Universal dnt cell public library, dnt cell preparation, and methods of making and using the same

Abstract

The application discloses a universal DNT cell public library, a DNT cell preparation and a preparation method thereof, 200 mL of peripheral blood of a healthy adult is separated to obtain mononuclear cells, the mononuclear cells are cultured for 12 days by using zoledronate in vitro, and cell surface markers are detected; when the proportion of CD3+CD4-CD8- is greater than 50%, the cells are frozen to obtain DNT public library cells; when needed, the DNT public library cells are co-cultured with zoledronate for 7 days, and a DNT cell preparation with high purity and high quality can be obtained. The application avoids the ethics and operation burden of repeatedly collecting blood of a donor, reduces production cost and risk of dependence on the donor, has a short preparation period, can support multi-batch production, and has stable quality between batches, and can provide sufficient raw material reserves for clinical trials and patients with diseases.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering technology, specifically referring to a universal DNT cell public bank for preparing peripheral blood from healthy adults, DNT cell preparations, and their preparation methods. Background Technology

[0002] DNT cells are a special subtype of T cells, most of which express CD3 molecules but not CD4 and CD8 molecules, accounting for approximately 0.5-5% of peripheral blood T cells in normal individuals. Activated DNT cells secrete large amounts of cytokines, such as IFN-γ and TNF-α. These cytokines can directly promote tumor cell apoptosis, improve the tumor microenvironment, further upregulate ligand expression on tumor cells, promote αβ-T cell activation, and achieve systemic tumor killing. DNT cells, through their highly expressed natural killer receptors (NKRs) such as NKG2D, DNAM-1, NKp30, and TRAIL, bind to ligands highly expressed on tumor cells, such as MICA, MICB, ULBP, CD112, and CD155, further activating DNT cells and achieving tumor cell clearance. Activated DNT cells initiate the granule efflux signaling pathway, releasing cell membrane perforating molecules such as perforin and pro-apoptotic granzymes, directly inducing tumor cell apoptosis.

[0003] Numerous preclinical studies and early clinical trials have demonstrated the unique application potential of DNT cells in tumor immunotherapy. Peripheral blood-derived DNT cells are in an immune-quiet state, unaffected by immunosuppression and functional exhaustion in the tumor microenvironment. Upon in vitro activation, they can recover and exhibit potent anti-tumor activity. Studies have confirmed that DNT cells expanded from healthy donor peripheral blood possess MHC-unrestricted broad-spectrum killing capabilities, exhibiting significant killing effects against various hematologic malignancies and solid tumor cells without inducing graft-versus-host disease, making them ideal candidate cells for allogeneic adoptive immunotherapy. Compared to traditional DNT cell preparation processes, this method eliminates the need for patients to provide shared cells, reducing patient suffering, shortens the preparation cycle, offers greater flexibility and convenience, and ensures batch-to-batch quality stability and control.

[0004] Existing technologies (such as CN119101648A) obtain DNT cells by sorting CD4+ / CD8+ magnetic beads from "filter residue". The DNT cells are then activated with anti-human CD3 monoclonal antibodies. However, a small number of CD4+ or CD8+ T cells often remain in the sorted DNT cells. In this case, the anti-human CD3 monoclonal antibody will non-specifically activate these residual CD4+ and CD8+ T cells, leading to their over-expansion during culture. This not only reduces the purity of DNT cells but may also increase the risk of graft-versus-host disease (GvHD) during allogeneic infusion, posing a safety hazard. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a universal DNT cell public bank, DNT cell preparations, their preparation methods and applications, which eliminates the need for patient blood collection, has a short preparation cycle, and ensures stable and controllable quality between batches.

[0006] This invention is achieved as follows: a method for preparing a universal DNT cell public library, comprising the following steps: (a) Peripheral blood was collected from healthy adults, and peripheral blood mononuclear cells were isolated; (b) The peripheral blood mononuclear cells obtained in step (a) were placed in a complete culture medium containing zoledronic acid, interleukin-2 and fetal bovine serum for a first induction culture until day 12. (c) Detect the proportion of CD3+CD4-CD8- DNT cells in the cells obtained in step (b). When the proportion is ≥50%, freeze the cells to obtain a universal DNT cell public library.

[0007] Furthermore, in step (b), the concentration of zoledronic acid is (1-5) nM / mL, the concentration of interleukin-2 is (200-1000) IU / mL, and the volume fraction of fetal bovine serum is 10%-15%.

[0008] Furthermore, in step (c), the cryopreservation conditions are as follows: 2 × 10⁻⁶ 8 Aliquoting cells into 4 mL portions and freezing them.

[0009] A universal DNT cell public library prepared by the above method, wherein the cells in the public library are cryopreserved cells and the proportion of CD3+CD4-CD8- DNT cells after thawing is ≥50%.

