Gamma delta T cell preparation as well as preparation method and application thereof

Abstract

The invention provides a gamma delta T cell preparation as well as a preparation method and application thereof, and belongs to the technical field of T cells. The liposome is prepared from the following raw materials in parts by weight: 15-20 parts of modified gamma delta T cell liposome, 1-3 parts of cell stimulating factors and 4-7 parts of culture medium freeze-dried powder, the modified gamma delta T cell lipidosome is prepared by embedding gamma delta T cells through lipidosome, coupling with acetylenic bond modified chitosan, and further mixing with MFAP4 protein and b4GALT1 protein. The culture medium freeze-dried powder is prepared by freeze-drying the culture medium in the engineering culture process of gamma delta T cells, and the cell stimulating factors are resveratrol and quercitrin. According to the gamma delta T cell preparation prepared by the invention, the survival ability and resistance of gamma delta T cells are improved, the anti-tumor activity of the gamma delta T cells is improved, and the gamma delta T cell preparation has relatively good antioxidant and anti-inflammatory effects, reduces the inhibition of inflammation on an immune system and has a very good treatment effect on pancreatic ductal adenocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of T cell technology, specifically to a γδT cell preparation, its preparation method, and its application. Background Technology

[0002] Based on the different compositions of the T cell receptor (TCR) double-chain peptide, human T lymphocytes can be divided into TCR αβ T cells (αβ T cells) and TCR γδ T cells (γδ T cells). γδ T cells directly recognize and bind to antigen molecules via a non-restrictive major histocompatibility complex (MHC) pathway, and kill tumor cells and virus-infected cells through perforin-granzyme, Fas / FasL, IFN-γ secretion, and TNF-related apoptosis-inducing ligand receptor TRAILR. In addition to effectively killing tumor cells, γδ T cells can also promote the activation of other immune cells by secreting cytokines such as IFN-γ. Therefore, they play an important role in immune surveillance and immunomodulation in both innate and adaptive immune responses, and are considered a crucial bridge between innate and adaptive immunity. γδ T cells possess dual characteristics of NK cells and T cells. Besides expressing TCRγδ, their surface also highly expresses the important NK cell activation receptor NKG2D. These two receptor molecules play a crucial role in the killing of tumor cells by γδ T cells. Among these, TCRγδ alone can activate γδT cells, while NKG2D acts as a co-stimulatory agent in this process, enabling γδT cells to rapidly activate upon antigen recognition and produce corresponding biological effects. This rapid activation during the body's defense against tumors or infections allows them to play a crucial role in anti-tumor or anti-infection efforts. Furthermore, studies have found that γδT cells possess MHC-free tumor-killing activity, exhibiting significant killing activity against various autologous, allogeneic, or xenogeneic tumor cells. Therefore, γδT cells have attracted increasing attention from domestic and international scholars as an important candidate cell type for adoptive immunotherapy in tumor treatment.

[0003] However, since γδT cells account for only 1-5% of human peripheral blood, obtaining a large number of γδT cells with high cytotoxic activity is extremely difficult, thus limiting their clinical application. Although existing techniques mostly use anti-TCRγδ antibodies or non-peptide phosphonate antigens to obtain large numbers of γδT cells, they have not been widely used due to their relatively high cost and limited expansion fold. Summary of the Invention

[0004] The purpose of this invention is to propose a γδT cell preparation, its preparation method, and its application, which improves the survival and resistance of γδT cells, enhances their antitumor activity, has good antioxidant and anti-inflammatory effects, reduces the suppression of the immune system by inflammation, and has a good therapeutic effect on pancreatic ductal adenocarcinoma.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a γδT cell preparation, which is prepared from the following raw materials in parts by weight: 15-20 parts of modified γδT cell liposomes, 1-3 parts of cell stimulating factors, and 4-7 parts of lyophilized culture medium powder; the modified γδT cell liposomes are prepared by embedding γδT cells in liposomes, then coupling them with chitosan modified by alkyne bonds, and further combining them with MFAP4 protein and b4GALT1 protein; the lyophilized culture medium powder is prepared by freeze-drying the culture medium used in the engineered culture of γδT cells; and the cell stimulating factors are resveratrol and quercetin.

[0007] As a further improvement of the present invention, the method for preparing the modified γδT cell liposomes is as follows:

[0008] S1. Membrane proteins of PANC02 cells were extracted using a membrane protein extraction kit and prepared into a membrane protein solution. Lecithin and cholesterol were dissolved in chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes. The membrane protein solution and engineered γδT cells were added, mixed evenly, and repeatedly extruded using a polycarbonate membrane liposome extruder to obtain biomimetic γδT cell liposomes.

[0009] S2. Add biomimetic γδT cell liposomes to water, add NHS and EDC, stir to activate, add modified chitosan, stir to react, centrifuge, wash, dry, and obtain modified chitosan biomimetic γδT cell liposomes;

[0010] S3. Mix MFAP4 protein, b4GALT1 protein and modified chitosan biomimetic γδT cell liposomes and add them to DMF solvent. Irradiate with ultraviolet light, centrifuge, wash and dry to obtain modified γδT cell liposomes.

