JP2023085151A - Stem cell fractionation method using in vitro boron neutron reaction - Google Patents

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 The present invention relates to the field of regenerative medicine and cell therapy in which radiation-sensitive cells having "stemness" are used. These cell culture techniques are particularly suitable for bone marrow and hematopoietic stem cell transplantation.

Radiation-sensitive 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. is mentioned. In regenerative medicine, pluripotent stem cells are induced to differentiate into living organs and tissues under appropriate conditions, and are utilized in regenerative medicine and transplantation medicine. At this time, there is a risk that undifferentiated cells may become teratoma. Although there is a technical guidance (June 2016) that describes the concept and points to note when doing so, no technical method for positive removal has been reported.

In the use cases of pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, the focus has been on the development of differentiation-inducing methods to reduce residual undifferentiated cells, so 'removal' methods have not been established. not In general, methods to remove "undesired" xenogeneic or atypical cells include the use of radiation such as gamma rays and exposure to cytotoxic agents such as anticancer agents. However, it is difficult to select any of them because of the concern that they may strongly damage useful pluripotent stem cells.

Technologies and methods that lead to "removal" of "undesirable" xenogeneic and atypical cells such as hematopoietic stem cells that exist in the bone marrow and have "stemness" and cancerous cells mixed in bone marrow grafts. has not been reported. Conventionally, only healthy transplant donors or remission bone marrow or hematopoietic stem cell transplantation (allogeneic or autologous) are assumed, and only "risks are reduced or avoided."

In the Boron Neutron Capture Reaction (BNCR), boron atoms ( ¹⁰ B) are irradiated with thermal neutrons or low-energy epithermal neutron beams to induce a nuclear reaction (α-decay reaction), resulting in boron atoms. BNCT is a chemical reaction that selectively destroys and kills only cells containing more than a certain amount on a cell-by-cell basis. This method may be used for this purpose. That is, using a boron drug that has relatively little effect on cells, after accumulating boron element ( ¹⁰ B) in “undesirable” heterogeneous/heteromorphic cells, we focused on “thermal neutrons” that have little effect on normal cells. Controlled irradiation of low-energy neutron beams to induce an "alpha decay reaction (nuclear reaction)", designated BNCR, in "undesired" heterologous and atypical cells, without affecting normal cells A method of destroying and removing only cells from the inside is envisioned. However, there have been no reports of the use of BNCR for such purposes. As a BNCT facility, in March 2020, the world's first accelerator-driven BNCT system that combines a cyclotron-type accelerator neutron source (NeuCure) and a boron drug steboronine (borofarane BPA and a solubilizing agent D-sorbitol intravenous drip) was unresectable. It is approved by the Ministry of Health, Labor and Welfare for the treatment of locally advanced or recurrent head and neck cancer. In addition, clinical trials targeting melanoma and angiosarcoma are underway with a system that combines the RFQ Linac Neutron Source (CICS-1) and steboronine installed at the National Cancer Center and Edogawa Hospital. The second accelerator neutron source (nuBeam) using an electrostatic accelerator and a rotating Li target installed at Helsinki Hospital in Finland has been introduced at Shonan Kamakura Hospital. At the University of Tsukuba, the development of the RFQ/DTL linac neutron source (iBNCT) is underway, and preparations are underway for non-clinical tests for practical use. The accelerator neutron source used in these BNCT facilities consists of a "proton beam accelerator," a "neutron generation target," and a "moderator (BSA) that slows down neutrons to an energy range suitable for treatment." Until now, multiple systems have been developed by changing the combination of accelerators (cyclotron, linac, electrostatic accelerator) and target materials (Be, Li) (Fig. 1), all of which are aimed at treating deep solid tumors. , the specifications are suitable for generating neutrons with energies in the epithermal region (0.5 eV to 10 keV).

Among them, the Li target can efficiently generate neutrons under a low-energy proton beam, and the generated neutron energy is lower than that of the Be target. It has the characteristic that neutrons are easily slowed down (Table 1).
Table 1 Neutron energy reduction ratio required for epithermal neutron generation in each system

Development of BNCT (Boron Neutron Capture Therapy), Isotope News, 755, 42-47 (2018) Future Prospects of BNCT, Isotope News, 756, 24-27 (2018) Development of accelerator neutron source for medical use and its application to industry, Isotope News, 757, 22-25 (2018)

Patent application 2016-062865 Patent application 2017-61979 Patent application 2017-105099

The object of the present invention is to provide undesired cells such as teratoma, atypical cells, cancer cells (leukemia, malignant cells) mixed with radiation-sensitive stem cells having "stemness" used in the fields of transplantation medicine and regenerative medicine. Blood cancer cells such as lymphoma and multiple myeloma) are removed to provide a cell sorting method for safely performing such transplantation medicine and regenerative medicine, and the cells obtained by the method.

Means for Invention to Solve

"Stemness" necessary for transplantation medicine and regenerative medicine, without affecting the function of stem cells, "unfavorable cells such as teratoma, atypical cells, etc. Cancer cells (blood cancer cells such as leukemia, malignant lymphoma, and multiple myeloma) are treated with the Boron Neutron Capture reaction (BNCR), which has little effect on the radiosensitivity of stem cells, outside the patient's body. By applying it, we provide a method (Ex. Vivo BNCR) to remove them.
That is, the present invention relates to the following items.

