Method for improving activity and function of CAR-T cells

Abstract

The invention relates to the technical field of cell drugs, in particular to a method for improving activity and functions of CAR-T cells. According to the method, different cell factor combined culture schemes are selected in the cell culture stage, so that the tolerance of the cells to cryopreservation is improved, and the activity and the anti-tumor function of the recovered CAR-T cells are improved. The method for culturing the CAR-T cells is simple to operate and economical in cost, and the stable motility rate and the better tumor killing ability can be obtained while the cell preparation period is shortened. The culture scheme is suitable for CAR-T cell products cultured by different cell factors and combinations, has feasibility in cell culture schemes of different CAR structures, and has wide application value in the field of cell therapy drug development.

Description

Technical Field

[0001] This invention relates to the field of cell drug technology, and in particular to a method for improving the viability and function of CAR-T cells. Background Technology

[0002] CAR-T therapy, or Chimeric Antigen Receptor T-Cell Immunotherapy, is a novel, precise, and targeted therapy for treating tumors. In recent years, through optimization and improvement, it has achieved excellent results in clinical tumor treatment, making it a very promising new immunotherapy method that is precise, rapid, efficient, and potentially curative for cancer. With the application and development of cell therapy, CAR-T cell therapy has become one of the most clinically promising tumor treatment methods. CAR-T cell therapy targeting CD19 has already achieved remarkable results in the treatment of relapsed / refractory acute lymphoblastic leukemia (ALL) and other hematological malignancies, and it also shows great potential in the treatment of solid tumors.

[0003] The preparation of CAR-T cells typically involves activating T lymphocytes with antibodies such as CD3 and CD28, and using one or more cytokines such as IL-2, IL-7, IL-15, and IL-21 to assist cell growth and maintain function. The prepared CAR-T cells usually need to be cryopreserved and kept at low temperatures to ensure sufficient time for cell quality control, facilitate long-distance transportation, and meet the needs of various clinical applications.

[0004] The quality of cryopreserved cells is a key aspect of CAR-T cell quality control. However, due to the effects of cryopreservation, the viability of CAR-T cells after thawing is compromised to some extent. Even if cells maintain good viability immediately after thawing, long-term cell survival and viability maintenance remain challenging, significantly impacting cell viability and function after thawing. Therefore, improving the cryoprotection of CAR-T cells is a key focus in the development and upgrading of cell therapy products. Besides improving the cryoprotection of CAR-T cells, the high cost of CAR-T cell preparation, involving the extensive use of multiple cytokines, necessitates addressing the issue of reducing CAR-T cell preparation costs through optimizing production processes and workflows. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for improving the viability and function of CAR-T cells.

[0006] The method for improving the viability and function of CAR-T cells provided by the present invention includes: culturing CAR-T cells in a basal medium containing cytokines until 12-120 hours before harvest, and then culturing them in a basal medium without cytokines until the cells are harvested.

[0007] Previous research in this invention revealed that while the addition of cytokines can ensure high cell activity during the culture phase, it can lead to a decrease in cell viability and activity during cryopreservation. This invention improves cell tolerance to cryopreservation and enhances the viability and anti-tumor function of CAR-T cells after resuscitation by selecting different cytokine combinations during the cell culture phase. In this invention, the cytokines used in cell culture are at least one of IL-1β, IL-6, IL-17, IL-12, IL-18, or IL-23. In a specific embodiment, the cytokines are at least one of IL-2, IL-7, IL-15, or IL-21. For example, the cytokine is any one of IL-2, IL-7, IL-15, or IL-21, or IL-2 and IL-7, or IL-2 and IL-15, or IL-2 and IL-21, or IL-7 and IL-15, or IL-7 and IL-21, or IL-15 and IL-21, or IL-2, IL-7, and IL-15, or IL-7, IL-15, and IL-21, or IL-2, IL-7, and IL-21, or IL-2, IL-15, and IL-21. Compared to other cytokines, the histochemistry of IL-7 and IL-15 is more beneficial in improving the viability and function of CAR-T cells.

[0008] In this invention, the basal culture medium can be any culture medium suitable for CAR-T cell culture, preferably T cell culture medium or immune cell culture medium. Examples include SCGM medium, RPMI 1640, DMEM, X-VIVO-15, and ImmunoCult. TM -XF, PRIME-XV, TexMACS TM HIPP TM -T009、SuperCulture TM L500, CTS TM OpTmizer TM T Cell Expansion SFM, KBM 581, GT-T561, Nobimpex, The culture medium for CAR-T cell expansion includes AIM-V and other similar media. In some embodiments, the CAR-T culture medium may be a medium comprising serum or a serum substitute, such as plasma, serum albumin, or a complex serum substitute. In other embodiments, the CAR-T culture medium may also be a serum-free medium containing other components. The culture medium described in this invention may also include other additives, such as non-essential amino acids, sodium pyruvate, glutamine, HEPES buffer, β-mercaptoethanol, or antibiotics. The antibiotics may be penicillin, streptomycin, or neomycin, etc.

