Method for enhancing activity of CIK (Cytokine-Induced Killer) cells and application of method in tumor treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for enhancing the activity of CIK cells and application of the CIK cells in tumor treatment. The method comprises the following steps: separating exosomes (the particle size is 50-150 nm, TSG101 and Alix are expressed, and CD9 and Calnexin are not expressed) from a CIK cell culture supernatant through an ultracentrifugation method, and transfusing back according to the proportion that every 2 * 10 < 7 > CIK cells are combined with 100-500 [mu] g of exosomes. The exosome can significantly improve the IFN-gamma expression level of CIK cells and promote PBMC proliferation, and in a liver cancer mouse model, it is proved that the tumor inhibition rate (70.7%) of a combined group (2 * 107 CIK cells + 400 micrograms of exosome) is significantly superior to that of a single treatment group. Waste exosome resources are utilized, the anti-tumor activity of the CIK cells is safely enhanced, and the CIK cells are suitable for treatment of solid tumors such as liver cancer, lung cancer and breast cancer and hematologic tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a method for enhancing CIK cell activity and an application thereof in tumor treatment. Background Art

[0002] Adoptive immune cell therapy is an important form of cancer immunotherapy. Among various immune cell therapies, chimeric antigen receptor T cell (CAR-T) therapy currently garners the most attention. However, its preparation process is complex, its application is limited to hematologic malignancies, and most importantly, its high cost restricts its applicable population. Tumor-infiltrating lymphocyte (TIL) therapy is also gaining attention, but its preparation process is complex, its operating conditions are demanding, and its yield is low, similarly limiting its scope of application. Cytokine-induced killer (CIK) cell therapy is one of the most established immune cell therapies. Due to its low cost and mature and simple preparation process, it is widely used in the treatment of various tumors and is now recommended as a postoperative adjuvant therapy in the "CSCO Guidelines for the Diagnosis and Treatment of Primary Liver Cancer 2022 Edition." CIK cells are derived from human peripheral blood mononuclear cells (PBMCs) stimulated with cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ), followed by expansion and culture. Due to their robust proliferation capacity, excellent cytotoxicity, broad antitumor spectrum, and minimal side effects, CIK cells can serve as an effective adjunctive anti-cancer treatment, eradicating residual cancer cells, preventing recurrence, and improving survival. As the core effector cells in adoptive immunotherapy, CIK cells directly participate in the effector phase of immune surveillance by releasing cytotoxic granules such as perforin and granzyme B. Their T / NK cell phenotype also enables them to recognize and eliminate malignant cells without MHC restriction. However, during the application of CIK cell therapy, some patients have been found to have insufficient anti-tumor immune responses and insignificant anti-tumor effects, necessitating further improvements. Some studies have explored the addition of adjunctive therapeutic agents, such as cytokines, immunoreactive peptides, and immunoreactive antibodies, to CIK cell products. However, these additives often carry additional risks, such as inducing cytokine storms, allergic reactions, and unpredictable side effects. Therefore, the search for safe and effective methods to enhance the anti-tumor effects of CIK cell therapy has become a pressing issue.

[0003] Exosomes are a class of extracellular vesicles with a diameter ranging from 30 to 150 nm and a bilayer phospholipid membrane. They were first described by Wolf and later discovered by Harding in mature reticulocytes. Current research indicates that the vast majority of cells secrete exosomes, and most cells, including tumor cells and immune cells, readily take up exosomes. Exosomes can unload markers from parent cells and deliver them to other cells, thereby exerting regulatory functions. The intercellular communication processes of exosomes significantly influence tumor progression, metastasis, angiogenesis, and drug resistance. In addition to their direct antitumor effects, exosomes can also serve as drug delivery systems for the delivery of antitumor drugs. Due to their potential to influence the tumor microenvironment, efforts are ongoing to develop and utilize exosomes that target the tumor microenvironment and modulate cancer progression. Therefore, exosomes exhibit multidimensional interventional potential in cancer treatment and hold significant clinical translational research value.

