Chimeric antigen receptor and use thereof

Abstract

Disclosed herein is a novel chimeric antigen receptor and use thereof. The novel chimeric antigen receptor consists of a signal peptide, an antigen binding domain, a transmembrane region, and an intracellular signal domain, and comprises a 4-1BB signal peptide and/or a 4-1BB molecular transmembrane region. Nucleic acid sequences of various chimeric antigen receptors are separated and purified and provided is a chimeric antigen receptor and a CAR-T cell which are specific for a CD19 malignant tumor antigen. In the malignant tumor killing test of hematological cell lines, the ability of immune cells to target and recognize tumor cells is significantly enhanced, and the killing activity against tumor cells is also enhanced.

Claims

1. A chimeric antigen receptor, comprising an extracellular signal peptide, an antigen binding domain, a transmembrane domain and an intracellular signal domain, wherein said extracellular signal peptide is selected from one of a 4-1BB signal peptide, a CD8α signal peptide, a GM-CSFRα signal peptide or a CD4 signal peptide, and the transmembrane domain is a 4-1BB molecular transmembrane region sequence, wherein the antigen bound by the antigen binding domain is CD19, and wherein the antigen binding domain comprises an amino acid sequence as set forth in SEQ ID NO. 9.

2. The chimeric antigen receptor according to claim 1, wherein an amino acid sequence of a 4-1BB molecular transmembrane region as said transmembrane domain is as set forth in SEQ ID NO. 1, or a polypeptide having 85%-99% identity with the amino acid sequence of SEQ ID NO. 1.

3. The chimeric antigen receptor according to claim 1, wherein said extracellular signal peptide is selected from a 4-1BB signal peptide with an amino acid sequence as set forth in SEQ ID NO. 2, or a polypeptide having 85%-99% identity with the amino acid sequence of SEQ ID NO. 2.

4. The chimeric antigen receptor according to claim 1, wherein an antibody from which the antigen binding domain is derived is selected from a monoclonal antibody, Fab, or scFv.

5. The chimeric antigen receptor according to claim 1, wherein the intracellular signal domain comprises an intracellular signaling domain and/or a co-stimulatory signal domain and is selected from intracellular domains of the following signal molecules: CD3ζ, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma, FcR beta, CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, lymphocyte function-associated antigen-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM, SLAMF7, CD7, NKp80, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, a ligand specifically binding to CD83, or any combination thereof.

6. The chimeric antigen receptor according to claim 5, wherein the intracellular signal domain comprises a CD3ζ, 4-1BB and/or CD28 signal domain.

7. The chimeric antigen receptor according to claim 6, wherein the intracellular signal domain comprises a 4-1BB intracellular co-stimulatory signal domain with an amino acid sequence as set forth in SEQ ID NO. 6.

8. The chimeric antigen receptor according to claim 6, wherein the intracellular signal domain comprises a CD28 intracellular co-stimulatory signal domain with an amino acid sequence as set forth in SEQ ID NO. 7.

9. The chimeric antigen receptor according to claim 6, wherein the intracellular signal domain comprises a CD3ζ intracellular signalling domain with an amino acid sequence as set forth in SEQ ID NO. 8.

10. The chimeric antigen receptor according to claim 1, wherein said chimeric antigen receptor comprises a 4-1BB signal peptide or CD8α signal peptide, an antigen binding domain, a 4-1BB transmembrane domain, a 4-1BB and/or CD28 intracellular co-stimulatory binding domain, and a CD3 intracellular signalling domain which are sequentially connected.

11. The chimeric antigen receptor according to claim 10, wherein said chimeric antigen receptor further comprises a hinge region between an extracellular antigen binding domain and the transmembrane domain.

12. The chimeric antigen receptor according to claim 10, wherein said chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID NO. 10 or SEQ ID NO. 12, or an amino acid sequence having 85%-99% identity with SEQ ID NO. 10 or SEQ ID NO. 12.

13. A nucleic acid encoding the chimeric antigen receptor of claim 1.

14. The nucleic acid according to claim 13, wherein the nucleic acid is selected from a nucleotide sequence as set forth in SEQ ID NO. 22 or SEQ ID NO. 24.