[0009] In some embodiments, the basal medium is SCGM medium containing 10% FBS, or SCGM medium containing 10% serum substitute. In some specific embodiments, the cytokine-free basal medium is SCGM medium containing 10 vol% FBS. This medium is more effective than other media.

[0010] In other embodiments, the basal medium is 1640 medium containing 10% FBS, or 1640 medium containing 10% serum substitute. In some specific embodiments, the cytokine-free basal medium is 1640 medium containing 10 vol% FBS. This medium is more effective than other media.

[0011] In some embodiments, the cytokine-containing culture medium is: a basal medium containing 100–1000 IU / ml IL-2; or a basal medium containing 1–100 ng / ml IL-15; or a basal medium containing 1–100 ng / ml IL-15 and 100–1000 IU / ml IL-2; or a basal medium containing 1–10 ng / ml IL-7 and 1–100 ng / ml IL-15; or a basal medium containing 1–10 ng / ml IL-7 and 1–100 ng / ml IL-21.

[0012] In some specific embodiments, the culture medium containing cytokines is: a basal medium containing 500 IU / ml IL-2; or a basal medium containing 10 ng / ml IL-15; or a basal medium containing 10 ng / ml IL-15 and 500 IU / ml IL-2; or a basal medium containing 5 ng / ml IL-7 and 10 ng / ml IL-15; or a basal medium containing 5 ng / ml IL-7 and 25 ng / ml IL-21.

[0013] In this invention, the cells are cultured for a total of 2 to 16 days. Before replacing the basal culture medium that does not contain cytokines, fresh culture medium is added every 1 to 3 days.

[0014] The present invention includes the following cell culture process: First, the cells are cultured in a medium containing plasma or serum substitutes or FBS, and containing cytokines IL-2, IL-7, IL-15, IL-21, and combinations thereof, with the above-mentioned complete medium containing cytokines added every 1 to 3 days; then, cytokines are removed from the culture system 6 to 120 hours before cell harvest, and thereafter only plasma or serum substitutes or FBS are provided until cell harvest; the total culture time can be any time greater than 24 hours, such as 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, etc. For example, the total culture time is 2 to 16 days. In the embodiments of the present invention, the total culture time is 8 days.

[0015] In this embodiment of the invention, cells are cultured in a basal medium containing cytokines until 12-120 hours before harvest, then collected, washed with a basal medium without cytokines, and then cultured in a basal medium without cytokines.

[0016] In some embodiments, the culture medium is changed between 12 and 72 hours before harvest, for example, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours before harvest. Experiments show that changing the culture medium at any time point within the 12-120 hour period before harvest is more effective than other treatments.

[0017] In the steps of this invention, cells are collected by centrifugation. Centrifugation conditions can be 300-500g for 5-15 minutes, or for example, centrifugation at 1000 rpm for 5 minutes, 1000 rpm for 10 minutes, 1000 rpm for 15 minutes; 1500 rpm for 5 minutes, 1500 rpm for 10 minutes, 1500 rpm for 15 minutes; 3000 rpm for 5 minutes, 3000 rpm for 10 minutes, 3000 rpm for 15 minutes; 800g-1000g for 5-15 minutes, and so on—any other centrifugation conditions suitable for cell collection. Of course, any other method of cell collection is also applicable to this invention, such as cell separation by filtration.

[0018] After centrifugation, the sample needs to be washed at least once with a cytokine-free basal medium, or any suitable number of washes, such as 1 to 5 times. The purpose of this step is to remove any residual cytokines. Therefore, any method in the art for removing residual cytokines is applicable to this method.

[0019] To meet the needs of remote cell use or cell use after a certain period of time, this invention further includes a step of mixing the harvested cells with a cryopreservation solution and then freezing them in liquid nitrogen. The cryopreservation solution can be any commercial cryopreservation solution or a self-prepared cryopreservation solution, such as CS10, PRIME-XV, FreezIS, Cellbanker2, etc. GibcoRecovery Commercially available cryopreservation solutions can be used, or self-prepared or commercially available cryoprotectants containing or without DMSO, formulated with compound electrolytes, glucose injection, dextran, and human serum albumin. The cryopreservation solution does not affect the superior characteristics of CAR-T cells cultured using the method described in this invention compared to traditional methods.

[0020] The cryopreservation solution of this invention comprises compound electrolytes, glucose sodium chloride, human serum albumin, glucose injection, dextran, and dimethyl sulfoxide. In some embodiments, the cryopreservation solution comprises 31.25 vol% compound electrolytes, 15.63 vol% glucose sodium chloride, 20 vol% human serum albumin, 8.96 vol% glucose injection, 16.67 vol% dextran 40, 7.5 vol% dimethyl sulfoxide, with the balance being water.

