CHIMERIC ANTIGEN RECEPTOR TARGETING CD7 AND USE THEREOF

Abstract

Provided is an engineered immune cell. The cell expresses a chimeric antigen receptor comprising an antigen-binding region. The antigen-binding region comprises an anti-CD7 antibody, and the expression of endogenous CD7, at least one TCR/CD3 gene, and at least one MHC-II related gene is suppressed or silenced. Further provided is the use of the engineered immune cell in the treatment of diseases associated with CD7 expression.

Claims

1. An engineered immune cell, (1) expressing a chimeric antigen receptor comprising an antigen-binding region, the antigen-binding region comprising an anti-CD7 antibody; and (2) having suppressed or silenced expression of endogenous CD7, at least one TCR/CD3 gene, and at least one MHC-II related gene.

2. The engineered immune cell according to claim 1, wherein the chimeric antigen receptor comprises the anti-CD7 antibody, a transmembrane domain, and an intracellular signaling domain.

3. The engineered immune cell according to claim 1, wherein the anti-CD7 antibody comprises CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 1, 2 and 3 respectively, and CDR-H1, CDR-H2 and CDR-H3 as set forth in SEQ ID NOs: 4, 5 and 6 respectively.

4. The engineered immune cell according to claim 1, wherein the antigen-binding region of the chimeric antigen receptor further comprises an antibody targeting a second antigen, or a functional fragment thereof, wherein the second antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal tract carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D and any combination thereof.

5. The engineered immune cell according to claim 1, wherein the chimeric antigen receptor comprises an anti-CD7 antibody and an anti-CD19 antibody.

6. The engineered immune cell according to claim 1, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

7. The engineered immune cell according to claim 1, wherein the intracellular signaling domain is an intracellular region of a protein selected from the group consisting of: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

8. The engineered immune cell according to claim 1, wherein the chimeric antigen receptor further comprises at least one co-stimulatory domain, which is an intracellular region of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70 and a combination thereof.

9. The engineered immune cell according to claim 1, wherein the TCR/CD3 gene is selected from the group consisting of TRAC, TRBC, CD3 γ, CD3 δ, CD3 ε, CD3 ζ and a combination thereof.

10. The engineered immune cell according to claim 1, wherein the MHC-II related gene is selected from the group consisting of: HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK, CIITA and a combination thereof.

11. The engineered immune cell according to claim 1, wherein the engineered immune cell has suppressed or silenced expression of endogenous CD7, at least one TCR/CD3 gene selected from the group consisting of TRAC and TRBC and at least one MHC class II gene selected from the group consisting of RFX5, RFXAP, RFXANK and CIITA.

12. The engineered immune cell according to claim 1, wherein the engineered immune cell further expresses a NK inhibitory molecule, and the NK inhibitory molecule comprises one or more NK inhibitory ligands, a transmembrane domain and at least one co-stimulatory domain.

13. The engineered immune cell according to claim 12, wherein the NK inhibitory ligand is an antibody targeting a NK inhibitory receptor or a functional fragment thereof.

14. The engineered immune cell according to claim 12, wherein the NK inhibitory ligand is HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1/PD-L2, CTLA-4, CD155, CD112, CD113, Gal-9, FGL1, or a NK inhibitory receptor binding region thereof.

15. The engineered immune cell according to claim 12, wherein the NK inhibitory molecule further comprises a CD3 ζ intracellular region as the intracellular signaling domain.

16. The engineered immune cell according to claim 1, wherein the engineered immune cell is a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell.

17. A pharmaceutical composition comprising the engineered immune cell according to claim 1, and one or more pharmaceutically acceptable excipients.

18. A method for treating a subject with a disease associated with CD7 expression, comprising administering to the subject an effective amount of the engineered immune cell according to claim 1.

19. The method according to claim 18, the disease associated with CD7 expression is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), T-lymphoblastic lymphoma (T-LBL), early pro-T lymphoblastic Leukemia (ETP-ALL) and extranodal NK/T cell lymphoma.

