WO2019107431A1

WO2019107431A1 - Haploidentical transplantation enhancer

Info

Publication number: WO2019107431A1
Application number: PCT/JP2018/043819
Authority: WO
Inventors: 賢市松岡; 真中村; 嘉信前田; 絵美川口; 秀俊秋元; 保之石井
Original assignee: 株式会社レグイミューン; 国立大学法人岡山大学
Priority date: 2017-11-28
Filing date: 2018-11-28
Publication date: 2019-06-06
Also published as: JP2021028296A

Abstract

The purpose of the present invention is to provide a haploidentical transplantation enhancer capable of maintaining or enhancing an antitumor effect of haploidentical transplantation and at the same time preventing the onset of graft-versus-host disease. The onset of graft-versus-host disease can be prevented while enhancing the antitumor effect of haploidentical donor hematopoietic cell transplantation by using liposomes containing α-galactosylceramide in combination with cyclophosphamide.

Description

Haplo transplantation enhancer

The present invention relates to a haplo transplantation enhancer that prevents the development of graft-versus-host disease while enhancing the anti-tumor effect of Leukocytes Antigen (HLA) semi-conforming (haplo) transplantation.

Allogeneic hematopoietic stem cell transplantation is useful as a treatment for hematologic diseases such as leukemia and other malignant hematologic cancer and aplastic anemia etc. However, donors compatible with human HLA type are found from relatives or unrelated bone marrow bank registrants. There is a need. Ideally, transplantation of bone marrow cells or peripheral blood stem cells from a HLA-matched donor is desired, but in the case of blood cancer, there is insufficient graft versus leukemia (GVL) reaction to prevent recurrence Is the problem. One way to solve this problem is to transplant hematopoietic stem cells from HLA single-locus mismatched donors to enhance GVL effect, but at the same time increase the risk of causing graft versus host disease (GVHD). As the prevention, the standard therapy which combines a calcineurin inhibitor (tacrolimus and cyclosporin) and methotrexate is given.

On the other hand, if no HLA-matched donor is found or remission is not achieved after transplantation, HLA half-match (haplo) transplantation is performed. Although the advantages of haplo transplantation are cancer recurrence prevention and remission induction by strong GVL effect, high survival failure and acute GVHD incidence are disadvantageous, and a method different from standard therapy is required.

In recent years, post-transplant cyclophosphamide (PTCy) therapy has attracted attention as a method for preventing GVHD while maintaining the GVL effect of haplo transplantation. The immunosuppressive action of cyclophosphamide has been reported by Berenbaum et al. After administration of MHC mismatched skin grafts (Non-patent Document 1). Subsequently, Eto et al. Revealed that the mechanism of action of PTCy is due to the selective cytotoxic activity of alloresponsive T cells (Non-patent Document 2). Also, in the clinical application of haplo transplantation, it is said that the method reported by Luznik et al. Is the starting point of the current PTCy therapy (Non-patent Document 3).

While PTCy therapy by Luznik et al. Has substantially resolved engraftment failure and GVHD after haplo transplantation, the GVL effect, which is an advantage of haplo transplantation, is reduced, and the recurrence rate of cancer is rising. It is a problem.

On the other hand, in the group including the present inventors, liposomal α-GalCer (liposomal α-GalCer) is presented to iNKT cells via CD1d of antigen-presenting cells, compared to α-GalCer in aqueous solution state. It has been reported that the effect on immune regulation is high (Non-patent documents 4 and 5), and when liposomal α-GalCer is administered during myeloablative bone marrow transplantation, Treg grows even when Full chimera is formed. It has been reported that GVHD is suppressed by doing this (Non-Patent Documents 6 to 9, Patent Document 2). Furthermore, the liposome containing KRN7000 as α-GalCer has an induction action of IL-10 producing T cells which is not exerted by α-GalCer alone in aqueous solution and an action of suppressing IgE antibody production, and allergic diseases, autoimmune diseases and It is reported that it is also useful as a preventive or therapeutic agent for GVHD (Patent Document 1). However, hitherto, the effect of a liposome preparation containing α-galactosylceramide has not been studied in PTCy therapy after haplo transplantation.

WO 2005/120574 pamphlet WO 2014/696554 brochure

An object of the present invention is to create a haplograft enhancer capable of preventing or developing graft vs. host disease while maintaining or enhancing the antitumor effect of haplograft.

The inventors of the present invention conducted intensive studies to solve the above problems and found that the combination of α-GalCer-containing liposome and cyclophosphamide is used to enhance the antitumor effect of haplodonor hematopoietic cell transplantation. Meanwhile, it was found that it is possible to prevent the onset of graft versus host disease. The present invention has been completed by further studies based on such findings.