15. A recombinant expression vector, comprising the nucleic acid molecule of claim 13.

16. A cell expressing the chimeric antigen receptor of claim 1.

17. The cell according to claim 16, wherein said cell is an immune cell.

18. The cell according to claim 17, wherein said cell is selected from T lymphocyte, NK cell, or an immune cell differentiated by culturing a hematopoietic stem cell, a pluripotent stem cell or an embryonic stem cell.

19. A method for preparing a T cell modified by the chimeric antigen receptor, wherein the method comprises separating and activating the T cell to be modified, and then transducing the T cell with the recombinant expression vector of claim 15.

20. A pharmaceutical composition, comprising an effective amount of the cell of claim 16 and a pharmaceutically acceptable carrier.

21. A method of treating a tumor that expresses CD19 in a patient, the method comprising administering to the patient an effective amount of the cell of claim 16.

22. A method of treating a tumor that expresses CD19 in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 20.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a vector structure graph for a chimeric antigen receptor, showing the structure of the vectors for the CAR1 (panel A) and CAR3 (panel B) chimeric antigen receptors in the present invention.

(2) FIG. 2 shows the detection of CD19 antigen expressed by a stable cell line constructed in the present invention. A. Detection of the expression of CD19 on the constructed Raji.Luc cell line and the isotype control Raji cell line respectively by using a CD19-specific antibody. B. Detection of the expression level of CD19 antigen in the constructed K562.CD19.Luc cell line and the control cell line respectively by using a CD19-specific antibody.

(3) FIG. 3 is a graph showing the effect of CAR-T killing CD19-positive cells in vitro. A. Under the condition of E:T ratio 1, the relative proportion of the remaining Raji.Luc cells was 20.2%±1.23% after the co-culture of CAR1-T with Raji.Luc, while the relative proportion of the remaining Raji.Luc cells was 98.84%±1.60% in the CAR2-T group and 100%±3.54% in the UnT group; under the condition of E:T ratio 2, the relative proportion of the remaining Raji.Luc cells was 50.09%±2.17% after the co-culture of CAR1-T with Raji.Luc, while the relative proportion of the remaining Raji.Luc cells was 107.07%±3.04% in the CAR2-T group and 100%±3.50% in the UnT group. B. The relative proportion of the remaining Raji.Luc cells was 26.83%±1.97% after the co-culture of CAR1-T with Raji.Luc, while the relative proportion of the remaining target cells was 36.86%±3.46% in the CAR3-T group and 100%±1.78% in the UnT group.

(4) FIG. 4 shows the detection OF IFNγ release level in CAR-T cells. A. The amount of IFNγ released after co-incubation with Raji.Luc cells for 4 h and 20 h in the CAR1-T, CAR2-T and UnT, Luc groups. B. The amount of IFNγ released after co-incubation with the B lymphocytes from patients with B cell acute lymphoblastic leukemia for 4 h and 20 h in the CAR1-T, CAR2-T and UnT, Luc groups.

(5) FIG. 5 shows the detection of tumor killing effect of the autologous T cells transduced with CAR1. CAR1 was transduced into T cells from 5 different B-ALL patients, respectively, and the transduced cells were named ALLCT01, ALLCT02, ALLCT03, ALLCT04 and ALLCT05, respectively. After the co-incubation of the CAR1 transduced cells with Raji.Luc target cells, the relative number of the remaining Raji.Luc cells was 9.33%-37.82%.

(6) FIG. 6 is a graph showing the release level of human interleukin 6. After the ALLCT01 patients were re-infused with CD19 CAR-T cells, the body temperature began to rise at day 6 after the last re-infusion, and the cytokine test results showed that the secretion level of interleukin-6 (IL-6) was significantly increased.

DETAILED DESCRIPTION OF EMBODIMENTS

(7) The present invention provides a novel chimeric antigen receptor, immune effector cells and their use in inhibiting tumor activity. The present invention is further illustrated in conjunction with the following specific examples.

(8) The term “transmembrane domain” (abbreviated as TM) as used herein may be used interchangeably with “transmembrane region” and refers to a protein structure region which has thermodynamical stability and is anchored within the cell membrane. The transmembrane region can be obtained from a native protein selected from the 4-1BB molecule.

(9) The novel chimeric antigen receptor of the present invention comprises an extracellular signal peptide structure, such as a 4-1BB signal peptide, a CD8α signal peptide, a GM-CSFRα signal peptide or a CD4 signal peptide, preferably a 4-1BB signal peptide.

