A method for preparing CIK cells and its application in treating tumors

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing CIK cells and their application in treating tumors. The present invention also provides a polypeptide that inhibits the activity of USP7. The performance of the polypeptide is tested, and it is found that the polypeptide has a strong inhibitory effect on the cloning ability and migration ability of gastric cancer cells. Furthermore, the present invention also uses the polypeptide in combination with CIK cells. The experimental results show that the combined use of the two can significantly inhibit the growth of tumor cells and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a method for preparing CIK cells and an application thereof in treating tumors. Background Art

[0002] CIK (cytokine-induced killer) cells are heterogeneous cells with non-major histocompatibility antigen (MHC)-restricted tumoricidal activity obtained by inducing and culturing peripheral blood mononuclear cells under conditions of multiple cytokines, such as interleukin-2 (IL-2) and interferon-γ (IFN-γ). Studies have found that CIK cells can simultaneously express two membrane protein molecules, CD3+ and CD56+, and are therefore also called NK cell-like T lymphocytes. CIK cells have the advantages of rapid proliferation, high tumoricidal activity, and a wide spectrum of tumorigenicity, and are considered to be the first choice for the new generation of anti-tumor adoptive cell immunotherapy. CIK cells kill tumor cells through three pathways: (1) CIK cells release cytokines that can directly inhibit or indirectly kill tumor cells by regulating the body's immune system; (2) CIK cells can recognize tumor cells through different mechanisms and release toxic particles to kill tumor cells; (3) CIK cells can also induce apoptosis of tumor cells.

[0003] Ubiquitination plays an important role in many physiological and pathological processes such as cell proliferation, apoptosis, migration, and invasion. Deubiquitinating enzymes can reverse this process and protect the target substrate. Deubiquitinating enzymes are divided into seven families: ubiquitin-specific proteases (USPs), ubiquitin carboxyl-terminal hydrolases (UCHs), ovarian tumor-associated proteases (OTUs), zinc-finger ubiquitin peptidases 1 (ZUP1), MPN(+) / JAMM metalloproteases (JAMMs), Machado-Josephin domain proteases (MJDs), and new DUB family proteases containing sequences that bind to ubiquitin molecules (MINDYs). Among them, the USP family is the largest.

[0004] Ubiquitin specific peptidase 7 (USP7) is a member of the ubiquitin specific protease family. USP7 is mainly distributed in the cell nucleus. It regulates multiple cellular physiological functions such as immune response, DNA damage repair, and DNA replication by specifically binding to substrates. Scholars have analyzed and summarized a large number of literature and found that USP7 is highly expressed in various tumor tissues such as gastric cancer cell lines and prostate cancer cell lines. It can be seen that USP7 is involved in the occurrence and development of various tumors such as gastric cancer, colon cancer, and cervical cancer. In addition, USP7 is also involved in regulating physiological processes such as the cellular immune system, cell proliferation, and migration. Based on the multiple biological functions of USP7, USP7 can be used as a target for tumor treatment.

[0005] USP7 inhibitors have great potential in the field of tumor treatment. Currently, various USP7 inhibitors have been reported, but they generally have problems such as poor therapeutic effects and poor drug resistance. Based on this, it is urgent to conduct deeper exploration and research on USP7 inhibitors. Summary of the invention

[0006] In order to overcome the above problems, the first object of the present invention is to provide a polypeptide that inhibits the activity of USP7, which has a strong inhibitory effect on the cloning ability and migration ability of gastric cancer cells.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A polypeptide that inhibits USP7 activity, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0009] The second object of the present invention is to provide a pharmaceutical composition which can significantly inhibit the growth of tumor cells.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] A pharmaceutical composition, which consists of the above-mentioned polypeptide inhibiting USP7 activity and CIK cells.

[0012] Preferably, the CIK cells are obtained as follows:

[0013] (1) Extracting mononuclear cells from peripheral blood;

[0014] (2) The mononuclear cells of step (1) were resuspended in RPMI1640 complete medium supplemented with INF-γ 700-800 U / mL and 8-10% FBS, and then rhlL-2 400-500 U / mL, IL-2 500-600 U / mL, IL-21 100-200 U / mL, and human CD3 monoclonal antibody 90-100 ng / mL were added and cultured continuously. The medium was changed every 2-3 days to obtain CIK cells.

[0015] Preferably, the density of the mononuclear cells in step (2) in RPMI 1640 complete medium is 1-4×10 6 Pieces / mL.

[0016] Preferably, the mononuclear cells in step (2) are cultured in RPMI 1640 complete medium for 1 day; and the continued culture in step (2) is for 14-15 days.

[0017] Preferably, the culture is carried out at 37°C and 5% CO2.

[0018] The third purpose of the present invention is to provide an application of a pharmaceutical composition with broad application prospects.