That is, the present invention provides the invention of the aspects listed below.
Item 1. It is a haplo-graft enhancing agent used for a therapy for enhancing the anti-tumor effect on recipient cancer cells and for preventing graft-versus-host disease by transplanting haploido hematopoietic cells into the recipient,
A haplo transplantation enhancer comprising a combination of a liposome containing α-galactosylceramide and cyclophosphamide.
Item 2. The haplo-transplant enhancer according to item 1, wherein the haplo donor hematopoietic cells to be transplanted are hematopoietic cells derived from bone marrow, umbilical cord blood or peripheral blood.
Item 3. The haplo transplantation enhancing agent according to

Item

1 or 2, wherein the haplo donor hematopoietic cells to be transplanted are bone marrow.
Item 4. The haplo transplantation enhancer according to any one of Items 1 to 3, wherein the α-galactosylceramide is KRN7000.
Item 5. The haplo transplantation enhancer according to any one of Items 1 to 4, which is a two-drug form comprising a first preparation comprising a liposome containing α-galactosylceramide and a second preparation comprising cyclophosphamide.
Item 6. Transplanting haplodonor hematopoietic cells into a recipient to enhance the anti-tumor effect on recipient cancer cells and to produce a haplograft enhancer for use in therapy to prevent graft-versus-host disease Use of liposomes containing galactosylceramide and cyclophosphamide.
Item 7. A method for enhancing the antitumor effect on recipient cancer cells transplanted with haplo donor hematopoietic cells and preventing graft-versus-host disease,
Administering a liposome containing α-galactosylceramide and cyclophosphamide to a recipient transplanted with haplo donor hematopoietic cells.
Item 8. A haplo-transplant-enhancing agent for use in a therapy that enhances anti-tumor activity and prevents graft-versus-host disease in cyclophosphamide therapy treated for a recipient transplanted with haplo donor hematopoietic cells,
Haplo transplantation enhancer comprising liposomalized α-GalCer.
Item 9. In the cyclophosphamide therapy to be treated for recipients transplanted with haplo donor hematopoietic cells, for the production of haplo transplantation enhancing agent to be used as a therapy for enhancing the antitumor effect and preventing graft-versus-host disease Use of Liposomal α-Galactosylceramide.
Item 10. A method for enhancing anti-tumor activity and preventing graft-versus-host disease in cyclophosphamide therapy to be treated for a recipient transplanted with haplo donor hematopoietic cells,
A method comprising the step of administering liposomalized α-galactosylceramide to a recipient who received cyclophosphamide after transplantation of haplo donor hematopoietic cells.

According to the haplo transplantation enhancing agent of the present invention, transplantation of haplo donor hematopoietic cells into a recipient enhances anti-tumor activity, and at the same time induces immune tolerance to the recipient cells, tissues and / or organs to the donor cells. Anti-tumor activity can be enhanced while preventing graft-versus-host disease. In addition, the haplo transplantation enhancer of the present invention can facilitate the expansion of regulatory T cells in the recipient, which also contributes to the effective prevention of graft-versus-host disease. It is thought that

Furthermore, although treatment with cyclophosphamide conventionally administered after haplo transplantation has had a problem with cancer recurrence, when the haplo transplantation enhancer of the present invention is used, immune tolerance can be effectively induced. The dose of phosphamide can be reduced to enhance the anti-tumor effect while reducing the burden on the recipient.

In Test Example 1, clophosfamide is administered to bone marrow transplanted mice (GVHD onset model mice), and the results of evaluating the survival rate for 100 days and the GVHD score for 70 days are shown. In Test Example 2, cyclophosphamide and / or KRN7000-containing liposomes are administered to bone marrow transplanted mice (GVHD onset model mice), and the results of evaluation of 100 day survival rate and GVHD score are shown. In Test Example 3, CD4 positive cells were isolated from spleen and mesenteric lymph nodes 14 days after bone marrow transplantation using bone marrow transplanted mice (GVHD onset model mice) administered with liposomes containing cyclophosphamide and KRN7000. The result of observing the expression of CD25 and Foxp3 is shown. In Test Example 3, CD4 positive cells were isolated from spleen and mesenteric lymph nodes 28 days after bone marrow transplantation using bone marrow transplanted mice (GVHD onset model mice) administered with liposomes containing cyclophosphamide and KRN7000. The result of observing the expression of CD25 and Foxp3 is shown. In Test Example 4, cyclophosphamide is administered to a mouse transplanted with bone marrow with a luciferase expression leukemia strain, and the results of evaluating the survival rate for 100 days and the distribution of the luciferase expression leukemia strain in the mouse body are shown. In Test Example 5, cyclophosphamide and / or KRN7000-containing liposomes are administered to mice transplanted with bone marrow together with a luciferase expression leukemia strain, and the results of evaluation of the survival rate for 100 days are shown.

