CN115282280A - New use of TGF-beta 1 signal inhibitor - Google Patents

Abstract

The invention relates to a new application of a TGF-beta 1 signal inhibitor, in particular to an application of the TGF-beta 1 signal inhibitor (such as Galunertib) in preparing a medicament or a kit for treating acute myeloid leukemia. As a result, the Galunertib can restore the capability of damaged AML patient bone marrow NK cells to kill tumor cells, prolong the survival period of acute myeloid leukemia and inhibit the load of leukemia cells. This provides a basis for clinical treatment of relapsed AML using TGF- β 1 signalling inhibitors.

Description

New use of TGF-beta 1 signal inhibitor

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of a TGF-beta 1 signal inhibitor Galunertib in reversing the damaged NK cell anti-tumor function of a patient with recurrent leukemia after hematopoietic stem cell transplantation and treating acute myeloid leukemia in a mouse animal model.

Background

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy, with a high incidence especially in the elderly, and has been a challenge for haematologists over the past few decades (Liu H. Emulsifying agents and registers for AML. J Hematol Oncol.2021Mar23;14 (1): 49. Doi. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment and is also the only treatment option for the majority of AML patients (Romero D. Sorafenib prevents AML relapse after allo-HSCT. Nat Rev Clin Oncol.2020Oct;17 (10): 591. Doi. Even with current treatment methods, recurrence of the primary disease after transplantation remains frequent and is associated with poor prognosis. Particularly, the recurrence of allogeneic hematopoietic stem cells 100 days after transplantation is a leading cause of death.

AML relapse is associated with the ability of AML cells to evade immune surveillance. Natural Killer (NK) cells play a crucial role in immune surveillance of cancer. Recent studies have shown that down-regulation of NK cell killing of AML cells following allogeneic hematopoietic stem cell transplantation may contribute to escape immune surveillance by AML cells. NK cells are the first to be reconstituted lymphocytes and may account for 80% of peripheral blood lymphocytes within the first 100 days after transplantation (Limongello R, marra a, manucus a, bonato S, hoxha E, ruggeri L, hui S, velardi a, pierce a. Novel Immune Cell-Based therapeutics to Eradicate High-rise enzyme myelid leukemia patent. Front immune.2021aug 3. NK cells evade NK Cell-mediated cytotoxicity and Immune surveillance by secreting particles containing proteins associated with cytolysis (e.g. granzymes), secreting effector cytokines (e.g. interferon-gamma (IFN- γ) and the like killing leukemia cells (Limongello R, marra a, manucus a, bonato S, hoxha E, ruggeri L, hui S, veirdi a, pierini a. Novel Immune Cell-Based therapeutics to artificial High-rise ace assay my leukemia.

TGF-. Beta.1 plays an essential role in regulating immune responses and triggering signals, primarily by binding to the TGF-. Beta.receptor (TGF-. Beta.R) complex. It consists of two TGF-beta receptor type I subunits. Both receptors are serine/threonine kinases. TGF-. Beta.1 may inhibit the effector functions of NK cells and CD8+ T cells. Blockade of the TGF-. Beta.1 pathway has become a method of restoring anti-tumor immunity (registers S, donderro A, caliendo F, bottino C, castriconi R.NK Cell Function Regulation by TGF-. Beta. -Induced Epigenetic mechanisms.Front Immunol.2020Feb 25, 311.doi. Galunertib is an oral TGF-beta R1 kinase small molecule inhibitor. Their ability to inhibit the proliferation of tumor cells has been demonstrated in several models of solid tumors, such as liver and pancreatic cancers (Melisi D, oh DY, hollebbecque A, calvo E, varghese A, borazanci E, macarulla T, merz V, zecchetto C, zhao Y, gueorguiova I, man M, gandhi L, estrem ST, benhadji KA, lanasa MC, avsar E, guba SC, garcia-Carnero R.safety and activity of the TGF receptor I kinase inhibitor promoter gene promoter plasmid plus the anti-PD-L1 antibody vector specific antibody marker plus, metagenic tissue engineering center, J. Center. 202069; 002898: 0028/898). However, its effect on AML disease is unclear, and it is unclear whether inhibition of the TGF- β 1 signaling pathway restores antitumor activity in AML relapsers. Therefore, molecular targeted therapeutic drugs against TGF- β 1 signaling pathway or its receptor are the focus of the research on drug therapy of acute myeloid leukemia. Also has a higher application prospect of transforming the basis into the clinic.

