A pharmaceutical combination against acute t-cell lymphoblastic leukemia and use thereof

Abstract

The application discloses a drug combination for resisting acute T lymphocyte leukemia and application thereof, and belongs to the technical field of medicines. The drug combination provided by the application comprises daunorubicin and CpG oligodeoxynucleotide, and the two are sequentially administered, wherein daunorubicin is administered first, and CpG oligodeoxynucleotide is administered after 10 hours. Daunorubicin can induce immunogenic death of tumor cells, release tumor antigens, CpG can promote maturation of dendritic cells, enhance antigen cross-presentation, and further activate CD8 + T cell-mediated specific anti-tumor immune response. Animal experiment results show that the drug combination can effectively delay disease progression, reduce multi-organ tumor load, prolong survival period in a NOTCH1-driven T-ALL mouse model, and can induce production of effector memory T cells, so that the mouse is completely protected after a second tumor attack. The drug combination does not need to be genetically modified in vitro, and has a good application prospect.

Description

Technical Field

[0001] This invention discloses a drug combination for treating acute T-lymphoblastic leukemia and its application. Background Technology

[0002] Acute T-lymphoblastic leukemia (T-ALL) is a highly heterogeneous, relapse-prone, and poorly prognostic hematologic malignancy. Current treatment primarily relies on high-intensity chemotherapy and hematopoietic stem cell transplantation (HSCT), with a 5-year overall survival rate of only 40%-60% for adult patients. New and effective treatment strategies are urgently needed. In recent years, immunotherapy, represented by CAR-T therapy, has emerged as a highly promising strategy for curing leukemia, achieving breakthrough efficacy in the treatment of acute B-lymphoblastic leukemia. However, its clinical application in T-ALL still faces multiple challenges. First, T-ALL tumor cells co-express lineage antigens such as CD3 / CD5 / CD7 with normal T cells, and targeting these antigens easily leads to exhaustion and impaired regeneration of normal T cells. Second, T-ALL exhibits significant clonal heterogeneity, making it difficult for single-target immunotherapy to cover all pathogenic subclones, resulting in persistent minimal residual disease and driving early relapse. Furthermore, the bone marrow microenvironment of T-ALL exhibits immunosuppressive characteristics, manifested as impaired dendritic cell (DC) maturation and antigen-presenting function, which in turn weakens T cell activation, proliferation, and cytotoxic function, ultimately leading to a low and unsustainable anti-tumor immune response rate. Therefore, developing immunomodulatory intervention strategies that can efficiently eliminate polyclonal T-ALL cells while selectively preserving normal T cell function is a core issue in current T-ALL immunotherapy research. Summary of the Invention

[0003] Dendritic cells (DCs) are the hub of innate and adaptive anti-tumor immunity, but their function is significantly suppressed in T-ALL. This invention aims to propose a drug combination for treating acute T-lymphoblastic leukemia and its application, which is essentially an anti-T-ALL strategy targeting and activating DCs. First, daunorubicin (DNR) induces immunogenic death (ICD) in T-ALL cells, releasing a full spectrum of tumor antigens to form an "in situ vaccine." Then, CpG oligodeoxynucleotides specifically activate DCs, promoting their maturation and enhancing their cross-presentation capacity, thereby efficiently activating CD8+. + T cell-mediated specific anti-leukemia immune responses. This strategy significantly enhanced DC maturation levels and CD8+ in a NOTCH1-induced T-ALL mouse model. + T cell killing ability. Compared with directly modifying T cells, this method combines safety, efficacy, and durability by reshaping the endogenous immune response.

[0004] To address the problems in the existing technology, the technical solution adopted by this invention is as follows: A drug combination for treating acute T-lymphoblastic leukemia, the drug combination comprising daunorubicin (DNR) and CpG oligodeoxynucleotides of type B, wherein the daunorubicin and the CpG oligodeoxynucleotides are each independently formulated for sequential administration.

[0005] Preferably, the sequential administration involves first administering a daunorubicin preparation, followed by an interval of a certain time before administering a CpG oligodeoxynucleotide preparation.

[0006] Preferably, the interval is 10 hours.

[0007] Preferably, the sequence of the class B CpG oligodeoxynucleotide is ODN 1826, as shown in SEQ ID NO:1.

[0008] Among them, SEQ ID NO:1:5'-TCCATGACGTTCCTGACGTT-3'.