(Item 1)
(1) culturing a non-target cell culture containing target cells under conditions in which boron ( ¹⁰ B) is specifically incorporated into the target cells, and (2) the boron ( ¹⁰ B) obtained in (1) A method of selectively killing non-target cells by irradiating the target cells into which the non-target cells have been taken up with a neutron beam that has little effect on non-target cells.
(Item 2)
The sorting method according to item 1, wherein in step (1), the cells are cultured under culture conditions in which the boron ( ¹⁰ B) drug is easily taken up by target cells and the boron ( ¹⁰ B) drug is difficultly taken up by non-target cells. .
(Item 3)
Item 1, 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 in order to selectively fractionate non-target cells. or the preparative method according to 2.
(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 sorting method according to any one of items 1 to 4, wherein the non-target cells are stem cells.
(Item 6)
The sorting method according to item 5, wherein the stem cells are pluripotent stem cells or hematopoietic stem cells.
(Item 7)
The sorting method according to item 6, wherein the pluripotent stem cells are human pluripotent stem cells.
(Item 8)
The sorting method according to item 6, wherein the pluripotent stem cells are human iPS cells.
(Item 9)
The sorting method according to item 6, wherein the hematopoietic stem cells are CD34-positive cells.
(Item 10)
10. The sorting method according to any one of items 1 to 9, wherein the target cells are one or more selected from the group consisting of heterologous cells, atypical cells and cancer cells.
(Item 11)
The sorting method according to any one of items 1 to 9, wherein the target cells are cancer cells.
(Item 12)
Any of items 1 to 11, wherein the boron ( ¹⁰ B) drug is a compound having a closo structure such as p-boronophenylalanine (BPA), mercaptoundecahydrodecaborate (BSH), or a mixture thereof. The preparative collection method according to item 1.
(Item 13)
The boron ( ¹⁰ B) drug is a complex 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, wherein the peptide is A6K or A6R. A fractionation method according to any one of items 1 to 11.
(Item 14)
14. The method of sorting according to any one of items 1 to 13, further comprising evaluating uptake of the boron ( ¹⁰ B) drug by target cells and non-target cells.
(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)
14. The sorting method according to any one of items 1 to 13, further comprising evaluating a boron ( ¹⁰ B) drug that has less effect on target cells and non-target cells.
(Item 17)
As a neutron irradiation field that has little effect on non-target cells, there is little contamination such as gamma rays, and the effect of fast neutrons (especially the hydrogen dose given to cells by neutrons reacting with hydrogen) is small. Maximum neutron energy is 50 eV or less. A preparative separation method according to item 3, characterized by a neutron moderator of
(Item 18)
As a neutron irradiation field that has little effect on non-target cells, the equipment that satisfies the standard value of the International Atomic Energy Agency (IAEA TECDOC-1223) and can generate low-energy epithermal neutrons (0.5ev-10keV) and the above-mentioned 4. A method of sorting according to item 3, characterized in that the water phantom is combined with a neutron irradiation field that has the effect of reducing the viability of the target cells.
(Item 19)
As a neutron irradiation field that has little effect on non-target cells, 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. A non-target cell culture containing target cells is irradiated with neutrons by moderating fast neutrons emitted from a plurality of small neutron generators installed on the side of the neutron moderator to 50 eV or less. Fractionation method as described.
(Item 20)
Non-target cells obtained by the sorting method according to any one of items 1 to 19.
(Item 21)
A pharmaceutical composition comprising the non-target cells of item 20.
(Item 22)
A preventive or therapeutic agent for cancer, comprising the non-target cell according to item 20.
(Item 23)
A preventive or therapeutic agent for blood cancer, comprising the non-target cell according to item 20.
(Item 24)
A method for preventing or treating cancer in a cancer patient, comprising administering an effective amount of the pharmaceutical composition according to item 21 to the patient.
(Item 25)
prior to anti-cancer treatment with boron neutron capture therapy (BNCT) for blood cancer patients, collecting hematopoietic stem cells from said patient;
A step of producing the collected hematopoietic stem cells by the sorting method according to any one of items 1 to 19;
a step of autotransplanting the hematopoietic stem cells obtained by the sorting method into the patient;
A method of treating blood cancers comprising:

Boron neutron capture reaction (BNCR) is utilized for sorting cells useful for regenerative medicine and cell therapy.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, are known to be highly radiation sensitive. For this reason, it is difficult to apply irradiation, which has been widely used in the past, to heterologous/mutant cells for the purpose of "removal". In this respect, if we can apply BNCR, which is a combination of a highly selective boron agent and neutrons with an extremely low contamination rate of gamma rays and high-energy neutrons, we will be able to provide a practical method that is almost unique.

If it is possible to selectively remove "cancer cells" that may be mixed with BNCR from the hematopoietic stem cells present in the bone marrow and the bone marrow graft itself, the scope of application of "autologous transplantation" may be greatly expanded. There is This is expected to increase the overall treatment options for blood cancers. In particular, in cases where the number of cancer cells remaining in the patient's body is zero or significantly reduced due to the improvement of the effect of "pretreatment for transplantation" by prescription of new drugs, latent cancer cells in autologous hematopoietic stem cells collected at the time of remission before treatment It cannot be ruled out that the amount of cancer may lead to recurrence or relapse after autologous transplantation. For myeloid hematologic cancers, for which autologous bone marrow transplantation or autologous hematopoietic stem cell transplantation is a promising treatment method, the creation of transplant materials by this method will provide an effective method.

We will realize a "paradigm shift" by expanding regenerative medicine, cell therapy, cancer treatment, for example, expanding the scope of "transplantation" in blood cancer treatment and improving selectivity. It has the potential to increase the number of cured patients and at the same time reduce the total medical costs, indicating a desirable direction for cancer treatment in Japan.

Ex. Vivo BNCT is a treatment method for blood cancers (leukemia, malignant lymphoma, multiple myeloma, etc.) that completely eliminates existing or latent cancer cells in the patient's body with high-dose chemotherapy or radiotherapy. (Transplant pretreatment) leads to safe autologous hematopoietic stem cell transplantation for the treatment of blood cancer. Alternatively, in regenerative medicine, removing cells that are at risk of becoming cancerous in the future, such as teratoma and atypical cells, will lead to safe regenerative medicine.

Radiation-sensitive cells with “stemness” are important biomaterials for regenerative medicine and cell therapy. There is no effective method, which is a major limiting factor. The present invention leads to fundamental solutions for regenerative medicine and cell therapy.

The method shown in the present invention can be applied to the treatment of hematologic cancers (malignant lymphoma, multiple myeloma, etc.). This is expected to improve the safety of "(autologous) hematopoietic stem cell" transplantation, which may lead to a radical cure.

Inventions in the chemical field related to regenerative medicine, cell therapy, and the provision of materials for these treatments will lead to new inventions and development of treatment methods in these medical fields. It is expected to shorten the period until approval and reduce costs.

Conventionally, transplantation of hematopoietic tissue (bone marrow, hematopoietic stem cells, etc.) has been recommended by the Japanese Society for Hematopoietic Cell Transplantation, etc., as shown in the guidelines. (e.g., tumor). However, the hematopoietic tissue transplantation shown in the guidelines only allows allogeneic transplantation, and autologous transplantation using the patient's own hematopoietic tissue is not assumed. This is because autologous transplantation involves transplanting cancer cells present in the hematopoietic tissue into the patient, which may cause the cancer to recur. Therefore, whether or not a patient can be rescued depends on whether or not a transplant donor (donor) can be secured, and the number of patients that can be treated is also limited.

According to the present invention, before performing anticancer drug treatment, by applying boron neutron capture therapy to hematopoietic stem cells such as bone marrow taken out of the patient's body, cancer cells are expelled and hematopoiesis A hematopoietic tissue that maintains stem cell function can be obtained. The use of such hematopoietic tissue enables autologous transplantation that avoids reintroduction of cancer cells. As a result, hematological cancer can be treated without being restricted to allotransplantation, which requires the securing of donors, and the number of rescued patients increases.