[0010] A method for preparing a universal DNT cell preparation using the aforementioned universal DNT cell public library includes the following steps: (i) Resuscitate cells from the aforementioned universal DNT cell bank; (ii) The revived cells were placed in a complete culture medium containing zoledronic acid, interleukin-2 and fetal bovine serum for a second induction culture for 7 days; (iii) Harvest the cells obtained in step (ii) to obtain a universal DNT cell preparation.

[0011] Furthermore, in step (i), four frozen cell lines are thawed, resulting in a total cell count of 8 × 10⁻⁶ cells after thaw. 8 In step (ii), the resuscitated cells are arranged at 2 × 10⁻⁶. 6Inoculate with cells at a concentration of 1 nM / mL, zoledronic acid at 1 nM / mL, interleukin-2 at 500 IU / mL, and fetal bovine serum at 10% (v / v). On day 3, add complete culture medium to a total volume of 1 L and continue culturing until day 7, when the total cell count reaches (3-4) × 10⁻⁴ cells / mL. 9 When there are only a few cells, all cells are recycled.

[0012] Furthermore, in step (iii), after harvesting the cells, they are washed with sodium chloride injection containing human serum albumin at a mass-volume fraction of (0.2-0.4) g / mL, and then resuspended with sodium chloride injection containing human serum albumin at a mass-volume fraction of (1-3) g / 100mL to prepare the formulation.

[0013] A universal DNT cell preparation prepared by the above method.

[0014] The above-mentioned universal DNT cell public library is used in the preparation of DNT cell preparations for the treatment of tumors.

[0015] The above-mentioned general-purpose DNT cell preparations are used in the preparation of drugs for treating tumors.

[0016] The advantages and technical effects of this invention are as follows: Mononuclear cells are obtained by isolating 200 mL of peripheral blood from healthy adults in a single procedure. After 12 days of in vitro induction and culture with zoledronic acid, cell surface markers are detected. When the ratio of CD3+CD4-CD8- is greater than 50%, the cells are cryopreserved to obtain a DNT cell public bank. When needed, the DNT public bank cells are co-cultured with zoledronic acid for 7 days to obtain a high-purity, high-quality DNT cell preparation. This invention avoids the ethical review burden and blood donation compliance issues caused by the need for repeated collection of blood from the same or different donors in traditional methods. A single collection can meet long-term needs, greatly simplifying the clinical trial and treatment initiation process. Furthermore, the shorter single culture time supports multiple batches of production, and the cell quality is stable and controllable between batches, providing sufficient raw material reserves for clinical trials and patients. Attached Figure Description

[0017] Figure 1 This is a flow cytometry result of DNT public library cells cultured on day 12 of Example 1 of the present invention.

[0018] Figure 2 This is a flow cytometry result of the DNT cell preparation library in Example 1 of the present invention.

[0019] Figure 3 This is a diagram showing the cell-killing experiment results of the universal DNT preparation in Example 1 of this invention.

[0020] Figure 4 This is a cell-killing experiment diagram of the universal DNT preparation in Example 1 of the present invention.

[0021] Figure 5 This is a flow cytometry result of DNT public library cells cultured on day 12 of Example 2 of the present invention.

[0022] Figure 6 This is a flow cytometry result of the DNT cell preparation library in Example 2 of the present invention.

[0023] Figure 7 This is a diagram showing the cell-killing experiment results of the universal DNT preparation in Example 2 of this invention.

[0024] Figure 8 This is a cell-killing experiment diagram of the universal DNT preparation in Example 2 of the present invention.

[0025] Figure 9 This is a flow cytometry plot of DNT public library cells cultured in the experimental group on day 12 in Example 3 of this invention.

[0026] Figure 10 This is a flow cytometry result of the DNT cell preparation library in the experimental group of Example 3 of the present invention.

[0027] Figure 11 This is a flow cytometry result of the control group DNT cell preparation in Example 3 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] (1) Prepare complete culture medium: containing 10% fetal bovine serum and interleukin-2 at a final concentration of 500 IU / mL.

[0030] (2) Preparation of DNT cell public bank: 200 mL of peripheral blood from healthy adults was collected, and PBMCs were obtained by Ficoll density gradient centrifugation. The PBMCs were then processed at a rate of 1 × 10⁻⁶. 6 PBMCs were seeded into multiple T182 cell culture flasks at a concentration of 1 nM / mL, with a total volume of 30 mL. Zoledronic acid (1 nM / mL) was added to each flask for induction culture. During culture, complete culture medium was added every 2-3 days to maintain a cell concentration of (0.4-0.6) × 10⁻⁶ cells / mL. 9 Cells / L; after 12 days of culture, the total cell count reached (2.8-3) × 10⁻⁶. 10 When the number of cells is ≥50% and the proportion of DNT cell surface markers (CD3+CD4-CD8-) is ≥50%, the cells are resuspended in 1640 medium to adjust the cell concentration to 1×10⁻⁶. 8 / mL, mix cell suspension with cryopreservation solution 1:1, and repeat at 2×108 Cells were cryopreserved at the DNT public bank at a rate of 1 cell / 4 mL.