[0011] As a further improvement of the present invention, in step S1, the concentration of the membrane protein solution is 8-15 mg / mL, the mass ratio of lecithin, cholesterol, engineered γδT cells, and membrane protein solution is 10-15:5-10:4-8:20-30, the number of repeated extrusions is 10-20 times, and the diameter of the liposome extruder of the polycarbonate membrane is 1-2 μm; in step S2, the mass ratio of biomimetic γδT cell liposomes, NHS, EDC, and modified chitosan is 15-20:2-4:2-4:3-5; in step S3, the mass ratio of MFAP4 protein, b4GALT1 protein, and modified chitosan biomimetic γδT cell liposomes is 1-2:1-2:8-10, and the ultraviolet lamp irradiation time is 1-2 min.

[0012] As a further improvement of the present invention, the method for preparing the engineered γδT cells and culture medium lyophilized powder is as follows:

[0013] T1. Preparation of fermentation extract: Lactobacillus plantarum seed liquid was inoculated into birch sap, fermented, filtered, and the filtrate was freeze-dried to obtain fermentation extract;

[0014] T2. Preparation of nanoparticles: Nanomagnetic iron oxide nanoparticles were added to water, along with lanthanum salt and cerium salt. The mixture was stirred and adsorbed, separated by a magnet, dried, and calcined to obtain nanoparticles.

[0015] T3. Preparation of culture medium: Nanoparticles, zoledronic acid and fermentation extract were added to RPMI1640 culture medium containing fetal bovine serum, ultrasonically dispersed evenly, and sterilized to obtain the culture medium;

[0016] T4. In vitro stimulation and expansion culture: The isolated peripheral blood mononuclear cells were added to the culture medium, the cell concentration was adjusted, and the cells were stimulated to proliferate under an external magnetic field. The cells were cultured, collected, separated, and engineered γδT cells were obtained. The nanoparticles were separated by a liquid magnet, and the remaining liquid was freeze-dried to obtain the lyophilized culture medium powder.

[0017] As a further improvement of the present invention, the bacterial count of the *Lactobacillus plantarum* seed solution in step T1 is 10. 8 -10 9 The fermentation culture temperature is 36-38℃, 100-200 r / min, and the time is 36-48 h; in step T2, the mass ratio of nano-magnetic iron oxide, lanthanum salt, and cerium salt is 10-20:1-3:2-4, the lanthanum salt is lanthanum chloride or lanthanum nitrate, and the cerium salt is cerium chloride and cerium nitrate; the calcination temperature is 500-700℃, and the time is 1-3 h; in step T3, the concentration of fetal bovine serum in the RPMI1640 culture medium is 5-10 wt%, and the mass ratio of the RPMI1640 culture medium containing fetal bovine serum, nanoparticles, zoledronic acid, and fermentation extract is 100:3-5:1-3:5-8; in step T4, the cell concentration is adjusted to 3-6 × 10⁻⁶. 6 The number of cells / mL, the strength of the external magnetic field is 0.2-0.5T, the culture conditions are 36-38℃, 4-6v / v%CO2, the time is 3-6d, and the separation is performed by flow cytometry.

[0018] As a further improvement of the present invention, the method for preparing the modified chitosan is as follows:

[0019] BOC-L-propargylglycine was added to acetone, chitosan was dissolved in dilute acid solution, and the above solution was added. EDC and NHS were added, and the mixture was stirred at room temperature. The product was added to ethanol, precipitated, filtered, and washed with the solid. The product was added to trifluoroacetic acid solution, heated and stirred, added to ethanol, precipitated, washed with the solid, and dried to obtain modified chitosan.

[0020] As a further improvement of the present invention, the mass ratio of BOC-L-propargylglycine, chitosan, NHS and EDC is 0.5-1:3-5:0.3-0.5:0.4-0.7, the dilute acid solution is a 1-3wt% hydrochloric acid or sulfuric acid solution, the stirring reaction time at room temperature is 10-15h, the concentration of the trifluoroacetic acid solution is 30-40wt%, and the heating and stirring reaction time is 30-35℃ for 20-40min.

[0021] As a further improvement of the present invention, the mass ratio of resveratrol to quercetin in the cell stimulating factor is 3-5:7-10.

[0022] The present invention further protects a method for preparing the above-mentioned γδT cell preparation, comprising the following steps: mixing modified γδT cell liposomes, cell stimulating factors, and lyophilized culture medium powder evenly to obtain the γδT cell preparation.

[0023] This invention further protects the use of the above-mentioned γδT cell preparation in the preparation of a treatment for pancreatic ductal adenocarcinoma.

[0024] The present invention has the following beneficial effects:

[0025] Birch sap contains abundant stimulating substances, including betulinic acid, which can promote the proliferation of γδT cells and the expression of perforin and granzyme B. It also provides sufficient nutrients for the proliferation of lactobacilli, including carbon sources, nitrogen sources, and trace elements, enabling them to produce abundant short-chain fatty acids and other cytokines. Propionic acid can directly act on γδT cells, inhibiting IL-17 production through histone deacetylase-dependent inhibition. Bacterial metabolites such as phosphoantigens, like (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP), are direct upstream metabolites of isopentenyl diphosphate (IPP) and have a strong stimulating effect on γδT cells. HMBPP can bind to the intracellular domain of butyrate protein 3A1, thereby enhancing the activation efficiency of γδT cells and strengthening their anti-cancer effects.

