Method for enhancing T cell amplification
By co-culturing feeder cells with T cells with short-term expression and IL-12/IL-18 pulse activation, the problem of balancing expansion efficiency and function during T cell expansion was solved, achieving efficient and safe T cell expansion and functional enhancement. It is suitable for modular expansion of different T cell subsets and is suitable for GMP production.
Patent Information
- Application Number
- CN202511599234.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2025-12-12
AI Technical Summary
Existing technologies struggle to balance efficient expansion and effector function during T-cell expansion. They suffer from high costs due to high doses of cytokines, risks of cell differentiation depletion, regulatory and safety concerns related to long-term cell feeding, a lack of modular stimulation platforms, and the different responses of different T-cell subsets to stimulation, as well as a lack of a unified parameter system, all of which affect process reproducibility and clinical translation efficiency.
Feeder cells expressing short-term expression were co-cultured with T cells. Human mesenchymal stem cells were transduced with a recombinant vector within 48-96 hours to express proliferation/co-stimulatory molecules. Feeder cells were then removed and expansion was maintained with low-dose cytokines. Combined with short-pulse functional activation stimulation in the later stages of expansion, combined pulses of IL-12 and IL-18 were used to enhance cell effector function.
It significantly increases the amplification rate, enhances cytotoxicity and secretion levels of IFN-γ and TNF-α, reduces exhaustion markers, maintains the memory/stem phenotype, achieves process standardization and GMP conversion, and avoids the deficiencies of long-term high-dose factors and stable feeder cells.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and cell biology, and relates to a method for enhancing T cell proliferation. Background Technology
[0002] In vitro expansion and functional maintenance of T cells are key technological steps in cell immunotherapy, such as tumor immunotherapy, chronic infection treatment, and CAR-T / CAR-T concomitant formulations. Existing publicly available technologies mainly fall into two categories: one focuses on optimizing cytokine combinations and stimulation rhythms to enhance the expansion efficiency of specific T cells (e.g., Vγ2Vδ2 γδT cells) and subsequently strengthening effector function with factors such as IL-12 / IL-18; related technologies have disclosed that the combination of IL-2 and IL-15 has a synergistic proliferative effect on Vγ2Vδ2 expansion, and the addition of IL-12 / IL-18 after expansion can enhance antibacterial / effector function. Examples of existing technologies can be found in the disclosures of Vγ2Vδ2 expansion methods in relevant patents.
[0003] Another approach employs a "feeder cell" platform—that is, by expressing cytokines or co-stimulatory molecules on the surface of feeder cells or modified stimulatory cells, a microenvironment rich in proliferative / co-stimulatory signals is constructed to achieve high-expansion and functional induction. For example, by transducing MSCs with recombinant vectors encoding multiple cytokines (such as baculoviruses), MSCs can release or present molecules such as IL-2, IL-15, IL-21, IFN-γ, membrane-bound IL-15 / mIL-15, and 4-1BBL during short-term co-culture, significantly increasing the expansion fold and effector receptor expression of targeted effector cells, such as NK or T cells, while maintaining good GMP operability. The corresponding feeder cell platforms have been used in the expansion of NK cells and CAR / NK, CAR-T cells with positive results.
[0004] However, both strategies have their advantages and disadvantages: simply administering high doses of free cytokines can lead to high costs, risks of early cell differentiation / exhaustion, and GMP operational burdens; while stable transgenic feeder cells or long-term high-expression systems can continuously supply stimulating signals, they may raise regulatory and safety concerns (problems such as long-term gene expression and feeder cell residue). Therefore, there is an urgent need for a universal methodology that can balance efficient expansion and effector function, while also being controllable / transient, safe, and easily scaled up for GMP production, applicable to the expansion and clinical translation of T cells from different sources—peripheral blood T, γδT, CAR-T, etc. The aforementioned problems have been raised in existing patent publications and partially addressed through transient expression of "factor feeder" or modified stimulating cell strategies, but a universal, modular, and clearly defined system method with specific time windows / parameter coupling schemes has not yet been disclosed.