[0031] (3) Preparation of cryopreservation solution: Mix 40% 1640 culture medium, 20% DMSO (ORIGEN / CP70) and 40% fetal bovine serum, and store under cold.

[0032] (4) Preparation of universal DNT cell preparation: Resuscitate 4 of the above-mentioned DNT common bank cells in a metal bath, according to 2×10 6 Cells were seeded at a concentration of 100 cells / mL in a permeable culture flask, and 1 nM / mL zoledronic acid was added for induction. The flasks were then incubated at 37°C in a 5.0% CO2 incubator. On day 3, complete culture medium was added to bring the total volume to 1 L. On day 7, the total cell count reached (3-4) × 10⁻⁶ cells / mL. 9 When the cells are in the range of 1, all cells are transferred to multiple 500mL centrifuge cups and centrifuged at 500g for 10min. After centrifugation, the supernatant is discarded, and the cells are resuspended in sodium chloride injection containing 0.2%-0.4% human serum albumin by volume. The cells are then washed three times by centrifugation at 500g for 10min. The cells are then resuspended in sodium chloride injection containing (1-3)g / 100mL human serum albumin by mass to prepare a universal DNT cell preparation.

[0033] Specifically, the relevant preparation and detection examples are as follows: Example 1: Preparation of DNT Cell Public Bank and Universal DNT Cell Preparation (1) Preparation of complete culture medium: The serum-free immune culture medium (Youkang / NC0101) contains 15% fetal bovine serum (EXCELL / FSP500) and interleukin-2 (nearshore / GMP-CD66) with a final concentration of 500 IU / mL.

[0034] (2) Isolation and extraction of peripheral blood mononuclear cells (PBMCs): The peripheral blood sample was transferred to a 500mL centrifuge tube (tube 1) via a transfusion set, resulting in a net blood volume of 210mL. 150mL of sodium chloride injection solution was added to tube 1 and thoroughly diluted. The diluted sample was then slowly added at a rate of 30mL / tube to 12 separate 50mL centrifuge tubes containing 15mL of lymphocyte separation medium (Dakewei / 7912011). The tubes were balanced and centrifuged using the following program: 500g, 19℃, 23min, start slow. After centrifugation, the white membrane cells were transferred to a new 250mL centrifuge tube. Sodium chloride injection solution was added to a total volume of 220mL. The tubes were balanced and centrifuged (500g, 19℃, 10min, start max, stop 3). The supernatant was discarded, and the cells were resuspended in 9mL of complete culture medium, resulting in a suspension volume of 9.4mL. 100μL of the cell suspension was diluted 5-fold, and the cell count was 4.7×10⁻⁶. 9 / L, total cell count 2.209×108 .

[0035] (3) DNT common bank cell preparation: Based on the counting results, the cell suspension was evenly seeded into 7 new T182 culture flasks. 1 nM / mL zoledronic acid was added to each flask for induction, and complete culture medium was added to a total volume of 30 mL. After mixing, the flasks were incubated at 37℃ in a 5% CO2 incubator. Cell status was observed daily. On day 3, one flask of cells was taken, mixed thoroughly, and 200 μL of cell suspension was counted. The result was: 0.5 × 10⁻⁶. 9 / L, add 50mL of complete culture medium to each bottle, and continue culturing in a 37℃, 5% CO2 incubator. On day 5, take one bottle of cells, pipette to mix, and take 200μL of the cell suspension for cell counting. Result: 1.1×10 9 / L, add 100mL of complete culture medium to each bottle, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 7, take one bottle of cells, pipette to mix, and take 200μL of cell suspension for cell counting. Result: 2.6×10 9 Seven flasks of cells were transferred to seven separate aerated culture flasks, each topped with 220 mL of complete culture medium. After mixing, the flasks were incubated at 37°C in a 5% CO2 incubator. On day 9, one flask of cells was taken, remixed by pipetting, and 200 μL of the cell suspension was collected for cell counting. The result was 3.7 × 10⁶ cells / L. 9 / L, add 600mL of complete culture medium to each bottle to a total volume of 1L, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 12, take one bottle of cells, pipette to mix, and take 200μL of the cell suspension for cell counting. Result: 5.1×10 9 / L, take 1mL of cell suspension and send it for flow cytometry analysis ( Figure 1 Results: The proportion of CD3+CD4-CD8- cells was 78.17%. All cell suspension was transferred to a 500mL centrifuge cup, balanced, and centrifuged using the following program: 500g, 19℃, 10min, start at max, stop at 3. After centrifugation, the supernatant was discarded, and the cells were resuspended in 89mL of 1640 medium (Gibco / C22400500BT). 89mL of cryopreservation buffer (composed of 40% 1640 medium, 20% DMSO (ORIGEN / CP70), and 40% fetal bovine serum) was added, and the mixture was stirred until a cell count of 2 × 10⁻⁶ cells was reached. 8 4 mL / tube was filled into 178 5 mL cryovials, placed in a programmable cooling box, and then transferred to a -80°C freezer. After 24 hours, the tubes were transferred to liquid nitrogen at -196°C for storage.