[0026] In addition, nanoparticles are added to the culture medium of this invention to deposit rare earth oxides such as lanthanum oxide and cerium oxide on magnetic iron oxide. On the one hand, these nanoparticles can generate heat energy under the action of an external magnetic field. At the same time, the rare earth oxides can also generate far-infrared rays, which can stimulate peripheral blood mononuclear cells to produce abundant γδT cells. The addition of zoledronic acid can promote the proliferation of γδT cells and the expression of perforin and granzyme B, thereby greatly improving the efficiency of in vitro engineered culture of γδT cells. The obtained γδT cells have better resistance and better anti-tumor activity.

[0027] This invention utilizes engineered γδT cell liposomes prepared with the participation of membrane proteins on the surface of PANC02 cancer cells to create biomimetic liposomes. This mediated ordered targeting increases the efficiency of sequential delivery of γδT cells into the tumor stroma and tumor cells, while also providing excellent protection for γδT cells, preventing their inactivation during transportation and storage. Furthermore, modified chitosan with alkyne bonds is coupled in, allowing for click chemistry-based coupling with MFAP4 and b4GALT1 proteins. Chitosan also induces polarization and rearrangement of the α-tubulin cytoskeleton in γδT cells, enhancing their cytotoxic ability. Additionally, the modified γδT cell liposomes prepared by this invention reduce the surface roughness of γδT cells, making the cell membrane smoother and improving γδT cell motility.

[0028] MFAP4, an extracellular glycoprotein, may play a role in the interaction between tumor cells and stromal cells, influencing the tumor microenvironment and thus participating in tumor invasion and metastasis. b4GALT1 (beta-1, 4-galactosyltransferase 1), an enzyme involved in intercellular communication and cell adhesion, exhibits altered expression levels in certain cancers, potentially related to tumor initiation and development. Studies have shown that changes in b4GALT1 expression levels in pancreatic ductal adenocarcinoma may affect tumor cell proliferation, migration, and invasion, with both having a synergistic effect.

[0029] Cell-stimulating factors include resveratrol and quercetin. Resveratrol is an antioxidant that can reduce the levels of oxidative stress markers, thereby indirectly supporting the normal function of γδT cells. It can also regulate the activation and function of γδT cells by activating the Sirt1 signaling pathway, and can directly inhibit tumor growth by inhibiting the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) and cutting off the nutrient supply to the tumor. Quercetin has antioxidant and anti-inflammatory effects, reducing DNA damage caused by oxidative stress and lowering the risk of gene mutations. It also indirectly affects the activity of γδT cells by inhibiting inflammatory signaling pathways such as NF-κB and reducing the production of inflammatory mediators. Inhibiting the production of inflammatory factors such as TNF-α, IL-6, and IL-8, thereby reducing the suppression of the immune system by inflammation, may indirectly promote the activity of γδT cells; the two have a synergistic effect.

[0030] The γδT cell preparation prepared by this invention improves the survival and resistance of γδT cells, enhances their anti-tumor activity, has good antioxidant and anti-inflammatory effects, reduces the suppression of the immune system by inflammation, and has a good therapeutic effect on pancreatic ductal adenocarcinoma. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Preparation of Lactobacillus plantarum seed culture: Lactobacillus plantarum (20 billion CFU / g) was inoculated into Gao's medium and activated at 37°C and 100 rpm for 24 h to obtain a culture with a bacterial count of 10. 8 -10 9 CFU / mL Lactobacillus plantarum seed solution.

[0033] The MFAP4 protein, Cynomolgus (HEK293, His), has a full length of 234 amino acids and a molecular weight of 35-40 kDa.

[0034] b4GALT1 protein, Human (HEK293, Y285L, His), molecular weight 50-60kDa.

[0035] The average particle size of the nano-magnetic iron oxide is 300-500 nm.

[0036] Preparation Example 1: Preparation of engineered γδT cells and lyophilized culture medium powder

[0037] The method is as follows:

[0038] T1. Preparation of fermentation extract: Lactobacillus plantarum seed liquid was inoculated into birch sap at an inoculation amount of 4v / v%, fermented at 36℃ and 100r / min for 36h, filtered, and the filtrate was freeze-dried to obtain fermentation extract.

[0039] T2. Preparation of nanoparticles: 10g of nano-magnetic iron oxide was added to 200mL of water, along with 1g of lanthanum chloride and 2g of cerium chloride. The mixture was stirred to adsorb the adsorption, separated by a magnet, dried, and calcined at 500℃ for 1h to obtain nanoparticles.

[0040] T3. Preparation of culture medium: Add 3g of nanoparticles, 1g of zoledronic acid and 5g of fermentation extract to 100g of RPMI1640 culture medium containing 5wt% fetal bovine serum, disperse by ultrasonication at 400W for 20min, sterilize, and obtain the culture medium.

[0041] T4. In vitro stimulation and expansion culture: The isolated peripheral blood mononuclear cells were added to the culture medium, and the cell concentration was adjusted to 3 × 10⁻⁶. 6Cells were stimulated to proliferate under an external magnetic field of 0.2T at a density of 1 / mL, and cultured at 36℃ with 4v / v%CO2 for 3 days. Cells were then collected, separated by flow cytometry to obtain engineered γδT cells, and nanoparticles were separated by a liquid magnet. The remaining liquid was freeze-dried to obtain lyophilized culture medium powder.

