ENGINEERED IMMUNE CELL EXPRESSING NK INHIBITORY MOLECULE AND USE THEREOF

Abstract

Provided is an NK inhibitory molecule, which includes one or more NK inhibitory ligands, a transmembrane domain and a co-stimulatory domain, wherein the NK inhibitory ligand specifically binds to NK inhibitory receptors to inhibit the killing of NK cells against the engineered immune cells expressing the NK inhibitory molecule. Also provided is an engineered immune cell expressing the NK inhibitory molecule of the present invention, wherein the expression of at least one MHC-related gene is suppressed or silenced. Further provided is the use of the engineered immune cell in the treatment of cancers, infections or autoimmune diseases. Compared to the traditional engineered immune cells, the engineered immune cell can significantly inhibit the killing effect of NK cells in a subject, thereby reducing the risk of HvGD.

Claims

1. An NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a co-stimulatory domain, wherein the one or more NK inhibitory ligands specifically bind to NK inhibitory receptors (NKIR) to inhibit killing of NK cells against an engineered immune cell expressing the NK inhibitory molecule.

2. The NK inhibitory molecule according to claim 1, wherein the NK inhibitory ligand is an antibody targeting NKIR, or a natural ligand of NKIR or an NKIR binding region contained therein.

3. The NK inhibitory molecule according to claim 1, wherein the NKIR is selected from the group consisting of an NKG2/CD94 component, a killer cell Ig-like receptor (KIR) family member, a leukocyte Ig-like receptor (LIR) family member, an NK cell receptor protein 1 (NKR-P1) family member, an immune checkpoint receptor, a carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), a sialic acid-binding immunoglobulin-like lectin (SIGLEC) family member, an leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), an Ly49 family member and a killer cell lectin-like receptor G1 (KLRG1).

4. The NK inhibitory molecule according to claim 3, wherein the NKG2/CD94 component is selected from the group consisting of NKG2A, NKG2B, and CD94; the KIR family member is selected from the group consisting of KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3; the LIR family member is selected from the group consisting of LIR1, LIR2, LIR3, LIR5, and LIR8; the NKR-P1 family member is selected from the group consisting of NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from the group consisting of PD-1, TIGIT, CD96, TIM3, and LAG3; the SIGLEC family member is selected from the group consisting of SIGLEC7 and SIGLEC9; and the Ly49 family member is selected from the group consisting of Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4.

5. The NK inhibitory molecule according to claim 3, wherein the NKIR is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, PD1, LAIR1, SIGLEC7, SIGLEC9, and KLRG1.

6. The NK inhibitory molecule according to claim 1, wherein the NK inhibitory ligand is an antibody targeting NKIR or a functional fragment thereof, and the antibody or the functional fragment thereof is selected from the group consisting of an intact antibody, Fab, Fab′, F(ab′)2, an Fv fragment, an scFv antibody fragment, a linear antibody, sdAb or a nanobody.

7. The NK inhibitory molecule according to claim 1, wherein the NK inhibitory ligand is an antibody targeting PD1, NKG2A, LIR1, KIR, SIGLEC7, SIGLEC9 and/or KLRG1.