[0036] (4) Preparation of universal DNT cell preparation: Prepare complete culture medium for later use. Resuscitate 4 tubes of DNT common bank cells in a metal bath and seed them into one aerated culture flask. Add zoledronic acid (Yangtze River / NA) to a final concentration of 1 nM / mL for induction. Add complete culture medium to a final volume of 400 mL, mix well, and incubate at 37°C in a 5% CO2 incubator. On day 3, resuspend the cells by pipetting and count 200 μL of the cell suspension. Result: 2.3 × 10⁻⁶ 9 / L, add complete culture medium to 1L, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 7, resuspend the cells by pipetting and take 200uL of the cell suspension for counting. Result: 3.5×10 9 / L, total cell count 3.5×10 9 Take 1 mL of cell suspension and send it for flow cytometry analysis. Figure 2 Results: The proportion of CD3+CD4-CD8- cells was 87.54%. All cells were harvested into two 500mL centrifuge cups and centrifuged (centrifugation program: 500g, 19℃, 10min, start max, stop 3). After centrifugation, the supernatant was discarded, and the cells were resuspended in washing buffer (sodium chloride injection containing 0.2-0.4% human serum albumin (Green Cross / SJ20160022) by volume), centrifuged again, and the cells were washed a total of 3 times. A universal DNT cell preparation was prepared using 200mL of preservation solution (preservation solution is 190mL sodium chloride injection plus 10mL human serum albumin (20g / 100mL, final concentration of human serum albumin is 1% (mass ratio)).

[0037] Related testing: CCK8 assay for the killing activity of universal DNT cell preparations (1) HeLa cells were prepared at a ratio of 1 × 10⁻⁶ cells per well. 5 Cells were inoculated into 24-well plates. After 24 hours, the culture medium was aspirated, and universal DNT cell preparations were added at effector-target ratios of 2:1 and 10:1. Each experimental group was divided into 3 replicates. No cells were added to the zero wells, and only 1640 medium containing 10% FBS was added.

[0038] (2) After 24 hours, the OD values ​​of each group were measured using an ELISA reader according to the CCK8 reagent instructions. The universal DNT cell preparation has a strong killing ability (see results below). Figure 3 , Figure 4 The kill efficiency of the 2:1 effective-to-target ratio group was 50.71%±3.92%, and the kill efficiency of the 10:1 effective-to-target ratio group was 91.09%±2.31%.

[0039] Example 2: Preparation of DNT Cell Public Bank and Universal DNT Cell Preparation (1) Preparation of complete culture medium: The serum-free immune culture medium contains 15% fetal bovine serum and interleukin-2 at a final concentration of 500 IU / mL.

[0040] (2) Isolation and extraction of peripheral blood mononuclear cells (PBMCs): A peripheral blood sample was transferred via transfusion set into a 500mL centrifuge tube (tube 1), with a net blood volume of 198mL. 162mL of sodium chloride injection solution was added to tube 1 and thoroughly diluted and mixed. The diluted sample was then slowly added at a rate of 30mL / tube to 12 50mL centrifuge tubes containing 15mL of lymphocyte separation solution. The tubes were balanced and centrifuged using the following program: 500g, 19℃, 23min, start slow. After centrifugation, the white membrane cells were transferred to a new 250mL centrifuge tube, and sodium chloride injection solution was added to a total volume of 220mL. The tubes were balanced and centrifuged (500g, 19℃, 10min, start max, stop 3). The supernatant was discarded, and the cells were resuspended in 10mL of complete culture medium, resulting in a suspension volume of 10.5mL. 100μL of the cell suspension was diluted 5-fold, and the cell count was 4.7×10⁻⁶. 9 / L, total cell count 2.415×10 8 .

