Genetically modified immune cells expressing NK inhibitory molecules and uses thereof

Abstract

To provide an NK inhibitory molecule that can significantly suppress the killing action of NK cells in the subject's body, thereby reducing the risk of HvGD, compared to conventional genetically modified immune cells, and to provide a genetically modified immune cell that expresses the molecule.SOLUTION: Provided is an NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain and a co-stimulatory domain, the NK inhibitory ligand specifically binding to an NK inhibitory receptor and inhibiting NK cells from killing genetically modified immune cells that express the NK inhibitory molecule. Provided is a genetically modified immune cell that expresses an NK inhibitory molecule of the invention, the expression of at least one MHC-associated gene being inhibited or silenced. Provided is use of the genetically modified immune cells in the treatment of cancer, infectious disease or autoimmune disease.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy. More specifically, the present invention relates to an NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain. The NK inhibitory ligand specifically binds to an NK inhibitory receptor and inhibits NK cells from killing genetically modified immune cells that express the NK inhibitory molecule. [Background technology]

[0002] In recent years, cancer immunotherapy technology has developed rapidly, and chimeric antigen receptor T cell (CAR-T)-related immunotherapy in particular has achieved excellent clinical efficacy in the treatment of hematological malignancies. CAR-T cell immunotherapy involves genetically modifying T cells in vitro to enable them to recognize tumor antigens, expanding them to a certain number, and then injecting them back into the patient to kill cancer cells, thereby achieving the goal of tumor treatment.

[0003] In 2017, two autologous CAR-T therapies received FDA approval in the United States. One was for B-cell acute leukemia, and the other was for diffuse B-cell non-Hodgkin's lymphoma. While these two CAR-T cell types have demonstrated excellent clinical efficacy, their high cost and lengthy preparation period make their large-scale use extremely difficult. Therefore, to address these issues, it is necessary to develop a universal CAR-T product. Universal CAR-T can be prepared using T cells isolated from the peripheral blood of healthy donors, enabling allogeneic reinfusion and significantly shortening patient waiting times. Furthermore, the viability and functionality of T cells derived from healthy donors are superior to those of patient-derived T cells, potentially increasing CAR infection rates and improving therapeutic efficacy.

[0004] However, the development of universal CAR-T cells still faces two challenges: (1) After artificial CAR-T cells enter the patient and proliferate to a certain extent, they may attack the patient's normal cells or tissues, causing graft-versus-host disease (GvHD). (2) The patient's normal immune system may reject the allogeneic CAR-T cells, causing host-versus-graft disease (HvGD). Currently, HvGD is suppressed or avoided primarily by knocking out CD52 or HLA. Specifically, knocking out CD52 can make universal CAR-T cells resistant to alemtuzumab (a CD52 antibody), preventing the introduced CAR-T cells from being killed when the patient's T cells are depleted using alemtuzumab. However, using alemtuzumab increases the manufacturing and treatment costs of universal CAR-T products. On the other hand, knocking out HLA molecules can prevent CAR-T cells from being eliminated by the patient's T cells without the use of antibodies or other treatments, but the cells with knocked-out HLA molecules can be recognized by the patient's NK cells, causing a rejection response.

[0005] Therefore, conventional universal CAR cell therapy needs to be further improved, especially to reduce the killing effect of NK cells on CAR cells, thereby further reducing or avoiding the risk of HvGD. Summary of the Invention

[0006] In a first aspect, the present invention provides an NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain. The NK inhibitory ligands specifically bind to NK inhibitory receptors (NKIRs) and inhibit NK cell killing of genetically modified immune cells expressing the NK inhibitory molecule.

[0007] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting an NKIR, or a natural ligand of an NKIR or an NKIR-binding fragment thereof. In one embodiment, the NKIR is an NKG2 / CD94 component (e.g., NKG2A, NKG2B, CD94), a killer cell Ig-like receptor (KIR) family member (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3), a leukocyte Ig-like receptor (LIR) family member (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8), an NK cell receptor protein 1 (NKR-P1) family member (e.g., NKR-P1B and NKR-P1C), or a NK cell receptor protein 2 (NKR-P1D) family member (e.g., NKR-P1E). R-P1D), immune checkpoint receptors (e.g., PD-1, TIGIT and CD96, TIM3, LAG3), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), sialic acid-binding immunoglobulin-like lectin (SIGLEC) family members (e.g., SIGLEC7 and SIGLEC9), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Ly49 family members (e.g., Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4), and killer cell lectin-like receptor G1 (KLRG1). Preferably, the NKIR is selected from PD1, NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9, and KLRG1. More preferably, said NKIR is selected from PD1, NKG2A, CD94, KIR2DL1, KIR2DL2 / 3, KIR3DL1, LIR1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9, and KLRG1.

[0008] In one embodiment, the NK inhibitory ligand is an antibody that targets an NKIR, wherein the antibody is an intact antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody, sdAb, or nanobody. In a preferred embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof that targets PD1, NKG2A, LIR1, KIR, SIGLEC7, SIGLEC9, and / or KLRG1.

[0009] In one embodiment, the NK inhibitory ligand is a natural ligand of NKIR or an NKIR-binding fragment thereof. Preferably, the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (e.g., PD-L1 / PD-L2, CD155, CD112, CD113, Gal-9, FGL1, etc.), and NKIR-binding regions contained therein. More preferably, the NK inhibitory ligand is sialic acid, HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, and NKIR-binding regions contained therein. More preferably, the NK inhibitory ligand is selected from sialic acid, the extracellular region of HLA-E, the extracellular region of HLA-G, the extracellular region of E-cadherin, the extracellular region of PD-L1, and the extracellular region of PD-L2. More preferably, the NK inhibitory ligand is the E-cadherin extracellular domain comprising EC1 and EC2, more preferably the E-cadherin extracellular domain comprising EC1, EC2, EC3, EC4, and EC5.

[0010] In one embodiment, the transmembrane domain contained in the NK inhibitory molecule is selected from the transmembrane domains of the proteins 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, and CD154, and the transmembrane domains of NKIR natural ligands, e.g., HLA-E, HLA-F, HLA-G, cadherin, collagen, and OCIL. In a preferred embodiment, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28, and CD278.

[0011] In one embodiment, the transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 or 11.

[0012] In one embodiment, the costimulatory domain comprised in the NK inhibitory molecule is selected from the costimulatory signaling domains of the following proteins: 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 combinations thereof. Preferably, the costimulatory domain of the present invention is derived from 4-1BB, CD28, CD27, OX40, CD278, or a combination thereof.

[0013] In one embodiment, the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13 or 15.

[0014] In one embodiment, the NK inhibitory molecule does not comprise an intracellular signaling domain, hi another embodiment, the NK inhibitory molecule further comprises an intracellular signaling domain.

[0015] In one embodiment, the intracellular signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling domain comprises the signaling domain of CD3ζ.

[0016] In one embodiment, the intracellular signaling domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17 or 19.

[0017] The present invention further provides a nucleic acid encoding the above-mentioned NK inhibitory molecule, and a vector comprising said nucleic acid.

[0018] In a second aspect, the present invention provides a genetically modified immune cell. The genetically modified immune cell is characterized in that (1) it expresses an NK inhibitory molecule of the present invention, and (2) the expression of at least one MHC-associated gene is inhibited or silenced. In one embodiment, the genetically modified immune cell of the present invention further expresses a chimeric antigen receptor comprising a ligand-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0019] In one aspect, the present invention provides genetically modified immune cells, characterized in that (1) they express a fusion protein of an NK inhibitory molecule of the present invention and a chimeric antigen receptor, the fusion protein comprising an NK inhibitory ligand, a ligand-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, and (2) the expression of at least one MHC-associated gene is inhibited or silenced.

[0020] In one embodiment, the MHC-related gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof, preferably HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0021] In one embodiment, the genetically modified immune cells have inhibited or silenced expression of at least one TCR / CD3 gene selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

[0022] In a preferred embodiment, the genetically modified immune cells have inhibited or silenced expression of at least one TCR / CD3 gene and at least one MHC-related gene. The at least one TCR / CD3 gene is selected from TRAC, TRBC, and a combination thereof. The at least one MHC-related gene is B2M, RFX5, RFXAP, RFXANK, CIITA, and a combination thereof. In one embodiment, the genetically modified immune cells have inhibited or silenced expression of TRAC or TRBC, and B2M. In one embodiment, the genetically modified immune cells have inhibited or silenced expression of TRAC or TRBC, and CIITA. In a preferred embodiment, the genetically modified immune cells have inhibited or silenced expression of TRAC or TRBC, B2M, and CIITA. In a preferred embodiment, the genetically modified immune cells have inhibited or silenced expression of TRAC or TRBC, B2M, and RFX5.

[0023] In one embodiment, the genetically modified immune cells of the present invention express genes encoding CD52, GR, dCK, and immune checkpoint genes, such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, T The expression of one or more genes selected from GFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3 is inhibited or silenced. Preferably, the expression of one or more genes selected from CD52, dCK, PD1, LAG3, TIM3, CTLA4, TIGIT, or a combination thereof is inhibited or silenced in the genetically modified immune cells.

[0024] In one embodiment, the ligand binding domain is 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, tEGFR, IL-22Ra, IL-2 , ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, 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 GMl, 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-la, MAGE-A1, 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-related 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, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut Binds to a target selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and any combination thereof.

[0025] In one embodiment, the genetically modified immune cells are B cells, T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells. Preferably, the genetically modified immune cells are T cells, such as CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, and αβ-T cells.

