Stem cell fractionation method using in vitro boron neutron reaction

Abstract

To provide cell fractionation methods for removing unfavorable cells mixed with radiation-sensitive stem cells with "stemness" used in the fields of transplantation medicine and regenerative medicine, e.g., teratoma, atypical cells, cancer cells (blood cancer cells such as leukemia, malignant lymphoma, multiple myeloma) to safely perform such transplantation medicine and regenerative medicine; and to provide cells obtained by the method.SOLUTION: The present invention provides a fractionation method of a non-target cell, comprising: (1) culturing a cell culture containing a target cell in the presence of a boron (10B) drug; and (2) irradiating the cell culture obtained in (1) with a neutron beam.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of regenerative medicine and cell therapy, where radiosensitive cells with "stemness" are utilized, particularly in cell culture techniques for bone marrow and hematopoietic stem cell transplantation. [Background technology]

[0002] Highly radiosensitive cells with "stemness" include pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells present in bone marrow, and bone marrow grafts themselves. Pluripotent stem cells are induced to differentiate into living organs and tissues under appropriate conditions in regenerative medicine and transplantation. However, there is a risk that undifferentiated cells may develop into teratomas. While there is guidance for testing for the presence of these cells (Pharmaceuticals and Medical Devices Agency: "Technical Guidance on Considerations and Points to Note When Developing Human Cell-Processed Products Among Regenerative Medicine Products (June 2016)"), no technical methods for actively removing them have been reported.

[0003] In the case of utilizing pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells, the focus has been on developing differentiation-inducing methods to reduce remaining undifferentiated cells, so no "removal" method has been established. Generally, methods for removing "undesirable" heterogeneous or dysmorphic cells include the use of radiation such as gamma rays and exposure to cytotoxic drugs such as anticancer drugs. However, both of these methods are difficult to choose due to concerns that they may severely damage useful pluripotent stem cells.

[0004] Even in the case of "undesirable" heterologous or atypical cells, such as hematopoietic stem cells that exist in the bone marrow and have "stem-like properties" or cancerous cells mixed in with bone marrow grafts, there have been no reported technologies or methods for "removal." Conventionally, only bone marrow or hematopoietic stem cell transplants (allogeneic or autologous transplants) from healthy donors or those in a state of remission have been considered, and "risk reduction or avoidance" has been achieved.

[0005] Boron Neutron Capture Reaction (BNCR) is a reaction in which a boron atom ( 10 B) with thermal neutrons or low-energy epithermal neutrons to induce a nuclear reaction (alpha decay reaction), which selectively destroys and kills only cells that contain a certain amount of boron atoms on a cell-by-cell basis. BNCT is a treatment method that utilizes this. This method may be applicable to this purpose. In other words, a boron drug that has a relatively small effect on cells is used to introduce boron element ( 10One potential method is to accumulate B (B) in the target cells and then irradiate them with controlled low-energy neutrons, primarily thermal neutrons, which have little effect on normal cells, to induce an alpha decay reaction (nuclear reaction) known as BNCR within the undesirable heterogeneous or dysplastic cells, destroying and eliminating the cells from within without affecting normal cells. However, there have been no reports of BNCR being used for this purpose. In March 2020, the Ministry of Health, Labour and Welfare approved the world's first accelerator-driven BNCT system, combining a cyclotron-type accelerator neutron source (NeuCure) with the boron drug steboronine (an intravenous infusion of borophalan BPA and the solubilizer D-sorbitol) for the treatment of unresectable locally advanced or recurrent head and neck cancer. Clinical trials are also underway for melanoma and angiosarcoma using a system combining steboronine with the RFQ linac neutron source (CICS-1) installed at the National Cancer Center and Edogawa Hospital. The second accelerator neutron source (nuBeam), which uses an electrostatic accelerator and a rotating Li target, was installed at Helsinki Hospital in Finland and has been installed at Shonan Kamakura Hospital. At the University of Tsukuba, development of an RFQ / DTL linac neutron source (iBNCT) is underway, and preparations for non-clinical trials are underway for practical application. The accelerator neutron sources used in these BNCT facilities consist of a proton accelerator, a neutron generation target, and a moderator (BSA) that slows neutrons to an energy range suitable for treatment. Several systems have been developed to date, using different combinations of accelerators (cyclotrons, linacs, electrostatic accelerators) and target materials (Be, Li) (Figure 1). All of these systems are designed to generate neutrons with energies in the epithermal range (0.5 eV to 10 keV) for the treatment of deep-seated solid tumors.

[0006] Among these, the Li target is capable of efficiently generating neutrons under a low-energy proton beam, and because the neutron energy generated is lower than that of a Be target, it has the advantage of being easy to slow down the neutrons to the epithermal neutron range suitable for BNCT treatment (Table 1). Table 1 Neutron energy moderation ratio required for epithermal neutron production in each system JPEG2023085151000001.jpg64156 [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] History of the development of BNCT (Boron Neutron Capture Therapy), Isotope News, 755, 42-47 (2018) [Non-patent document 2] Future Prospects of BNCT, Isotope News, 756, 24-27 (2018) [Non-patent document 3] Development of Accelerator-Based Neutron Source for Medical Use and Its Applications in Industrial and Commercial Fields, Isotope News, 757, 22-25 (2018) [Patent documents]

[0008] [Patent Document 1] Patent application 2016-062865 [Patent Document 2] Patent application 2017-61979 [Patent Document 3] Patent application 2017-105099 Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to provide a method for separating cells to safely perform transplantation medicine and regenerative medicine by removing undesirable cells, such as teratomas, dysplastic cells, and cancer cells (blood cancer cells such as leukemia, malignant lymphoma, and multiple myeloma), mixed in with highly radiosensitive stem cells with "stemness" used in the fields of transplantation medicine and regenerative medicine, and to provide cells obtained by said method.

[0010] We provide a method (Ex. Vivo BNCR) to remove undesirable cells, such as teratomas, atypical cells, and cancer cells (blood cancer cells such as leukemia, malignant lymphoma, and multiple myeloma), that are mixed into stem cells, which are necessary for transplantation and regenerative medicine, without affecting their function. The method uses a boron neutron capture reaction (BNCR) outside the patient's body, which has little effect on the high radiosensitivity of stem cells. That is, the present invention relates to the following items.

