Method for low-density amplification of T cells

Abstract

The invention discloses a method for low-density amplification of T cells and an obtained product, and belongs to the technical field of cell culture. The method comprises the following steps: activating a cell population containing T cells, and co-culturing with a lentiviral vector carrying a target gene for transduction; the method comprises the following steps: transduction is carried out on cells, then the transduction cells are inoculated into a shake flask at an inoculation density of not higher than 2 * 10 < 5 > cells / mL, shake culture is carried out to realize amplification, and amplification of 30-120 times can be realized. According to the method, a low-density cell microenvironment is optimized through dynamic oscillation, the problems of signal insufficiency, factor dilution and metabolism imbalance are solved, the efficiency bottleneck of traditional low-density amplification is broken through, and the method is suitable for large-scale production. The method has the beneficial effects of simplicity in operation, high cell product viability, high purity, low cost, wide scale adaptability and the like. The invention also provides a T cell product prepared by the method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to cell culture and immune cell therapy technology. More particularly, the present application relates to a method for expanding T cells at low density. BACKGROUND

[0002] In vitro expansion of T cells is a key preparation step for their application in immunotherapy. This process is highly dependent on a suitable cell microenvironment, which involves complex biological regulation mechanisms.

[0003] Firstly, activation and proliferation of T cells not only require specific signals from antigen-presenting cells or artificial activators, but also benefit from various intercellular interactions. For example, intercellular contact signals mediated by membrane surface proteins can provide important co-stimulatory or proliferation regulation signals. Secondly, activated T cells secrete growth factors, which form a concentration gradient locally to continuously activate downstream proliferation pathways in a paracrine or autocrine manner. In addition, the "population effect" of cell populations through vesicles such as exosomes for material and information exchange also has a positive effect on maintaining synchronous expansion of the population. Based on these mechanisms, in order to ensure that the above intercellular interactions and local factor microenvironment can be effectively established, conventional T cell in vitro expansion processes usually require maintaining a relatively high initial cell seeding density.

[0004] To meet the needs of large-scale expansion, the industry has developed various culture platforms. Static culture bags are commonly used due to their simple operation, but their material transfer efficiency is limited. In order to achieve the desired expansion effect, a relatively high seeding density (e.g., 5 x 10 5 to 1 x 10 6 cells / mL) is often required, which puts a higher requirement on the initial cell quantity. G-Rex and other culture systems using gas-permeable membrane technology improve gas exchange, which can theoretically support lower cell density for expansion, but the cost of this system and special consumables is significantly higher than that of conventional methods. Stirred or wave bioreactors can provide good mixing and mass transfer efficiency, but they usually require a larger minimum working volume, resulting in a greater demand for the total quantity of starting cells during process development or small-scale production stages, and limited flexibility.

[0005] In contrast, shake-flask culture is a low-cost, easily scaled-up dynamic culture tool. However, when attempting low-density amplification in shake-flask systems, inherent challenges are exacerbated by the reduced cell density: increased intercellular spacing leads to a sharp decline in contact-dependent signaling; secreted growth factors are rapidly diluted by the culture medium, making it difficult to maintain effective stimulatory concentrations around the cells; simultaneously, insufficient shaking can lead to localized accumulation of cell metabolites and toxicity, while excessive mixing may introduce harmful shear stress. Currently, shake-flask platforms lack a systematically validated, comprehensive process that clearly guides how to synergistically overcome these low-density limitations through precise control of shaking parameters, inoculation timing, and culture medium systems to achieve stable and efficient amplification.

[0006] Therefore, there is a need in the art for a detailed and reproducible method for low-density T-cell expansion specifically designed for shake-flask culture systems to address the problems in the prior art. Summary of the Invention

[0007] To address at least one of the aforementioned technical problems, a first aspect of the present invention provides a method for low-density expansion of T cells, comprising the following steps: (a) Activation: A cell population containing T cells is contacted with a T cell activator and cultured to obtain activated T cells; (b) Transduction: The activated T cells obtained in step (a) are co-cultured with a lentiviral vector to introduce the target gene carried by the lentiviral vector into the T cells, thereby obtaining transduced T cells; (c) Amplification: The transduced T cells obtained in step (b) are amplified at a rate not exceeding 2 × 10⁻⁶. 5 T cells were inoculated at a density of cells / mL in shake flasks and cultured with shaking to achieve T cell expansion.