[0009] Preferably, the unit dose of daunorubicin is 3 mg / kg, and the unit dose of CpG oligodeoxynucleotide is 1 mg / kg.

[0010] The use of any of the drug combinations described above in the preparation of a drug for treating acute T-lymphoblastic leukemia.

[0011] Preferably, the drug combination is used in the preparation of drugs for enhancing CD8. + Application in drugs that induce T-cell-mediated immune responses against acute T-lymphoblastic leukemia.

[0012] Preferably, the drug combination is used in the preparation of a drug for inducing immune memory against acute T-lymphoblastic leukemia. Beneficial effects

[0013] Compared with existing technologies, the present invention provides a drug combination for treating acute T-lymphoblastic leukemia and its application, which has the following advantages: (1) This invention employs sequential administration of daunorubicin and CpG oligodeoxynucleotides. Daunorubicin induces immunogenic death in tumor cells, releasing tumor antigens. CpG then promotes dendritic cell maturation, enhances antigen cross-presentation, and thereby activates CD8. + This regimen targets T-cell-mediated specific anti-tumor immune responses. It does not involve in vitro genetic modification of T cells, and its mechanism of action differs from CAR-T or bispecific antibody therapies that directly target T-cell surface antigens. In vitro and in vivo experiments have demonstrated that this combination is suitable for human MOLT-4 cells and NOTCH1-driven T-ALL mouse models, promoting the upregulation of CD80 and CD86 molecules on the surface of dendritic cells (DCs) and driving CD8+-mediated specific anti-tumor immune responses. + T cell activation and tumor cell-specific killing.

[0014] (2) The drug combination of the present invention effectively inhibited tumor progression in a NOTCH1-driven T-ALL mouse model. Compared with the PBS control group and each single-drug group, the peripheral blood GFP in the combination drug group was significantly higher. + The rate of increase in leukemia cells was significantly slowed (P<0.001); the leukemia burden in bone marrow, spleen, liver, and peripheral blood was significantly reduced (P<0.001); the median survival time of mice was significantly prolonged (P<0.001), and 62.5% (5 / 8) of mice survived to 80 days post-transplantation with no detectable GFP in peripheral blood. + Leukemia cells.

[0015] (3) The drug combination of the present invention can induce immunogenic death of in situ tumor cells, releasing a full spectrum of tumor antigens, thus overcoming immune escape against single antigens. In the NOTCH1-T-ALL mouse model, tumor cell residue was barely detectable after combined drug administration: GFP in peripheral blood, bone marrow, spleen, and liver on day 24. + The cell percentage was (0.00% ± 0.00%), which was significantly lower than that of the control group and the single-drug group (P<0.001).

[0016] (4) The drug combination of the present invention can promote the maturation of dendritic cells in vivo. In the NOTCH1-T-ALL mouse model, CD11c in the lymph nodes of the combined drug group was significantly increased. + CD80 + CD86 + The proportion of mature DCs was significantly higher than that of the control group (P<0.01).

[0017] (5) The drug combination described in this invention can enhance CD8 + T cell-mediated anti-tumor immune response. In the NOTCH1-T-ALL mouse model, CD8+ in the bone marrow of the combination therapy group... + T cell frequency and CD69 + The activation rates were significantly higher than those in the control group (P<0.01), and serum TNF-α and IFN-γ levels were simultaneously significantly increased (P<0.001).

[0018] (6) In mice that survived long-term after combined treatment according to the present invention, GFP was not detected in peripheral blood throughout the entire process after a second homologous tumor attack. + Cells; no GFP was detected in bone marrow, spleen, or liver on day 24 post-infection. + Cells (detection limit <0.001%), while GFP was present in all tissues of the control group. + The proportion of cells was greater than 80% in all groups. CD8+ levels were found in the bone marrow and spleen of mice in the combined treatment group. + The proportion of effector memory T cells (TEM; CD44hiCD62Llow) was significantly higher than that of the control group (P<0.05). Attached Figure Description

[0019] Figure 1 MOLT-4 cells underwent immunogenic death after DNR treatment.

[0020] Figure 2 CpG promotes the maturation of mouse bone marrow-derived dendritic cells (BMDCs) (CD11c). + CD80 + CD86 + (Percentage of triple-positive cells).

[0021] Figure 3 To delay the progression of peripheral blood leukemia in NOTCH1-T-ALL mice by combining DNR with CpG.

