An additive composition, culture medium, and application for enhancing the killing activity of CAR-CD19 T cells against B-cell malignant tumors.

Abstract

The application belongs to the field of biotechnology and immunotherapy, and particularly relates to an additive composition for enhancing the killing activity of CAR-CD19 T cells on B cell malignancies, a culture medium and application thereof. The composition is composed of SIRT1 activator SRT2104 and antioxidant ergothioneine, and the final concentrations of the two in the T cell culture medium are 1-10 muM and 5-50 muM respectively. In vitro killing experiments show that the killing efficiency of the composition on Nalm6 tumor cells is significantly better than that of SRT2104 or ergothioneine alone, and the composition has a significant synergistic effect, thereby providing a new solution for improving the CAR-T cell immunotherapy effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and immunotherapy, specifically relating to an additive composition, culture medium and its application that enhances the killing activity of CAR-CD19 T cells against B-cell malignant tumors. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) immunotherapy has been a significant breakthrough in cancer treatment in recent years. Among them, CAR-T cells targeting the CD19 antigen have been successfully applied to the treatment of B-cell malignancies, demonstrating significant efficacy in relapsed / refractory B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and other hematological malignancies. However, despite significant clinical efficacy, CAR-T therapy still faces many challenges in industrial-scale production and in vivo persistence. Among these, T-cell functional exhaustion and oxidative stress damage during in vitro expansion are major reasons for poor treatment outcomes. Currently, conventional CAR-T cell expansion media (such as X-vivo15 and TexMACS) mainly focus on nutrient supply and the addition of cytokines (such as IL-2) to maintain basic cell survival and proliferation. Although IL-2 can strongly drive T-cell expansion, a long-term high-concentration IL-2 culture environment can accelerate T-cell differentiation into terminal effector cells, which is detrimental to the maintenance of memory phenotypes such as stem cell-like memory T cells (Tscm) and central memory T cells (Tcm). Furthermore, T cells generate significant amounts of reactive oxygen species (ROS) and oxidative stress during activation and expansion. Existing culture media lack specific mitochondrial protection mechanisms, leading to a large number of T cells undergoing apoptosis or functional impairment in the later stages of culture. Recent studies have revealed that metabolic reprogramming of T cells has a decisive impact on their functional durability. Mitochondrial dysfunction is one of the core mechanisms of T cell exhaustion. During in vitro expansion of CAR-T cells, with increasing passage numbers, mitochondrial membrane potential decreases, ROS levels increase, and NAD+ pools are depleted, ultimately leading to cellular functional decline. NAD+ is a key cofactor in cellular energy metabolism and epigenetic regulation, and its level directly affects the activity of deacetylases such as SIRT1. SIRT1 activation promotes mitophagy and biosynthesis, maintains T cell metabolic homeostasis, and reduces exhaustion differentiation. However, the effectiveness of SIRT1 activation is constrained by intracellular redox status; excessively high ROS levels deplete NAD+ pools and reduce SIRT1 activity. Oxidative stress is a significant factor leading to T cell mitochondrial dysfunction and exhaustion. Traditional antioxidants (such as vitamin C and vitamin E) can scavenge ROS, but they have limitations such as low cellular uptake efficiency and short half-life. Therefore, there is an urgent need to develop a novel combination of CAR-T culture medium additives that can synergistically enhance mitochondrial function and reduce depletion of CAR-T cells by simultaneously regulating the SIRT1 pathway and providing antioxidant protection, thereby increasing the killing efficiency against B-cell malignancies (such as Nalm6 cells) and providing a more efficient and durable solution for CD19 CAR-T cell therapy. Summary of the Invention

[0003] Based on the above background, the present invention provides an additive composition, culture medium and application of enhancing the killing activity of CAR-CD19 T cells against B cell malignant tumors.

[0004] An additive composition for enhancing the killing activity of CAR-CD19 T cells against B-cell malignant tumors, comprising SIRT1 activator SRT2104 at a final concentration of 1-10 μM and antioxidant ergothioneine at a final concentration of 5-50 μM.

[0005] Preferably, it consists of SIRT1 activator SRT2104 at a final concentration of 3 μM and antioxidant ergothioneine at a final concentration of 25 μM.

