Application of combined use of NK cell and GD2 antibody in preparation of medicine for treating neuroblastoma

Abstract

The invention relates to the technical field of medicines, in particular to application of combined use of NK cells and a GD2 antibody in preparation of a medicine for treating neuroblastoma. The NK cell and the GD2 antibody are jointly used to prepare a medicine for treating neuroblastoma. In-vivo and in-vitro experiments find that the umbilical cord blood NK cell combined with the GD2 antibody can significantly enhance the tumor killing ability and inhibit the growth of neuroblastoma cells, provides a new therapeutic medication scheme for the treatment of neuroblastoma, and has a good application prospect in the prevention and treatment of neuroblastoma.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of the combined use of NK cells and GD2 antibodies in the preparation of drugs for treating neuroblastoma. Background Art

[0002] Neuroblastoma is the most common extracranial solid tumor in childhood and the most common and fatal tumor in infancy, accounting for about 10% of childhood malignancies and 15% of all childhood cancer-related deaths. Neuroblastoma is characterized by a high degree of heterogeneity, which is reflected in the clinical manifestations from spontaneous regression or differentiation to extensive metastatic dissemination. Even with intensive multimodal treatment, the long-term survival rate of high-risk neuroblastoma is currently still less than 50%. Despite various changes in the treatment strategies for high-risk neuroblastoma patients over the years, the prognosis of this group remains poor and the treatment is still challenging.

[0003] Currently, in neuroblastoma patients, GD2 antibodies can achieve complete remission in patients with bone marrow residues and can improve the survival rate of high-risk neuroblastoma patients. However, the dose-intensive chemotherapy for high-risk neuroblastoma consumes endogenous natural killer (NK) cells and impairs antibody-dependent cell-mediated cytotoxicity, which affects the efficacy of GD2 antibodies and their single use has poor efficacy.

[0004] Immune cell therapy is the fourth tumor treatment method after surgery, radiotherapy, and chemotherapy, with advantages such as strong specificity and few side effects. Among them, NK cells have the ability to recognize cancer and mediate killing, and do not induce graft-versus-host disease, cytokine release syndrome, or immune effector cell-related neurotoxicity syndrome. Therefore, their use as allogeneic cell therapy has potential prospects. Currently, most studies focus on the treatment with NK cells from parental haploid or patient autologous sources, but there are situations such as insufficient NK cell expansion and impaired NK cell function, which may lead to poor efficacy of NK cell therapy. Umbilical cord blood is one of the sources of hematopoietic stem cell transplantation. The low requirement for HLA matching and the low incidence of graft-versus-host disease have made umbilical cord blood increasingly a focus of cell therapy. Given the poor efficacy of GD2 antibodies, therefore, there is an urgent need for new methods to improve the therapeutic effect of GD2 antibodies in neuroblastoma. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of the combined use of NK cells and GD2 antibodies in the preparation of drugs for treating neuroblastoma.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the application of GD2 antibodies in the preparation of NK cell potentiators.

[0008] Preferably, the NK cells are used for preparing a medicament for treating neuroblastoma.

[0009] The present invention also provides the use of NK cells and GD2 antibody in combination in the preparation of a medicament for treating neuroblastoma.

[0010] The present invention also provides a medicament for treating neuroblastoma, comprising NK cells and GD2 antibody.

[0011] Preferably, it further comprises a pharmaceutically acceptable excipient or carrier.

[0012] Preferably, the NK cells are differentiated from human umbilical cord blood mononuclear cells.

[0013] Preferably, the pathogenic cancer cells of the neuroblastoma are Kelly cells.

[0014] Preferably, the dosage of the NK cells is the same as the number of Kelly cells.

[0015] Preferably, the final concentration of the GD2 antibody in the medicament for treating neuroblastoma is 0.8 - 1.2 μg / mL.

[0016] Preferably, the dosage form of the medicament is an injection or an infusion.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] In in vitro and in vivo experiments of the present invention, it is found that umbilical cord blood NK cells combined with GD2 antibody can significantly enhance the ability to kill tumors and inhibit the growth of neuroblastoma cells, providing a new treatment drug plan for the treatment of neuroblastoma and having good application prospects in the prevention and treatment of neuroblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0020] Figure 1 It is an immunofluorescence image (×200) of co - culture of umbilical cord blood NK cells combined with GD2 antibody and neuroblastoma Kelly cells at different time points.

[0021] Figure 2Flow cytometry apoptosis analysis of co-culture of umbilical cord blood NK cells combined with GD2 antibody and neuroblastoma Kelly cells (A Flow cytometry apoptosis detection chart; B Percentage of apoptotic cells).

