An active peptide that enhances the tumor-killing ability of NK cells and a method for in vitro culture of NK cells.

Abstract

This invention discloses an active peptide that enhances the tumor-killing ability of NK cells and a method for in vitro NK cell culture. The invention provides an octapeptide with the sequence GWLESWYP, which can be synthesized using conventional solid-phase synthesis methods with a purity exceeding 99%. The octapeptide provided by this invention can be used as an active peptide for in vitro NK cell culture; using this active peptide as a culture additive can effectively enhance the tumor-killing ability of NK cells.

Description

Technical Field

[0001] This invention belongs to the field of NK cells and relates to the in vitro culture of NK cells. Specifically, it relates to an active peptide that enhances the tumor-killing ability of NK cells and a method for in vitro culture of NK cells. Background Technology

[0002] Malignant tumors have become one of the leading causes of death in my country, with the number of cases increasing year by year. Currently, there is no ideal treatment method. In recent years, clinical research on immunotherapy has attracted much attention from scholars, especially the clinical research on natural killer (NK) cell therapy for malignant tumors, which has achieved good results.

[0003] NK cells are large granular lymphocytes derived from the bone marrow, accounting for 10%–15% of the total peripheral blood lymphocytes. They play a crucial role in both innate and adaptive immunity. NK cells are a subset of lymphocytes capable of eliminating cancer cells, but they do not present MHC class I molecules. As important innate immune cells for anti-tumor activity, they exert anti-tumor and antiviral effects by producing lysozyme granules and interferon-gamma (IFNg). Their function of recognizing and killing malignant tumor cells is mainly regulated by the balance between activating and inhibitory receptors. Because NK cells do not attack healthy tissues or induce T cell-driven inflammatory cytokine storms, many researchers believe that NK cell immunotherapy for malignant tumors holds great promise.

[0004] Tumor immunotherapy achieves long-term anti-tumor effects by improving the patient's own immune system, providing a new treatment strategy and powerful support for patients who have not responded well to traditional anti-tumor therapies.

[0005] Peptide drugs typically have molecular weights between small molecule drugs (less than 500 Da) and protein drugs (greater than 5000 Da), combining the advantages of both types. In terms of preparation methods, peptide drugs are similar to small molecule drugs, primarily synthesized through chemical methods, offering advantages such as precise structure, easy quality control, and lower production costs. In the early stages of drug development, chemical modifications can further optimize the affinity, solubility, and pharmacokinetic properties of candidate drugs. Their therapeutic effects are similar to protein drugs, with advantages such as fewer side effects, high specificity, and good efficacy. In terms of indications, peptide drugs can be used to treat a variety of diseases, including immunomodulation, tumors, and cardiovascular diseases. In recent years, interdisciplinary and cross-domain integration has driven advancements in key technologies such as drug screening, synthesis, and modification, improving the efficiency and cost-effectiveness of peptide drug development and promoting its large-scale production and application. Therefore, peptide drugs have become one of the key directions in new drug research and development.

[0006] This invention is proposed to provide a polypeptide that enhances the tumor-killing ability of NK cells. Summary of the Invention

[0007] The first objective of this invention is to provide an active peptide that enhances the tumor-killing ability of NK cells, and the second objective is to provide a method for using the active peptide in the in vitro culture of NK cells.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] An active peptide that enhances the tumor-killing ability of NK cells, the sequence of which is shown in Sequence NO.1.

[0010] A method for enhancing the tumor-killing ability of NK cells in vitro by using the active peptide shown in Sequence NO.1 of claim 1 as a culture additive.

[0011] Beneficial effects:

[0012] 1. This invention provides an octapeptide with the sequence GWLESWYP, which can be synthesized using conventional solid-phase synthesis methods with a purity of over 99%.

[0013] 2. The octapeptide provided by the present invention can be used as an active peptide for in vitro culture of NK cells. Using this active peptide as a culture additive can effectively enhance the killing power of NK cells against tumors. Attached Figure Description

[0014] Figure 1 This includes the HPLC chromatogram and peak integral area of ​​peptide 3;

[0015] Figure 2 This is a mass spectrometry image of peptide 3. Detailed Implementation

[0016] The following detailed description of the invention, in conjunction with specific embodiments, does not limit the scope of protection of the invention. In the following embodiments, instruments and reagents not specifically described are all commercially available conventional instruments and reagents.