20. The engineered immune cell according to claim 13, wherein the NK inhibitory receptor is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0099] FIG. 1 shows the ratio of CD7+ cells and CD7−cells in peripheral blood lymphocytes of patients.

[0100] FIG. 2 shows the expression levels of scFv on the CAR7-dKO T cells and CAR7-tKO T cells.

[0101] FIG. 3 shows the killing ability of CAR7-dKO T cells and CAR7-tKO T cells on target cells.

[0102] FIG. 4 shows the cytokine release levels after CAR7-dKO T cells and CAR7-tKO T cells were co-cultured with target cells.

[0103] FIG. 5 shows the inhibitory effect of NK inhibitory molecules on NK cell killing ability. A: a NK inhibitory molecule comprises anti-KIR scFv; B: a NK inhibitory molecule comprises anti-LIR scFv; C: a NK inhibitory molecule comprises anti-NKG2A scFv, a HLA-G extracellular region, a HLA-E extracellular region or an E-Cad extracellular region.

[0104] FIG. 6 shows the killing effect of CD4+ T cells (A) and CD8+ T cells (B) expressing NK inhibitory molecules comprising CD3 ζ on NK cells.

[0105] FIG. 7 shows the expression levels of scFv on CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0106] FIG. 8 shows the expression levels of NK inhibitory molecules of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0107] FIG. 9 shows the killing ability of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells on target cells.

[0108] FIG. 10 shows the cytokine release levels after CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells were co-cultured with target cells.

[0109] FIG. 11 shows the expression levels of scFv on CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells.

[0110] FIG. 12 shows the expression level of HLA-E in CAR7-E T cells and the expression level of PDL1 in CAR7-PDL1 T cells.

[0111] FIG. 13 shows the expression levels of HLA-E and PDL1 in CAR7-EPDL1 T cells.

[0112] FIG. 14 shows the killing ability of CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells on target cells.

[0113] FIG. 15 shows the cytokine release levels after CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells were co-cultured with target cells.

[0114] FIG. 16 shows the expression levels of CD7 scFv and CD19 scFv in CAR7-19 T cells.

[0115] FIG. 17 shows the killing ability of CAR7-19 T cells on two target cells.

[0116] FIG. 18 shows the cytokine release levels after CAR7-19 T cells were co-cultured with two kinds of target cells.

EXAMPLE

Example 1. Preparation of Universal CAR T Cells Against CD7

[0117] Sequences encoding the following proteins were synthesized and cloned sequentially into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 28), CD3 ζ intracellular signaling domain (SEQ ID NO: 30), and the correct insertion of the target sequences was confirmed by sequencing.

[0118] Three ml Opti-MEM (Gibco, Cat. No. 31985-070) was added to a sterile tube to dilute the above plasmid, and then packaging vector psPAX2 (Addgene, Cat. No. 12260) and envelope vector pMD2.G (Addgene, Cat. No. 12259) were added according to the ratio of plasmid:viral packaging vector:viral envelope vector=4:2:1. Then, 120 μl X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001) was added, mixed immediately, and incubated at room temperature for 15 min, and then the plasmid/vector/transfection reagent mixture was added dropwise to the culture flask of 293T cells. Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4° C., 2.5 hours) to obtain concentrated lentiviruses.

[0119] Wild-type T cells were activated with DynaBeads CD3/CD28 CTS™ (Gibco, Cat. No. 40203D) and cultured at 37° C. and 5% CO.sub.2 for 1 day. Then the TCR/CD3 component (specifically TRAC gene), CD7 gene and optional MHC-II related gene (specifically RFX5) in the wild-type T cells were knocked out using the CRISPR system to obtain TCR/CD7 double knockout dKO-T cells and TCR/CD7/RFX5 triple knockout tKO-T cells. Wild-type T cells without gene knockout (i.e., NT cells) were used as controls.

[0120] Using FITC Mouse Anti-Human CD3 ζ BD Pharmingen, Cat. No. 555916) antibody, PE mouse anti-human CD7 (biolegend Cat. No. 395604) and APC anti-human DR, DP, DQ (biolegend, Cat. No. 361714) antibodies, the gene editing efficiency of TCR/CD7/RFX5 in T cells was detected by flow cytometry, and the results are shown in Table 1.

