Universal car-t feeder cells and methods of making and using same

Abstract

The application provides a preparation method of a general CAR-T feeder cell, and is used for preparing a feeder cell of a CAR-T cell aiming at a target antigen, and has the characteristics that the method comprises the following steps: an erythroblast obtaining step, single nuclear cells are separated from donor blood, and are induced and differentiated to obtain erythroblasts; a vector obtaining step, a lentivirus vector at least expressing a target antigen is constructed, and a recombinant virus supernatant of the lentivirus vector is prepared; a transfection step, the recombinant virus supernatant is used for transfecting the erythroblasts; an amplification step, the transfected erythroblasts are amplified to obtain amplified erythroblasts; and an irradiation treatment step, the amplified erythroblasts are subjected to irradiation treatment to obtain the feeder cell. The feeder cell prepared by the method can promote antigen-dependent specific activation and amplification of the CAR-T cell aiming at the target antigen, can reduce the preparation difficulty and cost of the feeder cell, and can realize the generalization of the feeder cell.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a universal CAR-T feeder cell, its preparation method, and its application in the preparation of CAR-T cells. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has become a breakthrough treatment for relapsed / refractory B-cell malignancies (B-cell acute lymphoblastic leukemia, lymphoma), among which CD19-targeted CAR-T cell therapy has shown significant efficacy in acute lymphoblastic leukemia.

[0003] Clinical practice has shown that antigen-dependent activation of CAR-T cells in vivo is key to maintaining their long-term efficacy. However, tumor antigen loss or insufficient antigen load in vivo can prevent CAR-T cells from maintaining a sustained anti-tumor effect, thus limiting their long-term efficacy. Furthermore, the in vitro culture of CAR-T cells often requires activation and expansion using CD3 / CD28 magnetic beads. Since the latter lacks selectivity for both positive and negative CAR-T cells and needs to be removed after successful CAR-T cell preparation, it increases the complexity of the cell preparation process, making automated and large-scale preparation difficult to achieve.

[0004] To enhance the expansion and persistence of CAR-T cells, some existing technologies employ methods that provide continuous antigen stimulation by infusing "feeder cells" expressing the CD19 antigen. For example, Chen et al. (CHEN LY, GONG WJ, LI MH, et al. Anti-CD19 CAR T-cell consolidation therapy combined with CD19+ feeding Tcells and TKI for ph+ acute lymphoblastic leukemia[J / OL]. Blood Advances, 2023, 7(17): 4913-4925.) used autologous CD19⁺-fed T cells combined with CAR-T cell infusion. Annesley et al. (ANNESLEY C, SEIDEL K, WU Q, et al. Outcomes of PLAT-02 and PLAT-03: Evaluating CD19 CAR T-cell therapy and CD19-expressing T-APC support in pediatric B-ALL[J / OL]. Blood, 2025, 146(7): 789-801.) uses autologous truncated CD19 modified T cells as artificial antigen-presenting cells (CD19t-APC). Both of these existing technologies can improve the in vivo survival of CAR-T cells to some extent.

[0005] However, the feeder cells used in the aforementioned existing technologies are all autologous T cells, which need to be prepared through processes such as collecting peripheral blood from the patient, isolating T cells, lentiviral transduction, and in vitro expansion. This not only has drawbacks such as long preparation cycles, complex processes, high quality control requirements, and extremely high costs, but also suffers from the problem that T cells used as feeder cells have weak stimulation, activation, and expansion capabilities for CAR-T cells. Furthermore, even feeder cells derived from the patient's own T cells still carry the risk of non-specific viral insertion and long-term tumorigenesis due to the need for gene editing or modification.

[0006] Furthermore, because T cells are MHC-restricted, they cannot be used interchangeably between different patients. Therefore, it is impossible to prepare them in advance as "off-the-shelf" products for use by multiple patients, which further increases treatment costs and time costs, and limits clinical promotion.

[0007] Therefore, there is an urgent need to develop a universal feeder cell that is stable in origin, easy to prepare, and can be mass-produced to simplify the treatment process and improve its availability. Summary of the Invention

[0008] To address the aforementioned issues, the inventors of this invention, through research and analysis, discovered that human red blood cells possess advantages such as abundant source, lack of nucleus, no cross-reactivity between individuals with the same blood type, and the ability to be cultured on a large scale. Furthermore, they can express CD19 through lentiviral transfection, thus possessing the potential to become the most ideal feeder cells for the in vitro and in vivo application of CAR-T cells.

[0009] Specifically, the inventors first established a culture system for the directed induction of erythroblasts from umbilical cord blood mononuclear cells (UBMCs). Through co-transfection of CD19 antigen and the BMI1 gene, they simultaneously achieved large-scale expansion of erythroblasts and stable expression of membrane-anchored CD19 antigen, ultimately obtaining a chimeric, universal CAR-T feeder cell (CD19Ag-Ery) derived from erythrocytes that expresses CD19 antigen. They also demonstrated that this feeder cell can promote antigen-dependent, specific activation and expansion of CD19 CAR-T cells. Furthermore, to meet clinical safety requirements, the inventors attempted to eliminate the proliferative potential of CD19Ag-Ery through gamma-ray irradiation treatment to avoid the risk of ectopic proliferation in vivo. They found that other properties of irradiated CD19Ag-Ery were not significantly different from those of unirradiated CD19Ag-Ery, thus improving safety.

[0010] Based on the above findings, this invention provides a method for preparing universal CAR-T feeder cells, used to prepare feeder cells for CAR-T cells targeting a target antigen. The method is characterized by comprising: an erythroblast acquisition step, in which mononuclear cells are isolated from donor blood and induced to differentiate into erythroblasts; a vector acquisition step, in which a lentiviral vector expressing at least the target antigen is constructed, and a recombinant viral supernatant of the lentiviral vector is prepared; a transfection step, in which the erythroblasts are transfected using the recombinant viral supernatant; an amplification step, in which the transfected erythroblasts are amplified to obtain amplified erythroblasts; and an irradiation treatment, in which the amplified erythroblasts are irradiated to obtain feeder cells.

[0011] Preferably, in the method for preparing universal CAR-T feeder cells provided by the present invention, the lentiviral vector also co-expresses the BMI1 gene.

[0012] Preferably, in the method for preparing universal CAR-T feeder cells provided by the present invention, the target antigen is any one or a combination of several of CD19, CD20, CD22, CD79b, CD33, CD123, CLL1, CD7, CD5, CD2, CD1a, CD99, CD9, BCMA, GPRC5D, B7H3, GD2, CD171, MSLN, GPC2, GPC3, and ROR1.