[0026] In one aspect, the present invention further provides a pharmaceutical composition comprising an NK inhibitory molecule, nucleic acid molecule, vector or genetically modified immune cell of the present invention as an active agent, and one or more pharmaceutically acceptable excipients.

[0027] In one aspect, the present invention further provides a method for treating a subject suffering from cancer, an infectious disease, or an autoimmune disease. The method comprises administering to the subject an effective amount of an NK inhibitory molecule, nucleic acid molecule, vector, genetically modified immune cell, or pharmaceutical composition according to the present invention. Thus, the present invention encompasses the use of an NK inhibitory molecule, nucleic acid molecule, vector, or genetically modified immune cell in the manufacture of a medicament for treating cancer, an infectious disease, or an autoimmune disease.

[0028] In one embodiment, the cancer is a solid tumor or a blood tumor. More specifically, the cancer is brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, osteosarcoma, brain cancer and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon cancer and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system malignancies, vulvar cancer, and other cancers. and sarcomas, as well as B-cell lymphoma, T-cell lymphoma, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell neoplasm, and post-transplant lymphoproliferative disorder (PTLD).

[0029] In one embodiment, the infectious disease includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0030] In one embodiment, the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vascular inflammation, pernicious anemia, and systemic lupus erythematosus.

[0031] An advantage of the present invention is that the NK inhibitory molecule of the present invention further comprises a costimulatory domain compared to expressing only an NK inhibitory ligand, which can further reduce / inhibit the killing of the genetically modified immune cells by the subject's NK cells, and even if the NK inhibitory molecule comprises an intracellular signaling domain, it can enhance the killing of the subject's NK cells by the genetically modified immune cells, thereby better reducing the risk of HvGD and achieving true allogeneic reinfusion. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows the expression levels of HLA-E in E0-UNKi-T and E28-UNKi-T cells. [Figure 2] 1 shows the expression levels of HLA-G in G0-UNKi-T and G28-UNKi-T cells. [Figure 3] 1 shows the expression levels of E-cadherin in ECad0-UNKi-T and ECad28-UNKi-T cells. [Figure 4] 1 shows the expression level of NKG2A scFv in A28-UNKi-T cells. [Figure 5] 1 shows the expression level of KLRG1 in NK92-KLRG1 cells. [Figure 6] 1 shows the inhibitory effect of UNKi-T cells of the present invention on NK cell killing. Statistical analysis was performed using two-way ANOVA and T-test. * indicates P value less than 0.05, ** indicates P value less than 0.01, and *** indicates P value less than 0.001, reaching the significance level. [Figure 7] 1 shows the expression level of NKG2A ScFv in A28z-UNKi-T cells. [Figure 8] 1 shows the expression level of HLA-E in E28z-UNKi-T cells. [Figure 9]1 shows the inhibitory effect of UNKi-T cells of the present invention on NK cell killing. Statistical analysis was performed using two-way ANOVA and T-test. * indicates P value less than 0.05, ** indicates P value less than 0.01, and *** indicates P value less than 0.001, reaching the significance level. [Figure 10] 1 shows the level of IFN-γ release after co-culture of UNKi-T cells of the present invention with NK cells. Statistical analysis was performed using a two-way ANOVA and a T-test. *** indicates that the P value is less than 0.001 and reaches the significance level. [Figure 11] 1 shows the expression level of KIR scFv in the KIRG4-UNKi-T cells of the present invention. [Figure 12] 1 shows the expression levels of LIR1 scFv in LIRG4-UNKi-1-T and LIRG4-UNKi-2-T cells of the present invention. [Figure 13] 1 shows the inhibitory effect of the UNKi-T cells of the present invention on NK cell killing. [Figure 14] This shows the expression levels of scFv in SC7G4-T cells, SC7 / SC9G4-T cells, and K1G4-T cells. [Figure 15] This shows the inhibitory effects of SC7G4-T cells, SC7 / SC9G4-T cells, and K1G4-T cells on NK cell killing. [Figure 16] This shows the expression level of PDL1 in PDL1-T cells. [Figure 17] This shows the inhibitory effect of PDL1-T cells on NK cell proliferation. [Figure 18] 1 shows the expression levels of KIR scFv in NKi-B cells and NKi-Huh7 cells. [Figure 19] This shows the inhibitory effect of NKi-B cells and NKi-Huh7 cells on NK cell killing. DETAILED DESCRIPTION OF THE INVENTION

[0033] Unless otherwise defined, all scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0034] NK inhibitory molecules It has been reported that cells with reduced or eliminated expression of one or more HLA class I molecules can be recognized as non-self by NK cells and thus targeted for killing. Thus, expression of one or more NK inhibitory molecules on such cells can protect them from being killed by NK cells.

[0035] Thus, in a first aspect, the present invention provides an NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain, wherein the NK inhibitory ligands specifically bind to NK inhibitory receptors (NKIRs) and inhibit NK cell killing of genetically modified immune cells expressing the NK inhibitory molecule.

[0036] As used herein, the term "NK inhibitory ligand" refers to a molecule capable of binding to NKIR and inhibiting NK cell function (e.g., killing function). In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof that targets NKIR, or a natural ligand of NKIR or an NKIR-binding fragment thereof. Non-limiting examples of NKIR include NK cell surface receptors that have or bind to an immunoreceptor tyrosine-based inhibitory motif (ITIM). Such receptors include NKG2 / CD94 components (e.g., NKG2A, NKG2B, CD94), killer cell Ig-like receptor (KIR) family members (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3), leukocyte Ig-like receptor (LIR) family members (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8), NK cell receptor protein 1 (NKR-P1) family members (e.g., NKR-P1B and NKR-P1D), These include, but are not limited to, immune checkpoint receptors (e.g., PD-1, TIGIT and CD96, TIM3, LAG3), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), sialic acid-binding immunoglobulin-like lectin (SIGLEC) family members (e.g., SIGLEC7 and SIGLEC9), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Ly49 family members (e.g., Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4), and killer cell lectin-like receptor G1 (KLRG1).

[0037] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof that targets NKIR, such as a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a murine antibody, a chimeric antibody, and functional fragments thereof. Examples of antibodies or functional fragments thereof include, but are not limited to, intact antibodies, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, linear antibodies, sdAb (VH or VL), nanobodies (Nb), etc. Preferably, the antibody is selected from Fab, scFv, sdAb, and nanobody.

[0038] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting the NKG2A / CD94 component. In a preferred embodiment, the NK inhibitory ligand is an antibody targeting NKG2A, NKG2B, or CD94. NKG2 / CD94 is a heterodimer formed by CD94 linking to other NKG2 subunits via disulfide bonds. The cytoplasmic region of CD94 contains only seven amino acid residues and does not possess a signal-transducing structure. The NKG2 family includes members such as NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, and NKG2F, among which NKG2A and NKG2B are different splices of the same highly homologous gene. The cytoplasmic tail of NKG2A / 2B contains two ITIMs that transmit inhibitory signals by recruiting SHP1 or SHP-2. The natural ligand of NKG2A / 2B is HLA-E. NKG2A / 2B binds to its ligand with higher affinity than the activating receptor NKG2C. Therefore, when both the inhibitory and activating receptors of NK cells can bind to target cells expressing HLA-E, the inhibitory NKG2A / CD94 becomes dominant, ultimately inhibiting NK cell activity. NKG2A antibodies known to those skilled in the art can be used in the present invention. Examples include Z270 (available from Immunotech, France), Z199 (available from Beckman Coulter, USA), 20D5 (available from BD Biosciences Pharmingen, USA), and P25 (available from Moretta et al., Univ. Genova, Italy).

[0039] In one embodiment, the NK inhibitory ligand is an antibody that targets NKG2A and comprises (1) CDR-L1 set forth in SEQ ID NO: 72, CDR-L2 set forth in SEQ ID NO: 73, CDR-L3 set forth in SEQ ID NO: 74, CDR-H1 set forth in SEQ ID NO: 75, CDR-H2 set forth in SEQ ID NO: 76, and CDR-H3 set forth in SEQ ID NO: 77, or (2) CDR-L1 set forth in SEQ ID NO: 78, CDR-L2 set forth in SEQ ID NO: 79, CDR-L3 set forth in SEQ ID NO: 80, CDR-H1 set forth in SEQ ID NO: 81, CDR-H2 set forth in SEQ ID NO: 82, and CDR-H3 set forth in SEQ ID NO: 83. In one embodiment, the NK inhibitory ligand is an antibody that targets NKG2A and comprises a light chain variable region and a heavy chain variable region. The light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 7, or 68. The heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 5, or 67. In preferred embodiments, the NK inhibitory ligand is an anti-NKG2A antibody comprising SEQ ID NOs: 1 and 3, an anti-NKG2A antibody comprising SEQ ID NOs: 5 and 7, or an anti-NKG2A antibody comprising SEQ ID NOs: 67 and 68.