[0011] (Item 1) (1) A non-target cell culture containing target cells is specifically administered boron ( 10 B) is cultured under conditions in which it is incorporated; and (2) Boron (obtained in (1) 10 B) A method for separating non-target cells by irradiating target cells into which the compound has been taken up with neutron rays that have little effect on non-target cells, thereby selectively killing the target cells. (Item 2) In step (1), boron ( 10 B) The drug is easily taken up and boron ( 10 B) The separation method according to Item 1, in which the cells are cultured under culture conditions that make it difficult for the drug to be taken up. (Item 3) 3. The separation method according to item 1 or 2, wherein in step (2), a neutron irradiation field configured so that the maximum energy of the irradiated neutron beam is 50 eV or less is used to selectively separate non-target cells. (Item 4) 4. The method according to any one of items 1 to 3, wherein the cell culture obtained in step (2) is substantially free of target cells. (Item 5) 5. The method according to any one of items 1 to 4, wherein the non-target cells are stem cells. (Item 6) 6. The method of claim 5, wherein the stem cells are pluripotent stem cells or hematopoietic stem cells. (Item 7) 7. The method of claim 6, wherein the pluripotent stem cells are human pluripotent stem cells. (Item 8) 7. The method of claim 6, wherein the pluripotent stem cells are human iPS cells. (Item 9) 7. The method of claim 6, wherein the hematopoietic stem cells are CD34-positive cells. (Item 10) 10. The method according to any one of items 1 to 9, wherein the target cells are one or more types selected from the group consisting of heterologous cells, dysplastic cells, and cancer cells. (Item 11) 10. The method according to any one of items 1 to 9, wherein the target cells are cancer cells. (Item 12) The boron ( 10 B) The method for preparative separation according to any one of items 1 to 11, wherein the drug is a compound having a closo structure such as p-boronophenylalanine (BPA) or mercaptoundecahydrodecaborate (BSH), or a mixture thereof. (Item 13) The boron ( 10 B) The method according to any one of items 1 to 11, wherein the drug is a conjugate of a boron compound having a closo structure, such as BSH, and a peptide containing a hydrophobic amino acid residue and a basic amino acid residue, and the peptide is A6K or A6R. (Item 14) Furthermore, boron ( 10 B) The method for fractionation according to any one of items 1 to 13, further comprising a step of evaluating drug uptake. (Item 15) 14. The sorting method according to any one of items 1 to 13, further comprising a step of isolating non-target cells. (Item 16) Furthermore, boron ( 10 B) The method for fractionation according to any one of items 1 to 13, further comprising a step of evaluating a drug. (Item 17) Item 4. The separation method according to item 3, characterized in that the neutron irradiation field has little influence on non-target cells, and is characterized by a neutron moderator with a maximum neutron energy of 50 eV or less, which has little contamination by gamma rays and the like, and reduces the influence of fast neutrons (particularly, the hydrogen dose imparted to cells when neutrons react with hydrogen). (Item 18) The separation method according to item 3, characterized in that the neutron irradiation field has little effect on non-target cells and is formed by combining the water phantom with a device capable of generating low-energy epithermal neutrons (0.5 eV-10 keV) that meets the standard values ​​(IAEA TECDOC-1223) of the International Atomic Energy Agency (IAEA), thereby reducing the viability of target cells. (Item 19) Item 4. The separation method according to Item 3, characterized in that a non-target cell culture containing target cells is placed at the center of the axis of a cylindrical neutron moderator (water, heavy water, Be, C, etc.) with a radius of 5 cm or more as a neutron irradiation field that has little effect on non-target cells, and fast neutrons emitted from multiple small neutron generators installed beside the cylindrical neutron moderator are moderated to 50 eV or less and the neutrons are used to irradiate the non-target cell culture containing target cells. (Item 20) 20. Non-target cells obtained by the separation method according to any one of items 1 to 19. (Item 21) A pharmaceutical composition comprising the non-target cells according to Item 20. (Item 22) 21. A cancer preventive or therapeutic agent comprising the non-target cells according to Item 20. (Item 23) 21. A preventive or therapeutic agent for blood cancer, comprising the non-target cells according to Item 20. (Item 24) 22. A method for preventing or treating cancer in a cancer patient, comprising administering to the cancer patient an effective amount of the pharmaceutical composition according to Item 21. (Item 25) collecting hematopoietic stem cells from a patient with blood cancer prior to anti-cancer treatment with boron neutron capture therapy (BNCT) from the patient; A step of purifying the collected hematopoietic stem cells by the separation method described in any one of items 1 to 19; and autologous transplantation of the hematopoietic stem cells obtained by the separation method into the patient; A method for treating blood cancer, including: [Effects of the Invention]

[0012] The boron neutron capture reaction (BNCR) is utilized to separate cells useful for regenerative medicine and cell therapy.

[0013] Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, are known to be highly radiosensitive. Therefore, it is difficult to apply conventional radiation therapy to heterologous or mutant cells for the purpose of "removal." In this regard, the application of BNCR, which combines highly selective boron compounds with neutrons with extremely low levels of gamma rays or high-energy neutron contamination, would provide the only practical method.

[0014] If we could selectively remove cancer cells that may be present in bone marrow hematopoietic stem cells or bone marrow grafts themselves using BNCR, the scope of application of autologous transplantation could be greatly expanded. This would be expected to increase the overall treatment options for blood cancers. In particular, in cases where the effectiveness of pre-transplantation treatments, such as new drug prescriptions, has improved and the number of remaining cancer cells in the patient's body has been significantly reduced, the possibility cannot be ruled out that the number of latent cancer cells in the autologous hematopoietic stem cells collected during remission before treatment could lead to relapse or recurrence after autologous transplantation. For myeloid blood cancers for which autologous bone marrow transplantation or autologous hematopoietic stem cell transplantation is the primary treatment, the creation of transplantation materials using this method would provide an effective method.

[0015] This will bring about a paradigm shift through the expansion of regenerative medicine and cell therapy, and the expansion of the scope and selectivity of transplants in cancer treatment, such as blood cancer treatment. This could potentially increase the number of patients cured while simultaneously reducing total medical costs, indicating a desirable direction for cancer treatment in Japan.

[0016] In the case of blood cancers (leukemia, malignant lymphoma, multiple myeloma, etc.), Ex. Vivo BNCT can be combined with a treatment method (conditioning treatment) that completely eradicates existing or latent cancer cells in the patient's body using high-dose chemotherapy and / or radiation therapy, which can lead to the safe performance of autologous hematopoietic stem cell transplants for the treatment of blood cancers. It can also lead to the safe performance of regenerative medicine by removing cells that pose a risk of becoming cancerous in the future, such as teratomas and atypical cells.

[0017] Highly radiosensitive cells with "stem-like" properties are important biomaterials for regenerative medicine and cell therapy, but there is a major limitation in that there is no effective method for removing heterogeneous or mutated cells, which pose a risk of being mixed in, as well as undifferentiated cells. This invention will lead to a fundamental solution for regenerative medicine and cell therapy.

[0018] The method described in this invention can be applied to the treatment of blood cancers (malignant lymphoma, multiple myeloma, etc.), which is expected to improve the safety of (autologous) hematopoietic stem cell transplantation, which may lead to a cure.

[0019] The invention will be in the field of chemistry related to regenerative medicine, cell therapy, and the provision of materials for these, which will lead to new inventions and the development of new treatments in these medical fields. It is expected that the time and cost required for approval will be shortened.

[0020] Traditionally, hematopoietic tissue (bone marrow, hematopoietic stem cells, etc.) transplants have been performed on patients under 65 years of age with hematologic cancers (malignant lymphoma, multiple myeloma, etc.) who respond to chemotherapy, as outlined in guidelines from the Japan Society for Hematopoietic Cell Transplantation and other organizations. However, the guidelines only allow allogeneic hematopoietic tissue transplants, and do not contemplate autologous transplants using the patient's own hematopoietic tissue. This is because autologous transplants would involve transplanting cancer cells present in the hematopoietic tissue into the patient, which could lead to cancer recurrence. Therefore, whether or not patients can be saved depends on whether transplant cell donors can be secured, and the number of patients who can be treated is limited.

