ADJUVANT CAPABLE OF PROMOTING EXPANSION OF IMMUNE CELLS IN VIVO

Abstract

The present disclosure provides an adjuvant that can boost the quantitative expansion of immune cells in vivo, and a combination comprising the adjuvant and immune cells. The present disclosure also provides a cascade booster system comprising the adjuvant and modified immune cells. The present disclosure also provides a treatment method using the adjuvant and the immune cells of the present disclosure.

Claims

1-33. (canceled)

34. A method for assisting immune cell therapy in a subject comprising administering to the subject an adjuvant comprising a booster antigen (BA) which is capable of activating modified therapeutic immune cells in vivo, and boosting the expansion of the immune cells in vivo, wherein the booster antigen is a marker or membrane protein on the surface of peripheral blood cells or tumor cells or pathogens.

35. The method according to claim 34, wherein the peripheral blood cells are selected from B cells, T cells, NK cells, granulocytes, red blood cells, platelets, and the pathogen is selected from viruses, bacteria, and fungus.

36. The method according to claim 34, characterized in that the booster antigen is a wild type or variant of complete or partial fragment of a human natural protein, or a wild type or variant of complete or partial fragment of a plurality of human natural proteins.

37. The method according to claim 34, characterized in that the booster antigen is CD19 or EGFRviii, or a composition comprising CD19 and/or CD86 and/or CD137L, or a composition containing EGFRviii and/or CD86 and/or CD137L.

38. The method according to claim 34, characterized in that the adjuvant is selected from protein, compound, complex, booster cell or artificial nanomaterial.

39. The method according to claim 38, characterized in that the booster cells are one or more of peripheral blood mononuclear cells (PBMC) or B cells, T cells, and NK cells, and are autologous or heterologous to the subject.

40. The method according to claim 39, characterized in that the booster cells are unmodified natural cells, or modified cells, or immortalized B cells or NK cells such as NK92, or K562 cells.

41. The method according to claim 38, characterized in that the adjuvant is a booster cell, and the booster antigen is a transmembrane fusion protein expressed on the booster cell, wherein said booster antigen comprises an extracellular binding domain which can be recognized by the therapeutic immune cell, and a transmembrane region which is a partial or complete structure of a protein naturally expressed by the booster cell.

42. The method according to claim 41, characterized in that the booster cells are booster T cells, the extracellular binding domain is a partial region of EGFRviii or CD19, and the transmembrane region is a partial region of CD3.

43. The method according to claim 34, wherein the therapeutic immune cell has dual recognition specificity to specifically recognize a target cell and the booster antigen of the adjuvant, wherein the target cell is a cell harmful to human body, such as tumor cells, or pathogen cells such as viruses, bacteria, fungus, such that the therapeutic immune cells can be activated and expanded in vivo by the adjuvant, wherein the target of target cell recognized by the immune cell and the target of the booster antigen are different or the same.

44. The method according to claim 43, wherein the therapeutic immune cell expresses a chimeric antigen receptor (aBA-CAR) capable of specifically binding to the booster antigen, characterized in that the aBA-CAR comprises an antigen binding domain of an antibody specifically binding to the booster antigen.

45. The method according to claim 44, wherein the aBA-CAR comprises CD3ζ, a variable region of an antibody targeting the booster antigen, and a co-stimulatory molecule.

46. The aBA-CAR according to claim 43, characterized in that the co-stimulatory molecule is selected from one or more of CD28, 41BBz and ICOS.

47. The method according to claim 43, wherein the therapeutic immune cell is any one of T cell or NK cell, or a mixture thereof.

48. The method according to claim 46, characterized in that the T cell is selected from following (a)-(e): (a) tumor-recognizing T cells or tumor antigen-reactive T cells, which recognizes a. tumor or tumor antigen by means of unmodified natural TCRs to recognize tumor neoantigens, b. tumor-associated antigens (TAA), c. cancer testis antigens, or d. viral antigens (such as viral antigens on the surface of cells infected by HPV or EBV and become cancerous), wherein the tumor-recognizing T cell or tumor antigen-reactive T cell is obtained by vaccine induction, or is obtained from tumor infiltrating lymphocytes (TIL), or is obtained by positive or negative screening with markers of peripheral blood such as PD1, TIM3, CD137, CD39, CD28, (b) T cells derived from tumor-infiltrating T lymphocytes (TIL), (c) TIL that has been subjected to positive or negative screen with one or more of markers such as PD1, TIM3, CD137, CD39, CD28, (d) T cells are derived from peripheral blood mononuclear cells (PBMC), or derived from PBMC that has been subjected to positive or negative screen with one or more of markers such as PD1, TIM3, CD137, and CD39.

