emulsifying it in distilled water, physiological saline, Ringer's solution etc.) or oily solvent (eg olive oil, sesame oil, cottonseed oil, vegetable oil such as corn oil etc., propylene glycol etc.) etc. At this time, additives such as a solubilizing agent (eg, sodium salicylate, sodium acetate etc.), a stabilizer (eg, human serum albumin etc.), a soothing agent (eg, benzyl alcohol etc.) and the like may be used if desired. Furthermore, antioxidants, coloring agents, etc. and other additives may be added as required.

Also, a "pharmaceutically acceptable carrier" can be used. Such substances include solvents, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like in liquid formulations. In addition, if necessary, formulation additives such as preservatives, antioxidants, adsorbents, gelling agents and the like can be used according to a conventional method.

Preferred examples of the "antioxidant" include sulfites, ascorbic acid and the like. Preferred examples of the "isotonizing agent" include glucose, sodium chloride, glycerin, D-mannitol and the like.

Preferred examples of the "solubilizing agent" include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

As preferable examples of the "solvent", for example, water for injection, alcohol, propylene glycol, macrogol and the like are used.

Preferred examples of the "suspending agent" include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and the like.

Examples of the "surfactant" include sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate and the like.
As a preferable example of "the soothing agent", benzyl alcohol etc. are mentioned, for example.

Preferred examples of the "preservative" include p-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Alternatively, the composition for treating cancer thus obtained can be incubated with blood-derived cells contained in blood collected from the patient, and the activated blood-derived cells can be returned to the patient's body again as a result. . Alternatively, human T cells are cultured in vitro with said dendritic cells and subsequently in vitro activated T cells are administered to a cancer patient in need thereof.

At this time, in order to isolate blood-derived cells contained in blood, known methods such as magnetic bead method can be used, for example, from collected blood.

More specifically, for example, blood-derived cells contained in the blood are T lymphocytes, and at least major histocompatibility antigen (MHC) class I and a costimulatory molecule are used as a method for inducing cytotoxic T lymphocytes. Cells (for example, dendritic cells) can be contacted with the cancer tissue-derived cell mass or cancer cell aggregate of the present invention, and then cocultured with lymphocytes. Alternatively, co-culture with lymphocytes can be performed while contacting dendritic cells with the cancer tissue-derived cell mass or cancer cell aggregate of the present invention or a composition obtained by treating them.

The method of administration when returning to the body is preferably parenteral administration such as injection. Included is any form of administration in which the composition is absorbed into the subject without absorption through the intestine. Exemplary parenteral administrations include, but are not limited to, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, or intraarticular administration.

Furthermore, the cancer tissue-derived cell mass or cancer cell aggregate of the present invention can be used in an immunotherapeutic effect evaluation method for a patient who has been subjected to immunotherapy.

That is, in this evaluation method, blood-derived cells derived from an immunotherapy-treated patient are brought into contact with cancer tissue-derived cell masses or cancer cell aggregates. Here, typically, evaluation can be made by measuring the cytotoxicity of the blood-derived cells. Preferably, the blood-derived cells are isolated cytotoxic T cells. Alternatively, if the immunotherapy is antibody-based therapy, it is a natural killer cell. In the case of evaluating a therapy using an antibody, it is preferable to further allow the antibody used for treatment to coexist in this contacting step.

More specifically, it is possible to measure in vitro cytotoxicity using, for example, a 51-chromium-loaded cancer tissue-derived cell mass or a cancer cell aggregate as a target.

The patient who has been subjected to immunotherapy is a patient who has been subjected to immunotherapy using a cancer tissue-derived cell mass or a cancer cell aggregate, and the process since blood-derived cells derived from the patient have been administered immunotherapy It is also preferred that it be provided as a plurality of samples, collected at different times.

In particular, the cancer tissue-derived cell mass or cancer cell aggregate of the present invention has the effect of immunotherapy as necessary when necessary, in order to maintain its growth ability and survival well even after being refrigerated or frozen. Can be measured.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited by these examples. All parts and% in each example are based on weight. The culture conditions not particularly specified below are all 37 ° C. and 5% CO ₂ incubator conditions. The conditions for centrifugation are 4 ° C., 1000 rpm, 5 minutes unless otherwise stated.