1. Haplo transplantation enhancer (1)
The haplo transplantation enhancing agent of the present invention is used as a therapy for enhancing the antitumor effect on recipient cancer cells by transplanting haplo donor hematopoietic cells into the recipient and for preventing graft-versus-host disease. The enhancer is characterized in that it comprises a liposome containing α-galactosylceramide, and cyclophosphamide. Hereinafter, the present invention will be described in detail.

[Liposome containing α-galactosylceramide]
The α-galactosylceramide (α-GalCer) used in the present invention is a glycosphingolipid in which galactose and ceramide are linked by α coordination, and specifically, WO94 / 09020, WO94 / 02168, WO94 Examples thereof are disclosed in U.S. Pat. No. 4,824,142, WO 98/44928, Science, 278, p. 1626-1629, 1997 and the like. In the present invention, so-called KRN7000, that is, (2S, 3S, 4R) -1-O- (α) as α-GalCer, from the viewpoint of further improving the antitumor effect and the GVHD preventing effect on cancer cells of the recipient. -D-Galactopyranosyl) -2-hexacosanoylamino-1,3,4-octadecanetriol is preferably used.

In the present invention, α-GalCer is used in the state of being contained in a liposome. That is, in the present invention, a liposome containing α-GalCer is used. Since the ceramide portion of α-GalCer exhibits lipid solubility, α-GalCer contained in the liposome usually has a structure in which α-GalCer is localized in the lipid bilayer membrane layer of the liposome. Hereinafter, the liposome containing α-GalCer may be referred to as liposomalized α-GalCer.

As a liposomal-ized alpha-GalCer used for this invention, the KRN7000 containing liposome manufactured using KRN7000 as alpha-GalCer can be mentioned as a preferable example. In addition, a KRN7000-containing liposome preparation RGI-2001 (manufactured by Leguimune Co., Ltd.) prepared by the same method as described in Biol Blood Marrow Transplant. 1-15 (2011) can be mentioned as a particularly preferable example.

In the liposomal α-GalCer, the ratio of the lipid component of the liposome to the α-GalCer can be appropriately set by the person skilled in the art according to the application, and is not particularly limited. For example, 0.05 to 100 parts by weight, preferably 0.5 to 20 parts by weight of α-GalCer is exemplified per part.

The lipid used for the liposomal α-GalCer (liposome-constituting lipid) is not particularly limited as long as it can form a bilayer membrane structure. Specific examples of liposome-constituting lipids include diacyl phosphatidyl cholines such as dipalmitoyl phosphatidyl choline (DPPC), dioleoylphosphatidyl choline (DOPC), dimyristoyl phosphatidyl choline (DMPC), and disteroyl phosphatidyl choline (DSPC); dipalmitoyl phosphatidyl glycerol (DPPG), dioleoylphosphatidyl glycerol (DOPG), dimyristoyl phosphatidyl glycerol (DMPG), diacyl phosphatidyl glycerols such as disteroyl phosphatidyl glycerol (DSPG); cholesterol, 3β- [N- (dimethylaminoethane) carbamoyl] Cholesterol (DC-Chol), N- (trimethylammonioethyl) carbamoyl cholesterol (TC-Chol), tocopherol, cholesterol succinate, rano Sterols such as sterols, dihydrolanosterol, desmosterol, dihydrocholesterol, timosterol, ergosterol, stigmasterol, sitosterol, campesterol, brashicosterol; dioleoylphosphatidylethanolamine (DOPE), disteroylphosphatidylethanolamine ( DSPE), phosphatidyl ethanolamines such as polyethylene glycol phosphatidyl ethanolamine (PEG-PE); phosphatidines such as dimyristoyl phosphatidic acid; gangliosides such as ganglioside GM1; polyethylene glycols such as polyethylene glycol palmitate and polyethylene glycol myristylate Fatty acid esters and the like can be mentioned.

Although the said liposome structure lipid may be used individually by 1 type, it is preferable to use in combination of 2 or more types. Preferred examples of combinations of liposome-constituting lipids include a combination of diacylphosphatidylcholines, diacylphosphatidylglycerols and sterols, and a combination of diacylphosphatidylcholines and sterols; more preferably, DPPC, DOPC, DPPG, DPPG, DOPG, cholesterol and / or Or a combination with DC-Chol.