[0019] In order to achieve the above object, the technical solution adopted by the present invention is:

[0020] The above-mentioned pharmaceutical composition is used to prepare drugs for treating tumors.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0022] (1) The polypeptide of the present invention has a strong inhibitory effect on the cloning ability and migration ability of gastric cancer cells.

[0023] (2) The present invention uses a polypeptide that inhibits USP7 activity in combination with CIK cells and finds that the pharmaceutical composition can significantly inhibit the growth of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the morphological diagram of CIK cells;

[0025] Figure 2 This is the result diagram of the effect of peptide 1 on the cloning ability of gastric cancer cell line MGC-803;

[0026] Figure 3 This is a graph showing the effect of polypeptide 1 on the cloning ability of gastric cancer cell line BGC-823.

[0027] Figure 4 This is the result diagram of the effect of peptide 1 on the migration ability of gastric cancer cell line MGC-803;

[0028] Figure 5 This is the result diagram of the effect of peptide 1 on the migration ability of gastric cancer cell line BGC-823;

[0029] Figure 6 It is a graph showing the inhibition of tumor cell growth by the drug composition in each experimental group. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments 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 many different forms and is not limited to the embodiments described below. Those skilled in the art can make various changes and modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall into the protection scope 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.

[0031] The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents and instruments used were conventional products obtained from commercial channels unless otherwise specified.

[0032] 1. Implementation

[0033] Example 1

[0034] Example 1 provides a polypeptide that inhibits USP7 activity. The process of obtaining the polypeptide is as follows:

[0035] Using the Ph.D.-12 phage display peptide library kit, the USP7 recombinant protein was used as the target molecule. After three rounds of panning, a high-titer eluate was obtained. The high-titer plaques were sequenced to obtain the amino acid sequence of the polypeptide specifically binding to the USP7 recombinant protein as shown in SEQ ID NO: 1-4. The specific amino acid sequence is as follows:

[0036] HYVLTITGRVTYLKNP (SEQ ID NO: 1, denoted as polypeptide 1);

[0037] HDGYVLTITSTQYLKNP (SEQ ID NO: 2, denoted as polypeptide 2);

[0038] HYVLTAQRLVTYSGLRNP (SEQ ID NO: 3, denoted as polypeptide 3);

[0039] HFMLFAQGRVTYSGLLSP (SEQ ID NO: 4, denoted as polypeptide 4).

[0040] Example 2

[0041] Example 2 provides a method for preparing CIK cells, and the specific preparation process is as follows:

[0042] (1) Extraction of mononuclear cells from peripheral blood

[0043] i. Collect venous peripheral blood from volunteers using anticoagulant tubes with heparin added, transfer the blood into centrifuge tubes, and dilute the venous peripheral blood with normal saline at a volume ratio of 1:1 to obtain a diluent;

[0044] ii. Add lymphocyte separation solution to a new centrifuge tube, and then slowly add the above diluent. The volume ratio of diluent to lymphocyte separation solution is 2:1.

[0045] iii. Place the above centrifuge tube in a centrifuge for centrifugation, set the centrifuge speed to 1000g, centrifuge for 20 minutes, and after centrifugation, the liquid in the centrifuge tube is layered, from bottom to top, respectively, red blood cells and granulocytes layer, lymphocyte separation liquid layer, mononuclear cell layer, plasma layer;

[0046] iv. The mononuclear cell layer was aspirated and transferred to a new centrifuge tube, and the cells were washed with RPMI 1640 medium and centrifuged to obtain mononuclear cells.

[0047] (2) Obtaining CIK cells

[0048] The mononuclear cells obtained in step (1) were resuspended in RPMI 1640 complete medium supplemented with INF-γ 800 U / mL and 8% FBS at a density of 3×10 6 The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 1 day, and then rhlL-2 500U / mL, IL-2 600U / mL, IL-21 100U / mL, and human CD3 monoclonal antibody 93ng / mL were added and cultured for 14 days to obtain CIK cells (see Figure 1 ).

[0049] 2. Experimental Examples

[0050] Experimental Example 1

[0051] Experimental Example 1 The four polypeptides obtained in Example 1 were artificially synthesized and the polypeptide activity was detected. The specific experimental process is as follows:

[0052] Experimental group: 1.5 μL of the above four peptides, 0.8 μL of USP7 recombinant protein, and 95.6 μL of Hepes buffer were added to a 96-well plate;

[0053] Protein group: 0.8 μL USP7 recombinant protein and 95.6 μL Hepes buffer were added to a 96-well plate;

[0054] The above-mentioned 96-well plates were placed in a constant temperature (25°C) shaker and incubated for 15 min, 1.5 μL of substrate PHF8 with a final concentration of 12 μM was added, 1.5 μL of Hepes buffer was added to the blank group, and incubated in a constant temperature (37°C) shaker for 70 min, 10 μL of detection reagent was added, and incubated in a constant temperature (37°C) shaker for 60 min, and the fluorescence value of 665 / 615 nm was detected using an enzyme marker. The activity of the polypeptide is characterized by the inhibition rate, inhibition rate = (protein group fluorescence intensity-experimental group fluorescence intensity) / (protein group fluorescence intensity-blank group fluorescence intensity), and the results are shown in Table 1.

