Application of preparation in preparation of medicine for treating allogenic hematopoietic stem cell transplantation complications

Abstract

The invention provides application of a preparation in preparation of a medicine for treating allogenic hematopoietic stem cell transplantation complications, and relates to the technical field of biological medicine, and the preparation comprises an autophagy inhibitor. The autophagy inhibitor is applied to preparation of the medicine for treating the allogenic hematopoietic stem cell transplantation complications, and related symptoms of the allogenic hematopoietic stem cell transplantation complications can be effectively relieved, so that the lifetime of a patient is remarkably prolonged, and the life quality of the patient is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of a preparation in preparing a medicine for treating complications of allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Background Art

[0002] Allo-HSCT (allogeneic hematopoietic stem cell transplantation) is an important means of treating a variety of blood tumors and hematopoietic system disorders. Chronic Graft-Versus-Host Disease (cGVHD) is the main fatal complication in its later stage, and the incidence rate of patients can reach 30% to 70%. Although studies have shown that the occurrence of cGVHD can inhibit the late recurrence of tumors in patients, it only targets chronic myeloid leukemia (CML) and does not affect the recurrence of leukemias such as acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The occurrence of cGVHD and tumor recurrence after Allo-HSCT greatly affect the survival and quality of life of patients. Summary of the invention

[0003] The technical problem solved by the present invention is how to provide an application of a preparation in the preparation of a drug for treating Allo-HSCT complications. The application of autophagy inhibitors in the preparation of a drug for treating Allo-HSCT complications can inhibit Allo-HSCT complications, thereby significantly prolonging the survival of patients and improving the quality of life of patients.

[0004] In order to solve the above problems, the present invention provides an application of a preparation in the preparation of a drug for treating complications of allogeneic hematopoietic stem cell transplantation, wherein the preparation comprises an autophagy inhibitor.

[0005] Preferably, the autophagy inhibitor is 3-methyladenine (3-Methyladenine, 3-MA).

[0006] Preferably, the dosage of 3-methyladenine is 10-20 mg / kg / day or 20-40 mg / kg / two days.

[0007] Preferably, the formulation further comprises a PD-L1 neutralizing antibody.

[0008] Preferably, the PD-L1 neutralizing antibody comprises an anti-mouse PD-L1 antibody, or an anti-human PD-L1 antibody.

[0009] Preferably, the anti-mouse PD-L1 antibody comprises 10F.9G2;

[0010] and / or, anti-human PD-L1 antibodies including atezolizumab

[0011] Preferably, the dosage of the 3-methyladenine is 10-20 mg / kg / day or 20-40 mg / kg / two days; the dosage of the PD-L1 neutralizing antibody is 10-15 mg / kg / 2 times / week or 20-30 mg / kg / time / week.

[0012] Preferably, the preparation is used as an intravenous injection.

[0013] Preferably, the complications of allogeneic hematopoietic stem cell transplantation include chronic graft-versus-host disease and / or recurrence of the primary tumor.

[0014] The present invention provides an application of a preparation in the preparation of a drug for treating complications of allogeneic hematopoietic stem cell transplantation, wherein the preparation comprises an autophagy inhibitor; after the autophagy inhibitor is applied to the preparation of a drug for treating complications of allogeneic hematopoietic stem cell transplantation, the relevant symptoms of the complications of allogeneic hematopoietic stem cell transplantation can be effectively alleviated, thereby significantly prolonging the survival period of patients and improving the quality of life of patients.

[0015] The present invention also provides a pharmaceutical composition, comprising an autophagy inhibitor and a pharmaceutical carrier.

[0016] The beneficial effects of the pharmaceutical composition provided by the present invention relative to the prior art are substantially the same as those of the above-mentioned applications and will not be described in detail herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is an in vitro experimental analysis diagram of the effect of B cell autophagy on cGVHD in Example 1 of the present invention;

[0018] Figure 2 This is an in vivo experimental analysis diagram of the effect of B cell autophagy on cGVHD in Example 1 of the present invention;

[0019] Figure 3 This is an analysis diagram of the effect of autophagy in B cells on cGVHD in Example 1 of the present invention;

[0020] Figure 4 This is an analysis diagram of the effect of autophagy inhibitors on cGVHD in Example 2 of the present invention;