[0022] Figure 4 To reduce the leukemia burden in peripheral blood, bone marrow, spleen, and liver of NOTCH1-T-ALL mice by combining DNR with CpG.

[0023] Figure 5 To prolong the overall survival of NOTCH1-T-ALL mice by combining DNR with CpG.

[0024] Figure 6 The combination of DNR and CpG increased the proportion of mature dendritic cells (DCs) in the lymph nodes of NOTCH1-T-ALL mice.

[0025] Figure 7 To increase CD8 in the bone marrow of NOTCH1-T-ALL mice by combining DNR with CpG + T cell ratio, activation (CD69) + (and serum TNF-α / IFN-γ levels).

[0026] Figure 8 The dynamic tumor burden in peripheral blood of mice in the DNR combined with CpG treatment group after tumor re-attack.

[0027] Figure 9 The tumor burden in the bone marrow, spleen, and liver of mice in the DNR combined with CpG treatment group was measured 24 days after tumor re-attack.

[0028] Figure 10 To increase CD8 levels in the bone marrow and spleen of NOTCH1-T-ALL mice by combining DNR with CpG + Effector memory T cells. Detailed Implementation

[0029] The reagents and experimental animals used in this invention were obtained as follows: daunorubicin (DNR) and CpG oligodeoxynucleotides (ODN1826) were purchased from MedChemExpress (MCE), catalog numbers HY-13062 and HY-146245, respectively; anti-calreticulin antibody was purchased from Abcam (ab2907); ATP assay kit (enhanced version) was purchased from Beyotime Biotechnology Co., Ltd. (S0027); human HMGB1 / HMG-1 ELISA kit was purchased from Novus Biologicals (NBP2-62766); C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SPF grade, half male and half female, 6-8 weeks old).

[0030] Example 1

[0031] In vitro validation of immunogenic cell death (ICD) induced by daunorubicin (DNR) in the human acute T-lymphoblastic leukemia cell line MOLT-4.

[0032] Experimental Materials and Grouping Human T-ALL cell line MOLT-4 (ATCC, CRL-1582) in logarithmic growth phase was used at a concentration of 2 × 10⁻⁶. 4 The wells were seeded at a density of / wells in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0033] Cells were randomly divided into three groups: a blank control group (with an equal volume of phosphate-buffered saline PBS), a low-dose DNR group (with daunorubicin added to a final concentration of 100 nM), and a high-dose DNR group (with daunorubicin added to a final concentration of 200 nM), with three replicates in each group.

[0034] ICD characteristic damage-associated molecular patterns (DAMPs) detection ATP release assay: Six hours after administration, the culture supernatant of each group was aseptically collected, and the extracellular ATP concentration was detected by chemiluminescence using an enhanced ATP assay kit (Beyotime, S0027).

[0035] Simultaneous multi-index detection: 24 hours after drug administration, the following three tests were performed simultaneously: (1) Cell viability detection: 10 μL of CCK-8 solution (Beyotime, B34304) was added to each well, gently shaken to mix, and then incubated at 37°C for 2 hours. The absorbance (OD) was then measured at a wavelength of 450 nm. 450(2) Calculation of cell viability in each group: Collect cells, add Fc blocking antibody (1:500) and incubate at 4℃ for 10 minutes, add FITC-labeled anti-Calreticulin antibody (Abcam, ab2907) and stain at 4℃ in the dark for 30 minutes, and detect CRT by flow cytometry. + Cell percentage. (3) ATP detection: Collect the culture supernatant, add 100 μL of ATP detection working solution to the detection well, and let stand at room temperature for 3–5 min to eliminate background ATP; then add 100 μL of the sample to be tested or the standard, mix gently, and let stand for 2 s before immediately reading the RLU value with an ELISA reader (chemiluminescence mode). (4) HMGB1 release detection: Collect the culture supernatant, use the human HMGB1 ELISA kit (Novus Biologicals, NBP2-62766), follow the instructions, plot the standard curve and calculate the HMGB1 protein content in the supernatant.

[0036] Statistical analysis All experiments had three replicates per group. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0037] Experimental results like Figure 1 As shown, compared with the PBS control group, the DNR treatment group showed a significant dose-dependent decrease in MOLT-4 cell viability (P<0.01); a significant increase in the rate of CRT-positive cells (P<0.001); and a significant dose-dependent increase in the release of extracellular ATP and HMGB1 (P<0.01). The synergistic upregulation of these three DAMPs (CRT exposure, ATP secretion, and HMGB1 release) meets the generally accepted criteria for immunogenic cell death in this field, confirming that DNR can successfully induce immunogenic cell death in MOLT-4 cells, providing in vitro evidence for subsequent in vivo combination therapy.