8. The NK inhibitory molecule according to claim 7, wherein (i) the antibody targeting NKG2A comprises (1) CDR-L1 as represented by SEQ ID NO: 72, CDR-L2 as represented by SEQ ID NO: 73, CDR-L3 as represented by SEQ ID NO: 74, CDR-H1 as represented by SEQ ID NO: 75, CDR-H2 as represented by SEQ ID NO: 76, and CDR-H3 as represented by SEQ ID NO: 77, or (2) CDR-L1 as represented by SEQ ID NO: 78, CDR-L2 as represented by SEQ ID NO: 79, CDR-L3 as represented by SEQ ID NO: 80, CDR-H1 as represented by SEQ ID NO: 81, CDR-H2 as represented by SEQ ID NO: 82, and CDR-H3 as represented by SEQ ID NO: 83; (ii) the antibody targeting LIR1 comprises (1) CDR-L1 as represented by SEQ ID NO: 90, CDR-L2 as represented by SEQ ID NO: 91, CDR-L3 as represented by SEQ ID NO: 92, CDR-H1 as represented by SEQ ID NO: 93, CDR-H2 as represented by SEQ ID NO: 94, and CDR-H3 as represented by SEQ ID NO: 95, or (2) CDR-L1 as represented by SEQ ID NO: 96, CDR-L2 as represented by SEQ ID NO: 97, CDR-L3 as represented by SEQ ID NO: 98, CDR-H1 as represented by SEQ ID NO: 99, CDR-H2 as represented by SEQ ID NO: 100, and CDR-H3 as represented by SEQ ID NO: 101; (iii) the antibody targeting KIR comprises CDR-L1 as represented by SEQ ID NO: 84, CDR-L2 as represented by SEQ ID NO: 85, CDR-L3 as represented by SEQ ID NO: 86, CDR-H1 as represented by SEQ ID NO: 87, CDR-H2 as represented by SEQ ID NO: 88, and CDR-H3 as represented by SEQ ID NO: 89; (iv) the antibody targeting SIGLEC7, SIGLEC9 or both comprises (1) CDR-L1 as represented by SEQ ID NO: 102, CDR-L2 as represented by SEQ ID NO: 103, CDR-L3 as represented by SEQ ID NO: 104, CDR-H1 as represented by SEQ ID NO: 105, CDR-H2 as represented by SEQ ID NO: 106, and CDR-H3 as represented by SEQ ID NO: 107, (2) CDR-L1 as represented by SEQ ID NO: 122, CDR-L2 as represented by SEQ ID NO: 123, CDR-L3 as represented by SEQ ID NO: 124, CDR-H1 as represented by SEQ ID NO: 125, CDR-H2 as represented by SEQ ID NO: 126 and CDR-H3 as represented by SEQ ID NO: 127, (3) CDR-L1 as represented by SEQ ID NO: 131, CDR-L2 as represented by SEQ ID NO: 132, CDR-L3 as represented by SEQ ID NO: 133, CDR-H1 as represented by SEQ ID NO: 134, CDR-H2 as represented by SEQ ID NO: 135 and CDR-H3 as represented by SEQ ID NO: 136, (4) CDR-L1 as represented by SEQ ID NO: 140, CDR-L2 as represented by SEQ ID NO: 141, CDR-L3 as represented by SEQ ID NO: 142, CDR-H1 as represented by SEQ ID NO: 143, CDR-H2 as represented by SEQ ID NO: 144, and CDR-H3 as represented by SEQ ID NO: 155, (5) CDR-L1 as represented by SEQ ID NO: 176, CDR-L2 as represented by SEQ ID NO: 177, CDR-L3 as represented by SEQ ID NO: 178, CDR-H1 as represented by SEQ ID NO: 179, CDR-H2 as represented by SEQ ID NO: 180 and CDR-H3 as represented by SEQ ID NO: 181, or (6) CDR-L1 as represented by SEQ ID NO: 188, CDR-L2 as represented by SEQ ID NO: 189, CDR-L3 as represented by SEQ ID NO: 190, CDR-H1 as represented by SEQ ID NO: 191, CDR-H2 as represented by SEQ ID NO: 192, and CDR-H3 as represented by SEQ ID NO: 193; and/or (v) the antibody targeting KLRG1 comprises (1) CDR-L1 as represented by SEQ ID NO: 111, CDR-L2 as represented by SEQ ID NO: 112, CDR-L3 as represented by SEQ ID NO: 113, CDR-H1 as represented by SEQ ID NO: 114, CDR-H2 as represented by SEQ ID NO: 115, and CDR-H3 as represented by SEQ ID NO: 116, (2) CDR-L1 as represented by SEQ ID NO: 149, CDR-L2 as represented by SEQ ID NO: 150, CDR-L3 as represented by SEQ ID NO: 151, CDR-H1 as represented by SEQ ID NO: 152, CDR-H2 as represented by SEQ ID NO: 153 and CDR-H3 as represented by SEQ ID NO: 154, (3) CDR-L1 as represented by SEQ ID NO: 158, CDR-L2 as represented by SEQ ID NO: 159, CDR-L3 as represented by SEQ ID NO: 160, CDR-H1 as represented by SEQ ID NO: 161, CDR-H2 as represented by SEQ ID NO: 162, and CDR-H3 as represented by SEQ ID NO: 163, or (4) CDR-L1 as represented by SEQ ID NO: 167, CDR-L2 as represented by SEQ ID NO: 168, CDR-L3 as represented by SEQ ID NO: 169, CDR-H1 as represented by SEQ ID NO: 170, CDR-H2 as represented by SEQ ID NO: 171, and CDR-H3 as represented by SEQ ID NO: 172.

9. The NK inhibitory molecule according to claim 1, wherein the NK inhibitory ligand is selected from the group consisting of HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1/PD-L2, CD155, CD 112, CD113, Gal-9, FGL1, and an NKIR binding region contained therein.

10. The NK inhibitory molecule according to claim 9, wherein the NK inhibitory ligand is selected from the group consisting of sialic acid, HLA-E extracellular region, HLA-F extracellular region, HLA-G extracellular region, E-cadherin extracellular region, PD-L1 extracellular region, and PD-L2 extracellular region.

11. The NK inhibitory molecule according to claim 10, wherein (i) the HLA-E extracellular region has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 31 or 33; (ii) the HLA-G extracellular region has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 35; (iii) the E-cadherin extracellular region has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 39 or 41; (iv) the PD-L1 extracellular region has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 70; and (v) the PD-L2 extracellular region has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 71.

12. The NK inhibitory molecule 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, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HLA-E, HLA-F, HLA-G, cadherin, collagen, and OCIL.

13. The NK inhibitory molecule according to claim 1, wherein the co-stimulatory domain is a co-stimulatory signaling domain of a protein selected from the group consisting of LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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.