TABLE-US-00001 TABLE 1 Gene expression efficiency in T cells Name TCR/CD3 CD7 RFX5/MHC-II dKO-T 2.2%  5.3% 92% tKO-T  3% 8.9% 11.2%.sup.  NT 98%  94% 92%

[0121] It can be seen from Table 1 that the expression of related genes in dKO-T cells and tKO-T cells prepared in the present disclosure is effectively suppressed or silenced.

[0122] The concentrated lentivirus was added to the dKO-T cells and tKO-T cells to obtain CAR7-dKO T cells and CAR7-tKO T cells. Using Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab′).sub.2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) as the primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, the expression levels of scFv on the CAR7-dKO T cells and CAR7-tKO T cells were detected by flow cytometry, and the results are shown in FIG. 2.

[0123] It can be seen that the scFv in the CAR T cells prepared by the present disclosure can be effectively expressed.

Example 2. Killing Effect of CAR T Cells on Target Cells and Release of Cytokines Thereof

[0124] 2.1 Killing Effect of CAR-T Cells on Target Cells

[0125] In order to detect the killing ability of CAR-T cells on target cells, firstly Jurkat target cells carrying the fluorescein gene were plated into a 96-well plate at 1×10.sup.4/well, and then CAR T cells and NT cells were plated into the 96-well plate with effector-target ratios (i.e., the ratio of effector T cells to target cells) of 0.5:1, 0.25:1 and 0.125:1 for co-culture, and the fluorescence value was measured with a microplate reader after 16-18 hours. According to the calculation formula: (average fluorescence value of target cells−average fluorescence value of samples)/average fluorescence value of target cells×100%, the killing efficiency was calculated, and the results are shown in FIG. 3.

[0126] It can be seen that compared with NT, both CAR7-dKO T cells and CAR7-tKO T cells have specific killing ability on target cells, and the killing ability of CAR7-tKO T cells is higher than that of CAR7-dKO T cells.

[0127] 2.2 Cytokine Release of CAR-T Cells

[0128] When T cells kill target cells, cytokines are released while the number of target cells is reduced. According to the following steps, enzyme-linked immunosorbent assay (ELISA) was used to measure the release level of cytokine IFNγ when the CAR T cells of the present disclosure kill target cells.

[0129] (1) Collection of Cell Co-Culture Supernatant

[0130] Jurkat target cells were plated in a 96-well plate at 1×10.sup.5/well, and then CAR T and NT cells (negative control) were co-cultured with the target cells at a ratio of 0.125:1, and the cell co-culture supernatant was collected after 18-24 hours.

[0131] (2) Detection of the Secretion of IFN γ in the Supernatant by ELISA

[0132] A 96-well plate was coated with Purified anti-human IFN-γ Antibody (Biolegend, Cat. No. 506502) as capture antibody and incubated overnight at 4° C., and then the antibody solution was removed. 250 μL of PBST (1×PBS comprising 0.1% Tween) solution comprising 2% BSA (sigma, Cat. No. V900933-1 kg) was added, and incubated at 37° C. for 2 hours. The plate was then washed 3 times with 250 μL of PBST (1×PBS comprising 0.1% Tween). 50 μL of cell co-culture supernatant or standard per well was added and incubated at 37° C. for 1 hour, then the plate was washed 3 times with 250 μL of PBST (1×PBS comprising 0.1% Tween). Then 50 μL of Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, Cat. No. ab25017) as detection antibody, was added to each well, incubated at 37° C. for 1 hour, and the plate was washed 3 times with 250 μL of PBST (1×PBS comprising 0.1% Tween). Then HRP Streptavidin (Biolegend, Cat. No. 405210) was added, incubated at 37° C. for 30 minutes, and the supernatant was discarded. 250 μL of PBST (1×PBS comprising 0.1% Tween) was added for washing 5 times. 50 μL of TMB substrate solution was added to each well. Reactions were allowed to occur at room temperature in the dark for 30 minutes, after which 50 μL of 1 mol/L H.sub.2SO.sub.4 was added to each well to terminate the reaction. Within 30 minutes after the reaction termination, a microplate reader was used to detect the absorbance at 450 nm, and the content of cytokines was calculated according to the standard curve (drawn according to the reading value and concentration of the standard), the results are shown in FIG. 4.