[0004] During the production and preparation of CIK cells, a massive, multi-liter culture system is employed. The CIK cell components within these cultures are recovered, cleaned, and then returned to the patient for treatment. Conventional CIK therapy, however, discards the culture medium and the numerous exosomes secreted by CIK cells within them. However, exosomes secreted by CIK cells may carry numerous immunoreactive substances, such as cytokines, and preliminary studies have confirmed their potential for immune activation. CIK cells rely on their potent immune activity to exert their anti-tumor effects. If these exosomes are further combined with CIK cells to develop modified CIK therapies, this could potentially enhance CIK immune activity and strengthen the immune activity of recipient lymphocytes, thereby improving the anti-tumor efficacy of cell therapy. However, there is currently no publicly available information on the recovery of CIK cell-derived exosomes and their combined use with CIK cells. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides a method for enhancing CIK cell activity and its application in tumor treatment. The method for enhancing CIK cell activity is based on conventional CIK cell culture and is combined with the application of exosomes secreted by CIK cells to enhance CIK immune activity and enhance 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 object of the invention, the present invention provides the following technical solutions.

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

[0008] Furthermore, the exosomes are separated from the CIK culture supernatant by ultracentrifugation.

[0009] The present invention also discloses a method for enhancing CIK cell activity, which is characterized in that exosomes derived from CIK cells are used in combination with CIK cells.

[0010] Furthermore, the combined application ratio of exosomes and CIK cells was 2×10 7 CIK cells correspond to 100-500µg exosomes.

[0011] Furthermore, the ratio is 2×10 7 CIK cells correspond to 400 μg of exosomes.

[0012] The present invention also discloses an application of any of the above methods in preparing anti-tumor drugs.

[0013] Furthermore, the tumors include liver cancer, lung cancer, breast cancer, colorectal cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, bile duct cancer, thyroid cancer, glioma, lymphoma, and leukemia.

[0014] The present invention also discloses an anti-tumor pharmaceutical composition, characterized by comprising CIK cells, exosomes derived from CIK cells and a pharmaceutically acceptable carrier.

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

[0016] CIK cell-derived exosomes have potential immune activation. This invention, for the first time, combines CIK cell-derived exosomes with CIK cells to create a modified CIK cell product with enhanced immune activity and anti-tumor properties compared to conventional CIK cells. This modified CIK cell product can be applied to a variety of malignant tumor indications used in conventional CIK cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 CIK cells were prepared using conventional methods.

[0018] Figure 2 This is the extraction and identification of CIK cell-derived exosomes. Figure A shows the morphological characteristics of CIK exosomes under electron microscopy; Figure B shows the particle size of CIK exosomes; and Figure C shows the expression of exosome marker proteins in CIK cells by Western blot.

[0019] Figure 3 CIK cell-derived exosomes enhance the expression of IFN-γ in CIK cells. Figure A is a flow cytometer graph; Figure B is a bar graph.

[0020] Figure 4It is an improved CIK cell therapy (i.e. CIK cells combined with CIK cell-derived exosomes) for the anti-liver cancer effect. * 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 modified CIK cells is significantly enhanced. * Compared with the untreated CIK group, P <0.05. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention are further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. It should also be understood that the present invention can be implemented in other different forms and is not limited to the following examples. Those skilled in the art can make various changes and modifications without violating the connotation of the present invention, and the resulting equivalent forms also fall within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs.

[0023] Specific conditions not specified in the examples were carried out according to conventional conditions or the conditions recommended in the instructions. All reagents and instruments used were conventional products purchased through conventional sales channels unless otherwise specified.

[0024] Example 1 Preparation of CIK cells.

[0025] (1) Draw 100 mL of human peripheral venous blood or collect umbilical cord venous blood, dilute it with normal saline according to an equal volume ratio, and use human lymphocyte separation medium to separate and extract peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation.

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

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

[0028] (4) Observe the cells daily and transfer the cells to the culture bag according to the cell growth status. Replenish the culture medium to a final volume of 200 mL, add 5 mL of autologous serum, and supplement IL-2.

[0029] (5) From day 6 to day 12, double the amount of culture medium and IL-2 every 1 to 2 days to bring the final culture volume to 2 L.

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

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

[0032] Exosomes from CIK cell culture supernatant were extracted by ultracentrifugation. The specific process is as follows.

[0033] (1) After culturing CIK cells for 13 days 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) Centrifuge at 12,000 rpm for 30 min to separate high-purity exosomes, which were then stored at -80°C.