When two or more types of liposome-constituting lipids are used in combination, the blending ratio of each lipid is appropriately set in consideration of the size, fluidity, etc. required for the liposome. For example, when a combination of diacylphosphatidylcholines, diacylphosphatidylglycerols and sterols is employed, the diacylphosphatidylcholines: diacylphosphatidyl glycerols: sterols in a molar ratio of 1: 0.125 to 0.75: 0.125-1, Preferably, 1: 0.14 to 0.4: 0.14 to 0.6 can be mentioned. Further, for example, in the case of employing a combination of DPPC, DOPC, DPPG and cholesterol, DPPC: DOPC: DPPG: cholesterol in a molar ratio of 1: 0.16 to 1.65: 0.16 to 1.0: 0.16 to 1.3, preferably 1: 0.4 to 0.75: 0.2 to 0.5: 0.3 to 0.75. Also, for example, in the case of employing a combination of diacylphosphatidylcholines (preferably DOPC) and sterols (preferably cholesterol and / or DC-Chol), the diacylphosphatidylcholines: sterols are in a molar ratio of 1: 1: 0.05 to 4, preferably 1: 0.1 to 1 can be mentioned.

The liposomalized α-GalCer may contain cationic compounds such as stearylamine and oleylamine; anionic compounds such as dicetyl phosphate; and membrane proteins, if necessary. The blending ratio of these may be appropriately set. be able to.

The liposome size of the liposomal α-GalCer is not particularly limited, but the average particle size is usually 5 to 1000 nm, preferably 100 to 400 nm. The mean particle size of the liposomes is measured by dynamic light scattering. Further, the structure of liposomalized α-GalCer is also not particularly limited, and any of MLV (multilamellar vesicles), DRV (dehydration-rehydration vesicles), LUV (large umilamella vesicles) or SUV (small unilamellar vesicles) may be used. Good.

Examples of the solution to be encapsulated in the liposomal α-GalCer include pharmaceutically acceptable aqueous carriers such as water, buffer and physiological saline.

Liposomeized α-GalCer is prepared by using known liposome production methods such as hydration method, sonication method, ethanol injection method, ether injection method, ether injection method, reverse phase evaporation method, surfactant method, freeze / thaw method Be done. In addition, the particle size distribution of the liposome can be adjusted by passing through a filter of a predetermined pore size. In addition, conversion of MLV to unilamellar liposome, or conversion of MLV to unilamellar liposome can also be performed according to a known method.

[Cyclophosphamide]
In the haplo transplantation enhancer of the present invention, cyclophosphamide is used together with the liposomalized α-GalCer. Cyclophosphamide is a compound known as an antitumor agent classified as an alkylating agent and an immunosuppressant.

In the haplo transplantation enhancing agent of the present invention, cyclophosphamide is used in combination with liposomal α-GalCer and cyclophosphamide to suppress survival failure after haplo transplantation and to prevent GVHD as well as liposomal alpha -It becomes possible to enhance anti-tumor action (GVL action etc.) while maintaining or enhancing the GVHD preventive effect by GalCer.

[Formulation form]
In the haplo transplantation enhancer of the present invention, liposomalized α-GalCer and cyclophosphamide are usually administered separately, but in the case of coadministration, liposomalized α-GalCer and cyclophosphamide are in the same preparation. It may be contained, and may be contained in two different formulations. That is, one embodiment of the haplo transplantation enhancing agent of the present invention includes liposomalized α-GalCer, and a one-part form haplo transplantation enhancing agent containing cyclophosphamide, and in another embodiment, liposomalized α-GalCer Examples include haplograft enhancers in a two-drug form consisting of a preparation containing GalCer and a preparation containing cyclophosphamide.

The dosage form of the haplo transplantation enhancer of the present invention can be appropriately set according to the administration form, and may be any dosage form such as liquid, powder, granule, tablet, capsule and the like. In addition, when the haplo transplantation enhancer of the present invention is a two-drug form, the preparation containing liposomal α-GalCer and the preparation containing cyclophosphamide may be the same dosage form or different dosage forms. It may be

[Other compounding ingredients]
The haplo-graft enhancing agent of the present invention comprises a pharmaceutically acceptable carrier, as required, in addition to liposomal α-GalCer and cyclophosphamide, and is prepared in a desired dosage form. Examples of the pharmaceutically acceptable carrier include aqueous carriers such as distilled water, physiological saline, phosphate buffer, citrate buffer, acetate buffer and the like; sucrose, fructose, sucrose, glucose, lactose, mannitol, sorbitol Saccharides such as glycerin; polyhydric alcohols such as glycerin, propylene glycol and butylene glycol; nonionic surfactants, cationic surfactants, anionic surfactants, surfactants such as amphoteric surfactants; hydroxypropyl methylcellulose Cellulose derivatives such as hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, carboxymethyl ethyl cellulose; antioxidants; pH adjusters and the like.

[Use, dose, usage]
The haplo transplantation enhancer of the present invention is administered to a patient (recipient) who has been transplanted with haplo donor hematopoietic cells for the purpose of enhancing the antitumor action (GVL action and the like) while preventing GVHD.

Recipients to whom the haplograft enhancer of the present invention is administered suffer from hematopoietic diseases such as hematologic malignancies (eg, acute myeloid leukemia, chronic leukemia, malignant lymphoma, multiple myeloma, etc.), myelodysplastic syndrome, etc. And those who have transplanted haplodonor hematopoietic cells.