[0133] It can be seen that the cytokine release of CAR7-dKO T cells and CAR7-tKO T cells to target cells was significantly higher than that of the control NT group, and the release level of CAR7-tKO T cells was higher than that of CAR7-dKO T cells.

[0134] From the above results, it can be seen that the killing ability of CAR7-tKO T cells on target cells and the release level of cytokines are higher than those of CAR7-dKO T cells, indicating that knockout of MHC-II genes enhances CAR-T cell killing activity. This is unexpected, because existing reports generally believe that the expression of MHC-II genes is related to immune rejection, and there has not been any report on the relationship between MHC-II genes and the activity of CAR-T cells themselves.

Example 3. Inhibitory Effect of NK Inhibitory Molecules on NK Cell Killing Activity

[0135] The coding sequences of the following proteins were synthesized and cloned sequentially into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), wherein the NK inhibitory ligand is an extracellular region of E-cadherin (SEQ ID NO: 47, corresponding to the ECad0 plasmid), a fusion molecule of B2M and HLA-E extracellular region (comprising presenting peptide SEQ ID NO: 75, B2M SEQ ID NO: 74 and HLA-E extracellular region mutant SEQ ID NO: 51, wherein the coding sequence of B2M, SEQ ID NO: 82, comprises a synonymous mutation, corresponding to the E0 plasmid) or a fusion molecule of B2M and HLA-G extracellular region (comprising B2M SEQ ID NO: 74 and HLA-G extracellular region SEQ ID NO: 49, wherein the coding sequence of B2M, SEQ ID NO: 82, comprises a synonymous mutation, corresponding to the G0 plasmid). The CD28 co-stimulatory domain (SEQ ID NO: 26) was further included in the ECad0, E0 and G0 plasmids to obtain the ECad28, E28 and G28 plasmids, respectively. The correct insertion of the target sequences in the plasmid was confirmed by sequencing.

[0136] The coding sequences of the following proteins were synthesized and cloned sequentially into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, IgG4 hinge region (SEQ ID NO: 42), CD8α transmembrane region (SEQ ID NO: 22), CD28 co-stimulatory domain (SEQ ID NO: 26), wherein the NK inhibitory ligand is anti-NKG2A scFv (SEQ ID NO: 63, corresponding to A28 plasmid), anti-KIR scFv (SEQ ID NO: 67, corresponding to KIRG4 plasmid) or anti-LIR1 scFv (SEQ ID NO: 70, corresponding to LIR1-1 plasmid). The correct insertion of the target sequences in the plasmid was confirmed by sequencing.

[0137] The coding sequences of the following proteins were synthesized and cloned sequentially into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 36), anti-LIR1 scFv (SEQ ID NO: 73), CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), 4-1BB co-stimulatory domain (SEQ ID NO: 28), to obtain a LIR1-2 plasmid. The correct insertion of the target sequences in the plasmid was confirmed by sequencing.

[0138] Three ml Opti-MEM (Gibco, Cat. No. 31985-070) was added to a sterile tube to dilute the above plasmid, and then packaging vector psPAX2 (Addgene, Cat. No. 12260) and envelope vector pMD2.G (Addgene, Cat. No. 12259) were added according to the ratio of plasmid:viral packaging vector:viral envelope vector=4:2:1. Then, 120 μl X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001) was added, mixed immediately, and incubated at room temperature for 15 min, and then the plasmid/vector/transfection reagent mixture was added dropwise to the culture flask of 293T cells. Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4° C., 2.5 hours) to obtain concentrated lentiviruses.