[0021] According to the present invention, by applying boron neutron capture therapy to hematopoietic stem cells, such as bone marrow, extracted from a patient's body prior to anticancer drug therapy, it is possible to eliminate cancer cells and obtain hematopoietic tissue that maintains hematopoietic stem cell function. The use of such hematopoietic tissue enables autologous transplantation, which avoids the reintroduction of cancer cells. As a result, treatment of blood cancers becomes possible without being limited to allogeneic transplantation, which requires securing a donor, and an increasing number of patients can be saved. [Brief explanation of the drawings]

[0022] [Figure 1] A comparison of the characteristics of accelerator neutron sources is shown. [Figure 2] The results of measuring the OKD particles after ultrafiltration are shown. [Figure 3] The graph shows the results of measuring the boron concentration taken up by CD34-positive hematopoietic stem cells and multiple myeloma-derived cell lines when using cancer cell culture media. [Figure 4] The results of evaluating the intracellular internalization of BPA and BSH are shown. [Figure 5] This shows the concept of dose delivered to cancer cells and normal cells. [Figure 6] The neutron energy dependence of the kerma coefficient for biological tissue is shown. [Figure 7] The gamma-ray kerma coefficient is dependent on the gamma-ray energy. [Figure 8] Photographs of the electrostatic accelerator system and the beamline are shown. [Figure 9] The neutron deceleration irradiation device and the emitted neutron beam quality are shown. [Figure 10] The energy distribution of neutrons emitted from the BSA nozzle is shown. [Figure 11] A neutron moderator for stem cell irradiation is shown. [Figure 12] The structure of a neutron moderator for stem cell irradiation is shown. [Figure 13] Various dose distributions in a neutron moderator for stem cell irradiation (light water moderator) are shown. [Figure 14] Various dose distributions in neutron moderators for stem cell irradiation (light water moderator + lead shielding) are shown. [Figure 15] Various dose distributions in neutron moderators for stem cell irradiation (heavy water moderator) are shown. [Figure 16] The relationship between neutron irradiation time and cell survival rate of cancer cells (MM cells) and normal cells (hematopoietic stem cells) after treatment with a boron drug (OKD) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] In one embodiment of the present invention, (1) Non-target cell cultures containing target cells were treated with boron ( 10 B) culturing in the presence of a drug; and (2) Boron (obtained in (1) 10 B) is provided as a method for separating non-target cells, the method comprising the step of irradiating target cells into which the compound B has been taken up with neutrons to selectively separate non-target cells. In step (1), boron ( 10 B) It is preferable to culture the cells under culture conditions that facilitate drug uptake. 10 B) Culture conditions that facilitate drug uptake by target cells include optimizing the boron agent, type of cell culture medium, culture temperature, carbon dioxide concentration, etc. In addition, in step (2), in order to selectively separate non-target cells, it is preferable to use a neutron irradiation field in which the maximum energy of the irradiated neutron beam is 50 eV or less and which is configured to suppress gamma rays contaminating the neutrons.

[0024] Target cells refer to cells to be removed, and examples include "undesirable" heterologous or dysmorphic cells such as cancerous cells that are mixed with cells used in cell preparations.

[0025] Non-target cells refer to cells to be separated, and examples thereof include cells that have stem cell properties and are highly sensitive to radiation.

[0026] Examples of cells that have stem cell properties and are highly sensitive to radiation include pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells), multipotent stem cells (differentiated multipotent stem cells: MUSE cells, mesenchymal stem cells, and hematopoietic stem cells), and normal stem cells that differentiate from hematopoietic stem cells (Radiation Biology Research Communications 54(4), 266-280, 2019).

[0027] Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer embryonic stem cells (ntES cells)), multipotent germline stem cells (mGS cells), embryonic germ stem cells (EG cells), and Muse cells (multi-lineage differentiating stress enduring cells), with iPS cells (more preferably human iPS cells) being preferred. When the pluripotent stem cells are ES cells or any cells derived from a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, but are preferably cells produced without destroying an embryo.

[0028] Hematopoietic stem cells are primarily present in bone marrow. Hematopoietic stem cells differentiate into all blood cell lineages in the bone marrow, developing into red blood cells, white blood cells, and platelets. Hematopoietic stem cells can also proliferate through cell division and self-replicate. The cells for autologous transplantation used in the present invention include hematopoietic stem cells and are preferably collected from the bone marrow of a cancer patient. The cancer patient is a patient with a blood cancer such as leukemia, malignant lymphoma, or multiple myeloma who is eligible for hematopoietic stem cell transplantation. Bone marrow can be collected from a patient, for example, according to the bone marrow collection method published by the Japanese Society for Hematopoietic Cell Transplantation.

[0029] CD34 is a surface antigen that penetrates the cell membrane and is expressed on hematopoietic stem cells in bone marrow and peripheral blood, vascular endothelial progenitor cells, skeletal muscle satellite cells, epithelial hair follicle stem cells, and adipose tissue mesenchymal stem cells. Specifically, CD34 is a surface marker for various somatic stem cells, and CD34-expressing cells have the ability to differentiate into all blood cells and endothelial cells, but disappear as the cells mature and differentiate. Therefore, the hematopoietic stem cells used in the present invention are preferably CD34-positive cells. Whether the cells used are CD34-positive can be confirmed, for example, using a flow cytometer.

[0030] In Ex. Vivo BNCR, when applying BNCR to radiation-sensitive cells, (1) boron atoms ( 10 This is achieved by combining (B) a "boron agent" that selectively and effectively absorbs boron, and (2) a "neutron source" that stably generates epithermal neutrons of 50 eV or less with little contamination by radiation such as gamma rays.

[0031] Boron neutron capture therapy (BNCT) is a method of ionizing radiation using boron isotopes ( 10 B) into cancer cells and then irradiate them with neutrons to selectively kill the cancer cells. 10 B) The step of culturing in the presence of a drug is, for example, culturing autologous transplant cells containing hematopoietic stem cells collected from the bone marrow of a cancer patient in the presence of boron ( 10 B) A step of culturing the cells in the presence of a drug to introduce a sufficient amount of the boron drug into the cells. 10 B) As the drug, boron compounds having a closo structure such as p-boronophenylalanine (BPA) and mercaptoundecahydrodecaborate (BSH) and mixtures thereof can be used.

[0032] BPA is an amino acid derivative containing one boron atom in its molecule and is taken up into cells by amino acid transporters present in the cell membrane. BSH, on the other hand, is a crystalline form containing 12 boron atoms in its molecule. BPA and BSH are known compounds. Boron compounds with a closo structure, such as BSH, may be in the free form or in the form of a salt. Salts of boron compounds with a closo structure, such as BSH, include, but are not limited to, sodium salts, ammonium salts, and tetramethylammonium salts. Furthermore, boron compounds with a closo structure, such as BSH, may be modified or derivatized. Modified and derivatized BSH are known, including, but not limited to, peptide-bound BSH, sugar-bound BSH, and BSH with thiol, hydroxyl, carboxyl, amino, amide, azide, halogen, and phosphate groups. Methods for producing modified and derivatized BSH are known.

[0033] Boron compounds with a closo structure, such as BSH, have low cell membrane permeability and may not be adequately taken up by cells as they are. However, they can be encapsulated in liposomes and added to the culture medium for cellular uptake. If the amount of BSH taken up by cells is low, the amount of BSH taken up by cells can be increased by conjugating the closo structure boron compound, such as BSH, with a peptide containing hydrophobic and basic amino acid residues.