[0040] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting KIR. KIR molecules are type I transmembrane proteins belonging to the immunoglobulin superfamily, and their structure includes an extracellular region, a transmembrane domain, and a cytoplasmic region. KIRs can be divided into the KIR2D subfamily and the KIR3D subfamily based on the number of Ig-like domains in the extracellular region. Based on the length of their cytoplasmic regions, KIRs can also be divided into long (L) and short (S) types, such as KIR2DL, KIR2DS, KIR3DL, and KIR3DS. The cytoplasmic regions of KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, and KIR3DL3 contain two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), while the cytoplasmic region of KIR2DL5 contains one ITIM, which is an inhibitory KIR receptor. Specifically, upon phosphorylation of ITIMs contained in the cytoplasmic domain of inhibitory KIR receptors, they recruit the phosphatases SHP1 and SHP2, leading to the dephosphorylation of cytosolic substrates and ultimately inhibiting or terminating NK cell effector functions, such as cytotoxicity and cytokine secretion. KIRs are expressed on the majority of NK cells, although expression levels vary among individuals. Even within the same individual, different NK cells express different types of KIRs, and the same NK cell may express several different KIR molecules. KIRs recognize classical HLA class I molecules, including specific polymorphic epitopes of HLA-A, HLA-B, and HLA-C. For example, KIR3DL2 recognizes HLA-A alleles -A3 and -A11, KIR3DL1 recognizes HLA-Bw-4, and KIR2DL1 recognizes HLA-Cw2, HLA-Cw4, and HLA-Cw6 isoforms. The mouse homologue of KIR is gp49B1, which is 335 amino acids in length and contains two ITIM structures in its cytoplasmic domain.In a preferred embodiment, the antibody targeting KIR is an antibody targeting one or more selected from the group consisting of KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL5, and gp49B1. KIR antibodies known to those skilled in the art can be used in the present invention. Examples include GL183 (targeting KIR2DL2 / L3, available from Immunotech, France and Beckton Dickinson, USA), EB6 (targeting KIR2DL1, available from Immunotech, France and Beckton Dickinson, USA), AZ138 (targeting KIR3DL1, available from Moretta et al., Univ. Genova, Italy), Q66 (targeting KIR3DL2, available from Immunotech, France), and Z27 (targeting KIR3DL1, available from Immunotech, France and Beckton Dickinson, USA).

[0041] In one embodiment, the NK inhibitory ligand is an antibody targeting KIR and comprises CDR-L1 set forth in SEQ ID NO: 84, CDR-L2 set forth in SEQ ID NO: 85, CDR-L3 set forth in SEQ ID NO: 86, CDR-H1 set forth in SEQ ID NO: 87, CDR-H2 set forth in SEQ ID NO: 88, and CDR-H3 set forth in SEQ ID NO: 89. In one embodiment, the NK inhibitory ligand is an antibody targeting KIR and comprises a light chain variable region and a heavy chain variable region. The light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58. The heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59. In a preferred embodiment, the NK inhibitory ligand is an anti-KIR antibody, including SEQ ID NO: 58, and SEQ ID NO: 59, the amino acid sequences of which are set forth in, for example, SEQ ID NO: 57 or 60.

[0042] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting an LIR. LIRs are also known as immunoglobulin-like transcripts (ILTs) or monocyte-macrophage inhibitory receptors (MIRs). The LIR family includes eight members, of which LIR1 (also known as ILT2), LIR2 (also known as ILT4), LIR3 (also known as ILT5), LIR5 (also known as ILT3), and LIR8 contain two to four ITIM structures in their cytoplasmic regions. At least one of these ITIM structures is a VXYXXL / V motif, which is an inhibitory LIR receptor. LIR-1 has been reported to inhibit the killing of target cells expressing HLA class I molecules by the NK cell line NKL and the activation of NKL via CD16. Mouse homologs of LIRs are PIRs (paired Ig-like receptors), which contain PIR-A and PIR-B, where PIR-A transmits activating signals in cooperation with the FcRγ homodimer, and PIR-B transmits inhibitory signals via four ITIM structures contained in its cytoplasmic region. In a preferred embodiment, the NK inhibitory ligand is an antibody targeting LIR1, LIR2, LIR3, LIR5, LIR8, or PIR-B.

[0043] In one embodiment, the NK inhibitory ligand is an antibody targeting LIR1 and comprises (1) CDR-L1 set forth in SEQ ID NO: 90, CDR-L2 set forth in SEQ ID NO: 91, CDR-L3 set forth in SEQ ID NO: 92, CDR-H1 set forth in SEQ ID NO: 93, CDR-H2 set forth in SEQ ID NO: 94, and CDR-H3 set forth in SEQ ID NO: 95, or (2) CDR-L1 set forth in SEQ ID NO: 96, CDR-L2 set forth in SEQ ID NO: 97, CDR-L3 set forth in SEQ ID NO: 98, CDR-H1 set forth in SEQ ID NO: 99, CDR-H2 set forth in SEQ ID NO: 100, and CDR-H3 set forth in SEQ ID NO: 101. In one embodiment, the NK inhibitory ligand is an antibody targeting LIR1 and comprises a light chain variable region and a heavy chain variable region. The light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61 or 65. The heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 62 or 64. In a preferred embodiment, the NK inhibitory ligand is an anti-LIR1 antibody, the amino acid sequence of which is set forth in SEQ ID NO: 63 or 66. Other antibodies known in the art that target LIR family members can also be used in the present invention. In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof that targets an immune checkpoint receptor (e.g., PD-1, TIGIT, CD96, TIM3, LAG3). In a preferred embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof that targets PD-1. PD-1 is primarily expressed on activated NK cells and is a type I transmembrane glycoprotein of 268 amino acids belonging to the CD28 family. Its structure mainly includes an extracellular immunoglobulin variable region (IgV)-like structure, a hydrophobic transmembrane region, and an intracellular region.The intracellular tail contains two independent tyrosine residues: the N-terminal tyrosine residue is involved in the formation of the ITIM, and the C-terminal tyrosine residue is involved in the formation of the immunoreceptor tyrosine-based switch motif (ITSM). After PD-1 binds to its ligands (e.g., PD-L1 and PD-L2), tyrosine phosphorylation in the ITSM domain of PD-1 is promoted, leading to the dephosphorylation of the downstream protein kinases Syk and PI3K, which inhibit the activation of downstream pathways such as AKT and ERK, resulting in the inhibition of NK cell activity.

[0044] In a preferred embodiment, the NK inhibitory ligand is an antibody or functional fragment that targets TIGIT. TIGIT is a member of the immunoglobulin superfamily and is composed of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular region containing ITIM and immunoglobulin tyrosine tail (ITT) motifs. Upon binding to a ligand (e.g., CD155, CD112, or CD113), it induces the transduction of intracellular inhibitory signals, inhibiting NK cell activity.

[0045] In a preferred embodiment, the NK inhibitory ligand is an antibody or functional fragment targeting LAG3. LAG3 is a member of the Ig superfamily of proteins and a type 1 transmembrane protein expressed in activated T cells, NK cells, B cells, and plasmacytoid dendritic cells. The four IgG domains of LAG3 share high structural homology with the CD4 molecule, but their amino acid homology is less than 20%. Research has shown that LAG3 negatively regulates the proliferation and persistent memory of T cells and NK cells. When activated by its ligand (e.g., FGL1), it can promote the escape of "bad cells," such as tumor cells, from the immune system.

[0046] In a preferred embodiment, the NK inhibitory ligand is an antibody or functional fragment targeting TIM3. TIM3 is a receptor protein of the TIM family and is expressed on the surface of T cells, Treg cells, and innate immune cells (dendritic cells, natural killer cells, and monocytes). Members of the TIM family are encoded by three genes. Specifically, HAVCR1 encodes TIM1, HAVCR2 encodes TIM3, and TIMD4 encodes TIM4. TIM3 has multiple ligands, including phosphatidylserine, galectin-9 (also known as Gal-9), HMGB1, and CEACAM-1. When expressed on NK cells, TIM3 is thought to be a potential marker of dysfunctional NK cells, and blocking TIM3 has been shown to reverse NK cell dysfunction.

[0047] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting NKR-P1. NKR-P1 is a type II transmembrane glycoprotein expressed in human, mouse, and rat NK cells. Currently, six NKR-P1 members have been detected in mice: NKR-P1A, NKR-P1B, NKR-P1C, NKR-P1D, NKR-P1E, and NKR-P1F. However, only NKR-P1A (also known as CD161) has been detected in humans. The extracellular region of the NKR-P1 molecule is an NK receptor domain (NKD) of the C-type lectin-like superfamily, which is structurally similar to Ly49, Cd69, and CD94 / NKG2 molecules. NKR-P1 exists primarily in the form of a homodimer, but human NKR-P1A may exist in a monomeric form. The cytoplasmic regions of NKR-P1 family molecules have different structures in different species. For example, the cytoplasmic regions of NKR-P1B and NKR-P1D contain ITIM motifs, which recruit SHP-1 upon tyrosine phosphorylation to transmit inhibitory signals. On the other hand, NKR-P1C binds to Fc receptors via positively charged amino acids in its transmembrane domain, thereby recruiting Syk to transmit activating signals. Therefore, in a preferred embodiment, the NK inhibitory ligand is an antibody targeting NKR-P1B or NKR-P1D.

[0048] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting CEACAM1. CEACAM1, also known as CD66a, biliary glycoprotein (BGP), or C-CAM1, is a member of the carcinoembryonic antigen (CEA) gene family and belongs to the immunoglobulin (Ig) superfamily. In activated NK cells, CEACAM1 expression is upregulated, and its homophilic interaction leads to inhibition of the cytotoxic effect of lymphocytes. CEACAM1 interacts with other known CEACAM proteins, including CD66a (CEACAM1), CD66c (CEACAM6), and CD66e (CEACAM5, CEA). To date, 11 different CEACAM1 splice variants have been detected in humans. CEACAM1 isoforms are named according to the number of extracellular immunoglobulin-like domains (e.g., CEACAM1 with four extracellular immunoglobulin-like domains is called CEACAM1-4) and the length of their cytoplasmic tails (e.g., CEACAM1-4 with a long cytoplasmic tail is called CEACAM1-4L, and CEACAM1-4 with a short cytoplasmic tail is called CEACAM1-4S). The N-terminal domain of CEACAM1 begins immediately after the signal peptide, and its structure is considered to be IgV-type.