[0017] Example 1: Peptide Preparation Example

[0018] Peptide 3 sequence: GWLESWYP (Sequence NO.1).

[0019] I. Polypeptide Synthesis

[0020] Peptide 3 was synthesized by Synpeptide Co., Ltd. (http: / / www.synpeptide.com) using conventional solid-phase synthesis from the C-terminus to the N-terminus, specifically including the following steps:

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

[0022] 2. After 2 hours, wash the resin with 3 times the volume of N,N-dimethylacetamide (DMF), then dry it. Repeat this process four times until the resin is completely dry and ready for 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 groups on the resin. After deprotection, wash four times with DMF at a volume of 3 times the resin volume, and then dry it.

[0024] 4. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, it indicates that the deprotection was 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 well for 1 min. After the solution becomes clear, add it to the reactor, and then place the reactor in a shaker at 30 °C to react.

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

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

[0028] 8. Take a small amount of resin and test it using the ninhydrin (Ninhydrin hydrate) method (two drops each of test A and test B, react at 100℃ for 1 min). If the resin turns colored, it indicates that the deprotection was successful.

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

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

[0031] 11. After the reaction is complete, wash the resin four times with DMF, then dry it under vacuum. 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 groups on the resin. After deprotection, wash the resin four times with DMF, then dry it under vacuum to check whether the protection has been removed.

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

[0033] 13. After the last amino acid is added, the protection is removed, and the resin is washed four times with DMF. Then, the resin is dried with methanol. The peptide is then cleaved from the resin using a 95% cleavage buffer (trifluoroacetic acid: 1,2-ethylenedithiol: triisopropylsilane: water = 95:2:2:1) (10 mL of cleavage buffer per gram of resin), and precipitated four times by centrifugation with ice-cold diethyl ether (cleavage buffer: diethyl ether = 1:9). Finally, the peptide is purified by HPLC and then lyophilized to obtain the final product.

[0034] II. 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% Acetonide;

[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, 25%-85% B; 25.00-25.01 min, 85%-100% B; 25.01-30.00 min, 100%-100% B; 30.01 min, Stop.

[0043] 2. Test Results

[0044] Information such as the HPLC chromatogram and peak integral area of ​​peptide 3, etc. Figure 1 As shown, the purity of polypeptide 3 is 99.31%.

[0045] III. Mass Spectrometry Detection

[0046] Mass spectrometry detection image of peptide 3 as shown Figure 2 As shown, the molecular weight of [M+H]H+ is 1038.27, which is consistent with the theoretical molecular weight of polypeptide 3, 1037.128 (the theoretical molecular weight was calculated using the polypeptide molecular weight calculator of Hefei Guotai Biotechnology Co., Ltd.).

[0047] Example 2: Activity Test Example

[0048] I. Experimental Materials

[0049] Primary NK cell culture medium (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] II. Experimental Methods

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

[0053] PBMCs were isolated from peripheral blood using density gradient centrifugation, a standard technique in this field, for NK cell isolation and purification. The procedure was as follows: 20 mL of venous blood was drawn from healthy volunteers aged 18–35 years, and density gradient centrifugation was performed using Ficoll lymphocyte separation medium to separate PBMCs. The PBMCs were then resuspended in PBS buffer and counted.

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

[0055] NK cells were isolated using a conventional magnetic bead sorting method. The procedure was as follows: the PBMCs obtained above were sorted using a human NK cell isolation kit (Miltenyi NK Cell Isolation Kit) to obtain human NK cells (CD3-CD56+), which were resuspended in PBS and counted.