Disclosure of Invention

The ability of TGF- β 1 signaling inhibitors (e.g., gallunertib) to inhibit tumor cell proliferation has been demonstrated in several models of solid tumors (e.g., liver and pancreatic cancers), but because of the wide differences in tumor microenvironment and the like between solid tumors and leukemias, they cannot be extrapolated to the treatment of leukemia, and there is no motivation for those skilled in the art to use drugs for treating solid tumors for the treatment of leukemia.

Specifically, the currently preferred treatment scheme for solid tumors such as liver cancer is surgical resection, radiotherapy/chemotherapy and a few targeted drug therapies, and it is difficult to adopt a strategy of changing the tumor microenvironment to achieve the treatment effect. The etiology of myelodysplastic syndrome is currently unclear, and because bone marrow exhibits an abnormally proliferative state, immunosuppressive agents are often used for treatment, rather than strategies related to leukemia treatment that enhance the anti-tumor immune response. In addition, the part where the glioma occurs is the brain, and the research on how the microenvironment of the local tissues changes is unknown.

The generation site of leukemia is bone marrow, which is far from the generation sites of solid tumors and gliomas; meanwhile, due to the characteristics of tissues and organs and the existence of blood brain barrier, the immune cell composition and the immune response state in the microenvironment of the solid tumor or glioma are significantly different from those in the microenvironment of bone marrow. Thus, treatment strategies for both solid and glioma tumors are more difficult to use in leukemia therapy.

In addition, the leukemia is mainly treated according to the guidelines, including chemotherapy, targeted therapy, and hematopoietic stem cell transplantation. It is more difficult to consider the treatment of leukemia by improving the tumor microenvironment, such as by using TGF-beta 1 signaling inhibitors, to restore or enhance the anti-tumor function of immune cells.

In response to the above problems, the present inventors have for the first time used TGF- β 1 signalling inhibitors (e.g. gallunertib) in the treatment of acute myeloid leukaemia, in particular relapsed acute myeloid leukaemia. The result shows that the Galunertib can restore the capability of the NK cells of the relapsing AML patients, particularly the capability of the NK cells of the microenvironment of bone marrow to kill tumor cells, can enhance the capability of an organism to eliminate leukemia cells, and provides a theoretical basis of a new treatment strategy for the AML relapsing patients after transplantation.

Specifically, the invention provides the following technical scheme:

use of a tgf- β 1 signalling inhibitor (e.g. gallunertib) in the manufacture of a medicament or kit for the treatment of acute myeloid leukaemia.

2. The use according to item 1, wherein the acute myeloid leukemia is relapsed acute myeloid leukemia.

3. The use according to item 1, wherein the acute myeloid leukemia is an acute myeloid leukemia that recurs after chemotherapy, radiotherapy and/or stem cell transplantation.

Use of a tgf- β 1 signalling inhibitor (e.g. gallunertib) in the manufacture of a medicament or kit for restoring NK cell activity in patients with acute myeloid leukaemia.

5. The use according to item 4, wherein the acute myeloid leukemia is relapsed acute myeloid leukemia.

6. A kit (preferably for use in the treatment of acute myeloid leukemia), comprising a chemotherapeutic/targeting agent, and a TGF- β 1 signalling inhibitor (e.g. gallunertib).

7. The kit of item 6, wherein the chemotherapeutic agent comprises daunorubicin, cytarabine, or the like.

8. The kit of item 6, wherein the targeted drug comprises gemtuzumab ozogamicin, enzipine, na Wu Liyou mab, gelitinib, or the like.

9. The kit of item 6, wherein the patient is first administered a chemotherapeutic/targeting agent and then administered a TGF- β 1 signaling inhibitor (e.g., gallunertib); preferably, the TGF- β 1 signaling inhibitor (e.g., gallunertib) is administered after the chemotherapeutic/targeted drug treatment is ineffective.

In the present invention, gallunesertib is a novel TGF- β receptor I inhibitor developed by li pharmaceutical companies to inhibit the growth, invasion and metastasis processes of tumors by blocking TGF- β signaling pathway, which is commercially available with the following chemical formula:

technical effects of the invention

The inventor reports the application of a TGF-beta 1 signal inhibitor Galunertib in acute myeloid leukemia for the first time. The experimental result shows that the Galunertib can restore the capability of the damaged NK cells of the AML patient bone marrow to kill the tumor cells, and the results of in vivo experiments further show that the Galunertib treatment prolongs the survival period of the acute myeloid leukemia model mouse and inhibits the load of leukemia cells. The TGF-beta 1 signal inhibitor provides a certain experimental basis for clinically treating relapse refractory AML.