(10) The term “intracellular signal domain” as used herein refers to a protein structure region that is capable of transducing the cell effector function signal and directing the cell to perform a particular function. The intracellular signal domain can comprise a signalling domain and/or a co-stimulatory signal domain.

(11) As used herein, the term “identity” of amino acid sequences can be used interchangeably with “similarity” to refer to the degree of similarity between amino acid sequences determined by a sequence alignment software such as BLAST. Methods and software for alignment of amino acid sequences are well known to those skilled in the art. A modified amino acid sequence can be obtained by making substitutions, deletions, and/or additions of one or more (e.g., 1-15, such as, 2, 3, 5, 8, 10 or 12) amino acid residues on the basis of a known amino acid sequence. For example, a variant sequence having at least 85% (e.g., 85%-99% or 90%-99% or 95%-99%) sequence identity with SEQ ID NO. 1 and having a substantially same transmembrane function can be obtained by modifying the 4-1BB transmembrane domain as set forth in SEQ ID NO. 1 of the present invention through conventional protein engineering means (e.g., conservative substitution of amino acids, etc.).

(12) The term “antibody fragment” as used herein is a portion of an antibody having functions, preferably a variable region for antigen binding and/or a variable region of an intact antibody. Antibody fragments comprise Fab, Fab′, F(ab′).sub.2, Fv fragments, single chain antibody scFv, single domain antibody sdAb/V.sub.HH and multispecific antibodies.

(13) The term “scFv” or “scFv antibody” as used herein refers to a single chain antibody fragment (scFv) which is formed by connecting a heavy chain variable region VH and a light chain variable region VL of an antibody with a short peptide of 15-20 amino acids. As used herein, the term “Linker”, also referred to as a connecting peptide or adapter, refers to a flexible amino acid sequence used to connect two domains. Selection and preparation of a connecting peptide is readily available to those skilled in the art.

(14) The present invention designs a chimeric antigen receptor using the human 4-1BB molecular domain through optimization and is further illustrated in conjunction with the following specific examples.

Example 1 Preparation of a Chimeric Antigen Receptor

(15) (I) Preparation of the Gene Fragment of a Chimeric Antigen Receptor

(16) The present invention provides a novel chimeric antigen receptor comprising a CD19 antigen binding domain. The antibody from which the antigen binding domain is derived, provided by the present invention, consists of a heavy chain variable region (VH), a light chain variable region (VL) of a single chain antibody and a connecting peptide (G3S).sub.4 Linker. The heavy chain variable region and the light chain variable region sequence of the single chain antibody were derived from GenBank:Y14283.1 and GenBank:Y14284.1, respectively. Their codons and sequences were optimized to ensure that they were more suitable for expression in human cells.

(17) The novel chimeric antigen receptor provided by the present invention can be designed according to the order of the following coding genes and the fusion gene fragments for the chimeric antigen receptor can be changed: the 4-1BB signal peptide, V.sub.H-(G3S).sub.4-V.sub.L, the 4-1BB transmembrane region, the 4-1BB or CD28 intracellular co-stimulatory signal domain or the CD3ζ intracellular signalling domain. The gene synthesis technology service was provided by Nanjing Genscript Biotechnology Co., Ltd. The following coding gene sequences were selected by gene synthesis technology to perform the fusion gene synthesis:

(18) the nucleotide sequence encoding the 4-1BB transmembrane region as set forth in SEQ ID NO. 13;

(19) the nucleotide sequence encoding the 4-1BB signal peptide as set forth in SEQ ID NO. 14;

(20) the nucleotide sequence encoding the CD8α signal peptide as set forth in SEQ ID NO. 15;

(21) the nucleotide sequence encoding the CD8α hinge region as set forth in SEQ ID NO. 16;

(22) the nucleotide sequence encoding the CD8α transmembrane region as set forth in SEQ ID NO. 17;

(23) the nucleotide sequence encoding the 4-1BB intracellular co-stimulatory signal domain as set forth in SEQ ID NO. 18;

(24) the nucleotide sequence encoding the CD28 intracellular co-stimulatory signal domain as set forth in SEQ ID NO. 19;

(25) the nucleotide sequence encoding the CD3ζ intracellular signalling domain as set forth in SEQ ID NO. 20; and

(26) the nucleotide sequence encoding the CD19 antigen binding domain as set forth in SEQ ID NO. 21.