[0008] In some implementations, in step (c), the inoculation density is 5 × 10⁻⁶. 4 cells / mL to 2×10 5 The amplification step (c) is performed at cells / mL for 4 to 5 days.

[0009] In some embodiments, in step (a), the cell population containing T cells is peripheral blood mononuclear cells (PBMCs), preferably in a concentration of 2 × 10⁻⁶ cells. 6 cells / mL to 4×10 6 The initial density of cells / mL was mixed with the T cell activator.

[0010] In some embodiments, in step (a), the T-cell activator is a magnetic bead coated with anti-CD3 and anti-CD28 antibodies; the magnetic beads may be selected from nano-magnetic beads or micro-magnetic beads. When using nano-magnetic beads, the ratio of the culture volume containing the T cells to the added volume of the nano-magnetic beads is 20:1 to 100:1; when using micro-magnetic beads, the ratio of the number of T cells to the number of micro-magnetic beads added is 1:1 to 1:3.

[0011] In some implementations, step (b) is performed on the second day after the start of culture in step (a), and wherein 5 × 10⁻⁶ is taken. 6 Up to 2×10 7 The activated T cells were transduced.

[0012] In some implementations, in step (b), the lentiviral vector is used at a dose with a multiplicity of infection (MOI) of 0.025 to 0.5.

[0013] In some implementations, during step (b) transduction, the density of activated T cells is adjusted to 1 × 10⁻⁶. 6 cells / mL.

[0014] In some implementations, step (c) is performed on day 3 or day 4 after the start of co-cultivation in step (b), preferably on day 3.

[0015] In some embodiments, in step (c), the shaking rotation speed is 40 rpm to 145 rpm. Further, when the culture volume of the shake flask is no more than 250 mL, the rotation speed is preferably 80 rpm to 145 rpm; when the culture volume is no less than 1000 mL, the rotation speed is preferably 40 rpm to 100 rpm.

[0016] In some embodiments, in step (c), the shaking culture is performed using a cell culture medium with a chemical composition determined by the addition of interleukin-2 (IL-2), wherein the chemical composition determined by the cell culture medium is one of Optmizer™ medium, Optivitro™ medium, or T-VIVO™ medium. Preferably, the concentration of interleukin-2 in the culture medium is from 100 IU / mL to 1000 IU / mL.

[0017] In some embodiments, the shake flask can be selected from 125 mL shake flasks, 250 mL shake flasks, or 3 L shake flasks. Specifically, when using 3 L shake flasks and the culture volume is 1000 mL, the inoculation density in step (c) is preferably 8 × 10⁻⁶. 4 cells / mL up to 1.5 × 10⁻⁶ 5 cells / mL.

[0018] In some implementations, the target gene is the gene encoding a chimeric antigen receptor (CAR).

[0019] A second aspect of the present invention provides a T-cell product prepared by the method according to any one of the first aspects.

[0020] In some implementation schemes, the viability of T cell products is not less than 92%, and the purity of T cells is not less than 96%.

[0021] In this specification, unless otherwise expressly stated, the following terms shall be understood to have the following meanings: 1. "T cells": refers to T lymphocytes, which are one of the key effector cells of the adaptive immune system and can mediate cellular immune responses. In this invention, they are the target cells of the in vitro expansion method.

[0022] 2. "Peripheral blood mononuclear cells" or "PBMCs": refers to a population of cells with a single nucleus obtained from mammalian peripheral blood by density gradient centrifugation. This population mainly includes lymphocytes (such as T cells, B cells, and NK cells) and monocytes. In this invention, it is the preferred starting cell source for obtaining the T cells.

[0023] 3. "T-cell activator": This refers to a substance that can specifically stimulate T cells to enter an activated and proliferative state from a resting state. In this invention, it specifically refers to magnetic beads coated with anti-CD3 and anti-CD28 antibodies to mimic the dual-signal activation provided by antigen-presenting cells in vivo. Representative products include nanoscale TransACT magnetic beads and micron-scale Dynabeads magnetic beads.

[0024] 4. "Lentviral vector": refers to a gene delivery tool constructed based on lentiviruses and modified for safety, capable of efficiently introducing and integrating the target gene sequence into the host cell's genome. In this invention, it is used to introduce genes encoding target proteins (such as chimeric antigen receptors) into T cells.

