CN113082211A - Pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia and application thereof - Google Patents
Pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia and application thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia and application thereof, wherein the pharmaceutical composition comprises a delta NPM1 expression promoter and a tumor vaccine targeting delta NPM1, and the delta NPM1 expression promoter comprises decitabine or pharmaceutically acceptable salt or hydrate thereof. The decitabine or pharmaceutically acceptable salt or hydrate thereof and the tumor vaccine targeting delta NPM1 are combined, and the combined pharmaceutical composition successfully combines chemotherapy and tumor vaccine immunotherapy, has higher biological safety and stronger anti-tumor activity than single chemotherapy or single tumor vaccine immunotherapy; meanwhile, the dosage of the chemotherapeutic drug can be reduced, the toxic and side effects generated by the chemotherapeutic drug can be reduced, and the treatment cost of tumor patients can be reduced; meanwhile, the chemotherapy drugs induce stronger anti-tumor specific immunoreaction by up-regulating tumor cell surface tumor antigens, and exert the synergistic effect of continuous immunotherapy and chemotherapy.
Description
Technical Field
The invention belongs to the technical field of tumor treatment, and relates to a pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia and application thereof.
Background
Acute myeloid leukemia or Acute Myeloid Leukemia (AML) is a common invasive hematological malignancy, characterized primarily by abnormal clonal proliferation of myeloid blasts in the bone marrow and peripheral blood, the most common acute leukemia type in adults, especially the elderly. According to the statistics of the National Cancer Institute (NCI), the incidence rate of leukemia is 14.1/ten thousand per year and the death rate is 6.3/ten thousand per year, which accounts for 3.4% of new tumor diseases in recent 10 years. Prognosis of AML is poor, with a five-year survival rate of 34.4% in patients <65 years and only 5% in patients >65 years.
Chemotherapy is the primary treatment for AML, and guidelines suggest that the standard induction chemotherapy regimen for AML (non-APL) is an anthracycline in combination with a standard dose of cytarabine (Ara-C) (i.e., a 3+7 regimen), a consolidation treatment based on a high dose of Ara-C after clinical remission and restoration of the blood status in a bone marrow examination are achieved. Remission is obtained in 50-70% of patients after treatment with standard chemotherapy regimens, but recurrence occurs in more than half of young adults and 80-90% of elderly patients. Although the pathogenesis of leukemia is not clear, it is well recognized that AML is derived from tumorigenic transformation of hematopoietic stem cells or progenitor cells with self-renewal potential stem cell-like properties, which easily results in high recurrence rate of AML due to the ability of such leukemia cells with stem cell properties to tolerate chemotherapy, and also results in poor overall survival of AML. Although induction of post-chemotherapy Hematopoietic Stem Cell Transplantation (HSCT) has the potential to cure AML, this treatment modality is not feasible for elderly patients. The search for other cure modalities is an urgent need for AML treatment.
Immunotherapy aims at initiating or restarting the immunity of an individual against tumors, and this method is expected to cure various types of tumors. There are currently a variety of immunotherapies in which tumor vaccines can induce long-lasting immunity, with safety and reliability. The mechanism of action of tumor vaccines is to induce the generation and expansion of tumor-specific T cells, while reactivating tumor-specific T cells already present in the body itself, in a dormant or unresponsive state. Despite the great potential of tumor vaccines for tumor immunotherapy, the clinical outcome of some patients is still poor, which in the past may be due to the choice of tumor antigens, since traditional tumor vaccines are directed mainly against tumor-associated antigens, which may be present in normal cells as well as in tumor cells. With the development of sequencing technology, more and more tumor mutant genes are discovered at present, which is beneficial to the construction of new antigen tumor vaccines.
In AML, the nucleolar phosphoprotein 1(NPM1) mutation (Δ NPM1 or mutNPM1) is one of the most common mutations, with an incidence of 30-35%. NPM1 is widely expressed in nucleolus, and it can shuttle between nucleus and cytoplasm, participate in assembly and transport of ribosomal protein, regulate replication of centrosome and expression of tumor suppressor ARF, and play an important role in maintaining normal biological activity of cells. Mutation of NPM1 results in the transfer of the protein from the nucleus to the cytoplasm, leading to the development of AML. NPM1 has different mutation types, however, most of the mutations generate the same amino acid sequence at the C-terminus of the protein despite the different mutation types, and the generation of such a stable amino acid sequence means that the mutated NPM1 can be a good target for immunotherapy.