[0034] Examples of peptides containing the hydrophobic amino acid residues and basic amino acid residues include AAAAAK, AAAAAAK, AAAAAAAK, AAAAAKK, AAAAAAKK, AAAAAAAKK, AAAAAR, AAAAAAR, AAAAAAAR, AAAAAARR, AAAAAARR, and AAAAAAARR. More preferred examples of the peptides include AAAAAA-homoarginine, AAAAAA-ornithine, AAAAAA-2,7-diaminoheptanoic acid, AAAAAA-2,4-diaminobutanoic acid, and AAAAAA-2-amino-4-guanidinobutanoic acid, and even more preferred examples include, but are not limited to, AAAAAAK (abbreviated as A6K) and AAAAAAR (abbreviated as A6R). The above-mentioned peptides are represented by conventional one-letter amino acid symbols. Therefore, A represents alanine, K represents lysine, and R represents arginine.

[0035] The peptides used in the present invention may be in the free form, in the form of a salt, in the form of a solvate, or may be modified or derivatized. A variety of peptide salts are known, and methods for their preparation are also known. Examples of peptide salts include, but are not limited to, hydrochloride, sulfate, nitrate, phosphate, acetate, trifluoroacetate (TFA salt), citrate, succinate, maleate, fumarate, malate, tartrate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, alkali metal salts, and alkaline earth metal salts. Peptide solvates are also known, and methods for their preparation are also known. Examples of peptide solvates include, but are not limited to, solvates with water, methanol, ethanol, isopropanol, tetrahydrofuran, dimethyl sulfoxide, ethylene glycol, propylene glycol, acetamide, and the like. A variety of modified peptides and peptide derivatives are known. Methods for modifying and derivatizing peptides are also known. Examples of peptide modifications and derivatizations include, but are not limited to, acetylation, amidation, biotinylation, alkylation such as maleimidation and methylation, maleimidation, myristoylation, esterification, phosphorylation, and labeling with fluorescent labeling, radioactive labeling, etc. The N-terminus of the peptide is preferably acetylated. Furthermore, the amino acids constituting the peptide may be L- or D-isomers as optical isomers. Furthermore, one peptide molecule may contain both L- and D-amino acids. In the present invention, the term "peptide" encompasses the above-described modified peptides, derivatized peptides, peptides in the form of salts, and peptides containing D-amino acids.

[0036] The complex of a boron compound having a closo structure, such as BSH, with a peptide containing a hydrophobic amino acid residue and a basic amino acid residue, used in the present invention, can be prepared by mixing the peptide with the boron compound having a closo structure, such as BSH, in an aqueous solution. During mixing, stirring or ultrasonic treatment may be performed to promote dispersion and complex formation. The concentration of the peptide in the aqueous solution in which the peptide is mixed with the boron compound having a closo structure, such as BSH, is not particularly limited and is generally several μM to several mM. The concentration of BSH in the aqueous solution is also not particularly limited and is generally several tens of μM to several mM. The molar ratio of the peptide to BSH during mixing is not particularly limited, but a mixing ratio of about 1 mole to about 1,000 moles of BSH per mole of peptide is preferred. For example, a ratio of about 1 to about 100 moles of BSH per mole of peptide, or a ratio of about 100 to about 1,000 moles of BSH per mole of peptide may be used.

[0037] The aqueous solution in which the peptide is mixed with a boron compound having a closo structure, such as BSH, is a solution containing water as a medium. The aqueous solution may consist of water alone, or may contain other substances such as buffers and salts. Those skilled in the art can determine appropriate conditions for the mixing, such as temperature, pH, and mixing time. For example, when mixing BSH with A6K or A6R, it is preferable to mix them under conditions such that the lysine residue in A6K or the arginine residue in A6R is positively charged and BSH is negatively charged. The pH of the aqueous solution may be adjusted to a desired value using a buffer such as phosphate-buffered saline.

[0038] The complex of a boron compound having a closo structure, such as BSH, with a peptide containing a hydrophobic amino acid residue and a basic amino acid residue is spherical, either with or without protrusions on its surface. A spherical shape includes not only true spheres but also nearly spherical shapes. Specifically, a particle can be considered spherical if the ratio of its minor axis to its major axis is about 0.5 or greater, preferably about 0.6 or greater, and more preferably about 0.7 or greater. The particle size and particle size distribution of the particles formed from the complex can be measured by dynamic light scattering (DLS). The particle size of the complex can be adjusted by the mixing ratio of the boron compound having a closo structure, such as BSH, with the peptide during preparation of the complex. When the ratio of BSH to the peptide is low, the resulting complex tends to have a smaller diameter, while when the ratio of BSH to the peptide is high, the resulting complex tends to have a larger diameter.

[0039] In one embodiment of the present invention, furthermore, boron ( 10 B) A method for sorting non-target cells is provided, which includes a step of evaluating drug uptake.

[0040] In one embodiment of the present invention, there is provided a method for sorting non-target cells, further comprising the step of isolating target cells.

[0041] In one embodiment of the present invention, boron ( 10 B) A method for sorting non-target cells, including a step of evaluating a drug, is provided.

[0042] In one embodiment of the present invention, a method for separating non-target cells is provided, characterized in that the neutron irradiation field has little effect on non-target cells, and has a maximum neutron energy of 50 eV or less, which minimizes contamination by gamma rays and the like and reduces the effects of fast neutrons (particularly, the hydrogen dose imparted to cells when neutrons react with hydrogen).

[0043] In one embodiment of the present invention, a method for separating non-target cells is provided, which is characterized by a neutron irradiation field that has little effect on non-target cells, and that satisfies the standard values ​​of the International Atomic Energy Agency (IAEA) (IAEA TECDOC-1223) and is capable of generating low-energy epithermal neutrons (0.5 eV-10 keV) in combination with a water phantom.

[0044] In one embodiment of the present invention, a method for separating non-target cells is provided, which comprises placing a non-target cell culture containing target cells at the center of the axis of a cylindrical neutron moderator (water, heavy water, Be, C, etc.) with a radius of 5 cm or more as a neutron irradiation field that has little effect on non-target cells, and moderating high-speed neutrons emitted from multiple small neutron generators installed beside the cylindrical neutron moderator to 50 eV or less, and irradiating the neutrons onto the non-target cell culture containing target cells.

[0045] The autologous transplant cells are treated with the boron ( 10 B) In the step of culturing in the presence of a drug, the type of culture medium, the culture time, the boron ( 10 B) The culture conditions such as drug concentration and cell density in the culture medium are determined by the above-mentioned boron ( 10 B) It can be determined by the amount of drug taken up into cells and its intracellular distribution. 10 B) When the autologous transplant cells incorporating the drug are irradiated with neutrons, a nuclear reaction occurs between boron and thermal neutrons inside the cells, and the particle beams (alpha rays and 7Li particles) generated by the nuclear reaction damage the cells. 10 B) Cells with higher drug uptake are more susceptible to cytotoxicity due to neutron irradiation. The culture conditions in the isolation method of the present invention are set to minimize the boron ( 10 B) Minimizing drug uptake while promoting the delivery of the boron ( 10 B) It maximizes drug uptake. As a method to reduce uptake into non-target cells, Use OKD-001 as the boron agent. The final concentrations are A6K:BSH=0.2 mM / 2 mM. The medium used is RPMI1640 supplemented with 1% human serum albumin (HSA). -Culture temperature: 37℃ Cultured under 5% CO2 conditions Examples include: As a result, the number of viable cancer cells present after neutron irradiation is reduced compared to the number of viable cancer cells present before neutron irradiation. Preferably, viable cancer cells disappear after neutron irradiation. On the other hand, normal cells remain viable even after neutron irradiation, and preferably, there is almost no change in the number of viable normal cells before and after neutron irradiation. 10 B) Neutron irradiation of the autologous transplant cells incorporating the drug can be performed using a nuclear reactor or accelerator-type neutron generator, and conditions such as neutron dose, neutron spectrum, irradiation time, and irradiation angle can be determined while examining the cytotoxicity to cancer cells and normal cells.

