Oligopeptide and application thereof in improving proliferation activity and lethality of human natural killer cells

Abstract

The invention discloses oligopeptide and application thereof in improving proliferation activity and lethality of human natural killer cells. When the polypeptide with the sequence of RVHGLPRV provided by the invention is used for in-vitro culture of NK cells, not only can the proliferation activity of the NK cells be improved, but also the killing power of the NK cells to tumor cells can be improved. Therefore, the polypeptide can be used for in-vitro culture of NK cells.

Description

Technical Field

[0001] The present invention belongs to the field of NK cells, relates to in vitro culture of NK cells, and specifically relates to an oligopeptide and its use in improving the proliferation activity and lethality of human natural killer cells. Background Art

[0002] Natural killer (NK) cells are an important type of innate lymphocytes that have the ability to recognize stressed cells (such as tumor cells and certain infected cells) and can spontaneously kill tumor cells. NK cells recognize and kill tumor cells by expressing a variety of activating and inhibitory receptors and recognizing tumor-related ligands. Compared with CD8+T cells, the recognition mechanism of NK cells is more flexible and does not depend on MHC-I molecules. NK cells can kill tumor cells that lack MHC-I, and tumor cells that highly express activating ligands are usually killed by NK cells. This non-specific recognition mechanism and efficient anti-tumor activity make NK cells have the potential to be used as anti-cancer "living drugs".

[0003] As an emerging cancer treatment modality, immunotherapy has developed rapidly in recent years. Although immune checkpoint blockade and CAR-T cell therapy have achieved remarkable success, they have also brought new challenges. Loss of MHC-I expression is an important mechanism for tumor cells to acquire resistance to anti-tumor CD8+T cell responses. NK cells have the ability to recognize and rapidly kill tumor cells without MHC-I restriction, so the use of NK cells for treatment may supplement the deficiencies of anti-tumor T cell therapy. In addition, allogeneic NK cell infusion does not cause graft-versus-host disease (GVHD), and NK cells can be used as a "ready-to-use" cell therapy product and reduce the toxic effects associated with CAR-T cell therapy.

[0004] In this context, NK cells have become an important research target in the field of cancer treatment. Immunotherapy strategies to enhance the anti-tumor response of NK cells have been rapidly developed and entered clinical trials.

[0005] How to improve the proliferation activity and killing power of NK cells is an important research direction for using NK cells for anti-tumor treatment. Summary of the invention

[0006] The purpose of the present invention is to provide an oligopeptide and its use in improving the proliferation activity and lethality of human natural killer cells.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] An oligopeptide, the sequence of which is shown in Sequence NO.1.

[0009] The oligopeptide shown in the above-mentioned Sequence NO.1 is used for in vitro culture of NK cells to enhance their proliferation activity.

[0010] The oligopeptide shown in the above Sequence NO.1 is used for in vitro culture of NK cells to enhance their killing ability.

[0011] The oligopeptide shown in the above Sequence NO.1 is used for in vitro culture of NK cells to enhance their proliferation activity and killing ability.

[0012] Beneficial effects:

[0013] When the polypeptide with the sequence of RVHGLPRV provided by the present invention is used for NK cell in vitro culture, it can not only improve the proliferation activity of NK cells, but also improve their killing power against tumor cells. Therefore, the polypeptide can be used for NK cell in vitro culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The HPLC chromatogram and chromatographic peak integrated area of ​​peptide 2;

[0015] Figure 2 This is the mass spectrometry detection diagram of peptide 2. DETAILED DESCRIPTION

[0016] The following is a detailed description of the substantive content of the present invention in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. In the following embodiments, the instruments and reagents not specifically explained are all commercially available conventional instruments and reagents.

[0017] Example 1: Polypeptide Preparation Example

[0018] Peptide 2 sequence: RVHGLPRV (Sequence NO.1).

[0019] 1. Peptide Synthesis

[0020] Peptide 2 was commissioned to be synthesized by Synpeptide Co., Ltd. (http: / / www.synpeptide.com) and synthesized from the C-terminus to the N-terminus according to conventional solid phase synthesis method, specifically including the following steps:

[0021] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM).

[0022] 2. After 2 hours, wash the resin with 3 times the volume of N,N-dimethylacetamide (DMF) and then drain it. Repeat this four times and drain the resin for later use.

[0023] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.

[0024] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 minute). If the resin has color, it means that the deprotection is successful.

[0025] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30 °C for reaction.