[0013] Preferably, in the method for preparing universal CAR-T feeder cells provided by the present invention, the irradiation treatment is performed by gamma ray irradiation, the irradiation dose is 5~100Gy, and the treatment time is 10min~4h.

[0014] Preferably, in the method for preparing universal CAR-T feeder cells provided by the present invention, the donor blood is umbilical cord blood or peripheral blood.

[0015] As another embodiment, the present invention also provides a universal CAR-T feeder cell, characterized in that it is prepared by the preparation method of any one of the claims above.

[0016] As another embodiment, the present invention also provides a method for preparing CAR-T cells, which is used to prepare CAR-T cells targeting a target antigen, characterized in that the CAR-T cells are co-cultured with the above-mentioned universal CAR-T feeder cells.

[0017] Preferably, in the CAR-T cell preparation method provided by the present invention, the co-culture ratio of universal CAR-T feeder cells to CAR-T cells is 8:1 to 1:8.

[0018] Preferably, in the method for preparing universal CAR-T feeder cells provided by the present invention, the donor blood is provided by a donor with blood type O or Rh negative, or by a donor with the same blood type as the patient.

[0019] The universal CAR-T feeder cells and their preparation method provided by this invention, as well as the method for preparing CAR-T cells targeting a target antigen based on these universal CAR-T feeder cells, utilize the method of transfecting and amplifying erythroblasts induced by lentiviral vectors expressing the target antigen. This allows for the acquisition of chimeric CAR-T feeder cells derived from erythrocytes that express the target antigen. These feeder cells can promote antigen-dependent specific activation and amplification of CAR-T cells targeting the target antigen. Since erythrocytes are abundant, enucleated, and do not exhibit cross-reactivity among individuals with the same blood type, and because erythrocytes are easier to culture on a large scale compared to other cell types, enucleation not only reduces the difficulty and cost of feeder cell preparation but also achieves universality, enabling the preparation of feeder cells as "off-the-shelf" products for use by multiple patients.

[0020] Furthermore, since the feeder cell preparation method of the present invention also involves irradiation treatment of the transfected and expanded erythroblasts, their proliferative potential is eliminated, thus avoiding the risk of ectopic proliferation in vivo.

[0021] Therefore, this invention forms an integrated technical solution of "specific activation - large-scale preparation - safety optimization", providing a "partner" tool for the efficient in vitro and in vivo application of CAR-T cells, and helping CAR-T therapy achieve clinical translation upgrades and efficacy breakthroughs. This solution has the following advantages:

[0022] (1) By overexpressing CD19 on the surface of red blood cell (RBC) membranes, the corresponding RBCs are obtained by transdifferentiation of mononuclear cells from healthy donors of type O or Rh negative or umbilical cord blood, and by overexpressing BMI-1, large quantities of CD19-expressing RBCs can be obtained in vitro.

[0023] (2) The above RBCs can be used in the human body to simulate CD19 positive leukemia cells to activate the expansion of CD19-CART cells in the patient's body, thereby prolonging the duration and peak expression level of CAR-T cells in the patient's body, which can greatly improve the efficacy of CD19-CART, and of course can also improve the clinical efficacy of dual-target and triple-target CAR-T cells that simultaneously target CD19 and other targets.

[0024] (3) The above-mentioned RBCs can also be used for in vitro culture of CD19 positive CAR-T cells, thereby replacing CD3 / CD28 magnetic beads, greatly reducing the cost of CAR-T preparation, and simplifying the preparation process of CAR-T cells.

[0025] (4) In addition to being used on CD19-positive CAR-T cells, the present invention can also overexpress other targets on the surface of RBCs to replicate the above-mentioned effects of promoting CAR-T cells in vitro and in vivo. Attached Figure Description

[0026] Figure 1 This is a graph showing the RNA expression level of the BMI1 gene in bone marrow-derived blood immune cells and hematopoietic stem cells.

[0027] Figure 2 This is a schematic diagram of the lentivirus vector in Embodiment 1 of the present invention.

[0028] Figure 3 This is a graph showing the change (%) in the expression levels of erythroid cell surface markers CD71 and CD235 in Test Example 1 of the present invention.

[0029] Figure 4 It is the erythroblasts (CD71) cultured during the test example 1 of this invention. + Amplification factor plot of ).

[0030] Figure 5 This is a graph showing the changes (%) in the expression levels of erythroid cell surface markers CD71, CD235, and CD36 on days 16 and 23 during the culture process of Test Example 1 of this invention.

[0031] Figure 6 These are pro-erythrocytes (CD71) cultured on days 16 and 23. + CD235 - ) and erythroblasts (Ery, CD71) + CD235 + The percentage of cells in the graph is %.

[0032] Figure 7 These are photographs of the morphology of erythrocytes (observed under a confocal microscope) on days 16 and 23 of Test Example 1 of this invention.

[0033] Figure 8 This is a graph showing the expression levels (%) of 19Ag and BMI1 in erythroblasts on days 2 and 11 after transfection in Test Example 2 of this invention.

[0034] Figure 9 This is a graph showing the results of flow cytometry analysis of the expression levels (%) of CD19Ag-Ery erythroid and T-cell markers 12 days after transfection in Test Example 2 of this invention.

[0035] Figure 10 This is a cell morphology photograph of 19Ag feeder cells on day 10 after transfection in Test Example 2 of this invention.

[0036] Figure 11 This is a proliferation curve of CD19Ag-Ery after transfection in Test Example 2 of this invention.

[0037] Figure 12 This is a graph showing the changes (%) of the proportion of CAR-positive cells on day 0 and day 2 of co-culture with different proportions of CD19Ag-Ery in Test Example 3 of this invention and the Beads control group.

[0038] Figure 13 This is a bar chart showing the absolute number (n=3) of CD19-CAR-T cells in each group on day 6 of co-culture in Test Example 3 of this invention.

[0039] Figure 14 This is a graph showing the changes (%) of CAR positivity rates in each group on day 0 and day 2 after co-culture under different feeding systems in Test Example 3 of this invention.

[0040] Figure 15 This is a line graph showing the fold increase of CAR-T cells in different groups at different time points under different feeding systems in Test Example 3 of this invention (based on D0, n=3).

[0041] Figure 16This is a graph showing the change (%) of the proportion of 19Ag feeder cells on day 0 and day 4 in the two co-culture systems of CD19Ag-Ery+CD19CAR and CD19Ag-Ery+CD22CAR in Test Example 3 of the present invention.

[0042] Figure 17 This is a graph showing the absolute change in the number of 19Ag feeder cells on days 0, 2, and 4 in the two co-culture systems of CD19Ag-Ery+CD22CAR and CD19Ag-Ery+CD19CAR in Test Example 3 of the present invention.