[0046] In one embodiment of the present invention, non-target cells obtained by the separation method of the present invention are provided. The cells are, for example, cells for autologous transplantation, and are provided in a state suspended in a culture medium or frozen in a cell cryopreservation solution. Examples of cell cryopreservation solutions include a cell culture medium containing 10% dimethyl sulfoxide and 10% serum, and Cellbanker (registered trademark, Takara Bio).

[0047] In one embodiment of the present invention, a pharmaceutical composition is provided, comprising non-target cells obtained by the sorting method of the present invention.

[0048] Pharmaceutical compositions containing the non-target cells of the present invention as an active ingredient can be used to treat cancer patients. The pharmaceutical compositions of the present invention can be produced by conventional methods in the pharmaceutical technology field, such as those described in the Japanese Pharmacopoeia. The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable additives. Examples of such additives include cell culture medium, physiological saline, and appropriate buffer solutions (e.g., phosphate buffer solutions).

[0049] The pharmaceutical composition of the present invention can be produced by suspending non-target cells in physiological saline or an appropriate buffer solution (e.g., phosphate buffer solution). The amount of the non-target cells per dose is, for example, 1 x 10 7 It is preferable to contain 1×10 or more cells. More preferably, it contains 1×10 8 or more, more preferably 1×10 9 The number of cells is at least 1. The cell content can be adjusted appropriately taking into consideration the gender, age, weight, condition of the affected area, and cell condition of the subject. In addition to non-target cells, the pharmaceutical composition of the present invention may contain dimethyl sulfoxide (DMSO), serum albumin, and the like for the purpose of protecting the cells. It may also contain antibiotics, etc. for the purpose of preventing bacterial contamination, and vitamins, cytokines, and the like for the purpose of promoting cell activation and differentiation. Furthermore, the pharmaceutical composition of the present invention may contain other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.).

[0050] Pharmaceutical compositions containing non-target cells of the present invention as active ingredients can be cryopreserved. When cryopreserving, the temperature is not particularly limited as long as it is suitable for storing cells. Examples include -20°C, -80°C, and -120 to -196°C. When cryopreserving, cells can be stored in an appropriate container such as a vial. Procedures for minimizing the risk of cell damage during freezing and thawing of non-target cells are well known to those skilled in the art.

[0051] In cryopreservation of non-target cells, the non-target cells are collected from the culture medium, washed with a buffer solution or culture medium, counted, concentrated by centrifugation or the like, suspended in a freezing medium (e.g., a culture medium containing 10% DMSO), and then cryopreserved. Non-target cells can be cultured in multiple culture vessels and combined to form a single lot. The pharmaceutical composition containing the non-target cells of the present invention can be stored in a container such as a vial at a concentration of, for example, 5 x 10 cells. 4 pieces~9×10 10 The number of non-target cells included in the antibody may vary depending on the type of cancer to be treated, the target of administration, the route of administration, etc.

[0052] Examples of routes of administration for pharmaceutical compositions containing non-target cells of the present invention include infusion, intratumoral injection, arterial injection, portal vein injection, and intraperitoneal administration. However, the administration route is not limited to these, as long as the non-target cells, which are the active ingredient in the pharmaceutical composition of the present invention, are delivered to the affected area. The administration schedule can be a single administration or multiple administrations. The multiple administration period can be, for example, a method in which administration is repeated once every 2 to 4 weeks or a method in which administration is repeated once every six months to one year. When creating an administration schedule, the gender, age, weight, and pathological condition of the target patient can be taken into consideration.

[0053] In one embodiment of the present invention, there is provided a preventive or therapeutic agent for cancer, which comprises non-target cells obtained by the sorting method of the present invention.

[0054] In one embodiment of the present invention, there is provided a preventive or therapeutic agent for blood cancer, including, but not limited to, leukemia, malignant lymphoma, multiple myeloma, etc., comprising non-target cells obtained by the culture method of the present invention.

[0055] In one embodiment of the present invention, there is provided a method for preventing or treating cancer in a cancer patient, which comprises administering an effective amount of a pharmaceutical composition comprising non-target cells.

[0056] In one embodiment of the present invention, there is provided a method for treating blood cancer, comprising the steps of collecting hematopoietic stem cells from a patient with blood cancer before anticancer treatment using boron neutron capture therapy (BNCT) for the patient, producing the collected hematopoietic stem cells using the sorting method of the present invention, and autologously transplanting the hematopoietic stem cells obtained by the sorting method into the patient.

[0057] In one embodiment of the present invention, there is provided a method for treating blood cancer, comprising the steps of: collecting hematopoietic stem cells from a patient with blood cancer before anticancer treatment using boron neutron capture therapy (BNCT) for the patient; purifying the collected hematopoietic stem cells using the sorting method of the present invention; and autologously transplanting an effective amount of the hematopoietic stem cells obtained by the sorting method into a patient in need of blood cancer treatment.

[0058] The present invention will be explained in more detail and specifically below by showing examples, but the examples should not be construed as limiting the scope of the present invention. [Example]

[0059] <Target cells and boron drugs> Ex. Vivo BNCR is a boron drug that is expected to be used to remove heterogeneous and mutant cells from highly radiosensitive cells with "stemness" [ 10 The combinations of [Drug B] are shown in the table below. Heterogeneous and mutated cells include cancer cells, blood cancers (multiple myeloma, malignant lymphoma, myeloid leukemia (AML, CML, etc.)), sarcoma, teratoma, and other neoplastic cells, as well as remaining pluripotent stem cells. a (e.g., embryonic stem cells and induced pluripotent stem cells) and mutations that cause neurodegenerative diseases

[0060] Table 2 Boron drugs that are expected to be used to remove heterologous and mutant cells [ 10 B drug combination JPEG2023085151000002.jpg116138 [Example]

[0061] <Production of New Boron Agent OKD> Production of Complex Containing BSH (Tetramethylammonium Salt) and A6K Preparation of BSH aqueous solution The weighed BSH tetramethylammonium salt was transferred to a 15 mL tube. To it, water for injection (Otsuka distilled water, Otsuka Pharmaceutical Factory, Inc.) was added. The resulting solution was sonicated using a ultrasonic cleaner (CPX1800H-J, BRANSON). Then, it was stirred by vortex to confirm that the BSH tetramethylammonium salt was completely dissolved. Preparation of A6K aqueous solution The weighed A6K hydrochloride was transferred to a 1.5 mL tube. To it, water for injection (Otsuka distilled water, Otsuka Pharmaceutical Factory, Inc.) was added. The resulting solution was sonicated using a ultrasonic cleaner (AUC-1L, AS ONE Corporation). Then, it was stirred by vortex to confirm that the A6K hydrochloride was completely dissolved. Mixture of BSH and A6K Into a 1.5 mL tube, an aqueous solution of A6K, an aqueous solution of BSH, and water for injection (Otsuka distilled water, Otsuka Pharmaceutical Factory, Inc.) were mixed so that the final concentration of BSH was 20 mM and the final concentration of A6K was 2 mM. Properties of the resulting mixture The resulting mixture was a colorless and transparent aqueous solution. Analysis by dynamic light scattering (DLS) confirmed that a complex composed of BSH and A6K existed as particles in the aqueous solution. The measurement conditions of DLS were: temperature 25 °C, equilibrium 60 seconds, measured 3 times at 173 Å, and a general-purpose model was used (actual measurement time 70 seconds, count rate 192.6 kcps, measurement position 3.00 mm, attenuator 7). Most of the complex particles had a particle size in the range of 1000 - 6000 nm, and a peak was observed around a particle size of 2000 nm.