[0055] Table 1

[0056] Peptides Inhibition rate (%) Peptide 1 71.38 Peptide 2 66.05 Peptide 3 55.63 Peptide 4 49.92

[0057] It is not difficult to find from Table 1 that although peptides 2-4 can inhibit USP7 activity, their inhibitory ability is poor, while peptide 1 can inhibit USP7 activity well.

[0058] Based on the fact that peptide 1 has good performance in inhibiting USP7 activity, peptide 1 was used in the following experimental examples.

[0059] Experimental Example 2

[0060] Experimental Example 2 The effect of the polypeptide 1 of Example 1 on the cloning ability of gastric cancer cells was detected. The specific experimental process is as follows:

[0061] Culture medium: The culture medium used for cell culture was RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin.

[0062] a. The thawed gastric cancer cell lines MGC-803 and BGC-823 were resuspended in the above culture medium and inoculated into a 96-well plate, and cultured in a cell culture incubator at 37°C and 5% CO2 until the cells were confluent, the culture medium was discarded, the cells were washed with PBS buffer, 0.2% trypsin was added for digestion, the above culture medium was used to terminate the digestion process, and the cultured cells were centrifuged to obtain a cell pellet;

[0063] b. The cell pellet was inoculated into a 6-well plate with the above culture medium at a density of 500 cells / well, and the plate was placed in a cell culture incubator at 37°C and 5% CO2 until the cells adhered to the wall. 0 μM, 2 μM, 5 μM, 9 μM, and 15 μM of polypeptide 1 were added, and the positive control was 15 μM USP7 inhibitor (P5091). The culture was continued for 12 days, and the medium was changed every 2 days;

[0064] c. After the culture is completed, add 1 mL of 4% paraformaldehyde to each well and fix the cells at 25°C for 30 minutes. Then remove the fixative and add 1 mL of 0.1% crystal violet to stain at 25°C for 10 minutes; remove the staining solution, wash twice with PBS, and let stand at room temperature for 10 minutes; use a microscope to count and calculate the number of cell clones in each group. The results are as follows: Figure 2-3 shown.

[0065] Figure 2 This is a graph showing the effect of polypeptide 1 on the cloning ability of gastric cancer cell line MGC-803. Figure 3 This is the result of the effect of peptide 1 on the cloning ability of gastric cancer cell line BGC-823. Figure 2-3 It can be seen that the number of gastric cancer cell lines MGC-803 and BGC-823 was significantly reduced after adding polypeptide 1 or USP7 inhibitor (P5091). The number of gastric cancer cell lines MGC-803 and BGC-823 clones was dose-dependent. As the concentration of polypeptide 1 increased, the number of cell clones decreased. When the concentration was 15 μM, the inhibitory effect of polypeptide 1 was better than that of USP7 inhibitor (P5091).

[0066] Experimental Example 3

[0067] Experimental Example 3 The effect of polypeptide 1 of Example 1 on the migration ability of gastric cancer cells was detected. The specific experimental process is as follows:

[0068] a. The thawed gastric cancer cell lines MGC-803 and BGC-823 were resuspended in RPMI 1640 medium containing 3% FBS and 1% penicillin-streptomycin to prepare a cell suspension, and the cell suspension was placed in the upper chamber of the Transwell chamber;

[0069] b. At the same time, RPMI 1640 medium containing 20% ​​FBS and 1% penicillin-streptomycin was added to the lower chamber of the Transwell chamber, and 0 μM, 2 μM, 5 μM, 9 μM, and 15 μM polypeptide 1 were added to the culture medium, and the positive control was 15 μM USP7 inhibitor (P5091);

[0070] c. Place the Transwell chamber in a cell culture incubator and culture for 1 day, then take out the Transwell chamber, remove the culture medium in the chamber, move the cells in the Transwell chamber to another 24-well plate, and wash twice; then place the Transwell chamber in a methanol solution for fixation for 30 minutes; after fixation, place the Transwell chamber in a 0.1% crystal violet solution for staining for 30 minutes, wash three times with PBS, wipe the cells inside the Transwell chamber with a cotton swab, count the number of cells that penetrate the Transwell chamber membrane, and analyze the relative migration number of gastric cancer cell lines. The results are shown in Figure 2. Figure 4-5 shown.