[0025] 5. "Multiple of Infection" or "MOI": This is a parameter that measures the intensity of transduction. It is defined as the average number of infectious viral particles that each cell comes into contact with during the transduction process.

[0026] 6. Interleukin-2 (IL-2): This is a cytokine produced by activated T cells, which plays a crucial role in promoting the clonal proliferation, differentiation, and survival of T cells. In this invention, it is added to the culture medium during the expansion phase to support the continuous growth of T cells.

[0027] 7. "Chimeric antigen receptor" or "CAR": Refers to a synthetic receptor constructed through genetic engineering technology, typically containing an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. It enables T cells to specifically recognize and attack target cells expressing specific antigens. In this invention, it is a preferred protein encoded by the target gene.

[0028] 8. "Shake flask": Refers to a conical or flat-bottomed culture vessel suitable for shaking culture, with sizes ranging from tens of milliliters to several liters. The method of this invention is adapted to shake flasks of different sizes, such as 125 mL, 250 mL, and 3 L.

[0029] 9. "rpm": is a unit of rotation speed, indicating the number of times the shaker rotates per minute.

[0030] 10. “cells / mL”: This is a unit of cell density, representing the number of cells contained in one milliliter of culture medium or cell suspension.

[0031] 11. "Apheresis": refers to a medical technique that uses a blood cell separator to continuously process peripheral blood, selectively collecting one or more blood components (such as platelets, plasma, or leukocytes), while returning the remaining components to the donor. In this invention, it specifically refers to the procedure used to collect blood components rich in leukocytes (including PBMCs).

[0032] 12. "Density gradient centrifugation": refers to a technique that uses the principle that cells settle at different rates in a medium of a specific density to separate and purify them.

[0033] 13. "Ficoll": A commonly used, sterile polysucrose-sodium diatrizoate mixed solution, widely used as a density gradient centrifugation medium for the separation of PBMCs in human and animal peripheral blood.

[0034] 14. "Immunomagnetic bead sorting": refers to the technique of using magnetic microspheres coated with specific antibodies (such as anti-CD3, CD4, CD8 antibodies) to separate or remove specific cell subpopulations from a mixed cell population through the action of a magnetic field.

[0035] 15. "Low density": In this invention, "low density" refers to the seeding cell density in the amplification step (c), which is no higher than 2 × 10⁻⁶ cells / day. 5 cells / mL, preferably 5 × 10⁻⁶ 4 cells / mL to 2×10 5 cells / mL.

[0036] 15. The descriptions of culture time, such as "Day 0", "Day 2", "Day 3", etc., shall be calculated from the time when the initial operation step (such as the resuscitation and activation inoculation of PBMCs) is completed, which is used to clearly define the specific time nodes of each subsequent process step.

[0037] Compared with existing technologies, the in vitro T cell expansion method provided by this invention has the following advantages: 1. A breakthrough in the mechanism of efficient amplification at low inoculation densities has been achieved. Traditional methods face technical bottlenecks at low cell densities, such as insufficient intercellular contact signals, rapid dilution of autocrine / paracrine factors, and local accumulation of metabolic waste leading to microenvironmental imbalance. Therefore, they heavily rely on high initial densities or expensive dedicated systems like G-Rex to ensure amplification. This invention constructs a dynamic physical microenvironment through optimized shake-flask culture. This dynamic environment effectively compensates for the lack of direct, continuous contact signals at low densities by inducing a cycle of "brief contact-separation-re-contact" between cells. Simultaneously, gentle and continuous shaking ensures uniform distribution of nutrients and growth factors (such as IL-2) in the culture system, maintains an effective factor concentration field around cells, and accelerates the diffusion and removal of metabolic waste such as lactic acid and reactive oxygen species, avoiding local acidification and oxidative stress. Combined with a chemically defined culture medium, precise IL-2 concentration, and an efficient magnetic bead activation system, amplification can be achieved at 5 × 10⁻⁶ cells / year. 4 Up to 2×10 5 Even with a low initial seeding density of cells / mL, stable cell expansion of 30 to 120 times can still be achieved, breaking through the efficiency bottleneck of traditional low-density culture.