49. The method according to claim 44, characterized in that the therapeutic immune cell is subjected to additional gene modification other than the aBA-CAR modification.

50. The method according to claim 48, characterized in that the T cell expresses (a) or (b): (a) a CAR or exogenous TCR in addition to aBA-CAR modification, and the CAR or exogenous TCR in addition to aBA-CAR confers specificity of recognizing target cells other than the natural TCR of the T cell, or (b) an enhanced receptor, wherein the enhanced receptor is a transmembrane protein comprising an extracellular domain (ECD) and an intracellular domain, the ECD is a complete sequence of any one of a receptor, a ligand, an antibody of a membrane protein of the target cell of the immune cell, or other protein structure that can bind to the membrane protein, or a partial sequence comprising a binding domain thereof, and the ICD is any one of co-stimulatory signal proteins such as CD28 or 41BB or ICOS, and does not comprise CD3; wherein the ECD of the enhanced receptor generates an inhibitory signal on the T cells upon binding to its ligand, while an intact enhanced receptor generates an activating signal on the T cells upon binding to its ligand.

51. The method according to claim 48, characterized in that the NK cell is CAR-NK cell modified to express a second CAR in addition to aBA-CAR modification, and the second CAR enables the NK cell to recognize and kill the target cell.

52. A cascade booster system, comprising n kinds of booster cells comprising booster antigen, wherein the booster antigen is a marker or membrane protein on the surface of peripheral blood cells or tumor cells or pathogens and n is a positive integer, wherein the first-grade booster cells express a first booster antigen (BA1), the second-grade booster cells express a second booster antigen (BA2) and aBA1-CAR specifically targeting the first booster antigen, and a third-grade booster cells express a third booster antigen (BA3) and aBA2-CAR specifically targeting the second booster antigen, . . . the n.sup.th grade booster cells express the n.sup.th booster antigen (BAn) and aBA.sub.n−1-CAR targeting the n−1 booster antigen, said n.sup.th booster antigen BAn can be recognized by the therapeutic immune cells, and booster antigens of different grades are different from each other, wherein the booster cells of different grades can be the cells of the same type, or cells of different types, or a mixture of multiple types of cells.

53. A pharmaceutical combination, comprising (a) an adjuvant and (b) a modified therapeutic immune cell, wherein the adjuvant comprises a booster antigen (BA) which activates the modified therapeutic immune cells in vivo, and boosts the expansion of the immune cells in vivo, wherein the booster antigen is a marker or membrane protein on the surface of peripheral blood cells or tumor cells or pathogens, and wherein the therapeutic immune cell has dual recognition specificity to specifically recognize a target cell and the booster antigen of the adjuvant, wherein the target cell is a cell harmful to human body, such that the therapeutic immune cells can be activated and expanded in vivo by the adjuvant.

Description

EXAMPLES

[0185] In order to more comprehensively understand and utilize the present invention, the present invention will be described in detail below with reference to examples and drawings. The examples are only intended to illustrate the present invention, without any intention of limiting the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

Example 1. Preparation of CAR-T Cells and Super TIL Cells from Peripheral Blood

[0186] In Examples 1-7, we used CD19 as the booster antigen, and CD19-targeting CAR (for the sequence of the CAR being used, see SEQ ID NO: 1 of Chinese Invention application Publication No. CN 110016465 A) as aBA-CAR.

[0187] Human CAR-T cells were prepared as follows: after collecting human PBMC cells, T cells in CD19-cells derived from PBMC were activated by using CD3/CD28 magnetic beads and transfected with CD19-CAR lentivirus.