[0041] (3) DNT public bank cell preparation: Based on the counting results, the cell suspension was evenly seeded into 8 new T182 culture flasks. 1 nM / mL zoledronic acid was added to each flask for induction, and complete culture medium was added to a total volume of 30 mL. After mixing, the flasks were incubated at 37℃ in a 5% CO2 incubator. Cell status was observed daily. On day 3, one flask of cells was taken, mixed thoroughly, and 200 μL of the cell suspension was counted. The result was: 0.4 × 10⁻⁶. 9 / L, add 50mL of complete culture medium to each bottle, and continue culturing in a 37℃, 5% CO2 incubator. On day 5 of culture, take one bottle of cells, pipette and mix well, and take 200μL of the cell suspension for cell counting. Result: 1.0×10 9 / L, add 100mL of complete culture medium to each bottle, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 7, take one bottle of cells, pipette to mix, and take 200μL of cell suspension for cell counting. Result: 2.4×10 9 Eight flasks of cells were transferred to eight separate aerated culture flasks, with 220 mL of complete culture medium added to each flask. After mixing, the flasks were incubated at 37°C in a 5% CO2 incubator. On day 9, one flask of cells was taken, remixed by pipetting, and 200 μL of the cell suspension was collected for cell counting. The result was 3.7 × 10⁶ cells / L. 9 / L, add 600mL of complete culture medium to each bottle to a total volume of 1L, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 12, take one bottle of cells, pipette to mix, and take 200μL of the cell suspension for cell counting. Result: 4.9×10 9 / L, take 1mL of cell suspension and send it for flow cytometry analysis ( Figure 5Results: The proportion of CD3+CD4-CD8- cells was 88.62%. All cell suspension was transferred to a 500mL centrifuge cup, balanced, and centrifuged using the following program: 500g, 19℃, 10min, start at max, stop at 3. After centrifugation, the supernatant was discarded, and the cells were resuspended in 98mL of 1640 medium. 98mL of cryopreservation buffer (composed of 40% 1640 medium, 20% DMSO, and 40% fetal bovine serum) was added, and the mixture was stirred until a cell count of 2 × 10⁻⁶ cells was reached. 8 4 mL / tube was filled into 196 5 mL cryopreservation tubes, placed in a programmable cooling box, and then transferred to a -80°C freezer. After 24 hours, the tubes were transferred to liquid nitrogen at -196°C for storage.

[0042] (4) Preparation of universal DNT cell preparation: Prepare complete culture medium for later use. Resuscitate 4 tubes of DNT common bank cells in a metal bath and seed them into one aerated culture flask. Add zoledronic acid to a final concentration of 1 nM / mL for induction. Add complete culture medium to a final volume of 400 mL, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 3, resuspend the cells by pipetting and count 200 μL of the cell suspension. Result: 2.4 × 10⁻⁶ 9 / L, add complete culture medium to 1L, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 7, resuspend the cells by pipetting and take 200uL of the cell suspension for counting. Result: 3.7×10 9 / L, total cell count 3.7×10 9 Take 1 mL of cell suspension and send it for flow cytometry analysis. Figure 6 Results: The proportion of CD3+CD4-CD8- cells was 91.51%. All cells were harvested into two 500mL centrifuge cups and centrifuged (centrifugation program: 500g, 19℃, 10min, start max, stop 3). After centrifugation, the supernatant was discarded, and the cells were resuspended in washing buffer (sodium chloride injection containing 0.2-0.4% human serum albumin by volume). The cells were centrifuged again and the mixture was balanced. The cells were washed a total of 3 times. A universal DNT cell preparation was prepared using 200mL of preservation solution (preservation solution consisted of 190mL sodium chloride injection plus 10mL human serum albumin (20g / 100mL), with a final human serum albumin concentration of 1% (mass ratio)).

[0043] Related testing: CCK8 assay for the killing activity of universal DNT cell preparations (1) HeLa cells were prepared at a ratio of 1 × 10⁻⁶ cells per well. 5 Cells were inoculated into 24-well plates. After 24 hours, the culture medium was aspirated, and universal DNT cell preparations were added at effector-target ratios of 2:1 and 10:1. Each experimental group was divided into 3 replicates. No cells were added to the zero wells, and only 1640 medium containing 10% FBS was added.

[0044] (2) After 24 hours, the OD values ​​of each group were measured using an ELISA reader according to the CCK8 reagent instructions. The universal DNT cell preparation has a strong killing ability (see results below). Figure 7 , Figure 8 The kill efficiency of the 2:1 effective-to-target ratio group was 53.38% ± 5.26%, and the kill efficiency of the 10:1 effective-to-target ratio group was 93.27% ± 2.81%.

[0045] Example 3: Comparative Study of the Method of the Present Invention and Existing Techniques 200 mL of fresh blood was collected from healthy donors, of which 100 mL was used in the experimental group using the method of this invention, and the other 100 mL was used in the control group according to patent CN119101648A.

[0046] Experimental group: (1) Preparation of complete culture medium: The serum-free immune culture medium contains 15% fetal bovine serum and interleukin-2 at a final concentration of 500 IU / mL.