Example

[0062] Treatment of Boron Agent on Cancer Cells and Normal Cells <Cell Uptake Test of OKD>

[0063] Preparation of complexes containing BSH and A6K and cell uptake test Preparation of BSH complex using aqueous solution of BSH (sodium salt) and A6K peptide Aqueous solutions of BSH and A6K were prepared by dissolving each in water for injection (Otsuka). A BSH complex (BSH:A6K = 20 mM:2 mM) was prepared by mixing the aqueous solutions of BSH and A6K and water for injection. Dynamic light scattering (DLS) revealed peaks similar to those observed for the complex of BSH (tetramethylammonium salt) and A6K.

[0064] Preparation of a drug by adding a stabilizer to an aqueous solution of BSH complex consisting of BSH and A6K peptide Aqueous solutions of BSH and A6K were prepared by dissolving each in water for injection (Otsuka). A BSH complex (BSH:A6K = 20 mM:2 mM) was prepared by mixing the aqueous solutions of BSH, A6K, and water for injection. The drug was prepared by adding an appropriate amount of stabilizer or dispersant to this aqueous solution. As a stabilizer or dispersant, when prepared OKD-001 was diluted 5-fold with 1% human serum albumin (HSA) (BSH:A6K=4 mM:0.4 mM, 0.8% HSA), it could be stored stably at 4°C. In this case, OKD-001 refers to the BSH complex (BSH:A6K=20 mM:2 mM). Immediately after preparation, OKD-001 was diluted 5-fold with ultrapure water and filtered through an ultrafiltration (Amicon Ultra, regenerated We confirmed that desalination of the solvent and concentration of the diluted solution using a cellulose membrane (KD) enabled stable storage at 4°C. Figure 2 shows the results of DLS measurement of the OKD aqueous solution after ultrafiltration. The average particle diameter was 385.9 nm, and the dispersion remained stable at 4°C even one week after preparation.

[0065] Cell seeding and drug exposure On the day of drug addition, 3 × 10 cells of each type were 5Cells were seeded onto a 6-well plate at 2.7 ml / well of medium (RPMI1640 10% FBS, P / S). 300 μl / well of the aforementioned BSH complex solution (final concentrations of BSH:A6K = 2 mM:0.2 mM) was added to the cells seeded on the plate.

[0066] Cell harvesting, elastase treatment, and boron determination Twenty-four hours after drug addition, cells were collected in a 15 ml tube and treated with elastase (Sigma) to remove extracellular complexes. After washing with PBS, the cells were counted. The 2 ml tube containing the PBS-suspended cells was centrifuged, the supernatant removed, and the resulting cell pellet was frozen at -20°C. The frozen cell pellet was suspended and dissolved in 300 μl of nitric acid, subjected to thermal nitric acid digestion, and then diluted 20-fold. The boron concentration was measured using an ICP-MS (Agilent 7500cx).

[0067] Figure 3 shows the amount of boron uptake in each cell. 6 The values ​​are shown as measured values ​​per 1000 mg / kg (B ng). Each treatment group was tested in triplicate (n=3), and the average values ​​and standard deviations are shown in the graph. The amount of boron taken up by the new boron drug OKD into autologous hematopoietic stem cell model cells (CD34+ cells) and blood cancer cell lines (two types of multiple myeloma model cells: IM-9 and RPMI8226) was measured using ICP-MS. 10 B) It was confirmed that the concentration ratio was large (Figure 3). RPMI8226 / CD34+: 1700pm / 70ppm IM-9 / CD34+: 3000pm / 70ppm This suggests that, for example, during autologous hematopoietic stem cell transplantation in the treatment of multiple myeloma, cancer cells that are at risk of contamination can be removed using BNCR. 10 Neutrons cm -2 Cancer cells can be killed by irradiating them with neutrons.

[0068] <Cell uptake test of existing boron agents BPA and BSH> Boron uptake into various cells by BPA and BSH aqueous solutions Cell seeding and drug exposure On the day of drug addition, 3 × 10 cells of each type were 5 Cells were seeded into a 6-well plate at 2.925 ml / well. The medium was RPMI1640 10% FBS, P / S. 75 μl / well of a solution containing BPA and BSH, adjusted to a final concentration of 2 mM and 0.5 mM, respectively, was added to the plate. The medium for CD34-positive hematopoietic stem cells was X-VIVO 20 (Lonza) 1% ITES.

[0069] Cell harvesting and boron quantification Twenty-four hours after drug addition, cells were collected in a 15 ml tube and centrifuged at 300 × g for 5 minutes, after which the supernatant was removed. The cells were resuspended in 4 ml of PBS and centrifuged again. After removing the PBS from the supernatant, the cells were resuspended in 1 ml of PBS and transferred to a 2 ml tube. Five μl of the cell suspension from the 2 ml tube was collected in 1.5 ml tubes for each treatment group (a total of 15 μl per treatment group), mixed, and the average cell count was calculated. The 2 ml tubes containing the PBS-suspended cells were centrifuged, the supernatant removed, and the resulting cell pellets were frozen at -20°C. The frozen cell pellets were suspended and dissolved in 300 μl of nitric acid, subjected to thermal nitric acid decomposition, and then diluted 20-fold to measure the boron concentration using an ICP-MS (Agilent 7500cx). The boron content in each treatment group was measured at 10 cells / mL. 6 The values ​​are shown as measured values ​​(B ng) per 100 cells. Each treatment group was tested in triplicate (n=3), and the average values ​​and standard deviations are shown in the graph. 11 Neutrons cm -2 Cancer cells can be killed by irradiating them with neutrons.

[0070] Cells used in the experiment RPMI8226 cells: a cell line derived from the peripheral blood of a human multiple myeloma patient IM-9 cells: a cell line derived from the bone marrow of patients with human multiple myeloma and B lymphoblastic leukemia Human CD34-positive hematopoietic stem cells: derived from human bone marrow (Lonza)

[0071] Figure 4 shows the results of an evaluation of the intracellular internalization of BPA and BSH. It was confirmed that the boron agents BPA and BSH had higher boron uptake into two types of blood cancer (multiple myeloma: MM) cells (RPMI-8226 and IM-9) than into hematopoietic stem cells (indicated by CD34+) (Figure 4). This suggests that, for example, during autologous hematopoietic stem cell transplantation in the treatment of multiple myeloma, cancer cells at risk of contamination can be eliminated by utilizing BNCR, which is a combination of these boron agents and neutron beams. 10 Neutrons cm -2 Cancer cells can be killed by irradiating them with neutrons. [Example]

[0072] Neutron irradiation (simulation test using a water phantom) <Neutron irradiation field> Stem cells are highly sensitive to radiation, and in the case of hematopoietic stem cells (stem / progenitor cells) in bone marrow, the radiation sensitivity D0 has been reported to be 0.8-1.2 Gy (see table below). In this case, if hematopoietic stem cells are given a dose of about 1 Gy, the survival rate of the stem cells drops to 0.34. Therefore, when using the BNCR reaction to remove cancer cells contained in bone marrow for autologous transplantation, it is preferable not to give hematopoietic stem cells a high radiation dose.