[0038] 2. Simplified Process Advantages Based on a Stable Microenvironment. Existing amplification processes often require frequent monitoring and adjustment of cell density due to metabolic stress or signal heterogeneity, which is cumbersome and introduces the risk of contamination. The dynamic culture method provided by this invention optimizes oxygen and nutrient delivery and maintains metabolic homeostasis, mechanistically supporting the continuous and stable expansion of cells for 2 to 4 days after low-density seeding without the need for density adjustment or passage. This significantly simplifies the production process, reduces operational complexity and the risk of human error, and is easier to standardize and automate.

[0039] 3. High stability of cell product quality. In existing technologies, static or insufficient mixing can easily lead to heterogeneity in the cell microenvironment, resulting in fluctuations in cell viability, purity, and functional state. This invention, through the aforementioned dynamic culture environment, reduces metabolic damage and ensures cell health. Simultaneously, the CD3 / CD28 antibody-coated magnetic beads used specifically activate T cells, achieving selective proliferation of target cells. Therefore, this method can stably prepare high-quality cell products with a viability of no less than 92% and T cell purity of no less than 96%, with stable gene transduction efficiency, achieving a dual guarantee of amplification efficiency and cell quality.

[0040] 4. Significant cost advantages and excellent economic efficiency. Compared to solutions relying on expensive equipment such as G-Rex, the core process of this invention is based on conventional shake-flask equipment, requiring no special or expensive devices. Simultaneously, the lower initial seeding density reduces the requirement for a large number of precious starting cells, while the defined IL-2 concentration range and magnetic bead usage ratio (20:1 to 200:1 volume ratio of nano-magnetic beads, and 1:3 to 3:1 quantity ratio of micron-magnetic beads) avoids excessive reagent use and waste, optimizing production costs from multiple dimensions and achieving excellent cost-effectiveness while ensuring efficient amplification.

[0041] 5. Wide process adaptability and flexible application scenarios. Existing technology platforms are often limited by specific equipment or culture volume, resulting in insufficient flexibility. Based on an understanding of the hydrodynamic characteristics of shake flasks of different sizes, this invention provides suitable process parameters: for small-scale cultures (e.g., 125-250 mL shake flasks), a relatively high rotation speed of 80-145 rpm is used to ensure mixing efficiency; for large-scale cultures (e.g., 3L shake flasks, 1000 mL working volume), a lower rotation speed of 40-100 rpm is used to avoid shear damage, and the inoculation density is further optimized to 8×10⁻⁶. 4 Up to 1.5×10 5 This method is compatible with various common shake flasks, including flat-bottomed and conical flasks, forming a complete, parameter-defined solution that covers process development and optimization from tens of milliliters to production preparation at the liter scale. This greatly expands its application scenarios and can meet the needs of different stages from basic research to clinical production. Attached Figure Description

[0042] Figure 1 This is a schematic diagram showing the statistical results of cell viability in each group in Example 1.

[0043] Figure 2 This is a schematic diagram showing the statistical results of the total cell expansion fold in each group in Example 1.

[0044] Figure 3This is a schematic diagram showing the statistical results of the CAR positivity rate (transduction rate) of cells in different experimental groups in Example 1 and the calculated theoretical number of CAR positive cells.

[0045] Figure 4 This is a schematic diagram showing the dynamic changes in cell viability from day 3 to day 8 during low-density amplification culture at different oscillation speeds (80 rpm, 90 rpm, 100 rpm, 145 rpm) starting from day 3 in Example 2.

[0046] Figure 5 This is a schematic diagram showing the statistical results of the total cell expansion fold in each group in Example 2.

[0047] Figure 6 This is a schematic diagram showing the statistical results of the CAR positivity rate (transduction rate) of each group of cells in Example 2 and the calculated theoretical number of CAR positive cells.

[0048] Figure 7 This is a schematic diagram showing the statistical results of the total cell expansion fold in each group in Example 3.

[0049] Figure 8 This is a schematic diagram showing the statistical results of the CAR positivity rate (transduction rate) of each group of cells in Example 3 and the theoretical number of CAR positive cells calculated based on the total amplification fold.

[0050] Figure 9 This is a schematic diagram showing the statistical results of T cell purity in each group of cell products in Example 3.

[0051] Figure 10 This is a schematic diagram of the daily cell concentration change curve monitored in Example 3. Detailed Implementation

[0052] To make the technical means, inventive features, objectives and effects of the invention easier to understand, the invention is further illustrated below with reference to specific figures. However, the invention is not limited to the following embodiments.