Immunotherapy is a novel treatment method for acute myeloid leukemia, wherein polypeptide vaccine can mobilize the immune system of the body, and is a safe and effective treatment method, so the tumor vaccine aiming at Δ NPM1 is an effective method for treating AML with NPM1 mutation. However, the tumor microenvironment is a complex environment in which tumor cell antigen immune escape is a significant cause of therapeutic failure with polypeptide vaccines alone. Therefore, improving the tumor microenvironment and enhancing the tumor cell immunogenicity through combined treatment with other compounds is one of the important factors for ensuring the success of tumor polypeptide vaccine treatment.
Disclosure of Invention
In order to solve the problems suggested in the background art described above, it is an object of the present invention to provide a pharmaceutical composition for treating AML mutated by NPM1 and use thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: on one hand, the invention provides application of a delta NPM1 expression promoter in preparing a medicine for enhancing the curative effect of a tumor vaccine targeting delta NPM1 on NPM1 mutant acute myeloid leukemia.
On the other hand, the invention provides application of the delta NPM1 expression promoter in preparing a medicine for enhancing the killing effect of a tumor vaccine targeting delta NPM1 on NPM1 mutant acute myeloid leukemia cells.
On the other hand, the invention provides application of the delta NPM1 expression promoter and a tumor vaccine targeting delta NPM1 in preparation of a medicine for treating NPM1 mutant acute myelogenous leukemia.
In a further aspect, the invention provides a pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia, which comprises a delta NPM1 expression promoter and a tumor vaccine targeting delta NPM 1.
In still another aspect, the present invention provides a kit comprising the above pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia.
Further, the Δ NPM1 expression promoter includes decitabine or a pharmaceutically acceptable salt thereof or a hydrate thereof.
Further, the tumor vaccine targeting Δ NPM1 is a CTL epitope peptide vaccine targeting Δ NPM 1.
Further, the CTL epitope peptide vaccine targeting delta NPM1 comprises a polypeptide with an amino acid sequence shown as CLAVEEVSL, AVEEVSLRK, CLAVEEVSLRK, VEEVSLRK, AVEEVSLR.
The invention has the beneficial effects that: applicants found in vitro that decitabine up-regulates the expression of Δ NPM1 on cell membranes. The decitabine or pharmaceutically acceptable salt or hydrate thereof and the tumor vaccine targeting delta NPM1 are combined for use, the combined pharmaceutical composition successfully combines chemotherapy and tumor vaccine immunotherapy, has higher biological safety, has stronger anti-tumor activity than single chemotherapy or single tumor vaccine immunotherapy, and provides a new strategy and thought for tumor treatment; meanwhile, the dosage of the chemotherapeutic drug can be reduced, the toxic and side effects generated by the chemotherapeutic drug can be reduced, and the treatment cost of tumor patients can be reduced; meanwhile, the chemotherapy drugs induce stronger anti-tumor specific immunoreaction by up-regulating tumor cell surface tumor antigens, and exert the synergistic effect of continuous immunotherapy and chemotherapy. The combination of decitabine (De citabine) or pharmaceutically acceptable salt or hydrate thereof and the tumor vaccine targeting NPM1 mutation has good treatment effect on patients suffering from NPM1 mutant Acute Myeloid Leukemia (AML).
Drawings
FIG. 1 is a graph showing the results of the proliferation capacity of OCI-AML 3/HLA-A1101 cells at various concentrations of decitabine;
FIG. 2 is a graph showing the apoptotic flow of decitabine on OCI-AML 3/HLA-A1101 cells;
figure 3 is a graph of the results of the synergistic killing of OCI-AML3/HLA-a 1101 by decitabine and specific CTLs.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The meanings of abbreviations used in the specification and claims are listed in the following table:
example 1
The experimental materials and reagents required for this example include:
cell line: OCI-AML 3;
chemotherapeutic drugs: decitabine (Decitabine, abbreviated DAC);
virus transfection: HLA-a 1101;
polypeptide: a polypeptide that targets Δ NPM1 that acts on HLA-a 1101, AVEEVSLRK.
1. Construction of HLA-A1101-positive OCI-AML3 cells
(1) The well-conditioned OCI-AML3 cells were harvested and resuspended in serum-free RPMI-1640 at 2X 10/well4Cells (200. mu.l volume) were seeded into 48-well plates;
(2) diluting virus with virus transfection enhancing solution Eni.S, adding virus solution with MOI of 100, 37 deg.C, and 5% CO2Culturing in an incubator for 48h, and adding 200 μ l of fresh culture medium;
(3) after 72h infection, GFP expression was observed under a fluorescent microscope.