[0035] (3) Morphological identification: Exosome samples were negatively stained with 2% uranyl acetate and placed on a copper grid to dry. The ultrastructure of exosomes was observed by transmission electron microscopy (accelerating voltage 80 kV). Under the electron microscope, CIK exosomes had obvious vesicle structure and intact membrane structure without rupture, which was consistent with the morphological characteristics of exosomes (see Figure 2 A).

[0036] (4) Particle size distribution analysis: A nanoflow cytometer was used to measure the particle size distribution of exosomes and estimate the particle concentration. Nanosilica fluorescent microspheres (250 nm) were used to calibrate the instrument and calculate the sample flow rate and particle concentration. Standard spheres (68 nm, 91 nm, 113 nm, 155 nm) were used to establish a standard curve of scattered light intensity and particle size to obtain the particle size distribution of the exosome sample. The test results showed that the average particle size of CIK exosomes was 75.77 nm, which was consistent with the exosome particle size range of 50-150 nm (see Figure 2 B).

[0037] (5) Determination of molecular markers: Western blot was used to verify the characteristic proteins of exosomes (CD9, TSG101 and Alix), and Calnexin was used as a negative control. Specific process: The exosome PBS resuspension was mixed with the lysis buffer (RIPA + 1% PMSF) at a volume ratio of 3:7 to extract the total protein. After quantification by BCA method, 27 μg / well was loaded, 10% SDS-PAGE electrophoresis was transferred to the membrane, 5% skim milk powder was blocked for 1 hour, the primary antibody was incubated at 4℃ overnight, HRP-labeled secondary antibody (1:5000) was reacted at room temperature for 1 hour, and ECL development was used to analyze the band specificity. Western blot results showed that the extracted CIK cell exosomes expressed cytosolic proteins TSG101 and Alix, but did not express the membrane protein marker CD9 and the negative cytoplasmic protein Calnexin (see Figure 2 C).

[0038] Based on the above tests, it was confirmed that the isolated and extracted CIK cell exosomes met the characteristics of exosomes.

[0039] Example 3 verifies the effect of CIK cell-derived exosomes on enhancing CIK cell activity.

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

[0041] (1) CIK cells treated with 200 μg / mL CIK exosomes for 72 h and untreated CIK cells in the blank control group were added with a stimulation mixture containing PMA (100 ng / mL), ionomycin (1 μg / mL) and brefeldin A (10 μg / mL) and incubated at 37°C and 5% CO2 for 4-6 h to activate the cytokine secretion pathway.

[0042] (2) Collect the above cells, wash them by centrifugation, and resuspend the cell pellet in 500 μL of 4% paraformaldehyde solution / nuclear fixation buffer. Fix the cells at room temperature in the dark for 20 minutes to maintain cell membrane integrity. After fixation, add 500 μL of permeabilization working solution / nuclear permeabilization buffer (Perm / Wash buffer) to the cells and centrifuge them at 1800 rpm for 5 minutes. Repeat three times to ensure full permeabilization. Discard the supernatant and collect the pellet.

[0043] (3) The cell pellet was resuspended in 100 μL PBS, and the fluorescent antibody IFN-γ-PE was added. The cells were stained at low temperature (4°C) in the dark for 20 min.

[0044] (4) Wash the stained cells twice with PBS (500 μL / tube, centrifuge at 1800 rpm for 5 min), discard the supernatant, and resuspend the cells for flow cytometry analysis.

[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 the improved CIK cell therapy (i.e., CIK cells combined with CIK cell-derived exosomes).

[0047] (1) Twenty-four 6-week-old female NOD / SCID mice were selected and the experiment began after one week of adaptive feeding.

[0048] (2) Human hepatoma cell line HepG2 (4×10 6 cells / 100 μL PBS), the day of subcutaneous injection was recorded as day 0. When the tumor grew to 100 mm 3 The tumor volume was recorded starting from around day 7. The mice were randomly divided into 4 groups according to the tumor volume: 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 In the model control group, 100 μL PBS was injected into each tail vein of each mouse; ② In the exosome group, 400 μg exosomes / 100 μL PBS was injected into each tail vein of each mouse; ③ In the CIK group, 2×10 7 CIK / 100 μL PBS was used for treatment; ④ CIK+exosome group mice were injected into the tail vein (2×10 7 CIK + 400 μg exosomes) / 100 μL PBS were treated once a week for 3 consecutive weeks.