A comparison of the characteristics of accelerator neutron sources is shown. OKD particle measurement results after ultrafiltration are shown. FIG. 4 shows the results of measuring the concentration of boron incorporated into CD34-positive hematopoietic stem cells and multiple myeloma-derived cell lines when using cancer cell culture media. Fig. 3 shows the results of evaluating the intracellular translocation of BPA and BSH. The concept of dose delivered to cancer cells and normal cells is shown. Neutron energy dependence of the kerma coefficient for biological tissue is shown. γ-ray energy dependence of the γ-ray kerma coefficient. Photographs of the electrostatic accelerator system and the beamline are shown. Neutron moderation irradiation equipment and emitted neutron beam quality are shown. The energy distribution of neutrons emitted from a BSA nozzle is shown. A neutron moderator for stem cell irradiation is shown. Neutron moderator structure for stem cell irradiation is shown. Various dose distributions in the neutron moderator for stem cell irradiation (light water moderator) are shown. Various dose distributions in the neutron moderator for stem cell irradiation (light water moderator + lead shielding) are shown. Various dose distributions in the neutron moderator for stem cell irradiation (heavy water moderator) are shown. The relational expression between the neutron irradiation time and the cell viability of cancer cells (MM cells) and normal cells (hematopoietic stem cells) after boron drug (OKD) treatment is shown.

In one embodiment of the invention,
(1) culturing a non-target cell culture containing target cells in the presence of a boron ( ¹⁰ B) ^drug ; A method for sorting non-target cells is provided, comprising the step of selectively sorting non-target cells by irradiating with radiation.
In step (1), the cells are preferably cultured under culture conditions that favor the uptake of the boron ( ¹⁰ B) drug into the target cells. In step (1), culture conditions that facilitate incorporation of the boron ( ¹⁰ B) drug into the target cells include, for example, optimization of the boron drug, type of cell culture medium, culture temperature, carbon dioxide gas concentration, and the like.
Further, in step (2), in order to selectively fractionate non-target cells, the maximum energy of the neutron beam to be irradiated is 50 eV or less, and the neutron irradiation field is configured to suppress γ rays mixed in the neutrons. is preferably used.

A target cell means a cell to be removed, and includes, for example, “undesirable” heterologous/atypical cells such as cancerous cells mixed in cells used as cell preparations.

A non-target cell means a cell to be sorted, and can be exemplified by a cell having stemness and high radiosensitivity.

Examples of highly radiosensitive cells with stemness include pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, etc.) or multipotent stem cells (differentiated multipotent stem cells, bone: MUSE cells and mesenchymal stem cells). Stem cells and hematopoietic stem cells) or normal stem cells differentiated from hematopoietic stem cells (Radiation Biology Research Communications 54(4), 266-280, 2019).

Pluripotent stem cells used in the present invention include, for example, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and embryos derived from cloned embryos obtained by nuclear transfer. stem cells (nuclear transfer Embryonic stem cells: ntES cells), multipotent germline stem cells ("mGS cells"), embryonic germline stem cells (EG cells), Muse cells (multi-lineage differentiating stress enduring cells) ), preferably iPS cells (more preferably human iPS cells). When the above-mentioned pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be cells produced by destroying embryos or those produced without destroying embryos. Although it may be, preferably the cell is produced without destroying the embryo.

Hematopoietic stem cells reside primarily in the bone marrow. Hematopoietic stem cells differentiate into all blood cell lineages in the bone marrow and develop into red blood cells, white blood cells and platelets. Hematopoietic stem cells can also proliferate by cell division and self-renew. Cells for autologous transplantation used in the present invention contain hematopoietic stem cells, and are preferably collected from the bone marrow of cancer patients. The cancer patient is a patient with blood cancer such as leukemia, malignant lymphoma, and multiple myeloma, who is a target for hematopoietic stem cell transplantation. Bone marrow collection from a patient is performed, for example, according to the method of bone marrow collection published by the Japan Society for Hematopoietic Cell Transplantation.

CD34 is a surface antigen that penetrates cell membranes, and is expressed on bone marrow and peripheral blood hematopoietic stem cells, vascular endothelial progenitor cells, skeletal muscle satellite cells, epithelial hair follicle stem cells, adipose tissue mesenchymal stem cells, and the like. That is, CD34 is a surface marker for various somatic stem cells, and cells expressing CD34 have the ability to differentiate into all blood cells and endothelial cells, but they disappear as the cells mature and differentiation progresses. Therefore, hematopoietic stem cells used in the present invention are preferably CD34-positive cells. Whether or not the cells to be used are CD34-positive can be confirmed by, for example, a flow cytometer.

In Ex. Vivo BNCR, when applying BNCR to radiation-sensitive cells, (1) boron atoms ( ¹⁰ B) are selectively and effectively applied to "target heterogeneous/atypical cells" including undifferentiated cells. It is carried out by combining a "boron drug" to be taken in, and (2) a "neutron beam source" that stably generates epithermal neutrons of 50 eV or less with little radiation such as gamma rays.

Boron neutron capture therapy (BNCT) is a method of selectively killing cancer cells by introducing boron isotopes ( ¹⁰ B) into cancer cells and irradiating them with neutron beams. In the present invention, the step of culturing cells for autologous transplantation in the ^presence of a boron ( ¹⁰ B) drug means, for example, autologous transplantation cells containing hematopoietic stem cells collected from the bone marrow of a cancer patient Culturing in the presence of a drug to introduce a sufficient amount of the boron drug into the cells. As the boron ( ¹⁰ B) drug, boron compounds having a closo structure such as p-boronophenylalanine (BPA), mercaptoundecahydrodecaborate (BSH), and mixtures thereof can be used.

BPA is an amino acid derivative containing one boron atom in the molecule, and is taken up into cells by amino acid transporters present in cell membranes. On the other hand, BSH is a crystal containing 12 boron atoms in the molecule. BPA and BSH are known compounds. A boron compound having a closo structure such as BSH may be in a free form or in a salt form. Salts of boron compounds having a closo structure such as BSH include, but are not limited to, sodium salts, ammonium salts, tetramethylammonium salts, and the like. In addition, boron compounds having a closo structure such as BSH may be modified or derivatized. Modified and derivatized BSH are known and include peptide-conjugated BSH, sugar-conjugated BSH, thiol groups, hydroxyl groups, carboxyl groups, amino groups, amide groups, azido groups, halogen groups and phosphate groups. are exemplified, but not limited to. Methods for making modified BSH and derivatized BSH are known.

Boron compounds with a closo structure, such as BSH, have low cell membrane permeability, so they may not be fully taken up into cells as they are. can. When the intracellular uptake of BSH is low, BSH can be absorbed into cells by complexing 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. Uptake can be increased.

Examples of peptides containing the hydrophobic amino acid residue and the basic amino acid residue include AAAAAK, AAAAAAK, AAAAAAK, AAAAAKK, AAAAAKK, AAAAAAKK, AAAAAAR, AAAAAAR, AAAAAAR, AAAAARR, AAAAAAARR, and AAAAAAARR. More preferably, the peptide includes AAAAA-homoarginine, AAAAA-ornithine, AAAAA-2,7-diaminoheptanoic acid, AAAAA-2,4-diaminobutanoic acid, AAAAA-2-amino-4-guanidinobutanoic acid, and the like. and more preferably AAAAAAK (abbreviated as A6K) and AAAAAAR (abbreviated as A6R), but are not limited thereto. The peptides described above are indicated by conventional one-letter amino acid symbols. Therefore, A represents alanine, K represents lysine, and R represents arginine.