[0021] In this invention, the CAR-T cells are T lymphocytes expressing chimeric antigen receptors; the chimeric antigen receptors include extracellular antigen-binding domains, transmembrane domains, and / or cytoplasmic signal transduction domains.

[0022] In some embodiments, the preparation of the CAR-T cells includes:

[0023] 1) Cell activation: Mononuclear cells or enriched T lymphocytes are activated using antibodies containing CD3 and CD28 or magnetic beads containing CD3 and CD28 antibodies;

[0024] 2) Viral transduction: Activated T lymphocytes are transduced with viruses and genetically modified to prepare CAR-T cells expressing chimeric antigen receptors.

[0025] Specifically, the preparation includes: in an activation and / or transduction medium, frozen or unfrozen mononuclear cells or enriched T lymphocytes are activated by an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the cell surface. Simultaneously or after activation, the target gene is transduced. The transduction scheme can be viral transduction, transposon transduction, or transduction of DNA, RNA, or other vectors via delivery methods such as liposomes, LNPs, LPX, exosome vesicles, nanocarriers, or electroporation to obtain CAR-T cells.

[0026] This invention involves culturing CAR-T cells in a medium containing cytokines IL-2, IL-7, IL-15, IL-21, or combinations thereof. 12-120 hours before cell harvest, the cytokines are removed from the culture system, and only a medium containing plasma or serum substitutes or FBS is provided until cell harvest. The culture medium is then centrifuged to remove the culture residue, and the harvested cells are cryopreserved. By selecting different cytokine combinations during the cell culture stage, the cell tolerance to cryopreservation is improved, enhancing the viability and anti-tumor function of CAR-T cells after resuscitation. This method for culturing CAR-T cells is simple to operate, economical, and can shorten the cell preparation cycle while achieving stable viability and superior tumor-killing ability. This culture protocol is applicable to CAR-T cell products cultured with different cytokines and combinations, and is feasible in cell culture protocols with different CAR structures, demonstrating broad application value in the field of cell therapy drug development.

[0027] The "chimeric antigen receptor" or "CAR" described in this invention refers to a group of engineered peptides or proteins that, when present in immune effector cells, bind to specific antigens contained on target cells and generate intracellular signals upon recognition of the specific antigen, activating downstream pathways in the cell containing the receptor to initiate the killing effect of the immune effector cells on the target cells. CARs typically include at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain specifically recognizes antigens, and non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments or natural ligands that bind to their homologous receptors, artificially designed target-specific recognition domains that recognize specific targets, such as combinations of fibronectin type III (FN3) domains, and designed ankyrin repeat proteins (DARPins) that recognize specific targets. In some embodiments, the extracellular antigen-binding region may contain scFv, Fab, or natural ligands, and any derivatives thereof. Extracellular antigen-binding regions can refer to molecules other than intact antibodies, which may contain a portion of the intact antibody and can bind to the antigen bound to the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv), where scFv can be murine antibodies, fully human antibodies, or human-mouse chimeric antibodies, or single-domain antibodies such as shark, alpaca, or camel antibodies; and multispecific antibodies formed from antibody fragments.

[0028] In some embodiments, the extracellular antigen-binding region of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumor or hematologic malignancy cells / tissues, said target molecules including, but not limited to: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, T rop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), or TAG-72, etc.

[0029] "Chimeric antigen receptors" or "CARs" can have various structures, such as those containing secretible or membrane-expressed cytokines or antibody gene sequences; and those containing structures that can be regulated for activation or inactivation, including: suicide switches such as inducible caspase-9 (iCasp9), thymidine kinase (HSV-TK) and suicide epitopes in herpes simplex virus, truncated EGFR (EGFRt), and Fas-FasL apoptosis structures; and induced CAR structures such as: peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv linked to antibody, streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structures, and biotin-biding immune receptors. The receptor (BBIR) system; the "logic gate" regulatory system that binds to the SynNotch receptor, etc.

[0030] In some embodiments, the structure described as a "chimeric antigen receptor" or "CAR" may further include a chimeric fusion protein, which includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular antigen recognition domain of the chimeric fusion protein may be a complete and continuous extracellular segment of the cell membrane of the expressed molecule / peptide, or it may be a fusion of the expressed molecule with other peptides such as human CD8 or CD4-derived peptides. Attached Figure Description

[0031] Figure 1 The study showed the survival rate of CAR-T cells cryopreserved and thawed at different times after premature removal of cytokines, and the survival rate at 24 hours after thaw.