[0043] Figure 18 This is a graph showing the expression rates (%) of CD19, CD71, and CD235a of CD19Ag-Ery before and after irradiation in Test Example 4 of this invention.

[0044] Figure 19 This is a graph showing the changes in the absolute number and survival rate (%, n=3) of Irra-19Ag-Ery after 3 days of in vitro culture in Test Example 4 of this invention.

[0045] Figure 20 The change in CAR positivity rate (%, n=3) on day 0 and day 2 of co-culture under different feeder cell systems.

[0046] Figure 21 This is a graph showing the IFN-γ level in the supernatant after co-culturing for 2 days in different feeder cell systems.

[0047] Figure 22 These are the expansion curves of CAR-T cells under different feeder cell systems.

[0048] Figure 23 shows the expansion curves of CAR-T cells under different feeder cell systems. Detailed Implementation

[0049] The specific embodiments of the present invention are described below with reference to the accompanying drawings and examples. In the following examples, reagents and materials not specified in the description were purchased through conventional commercial channels, and experimental procedures and conditions not specified in the description were performed in accordance with conventional techniques in the art, or by adopting the same experimental procedures and conditions as in other embodiments or test examples in this application specification.

[0050] Example 1

[0051] This embodiment provides a method for preparing feeder cells for CAR-T cells targeting a target antigen, specifically including the following steps:

[0052] Step 1: Erythroblast Acquisition Step. Unnuclear mononuclear cells (UBMCs) are isolated from donor blood and induced to differentiate into erythroblasts. The specific process is as follows:

[0053] Select healthy donors and collect umbilical cord blood. Based on the volume of umbilical cord blood obtained, prepare PBS in a clean 50ml centrifuge tube at a 1:1 volume ratio. Use a 50ml sterile syringe to draw up the umbilical cord blood and dilute it thoroughly in the PBS. Based on the diluted volume, prepare Ficoll solution in 15ml centrifuge tubes (5ml per tube) at a 1:1 volume ratio. Use a sterile pipette to slowly add the umbilical cord blood dropwise to the Ficoll solution, then centrifuge at a density gradient of 1400 rpm for 30 minutes with a constant acceleration / deceleration rate. Simultaneously, prepare 15ml centrifuge tubes at a 2:1 volume ratio and add 6ml of PBS to each. After centrifugation, use a sterile pipette to remove as much of the white membrane layer as possible and transfer the cells to 15ml centrifuge tubes to form a cell suspension. Mix well by shaking, then mix 20ml of the cell suspension with 20ml of AOPI dye and count the cells using a Count Star counter. Centrifuge the required amount of cells at 2000 rpm for 8 minutes with a speed of 9 for both vertical and horizontal acceleration. Freeze the remaining cells in liquid nitrogen.

[0054] After centrifugation, discard the supernatant as thoroughly as possible to obtain a cell pellet. Resuspend the cell pellet in serum-free SFEM medium supplemented with erythropoietin (EPO, 1-4 U / mL), human stem cell factor (SCF, 20-100 ng / mL), dexamethasone (DEX, 0.5-2 μmol / L), and insulin-like growth factor (IGF1, 20-80 ng / mL). Adjust the cell density to 2-6 mg / mL. 6 / ml, for in vitro culture to induce differentiation.

[0055] During in vitro culture and induced differentiation, cell morphology is observed regularly under a microscope, and samples are taken to assess cell density and cell viability using the cell activity dye AOPI. Fresh culture medium is added according to the cell density. The ideal cell density can usually be reached in about 5-10 days, which is to obtain the induced differentiated erythroblast population.

[0056] Step 2: Vector acquisition step, construct a lentiviral vector co-expressing the coding regions of CD19 antigen and BMI1 gene, and prepare the recombinant viral supernatant of the lentiviral vector.

[0057] Figure 1 This is a graph showing the RNA expression level of the BMI1 gene in bone marrow-derived blood immune cells and hematopoietic stem cells. Figure 2 This is a schematic diagram of the lentivirus vector in Embodiment 1 of the present invention.

[0058] like Figure 1As shown, when the inventors of this application analyzed the transcript expression profile of the BMI1 gene using The Human Protein Altas database (HPA), they found that the RNA expression levels in hematopoietic stem cells and erythrocyte progenitors were 23.8 nCPM and 28.3 nCPM, respectively, while in erythrocytes it was 5.3 nCPM. That is, the expression level of the BMI1 gene in hematopoietic stem cells and erythrocyte progenitors was significantly higher than that in mature erythrocytes. Furthermore, previous studies have shown that the BMI1 gene is expressed in CD34... + HSPCs play an important role in the differentiation of erythroid cells and can promote the proliferation of proerythrocytes and maintain their immature state.

[0059] To this end, the applicant designed a lentiviral vector co-expressing the CD19 antigen (19Ag) and the coding region of the BMI1 gene (CDS) for the modification of UBMC-derived erythroblasts. The structure of the lentiviral vector is as follows: Figure 2 As shown.

[0060] The specific process for constructing the above lentiviral vector and preparing recombinant virus supernatant is as follows:

[0061] (1) Search the database of the National Center of Biotechnology Information (NCBI) for the cDNA sequences of BMI1, CD19 antigen and P2A, and screen for suitable vector backbone sequences; complete molecular cloning design and sequence assembly using Snap Gene software, and design forward and reverse primers based on the restriction sites on the vector, and then entrust a commercial company to synthesize primers and target fragments.

[0062] (2) Centrifuge the synthesized primers at 12000 rpm for 2 minutes, add sterile water to dilute to 1x, vortex to mix, and store at 4℃; centrifuge the synthesized target fragment plasmid (4 μg) at 12000 rpm for 2 minutes, dissolve the plasmid dry powder in 40 μl of water (centrifuge to precipitate) to obtain a working solution of 100 ng / μl.

[0063] (3) Take 100 μL of working solution and perform double enzyme digestion with Xho I and Spe I. The reaction system is 50 μL. Digest the enzymes at 37°C for 2 h on a PCR instrument. The reaction system is as follows:

[0064]

[0065] (4) Weigh 0.35g of agarose powder and 30ml of 1×TAE using an electronic balance to prepare a 1% agarose gel. After solidification at room temperature for 30min, transfer the gel to an electrophoresis tank containing 1×TAE. After enzyme digestion, take out 50ul of the digestion product and add 5ul of 10×loading. Add all of the digestion product to the agarose gel wells. Add 5ul of 5000bp marker to another well. Perform DNA gel electrophoresis at 135V, 400mA for 30min. Use a gel imaging instrument to observe the position of the DNA marker. Cut the gel at the corresponding position of the band, weigh the gel, and then use a large volume agarose gel DNA recovery kit to recover the gel. Finally, use a Nanodrop instrument to measure the concentration of the target fragment recovered from the gel.