[0005] In addition to the above, the existing technology also has the following problems:
[0006] ① A balance needs to be struck between proliferation efficiency and cytotoxicity, cytokine secretion, and memory phenotypic effects. However, conventional high-dose, continuous free factor regimens often lead to functional exhaustion or differentiation deviation. ② While long-term or stable feeder cells can provide continuous stimulation, they bring regulatory and residual risks and process complexity. There is still a lack of sufficient methods to construct a transient, controllable, and GMP-compatible "peak-type" stimulation platform. ③ Different T cell subsets—conventional αβ T, γδ T, CAR-T, etc.—respond differently to different types of stimulation—TCR / CD3 combined stimulation, co-stimulatory molecules, different combinations of cytokines, and time windows. There is a lack of a modular and adjustable process that facilitates migration between different indications. ④ Existing amplification processes lack a unified and scalable parameter system for process rhythms such as half-medium replacement, maintenance concentration, and late functional pulses (e.g., IL-12 / IL-18), affecting process reproducibility and clinical translation efficiency.
[0007] Therefore, there is a need in the field for a reasonable method to enhance T cell expansion that can balance the above-mentioned technical problems. Summary of the Invention
[0008] To address the shortcomings of existing technologies, this invention provides a method for enhancing T cell proliferation.
[0009] To achieve the above objectives, the present invention adopts the following technical solution: a method for enhancing T cell proliferation, comprising the following steps:
[0010] 1) Collect peripheral blood or other lymphocyte-containing samples from the donor and isolate peripheral blood mononuclear cells (PBMCs) or the target starting cell population;
[0011] 2) Transducing / infecting human mesenchymal stem cells (MSCs) or other suitable feeder / stimulatory cells in vitro with a recombinant vector carrying a mammalian promoter, so that they express or secrete at least one proliferation / co-stimulatory molecule within 48-96 hours to form a short-term "factor-enriched" feeder cell population.
[0012] 3) The feeder cells with short-term expression obtained in step 2) are co-cultured with the PBMCs or target T cells obtained in step 1) for 24-60 h in the presence of T cell activation signals;
[0013] 4) Remove the feeder cells from step 3) and continue to expand and culture for 7-14 days without feeder cells, while periodically feeding with low doses of maintenance cytokines and changing half the medium to maintain a suitable proliferation environment.
[0014] 5) During any 48-72h window in the 5th-10th day of culture in the later stage of expansion, apply short-pulse functional activation stimulation to the expanded cells to induce / enhance cell effector function;
[0015] The starting cells are CAR-modified T cells or a population of T cells containing CAR, and the method is used to scale up the preparation of CAR-T cells and improve their in vitro functional phenotype.
[0016] Preferably, the recombinant vector is selected from baculovirus, adeno-associated virus, retrovirus, or lentivirus, and is preferably baculovirus.
[0017] Preferably, the MSCs are derived from umbilical cord-derived mesenchymal stem cells (hUC-MSCs) or bone marrow-derived MSCs.
[0018] Preferably, the combination of cytokines expressed in step 2) includes at least IL-2 and IL-15, wherein the expression of IL-2 and IL-15 is adequate or the amount of secretion is sufficient to significantly increase the proliferation rate of T cells during co-culture.
[0019] Furthermore, the molecules expressed or secreted in step 2) include, but are not limited to, IL-2, IL-7, IL-15, IL-21, membrane-bound IL-15 (mIL-15) and / or membrane-bound co-stimulatory molecule 4-1BBL.
[0020] Preferably, step 2) further expresses at least IL-21 or membrane-bound 4-1BBL to improve co-stimulatory receptor activation and memory / persistent phenotype of the expanded T cells.
[0021] Furthermore, the recombinant vector in step 2) is a combination of vectors carrying the genes of IL-2, IL-15, IL-21 and IFN-γ respectively, and the transcription units contained in the vector are driven by strong mammalian promoters.