[0032] Figure 2 The proportion of apoptotic cells in CD19 CAR-T cells after cryopreservation and thawing at different times after premature removal of cytokines was shown.

[0033] Figure 3 Demonstrates the tumor-killing function of CD19 CAR-T cells cryopreserved and thawed after premature removal of cytokines at different times;

[0034] Figure 4 The survival rate of CEACAR-T cells after cryopreservation and thawing was shown at different times after premature removal of cytokines.

[0035] Figure 5 This study demonstrates the survival rate of CEACAR-T cells after cryopreservation and thawing following the removal of cytokines in advance during culture in different culture media. Detailed Implementation

[0036] This invention provides a method for culturing CAR-T cells to improve their viability and function. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0037] In this invention, frozen or unfrozen mononuclear cells or enriched T lymphocytes are activated in an activation and / or transduction medium by an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the cell surface. Simultaneously or after activation, the target gene is transduced. The transduction protocol can be viral transduction, transposon transduction, or transduction using vectors such as DNA and RNA via liposomes, LNPs, LPX, exosome vesicles, nanocarriers, or electroporation. The transduced CAR-T cells are cultured in a medium containing cytokines IL-2, IL-7, IL-15, IL-21, and combinations thereof (the medium also contains plasma or serum substitutes or FBS). Cytokines are removed from the culture system 12-120 hours before cell harvest, and only a medium containing plasma or serum substitutes or FBS is provided until cell harvest. The culture medium is then centrifuged to remove the culture medium, and the harvested cells are cryopreserved.

[0038] The specific steps of the method described in this invention include:

[0039] 1) Cell activation: Mononuclear cells or enriched T lymphocytes are activated using antibodies containing CD3 and CD28 or magnetic beads containing CD3 and CD28 antibodies;

[0040] 2) Viral transduction: Activated T lymphocytes were transduced with chimeric antigen receptor genes and genetically modified to prepare CAR-T cells expressing chimeric antigen receptors.

[0041] 3) Cell culture: Cells are cultured for 5 to 14 days in a medium containing plasma or serum substitutes or FBS, and containing cytokines IL-2, IL-7, IL-15, IL-21 and combinations thereof. The above complete medium containing cytokines is added every 1 to 3 days. Cytokines are removed from the culture system 12 to 120 hours before cell harvest, and only medium containing plasma or serum substitutes or FBS is provided until cell harvest.

[0042] 4) Cell collection and cryopreservation: The harvested cells were cryopreserved in a cryoprotectant or commercial cryopreservation solution containing 31.25% compound electrolyte, 15.63% glucose sodium chloride, 20% human serum albumin, 8.96% glucose, 16.67% dextran 40, and 7.5% dimethyl sulfoxide, and stored in liquid nitrogen.

[0043] To meet the needs of remote cell use or cell use after a certain period of time, this invention further includes a step of mixing the harvested cells with a cryopreservation solution and then freezing them in liquid nitrogen. The cryopreservation solution can be any commercially available cryopreservation solution or a self-prepared cryopreservation solution, such as CryoStor. PRIME-XV FREE ZIS Cryopreservation solutions, etc., can also be self-prepared or commercially available cryoprotectants containing or without DMSO, formulated with compound electrolytes, glucose, dextran, and human serum albumin.

[0044] Preferably, the cryopreservation solution comprises 31.25 vol% compound electrolyte, 15.63 vol% glucose sodium chloride, 20 vol% human serum albumin, 8.96 vol% glucose injection, 16.67 vol% dextran 40, 7.5 vol% dimethyl sulfoxide, and water. In some embodiments, the cryopreservation solution may also be prepared by mixing glucose injection, sodium chloride, human serum albumin, dextran 40, and dimethyl sulfoxide in a weight ratio, and the composition of the prepared cryopreservation solution is the same as or similar to the above volume ratio.

[0045] The compound electrolyte is a solution containing electrolytes such as sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride. The compound electrolyte is a compound electrolyte injection solution, and the glucose sodium chloride solution is a glucose sodium chloride injection solution. The human serum albumin solution is a human serum albumin injection solution. The dextran 40 solution is a dextran 40 injection solution.

[0046] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0047] Example 1: Pre-harvest removal of cytokines from CAR-T cells can improve cell survival rate after thawing frozen cells.

[0048] T cells in PBMCs were treated with a solution containing CD3 / CD28 (Gibco). TM 40203D) magnetic beads were activated, and CAR-T cells were prepared by transducing lentiviral vectors targeting CD19 or CEA, respectively. The cells were cultured in SCGM medium (CellGenix, 20802-0500) containing 10% FBS or serum substitutes such as plasma, serum albumin, etc., and different combinations of factors. Complete medium containing the above cytokines was added every 1-2 days. All cytokines in the culture system were removed 24h, 48h, and 72h before cell collection. The cells were cultured in factor-free medium containing 10% FBS until harvest, and compared with 0h (representing the control without removal of cytokines). Harvested cells were cryopreserved using a cryoprotectant consisting of 31.25% compound electrolyte, 15.63% glucose sodium chloride, 20% human serum albumin, 8.96% glucose injection, 16.67% dextran 40, and 7.5% dimethyl sulfoxide, or commercial cryopreservation solution (FUJIFILM Irvine Scientific, 91139).