[0006] A culture medium for enhancing the killing activity of CAR-CD19 T cells against B-cell malignancies, comprising a basal culture medium, IL-2 at a final concentration of 300 IU / mL, and an additive composition as described in any of the preceding claims.

[0007] Preferably, the basal culture medium is either X-vivo15 medium or TexMACS medium.

[0008] The culture medium described in any of the preceding items is used for the culture of CAR-CD19 T cells.

[0009] Beneficial effects: This invention cultured CAR-CD19 T cells by adding SRT2104 and ergothionein to the basal culture medium. The T cells obtained by this invention have high cell viability, high expansion rate, and high transduction efficiency of CAR into T cells. The combined use of the two significantly improves the CAR-T memory phenotype, reduces exhaustion markers, and enhances the tumor cell killing efficiency, making it suitable for further scientific research and clinical application research.

[0010] The composition of this invention contains ergothioneine, a natural antioxidant that efficiently targets and enters cells and mitochondria via the OCTN1 transporter protein. It is both water-soluble and lipid-soluble, protecting mitochondrial membranes and DNA. Its ability to scavenge hydroxyl radicals is far stronger than that of vitamin C. This substance has a well-established safety profile and is widely used as a food / cosmetic ingredient. The composition of this invention also contains SRT2104, which acts as a SIRT1 activator. By directly activating the SIRT1 enzyme, it increases NAD+ levels and promotes mitochondrial function. However, when SIRT1 activators are used alone, intracellular ROS levels may limit their effectiveness, and oxidative stress can deplete NAD+, creating a negative feedback loop. Combining SIRT1 activation with antioxidant protection produces a synergistic effect: SRT2104 provides the SIRT1 activation signal, and ergothioneine protects the NAD+ pool from oxidative depletion, jointly maintaining T cell mitochondrial function and metabolic homeostasis, significantly improving the specific killing efficiency of CAR-CD19 T cells against Nalm6 cells. Attached Figure Description

[0011] Figure 1 This is a characterization graph of the CAR-CD19 T cell transduction positivity rate measured by flow cytometry in Example 1; Figure 2 This is a characterization diagram obtained by flow cytometry from the M1 and M2 experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 1. Figure 3 This is a characterization diagram obtained by flow cytometry from the M3 and M4 experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 1. Figure 4 This is a characterization diagram obtained by flow cytometry for the detection of memory phenotypic markers of CAR-CD19 T cells in Example 1. Figure 5 This is a characterization diagram obtained by flow cytometry for the CAR-CD19 T cell cytotoxicity assay in Example 1. Figure 6 This is a characterization graph of the CAR-CD19 T cell transduction positivity rate measured by flow cytometry in Example 2; Figure 7 These are characterization diagrams obtained by flow cytometry from the first and second experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 2. Figure 8 These are characterization diagrams obtained by flow cytometry from the third and fourth experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 2. Figure 9 This is a characterization diagram obtained by flow cytometry for the detection of memory phenotypic markers of CAR-CD19 T cells in Example 2. Figure 10 This is a characterization diagram obtained by flow cytometry for CAR-CD19 T cell cytotoxicity detection in Example 2; Figure 11 This is a characterization graph of the CAR-CD19 T cell transduction positivity rate measured by flow cytometry in Example 3; Figure 12 This is a characterization diagram obtained by flow cytometry from the M1 and M2 experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 3. Figure 13 This is a characterization diagram obtained by flow cytometry from the M3 and M4 experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 3. Figure 14 This is a characterization diagram obtained by flow cytometry for the detection of memory phenotypic markers of CAR-CD19 T cells in Example 3. Figure 15This is a characterization diagram obtained by flow cytometry for CAR-CD19 T cell cytotoxicity detection in Example 3; Figure 16 This is a characterization graph of the CAR-CD19 T cell transduction positivity rate measured by flow cytometry in Example 4; Figure 17 These are characterization diagrams obtained by flow cytometry from the first and second experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 4. Figure 18 These are characterization diagrams obtained by flow cytometry from the second and third experimental groups in the detection of CAR-CD19 T cell exhaustion markers in Example 4. Figure 19 This is a characterization diagram obtained by flow cytometry for the detection of memory phenotypic markers of CAR-CD19 T cells in Example 4. Figure 20 This is a characterization diagram obtained by flow cytometry for CAR-CD19 T cell toxicity detection in Example 4. Detailed Implementation

[0012] The technical solutions of the present invention will be further described below with reference to specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] To more clearly illustrate the purpose, technical solution, and advantages of this invention, the following detailed description is provided in conjunction with the accompanying drawings.