14. The NK inhibitory molecule according to claim 1, wherein the NK inhibitory molecule does not contain an intracellular signaling domain.

15. The NK inhibitory molecule according to claim 1, wherein the NK inhibitory molecule further contains an intracellular signaling domain.

16. The NK inhibitory molecule according to claim 14, wherein the intracellular signaling domain is a signaling domain of a protein selected from the group consisting of: FcR γ, FcR J, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

17.-19. (canceled)

20. An engineered immune cell, (1) expressing the NK inhibitory molecule according to claim 1, and (2) with the expression of at least one MHC-related gene being suppressed or silenced.

21. The engineered immune cell according to claim 20, wherein the engineered immune cell further expresses a chimeric antigen receptor, and the chimeric antigen receptor comprises a ligand binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

22. The engineered immune cell according to claim 21, wherein the NK inhibitory molecule is in the form of a fusion protein with a chimeric antigen receptor, wherein the fusion protein comprises an NK inhibitory ligand, a ligand binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

23. The engineered immune cell according to claim 20, wherein the at least one MHC-related gene is selected from the group consisting of HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and a combination thereof.

24. The engineered immune cell according to claim 23, wherein the at least one MHC-related gene is selected from the group consisting of B2M, RFX5, RFXAP, RFXANK, CIITA, and a combination thereof.

25. The engineered immune cell according to claim 24, wherein the MHC-related gene comprises B2M, wherein the NK inhibitory ligand is a fusion molecule of B2M and an extracellular region of non-classical HLA-class I molecule.

26. The engineered immune cell according to claim 25, wherein the non-classical HLA-class I molecule is HLA-E or HLA-G.

27. (canceled)

28. The engineered immune cell according to claim 20, wherein expression of at least one TCR/CD3 gene of the engineered immune cell is suppressed or silenced, and 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.

29. (canceled)

30. The engineered immune cell according to claim 21, wherein the ligand binding domain binds to a target selected from the group consisting of: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, tEGFR, GD2, GD3, BCMA, GPRC5D, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-22Ra, IL-2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, ber-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor R, 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, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, MAGE-A4, MART-1, WT-1, 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, TGFβ, APRIL, NKG2D, NKG2DL, and any combination thereof.

31. (canceled)

32. The engineered immune cell according to claim 20, wherein the engineered immune cell is a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.

33. The engineered immune cell according to claim 32, wherein the engineered immune cell is a CD4+/CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell, or an αβ-T cell.

34. A pharmaceutical composition, which contains the NK inhibitory molecule according to claim 1 and one or more pharmaceutically acceptable excipients.

35. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0143] FIG. 1: Expression levels of HLA-E in E0-UNKi-T and E28-UNKi-T cells are shown.

[0144] FIG. 2: Expression levels of HLA-G in G0-UNKi-T and G28-UNKi-T cells are shown.

[0145] FIG. 3: Expression levels of E-cadherin in ECad0-UNKi-T and ECad28-UNKi-T cells are shown.

[0146] FIG. 4: Expression level of NKG2A scFv in A28-UNKi-T cells is shown.

[0147] FIG. 5: Expression level of KLRG1 in NK92-KLRG1 cells is shown.

[0148] FIG. 6: Inhibitory effect of UNKi-T cells of the present disclosure on NK cell killing effect is shown. Two-way ANOVA is used for analysis, and T test is used for statistical analysis. * indicates that P value is less than 0.05, ** indicates that P value is less than 0.01, *** indicates that P value is less than 0.001, reaching significant levels.

[0149] FIG. 7: Expression level of NKG2A scFv in A28z-UNKi-T cells is shown.

[0150] FIG. 8: Expression level of HLA-E in E28z-UNKi-T cells is shown.

[0151] FIG. 9: Killing effect of UNKi-T cells of the present disclosure on NK cells is shown. Two-way ANOVA is used for analysis, and T test is used for statistical analysis. * indicates that P value is less than 0.05, ** indicates that P value is less than 0.01, *** indicates that P value is less than 0.001, reaching significant levels.

[0152] FIG. 10: IFN-γ release levels after co-culture of UNKi-T cells of the present disclosure with NK cells are shown. Two-way ANOVA is used for analysis, and T test is used for statistical analysis. *** indicates that P value is less than 0.001, reaching significant levels.

[0153] FIG. 11: Expression level of KIR scFv in KIRG4-UNKi-T cells of the present disclosure is shown.

[0154] FIG. 12: Expression levels of LIR1 scFv in LIRG4-UNKi-1-T and LIRG4-UNKi-2-T cells of the present disclosure are shown.

[0155] FIG. 13: Inhibitory effect of UNKi-T cells of the present disclosure on NK cell killing effect is shown.

[0156] FIG. 14: Expression levels of scFv in SC7G4-T cells, SC7/SC9G4-T cells, and K1G4-T cells are shown.