In the present invention, the recipient is subjected to myeloablative treatment prior to transplantation of haplo donor hematopoietic cells, but preferably weight loss strength pretreatment is desirable. The weight loss pretreatment is not particularly limited, and may be a combination of various anticancer agents and radiation. For example, the following two weight loss intensity pretreatments can be mentioned. The first was intravenous injection of fludarabine (20 to 50 mg / m ² ) 7 to 5 days prior to transplantation and cyclophosphamide (10 to 20 mg / kg) daily for 2 days before It is a treatment in which 100 to 400 cGy whole-body radiation of the day before transplantation is added to intravenous administration 1 to 3 times before and after 6 to 5 days before. The second is the administration of flufarabine (20 to 50 mg / m ² ) intravenously four times daily from 2 days to 5 days prior to transplantation with melfarfan (50 to 150 mg / m ² ) It is a treatment that adds 150 to 400 cGy whole-body radiation the day before transplantation to a single intravenous administration in the period of 5 to 6 days before transplantation. Specifically, intravenous administration of fludarabine (30 mg / m ² ) 5 days a day from 6 days to 2 days before transplantation and 2 days of veins of cyclophosphamide (14.5 mg / kg) 6 days before and 5 days before transplantation It is a treatment to which 200 cGy whole body irradiation of the day before transplantation is added to internal administration. Intravenous administration of fludarabine (40 mg / m ² ) 4 days daily from 5 days to 2 days before transplantation and melfarfan (100 mg / m ² ) on the day 6 days before transplantation with a single intravenous administration 200 cGy the day before transplantation It is a treatment that adds whole body radiation.

In the present invention, “donor hematopoietic cells” refer to hematopoietic cells on the donor side to be transplanted to a recipient, which are stem cells capable of differentiating into hematologic cells. The origin tissue of hematopoietic cells to be transplanted into the recipient is not particularly limited, and may be, for example, bone marrow, umbilical cord blood, or peripheral blood itself, or hematopoietic cells derived from bone marrow, umbilical cord blood, or peripheral blood It may be The hematopoietic cells to be transplanted into the recipient preferably include hematopoietic cells derived from bone marrow, umbilical cord blood or peripheral blood. The amount of donor hematopoietic cells to be transplanted may be appropriately set according to the condition of the recipient, age, etc., but it is usually about 1 × 10 ⁶ to 3 × 10 ⁸ cells / kg. Hematopoietic cells derived from peripheral blood are preadministered to donors with G-CSF to mobilize blood stem cells into peripheral blood. Preferably, CD34 positive cells are 4-5 × 10 ⁶ cells / kg, at least 2 × 10 ⁶ Preferably, cells / kg or more are present.

The administration mode of the haplo transplantation enhancer of the present invention may be any of parenteral administration and oral administration. Specifically, intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intraarticular administration And mucosal administration. Among these administration forms, parenteral administration, particularly intravenous administration, is preferable from the viewpoint of further improving induction of immune tolerance.

Examples of administration targets of the haplo transplantation enhancer of the present invention include humans, monkeys, mice, rats, dogs, rabbits, cats, mammals such as cats, cattle, horses, and goats; and birds such as chickens and ostrich. Preferably it is a human.

The administration timing of the haplo transplantation enhancer of the present invention is not particularly limited as long as it is performed after haploid donor hematopoietic cell transplantation. For example, cyclophosphamide is preferably administered twice on the third and fourth day after transplantation of haploid donor hematopoietic cells. In addition, liposomalized α-GalCer may be administered multiple times before reconstruction of donor bone marrow cells starts, and in animal studies, day 4 and day 9 of the day after transplantation of haplo donor hematopoietic cells (the day of final administration of cyclophosphamide) It is suggested that a total of 3 doses of eye and day 14 should be administered. In humans, it is preferable to continue intravenous administration once a week four times or more, preferably five times or more, more preferably six times or more from the fifth day after transplantation of haplo donor hematopoietic cells.

The dose of the haplo transplantation enhancer of the present invention may be appropriately set according to the degree of symptoms of the patient, age and the like. For example, cyclophosphamide is administered twice at a dose of 50 mg / kg or less per dose, but further reduced (for example, a dose of 25-40 mg / kg of cyclophosphamide per dose) It can be set. The amount of liposomal α-GalCer may be about 1 to 100 μg / kg in terms of the amount of α-GalCer to be administered.

Drugs that can be used in combination with the haplo transplantation enhancer of the present invention are limited to immunosuppressants that do not adversely affect Treg in the body. For example, rapamycin and mycophenolate mofetil may be used after haplo-transplantation, but steroids and calicheurinulin inhibitors tacrolimus and cyclosporin can not be used.