[0053] It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings of this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0054] Terms such as “comprising” and “including” indicate that, in addition to the components that are directly and explicitly stated in the specification and claims, the technical solution of the present invention does not exclude the presence of other components that are not directly or explicitly stated.

[0055] The method of this invention preferably uses peripheral blood mononuclear cells (PBMCs) as the starting cell population for the activation, transduction, and expansion of T cells. PBMCs are a type of cell isolated from peripheral blood, mainly comprising two categories of immune-related cells: lymphocytes (approximately 70%–90% of the total PBMCs), including T cells, B cells, and NK cells; and monocytes (approximately 10%–30%), which can differentiate into macrophages or dendritic cells in vitro. PBMCs are typically obtained from the circulating blood of a subject through apheresis, and further separated, enriched, and cryopreserved from the apheresis components using density gradient centrifugation (e.g., using Ficoll separation fluid).

[0056] Choosing PBMCs as the starting material offers significant advantages in process simplification and cost control compared to directly using T cells purified by immunomagnetic beads (such as CD3, CD4, or CD8 beads). This strategy avoids the additional reagents, specialized equipment, and consumable costs required for immunomagnetic bead sorting, simplifying the initial preparation process. Therefore, developing an efficient T cell preparation process starting with PBMCs, which have simple subsequent processing, is one of the important pathways to reduce the production costs of adoptive immunotherapy products such as CAR-T cells. This invention provides such an efficient and low-cost amplification method.

[0057] Example 1: Effects of different time points and seeding densities after transduction on T cell expansion This embodiment aims to evaluate the effects of low-density seeding at different time points after lentivirus transduction on T cell proliferation, viability, and transduction efficiency.

[0058] 1. Cell activation: On day 0, frozen peripheral blood mononuclear cells (PBMCs) were revived, washed, and then activated at 3.0 × 10⁻⁶ cells / mL. 6 T-cell activator was seeded at a density of 20 cells / mL in 125 mL shake flasks. Then, T-cell activator was added at a volume ratio of 100:1 (cell suspension to TransACT nanobeads). The shake flasks were placed in a shaking incubator and cultured with shaking at 90 rpm.

[0059] 2. Lentiviral transduction: On day 2 of culture, samples were taken for viable cell counting. Approximately 2.0 × 10⁶ cells were collected. 7 Add fresh culture medium to each live cell, adjusting the cell density to 1.0 × 10⁶ cells. 6The lentivirus volume was maintained at 20 mL, with cells / mL. The required lentivirus volume was calculated based on a multiplicity of infection (MOI) of 0.05 (viral volume = number of viable cells for transduction × MOI / viral titer), and the corresponding amount of lentivirus carrying the CAR gene was added. After adding the virus, the flask was incubated with shaking at 90 rpm.

[0060] 3. Low-density inoculation and amplification: Group 1 (inoculated 24 hours after transduction): 24 hours after the start of viral transduction (i.e., day 3), a portion of cells were harvested and their inoculation density was adjusted to 5.0 × 10⁶ cells / day. 4 cells / mL, 1.0×10 5 cells / mL and 2.0×10 5 Cells / mL were transferred to new 125 mL shake flasks, each with a culture volume of 20 mL, and then cultured by shaking at 90 rpm.

[0061] Group 2 (inoculated 48 hours after transduction): 24 hours after the start of viral transduction (day 3), the density of another portion of cells was adjusted to 5.0 × 10⁻⁶. 5 Cells / mL (as control density), 20 mL volume, and continued culture at 90 rpm. 48 hours after transduction began (day 4), this portion of cells was adjusted to 5.0 × 10⁶ cells / mL. 4 cells / mL, 1.0×10 5 cells / mL, 2.0×10 5 cells / mL and 5.0×10 5 The cells / mL were seeded into 125 mL shake flasks (20 mL / flask) and cultured with shaking at 90 rpm.

[0062] 4. Culture Maintenance and Monitoring: On days 5 and 6, sample and count cells from all culture flasks. If the cell concentration in any group exceeds 2.0 × 10⁻⁶, [further action will be taken]. 6 If the density is found to be 5.0 × 10⁻⁶ cells / mL, immediately adjust it back to 5.0 × 10⁻⁶. 5 Cells / mL (culture volume 20 mL), and continue to culture with shaking at 90 rpm.