[0047] (2) Isolation and extraction of peripheral blood mononuclear cells (PBMCs): A peripheral blood sample was placed into a 250mL centrifuge tube (tube 1) via a transfusion set, with a net blood volume of 100mL. 80mL of sodium chloride injection solution was added to tube 1 and thoroughly diluted and mixed. The diluted sample was then slowly added at a rate of 30mL / tube to six 50mL centrifuge tubes containing 15mL of lymphocyte separation medium. The tubes were balanced and centrifuged using the following program: 500g, 19℃, 23min, start slow. After centrifugation, the white membrane cells were transferred to a new 250mL centrifuge tube, and sodium chloride injection solution was added to a total volume of 220mL. The tubes were balanced and centrifuged (500g, 19℃, 10min, start max, stop 3). The supernatant was discarded, and the cells were resuspended in 5mL of complete culture medium, resulting in a suspension volume of 5.3mL. 100μL of the cell suspension was diluted 5-fold, and the cell count was 4.5×10⁻⁶. 9 / L, total cell count 1.1925×10 8 .

[0048] (3) DNT public bank cell preparation: Based on the counting results, the cell suspension was evenly seeded into 4 new T182 culture flasks. 1 nM / mL zoledronic acid was added to each flask for induction, and complete culture medium was added to a total volume of 30 mL. After mixing, the flasks were incubated at 37℃ in a 5% CO2 incubator. Cell status was observed daily. On day 3, one flask of cells was taken, mixed thoroughly, and 200 μL of the cell suspension was counted. The result was: 0.4 × 10⁻⁶. 9 / L, add 50mL of complete culture medium to each bottle, and continue culturing in a 37℃, 5% CO2 incubator. On day 5, take one bottle of cells, pipette to mix, and take 200μL of the cell suspension for cell counting. Result: 1.1×10 9 / L, add 100mL of complete culture medium to each bottle, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 7, take one bottle of cells, pipette to mix, and take 200μL of cell suspension for cell counting. Result: 2.3×10 9 Four flasks of cells were transferred to four separate aerated culture flasks, with 220 mL of complete culture medium added to each flask. After mixing, the flasks were incubated at 37°C in a 5% CO2 incubator. On day 9, one flask of cells was taken, remixed by pipetting, and 200 μL of the cell suspension was collected for cell counting. The result was 3.5 × 10⁶ cells / L. 9 / L, add 600mL of complete culture medium to each bottle to a total volume of 1L, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 12, take one bottle of cells, pipette to mix, and take 200μL of the cell suspension for cell counting. Result: 4.6×10 9 / L, take 1mL of cell suspension and send it for flow cytometry analysis ( Figure 9 Results: The proportion of CD3+CD4-CD8- cells was 72.09%. All cell suspension was transferred to a 500mL centrifuge cup, balanced, and centrifuged using the following program: 500g, 19℃, 10min, start at max, stop at 3. After centrifugation, the supernatant was discarded, and the cells were resuspended in 46mL of 1640 medium. 46mL of cryopreservation buffer (composed of 40% 1640 medium, 20% DMSO, and 40% fetal bovine serum) was added, and the mixture was stirred until a cell count of 2 × 10⁻⁶ cells was reached. 8 / 4mL / tube was filled into 92 5mL cryovials, placed in a programmable cooling box, and then transferred to a -80℃ freezer. After 24 hours, the tubes were transferred to liquid nitrogen at -196℃ for storage.

[0049] (4) Preparation of universal DNT cell preparation: Prepare complete culture medium for later use. Resuscitate 4 tubes of DNT common bank cells in a metal bath and seed them into one aerated culture flask. Add zoledronic acid to a final concentration of 1 nM / mL for induction. Add complete culture medium to a final volume of 400 mL, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 3, resuspend the cells by pipetting and count 200 μL of the cell suspension. Result: 2.2 × 10⁻⁶ 9 / L, add complete culture medium to 1L, mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 7, resuspend the cells by pipetting and take 200uL of the cell suspension for counting. Result: 3.6×10 9 / L, total cell count 3.6×10 9 Take 1 mL of cell suspension and send it for flow cytometry analysis. Figure 10Results: The proportion of CD3+CD4-CD8- cells was 94.22%. All cells were harvested into two 500mL centrifuge cups and centrifuged (centrifugation program: 500g, 19℃, 10min, start max, stop 3). After centrifugation, the supernatant was discarded, and the cells were resuspended in washing buffer (sodium chloride injection containing human serum albumin at a mass fraction of (0.2-0.4) g / mL). The cells were centrifuged again and the mixture was balanced. The cells were washed a total of 3 times. A universal DNT cell preparation was prepared using 200mL of preservation solution (preservation solution consisted of 190mL sodium chloride injection plus 10mL human serum albumin (20g / 100mL), with a final human serum albumin concentration of 1% (mass ratio)).