Example 1
(Preparation of cancerous tissue-derived cell mass from mouse colon cancer transplanted tumor)
Mouse colon cancer transplanted tumors were produced by xenograft as follows.

First, a surgically excised sample of human tumor (colorectal cancer) is cut into about 2 mm cubes under aseptic operation. Next, a small incision of about 5 mm is made on the back of severe immunodeficient mice (nude mice, preferably NOD / SCID mice) to detach the subcutaneous tissue. The prepared tumor piece is inserted subcutaneously and closed with a skin suture clip. Some transplanted tumors are observed as subcutaneous tumors after about 14 days to 3 months.

The obtained colon cancer mice are bred under specific pathogen free (SPF) breeding conditions, and when the tumors become 1 cm in size, the tumors are excised and 20 ml of DMEM (Gibco; 11965-092) + 1% Pen Strep ( Gibco; 15140-022) (both at a final concentration of 100 units / ml penicillin, 100 μg / ml) were collected in a 50 ml centrifuge tube (IWAKI; 2345-050).

Next, 20 ml HBSS (Gibco; 14025-092) was added and the tumor was washed by inversion mixing. Next, 20 ml of fresh HBSS was added, this operation was repeated twice, and the tumor tissue was transferred to a 10 cm tissue culture dish (tissue culture dish) (IWAKI; 3020-100). On this culture dish, necrotic tissue was removed using a surgical knife.

Debris-free tumor pieces were transferred to a fresh 10 cm dish containing 30 ml of HBSS. Next, the tumor pieces were cut into pieces of about 2 mm using a surgical knife.

After transferring tumor fragments together with HBSS to a new 50 ml centrifuge tube, they were centrifuged, the supernatant was discarded, and washed with 20 ml HBSS by inversion mixing.

Repeated centrifugation and washing. Thereafter, 20 ml of DMEM + 1% Pen Strep + 0.28 U / ml (final concentration) Blendzyme 1 (Roche; 11988417001) was added and mixed. This was transferred to a 100 ml Erlenmeyer flask, and treated with Liberase Blendzyme 1 (manufactured by Roche Diagnostics) for 2 hours while rotating the stirrer at low speed in a 37 ° C. thermostat.

Next, the enzyme-treated product was collected in a 50 ml centrifuge tube, centrifuged, the supernatant was discarded, and 20 ml HBSS was added and mixed. The material was passed through a stainless steel mesh (500 μm), the components passed through the filter were collected in a 50 ml centrifuge tube, and centrifugation was performed. Discard the supernatant, mix with 1 mg / ml DNase I solution (Roche; 1284932) (10 mg / ml stock 100 μl + PBS 900 μl), mix and leave at 4 ° C for 5 minutes, add 20 ml HBSS and mix. Centrifugation was performed and the supernatant was discarded. After mixing with 20 ml HBSS, it was sieved stepwise with 500-250-100 μm and then passed through a 40 μm cell strainer (BD; 352340). The cell strainer was dipped in a 10 cm tissue culture dish (tissue culture dish) containing 30 ml of HBSS and gently shaken to remove single cells, small cell clusters of 40 μm or less, and debris. The cell strainer was transferred to another 10 cm tissue culture dish (tissue culture dish) containing 30 ml of HBSS, and the cell mass captured by the cell strainer was collected by pipetting.

Furthermore, the same centrifugation operation as described above is performed several times, and the component obtained is 4 ml StemPro hESC SFM (Gibco; A10007-01) + 8 ng / ml bFGF (Invitrogen; 13256-029) + 0.1 mM 2-mercapto Ethanol (Wako; 137-06862) + 1% PenStrep + 25 μg / ml Amphotericin B (Wako; 541-01961) was added and mixed, and transferred to a 6 cm non-treated dish (EIKEN CHEMICAL; AG 2000).

This was cultured at 37 ° C. in a 5% CO ₂ incubator (MCO-17AIC manufactured by Sanyo Co., Ltd.) for 36 hours.