[0073] Table 3. Radiation sensitivity of various cells in bone marrow JPEG2023085151000003.jpg55124

[0074] Neutron fields suitable for Ex. Vivo BNCT are discussed below. In BNCR, the radiation dose D(x) received by tumor or normal cells is calculated as the dose from neutrons Dn(x), the dose from gamma rays Dγ(x), and the dose given when boron undergoes nuclear fission by thermal neutrons D B The neutron dose Dn at location x is calculated using the neutron energy distribution Φn, the neutron kerma factor Kn, and the neutron quality factor RBE. n The gamma ray dose Dγ due to gamma rays contained in neutrons is calculated by the product of the gamma ray energy distribution Φγ, the gamma ray kerma coefficient Kγ, and the radiation quality factor RBE γ (=1.0). Boron dose D B is the neutron energy distribution Φn and the boron kerma factor K B , boron quality factor RBE B It can be calculated by multiplying C B is the boron concentration (ppm) accumulated in the cells. JPEG2023085151000004.jpg93145When neutrons are irradiated to cancer cells and stem cells (normal cells), the neutron dose Dn and gamma ray dose Dγ are equal to each other, and the boron dose D, which is proportional to the boron concentration, is B will be granted. To kill cancer cells while sparing stem cells, (i) the boron dose D between the cancer cells and stem cells is B It is essential to increase the difference between the neutron dose Dn and the gamma ray dose Dγ as much as possible.

[0075] Figure 5 shows the concept of the dose delivered to cancer cells and normal cells. Looking at the energy dependence of the kerma coefficient Kn(E)* for biological tissue, which is a factor that determines the neutron dose Dn, we can see that when the neutron energy is 50 eV or less, the contribution from ionization of nitrogen elements is large, and when the neutron energy is 50 eV or more, the energy transfer from recoil of hydrogen elements is large. For this reason, in order to reduce the neutron dose Dn, it is preferable to irradiate with neutrons with an energy distribution of 50 eV or less. (*Kerma coefficient: The kinetic energy dE of charged particles generated by indirect ionization particles in a given unit weight of material dm. K=dE / dm.) The main way to reduce the contribution of the gamma ray dose Dγ is to reduce the intensity of the gamma rays emitted from the BSA. For this purpose, an accelerator neutron source using a Li target is preferred.

[0076] Figure 6 shows the neutron energy dependence of the kerma coefficient for biological tissue. FIG. 7 shows the gamma-ray energy dependence of the gamma-ray kerma coefficient.

[0077] The figure below shows an example of a system that combines a Li target and an electrostatic accelerator (proton energy: 2.8 MeV to 1.9 MeV) as an accelerator neutron source. This system has the advantage that the accelerator body and beam line are hardly activated by protons because the maximum beam energy is low at 2.8 MeV, making it possible to perform accelerator maintenance and repairs safely and quickly, and reducing the electricity costs for accelerator operation. On the other hand, Li is chemically active and mechanically weak, and radioactive materials are generated when neutrons are generated. 7 There are some issues, such as Be being generated within Li, but Li and 7 Be is sealed in a titanium thin film (Li sealed target), enabling stable operation and maintenance. Figure 8 shows a photograph of the electrostatic accelerator system and the beamline.

[0078] A Beam Shaping Assembly (BSA) is used to slow down the high-speed neutrons generated in the target to an epithermal energy range (0.5 eV to 10 keV) suitable for cancer treatment of brain tumors, head and neck, etc. The energy spectrum of the emitted neutrons is shown in the figure below. The radiation quality of the epithermal neutrons emitted from this BSA conforms to the characteristics recommended by the IAEA, and is characterized by high-speed neutrons of 10 keV or more that have a large effect on normal tissue, and a low gamma ray contamination rate in the neutrons, but also satisfies the neutron energy requirement of "50 eV or less" that is suitable for stem cell irradiation. Figure 9 shows the neutron moderation irradiation device and the quality of the emitted neutron beam.

[0079] In order to obtain low-energy neutrons suitable for neutron irradiation of hematopoietic stem cells, a neutron moderator for cell irradiation was installed at the exit of the BSA. Two types of moderator systems were selected, and the results of an evaluation of the system suitable for hematopoietic stem cell irradiation using Monte Carlo simulation analysis PHITS (nuclear data JENDEL-4) are shown in the figure below. (Moderator dimensions: 20cm x 20cm x 20cm, acrylic wall thickness 3mm) (1) Light water moderator Light water (H2O) is ideal for slowing down neutrons because it contains H, which has the same mass as a neutron. On the other hand, gamma rays are produced. (2) Light water moderator and gamma ray lead shielding (1) System in which gamma rays are shielded with lead. (Reference) Heavy water moderator Neutrons can be moderated using heavy water (D2O), which does not emit gamma rays, but the mass ratio of deuterium D to neutrons is doubled, so neutron moderation does not proceed quickly.

[0080] Figure 11 shows a neutron moderator for stem cell irradiation. Figure 12 shows the structure of a neutron moderator for stem cell irradiation.

[0081] Figure 13 shows an example of the dose distribution within a neutron moderator for stem cell irradiation. The vertical axis represents the dose (Gy-eq / mA / sec) calculated by PHITS analysis. The expected dose (Gy-eq) delivered to cells can be calculated by multiplying the beam current (maximum 2 mA) and irradiation time. When epithermal neutrons enter the moderator from the BSA exit, they react with nitrogen and hydrogen in the body, delivering "nitrogen dose" and "hydrogen dose" to all cells in the body. Furthermore, gamma rays cause "gamma ray dose" to be delivered to all cells in the body. B-10 accumulated in cancer cells undergoes nuclear fission with thermal neutrons, delivering "boron dose" exclusively to the cancer cells. When using Ex. Vivo BNCR to remove only cancer cells, it is preferable to set the neutron irradiation conditions so that the dose to hematopoietic stem cells is kept below a certain level (1 Gy-eq or less) while the dose to the cancerous area is above a certain level (e.g., 30 Gy or more).

[0082] When light water is used as a neutron moderator for stem cell irradiation, neutron components of 50 eV or more are sufficiently reduced after passing through light water for about 4 cm, and the hydrogen dose D H It can be seen that the "gamma ray dose" from the BSA exit is small, but the dose due to gamma rays increases when neutrons collide with light water (H), emitting gamma rays. When operated for 40 minutes with a proton beam current of 2 mA, the expected dose at 4 cm from the moderator surface is nitrogen dose (3.8E-5 Gy-Eq / mA / s x 2 mA x 40x60 sec), hydrogen dose (4E-6 Gy-Eq / mA / s x 2 mA x 40x60 sec), and gamma ray dose (6.5E-5 Gy-Eq / mA / s x 2 mA x 40x60 sec), and the dose other than the boron dose is evaluated to be a total of 0.5 Gy-eq. The concentration of boron that accumulates in cancer cells varies depending on the boron agent used and the type of cancer cells, but if 1500 ppm of boron is taken up by cells 4 cm from the body surface, the "boron dose" is estimated to be 165 Gy-eq (extrapolated from 9.6 Gy-eq when the boron concentration is 87.5 ppm). A certain percentage of boron agents may also be taken up by hematopoietic stem cells; if 5 ppm or 50 ppm of boron is taken up by hematopoietic stem cells, the "boron dose" would be 0.5 Gy-eq and 5 Gy-eq, respectively. If a boron agent that is less easily taken up by hematopoietic stem cells than cancer cells can be used, and combined with a neutron moderator using light water, it is expected that cancer cells can be eliminated with less impact on hematopoietic stem cells.