The haplotransplant enhancer of the present invention may be practiced to reduce the relapse rate, which is a drawback, without losing the benefits of PTCy therapy. The inclusion of liposomal α-GalCer as an active ingredient of the haplo transplantation enhancer having such a remarkable effect has not been described in any of the known non-patent documents, and it is easily possible even by those skilled in the art. I can not think.

2. Haplo transplantation enhancer (2)
The strong haplo-graft agent of the present invention is used in a therapy that enhances anti-tumor activity and prevents graft-versus-host disease in cyclophosphamide therapy to be treated for recipients transplanted with haplo donor hematopoietic cells. Haplo-graft enhancing agent, characterized in that it comprises liposomalized α-GalCer.

The strong haplo-graft agent of the present invention contains liposomalized α-GalCer as an active ingredient, and in the therapy by administration of cyclophosphamide, which is performed after haplo-grafting, the antitumor effect (GVHD suppression effect is maintained) ( It is used to enhance GVL action etc.).

In the haplo-graft strong drug of the present invention, the type and composition of liposomalized α-GalCer as an active ingredient, formulation form, application subject, administration form, administration timing, dose and the like of liposomalized α-GalCer are described in “1. As described in the column "Haplo transplantation enhancer (1)".

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to the examples.

Test Example 1: The graft-versus-host disease (GVHD) inhibitory effect by cyclophosphamide (PTCy) administration after bone marrow transplantation The mouse is a female B6 mouse (C57BL / 8 to 12 weeks old from (NSC)). 6JJmsSlc, H-2 ^b ), 8 weeks old (10 or 11 weeks old at transplantation) female BDF1 mice (C57BL / 6NCrScm female x DBA / 2CrSlc male, F1, H-2 ^{b / d} ) purchased did. The mice were bred under the Specific Pathogen Free (SPF) environment at the Central Research Facility of Okayama University in accordance with the NIH animal management guidelines.

After euthanasia of donor B6 mice, femurs and tibiae were harvested and bone marrow was washed out of the marrow cavity using a 21 gauge needle. The bone marrow cells were suspended in 4% FCS (RPMI medium containing fetal bovine serum (4% FCS-RPMI) and passed through a 70 μm nylon mesh and centrifuged at 1800 rpm for 5 minutes. The supernatant was removed, and again, 1800 rpm, After centrifuging for 5 minutes and resuspending in 2% FCS-PBS to count the number of cells, bone marrow cells from which T cells have been removed (TCD-BM) are prepared using CD 90. 2 Micro Beads (Miltenyi). Also, the spleen was isolated from the same mouse and squeezed between 2 slide glasses, then the spleen cells were suspended with 4% FCS-RPMI, passed through a 70 μm nylon mesh, and further centrifuged at 1800 rpm for 5 minutes. The supernatant was removed, centrifuged again at 1800 rpm for 5 minutes, and the number of cells was counted The cell suspension containing 5 × 10 ⁶ TCD-BMs and 1 × 10 ⁷ spleen cells suspended in 250 μL is a recipe Intravenous injection was performed from the tail vein of an enter BDF1 mouse to prepare a GVHD onset model mouse.

Total body Irradiation (TBI) 12 Gy (X-ray generator (MBR-1520R-3, Hitachi medical Corp), divided into recipient's BDF1 mice 6 times before bone marrow transplantation and twice before transplantation Then, three days after bone marrow transplantation, cyclophosphamide (Sigma Aidrich) was diluted in saline and administered intraperitoneally at a dose of 25 mg / kg or 50 mg / kg.

FIG. 1 shows the survival rate and GVHD (graft versus host disease) scores for 100 days after bone marrow transplantation. In FIG. 1, "Vehicle" is a group to which only saline was administered, "PTCy 25 mg / kg" is a group to which cyclophosphamide was administered at a dose of 25 mg / kg, and "PTCy 50 mg / kg" is 50 mg of cyclophosphamide. The group administered by the / kg dose is shown.

The GVHD score was calculated from the observation results according to the following criteria. The GVHD score criteria were evaluated on a total of 7 points for the following 5 items.
Body weight change after transplantation 0.5 points: body weight change after transplantation is 95% or less and over 90% 1 point: body weight change after transplantation is 90% or less over 85% 1.5 points: body weight change after transplantation is 85% or less and over 75% 2 points: body weight change less than 75% after transplantation
The area of hair loss occupied on the body surface 0.5 point: The ratio of the area of hair loss occupied on the body surface is 0% to less than 25% 1 point: the ratio of the area of hair loss occupied on the body surface is 25% or more but less than 50% 1.5 points: body surface 50% or more of the area of hair loss in
Posture 0.5 point: backrest at body rest 1 point: backbone also at movement

Movement

point 1 point: Resting until stimulation occurs 1.5 points: Resting even if stimulation occurs
One line of hair : standing upside down

As can be seen from FIG. 1, the cyclophosphamide (PTCy) 25 mg / kg-administered group after bone marrow transplantation showed almost the same change in survival rate and GVHD score as the non-administered group. On the other hand, PTCy 50 mg / kg group showed high survival rate and suppression of GVHD onset.