[0063] 5. Endpoint Analysis: All cells were cultured until day 7. After sampling, the percentage of CAR-positive cells (CAR+%) was determined by flow cytometry. Cell viability was recorded throughout the process, and the total fold increase was calculated. The total fold increase was the product of the daily cell expansion fold increases from day 2 to day 7. The absolute number of CAR-positive cells was calculated using the following formula: CAR+ cell count = CAR+% × (number of viable cells used for transduction on day 2) × total fold increase.

[0064] 6. Results: Cells diluted and passaged on day 3 or 4 maintained a viability of over 92% under shaking culture conditions (see...). Figure 1 Under shaking culture conditions, the cell concentration was adjusted to 5 × 10⁶ cells / mL on day 3 or 4. 4 2×10 per milliliter 5 Within a range of individual cells, cells can achieve effective expansion (see...). Figure 2 Data showed that the CAR transduction efficiency was higher in the group whose density was adjusted on day 3 than in the group whose density was adjusted on day 4 (see...). Figure 3 ).

[0065] Specifically, 5 × 10 per milliliter 4 2×10 per milliliter 5 The low-density seeding group of cells achieved a 31.5 to 50.9-fold expansion within 7 days, with the viability consistently maintained above 92%. This result breaks through the understanding of traditional static culture or high-density seeding, and confirms that the dynamic oscillation environment can effectively compensate for the loss of intercellular contact signals caused by the increase in intercellular spacing. Furthermore, continuous mixing avoids the local accumulation of metabolic waste (such as lactic acid) and oxidative stress (ROS), thereby maintaining the metabolic homeostasis and high proliferative potential of cells even at low density.

[0066] Example 2: Optimization of Oscillation Speed ​​on Low-Density T Cell Expansion. This example aims to explore the effects of different oscillation speeds on the expansion kinetics and yield of T cells at low seeding densities.

[0067] 1. Cell activation and transduction: The basic steps are the same as in Example 1. The difference is that during activation, the volume ratio of cell suspension to TransACT nanobeads is between 20:1 and 100:1; during transduction, the MOI used is 0.025.

[0068] 2. Rotation grouping and low-density culture: 24 hours after the start of virus transduction (day 3), the cells were uniformly diluted to 1.0 × 10⁶ cells / day. 5The cells / mL culture volume was adjusted to 25 mL and aliquoted into four 250 mL transfer bottles. These four bottles of cells were then cultured with shaking at 80 rpm, 90 rpm, 100 rpm, and 145 rpm, respectively.

[0069] 3. Culture maintenance: Take samples and count cells daily from day 4 to day 7. If the cell density exceeds 2.0 × 10⁻⁶... 6 If the number of cells / mL is 5.0 × 10⁻⁶, then use 5.0 × 10⁻⁶. 5 The cells / mL density was used for passage at a volume of 25 mL, and the cells were cultured at the original rotation speed.

[0070] 4. Endpoint Analysis: Cell counting, viability testing, and flow cytometry CAR positivity rate analysis were performed at the end of day 8. The total amplification fold and the total number of CAR-positive cells from day 3 to day 8 were calculated (calculation method is the same as in Example 1).

[0071] 5. Results: Within the rotation speed range of 80 rpm to 145 rpm, the higher the rotation speed in the initial stage of culture (days 3-4), the higher the cell viability; by day 6, the cell viability in all groups could recover to over 95% (see...). Figure 4 In a culture system of 20-30 mL, an oscillation speed of 80-145 rpm is recommended; higher speeds are more conducive to cell proliferation (see [link to culture system]). Figure 5 Different rotation speeds in this culture system did not significantly affect the final CAR transduction efficiency (see...). Figure 6 Specific data showed that the amplification fold in the 145 rpm group reached 113.9-fold, and the number of CAR-positive cells was 1.08 × 10⁻⁶. 7 The number of amplifications was significantly higher than the 68.9-fold increase and 6.26 × 10⁻⁶ increase in the 80 rpm group. 6 Number of CAR-positive cells.

[0072] Specifically, within the 80-145 rpm range, higher oscillation speeds resulted in higher cell viability and a greater overall amplification fold. The 145 rpm group achieved a 113.9-fold amplification and 1.08 × 10⁻⁶ cells / mL. 7 The CAR+ cell group showed significantly better performance than the 80 rpm group. This experimentally verifies that increasing the rotation speed enhances the mixing efficiency of the culture system, ensuring that low-density cells can uniformly and adequately acquire oxygen and nutrients, and promoting the timely diffusion and removal of metabolic products. This improved mass transfer condition directly enhances cellular energy metabolism and biosynthesis, resulting in higher viability and stronger proliferation capacity.