[0014] The experimental method used in this invention for CAR-CD19 T cells is specifically operated as follows: 1. Experimental Materials: X-vivo15 medium (Lonza, catalog number 20802-0500), TexMACS medium (Mitten, catalog number 130-097-196), rhIL-2 (nearshore protein, catalog number GMP-C013), SRT2104 (Aladdin, catalog number S420565), ergothioneine (Aladdin, catalog number E1499876), CD3 antibody (Baijin Biotechnology, catalog number 344818), PD-1 antibody (Baijin Biotechnology, catalog number 379206), TIM-3 antibody (Baijin Biotechnology, catalog number 345022), CD95 antibody (Baijin Biotechnology, catalog number 305630), CCR7 antibody (Baijin Biotechnology, catalog number 353214), CD45RA antibody (Baijin Biotechnology, catalog number 304108).

[0015] II. Experimental Methods: (1) CAR-CD19 T cell culture and lentivirus transduction: PBMCs were freshly isolated from human whole blood using density centrifugation for T cell activation and expansion. On day 0, the isolated PBMCs were adjusted to a cell density of 2 × 10⁶ cells / year using T cell culture medium supplemented with the composition of this invention and 300 IU / mL IL-2. 6 Lentiviral cells were seeded at a density of 1 / mL into 12-well plates and incubated at 37°C in a 5% CO2 incubator for cell activation. On day 1, lentivirus was added at an MOI of 3 (3 viruses per cell). On day 3, cells were transferred to 6-well plates and 2 mL of culture medium was added. Cells were then counted every 2 days thereafter, maintaining a density of 1×10⁶ cells / mL. 6 Expand the culture by cell density of cells / mL.

[0016] (2) Detection of CAR-CD19 T cell viability and proliferation efficiency On days 5 (D5), 7 (D7), and 9 (D9) of culture, samples were taken for counting. During the culture process, the cells were mixed evenly, sampled, stained with AO / PI, and counted to obtain cell viability and cell expansion fold.

[0017] (3) CAR-CD19 T cell transduction efficiency detection: Under the above culture conditions, cell transduction efficiency was assessed on day 9 of culture. Approximately 1.0 × 10⁶ cells were used. 6 Centrifuge at 400 g for 5 minutes, discard supernatant, wash once with 1 mL PBS, resuspend in 0.5 mL PBS, and analyze by flow cytometry. The negative control is 1.0 × 10⁻⁶ cells without transduction. 6 Each cell was analyzed by flow cytometry after the same procedure.

[0018] (4) Detection of CAR-CD19 T cell surface markers: Under the above culture conditions, the expression of T cell surface markers was detected on day 9 of culture. CD3 antibody, PD-1 antibody, TIM-3 antibody, CD95 antibody, CCR7 antibody, and CD45RA antibody were used to detect the proportion of T cell exhaustion markers and memory phenotype expression.

[0019] (5) CAR-CD19 T cell toxicity assay: Under the above culture conditions, cytotoxicity was assessed on day 9 of culture. Using the B-cell malignant tumor cell line Nalm6 as target cells, co-culture systems of CAR-T cells and targeted tumor cells were established according to different E:T (effective cell: target cell) ratios. The biological efficacy of CAR-T was evaluated by detecting the tumor cell killing rate. At the same time, a control system of co-culturing untransduced CAR-CD19 T cells with tumor cells was also established.

[0020] The present invention will now be described in detail with reference to specific embodiments.

