Method for preparing autologous CD19 CAR-T (chimeric antigen receptor T) cells by utilizing PBMC (peripheral blood mononuclear cells) separated from healthy human peripheral blood and cryopreserved in isolator

Abstract

The invention relates to the technical field of cellular immunotherapy, in particular to a method for preparing autologous CD19 CAR-T cells by utilizing PBMC separated from healthy human peripheral blood and cryopreserved in an isolator, the method is carried out in a C-level isolator, firstly, the PBMC is separated from the healthy human peripheral blood and cryopreserved, after recovery, a complete culture medium-A containing DNA enzyme is used for static culture, and the autologous CD19 CAR-T cells are obtained. Adding magnetic beads to activate T cells on the next day, performing CD19 CAR transduction with lentivirus on the second day, replacing with a complete medium-B containing no DNA enzyme, maintaining the cell density with the medium-B every day from the third day to the tenth day to perform multiplication culture, and finally obtaining a high-activity CD19 CAR-T cell product. The whole process is completed in a closed environment, the safety and quality of products are ensured, and the closed operating system not only improves the safety of the products, but also lays a foundation for automatic and standardized production.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy technology, specifically to a method for preparing autologous CD19 CAR-T cells using PBMCs isolated from and cryopreserved from peripheral blood of healthy individuals in an isolator. Background Technology

[0002] Chimeric antigen receptor T-cell therapy, as a novel immunotherapy approach, has demonstrated remarkable clinical efficacy in the treatment of hematologic malignancies. Since the FDA approved the first CD19-targeted CAR-T cell product in the United States in 2017, research and clinical applications in this field have developed rapidly. Currently, several CAR-T products have been approved for marketing both domestically and internationally, bringing new hope to patients with relapsed or refractory acute lymphoblastic leukemia, large B-cell lymphoma, and other diseases.

[0003] However, existing CAR-T cell preparation technologies still face many challenges. Currently, commercially produced CAR-T cell products mainly use the patient's own peripheral blood as the starting material. This involves collecting peripheral blood mononuclear cells through apheresis, followed by a series of complex processes including T cell isolation, activation, gene transduction, and in vitro expansion. This preparation method presents several technical problems.

[0004] First, when patients are in a disease state, the quantity, quality, and functional status of their T cells are often poor due to the disease itself and the effects of previous treatments. Studies have shown that patients have a high proportion of exhausted phenotype cells in their T cells, and their proliferative capacity is reduced, which directly affects the quality of the final CAR-T cell product and the therapeutic effect. The study "CAR-T manufactured from frozen PBMC yield efficient function with prolonged in vitro production" published in Frontiers in Immunology in 2022 explored in detail the feasibility of preparing CAR-T cells from frozen PBMCs, but the study also pointed out the problem of slow expansion rate of frozen PBMCs after thawing.

[0005] Secondly, existing preparation processes are typically carried out in Class C or Class B biosafety cabinet environments. While these meet basic aseptic requirements, a certain risk of contamination still exists. Cell therapy products cannot undergo terminal sterilization filtration; if contamination occurs during preparation, the entire batch will be scrapped, resulting not only in economic losses but, more importantly, delaying patients' treatment.

[0006] Furthermore, while using PBMCs collected and cryopreserved from healthy individuals before the onset of disease as starting material theoretically yields higher-quality T cells, significant technical challenges arise in practice. The proportion of T cells in the peripheral blood of healthy individuals is relatively low. Cryopreserved and thawed cells require multiple intensive steps, including washing, sorting, activation, and transduction. These frequent processes further deteriorate the cell state, increasing the number of dead cells and causing them to aggregate, severely impacting subsequent cell expansion efficiency. Current technologies perform cell sorting and activation on the day of thawing, failing to provide sufficient recovery time, resulting in a strong cellular stress response, decreased viability, and ultimately, low expansion rates and prolonged preparation cycles.

[0007] Furthermore, existing culture systems also have shortcomings. Traditional complete culture medium formulations are relatively fixed and have not been optimized for the specific physiological state of cells after cryopreservation and thawing. Especially in the early stages of culture, a large number of dead cells and their released DNA fragments can form sticky clumps. These clumps not only physically hinder the proliferation of living cells but may also affect the exchange of nutrients and gases, further inhibiting cell growth.

[0008] Therefore, there is an urgent need to develop an improved preparation method that can fully leverage the advantages of cryopreserved PBMCs from healthy individuals. By optimizing the operating environment, improving the process flow and culture medium formulation, the cell expansion efficiency and final product quality can be enhanced, providing more reliable technical support for the clinical application of CAR-T cell therapy. Summary of the Invention

[0009] To address the aforementioned problems in existing technologies, the present invention aims to provide a method for preparing autologous CD19 CAR-T cells using peripheral blood mononuclear cells (PBMCs) isolated from and cryopreserved in an isolator. This method effectively solves the technical challenges of poor cell state and slow expansion during the preparation of CAR-T cells from cryopreserved PBMCs by adopting a Class C isolator environment, optimizing the cell processing procedure, and improving the culture medium formulation, thereby significantly increasing the cell count and CAR positivity rate of the final product.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A method for preparing autologous CD19 CAR-T cells using PBMCs isolated from and cryopreserved from healthy human peripheral blood in an isolator includes the following steps:

[0012] 50ml-200ml of peripheral blood from healthy individuals was transferred into a Class C isolator and diluted with physiological saline at a volume ratio of 1:1-1:2. PBMCs were obtained by Ficoll density gradient centrifugation, washed with physiological saline containing 0.1%-0.5% HSA, and counted at a ratio of 1×10⁻⁶. 7pcs / ml - 1×10 8 The PBMCs with a specification of 1 unit / ml were cryopreserved using cryopreservation solution;

[0013] Prepare complete culture medium-A and complete culture medium-B. Complete culture medium-A includes basal culture medium, 1%-10% SR additive, and 1‰-5‰ DNase. Complete culture medium-B includes basal culture medium and 1%-10% SR additive.