[0021] Figure 5 This is an analysis diagram of the effect of the autophagy inhibitor on B cell proliferation and migration in Example 3 of the present invention;

[0022] Figure 6This is a diagram analyzing the effect of the autophagy inhibitor on the pathological changes of B cells in Example 3 of the present invention;

[0023] Figure 7 This is a scheme diagram for verifying the combined therapeutic effect of autophagy inhibitor and PD-L1 neutralizing antibody through a haploidentical mouse transplantation model in Example 4 of the present invention;

[0024] Figure 8 Analysis of the effect of the combination of autophagy inhibitor 3-MA and PD-L1 neutralizing antibody 10F.9G2 on cGVHD and tumor recurrence in Example 4 of the present invention Figure 1 ;

[0025] Fig. 9 Analysis of the effect of the combination of autophagy inhibitor 3-MA and PD-L1 neutralizing antibody 10F.9G2 on cGVHD and tumor recurrence in Example 4 of the present invention Figure 2 ;

[0026] Fig.10 This is a scheme diagram for verifying the combined therapeutic effect of the autophagy inhibitor 3-MA and the PD-L1 neutralizing antibody through a xenograft model in Example 4 of the present invention;

[0027] Fig.11 This is an analysis chart of the effect of the combined use of the autophagy inhibitor 3-MA and the PD-L1 neutralizing antibody Atezolizumab on cGVHD and tumor recurrence in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. In addition, the meanings of the terms "comprise", "include", "contain", and "have" are non-restrictive, and other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are all commercially available.

[0029] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in the present invention to express the dosage, mass fraction, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0031] According to statistics, about 50,000 patients receive HSCT every year worldwide, and 40% of them are allogeneic transplants. GVHD, as one of its serious complications, has a very high mortality rate and has become the main factor limiting the widespread clinical application of Allo-HSCT. According to the differences in the onset time and symptoms, GVHD can be divided into acute (acute GVHD, aGVHD) and chronic. aGVHD often occurs within 100 days after transplantation, involving the patient's intestines, liver, skin and other organs. Clinical studies have shown that the overall mortality rate of aGVHD patients can reach about 40% within 12 months after the onset of the disease. Therefore, after Allo-HSCT, the preventive treatment of aGVHD has become a routine means of clinical treatment. However, the preventive treatment of aGVHD cannot completely suppress the allogeneic immune response of the donor immune cells. At least 40% of patients still develop cGVHD in the late stage of transplantation, involving the patient's intestines, liver, lungs, skin, central and reproductive systems and other systemic organs, and the 10-year survival rate is only about 42%. Moreover, in the late stage of transplantation, some patients suffer from cGVHD and recurrence of the primary tumor.

[0032] The present invention is mainly aimed at treating complications of patients who receive allogeneic hematopoietic stem cell transplantation (Allo-HSCT) due to malignant tumors of the blood system.

[0033] An embodiment of the present invention provides an application of a preparation in preparing a drug for treating complications of Allo-HSCT, wherein the preparation includes an autophagy inhibitor.

[0034] Allo-HSCT is one of the effective means to treat hematological malignancies, but the occurrence of fatal complications such as GVHD limits its widespread clinical application. cGVHD and primary tumor recurrence are two major complications in the late stage of transplantation, and exploring effective treatment methods has always been an important focus in this field.

[0035] The pathological process of cGVHD is complex and involves a variety of immune cells, among which donor B cells are one of the important pathogenic cells. After the graft is infused, the recipient antigen-presenting cells (APCs) activated by conditional pretreatment such as radiotherapy and chemotherapy can present self- and allogeneic antigens to donor B cells, promoting their proliferation. Subsequently, Tfh (Tfollicular helper cell) can secrete IL-21 (Interleukin-21) and cell co-stimulatory factors to promote the formation of germinal centers (GCs); CD4 +T cells can secrete IL-4 to mediate antibody class conversion on the surface of B cells; stromal cells can produce B-cell activating factor (BAFF) to promote its activation. Ultimately, B cells can differentiate into plasma cells and plasmablasts, producing a large amount of immunoglobulin (Ig), and ectopic Ig can mediate macrophage activation and organ damage. Therefore, targeting pathogenic B cells and exploring effective ways to inhibit B cell overactivation, differentiation and antibody production have been considered as potential therapeutic means to alleviate cGVHD. Studies have pointed out that autophagy is an important physiological process that regulates the development and maturation of B cells. The downregulation and loss of autophagy can inhibit the formation of GC B cells, but the effect of B cell autophagy on cGVHD has not been reported.

