A method for enhancing activity of cik cells and its application in tumor treatment

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a method for enhancing CIK cell activity and application thereof in tumor treatment. The method separates exosomes (particle size 50-150 nm, expressing TSG101 and Alix, not expressing CD9 and Calnexin) from CIK cell culture supernatant by ultracentrifugation, and reinfuses the exosomes at a proportion of 100-500 ug exosomes per 2x10 7 CIK cells. The exosomes can significantly improve the expression level of CIK cell IFN-gamma, promote PBMC proliferation, and in a liver cancer mouse model, it is proved that the tumor inhibition rate (70.7%) of the combination group (2x10 7 CIK cells+400 ug exosomes) is significantly better than that of the single treatment group. The application uses waste exosome resources to safely enhance the anti-tumor activity of CIK cells, and is suitable for the treatment of liver cancer, lung cancer, breast cancer and other solid tumors and hematological tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a method for enhancing the activity of CIK cells and application thereof in tumor treatment. BACKGROUND

[0002] Adoptive immunotherapy is one of the important forms of tumor immunotherapy. Among various immunotherapy methods, chimeric antigen receptor T cell (CAR-T) therapy is the most concerned therapy at present, but the preparation process of the therapy is complex, the application is limited to hematological tumors, and most importantly, the cost is high, and the applicable population is extremely limited. In addition, tumor infiltrating lymphocyte (TIL) therapy is also a relatively concerned therapy, but the preparation process of the therapy is also relatively complicated, the operation condition is required to be relatively high, the yield is low, and the application range is also relatively limited. Cytokine-induced killer (CIK) therapy is one of the most mature immunotherapy methods, which is widely used in the treatment of various tumors due to its low cost, mature and simple preparation process, and has been included in the CSCO Primary Liver Cancer Diagnosis and Treatment Guide 2022 Edition as a recommended postoperative adjuvant therapy. CIK cells are prepared by stimulating and expanding peripheral blood mononuclear cells with interleukin-2 (IL-2), interferon-γ (IFN-γ) and other cytokines. Due to the strong proliferation ability, good killing effect, wide killing spectrum and small toxic and side effects of CIK cells, CIK cells can be used as an effective adjuvant anticancer means for eradicating residual cancer cells, preventing recurrence and improving survival rate. As the core effector cell of adoptive immunotherapy, CIK cells directly participate in the effector phase of immune surveillance by releasing cytotoxic granules such as perforin and granzyme B, and have the treatment function of non-MHC restricted recognition and elimination of malignant transformed cells due to the combination of T / NK cell phenotypic characteristics. However, in the application process of CIK cell therapy, it is found that some patients have insufficient anti-tumor immune response intensity and insignificant anti-tumor effect, which needs to be further improved. Some studies have added adjuvant therapies such as cytokines, immunologically active polypeptides and immunologically active antibodies to CIK cell products, but these additives often increase additional risks such as inducing cytokine storm, causing allergic reactions, and triggering some unpredictable toxic side effects. Therefore, finding a safe and effective method to improve the anti-tumor effect of CIK cell therapy has become a current concern.

[0003] Exosomes are a class of extracellular vesicles with a diameter of 30-150 nm, double-layer phospholipid membrane structure, first reported by Wolf and then found in mature reticulocytes by Harding. Current studies have shown that most cells secrete exosomes, and most cells, including tumor cells and immune cells, are also prone to uptake exosomes. Exosomes can unload parent cell markers and deliver them to other cells, thereby playing a regulatory function. The intercellular communication process of exosomes greatly affects tumor progression, metastasis, angiogenesis and drug resistance. In addition to the direct anti-tumor effect of exosomes, exosomes can also be used as a drug delivery system to deliver anti-tumor drugs. Due to the potential of exosomes to affect the tumor microenvironment, efforts are still being made to develop exosomes that target the tumor microenvironment and regulate cancer progression. It can be seen that exosomes exhibit multi-dimensional intervention potential in the field of tumor treatment, and therefore have important clinical translation research value.

[0004] In the production and preparation process of CIK cells, a large culture system of several liters is used. After the CIK cell components in the several liter culture system are recovered and washed, they are then returned to the patient for treatment application. The culture medium and a large amount of exosomes secreted by CIK cells in the culture system are discarded in conventional CIK treatment. The exosomes secreted by CIK cells may carry a large amount of cytokines and other immunologically active substances, and preliminary studies have confirmed that they have the potential for immune activation. CIK cells rely on strong immune activity to exert an anti-tumor effect, and if further combined with the exosomes secreted by themselves, an improved CIK therapy can be established, which is expected to enhance the immune activity of CIK cells and the immune activity of lymphocytes in the recipient, thereby improving the anti-tumor effect of cell therapy. However, there is currently no public information on the recovery of exosomes from CIK cells and their combined application with CIK cells. SUMMARY

[0005] In view of the problems in the prior art, the present application provides a method for enhancing the activity of CIK cells and its application in tumor treatment. The method for enhancing the activity of CIK cells is based on conventional CIK cell culture, combined with exosomes secreted by CIK cells, to enhance the immune activity of CIK cells and the immune activity of lymphocytes in the recipient, thereby improving the anti-tumor effect of cell therapy.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0007] The present application discloses the application of CIK cell-derived exosomes in the preparation of a drug for enhancing the activity of CIK cells.