[0066] (5) Ligate the target fragment to the PALD vector using T4 ligase at room temperature for more than 2 hours. The reaction system is 10 μL, and the molar ratio of the target fragment to the vector is greater than 10. The reaction system is as follows:

[0067]

[0068] (6) Take 50 μL of competent DH5α cells into a 1.5 mL EP tube, add 10 μL of T4 ligation product, gently tap to mix, and incubate on ice for 30 min. Then place the 1.5 mL EP tube in a 42°C water bath for 90 s, and immediately incubate on ice for 2 min. Finally, in a biosafety cabinet, add 200 μL of LB without cannabinoids using a 1 mL pipette. Shake to mix, place the EP tube on a float plate, and incubate on a shaker at 37°C and 220 rpm for 50 min.

[0069] (7) Use a 1ml pipette to drop the transformed competent cells onto an LB culture dish containing cannabinoids, spread them evenly with an inoculation loop, and incubate upside down in a 37°C incubator for 12-16 hours.

[0070] (8) After the culture is completed, observe and select 5 single colonies. Pick the single colony with a 10 μL pipette and transfer it to a 500 μL EP tube containing LB resistant to kanamycin. Shake the colony in a shaker at 37°C for 4-5 h and then send it to Sanger for sequencing. Compare the sequencing results of the target sequence with those on SnapGene.

[0071] (9) Select the bacterial culture with correct sequencing and expand the culture volume to 5 ml. Shake the culture in a shaker at 37°C for 12-16 h.

[0072] (10) Take 500ul of bacterial culture into an EP tube, add 200ul of 16% glycerol, shake and store in a -80°C freezer. Then use a plasmid miniprep kit to extract the remaining plasmid and measure the plasmid concentration with Nanodrop. Store at -20°C for subsequent lentivirus packaging.

[0073] (11) Culture 293T cells, observe the growth of 293T cells under a microscope, ensure that the growth density reaches about 70%, remove 6ml of old culture medium with a pipette, add 4ml of fresh DMEM + 10% FBS culture medium, and put it back into the incubator for storage.

[0074] (12) Count the required volume according to the molar ratio of vsvg: gag: rev: target plasmid = 1: 1: 1: 1. Prepare solution 1: Add 200ul serum-free OPTI-MEN medium and 20ul Fugene6 (Promega) per 10cm dish; Prepare solution 2: Add 50ul serum-free OPTI-MEN medium and the required volume of each plasmid per 10cm dish, shake to mix, and let stand for 5min. Then transfer solution 1 to solution 2, mix, and let stand for 15min; Take out the prepared 293T cells, add the solution to the 293T cells with a 1ml pipette, put them back into the incubator to continue culturing, and add 4ml of fresh DMEM + 10% FBS after 4-6h.

[0075] (13) After culturing for 72 h, the growth status of 293T cells was observed under a microscope. Subsequently, virus concentration was performed in a biosafety cabinet: a concentration filter column and a 0.45 μm filter membrane were prepared in sequence, and the filter membrane was installed on the filter column; the supernatant of 293T cells was slowly aspirated using a 20 ml sterile syringe, filtered through the filter membrane, and transferred to the concentration filter column. The column was centrifuged at 4 ℃ and 4000 rpm for 30 min (speed setting 9); after centrifugation, the concentrated virus solution in the filter column was transferred to a sterile EP tube to obtain the recombinant virus supernatant of the lentiviral vector co-expressing the coding regions of CD19 antigen and BMI1 gene. The supernatant was stored in an ultra-low temperature freezer at -80 ℃ for later use.

[0076] Step 3: Transfection step. The recombinant virus supernatant obtained in Step 2 is used to transfect the erythroblasts obtained in Step 1. The specific process is as follows:

[0077] (1) Observe the growth status of erythroblasts after 6 days of induced differentiation culture under a microscope, and then perform virus transfection in a biosafety cabinet. Take 20 μL of cell suspension and 20 μL of AOPI dye and gently pipette to mix. Use a CountStar cell counter to count the cells and calculate the amount of cells required for transfection based on the counting results.

[0078] (2) Transfer the required cell suspension to a 15ml centrifuge tube, add PBS to 15ml, centrifuge at 1400rpm for 8min. After centrifugation, use a 1ml pipette to remove as much supernatant as possible, resuspend the cell pellet in SFEM medium, transfer it to a T25 cell culture flask, add transfection reagent at a ratio of 4ul / ml, and incubate at 37°C for 30min.

[0079] (3) Calculate the required amount of virus based on the number of cells, melt the required virus solution in a 37°C metal bath in advance, add the virus solution with a 200ul pipette for transfection, and add fresh SFEM medium after incubation for 4-6 hours.

[0080] (4) During the transfection period, observe the growth status of erythroblasts regularly with a microscope and supplement SFEM medium at an appropriate growth density.

[0081] Step 4. Amplification step: Amplify the transfected erythrocytes obtained in Step 3 until the target number is reached, obtaining amplified erythrocytes. The specific process is as follows:

[0082] (1) The transfected erythrocytes obtained in step 3 were amplified in vitro using SFEM medium. During the process, the cell density and survival rate were detected using AOPI live cell dye.

[0083] (2) Replenish fresh culture medium to the appropriate culture density in a timely manner. After the target number of cells is reached, collect the cells and irradiate them to obtain amplified red blood cells.

[0084] Step 5. Irradiation treatment: The expanded red blood cells obtained in Step 4 are subjected to irradiation treatment to obtain feeder cells. The specific process is as follows:

[0085] (1) Use a 50 ml sterile syringe to transfer the cell suspension obtained in step 4 to a 250 mL sterile blood bag, seal it, and then irradiate it with γ-rays at a dose of 25 Gy for 30 min.

[0086] (2) After irradiation, the cell suspension was transferred to a 50ml centrifuge tube using a 50ml sterile syringe and centrifuged at 2000rpm for 8min. After centrifugation, the supernatant was discarded, the cells were resuspended in PBS, and the total cell mass after irradiation was calculated using AOPI dye and a Count Star calculator. The appropriate cryopreservation density was calculated, and PBS was added for repeated washing and centrifugation.

[0087] (3) After centrifugation, discard the supernatant and gently resuspend the cell pellet with an appropriate amount of CS10 cryopreservation solution. Collect the cell pellet into the same centrifuge tube and rinse the tube wall again with CS10 cryopreservation solution to remove any remaining cells. Finally, transfer the cells to a cryopreservation tube using a pipette to obtain feeder cells. Store the feeder cells in liquid nitrogen for later use.