[0049] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting SIGLEC. Sixteen SIGLEC proteins have been identified in humans and nine in mice. The SIGLEC proteins consist of two to 17 extracellular Ig domains, including an amino-terminal V-type domain. The V-type domain contains a sialic acid-binding site. Siglecs are generally divided into two groups: a first subset consisting of Siglec1, Siglec2, Siglec4, and Siglec15, and a second CD33-associated subset consisting of Siglec3, Siglec5, Siglec6, Siglec7, Siglec8, Siglec9, Siglec10, Siglec11, Siglec12, Siglec14, and Siglec16. Siglec7, also known as p75, CD328, or AIRM, contains an extracellular N-terminal Ig-like V-type domain, two Ig-like C2-type domains, and an intracytoplasmic region containing one ITIM motif and one ITIM-like motif region. Siglec7 is constitutively expressed in NK cells, dendritic cells, monocytes, and neutrophils. It has been observed that Siglec7 has an inhibitory effect on NK cell-mediated tumor clearance. Siglec9 is structurally very similar to Siglec7, with their N-terminal V-group Ig domains sharing approximately 77% overall amino acid sequence identity and exhibiting distinct sialic acid binding specificities. NK cell functional studies have demonstrated that tumor cells expressing Siglec9-binding sialic acid ligands inhibit NK cell activation and tumor cell killing. Many human tumors strongly upregulate sialic acid ligands that bind to Siglec9, allowing tumors to evade the immune system and leading to cancer progression.

[0050] In a preferred embodiment, the NK inhibitory ligand is an antibody that targets Siglec7 or Siglec9, as known in the art. For example, anti-Siglec7 antibodies are derived from human Siglec7 / CD328 antibody (AF1138, R&D Systems), clone #194212 (MAB1138, R&D Systems), clone #194211 (MAB11381, R&D Systems), clone Z176 (A22330, Beckman Coulter), 6-434 (339202, Biolegend), REA214 (Miltenyl Biotec), S7.7 (MCA5782GA, BioRad), 10B2201 (MBS604764, MyBioSource), 8D8 (MBS690562, MyBioSource), 10B2202 (MBS608694, MyBioSource), and 5-386 (MBS214370, MyBioSource). Anti-Siglec9 antibodies were derived from MAB1139 (clone #191240, R&D Systems), AF1139 (R&D Systems), D18 (SC-34936, Santa Cruz Biotechnology), Y-12SC34938 (SC3-4938, Santa Cruz Biotechnology), AB 197981 (Abeam), AB96545 (Abeam), AB89484 (clone #MM0552-6K12, Abeam), AB 130493 (Abeam), AB117859 (clone #3G8, Abeam), and E10-286 (Becton Dickinson). Given the similarity in the extracellular structures of Siglec7 and Siglec9, antibodies targeting both can also function as NK inhibitory ligands in the present invention.

[0051] In one embodiment, the NK inhibitory ligand is an antibody targeting SIGLEC7, SIGLEC9, or both, and is selected from the group consisting of: (1) CDR-L1 set forth in SEQ ID NO: 102, CDR-L2 set forth in SEQ ID NO: 103, CDR-L3 set forth in SEQ ID NO: 104, CDR-H1 set forth in SEQ ID NO: 105, CDR-H2 set forth in SEQ ID NO: 106, and CDR-H3 set forth in SEQ ID NO: 107; (2) CDR-L1 set forth in SEQ ID NO: 122, CDR-L2 set forth in SEQ ID NO: 123, CDR-L3 set forth in SEQ ID NO: 124, CDR-H1 set forth in SEQ ID NO: 125, CDR-H2 set forth in SEQ ID NO: 126, and CDR-H3 set forth in SEQ ID NO: 127; (3) CDR-L1 set forth in SEQ ID NO: 131, CDR-L2 set forth in SEQ ID NO: 132, CDR-L3 set forth in SEQ ID NO: 133, CDR-H1 set forth in SEQ ID NO: 134, CDR-H2 set forth in SEQ ID NO: 135; (4) CDR-L1 shown in SEQ ID NO: 140, CDR-L2 shown in SEQ ID NO: 141, CDR-L3 shown in SEQ ID NO: 142, CDR-H1 shown in SEQ ID NO: 143, CDR-H2 shown in SEQ ID NO: 144, and CDR-H3 shown in SEQ ID NO: 155; (5) CDR-L1 shown in SEQ ID NO: 176, CDR-L2 shown in SEQ ID NO: 177, CDR-L3 shown in SEQ ID NO: 178 or (6) CDR-L1 set forth in SEQ ID NO: 188, CDR-L2 set forth in SEQ ID NO: 189, CDR-L3 set forth in SEQ ID NO: 190, CDR-H1 set forth in SEQ ID NO: 191, CDR-H2 set forth in SEQ ID NO: 192, and CDR-H3 set forth in SEQ ID NO: 193. The above antibodies (1)-(4) target SIGLEC7, and antibodies (5)-(6) target both SIGLEC7 and SIGLEC9. In one embodiment, the NK inhibitory ligand is an antibody that targets SIGLEC7, SIGLEC9, or both, and comprises a light chain variable region and a heavy chain variable region.The light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 108, 128, 137, 146, 182, 185, or 194. The heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 109, 129, 138, 147, 183, 186, or 195. In a preferred embodiment, the NK inhibitory ligand is an anti-SIGLEC7, SIGLEC9, or both, antibody, the amino acid sequence of which is set forth in SEQ ID NO: 110, 130, 139, 148, 184, 187, or 196. In one embodiment, the NK inhibitory ligand is an antibody targeting LAIR1 or a functional fragment thereof. LAIR1 contains 10 exons and encodes a 287-amino acid type I transmembrane glycoprotein containing a single extracellular C2-type Ig-like domain, followed by a stalk domain linked to a single transmembrane domain and two transmembrane ITIM motifs that inhibit signal transduction. LAIR1 shares some structural homology with members of the LIR and KIR families, suggesting that these molecules may have originated from the same ancestral gene. LAIR1 is expressed in hematopoietic progenitor cells, including T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, and human CD34+ cells. Due to the presence of the ITIM motifs, previous human and mouse studies have demonstrated that LAIR1 plays an immunosuppressive role. Further studies have shown that LAIR1 not only inhibits resting NK cells but also inhibits activated NK cell killing of target cells.

[0052] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting Ly49. Ly49 is a type II transmembrane glycoprotein that forms a homodimer via disulfide bonds and functions similarly to human KIR, i.e., interacts with MHC-I molecule ligands to transmit signals and regulate NK cell activity. To date, the mouse Ly49 family includes 11 members: Ly49A, Ly49B, Ly49C, Ly49D, Ly49E, Ly49F, Ly49G, Ly49H, Ly49I, Ly49P, and Ly49Q. Ly49A, Ly49C, Ly49F, Ly49G, and Ly49Q all contain ITIM motifs in their cytoplasmic regions, which bind to and activate the tyrosine kinase SHP-1, preventing the generation of phosphorylated tyrosine and inhibiting NK cell activation. Thus, in a preferred embodiment, the NK inhibitory ligand is an antibody that targets Ly49A, Ly49C, Ly49F, Ly49G, or Ly49Q.

[0053] In one embodiment, the NK inhibitory ligand is an antibody or functional fragment thereof targeting KLRG1. KLRG1 is a type II transmembrane protein surface co-inhibitory receptor that regulates the activity of T cells and NK cells. Its extracellular portion contains a C-type lectin domain, its known ligand is cadherin, and its intracellular portion contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain. It has been reported in the literature that expression of the KLRG1 receptor on NK cells in the peripheral blood of patients with hepatitis C may promote a decrease in the number and function of NK cells. The mechanism of action is primarily to inhibit NK cell proliferation, promote NK cell apoptosis, and reduce the release of proinflammatory cytokines from NK cells.

[0054] In one embodiment, the NK inhibitory ligand is an antibody targeting KLRG1, and is selected from the group consisting of: (1) CDR-L1 set forth in SEQ ID NO: 111, CDR-L2 set forth in SEQ ID NO: 112, CDR-L3 set forth in SEQ ID NO: 113, CDR-H1 set forth in SEQ ID NO: 114, CDR-H2 set forth in SEQ ID NO: 115, and CDR-H3 set forth in SEQ ID NO: 116; (2) CDR-L1 set forth in SEQ ID NO: 149, CDR-L2 set forth in SEQ ID NO: 150, CDR-L3 set forth in SEQ ID NO: 151, CDR-H1 set forth in SEQ ID NO: 152, CDR-H2 set forth in SEQ ID NO: 153, and CDR-H3 set forth in SEQ ID NO: 154; (3) CDR-L1 set forth in SEQ ID NO: 158, CDR-L2 set forth in SEQ ID NO: 159, CDR-L3 set forth in SEQ ID NO: 160, CDR-H1 set forth in SEQ ID NO: 161, CDR-H2 set forth in SEQ ID NO: 162, and CDR-H3 set forth in SEQ ID NO: 163, or (4) CDR-L1 set forth in SEQ ID NO: 167, CDR-L2 set forth in SEQ ID NO: 168, CDR-L3 set forth in SEQ ID NO: 169, CDR-H1 set forth in SEQ ID NO: 170, CDR-H2 set forth in SEQ ID NO: 171, and CDR-H3 set forth in SEQ ID NO: 172. In one embodiment, the NK inhibitory ligand is an antibody targeting KLRG1 and comprises a light chain variable region and a heavy chain variable region. The light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 117, 155, 164, or 173. The heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 118, 156, 165, or 174. In a preferred embodiment, the NK inhibitory ligand is an anti-KLRG1 antibody, the amino acid sequence of which is set forth in SEQ ID NO: 119, 157, 166, or 175.