[0021] Example 1: Using X-Vivo15 as the basal medium, different additives were added to the X-Vivo15 medium, and the experimental groups were set up as follows: M1 (SRT2104): Add SRT2104 to X-Vivo15 medium containing IL-2 at a final concentration of 300 IU / mL, with a concentration of 3 μM. M2 (ergothioneine): Ergothioneine was added to X-Vivo15 medium containing IL-2 at a final concentration of 300 IU / mL, and the concentration was set to 25 μM. M3 (blank control): IL-2 was added to X-Vivo15 medium at a final concentration of 300 IU / mL; M4 (complex combination): Add SRT2104 at a final concentration of 3 μM and ergothioneine at a final concentration of 25 μM to X-Vivo15 medium containing IL-2 at a final concentration of 300 IU / mL.

[0022] The test results are as follows: 1. Cell viability of CAR-CD19 T cells, data are shown in Table 1: Table 1 M1 83.16 92.2 94.33 M2 81.02 88.87 93.59 M3 79.84 83.92 90.27 M4 85.87 91.82 96.57 The addition of the composition of this invention maintained high cell viability. On day 9, the viability of experimental group M1 was 94.33%, experimental group M2 was 93.59%, experimental group M3 was 90.27%, and experimental group M4 was 96.57%. The addition of the composition maintained a high level of T cell viability at harvest.

[0023] 2. The fold increase in CAR-CD19 T cell proliferation is shown in Table 2: Table 2 M1 11.22 31.30 160.77 M2 10.15 30.30 165.75 M3 9.29 27.74 136.10 M4 12.28 40.77 177.52 The addition of the composition of this invention maintained the high cell proliferation capacity. On day 9, the T cells in the M1 experimental group increased by 160.77 times, the T cells in the M2 experimental group increased by 165.75 times, the T cells in the M3 experimental group increased by 136.10 times, and the T cells in the M4 experimental group increased by 177.52 times. Compared with the blank control group (M3) without the addition of active ingredients, the addition of the composition did not reduce the proliferation rate, but rather slightly increased it.

[0024] 3. The positive rate of CAR-CD19 T cell transduction is shown in Table 3: Table 3 M1 62.5 M2 64.0 M3 59.7 M4 70.7 The addition of the composition of this invention maintained high T cell transduction efficiency. On day 9, the transduction rate of the M1 experimental group was 62.5%, the transduction rate of the M2 experimental group was 64%, the transduction rate of the M3 experimental group was 59.7%, and the transduction rate of the M4 experimental group was 70.7%. The addition of the composition maintained a high positive rate of T cell transduction at harvest.

[0025] 4. Detection of CAR-CD19 T cell exhaustion markers; data are shown in Table 4: Table 4 M1 44.8 19.1 M2 41.9 19.4 M3 50.3 27.6 M4 30.4 12.2 Adding the composition of this invention significantly downregulated the exhaustion marker phenotype of harvested T cells. On day 9, the proportions of PD-1 and TIM-3 in T cells of the M1 experimental group were 44.8% and 19.1%, respectively; the proportions of PD-1 and TIM-3 in T cells of the M2 experimental group were 41.9% and 19.4%, respectively; the proportions of PD-1 and TIM-3 in T cells of the M3 experimental group were 50.3% and 27.6%, respectively; and the proportions of PD-1 and TIM-3 in T cells of the M4 experimental group were 30.4% and 12.2%, respectively. Compared with the blank control group (M3) without the addition of the composition, the proportions of PD-1 and TIM-3 cells in CAR-T cells of the group with the addition of the composition of this invention (M4) were significantly reduced.

[0026] 5. Detection of memory phenotypic markers of CAR-CD19 T cells; data are shown in Table 5: Table 5 M1 25.4% 21.4% 46.8% M2 29.7% 22.2% 51.9% M3 28.2% 15.4% 43.6% M4 42.0% 22.7% 64.7% The addition of the composition of this invention significantly enhanced the memory phenotype of harvested T cells. On day 9, the total proportion of Tscm+Tcm in T cells in the M1 experimental group was 46.8%, the total proportion of Tscm+Tcm in the M2 experimental group was 51.9%, the total proportion of Tscm+Tcm in the M3 experimental group was 43.6%, and the total proportion of Tscm+Tcm in the M4 experimental group was 64.7%. The total proportion of Tscm+Tcm in CAR-T cells in the group with the added composition of this invention (M4) was significantly higher than that in the blank control group (M3), indicating that the added composition can promote the differentiation of CAR-T cells into a younger memory phenotype with low differentiation and self-renewal potential.