[0036] The embodiment of the present invention provides an application of a preparation in the preparation of a drug for treating Allo-HSCT complications, wherein the preparation includes an autophagy inhibitor; after being used in the preparation of a drug for treating Allo-HSCT complications, the symptoms of Allo-HSCT complications can be significantly improved, Allo-HSCT complications can be inhibited, thereby significantly prolonging the patient's survival period and improving the patient's quality of life.

[0037] In some embodiments, the autophagy inhibitor is 3-MA.

[0038] The autophagy inhibitor 3-MA can inhibit autophagy by blocking the formation of autophagosomes and reducing the acidity of the autophagic environment. When used in the preparation of drugs for the treatment of Allo-HSCT complications, it can inhibit B cell signals, weaken B cell proliferation and migration, thereby reducing the migration of donor B cells to various target organs of cGVHD, while reducing the percentage of germinal center B cells and plasmablasts in target organs, and reducing the expression levels of donor B cell IgG1 and integrin protein α4β7 in interstitial lymph nodes, thereby achieving the effect of alleviating cGVHD.

[0039] In some embodiments, the autophagy inhibitor is 3-MA.

[0040] In some embodiments, the dosage of 3-MA is 15 mg / kg / day or 30 mg / kg / two days. By properly setting the dosage of 3-MA, the therapeutic effect can be better achieved.

[0041] In some embodiments, the preparation further comprises a PD-L1 neutralizing antibody. That is, the autophagy inhibitor 3-MA and the PD-L1 neutralizing antibody are used in combination. Compared with the use of the autophagy inhibitor alone, the combination of the two can promote each other, inhibit cGVHD and the malignant progression of the primary tumor, and prolong the patient's survival.

[0042] Among them, the PD-L1 neutralizing antibody preferably includes anti-mouse PD-L1 antibody and / or anti-human PD-L1 antibody.

[0043] Exemplarily, the mouse PD-L1 antibody includes 10F.9G2, and the anti-human PD-L1 antibody includes Atezolizumab.

[0044] In some embodiments, the dosage of 3-MA is 10-20 mg / kg / day or 20-40 mg / kg / two days; the dosage of the PD-L1 neutralizing antibody is 10-15 mg / kg / 2 times / week or 20-30 mg / kg / time / week. Among them, the dosage of 3-MA is preferably 15 mg / kg / day or 30 mg / kg / two days; the dosage of the PD-L1 neutralizing antibody is preferably 12.5 mg / kg / 2 times / week or 25 mg / kg / time / week. By reasonably setting the dosage of 3-MA and PD-L1 neutralizing antibody, the therapeutic effect can be better achieved.

[0045] In some embodiments, the preparation is used as an intravenous injection. Through intravenous injection, it can quickly enter the blood circulation system, so as to quickly reach the target site and take effect quickly, and avoid the metabolic decomposition that may be encountered in the gastrointestinal tract by oral administration, thereby improving bioavailability.

[0046] In some embodiments, the Allo-HSCT complications include cGVHD and / or primary tumor recurrence.

[0047] That is to say, the complications of Allo-HSCT in the embodiments of the present invention include not only cGVHD produced after Allo-HSCT treatment, but also the recurrence of the primary tumor before Allo-HSCT treatment.

[0048] Another embodiment of the present invention further provides a pharmaceutical composition, comprising an autophagy inhibitor and a drug carrier.

[0049] The beneficial effects of the pharmaceutical composition provided in the embodiments of the present invention relative to the prior art are substantially the same as those of the above-mentioned applications and will not be described in detail herein.

[0050] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on the conditions recommended by the manufacturer.

[0051] Example 1 Study on the effect of cell autophagy on cGVHD

[0052] (1) In vitro experiments:

[0053] Microtube-associated protein light chain 3 transgenic mice (LC3-Tg mice) were used as tool mice, and B cells in their spleen were extracted and stimulated with IgM and cluster of differentiation 40 (CD40) specific antibodies, or lipopolysaccharide (LPS) and interleukin 4 (IL-4) to simulate B cell surface receptor (B cell receptor, BCR) activation signals and inflammatory environment, respectively, to activate B cells. The results are as follows Figure 1 shown.