[0055] In one embodiment, the NK inhibitory ligand is an NKIR natural ligand or an NKIR-binding domain (e.g., extracellular domain) contained therein, and such natural ligands include, but are not limited to, non-classical HLA-I molecules (e.g., HLA-E, HLA-F, and HLA-G), cadherins, collagens, OCIL, sialic acid, immune checkpoint ligands (e.g., PD-L1 / PD-L2, CD155, CD112, CD113, Gal-9, FGL1), and the like.

[0056] In one embodiment, the NK inhibitory ligand is a non-classical HLA-I molecule or its extracellular region, more preferably the α1 and α2 domains of a non-classical HLA-I molecule. Non-classical HLA-I molecules are located in the same chromosomal region, 6p21.3, on the short arm of chromosome 6. They consist of a non-covalently bound heavy chain (α chain) and light chain (beta chain, encoded by the B2M gene). The α chain contains three regions: an extracellular region (including α1, α2, and α3 domains), a transmembrane domain, and a cytoplasmic region. The α1 and α2 domains form an antigen-binding groove and are involved in binding to antigenic peptides that fit into the groove. The α3 domain is homologous to the constant region domain of immunoglobulins and binds to the T cell surface molecule CD8. Non-classical HLA class I molecules include three members: HLA-E, HLA-F, and HLA-G. HLA-E regulates NK cell activity by binding to the CD94 / NKG2 receptor on the NK cell surface. The function of HLA-E is to bind peptides derived from the leader sequences of HLA class I molecules (HLA-A, -B, -C, and -G) and present them to NK cells through interaction with the inhibitory receptor CD94 / NKG2A, thereby inhibiting NK cell lysis of cells expressing normal levels of HLA class I molecules. Under physiological conditions, the affinity of HLA-E for the inhibitory receptor CD94 / NKG2A is significantly higher than its affinity for the activating receptor CD94 / NKG2C, so upregulation of HLA-E expression levels can protect target cells from the killing effects of NK cells. HLA-F can bind to the NK inhibitory receptors ILT2 and ILT4, and this binding can be effectively blocked by ILT2 and ILT4 antibodies. Although the function of HLA-F is still under investigation, it is speculated that its binding to ILT2 and ILT4 may have an immunosuppressive effect. HLA-G can recognize various NK inhibitory receptors, such as CD94 / NKG2A, LIR-1, LIR-2, and KIR2DL1. Studies have shown that HLA-G molecules on the surface of fetal cells can bind to KIR on the surface of maternal NK cells, thereby inhibiting the killing activity of NK cells and potentially conferring immune tolerance to the HLA-heterologous fetus in the mother.Additionally, HLA-G molecules, which are highly expressed on the cell surface of solid tumors such as melanoma, sarcoma, and lymphoma, can enable tumor cells to escape the killing and lytic effects of NK cells.

[0057] In one embodiment, the NK inhibitory ligand is an HLA-E extracellular domain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31, or the coding sequence of which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 32. In another embodiment, the NK inhibitory ligand is a mutant of the HLA-E extracellular domain (comprising a Y84C mutation) having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33, or the coding sequence of which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 34.

[0058] In one embodiment, the NK inhibitory ligand is the extracellular domain of HLA-G, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 36.

[0059] In one embodiment, when endogenous B2M is knocked out of genetically modified immune cells and a non-classical HLA class I molecule is expressed as an NK inhibitory ligand, a B2M gene with a synonymous mutation (i.e., a change in the nucleotide sequence but not the amino acid sequence) is introduced to form a complex with the non-classical HLA-I molecule and exert its inhibitory function. At the same time, the B2M gene with the synonymous mutation can also avoid knockout by gene editing tools that target endogenous B2M. In this embodiment, the NK inhibitory ligand comprises a fusion molecule of B2M and the extracellular domain of a non-classical HLA class I molecule. For example, the NK inhibitory ligand comprises a fusion molecule of B2M and the extracellular domain of HLA-E or HLA-G. In a specific embodiment, the fusion molecule comprises B2M and the extracellular domain of HLA-E, preferably the HLA-E extracellular domain contains the Y84C mutation (SEQ ID NO: 33). In a preferred embodiment, the NK inhibitory ligand comprises a presenting peptide selected from the group consisting of SEQ ID NOs: 46 to 53, and a fusion molecule of B2M and the extracellular domain of HLA-E. Methods for synonymous mutation of the B2M gene are well known to those skilled in the art. In a preferred embodiment, B2M has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37. The nucleotide sequence of the synonymously mutated B2M gene is, for example, that shown in SEQ ID NO: 38.

[0060] In one embodiment, the NK inhibitory ligand is osteoclast inhibitory lectin (OCIL) or its NKIR-binding domain. Mouse OCIL contains three members: OCIL (also known as Clr-b), OCILrP1 (also known as Clr-d), and mOCILrP2 (also known as Clr-g). OCIL is widely expressed in various tissues, and its expression pattern is similar to that of MHC-I molecules. OCIL is a ligand for NKR-P1B / D. Studies have shown that expression of OCIL in tumor cells can suppress the killing effect of NK cells against tumor cells, and OCIL-specific antibodies can reverse this suppressive effect.

[0061] In one embodiment, the NK inhibitory ligand is a cadherin or its extracellular domain, such as E-cadherin (E-cad), N-cadherin (N-cad), or R-cadherin (R-cad), preferably the extracellular domain of E-cadherin. Cadherins are a type of calcium-dependent transmembrane protein that primarily mediates uniform cell-cell adhesion and binds to the NK inhibitory receptor KLRG1. Cadherin molecules are type I membrane proteins consisting of approximately 723 to 748 amino acids and include an extracellular domain involved in ligand binding, a transmembrane domain, and a highly conserved cytoplasmic region. The extracellular domain contains several cadherin repeat domains (ECs), which contain repeat sequences of 4 to 5 amino acid residues involved in ligand binding. Human E-cadherin is encoded by the CDH1 gene and is currently the most studied member of the cadherin family. Therefore, in a preferred embodiment, the NK inhibitory ligand is the extracellular domain of E-cadherin, including EC1 and EC2. More preferably, the NK inhibitory ligand is the extracellular domain of E-cadherin, including EC1, EC2, EC3, EC4, and EC5. More preferably, the NK inhibitory ligand is E-Cad and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39 or 41, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 40 or 42.

[0062] In one embodiment, the NK inhibitory ligand is collagen or its NKIR-binding domain, which binds to LAIR1. Collagen molecules are trimers composed of three α chains, each containing a (glycine-proline-hydroxyproline) n repeat sequence. LAIR1 recognizes and interacts with the Gly-Pro-Hyp repeat sequence. Due to the widespread presence of this repeat sequence, LAIR1 has been shown to bind to a wide variety of collagen molecules, including, but not limited to, transmembrane collagens such as collagens XVII, XIII, and XXIII, and non-transmembrane collagens such as collagens I, II, and III. Tumor cells or tumor stromal cells often highly express a variety of collagen molecules, which may transmit inhibitory signals to immune cells by binding to the inhibitory receptor LAIR1 on the surface of immune cells, potentially achieving the goal of immune evasion.

[0063] In one embodiment, the NK inhibitory ligand is sialic acid or its NKIR-binding region, which binds to SIGLEC family members (e.g., SIGLEC7 and / or SIGLEC9). Sialic acid is an important component of the innate immune system of vertebrates, and the killing activity of NK cells is associated with sialylation on the surface of tumor cells. Sialylation on tumor cells not only prevents physical interactions between tumor cells and NK cells, but also masks activating ligands on the tumor cell surface that can bind to NK cells. Furthermore, sialylation on the surface of tumor cells inhibits the formation of an immune synapse between tumor cells and NK cells, thereby reducing the toxicity of NK cells against tumors. Research has shown that sialylation on the surface of tumor cells can inhibit the killing activity of NK cells by inducing Siglec-mediated immunosuppressive signals. Siglec7 is expressed on the surface of most NK cells, and after tumor cells bind to Siglec7 on the surface of NK cells via α-2,8 glycosidic linkages, it inhibits NK cell activation, allowing tumor cells to escape the killing function of NK cells.

[0064] In one embodiment, the NK inhibitory ligand is PD-L1 / PD-L2 or its extracellular domain, which binds to PD1. PD-L1 is constitutively expressed at low levels on antigen-presenting cells (APCs) and non-hematopoietic cells such as vascular endothelial cells, pancreatic islet cells, and immune-privileged sites (e.g., placenta, testis, and eye). Proinflammatory cytokines such as type I and type II interferons, TNF-α, and VEGF can induce PD-L1 expression. PD-L2 is expressed only on activated macrophages and dendritic cells. Tumor cells themselves can upregulate PD-L1 expression. Proinflammatory cytokines in the tumor microenvironment can also induce PD-L1 and PD-L2 expression. Upregulated expression of PD-L1 and PD-L2 on the tumor cell surface can trigger the transmission of immunosuppressive signals by PD-1, inhibiting the killing activity of NK cells. In a preferred embodiment, the NK inhibitory ligand is the PD-L1 extracellular domain or the PD-L2 extracellular domain, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70 or 71.