2. Preparation of a tumor therapeutic vaccine targeting Δ NPM1 that can act on HLA-a 1101:
(1) conventional polysucrose-flood separation of fresh human whole blood to obtain Peripheral Blood Mononuclear Cells (PBMC) adjusted to a cell concentration of 3.0X 10 with RPMI-1640 complete medium containing 10% fetal bovine serum6The solution is inoculated into a 24-well plate, 1ml of the solution is added with IL-2 until the final concentration is 50U/ml;
(2) adding polypeptide into each hole the next day until the final concentration is 10 μ g/ml;
(3) half-amount liquid change and IL-2 supplement are carried out every 2 to 3 days until the final concentration is 50U/ml;
(4) the PBMC were stimulated weekly with the polypeptide by adding the polypeptide separately to each group to a final concentration of 10. mu.g/ml;
(5) and collecting cells after the third round of stimulation to obtain effector cells CTL.
3. Assaying the ability of decitabine to inhibit proliferation of HLA-a 1101 positive OCI-AML3 cells:
(1) cell treatment: OCI-AML 3/HLA-A1101 cells were collected at the logarithmic growth phase at 1.0X 104Cell/well 96-well plate, using 10% fetal bovine serum containing different concentrations of decitabine RPMI-1640 complete medium culture, concentration of 0M, 0.01M, 0.025M, 0.05M, 0.075M, 0.1M, 0.25M, 0.5M, 0.75M, 1M, 1.25M, 1.5M, 1.75M, 2M, each hole set up 3 duplicate wells, and set up blank control hole (i.e. only adding culture medium, not adding cells);
(2) after 72 hours of cell treatment, 10. mu.l of CCK-8 was added per well;
(3) putting the 96-well plate in a cell culture box for incubation for 3 h;
(4) taking a wavelength of 450nm, and detecting the absorbance value (A) of each hole in a 96-hole plate by using an enzyme-labeling instrument;
(5) and (4) calculating a result: the cell proliferation rate was 1- (a-treatment group-a blank)/(a non-treatment group-a blank) × 100%.
The result of the inhibition of OCI-AML 3/HLA-A1101 by decitabine is shown in figure 1, the inhibition of OCI-AML 3/HLA-A1101 by decitabine is gradually increased with the increase of concentration, and the inhibition rate reaches 25.04 +/-2.36% at the concentration of 0.1. mu.M, thereby showing that the toxic effect of decitabine on OCI-AML 3/HLA-A1101 is gradually increased with the concentration, and 0.1. mu.M can be regarded as the effect of decitabine and the IC of OCI-AML 3/HLA-A110125And flow apoptosis experiments show that the concentration does not generate toxicity to cells, and the results are shown in figure 2. The subsequent experiments all adopt 0.1 mu M to treat the cells (because the flow apoptosis experiment shows that the decitabine under the concentration does not generate toxicity to the cells, the subsequent experiments can further show that the enhanced tumor killing effect generated by the combination of the decitabine and the tumor vaccine is not caused by the medicament, but is the synergistic effect caused by up-regulating the expression of the membrane NPM1, so the concentration and the treated cells are used subsequently).
4. Detection of specific CTL induced by CTL epitope peptide on specific killing effect of OCI-AML3/HLA-a 1101 after decitabine pretreatment:
(1) preparing target cells:
target cells: OCI-AML 3/HLA-A1101 cells
② culturing OCI-AML 3/HLA-A1101 cells by RPMI-1640 complete culture medium containing 10% fetal bovine serum, adjusting the concentration of decitabine to IC of decitabine acting on OCI-AML 3/HLA-A1101 cells25Concentration;
③ after 72 hours of pretreatment, collecting OCI-AML 3/HLA-A1101 cells, and washing twice with PBS;
(2) preparing effector cells: the effector cells CTL prepared in 2 were adjusted to 1X 10 by using RPMI-1640 medium containing 5% fetal bovine serum5Individual cells/well as effector cells;
(3) and (3) detecting the killing efficiency:
grouping: OCI-AML 3/HLA-A1101 cells and PBS group, decitabine pretreatment OCI-AML 3/HLA-A1101 cells group, OCI-AML 3/HLA-A1101 cells and specific CTL co-incubation group, and OCI-AML 3/HLA-A1101 cells and specific CTL co-incubation group after decitabine pretreatment;
② OCI-AML 3/HLA-A1101 cells treated by the above-mentioned treatment are pre-stained with cell membrane dye CFSE;
③ grouping OCI-AML 3/HLA-A1101 cells according to 1 × 104Adding each cell/well into a V-bottom 96-well plate;
fourthly, according to the effective target ratio of 10: 1 adding specific CTL, and supplementing RPMI-1640 culture medium containing 5% fetal calf serum in each well to a final volume of 100 μ l;
fifthly, after the cells are plated, the temperature is 37 ℃, and the CO content is 5 percent2Incubating for 12 hours under saturated humidity conditions;
sixthly, adding 5ul7-AAD dye into 50 mu l of 1 × assay Buffer, and mixing uniformly;
seventhly, collecting the grouped cells, adding the cells into a 7-AAD dye, and uniformly mixing; reacting at room temperature in dark for 15 min;
add 450. mu.l of 1 × Assays Buffer suspension cells and detect them in 1 hour with a flow cytometer.