(27) The extracellular domain and the intracellular signal domain used in the present invention have various combinations, and comprise the structures or combinations selected from the following items:

(28) the 4-1BB transmembrane region having an amino acid sequence as set forth in SEQ ID NO. 1;

(29) the 4-1BB signal peptide having an amino acid sequence as set forth in SEQ ID NO. 2;

(30) the CD8α signal peptide having an amino acid sequence as set forth in SEQ ID NO. 3;

(31) the CD8α hinge region having an amino acid sequence as set forth in SEQ ID NO. 4;

(32) the CD8α transmembrane region having an amino acid sequence as set forth in SEQ ID NO. 5;

(33) the 4-1BB intracellular co-stimulatory signal domain having an amino acid sequence as set forth in SEQ ID NO. 6;

(34) CD28 intracellular co-stimulatory signal domain having an amino acid sequence as set forth in SEQ ID NO. 7;

(35) the CD3ζ intracellular signalling domain having an amino acid sequence as set forth in SEQ ID NO. 8; and

(36) the CD19 antigen binding domain having an amino acid sequence as set forth in SEQ ID NO. 9.

(37) The composition of the chimeric antigen receptor in the examples of the present invention include, but not limited to, the following structures:

(38) CAR1: 4-1BB signal peptide-V.sub.H-(G3S).sub.4-VL-4-1BB TM-4-1BBcyto-CD3ζ, the amino acid sequence of which is as set forth in SEQ ID NO. 10 and the nucleotide coding sequence of which is as set forth in SEQ ID NO. 22; the gene sequence as set forth in SEQ ID NO. 22 was synthesized to construct a recombinant expression vector;

(39) CAR2: CD8α signal peptide-V.sub.H-(G3S).sub.4-V.sub.L-CD8α Hinge&TM-4-1BBcyto-CD3ζ, the amino acid sequence of which is as set forth in SEQ ID NO. 11 and the nucleotide coding sequence of which is as set forth in SEQ ID NO. 23; the gene sequence as set forth in SEQ ID NO. 23 was synthesized to construct a recombinant expression vector;

(40) CAR3: CD8α signal peptide-V.sub.H-(G3S).sub.4-V.sub.L-4-1BB TM-4-1BBcyto-CD3ζ, the amino acid sequence of which is as set forth in SEQ ID NO. 12, and the nucleotide coding sequence of which is as set forth in SEQ ID NO. 24; the gene sequence as set forth in SEQ ID NO. 24 was synthesized to construct a recombinant expression vector;

(41) wherein the antigen binding domain can be replaced with different tumor antigen targets within the technical scope of the art. The antigen-specific heavy chain variable region and light chain variable region of the antibody can be linked in the direction of V.sub.H-V.sub.L or V.sub.L-V.sub.H, and the connecting peptide Linker can be selected from (G3S).sub.4Linker, (G4S).sub.3Linker or other GS Linker or other protein linkers, such as Whitlow Linker: GSTSGSGKPGSGEGSTKG.

(42) (II) Construction of a Lentivirus Expression Vector for a Chimeric Antigen Receptor

(43) In one specific embodiment, the present invention uses a self-inactivated lentivirus expression vector to express the target CAR gene sequence. First, the expression plasmid and pCMV-ΔR-8.74 and pMD2.G helper plasmid were extracted and mixed in a certain ratio to co-transfect 293T cells. After transfection for 48 h and 96 h, the cell culture supernatant containing the virus was collected and centrifuged at 3000 rpm at 4° C. for 5 min. After the supernatant was filtered through a 0.45 μm filter, it was mixed with PEG6000/NaCl in a volume ratio of 4:1, and allowed to stand at 4° C. for 2-3 h, and then centrifuged at a high speed for 30 min. The supernatant was discarded, and the pellet was resuspended and dissolved in pre-cooled PBS to obtain a virus concentrate which was stored at −80° C. until use.

Example 2 Construction of the K562 Cell Line Expressing CD19 Antigen (K562.CD19.Luc)

(44) K562 cells hardly express CD19. For the gene synthesis of the nucleotide sequence encoding human CD19 molecule (the protein sequence thereof is NCBI number NP_001171569.1) in the present invention, the gene synthesis technology service was provided by Nanjing Genscript Biotechnology Co., Ltd. Under the action of T4 ligase, the synthesized CD19 nucleotide sequence was ligated at 20° C. overnight to the pLVX-Puro (Clontech, Cat #632164) lentivirus vector which was digested at the BamH1 and XbaI restriction sites in advance. The ligation product was transformed into DH5α competent cells and plated onto bacterial plates, and multiple clone spots were picked for the extraction of plasmids (Qiagen Endofree Mega kit). After enzyme digestion identification, sequencing and alignment, the successfully constructed vector was named as pLVX-CD19-Puro.