[0026] 6. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with DMF 3 times the volume of the resin, and drain it for later use.

[0027] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.

[0028] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 min). If the resin has color, it means that the deprotection is successful.

[0029] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50 mL centrifuge tube, add 25 mL of DMF to dissolve them, then add 2.5 mL of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0030] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, it means that the condensation is incomplete and the reaction should continue.

[0031] 11. After the reaction is complete, wash the resin four times with DMF, then drain, add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain to check whether the protection is removed.

[0032] 12. Follow steps 9-11 to connect the following amino acids.

[0033] 13. After the last amino acid is attached, remove the protection, wash with DMF four times, and then drain the resin with methanol. Then use 95 cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: triisopropylsilane: water = 95:2:2:1) to cut the polypeptide from the resin (add 10mL cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9). Finally, separate and purify with HPLC, and then freeze-dry to obtain.

[0034] 2. HPLC detection

[0035] 1. Chromatographic conditions

[0036] Mobile phase A: 0.1% Trifluoroacetic in 100% Water;

[0037] Mobile phase B: 0.1% Trifluoroacetic in 100% Acetonrtrile;

[0038] Flow rate: 1 mL / min;

[0039] Wavelength: 220nm;

[0040] Chromatographic column: SHIMADZU Inertsil ODS-SP (4.6*250mm*5um);

[0041] Injection volume: 30 μL;

[0042] Elution gradient: 0.00-25.00 min, 5%-65% B; 25.00-25.01 min, 65%-100% B; 25.01-30.00 min, 100%-100% B; 30.01 min, Stop.

[0043] 2. Test results

[0044] The HPLC chromatogram and chromatographic peak integrated area of ​​peptide 2 are as follows: Figure 1 As shown, the purity of polypeptide 2 was 95.82%.

[0045] 3. Mass spectrometry detection

[0046] Mass spectrometry detection of peptide 2 Figure 2 As shown, the [M+H]H+ molecular weight is 934.06, which is consistent with the theoretical molecular weight of peptide 2 of 933.118 (the theoretical molecular weight is calculated using the peptide molecular weight calculator of Hefei Guopeptide Biotechnology Co., Ltd.).

[0047] Example 2: Activity Test Example

[0048] 1. Experimental Materials

[0049] Special culture medium for primary NK cells (brand SAIOS, product number PM-032, product specification 500mL / set), Wuhan Saios Biotechnology Co., Ltd.

[0050] Human pancreatic cancer Capan-2 cells, Shanghai Yaji Biotechnology Co., Ltd.

[0051] 2. Experimental Methods

[0052] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0053] The conventional density gradient centrifugation method in the art is used to separate PBMC from peripheral blood for NK cell separation and purification. The operation method is: 20 mL of venous blood is drawn from healthy volunteers aged 18 to 35 years old, and Ficoll lymphocyte separation fluid is used for density gradient centrifugation to separate PBMC, which is then resuspended in PBS buffer and counted.

[0054] 2. NK cell sorting and flow cytometry identification

[0055] NK cells were separated by conventional magnetic bead separation methods in the art. The operation method is as follows: the PBMCs separated above were sorted using a human NK cell separation kit (Miltenyi cell separation magnetic bead NK Cell Isolation Kit), and human NK cells (CD3-CD56+) were separated and purified, resuspended in PBS, and counted.

[0056] Take 1×10 7 20 μL FTTC-labeled CD56 and PerCP-Cy5.5-labeled CD3 were added to each cell, and the proportion of NK cells (CD3-CD56+) was detected by flow cytometry.

[0057] 3. NK cell culture in vitro

[0058] The sorted NK cells were cultured in a primary NK cell-specific medium at 37°C and 5% CO. 2 The cells were cultured in a saturated humidity environment and the culture medium was added every 2-3 days. After 9 days of culture, the cells were harvested for subsequent experiments.

[0059] 3. NK cell proliferation activity assay

[0060] The NK cells cultured for 9 days were resuspended in RPMI 1640 medium containing 10% FBS and 1% double antibody and the concentration was 5×10 3 Each well was inoculated in a 96-well plate. After 12 hours, the culture medium was replaced with the following according to the different groups, with 5 replicate wells in each group:

[0061] Conventional group: RPMI 1640 medium containing 10% FBS and 1% double antibody;

[0062] Peptide 2 low-dose group: RPMI 1640 medium containing 25 μM peptide 2, 10% FBS and 1% double antibody;

[0063] Peptide 2 high-dose group: RPMI 1640 medium containing 50 μM peptide 2, 10% FBS and 1% double antibody.