[0065] In one embodiment, NK inhibitory ligand is CD155, CD112 or CD113 or its NKIR binding region, and all of them bind to TIGIT.CD155 is a high affinity ligand of TIGIT.When CD155, which is highly expressed on tumor surface, binds to TIGIT on NK surface, it inhibits the killing effect of NK cells against tumor cells.CD112 and CD113 also bind to TIGIT, but with relatively weak affinity.

[0066] In one embodiment, the NK inhibitory ligand is galectin-9 (also known as Gal-9) or its NKIR-binding domain, which binds to TIM3. Gal-9 is a C-type lectin widely expressed and secreted by many hematopoietic cells and binds to carbohydrate moieties on cell surface proteins. In TIM3, Gal-9 binds to carbohydrate motifs in its IgV domain, inducing calcium influx and cell death in TIM3-positive NK cells. It has been well documented that TIM3 / Gal-9 interaction plays a role in suppressing immune responses.

[0067] In one embodiment, the NK inhibitory ligand is FGL1 or its NKIR-binding domain, which binds to LAG3. FGL1 belongs to the fibrinogen family and is a newly discovered ligand for LAG3, but it lacks characteristic domains such as platelet-binding sites and thrombin-sensitive sites. FGL1 protein is distributed mainly in tumor cells, with low expression in the tumor stroma. The FGL1 / LAG3 interaction is another tumor immune evasion pathway independent of the B7-H1 / PD-1 pathway, and blocking this pathway exerts a synergistic effect with blocking the PD-1 pathway.

[0068] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric antigen receptor on the surface of an immune cell (e.g., a lymphocyte or NKT cell) and directs the cellular response of the immune cell against a target cell. The transmembrane domain can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can transmit signals when the chimeric antigen receptor binds to a target antigen. Transmembrane domains particularly useful in the present invention can be derived from the transmembrane domains of, for example, the 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, and NKIR natural ligands, including those of non-classical HLA-I molecules (e.g., HLA-E, HLA-F, and HLA-G), cadherin, collagen, and OCIL. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28, and CD278. Alternatively, the transmembrane domain can be synthetic and contain primarily hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8α or CD28 and more preferably has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 or 11, or the coding sequence for the transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10 or 12.

[0069] In one embodiment, the NK inhibitory molecule of the present invention may further comprise a hinge region located between the NK inhibitory ligand and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand-binding domain. Specifically, the hinge region provides greater flexibility and accessibility to the ligand-binding domain. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from a naturally occurring molecule, for example, derived in whole or in part from the extracellular region of CD8, CD4, or CD28, or derived in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a synthetic hinge sequence. In preferred embodiments, the hinge region comprises a portion of the hinge region of CD8α, CD28, an FcγRIIIα receptor, IgG4 or IgG1, more preferably a CD8α, CD28 or IgG4 hinge, and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25, 27 or 29, or the coding sequence for a CD28 hinge has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 26, 28 or 30.

[0070] The NK inhibitory molecules of the present invention contain a costimulatory domain. Therefore, when an NK inhibitory ligand binds to NKIR, it can transmit an inhibitory signal to NK cells via the signal transduction domain of NKIR, reducing killing of target cells (e.g., genetically modified immune cells of the present invention). On the other hand, it can transmit a stimulatory signal to target cells (e.g., genetically modified immune cells of the present invention) via the costimulatory domain contained in the NK inhibitory molecule, stimulating proliferation and survival of the target cells (e.g., genetically modified immune cells of the present invention), thereby better resisting killing by NK cells. Previous studies have shown that antibodies targeting inhibitory receptors on NK cells, such as NKG2A, KIR, and ILT2, can compete for binding to the binding sites of inhibitory molecules such as HLA-E and HLA-G or neutralize their inhibitory effects, thereby activating NK cells. Conversely, the present inventors have discovered for the first time that NK cells can be inhibited by expressing an NK inhibitory molecule that targets an NK inhibitory receptor. The present inventors have further discovered that NK inhibitory molecules containing a costimulatory domain have a more potent inhibitory effect on the killing of NK cells than NK inhibitory molecules lacking a costimulatory domain.

[0071] The costimulatory domain can be an intracellular functional signaling domain from a costimulatory molecule, including the entire intracellular portion of the costimulatory molecule or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that mediates a costimulatory response (e.g., proliferation) of T cells by specifically binding to a costimulatory ligand in T cells. Costimulatory molecules include, but are not limited to, class I MHC molecules, BTLA, and Toll ligand receptors. Non-limiting examples of costimulatory domains of the present invention include costimulatory signaling domains derived from proteins such as 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 combinations thereof. Preferably, the costimulatory domain of the invention is derived from 4-1BB, CD28, CD27, OX40, CD278, or a combination thereof, more preferably from 4-1BB, CD28, or a combination thereof. In one embodiment, the costimulatory domain of the invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13 or 15, or the coding sequence of the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 14 or 16.

[0072] In one embodiment, the NK inhibitory molecule does not contain an intracellular signaling domain. In another embodiment, the NK inhibitory molecule further contains an intracellular signaling domain. That is, the NK inhibitory molecule contains an NK inhibitory ligand, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In this embodiment, binding of the NK inhibitory ligand to an NKIR transmits an activation signal to a target cell (e.g., a genetically modified immune cell of the present invention) via the costimulatory domain and the intracellular signaling domain, promoting NK cell killing by the target cell, thereby further enhancing the inhibitory effect on NK cell killing.

[0073] As used herein, the term "intracellular signaling domain" refers to a protein portion that transmits an effector functional signal and instructs a cell to perform a specific function. The intracellular signaling domain is involved in primary intracellular signal transduction after the ligand-binding domain binds to an antigen, resulting in immune cell activation and an immune response. In other words, the intracellular signaling domain is involved in activating at least one of the normal effector functions of immune cells expressing NK inhibitory molecules. For example, the effector function of T cells can be cytolytic activity or helper activity, including cytokine secretion.

[0074] In one embodiment, the intracellular signaling domain of the present invention can be the cytoplasmic sequences of T cell receptors and co-receptors, which act together to initiate primary signal transduction after antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same or similar functions. The intracellular signaling domain can contain multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the present invention include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of a CAR of the invention comprises a CD3ζ signaling domain, which signaling domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17 or 19, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 18 or 20.

[0075] In one embodiment, the NK inhibitory molecule of the present invention may further comprise a signal peptide, which, when expressed in cells such as T cells, guides the nascent protein to the endoplasmic reticulum and then to the cell surface. The core of the signal peptide can contain a long hydrophobic amino acid segment and tends to form a single α-helix. The end of the signal peptide generally contains an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave the signal peptide during or after translocation to generate a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, and include signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, and the like. In one embodiment, a signal peptide useful in the invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 or 23, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 22 or 24.

[0076] Inhibition or silencing of at least one MHC-associated gene In one embodiment, the genetically modified immune cells that express an NK inhibitory molecule have inhibited or silenced expression of at least one MHC-related gene, e.g., inhibited or silenced expression of at least one MHC gene, or inhibited or silenced expression of a gene that interacts with or regulates the expression of at least one MHC gene.

[0077] The major histocompatibility complex (MHC) was originally characterized as a protein that plays a key role in transplantation responses. It is expressed on the surface of all higher vertebrates and is called H-2 in mice and HLA in humans. There are two main classes of MHC: class I and class II. Class I MHC proteins are heterodimers of two proteins: a transmembrane α chain encoded by the MHC1 gene and an extracellular beta-2 microsphere protein chain encoded by a gene outside the MHC gene cluster. The α chain contains three domains, and foreign peptides bind to the two most variable N-terminal domains, α1 and α2. Class II MHC proteins are also heterodimers, containing two transmembrane proteins encoded by genes within the MHC complex. Class I MHC / antigen complexes interact with cytotoxic T cells, while class II MHC presents antigens to helper T cells. Also, class I MHC proteins tend to be expressed on almost all nucleated cells and platelets (and red blood cells in mice), whereas class II MHC proteins are more selectively expressed: generally, they are expressed on B cells, some macrophages and monocytes, Langerhans cells, and dendritic cells.

[0078] The human class I HLA gene cluster contains three major loci: B, C, and A. HLA-A, HLA-B, and HLA-C are class I HLA heavy chain paralogs. Class I molecules are heterodimers composed of an MHC α heavy chain (encoded by HLA-A, HLA-B, or HLA-C) and a light chain (encoded by the β-2 microglobule protein, B2M). The heavy chain is membrane-anchored, approximately 45 kDa, and contains eight exons. Exon 1 encodes the leader peptide; exons 2 and 3 encode the α1 and α2 domains, both of which bind peptides; exon 4 encodes the α3 domain; exon 5 encodes the transmembrane region; and exons 6 and 7 encode the cytoplasmic tail. Polymorphisms within exons 2 and 3 confer peptide-binding specificity to each class of molecule. Thus, in one embodiment, inhibiting or silencing the expression of an MHC-associated gene refers to inhibiting or silencing the expression of one or more genes selected from the group consisting of HLA-A, HLA-B, HLA-C, and B2M.