[0027] 6. CAR-CD19 T cell cytotoxicity assay, data are shown in Table 6: Table 6 M1 17% 36.8% 58.6% M2 15.2% 30.7% 55.4% M3 16.2% 29.4% 47.7% M4 17.6% 43.4% 74% Under the condition of E:T=5:1, the T-kill rate of experimental group M1 was 58.6%, that of experimental group M2 was 55.4%, that of experimental group M3 was 47.7%, and that of experimental group M4 was 74%. Compared with the blank control group and the single-use group, the combination of the composition of the present invention produced a synergistic effect and significantly enhanced the in vitro killing activity of CAR-CD19 T cells against B cell malignant tumors.

[0028] Example 2: Using X-Vivo15 as the basal medium, different additives were added to the X-Vivo15 medium, and the experimental groups were set up as follows: The first group is the low-concentration additive complex combination of the present invention: SRT2104 with a final concentration of 1 μM and ergothioneine with a final concentration of 50 μM are added to X-Vivo15 medium containing IL-2 with a final concentration of 300 IU / mL.

[0029] The second group is the medium-concentration additive complex combination of the present invention: adding SRT2104 at a final concentration of 3 μM and ergothioneine at a final concentration of 25 μM to X-Vivo15 medium containing IL-2 at a final concentration of 300 IU / mL.

[0030] The third group is the high-concentration additive complex combination of the present invention: adding SRT2104 at a final concentration of 10 μM and ergothioneine at a final concentration of 50 μM to X-Vivo15 medium containing IL-2 at a final concentration of 300 IU / mL.

[0031] The fourth group was a control group without the added complex, consisting only of X-Vivo15 medium containing IL-2 at a final concentration of 300 IU / mL.

[0032] The test results are as follows: 1. Cell viability of CAR-CD19 T cells, the data are shown in Table 7 below: Table 7 Group 1 84.08 88.74 92.84 Group 2 85.87 91.82 96.57 Group 3 82.75 86.04 91.48 Group 4 79.84 83.92 90.27 At day 9, the cell viability was 92.84% in group 1, 96.57% in group 2, 91.48% in group 3, and 90.27% in group 4. Therefore, compared with the low-concentration group, high-concentration group, and control group, the medium-concentration combination in group 2 performed best in maintaining high cell viability. 2. The fold increase of CAR-CD19 T cells is shown in Table 8 below: Table 8 Group 1 10.89 36.67 154.22 Group 2 12.28 40.77 177.52 Group 3 9.15 31.31 150.74 Group 4 9.29 27.74 136.10 At day 9, the cell proliferation rate was 154.22 in group 1, 177.52 in group 2, 150.74 in group 3, and 136.10 in group 4. Therefore, compared with the low-concentration group, high-concentration group, and control group, the medium-concentration combination in group 2 performed best in maintaining a high cell proliferation rate. 3. The CAR-CD19 T cell transduction positivity rate is shown in Table 9 below: Table 9 Group 1 61.4 Group 2 70.7 Group 3 62.8 Group 4 59.7 At day 9, the T cell transduction rate was 61.4% in group 1, 70.7% in group 2, 62.8% in group 3, and 59.7% in group 4. Therefore, compared with the low-concentration group, high-concentration group, and control group, the medium-concentration combination in group 2 performed best in maintaining a high T cell transduction rate. 4. Detection of CAR-CD19 T cell exhaustion markers, data are shown in Table 10: Table 10 Group 1 42.6 21.6 Group 2 30.4 12.2 Group 3 42 17.7 Group 4 50.3 27.6 Adding the composition of this invention significantly downregulated the exhaustion marker phenotype of harvested T cells. On day 9, the proportions of PD-1 and TIM-3 in the first group of T cells were 42.6% and 21.6%, respectively; the proportions of PD-1 and TIM-3 in the second group of T cells were 30.4% and 12.2%, respectively; the proportions of PD-1 and TIM-3 in the third group of T cells were 42% and 17.7%, respectively; and the proportions of PD-1 and TIM-3 in the fourth group of T cells were 50.3% and 27.6%, respectively. Therefore, compared with the low-concentration group, the high-concentration group, and the control group, the medium-concentration combination in the second group was the most effective in downregulating the exhaustion marker phenotype.