[0022] Preferably, in step 3), the inoculation ratio of feeder cells to T cells is 1:8-1:12, based on the cell number ratio; after co-culturing for 24-60 hours, the feeder cells are removed by gentle centrifugation or washing.
[0023] Furthermore, the T cell activation signal in step 3) is CD3 / CD28 co-stimulation, pyrrole phosphate small molecules (such as zoledronic acid), or an equivalent TCR agonist;
[0024] Preferably, the maintenance cytokine used in step 4) is selected from one or a combination of IL-7 (1-10 ng / mL) and IL-15 (1-20 ng / mL), and the medium is replaced with 50% half volume on days 2, 3 and 5, and the same concentration of maintenance factor is added.
[0025] Preferably, in step 5), the pulse concentrations of IL-12 and IL-18 are 50-200 ng / mL, respectively, and the pulse duration is 12-72 h.
[0026] Furthermore, the short-pulse functional activation stimulation described in step 5) includes a combined pulse of IL-12 and IL-18.
[0027] Preferably, in step 3), CD3 / CD28 co-stimulatory microbeads are used simultaneously, with the ratio of microbeads to cells being 1:1 to 1:3.
[0028] The present invention also provides an expanded T cell, characterized in that the expanded T cell is used to prepare a cell therapy preparation for treating tumors, chronic infections or immunodeficiency-related diseases.
[0029] The beneficial effects of this invention are:
[0030] 1) The amplification fold is significantly improved, reaching 2–3 times that of the traditional IL-2 method.
[0031] 2) Enhanced effector function, with significantly increased cytotoxicity and secretion levels of IFN-γ, TNF-α, etc.
[0032] 3) Decreased exhaustion markers and increased memory / stem phenotype ratios help maintain long-lasting immune function.
[0033] 4) The transient MSC-factor platform balances amplification and safety, avoids the shortcomings of long-term high-dose factors / stable feeder cells, and the process is easier to standardize and GMP convert. Detailed Implementation
[0034] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.
[0035] Unless otherwise stated, all films and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, as are the instruments used in the examples.
[0036] Example 1: (Small-scale verification of the 96-well U-plate system)
[0037] 1. Experimental Materials
[0038] Starting cells: fresh peripheral blood PBMCs (Ficoll isolation).
[0039] Culture medium: X-VIVO15 (serum-free, suitable for clinical cell therapy); alternative: RPMI-1640 + 10% FBS (control).
[0040] Stimulant: zoledronic acid (ZOL) or CD3 / CD28 microbeads (subgroup selection). In this embodiment, ZOL is used for γδ T stimulation, preferably at 1.5 μg / mL (range 0.75–15 μg / mL).
[0041] Cytokines:
[0042] IL-2: Preferred 10 ng / mL (5–100 ng / mL is acceptable).
[0043] IL-15: Preferred 100 ng / mL (10–200 ng / mL is acceptable).
[0044] Late pulse: 100 ng / mL each of IL-12 and IL-18 (50–200 ng / mL can be used, lasting 12–72 h).
[0045] Feeder cells (for Example 2): See Example 2.
[0046] 2. Culture vessels and inoculation density (small-scale)
[0047] 96-well U-plate, 200 μL culture medium per well, initial PBMC density 0.5 × 10^6 cells / well (i.e. 2.5 × 10^6 cells / mL).
[0048] 3. Cultivation Process (Timeline)
[0049] Day 0 (Initiation): Inoculate PBMCs into each well and add the following stimulants: ZOL 1.5 μg / mL (or CD3 / CD28 microbeads), IL-2 10 ng / mL, and IL-15 100 ng / mL. Incubate at 37°C with 5% CO2.
[0050] Day 2, Day 3, Day 5: Replace half of the culture medium with 50% of the medium and add fresh culture medium containing the same concentration of cytokines (i.e., maintain a constant IL-2 / IL-15 concentration).