[0050] Control group: (1) Referring to patent CN119101648A, 100 mL of fresh blood was collected from a healthy donor, leukocyte residue was prepared, RosetteSep™ Human CD4 Depletion Cocktail and RosetteSep™ Human CD8 Depletion Cocktail were added, CD4+ / CD8+ cells were removed by centrifugation, and enriched DNT cells were obtained, totaling 3.12 × 10⁻⁶ cells. 6 Each cell.

[0051] (2) T25 culture flasks were coated with anti-human CD3 monoclonal antibody (10 μg / mL) and adjusted to 0.6 × 10⁻⁶ with serum-free medium (containing 10% serum substitute and 250 IU / mL recombinant human interleukin-2). 6 Cells were added to a coated T25 culture flask at a concentration of [number] cells / mL and cultured in a 5% CO2 incubator at 37°C. Cell status was observed daily. On day 3, the cells were resuspended and 200 μL of the cell suspension was used for cell counting. The result was 0.7 × 10⁻⁶ cells / mL. 9 / L, add 5mL of fresh serum-free culture medium (containing 10% serum substitute and 250IU / mL recombinant human interleukin-2), and continue culturing in a 37℃, 5% CO2 incubator. On day 5, mix the cells by pipetting, and take 200μL of the cell suspension for counting. Result: 1.1×10 9 / L, transferred to a T-182 culture flask, added 40mL of fresh serum-free culture medium (containing 10% serum substitute and 250IU / mL recombinant human interleukin-2), mixed well, and incubated at 37℃ in a 5% CO2 incubator. On day 7, the cells were resuspended and mixed by pipetting, and 200μL of the cell suspension was taken for cell counting. Result: 1.4×10 9 / L, add 50mL of fresh serum-free culture medium (containing 100ng / mL anti-human CD3 monoclonal antibody and 250IU / mL recombinant human interleukin-2), mix well, and continue culturing in a 37℃, 5% CO2 incubator. On day 9, the cells were remixed by pipetting, and 200μL of the cell suspension was taken for cell counting. Result: 2.0×10 9 / L, add 80mL of fresh serum-free culture medium (containing 100ng / mL anti-human CD3 monoclonal antibody and 500IU / mL recombinant human interleukin-2), mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 11, mix the cells by pipetting, take 200μL of cell suspension for counting, result: 2.0×10 9 / L, the cell suspension was transferred to a cell culture bag, and 220 mL of fresh serum-free culture medium (containing 100 ng / mL anti-human CD3 monoclonal antibody and 250 IU / mL recombinant human interleukin-2) was added. After mixing, the mixture was placed in a 37°C, 5% CO2 incubator for further culture. On day 13, 200 μL of cell suspension was taken for cell counting, and the result was: 2.3 × 10⁶ cells / mL. 9 / L, add 400mL of fresh serum-free culture medium (containing 100ng / mL anti-human CD3 monoclonal antibody and 250IU / mL recombinant human interleukin-2), mix well, and incubate at 37℃ in a 5% CO2 incubator. On day 15, take 200μL of cell suspension for cell counting. Result: 2.2×10 9 / L, add 800mL of fresh serum-free culture medium (containing 100ng / mL anti-human CD3 monoclonal antibody and 250IU / mL recombinant human interleukin-2), mix well, and continue culturing in a 37℃, 5% CO2 incubator. On day 18, take 200μL of cell suspension for cell counting, result: 2.3×10 9 / L, total cell count 3.68×10 9 Take 1 mL of cell suspension and send it for flow cytometry analysis. Figure 11 Results: The proportion of CD3+CD4-CD8- cells was 51.81%.

[0052] (3) All cells were harvested into four 500mL centrifuge cups, balanced and centrifuged (centrifugation program: 500g, 19℃, 10min, start max, stop 3). After centrifugation, the supernatant was discarded, and the cells were resuspended in washing buffer (sodium chloride injection containing human serum albumin at a mass-volume fraction of (0.2-0.4) g / mL). The cells were then balanced and centrifuged, and the cells were washed a total of 3 times. A universal DNT cell preparation was prepared using 200mL of preservation solution (preservation solution is 190mL sodium chloride injection plus 10mL human serum albumin (20g / 100mL), with a final concentration of human serum albumin of 1% (mass ratio)).