[0064] 37°C, 5% CO 2 After culturing for 48 h in a saturated humidity environment, 20 μL of CCK-8 solution was added to each well and the cells were placed at 37°C and 5% CO 2 After incubation for 4 hours under the same conditions, the OD value of each well was measured at a wavelength of 490 nm. The cell proliferation activity of the conventional group was taken as 100%, and the cell proliferation activity of the culture group containing polypeptide 2 was calculated (the ratio of the OD value of the conventional group, converted into percentage).

[0065] 4. NK cell killing activity assay

[0066] NK cells incubated with different culture media (as shown below) were used as effector cells, and human pancreatic cancer Capan-2 cells were used as target cells. Target cells were cultured at 5×10 3 Each well was inoculated in a 96-well plate. After 12 hours, the culture medium was gently removed, and effector cells were added at a ratio of 5:1, with 200 μL of culture medium (RPMI 1640 medium containing 10% FBS and 1% dual antibody) per well. At the same time, separate target cell wells and separate effector cell wells were set up, with 5 replicates each. After 24 hours of co-culture, 20 μL of CCK-8 solution was added to each well and placed at 37°C, 5% CO 2 After incubation for 4 h under the same conditions, the OD value of each well was measured at a wavelength of 490 nm, and the cytotoxic activity of NK cells incubated with different culture media against tumor cells was calculated according to the formula: killing rate (%) = [1-(OD value of the experimental group-OD value of the effector cell alone) / OD value of the target cell group alone] × 100%.

[0067] Conventional group: incubated with RPMI 1640 medium containing 10% FBS and 1% double antibody for 12 h;

[0068] Peptide 2 low-dose group: incubated with RPMI 1640 medium containing 25 μM peptide 2, 10% FBS and 1% double antibody for 12 h;

[0069] Peptide 2 high-dose group: incubated with RPMI 1640 medium containing 50 μM peptide 2, 10% FBS and 1% double antibody for 12 h.

[0070] 5. Statistical processing

[0071] The experimental results were expressed as mean ± standard deviation. GraphPad Prism 9.0 software was used for statistical analysis. One-way analysis of variance was used, and a significance level of P < 0.05 was considered statistically significant.

[0072] 3. Experimental Results

[0073] 1. NK cell sorting and flow cytometry identification results

[0074] After magnetic bead sorting, the proportion of CD3-CD56+ cells is as high as 94.39%. Those skilled in the art know that CD3-CD56+ cells are NK cells, indicating that the magnetic bead sorting is successful.

[0075] 2. Results of NK cell proliferation activity assay

[0076] The NK cell proliferation activity of each group is shown in Table 1. Compared with the conventional group, the NK cell proliferation activity of the low-dose and high-dose peptide 2 groups was significantly increased, and showed obvious dose dependence.

[0077] Table 1 NK cell proliferation activity in each group

[0078] Conventional group Peptide 2 low dose group Peptide 2 high dose group Proliferation activity (%) 100±2.33 142.85±2.68 195.09±2.51

[0079] The results showed that peptide 2 has the effect of promoting the proliferation of NK cells in vitro.

[0080] 3. NK cell killing activity assay

[0081] The killing rate of NK cells against tumor cells in each group is shown in Table 2. Compared with the conventional group, the killing activity of NK cells against Capan-2 cells in the low-dose and high-dose groups of peptide 2 was significantly increased, and showed obvious dose dependence.

[0082] Table 2 Killing rate of NK cells in each group on Capan-2 cells

[0083] Conventional group Peptide 2 low dose group Peptide 2 high dose group Kill rate (%) 18.05±1.97 30.51±2.22 49.76±2.08

[0084] The results showed that peptide 2 has the effect of enhancing the killing activity of NK cells.

[0085] In summary, when the polypeptide 2 provided by the present invention is used for NK cell in vitro culture, it can not only improve the proliferation activity of NK cells, but also improve their killing ability against tumor cells. Therefore, polypeptide 2 can be used for NK cell in vitro culture.

[0086] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. An oligopeptide, characterized in that: The sequence is shown as Sequence NO.

1.

2. Use of the oligopeptide according to claim 1 for increasing the proliferation activity of NK cells in vitro.

3. Use of the oligopeptide according to claim 1 for in vitro culture of NK cells to improve their lethality.

4. Use of the oligopeptide according to claim 1 for in vitro culture of NK cells to enhance their proliferation activity and lethality.

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