[0049] The method described in this invention is applicable to any seeding density, which can be 1E5 / ml-1E8 / ml, 1E5 / ml-1E7 / ml, or other cell culture densities. The preferred seeding density is 1E+05 / ml to 1E+07 / ml; this experiment uses 1E+05 / ml-1E+07 / ml as an example. The cryopreserved cell density can be adjusted according to the cryopreservation protocol and can be any density; this experiment uses 5E+05 / ml to 1E+08 / ml as an example.

[0050] The SCGM medium described above can be replaced by any T-cell culture medium or immune cell culture medium. For example, X-VIVO-15 or ImmunoCult can be used. TM -XF, PRIME-XV, TexMACS TM HIPP TM -T009、SuperCulture TM L500, CTS TM OpTmizer TM T Cell Expansion SFM, KBM 581, GT-T561, Nobimpex, In the inventors' early verification, the replacement of various culture media such as AIM-V and 1640 did not affect the effectiveness of the invention.

[0051] The specific culture protocols for the cytokines or combinations of cytokines mentioned above include:

[0052] 1) 500 IU / ml IL-2 (Quanzhou Port, Shandong, 2208013)

[0053] 2)10ng / ml IL-15 (Peprotech, AF-200-15)

[0054] 3) 500 IU / ml IL-2 + 10 ng / ml IL-15

[0055] 4)5ng / ml IL-7 (Peprotech, AF-200-7)+10ng / ml IL-15

[0056] 5)5ng / ml IL-7+25ng / ml IL-21 (Peprotech, AF-200-21)

[0057] Taking a total cultivation cycle of 8 days as an example, the specific implementation plan is as follows:

[0058] In the activated transduction medium, T cells from PBMCs were used with a solution containing CD3 / CD28 (Gibco). TMActivation of magnetic beads (40203D) was used to transduce lentiviral vectors targeting CD19 or CEA, respectively. CAR-T cells were cultured in SCGM medium (CellGenix, 20802-0500) containing 10% FBS and 500 IU / ml IL-2 or 10 ng / ml IL-15 or 500 IU / ml IL-2 + 10 ng / ml IL-15 or 5 ng / ml IL-7 + 10 ng / ml IL-15 or 5 ng / ml IL-7 + 25 ng / ml IL-21 or other feasible cytokines and combinations thereof. Complete medium containing the above cytokines was added every 1-2 days. On day 7, day 6, or day 5 of culture, the medium was replaced with SCGM medium (CellGenix, 20802-0500) containing 10% FBS and cultured for another 8 days. Cells were then collected and cryopreserved (all cytokines in the culture system were removed 24h, 48h, and 72h before cell collection).

[0059] Before cryopreservation, the cell viability was detected by AOPI staining. 10 μl of AOPI (Shanghai Ruiyu, RE010213) was used to stain 10 μl of cells, and the cell viability was detected by cell analyzer. The cell viability before cryopreservation is shown in the "Cell viability (%) before cryopreservation" section of Table 1.

[0060] The frozen cells were thawed after 3 months of cryopreservation. During thaw, the cells were rapidly thawed in a 37°C water bath. The cell viability at thaw was detected by AOPI staining. The cell viability after thaw is shown in the "Cell viability at thaw (%)" section of Table 1.

[0061] After resuscitation, cells were seeded in medium containing 10% FBS and cultured for 24 hours. The cell survival rate 24 hours after resuscitation was detected by AOPI staining. The survival rate is shown in Table 1, “Cell survival rate (%) 24 hours after resuscitation”.

[0062] Table 1 regarding 24h, 48h, and 72h is the same as below. Figure 1 The explanation is that different cytokines refer to different cytokine or cytokine combination culture protocols, in which the same cytokine or cytokine group was repeated multiple times. The data are presented in Table 1 as average values.

[0063] Table 1 also considers the CAR-T cell resuscitation viability after cryopreservation under different cryopreservation solution conditions. The data in Table 1 show that the CAR-T cells cultured using the present invention can improve the cell viability after cryopreservation in various cryopreservation solutions.