Said peptides for use in the invention may be in free form, in salt form, in solvate form, or may be modified or derivatized. Various salts of peptides are known, and methods for producing them are also known. Examples of salts of peptides include hydrochloride, sulfate, nitrate, phosphate, acetate, trifluoroacetate (TFA salt), citrate, succinate, maleate, fumarate, malate. Salts, tartrates, p-toluenesulfonates, benzenesulfonates, methanesulfonates, alkali metal salts, alkaline earth metal salts, and the like, but are not limited to these. Peptide solvates are also known, and methods for their preparation are also known. Examples of solvates of peptides include, but are not limited to, solvates of water, methanol, ethanol, isopropanol, tetrahydrofuran, dimethylsulfoxide, ethylene glycol, propylene glycol, acetamide, and the like. Various modified peptides and peptide derivatives are known. Methods for peptide modification and derivatization are also known. Examples of peptide modifications and derivatizations include acetylation, amidation, biotinylation, maleimidation, alkylation such as methylation, maleimidation, myristoylation, esterification, phosphorylation, fluorescent labeling, radiolabeling, etc. Examples include, but are not limited to, labeling. Preferably, the N-terminus of the peptide is acetylated. In addition, amino acids that constitute peptides may be L-isomers or D-isomers as optical isomers. Furthermore, one peptide molecule may contain L- and D-amino acids. In the present invention, the term "peptide" includes modified peptides, derivatized peptides, salt-form peptides, and peptides containing D-amino acids as described above.

The complex 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, which is used in the present invention, is obtained by combining a boron compound having a closo structure such as BSH with the peptide in an aqueous solution. It can be prepared by mixing Stirring or sonication may be used during mixing to facilitate dispersion and complex formation. In an aqueous solution in which a boron compound having a closo structure such as BSH is mixed with the peptide, the concentration of the peptide 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 and BSH when mixed is not particularly limited, but the mixing ratio is preferably about 1 mol to about 1000 mol of BSH per 1 mol of peptide, for example, about 1 mol of peptide and about 1000 mol of BSH. A ratio of 1 to about 100 moles, a ratio of about 100 to about 1000 moles of BSH to 1 mole of peptide, and the like.

The aqueous solution in which the boron compound having a closo structure such as BSH and the peptide are mixed is a solution containing water as a medium. The aqueous solution may be water only, or may have other substances added, such as buffers and salts. A person skilled in the art can appropriately set conditions such as the temperature, pH, and mixing time during the mixing. For example, when BSH is mixed with A6K or A6R, it is preferable to mix under conditions such that the lysine residue of A6K or the arginine residue of A6R has a positive charge and BSH has a negative charge. Buffers such as phosphate buffered saline may be used to bring the pH of the aqueous solution to the desired value.

The complex 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 has a spherical shape with horns on the surface or a spherical shape without projections. A spherical shape includes not only a perfect sphere but also a substantially spherical shape. Specifically, if the ratio of the minor axis to the major axis is about 0.5 or more, preferably about 0.6 or more, and more preferably about 0.7 or more, it can be said to be spherical. The particle size and particle size distribution of the particles formed from the composite can be measured by dynamic light scattering (DLS). The particle size of the complex can be adjusted by adjusting the mixing ratio of the boron compound having a closo structure such as BSH and the peptide during preparation of the complex. A low ratio of BSH to the peptides to be mixed gives rise to smaller diameter complexes, and a high ratio of BSH to the peptides gives rise to larger diameter complexes.

In one embodiment of the present invention, there is further provided a method for sorting non-target cells comprising assessing boron ( ¹⁰ B) drug uptake by target cells and non-target cells.

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

In one embodiment of the present invention, there is further provided a method for sorting non-target cells, comprising evaluating a boron ( ¹⁰ B) drug that has less effect on target cells and non-target cells.

In one embodiment of the present invention, as a neutron irradiation field that has little effect on non-target cells, there is little contamination such as gamma rays, and the influence of fast neutrons (especially, the hydrogen dose given to cells by the reaction of neutrons with hydrogen) is reduced. Provided is a method for sorting non-target cells, characterized in that the maximum neutron energy to be reduced is 50 eV or less.

In one embodiment of the present invention, the neutron irradiation field that has little effect on non-target cells satisfies the International Atomic Energy Agency (IAEA) standard (IAEA TECDOC-1223) and uses low-energy epithermal neutrons (0.5ev- 10 keV) and a water phantom are combined with a neutron irradiation field having the effect of reducing the viability of target cells.

In one embodiment of the present invention, as a neutron irradiation field that has little effect on non-target cells, a cylindrical neutron moderator (water, heavy water, Be, C, etc.) with a radius of 5 cm or more Non-target cells containing target cells at the center of the axis A culture is installed, and fast neutrons emitted from a small neutron generator installed multiple times on the side of a cylindrical neutron moderator are moderated to 50 eV or less, and neutrons are irradiated to a non-target cell culture including target cells. To provide a method for sorting non-target cells, characterized by

In the step of culturing the cells for autologous transplantation in the presence of the boron ( ¹⁰ B) drug, the type of culture medium, the culture time, the concentration of the boron ( ¹⁰ B) drug added to the culture medium, and the cell density in the culture medium Such culture conditions can be determined by the intracellular uptake amount and intracellular distribution of the boron ( ¹⁰ B) drug. When the cells for autologous transplantation that have taken up a boron ( ¹⁰ B) drug such as BPA are irradiated with neutron beams, a nuclear reaction between boron and thermal neutrons occurs inside the cells, and particle beams (alpha rays and 7Li particles) damage cells. Therefore, cells with higher uptake of the boron ( ¹⁰ B) drug are more susceptible to cytotoxicity due to neutron beam irradiation. The culture conditions in the sorting method of the present invention minimize the incorporation of the boron ( ¹⁰ B) drug into non-target cells (e.g., normal cells), while minimizing the incorporation of the boron ( 10 B) drug into target cells (e.g., cancer cells). Boron ( ¹⁰ B) drug uptake is maximized.
As a method to reduce uptake into non-target cells,
・OKD-001 is used as a boron agent. The final concentration is A6K:BSH=0.2 mM/2 mM.
・Use RPMI1640 medium supplemented with 1% human serum albumin (HSA).
・Incubation temperature is 37℃
・Culture under 5% CO ₂ conditions.
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 are extinguished after neutron beam irradiation. On the other hand, normal cells exist in a viable state even after neutron beam irradiation, and preferably, there is little change in the number of viable normal cells before and after neutron beam irradiation. Neutron irradiation of the autologous transplant cells incorporating the boron ( ¹⁰ B) drug is performed using a nuclear reactor or an accelerator type neutron generator, while examining cytotoxicity to cancer cells and normal cells, neutron dose, neutron spectrum , irradiation time, irradiation angle, etc. can be determined.