(45) The extracted pLVX-CD19-Puro expression plasmid and pCMV-ΔR-8.74 (J. Virol.-1998-Dull-A third-generation lentivirus vector with a conditional packaging system) and pMD2.G helper plasmid were mixed in a certain ratio to co-transfect 293FT cells. After transfection for 96 h, the cell culture supernatant containing the virus was collected and centrifuged at 3000 rpm at 4° C. for 5 min. After the supernatant was filtered through a 0.45 μm filter, it was centrifuged at a high speed of 25,000 rpm at 4° C. for 120 min. The virus concentrate was obtained after discarding the supernatant and resuspending the pellets to dissolve, and stored at −80° C. until use. The lentivirus vector carrying the firefly luciferase/Luciferase reporter gene was also prepared by the same method.

(46) The K562 cell line was purchased from ATCC (Cat No. CCL-243), and was subjected to conventional culture using 90% IMDM (Life technology, Cat No. A10491-01)+10% FBS (Life technology, Cat #10099-141)+1% penicillin/streptomycin (Life technology, Cat #15140-122) (hereinafter referred to as IMDM10 culture solution in short). The constructed lentivirus vector carrying the CD19 gene and the lentivirus vector carrying the firefly luciferase/Luciferase reporter gene were added to the cultured K562 cell supernatant to co-transduce the K562 cell line. After transduction for 24 h, puromycin was added at a final concentration of 10 μg/ml. The culture solution was exchanged every three days and puromycin at the same concentration was added. The screening of monoclones were further performed to obtain monoclonal cells, which were named as K562.CD19.Luc. Another stable cell line K562.CD123.Luc (CD123 gene source: GenBank:NM_002183) constructed simultaneously according to the above-mentioned method in the present invention was used as a CD19 negative control cell line.

(47) The CD19-positive human lymphoma cell line Raji was purchased from American ATCC (ATCC #CCL86™) and was cultured with RPMI1640+10% FBS+1% penicillin/streptomycin (hereinafter referred to as R10 culture solution in short). The firefly luciferase/Luciferase reporter gene was transduced into Raji cells using a method similar to the above-mentioned method. A cell line stably expressing the luciferase reporter gene was obtained by the screening under the puromycin stress, which was named as Raji.Luc.

(48) The monoclonal cells obtained above were identified for CD19 expression by flow cytometry. As shown in FIG. 2A, the expression of CD19 on the constructed Raji.Luc cell line and the isotype control Raji cell line (purchased from Miltenyi) was detected respectively by using a CD19-specific antibody. The flow cytometry results showed that the constructed Raji.Luc cell line had a higher CD19 expression level. FIG. 2B shows the expression level of CD antigen in the constructed K562.CD19.Luc cell line and the control cell line respectively detected by using a CD19-specific antibody. The flow cytometry results showed that the constructed K562.CD19.Luc cell line also had a very high CD19 expression level.

Example 3 Preparation of Immune Effector Cells Modified by the Chimeric Antigen Receptor and Detection of Killing Activity In Vitro

(49) (I) Preparation of T Lymphocytes

(50) 50 mL fresh blood from healthy human was collected, and peripheral blood mononuclear cells (PBMC) were separated by a lymphocyte separation solution and a density gradient centrifugation method. The cells were magnetically labelled using a Pan T Cell Isolation Kit (purchased from Miltenyi Biotech), and T lymphocytes were separated and purified. The purified T cells were then subjected to T lymphocyte activation and proliferation using CD3/CD28 magnetic beads.

(51) (II) Transduction of T Lymphocytes with Lentivirus

(52) The activated T lymphocytes were collected and resuspended in RPMI1640 culture medium. 1×10.sup.6 activated T lymphocytes were infected with lentivirus, the cell suspension was added to a 6-well plate and incubated overnight at 37° C. in a 5% CO.sub.2 incubator. On the next day, centrifugation was performed again and a fresh culture medium was replaced. A fresh culture medium was added every 3 days to continue the expansion culture.