[0053] This comparative example used 200 mL of fresh peripheral blood from the same healthy donor, divided into two equal portions (100 mL each). DNT cells were prepared using the method of this invention (experimental group) and the method of patent CN119101648A (control group), respectively, to eliminate the influence of individual differences on the experimental results. The experimental results (see Table 1) show that, in terms of DNT cell purity, the experimental group achieved a purity of over 80% in the final product, while the control group's purity was only about 50%. Furthermore, the experimental group achieved a DNT cell expansion fold of 16229.05 times, while the control group's expansion fold was 612.18 times, indicating that the method of this invention is significantly superior to existing technologies in terms of DNT cell purity and expansion fold. Regarding the culture period, the experimental group used a two-stage "public library + formulation" model, requiring only 7 days for the formulation preparation stage to obtain a clinical dose of DNT cells. In contrast, the control group used a single, long-cycle expansion, requiring 18 days to harvest cells. The method of this invention shortens the culture period by more than 60%. In terms of process complexity, the experimental group only required the addition of zoledronic acid to the culture medium, without the need for antibody coating, making the process simple; while the control group required anti-CD3 antibody coating, two-stage concentration control, multiple medium changes, and roller bottle operations, making the process complex. In terms of safety, residual CD4+ / CD8+ T cells in the control group after sorting could be non-specifically activated by anti-CD3 antibodies, increasing the risk of graft-versus-host disease.

[0054] Table 1. Phenotypic purity and amplification fold of the experimental and control groups in Example 3 of this invention.

[0055] This invention avoids the ethical review burden and blood donation compliance issues caused by the need for repeated collection of blood from the same or different donors in traditional methods. A single collection of peripheral blood can meet long-term needs, greatly simplifying the process of initiating clinical trials and treatments. In addition, the single culture time is short, supporting multiple batches of production. The cell quality is stable and controllable between batches, providing sufficient raw material reserves for clinical trials and patients.

[0056] The peripheral blood may be derived from one or more donors.

[0057] This invention uses zoledronic acid to activate DNT cells, completely avoiding the use of anti-human CD3 monoclonal antibodies, resulting in a simpler process, lower cost, and higher safety. Furthermore, numerous studies have shown that fetal bovine serum (FBS) provides the adhesion factors, growth hormones, and trace elements required for cell growth, effectively maintaining high viability and proliferative activity of DNT cells during in vitro expansion. In contrast, while serum-free culture systems avoid the introduction of animal-derived components, their formulations cannot fully mimic the complex components of serum, and the expansion effect of some cell subpopulations may be less than ideal.

[0058] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a universal DNT cell public library, characterized in that, Includes the following steps: (a) Peripheral blood was collected from healthy adults, and peripheral blood mononuclear cells were isolated; (b) The peripheral blood mononuclear cells obtained in step (a) were placed in a complete culture medium containing zoledronic acid, interleukin-2 and fetal bovine serum for a first induction culture until day 12. (c) Detect the proportion of CD3+CD4-CD8- DNT cells in the cells obtained in step (b). When the proportion is ≥50%, freeze the cells to obtain a universal DNT cell public library.

2. The preparation method according to claim 1, characterized in that, In step (b), the concentration of zoledronic acid is (1-5) nM / mL, the concentration of interleukin-2 is (200-1000) IU / mL, and the volume fraction of fetal bovine serum is 10%-15%.

3. The preparation method according to claim 1, characterized in that, In step (c), the freezing conditions are as follows: 2 × 10 8 Aliquoting cells into 4 mL portions and freezing them.

4. A universal DNT cell public library prepared by the method according to any one of claims 1-3, characterized in that, The cells in the public library are cryopreserved cells, and the proportion of CD3+CD4-CD8- DNT cells after thawing is ≥50%.

5. A method for preparing universal DNT cell preparations using the universal DNT cell public library according to claim 4, characterized in that, Includes the following steps: (i) Resuscitate cells in the universal DNT cell bank as described in claim 4; (ii) The revived cells were placed in a complete culture medium containing zoledronic acid, interleukin-2 and fetal bovine serum for a second induction culture for 7 days; (iii) Harvest the cells obtained in step (ii) to obtain a universal DNT cell preparation.

6. The method according to claim 5, characterized in that, In step (i), four frozen cell lines are thawed, resulting in a total cell count of 8 × 10⁻⁶. 8 In step (ii), the resuscitated cells are arranged at 2 × 10⁻⁶. 6 Inoculate with cells at a concentration of 1 nM / mL, zoledronic acid at 1 nM / mL, interleukin-2 at 500 IU / mL, and fetal bovine serum at 10% (v / v). On day 3, add complete culture medium to a total volume of 1 L and continue culturing until day 7, when the total cell count reaches (3-4) × 10⁻⁴ cells / mL. 9 When there are only a few cells, all cells are recycled.

7. The method according to claim 5, characterized in that, In step (iii), after harvesting the cells, they are washed with sodium chloride injection containing human serum albumin at a mass-volume fraction of (0.2-0.4) g / mL, and then resuspended with sodium chloride injection containing human serum albumin at a mass-volume fraction of (1-3) g / 100mL to prepare the formulation.

8. A universal DNT cell preparation prepared by the method according to any one of claims 5-7.

9. Use of the universal DNT cell public library as described in claim 4 in the preparation of DNT cell preparations for the treatment of tumors.

10. Use of the universal DNT cell preparation of claim 8 in the preparation of a medicament for treating tumors.

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