[0051] Days 3–15 (as needed, preferably Day 7–10): 3–15 days after the initial amplification (Day 7–10 is generally recommended as the priority window), add IL-12 and IL-18 (100 ng / mL each) to the culture system and continue culturing for 48 hours (can be adjusted by 12–72 hours). Record the sampling time points (Day 0, Day 3, Day 7, Day 10, Day 14).
[0052] 4. Endpoint processing
[0053] After expansion (e.g., Day 10 or Day 14): Collect cells, count them, and use trypan blue / viability staining to detect viability; separate target subpopulations as needed (e.g., Vγ2Vδ2T cells sorted by specific antibody magnetic beads).
[0054] 5. Testing and Evaluation Indicators (Small-scale)
[0055] 5.1 Flow Cytometry Cell Phenotypes (Panel Example)
[0056] Panel A (Universal T cell phenotype): CD3, CD4, CD8, CD45, CD45RA, CCR7, CD62L.
[0057] Panel B (exhaustion / memory / activation): PD-1, LAG-3, TIM-3, TIGIT, CD27, CD28, Ki-67 (proliferation).
[0058] Panel C (γδ specific): Vγ9, Vδ2 (or Vγ2 / Vδ2), CD107a (degranulation).
[0059] Flow cytometry staining and collection: 0.5–1×10^6 cells per sample, positive and negative control and FMO control, intracellular IFN-γ, TNF-α and Granzyme B were detected using standard lysis / fixation-permeabilization procedure.
[0060] 5.2 Functional Testing
[0061] Cytotoxicity: CFSE / 7-AAD or CFSE / PI cytotoxicity assay. Target cells (e.g., K562 or specific tumor cell lines) were labeled with CFSE and co-cultured for 4 h at different E:T ratios (1:1, 4:1, 10:1), and the target cell lysis rate (7-AAD positive / CFSE positive) was detected.
[0062] Degranulation: CD107a surface markers were used to detect the percentage of CD107a positivity after short-term PMA / ionomycin or target cell stimulation.
[0063] Cytokine secretion: Quantify IFN-γ, TNF-α, and IL-2 (pg / mL) using ELISA or Luminex.
[0064] Cell proliferation: CFSE dilution or Ki-67 positivity rate.
[0065] For Vγ2Vδ2 cell / antibacterial activity: the reduction rate of target bacterial CFU was determined using a macrophage-BCG / endomicrobial assay.
[0066] 5.3 Data Statistics and Representation
[0067] Each group has n≥3 donors (independent biological replicates), and data are expressed as mean ± standard deviation (mean ± SD); comparisons between groups are performed using Student's t-test or one-way ANOVA (multiple groups).
[0068] Example 2: (Transient expression platform for feeder cells, pilot-scale verification)
[0069] pFB-HU-IL2, pFB-HU-IL15, and pFB-HU-IL21 (or combined vectors) were constructed using recombinant baculovirus vectors (containing mammalian promoter CMV). The viruses were amplified in SF9 insect cell systems and used to infect hUC-MSCs (or bone marrow MSCs) for a short period of time. This resulted in short-term high expression and secretion / surface presentation of the required factors by MSCs within 48–96 hours after infection. The short-term expressed MSCs were then co-cultured with activated initiating T cells (or PBMCs) for 24–60 hours. After removing the MSCs, feederless cell expansion was continued and the procedure was performed according to the half-volume medium change / late-stage pulsed process in Example 1.
[0070] Preparation of recombinant baculoviruses: Recombinant baculoviruses (pFB-HU-IL2, pFB-HU-IL15, pFB-HU-IL21, pFB-HU-IFNγ, etc.) were constructed and prepared. The viral supernatant was collected and concentrated and stored at 4°C or -80°C.
[0071] MSC infection: hUC-MSCs (adherent culture) were placed at an appropriate density in T25 / T75 during the logarithmic growth phase, and recombinant baculovirus was added (infection conditions were optimized based on laboratory experience, aiming for significant expression and good MSC cell condition at 48–96 h; suitable conditions could be determined using parallel infection with a reporter gene). MSCs with peak expression / secretion were collected 48–96 h after infection and washed with PBS.