[0014] On day 0, the frozen PBMCs were thawed, transferred to the isolator, resuspended in the complete culture medium-A, and the cell density was adjusted to 1 × 10⁶ cells / day. 6 5 x 10 cells / ml 6 Cells / ml, incubated statically at 37℃ and 5% CO2 for 12-24 hours;

[0015] On day 1, activation magnetic beads were added to the PBMCs at a ratio of 1:2 to 1:1 with the cells, and the culture continued.

[0016] On day 2, lentivirus encoding CD19 CAR was added to the PBMCs for transduction, with an MOI of 5-20. The cell density was adjusted to 1×10⁶ cells / year using the complete culture medium-B. 6 3 x 10 cells / ml 6 pcs / ml;

[0017] From day 3 to day 10, the cell density was adjusted to 1×10⁶ cells / ml daily using the complete culture medium-B, and the cells were cultured continuously to obtain the CD19 CAR-T cells.

[0018] Preferably, the specific steps of the Ficoll density gradient centrifugation are as follows: take a gradient density centrifuge tube, add 10ml-20ml of Ficoll to it, centrifuge at 700g-1500g for 1min-2min, then add 10ml-20ml of diluted peripheral blood to the centrifuge tube and centrifuge at 700g-1500g for 20min-40min. After centrifugation, take the white membrane layer from each tube to harvest the PBMCs.

[0019] Preferably, the specific steps of washing with physiological saline containing 0.1%-0.5% HSA are as follows: add 10ml-200ml of physiological saline containing 0.1%-0.5% HSA to the collected PBMCs, centrifuge the cell suspension at 500g for 10min, discard the supernatant after centrifugation, resuspend the cells in 10ml-50ml of physiological saline containing 0.1%-0.5% HSA, centrifuge the cell suspension at 500g for 10min, discard the supernatant after centrifugation.

[0020] Preferably, day 1 further includes a step of sorting CD3-positive cells from the PBMCs, specifically: transferring the statically cultured PBMCs to centrifuge tubes containing 10ml-40ml of basal culture medium, centrifuging at 500g for 5-10 minutes, discarding the supernatant after centrifugation, and resuspending the cells in 1ml-5ml of physiological saline containing 0.1%-0.5% HSA; adding CD3 sorting magnetic beads to the cell suspension at a ratio of 1×10⁻⁶. 9 Each cell was incubated with 2 ml of magnetic beads at 2℃-8℃ for 15 min. After incubation, pre-cooled physiological saline containing 0.1%-0.5% HSA was added to the cell suspension to a final volume of 35 ml-45 ml. The cells were centrifuged at 500 g for 5-10 min, and the supernatant was discarded after centrifugation. The cell pellet was resuspended in 3 ml-10 ml of physiological saline containing 0.1%-0.5% HSA. The LS column was placed on a cell sorting magnetic rack, and the LS column was rinsed with 3 ml-10 ml of physiological saline containing 0.1%-0.5% HSA. The cell suspension was then passed through a cell sieve and added to the LS column. After the liquid had drained, the LS column was washed three times with 3 ml-10 ml of physiological saline containing 0.1%-0.5% HSA. The CD3-positive T cells in the LS column were then quickly rinsed into centrifuge tubes with 3 ml-10 ml of physiological saline containing 0.1%-0.5% HSA and collected.

[0021] Preferably, after collecting the CD3-positive T cells, the CD3-positive T cell suspension is centrifuged at 500g for 5-10 minutes. After centrifugation, the supernatant is discarded, and the CD3-positive T cells are resuspended in the complete culture medium A according to the counting results to achieve a cell density of 2 × 10⁻⁶ cells / mL. 6 4 x 10 cells / ml 6 Add the activated magnetic beads at a concentration of 1 / ml.

[0022] Preferably, on day 1, the CD3-positive cell sorting step is not performed. Instead, the cell density of the PBMCs after static culture is adjusted to 2 × 10⁻⁶ cells based on the counting results using the complete culture medium-A. 6 4 x 10 cells / ml 6 Add the activated magnetic beads at a concentration of 1 / ml.

[0023] Preferably, the basal culture medium is selected from one of X-VIVO 15 medium, AIM V medium, and OpTmizer medium, and the basal culture medium is also supplemented with cytokines and essential nutrients.

[0024] Preferably, the specific operating steps on the second day are as follows: Remove the PBMCs from the incubator, mix and count them, centrifuge the PBMCs at 500g for 5-10 minutes, discard the supernatant after centrifugation, and resuspend the PBMCs in the complete culture medium-B according to the counting results to achieve a cell density of 1×10⁻⁶ cells / mL. 6 3 x 10 cells / ml 6 The lentivirus encoding CD19 CAR was added to the PBMCs at a concentration of 1 cell / ml, and the cell suspension was mixed and cultured further.

[0025] Preferably, the specific operating steps on the third day are as follows: remove the PBMCs from the incubator, mix and count them, centrifuge the PBMCs at 500g for 5-10 minutes, discard the supernatant after centrifugation, resuspend the PBMCs in the complete culture medium-B and adjust the density to 1×10⁻⁶. 6 Cells / ml, continue culturing.

[0026] Preferably, on day 10, the number, viability, and CAR positivity of the CD19 CAR-T cells are detected, wherein the CD3 positivity rate is ≥98% and the CAR positivity rate is ≥40%.

[0027] The beneficial effects of this invention are as follows:

[0028] First, by employing a Class C isolator as the operating environment, a completely closed-loop operation was achieved from peripheral blood separation to the final production of CAR-T cells. The isolator provides a highly controllable sterile environment, effectively isolating external sources of contamination, and significantly reducing the risk of contamination compared to traditional biosafety cabinet operations. This closed-loop operating system not only improves product safety but also lays the foundation for automated and standardized production, meeting the quality control requirements for CAR-T cell products as live cell drugs.