In one embodiment of the present invention, non-target cells obtained by the sorting method of the present invention are provided. The cells are, for example, cells for autologous transplantation, and are provided in a state of being suspended in a culture medium or the like, or in a state of being frozen in a cell cryopreservation medium. Examples of the cell cryopreservation medium include cell culture medium containing 10% dimethylsulfoxide and 10% serum, Cellbanker (registered trademark, Takara Bio), and the like.

In one embodiment of the present invention, pharmaceutical compositions containing non-target cells obtained by the sorting method of the present invention are provided.

A pharmaceutical composition containing the non-target cells of the present invention as an active ingredient can be used to treat cancer patients. The pharmaceutical composition of the present invention can be produced by a method commonly used in the field of formulation technology, such as the method described in the Japanese Pharmacopoeia. The pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of such additives include cell culture media, physiological saline, and suitable buffers (eg, phosphate buffers).

The pharmaceutical composition of the present invention can be produced by suspending non-target cells in physiological saline, an appropriate buffer (eg, phosphate buffer), or the like. It is preferable to contain, for example, 1×10 ⁷ cells or more in a single dose so as to achieve the desired therapeutic effect. It is more preferably 1×10 ⁸ or more, still more preferably 1×10 ⁹ or more. The content of cells can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of cells, etc. of the subject. In addition to non-target cells, the pharmaceutical composition of the present invention may contain dimethylsulfoxide (DMSO), serum albumin and the like for the purpose of protecting cells. Antibiotics and the like may be added for the purpose of preventing bacterial contamination, and vitamins, cytokines, and the like may be included for the purpose of promoting activation and differentiation of cells. Furthermore, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) It may be contained in the pharmaceutical composition of the present invention.

A pharmaceutical composition containing the non-target cells of the present invention as an active ingredient can be cryopreserved. In the case of low-temperature cryopreservation, the temperature is not particularly limited as long as it is suitable for cell preservation. Examples include -20°C, -80°C and -120 to -196°C. For cryopreservation, cells can be stored in suitable containers such as vials. Manipulations to minimize the risk of cell damage during freezing and thawing of non-target cells are well known to those skilled in the art.

For cryopreservation of non-target cells, non-target cells are harvested from the culture medium, washed with buffer or culture medium, counted, concentrated by centrifugation, etc., and placed in a freezing medium (e.g., 10% DMSO After suspending it in the culture medium containing the cells, it is cryopreserved. Non-target cells can be combined into a single lot from cells cultured in multiple culture vessels. The pharmaceutical composition containing non-target cells of the present invention contains, for example, 5×10 ⁴ to 9×10 ¹⁰ non-target cells per container such as a vial. , route of administration, etc.

Examples of administration routes of the pharmaceutical composition containing the non-target cells of the present invention include infusion, intratumoral injection, arterial injection, portal vein injection, intraperitoneal administration and the like. However, the administration route is not limited to this as long as the non-target cells, which are the active ingredients in the pharmaceutical composition of the present invention, are delivered to the affected area. Dosage schedules can be single doses or multiple doses. For the period of multiple administrations, for example, a method of repeating administration once every 2 to 4 weeks, a method of repeating administration once every six months to a year, or the like can be adopted. In preparing the administration schedule, the sex, age, body weight, disease state, etc. of the target patient can be taken into consideration.

In one embodiment of the present invention, a preventive or therapeutic agent for cancer containing non-target cells obtained by the sorting method of the present invention is provided.

In one embodiment of the present invention, there is provided a preventive or therapeutic agent for blood cancer containing non-target cells obtained by the culture method of the present invention. Hematologic cancers include, but are not limited to, leukemia, malignant lymphoma, multiple myeloma, and the like.

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

In one embodiment of the present invention, a step of collecting hematopoietic stem cells from a blood cancer patient before anti-cancer treatment by boron neutron capture therapy (BNCT), and subjecting the collected hematopoietic stem cells to the fractionation method of the present invention. and autotransplanting the hematopoietic stem cells obtained by the sorting method into the patient.

In one embodiment of the present invention, a step of collecting hematopoietic stem cells from a blood cancer patient before anti-cancer treatment by boron neutron capture therapy (BNCT), and subjecting the collected hematopoietic stem cells to the fractionation method of the present invention. and autotransplanting an effective amount of the hematopoietic stem cells obtained by the fractionating method into a patient in need of hematologic cancer treatment.

The present invention will be described in greater detail and specificity with reference to the following examples, but the examples should not be construed as limiting the scope of the present invention.

<Target cells and boron drugs>
Combinations of boron agents [ ¹⁰ B agents] that are assumed to be applied in removing heterologous/mutant cells from radiation-sensitive cells having “stemness” by Ex. Vivo BNCR are shown in the table below.
Heterogeneous/mutated cells include cancer cells, hematologic cancers (multiple myeloma, malignant lymphoma, myeloid leukemia (AML, CML, etc.)), sarcoma, teratoma, and other neoplastic cells. pluripotent stem ^cellsa (including embryonic stem cells and induced pluripotent stem cells) and malformations that cause neurodegenerative diseases

Table 2 Combinations of boron agents [ ¹⁰ B agents] that are assumed to be applied when removing heterologous/mutant cells

<Production of new boron drug OKD>
Preparation of complexes containing BSH (tetramethylammonium salt) and A6K
Preparation of BSH aqueous solution
A weighed amount of BSH tetramethylammonium salt was transferred to a 15 mL tube. Water for injection (Otsuka distilled water, Otsuka Pharmaceutical Factory Co., Ltd.) was added thereto. The resulting solution was sonicated using an ultrasonic cleaner (CPX1800H-J, BRANSON). After that, the mixture was stirred by vortexing to confirm that the BSH tetramethylammonium salt was completely dissolved.
Preparation of A6K aqueous solution
A weighed amount of A6K hydrochloride was transferred to a 1.5 mL tube. Water for injection (Otsuka distilled water, Otsuka Pharmaceutical Factory Co., Ltd.) was added thereto. The resulting solution was subjected to ultrasonic treatment using an ultrasonic cleaner (AUC-1L, AS ONE Corporation). After that, it was confirmed that the A6K hydrochloride was completely dissolved by stirring with a vortex.
Mixing BSH and A6K
A6K aqueous solution, BSH aqueous solution, and water for injection (Otsuka distilled water, Otsuka Pharmaceutical Factory, Inc.) were mixed in a 1.5 mL tube 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 mixed liquid was a colorless and transparent aqueous solution. Analysis by dynamic light scattering (DLS) confirmed the presence of a complex of BSH and A6K as particles in the aqueous solution. DLS was measured three times at a temperature of 25° C., equilibrium of 60 seconds, and 173 Å, and was used as a general-purpose model (actual measurement time of 70 seconds, count rate of 192.6 kcps, measurement position of 3.00 mm, attenuator of 7). Most of the composite particles had a particle size in the range of 1000 to 6000 nm, with a peak around 2000 nm in particle size.