[0054] Figure 1 (a) is the relevant information of transgenic mice. C57BL / 6-Tg(CAG-RFP / EGFP / Map1lc3b)1Hill / J mice are LC3 transgenic mice (LC3-Tg mice for short), in which LC3 is coupled to enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP). When autophagy occurs and LC3 participates in the formation of autophagosomes, cells express EGFP and RFP at the same time. Because EGFP (pka5.9) cannot exist in a strong acid environment, after LC3 is esterified and participates in the formation of autophagolysosomes, EGFP is degraded and cells only express RFP (pka 4.5).

[0055] Figure 1 (b) is an analysis of the B cells extracted from the spleen of LC3-Tg mice, and the expression of EGFP and RFP levels of B cells was detected by flow cytometry after stimulating with IgM (10ug / mL) and CD40 (10ug / mL) activating antibodies, or LPS (10ng / mL) and IL-4 (10ng / mL) for 12hrs, 24hrs and 48hrs; Figure 1 As shown in (b), as the stimulation time increases, the double positive signals of EGFP and RFP in B cells (B220+) increase, indicating that autophagy is upregulated;

[0056] Figure 1(c) B cells were extracted from the spleen of LC3-Tg mice, and the expression of autophagy related gene 5 (Atg5) protein level in B cells was detected by flow cytometry after stimulating with IgM (10ug / mL) and CD40 (10ug / mL) activating antibodies, or LPS (10ng / mL) and IL-4 (10ng / mL) for 12hrs, 24hrs and 48hrs; Figure 1 As shown in (c), the level of Atg5 protein increased, indicating upregulation of autophagy.

[0057] (2) In vivo experiments:

[0058] CD45.2-LC3-Tg C57BL / 6 mouse spleen cells were collected and transplanted into CD45.1 C57BL / 6 or Balb / c mice to establish major histocompatibility complex (MHC)-matched and mismatched transplantation models. The recipient mice were killed 4 days after transplantation, and the expression of EGFP and RFP levels of donor B cells were detected. The results are as follows: Figure 2 As shown in (a).

[0059] from Figure 2 As can be seen in (a), compared with fully matched transplantation (C57BL / 6→C57BL / 6), allogeneic transplantation (C57BL / 6→Balb / c) can significantly upregulate the level of autophagy in donor B cells, as manifested by enhanced EGFP / RFP double-positive and RFP single-positive signals.

[0060] In the same experiment as above, recipient mice were killed 2 and 4 days after transplantation, and the expression of Atg5 in donor B cells was detected by flow cytometry. The expression of EGFP, RFP and Atg5 in allogeneically activated donor B cells and plasma cells was also detected 4 days after transplantation. The results are as follows: Figure 2 As shown in (b)-(d).

[0061] like Figure 2 In (b), it can be seen that the Atg5 protein level of allogeneically activated donor B cells increases, and the expression increases with the extension of transplantation time; Figure 2 As can be seen in (c) and (d), in allogeneic transplanted mice, compared with non-pathogenic donor B cells (CD45.2 + B220 + CD138 - ), donor pathogenic plasma cells (CD45.2 + B220 + CD138 + )The autophagy level was significantly upregulated.

[0062] It should be noted that Figure 2 In the figure, Isotype Con is a negative control staining; Syngeneic means syngeneic transplantation; Allogeneic means allogeneic transplantation; CD45.1 and CD45.2 are markers for distinguishing donor-recipient derived cells. Each experimental group consists of 4-5 mice; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0063] (3) Validation experiment of the effect of B cell autophagy on cGVHD

[0064] Using Atg5 conditional knockout mice in B cells as donors, MHC-mismatched and half-matched mouse bone marrow transplantation models were established.