[0139] T cells were activated with DynaBeads CD3/CD28 CTS™ (Gibco, Cat. No. 40203D) and cultured at 37° C. and 5% CO.sub.2 for 1 day. Then, the concentrated lentivirus was added, and after continuous culture for 3 days, T cells expressing NK inhibitory molecules, i.e., UNKi-T cells, were obtained.

[0140] Then the TCR/CD3 component (specifically TRAC gene) and MHC-related genes (specifically B2M and RFX5) in wild-type T cells (Mock T cells, used as a control) and the UNKi-T cells were knocked out using the CRISPR system, and it is confirmed that each gene was effectively knocked out by flow cytometry.

[0141] Then, the inhibitory effect of the UNKi-T cells prepared in the present disclosure on the killing effect of NK cells was detected according to the following method: the UNKi-T cells and Mock-T cells prepared in the present disclosure were labeled with Far-Red (invitrogen, Cat. No. C34564). Then the labeled UNKi-T cells and Mock T cells were plated into 96-well plates at a concentration of 1×10.sup.4 cells/well, and NK92 cells (for UNKi-T cells expressing HLA-E extracellular region, HLA-G extracellular region, anti-NKG2A scFv, anti-KIR scFv or anti-LIR1 scFv and Mock T cells) or NK92-KLRG1 cells (for UNKi-T cells expressing E-cadherin extracellular region, prepared by introducing the KLRG1 gene into NK92 cells) were added at an effector-target ratio of 2:1 for co-culture. After 16-18 hours, the proportion of T cells in the culture was detected by flow cytometry, and then the killing effect of NK cells on T cells was calculated. The results are shown in FIG. 5.

[0142] It can be seen from FIG. 5 that compared with Mock T cells that do not express NK inhibitory molecules, UNKi-T cells expressing inhibitory ligands such as anti-KIR scFv, anti-LIR1 scFv, anti-NKG2A scFv, HLA-G extracellular region, HLA-E extracellular region and E-cadherin extracellular region can significantly reduce the killing effect of NK cells on T cells. In addition, compared with T cells (G0, E0, Ecad0) expressing only an inhibitory ligand and a transmembrane domain (i.e., not comprising a co-stimulatory domain), the addition of the co-stimulatory domain can further significantly enhance the inhibition of killing effect of NK cell on T cells (G28, E28, ECad28). Therefore, the NK inhibitory molecules comprising an inhibitory ligand, a transmembrane domain and a co-stimulatory domain of the present disclosure can significantly reduce the killing effect of NK cells on UNKi-T cells, thereby effectively reducing the risk of HvGD.

[0143] In addition, in some cases, it is not only necessary to inhibit the killing effect of NK cells on CAR-T cells, but even further requires T cells to kill NK cells. Therefore, the inventors further included the CD3 ζ intracellular signaling domain (SEQ ID NO: 30) on the basis of the E28 plasmid and A28 plasmid cells, and packaged them as lentivirus according to the above method, and infected T cells in which TCR/CD3 components (specifically TRAC gene) and MHC-related genes (specifically B2M and RFX5) were effectively knocked out, to obtain E28z-UNKi-T cells and A28z-UNKi-T cells.

[0144] The killing of NK cells by UNKi-T cells was detected by the following method: the target cells (NK92 cells) were plated in a 96-well plate at a concentration of 1×10.sup.5 cells/well, and then Mock T cells, E28z-UNKi-T cells and A28z-UNKi-T cells were added to each well at a ratio of 1:1, and at the same time, 10 μl of PE-anti-human CD107a (BD Pharmingen, Cat. No. 555801) was added for co-culturing at 37° C. and 5% CO.sub.2. After 1 hour, Goigstop (BD Pharmingen, Cat. No. 51-2092KZ) was added to continue incubation for 2.5 hours. Then 5 μl APC-anti human CD8 (BD Pharmingen, Cat. No.: 555369) and 5 μl FITC-anti human CD4 (BD Pharmingen, Cat. No.: 561005) were added to each well, and incubated at 37° C. for 30 minutes. The expression of CD107a was detected by flow cytometry, and the results are shown in FIG. 6A (CD4+ T cell toxicity) and FIG. 6B (CD8+ T cell toxicity).