Drawings

FIG. 1 shows that TGF-. Beta.1 inhibits the anti-tumor effector function of human bone marrow NK cells. Wherein, fig. 1A shows that the killing function of NK cells on HL60 tumor cells is significantly reduced after TGF- β 1 stimulation; FIG. 1B shows that NK cells have a significantly reduced ability to produce the anti-tumor effector molecule Granzyme B, IFN- γ, CD107a following TGF-. Beta.1 stimulation.

FIG. 2 shows that Galunertib can restore the antitumor ability of NK cells in bone marrow of a patient who relapsed after hematopoietic stem cell transplantation. Wherein, fig. 2A and 2B show that gallunertib can reverse the killing function of damaged NK cells to tumor cells; figures 2C and 2D show that gallunertib is able to restore the ability of NK cells to produce anti-tumor effector molecules.

FIG. 3 shows that Galunertib can reverse the inhibitory effect of TGF-. Beta.1 signaling on NK cells. Among them, FIGS. 3A and 3B show that NK cells significantly restored the ability to produce anti-tumor effector molecules Granzyme B, IFN-. Gamma., CD107a after treatment with TGF-. Beta.1 inhibitors or Galunesertib.

FIG. 4 shows that Galunertib is able to restore the impaired antitumor ability of NK cells mediated by TGF-. Beta.1 in a mouse acute myeloid leukemia model. Among them, fig. 4A and 4B show that treatment of gallunertib can restore the therapeutic effect of NK cells and prolong the survival of mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the accompanying drawings in combination with the embodiments.

The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

Laboratory animal

Female NOD/ShiLtJGpt-Prkdcem26Cd52IL-2rgem26Cd22/Gpt (NCG) mice. Purchased from college drug kang corporation. A breeding environment: SPF grade.

Experimental procedure

Flow cytometry:

freshly isolated bone marrow mononuclear cells (NK) were purified using a separation and purification kit (purchased from Miltenyi Biotec, inc. # 130-092-657), added to 48-well cell culture plates, and HL60 (purchased from Shanghai cell Bank) or patient myeloid leukemia primary tumor cells were added, as effector cells: target cell = 5:1. Simultaneously, monensin (purchased from Sigma, with the product number # M5273) is added to block the secretion of the cell factors; and a fluorescent-labeled CD107a antibody (BD Co.) was added thereto, and the mixture was incubated at 37 ℃ for 5% CO2 for 5 hours; cells were then harvested by centrifugation. The cells collected after centrifugation were first subjected to mouse serum (purchased from ericsson) blocking for 15 minutes; then labeling the anti-human flow antibodies to be tested (anti-annexin V,7AAD, IFN-gamma, CD107a, granzyme B, CD56 antibodies; all purchased from BD company; respectively), and incubating for 30 minutes in a refrigerator at 4 ℃; cells were washed according to the instructions to remove non-specific antibodies. And then the obtained product is detected by a LSRII flow cytometer and analyzed by FlowJo software.

Wherein the used patient cells are obtained by separation at the blood station of Hefei city (ethical approval No.: first Hospital 2021-N (H) -120 affiliated to China university of science and technology).

Mouse model construction and treatment

Luciferase-labeled HL60 cells (purchased from Shanghai cell Bank) (5X 10) ⁵ ) NCG mice were injected via tail vein (6 weeks). After one week, tumor formation was confirmed, and 2.5X 10 humanized antibody was administered to the tail vein of the mouse ⁶ NK cells. And mice were injected intraperitoneally every two days with 50,000U IL-2 (purchased from Kingsle, jiangsu). One group of mice was intraperitoneally injected with 50 microliters of 5ng/ml TGF-. Beta.1 (purchased from R) once a week during NK cell therapy&D, cat # 240-B). Another group of mice was given 75mg/kg of Galunesertib (purchased from Selleck company under the designation LY 2157299) twice a day for 21 days, in addition to once weekly intraperitoneal injections of 50 microliters of 5ng/ml of TGF- β 1.

Tumor burden of AML was observed and counted by a small animal imager (purchased from Perkin Elmer).

Test compound preparation method: galunertib was dissolved in 1% sodium carboxymethylcellulose and fed to each mouse at 75 mg/kg. TGF-. Beta.1 was directly dissolved in physiological saline to a concentration of 5 ng/ml.

Data analysis

Data were analyzed using GraphPad Prism 8. Comparisons between groups were performed using student t-test. P values < 0.05 were considered statistically significant.

Example 1

After in vitro stimulation culture of bone marrow NK cells of AML patients who do not relapse after hematopoietic stem cell transplantation by TGF-beta 1 (10 ng/mL), the killing function of the NK cells on HL60 tumor cells (purchased from Shanghai cell bank) is remarkably reduced (figure 1A), and the capability of the NK cells for producing anti-tumor effector molecules Granzyme B, IFN-gamma and CD107a is remarkably reduced (figure 1B). The results show that TGF-beta 1 can inhibit the anti-tumor effect function of human bone marrow NK cells.