[0188] Human super TIL cells were prepared as follows: after collecting monocytes from patients with advanced tumors, magnetic beads were used to capture PD1+ T cells. Lentiviral vectors containing the following two coding sequences were constructed: the nucleotide sequence encoding the CD19 CAR as aBA-CAR, and the nucleotide sequence encoding the enhanced receptor-PD1-CD28. The constructed lentivirus was used to transfect the captured PD1+ T cells.

Example 2. Booster Adjuvant—Autologous B Cells Boost Expansion of Super TILs

[0189] B cells were isolated from the apheresis cells of patients and cultured to the order of 10.sup.7 cells. B cells naturally carry CD19 membrane antigen as booster antigen.

[0190] The super TIL cells prepared as in Example 1 were divided into two groups, A and B, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, and group B was treated as follows. The 10.sup.6-order autologous B cells were co-cultured with 10.sup.6-order super TIL cells, 10 days for each round, adding 10.sup.6-order autologous B cells to the system in each round, and for 3 consecutive rounds. It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round, while the number of super TILs in the control group, group A, did not significantly increase.

Example 3. Booster Adjuvant 2—Autologous CD19+ T Cells Boost Expansion of Super TILs

[0191] T cells were isolated from the apheresis cells of patients, and the isolated T cells were transfected by the lentivirus carrying CD19 coding sequence to obtain CD19+ T cells as booster cells, which were cultured to the order of 10.sup.7 cells by stimulation with CD3/CD28 magnetic beads.

[0192] The super TIL cells were divided into two groups, A and B, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, and group B was treated as follows. The 10.sup.6-order autologous CD19+ T cells were co-cultured with 10.sup.6-order super TIL cells, 10 days for each round, adding 10.sup.6-order autologous CD19+ T cells to the system in each round, and for 3 consecutive rounds. It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round, while the number of super TILs in the control group, group A, did not significantly increase.

Example 4. Booster Adjuvant 3—Patient Autologous NK Cells Boost Expansion of Super TILs

[0193] NK cells were isolated from the apheresis cells of patients, and the isolated NK cells were transfected by the lentivirus carrying CD19 coding sequence to obtain CD19+NK cells as booster cells, which were cultured to the order of 10.sup.7 cells by stimulation with CD3/CD28 magnetic beads.

[0194] The super TIL cells were divided into two groups, A and B, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, and group B was treated as follows. The 10.sup.6-order autologous CD19+NK cells were co-cultured with 10.sup.6-order super TIL cells, 10 days for each round, adding 10.sup.6-order autologous CD19+NK cells to the system in each round, and for 3 consecutive rounds. It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round, while the number of super TILs in the control group, group A, did not significantly increase.

Example 5. Booster Adjuvant 4—Heterologous B Cells Boost Expansion of Super TILs

[0195] B cells from another individual volunteer who was different from the subject was cultured to a number of the order of 10.sup.7. B cells naturally carry CD19 membrane antigen as booster antigen.

[0196] The super TIL cells were divided into two groups, A and B, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, and group B was treated as follows. The 10.sup.6-order heterologous B cells were co-cultured with 10.sup.6-order super TIL cells, 10 days for each round, adding 10.sup.6-order heterologous B cells to the system in each round, and for 3 consecutive rounds. It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round, while the number of super TILs in the control group, group A, did not significantly increase.

Example 6. Booster Adjuvant 5—Heterologous NK Cells Boost Expansion of Super TILs

[0197] NK cells were isolated from the apheresis cells of another individual (heterologous) volunteer different from the subject, and the isolated heterologous NK cells were transfected by the lentivirus carrying CD19 coding sequence to obtain heterologous CD19+NK cells as booster cells, which were cultured to the order of 10.sup.7.

[0198] The super TIL cells were divided into two groups, A and B, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, and group B was treated as follows. The 10.sup.6-order heterologous CD19+NK cells were co-cultured with 10.sup.6-order super TIL cells, 10 days for each round, adding 10.sup.6-order heterologous CD19+NK cells to the system in each round, and for 3 consecutive rounds. It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round, while the number of super TILs in the control group, group A, did not significantly increase.