[0065] Specifically, using Atg5 flox / flox C57BL / 6 mice were crossed with CD19-cre C57BL / 6 mice to establish mice with conditional knockout of Atg5 in B cells, which specifically downregulated the level of autophagy in B cells. These mice were used as donors to establish MHC mismatched and half-matched bone marrow transplantation models to induce cGVHD. Since homozygous deletion of CD19 can lead to dysplasia of mouse B cells, Atg5 was selected in the experiment. wt / wt CD19 wt / cre C57BL / 6 mice were used as control donors, Atg5 flox / flox CD19 wt / cre C57BL / 6 mice were used as donors for the experiment. wt / wt CD19 wt / cre C57BL / 6 and Atg5 flox / flox CD19 wt / cre The bone marrow and spleen cells of C57BL / 6 mice were mixed and transplanted into Balb / c mice that had been irradiated with γ-rays (950 cGy) to eliminate the immune system. The MHC-mismatched bone marrow transplantation model was established. The weight and clinical scores of the mice were recorded in real time (scoring was based on the arched back morphology, activity, fur integrity, skin damage, diarrhea, eye inflammation and weight loss of the mice, with 2 points for each item and a maximum of 12 points. When the weight of the mice was less than 65% of the original weight or the clinical score was higher than 9 points, they were killed and counted as the terminal stage of survival). The severity of cGVHD was evaluated (the amount of transplanted cells was 5×10 6 T cell-depleted bone marrow cells / recipient mouse, 0.35×10 6 splenocytes / recipient mice);

[0066] In another experiment, the extracted Atg5 wt / wt CD19 wt / cre C57BL / 6 and Atg5flox / flox CD19 wt / cre The bone marrow and spleen cells of C57BL / 6 mice were mixed and transplanted into B6D2F1 mice that had been irradiated with γ-rays (1200 cGy) to eliminate the immune system. The half-matched bone marrow transplantation model was established, and the weight and clinical scores of the mice were recorded in real time (the amount of transplanted cells was: 5×10 6 T cell-depleted bone marrow cells / recipient mouse, 2×10 6 splenocytes / recipient mice), the results were as Figure 3 shown.

[0067] It should be noted that in the above experiments, each experimental group consisted of 8-10 mice.

[0068] Figure 3 In the table, BMA (Bone marrow alone): transplantation of donor bone marrow cells with T cells removed into recipient mice; BMT (Bone marrow transplantation): transplantation of donor bone marrow cells and whole spleen cells with T cells removed into recipient mice; * indicates P < 0.05, ** indicates P < 0.01. Figure 3 As shown, downregulation of Atg5-dependent autophagy in B cells can significantly inhibit cGVHD.

[0069] Example 2 Verification of the therapeutic effect of autophagy inhibitors on Allo-HSCT complications

[0070] Using NSG-HLA-A2 + Humanized mice were used as donors and transfused with granulocyte colonystimulating factor (G-CSF) activated human HLA-A2 - Peripheral blood mononuclear cells (PBMC) were used to establish a xenograft model, induce cGVHD, and treat it with 3-MA.

[0071] Three days and two days before transplantation, NSG-HLA-A2 + Humanized mice were injected with cyclophosphamide (20 mg / kg, intraperitoneal injection) and NSG-HLA-A2 was transfected one day before transplantation. + Humanized mice were irradiated with gamma rays (200 cGy). On the day of transplantation, G-CSF-activated human HLA-A2-PBMC (1x10 6Cells / recipient mice), a xenograft model was established, cGVHD was induced, and phosphate buffered saline (PBS, Phosphate bufferedsaline) was injected, which was regarded as the blank treatment group (Vehicle) and 3-MA (15 mg / kg / day, intraperitoneal injection) treatment. The weight and clinical scores of mice were recorded in real time to evaluate the severity of cGVHD. Among them, Con (Control): refers to control mice, which refer to recipient mice that have been conditionally pretreated with radiotherapy and chemotherapy but have not been transplanted with PBMC. Each experimental group consists of 8-10 mice. * indicates P<0.05, ** indicates P<0.01. The results are shown in Figure 4 shown.

[0072] Figure 4 (a) is a comparative analysis of the body weight of mice treated with different methods. Figure 4 (b) is a comparison of clinical scores of mice treated with different methods. Figure 4 As shown, 3-MA could significantly improve cGVHD compared with the control group.