[0033] 5. Detection of memory phenotypic markers of CAR-CD19 T cells; data are shown in Table 11: Table 11 Group 1 33.7% 15.1% 48.8% Group 2 42.0% 22.7% 64.7% Group 3 36.1% 14.8% 50.9% Group 4 28.2% 15.4% 43.6% Adding the composition of this invention significantly enhanced the memory phenotype of harvested T cells. On day 9, the total proportion of Tscm+Tcm in the first group was 48.8%, the total proportion of Tscm+Tcm in the second group was 64.7%, the total proportion of Tscm+Tcm in the third group was 50.9%, and the total proportion of Tscm+Tcm in the fourth group was 43.6%. Therefore, compared with the low concentration group, the high concentration group and the control group, the medium concentration combination in the second group was the most effective in enhancing the memory biomarker phenotype.

[0034] 6. CAR-CD19 T cell cytotoxicity assay data are shown in Table 12: Table 12 Group 1 15.4% 32.9% 54.9% Group 2 17.6% 43.4% 74% Group 3 18.2% 32.8% 59.4% Group 4 16.2% 29.4% 47.7% Under the condition of E:T=5:1, the T cell killing rate of the first group was 54.9%, the T cell killing rate of the second group was 74%, the T cell killing rate of the third group was 59.4%, and the T cell killing rate of the fourth group was 47.7%. Therefore, compared with the low concentration group, the high concentration group and the control group, the medium concentration combination of the second group showed the most significant effect in improving the T cell killing efficiency.

[0035] Example 3: Using TexMACS as the basal medium, different additives were added to the TexMACS medium, and the experimental groups were set up as follows: M1 (SRT2104): Add SRT2104 to TexMACS medium containing IL-2 at a final concentration of 300 IU / mL, with a concentration of 3 μM. M2 (ergothioneine): Ergothioneine was added to TexMACS medium containing IL-2 at a final concentration of 300 IU / mL, and the concentration was set to 25 μM. M4 (blank control): IL-2 was added to TexMACS medium at a final concentration of 300 IU / mL; M5 (complex combination): Add SRT2104 at a final concentration of 3 μM and ergothioneine at a final concentration of 25 μM to TexMACS medium containing IL-2 at a final concentration of 300 IU / mL.

[0036] The test results are as follows: 1. Cell viability of CAR-CD19 T cells, data are shown in Table 13: Table 13 M1 85.45 90.85 93.04 M2 83.05 88.79 92.09 M3 78.51 84.02 89.41 M4 87.48 93.01 96.19 The addition of the composition of this invention maintained high cell viability. On day 9, the viability rates were 93.04% in group M1, 92.09% in group M2, 89.41% in group M3, and 96.19% in group M4. The addition of the composition maintained a high T cell viability level at harvest. 2. CAR-CD19 T cell proliferation fold, data are shown in Table 14: Table 14 M1 8.78 41.48 216.56 M2 7.74 43.87 192.74 M3 6.60 32.05 169.86 M4 9.54 50.11 236.08 The addition of the composition of this invention maintained the high cell proliferation capacity. On day 9, T cells in the M1 experimental group increased by 216.56-fold, those in the M2 experimental group by 192.74-fold, those in the M3 experimental group by 169.86-fold, and those in the M4 experimental group by 236.08-fold. Compared with the blank control group (M3) without the active ingredient, the addition of the composition did not reduce the proliferation rate, but rather slightly increased it. 3. The CAR-CD19 T cell transduction positivity rate is shown in Table 15: Table 15 M1 58.7 M2 55.8 M3 52.5 M4 64.0 The addition of the composition of this invention maintained high T cell transduction efficiency. On day 9, the transduction rate of the M1 experimental group was 58.7%, the transduction rate of the M2 experimental group was 55.8%, the transduction rate of the M3 experimental group was 52.5%, and the transduction rate of the M4 experimental group was 64%. The addition of the composition maintained a high positive rate of T cell transduction at harvest.