The detection result is shown in fig. 3, after the pretreated OCI-AML3/HLA-a 1101 cells and the specific CTLs are incubated for 12 hours, the 7-AAD positive proportion in the culture system is up-regulated, and the up-regulation amplitude is larger than that of the culture system in which decitabine is used alone and incubated with the specific CTLs alone, which indicates that the decitabine and the specific CTLs can generate a synergistic effect to kill tumor cells.
The experiments prove that the expression of delta NPM1 on the membrane of an NPM1 mutant AML cell line (OCI-AML3 cell) can enhance the tumor killing effect of a tumor vaccine targeting delta NPM 1; the decitabine and the tumor vaccine targeting the delta NPM1 have obvious synergistic effect on the aspect of tumor killing, and specifically, the combined use of the decitabine and the tumor vaccine targeting the delta NPM1 has stronger antitumor activity than that of a single use of the decitabine or a single tumor vaccine immunotherapy.
In conclusion, decitabine can enhance the tumor killing effect of the tumor vaccine by up-regulating the tumor specific antigen on the surface of the tumor cell, and the decitabine and the specific CTL can generate a synergistic effect to kill the tumor cell. Therefore, the decitabine and the tumor vaccine are combined for use, and the decitabine vaccine has great development and application potential in the field of tumor specific immunotherapy.
The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.
Claims (10)
1. Application of the delta NPM1 expression promoter in preparing a medicament for enhancing the curative effect of a tumor vaccine targeting delta NPM1 on NPM1 mutation acute myeloid leukemia.
2. Application of the delta NPM1 expression promoter in preparing a medicament for enhancing the killing effect of a tumor vaccine targeting delta NPM1 on NPM1 mutation acute myeloid leukemia cells.
3. The delta NPM1 expression promoter and the tumor vaccine targeting the delta NPM1 are used together to prepare the medicine for treating the NPM1 mutation acute myelogenous leukemia.
4. The use according to any one of claims 1 to 3, wherein the promoter of Δ NPM1 expression comprises decitabine or a pharmaceutically acceptable salt or hydrate thereof.
5. The use of any one of claims 1-3, wherein the tumor vaccine targeting Δ NPM1 is a CTL epitope peptide vaccine targeting Δ NPM 1.
6. The use of claim 5, wherein the CTL epitope peptide vaccine targeting Δ NPM1 comprises a polypeptide having an amino acid sequence represented by CLAVEEVSL, AVEEVSLRK, CLAVEEVSLRK, VEEVSLRK, AVEEVSLR.
7. A pharmaceutical composition for treating NPM1 mutant acute myeloid leukemia, which comprises a delta NPM1 expression promoter and a tumor vaccine targeting the delta NPM 1.
8. The pharmaceutical composition of claim 7, wherein the promoter of Δ NPM1 expression comprises decitabine or a pharmaceutically acceptable salt or hydrate thereof.
9. The pharmaceutical composition of claim 7, wherein the Δ NPM 1-targeted tumor vaccine is a CTL epitope peptide vaccine targeting Δ NPM 1;
preferably, the CTL epitope peptide vaccine targeting Δ NPM1 comprises a polypeptide having the amino acid sequence set forth in CLAVEEVSL, AVEEVSLRK, CLAVEEVSLRK, VEEVSLRK, AVEEVSLR.
10. A kit comprising a pharmaceutical composition according to any one of claims 7 to 9 for the treatment of NPM1 mutant acute myeloid leukemia.
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| US20200222462A1 (en) * | 2018-11-30 | 2020-07-16 | Mustang Bio, Inc. | Methods of treatment using decitabine and a cd123-targeted therapy |
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| WO2015127172A1 (en) * | 2014-02-20 | 2015-08-27 | Agios Pharmaceuticals, Inc. | Therapeutically active compounds and their methods of use |
| CN109462980A (en) * | 2016-03-15 | 2019-03-12 | 奥莱松基因组股份有限公司 | For treating the combination of the LSD1 inhibitor of hematologic malignancies |
| CN111032686A (en) * | 2017-06-30 | 2020-04-17 | 莱顿大学医学中心附属莱顿教学医院 | Treatment of hematologic malignancies |
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