[0073] Meanwhile, the results indicate that the rotational speed in the range of 80-145 rpm has no significant effect on the final CAR transduction efficiency (see...).Figure 6 This indicates that transduction efficiency mainly depends on the biological processes of virus-cell interaction (such as receptor binding, memory, and integration), and physical oscillation within this range does not negatively interfere with this process, further demonstrating the mildness and compatibility of the oscillation culture method.

[0074] Example 3: Validation of low-density amplification process under large-scale culture conditions This embodiment aims to verify the feasibility and cell quality of large-scale T cell expansion using the method described in this invention in 3L shake flasks.

[0075] 1. Cell activation: On day 0, PBMCs were resuscitated at a concentration of 2.5 × 10⁻⁶. 6 Initially seeded at a density of cells / mL, cells were cultured in 3L flat-bottomed shake flasks or 3L conical shake flasks, with a culture volume of 200 mL. T-cell activator was added at a 1:1 ratio of cell number to Dynabeads microbeads. The cells were then cultured with shaking at 40-60 rpm for activation.

[0076] 2. Lentiviral transduction: On day 2 of culture, mix the cells and count them. Based on the total number of viable cells obtained, calculate the MOI (Mean Intake) at 0.5 and add lentivirus. After adding the virus, continue culturing at 40-60 rpm.

[0077] 3. Large-scale low-density amplification: 24 hours after the start of viral transduction (day 3), fresh culture medium was added to adjust the cell concentration to 8.0 × 10⁻⁶. 4 cells / mL up to 1.5 × 10⁻⁶ 5 Set the cell / mL ratio between 1000 mL and bring the total culture volume to 1000 mL. Then, adjust the shaking speed to 50-100 rpm.

[0078] 4. Culture Maintenance: Monitor cell concentration daily from day 4 to day 7. If the cell density exceeds 3.0 × 10⁻⁶... 6 If the number of cells / mL is 5.0 × 10⁻⁶, then adjust it to 5.0 5 After reaching a density of cells / mL (volume 1000 mL), continue oscillating amplification.

[0079] 5. Endpoint analysis: The culture was completed on day 8 with shaking. Samples were taken for cell counting, viability determination, flow cytometry analysis of CAR positivity rate, and T cell purity.

[0080] 6. Results: Under large-scale culture conditions, the cell density was adjusted to 8 × 10⁸ cells / mL using different shaking speeds and different types of shake flasks. 4 1.5 × 10⁻⁶ per milliliter 5The density of individual cells is such that all cells can be effectively expanded, and a high purity of T cells can be obtained (see...). Figure 7 , Figure 8 , Figure 9 However, the hydrodynamics generated by oscillation differs in different shaking culture systems, resulting in varying degrees of mixing of nutrients and oxygen, and also different shear forces on T cells. In this experiment, the theoretical number of CAR+ cells obtained under 60 rpm culture conditions in conical shake flasks was higher than that under 50 rpm conditions, and the theoretical number of CAR+ cells obtained under 80 rpm expansion conditions in flat-bottomed high-efficiency shake flasks was higher than that under other culture conditions. In large-scale culture systems, the cell density was adjusted to the above range on day 3 for shaking culture, and by day 7, the cell concentration reached 1.0 × 10⁶ cells / mL. 6 1.8 × 10⁻⁶ per milliliter 6 Cells, reaching 2.0 × 10⁶ per ml by day 8. 6 3.0 × 10⁶ per milliliter 6 Cells (see) Figure 10 This lays the foundation for diluting cells to a low concentration and culturing them with shaking on Day 3 in actual production, without any further treatment for 2-4 days. Specific data analysis shows that 8 × 10⁸ cells per milliliter in a 3L shake flask... 4 1.5 × 10⁻⁶ per milliliter 5 Cell density groups were cultured until day 8, at which point the cell concentration reached 2 × 10⁶ cells / mL. 6 3×10 per milliliter 6 Each cell type has a T cell purity of no less than 96.8%.