[0073] Example 3 Study on the mechanism of B cell autophagy promoting cGVHD and autophagy inhibitors in treating cGVHD

[0074] Luciferase transgenic C57BL / 6 mice and wild-type C57BL / 6 mice were used as donors to establish an MHC-mismatched bone marrow transplantation model. In the experiment, the bone marrow and spleen cells of Luciferase mice were collected, and the B cells in the mouse bone marrow and spleen were collected by flow sorting; the bone marrow and spleen cells of wild-type C57BL / 6 mice were collected simultaneously, and the B cells were removed; according to the normal percentage of B cells in the bone marrow and spleen, they were mixed as donor cells for the establishment of the transplantation model. After transplantation, Luciferin was injected into the recipient mice, and the Luciferase in the B cells could degrade them and excite fluorescence, which was detected by a small animal imaging device to show the proliferation and migration of donor B cells in the recipient.

[0075] Bone marrow and spleen of Luciferase mice were extracted, and B cells (B220 + ); extract the bone marrow and spleen of wild-type C57BL / 6 mice, remove the B cells; according to the normal distribution of B cells in the bone marrow (~25%) and spleen (~60%), mix them separately and use them as transplant donor cells. Balb / c mice were irradiated with gamma rays (950cGy) to eliminate the immune system, and then the above cells were transplanted into the body. Luciferin was injected into the recipient mice 7, 14, 21 and 28 days after transplantation, and the fluorescence signal was collected using a small animal imager. The results are as follows Figure 5As shown in (a)-(b).

[0076] In the above experiment, the recipient mice were killed 28 days after transplantation, and the spleen, liver, intestine, mesenteric lymph node (mLN) and lung were collected. The fluorescence signal was collected using a small animal imaging device. The results are as follows: Figure 5 As shown in (c)-(d).

[0077] The number of transplanted cells in the experiment was 5×10 6 T cell-depleted bone marrow cells / recipient mouse, 1×10 6 Splenocytes / recipient mouse. BMA (Bone marrow alone): mice transplanted with 5×10 6 Wild-type C57BL / 6 mouse bone marrow cells without T cells. Vehicle indicates control group. During treatment, PBS was injected intraperitoneally. Each experimental group consisted of 2-4 mice. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0078] In the above experiment, the intestinal tract and interstitial lymph nodes of the recipient mice were collected, and the lymphocytes were extracted. The donor B cells (H2Kb + B220 + ), plasmablasts (H2Kb + B220 + CD138 + ) and germinal center B cells (H2Kb + B220 + GL-7 + Fas + ) percentage; donor B cells (H2Kb + B220 + ) division, donor B cell immunoglobulin G1 (Immunoglobulin G1, IgG1; H2Kb + B220 + IgG1 + ) and integrin protein α4β7 (H2Kb + B220 + α4β71 + ) expression level. Each experimental group consisted of 4 mice. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Figure 6 shown.

[0079] from Figure 5-6As shown in Figure 2, 3-MA treatment not only significantly reduced the migration of donor B cells into the recipient intestine, but also reduced the number of germinal center B cells (H2Kb + B220 + GL-7 + Fas+ and plasmablasts (H2Kb + B220 + CD138 + ) percentage, and donor H2Kb in interstitial lymph nodes + B220 + IgG1 + and H2Kb + B220 + α4β71 + The above results confirmed the cellular mechanism by which 3-MA inhibits B cell autophagy and alleviates cGVHD.

[0080] Example 4 Verification of the effect of combined use of 3-MA and PD-L1 neutralizing antibody on cGVHD and primary tumor recurrence

[0081] cGVHD was induced in a C57BL / 6→B6D2F1 mouse haploidentical bone marrow transplantation model, and a tumor model was established by injecting P815 mouse mastocytoma cell line to verify the effect of combined use of 3-MA and PD-L1 neutralizing antibody (Clone: ​​10F.9G2) on cGVHD and tumor proliferation. The specific experimental plan is as follows Figure 7 As shown;

[0082] C57BL / 6 mice were used as donors and B6D2F1 mice whose immune system was eliminated by γ-ray (1200 cGy) were used as recipients to establish a half-matched bone marrow transplantation model, inducing cGVHD, and simultaneously infusing Luciferase transgenic P815 mastocytoma to simulate tumor recurrence after Allo-HSCT. Figure 7 The shown scheme treated the recipient mice with 3-MA and PD-L1 neutralizing antibody (10F.9G2) at different time points, recorded the mouse body weight, clinical score, survival period, and tumor mortality in real time, and injected Luciferin intraperitoneally. The tumor cell growth was evaluated by detecting the bioluminescence intensity in the mice.