[0157] FIG. 15: Inhibitory effect of SC7G4-T cells, SC7/SC9G4-T cells, and K1G4-T cells on NK cell killing effect is shown.

[0158] FIG. 16: Expression level of PDL1 in PDL1-T cells is shown.

[0159] FIG. 17: Inhibitory effect of PDL1-T cells on NK cell proliferation is shown.

[0160] FIG. 18: Expression levels of KIR scFv in NKi-B cells and NKi-Huh7 cells are shown.

[0161] FIG. 19: Inhibitory effects of NKi-B cells and NKi-Huh7 cells on NK cell killing effect are shown.

DETAILED DESCRIPTION OF EMBODIMENTS

[0162] The T cells used in all the examples of the present disclosure are primary human CD4+CD8+ T cells isolated from healthy donors by Ficoll-Paque™ PREMIUM (GE Healthcare, Lot No. 17-5442-02) using leukapheresis.

Example 1: Construction of UNKi-T Immune Cells Expressing NK Inhibitory Molecules and with TCR/HLA-I/HLA-H being Knocked Out

[0163] The following encoding sequences were synthesized, and sequentially cloned into a pGEM-T Easy vector (Promega, Lot No. A1360): B2m signal peptide (SEQ ID NO: 21), NK inhibitory ligand, CD28 hinge region (SEQ ID NO: 27), CD28 transmembrane region (SEQ ID NO: 11), wherein the NK inhibitory ligand was an extracellular region of E-cadherin (SEQ ID NO: 41, corresponding to ECad0 plasmid), a fusion molecule of B2M and HLA-E extracellular region (containing presenting peptide of SEQ ID NO: 46, B2M of SEQ ID NO: 37, and HLA-E extracellular region mutant of SEQ ID NO: 33, wherein a nucleic acid sequence of B2M was synonymously mutated SEQ ID NO: 38, corresponding to E0 plasmid), or a fusion molecule of B2M and HLA-G extracellular region (containing B2M of SEQ ID NO: 37 and HLA-G extracellular region of SEQ ID NO: 35, wherein the nucleic acid sequence of B2M was the synonymously mutated SEQ ID NO: 38, corresponding to G0 plasmid). A CD28 co-stimulatory domain (SEQ ID NO: 13) was further included in the ECad0, E0, and G0 plasmids to obtain ECad28, E28, and G28 plasmids, respectively. Correct insertion of the target sequences into the plasmids was confirmed by sequencing.

[0164] The following encoding sequences were synthesized, and sequentially cloned into pGEM-T Easy vector (Promega, Lot No. A1360): B2m signal peptide (SEQ ID NO: 21), anti-NKG2A-scFv (containing SEQ ID NOS: 5 and 7), IgG4 hinge region (SEQ ID NO: 29), CD28 transmembrane region (SEQ ID NO: 11), CD28 co-stimulatory domain (SEQ ID NO: 13), to obtain A28 plasmid, and correct insertion of the target sequence into the plasmid was confirmed by sequencing.

[0165] After 3 ml of Opti-MEM (Gibco, Lot No. 31985-070) was added to a sterile tube to dilute the above plasmids, a packaging vector psPAX2 (Addgene, Lot No. 12260) and an envelope vector pMD2.G (Addgene, Lot No. 12259) were then added according to a ratio of plasmid:virus packaging vector:virus envelope vector=4:2:1. Then, 120 μl of X-treme GENE HP DNA transfection reagent (Roche, Lot No. 06366236001) was added, well mixed immediately, followed by incubation at room temperature for 15 min, and then the plasmid/vector/transfection reagent mixture was added dropwise into a culture flask of 293T cells. The viruses were collected at 24 and 48 hours and combined, and then subjected to ultracentrifugation (25000 g, 4° C., 2.5 hours) to obtain a concentrated lentivirus.

[0166] T cells were activated with DynaBeads CD3/CD28 CTS™ (Gibco, Lot No. 40203D), and cultured for 1 day at 37° C. and 5% CO.sub.2. Then, the concentrated lentivirus was added, and after 3 days of continuous culture, T cells expressing NK inhibitory molecule were obtained.

[0167] TCR/CD3 component (specifically TRAC gene) and MHC-related genes (specifically B2M and RFX5) in the T cells expressing the NK inhibitory molecule were then knocked out using the CRISPR system. Specifically, 10 μg of Cas9 protein and 10 μg of sgRNA (3.3 μg TRAC sgRNA (SEQ ID NO: 43)+3.3 μg of B2m sgRNA (SEQ ID NO: 44)+3.3 μg of RFX5 sgRNA (SEQ ID NO: 45)) were electrically transfected into activated NKi-T cells at 400 V, 0.7 ms using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX). Immediately after electrotransfection, the NKi-T cells were placed in 1 ml of pre-heated medium, and cultured in the presence of IL-2 (300 IU/ml) at 37° C. and 5% CO.sub.2 to obtain TCR/B2M/RFX5 triple knockout UNKi-T cells. The wild-type T cell with TCR/B2M/RFX5 being knocked out by the CRISPR system (i.e., Mock T cell) and the wild-type T cell without gene knockout (i.e., NT cell) were used as control.