Example 7. Booster Adjuvant 6—Nanoparticles Carrying Booster Antigen Boost Expansion of Super TILs

[0199] CD19 protein was attached to nanoparticles to construct nanoparticles carrying the booster antigen.

[0200] The super TIL cells were divided into two groups, A and B, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, and group B was treated as follows. The nanoparticles with a total of about 1 microgram of CD19 protein loaded were co-cultured with 10.sup.6-order super TIL cells, 7 days for each round, adding nanoparticles with a total of about 1 microgram of CD19 protein loaded to the system in each round, and for 3 consecutive rounds. It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round, while the number of super TILs in the control group, group A, did not significantly increase.

Example 8. Two-Trade Boosting System of Autologous T Cells Boosts Expansion of Super TILs

[0201] Two types of booster cells were constructed with patient's own T cells, constructing a two-grade boosting system.

[0202] First grade boosting cells: T cells were transfected with a lentivirus loaded with EGFRviii coding sequence, resulting in EGFRviii+ T cells as booster cells, and the cells were cultured to the order of 10.sup.7 with stimulation by CD3/CD28 magnetic beads.

[0203] Second grade boosting cells: T cells were transfected with a lentivirus loaded with both the CD19 coding sequence and the coding sequence of EGFRviii-targeting CAR (the coding nucleotide sequence is shown in SEQ ID NO: 1), resulting in CD19.sup.+ EGFRviii+-CAR-T cells, and the cells were cultured to the order of 10.sup.7 with stimulation by CD3/CD28 magnetic beads.

[0204] Super TIL cells were divided into three groups, A, B, and C, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control, group B and group C were treated as follows.

[0205] In group B, 10.sup.6-order of the second grade booster cells (.sup.CD19.sup.+ EGFRviii-CAR-T cells) were co-cultured with super TILs, as positive control for using single grade booster cells. That is, 10.sup.6-order of the second grade booster cells were co-cultured with 10.sup.6-order of super TILs, 10 days for each round, adding 10.sup.6-order second grade booster cells to the system in each round, and for 3 consecutive rounds.

[0206] In group C, 10.sup.6-order of the first grade booster cells (EGFRviii.sup.+ T cells) were co-cultured with the second grade booster cells (.sup.CD19.sup.+ EGFRviii-CAR-T cells) for 10 days, followed by addition of 10.sup.6-order of super TIL cells, and simultaneous addition of 10.sup.6-order of the first grade booster cells; super TILs were co-cultured under this condition, 10 days for each round, adding 10.sup.6-order first grade booster cells every 10 days, and for 3 consecutive rounds.

[0207] It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round in group B, while group C had more multiples of amplification in each round than group B. The number of super TILs in group A did not significantly increase.

Example 9. Two-Trade Boosting System of Heterologous T Cells Boosts Expansion of Super TILs

[0208] Two types of booster cells were constructed with heterologous NK cells and patient's own T cells, constructing a two-grade boosting system.

[0209] First grade boosting cells: heterologous NK cells were transfected with a lentivirus loaded with EGFRviii coding sequence, resulting in EGFRviii+NK cells as booster cells, and the cells were cultured to the order of 10.sup.7 with stimulation by CD3/CD28 magnetic beads.

[0210] Second grade boosting cells: autologous T cells were transfected with a lentivirus loaded with both the CD19 coding sequence and the coding sequence of EGFRviii-targeting CAR (SEQ ID NO: 1), resulting in CD19+ EGFRviii+-CAR-T cells, and the cells were cultured to the order of 10.sup.7 with stimulation by CD3/CD28 magnetic beads.

[0211] Super TIL cells were divided into three groups, A, B, and C, each having the order of 10.sup.6 cells, in which group A was subjected to natural growth as a control.

[0212] Group B was treated as follows: 10.sup.6-order of the second grade booster cells (CD19.sup.+ EGFRviii-CAR-T cells) were co-cultured with super TILs, as positive control for using single grade booster cells. That is, 10.sup.6-order of the second grade booster cells were co-cultured with 10.sup.6-order of super TILs, 10 days for each round, adding 10.sup.6-order second grade booster cells to the system in each round, and for 3 consecutive rounds

[0213] Group C was treated as follows: 10.sup.6-order of the first grade booster cells (EGFRviii.sup.+ NK cells) were co-cultured with the second grade booster cells (CD19.sup.+ EGFRviii-CAR-T cells) for 10 days, followed by addition of 10.sup.6-order of super TIL cells, and simultaneous addition of 10.sup.6-order of the first grade booster cells; super TILs were co-cultured under this condition, 10 days for each round, adding 10.sup.6-order first grade booster cells every 10 days, and for 3 consecutive rounds.