Boron drug treatment to cancer cells and normal cells <OKD cell uptake test>

Preparation of conjugates containing BSH and A6K and cell uptake studies
Preparation of BSH complex using BSH (sodium salt) aqueous solution and A6K peptide aqueous solution
A BSH aqueous solution and an A6K aqueous solution were each dissolved in water for injection (Otsuka) and prepared. A BSH aqueous solution, an A6K aqueous solution, and water for injection were mixed to prepare a BSH complex (BSH:A6K=20 mM:2 mM). Dynamic light scattering (DLS) showed peaks similar to those exhibited by a complex consisting of BSH (tetramethylammonium salt) and A6K.

Preparation of drug by adding stabilizer to BSH complex aqueous solution consisting of BSH and A6K peptide
A BSH aqueous solution and an A6K aqueous solution were each dissolved in water for injection (Otsuka) and prepared. A BSH aqueous solution, an A6K aqueous solution, and water for injection were mixed to prepare a BSH complex (BSH:A6K=20 mM:2 mM). A suitable amount of stabilizer or dispersant was added to this aqueous solution to prepare a drug.
As a stabilizer or dispersant, OKD-001 after preparation is diluted 5-fold with 1% human serum albumin (HSA) (BSH: A6K = 4 mM: 0.4 mM, 0.8% HSA) and is stable at 4°C. and saved. In this case, OKD-001 represents a BSH complex (BSH:A6K=20 mM:2 mM).
In addition, OKD-001 was diluted 5-fold with ultrapure water immediately after preparation, followed by ultrafiltration (Amicon Ultra, regenerated
By desalting the solvent and concentrating the diluted solution with a cellulose membrane, it was confirmed that it could be stably stored at 4°C. FIG. 2 shows the results of DLS measurement of the OKD aqueous solution after ultrafiltration. The average particle diameter was 385.9 nm, and it was stably dispersed at 4°C even one week after preparation.

Cell Seeding and Drug Exposure On the day of drug addition, each cell was seeded in a 6-well plate at 3×10 ⁵ cells/well in a medium volume of 2.7 ml/well. RPMI1640 10% FBS, P/S was used as the medium. 300 μl/well of the aforementioned BSH complex aqueous solution (final concentration: BSH:A6K=2 mM:0.2 mM) was added to the cells seeded on the plate.

Collection of cells, elastase treatment, addition of boron quantification agents 24 hours later, cells were collected in a 15 ml tube and treated with elastase (Sigma) to remove extracellular complexes. After washing with PBS, cell numbers were counted. The 2 ml tube into which the PBS-suspended cells were transferred was centrifuged, the supernatant was removed, and the cell pellet was cryopreserved at -20°C. The cryopreserved cell pellet was suspended and dissolved in 300 μl of nitric acid, subjected to thermal nitric decomposition, diluted 20-fold, and measured for boron concentration by ICP-MS (Agilent 7500cx).

FIG. 3 shows the amount of boron uptake in each cell as a measured value (B ng) per 10 ⁶ cells. Each treatment plot was tested in triplicate (n=3), and the mean and standard deviation are shown in the graph.
ICP-MS was used to measure the uptake of 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). It was confirmed that the boron ( ¹⁰ B) concentration ratio taken up by the cells was large (Fig. 3).
RPMI8226/CD34+: 1700pm/70ppm
IM-9/CD34+: 3000pm/70ppm
This suggests that, for example, BNCR can be used to remove cancer cells at risk of contamination during autologous hematopoietic stem cell transplantation in the treatment of multiple myeloma. That is, cancer cells can be killed by irradiating cells exposed to boron agents with 1 to 2×10 ¹⁰ neutrons·cm ⁻² neutrons.

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

Cell recovery and boron quantification 24 hours after the addition of the drug, the cells were recovered in a 15 ml tube, subjected to 300×g for 5 minutes, and then the supernatant was removed. Resuspend in 4 ml PBS and centrifuge again. After removing the PBS from the supernatant, the cells were resuspended in 1 ml of PBS and transferred to a 2 ml tube. From the cell suspension transferred to a 2-ml tube, 5 μl of each treatment group was collectively collected in a 1.5-ml tube (total of 15 μl for each treatment group), mixed and counted as the average cell number.
The 2 ml tube into which the PBS-suspended cells were transferred was centrifuged, the supernatant was removed, and the cell pellet was cryopreserved at -20°C. The cryopreserved cell pellet was suspended and dissolved in 300 μl of nitric acid, subjected to thermal nitric decomposition, diluted 20-fold, and measured for boron concentration by ICP-MS (Agilent 7500cx). The amount of boron in each treatment group was shown as the measured value (B ng) per 10 ⁶ cells. Each treatment plot was tested in triplicate (n=3), and the mean and standard deviation are shown in the graph. Cancer cells can be killed by irradiating cells exposed to boron agents with 1 to 2×10 ¹¹ neutrons·cm ⁻² neutrons.

Cells used in experiments
RPMI8226 cells: peripheral blood-derived cell line of human multiple myeloma patients
IM-9 cells: human multiple myeloma and B lymphoblastic leukemia (myeloma, multiple, B lymphoblastic) patient bone marrow-derived cell line Human CD34-positive hematopoietic stem cells: human bone marrow-derived (Lonza)

FIG. 4 shows the results of evaluating the intracellular translocation properties of BPA and BSH. Boron drugs BPA and BSH showed higher boron uptake into two hematologic cancer (multiple myeloma: MM) cells (RPMI-8226 and IM-9) compared to hematopoietic stem cells (indicated by CD34+). confirmed (Fig. 4).
This suggests that BNCR, which is a combination of these boron agents and neutron beams, can be used to remove cancer cells that are at risk of contamination during autologous hematopoietic stem cell transplantation in the treatment of multiple myeloma, for example. That is, cancer cells can be killed by irradiating cells exposed to boron agents with 1 to 2×10 ¹⁰ neutrons·cm ⁻² neutrons.

Neutron beam irradiation (simulation test using water phantom)
<Neutron irradiation field>
Stem cells are highly radiosensitive, and for "hematopoietic stem cells (stem/progenitor cells)" in the bone marrow, the radiosensitivity _D0 is reported to be "0.8-1.2 Gy" (see table below). In this case, when a dose of about 1 Gy is given to hematopoietic stem cells, the survival rate of stem cells decreases to 0.34. It is preferred that the dose of radiation applied to the is not strong.

Table 3 Radiosensitivity of various cells in bone marrow

中性子をがん細胞および幹細胞（通常細胞）に照射すると、中性子線量Dn、γ線線量Dγは両細胞に等量で、ホウ素濃度に比例したホウ素線量D_Bが付与される。
幹細胞を残してがん細胞を殺傷するためには、（i）がん細胞と幹細胞間のホウ素線量D_Bの差を大きくすると共に、(ii)中性子線量Dn、γ線線量Dγを極力少なくすることが必須となる。 Neutron fields suitable for Ex. Vivo BNCT are discussed below.
In BNCR, the radiation dose D(x) received by tumor or normal cells is the dose Dn(x) due to neutron beams, the dose Dγ(x) due to gamma rays, and the dose D _B given when boron undergoes nuclear fission by thermal neutrons. It can be expressed by the sum of (x) (the following formula). The neutron dose Dn at the location x is determined by the product of the neutron energy distribution Φn, the neutron kerma coefficient Kn, and the neutron beam quality coefficient RBE _n . The γ-ray dose Dγ due to the γ-rays contained in the neutrons is determined by the product of the γ-ray energy distribution Φγ, the γ-ray kerma coefficient Kγ, and the radiation quality coefficient RBE _γ (=1.0). The boron dose D _B is obtained by multiplying the neutron energy distribution Φn, the boron kerma coefficient K _B and the boron quality coefficient RBE _B . _CB is the boron concentration (ppm) accumulated in the cell.