[0168] The structure of the NK inhibitory molecule contained in the UNKi-T cells prepared in the present example is as shown in Table 1 below.

TABLE-US-00001 TABLE 1 UNKi-T cell comprising NK inhibitory molecule co- signal hinge transmembrane stimulatory Cell Name peptide NK inhibitory ligand region domain domain Ecad0-UNKi-T B2M E-cadherin extracellular region CD28 CD28 none Ecad28-UNKi-T B2M E-cadherin extracellular region CD28 CD28 CD28 E0-UNKi-T B2M presenting peptide + none CD28 none synonymously mutated B2M+ HLA-E extracellular region mutant E28-UNKi-T B2M presenting peptide + none CD28 CD28 synonymously mutated B2M+ HLA-E extracellular region mutant G0-UNKi-T B2M synonymously mutated B2M+ none CD28 none HLA-G extracellular region G28-UNKi-T B2M synonymously mutated B2M+ none CD28 CD28 HLA-G extracellular region A28-UNKi-T B2M anti-NKG2A-scFv IgG4 CD28 CD28

[0169] After 11 days, expression efficiencies of TCR/HLA-I/HLA-II in UNKi-T cell, Mock T cell, and NT cell were measured by a flow cytometer using FITC Mouse Anti-Human CD3 (BD Pharmingen, Lot No. 555916) antibody, PE mouse anti-human HLA-I (R&D, Lot No. FAB7098P) and APC anti-human DR, DP, DQ (biolegend, Lot No. 361714) antibody, and results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Gene expression efficiency in UNKi-T cell Cell Name TCR/CD3 B2M/HLA-I RFX5/HLA-II Ecad0-UNKi-T 3.6%   19%   11% Ecad28-UNKi-T 2.9% 18.3% 10.4% E0-UNKi-T 4.8% 18.9% 11.3% E28-UNKi-T 3.4%   20%   12% G0-UNKi-T   4%   19%  9.8% G28-UNKi-T 2.9% 19.7%   10% A28-UNKi-T 3.8% 21.1% 12.1% Mock T 2.7% 17.8%  9.8% NT  97%   98%   60%

[0170] As can be seen from Table 2, expression of TCR/B2M/RFX5 in UNKi-T cell prepared in the present disclosure and Mock T cell is effectively suppressed or silenced.

[0171] Furthermore, HLA-E expression in UNKi-T cell and Mock T cell was detected by a flow cytometer using PE mouse anti-human HLA-E (biolegend, Lot No. 342604) (FIG. 1), HLA-G expression in UNKi-T cell and Mock T cell was detected using PE mouse anti-human HLA-G (biolegend, Lot No. 335906) (FIG. 2), E-cadherin expression in UNKi-T cell and Mock T cell was detected using E-cadherin monoclonal antibody (invitrogen, Lot No. 13-5700) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, Lot No. A-11001) (FIG. 3), and anti-NKG2A scFv expression in UNKi-T cell and Mock T cell was detected using Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, F(ab′) fragment specific antibody (Jackson ImmunoResearch, Lot No. 109-065-097) and APC Streptavidin (BD, Lot No. 554067) (FIG. 4).

[0172] It can be seen from FIGS. 1-4 that the NK inhibitory molecules in the UNKi-T cell prepared in the present disclosure are all effectively expressed.

Example 2. Inhibitory Effect of UNKi-T Cell on NK Cell Killing Effect

[0173] The effector cells used in the present example are NK92 cells. Because the NK92 cell line does not express the receptor KLRG1 of E-cadherin, NK92 cell overexpressing KLRG1 was prepared first.

[0174] Nucleic acid sequences encoding KLRG1 (SEQ ID NO: 54) were synthesized, and sequentially cloned into pGEM-T Easy vector (Promega, Lot No. A1360), and the correct insertion of the target sequence was confirmed by sequencing. The vector was subjected to enzyme digestion with SpeI enzyme, purified, and recovered to obtain a linearized vector. Then mRNA was prepared using mMESSAGE mMACHINE@T7 Ultra Kit (Invitrogen, Lot No. AM1345) with a linearized vector as a template according to the manufacturer's recommendations, and purified with Fastpure cell/Tissue total RNA isolation kit (Vazyme, Lot No. RC101-01) to obtain purified mRNA. Then 20 μg of purified mRNA prepared above was electrically transfected into NK92 cells at 200 V and 2 ms using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX) to obtain NK92-KLRG1 cells. 16 h later, the expression of KLRG1 was detected, and result is as shown in FIG. 5. NK92 cells not transfected with KLRG1 served as control.

[0175] As can be seen from FIG. 5, NK92-KLRG1 cells can effectively express KLRG1.