[0081] Specifically, cells were cultured in 3L shake flasks at a rate of 8.0 × 10⁻⁶. 4 Up to 1.5×10 5 By seeding at a density of cells / mL and optimizing the rotation speed, the cells successfully expanded. The density reached 2.0-3.0 × 10⁶ cells / mL by day 8. 6 cells / mL (see cells / mL) Figure 10 This result demonstrates that by adapting to the hydrodynamic characteristics of large-scale containers (such as reducing rotational speed to decrease shear force), the core principle of the invention, "low-density oscillatory amplification," is equally effective on different scales, achieving a seamless transition from process principle to production practice.

[0082] Under large-scale culture conditions, the obtained T cell products had a purity of no less than 96.8% and maintained a high viability. This demonstrates that the activation method selectively stimulates T cells, rather than other cells in PBMCs (such as B cells and monocytes), thereby enriching the target T cells during expansion and ensuring the high purity of the final product.

[0083] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for low-density expansion of T cells, characterized in that, Includes the following steps: (a) Activation: A cell population containing T cells is contacted with a T cell activator and cultured to obtain activated T cells; (b) Transduction: The activated T cells obtained in step (a) are co-cultured with a lentiviral vector to introduce the target gene carried by the lentiviral vector into the T cells to obtain transduced T cells; (c) Amplification: The transduced T cells obtained in step (b) are amplified at a rate not exceeding 2 × 10⁻⁶. 5 T cells were inoculated at a density of cells / mL in shake flasks and cultured with shaking to achieve T cell expansion.

2. The method according to claim 1, characterized in that, In step (c), the inoculation density is 5 × 10⁻⁶. 4 cells / mL to 2×10 5 The amplification step (c) is performed at cells / mL for 4 to 5 days.

3. The method according to claim 1 or 2, characterized in that, In step (a), the cell population containing T cells is peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are divided into groups of 2 × 10⁻⁶ cells. 6 cells / mL to 4×10 6 The initial density of cells / mL was mixed with the T cell activator.

4. The method according to claim 1, characterized in that, In step (a), the T cell activator is a magnetic bead coated with anti-CD3 antibody and anti-CD28 antibody; the magnetic bead is a nano magnetic bead or a micro magnetic bead, wherein, when the nano magnetic bead is used, the ratio of the culture volume containing the T cell to the added volume of the nano magnetic bead is 20:1 to 100:1; when the micro magnetic bead is used, the ratio of the number of T cells to the number of micro magnetic beads added is 1:1 to 1:

3.

5. The method according to claim 1, characterized in that, Step (b) is performed on the second day after the start of cultivation in step (a), and 5 × 10⁻⁶ ppm is taken. 6 Up to 2×10 7 The activated T cells perform the transduction.

6. The method according to claim 1 or 5, characterized in that, In step (b), the lentiviral vector is used at a dose with a multiplicity of infection of 0.025 to 0.

5.

7. The method according to claim 1, 5, or 6, characterized in that, During step (b) transduction, the density of activated T cells is 1 × 10⁻⁶. 6 cells / mL.

8. The method according to claim 1, characterized in that, Step (c) is performed on day 3 or day 4 after co-culturing begins in step (b).

9. The method according to claim 1, characterized in that, In step (c), the rotation speed of the shaking culture is 40 rpm to 145 rpm; and when the culture volume of the shaking flask is not greater than 250 mL, the rotation speed is 80 rpm to 145 rpm, and when the culture volume is not less than 1000 mL, the rotation speed is 40 rpm to 100 rpm.

10. The method according to claim 1, characterized in that, In step (c), the shaking culture is performed using a cell culture medium with added interleukin-2.

11. The method according to claim 10, characterized in that, The concentration of interleukin-2 in the culture medium is from 100 IU / mL to 1000 IU / mL.

12. The method according to claim 1, characterized in that, The shake flask is a 125 mL shake flask, a 250 mL shake flask, or a 3 L shake flask; wherein, when a 3 L shake flask is used and the culture volume is 1000 mL, the inoculation density in step (c) is 8 × 10⁻⁶. 4 cells / mL up to 1.5 × 10⁻⁶ 5 cells / mL.

13. The method according to claim 1, characterized in that, The target gene encodes a chimeric antigen receptor.

14. A T cell product, characterized in that, It is prepared by any one of claims 1 to 13.

15. The T cell product according to claim 14, characterized in that, The viability of the T cell products is not less than 92%, and the purity of the T cells is not less than 96%.

Images