When cancer cells and stem cells (normal cells) are irradiated with neutrons, the neutron dose Dn and the γ-ray dose Dγ are equal to both cells, and the boron dose D _B is given in proportion to the boron concentration.
To kill cancer cells while leaving stem cells, (i) increase the difference in boron dose D _B between cancer cells and stem cells, and (ii) minimize neutron dose Dn and γ-ray dose Dγ. is essential.

FIG. 5 shows the concept of dose given to cancer cells and normal cells.
Looking at the energy dependence of the kerma coefficient Kn(E)* for living tissue, which is the factor that determines the neutron dose Dn, when the neutron energy is 50 eV or less, the ionization of nitrogen elements contributes greatly, and when the neutron energy is 50 eV or more, the recoil energy of hydrogen elements It turns out that the grant is large. For this reason, it is preferable to irradiate neutrons having an energy distribution of 50 eV or less in order to reduce the neutron dose Dn.
(*Kerma coefficient: "Kinematic energy dE of charged particles generated by indirectly ionized particles" in "substance dm per unit weight" of interest. K = dE/dm.)
In order to reduce the contribution of the γ-ray dose Dγ, the main means is to reduce the intensity of γ-rays emitted from the BSA. For this purpose, an accelerator neutron source using a Li target is preferred.

FIG. 6 shows the neutron energy dependence of the Kerma coefficient for biological tissue.
FIG. 7 shows the γ-ray energy dependence of the γ-ray kerma coefficient.

The figure below shows an example of a system that combines a Li target and an electrostatic accelerator (proton energy: 2.8MeV to 1.9MeV) as an accelerator neutron source. Since the system has a low maximum beam energy of 2.8 MeV, activation of the accelerator itself and the beam line by protons hardly occurs, and maintenance and repair of the accelerator can be performed safely and quickly. It has the advantage of being cheaper. On the other hand, Li is chemically active ^and mechanically weak, and there are issues such as the generation of radioactive ^7Be in Li as a result of neutron generation. Li-filled target), enabling stable operation and maintenance. FIG. 8 shows the appearance of the electrostatic accelerator system and the beamline.

A slow neutron irradiation apparatus (Beam Shaping Assembly: BSA) is used to slow down the high-speed neutrons generated by the target to the exothermic energy range (0.5 eV to 10 keV) suitable for cancer treatment such as brain tumors and head and neck. ing. The figure below shows the energy spectrum of emitted neutrons. The radiation quality of epithermal neutrons emitted from this BSA conforms to the characteristics recommended by the IAEA, so fast neutrons of 10 keV or higher, which have a large effect on normal tissue, and gamma rays contained in neutrons are low. Although it has characteristics, it does not satisfy the neutron energy "50 eV or less" suitable for stem cell irradiation. FIG. 9 shows the neutron moderation irradiation device and the emitted neutron beam quality.

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. The figure below shows the results of evaluation by simulation analysis PHITS (nuclear data JENDEL-4). (Moderator dimensions: 20cm x 20cmx20cm, acrylic wall thickness 3mm)
(1) Light water moderator Light water (H ₂ O) is most suitable for moderating neutrons because it contains H which has the same mass as neutrons. On the other hand, gamma rays are produced.
(2) The system of light water moderator and gamma ray lead shielding (1), where γ rays are shielded by lead.
(Reference) Heavy water moderator Neutrons can be moderated by using heavy water (D ₂ O), which does not generate gamma rays during moderation.

FIG. 11 shows a neutron moderator for stem cell irradiation.
FIG. 12 shows the neutron moderator structure for stem cell irradiation.

FIG. 13 shows an example of dose distribution in the neutron moderator for stem cell irradiation. The vertical axis is the dose (Gy-eq/mA/sec) obtained by PHITS analysis, and the expected dose (Gy-eq) given to the cell can be evaluated by multiplying the beam current (maximum 2 mA) and the irradiation time. When epithermal neutrons enter the moderator from the exit of the BSA, they react with nitrogen and hydrogen in the body to give "nitrogen dose", "hydrogen dose", and "γ-ray dose" by γ-rays to all cells in the body. be. Since B-10 accumulated in cancer cells undergoes nuclear fission by thermal neutrons, it imparts a "boron dose" only to cancer cells. Ex. When only cancer cells are removed with Ex. Vivo BNCR, the dose to the hematopoietic stem cells is kept below a certain level (1 Gy-Eq or below), while the dose at the cancer-affected area is above a certain standard (e.g., 30 Gy or above). It is preferable to set the neutron irradiation conditions as follows.

When light water is used as a neutron moderator for stem cell irradiation, neutron components of 50 eV or more are sufficiently reduced when passing through about 4 cm of light water, and the "hydrogen dose D _H " becomes small. Although the "γ-ray dose" from the BSA outlet is small, the dose given by γ-rays increases when neutrons collide with light water (H) and emit γ-rays. When the proton beam current is 2mA for 40 minutes, nitrogen dose (3.8E-5 Gy-Eq/mA/sx 2 mA x 40x60 seconds), hydrogen dose (4E-6 Gy-Eq/mA) at 4cm from the moderator surface /sx 2 mA x 40x60 s), gamma-ray dose (6.5E-5 Gy-Eq/mA/sx 2 mA x 40x60 s), and a total non-boron dose estimated at 0.5 Gy-eq. The concentration of boron accumulated in cancer cells varies depending on the boron drug used and the type of cancer cell, but when 1500 ppm of boron is taken into a cell located 4 cm from the body surface, the "boron dose" is 165 Gy-eq. (extrapolated from 9.6 Gy-eq at 87.5 ppm boron concentration). There is a possibility that hematopoietic stem cells also take up a certain percentage of boron drugs, and if hematopoietic stem cells take up 5 ppm or 50 ppm of boron, the "boron dose" will be 0.5 Gy-Eq and 5 Gy-eq, respectively. If a boron drug that is less likely to be taken up by hematopoietic stem cells compared to cancer cells can be used, and in combination with a neutron moderator using light water, cancer cells can be removed with less effect on hematopoietic stem cells. There is expected.