[0029] Secondly, this invention innovatively incorporates a 12-24 hour static culture phase after PBMC thawing. This crucial step fully considers the physiological impact of cryopreservation and thawing on cells. During cryopreservation and thawing, cell membrane integrity, mitochondrial function, and enzyme activity are all affected to varying degrees, placing cells in a state of stress. Through static culture, cells gain sufficient time for self-repair and functional recovery, membrane structure restabilizes, and metabolic activity gradually returns to normal. Experimental data shows that PBMCs cultured statically exhibit stronger proliferation capacity and higher cell viability in the subsequent activation and expansion phases. This "time-for-space" strategy, although slightly reducing the cell count on day 1, creates favorable conditions for subsequent efficient expansion.

[0030] Third, this invention designs two complementary culture medium formulations, embodying the concept of phased, refined culture. Complete medium-A is specifically designed for the early stages of culture, uniquely featuring the simultaneous addition of SR additives and DNase. SR additives are rich in various growth factors and nutrients, providing comprehensive nutritional support to cells and promoting cell metabolism and proliferation. The addition of DNase is an innovative measure designed to address the specific problem of a large number of dead cells after cryopreservation and thawing. During cryopreservation and thawing, some cells inevitably die, releasing DNA that mixes with cell debris, forming viscous clumps in the culture medium. These clumps physically encapsulate living cells, hindering nutrient exchange and cell movement, and may also trigger inflammatory responses, inhibiting cell proliferation. DNase, by specifically degrading free DNA, effectively prevents clump formation, maintains a uniform dispersion of the cell suspension, and significantly improves the cell growth microenvironment. By day 2, after transduction, the number of dead cells has been significantly reduced. At this point, switching to complete medium-B, which does not contain DNase, ensures a continuous supply of nutrients while avoiding the potential adverse effects of long-term DNase use.

[0031] Fourth, this invention provides a flexible process route selection. Depending on the quality of the initial PBMCs and the requirements of the target product, it is possible to choose whether or not to perform CD3-positive cell sorting. When the PBMC quality is poor and the proportion of T cells is low, the sorting step can be omitted, and activation can be performed directly. This not only simplifies the process and reduces the number of steps, but more importantly, it avoids the additional stress and loss to cells caused by the sorting process. Experimental results show that the PBMC-3 group without sorting achieved 8.2 × 10⁻⁶ cells on day 10. 7 The CAR positivity rate reached 60.5% in 100 cells, a further improvement compared to the sorted PBMC-2 group. This result demonstrates that, under the technical system of this invention, by optimizing culture conditions, high-quality CAR-T cell products can be obtained even without sorting, and the purity of T cells can still reach over 99%.

[0032] Fifth, from the perspective of overall process effectiveness, this invention significantly improves the preparation efficiency of CAR-T cells. Comparative experiments clearly demonstrate the technical advantages of this invention: the control group PBMC-1 prepared using existing technology showed a reduction in CAR-T cell production from 3.4 × 10⁻⁶ cells / year. 7 Starting from [number] initial cells, only 4.2 × 10 [units] were obtained by day 10. 4 The initial cell count not only failed to expand but also showed a significant decrease, with a CAR positivity rate of only 40.5%. In contrast, the PBMC-3 group, employing the complete technical solution of this invention, ultimately obtained 8.2 × 10⁸ cells. 7The study achieved a net increase of approximately 22.78 times in cell proliferation, with a CAR positivity rate as high as 60.5%, nearly 50% higher than the control group. More importantly, the obtained cells were of excellent quality, with a CD3 positivity rate of 99.3%, indicating extremely high T cell purity. These data fully demonstrate the significant effect of this invention in improving cell proliferation and CAR positivity rate. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0034] The main raw materials and reagents used in the examples are as follows:

[0035] Basic culture medium: X-VIVO 15 medium, purchased from Lonza;

[0036] SR Additive: A clinical-grade cell expansion additive containing multiple growth factors and nutrients;

[0037] DNA enzyme: Deoxyribonuclease I, purchased from Sigma, catalog number DN25;

[0038] Ficoll separation solution: Ficoll-Paque PLUS, purchased from GE Healthcare;

[0039] Human serum albumin: Clinical grade HSA, 20% concentration, diluted to the required concentration before use;

[0040] CD3 / CD28 activation beads: Dynabeads Human T-Activator CD3 / CD28, purchased from ThermoFisher.

[0041] CD3 sorting beads: CD3 MicroBeads, purchased from Miltenyi Biotec;

[0042] LS Columns: Purchased from Miltenyi Biotec.

[0043] Lentiviral vector: A lentiviral vector encoding the CD19 CAR gene. The CAR structure includes a CD19 single-chain antibody, a CD8 hinge region and a transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain.

[0044] Cell cryopreservation solution: CryoStor CS10, purchased from BioLife Solutions;

[0045] Class C isolator: A custom-designed GMP-grade cell preparation isolator system.

[0046] Example 1: Preparation of CD19 CAR-T cells using a completely improved protocol

[0047] This embodiment employs the complete technical solution of the present invention, including static culture, improved culture medium formulation, and no CD3 sorting, demonstrating the best implementation of the present invention.

[0048] Preparation of complete culture medium-A: Take 1000ml of X-VIVO 15 basal culture medium, add 100ml of SR additive to make the final concentration of SR additive 10%, add 2ml of DNase solution to make the final concentration of DNase 2‰, and add 500IU / ml IL-2 and 5ng / ml IL-7 as cytokines. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ for later use.

[0049] Preparation of complete culture medium-B: Take 1000ml of X-VIVO 15 basal culture medium, add 100ml of SR additive to make the final concentration of SR additive 10%, add 500IU / ml IL-2 and 5ng / ml IL-7, mix well, filter through a 0.22μm filter membrane for sterilization, and store at 4℃ for later use.