[0079] The human class II HLA cluster contains three major loci: DP, DQ, and DR. Both class I and class II clusters are polymorphic. HLA-DPA1, HLA-DQA1, and HLA-DRA belong to the class II HLA α chain paralogs. Class II molecules play a key role in the immune system by presenting exogenous peptides and are primarily expressed in antigen-presenting cells (e.g., B lymphocytes, dendritic cells, and macrophages). Class II molecules are heterodimers consisting of a membrane-anchored α chain and a beta chain. The α chain is approximately 33–35 kDa and contains five exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode two extracellular domains, exon 4 encodes the transmembrane domain, and exon 5 encodes the cytoplasmic tail. Therefore, in one embodiment, inhibiting or silencing the expression of an MHC-associated gene refers to inhibiting or silencing the expression of one or more genes selected from the group consisting of HLA-DPA, HLA-DQ, and HLA-DRA.

[0080] Expression of MHC class I and II also depends on various accessory proteins. For example, Tap1 and Tap2 subunits are part of the TAP transporter complex, which is required for loading peptide antigens into class I HLA complexes. LMP2 and LMP7 proteasome subunits play a role in the proteolysis of antigens into peptides for display on HLA. Reduction of LMP7 has been shown to reduce cell surface MHC class I expression. MHC class II expression is induced and expressed by several positive regulators, including the RFX complex and CIITA. The RFX complex is composed of three subunits: RFXANK (also known as RFXB), RFX5, and RFX accessory protein (also known as RFXAP). The RFX complex promotes MHC class II molecule expression by facilitating the binding of other transcription factors to the promoter of MHC class II molecules and enhancing promoter binding specificity. CIITA is the master controller of MHC class II expression. CIITA comprises an N-terminus rich in acidic amino acids, a PST region rich in Pro, Ser, and Thr, a central GTP-binding region, and a C-terminus rich in Leu repeat sequences (LRR). The terminal acidic region and the PST region are transcriptional activation regions. Therefore, in one embodiment, inhibiting or silencing the expression of MHC-related genes refers to inhibiting or silencing the expression of one or more genes selected from the group consisting of TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, and CIITA.

[0081] Therefore, in one embodiment, inhibiting or silencing the expression of an MHC-related gene refers to inhibiting or silencing the expression of one or more genes selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof, and preferably inhibiting or silencing the expression of one or more genes selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0082] In one embodiment, the genetically modified immune cells expressing an NK inhibitory molecule have the expression of at least one TCR / CD3 gene inhibited or silenced.

[0083] The T cell receptor (TCR) is a distinctive marker on the surface of all T cells. It binds non-covalently to CD3 to form the TCR / CD3 complex, which binds to specific MHC-antigen peptide complexes on the surface of antigen-presenting cells to generate specific antigen-stimulating signals, activating the T cell and playing a role in killing. TCRs are heterodimers composed of two distinct peptide chains. They are typically divided into two categories: α / β and γ / δ. More than 95% of peripheral T lymphocytes express TCRα / β. The TCRα chain is encoded by the TRAC gene, and the β chain is encoded by the TRBC gene. Each TCR peptide chain contains a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. The cytoplasmic region is very short and does not transmit antigen-stimulating signals. TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity is determined by the V region. The V region contains three hypervariable regions, CDR1, CDR2, and CDR3, of which CDR3 exhibits the greatest degree of variability, directly determining the antigen-binding specificity of the TCR. When the TCR recognizes an MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the MHC molecule, while CDR3 directly binds to the antigen peptide. CD3 contains four subunits: γ, δ, ε, and ζ, and typically exists in the form of dimers: εγ, εδ, and ζζ. All four subunits contain a conserved immunoreceptor tyrosine-based activation motif (ITAM), two of which are phosphorylated by tyrosine protein kinases to transmit activation signals to T cells. Therefore, in one embodiment, inhibiting or silencing the expression of at least one TCR / CD3 gene refers to inhibiting or silencing the expression of one or more genes selected from the group consisting of TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0084] In a preferred embodiment, the genetically modified immune cells expressing the NK inhibitory molecule have inhibited or silenced expression of at least one TCR / CD3 gene and at least one MHC-related gene. The at least one TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and a combination thereof. The at least one MHC-related gene is selected from 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, preferably HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and a combination thereof.

[0085] In a preferred embodiment, the at least one TCR / CD3 gene is selected from TRAC, TRBC, and a combination thereof. The at least one MHC-related gene is selected from B2M, RFX5, RFXAP, RFXANK, CIITA, and a combination thereof. In one embodiment, the expression of TRAC or TRBC, and B2M in the genetically modified immune cells is inhibited or silenced. In one embodiment, the expression of TRAC or TRBC, and CIITA in the genetically modified immune cells is inhibited or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M, and CIITA in the genetically modified immune cells is inhibited or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M, and RFX5 in the genetically modified immune cells is inhibited or silenced.

[0086] In one embodiment, in addition to the MHC-related genes and any TCR / CD3 genes, the genetically modified immune cells of the present invention have inhibited or silenced expression of at least one gene selected from the group consisting of CD52, GR, dCK, and immune checkpoint genes. Examples include PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

[0087] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art and include, but are not limited to, DNA fragmentation via meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, or gene inactivation via techniques such as antisense oligonucleotides, RNAi, shRNA, etc.

[0088] Chimeric Antigen Receptor

[0089] In another aspect, the genetically modified immune cells of the present invention that express an NK inhibitory molecule may further express a chimeric antigen receptor. That is, in this embodiment, the genetically modified immune cells express an NK inhibitory molecule and a chimeric antigen receptor, and preferably, the expression of at least one MHC-related gene of the genetically modified immune cells is inhibited or silenced. In a preferred embodiment, the expression of at least one TCR / CD3 gene of the genetically modified immune cells is inhibited or silenced.

[0090] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide, which typically contains one or more ligand-binding domains (e.g., the antigen-binding portion of an antibody), a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, each connected by a linker. CARs utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition can circumvent a major mechanism of tumor escape by endowing CAR cells with antigen recognition capabilities independent of antigen processing. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0091] As used herein, the term "ligand-binding domain" refers to any structure or functional variant thereof that can bind to a ligand (e.g., an antigen). The ligand-binding domain may be an antibody structure, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and functional fragments thereof. For example, the ligand-binding domain may be, but is not limited to, an intact antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody, sdAb (VH or VL), nanobody (Nb), recombinant fibronectin domain, anticalin, and DARPIN. Preferably, the ligand-binding domain is selected from Fab, scFv, sdAb, and nanobody. In the present invention, the ligand-binding domain may be monovalent or bivalent, and may be a monospecific, bispecific, or multispecific antibody.

[0092] "Fab" refers to either of two identical fragments produced upon cleavage of an immunoglobulin molecule with papain, consisting of an intact light chain and the N-terminal portion of a heavy chain linked by a disulfide bond, with the N-terminal portion of the heavy chain containing the heavy chain variable region and CH1. Compared to intact IgG, Fab lacks the Fc fragment, has increased mobility and tissue penetration, and can bind monovalently to antigens without mediating antibody effects.

[0093] A "single-chain antibody" or "scFv" is an antibody composed of an antibody heavy chain variable region (VH) and light chain variable region (VL) connected by a linker. The optimal length and / or amino acid composition of the linker can be selected. Linker length significantly affects the folding and interaction of the variable regions of the scFv. Indeed, the use of a relatively short linker (e.g., 5-10 amino acids) can prevent intrachain folding. For selection of linker size and composition, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448; U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794; and PCT Application Publications WO2006 / 020258 and WO2007 / 024715, the entire contents of which are incorporated herein by reference. An scFv can comprise a VH and a VL linked in any order, for example, VH-linker-VL, or VL-linker-VH.

[0094] "Single domain antibodies" or "sdAbs" refer to antibodies that naturally lack light chains and contain only one variable heavy chain (VHH) and two common CH2 and CH3 regions; they are also called "heavy chain antibodies."

[0095] "Nanobody" or "Nb" refers to a single cloned and expressed VHH structure that has the same structural stability and antigen-binding activity as the original heavy chain antibody and is the smallest known unit capable of binding to a target antigen.

[0096] The term "functional variant" or "functional fragment" refers to a variant that essentially contains a parent amino acid sequence but contains at least one amino acid modification (i.e., substitution, deletion, or insertion) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. For example, in the case of an antibody, the functional fragment is its antigen-binding portion. In one embodiment, preferably, the amino acid modification is a conservative modification.

[0097] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0098] Thus, a "functional variant" or "functional fragment" is at least 75% identical to the parent amino acid sequence, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and retains the biological activity, e.g., binding activity, of the parent amino acid.

[0099] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of the exact same sequence would have 100% identity. Those skilled in the art will appreciate that several algorithms can determine sequence identity using standard parameters. Examples include Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197), and ClustalW.

[0100] The choice of ligand-binding domain will depend on the cell surface marker on the target cell that is relevant to a particular pathology, e.g., a tumor-specific or tumor-associated antigen, that it recognizes. Thus, in one embodiment, the ligand-binding domain of the invention is selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, 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, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, and Ephrin. B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, 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-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe antibodies bind to one or more targets selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, Claudin18.2, NKG2D, and any combination thereof. Preferably, the targets are selected from CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2 / neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D, and any combination thereof. Depending on the antigen to be targeted, the CAR of the present invention can be designed to include a ligand-binding domain specific to that antigen. For example, if CD19 is the target antigen, a CD19 antibody can be used as the ligand-binding domain of the present invention.