[0042] Preparation Example 2: Preparation of engineered γδT cells and lyophilized culture medium powder

[0043] The method is as follows:

[0044] T1. Preparation of fermentation extract: Lactobacillus plantarum seed liquid was inoculated into birch sap at an inoculation amount of 4v / v%, fermented at 38℃ and 200r / min for 48h, filtered, and the filtrate was freeze-dried to obtain fermentation extract;

[0045] T2. Preparation of nanoparticles: 20g of nano-magnetic iron oxide was added to 200mL of water, along with 3g of lanthanum nitrate and 4g of cerium nitrate. The mixture was stirred to adsorb the particles, separated by a magnet, dried, and calcined at 700℃ for 3h to obtain nanoparticles.

[0046] T3. Preparation of culture medium: Add 5g of nanoparticles, 3g of zoledronic acid and 8g of fermentation extract to 100g of RPMI1640 culture medium containing 10wt% fetal bovine serum, disperse by ultrasonication at 400W for 20min, sterilize, and obtain the culture medium.

[0047] T4. In vitro stimulation and expansion culture: The isolated peripheral blood mononuclear cells were added to the culture medium, and the cell concentration was adjusted to 6 × 10⁻⁶. 6 Cells were stimulated to proliferate under an external magnetic field of 0.5T at 38°C and 6v / v%CO2 for 6 days. Cells were collected, separated by flow cytometry to obtain engineered γδT cells, and nanoparticles were separated by a liquid magnet. The remaining liquid was freeze-dried to obtain lyophilized culture medium powder.

[0048] Preparation Example 3: Preparation of engineered γδT cells and lyophilized culture medium powder

[0049] The method is as follows:

[0050] T1. Preparation of fermentation extract: Lactobacillus plantarum seed liquid was inoculated into birch sap at an inoculation amount of 4v / v%, fermented at 37℃ and 150r / min for 42h, filtered, and the filtrate was freeze-dried to obtain fermentation extract.

[0051] T2. Preparation of nanoparticles: 15g of nano-magnetic iron oxide was added to 200mL of water, along with 2g of lanthanum nitrate and 3g of cerium nitrate. The mixture was stirred to adsorb the adsorption, separated by a magnet, dried, and calcined at 600℃ for 2h to obtain nanoparticles.

[0052] T3. Preparation of culture medium: Add 4g of nanoparticles, 2g of zoledronic acid and 6.5g of fermentation extract to 100g of RPMI1640 culture medium containing 8wt% fetal bovine serum, disperse by ultrasonication at 400W for 20min, sterilize, and obtain the culture medium;

[0053] T4. In vitro stimulation and expansion culture: The isolated peripheral blood mononuclear cells were added to the culture medium, and the cell concentration was adjusted to 4 × 10⁻⁶. 6 Cells were stimulated to proliferate under an external magnetic field of 0.3T at 37°C and 5v / v%CO2 for 5 days. Cells were collected, separated by flow cytometry to obtain engineered γδT cells, and nanoparticles were separated by a liquid magnet. The remaining liquid was freeze-dried to obtain lyophilized culture medium powder.

[0054] Comparative Preparation Example 1

[0055] The difference from Preparation Example 3 is that no fermentation extract was added in step T3.

[0056] The details are as follows:

[0057] T3. Preparation of culture medium: Add 4g of nanoparticles and 2g of zoledronic acid to 100g of RPMI1640 culture medium containing 8wt% fetal bovine serum, disperse by ultrasonication at 400W for 20min, sterilize, and the culture medium is obtained.

[0058] Comparative Preparation Example 2

[0059] The difference from Preparation Example 3 is that lanthanum nitrate and cerium nitrate were not added in step T2.

[0060] The details are as follows:

[0061] T3. Preparation of culture medium: Add 4g of nano-magnetic iron oxide, 2g of zoledronic acid and 6.5g of fermentation extract to 100g of RPMI1640 culture medium containing 8wt% fetal bovine serum, disperse by ultrasonication at 400W for 20min, sterilize, and obtain the culture medium.

[0062] Comparative preparation example 3

[0063] The difference from Preparation Example 3 is that no nanoparticles were added in step T3.

[0064] The details are as follows:

[0065] T3. Preparation of culture medium: Add 2g of zoledronic acid and 6.5g of fermentation extract to 100g of RPMI1640 culture medium containing 8wt% fetal bovine serum, disperse by ultrasonication at 400W for 20min, sterilize, and the culture medium is obtained.

[0066] Preparation Example 4: The preparation method of modified chitosan is as follows:

[0067] 0.5 g of BOC-L-propargylglycine was added to 50 mL of acetone. 3 g of chitosan was dissolved in 200 mL of 1 wt% hydrochloric acid solution. The above solution was added, along with 0.3 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 0.4 g of NHS (N-hydroxysuccinimide). The mixture was stirred at room temperature for 10 h. The product was added to ethanol and precipitated for 1 h. After filtration, the solid was washed and added to 30 wt% trifluoroacetic acid solution. The mixture was heated to 30 °C and stirred for 20 min. After precipitation, the solid was added to ethanol and precipitated for 1 h. The solid was washed and dried to obtain modified chitosan.