[0038] Example 2

[0039] In vitro validation of CpG oligodeoxynucleotides (ODN 1826) promoting the maturation of mouse bone marrow-derived dendritic cells (BMDCs). Laboratory animals and materials: SPF-grade C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were euthanized with carbon dioxide, and the bilateral femurs, tibias, and iliac bones were aseptically separated.

[0040] Induction culture of bone marrow-derived dendritic cells (BMDCs) Step 1: Use a sterile syringe (1 mL) to draw up RPMI 1640 basal culture medium and repeatedly flush the bone marrow cavity until the bone ends turn white. Collect the bone marrow cells in the flushing fluid. Step 2: After filtering the cell suspension through a 70 μm cell sieve to remove bone fragments and tissue clumps, add red blood cell lysis buffer (Solarbio, R1010) and lyse at 37°C for 1 minute. Then add 3 times the volume of PBS to stop the lysis reaction. Centrifuge at 400×g for 5 minutes at room temperature, discard the supernatant, and collect the bone marrow mononuclear cell pellet. Step 3, the above cells are divided into 1×10 6 The solution was resuspended at a density of 20 ng / mL in BMDC induction medium, the medium formulation of which was: RPMI 1640 basal medium (Gibco, C22400500CP) + 10% fetal bovine serum (Eallbio, 03.U16001DC) + 20 ng / mL recombinant mouse GM-CSF (Novoprotein, CK02) + 1% penicillin-streptomycin-glutamine (Gibco, 10378016). Step 4: Seed cells into 6-well cell culture plates and incubate at 37°C with 5% CO2 for induction culture. On day 3 of culture, replace half of the BMDC induction medium with fresh medium and continue culturing until day 7.

[0041] Flow cytometry to determine BMDC maturity On day 7 of cultivation, all suspended and loosely attached immature BMDCs were collected, counted, and then divided into 5×10⁻⁶ cells. 5 Cells were seeded at a density of / wells in 12-well plates. Cells were randomly divided into two groups (three replicates per group): a PBS control group (with an equal volume of phosphate-buffered saline) and a CpG treatment group (with CpG oligodeoxynucleotides (ODN 1826, MedChemExpress, HY-146245) added to a final concentration of 1 μg / mL). Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, cells from each group were collected and stained with the following fluorescently labeled antibodies at a 1:100 ratio (antibodies: CD11c-BV510, BioLegend, 117338; CD80-PE, BioLegend, 104708; CD86-APC, BD, 558703). After incubation at 4°C in the dark for 30 minutes, cells were washed twice with PBS and resuspended in 200 μL of PBS. Flow cytometry was used to collect 10,000 viable cells per tube to detect CD11c. + CD80 + CD86 + Cells account for CD11c + The proportion of cells.

[0042] Statistical analysis All experiments were independently repeated at least three times. Data are expressed as mean ± standard deviation (Mean ± SD). Unpaired t-tests were used to compare two groups, and P < 0.05 was considered statistically significant.

[0043] Experimental results like Figure 2 As shown, compared with the PBS control group, the CpG-treated group had significantly lower CD11c levels. + CD80 + CD86 + The proportion of mature BMDCs increased significantly (P<0.001), indicating that CpG can effectively promote the differentiation of BMDCs into mature phenotypes. Example 3

[0044] In vivo therapeutic effect evaluation of DNR combined with CpG in NOTCH1-driven T-ALL mouse model Establishment and passage of the NOTCH1-T-ALL mouse model SPF-grade C57BL / 6 mice were pretreated with 5-fluorouracil (5-FU, Sigma-Aldrich, F6627) via intraperitoneal injection at a dose of 150 mg / kg for 5 consecutive days. After pretreatment, the mice were euthanized with carbon dioxide, and the femur and tibia were aseptically isolated to collect nucleated bone marrow cells. These nucleated bone marrow cells were placed in RPMI 1640 medium containing 10% fetal bovine serum, and lentiviral particles carrying the NOTCH1 intracellular domain (ICN1) mutant were added for in vitro transfection at an MOI of 100. After 48 hours of culture, GFP was sorted by flow cytometry. + Cells are sorted to ensure a purity of ≥95%.