[0088] Test Example 1

[0089] This test case is a detection and analysis experiment in step 1 of Example 1 during the erythroblast induction and differentiation process.

[0090] 1. Flow cytometry was used to detect and analyze the phenotype of erythroid cells.

[0091] During the in vitro culture and differentiation induction period in step 1, starting from day 0, the expression of erythroid cell surface markers was detected by flow cytometry every 4 days, as follows:

[0092] (1) Take 100ul of culture sample into an EP tube, add 1ml of PBS, centrifuge at 500rcf for 2min; after centrifugation, discard the supernatant to 100ul, add 1ul of CD33 (FITC), CD235a (PE), CD34 (PerCP), CD117 (PEcy7), CD71 (APC), CD19 (APCH7), CD3 (V450) and CD45 (V500) flow cytometry antibodies respectively, vortex to mix, and incubate in the dark for 15min.

[0093] (2) After incubation, add 1 ml of PBS, centrifuge at 500 rcf for 2 min, discard the supernatant as much as possible, resuspend the cells in 200 μl of PBS and transfer them to flow cytometry tubes, and use flow cytometry to detect and analyze the positive expression rate of each erythroid cell surface marker.

[0094] (3) On the day the target quantity is reached, take another 100ul sample for flow cytometry detection. The detection antibody panel is CD36 (FITC), CD235a (PE), CD3 (PerCP), CD117 (PEcy7), CD71 (APC), CD49d (APCH7) and CD45 (V500).

[0095] 2. Wright-Giemsa staining and blood cell smears

[0096] The cell samples taken in step 1 were stained using the Wright-Giemsa staining method to prepare the corresponding cell smears (i.e., blood cell smears). The specific process is as follows:

[0097] (1) Take 50 μL of sample into an EP tube, add 1 mL of PBS, and centrifuge at 800 rpm for 10 min.

[0098] (2) Discard the supernatant to 100ul, resuspend the cells with a 1ml pipette and transfer them to the sample holder of the TXD3 centrifuge. Centrifuge at 1000rpm for 6min.

[0099] (3) After centrifugation, remove the sample holder, carefully remove the filter paper, and place the glass slide on absorbent paper to dry.

[0100] (4) After drying, use a Pasteur pipette to draw 1 ml of Wright-Giemsa stain solution A and add it evenly to the cell smear on the slide. Let it stand for 1 min 30 s. Then use a Pasteur pipette to draw 1 ml of buffer solution B and slowly wash the slide. After standing for 6 min, rinse off the stain under running water and let it dry.

[0101] (5) Start the confocal microscope according to the instrument operation guide, open the ZEN System software, and wait for the system to self-test and initialize.

[0102] (6) Place the cell smear under the microscope, find the Locate button in the software interface, switch to human eye observation mode, find the focal plane; switch to Acquisition, select mode, automatically match the light path settings, select the appropriate pixel resolution in live preview mode, adjust the fine focus knob, click the Snap button, take a picture and save it.

[0103] Figure 3 This is a graph showing the change (%) in the expression levels of erythroid cell surface markers CD71 and CD235 in Test Example 1 of the present invention.

[0104] like Figure 3 As shown, flow cytometry analysis based on the erythroid surface markers CD71 (transferrin receptor) and CD235a (blood group glycoprotein A) revealed a progressive differentiation trajectory: on day 0, only a small number of cells expressed low levels of CD71 and CD235a; by day 8, 72.1% of the cells expressed CD71. + CD235a - The phenotype is consistent with early erythroid precursor cells (Pro-Erythroblast), gradually transforming into a more mature intermediate-to-late erythroblast stage (Erythroblast, CD71) by day 16. + CD235a + (86.4%), with only a very small number of non-red series (CD71) detected. - CD235a - )cell.

[0105] Figure 4It is the erythroblasts (CD71) cultured during the test example 1 of this invention. + The amplification fold map was generated, with the amplification fold on day 0 of culture being defined as 1. At different time points, viable cell counts were performed using AOPI staining and a Countstar counter to calculate the amplification fold.

[0106] like Figure 4 As shown, erythroblasts enter a rapid expansion phase from day 5 to day 13. From day 13 to day 16, cell expansion slows down, ultimately resulting in an erythroblast expansion fold of nearly 200-fold. This expansion trend is consistent with... Figure 3 The cell differentiation trajectories shown are basically synchronous.

[0107] Figure 5 This is a graph showing the changes (%) in the expression levels of erythroid cell surface markers CD71, CD235, and CD36 on days 16 and 23 during the culture process of Test Example 1 of this invention.

[0108] like Figure 5 As shown, the expression of CD36, CD71, and CD235a was analyzed by flow cytometry on days 16 and 23. The results indicated that erythroid cells (CD71) showed the expression of CD36, CD71, and CD235a. + CD36 + The proportion of red blood cells can reach over 95%, indicating that the culture system has extremely high red purity.

[0109] Figure 6 These are pro-erythrocytes (CD71) cultured on days 16 and 23. + CD235 - ) and erythroblasts (Ery, CD71) + CD235 + The percentage of cells (%) is plotted, where n=3, and the data are expressed as mean ± standard deviation (Mean±SEM), with error bars representing standard errors (SEM).

[0110] like Figure 6 As shown, on day 16, the erythroid cell population was mainly composed of intermediate and late erythroblasts (72.7%); by day 23, the proportion of this cell further increased to 81.4%, suggesting that the cells gradually transformed into terminal differentiation.

[0111] Figure 7 These are photographs of the morphology of erythroblasts (observed under a confocal microscope) on days 16 and 23 of Test Example 1 of this invention, with the scale bar set to 20 μm.

[0112] like Figure 7As shown, compared to cells on day 16, the nuclear chromatin of cells on day 23 was denser, and some cells exhibited nuclear pyknosis, consistent with the characteristics of gradual maturation of erythroid cells. This morphological evolution also aligns with... Figure 6 The differentiation and transformation conclusions shown.

[0113] Based on the above test results, it can be concluded that the culture system established in this invention can effectively support the expansion and stepwise differentiation of erythroblasts induced from UBMCs, producing a highly purified erythroblast population, and can maintain the cell state over a relatively long culture period.

[0114] Test Example 2

[0115] This test case is an experimental analysis of the transfection and amplification of erythroblasts in steps 3-4 of Example 1.