[0022] Figure 3 Effect of umbilical cord blood NK cells combined with GD2 antibody on the growth of orthotopic tumors in neuroblastoma mice (A Flow chart of mouse tumor-bearing experiment; B Bioluminescence imaging of mouse orthotopic tumors; C Overall survival; D Disease-free survival). Detailed implementation manners

[0023] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Example 1

[0025] 1. Experimental materials

[0026] (1) Drugs: GD2 antibody (from BD Pharmingen), IgG antibody (from BD Pharmingen);

[0027] (2) NK cells: NK cells expanded from human umbilical cord blood mononuclear cells (from Shandong Cord Blood Bank);

[0028] (3) Cancer cells: Neuroblastoma Kelly cells from ECACC (from European Collection of Authenticated Cell Cultures).

[0029] 2. Experimental grouping

[0030] (1) Control group: Blank control, that is, neuroblastoma cells are only treated with IgG antibody;

[0031] (2) GD2 antibody group: Only treat neuroblastoma cells with 1 μg / mL GD2 antibody;

[0032] (3) NK cell group: Only treat neuroblastoma cells with umbilical cord blood NK cells;

[0033] (4) NK cell + GD2 antibody group: Treat neuroblastoma cells with umbilical cord blood NK cells and GD2 antibody simultaneously.

[0034] 3. Detection of the effect of umbilical cord blood NK cells combined with GD2 antibody on killing neuroblastoma cells by in vitro immunofluorescence experiment

[0035] (1) Digest, centrifuge, resuspend and count the neuroblastoma tumor cells with GFP, and then plate them in a six-well plate overnight, adding 2×10 5 tumor cells per well.

[0036] (2) Centrifuge the umbilical cord blood NK cells to obtain cell pellets, count the required cell amount according to the effector-to-target ratio, then add 1 μl of 1 mM cell tracker (from Thermo Fisher Scientific) to 1 ml of serum-free lymphocyte medium to keep the final concentration at 1 μM. Keep this mixed solution at 37°C, add it to the cell pellets, mix well to resuspend the cells, and place them in a cell incubator for dark incubation for 30 minutes (37°C, 5% CO2).

[0037] (3) After incubation, add complete medium to centrifuge and wash the cells, then add the corresponding amount of NK cells and GD2 antibody to a six-well plate, and immediately perform continuous culture and photographing with a live cell fluorescence microscope.

[0038] 4. Experimental results

[0039] The experimental results are as Figure 1 shown. When NK cells and neuroblastoma tumor cells were co-cultured at a ratio of 1:1, after adding NK cells, the NK cells began to approach the tumor cells for killing around 4 hours, and it was clearly observable that the tumor cells were surrounded by NK cells and the death of tumor cells occurred at 8 hours. The addition of GD2 antibody further increased the approach and contact of NK cells to tumor cells, thus more effectively lysing tumor cells. This demonstrated that NK cells have certain killing activity against neuroblastoma tumor cells, and it also proved the promoting effect of GD2 antibody on the killing activity of NK cells.

[0040] Example 2

[0041] 1. Experimental materials

[0042] (1) Drugs: GD2 antibody (from BD Pharmingen), IgG antibody (from BD Pharmingen);

[0043] (2) NK cells: NK cells amplified from human umbilical cord blood mononuclear cells;

[0044] (3) Cancer cells: Neuroblastoma Kelly cells.

[0045] 2. Experimental groups

[0046] (1) Control group: Blank control, that is, neuroblastoma cells were only treated with IgG antibody;

[0047] (2) GD2 antibody group: Only treat neuroblastoma cells with 1 μg / mL GD2 antibody;

[0048] (3) NK cell group: Only treat neuroblastoma cells with umbilical cord blood NK cells;

[0049] (4) NK cell + GD2 antibody group: Cord blood NK cells and 1 μg / mL GD2 antibody were used to treat neuroblastoma cells simultaneously.

[0050] 3. Detection of the effect of cord blood NK cells combined with GD2 antibody on the killing of neuroblastoma cells by flow cytometry apoptosis assay in vitro

[0051] (1) Digest, centrifuge, resuspend and count the tumor cells, then inoculate a certain number of cells into a 6-well plate and culture overnight in an incubator until they adhere to the wall. Then add NK cells and GD2 antibody according to different groups and continue to culture for 2 hours to collect the cells.

[0052] (2) Aspirate the cell culture medium into a flow tube, then gently wash the cells twice with PBS buffer and discard the PBS solution. Add trypsin without EDTA to digest the cells. When the cells become deformed and detached, neutralize the trypsin with the medium in the flow tube and aspirate the digested cell suspension into the flow tube.

[0053] (3) Set the centrifuge speed to 1000 revolutions per minute, centrifuge for 5 minutes, discard the upper layer of the culture medium, wash the cell pellet twice with PBS buffer, then add CD45 dye (from BD Biosciences) and mix well, incubate in the dark for 30 minutes, and then wash with PBS buffer.

[0054] (4) Aspirate 100 μl of 1×binding buffer to resuspend the cells in the flow tube. Under dark conditions, add 5 μl of AnnexinV-FITC dye and 5 μl of PI dye (from BD Biosciences) to the flow tube, mix well, and incubate in the dark at room temperature for 15 minutes.