[0064] After performing statistical analysis on Table 1, the results are as follows: Figure 1As shown, the horizontal axis represents different cell states during cryopreservation, thawing, and seeding culture, while the vertical axis represents cell viability. In the legend, 24h, 48h, and 72h refer to the following times: 24 hours, 48 ​​hours, and 72 hours before cell collection (using basal medium, specifically SCGM medium containing 10% FBS in this example), after removing cytokines on day 7, day 6, or day 5 of culture, respectively, and continuing culture until the culture endpoint or the cell collection time point (day 8 in this example), followed by cell collection for cryopreservation. Combining the horizontal and vertical axes... Figure 1 The cell viability of cells prepared using the above preparation method from multiple different donor sources was measured at different stages of cell cryopreservation, thawing after 3 months of cryopreservation, and 24 hours after thawing after 3 months of cryopreservation.

[0065] Two-way ANOVA showed that, compared with the group without cytokine removal, removing cytokines from the culture system 24-72 hours before collection significantly improved cell viability after cryopreservation and thawing. The cell viability 24 hours after thawing was significantly better than the control group. In different cytokine combinations, the cells cultured using the method of this invention exhibited superior cryopreservation tolerance. (2-way ANOVA, *p<0.05, **p<0.001, ***p<0.0001, ****p<0.0001) Table 1: Cryopreservation and thawing viability of CAR-T cells with different cytokine removal times and 24-hour survival rate after thawing.

[0066]

[0067] In addition to the above-mentioned factor combinations, there are also various combinations such as IL-2+IL7, IL-2+IL-15+IL-7, IL-7+IL-15+IL21, and IL21+IL-15. The preferred concentrations of the cytokines are: IL-2 500 IU / ml, IL-15 10 ng / ml, IL-7 5 ng / ml, and IL-21 25 ng / ml.

[0068] In addition to the cytokines mentioned above, other cytokines may include IL-1β, IL-6, IL-17, IL-12, IL-18, IL-23, or combinations thereof. Different combinations of factors and cytokine concentrations do not affect the technical effect of this invention. In some embodiments, the concentration of IL-2 may be 100–1000 IU / ml, the concentration of IL-15 may be 1–100 ng / ml, the concentration of IL-7 may be 0.25–50 ng / ml, and the concentration of IL-21 may be 1.25–250 ng / ml. These factors, alone or in combination, can be adapted to the scheme described in this invention to achieve the corresponding technical effect of increasing cell viability.

[0069] Example 2: Pre-harvest removal of cytokines from CAR-T cells to reduce the proportion of apoptosis

[0070] CAR-T cells prepared and cryopreserved according to the cell culture protocol described in Example 1 of this invention were rapidly thawed and revived in a 37°C water bath. Cells washed with PBS were subjected to flow cytometry to detect apoptosis: cells were resuspended in 200 μlbuffer (KGI Biotech, KGA1021), and Annexin V (KGI Biotech, KGA1021) and 7-AAD Viability Staining Solution (420404) were added for cell staining. Cells were incubated at 2–8°C in the dark for 15–60 min. Cell apoptosis was detected by flow cytometry. Annexin V-7-AAD- indicated live cells, Annexin V+7-AAD- indicated early-regulation cells, and Annexin V+7-AAD+ indicated dead cells.

[0071] Apoptotic cells (%) = Early-regulation cells (%) + Dead cells (%)

[0072] CAR-T cells targeting CD19, cultured using different protocols, were cultured for 24, 48, and 72 hours prior to cell collection after the removal of cytokines (using cytokine-free basal medium) until the cell collection endpoint, at which point the cells were collected and cryopreserved. The apoptosis profile of these CD19-targeting CAR-T cells after resuscitation is shown in Table 2, with the apoptosis rates at 24h, 48h, and 72h listed below. Figure 2 The explanation states that different cytokines refer to different cytokine or cytokine combination culture protocols. Multiple data points for the same cytokine or cytokine combination indicate that the same experiment was repeated multiple times. Statistical analysis of the data from benchmark 2 yielded the following results: Figure 2 As shown, the horizontal axis represents different culture protocols using different combinations of cytokines, and the vertical axis represents the proportion of apoptotic cells after cell resuscitation. In the legend, 24h, 48h, and 72h refer to the removal of cytokines (using basal medium, specifically SCGM medium containing 10% FBS in this example) 24 hours, 48 ​​hours, and 72 hours before cell collection on day 7, day 6, or day 5 of culture, respectively. Cells were then cultured until the culture endpoint or the cell collection point (day 8 in this example) before being collected for cryopreservation. Combining the horizontal and vertical axes... Figure 2 Cells collected for the above-mentioned culture protocols with cytokine removal at different time points were cryopreserved. The apoptosis of the cryopreserved cells after thawing was compared with that of the traditional culture protocol (0h group without cytokine removal).