Example 2

Bone marrow NK cells of AML patients who recurred after hematopoietic stem cell transplantation were isolated and purified, co-culture stimulated with Galunertib (10. Mu.M) in vitro, and then their functions were examined. The killing function of the NK cells of the relapsed patients on the original tumor cells in the bone marrow of the patients is low, but the Galunertib can well reverse the killing function of the damaged NK cells on the tumor cells (figure 2A, 2B); meanwhile, the capability of the NK cells of the bone marrow of the relapsed patients to generate anti-tumor effector molecules Granzyme B, IFN-gamma and CD107a is found to be remarkably low, but the Galunertib can restore the capability of the NK cells to generate the anti-tumor effector molecules (figure 2C, 2D). The above results indicate that gallunertib can restore the antitumor ability of bone marrow NK cells in AML relapsing patients.

Example 3

After in vitro TGF-beta 1 (10 ng/mL) stimulation culture is carried out on bone marrow NK cells of an AML patient which do not relapse after hematopoietic stem cell transplantation, and then combined treatment is carried out on the bone marrow NK cells by using a TGF-beta 1 inhibitor (such as Galunertib), the fact that the killing function of the NK cells on original tumor cells in the bone marrow of the patient is remarkably reduced after the treatment by using the TGF-beta 1, but the killing function can be recovered after the treatment by using the TGF-beta 1 inhibitor (such as Galunertib) is found; meanwhile, the capability of NK cells for generating anti-tumor effector molecules Granzyme B, IFN-gamma and CD107a is found to be remarkably reduced after treatment by TGF-beta 1, but is remarkably recovered after treatment by a TGF-beta 1 inhibitor or Galunertinib (figure 3A, 3B), and the results show that the Galunertiib can reverse the inhibition effect of TGF-beta 1 signals on the NK cells.

Example 4

After tail vein transfusion of HL60 cells (purchased from Shanghai cell bank) with luciferase is carried out on NCG mice, the tumor growth can be remarkably inhibited after NK cell treatment, and the survival period of the mice is longer; after TGF-beta 1 is injected into the abdominal cavity during NK cell treatment, the tumor load of a mouse is found to be remarkably increased, namely, the killing function of NK cells on tumor cells is remarkably reduced after TGF-beta 1 stimulation; but treatment with gallunertiib restored therapeutic effect of NK cells and prolonged survival of mice (fig. 4a,4 b).

Conclusion

Galunertib can obviously restore the anti-tumor capability of NK cells in marrow of a relapsed AML patient, can obviously reverse the inhibition effect of TGF-beta 1 on the anti-tumor function of the NK cells in the in vitro and in vivo treatment process, reduces the tumor load of a mouse and prolongs the life cycle of the mouse. The results suggest that Galunesertib can effectively assist in the treatment of acute myeloid leukemia.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. Use of a tgf- β 1 signalling inhibitor (e.g. gallunertib) in the manufacture of a medicament or kit for the treatment of acute myeloid leukaemia.
2. 2. The use of claim 1, wherein the acute myeloid leukemia is relapsed acute myeloid leukemia.
3. 3. The use according to claim 1, wherein the acute myeloid leukemia is an acute myeloid leukemia that recurs after chemotherapy, radiotherapy and/or stem cell transplantation.
4. Use of a tgf- β 1 signalling inhibitor (e.g. gallunertib) in the manufacture of a medicament or kit for restoring NK cell activity in patients with acute myeloid leukaemia.
5. 5. The use of claim 4, wherein the acute myeloid leukemia is relapsed acute myeloid leukemia.
6. 6. A kit (preferably for use in the treatment of acute myeloid leukemia), comprising a chemotherapeutic/targeting agent, and a TGF- β 1 signalling inhibitor (e.g. gallunertib).
7. 7. The kit of claim 6, wherein the chemotherapeutic agent comprises daunorubicin, cytarabine, or the like.
8. 8. The kit of claim 6, wherein the targeted drug comprises gemtuzumab ozogamicin, enzipine, na Wu Liyou mab, gelitinib, or the like.
9. 9. The kit of claim 6, wherein the patient is first administered a chemotherapeutic/targeting agent and then administered a TGF- β 1 signaling inhibitor (e.g., gallunertib); preferably, the TGF- β 1 signaling inhibitor (e.g., gallunertib) is administered after the chemotherapeutic/targeted drug treatment is ineffective.

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