[0068] Preparation Example 5: The preparation method of modified chitosan is as follows:

[0069] 1 g of BOC-L-propargylglycine was added to 50 mL of acetone. 5 g of chitosan was dissolved in 200 mL of 3 wt% hydrochloric acid solution. The above solution was added, along with 0.5 g of EDC and 0.7 g of NHS. The mixture was stirred at room temperature for 15 h. The product was added to ethanol and precipitated for 1 h. The mixture was filtered, washed with the solid, and added to 40 wt% trifluoroacetic acid solution. The mixture was heated to 35 °C and stirred for 40 min. The mixture was then added to ethanol and precipitated for 1 h. The solid was washed and dried to obtain modified chitosan.

[0070] Preparation Example 6: The preparation method of modified chitosan is as follows:

[0071] 0.7 g of BOC-L-propargylglycine was added to 50 mL of acetone. 4 g of chitosan was dissolved in 200 mL of 2 wt% hydrochloric acid solution. The above solution was added, along with 0.4 g of EDC and 0.55 g of NHS. The mixture was stirred at room temperature for 12 h. The product was added to ethanol and precipitated for 1 h. After filtration and washing of the solid, the product was added to 35 wt% trifluoroacetic acid solution. The mixture was heated to 32 °C and stirred for 30 min. After precipitation, the product was added to ethanol and precipitated for 1 h. The solid was washed and dried to obtain modified chitosan.

[0072] Preparation Example 7: Preparation of modified γδT cell liposomes

[0073] The method is as follows:

[0074] S1. Membrane proteins of PANC02 cells in the logarithmic growth phase were extracted using a membrane protein extraction kit (brand: Solarbio, catalog number EX1500) to prepare an 8 mg / mL membrane protein solution; 10 g of lecithin and 5 g of cholesterol were dissolved in 200 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes; 20 g of membrane protein solution and 4 g of engineered γδT cells prepared in Example 1 were added, mixed evenly, and repeatedly extruded 10 times using a polycarbonate membrane liposome extruder with a diameter of 1 μm to obtain biomimetic γδT cell liposomes;

[0075] S2. Add 15g of biomimetic γδT cell liposomes to 200mL of water, add 2g of NHS and 2g of EDC, stir and activate for 30min, add 3g of modified chitosan obtained in Preparation Example 4, stir and react for 10h, centrifuge, wash, dry, and obtain modified chitosan biomimetic γδT cell liposomes.

[0076] S3. Mix 1g MFAP4 protein, 1g b4GALT1 protein and 8g modified chitosan biomimetic γδT cell liposomes and add them to 100mL DMF (N,N-dimethylformamide) solvent. Irradiate with UV lamp for 1min, centrifuge, wash and dry to obtain modified γδT cell liposomes.

[0077] Preparation Example 8: Preparation of modified γδT cell liposomes

[0078] The method is as follows:

[0079] S1. Membrane proteins of PANC02 cells in the logarithmic growth phase were extracted using a membrane protein extraction kit and prepared into a 15 mg / mL membrane protein solution. 15 g of lecithin and 10 g of cholesterol were dissolved in 200 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes. 30 g of the membrane protein solution and 8 g of the engineered γδT cells prepared in Example 2 were added, mixed evenly, and repeatedly extruded 20 times using a polycarbonate membrane liposome extruder with a diameter of 2 μm to obtain biomimetic γδT cell liposomes.

[0080] S2. Add 20g of biomimetic γδT cell liposomes to 200mL of water, add 4g of NHS and 4g of EDC, stir and activate for 30min, add 5g of modified chitosan obtained in Preparation Example 4, stir and react for 10h, centrifuge, wash, dry, and obtain modified chitosan biomimetic γδT cell liposomes.

[0081] S3. Mix 2g MFAP4 protein, 2g b4GALT1 protein and 10g modified chitosan biomimetic γδT cell liposomes into 100mL DMF solvent, irradiate with UV lamp for 2min, centrifuge, wash and dry to obtain modified γδT cell liposomes.

[0082] Preparation Example 9: Preparation of modified γδT cell liposomes

[0083] The method is as follows:

[0084] S1. Membrane proteins of PANC02 cells in the logarithmic growth phase were extracted using a membrane protein extraction kit and prepared into a 12 mg / mL membrane protein solution. 12 g of lecithin and 7 g of cholesterol were dissolved in 200 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes. 25 g of the membrane protein solution and 6 g of the engineered γδT cells prepared in Example 3 were added, mixed evenly, and repeatedly extruded 15 times using a polycarbonate membrane liposome extruder with a diameter of 2 μm to obtain biomimetic γδT cell liposomes.

[0085] S2. Add 17g of biomimetic γδT cell liposomes to 200mL of water, add 3g of NHS and 3g of EDC, stir and activate for 30min, add 4g of modified chitosan obtained in Preparation Example 4, stir and react for 10h, centrifuge, wash, dry, and obtain modified chitosan biomimetic γδT cell liposomes.

[0086] S3. Mix 1.5g MFAP4 protein, 1.5g b4GALT1 protein and 9g modified chitosan biomimetic γδT cell liposomes and add them to 100mL DMF solvent. Irradiate with UV lamp for 90s, centrifuge, wash and dry to obtain modified γδT cell liposomes.