[0008] Further, the exosomes are obtained by ultracentrifugation from the CIK culture supernatant.

[0009] The application also discloses a method for enhancing CIK cell activity, characterized in that CIK cell-derived exosomes are combined with CIK cells.

[0010] Further, the combined application ratio of the exosomes and the CIK cells is 100-500 µg of the exosomes corresponding to 2×10 7 CIK cells.

[0011] Further, the combined application ratio of the exosomes and the CIK cells is 100-500 µg of the exosomes corresponding to 2×10 7 CIK cells.

[0012] The application also discloses application of any of the above methods in preparation of an antitumor drug.

[0013] Further, the tumor includes liver cancer, lung cancer, breast cancer, colorectal cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, cholangiocarcinoma, thyroid cancer, glioma, lymphoma and leukemia.

[0014] The application also discloses an antitumor drug composition, characterized in that the composition comprises CIK cells, CIK cell-derived exosomes and a pharmaceutically acceptable carrier.

[0015] Compared with the prior art, the application has the following beneficial effects.

[0016] The CIK cell-derived exosomes have potential immune activation functions. The application combines the CIK cell-derived exosomes with the CIK cells for the first time, and obtains a modified CIK cell product, which has stronger immune activity and antitumor function than conventional CIK cells; the modified CIK cell product can be applied to various malignant tumor indications of conventional CIK cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 CIK cells prepared by a conventional method.

[0018] Figure 2 Extraction and identification of CIK cell-derived exosomes. A is the morphological characteristics of the CIK exosomes under an electron microscope; B is CIK exosome particle size detection; and C is Western blot detection of CIK cell exosome marker protein expression characteristics.

[0019] Figure 3 CIK cell-derived exosomes enhance the expression level of IFN-γ of CIK cells. A is a flow cytometry graph; and B is a columnar graph.

[0020] Figure 4The effect of the improved CIK cell therapy (i.e. CIK cells combined with CIK cell-derived exosomes) on liver cancer is improved. * Compared with the model control group, P <0.05. # Compared with the exosome group, P <0.05. & Compared with the CIK group, P <0.05.

[0021] Figure 5 The broad-spectrum tumor killing effect of the improved CIK cells is significantly enhanced. Among them, * Compared with the untreated CIK group, P <0.05. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be further described below in combination with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present application and not to limit the scope of the present application. It should also be understood that the present application can be implemented in other different forms and is not limited to the following embodiments. Those skilled in the art can make various modifications and modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by those skilled in the art.

[0023] The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended in the specification. The reagents or instruments used, unless otherwise specified, are conventional products purchased through conventional sales channels.

[0024] Example 1 Preparation of CIK cells.

[0025] (1) Extract 100 mL of human peripheral venous blood or collect umbilical cord venous blood, dilute with an equal volume of physiological saline, and then use human lymphocyte separation medium to separate and extract peripheral blood mononuclear cells (PBMC) by density gradient centrifugation.

[0026] (2) Add the mononuclear cells extracted in step (1) to the cell culture solution, adjust the cell concentration to (0.5-1) × 10 7 / mL, and add 5% autologous serum; in addition, add IFN-γ (650 U / mL), anti-CD3 (88 ng / mL), and IL-1α (560 U / mL); then, place in a 75 cm 2 culture bottle and incubate at 37°C with 5% CO2.

[0027] (3) Observe the cells after 24 hours and add IL-2 (580 U / mL).

[0028] (4) Daily observation of cells, according to the cell growth state, the cells were transferred to the culture bag, the culture solution was supplemented to a final volume of 200 mL, 5 mL of autologous serum was added, and IL-2 was supplemented.

[0029] (5) The culture solution and IL-2 were supplemented every 1 to 2 days from the 6th to 12th day, and the final volume was 2 L.

[0030] (6) CIK cells were obtained on the 13th day (see Figure 1 ).

[0031] Example 2: Recovery of CIK cell-derived exosomes.

[0032] The exosomes in the supernatant of CIK cell culture were extracted by ultracentrifugation, and the specific process was as follows.

[0033] (1) After 13 days of CIK cell culture in Example 1, the supernatant was collected and centrifuged at 800 rpm for 5 min to remove dead cells, cell debris, and extracellular particles larger than 200 nm.