[0071] Figure 4 This is a graph showing the effect of polypeptide 1 on the migration ability of gastric cancer cell line MGC-803. Figure 5 This is the result of the effect of peptide 1 on the migration ability of gastric cancer cell line BGC-823. Figure 4-5 It can be seen that the relative migration number of gastric cancer cell lines MGC-803 and BGC-823 was significantly reduced after adding polypeptide 1 or adding USP7 inhibitor (P5091). At a concentration of 15 μM, polypeptide 1 can significantly inhibit the migration ability of gastric cancer cell lines MGC-803 and BGC-823 compared with USP7 inhibitor (P5091).

[0072] A pharmaceutical composition, comprising the polypeptide of SEQ ID NO: 1 in Example 1 and CIK cells.

[0073] Experimental Example 4

[0074] Experimental Example 4 The anti-tumor activity of the pharmaceutical composition composed of the polypeptide 1 prepared in Example 1 and the CIK cells prepared in Example 2 was experimentally verified. The specific experimental process is as follows:

[0075] A. Construction of nude mouse transplant tumor model

[0076] Fifty nude mice with similar physiological conditions were randomly divided into five groups, with 10 mice in each group. The gastric cancer cell lines MGC-803 and BGC-823 were resuspended in RPMI 1640 medium and the cell density was adjusted to 5×10 7 / mL, and inoculated subcutaneously on the back of nude mice to establish a nude mouse tumor transplantation model.

[0077] B. Experimental Setup

[0078] CIK cell group: only 1 mL CIK cells (1×10 8 / mL), injected once a week, for a total of 4 injections;

[0079] Peptide 1 group: Peptide 1 (15 mg / kg) was administered orally every day for 28 days;

[0080] Peptide 1 + CIK cell group: Peptide 1 (15 mg / kg) was administered orally every day for 28 days, and CIK cells (1 mL) were injected once a week for a total of 4 times;

[0081] USP7 inhibitor (P5091) + CIK cell group: USP7 inhibitor (P5091, 15 μM) was gavaged daily for 28 days, and CIK cells (1 mL) were injected once a week for a total of 4 times, and a blank control group was set up;

[0082] C. After 28 days, the nude mice were killed, tumor tissues were obtained, and the weight of the tumor tissues was recorded. The tumor inhibition rate was statistically analyzed. The tumor weight results are shown in Table 2. The tumor inhibition rate is shown in Table 2. Figure 6 shown.

[0083] Table 2

[0084] Grouping Average tumor weight (g) Average tumor weight (g) Peptide 1+CIK cell group 1.12±0.24 0.67±0.15 P5091+CIK cell group 3.80±0.53 3.45±0.36 Peptide 1 group 6.57±0.29 6.28±0.44 CIK cell group 17.26±1.68 18.43±2.15 Blank control group 23.20±1.45 25.21±1.97

[0085] Figure 6 It is a diagram showing the inhibition of tumors by each group of drug compositions. From Table 2, Figure 6 It can be seen that both the CIK cell group and the polypeptide 1 group can inhibit the growth of tumor cells, but the inhibitory effect is poor when used alone. When polypeptide 1 is used in combination with CIK cells, it is found that it can significantly inhibit the growth of tumor cells.

[0086] In summary, the polypeptide of the present invention has a strong inhibitory effect on the cloning ability and migration ability of gastric cancer cells. Furthermore, the present invention also uses the polypeptide in combination with CIK cells. The experimental results show that the combination of the two can significantly inhibit the growth of tumor cells and has broad application prospects.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A polypeptide for inhibiting USP7 activity, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A pharmaceutical composition, characterized in that The pharmaceutical composition consists of the polypeptide for inhibiting USP7 activity according to claim 1 and CIK cells.

3. The pharmaceutical composition according to claim 2, characterized in that The CIK cells are obtained as follows: (1) Extracting mononuclear cells from peripheral blood; (2) The mononuclear cells of step (1) were resuspended in RPMI 1640 complete medium supplemented with INF-γ 700-800 U / mL and 8-10% FBS, and then rhlL-2 400-500 U / mL, IL-2 500-600 U / mL, IL-21 100-200 U / mL, and human CD3 monoclonal antibody 90-100 ng / mL were added and cultured continuously. The medium was changed every 2-3 days to obtain CIK cells.

4. The pharmaceutical composition according to claim 3, characterized in that The density of the mononuclear cells in step (2) in RPMI 1640 complete medium is 1-4×10 6 Pieces / mL.

5. The pharmaceutical composition according to claim 3, characterized in that: The mononuclear cells in step (2) are cultured in RPMI 1640 complete medium for 1 day; and the continued culture in step (2) is for 14-15 days.

6. The pharmaceutical composition according to claim 3, characterized in that The culture was carried out at 37°C and 5% CO2.

7. The use of the pharmaceutical composition according to any one of claims 2 to 6, characterized in that: Used in preparing medicine for treating gastric cancer.

Images