[0050] Day 0 Procedure: Retrieve the cryopreserved PBMCs from the healthy individual from liquid nitrogen. These PBMCs were collected from 150ml of peripheral blood during a health check-up, separated by Ficoll density gradient centrifugation, and then centrifuged at 5×10⁻⁶ ppm. 7 Cryopreservation was performed at a specification of cells / ml. The cryovials were rapidly thawed in a 37°C water bath, and removed immediately after approximately 2 minutes when the contents were completely thawed. The thawed PBMCs were transferred to a 50ml centrifuge tube containing 30ml of pre-warmed X-VIVO 15 basal medium at 37°C, gently mixed, and centrifuged at 500g for 10 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1.5ml of complete culture medium-A, mixed, and then sampled for counting. The counting results showed a total of 3.4 × 10⁴ cells. 7 The cell count yielded 78.3% viable cells. Based on the count results, an appropriate amount of complete culture medium-A was added to adjust the cell density to 3 × 10⁶ cells / year. 6 Cells / ml. The cell suspension was transferred to cell culture flasks in a honeycomb incubator and incubated statically at 37°C with 5% CO2 for 20 hours. During this stage, the cells were not disturbed by any physical manipulation and were able to fully recover from the stress caused by cryopreservation and thawing.

[0051] Day 1 Procedure: Remove the culture flask from the incubator and gently mix the cell suspension. Take a sample for cell counting; the result showed a cell count of 3.6 × 10⁻⁶. 6The cell viability increased to 89.5%. Observations showed that compared to the cell suspension at day 0, cell dispersion was significantly improved, clumping was significantly reduced, and cell morphology was fuller. Based on the counting results, the cell density was adjusted to 3 × 10⁶ cells / day using complete culture medium-A. 6 Cells / ml. Add CD3 / CD28 activation magnetic beads at a 1:1 ratio to cells, mix gently, and return the culture flask to the incubator for further culture. The activation magnetic beads mimic the function of antigen-presenting cells, simultaneously stimulating CD3 and CD28 of T cells, thus initiating the activation and proliferation process of T cells.

[0052] Day 2 procedure: Remove the culture flask, gently mix the cell suspension, and then take a sample for counting. The count showed a cell count of 2.0 × 10⁶ cells / year. 6 The cell count was 91.2%. The cell suspension was transferred to a 50ml centrifuge tube and centrifuged at 500g for 10 minutes, discarding the supernatant. The cells were resuspended in complete culture medium-B and the cell density was adjusted to 2×10⁶ cells / mL. 6 Cells / ml. Add lentivirus encoding CD19 CAR to the cell suspension, setting the MOI to 10. Gently mix and transfer the cells to a new culture flask, then return it to the incubator. At this point, switch the culture medium from complete medium-A to complete medium-B, as dead cells have been largely removed after the first two days of culture, and DNase is no longer needed.

[0053] Day 3 procedure: The culture flask was removed for cell counting, and the result showed a cell count of 2.6 × 10⁶. 6 The cell count was [number], with a cell viability of 93.8%. Cells were observed to be entering a rapid proliferation phase. After centrifugation, the supernatant was discarded, and the cells were resuspended in complete culture medium-B, adjusting the density to 1×10⁶. 6 Cells / ml were transferred to larger culture flasks to provide ample growth space.

[0054] Procedures from Day 4 to Day 9: Cells were removed from the incubator daily for counting and viability testing, and the cell density was adjusted to 1×10⁶ cells / day using complete culture medium-B. 6 Cells / ml. At this stage, cells exhibit stable logarithmic growth, proliferating approximately 1.5-2 times every two days. The SR supplement in the culture system continuously provides rich nutritional support to the cells, promoting efficient cell proliferation.

[0055] Day 10 Procedure: The culture flask was removed for final cell counting and quality testing. The counting results showed 8.2 × 10⁻⁶ cells. 7The cell count was 95.1%. Flow cytometry analysis showed a CD3 positivity rate of 99.3%, indicating extremely high T cell purity; the proportion of CD3+CAR+ double-positive cells was 60.5%, indicating excellent CAR gene transduction efficiency and expression level. These data demonstrate that this embodiment successfully prepared a high-quality, high-quantity CD19 CAR-T cell product.

[0056] Example 2: Preparation method using static culture and CD3 sorting

[0057] This embodiment adds a CD3-positive cell sorting step to the static culture and improved culture medium to verify the impact of sorting on the final product.

[0058] Day 0 was performed the same as in Example 1. After reviving the PBMCs, they were resuspended in complete culture medium-A and the cell density was adjusted to 3 × 10⁶ cells / day. 6 Cells / ml, incubated statically for 20 hours.

[0059] Day 1 Procedure: Remove the culture flask from the incubator, mix well, take a sample and count, obtaining 2.5 × 10⁻⁶. 6 The cell count was 88.7%. The cell suspension was transferred to a 50ml centrifuge tube and centrifuged at 500g for 10 minutes, discarding the supernatant. The cells were resuspended in 2ml of physiological saline containing 0.3% HSA. 50μl of CD3 sorting beads were added to the cell suspension, mixed well, and the centrifuge tube was incubated at 4°C on ice for 15 minutes, gently mixing every 5 minutes to ensure adequate contact between the beads and cells. After incubation, pre-cooled physiological saline containing 0.3% HSA was added to a final volume of 40ml, and the tube was centrifuged at 500g for 10 minutes, discarding the supernatant.

[0060] Resuspend the cell pellet in 5 ml of physiological saline containing 0.3% HSA. Mount the LS column on a magnetic rack and rinse the column with 5 ml of wash buffer. Filter the cell suspension through a 40 μm cell sieve and add it to the LS column, allowing the liquid to flow naturally. After the liquid has drained, rinse the LS column with 5 ml of wash buffer, repeating this process three times. Remove the magnetic rack, place the LS column over a new collection tube, add 5 ml of wash buffer, and quickly flush the CD3-positive cells from the column into the collection tube using the plunger, repeating once. Centrifuge at 500 g for 10 min and discard the supernatant. Resuspend the cells in complete culture medium A, count them, and adjust the density to 3 × 10⁻⁶ cells / mL. 6 Activating magnetic beads are added at a 1:1 ratio, mixed well, and then transferred to a culture flask for incubation.