[0034] (2) High-purity exosomes were obtained by centrifugation at 12000 rpm for 30 min and stored at -80°C.

[0035] (3) Morphological identification: After being negatively stained with 2% uranyl acetate and dried on a copper grid, the exosome sample was observed under a transmission electron microscope (accelerating voltage 80 kV) to observe the ultrastructure of the exosomes. Under the electron microscope, CIK exosomes had obvious vesicular structures, and the membrane structure was complete without rupture, which was consistent with the morphological characteristics of exosomes (see Figure 2 A).

[0036] (4) Particle size distribution analysis: The particle size distribution of exosomes was measured and the particle concentration was estimated using a nano-flow detector. Nano-silicon dioxide fluorescent microspheres (250 nm) were used to calibrate the instrument, and the sample flow rate and particle concentration were calculated. The standard curve of scattering light intensity and particle size was established using standard spheres (68 nm, 91 nm, 113 nm, 155 nm), and the particle size distribution of the exosome sample was obtained. The detection results showed that the average particle size of CIK exosomes was 75.77 nm, which was consistent with the range of 50-150 nm of exosomes (see Figure 2 B).

[0037] (5) Molecular marker detection: Western blot was used to verify the exosome characteristic proteins (CD9, TSG101 and Alix), with Calnexin as a negative control. The specific process is as follows: after mixing the exosome PBS resuspension and the lysis buffer (RIPA+1% PMSF) at a volume ratio of 3:7, total protein was extracted. After quantification by BCA method, 27 μg / well was loaded, 10% SDS-PAGE electrophoresis was carried out, and the membrane was blocked with 5% skim milk for 1 h. The primary antibody was incubated at 4°C overnight, the HRP-labeled secondary antibody (1:5000) was reacted at room temperature for 1 h, and the ECL strip was developed and analyzed for specific bands. The Western blot results show that the extracted CIK cell exosomes express cytosolic proteins TSG101 and Alix, and do not express membrane protein marker CD9 and negative cytoplasmic protein Calnexin (see Figure 2 C).

[0038] According to the above detection, it is confirmed that the CIK cell exosomes separated and extracted meet the characteristics of exosomes.

[0039] Example 3 verifies that CIK cell-derived exosomes enhance the activity of CIK cells.

[0040] Flow cytometry experiment was used to detect the immune activation effect of exosomes on promoting CIK cells to express functional molecules IFN-γ. The specific process is as follows.

[0041] (1) Take the CIK cells treated with 200 μg / mL CIK exosomes for 72 h, and the blank control group without treatment, add the stimulation mixture containing PMA (100 ng / mL), ionomycin (1 μg / mL) and brefeldin A (10 μg / mL), incubate at 37°C, 5% CO2for 4-6 h, activate the cytokine secretion pathway.

[0042] (2) Collect the above cells, centrifuge and wash, and resuspend the cell precipitate with 500 μL 4% paraformaldehyde solution / nuclear fixation buffer. Fix at room temperature for 20 min in the dark. Maintain cell membrane integrity. After fixation, add 500 μL membrane-breaking working solution / nuclear permeabilization buffer (Perm / Wash buffer) to the cells, centrifuge at 1800 rpm for 5 min, repeat 3 times for sufficient permeation, and discard the supernatant and collect the precipitate.

[0043] (3) Resuspend the cell precipitate with 100 μL PBS, add fluorescent antibody IFN-γ-PE, and stain at low temperature (4°C) in the dark for 20 min.

[0044] (4) Add the stained cells to PBS and wash twice (500 μL / tube, 1800 rpm centrifugation for 5 min), discard the supernatant, resuspend and perform flow cytometry detection.

[0045] (5) Flow cytometry results showed that compared with the blank control group, the level of IFN-γ expressed by CIK cells treated with exosomes was significantly increased (see Figure 3 ).

[0046] Example 4 confirmed the anti-tumor effect of modified CIK cell therapy (i.e. CIK cells combined with CIK cell-derived exosomes).

[0047] (1) 24 six-week-old female NOD / SCID mice were selected, and after one week of adaptive feeding, the experiment began.

[0048] (2) Human liver cancer cells HepG2 (4 x 10 6 cells / 100 µL PBS) were injected subcutaneously into the right axillary of the mice, and the day of subcutaneous injection was recorded as day 0. When the tumor grew to 100 mm 3 (approximately day 7), the tumor volume was recorded, and the mice were grouped according to the tumor volume, and randomly divided into 4 groups: model control group, exosome group, CIK group, and CIK+exosome group, with 6 mice in each group.