Test Example 2: Graft-versus-host disease (GVHD) suppressive effect by PTCy and RGI-2001 administration after bone marrow transplantation to a GVHD onset model mouse prepared under the same conditions as in Test Example 1 above, bone marrow transplantation with 25 mg / kg of bone marrow transplantation for 3 days Thereafter, it was intraperitoneally administered once, and 1 μg / kg of KRN7000-containing liposome (RGI-2001) was administered once a total of 3 times via tail vein after 3, 5 and 7 days of bone marrow transplantation. The KRN7000-containing liposome contains 10% of KRN7000 in terms of lipid weight, and was prepared by the same method as in WO 2005/120574.

FIG. 2 shows the survival rate and GVHD score for 100 days after bone marrow transplantation. In FIG. 2, "Allo NS + NS" is a group to which only saline was administered, "Allo NS + PTCy 50 mg / kg" is a group to which only cyclophosphamide was administered, and "Allo α GC + NS" is a liposome containing KRN7000. “Alo α GC + PTCy 50 mg / kg” represents a group administered with cyclophosphamide and KRN7000-containing liposomes. As apparent from FIG. 2, in the group to which both PTCy and RGI-2001 were administered, an increase in survival rate and a decrease in GVHD score were observed.

Test Example 3: Increase effect of regulatory T cells by administration of PTCy and RGI-2001 after bone marrow transplantation A GVHD onset model mouse is prepared under the same conditions as in Test Example 1 except that the radiation dose at the time of bone marrow transplantation is changed to 10 Gy. did. PTCy 25 mg / kg was administered once intraperitoneally 3 days after bone marrow transplantation to the GVHD onset model mice prepared, and 1 μg / kg of KRN7000-containing liposome (RGI-2001) per bone marrow transplantation 3,5, And after 7 days, a total of 3 doses were given via the tail vein. Spleen and mesenteric lymph nodes were collected 14 and 28 days after bone marrow transplantation of GVHD onset model mice. 3 x 10 ⁶ cells from the obtained cells are suspended in 100 μl of 2% FCS-PBS, and eFluor 450 labeled anti-CD4 antibody (eBioscience), APC eFluor 780 labeled anti CD8a antibody (eBioscience) and PE-Cy7 labeled anti CD25 antibody After addition of (eBioscience), the reaction was performed at 4 ° C. for 30 minutes. Furthermore, cells are fixed and permeabilized using FoxP3 staining kit (eBioscience, San Diego, CA, USA), and after intranuclear staining with APC labeled anti-FoxP3, measured by MACS Quant (Myltenyi), Flowjo ver .10 (Tomy Digital Biology).

The analysis results are shown in FIGS. In FIGS. 3 and 4, “NS + NS” and “PTCy (−), αGalCer (−)” are groups to which only saline was administered, “PTCy + NS” and “PTCy 25 mg / kg, αGalCer (−)”. Group treated with cyclophosphamide only, "NS + α GC" and "PTCy (-), αGalCer (+)" group treated with KRN7000 containing liposome only, "PTCy + α GC" and PTCy 25 mg / kg, αGal Cer (+) Indicates a group to which cyclophosphamide and KRN7000-containing liposomes were administered. The percentage of regulatory T cell (Treg) populations copositive for CD25 and Foxp3 among CD4 positive cells was increased in the RGI-2001 group regardless of whether or not PTCy was administered. In particular, the rate of Treg increase in mesenteric lymph nodes was more pronounced than in the spleen.

Test Example 4: Verification of graft-leukemia (GVL) effect by PTCy administration after bone marrow transplantation The radiation dose at the time of bone marrow transplantation is changed to 8 Gy, and 5 × 10 ⁵ luciferase expression leukemia strains (P815L) are simultaneously treated with bone marrow transplantation. Tests were conducted under the same conditions as in Test Example 1 except that administration was performed from the tail vein (allo transplantation). In addition, tests were performed under the same conditions as described above except that syngeneic transplantation was performed using BDF1 mice as donor mice and recipient mice (syn transplantation). The distribution of P815L cells in the mouse body was measured by luminescence imaging of luciferase protein with an electron multiplying CCD (Electron Multiplying-CCD: EM-CCD) camera.