[0116] 1. Detection of positive transfection rate

[0117] The transfection positivity rate was tested on the second day after transfection, and the specific procedure is as follows:

[0118] ① Take 100 μL of culture medium sample and control into an EP tube, add 1 mL of PBS, centrifuge at 500 rcf for 2 min; after centrifugation, discard the supernatant to 100 μL, add 1 μL of APC-H7 Mouse Anti-Human CD19 flow cytometry antibody, vortex, and incubate in the dark for 15 min.

[0119] ② Add 1 ml PBS, centrifuge at 500 rcf for 2 min; after centrifugation, discard the supernatant to 100 μl, add 20 μl of fixation solution A, vortex to mix, and incubate in the dark for 10 min; add 1 ml PBS, centrifuge at 500 rcf for 2 min; after centrifugation, try to remove the supernatant, add 20 μl of fixation solution B, shake slowly by hand for 2-3 seconds, and incubate in the dark for 5 min.

[0120] ③ After incubation, add 1 μL of HA-Tag (6E2) Mouse mAb (Alexa Fluor® 488 Conjugate) antibody directly, gently pipette to mix, and incubate in the dark for 15 min. Then add 1 ml of PBS, centrifuge at 500 rcf for 2 min, aspirate the supernatant as thoroughly as possible, resuspend the cells in 200 μL of PBS and transfer them to flow cytometry tubes. Detect the transfection positivity rate by flow cytometry.

[0121] Figure 8This is a graph showing the expression levels (%) of 19Ag and BMI1 in erythroblasts on days 2 and 11 after transfection in Test Example 2 of this invention. 19Ag is in the top row of the graph, and BMI1 is in the bottom row. The right side is a quantitative bar chart. n=3. Data are expressed as mean ± standard error (Mean±SEM). Error bars represent standard errors. ****P < 0.0001.

[0122] like Figure 8 As shown, on the second day after transfection, the positive expression rates of both CD19 antigen and BMI1 exceeded 50%; when cultured until amplification was completed (approximately day 11), the positive rate of CD19 antigen remained stable at over 95%.

[0123] Figure 9 This is a graph showing the results of flow cytometry analysis of the expression levels (%) of erythroid and T-cell markers of CD19Ag-Ery 12 days after transfection in Test Example 2 of this invention. The detection process for erythroid markers was the same as in Test Example 1, and the detection process for T-cell markers was the same as conventional detection procedures in the art, and will not be repeated here.

[0124] like Figure 9 As shown, on day 12 post-transfection, flow cytometry analysis revealed that the expression rates of erythroid markers (CD71, CD235a, CD36, and CD49d) were all above 95%, and the proportion of CD3-positive T cells was 0, confirming that the co-expression of BMI1 and 19Ag did not interfere with the differentiation process of erythroblasts. Further analysis showed that CD19Ag-Ery was still predominantly composed of mid-to-late stage erythroblasts (CD71...). + CD117 - (51.3%)

[0125] Figure 10 This is a cell morphology photograph of 19Ag feeder cells on day 10 after transfection in Test Example 2 of this invention. The cells were stained with Wright-Giemsa and the scale bar was 20µm.

[0126] like Figure 10 As shown, the cells on day 10 after transfection mainly exhibited the typical characteristics of intermediate erythroblasts: large cell bodies, basophilic cytoplasm, and coarse, wheel-shaped condensation of nuclear chromatin.

[0127] Figure 11 This is a proliferation curve of CD19Ag-Ery after transfection in Test Example 2 of this invention.

[0128] like Figure 11 As shown, by scaling up the transfected CD19Ag-Ery cells into single-batch cultures, the total cell count reached 1×10⁻⁶ cells by day 12 post-transfection. 9The above meets the cell quantity requirements for clinical applications.

[0129] Example 2

[0130] This embodiment provides a method for preparing CAR-T cells using the feeder cells prepared in Example 1. The CAR-T cells are T cells isolated from healthy donor PBMCs, transfected with CD19 car virus, expanded to a sufficient number, and then cryopreserved in liquid nitrogen for later use.

[0131] Specifically, this embodiment uses a method of co-culturing feeder cells with the above-mentioned CAR-T cells to prepare CD19CAR-T cells, including the following steps:

[0132] (1) Remove the cryovials of CAR-T cells from liquid nitrogen and place them in a 37 ℃ constant temperature water bath for rapid thawing. Gently shake the cryovials during the process to accelerate thawing.

[0133] (2) Transfer the completely thawed cryovials to the biosafety cabinet, gently invert to mix, and then dilute 20 μL of cell suspension to 180 μL of CS10 cryopreservation solution; gently pipette to mix, and then mix 20 μL of this diluent with 20 μL of trypan blue dye. Detect cell density using a CountStar cell counter, based on a density of 4 × 10⁻⁶ cells / mL. 5 The required CAR-T cell suspension volume is calculated based on the seeding rate of cells / well; at the same time, feeder cells are counted, and the required number of feeder cells is calculated based on the preset co-culture ratio.

[0134] (3) Take the calculated amounts of CAR-T cell suspension and feeder cell suspension respectively, transfer them to 15mL centrifuge tubes, add PBS to the total volume of 5mL, and centrifuge at 2000rpm for 8min.

[0135] (4) After centrifugation, discard the supernatant, resuspend the CAR-T cells in T cell culture medium and transfer them to 12-well plates; separately resuspend the feeder cells in SFEM culture medium and add them to the same 12-well plate, adjusting the system to a final cell density of 1×10⁻⁶. 6 Add SFEM medium to the appropriate co-culture volume, gently shake well, and co-culture.

[0136] During the co-culture period, starting from day 0, cell density and viability were periodically detected using AOPI live cell dye combined with CountStar cell counter, and the ratio of CAR-T cells to feeder cells was detected by flow cytometry to dynamically plot CAR-T cell expansion curves.

[0137] Test Example 3

[0138] This test case demonstrates the specific activation and expansion of CD19 CAR-T cells promoted by feeder cells. The expanded erythrocytes (denoted as CD19Ag-Ery) prepared in Example 1 were used in this test case.

[0139] Specifically, this test case set up a gradient ratio co-culture system of CD19Ag-Ery and CD19 CAR-T (ratios of 1:0, 1:1, 2:1, 4:1, 10:1, and 20:1), with existing CD3 / CD28 Dynabeads (Beads group) as a positive control and pure erythroblasts (Con group) as a negative control. The specific co-culture conditions and procedures were performed as described in Example 2, and will not be repeated here.

[0140] Figure 12 This is a graph showing the changes (%) of the proportion of CAR-positive cells on day 0 and day 2 of co-culture with different proportions of CD19Ag-Ery in Test Example 3 of this invention and the Beads control group. Figure 13 This is a bar chart showing the absolute number (n=3) of CD19-CAR-T cells in each group on day 6 of co-culture in Test Example 3 of this invention.