[0072] Co-culture: Infected MSCs and activated PBMCs / T cells were co-cultured at a ratio of 1:8–1:12 (MSC:T cell number) for 24–60 h. Co-culture conditions included CD3 / CD28 activation or IL-2 pre-activation.
[0073] Removal of feeder cells and continued expansion: After co-culture, MSCs were removed by gentle centrifugation / washing (or by removing them with anti-MSC antibody magnetic beads), and then transferred to G-REX, T-75 or shake flasks for continued expansion for 7–14 days. Half the medium was changed (Day 2 / 3 / 5) and low doses of IL-7 / IL-15 (or IL-2 / IL-15) were maintained to sustain expansion and phenotype.
[0074] Late pulse: Short pulses of IL-12 / IL-18 (50–200 ng / mL each, preferably 100 ng / mL, duration 12–72 h) are administered during the 5–10 day window of amplification to enhance the effector function.
[0075] 3. Scaling-up / Scale-up Recommendations (Process Engineering Tips)
[0076] For pilot-scale operations, G-REX or gas-permeable bags / wave bioreactors can be used to maintain high-density culture. The initial cell density can be set at 0.5–2 × 10^6 cells / mL. During each half-volume medium change, the cell density should be kept below 5 × 10^6 cells / mL to avoid excessive nutrient depletion. The above density range is a commonly used range in the industry and takes into account the parameters for small-scale trials (0.5 × 10^6 / well) and recommendations in the literature (please describe the process optimization process in the examples).
[0077] We recommend establishing a scaled-up SOP: Virus preparation → MSC infection condition confirmation (transduction rate, viability) → MSC batch-to-batch quality control (secretion amount / surface molecule detection) → Co-culture and MSC removal → Amplification culture → Final product quality release (cell count, viability, cell phenotype, microbial detection).
[0078] Control group setup (n≥3 independent donors per group)
[0079] Evaluation indicators and time points
[0080] Time points: Day 0, Day 3, Day 7, Day 10, Day 14.
[0081] Indicators: Total cell number (expansion fold), viability (%), percentage of target subset (Vδ2%), memory / exhaustion markers (PD-1%, CD62L%, CD45RA / RO), proliferation (Ki-67 or CFSE), function (IFN-γ pg / mL, TNF-α, CD107a%, cytotoxicity at E:T=1:1 / 4:1 / 10:1).
[0082] Statistics and Judgment
[0083] Statistics: mean ± SD; ANOVA was used for comparisons among multiple groups, and Tukey test was used for pairwise comparisons between groups. Significance p < 0.05.
[0084] Determining the superiority of the invention: If the amplification fold of group C on Day 7 / 10 is significantly higher than that of A / B (e.g., p<0.05), and at the same time it is not inferior to or superior to the control group in functional / phenotypic indicators such as CD107a%, IFN-γ secretion and PD-1 expression, then it proves the beneficial effect of both improving amplification and maintaining / enhancing function.
[0085] Comparative Example 1: Group A (conventional high-dose IL-2): PBMC + ZOL (or CD3 / CD28) + IL-2 (high dose, e.g., 50 ng / mL) for continuous culture (half-volume medium replacement as usual).
[0086] Comparative Example 2: Group B (IL-2 + IL-15 continuously supplemented): PBMC + ZOL + IL-2 (10 ng / mL) + IL-15 (100 ng / mL), half-volume solution change Day 2 / 3 / 5.
[0087] Comparative Example 3: Group D (MSCs without infection only): Same as Group C, but MSCs were not infected with recombinant virus to exclude the influence of physical contact with MSCs.
[0088] Group C (Invention—Transient MSC Factor Enrichment Platform): MSC infection and co-culture for 24–48 h as in Example 2 → MSC removal → low-dose maintenance (IL-7 / IL-15 or IL-2 / IL-15) → late-stage IL-12 / IL-18 pulse.