[0061] The procedures from day 2 to day 10 were the same as in Example 1. On day 2, complete culture medium-B was used for virus transduction with an MOI of 10. The cell density was then adjusted to 1×10⁻⁶ cells per day thereafter. 6 per ml.

[0062] Day 10 test results: 7.5 × 10⁻⁶ 6 The cell count was 94.3%, the CD3 positivity rate was 98.9%, and the CAR positivity rate was 45.5%. Compared with Example 1, although the sorting step improved the initial purity of T cells, the final cell count and CAR positivity rate were slightly lower. This may be due to the mechanical stress and loss of cells caused by the sorting process.

[0063] Example 3: Verification of Parameter Endpoint Values

[0064] This embodiment uses the endpoint values ​​of each parameter of the technical solution of the present invention for verification to ensure the validity of the entire parameter range.

[0065] 50 ml of peripheral blood from healthy individuals was diluted 1:1 with physiological saline. During Ficoll density gradient centrifugation, 10 ml of Ficoll was added to the centrifuge tube. The tube was pre-centrifuged at 500 g for 1 min, then 10 ml of diluted peripheral blood was added, and the tube was centrifuged at 1500 g for 40 min. The separated PBMCs were washed with physiological saline containing 0.5% HSA, and centrifuged at 1×10⁻⁶ ml. 7 Frozen at a specification of 1 piece / ml.

[0066] After reviving PBMCs, they were resuspended in complete medium-A, with SR supplementation at 1% and DNase concentration at 1‰. The cell density was then adjusted to 5 × 10⁶ cells / year. 6 Cells / ml, incubated statically for 12 hours.

[0067] On day 1, no sorting is performed; instead, activation magnetic beads are added directly at a ratio of 1:2 between the magnetic beads and the cells.

[0068] On day 2, the MOI was set to 5 during viral transduction, and the cell density was adjusted to 3 × 10⁶ cells / year using complete culture medium-B. 6 The concentration of SR additive is 1% per ml.

[0069] From day 3 to day 10, adjust the cell density to 1×10⁻⁶ cells per day. 6 per ml.

[0070] Day 10 test results: 3.8 × 10⁻⁶ 7 The cell count was 92.6%, the CD3 positivity rate was 98.5%, and the CAR positivity rate was 48.3%. The results indicate that even with the endpoint values ​​of the parameters, the method of this invention can still achieve good preparation results.

[0071] Example 4: Verification of another set of parameter endpoint values

[0072] 200 ml of peripheral blood from healthy individuals was diluted with physiological saline at a 1:2 ratio. During Ficoll density gradient centrifugation, 20 ml of Ficoll was added to the centrifuge tube. The tube was pre-centrifuged at 300 g for 2 min, then 20 ml of diluted peripheral blood was added, and the tube was centrifuged again at 700 g for 20 min. The separated PBMCs were washed with physiological saline containing 0.1% HSA, and centrifuged at 1×10⁻⁶ ml. 8 Frozen at a specification of 1 piece / ml.

[0073] After reviving PBMCs, they were resuspended in complete medium-A, with SR supplementation at 10% and DNase concentration at 5‰. The cell density was then adjusted to 1×10⁶ cells / year. 6 Cells / ml, incubated statically for 24 hours.

[0074] CD3 sorting was performed on day 1, and the samples were resuspended in complete culture medium-A to a final volume of 4 × 10⁻⁶. 6 Add activation magnetic beads at a ratio of 1:1 (number of beads per ml).

[0075] On day 2, the MOI was set to 20 for virus transduction, and the cell density was adjusted to 1×10⁻⁶ using complete culture medium-B. 6 The concentration of SR additive is 10%, with a concentration of 10% per ml.

[0076] From day 3 to day 10, adjust the cell density to 1×10⁻⁶ cells per day. 6 per ml.

[0077] Day 10 test results: 1.1 × 10⁻⁶ 7 The cell count was 94.8%, the CD3 positivity rate was 99.1%, and the CAR positivity rate was 58.2%. These results further validate the rationality of the parameter range in this invention.

[0078] Example 5: Optimization of Intermediate Parameter Values

[0079] 120 ml of peripheral blood from healthy individuals was diluted with physiological saline at a ratio of 1:1.5. During Ficoll density gradient centrifugation, 15 ml of Ficoll was added to the centrifuge tube. The tube was pre-centrifuged at 700 g for 1.5 min, then 15 ml of diluted peripheral blood was added, and the tube was centrifuged at 1500 g for 30 min. The separated PBMCs were washed with physiological saline containing 0.3% HSA and centrifuged at 5 × 10⁻⁶ ml / mL. 7 Frozen at a specification of 1 piece / ml.

[0080] After reviving PBMCs, they were resuspended in complete culture medium-A, with SR supplementation at 5% and DNase concentration at 3‰. The cell density was then adjusted to 2 × 10⁶ cells / year. 6 Cells / ml, incubated statically for 18 hours.

[0081] No sorting was performed on day 1; the complete culture medium-A was directly adjusted to 2.5 × 10⁻⁶. 6 Add activation magnetic beads at a ratio of 1:1.5, with the number of beads per ml.

[0082] On day 2, the MOI was set to 10 during virus transduction, and the cell density was adjusted to 1.5 × 10⁻⁶ using complete culture medium-B. 6 The concentration of SR additive is 5% per ml.

[0083] From day 3 to day 10, adjust the cell density to 1×10⁻⁶ cells per day. 6 per ml.