(53) (III) Detection of CAR-T Expression

(54) The CAR-T cells prepared according to the above-mentioned protocol were collected by centrifugation, washed respectively three times with DPBS, and then genomic DNA was prepared using a human genome extraction kit (Gentra Puregene Cell Kit (purchased from Qiagen)). The prepared DNA was detected for the absorbance of OD.sub.260 nm and OD.sub.280 nm and then the concentration was calculated. The appropriate DNA concentration was adjusted. The Q-PCR reaction system was configured according to the kit SYBR Green Real time PCR Master mix plus (purchased from Toyobo). Then the gene copy number was detected on a fluorescence quantitative PCR machine (ABI #7300). The Q-PCR detection used accurately quantified plasmids containing the target fragments as positive control and for a standard curve. The CT value of each copy number concentration obtained by the Q-PCR and the corresponding copy number were plotted as a straight line and fit into a standard curve. The relative copy number for other test samples were calculated based on the fitted equation of the standard curve.

(55) For the detection of the chimeric antigen receptor expressed in CAR-T cells in the present invention, the untransduced T lymphocyte (UnT) was used as a blank control, and the CD19 CAR-T (CAR2-T) comprising a CD8α signal peptide, a hinge region and a transmembrane region structures in the U.S. Pat. No. 8,399,645 B2 was used as a control.

(56) The results of the detection of CAR integration copy number are shown in Table 1. The results showed that the integration of CAR1 gene was detected in the cell genome in the CAR1-T group, with the copy number of 7.46×10.sup.5 copies/ng genomic DNA; the copy number of the transduced CAR2 gene was 12.90×10.sup.5 copies/ng genomic DNA in the CAR2-T group; the measurements were extremely low (about 30 copies/ng genomic DNA) in the blank control UnT group and the H.sub.2O, which belong to the background of the test.

(57) TABLE-US-00001 TABLE 1 The copy number of CAR gene integrated into the donor T cell genome detected by the Q-PCR method Integrated copy Test Ct value Ct value Average Ct number/ng genomic samples repeat 1 repeat 2 value DNA CAR1-T 21.14 20.88 21.01 74643.34 CAR2-T 20.06 20.18 20.12 129031.70 UnT 34.10 33.41 33.76 30.31 H.sub.2O 33.70 33.06 33.38 38.16

Example 4 Detection of Killing Activity In Vitro

(58) The CAR-T cells prepared above were collected, adjusted to a suitable density with R10 culture solution, and inoculated in a 384-well plate. CAR-T cells was co-cultured with the target Raji-Luc cells at an effector-to-target ratio (E:T) of 50:1 or 20:1 at 37° C. for 20 h. Then an equal amount of luciferase activity detection reagent One-Glo™ Luciferase Assay (purchased from Promega) was added. After the completion of the co-culture, the relative luciferase activity (RLU, relative light unit) remained in the reaction wells corresponds to the relative amount of viable cells in the wells. If the revealed luciferase RLU value is high, it means that there are many target cells which are not killed remained in the reaction wells, indicating that the cell killing activity in the wells is weak; conversely, if the revealed luciferase RLU value is low, it means that there are few target cells which are not killed in the reaction wells, indicating the cell killing activity in the well is strong. For the killing test of the CAR-T cells of the present invention against tumor cells, the untransduced T lymphocyte (UnT) was used as a blank control and CAR2 was used as a control.

(59) As shown in FIG. 3A, under the conditions of equivalent copy number of integration and different effector-to-target ratios (E:T ratio 1=50:1, E:T ratio 2=20:1), CAR1-T can significantly kill Raji. Luc cells (the relative luciferase activity RLU % was reduced), which was significantly better than the killing effect of CAR2-T in the control group. Under the condition of E:T ratio 1, the relative proportion of the remaining Raji.Luc cells was 20.2%±1.23% after the co-culture of CAR1-T with Raji.Luc, while the relative proportion of the remaining Raji.Luc cells was 98.84%±1.60% in the CAR2-T group and 100%±3.54% in the UnT group; under the condition of E:T ratio 2, the relative proportion of the remaining Raji.Luc cells was 50.09%±2.17% after the co-culture of CAR1-T with Raji.Luc, while the relative proportion of the remaining Raji.Luc cells was 107.07%±3.04% in the CAR2-T group and 100%±3.50% in the UnT group. The above results can reveal that CAR1-modified immune cells comprising the 4-1BB signal peptide and the transmembrane region domain and lacking the hinge region have a better in vitro tumor killing effect than the CAR2-modified immune cells comprising the CD8α signal peptide and the transmembrane region domain.