[0083] Placing a 1cm thick layer of lead shielding around a container containing hematopoietic stem cells can reduce the gamma ray dose by approximately half, but at the same time, the boron dose also decreases, so there is no significant improvement in the ratio of non-boron dose to boron dose (right diagrams in Figures 13 and 14). However, since the boron dose can be made more uniform between 2 and 6cm than without lead shielding, there is the advantage that a uniform boron dose can be imparted to a thicker bone marrow bag. Furthermore, the gamma ray dose can be reduced by reducing the thickness of the light water from 20cm to 10cm.

[0084] When heavy water is used for neutron moderation, γ-rays generated by collisions with H can be reduced, but there are problems in that a long distance is required for neutron moderation, the peak intensity of thermal neutrons decreases, and the irradiation time significantly increases. These can be solved by performing boron dose calculations. That is, in cells that contain little boron agent (non-target cells), when the boron dose from neutrons (thermal neutrons, epithermal neutrons) from the accelerator-type neutron generator shown in this patent is calculated to have a limited effect, direct irradiation can be performed without using such a moderator. It is considered that the influence of gamma rays generated due to the irradiated neutron beam can also be regarded as limited. Even in cells that contain a large amount of boron agent (target cells), its influence can be estimated from the boron dose. Thus, appropriate selection of irradiation conditions etc. according to the combination of target cells and non-target cells etc. leads to a solution.

Example

[0085] Neutron beam irradiation test on cancer cells and normal hematopoietic stem cells after boron agent OKD treatment

[0086] <Confirmation of BNCR effect> The results of confirming the BNCR effect by neutron irradiation are summarized below. The uptake amount of the new boron agent OKD-001 into autologous hematopoietic stem cell model cells (CD34+ cells) and blood cancer cells (two types of multiple myeloma model cells: IM-9, RPMI8226) was measured by ICP-MS, and the boron ( 10 B) concentration ratio was large, confirming that the cancer cell accumulation property is high. RPMI8226 / CD34+: 1700 pm / 70 ppm IM-9 / CD34+ : 3000 pm / 70 ppm

[0087] Cells incorporated with boron at the concentration shown in Figure 3 were placed in the "neutron moderator for stem cell irradiation" and neutrons (thermal neutron flux: 1x10 8 n / cm 2Neutron irradiation was performed on light water blood cancer cells (MM model cells IM-9: highly expressing the mechanism for uptake of the boron drug used (denoted as OKD)), and cell viability was evaluated three days after irradiation using an absorption spectrophotometer (WST-8). It was found that BNCR (alpha decay reaction) occurred upon neutron irradiation, resulting in significant damage to the cancer cells. This enabled the removal of target cells (in this case, MM model cells) and the isolation of non-target cells (here, model cells of autologous hematopoietic stem cells).

[0088] Figure 16 shows the relationship between neutron irradiation time and cell survival rate of target cancer cells (MM cells) and non-target normal cells (hematopoietic stem cells) after treatment with a boron drug (OKD). The two relationships are significantly different, proving the concept of this patent. FIG. 16 shows the neutron irradiation time and cell survival rate transition of target cells (MM cells) and non-target cells (hematopoietic stem cells) irradiated with neutrons in the presence of a drug (OKD-001). The cell survival rates of target and non-target cells showed an increasing discrepancy depending on the irradiation time. OKD-001 represents a boron drug that is a complex of BSH and the peptide A6K.

Claims

1. (1) A non-target cell culture containing target cells is treated with boron ( １０ B) is cultured under conditions in which it is incorporated; and (2) The boron ( １０ A method for isolating non-target cells by a step of selectively killing the target cells by irradiating the target cells into which B) has been taken up with neutron rays that have little effect on non-target cells.

2. In step (1), boron ( １０ B) The drug is easily taken up and boron ( １０ B) The method for isolating the cells according to claim 1, wherein the cells are cultured under culture conditions that make it difficult for the cells to take up the drug.

3. 3. The separation method according to claim 1 or 2, characterized in that in step (2), a neutron irradiation field configured so that the maximum energy of the irradiated neutron beam is 50 eV or less is used to selectively separate non-target cells.

4. The method according to any one of claims 1 to 3, wherein the cell culture obtained in step (2) is substantially free of target cells.

5. The method according to any one of claims 1 to 4, wherein the non-target cells are stem cells.

6. The method according to claim 5 , wherein the stem cells are pluripotent stem cells or hematopoietic stem cells.

7. The separation method according to claim 6 , wherein the pluripotent stem cells are human pluripotent stem cells.

8. The separation method according to claim 6 , wherein the pluripotent stem cells are human iPS cells.

9. The method according to claim 6 , wherein the hematopoietic stem cells are CD34 positive cells.

10. The method according to any one of claims 1 to 9, wherein the target cells are one or more types selected from the group consisting of heterologous cells, dysplastic cells, and cancer cells.

11. The method according to any one of claims 1 to 9, wherein the target cells are cancer cells.

12. The boron ( １０ The method for separation according to any one of claims 1 to 11, wherein the drug B) is p-boronophenylalanine (BPA), mercaptoundecahydrodecaborate (BSH), or a mixture thereof.

13. The boron ( １０ The method for separating the drug according to any one of claims 1 to 11, wherein B) the drug is a complex of BSH and a peptide containing a hydrophobic amino acid residue and a basic amino acid residue, and the peptide is A6K or A6R.

14. In addition, boron uptake by target and non-target cells ( １０ The method for fractionation according to any one of claims 1 to 13, further comprising a step B) of evaluating drug uptake.

15. The method according to any one of claims 1 to 13, further comprising a step of isolating target cells.

16. In addition, boron ( １０ The method for fractionation according to any one of claims 1 to 13, further comprising a step B) of evaluating a drug.

17. The separation method according to claim 3, characterized in that the neutron irradiation field has little effect on non-target cells, has little contamination with gamma rays and the like, has a maximum neutron energy of 50 eV or less, and has small effects of fast neutrons (particularly, the hydrogen dose imparted to cells by neutrons reacting with hydrogen).

18. The separation method according to claim 3, characterized in that the neutron irradiation field which has little effect on non-target cells is a neutron field which has the effect of reducing the survival rate of target cells by combining a water phantom with a device capable of generating low-energy epithermal neutrons (0.5ev-10keV) which satisfies the standard values ​​(IAEA TECDOC-1223) of the International Atomic Energy Agency (IAEA).

19. The separation method according to claim 3, characterized in that a non-target cell culture including target cells is placed at the center of an axis of a cylindrical neutron moderator (water, heavy water, Be, C, etc.) having a radius of 5 cm or more as a neutron irradiation field that has little effect on non-target cells, and high-speed neutrons emitted from multiple small neutron generators installed beside the cylindrical neutron moderator are slowed down to 50 eV or less and the neutrons are irradiated onto the non-target cell culture including target cells.

20. A non-target cell obtained by the method according to any one of claims 1 to 19.

21. A pharmaceutical composition comprising the non-target cell of claim 20.

22. A preventive or therapeutic agent for cancer, comprising the non-target cell according to claim 20.

23. A preventive or therapeutic agent for blood cancer, comprising the non-target cell according to claim 20.