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

[0057] 3. In vitro culture of NK cells

[0058] The sorted NK cells were cultured in a primary NK cell culture medium at 37°C, 5% CO2, and saturated humidity, with culture medium 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] NK cells cultured for 9 days were resuspended in RPMI 1640 medium containing 10% FBS and 1% penicillin antibiotics, and cultured at 5 × 10⁻⁶ cells / day. 3 Inoculate one culture medium per well into a 96-well plate. After 12 hours, replace the medium with the following media according to different groups, with 5 replicates per group:

[0061] Standard group: RPMI 1640 medium containing 10% FBS and 1% penicillin antibiotics;

[0062] Low-dose peptide 3 group: RPMI 1640 medium containing 25 μM peptide 3, 10% FBS and 1% penicillin antibiotics;

[0063] High-dose peptide 3 group: RPMI 1640 medium containing 50 μM peptide 3, 10% FBS and 1% penicillin antibody.

[0064] After culturing at 37℃, 5% CO2, and saturated humidity for 48 hours, 20 μL of CCK-8 solution was added to each well, and the cells were incubated at 37℃ and 5% CO2 for 4 hours. The OD value of each well was then 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 3 was calculated (the ratio of the OD value of the conventional group to that of the conventional group, converted to a percentage).

[0065] 4. Assay for NK cell killing activity

[0066] NK cells incubated in different culture media (as shown below) were used as effector cells, and human pancreatic cancer Capan-2 cells were used as target cells. The target cells were cultured in a 5×10⁻⁶ culture medium. 3 NK cells were seeded per well in a 96-well plate. After 12 hours, the culture medium was gently aspirated, and effector cells were added at an effector-to-target ratio of 5:1, with 200 μL of culture medium (RPMI 1640 medium containing 10% FBS and 1% penicillin antibiotics) per well. Five replicates of each were also prepared, one for target cells and one for effector cells. After co-culturing for 24 hours, 20 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 4 hours. The OD value of each well was measured at 490 nm. The cytotoxic activity of NK cells against tumor cells incubated in different culture media was calculated using the formula: Kill rate (%) = [1 - (OD value of experimental group - OD value of effector cells only) / OD value of target cell group only] × 100%.

[0067] Conventional group: Incubated for 12 h in RPMI 1640 medium containing 10% FBS and 1% penicillin antibiotics;

[0068] Low-dose peptide 3 group: incubated for 12 h in RPMI 1640 medium containing 25 μM peptide 3, 10% FBS and 1% penicillin antibiotics;

[0069] High-dose peptide 3 group: incubated for 12 h in RPMI 1640 medium containing 50 μM peptide 3, 10% FBS and 1% penicillin antibody.

[0070] 5. Statistical processing

[0071] Experimental results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.0 software and one-way ANOVA was used. A significance level of P < 0.05 was considered statistically significant.

[0072] III. Experimental Results

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

[0074] After magnetic bead sorting, the proportion of CD3-CD56+ cells reached as high as 95.62%. Those skilled in the art know that CD3-CD56+ cells are NK cells, indicating that the magnetic bead sorting was successful.

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

[0076] The proliferation activities of NK cells in each group are shown in Table 1. Compared with the conventional group, the proliferation activities of NK cells in the low-dose and high-dose groups of peptide 3 showed a slight decreasing trend, but the decrease was not significant, and the proliferation activities remained above 95%.

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

[0078] Regular group Low-dose group of peptide 3 High-dose group of peptide 3 Proliferative activity (%) 100±2.29 98.35±2.45 96.57±2.36

[0079] The results indicate that when peptide 3 is used to culture NK cells in vitro, it does not significantly promote their proliferation activity.

[0080] 3. Assay of NK cell killing activity

[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 3 was significantly increased, and showed a clear dose-dependent effect.

[0082] Table 2. Kill rate of NK cells against Capan-2 cells in each group

[0083] Regular group Low-dose group of peptide 3 High-dose group of peptide 3 lethality (%) 19.65±2.14 41.08±1.98 68.63±2.52

[0084] These results indicate that peptide 3 can significantly enhance the killing activity of NK cells against tumor cells.

[0085] In summary, the polypeptide 3 provided by this invention can be used as an active peptide for in vitro culture of NK cells, and using this active peptide as a culture additive can effectively enhance the killing power of NK cells against tumors.

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

Claims

1. An active peptide that enhances the tumor-killing ability of NK cells, characterized in that: The sequence is shown as Sequence NO.

1.

2. A method for enhancing the tumor-killing ability of NK cells in vitro by using the active peptide shown in Sequence NO.1 of claim 1 as a culture additive.

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