[0087] Comparative preparation example 4

[0088] The difference from Preparation Example 3 is that no membrane protein solution was added in step S1.

[0089] The details are as follows:

[0090] S1. Dissolve 12g of lecithin and 7g of cholesterol in 200mL of chloroform, remove the solvent by rotary evaporation under reduced pressure to obtain liposomes, add 6g of engineered γδT cells prepared in Preparation Example 3, mix well, and repeatedly extrude 15 times using a polycarbonate membrane liposome extruder with a diameter of 2μm to obtain γδT cell liposomes.

[0091] Comparative preparation example 5

[0092] The difference from preparation example 3 is that step S2 was not performed.

[0093] The details are as follows:

[0094] S1. Membrane proteins of PANC02 cells in the logarithmic growth phase were extracted using a membrane protein extraction kit and prepared into a 12 mg / mL membrane protein solution. 12 g of lecithin and 7 g of cholesterol were dissolved in 200 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes. 25 g of the membrane protein solution and 6 g of the engineered γδT cells prepared in Example 3 were added, mixed evenly, and repeatedly extruded 15 times using a polycarbonate membrane liposome extruder with a diameter of 2 μm to obtain biomimetic γδT cell liposomes.

[0095] S2. Mix 1.5g MFAP4 protein, 1.5g b4GALT1 protein and 9g biomimetic γδT cell liposomes and add them to 100mL DMF solvent. Irradiate with UV lamp for 90s, centrifuge, wash and dry to obtain modified γδT cell liposomes.

[0096] Comparative preparation example 6

[0097] The difference from Preparation Example 3 is that MFAP4 protein was not added in step S3.

[0098] The details are as follows:

[0099] S3. Mix 3g of b4GALT1 protein and 9g of modified chitosan biomimetic γδT cell liposomes and add them to 100mL of DMF solvent. Irradiate with UV lamp for 90s, centrifuge, wash, and dry to obtain modified γδT cell liposomes.

[0100] Comparative preparation example 7

[0101] The difference from Preparation Example 3 is that b4GALT1 protein was not added in step S3.

[0102] The details are as follows:

[0103] S3. Mix 3g of MFAP4 protein and 9g of modified chitosan biomimetic γδT cell liposomes into 100mL of DMF solvent, irradiate with UV lamp for 90s, centrifuge, wash, and dry to obtain modified γδT cell liposomes.

[0104] Comparative Preparation Example 8

[0105] The difference from preparation example 3 is that step S3 was not performed.

[0106] The details are as follows:

[0107] S1. Membrane proteins of PANC02 cells in the logarithmic growth phase were extracted using a membrane protein extraction kit and prepared into a 12 mg / mL membrane protein solution. 12 g of lecithin and 7 g of cholesterol were dissolved in 200 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes. 25 g of the membrane protein solution and 6 g of the engineered γδT cells prepared in Example 3 were added, mixed evenly, and repeatedly extruded 15 times using a polycarbonate membrane liposome extruder with a diameter of 2 μm to obtain biomimetic γδT cell liposomes.

[0108] S2. Add 17g of biomimetic γδT cell liposomes to 200mL of water, add 3g of NHS and 3g of EDC, stir and activate for 30min, add 4g of modified chitosan obtained in Preparation Example 4, stir and react for 10h, centrifuge, wash, dry, and obtain modified chitosan biomimetic γδT cell liposomes.

[0109] Comparative preparation example 9

[0110] The difference from Preparation Example 3 is that the engineered γδT cells were prepared by Comparative Preparation Example 1.

[0111] Comparative Preparation Example 10

[0112] The difference from Preparation Example 3 is that the engineered γδT cells were prepared by Comparative Preparation Example 2.

[0113] Comparative Preparation Example 11

[0114] The difference from Preparation Example 3 is that the engineered γδT cells were prepared by Comparative Preparation Example 3.

[0115] Example 1

[0116] This embodiment provides a γδT cell preparation.

[0117] Raw material composition (parts by weight): 15 parts of modified γδT cell liposomes obtained in Preparation Example 7, 1 part of cell stimulating factor, and 4 parts of lyophilized culture medium powder obtained in Preparation Example 1. The mass ratio of resveratrol to quercetin in the cell stimulating factor is 3:7.

[0118] The preparation method includes the following steps: mixing modified γδT cell liposomes, cell stimulating factors, and lyophilized culture medium powder for 10 min to obtain γδT cell preparation.

[0119] Example 2

[0120] This embodiment provides a γδT cell preparation.

[0121] Raw material composition (parts by weight): 20 parts of modified γδT cell liposomes prepared in Preparation Example 8, 3 parts of cell stimulating factor, and 7 parts of lyophilized culture medium powder prepared in Preparation Example 2. The mass ratio of resveratrol to quercetin in the cell stimulating factor is 5:10.

[0122] The preparation method includes the following steps: mixing modified γδT cell liposomes, cell stimulating factors, and lyophilized culture medium powder for 10 min to obtain γδT cell preparation.

[0123] Example 3

[0124] This embodiment provides a γδT cell preparation.

[0125] Raw material composition (parts by weight): 17 parts of modified γδT cell liposomes obtained in Preparation Example 9, 2 parts of cell stimulating factor, and 5.5 parts of lyophilized culture medium powder obtained in Preparation Example 3. The mass ratio of resveratrol to quercetin in the cell stimulating factor is 4:8.