[0145] It can be seen that Mock T cells that do not express NK inhibitory molecules hardly kill target cells. On the contrary, after the E28z-UNKi-T cells and A28z-UNKi-T cells prepared by the present disclosure were co-cultured with the target cells, the expression rate of CD107a was significantly increased, indicating that the UNKi-T cells of the present disclosure can significantly kill NK cells.

Example 4. Preparation of Universal CAR T Cells Expressing NK Inhibitory Molecules

[0146] Sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), CD3 ζ intracellular signaling domain (SEQ ID NO: 30), F2A, E-cadherin extracellular region (SEQ ID NO: 48), CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24) and CD28 intracellular region (SEQ ID NO: 26), and the correct insertion of the target sequences was confirmed by sequencing.

[0147] Three ml Opti-MEM (Gibco, Cat. No. 31985-070) was added to a sterile tube to dilute the above plasmid, and then packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029) was added according to the ratio of plasmid:viral packaging vector=3:1. Then, 120 μl X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001) was added, mixed immediately, and incubated at room temperature for 15 min, and then the plasmid/vector/transfection reagent mixture was added dropwise to the culture flask of 293 GP cells. Viruses were collected at 72 hours and 96 hours, pooled, centrifuged (2000 rpm, 4° C., 10 min) to remove fragments and obtain retroviral supernatant.

[0148] TCR/CD7 double-knockout dKO T cells and TCR/CD7/RFX5 triple-knockout tKO T cells were prepared according to the knockout method in Example 1. Wild-type T cells without gene knockout (i.e., NT cells) were used as controls.

[0149] A 24-well plate was coated with Retronectin and incubated overnight at 4° C. Then the solution was removed, and 300 μL of PBS solution comprising 5% FBS (Gibco, Cat. No.) was added and left at room temperature for 30 min. The supernatant was then removed and the plate was washed 2 times with 1 mL of PBS. 2 mL retrovirus supernatant and 0.5M dKO-T cells or tKO-T cells were added to each well, centrifuged at 2000 g and 32° C. for 2h and cultured in a carbon dioxide incubator to obtain CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0150] After 7 days of culture, using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab′).sub.2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) as the primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, the expression levels of CD7 scFv in the CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells were detected by flow cytometry, and the results are shown in FIG. 7. The expression of E-Cadherin in CAR T cells was detected using E-cadherin monoclonal antibody (Invitrogen, Cat. No. 13-5700) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, Cat. No. A-11001), and the results are shown in FIG. 8.

[0151] It can be seen that both anti-CD7 scFv and NKi inhibitory molecules can be effectively expressed in the CAR T cells prepared in the present disclosure.

[0152] The killing effect of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells on Jurkat target cells was detected according to the method described in 2.1 of Example 2, and the results are shown in FIG. 9. It can be seen that the two CAR-T cells can significantly kill target cells at various effector-target ratios, and at the effector-target ratio of 0.125:1, the killing effect of CAR7-NKi-tKO T cells is better than that of CAR7-NKi-dKO T cells.

[0153] The cytokine release levels after CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells were co-cultured with Jurkat target cells were detected according to the method described in 2.2 of Example 2, and the results are shown in FIG. 10. It can be seen that the cytokine release levels of the CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells of the present disclosure were significantly higher than those of the control NT cells, and the release level of CAR7-NKi-tKO T cell group was significantly higher than that of CAR7-NKi-dKO T cell group.

Example 5. Preparation of Universal CAR T Cells Expressing NK Inhibitory Molecules and Verification of their Functions

[0154] Sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD28 hinge region (SEQ ID NO: 40), CD8α transmembrane region (SEQ ID NO: 22), CD28 intracellular region (SEQ ID NO: 26), CD3 ζ intracellular signaling domain (SEQ ID NO: 32), F2A, PD-L1 signal peptide (SEQ ID NO: 44), PD-L1 extracellular region (SEQ ID NO: 45), CD28 transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 28), and the correct insertion of the target sequences was confirmed by sequencing (plasmid designation: CAR7-PDL1).