(60) In another set of experiments, as shown in FIG. 3B, the relative proportion of the remaining Raji.Luc cells was 26.83%±1.97% after the co-culture of CAR1-T with Raji.Luc, while the relative proportion of the remaining Raji.Luc cells was 36.86%±3.46% in the CAR3-T group and 100%±1.78% in the UnT group. CAR1 differs from CAR3 in that the extracellular signal peptide is different, and CAR3 comprises the CD8α signal peptide. From the results of FIG. 3B, it can be inferred that CAR1-modified immune cells comprising the 4-1BB signal peptide and the transmembrane region have a better in vitro tumor killing effect than the CAR3-modified immune cells comprising the CD8α signal peptide.

(61) Combining the data from FIGS. 3A and 3B, it can be inferred that the immune cells modified by the chimeric antigen receptors comprising different signal peptides or transmembrane region structures have different in vitro killing ability, and the transmembrane region structure may have more significant impacts on the performance of the chimeric antigen receptor.

(62) Further, in the target-specific killing experiments, the present invention used the constructed K562.CD19.Luc expressing CD19 and the K562.CD123.Luc stable cell line not expressing CD19 but expressing CD123 (Example 2) to evaluate the specificity of the killing effect of CAR1. As shown in FIG. 3C, the relative proportion of the remaining K562.CD19.Luc cells was 6.77%±0.84% after the co-culture of CAR1-T with K562.CD19.Luc; while the relative proportion of the remaining K562.CD123.Luc cells was 107.06%±14.39% after the co-culture of CAR1-T with K562.CD123.Luc; however, the UnT cells had no obvious killing effect on both target cells; and CAR1-T had an obvious target-selective killing effect on K562.CD19.Luc cells.

Example 5 Detection of IFNγ Release Level in CAR-T Cells

(63) The CAR-T cells prepared above were collected, and adjusted to an appropriate density with R10 culture solution and inoculated in a 96-well plate. The release of IFNγ is a hallmark of T cell activation. In this example, different CAR-T cells were co-cultured with target cells at E:T=20:1 at 37° C. Then the co-cultured supernatant was removed. Using a real-time fluorescence resolution technology kit (Homogeneous Time Resolved Fluorescence (HTRF), Cisbio #64IL2PEB), the amount of IFN-γ released in the supernatant was detected. At the same time, CAR2-T was used as a control, the untransduced T lymphocyte (UnT) and the T lymphocyte (Luc) transduced only with viral vector were used as blank controls.

(64) As shown in FIG. 4A, after CAR-T cells or UnT cells were co-incubated with CD19-positive Raji.Luc cells for 4 h, the amount of IFNγ released in the CAR1-T group was 894.49±101.64 pg/mL, the amount of IFNγ released in the CAR2-T group was 343.88±44.30 pg/mL, the amount of IFNγ released in the UnT group was 59.49±6.52 pg/mL, and the amount of IFNγ released in the Luc group was 67.43±1.52 pg/mL; after co-incubation for 20 h, the amount of IFNγ released in the CAR1-T group was 1572.23±0.60 pg/mL, the amount of IFNγ released in the CAR2-T group was 808.67±21.42 pg/mL, the amount of IFNγ released in the UnT group was 240.82±34.11 pg/mL, and the amount of IFNγ released in the Luc group was 239.82±83.47 pg/mL. Compared with the UnT and Luc controls, CAR1-T and CAR2-T can significantly release IFNγ, and CAR1-T has a more prominent antigen-dependent IFNγ release level than CAR2-T.

(65) As shown in FIG. 4B, after CAR-T cells or UnT cells were co-incubated with the B lymphocytes from patients with B cell acute lymphoblastic leukemia (B-ALL) for 4 h, the amount of IFNγ release in the CAR1-T group was 411.73±16.14 pg/mL, the amount of IFNγ released in the CAR2-T group was 349.41±43.09 pg/mL, the amount of IFNγ released in the UnT group was 88.66±2.42 pg/mL, and the amount of IFNγ released in the Luc group was 82.87±14.24 pg/mL; after co-incubation for 20 h, the amount of IFNγ released in the CAR1-T group was 1119.37±18.045 pg/mL, the amount of IFNγ released in the CAR2-T group was 1099.93±75.93 pg/mL, the amount of IFNγ released in the UnT group was 146.04 pg/mL, and the amount of IFNγ released in the Luc group was 236.45±57.50 pg/mL. Compared with the UnT and Luc control groups, both CAR1-T and CAR2-T can have a significant IFNγ release amount, and CAR1-T have a slightly higher IFNγ release level than the CAR2-T control group.