[0045] The above GFP + Transfected cells and 1×10 5 A mixture of normal homologous C57BL / 6 mouse bone marrow cells (as supporting cells) was resuspended in sterile PBS and injected via tail vein into recipient mice receiving 9 Gy lethal whole-body irradiation (X-ray irradiation). Each mouse received a total of 5 × 10⁶ cells. 5 Each cell.

[0046] The survival status of recipient mice was observed daily. Starting from day 7 post-transplantation, body weight and activity levels were recorded every 3 days. Modeling was considered successful when mice exhibited at least two of the following indicators: ① Body weight decreased by ≥15% compared to pre-transplantation levels; ② Hair frizz, arched back, and reduced activity (≥50% reduction in activity level compared to normal); ③ Peripheral blood GFP levels. + The cell proportion is greater than 1%.

[0047] After successful modeling, bone marrow and spleen were harvested from mice, and single-cell suspensions were prepared. GFP was then purified by flow cytometry. + Cells (purity ≥95%), at 1×10 6 A dose of 4.5 Gy was injected via the tail vein into a new batch of C57BL / 6 receptor mice to establish a stable and continuously passaged NOTCH1-T-ALL model.

[0048] Treatment grouping and dosing regimen Mice that successfully underwent secondary transplantation were randomly divided into four groups (n=5-8): PBS control group: administered an equal volume of phosphate-buffered saline via tail vein; DNR monotherapy group: administered daunorubicin via tail vein at a single dose of 3 mg / kg; CpG monotherapy group: administered CpG oligodeoxynucleotide (ODN 1826) via tail vein at a single dose of 1 mg / kg; DNR+CpG combination group: administered DNR via tail vein on the morning of administration (single dose of 3 mg / kg), followed by CpG via tail vein at a 10-hour interval (single dose of 1 mg / kg). All groups received five doses on days 3, 6, 9, 16, and 23 post-secondary transplantation. The timing, route of administration, and single dose were strictly consistent across groups. It should be noted that the day of the second transplantation was day 0, and the doses on days 3, 6, and 9 were standard. To enhance the immune response, booster doses were administered every 7 days thereafter.

[0049] Disease dynamic monitoring Blood was collected starting on day 15 after the second transplant, and every 2-10 days thereafter (days 15, 17, 20, 22, 24, 26, 28, 30, 32, 35, 37, 40, 45, 50, 60, and 70). After lysing the red blood cells with erythrocyte lysis buffer, GFP was detected by flow cytometry. + The percentage of cells in nucleated cells in peripheral blood (n=8) was used to dynamically monitor the changing trend of peripheral blood leukemia burden.

[0050] Tissue tumor burden analysis Within 48 hours of the last administration, mice in each group (n=5) were sacrificed, and peripheral blood, bone marrow, spleen, and liver were collected. Single-cell suspensions of each tissue were prepared, treated with erythrocyte lysis buffer, and then flow cytometry was used to detect GFP in each tissue. + Cell percentage, quantifying the degree of leukemia infiltration in various organs.

[0051] Survival Analysis The remaining mice (n=8) were continuously monitored until death or the experimental endpoint (80 days post-transplantation). The overall survival (OS) of mice in each group was recorded, and Kaplan-Meier survival curves were plotted. Mice exhibiting a near-death state (e.g., weight loss ≥20%, inability to eat or drink independently, respiratory distress, or complete paralysis of the hind limbs, or peripheral blood tumor burden greater than 50%) were humanely euthanized and recorded as death events.

[0052] Statistical analysis Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons among multiple groups, and Tukey or Bonferroni post-hoc tests were used for pairwise comparisons between groups. Survival analysis used the Kaplan-Meier method to plot survival curves, and the Log-rank test was used to compare differences between groups. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism software (version 9.3.0).