The results are shown in FIG. In FIG. 5, "allo NS + NS" and "NS + NS" are groups to which only saline was administered in allo transplantation, "allo NS + PTCy 25 mg / kg" and "NS + PTCy 25 mg / kg" allo transplantation "Allo NS + PTCy 50 mg / kg" and "NS + PTCy 50 mg / kg" received cyclophosphamide at a dose of 50 mg / kg in allo transplantation The group "syn NS + NS" received saline alone in syn transplantation, the group "syn NS + PTCy 25 mg / kg" received cyclophosphamide in syn transplantation at a dose of 25 mg / kg. Show.

In the 50 mg / kg PTCy administration group of allo transplantation, the survival rate was rather deteriorated as compared with the 25 mg / kg PTCy administration group, although the GVHD inhibitory effect was exceeded. As a result of luminescence imaging measurement on day 38 after transplantation, weak luminescence was observed in 1 of 5 mice surviving in the 25 mg / kg PTCy administration group of allo transplantation, but luminescence was observed in the remaining 4 mice. I was not able to admit. On the other hand, the maximum light emission was observed in one animal surviving in the 50 mg / kg PTCy administration group of allo transplantation. These results suggest that the GVL effect might be attenuated following allo-responsive T cell removal by high dose PTCy.

On the other hand, in the negative control non-administration group, it was suggested that the survival rate might be deteriorated due to the GVHD exacerbation over the GVL effect as compared with the 25 mg / kg PTCy administration group.

Test Example 5: Graft vs. leukemia (GVL) effect by co-administration of PTCy and RGI-2001 after bone marrow transplantation PTCy 25 mg / kg once in the same experimental system as in Test Example 4 (3 days after bone marrow transplantation) peritoneal cavity In addition, 1 μg / kg of KRN7000-containing liposome (RGI-2001) was administered 3 times (3, 5 and 7 days after bone marrow transplantation) from the tail vein.

The results are shown in FIG. In FIG. 5, "allo NS + NS" is a group to which only saline was administered, "allo NS + PTCy 25 mg / kg" is a group to which only cyclophosphamide was administered, and "allo α GC + NS" is a liposome containing KRN7000. The group administered only, "allo α GC + PTCy 25 mg / kg", represents a group administered cyclophosphamide- and KRN7000-containing liposomes. As a result, it was found that the survival rate of the group receiving both PTCy and RGI-2001 was similar to or slightly improved as that of the PTCy single administration group.

From this result, co-administration of low dose PTCy and RGI-2001 of the present invention can significantly improve the serious defect anti-tumor effect while maintaining the suppressive effect of GVHD which is an advantage of high dose PTCy. confirmed.

Claims

Alpha-galactosyl, which is a haplograft enhancing agent for use in therapy to enhance the antitumor effect on recipient cancer cells and to prevent graft-versus-host disease by transplanting haploido hematopoietic cells into the recipient. A haplo transplantation enhancer comprising a combination of a ceramide-containing liposome and cyclophosphamide.
The haplo-transfer enhancing agent according to claim 1, wherein the haplo donor hematopoietic cells to be transplanted are hematopoietic cells derived from bone marrow, umbilical cord blood or peripheral blood.
The haplo transplantation enhancing agent according to claim 1 or 2, wherein the haplo donor hematopoietic cells to be transplanted are bone marrow.
The haplo transplantation enhancer according to any one of claims 1 to 3, wherein the α-galactosylceramide is KRN7000.
The haplo transplantation enhancer according to any one of claims 1 to 4, which is a two-drug form comprising a first preparation containing a liposome containing α-galactosylceramide and a second preparation containing cyclophosphamide.
Transplanting haplodonor hematopoietic cells into a recipient to enhance the anti-tumor effect on recipient cancer cells and to produce a haplograft enhancer for use in therapy to prevent graft-versus-host disease Use of liposomes containing galactosylceramide and cyclophosphamide.
A method for enhancing the antitumor effect on recipient cancer cells transplanted with haplo donor hematopoietic cells and preventing graft-versus-host disease,
Administering a liposome containing α-galactosylceramide and cyclophosphamide to a recipient transplanted with haplo donor hematopoietic cells.
A haplo-transplant-enhancing agent for use in a therapy that enhances anti-tumor activity and prevents graft-versus-host disease in cyclophosphamide therapy treated for a recipient transplanted with haplo donor hematopoietic cells,
A haplo transplantation enhancer comprising liposomalized α-galactosylceramide.
In the cyclophosphamide therapy to be treated for recipients transplanted with haplo donor hematopoietic cells, for the production of haplo transplantation enhancing agent to be used as a therapy for enhancing the antitumor effect and preventing graft-versus-host disease Use of Liposomal α-Galactosylceramide.
A method for enhancing anti-tumor activity and preventing graft-versus-host disease in cyclophosphamide therapy to be treated for a recipient transplanted with haplo donor hematopoietic cells,
A method comprising the step of administering liposomalized α-galactosylceramide to a recipient who received cyclophosphamide after transplantation of haplo donor hematopoietic cells.