[0084] Day 10 test results: 7.8 × 10⁻⁶ 7 The cell count was 95.3%, the CD3 positivity rate was 99.2%, and the CAR positivity rate was 59.8%. This combination of parameters showed excellent preparation results.

[0085] Comparative Example 1: Prior Art Control Group

[0086] This comparative example uses existing technical methods, sorting and activating PBMCs immediately after revival on day 1, without static culture, and using traditional complete culture medium.

[0087] No action is taken on day 0.

[0088] Day 1: Healthy human PBMCs were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. The thawed PBMCs were transferred to centrifuge tubes containing 30 ml of X-VIVO 15 basal medium and centrifuged at 500 g for 10 min. The supernatant was discarded, and the cells were resuspended in 2 ml of physiological saline containing 0.3% HSA. CD3-positive T cells were immediately sorted using CD3-sorting magnetic beads, following the same sorting procedure as in Example 2. The sorted CD3-positive T cells were resuspended in conventional complete culture medium containing only X-VIVO 15 basal medium, IL-2, and IL-7, without SR additives or DNase. The cell density was adjusted to 3 × 10⁶ cells / mL. 6 Cells / ml, add activating magnetic beads and then culture.

[0089] Days 2 to 10: Cells were cultured and their density adjusted using conventional complete culture medium. Other procedures were similar to those in Example 1.

[0090] Day 10 test results: Only 4.2 × 10⁻⁴ 4 The cell viability was 86.5%, the CD3 positivity rate was 98.3%, and the CAR positivity rate was only 40.5%. These results are significantly lower than those of the embodiments of the present invention, which fully illustrates the problems of the prior art.

[0091] Comparative Example 2: Static culture was used without any modification to the culture medium.

[0092] This comparative example was statically cultured on day 0, but using conventional culture medium without the addition of SR additives and DNase, to verify the importance of improving the culture medium formulation.

[0093] Day 0: After reviving PBMCs, resuspend them in conventional complete culture medium and adjust the density to 3 × 10⁻⁶. 6 Cells / ml, incubated statically for 20 hours.

[0094] Day 1: Do not sort, add the activation magnetic beads directly.

[0095] Day 2 to Day 10: Cultured using conventional complete culture medium.

[0096] Day 10 test results: 8.5 × 10⁻⁶ 6 The cell count was 90.2%, with a CD3 positivity rate of 98.7% and a CAR positivity rate of 46.8%. Although this was an improvement over control example 1, it was still significantly lower than the example using the modified culture medium, indicating that the SR additive and DNase play an important role in improving cell expansion efficiency.

[0097] Comparative Example 3: Using only the improved culture medium but without static incubation.

[0098] This comparative experiment was performed immediately after recovery on day 1, but an improved medium containing SR additive and DNase was used to verify the necessity of the static culture step.

[0099] Day 1: Immediately after reviving PBMCs, resuspend them in complete medium-A without static culture, and directly perform CD3 sorting and activation.

[0100] Day 2 to Day 10: Cultured using complete culture medium-B.

[0101] Day 10 test results: 5.2 × 10⁻⁶ 6 The cell viability was 88.9%, the CD3 positivity rate was 98.8%, and the CAR positivity rate was 49.2%. This result is better than Comparative Example 1, but still lower than Example 1 and Example 2, indicating the importance of static culture for cell state recovery.

[0102] Comparative Example 4: Class C Biosafety Cabinet Environmental Control

[0103] This comparative example uses the same process flow and culture medium formulation as Example 1, but is operated in a Class C biosafety cabinet environment instead of an isolator environment, to verify the impact of the operating environment on product quality.

[0104] The operating procedure is exactly the same as in Example 1, but all operations are performed in a Class C biosafety cabinet.

[0105] Day 10 test results: 7.5 × 10⁻⁶ 7 The cell count was 94.5%, with a CD3 positivity rate of 98.9% and a CAR positivity rate of 57.8%. Although the final product quality was good, a slight bacterial contamination was observed during the preparation process (two colonies were found on the settling plate, indicating a risk of environmental contamination). However, the final product was not contaminated, highlighting the risk of contamination in an open operating environment.

[0106] Test method description:

[0107] Cell counting and viability assay: Trypan blue staining was used. 10 μl of cell suspension was mixed with 10 μl of 0.4% trypan blue solution, and the cells were counted using a hemocytometer under an inverted microscope. Live cells remained unstained and appeared transparent; dead cells were stained blue. The percentage of live cells was calculated as cell viability.

[0108] Flow cytometry detection: Take 1×10 6 Cells were washed with PBS and resuspended in 100 μl PBS. CD3-APC-Cy7 antibody and Protein L-PE reagent were added, and the cells were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice with PBS and resuspended in 300 μl PBS for flow cytometry analysis. The expression of CD3 and CAR was analyzed to calculate the CD3 positivity rate and CAR positivity rate.

[0109] The key data of each embodiment and comparative example are summarized in the following table:

[0110] Group static culture Improved culture medium CD3 sorting Operating environment Cell count on day 10 Cell viability CD3 positivity rate CAR positivity rate Example 1 have have none isolator <![CDATA[8.2×10 7 ]]> 95.1% 99.3% 60.5% Example 2 have have have isolator <![CDATA[7.5×10 6 ]]> 94.3% 98.9% 45.5% Example 3 have have none isolator <![CDATA[3.8×10 7 ]]> 92.6% 98.5% 48.3% Example 4 have have have isolator <![CDATA[1.1×10 7 ]]> 94.8% 99.1% 58.2% Example 5 have have none isolator <![CDATA[7.8×10 7 ]]> 95.3% 99.2% 59.8% Comparative Example 1 none none have isolator <![CDATA[4.2×10 4 ]]> 86.5% 98.3% 40.5% Comparative Example 2 have none none isolator <![CDATA[8.5×10 6 ]]> 90.2% 98.7% 46.8% Comparative Example 3 none have have isolator <![CDATA[5.2×10 6 ]]> 88.9% 98.8% 49.2% Comparative Example 4 have have none Biosafety cabinet <![CDATA[7.5×10 7 ]]> 94.5% 98.9% 57.8%