[0214] It could be observed that super TILs of each round has increased by more than 5 times as compared with the previous round in group B, while group C had more multiples of amplification in each round than group B. The number of super TILs in group A did not significantly increase.

Example 10. Comparison of the Effects of B Cells and Two Artificial Booster Cells with CD19-CD86 as Booster Antigen in Boosting Expansion of CAR-T Cells

[0215] This example compares the booster expansion effects of three booster cells on specific immune cells—CAR-T cells targeting CD19, in which the three booster cells are: 1) natural B cells expressing CD19; 2) artificial booster cells with a combination of CD19 and CD86 as the booster antigen and T cell as the booster cell (abbreviated as T/CD19-86); and 3) artificial booster cells with a combination of CD19 and CD86 as the booster antigen and K562 cell as the booster cell (abbreviated as K562/CD19-86).

[0216] The experiment protocol of this example is as follows.

[0217] 1. Preparation of PBMC and B Cells [0218] 1) PBMCs were isolated and purified from fresh bloods; [0219] 2) CD19+ cells (B cells) and CD19− cells were isolated by cell sorting.

[0220] 2. Preparation of CAR-T Cells Targeting CD19 (Referred as CART19) [0221] 1) 293T cells were transfected to prepare a lentivirus comprising CD19-CAR cDNA; [0222] 2) T cells in the CD19-cells derived from PBMC were stimulated by CD3/CD28 magnetic beads; [0223] 3) T cells in step 2) were transfected by the lentivirus loaded with CD19-CAR; [0224] 4) Expression of CD19-CAR on the transfected T cells was analyzed.

[0225] 3. Preparation of Booster Cell T/CD19-86 [0226] 1) 293T cells were transfected to prepare a lentivirus comprising CD19-CD86 cDNA; [0227] 2) T cells in the CD19-cells derived from PBMC were stimulated by CD3/CD28 magnetic beads; [0228] 3) T cells in step 2) were transfected by the lentivirus loaded with CD19-CD86; [0229] 4) Expression of CD19-CD86 on the transfected T cells was analyzed.

[0230] 4. Preparation of Booster Cell K562/CD19-86 [0231] 1) 293T cells were transfected to prepare a lentivirus comprising CD19-CD86 cDNA; [0232] 2) K562 cells were transfected by the lentivirus loaded with CD19-CD86; [0233] 3) Expression of CD19-CD86 was analyzed and CD19+CD86+ cells were sorted; [0234] 4) Conduct expanded culture, 100 Gray γ-ray irradiation, aliquoted, and frozen.

[0235] 5. Co-Culture of CART19 with Three Booster Cells, Respectively [0236] 1) CART19 were divided into three equal parts, and each was mixed with one of the three booster cells as prepared in above 2-4, namely B cells, T/CD19-CD86, K562/CD19-CD86 blends, with the ratio of the CART19 cells and booster cells being 1:1; [0237] 2) The cell concentration was adjusted to 1×10.sup.6/ml, and 2 U/ml Tscm and 100 U/ml IL-2 were supplemented into the medium (same below); [0238] 3) Cells were cultured in an incubator, with half of the medium being exchanged every Monday and Wednesday (reserve half the volume of the old medium after exchanging the medium, add half the volume of fresh medium, and maintain the cell concentration at 1×10.sup.6/ml); flow cytometry analysis was conducted on Friday, the booster cells were calculated and added according to the proportion of CART19 cells to maintain the ratio of CART19 cells to booster cells at 1:1, and all the medium was exchanged with fresh medium; [0239] 4) After adding booster cells for the third time, culture was continued for a week, and the proportion of CART19 cells was analyzed by flow cytometry.