As a result, as shown in FIG. 1, with the passage of time, it changes from amorphous to well-shaped spheres, is approximately spherical shape after at least 3 to 6 hours, and is perfectly spherically shaped cancer tissue after 24 hours Derived cell mass was obtained.

(Example 2)
(Preparation of cancerous tissue-derived cell mass from human colorectal cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 1 except that a colorectal cancer surgical specimen was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 3)
(Preparation of cancer tissue-derived cell mass from human ovarian cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that an ovarian cancer surgical specimen was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 4)
(Preparation of cancerous tissue-derived cell mass from human pancreatic cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a pancreatic cancer surgical specimen was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 5)
(Preparation of cancer tissue-derived cell mass from human small cell carcinoma surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a small cell cancer surgical specimen which is a type of lung cancer was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 6)
(Preparation of cancer tissue-derived cell mass from human renal cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a renal cancer surgical specimen was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 7)
(Preparation of cancerous tissue-derived cell mass from human bladder cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a bladder cancer surgery sample was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 8)
(Preparation of cancer tissue-derived cell mass from human breast cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a breast cancer surgical specimen was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 9)
(Preparation of cancer tissue-derived cell mass from human prostate cancer surgical specimens)
A tissue-derived cell mass was obtained in the same manner as in Example 2 except that a prostate cancer surgical specimen was used. To the culture medium, dihydrotestosterone (DHT) at a concentration of 10 ^-8 mol / L was added and cultured as in Example 1. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 10)
(Preparation of cancer tissue-derived cell mass from human pharyngeal cancer surgical specimens)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a pharyngeal cancer surgical specimen was used. As a result, as shown in FIG. 4, a substantially spherical cancer tissue-derived cell mass similar to FIG. 1 was obtained after at least 12 hours.

(Example 11)
The cancer tissue-derived cell mass obtained in Example 2 and shown in FIG. 4 was taken out together with the medium at 24 hours after culture, and 5 ml was taken out with the medium, centrifuged at 1000 rpm at 4 ° C. The collected cancer tissue-derived cell mass is suspended in a selbunker (BLC-1, manufactured by Mitsubishi Chemical Medicine Co., Ltd.), 10 μM of Y27632 (manufactured by Wako Pure Chemical Industries, Ltd.) is added, and cryopreservation tube (Cryogenic vials 2.0) The solution was transferred to ml, Nalge Nunc) and stored in -80.degree. C. deep freezer.

After 7 days after storage, it was briefly rewarmed in a 37 ° C. water bath. This was suspended in PBS, further centrifuged at 1000 rpm and 4 ° C., and the supernatant was discarded. The obtained precipitate was suspended in StemPro (manufactured by in vitro) and cultured. As shown in FIG. 5, the cell condition for 24 hours after thawing was good.

Furthermore, the survival of the obtained cancer tissue-derived cell mass was confirmed by transplantation into NOD-SCID mice as a mass containing about 1000 cells.

(Example 12)
(Preparation of cancer cell aggregate from cancer tissue-derived cell mass)
The following treatment was performed using the cancer tissue-derived cell mass obtained by the same method as in Example 2. First, 50 μL / well of collagen gel (Cell Matrix type IA: 5 × DMEM: buffer for gel reconstitution = 7: 2: 1) was placed in the center of a 24-well plate (untreated dish). The collagen gel was solidified by standing for 30 minutes at 37 ° C. Cancer tissue-derived cell masses in suspension culture (100 cells per well) are collected in 1.5 mL tubes. This was centrifuged for about 5 seconds, and the supernatant was removed. The cancer tissue-derived cell mass was suspended in collagenase gel (30 μL per well), and 30 μL was loaded on the previously solidified gel. The mixture was allowed to stand at 37 ° C. for 30 minutes to solidify, and 600 μL / well of StemPro (EGF 50 ng / mL) was added. The cells were cultured for 10 days while changing the medium once every 2 to 3 days.
Next, the medium was replaced with 1 mL / well of DMEM (Gibco; 11965-092, containing collagenase IV 200 mg / mL) and cultured at 37 ° C. for about 5 hours.
After incubation, transfer to a 1.5 mL eppen tube, centrifuge (approximately 5 seconds), remove the supernatant, suspend by adding 1 mL of PBS, and remove the supernatant after centrifugation (Chibitan, approximately 5 seconds) I repeated twice. One mL of Trypsin / EDTA (0.05%) was added and suspended, and the mixture was allowed to stand at 37 ° C. for 8 minutes. The suspension was repeated several times to confirm that the cancer tissue-derived cell mass was removed. This was transferred to a 15 mL tube and suspended by adding 2 mL of DMEM (Gibco; 11965-092).
The suspension was then centrifuged (1000 rpm, 5 minutes) and the supernatant removed. The cells were suspended in 2 mL of StemPro (EGF 50 ng / mL, Y-27632 10 μM), and transferred to a φ35 mm non-treated dish (Iwaki: 1000-035). This was cultured at 37 ° C. overnight.
After 12 hours, formation of a cancer tissue-derived cell mass having a diameter of about 40 μm was confirmed. The medium was changed to StemPro (EGF 50 ng / mL).