[0111] Through in-depth analysis of the above data, the following important conclusions can be drawn:

[0112] First, the synergistic effect of static incubation and the improved culture medium is the core of the excellent results achieved in this invention. Comparative Example 1, using only existing technology, ultimately yielded only 4.2 × 10⁻⁴ ppm. 4 10 cells, compared to the initial 3.4 × 10 7 The number of cells decreased by nearly a thousandfold, which fully exposes the serious shortcomings of existing technology. Comparative Example 2, which only underwent static culture, ultimately yielded 8.5 × 10⁸ cells. 6 Compared to control example 1, this showed a significant improvement, but the CAR positivity rate was only 46.8%. Control example 3 used only the improved culture medium but did not allow for static culture, yielding 5.2 × 10⁶ cells. 6The CAR positivity rate was 49.2% for 100 cells. Example 1, using both static culture and an improved culture medium, ultimately yielded 8.2 × 10⁸ cells. 7 The CAR positivity rate was as high as 60.5% in 100 cells, which is significantly better than the simple sum of the results of Comparative Example 2 and Comparative Example 3, fully demonstrating the significant synergistic effect between the two techniques.

[0113] Secondly, this invention reveals the crucial role of static culture in cell state recovery. The damage to cells during cryopreservation and thawing is multifaceted, including physical damage to the cell membrane, mitochondrial dysfunction, and oxidative stress. Static culture provides a low-stress recovery environment for cells, allowing the lipid bilayer of the cell membrane to reorganize and repair, the conformation of membrane proteins to return to normal, and the function of cell surface receptors to be restored. After the cold shock of cryopreservation and thawing, mitochondria require time to recover their membrane potential and ATP synthesis capacity. Static culture allows mitochondrial function to gradually normalize, providing sufficient energy for subsequent cell activation and proliferation. Furthermore, the intracellular metabolic enzyme system also needs time to rebalance, and protein synthesis mechanisms need to be restarted. Experimental data show that after 12-24 hours of static culture, cell viability increased from approximately 78% to nearly 90%, a significant improvement that has a decisive impact on subsequent expansion efficiency.

[0114] Furthermore, the improved culture medium formulation reflects a precise understanding of cellular physiological needs. The various growth factors contained in the SR supplement can activate growth factor receptors on the cell surface, initiate intracellular signal transduction pathways, promote cell cycle progression, and increase cell proliferation rate. Simultaneously, the nutrients in the SR supplement provide a sufficient material basis for rapid cell division. The mechanism of action of DNase is even more unique. In the cell culture system after cryopreservation and thawing, a large amount of DNA released from dead cells exists. This DNA is highly viscous and forms complexes with proteins and lipids, aggregating into clumps in the culture medium. These clumps not only physically encapsulate living cells, hindering their contact with the culture medium and nutrient uptake, but may also be recognized by cells as pathogen-associated molecular patterns, activating inflammatory response pathways and inhibiting cell proliferation. DNase, by specifically degrading free DNA, cuts long-chain DNA into small fragments, fundamentally preventing clumping. Experimental observations showed that cells cultured in complete medium-A containing DNase exhibited significantly better suspension homogeneity and cell free state than the control group without DNase. This favorable cell dispersion creates the necessary conditions for efficient expansion.

[0115] Furthermore, the optional CD3 sorting step demonstrates the flexibility of the process and a deep understanding of cell characteristics in this invention. Traditionally, it is believed that the proportion of T cells in healthy human PBMCs is low, necessitating sorting to obtain a high-purity T cell population. However, the comparative results of Examples 1 and 2 show that, under the technical system of this invention, skipping sorting yields better results. This is because CD3 / CD28 activating magnetic beads themselves selectively activate T cells. Under suitable culture conditions, T cells proliferate rapidly, while non-T cells gradually undergo apoptosis or growth arrest, ultimately resulting in T cells dominating the cell population. Although no sorting was performed in Example 1, the final CD3 positivity rate still reached 99.3%, proving the effectiveness of this mechanism. More importantly, omitting the sorting step avoids additional operations such as centrifugation, magnetic bead incubation, and column chromatography, reducing cell loss and stress responses, which significantly impacts the final cell yield.

[0116] Finally, the advantage of a Class C isolator environment compared to a traditional biosafety cabinet lies in providing a higher level of aseptic protection and better process control. The isolator, through positive pressure and a high-efficiency filtration system, ensures complete isolation between the internal environment and the external environment, maintaining an extremely low risk of contamination even during prolonged operation. Although the results of Comparative Example 4 are numerically close to those of Example 1, the signs of contamination observed during the preparation process cannot be ignored. In actual production, any contamination event could lead to product scrapping and delays in patient treatment. The adoption of an isolator environment is a key manifestation of this invention's pursuit of high product quality standards.

[0117] Based on the combined results of all experiments, this invention successfully solves the technical challenges of preparing CAR-T cells from cryopreserved PBMCs through systematic process innovation. The static culture strategy creates conditions for cell recovery, the improved culture medium formulation precisely meets the nutritional and environmental needs of the cells, the flexible process route selection balances efficiency and quality, and the isolator environment ensures product safety. The organic combination of these technical elements enables this invention to efficiently prepare high-quality CD19 CAR-T cell products from cryopreserved PBMCs from healthy individuals, providing a more reliable and optimized technical solution for the clinical application of CAR-T cell therapy, and possessing significant application value and broad application prospects.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing autologous CD19 CAR-T cells from peripheral blood mononuclear cells (PBMCs) isolated and cryopreserved in an isolator, comprising the following steps: 50ml-200ml of peripheral blood from healthy individuals was transferred into a Class C isolator and diluted with physiological saline at a volume ratio of 1:1-1:

2. PBMCs were obtained by Ficoll density gradient centrifugation, washed with physiological saline containing 0.1%-0.5% HSA, and counted at a ratio of 1×10⁻⁶. 7 pcs / ml - 1×10 8 The PBMCs were cryopreserved using cryopreservation solution at a specification of 1 unit / ml. Prepare complete culture medium-A and complete culture medium-B. Complete culture medium-A includes basal culture medium, 1%-10% SR additive, and 1‰-5‰ DNase. Complete culture medium-B includes basal culture medium and 1%-10% SR additive. On day 0, the frozen PBMCs were thawed, transferred to the isolator, resuspended in the complete culture medium-A, and the cell density was adjusted to 1 × 10⁶ cells / day. 6 5 x 10 cells / ml 6 Cells / ml, incubated statically at 37℃ and 5% CO2 for 12-24 hours; On day 1, activation magnetic beads were added to the PBMCs at a ratio of 1:2 to 1:1 with the cells, and the culture continued. On day 2, lentivirus encoding CD19 CAR was added to the PBMCs for transduction, with an MOI of 5-20. The cell density was adjusted to 1×10⁶ cells / year using the complete culture medium-B. 6 3 x 10 cells / ml 6 pcs / ml; From day 3 to day 10, adjust the cell density to 1×10⁻⁶ cells daily using the complete culture medium-B described above. 6 CD19 CAR-T cells were obtained by continuously culturing cells at a density of 1 cell / ml.

2. The method according to claim 1, wherein the specific steps of the Ficoll density gradient centrifugation are as follows: take a gradient density centrifugation tube, add 10ml-20ml of Ficoll to it, centrifuge at 700g-1500g for 1min-2min, add 10ml-20ml of diluted peripheral blood to the centrifugation tube and centrifuge at 700g-1500g for 20min-40min, and after centrifugation, take the white membrane layer from each tube to harvest the PBMCs.

3. The method according to claim 1, wherein the specific steps of washing with physiological saline containing 0.1%-0.5% HSA are as follows: add 10ml-200ml of physiological saline containing 0.1%-0.5% HSA to the collected PBMCs, centrifuge the cell suspension at 500g for 10min, discard the supernatant after centrifugation, resuspend the cells in 10ml-50ml of physiological saline containing 0.1%-0.5% HSA, centrifuge the cell suspension at 500g for 10min, discard the supernatant after centrifugation.

4. The method according to claim 1, wherein the first day further includes a step of sorting the PBMCs for CD3-positive cells, specifically: After static culture, the PBMCs were transferred to centrifuge tubes containing 10ml-40ml of basal culture medium, centrifuged at 500g for 5-10 minutes, and the supernatant was discarded. The cells were then resuspended in 1ml-5ml of physiological saline containing 0.1%-0.5% HSA. CD3 sorting magnetic beads were added to the cell suspension at a ratio of 1×10⁻⁶. 9 Each cell was treated with 2 ml of magnetic beads, mixed well, and incubated at 2℃-8℃ for 15 min. After incubation, add pre-cooled physiological saline containing 0.1%-0.5% HSA to the cell suspension to a volume of 35-45 ml, centrifuge at 500g for 5-10 minutes, and discard the supernatant after centrifugation. Resuspend the cell pellet in 3-10 ml of physiological saline containing 0.1%-0.5% HSA. Place the LS column on a cell sorting magnetic rack and rinse the LS column with 3-10 ml of physiological saline containing 0.1%-0.5% HSA. After passing the cell suspension through a cell sieve, add it to the LS column. After the liquid has drained, wash the LS column three times with 3-10 ml of physiological saline containing 0.1%-0.5% HSA. Quickly rinse the CD3-positive T cells in the LS column with 3-10 ml of physiological saline containing 0.1%-0.5% HSA into a centrifuge tube and collect the CD3-positive T cells.

5. The method according to claim 4, wherein after collecting the CD3-positive T cells, the CD3-positive T cell suspension is centrifuged at 500g for 5-10 minutes, the supernatant is discarded after centrifugation, and the CD3-positive T cells are resuspended in the complete culture medium-A according to the counting results to achieve a cell density of 2×10⁻⁶ cells / mL. 6 4 x 10 cells / ml 6 Add the activated magnetic beads at a concentration of 1 / ml.

6. The method according to claim 1, wherein on day 1, the CD3-positive cell sorting step is not performed, but the cell density of the statically cultured PBMCs is directly adjusted to 2 × 10⁻⁶ cells using the complete culture medium-A based on the counting results. 6 4 x 10 cells / ml 6 Add the activated magnetic beads at a concentration of 1 / ml.

7. The method according to claim 1, wherein the basal culture medium is selected from one of X-VIVO 15 medium, AIM V medium, and OpTmizer medium, and the basal culture medium is further supplemented with cytokines and essential nutrients.

8. The method according to claim 1, wherein the specific steps on the second day are as follows: Remove the PBMCs from the incubator, mix and count them; centrifuge the PBMCs at 500g for 5-10 minutes; discard the supernatant after centrifugation; resuspend the PBMCs in the complete culture medium-B according to the counting results to achieve a cell density of 1×10⁻⁶ cells / mL. 6 3 x 10 cells / ml 6 The lentivirus encoding CD19 CAR was added to the PBMCs at a concentration of 1 cell / ml, and the cell suspension was mixed and cultured further.

9. The method according to claim 1, wherein the specific steps on the third day are as follows: remove the PBMCs from the incubator, mix and count them, centrifuge the PBMCs at 500g for 5-10 minutes, discard the supernatant after centrifugation, resuspend the PBMCs in the complete culture medium-B and adjust the density to 1×10⁻⁶. 6 Cells / ml, continue culturing.

10. The method according to claim 1, wherein the number, viability and CAR positivity of the CD19 CAR-T cells are detected on day 10, wherein the CD3 positivity rate is ≥98% and the CAR positivity rate is ≥40%.