[0037] 4. Detection of CAR-CD19 T cell exhaustion markers; data are shown in Table 16: Table 16 M1 34.6 14.9 M2 37 15.3 M3 48.6 23 M4 29.6 8.2 Adding the composition of this invention significantly downregulated the exhaustion marker phenotype of harvested T cells. On day 9, the proportions of PD-1 and TIM-3 in T cells of the M1 experimental group were 34.6% and 14.9%, respectively; the proportions of PD-1 and TIM-3 in T cells of the M2 experimental group were 37% and 15.3%, respectively; the proportions of PD-1 and TIM-3 in T cells of the M3 experimental group were 48.6% and 23%, respectively; and the proportions of PD-1 and TIM-3 in T cells of the M4 experimental group were 29.6% and 8.2%, respectively. Compared with the blank control group (M3) without the addition of the composition, the proportions of PD-1 and TIM-3 cells in CAR-T cells of the group with the addition of the composition of this invention (M4) were significantly reduced.

[0038] 5. Detection of memory phenotypic markers of CAR-CD19 T cells; data are shown in Table 17: Table 17 M1 47.1% 15.9% 63.0% M2 42.5% 14.9% 57.4% M3 31.7% 9.2% 40.9% M4 61.3% 21.8% 83.1% The addition of the composition of this invention significantly enhanced the memory phenotype of harvested T cells. On day 9, the total proportion of Tscm+Tcm in T cells in the M1 experimental group was 63.0%, in the M2 experimental group it was 57.4%, in the M3 experimental group it was 40.9%, and in the M4 experimental group it was 83.1%. The total proportion of Tscm+Tcm cells in CAR-T cells in the group with the added composition of this invention (M4) was significantly higher than that in the blank control group (M3), indicating that the added composition can promote the differentiation of CAR-T cells into a younger memory phenotype with low differentiation and self-renewal potential. 6. CAR-CD19 T cell toxicity detection, data are shown in Table 18: Table 18 M1 13.1% 30% 61.1% M2 14.7% 26.9% 59.2% M3 9.5% 22.2% 48.6% M4 17.7% 41.7% 69.8% Under the condition of E:T=5:1, the T-kill rate of experimental group M1 was 61.1%, that of experimental group M2 was 59.2%, that of experimental group M3 was 48.6%, and that of experimental group M4 was 69.8%. Compared with the blank control group and the single-use group, the combination of the composition of the present invention produced a synergistic effect and significantly enhanced the in vitro killing activity of CAR-CD19 T cells against B-cell malignant tumors.

[0039] Example 4: Using TexMACS as the basal medium, different additives were added to the TexMACS medium, and the experimental groups were set up as follows: The first group is the low-concentration additive complex combination of the present invention: SRT2104 with a final concentration of 1 μM and ergothioneine with a final concentration of 50 μM are added to TexMACS medium containing IL-2 with a final concentration of 300 IU / mL.

[0040] The second group is the medium-concentration additive complex combination of the present invention: SRT2104 with a final concentration of 3 μM and ergothioneine with a final concentration of 25 μM are added to TexMACS medium containing IL-2 with a final concentration of 300 IU / mL.

[0041] The third group is the high-concentration additive complex combination of the present invention: SRT2104 with a final concentration of 10 μM and ergothioneine with a final concentration of 50 μM are added to TexMACS medium containing IL-2 with a final concentration of 300 IU / mL.

[0042] The fourth group was a control group without the added complex, containing only TexMACS medium with IL-2 added to a final concentration of 300 IU / mL.