[0126] The preparation method includes the following steps: mixing modified γδT cell liposomes, cell stimulating factors, and lyophilized culture medium powder for 10 min to obtain γδT cell preparation.

[0127] Comparative Example 1

[0128] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 4.

[0129] Comparative Example 2

[0130] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 5.

[0131] Comparative Example 3

[0132] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 6.

[0133] Comparative Example 4

[0134] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 7.

[0135] Comparative Example 5

[0136] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 8.

[0137] Comparative Example 6

[0138] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 9.

[0139] Comparative Example 7

[0140] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 10.

[0141] Comparative Example 8

[0142] The difference from Example 3 is that the modified γδT cell liposomes were prepared by Comparative Preparation Example 11.

[0143] Test Example 1

[0144] PANC02 cells in the logarithmic growth phase were digested and collected to prepare a tumor cell suspension (10). 6 (units / mL).

[0145] C57BL / 6 mice, weighing 18-22g, were selected. 0.3mL of mouse pancreatic cancer PANC02 tumor cell suspension was subcutaneously injected into the left axillary dorsal region of each mouse. The day of injection was recorded as D0. The mice were randomly divided into three groups: a PBS (solvent control) group, a gemcitabine group, and groups of Examples 1-3 or Comparative Examples 1-8, with 10 mice in each group. On day 1 (D1), the gemcitabine group received an intraperitoneal injection of 50.0 mg / kg, administered once every 4 days for a total of 2 doses. The Examples 1-3 or Comparative Examples 1-8 groups received an intraperitoneal injection of the corresponding prepared product, 10.0 mg / kg, administered once every 2 days for a total of 3 doses. The PBS group received an intraperitoneal injection of an equal volume of PBS buffer, 10.0 mg / kg, administered once every 2 days for a total of 3 doses.

[0146] At the end of the experiment (D7), the tumor volume was measured three times using vernier calipers, and the average value was taken. This included both the major and minor diameters of the tumor. The volume was calculated using the formula: Volume = 0.5 × Major Diameter × Minor Diameter 2 .

[0147] Tumor inhibition rate (%) = [1 - tumor volume in the treatment group / tumor volume in the PBS group] × 100%.

[0148] The results are shown in Table 1.

[0149] Table 1

[0150] Group Tumor inhibition rate (%) PBS group / Giscitabine Group 85.7 Example 1 90.4 Example 2 90.9 Example 3 91.2 Comparative Example 1 80.1 Comparative Example 2 86.9 Comparative Example 3 87.0 Comparative Example 4 86.4 Comparative Example 5 83.1 Comparative Example 6 84.3 Comparative Example 7 87.4 Comparative Example 8 85.2

[0151] As can be seen from the table above, the γδT cell preparations obtained in Examples 1-3 of this invention have good tumor suppression effects.

[0152] At the end of the experiment (D7), the mice were weighed, anesthetized and euthanized, and their spleens and thymuses were removed, washed, weighed and recorded, and the corresponding organ indices were calculated.

[0153] Spleen index = spleen mass (mg) / body mass (g) × 10;

[0154] Thymus index = thymus mass (mg) / body mass (g) × 10.

[0155] The results are shown in Table 2.

[0156] Table 2

[0157] Group Spleen index (mg / 10g) Thymus index (mg / 10g) PBS group 46.82±10.35 12.09±2.11 GEM group 28.95±6.19 6.89±1.84 Example 1 45.18±7.25 6.46±1.72 Example 2 45.01±6.89 6.55±1.80 Example 3 44.48±7.39 6.62±1.69 Comparative Example 1 44.82±6.82 6.44±1.72 Comparative Example 2 44.18±8.94 6.32±1.67 Comparative Example 3 45.28±6.38 6.67±1.82 Comparative Example 4 44.81±6.18 6.51±1.88 Comparative Example 5 45.32±5.59 6.60±2.05 Comparative Example 6 44.39±6.79 6.43±1.82 Comparative Example 7 45.73±7.79 6.33±1.75 Comparative Example 8 45.10±8.39 6.49±1.84

[0158] As can be seen from the table above, the γδT cell preparations obtained in Examples 1-3 of this invention do not cause damage to organs.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A γδT cell preparation, characterized in that, The product is prepared from the following raw materials in parts by weight: 15-20 parts of modified γδT cell liposomes, 1-3 parts of cell stimulating factors, and 4-7 parts of lyophilized culture medium powder. The modified γδT cell liposomes are prepared by embedding γδT cells in liposomes, then coupling them with chitosan modified by alkyne bonds, and further combining them with MFAP4 protein and b4GALT1 protein. The lyophilized culture medium powder is prepared by freeze-drying the culture medium used in the engineered culture of γδT cells. The cell stimulating factors are resveratrol and quercetin.