As a result, as shown in FIG. 6, completely spherical-shaped cancer cell aggregates were obtained after 4 days.

(Example 13)
(Preparation of cancer cell clumps from human colorectal cancer surgical specimens)
Cancer cell aggregates were obtained in the same manner as in Example 12 except that a colorectal cancer surgical specimen was used. As a result, as shown in FIG. 7, substantially spherical cancer cell aggregates similar to FIG. 1 were obtained after at least 12 hours.

(Example 14)
Cell preservation of the cancer tissue origin cell mass obtained by the same method as Example 2 was performed. The cancer tissue-derived cell mass was treated with trypsin in the same manner as in Example 14 to perform unicellularization. As a cryopreservation solution, a solution obtained by adding Y-27632 to Selvanker 1 (Junji field) was used.

What was single-celled and stored frozen for 10 days was then briefly rewarmed in a 37 ° C. water bath. This was suspended in PBS, further centrifuged at 1000 rpm and 4 ° C., and the supernatant was discarded. The obtained precipitate was suspended in StemPro (manufactured by in vitro) and cultured. As shown in FIG. 8, the condition of the cells for 24 hours after thawing was good, and after thawing, the cancer tissue-derived cell mass was reformed.

(Example 15)
Immunotherapy using the cancer tissue-derived cell mass of the present invention is examined. The cancer tissue-derived cell mass and cancer cell aggregate obtained in the same manner as in Examples 2 and 12 are each treated with trypsin (37 ° C. for 5 minutes). The resulting product contains many single cells, and when left for 24 hours, the cells die. After trypsinization (5 minutes at 37 ° C.) of the cancer tissue-derived cell mass obtained in Example 2, double staining with Annexin-V and PI was analyzed with a flow cytometer. Untreated cancer tissue-derived cell masses were cultured for 24 hours and digested with trypsin immediately before analysis (intact CTOS). With regard to the degraded cancer tissue-derived cell mass, the cancer tissue-derived cell mass was first separated into single cells, cultured, and then analyzed (distributed CTOS). The results are shown in FIG. Furthermore, the ratio of the result of this staining was graphed (FIG. 10). The double-stained Annexin-V and PI are shown in black, the single-stained Annexin-V in gray, and the unstained sample in white. As a result, it was confirmed that apoptosis was progressing smoothly in the treated cancer tissue-derived cell mass. Furthermore, the induction of apoptosis was confirmed by western blotting of caspase-3, cleaved caspase-3 (cl-casp-3) and PARP (FIG. 11). The culture was performed for the time indicated in FIG. 11 and then analyzed.
Next, the tumor tissue-derived cell mass or cancer cell aggregate trypsin treated product is suspended in 2 ml of distilled water and sterilized by passing through a filter having a pore diameter of 0.22 microns, 1-ethyl at a concentration of 20 mg / ml -3- (3-dimethylaminopropyl) carbodiimide (hereinafter abbreviated as "EDC") solution is added to a concentration of 0.8 mg / ml. This is stored at 25 ° C. for 15 minutes, and then sterile 2 ml of 0.1 M glycine solution is added. After storage at 25 ° C. for 30 minutes, physiological saline is added to prepare a raw material solution for vaccine.