[0043] The test results are as follows: 1. Cell viability of CAR-CD19 T cells, the data are shown in Table 19 below: Table 19 Group 1 84.94 87.09 93.79 Group 2 87.48 93.01 96.19 Group 3 84.11 88.53 92.45 Group 4 78.51 84.02 89.41 At day 9, the cell viability was 93.79% in group 1, 96.19% in group 2, 92.45% in group 3, and 89.41% in group 4. Therefore, compared with the low-concentration group, the high-concentration group, and the control group, the medium-concentration combination in group 2 showed the best performance in maintaining high cell viability. 2. The fold increase of CAR-CD19 T cells is shown in Table 20 below: Table 20 Group 1 8.13 41.88 191.28 Group 2 9.54 50.11 236.08 Group 3 6.83 39.41 184.42 Group 4 6.60 32.05 169.86 At day 9, the cell proliferation rate was 191.28 in group 1, 236.08 in group 2, 184.42 in group 3, and 169.86 in group 4. Therefore, compared with the low-concentration group, high-concentration group, and control group, the medium-concentration combination in group 2 showed the best performance in maintaining a high cell proliferation rate. 3. The CAR-CD19 T cell transduction positivity rate is shown in Table 21 below: Table 21 Group 1 59.9 Group 2 64.0 Group 3 59.4 Group 4 52.5 At day 9, the T cell transduction rate was 59.9% in group 1, 64.0% in group 2, 59.4% in group 3, and 52.5% in group 4. Therefore, compared with the low-concentration group, high-concentration group, and control group, the medium-concentration combination in group 2 showed the best performance in maintaining a high T cell transduction rate. 4. Detection of CAR-CD19 T cell exhaustion markers, data are shown in Table 22: Table 22 Group 1 35.9 14.6 Group 2 29.6 8.2 Group 3 37.8 12.5 Group 4 48.6 23 Adding the composition of this invention significantly downregulated the exhaustion marker phenotype of harvested T cells. On day 9, the proportions of PD-1 and TIM-3 in the first group of T cells were 35.9% and 14.6%, respectively; the proportions of PD-1 and TIM-3 in the second group of T cells were 29.6% and 8.2%, respectively; the proportions of PD-1 and TIM-3 in the third group of T cells were 37.8% and 12.5%, respectively; and the proportions of PD-1 and TIM-3 in the fourth group of T cells were 48.6% and 23.0%, respectively. Therefore, compared with the low concentration group, the high concentration group and the control group, the medium concentration combination in the second group was the most effective in downregulating the exhaustion marker phenotype.

[0044] 5. Detection of memory phenotypic markers of CAR-CD19 T cells; data are shown in Table 23: Table 23 Group 1 45.3% 17.8% 63.1% Group 2 61.3% 21.8% 83.1% Group 3 46.4% 16.7% 63.1% Group 4 31.7% 9.2% 40.9% Adding the composition of this invention significantly enhanced the memory phenotype of harvested T cells. On day 9, the total proportion of Tscm+Tcm in the first group was 63.1%, the total proportion of Tscm+Tcm in the second group was 83.1%, the total proportion of Tscm+Tcm in the third group was 63.1%, and the total proportion of Tscm+Tcm in the fourth group was 40.9%. Therefore, compared with the low concentration group, the high concentration group and the control group, the medium concentration combination in the second group was the most effective in enhancing the memory biomarker phenotype.

[0045] 6. CAR-CD19 T cell cytotoxicity assay data are shown in Table 24: Table 24 Group 1 11.3% 26.6% 54.6% Group 2 17.7% 41.7% 69.8% Group 3 16.1% 28% 60.9% Group 4 9.5% 22.2% 48.6% Under the condition of E:T=5:1, the T cell killing rate of the first group was 54.6%, the T cell killing rate of the second group was 69.8%, the T cell killing rate of the third group was 60.9%, and the T cell killing rate of the fourth group was 48.6%. Therefore, compared with the low concentration group, the high concentration group and the control group, the medium concentration combination of the second group showed the most significant effect in improving the T cell killing efficiency. Compared with the blank control group and the single use group, the combination of the composition of the present invention produced a synergistic effect and significantly enhanced the in vitro killing activity of CAR-CD19 T cells against B cell malignant tumors.

[0046] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An additive composition for enhancing the killing activity of CAR-CD19 T cells against B-cell malignant tumors, characterized in that, It consists of SIRT1 activator SRT2104 at a final concentration of 3 μM and antioxidant ergothioneine at a final concentration of 25 μM.

2. A culture medium that enhances the killing activity of CAR-CD19 T cells against B-cell malignant tumors, characterized in that, It includes a basal culture medium, IL-2 at a final concentration of 300 IU / mL, and the additive composition as described in claim 1.

3. The culture medium as described in claim 2, characterized in that, The basal culture medium is either X-vivo15 medium or TexMACS medium.

4. The application of the culture medium according to any one of claims 2 to 3, characterized in that, For the culture of CAR-CD19 T cells.

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