[0073] Statistical analysis of the results was performed using a paired t-test. The results showed that compared with the 0h group where cytokines were not removed, removing cytokines from the culture system 24h to 72h before collection significantly reduced the proportion of apoptotic cells after cryopreservation and thawing. Cells prepared using the culture protocol of this invention exhibited a lower level of apoptosis after thawing. (Paired t test, *p=0.0119, *p=0.0463)

[0074] Table 2. Proportion of apoptotic cells in CD19CAR-T cells after cryopreservation and thawing at different times after premature removal of cytokines.

[0075]

[0076] Example 3: Pre-harvest removal of cytokines from CAR-T cells enhances their anti-tumor function.

[0077] CAR-T cells targeting the CD19 chimeric antigen receptor were prepared according to the cell culture protocol described in Example 1 of this invention, and collected and cryopreserved. The cryopreserved cells were rapidly thawed and thawed in a 37°C water bath, and co-cultured with the Nalm-6 tumor cell line expressing the CD19 antigen at an effector-to-target ratio of 2:1 for 24 hours. The tumor-killing ability of the CAR-T cells was detected, and the culture supernatant after 24 hours of killing was collected. The IFN-γ secretion level in the supernatant was detected by ELISA (BD Biosciences, 555142). For specific detection methods, please refer to the instruction manual.

[0078] In Table 3, 24h, 48h, and 72h refer to the removal of cytokines (using basal medium, which in this example refers to SCGM medium containing 10% FBS) 24 hours, 48 ​​hours, and 72 hours before cell collection on the 7th, 6th, or 5th day of culture, respectively, and continued culturing until the culture endpoint or cell collection time point (the 8th day of culture in this example), after which cells are collected for cryopreservation. Different cytokines refer to different cytokines or cytokine combination culture protocols. Multiple data for the same cytokine or cytokine combination indicate that the same experiment was repeated multiple times.

[0079] Statistical analysis was performed on the data in Table 3, and the results are as follows: Figure 3 As shown in the figure, the horizontal axis represents the time before collection when cytokines were removed, and the vertical axis represents the tumor-killing ratio of cells. The 24h, 48h, and 72h values ​​in the legend are explained in Table 3 above. A paired t-test was used to statistically analyze the results. The results showed that compared with the 0h group where cytokines were not removed, removing cytokines from the culture system 24h to 72h before collection significantly enhanced the tumor-killing function of CAR-T cells. Cells prepared using the culture protocol of this invention exhibited superior anti-tumor activity after resuscitation. (Paired t test, *p = 0.0172).

[0080] Table 3. Tumor-killing function of CD19 CAR-T cells after cryopreservation and thawing following premature removal of cytokines at different time points.

[0081]

[0082] Example 4: Expanded application of cytokine removal at different times

[0083] To further determine the time range for early removal of cytokines in the cell culture protocol described in this invention, and its feasibility in CAR-T cell culture targeting solid tumors, T cells from PBMCs were cultured with a solution containing CD3 / CD28 (Gibco). TM CAR-T cells were prepared by activating magnetic beads (40203D) and transducing lentiviral vectors targeting CEA. Cells were cultured for 8 days in SCGM medium (CellGenix, 20802-0500) containing 10% FBS (or serum substitutes such as plasma or serum albumin) and cytokines 5 ng / ml IL-7 (Peprotech, AF-200-7) and 25 ng / ml IL-21 (Peprotech, AF-200-21). Complete medium containing the above cytokines was added every 1-3 days. Before cell harvest, all cytokines in the culture system were removed by centrifugation at 0 h (representing the control group without cytokine removal), 6 h, 12 h, 24 h, 48 h, 72 h, and 120 h. Cells were then cultured in factor-free medium containing 10% FBS until harvest. The harvested cells were cryopreserved using a cryoprotectant consisting of 31.25% compound electrolyte, 15.63% glucose sodium chloride, 20% human serum albumin, 8.96% glucose injection, 16.67% dextran 40, and 7.5% dimethyl sulfoxide.

[0084] Cells were rapidly thawed in a 37°C water bath during resuscitation. They were then seeded in medium containing 10% FBS and cultured for 24 hours. Cell staining was performed using AOPI (Shanghai Ruiyu, RE010213), and cell viability was assessed using a cell analyzer. Results are as follows: Figure 4 As shown in Table 4, the horizontal axis represents the different cytokine removal times before collection (see Examples 1-3 for detailed explanation), and the vertical axis represents the cell survival rate 24 hours after resuscitation. The results show that when CAR-T cells targeting CEA are cultured, removing cytokines from the culture system 12h to 120h before cell collection improves the cell survival rate after cryopreservation and resuscitation compared to the control group (0h) without cytokine removal.