[0155] Sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD28 transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 28), CD3 ζ intracellular signaling domain (SEQ ID NO: 32), F2A, B2M signal peptide (SEQ ID NO: 34), HLA-E extracellular region (SEQ ID NO: 50), CD28 transmembrane region (SEQ ID NO: 24), CD28 intracellular region (SEQ ID NO: 26), and the correct insertion of the target sequences was confirmed by sequencing (Plasmid designation: CAR7-E).

[0156] Sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), CD3 ζ intracellular signaling domain (SEQ ID NO: 32), F2A, B2M signal peptide (SEQ ID NO: 34), HLA-E extracellular region (SEQ ID NO: 50), linker peptide (SEQ ID NO: 64), PD-L1 extracellular region (SEQ ID NO: 45), CD28 transmembrane region (SEQ ID NO: 24), CD28 intracellular region (SEQ ID NO: 26), and the correct insertion of the target sequences was confirmed by sequencing (plasmid designation: CAR7-EPDL1).

[0157] According to the method described in Example 3, the above plasmids were packaged into retroviruses and infected into tKO-T cells to obtain CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells, respectively.

[0158] After 7 days of culture, using Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab′).sub.2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) as the primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, the expression levels of scFv in the three cells were detected by flow cytometry, and the results are shown in FIG. 11. The expression of HLA-E and PDL1 in CAR T cells was detected using PE mouse anti-human HLA-E (biolegend, Cat. No. 342604) and PE anti-human PDL1 (biolegend, Cat. No. 329706), respectively, and the results are shown in FIG. 12 and FIG. 13.

[0159] It can be seen that both anti-CD7 scFv and NK inhibitory molecules (HLA-E and PD-L1) can be effectively expressed in the CAR T cells prepared in the present disclosure.

[0160] According to the method described in 2.1 of Example 2, the killing effect of three CAR-T cells on Jurkat target cells was detected, and the results are shown in FIG. 14. It can be seen that all three CAR-T cells can significantly kill target cells.

[0161] According to the method described in 2.2 of Example 2, the cytokine release levels after the three CAR-T cells were co-cultured with Jurkat target cells were detected, and the results are shown in FIG. 15. Compared with the NT group, the cytokine release levels of the three CAR-T cells were significantly increased.

Example 6. Preparation of Dual-Target CAR-T Cells and Verification of their Functions

[0162] Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 20), linker peptide (SEQ ID NO: 64), anti-CD19 scFv (SEQ ID NO: 54), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), CD3 ζ intracellular signaling domain (SEQ ID NO: 32), and the correct insertion of the target sequences was confirmed by sequencing.

[0163] According to the method of Example 1, the above plasmid vectors were packaged into lentivirus, and infected into tkO-T cells to obtain CAR7-19 T cells. Unmodified wild-type T cells were used as negative controls (NT).

[0164] Using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab′).sub.2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) or Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab′).sub.2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) as the primary antibody, FITC Streptavidin (BD Pharmingen, Cat. No. 554060) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, the expression level of scFv in CAR7-19 T cells was detected by flow cytometry, and the results are shown in FIG. 16.

[0165] It can be seen that both CD7 scFv and CD19 scFv can be effectively expressed in the CAR T cells prepared in the present disclosure.

[0166] According to the method in Example 2, the killing function and cytokine release level of CAR7-19 T cells were detected, and the results are shown in FIG. 17 and FIG. 18, respectively. It can be seen that CAR7-19 T cells have specific killing effect on both Nalm6 and Jurkat target cells and significantly increased cytokine release.

[0167] It should be noted that the above-mentioned are merely for preferred examples of the present disclosure and not used to limit the present disclosure. For one skilled in the art, various modifications and changes may be made to the present disclosure. Those skilled in the art should understand that any amendments, equivalent replacements, improvements, and so on, made within the spirit and principle of the present disclosure, should be covered within the scope of protection of the present disclosure.