[0049] (3) The treatment measures for each group of mice were as follows: ① Model control group mice, each tail vein injected with 100 µL PBS; ② Exosome group mice, each tail vein transfused with 400 µg exosomes / 100 µL PBS for treatment; ③ CIK group mice, each tail vein transfused with 2 x 10 7 CIK / 100 µL PBS for treatment; ④ CIK+exosome group mice, each tail vein transfused with (2 x 10 7 CIK+400 µg exosomes) / 100 µL PBS for treatment; the above treatments were performed once a week for 3 consecutive weeks.

[0050] (4) The body weight of the mice was recorded every 2 days, the long diameter and short diameter of the tumor were measured, and the tumor volume was calculated. Tumor volume (mm 3 ) = [length (mm) x width (mm) 2 ] / 2. The tumor growth curve was drawn, and the experiment was terminated when the largest subcutaneous transplanted tumor reached 1500 mm 3 .

[0051] (5) The experimental results showed that compared with the model control group mice, the growth trend of the subcutaneously transplanted tumors in the exosome group, CIK group, and CIK+exosome group mice was significantly slowed down; compared with the exosome group and CIK group, the growth trend of the subcutaneously transplanted tumors in the CIK+exosome group mice was further significantly slowed down (see Figure 4) ; and the tumor inhibition effect of the CIK + exosome group was stronger than the sum of the tumor inhibition effects of the exosome group and the CIK group (see Table 1). This shows that the combined use of CIK-derived exosomes significantly enhances the anti-tumor effect of CIK cell therapy, and the two have a synergistic effect, which is a new improved CIK cell therapy.

[0052] Table 1. Enhanced anti-mouse hepatocarcinoma transplanted tumor effect of improved CIK cell therapy.

[0053]

[0054] Example 5 verifies that CIK cell-derived exosomes enhance the broad-spectrum anti-tumor effect of CIK cells.

[0055] The calcium calcein release experiment was used to detect the immune activation effect of exosomes on CIK cells expressing lytic killing of tumor cells, and the specific process is as follows.

[0056] (1) The following human tumor cell lines were selected as target cells: lung cancer A549, breast cancer MCF7, colorectal cancer SW480, gastric cancer NCI-N87, prostate cancer DU145, ovarian cancer SKOV3, cervical cancer SiHa, endometrial cancer ECC-1, pancreatic cancer PANC-1, cholangiocarcinoma HuH-28, thyroid cancer TPC1, glioma U251, lymphoma SNK-6, and leukemia K562. Collect the above tumor target cells in the logarithmic growth phase, label with 25 uM calcein-AM at 37°C, 5% CO2 for 25 min, and then wash with PBS for 3 times for standby.

[0057] (2) Take CIK cells treated with 200 ug / mL CIK exosomes for 72 h, and untreated CIK cells as control group, and co-culture the cells at a ratio of 20:1 with the above various target cells in a 96-well plate, each well containing 20000 CIK cells and 1000 tumor cells of any kind. Another spontaneous release group, in which the target cells are only incubated with the culture medium; and the maximum release group, in which the target cells are incubated with 2% Triton X-100. After incubation at 37°C, 5% CO2 for 4 hours, the fluorescence microplate reader detects the cell supernatant, the excitation wavelength is 485 nm, and the emission wavelength is 535 nm. Each determination is performed in triplicate. The specific lytic killing percentage is: (OD 实验释放 -OD 自发释放 ) / (OD 最大释放 -OD 自发释放 ) ×100%.

[0058] (3) The killing rates of CIK cells in different treatment groups on the above 14 kinds of tumor cells are shown in Table 2 and Figure 5, and the killing efficiency of the CIK cells treated by the CIK cell-derived exosomes on 14 kinds of tumor cells was significantly higher than that of the untreated CIK cells P <0.05), indicating that the improved CIK cells treated by the CIK cell-derived exosomes have a significantly enhanced anti-tumor effect and have a broad spectrum.

[0059] Table 2 The improved CIK cells have a significantly enhanced broad-spectrum tumor killing effect (killing rate, %).

[0060]

[0061] In summary, compared with the conventional CIK cell therapy, the improved CIK cell therapy of the present application further combines the exosomes secreted by the CIK cells, effectively improves the activity of the immune cells such as CIK cells, and enhances the anti-tumor effect of the cell therapy, and has a broad application prospect.

[0062] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them. The basic principles and main features of the present application have been described above with specific embodiments, and some modifications or replacements can be made on the basis of the present application, but these modifications or replacements do not make the corresponding technical solutions deviate from the scope of the present application.

Claims

1. A method for enhancing the activity of CIK cells in vitro, characterized in that: CIK cells were pretreated with CIK cell-derived exosomes.

2. The use of CIK cells obtained by the method of claim 1 in the preparation of antitumor drugs.

3. The application as described in claim 2, characterized in that, The tumors include liver cancer, lung cancer, breast cancer, colorectal cancer, stomach cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, bile duct cancer, thyroid cancer, glioma, lymphoma, and leukemia.

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