[0141] like Figures 12-13 As shown, flow cytometry analysis revealed that on day 2 of co-culture, the CD19-CAR-T positivity rate significantly increased in the 1:1 and 2:1 ratio groups; by day 6 of co-culture, the absolute number of CD19-CAR-T in the 1:1 ratio group was significantly higher than that in the Beads positive control group (**P < 0.01), demonstrating superior amplification efficiency.

[0142] To further clarify the functional differences between CD19Ag-Ery and CD3 / CD28-coupled feeder cells (CD3 / CD28-RBC), this test case was also co-incubated with CD19- or CD22-CAR-T cells at a 1:1 ratio, with CD3 / 28 Beads+19CAR and PBS+19CAR set as controls.

[0143] Figure 14 This is a graph showing the changes (%) of CAR positivity rates in each group on day 0 and day 2 after co-culture under different feeding systems in Test Example 3 of this invention. Figure 15 This is a line graph showing the fold increase of CAR-T cells in different groups at different time points under different feeding systems in Test Example 3 of this invention (based on D0, n=3).

[0144] like Figures 14-15As shown, on day 2 of co-culture, the CAR positivity rate of the CD 19Ag-Ery+ CD 19CAR group increased from 42.9% to 71.7%, which was significantly higher than that of the CD3 / CD28-RBC+19CAR group; by day 8, the amplification fold of the CD 19Ag-Ery+ CD 19CAR group reached more than 80-fold, which was significantly better than other control groups (*P < 0.05).

[0145] Figure 16 This is a graph showing the change (%) of the percentage of 19Ag feeder cells on day 0 and day 4 in the co-culture systems of CD 19Ag-Ery+ CD 19CAR and CD 19Ag-Ery+ CD 22CAR in Test Example 3 of this invention. Figure 17 This is a graph showing the absolute change in the number of 19Ag feeder cells on days 0, 2, and 4 in the two co-culture systems of CD 19Ag-Ery+ CD 22CAR and CD 19Ag-Ery+ CD 19CAR in Test Example 3 of the present invention. Figure 16 , 17 In this study, data are expressed as mean ± standard deviation (Mean ± SD), and error bars represent standard deviation. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

[0146] like Figures 16-17 As shown, CD19Ag-Ery has a specific activating effect only on CD19-CAR-T cells, but no activating effect on non-target CD22-CAR-T cells: On day 4 of co-culture, the proportion and absolute number of CD19Ag-Ery decreased sharply when co-incubated with CD19-CAR-T cells (P < 0.0001); while when co-incubated with CD22-CAR-T cells, its number increased (P < 0.05).

[0147] Based on the above test results, it can be seen that the antigen-specific feeder cells (CD19Ag-Ery) of the present invention can specifically activate and expand CAR-T cells targeting CD19, while having no activating effect on CAR-T cells targeting non-CD19, demonstrating good target specificity and application potential.

[0148] Test Example 4

[0149] To meet the safety requirements for clinical applications, CD19Ag-Ery was subjected to gamma irradiation inactivation in step 5 of Example 1 to obtain irradiated feeder cells, namely Irra-19Ag-Ery. This test case is a characterization and functional verification experiment of Irra-19Ag-Ery.

[0150] Figure 18 This is a graph showing the expression rates (%) of CD19, CD71, and CD235a of CD19Ag-Ery before and after irradiation in Test Example 4 of this invention.

[0151] like Figure 18 As shown, flow cytometry analysis of the CD19Ag-Ery phenotype (CD19, CD71, CD235a) before and after irradiation revealed a slight decrease in the proportion of CD19+CD71+ double-positive feeder cells after irradiation (P < 0.01), but the erythroid markers and CD19 antigen phenotype remained relatively stable, suggesting that irradiation has a limited effect on cell phenotype.

[0152] Figure 19 This is a graph showing the changes in the absolute number and survival rate (%, n=3) of Irra-19Ag-Ery after 3 days of in vitro culture in Test Example 4 of this invention.

[0153] like Figure 19 As shown, after culturing Irra-19Ag-Ery in vitro for 2 days, dynamic monitoring revealed a gradual decrease in the absolute number of cells, and cell viability initially fluctuated slightly before significantly decreasing. This confirms that irradiation effectively eliminated the proliferative capacity of CD19Ag-Ery and significantly improved the safety of clinical applications.

[0154] Figure 20 The change in CAR positivity rate (%, n=3) on day 0 and day 2 of co-culture under different feeder cell systems.

[0155] like Figure 20 As shown in Example 2, Irra-19Ag-Ery was co-cultured with two types of CAR-T cells (i.e., CD19-CAR-T cells and non-CD19-target 72 CAR-T cells). On the second day of co-culture, the CAR positivity rates of both CD19Ag-Ery and Irra-19Ag-Ery co-incubated with CD19-CAR-T cells were significantly increased (P < 0.001), while the CAR positivity rate of the control group of non-CD19-target CAR-T cells showed no significant change, suggesting that irradiation did not change the target specificity of CD19Ag-Ery.

[0156] This test case also detected cytokines during the co-culture process described above. Specifically, on day 2 of co-culture, cell supernatant from each group was collected, centrifuged at 500 rcf for 2 min to remove cell debris, and cytokine levels were detected using the BD Cytokine Microsphere Array (CBA) kit. The procedure is as follows:

[0157] (1) A standard curve was established using the setup beads that come with the kit, which was used for subsequent quantitative analysis of cytokine concentrations.

[0158] (2) Vortex the microspheres for detecting 7 cytokines (IL-2 / 4 / 6 / 10 / 17A / TNF / IFN-γ) and calculate the total amount of microspheres required based on the total number of samples, according to the amount of 4ul microspheres per test well. Mix the corresponding volume of microspheres in the same EP tube; centrifuge at 200 g at room temperature for 5 min, carefully aspirate all supernatant (be careful to avoid aspirating microspheres), add serum enhancement agent of the same volume as microspheres, and incubate at room temperature in the dark for 30 min.

[0159] (3) Dilute the supernatant of the cells to be tested 5 times with the reagent kit diluent. Take 25 μL of the diluted supernatant, add 25 μL of the above mixed microspheres and 25 μL of PE-labeled detection antibody, vortex to mix, and incubate at room temperature in the dark for 3 h.

[0160] (3) After incubation, add 1 mL of the washing buffer (water buffer) provided with the kit, centrifuge at 200 g at room temperature for 5 min, discard the supernatant and place it on a flow cytometer for detection; delineate the microsphere region by FSC / SSC scatter plot, collect 3000 microsphere events for each sample; use the APC channel to distinguish different types of detection microspheres, and use the PE channel to detect fluorescence intensity, which is positively correlated with cytokine concentration.