[0089] The results are shown in the table below:
[0090] Table 1. Amplification and Survival
[0091] Group Time Total cell count (×10^6) Amplification factor (relative to Day 0) vitality(%) A (IL-2) Day 7 40 80 92.1 B (IL-2+IL-15) Day 7 65 130 90.5 C Day 7 150 300 93.8
[0092] Note that the expansion fold (Total cell yield / initial cell number), survival rate (%), major subset % (CD 8%, Vδ 2%), and time points Day 0 / 3 / 7 / 10 / 14 are listed in Table 1.
[0093] Table 2. Main phenotypes
[0094] Group Time CD3+ (%) CD8+ (%) Vδ2+(%) PD-1 (%) CD62L+(%) A Day 10 92.3 62.5 — 18.7 20.2 B Day 10 90.8 60.1 — 15.4 24.1 C Day 10 94.0 64.2 — 9.8 38.5
[0095] Note that in Table 2, the flow cytometry phenotypes (PD-1%, CD62L%, CD45RA%, etc.) are compared with the MFI (median fluorescence intensity).
[0096] Table 3. Cytotoxicity (E:T) and Cytokines
[0097] Group E:T 4h cell lysis (%) IFN-γ (pg / mL) A 4:1 45.2 1200 B 4:1 60.8 2100 C 4:1 78.5 5200
[0098] Note: Table 3: Functional assays (E:T ratio at each point:% cell lysis, IFN-γ pg / mL, CD107a%).
[0099] Analysis: Under the same donor source and initial PBMC quantity conditions, in vitro amplification was carried out according to the methods of Example 1, Example 2, and Comparative Examples A, B, and D, respectively;
[0100] 1) Amplification efficiency: Comparative Example A (continuous high-dose IL-2 supplementation) showed an amplification fold of approximately 50–100 times, with cell numbers plateauing after 7–10 days; Comparative Example B (continuous IL-2 + IL-15 supplementation) showed an amplification fold of 100–150 times, but exhibited a significant exhaustion phenotype (increased PD-1 and TIM-3) in the later stages of culture (10–14 days); Example 1 (small-scale process, including late-stage IL-12 / IL-18 pulse) achieved an amplification fold of 200–250 times; Example 2 (transient MSC-factor plateau + maintenance + pulse) achieved an amplification fold of 250–300 times, significantly better than the comparative example.
[0101] 2) Functional phenotype and memory characteristics: Comparative Example A: After expansion, the PD-1 positivity rate of T cells was >20%, and the proportion of CD62L+ memory phenotype cells was <25%; Comparative Example B: Despite the high expansion rate, the PD-1 positivity rate remained at around 15%, and the proportion of CD62L+ was 20–25%; Example 1: After IL-12 / IL-18 pulses, the PD-1 positivity rate decreased to below 10%, and the proportion of CD62L+ memory / stem cells increased to 35–40%; Example 2: Under the combination of transient MSC-factor peak stimulation and subsequent pulses, the PD-1 positivity rate was the lowest (approximately 8–10%), and the proportion of CD62L+ was the highest (40–45%).
[0102] 3) Functional phenotype and memory characteristics: Comparative Example A: After expansion, the PD-1 positivity rate of T cells was >20%, and the proportion of CD62L+ memory phenotype cells was <25%; Comparative Example B: Despite the high expansion rate, the PD-1 positivity rate remained at around 5%, and the proportion of CD62L+ was 20–25%; Example 1: After IL-12 / IL-18 pulses, the PD-1 positivity rate decreased to below 10%, and the proportion of CD62L+ memory / stem cells increased to 35–40%; Example 2: Under the combination of transient MSC-factor peak stimulation and subsequent pulses, the PD-1 positivity rate was the lowest (approximately 8–10%), and the proportion of CD62L+ was the highest (40–45%).