[0050] (4) Record the weight of mice every 2 days, measure the long and short diameters of the tumor, and calculate the tumor volume (mm). 3 ) = [length (mm) × width (mm) 2 ] / 2. Draw a tumor growth curve and observe until the maximum volume of the subcutaneous transplanted tumor reaches 1500 mm 3 When , terminate the experiment.

[0051] (5) The experimental results showed that compared with the model control group, the growth trend of subcutaneous transplanted tumors in the exosome group, CIK group and CIK+exosome group was significantly slowed down; compared with the exosome group and CIK group, the growth trend of subcutaneous transplanted tumors in the CIK+exosome group was further significantly slowed down (see Figure 4Furthermore, the tumor-suppressing effect of the CIK+exosome group was even greater than the combined tumor-suppressing effects of the exosome and CIK groups (see Table 1). This suggests that the combined use of CIK-derived exosomes significantly enhances the anti-tumor efficacy of CIK cell therapy, demonstrating a synergistic effect, representing a novel and improved CIK cell therapy.

[0052] Table 1 Improved CIK cell therapy has enhanced anti-tumor effect against mouse liver cancer xenografts.

[0053] Example 5 verifies that CIK cell-derived exosomes enhance the broad-spectrum anti-tumor effect of CIK cells.

[0054] Calcein release assay was used to detect the immune activation effect of exosomes in enhancing the expression of CIK cells to lyse and kill tumor cells. The specific process is as follows.

[0055] (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, bile duct cancer HuH-28, thyroid cancer TPC1, glioma U251, lymphoma SNK-6, and leukemia K562. The above tumor target cells were collected in the logarithmic growth phase and labeled with 25 μM calcein-AM at 37°C, 5% CO2 for 25 min, and then washed three times with PBS for use.

[0056] (2) CIK cells treated with 200 μg / mL CIK exosomes for 72 h and untreated CIK cells in the control group were co-cultured with the above-mentioned target cells at a ratio of 20:1 in 96-well plates, with each well containing 20,000 CIK cells and 1,000 tumor cells of any type. A spontaneous release group was set up, in which the target cells were co-incubated with culture medium only; and a maximum release group was set up, in which the target cells were co-incubated with 2% Triton X-100. After mixed culture at 37°C and 5% CO2 for 4 hours, the cell supernatant was detected by fluorescence microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Each assay was performed in triplicate. The percentage of specific lysis killing was: (OD 实验释放 -OD 自发释放 ) / (OD 最大释放 -OD 自发释放 ) ×100%.

[0057] (3) The killing rates of CIK cells in different treatment groups against the above 14 tumor cells are shown in Tables 2 and Figure 5The results showed that compared with untreated CIK cells, the killing efficiency of CIK cells treated with CIK exosomes against 14 types of tumor cells was significantly increased ( P <0.05), indicating that CIK cells modified by treatment with CIK cell-derived exosomes have significantly enhanced anti-tumor effects and are broad-spectrum.

[0058] Table 2 The broad-spectrum tumor killing effect of modified CIK cells was significantly enhanced (killing rate, %).

[0059] In summary, compared with conventional CIK cell therapy, the improved CIK cell therapy of the present invention further combines the use of exosomes secreted by CIK cells to effectively enhance the activity of CIK cells and other immune cells, thereby enhancing the anti-tumor effect of cell therapy and has broad application prospects.

[0060] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. An antitumor pharmaceutical composition, characterized in that: The invention comprises CIK cells, CIK cell-derived exosomes and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein: The ratio of CIK cell-derived exosomes to CIK cells was 2×10 7 CIK cells correspond to 100-500 μg of exosomes.

3. The pharmaceutical composition according to claim 2, wherein: The ratio is per 2×10 7 CIK cells correspond to 400 μg of exosomes.

4. A method for enhancing CIK cell activity in vitro, characterized by: CIK cells were pretreated with CIK cell-derived exosomes.

5. Use of the method according to claim 4 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that The tumors include liver cancer, lung cancer, breast cancer, colorectal cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, bile duct cancer, thyroid cancer, glioma, lymphoma, and leukemia.

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