Example 6 Clinical Trial Treatment Effect of the CAR1 Transduced Autologous T Cells on the Patients with CD19-Positive B Cell Acute Lymphoblastic Leukemia

(66) The peripheral blood of patients with acute B cell lymphoblastic leukemia was collected. The T cells were separated and purified (Example 2), and were transduced with CAR1 lentivirus vectors after 2 days of in vitro activation. After transduction, centrifugation was performed, and the CAR1 lentivirus vector that did not enter the cells in the culture supernatant was washed away. The transduced T cells were resuspended in the culture medium containing IL-2 at a final concentration of 100 IU/mL and CD3/CD28 magnetic beads. On the next day, the expansion culture was performed at a culture density of 0.5-2×10.sup.6/ml for 11-14 days.

(67) CAR1 was transduced into T cells from 5 different B-ALL patients, respectively, and the transduced cells were named ALLCT01, ALLCT02, ALLCT03, ALLCT04 and ALLCT05, respectively. The results in FIG. 5 showed that all the cells transduced with CAR1 demonstrated strong ability of killing Raji.Luc target cells in vitro, and the relative number of the remaining Raji.Luc cells was 9.33%-37.82% after co-incubation with the Raji.Luc target cells.

(68) The following contents exemplify the CD19 CAR-T treatment in ALLCT01 patient with acute B lymphoblastic leukemia.

(69) ALLCT01 patient: through the examination in the hospital, the disease was manifested as active bone marrow hyperplasia, abnormal lymphocyte system hyperplasia, 65% of original lymphocytes, dominant mast cells, irregular edges, relative more cytoplasm, irregular karyotype, visible depression and folding, reduced granulocyte system proliferation, reduced red blood cell system proliferation, and rare platelets, which was clinically diagnosed as B-ALL (NR). After the hospital has made the recommendation and the ethical review has passed, the patient signed the informed consent form, and then the clinical trial study of CAR-T cell treatment was performed.

(70) The process of the CAR-T cell treatment was: collecting the peripheral blood of the patient, separating and purifying T cells in vitro, preparing CD19 CAR-T cells in vitro, autologously re-infusing into the patient intravenously at three time points. About 1.5×10.sup.8 cells was infused totally.

(71) After the ALLCT01 patients were re-infused with CD19 CAR-T cells, a relatively mild response occurred: the body temperature began to rise at day 6 after the last re-infusion; the cytokine test results showed that the secretion level of interleukin-6 (IL-6) was significantly increased (FIG. 6); and fever lasted for 6 days and the body temperature did not exceed 40° C. The symptomatic treatment was performed by infusing a small amount of IL-6R monoclonal antibody Tocilizumab 3 days after the fever, and then the body temperature gradually decreased and reached the normal body temperature after 4 days. However, generally the B-ALL patients who had received the CD19 CAR-T treatment had high fever within 3 days in the foreign reports. The results of this test indicate that the CD19 CAR (CAR1) of the present invention has a milder response in the treatment of B-ALL and has less side effects in patients.

(72) The peripheral blood of the patients was tested 14 days after the last re-infusion. As shown in the results of Table 2, the percentage of CD19-positive (CD19+) cells in peripheral blood decreased from 63% before treatment to 0%, and 83.59% of the cells were CD3-positive (CD3+) cells. Combined with the results of hematological detection, it was shown that the CD19-positive leukemia cells in the peripheral blood were completely eliminated after the patient was treated with the CD19 CAR-T cells provided in the present invention, and the clinical complete remission was achieved.

(73) TABLE-US-00002 TABLE 2 Changes of T and B lymphocytes in the hemogram before and after the CAR-T treatment in B-ALL patients Detection item Before Detection at day 14 CD19+ cells 62.80%    0% CD3+ cells   22% 83.59% CD3+ CD4+ cells 14.30% 54.01% CD3+ CD8+ cells 14.20% 22.72%

(74) The experimental methods not recorded in detail in the present application document are all conventional techniques in the art, and can be realized by the literature or technical means before the filing date.