[0176] The inhibitory effect of UNKi-T cells prepared in the present disclosure on NK cell killing effect was then detected according to the following method: UNKi-T cells prepared in the present disclosure and Mock-T cells were labeled with Far-Red (invitrogen, Lot No. C34564). Labeled UNKi-T cells and Mock T cells were then plated into a 96-well plate at a concentration of 1×10.sup.4 cells per well, and were co-cultured with NK92 cells (for Mock T cells and UNKi-T cells expressing HLA-E, HLA-G or NKG2A scFv) or NK92-KLRG1 cells (for UNKi-T cells expressing E-cadherin) at an effector-target ratio of 2:1. After 16-18 hours, the ratio of T cells in the culture was detected by a flow cytometer, and the killing rate of the NK cells to the T cells was further calculated, and the results are shown in FIG. 6.

[0177] It can be seen from FIG. 6 that compared with Mock T cells not expressing NK inhibitory molecules, UNKi-T cells expressing NK inhibitory molecules containing inhibitory ligands such as NKG2A scFv, HLA-G, HLA-E, and E-cadherin can significantly reduce the killing effect of NK cells on T cells. Moreover, compared with T cells only expressing inhibitory ligands and transmembrane domain, the addition of co-stimulatory domain may further significantly enhance inhibition of NK cell killing against T cells (see G0 vs G28; E0 vs E28; ECad0 vs ECad28). Therefore, the NK inhibitory molecule comprising an inhibitory ligand, a transmembrane domain, and a co-stimulatory domain prepared in the present disclosure can significantly reduce the killing effect of NK cells on UNKi-T cells, thereby being capable of effectively reducing the risk of HvGD.

Example 3. Killing Effect of UNKi-T Cell on NK Cell

[0178] E28z-UNKi-T and A28z-UNKi-T cells were prepared according to the method in Example 1, which differ from E28-UNKi-T and A28-UNKi-T cells only in that they further comprise a CD3 ζ intracellular signaling domain (SEQ ID NO: 17).

(1) Detecting Expression of NK Inhibitory Molecule

[0179] The anti-NKG2A scFv expression in A28z-UNKi-T cells and Mock T cells was detected with Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, F(ab′) fragment specific antibody (Jackson ImmunoResearch, Lot No. 109-065-097) and APC Streptavidin (BD, Lot No. 554067) (FIG. 7). The HLA-E expression in E28z-UNKi-T cells and Mock T cells was detected with PE mouse anti-human HLA-E (biolegend Lot No. 342604) by a flow cytometer (FIG. 8).

[0180] It can be seen that the NK inhibitory molecules in the E28z-UNKi-T cells and A28z-UNKi-T cells prepared in the present disclosure can be effectively expressed.

(2) Detecting Expression of CD107a

[0181] The cytotoxic T lymphocyte (CTL cell) cytoplasm contains a high concentration of cytotoxic particles in the form of vesicles, and a lysosome associated membrane protein I (CD107a) is a main component of the vesicle membrane protein. When the CTL cell kills the target cell, toxic particles will reach the cell membrane and fuse with it (at this time, the CD107a molecule is transported to the surface of the cell membrane), causing release of particle content, and finally death of the target cell. Therefore, CD107a molecule is a sensitive marker for degranulation of CTL cells, and can reflect the cell killing activity.

[0182] Target cells (NK92 cells) were plated in a 96-well plate at a concentration of 1×10.sup.5 cells/well, then Mock T cells, E28z-UNKi-T cells, and A28z-UNKi-T cells were added at a ratio of 1:1 per well, meanwhile 10 μl of PE-anti-human CD107a (BD Pharmingen, Lot No. 555801) was added for co-culture at 37° C. and 5% CO.sub.2 culture condition. After 1 h, Goigstop (BD Pharmingen, Lot No. 51-2092KZ) was added for further incubation of 2.5 h. 5 μl of APC-anti human CD8 (BD Pharmingen, Lot No.: 555369) and 5 μl of FITC-antihuman CD4 (BD Pharmingen, Lot No.: 561005) were then added to each well, after 30 minutes of incubation at 37° C., the expression level of CD107a was detected by a flow cytometer, and results are as shown in FIG. 9A (CD4+ T cell toxicity) and FIG. 9B (CD8+ T cell toxicity).

[0183] It can be seen that Mock T cells not expressing NK inhibitory molecules seldom kill target cells. On the contrary, after E28z-UNKi-T cells and A28z-UNKi-T cells prepared in the present disclosure were co-cultured with target cells, the expression rate of CD107a was significantly improved, indicating that the UNKi-T cells of the present disclosure can significantly kill the NK cells.

(3) Detecting IFN-γ Secretion

[0184] Target cells (NK92 cells) were plated in a 96-well plate at 1×10.sup.5 cells/well, Mock T cells, E28z-UNKi-T cells, and A28z-UNKi-T cells were added at a ratio of 1:1 per well, and co-cultured at 37° C. and 5% CO.sub.2 culture condition. After 18-24 hours, cell co-culture supernatant was collected.