[0083] In the experiment, the amount of transplanted cells was: 5×10 6 T cell-depleted bone marrow cells / recipient mouse, 0.35×10 6 Splenocytes / recipient mouse, 5×10 3Luciferase-P815 cells / recipient mice. Mice were injected with PBS (Vehicle: blank treatment) or 3-MA (15 mg / kg / day, intraperitoneal injection, 0-14 days after transplantation) and 10F.9G2 (12.5 mg / kg / twice / week, intraperitoneal injection, 14-42 days after transplantation), with 6-10 mice in each group. ** indicates P<0.01, *** indicates P<0.001. The results are shown in Figure 8 and 9 shown.

[0084] Figure 8 (a)-(d) are the combined data of two independent experiments. Fig. 9 This is a picture recorded during one of the experiments.

[0085] like Figure 8 As shown in (a)-(c), 3-MA treatment can significantly inhibit cGVHD; Figure 8 Middle (d) and Fig. 9 As shown, 10F.9G2 (PD-L1 neutralizing antibody) treatment can significantly inhibit tumorigenesis.

[0086] Using HLA-A2 + NSG humanized mice were used as recipients and G-CSF-activated human HLA-A2 was infused - Xenograft / tumor models were established with PBMC and human Raji lymphoma cell lines to verify the effects of combined use of 3-MA and PD-L1 neutralizing antibody (Clone: ​​Atezolizumab) on cGVHD and tumor proliferation. The specific experimental plan is as follows: Fig.10 As shown;

[0087] Three days and two days before transplantation, NSG-HLA-A2 + Humanized mice were injected with cyclophosphamide (20 mg / kg, intraperitoneal injection) and NSG-HLA-A2 was transfected one day before transplantation. + Humanized mice were irradiated with γ-rays (200 cGy). On the day of transplantation, G-CSF-activated human HLA-A2-PBMC (1×10 6 Cells / recipient mice) were used to establish a xenograft model, induce cGVHD, and simultaneously infuse Luciferase transgenic human Raji lymphoma cells to simulate tumor recurrence after Allo-HSCT. Fig.10The scheme shown in the figure, the recipient mice were treated with 3-MA and PD-L1 neutralizing antibody (Atezolizumab) at different time points, the weight, survival and tumor mortality of the mice were recorded in real time, and Luciferin was injected intraperitoneally. The growth of tumor cells was evaluated by detecting the bioluminescence intensity in the mice. * indicates P < 0.05, ** indicates P < 0.01. The results are shown in Fig.11 shown.

[0088] in, Fig.11 (a)-(c) are the combined data of two independent experiments.

[0089] like Fig.11 As shown, the combination of 3-MA and PD-L1 neutralizing antibody significantly prolonged the survival of recipient mice compared with the blank treatment group.

[0090] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. Use of a preparation in the preparation of a drug for treating complications of allogeneic hematopoietic stem cell transplantation, characterized in that: The formulation includes an autophagy inhibitor.

2. The use according to claim 1, characterized in that: The autophagy inhibitor is 3-methyladenine.

3. The use according to claim 2, characterized in that: The dosage of 3-methyladenine is 10-20 mg / kg / day or 20-40 mg / kg / two days.

4. The use according to claim 2, characterized in that: The formulation also includes a PD-L1 neutralizing antibody.

5. The use according to claim 4, characterized in that: The PD-L1 neutralizing antibodies include anti-mouse PD-L1 antibodies and / or anti-human PD-L1 antibodies.

6. The use according to claim 5, characterized in that: The anti-mouse PD-L1 antibodies include 10F.9G2; And / or, the anti-human PD-L1 antibody includes atezolizumab.

7. The use according to claim 5, characterized in that: The dosage of the 3-methyladenine is 10-20 mg / kg / day or 20-40 mg / kg / two days; the dosage of the PD-L1 neutralizing antibody is 10-15 mg / kg / 2 times / week or 20-30 mg / kg / time / week.

8. The use according to any one of claims 3 to 7, characterized in that: The preparation is used as an intravenous injection.

9. The use according to claim 1, characterized in that: The complications of allogeneic hematopoietic stem cell transplantation include chronic graft-versus-host disease and / or recurrence of the primary tumor.

10. A pharmaceutical composition, characterized in that Including autophagy inhibitors and drug carriers.

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