The vaccine stock solution thus obtained and TiterMax Gold (CytRX, Atlanta, Norcross, GA) marketed as an adjuvant are mixed to form a tumor vaccine.

The vaccine obtained is injected 1 ml into cancer patients from which it originates, as a preventive measure or as a treatment after recurrence. Seven days later, another injection is given. Regularly monitor to see if the cancer has not recurred, or check the size of the tumor after relapse to see the effect of the treatment.

(Example 16)
Regularly monitor immunotherapy for colorectal cancer patients and evaluate the effects of immunotherapy. Specifically, the blood of a patient who has been given general immunotherapy (for example, WT1 peptide therapy) is collected, and the cytotoxic T cells are collected targeting CD8 positive. It is examined whether patient-derived CD8 positive T cells can damage the cancer tissue-derived cell mass or cancer cell aggregate of the present invention. The cell mass derived from cancer tissue derived from colon cancer of the patient obtained in the same manner as in Example 2 is collected in a 50 ml centrifuge tube, 100 μCi of chromium 51 is added and the mixture is incubated at 37 ° C. for 2 hours. Thereafter, the plate is washed three times with AIM-V medium containing 10% human AB serum, and 100 μl is added to each well of a 96-well V-bottom plate. To this were added 10 ⁵ CD8 positive T cells (taken for multiple times during the immunotherapy period) suspended in AIM-V medium containing 10% human AB serum, respectively, at 37 ° C. Incubate for 4 hours under 5% CO ₂ conditions. Calculating the cytotoxic activity of CD8 positive T cell cells of a patient receiving immunotherapy by measuring the amount of chromium 51 in the culture supernatant released from damaged tumor cells after culture it can.

The evaluation items in the examples and the like were measured as follows.

<Confirmation of basement membrane-like material>
The cancer tissue-derived cell mass obtained in Example 1 was cultured for 3 days in 1 cc of STEMPRO human ES cell serum-free medium (Gibco) under culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. This was formalin-fixed, paraffin-embedded, sliced, and subjected to anti-laminin antibody staining (Sigma-Aldrich, mouse laminin-derived rabbit antibody) according to the manufacturer's instructions. The antigenicity of laminin was observed in the cytoplasm of cells close to the periphery. This revealed that the cancer tissue-derived cell mass of the present invention was surrounded by laminin at the periphery of the cancer cell aggregate. On the other hand, the expression of laminin could not be confirmed 24 hours after the treatment of the surgical specimen.

<Detection of hypoxia>
Example of detection of hypoxia using pimonidazole The nitroimidazole compound pimonidazole has the property of forming Adduct with proteins and nucleic acids in the absence of oxygen. The hypoxic region of pimonidazole-treated tissues under hypoxia can be recognized using an antibody that specifically recognizes pimonidazole. In cancer tissues, a hypoxic region appears about 100 micrometers away from blood vessels. However, even in the cancer tissue-derived cell mass obtained in Example 1, the inside is a hypoxic region at a boundary of about 100 micrometers from the outer edge. Cell death was observed.

<Evaluation of proliferation ability in vitro>
The proliferation ability of the cancer tissue-derived cell mass in vitro was verified as follows. Collagen tissue (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): buffer solution for gel reconstitution (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) = 10: embedded in 7: 2: 1), and culture was performed with 1 cc of serum-free medium (Gibco) for STEMPRO human ES cells under culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. The state of cells was periodically observed, and the size was measured with a phase contrast microscope (40 × magnification) equipped with a CCD camera. As a result, it was possible to maintain the proliferation ability for at least 13 days without mechanical division. Furthermore, when mechanical division was performed on the 13th day, it was confirmed that the proliferation ability was maintained for at least 13 days (FIG. 2). The mechanical division was performed by dividing a cancer tissue-derived cell mass having a diameter of 500 micrometers into four parts with an eye knife.