[0185] The 96-well plate was coated with capture antibody Purified anti-human IFN-γ Antibody (Biolegend, Lot No. 506502) and incubated overnight at 4° C., then the antibody solution was removed. 250 μL of PBST (1×PBS containing 0.1% Tween) solution containing 2% BSA (sigma, Lot No. V90093-1kg) was added, and incubated at 37° C. for 2 hours. The plate was then washed 3 times with 250 μL of PBST (1×PBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or a standard was added to each well, followed by incubation at 37° C. for 1 hour, then the plate was washed 3 times with 250 μL of PBST (1×PBS containing 0.1% Tween). Then 50 μL of detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, Lot No. ab25017) was added to each well, and after 1 hour of incubation at 37° C., the plate was washed 3 times with 250 μL of PBST (1×PBS containing 0.1% Tween). HRP Streptavidin (Biolegend, Lot No. 405210) was then added, and after 30 minutes of incubation at 37° C., the supernatant was discarded, 250 μL of PBST (1×PBS containing 0.1% Tween) was added, and the plate was washed 5 times. 50 μL of TMB substrate solution was added to each well. The reaction was carried out in the dark at room temperature for 30 minutes, after which 50 μL of 1 mol/L H.sub.2SO.sub.4 was added to each well to stop the reaction. Within 30 minutes after the stop of the reaction, the absorbance at 450 nm was detected by a plate reader, and the content of cytokines was calculated according to a standard curve (drawn according to read value and concentration of the standard), and the result is as shown in FIG. 10.

[0186] It can be seen that cytokine IFN-γ release levels of E28z-UNKi-T cell and A28z-UNKi-T cell of the present disclosure are much higher than that of Mock T cell, which also indicates that their killing against the NK92 target cell is significantly increased.

Example 4. UNKi-T Cell Targeting KIR or LIR1 and Inhibitory Effect Thereof on NK Cell Killing Effect

[0187] The following encoding sequences were synthesized, and sequentially cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Lot No.: PPL00157-4a): B2m signal peptide (SEQ ID NO: 21), anti-KIR-scFv (containing SEQ ID NOS: 55 and 56) or anti-LIR1 scFv (containing SEQ ID NOS: 57 and 58, or containing SEQ ID NOS: 59 and 60), IgG4 hinge region (SEQ ID NO: 29), CD8 α transmembrane region (SEQ ID NO: 9), CD28 co-stimulatory domain (SEQ ID NO: 13), to obtain KIRG4, LIRG4-1, and LIRG4-2 plasmids, and the correct insertion of the target sequence into the plasmids was confirmed by sequencing.

[0188] UNKi-T cells were prepared according to knockout and infection method in Example 1, and expression efficiencies of CD3/HLA-I/HLA-II in UNKi-T cell, Mock T cell, and NT cell were measured by a flow cytometer using FITC Mouse Anti-Human CD3 (BD Pharmingen, Lot No. 555916) antibody, PE mouse anti-human HLA-I (R&D, Lot No. FAB7098P) and APC anti-human DR, DP, DQ (biolegend, Lot No. 361714) antibody, and results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Gene expression efficiency in UNKi-T cell Cell Name TCR/CD3 B2M/HLA-I RFX5/HLA-II KIRG4-UNKi-T 4.5%   16% 10.8% LIRG4-UNKi-1-T 3.5% 17.6% 10.9% LIRG4-UNKi-2-T 4.3% 16.9% 10.3% Mock T 2.6% 15.8%  9.3% NT  98%   98%   83%

[0189] As can be seen from Table 3, expression of CD3/HLA-I/HLA-II in UNKi-T cell prepared in the present disclosure and Mock T cell is effectively suppressed or silenced.

[0190] The scFv expression in KIRG4-UNKi-T cell was detected with Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, F(ab′) fragment specific antibody (Jackson ImmunoResearch, Lot No. 109-065-097) and APC Streptavidin (BD, Lot No. 554067), the results are as shown in FIG. 11. The scFv expression in LIRG4-UNKi-1 T cell and LIRG4-UNKi-2 T cell was detected with recombinant human LILRB1 protein (sino biological, Lot No. 16014-H02H) as primary antibody, APC anti-human IgG Fc (biolegend, Lot No. 409306) as secondary antibody, the results are as shown in FIG. 12. It can be seen from FIG. 11 and FIG. 12 that the scFvs of the UNKi-T cell prepared in the present disclosure are all effectively expressed.

[0191] UNKi-T cell and NK92 cell were co-cultured according to the method of Example 2 to detect the inhibitory effect of UNKi-T cell on NK cell killing effect, and the results are as shown in FIG. 13. It can be seen that compared with Mock T, all the UNKi-T cells targeting KIR (FIG. 13A) or LIR1 (FIG. 13B) prepared in the present example can significantly reduce the killing effect of NK cells on T cells.