[0161] (4) Open the FCAP.GUI software, import the flow cytometry data, and calculate the specific concentrations of different cytokines in the cell supernatant of each group based on the standard curve.

[0162] Figure 21 This is a graph showing the IFN-γ level in the supernatant after co-culturing for 2 days in different feeder cell systems.

[0163] like Figure 21 As shown, cytokine detection of the supernatant on day 2 of co-culture revealed that the IFN-γ secretion level in the Irra-19Ag-Ery+CD 19CAR 2:1 group was higher than that in the Irra-19Ag-Ery+CD 19CAR 1:1 group and the control group, but there was no significant difference compared with the unirradiated 19Ag-Ery+CD 19CAR group. This indicates that irradiation did not affect the ability of CD 19Ag-Ery to induce CD 19CAR-T cells to secrete effector cytokines.

[0164] Figure 22These are the expansion curves of CAR-T cells under different feeder cell systems.

[0165] like Figure 22 As shown, on day 8 of co-culture, the expansion fold of CD19-CAR-T cells in the Irra-19Ag-Ery+19CAR group was comparable to that in the unirradiated CD19Ag-Ery+CD19CAR group and superior to that in the control group (P < 0.05), confirming that irradiated feeder cells can still effectively support the specific expansion of CD19-CAR-T cells.

[0166] Figure 23 shows the expansion curves of CAR-T cells in different feeder cell systems. The figure uses T cell subset distribution and CD4 / CD8 ratio as plotting parameters. + The ratio of T cells (%, n=3) is used to characterize the data. Data are expressed as mean ± standard error (Mean ± SEM). Error bars represent standard errors. ***P < 0.001, **P < 0.01, *P < 0.05, ns = no significant. Symbol meaning: TN: Naïve T cells (CD45RA) + CD62L + TCM: Central Memory T Cells (CD45RA) - CD62L + ); TEM: effector memory T cells (CD45RA) - CD62L - ); TEMRA: Terminal differentiation effector memory T cells (CD45RA) + CD62L - ).

[0167] like Figure 23 As shown, T cell subset analysis on day 10 of co-culture revealed that CAR-T cells in each group were predominantly naïve T cells (TN, CD45RA). + CD62L + The main component was CD19-CAR-T cells; compared to the control group, CD4+ in specifically activated CD19-CAR-T cells was the most abundant. + / CD8 + The T cell ratio decreased, suggesting that CD8 cells are more cytotoxic. + The proportion of T cells is relatively increased.

[0168] The test results above show that CD19 antigen-specific feeder cells (Irra-19Ag-Ery) irradiated with gamma rays can still specifically activate and expand CD19-targeting CAR-T cells while eliminating their proliferative capacity and improving clinical safety. Furthermore, they do not affect the subset characteristics and cytokine secretion function of CAR-T cells, demonstrating good potential for clinical translation.

[0169] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0170] For example, the feeder cells prepared in Example 1 express the CD19 target. In this invention, other targets such as CD20, CD22, CD79b, CD33, CD123, CLL1, CD7, CD5, CD2, CD1a, CD99, CD9, BCMA, GPRC5D, B7H3, GD2, CD171, MSLN, GPC2, GPC3, and ROR1, which are common target antigens in the field of cell therapy, can also be applied to the feeder cell preparation method of this invention, and CAR-T cells targeting the corresponding target antigens can be prepared from the feeder cells.

[0171] Furthermore, the number of target points expressed in the same feeder cell is not limited to one, and CAR-T feeder cells that express two or more of the above-mentioned target points can be easily obtained according to the preparation method of the present invention.

[0172] Example 1 uses umbilical cord blood to induce differentiation to obtain erythroblasts. However, other mononuclear cells obtained from peripheral blood can also obtain the same erythroblasts through the same induction differentiation process, and can also be used for subsequent transfection, expansion and irradiation to obtain feeder cells with the same effect as in Example 1.

[0173] Furthermore, for donors, because red blood cells from type O and Rh-negative donors (peripheral blood, cord blood, etc.) are unresponsive to patients with other blood types, donor blood from "universal donors" such as type O and Rh-negative donors has greater application potential. Of course, donor blood from other blood types (type A, type B, or special blood types) can also be collected specifically to prepare feeder cell products suitable for patients with the corresponding blood types, for clinical use.

[0174] Example 2 describes the in vitro preparation of CAR-T cells using a co-culture method with the feeder cells of this invention. However, due to the non-cross-reactivity of erythrocytes, these feeder cells can also be used in vivo, for example, by stimulating and activating CAR-T cells using existing methods, such as infusion into the patient during treatment.

Claims

1. A method for preparing universal CAR-T feeder cells, used to prepare feeder cells for CAR-T cells targeting a target antigen, characterized in that, include: The erythroblast acquisition step involves isolating mononuclear cells from donor blood and inducing their differentiation to obtain erythroblasts; The vector acquisition step involves constructing a lentiviral vector that expresses at least the target antigen and preparing a recombinant viral solution of the lentiviral vector. The transfection step involves transfecting the erythroblasts with the recombinant virus solution. The amplification step involves amplifying the transfected erythroblasts to obtain amplified erythroblasts; The expanded red blood cells are subjected to irradiation treatment to obtain the feeder cells. The target antigen is CD19 or CD7.

2. The method for preparing universal CAR-T feeder cells according to claim 1, characterized in that: in, The lentiviral vector also co-expresses the BMI1 gene.

3. The method for preparing universal CAR-T feeder cells according to claim 1, characterized in that: in, The irradiation treatment uses gamma rays, with an irradiation dose of 5-100 Gy and a treatment duration of 10 min-4 h.

4. The method for preparing universal CAR-T feeder cells according to claim 1, characterized in that: in, The donor blood is umbilical cord blood or peripheral blood.

5. A universal CAR-T feeder cell, characterized in that, It is prepared by the preparation method described in any one of claims 1-4.

6. A method for preparing CAR-T cells, used to prepare CAR-T cells targeting a target antigen, characterized in that, It was obtained by co-culturing the universal CAR-T feeder cells as described in claim 5 with CAR-T cells.

7. The method for preparing CAR-T cells according to claim 6, characterized in that: in, The co-culture ratio of the universal CAR-T feeder cells to the CAR-T cells is 8:1 to 1:

8.

8. The method for preparing CAR-T cells according to claim 7, characterized in that: in, The donor blood is provided by a donor with blood type O or Rh negative, or by a donor with the same blood type as the patient.

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