[0055] (5) Add 400 μl of PBS buffer, mix well, and immediately perform flow cytometry apoptosis detection on the machine.

[0056] 4. Experimental results

[0057] The experimental results are as Figure 2 shown. When NK cells and neuroblastoma tumor cells were co-cultured at a ratio of 1:1, after adding NK cells and co-culturing for 2 hours, apoptosis molecule detection was performed, and it was found that cord blood NK cells combined with GD2 antibody had a significant killing effect on tumor cells, and GD2 antibody had a significant synergistic effect on NK cells.

[0058] Example 3

[0059] 1. Experimental materials

[0060] (1) Drugs: GD2 antibody (from BD Pharmingen), IgG antibody (from BD Pharmingen);

[0061] (2) NK cells: NK cells expanded from human umbilical cord blood mononuclear cells;

[0062] (3) Cancer cells: Neuroblastoma Kelly cells;

[0063] (4) Commercially available immunodeficient NSG mice.

[0064] 2. Experimental grouping

[0065] (1) Control group: Neuroblastoma-bearing mice were only treated with IgG antibody;

[0066] (2) GD2 antibody group: Neuroblastoma-bearing mice were only treated with GD2 antibody;

[0067] (3) NK cell group: Neuroblastoma-bearing mice were only treated with umbilical cord blood NK cells;

[0068] (4) NK cell + GD2 antibody group: Neuroblastoma-bearing mice were treated with both umbilical cord blood NK cells and GD2 antibody.

[0069] 3. Antitumor effect of the combination of umbilical cord blood NK cells and GD2 antibody in vivo on orthotopic xenograft mice with neuroblastoma

[0070] (1) Digest, centrifuge, resuspend, and count the well-grown neuroblastoma cell line LUC-Kelly cells. Wash the cells twice with PBS, then resuspend the cells with PBS, adjust the cell density to 1×10 6 cells / 20 μl, place on ice, and quickly transfer to the animal room for experiments.

[0071] (2) Anesthetize the mice intraperitoneally, then shave the area near the left kidney, find the lower edge of the left costal arch, gently make a small incision in the skin, then separate the muscle layer, find the left kidney, gently lift and expose it, then inject 20 μl of cell suspension under the renal capsule, rotate and withdraw the needle, and gently press with a sterile cotton ball to avoid cell leakage, then gradually suture the muscle layer and skin layer, and perform iodophor disinfection.

[0072] (3) Starting from the 7th day after inoculation, perform bioluminescence imaging once a week, and record the change in mouse body weight at the same time. A total of 20 mice were orthotopically xenografted and divided into 4 groups. After confirming tumor formation on the 7th day after tumor inoculation, start drug administration. The administration of umbilical cord blood NK cells was on D1, 4, 7, 11 (a total of 4 times), and each time 1×10 7The NK cells were resuspended in 200 μl of PBS and injected via the tail vein. The remaining groups were injected with 200 μl of PBS via the tail vein as a control. The GD2 antibody was 60 μg per mouse per injection, resuspended in 100 μl of PBS, and administered intraperitoneally on days 1, 3, 5, 7, and 9. According to the ethical principles, when the body weight of the mice decreased by more than 20% or the mice showed other unbearable conditions, the experiment was terminated prematurely and the mice were euthanized.

[0073] 4. Experimental results

[0074] The experimental results are as Figure 3 shown. In the IgG control group, the tumors grew significantly. In the GD2 antibody monotherapy group, although the tumors were somewhat controlled, they continued to grow after a short period of control. In the cord blood NK cell group, the tumors of some mice continued to grow. Compared with other groups, the tumors of the mice in the cord blood NK cell combined with GD2 antibody group were better controlled. These results are basically consistent with the in vitro results, demonstrating the effectiveness of NK cells prepared from cord blood in the treatment of neuroblastoma. The GD2 antibody has a synergistic effect on NK cells, and the combination of NK cells and GD2 antibody has a more significant anti-tumor effect.

[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of GD2 antibody in the preparation of NK cell potentiators.

2. The application according to claim 1, wherein The NK cells are used for the preparation of drugs for treating neuroblastoma.

3. Use of the combined use of NK cells and GD2 antibody in the preparation of drugs for treating neuroblastoma.

4. A drug for treating neuroblastoma, characterized in that, It includes NK cells and GD2 antibody.

5. The drug according to claim 4, characterized in that, It also includes pharmaceutically acceptable excipients or carriers.

6. The drug according to claim 4, characterized in that, The NK cells are differentiated from human umbilical cord blood mononuclear cells.

7. The drug according to claim 4, characterized in that, The pathogenic cancer cells of the neuroblastoma are Kelly cells.

8. The drug according to claim 7, characterized in that, The dosage of the NK cells is the same as the number of Kelly cells.

9. The drug according to claim 4, wherein The final concentration of the GD2 antibody in the drug for treating neuroblastoma is 0.8 - 1.2 μg / mL.

10. The drug according to claim 4, characterized in that, The dosage form of the drug is injection or infusion.

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