[0103] 4) Safety and process controllability: Comparative Example D (co-culture of uninfected MSCs) only provided physical support, with no significant improvement in amplification and function. This indicates that MSC co-culture alone is insufficient to achieve the technical effects of the invention;
[0104] Example 2, through the strategy of "transient expression + short-term co-culture", can achieve virtually no MSC residue after MSC removal, avoiding the risk of long-term residue of stable transgenic feeder cells and better meeting GMP compliance requirements.
[0105] In summary, the method of this invention is significantly superior to traditional methods with continuous free factor replenishment in terms of amplification efficiency, and can better preserve memory phenotypes during amplification, reduce the expression of depletion markers, and has significantly better cytotoxicity and cytokine secretion capacity than existing methods of the same type. It is also superior to existing methods in terms of safety and process controllability.
[0106] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for enhancing T cell proliferation, characterized in that, Includes the following steps: 1) Collect peripheral blood or other lymphocyte-containing samples from the donor and isolate peripheral blood mononuclear cells (PBMCs) or the target starting cell population; 2) Transducing / infecting human mesenchymal stem cells (MSCs) or other suitable feeder / stimulatory cells in vitro with a recombinant vector carrying a mammalian promoter, so that they express or secrete at least one proliferation / co-stimulatory molecule within 48-96 hours to form a short-term "factor-enriched" feeder cell population; 3) The feeder cells with short-term expression obtained in step 2) are co-cultured with the PBMCs or target T cells obtained in step 1) for 24-60 h in the presence of T cell activation signals; 4) Remove the feeder cells from step 3) and continue to expand and culture for 7-14 days without feeder cells, while periodically feeding with low doses of maintenance cytokines and changing half the medium to maintain a suitable proliferation environment. 5) During any 48-72h window in the 5th-10th day of culture in the later stage of expansion, short-pulse functional activation stimulation is applied to the expanded cells to induce / enhance cell effector function; The starting cells are CAR-modified T cells or a population of T cells containing CAR, and the method is used to scale up the preparation of CAR-T cells and improve their in vitro functional phenotype.
2. The method for enhancing T cell proliferation according to claim 1, characterized in that, The recombinant vector is selected from baculovirus, adeno-associated virus, retrovirus, or lentivirus, with baculovirus being the preferred option.
3. The method for enhancing T cell proliferation according to claim 1, characterized in that, The MSCs are derived from umbilical cord mesenchymal stem cells or bone marrow.
4. The method for enhancing T cell proliferation according to claim 3, characterized in that, The combination of expressed cytokines described in step 2) includes at least IL-2 and IL-15, wherein the expression of IL-2 and IL-15 is adequate or the amount of secretion is sufficient to significantly increase the proliferation rate of T cells during co-culture.
5. The method for enhancing T cell proliferation according to claim 4, characterized in that, Step 2) Further express at least IL-21 or membrane-bound 4-1BBL to improve co-stimulatory receptor activation and memory / persistent phenotype of expanded T cells.
6. The method for enhancing T cell proliferation according to claim 1, characterized in that, In step 3), the ratio of feeder cells to T cells is 1:8 to 1:12, based on the cell number ratio; after co-culturing for 24-60 hours, the feeder cells are removed by gentle centrifugation or washing.
7. A method for enhancing T cell proliferation according to claim 1, characterized in that, Step 4) The maintenance cytokines used are selected from IL-7 (1-10 ng / mL) and IL-15 (1-20 ng / mL) or a combination thereof, and the medium is replaced with 50% half volume on days 2, 3 and 5, and the same concentration of maintenance factors is added.
8. The method for enhancing T cell proliferation according to claim 1, characterized in that, In step 5), the pulse concentrations of IL-12 and IL-18 are 50-200 ng / mL, respectively, and the duration is 12-72 h.
9. The method for enhancing T cell proliferation according to claim 6, characterized in that, In step 3), CD3 / CD28 co-stimulatory beads are used simultaneously, with the ratio of beads to cells being 1:1 to 1:
3.
10. Expanded T cells prepared by the method according to any one of claims 1-9, characterized in that, These expanded T cells are used to prepare cell therapy agents for treating tumors, chronic infections, or immunodeficiency-related diseases.