Placing gamma-ray shielding lead (1 cm thick) around the container containing hematopoietic stem cells can reduce the gamma-ray dose by about half, but at the same time the boron dose is also reduced. ratio (Figs. 13 and 14, right figures) does not show much improvement. However, since the boron dose can be more uniform between 2 and 6 cm than when there is no lead shield, there is an advantage that a uniform boron dose can be given to a thick bone marrow bag. Furthermore, by reducing the thickness of light water from 20 cm to 10 cm, the amount of γ-rays can be reduced.

When heavy water is used to moderate neutrons, the amount of gamma rays generated by collisions with H can be reduced. be. These issues can be resolved by performing boron dose calculations. That is, in cells (non-target cells) that do not contain much boron agents, if the boron dose with neutrons (thermal neutrons, epithermal neutrons) from the accelerator-type neutron generator shown in this patent is calculated as a limited effect , can be directly irradiated without using such a moderator. It is thought that the influence of gamma rays generated due to irradiated neutron beams can also be regarded as limited. Even in cells containing a large amount of boron drugs (target cells), the effects can be inferred from the boron dose. In this way, appropriate selection of conditions such as irradiation according to the combination of target cells and non-target cells leads to a solution.

Neutron beam irradiation test on cancer cells and normal hematopoietic stem cells after boron drug OKD treatment

<Confirmation of BNCR effect>
The results of confirming the BNCR effect by neutron irradiation are summarized below.
ICP-MS was used to measure the uptake of the new boron drug OKD-001 into autologous hematopoietic stem cell model cells (CD34+ cells) and blood cancer cells (2 types of multiple myeloma model cells: IM-9 and RPMI8226). It was confirmed that cancer cell accumulation was high due to the high boron ( ¹⁰ B) concentration ratio taken up.
RPMI8226/CD34+: 1700pm/70ppm
IM-9/CD34+: 3000pm/70ppm

Cells with boron uptake at the concentration shown in Fig. 3 are placed in a neutron moderator for stem cell irradiation and irradiated with neutrons (thermal neutron flux: 1x10 ⁸ n/cm ² /s) for 40 minutes, three days after irradiation. Eye cell viability was assessed by spectrophotometric (WST-8) measurements. In light water blood cancer cells (MM model cell IM-9: high expression of the mechanism that takes up the boron drug (denoted as OKD) used)), BNCR (α-decay reaction) occurred due to neutron irradiation, and the cancer cells were significantly damaged. It turns out. This made it possible to remove target cells (MM model cells in this case) and separate non-target cells (autologous hematopoietic stem cell model cells in this case).

Fig. 16 shows the relationship between the neutron beam irradiation time and the cell survival rate for cancer cells (MM cells), which are target cells, and normal cells (hematopoietic stem cells), which are non-target cells, after boron drug (OKD) treatment. It is The two relational expressions diverge greatly, which proves the concept of this patent.
FIG. 16 shows changes in neutron beam irradiation time and cell viability of target cells (MM cells) and non-target cells (hematopoietic stem cells) by neutron beam irradiation in the presence of a drug (OKD-001).
The cell viability of target cells and non-target cells increased with increasing irradiation time.
OKD-001 represents a boron drug that is a complex of BSH and peptide A6K.

Claims (25)

(1) culturing a non-target cell culture containing target cells under conditions in which boron ( ¹⁰ B) is specifically incorporated into the target cells, and (2) the boron ( ¹⁰ B) obtained in (1) A method of selectively killing non-target cells by irradiating the target cells into which the non-target cells have been taken up with a neutron beam that has little effect on non-target cells. The fractionation according to claim 1, wherein in step (1), the cells are cultured under culture conditions in which the boron ( ¹⁰ B) drug is easily taken up by target cells and the boron ( ¹⁰ B) drug is difficultly taken up by non-target cells. Method. A neutron irradiation field configured such that the maximum energy of the neutron beam to be irradiated is 50 eV or less is used in step (2) to selectively separate non-target cells. 3. The preparative collection method according to 1 or 2. 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. The sorting method according to any one of claims 1 to 4, wherein the non-target cells are stem cells. The sorting method according to claim 5, wherein the stem cells are pluripotent stem cells or hematopoietic stem cells. The sorting method according to claim 6, wherein the pluripotent stem cells are human pluripotent stem cells. The sorting method according to claim 6, wherein the pluripotent stem cells are human iPS cells. The sorting method according to claim 6, wherein the hematopoietic stem cells are CD34-positive cells. The sorting method according to any one of claims 1 to 9, wherein the target cells are one or more selected from the group consisting of heterologous cells, atypical cells and cancer cells. The sorting method according to any one of claims 1 to 9, wherein the target cells are cancer cells. The preparative method according to any one of claims 1 to 11, wherein the boron ( ¹⁰ B) drug is p-boronophenylalanine (BPA), mercaptoundecahydrodecaborate (BSH) or a mixture thereof. . 12. Any of claims 1-11, wherein said boron ( ¹⁰ B) drug is a conjugate of BSH and a peptide comprising hydrophobic and basic amino acid residues, said peptide being A6K or A6R. The preparative collection method according to item 1. 14. The method of sorting according to any one of claims 1 to 13, further comprising assessing boron ( ¹⁰ B) drug uptake by target cells and non-target cells. The sorting method according to any one of claims 1 to 13, further comprising a step of isolating target cells. The sorting method according to any one of claims 1 to 13, further comprising evaluating a boron ( ¹⁰ B) drug that has less effect on target cells and non-target cells. As a neutron irradiation field that has little effect on non-target cells, there is little contamination such as gamma rays, the effect of fast neutrons (especially the hydrogen dose given to cells by neutrons reacting with hydrogen) is small, and the maximum neutron energy is 50 eV. The fractionation method according to claim 3, characterized by the following. As a neutron irradiation field that has little effect on non-target cells, a device that satisfies the standards of the International Atomic Energy Agency (IAEA) (IAEA TECDOC-1223) and can generate low-energy epithermal neutrons (0.5ev-10keV) and water 4. The sorting method according to claim 3, characterized in that the phantom is combined with a neutron field that has the effect of reducing the viability of target cells. As a neutron irradiation field that has little effect on non-target cells, 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. A non-target cell culture containing target cells is irradiated with neutrons by moderating fast neutrons to 50 eV or less emitted from a plurality of small neutron generators installed beside the neutron moderator. The fractionation method described in . Non-target cells obtained by the sorting method according to any one of claims 1 to 19. A pharmaceutical composition comprising the non-target cells of claim 20. A preventive or therapeutic agent for cancer, comprising the non-target cell according to claim 20. A preventive or therapeutic agent for blood cancer, comprising the non-target cell according to claim 20. A method for preventing or treating cancer in a cancer patient, which comprises administering an effective amount of the pharmaceutical composition according to claim 21 to the patient. collecting hematopoietic stem cells from a blood cancer patient prior to anti-cancer treatment with boron neutron capture therapy (BNCT) for said patient;
A step of producing the collected hematopoietic stem cells by the sorting method according to any one of claims 1 to 19;
a step of autotransplanting the hematopoietic stem cells obtained by the sorting method into the patient;
A method of treating blood cancers comprising:

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