[0240] 6. Data Analysis

[0241] The expansion factor of CART19 after co-culture of the three booster cells and CART19 was calculated, respectively, and the results are as follows: [0242] B cell group, CART19 was expanded 487 times; [0243] T/CD19-CD86 group, CART19 was expanded 134 times; [0244] K562/CD19-CD86 group, CART19 was expanded 2719 times.

Example 11. Comparison of the Effects of Artificial Booster Cells with Artificial Booster Cells Comprising a Composition of EGFRviii as Booster Antigen in Boosting Expansion of Two CAR-Ts

[0245] This example compares the boosting expansion effect of artificial booster cells expressing a composition containing EGFRviii on two specific immune cells, one of which comprises an enhanced receptor. Among them, the composition comprising EGFRviii is a composition of EGFRviiit (truncated EGFRviii with the intracellular segment removed), CD86, CD137L, and mbIL15-IL15Ra, the artificial booster cell is K562, and the cell expressing the composition is indicated as K562/EGFRviiit. The two immune cells being boosted are: 1) CAR-T cells targeting EGFRviii (expressed as EGFRviii-CAR-T); and 2) CAR-T cells targeting EGFRviii and additionally modified with PD1-CD28 enhanced receptor (expressed asEGFRviii-ECAR-T).

[0246] The experiment protocol of this example is as follows.

[0247] 1. Preparation of PBMC [0248] 1) PBMCs were isolated and purified from fresh bloods; [0249] 2) aliquoted and frozen.

[0250] 2. Preparation of EGFRviii-CART and EGFRviii-ECART [0251] 1) 293T cells were transfected to prepare lentivirus comprising EGFRviii-CAR and EGFRviii-CAR/PD1-CD28; [0252] 2) PBMCs were resuscitated, and T cells were stimulated with CD3/CD28 magnetic beads; [0253] 3) The PBMCs treated with CD3/CD28 magnetic bead were transfected with the three lentiviruses as prepared, respectively; [0254] 4) The expression of EGFRvIII-CAR in T cells was analyzed.

[0255] 3. Preparation of Booster Cell K562/EGFRviiit [0256] 1) 293T cells were transfected to prepare a lentivirus comprising a composition of EGFRviiit; [0257] 2) K562 cells were transfected by the lentivirus; [0258] 3) Expression of EGFRviiit, CD86, CD137L and mbIL15-IL15Ra was analyzed and EGFRviiit+CD86+CD137L+mbIL15+ cells were sorted; [0259] 4) Conduct expanded culture, 100 Gray γ-ray irradiation, aliquoted, and frozen.

[0260] 4. Co-Culture of Two CAR-Ts with Booster Cell K562/EGFRviiit [0261] 1) EGFRviii-CART and EGFRviii-ECART, respectively, were mixed with above described booster cell K562/EGFRviiit, wherein the ratio of the CAR+ T cells and the booster cells is 1:1; [0262] 2) The cell concentration was adjusted to 1×10.sup.6/ml, and 30 ng/ml IL-21, 50 U/ml IL-2, 10 ng/ml IL-15, 50 ng/ml IL-7 were supplemented into the medium (same below); [0263] 3) Cells were cultured in an incubator, with half of the medium being exchanged every Monday and Wednesday (reserve half the volume of the old medium after exchanging the medium, add half the volume of fresh medium, and maintain the cell concentration at 1×10.sup.6/ml); flow cytometry analysis was conducted on Friday, the booster cells were calculated and added according to the proportion of CAR+ T cells to maintain the ratio of CAR+ T cells to booster cells at 1:1, and all the medium was exchanged with fresh medium; [0264] 5) After adding booster cells for the fourth time, culture was continued for a week, and the proportion of CAR+ T cells was analyzed by flow cytometry.

[0265] 5. Data Analysis

[0266] The expansion factor of two CAR-Ts after co-culture of the booster cell K562/EGFRviiit with EGFRviii-CART and with EGFRviii-ECART, respectively, for three weeks was calculated, and the results are as follows: [0267] EGFRviii-CART group, the number of CAR-T cells were expanded 3.6 times; [0268] EGFRviii-ECARTgroup, the number of CAR-T cells were expanded 21.8 times.