[0053] Experimental results like Figure 3 –5 shows disease progression ( Figure 3 Compared with the PBS control group and each single-drug group, the peripheral blood GFP in the DNR+CpG combined treatment group was significantly higher. + The rate of cell ascent was significantly slowed (P<0.001), suggesting that combination therapy can effectively control disease progression; tissue leukemia burden ( Figure 4 ): GFP levels in bone marrow, spleen, liver, and peripheral blood of the DNR+CpG combined treatment group + The proportion of leukemia cells was significantly lower in all cases than in the control groups (P<0.001), indicating that the combined medication could systematically clear leukemia infiltration from various organs and prolong survival. Figure 5 The median survival of mice in the DNR+CpG combined treatment group was significantly longer than that in the control groups (P<0.001). At the follow-up endpoint (80 days post-transplantation), 62.5% (5 / 8) of the mice in the combined treatment group remained alive, and GFP was consistently undetectable in their peripheral blood. + Leukemia cells were detected, while mice in the PBS control group and each single-drug group died during the observation period.

[0054] The above results indicate that the combined use of DNR and CpG can synergistically exert anti-T-ALL effects, effectively control disease progression, reduce tumor burden in multiple organs, and significantly prolong survival. Example 4

[0055] DNR combined with CpG on dendritic cells and CD8 in NOTCH1-T-ALL mouse model + T cell regulation 1. Within 48 hours of the last administration, mice in each group (at least 3 mice per group) were sacrificed, and inguinal lymph nodes, bilateral femurs and tibias (for bone marrow collection) and peripheral blood were aseptically isolated. The preparation methods for lymph node, bone marrow and peripheral blood single-cell suspensions were the same as in Examples 2 and 3.

[0056] Lymph node maturation DC detection: Take a single-cell suspension of lymph nodes, add anti-mouse CD11c-BV510 (BioLegend, 117338), CD80-PE (BioLegend, 104708), and CD86-APC (BD, 558703) antibodies (dilution ratio 1:100), incubate at 4°C in the dark for 30 minutes, wash twice with PBS, and detect CD11c by flow cytometry. + CD80 + CD86 + Cells account for CD11c + The proportion of cells.

[0057] Bone marrow CD8 + T-cell activation assay: Bone marrow single-cell suspension was collected and anti-mouse CD3, CD4, CD8, and CD69 antibodies (CD3-APC, biolegend, 100236; CD4-BV421, biolegend, 116023; CD8-PE, biolegend, 100708; CD69-BV510, biolegend, 104531; all antibody dilutions were 1:100). The suspension was incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and analyzed by flow cytometry: (a) CD3 + CD8 + (a) The proportion of T cells among bone marrow GFP-nucleated cells; (b) CD69 + Activate CD8 + T cells account for CD8 + The proportion of T cells.

[0058] Serum cytokine detection: Peripheral blood was collected when mice were euthanized, serum was separated, and the concentrations of TNF-α (Invitrogen, 88-7324-22) and IFN-γ (Invitrogen, 88-7314-22) in the serum were detected by ELISA, following the instructions of the kit.

[0059] Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and P < 0.05 was considered statistically significant.

[0060] like Figure 6As shown, compared with the PBS control group and each single-drug group, CD11c in the lymph nodes of mice in the DNR+CpG combined treatment group was significantly higher. + CD80 + CD86 + The proportion of mature DCs increased significantly (P<0.01).

[0061] like Figure 7 As shown, compared with the PBS control group and each single-drug group, the CD8+ in the bone marrow of mice in the DNR+CpG combined treatment group was significantly higher. + T cell frequency and its CD69 + The activation rates of both groups were significantly increased (P<0.01); serum TNF-α and IFN-γ levels were also significantly increased (P<0.001). These results indicate that DNR+CpG combined therapy can promote DC maturation and CD8 activation. + T cell activation and expansion enhance the specific immune response against T-ALL. Example 5

[0062] Validation of secondary tumor attack induced by DNR combined with CpG therapy to induce long-term immune memory effect against T-ALL. Mice that had undergone DNR+CpG combined treatment in Example 3 and maintained disease-free survival for ≥57 days (i.e., mice that were still alive 80 days after transplantation at the follow-up endpoint in Example 3) were injected via tail vein with 1×10 6 GFP + NOTCH1-T-ALL cells (derived from passaged and preserved tumor cells from Example 3) were used for homologous tumor re-invasion. Age-matched, untreated C57BL / 6 mice of the same strain served as the control group and were also injected with 1×10⁻⁶ cells. 6 GFP + NOTCH1-T-ALL cells.

[0063] Peripheral blood was collected every two days starting from day 11 of the second tumor attack. Red blood cells were lysed using erythrocyte lysis buffer, and GFP was detected by flow cytometry. + The percentage of cells in the peripheral blood nucleated cells is used to dynamically monitor tumor recurrence.