2. The γδT cell preparation according to claim 1, characterized in that, The method for preparing the modified γδT cell liposomes is as follows: S1. Membrane proteins of PANC02 cells were extracted using a membrane protein extraction kit and prepared into a membrane protein solution. Lecithin and cholesterol were dissolved in chloroform, and the solvent was removed by rotary evaporation under reduced pressure to obtain liposomes. The membrane protein solution and engineered γδT cells were added, mixed evenly, and repeatedly extruded using a polycarbonate membrane liposome extruder to obtain biomimetic γδT cell liposomes. S2. Add biomimetic γδT cell liposomes to water, add NHS and EDC, stir to activate, add modified chitosan, stir to react, centrifuge, wash, dry, and obtain modified chitosan biomimetic γδT cell liposomes; S3. Mix MFAP4 protein, b4GALT1 protein and modified chitosan biomimetic γδT cell liposomes and add them to DMF solvent. Irradiate with ultraviolet light, centrifuge, wash and dry to obtain modified γδT cell liposomes.

3. The γδT cell preparation according to claim 2, characterized in that, In step S1, the concentration of the membrane protein solution is 8-15 mg / mL, the mass ratio of lecithin, cholesterol, engineered γδT cells, and membrane protein solution is 10-15:5-10:4-8:20-30, the number of repeated extrusions is 10-20, and the diameter of the liposome extruder for the polycarbonate membrane is 1-2 μm; in step S2, the mass ratio of biomimetic γδT cell liposomes, NHS, EDC, and modified chitosan is 15-20:2-4:2-4:3-5; in step S3, the mass ratio of MFAP4 protein, b4GALT1 protein, and modified chitosan biomimetic γδT cell liposomes is 1-2:1-2:8-10, and the UV irradiation time is 1-2 min.

4. The γδT cell preparation according to claim 2, characterized in that, The preparation method of the engineered γδT cells and culture medium lyophilized powder is as follows: T1. Preparation of fermentation extract: Lactobacillus plantarum seed liquid was inoculated into birch sap, fermented, filtered, and the filtrate was freeze-dried to obtain fermentation extract; T2. Preparation of nanoparticles: Nanomagnetic iron oxide nanoparticles were added to water, along with lanthanum salt and cerium salt. The mixture was stirred and adsorbed, separated by a magnet, dried, and calcined to obtain nanoparticles. T3. Preparation of culture medium: Nanoparticles, zoledronic acid and fermentation extract were added to RPMI1640 culture medium containing fetal bovine serum, ultrasonically dispersed evenly, and sterilized to obtain the culture medium; T4. In vitro stimulation and expansion culture: The isolated peripheral blood mononuclear cells were added to the culture medium, the cell concentration was adjusted, and the cells were stimulated to proliferate under an external magnetic field. The cells were cultured, collected, separated, and engineered γδT cells were obtained. The nanoparticles were separated by a liquid magnet, and the remaining liquid was freeze-dried to obtain the lyophilized culture medium powder.

5. The γδT cell preparation according to claim 4, characterized in that, The bacterial count of the *Lactobacillus plantarum* seed solution in step T1 is 10. 8 -10 9 The fermentation culture temperature is 36-38℃, 100-200 r / min, and the time is 36-48 h; in step T2, the mass ratio of nano-magnetic iron oxide, lanthanum salt, and cerium salt is 10-20:1-3:2-4, the lanthanum salt is lanthanum chloride or lanthanum nitrate, and the cerium salt is cerium chloride and cerium nitrate; the calcination temperature is 500-700℃, and the time is 1-3 h; in step T3, the concentration of fetal bovine serum in the RPMI1640 culture medium is 5-10 wt%, and the mass ratio of the RPMI1640 culture medium containing fetal bovine serum, nanoparticles, zoledronic acid, and fermentation extract is 100:3-5:1-3:5-8; in step T4, the cell concentration is adjusted to 3-6 × 10⁻⁶. 6 The number of cells / mL, the strength of the external magnetic field is 0.2-0.5T, the culture conditions are 36-38℃, 4-6v / v%CO2, the time is 3-6d, and the separation is performed by flow cytometry.

6. The γδT cell preparation according to claim 2, characterized in that, The modified chitosan is prepared as follows: BOC-L-propargylglycine was added to acetone, chitosan was dissolved in dilute acid solution, and the above solution was added. EDC and NHS were added, and the mixture was stirred at room temperature. The product was added to ethanol, precipitated, filtered, and washed with the solid. The product was added to trifluoroacetic acid solution, heated and stirred, added to ethanol, precipitated, washed with the solid, and dried to obtain modified chitosan.

7. The γδT cell preparation according to claim 6, characterized in that, The mass ratio of BOC-L-propargylglycine, chitosan, NHS, and EDC is 0.5-1:3-5:0.3-0.5:0.4-0.

7. The dilute acid solution is a 1-3 wt% hydrochloric acid or sulfuric acid solution. The stirring reaction at room temperature lasts for 10-15 hours. The concentration of the trifluoroacetic acid solution is 30-40 wt%. The heating and stirring reaction lasts for 20-40 minutes at 30-35°C.

8. The γδT cell preparation according to claim 1, characterized in that, The mass ratio of resveratrol to quercetin in the cell stimulating factor is 3-5:7-10.

9. A method for preparing a γδT cell preparation according to any one of claims 1-7, characterized in that, Includes the following steps: The modified γδT cell liposomes, cell stimulating factors, and lyophilized culture medium powder were mixed evenly to prepare the γδT cell preparation.

10. The use of a γδT cell preparation as described in any one of claims 1-7 in the preparation of a treatment for pancreatic ductal adenocarcinoma.