[0085] Table 4: Survival rate of CEA CAR-T cells after cryopreservation and thawing at different times after premature removal of cytokines

[0086] 0h 6h 12h 24h 48h 72h 120h 67.39％ 63.40％ 72.36％ 73.37％ 80.68％ 76.66％ 74.80％

[0087] To further determine the applicability of the cell culture protocol described in this invention in different commercial cell culture media, T cells from PBMCs were cultured using a medium containing CD3 / CD28 (Gibco). TM CAR-T cells were prepared by activating magnetic beads (40203D) and transducing lentiviral vectors targeting CEA. Cells were cultured for 8 days in a medium containing 10% FBS (or serum substitutes such as plasma or serum albumin), 5 ng / ml IL-7 (Peprotech, AF-200-7), and 25 ng / ml IL-21 (Peprotech, AF-200-21). Three commercially available media from different manufacturers were used: LONZA (LONZA, BP12-970Q), HIPP-T009 (Beiyanji, FG0103801), and SCGM (CellGenix, 20802-0500). Complete medium containing the aforementioned cytokines was added every 1–3 days. All cytokines were removed from the culture system 72 hours before cell harvest. Cells were then cultured in a cytokine-free medium containing 10% FBS until harvest. A control group (0 h) without cytokine removal served as the control. The harvested cells were cryopreserved using a cryoprotectant consisting of 31.25% compound electrolyte, 15.63% glucose sodium chloride, 20% human serum albumin, 8.96% glucose injection, 16.67% dextran 40, and 7.5% dimethyl sulfoxide.

[0088] Cells were rapidly thawed in a 37°C water bath and seeded in medium containing 10% FBS for 24 hours. Cell staining was performed using AOPI (Shanghai Ruiyu, RE010213), and cell viability was assessed using a cell analyzer. The cell viability of the prepared cells after cryopreservation and thawing is shown in Table 5. Statistical analysis of the data in Table 5 yielded the following results: Figure 5 As shown, the horizontal axis represents the two cell culture protocols with and without prior removal of cytokines, and the vertical axis represents the cell survival rate 24 hours after resuscitation. Paired t-test analysis revealed that for CEACAR-T cells cultured using commercially available culture media from three different manufacturers, removing cytokines from the culture system 72 hours before cell collection significantly improved the survival rate after cryopreservation and resuscitation compared to the control group (0h, without cytokine removal). (Paired t-test, **p = 0.0033).

[0089] Table 5: Survival rate of CAR-T cells after cryopreservation and thawing following premature removal of cytokine CEA during culture in different culture media.

[0090]

[0091]

[0092] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Methods to improve CAR-T cell viability and function include: CAR-T cells were cultured in basal medium containing cytokines until 12–120 hours before harvest, and then cultured in basal medium without cytokines until cell harvest.

2. The method according to claim 1, characterized in that, The cytokine is at least one of IL-1β, IL-6, IL-17, IL-12, IL-18 or IL-23.

3. The method according to claim 2, characterized in that, The culture medium containing cytokines is: Basic culture medium containing 100–1000 IU / ml IL-2; Or a basal culture medium containing 1–100 ng / ml IL-15; Or a basal culture medium containing 1–100 ng / ml IL-15 and 100–1000 IU / ml IL-2; Or a basal culture medium containing 0.25–50 ng / ml IL-7 and 1–100 ng / ml IL-15; Alternatively, a basal culture medium containing 0.25–50 ng / ml IL-7 and 1.25–250 ng / ml IL-21 may be used.

4. The method according to claim 1, characterized in that, Co-culture for 2–16 days. Before replacing with a basal medium that does not contain cytokines, replenish with fresh medium every 1–3 days.

5. The method according to claim 1, characterized in that, Cells were cultured in a basal medium containing cytokines until 12-120 hours before harvest. Cells were then collected, washed with a basal medium without cytokines, and then cultured in a basal medium without cytokines.

6. The method according to claim 5, characterized in that, The cells are collected by centrifugation or filtration, with the centrifugation speed not exceeding 3000 rpm and the centrifugation time not exceeding 15 minutes.

7. The method according to any one of claims 1 to 6, characterized in that, The basal culture medium is a T cell culture medium or an immune cell culture medium.

8. The method according to any one of claims 1 to 6, characterized in that, The harvested cells are then mixed with a cryopreservation solution and then frozen in liquid nitrogen.

9. The method according to claim 8, characterized in that, The cryopreservation solutions were CS10, PRIME-XV FreezIS, and Cellbanker2. Gibco Recovery or Alternatively, the cryopreservation solution contains 31.25 vol% compound electrolyte, 15.63 vol% glucose sodium chloride, 20 vol% human serum albumin, 8.96 vol% glucose injection, 16.67 vol% dextran 40, and 7.5 vol% dimethyl sulfoxide.

10. The method according to any one of claims 1 to 9, characterized in that, The CAR-T cells are T lymphocytes expressing chimeric antigen receptors; the chimeric antigen receptors include extracellular antigen-binding domains, transmembrane domains, and / or cytoplasmic signal transduction domains.

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