[0064] On day 24 after the second challenge, all surviving mice and control mice were sacrificed, and bone marrow, spleen, and liver were collected. Nucleated single-cell suspensions were prepared, and GFP levels in each tissue were detected by flow cytometry. + The proportion of leukemia cell infiltration.

[0065] Nucleated cells from the bone marrow and spleen of mice were collected on day 24 after the second challenge. Antibodies against mouse CD3-APC, CD4-BV421, CD8-PE, CD44-PE / Cy7 (eBioscience, 25-0441-82), and CD62L-APC / Cy7 (BioLegend, 104428) were added for surface staining. Flow cytometry analysis was performed on CD8 cells. + Effector memory T cells (TEM; CD44hiCD62Llow) subset constitutes CD8 + Regarding the proportion of T cells, it should be noted that, except for CD44 and CD62L, the colors and catalog numbers of the other antibodies are consistent with those in Example 4.

[0066] like Figure 8 As shown, GFP was not detected in the peripheral blood of mice in the DNR+CpG combined treatment group after the second tumor attack. + Cells (detection limit <0.001%); such as Figure 9 As shown, on day 24 post-attack, GFP levels in the bone marrow, spleen, and liver of control mice were significantly lower. + The cell proportions of all three groups were >80%, and GFP was not detected in the tissues of mice that survived long-term after combination therapy. + Cells (detection limit <0.001%); such as Figure 10 As shown, CD8+ levels in the bone marrow and spleen of mice in the combined treatment group were significantly increased. + CD44 in T cells hi CD62L low The proportion of effector memory T cells (TEM) was significantly higher than that of the control group (P<0.05).

[0067] The above results indicate that DNR+CpG combined therapy can not only clear the initial tumor burden, but also induce long-term, functional anti-T-ALL immune memory, giving the host complete protective immunity against re-attack from the same tumor.

[0068] In summary, this invention, through the sequential combined application of DNR and CpG, successfully constructed an "ICD-induced-DC-targeted activation-CD8" model in a NOTCH1-driven T-ALL mouse model. + The cascade immune response pathway of "T cell expansion and activation" involves: DNR inducing immunogenic death of T-ALL cells, releasing a full spectrum of tumor antigens and DAMPs; CpG specifically activates dendritic cells (DCs), driving their maturation and enhancing their cross-presentation capacity, thereby efficiently activating CD8+. + The specific activation, expansion, and effector function of T cells were enhanced. Experimental results confirmed that this strategy significantly increased the proportion of mature dendritic cells (DCs) in lymph nodes and CD8+ in bone marrow. +This approach modulates T-cell frequency and TEM subset proportions, and completely blocks leukemia relapse after secondary tumor attack. Compared to therapies that directly modify T cells, this approach relies on the reactivation of the endogenous immune system, avoiding potential risks such as T-cell depletion, cytokine storm, and off-target toxicity. It achieves a balance between safety, efficacy, and durable immune memory, providing a new strategy for the immunotherapy of T-ALL.

Claims

1. A drug combination for treating acute T-lymphoblastic leukemia, characterized in that, The drug combination comprises daunorubicin and CpG oligodeoxynucleotides of type B, wherein the daunorubicin and the CpG oligodeoxynucleotides are each independently formulated for sequential administration.

2. The drug combination according to claim 1, characterized in that, The sequential administration involves first administering a daunorubicin preparation, followed by a CpG oligodeoxynucleotide preparation after a certain interval.

3. The drug combination according to claim 2, characterized in that, The interval is 10 hours.

4. The drug combination according to claim 1, characterized in that, The sequence of the class B CpG oligodeoxynucleotide is shown in SEQ ID NO:

1.

5. The drug combination according to claim 1, characterized in that, The unit dose of the daunorubicin is 3 mg / kg, and the unit dose of the CpG oligodeoxynucleotide is 1 mg / kg.

6. Use of the pharmaceutical combination based on any one of claims 1-5 in the preparation of a medicament for treating acute T-lymphoblastic leukemia.

7. The application according to claim 6, characterized in that, The drug combination is used in the preparation of drugs to enhance CD8. + Application in drugs that induce T-cell-mediated immune responses against acute T-lymphoblastic leukemia.

8. The application according to claim 6, characterized in that, The use of the drug combination in the preparation of a drug for inducing immune memory against acute T-lymphoblastic leukemia.

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