<Confirmation of cell number>
In the same manner as in Example 1, 100 to 250 μm cancer tissue-derived cell masses were treated with 0.25% trypsin and 2.6 mM EDTA for 3 minutes and mechanically degraded by pipetting about 30 times. This was diluted and dispensed so that the cells were contained in a proportion of 1 per 96-well culture plate. With respect to non-unicellularized cell masses, the number of constituent cells was counted and recorded. Thereafter, the cells were cultured (conditions as described above), the increase in the number of cells in each well was recorded, and culture was observed for 30 days. As a result, it was confirmed that a cell mass can grow if there are eight cells.

<Transplantation test to foreign animals>
Ten cells of the cancer tissue-derived cell mass of about 100 micrometers in diameter of the present invention obtained in Example 2 and cultured for 3 days were suspended in Matrigel (BD Co.) and administered subcutaneously in the back of NOD-SCID mice . Evaluation of tumor formation was performed by measuring the size of the tumor over time. As a result, significant tumorigenesis was observed in a mouse individual to which the cancer tissue-derived cell mass of Example 2 of the present invention was transplanted, and it was confirmed that the cancer tissue-derived cell mass of the present invention has high tumorigenicity. . When this tissue was analyzed, it was found that similar tumor types were obtained for the tumor formed by transplantation into mice and the tumor existing in the living body (FIG. 3).

The cancer tissue-derived cell mass or cancer cell aggregate of the present invention can be cryopreserved in a culturable state in vitro, and can be used to provide an antigenic peptide, particularly for use in immunotherapy. Furthermore, it can also be used as an evaluation tool for measuring the effect of immunotherapy.

Claims

A composition for treating cancer, which is obtained by processing a cancer tissue-derived cell mass or a cancer cell aggregate.
The composition for treating cancer according to claim 1, wherein the treatment is a chemical treatment.
The composition for treating cancer according to claim 2, wherein the chemical treatment is enzyme treatment or formalin treatment.
The composition for treating cancer according to claim 1, wherein the treatment is a photodynamic treatment.
The composition for treating cancer according to claim 4, wherein the treatment is radiation treatment.
The composition for treating cancer according to any one of claims 1 to 5, which is a cancer vaccine for oral administration or parenteral administration to a patient derived therefrom.
The composition for treating cancer according to any one of claims 1 to 6, which is for contacting blood-derived cells obtained from blood from a patient in vitro.
The composition for treating cancer according to claim 7, wherein the blood is peripheral blood.
The composition for treating cancer according to claim 7 or 8, wherein the blood-derived cells are dendritic cells derived from monocytes.
Contacting the blood-derived cells collected from the blood from the patient with the composition for treating cancer according to any one of claims 1 to 6 from the patient; and the blood-derived blood activated by the contact A method of producing an immunotherapeutic agent for inoculating said patient, comprising the step of collecting cells.
The method for producing an immunotherapeutic agent according to claim 10, wherein the blood is peripheral blood.
The method for producing an immunotherapeutic agent according to claim 10, wherein the blood-derived cells are monocytes, and the method further comprises the step of inducing monocytes to dendritic cells.
A method of evaluating the effect of immunotherapy, which comprises contacting blood-derived cells derived from an immunotherapy-treated patient with a cancer tissue-derived cell mass or a cancer cell aggregate;
Methods for evaluating immunotherapeutic effects, including
The immunotherapeutic effect evaluation method according to claim 13, further comprising the step of measuring the cytotoxicity of the blood-derived cells.
The immunotherapeutic effect evaluation method according to claim 13 or 14, wherein the blood-derived cells are isolated cytotoxic T cells.
The immunotherapeutic effect evaluation method according to claim 13 or 14, wherein the blood-derived cells are isolated natural killer cells, and the contacting step further causes an antibody to coexist.
The patient who has been subjected to the immunotherapy is a patient who has been subjected to immunotherapy using a cancer tissue-derived cell mass or a cancer cell aggregate, and blood-derived cells derived from the patient have been administered immunotherapy. The immunotherapeutic effect evaluation method according to any one of claims 13 to 16, which is provided as a plurality of samples collected at different elapsed times.