Example 5. T Cell Targeting SIGLEC7, SIGLEC9 or KLRG1 and Inhibitory Effect Thereof on NK Cell Killing Effect

[0192] The following encoding sequences were synthesized, and sequentially cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Lot No.: PPL00157-4a): B2m signal peptide (SEQ ID NO: 21), anti-SIGLEC7-scFv (SEQ ID NO: 130), anti-SIGLEC7/SIGLEC9-scFv (SEQ ID NO: 184), or anti-KLRG1-scFv (SEQ ID NO: 119), IgG4 hinge region (SEQ ID NO: 29), CD8 α transmembrane region (SEQ ID NO: 9), CD28 co-stimulatory domain (SEQ ID NO: 13), to obtain SC7G4, SC7/SC9G4, and K1G4 plasmids, and the correct insertion of the target sequence into the plasmids was confirmed by sequencing.

[0193] The above plasmids were transferred into T cells according to the method in Example 1, and the B2M gene therein was knocked out to obtain NKi-T cells (namely, SC7G4-T cells, SC7/SC9G4-T cells, and K1G4-T cells) expressing NK inhibitory molecules and with B2M being knocked out. T cells with only B2M being knocked out served as negative control (NT).

[0194] The scFv expression in SC7G4, SC7/SC9G4, and K1G4T cell was detected with Biotin-SP (long spacer) AffiniPure Goat Anti-mouse IgG, F(ab′) fragment specific antibody (Jackson ImmunoResearch, Lot No. 115-066-072) and APC Streptavidin (BD, Lot No. 554067), the results are as shown in FIG. 14. It can be seen that the scFvs of the NKi-T cells prepared in the present disclosure are all effectively expressed.

[0195] According to the method of Example 2, the above NKi-T cell was co-cultured with NK92-KLRG1 cell to detect its inhibitory effect on the killing effect of NK cell, and results are as shown in FIG. 15. It can be seen that compared with NT, all the T cells targeting SIGLEC7, SIGLEC9 or KLRG1 prepared in the present example can significantly reduce the killing effect of NK cell on T cell.

Example 6 T Cell Targeting PD-1 and Inhibitory Effect Thereof on NK Cell Proliferation

[0196] The following encoding sequences were synthesized, and sequentially cloned into a pLVX vector (Public Protein/Plasmid Library (PPL), Lot No.: PPL00157-4a): PDL1 signal peptide (SEQ ID NO: 121), PDL1 extracellular region (SEQ ID NO: 70), PDL1 transmembrane region (SEQ ID NO: 120), CD28 co-stimulatory domain (SEQ ID NO: 13) to obtain a PDL1 plasmid, and correct insertion of the target sequence into the plasmid was confirmed by sequencing.

[0197] The above plasmid was transferred into T cell according to the method in Example 1 to obtain NKi-T cell expressing NK inhibitory molecule, namely, PDL1-T cell. Expression thereof was detected with Anti-PD-L1 Antibody (manufacturer: Solarbio, Lot No.: 10084-R312-A), and results are as shown in FIG. 16. It can be seen that PDL1 is effectively expressed. Untreated T cell served as negative control (NT).

[0198] The above NKi-T cell was cultured and treated with cell mitomycin C. Two kinds of PBMCs from foreign sources (donor 1 and donor 2) were labeled with Far-Red, and then were co-cultured with T cell at a ratio of T cell:PBMC=1:2. Half-medium-replacement treatment was performed every 2-3 days. After 8 days, cells were counted and stained with PE anti-human CD3 (manufacturer biolegend, Lot No.: 317308) and FITC anti-human CD56 (manufacturer: biolegend, Lot No.: 362546), and then NK cell population ratio was detected by flow cytometry. The number of NK cells was calculated by the total cell amount*NK cell population ratio, and results are as shown in FIG. 17. It can be seen that NKi-T cell targeting PD-1 can significantly inhibit the proliferation of NK cell.

Example 7. Inhibitory Effect of B Cell and Huh7 Cell Targeting KIR on NK Cell Killing Effect

[0199] The KIRG4 plasmid prepared in Example 4 was transducted into B cell and Huh7 cell (liver cancer cell) according to the method in Example 1, and the B2M gene therein was knocked out to obtain NKi-B cell and NKi-Huh7 cell expressing NK inhibitory molecule and with B2M being knocked out. B cell and Huh7 cell (NC cell), with only B2M gene being knocked out, served as negative control.

[0200] The scFv expression in NKi-B cell and NKi-Huh7 cell was detected with Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, F(ab′) fragment specific antibody (Jackson ImmunoResearch, Lot No. 109-065-097) and APC Streptavidin (BD, Lot No. 554067), and results are as shown in FIG. 18. It can be seen that the NK inhibitory molecules are effectively expressed in both B cell and Huh7 cell.

[0201] The cells prepared in the above and NK cells were co-cultured according to the method of Example 2 to detect the inhibitory effect of the above cell on the killing effect of NK cell, and results are as shown in FIG. 19. It can be seen that compared with the NC cell, both NKi-B cell and NKi-Huh7 cell prepared in the present example can significantly reduce the killing effect of NK cell on them.

[0202] 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.