[0101] The definitions of the transmembrane domain, costimulatory domain, intracellular signaling domain, and optional hinge region, signal peptide, etc. contained in a CAR useful in the present invention can be found above.

[0102] In one embodiment, the CAR of the present invention can further comprise a switch structure for regulating the expression time of the CAR. For example, the switch structure can take the form of a dimerization domain, which, upon binding to its corresponding ligand, induces a conformational change, exposing the extracellular binding domain to bind to the target antigen and thereby activating the signaling pathway. Alternatively, the switch domain can be used to connect the binding domain and the signaling domain, respectively, and only when the switch domains bind to each other (e.g., in the presence of an inducing compound), the binding domain and the signaling domain are linked by a dimer and can activate the signaling pathway. The switch structure can be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain to prevent binding to the target antigen, and when the masking peptide is cleaved, for example, by a protease, the extracellular binding domain is exposed, resulting in a "normal" CAR structure. Various switch structures known to those skilled in the art can also be used in the present invention.

[0103] In one embodiment, the CAR of the present invention may contain a suicide gene, i.e., express a cell death signal that can be induced by an exogenous substance, resulting in the elimination of CAR cells when necessary (e.g., when severe toxic side effects occur). For example, the suicide gene may take the form of an inserted epitope, such as the CD20 epitope or RQR8, and CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes as needed. The suicide gene may be herpes simplex virus thymidine kinase (HSV-TK), which can be induced by ganciclovir treatment to cause cell death. The suicide gene may be iCaspase-9, which can be induced to dimerize with chemical inducers such as AP1903 and AP20187, activating downstream Caspase 3 molecules and causing apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

[0104] If desired, when the NK inhibitory molecule includes an intracellular signaling domain and the cell expresses a CAR, the NK inhibitory molecule of the present invention and the CAR can share other structures other than the binding region, such as a costimulatory domain and an intracellular signaling domain. Thus, in this embodiment, the genetically modified immune cell of the present invention (1) expresses a fusion protein of the NK inhibitory molecule of the present invention and a chimeric antigen receptor, wherein the fusion protein contains an NK inhibitory ligand, a ligand-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, and (2) the expression of at least one MHC-associated gene is inhibited or silenced.

[0105] Nucleic acids and vectors The present invention further provides a nucleic acid molecule comprising a nucleic acid sequence encoding an NK inhibitory molecule of the present invention. Optionally, the nucleic acid molecule may further comprise a nucleic acid sequence encoding a chimeric antigen receptor.

[0106] As used herein, the term "nucleic acid molecule" includes a sequence of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in a linear or circular form in single-stranded and / or double-stranded form, or a mixture thereof (including hybrid molecules). Thus, nucleic acids according to the present invention include DNA (e.g., dsDNA, ssDNA, cDNA), RNA (e.g., dsRNA, ssRNA, mRNA, ivtRNA), and combinations or derivatives thereof (e.g., PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

[0107] Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds such as those found in peptide nucleic acids (PNAs)). The nucleic acids of the present invention may further contain one or more modified bases, such as tritylated bases and rare bases (e.g., inosine). Other modifications, including chemical, enzymatic, or metabolic modifications, are also possible, as long as the multi-chain CAR of the present invention can be expressed from the polynucleotide. The nucleic acid can be provided in an isolated form. In one embodiment, the nucleic acid may contain regulatory sequences such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, etc.

[0108] The nucleic acid sequence of the present invention may be codon-optimized for optimal expression in desired host cells (e.g., immune cells) or for use in expression in bacteria, yeast, or insect cells. Codon optimization refers to replacing rare codons in highly expressed genes of a particular species present in the targeting sequence with common codons in highly expressed genes of that species, so that the codons before and after the replacement code for the same amino acid. Therefore, the selection of optimal codons depends on the codon usage preference of the host genome.

[0109] The present invention further provides a vector comprising a nucleic acid molecule according to the present invention. Optionally, the nucleic acid sequence encoding the NK inhibitory molecule and the nucleic acid sequence encoding the chimeric antigen receptor are located on the same vector or on different vectors.

[0110] As used herein, the term "vector" refers to a nucleic acid molecule used as a vehicle to transfer (exogenous) genetic material into a host cell where it may, for example, be replicated and / or expressed.

[0111] Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a vehicle that delivers an isolated nucleic acid into a cell, for example, by homologous recombination or by using a hybrid recombinase that specifically targets a sequence at a specific site. An "expression vector" is a vector used to transcribe a heterologous nucleic acid sequence (e.g., a sequence encoding a chimeric antigen receptor polypeptide of the present invention) and translate its mRNA in an appropriate host cell. Vectors that can be used in the present invention are known in the art, and commercially available vectors can be used. In one embodiment, vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses (RSV, polyomaviruses, and adeno-associated viruses (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). Typically, the vector itself is a nucleotide sequence, comprising a DNA sequence containing an insert (transgene) and a relatively large sequence that functions as the "backbone" of the vector. Genetically modified vectors typically contain an origin of autonomous replication in a host cell (if stable expression of a polynucleotide is desired), a selection marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). Vectors may further contain elements such as a promoter, a polyadenylation tail (polyA), a 3' UTR, an enhancer, a terminator, an insulator, an operon, a selection marker, a reporter gene, a targeting sequence, and / or a protein purification tag. In a specific embodiment, the vector is an in vitro transcribed vector.

[0112] Genetically modified immune cells The present invention also provides genetically modified immune cells expressing the NK inhibitory molecules of the present invention, wherein the expression of at least one MHC-related gene is inhibited or silenced. In one embodiment, the genetically modified immune cells of the present invention further express a chimeric antigen receptor comprising one or more ligand-binding domains, transmembrane domains, costimulatory domains, and intracellular signaling domains. In a preferred embodiment, the genetically modified immune cells of the present invention have the expression of at least one TCR / CD3 gene inhibited or silenced.

[0113] In one embodiment, in addition to the MHC-related genes and any TCR / CD3 genes, the genetically modified immune cells of the present invention have inhibited or silenced expression of at least one gene selected from the group consisting of CD52, GR, dCK, and immune checkpoint genes. Examples include PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

[0114] Optionally, when the NK inhibitory molecule includes an intracellular signaling domain and the cell expresses a CAR, the NK inhibitory molecule of the present invention and the CAR can share other structures other than the binding domain, such as a costimulatory domain and an intracellular signaling domain. Thus, in this embodiment, the genetically modified immune cells of the present invention (1) express a fusion protein of the NK inhibitory molecule of the present invention and a chimeric antigen receptor, the fusion protein comprising an NK inhibitory ligand, a ligand-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, and (2) inhibit or silence the expression of at least one MHC-related gene. In a preferred embodiment, the genetically modified immune cells inhibit or silence the expression of at least one TCR / CD3 gene.

[0115] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, induction of ADCC and / or CDC). For example, the immune cell can be a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell, or an immune cell obtained from a stem cell source such as umbilical cord blood. Preferably, the immune cell is a T cell. The T cell can be any T cell, for example, an in vitro cultured T cell, e.g., a primary T cell, or an in vitro cultured T cell from a T cell line such as Jurkat or SupT1, or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumors, etc. T cells can also be enriched or purified. T cells can be at any stage of development and include, but are not limited to, CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, and the like. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using various techniques known to those skilled in the art, such as Ficoll separation.

[0116] The NK inhibitory molecule and optional chimeric antigen receptor can be introduced into immune cells using conventional methods known in the art (e.g., by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. One example is RNA transfection, i.e., the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into host cells. This term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of nucleic acid molecules or polynucleotides via viruses. Transfection of animal cells usually involves opening transient pores or "holes" in the cell membrane to allow uptake of substances. Transfection can be performed using calcium phosphate, electroporation, cell extrusion, or by mixing cationic lipids with materials to generate liposomes that fuse with the cell membrane and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and non-animal eukaryotic cells (including plant cells). Thus, transformation is the genetic alteration of bacteria or non-animal eukaryotic cells that occurs through direct uptake of exogenous genetic material (nucleic acid molecules) from the surrounding cell membrane. Transformation can be achieved by artificial means. For transformation to occur, the cells or bacteria must be in a competent state. For prokaryotic transformation, the techniques of heat shock-mediated uptake, fusion of intact cells with bacterial protoplasts, microinjection, and electroporation are available.

[0117] Kits and Pharmaceutical Compositions The present invention provides kits comprising the NK inhibitory molecules, nucleic acid molecules, vectors or genetically modified immune cells of the present invention.

[0118] In a preferred embodiment, the kit of the present invention further comprises instructions.

[0119] The present invention further provides pharmaceutical compositions comprising an NK inhibitory molecule, a genetically modified immune cell, a nucleic acid molecule or vector of the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Thus, the present invention encompasses the use of an NK inhibitory molecule, a nucleic acid molecule, a vector, or a genetically modified immune cell in the preparation of a pharmaceutical composition or medicament.

[0120] As used herein, the term "pharmaceutically acceptable excipient" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing undesirable local or systemic effects), and those known in the art can be used (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonicity agents, absorption delaying agents, stabilizers, and tonicity adjusters. It is well within the skill of those skilled in the art to select appropriate excipients to prepare the desired pharmaceutical composition of the present invention. Exemplary excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose, and water. Generally, the selection of an appropriate excipient will depend, inter alia, on the active agent used, the disease being treated, and the desired dosage form of the pharmaceutical composition.

[0121] The pharmaceutical compositions of the present invention are suitable for administration by a variety of routes. Typically, administration is parenteral. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0122] The pharmaceutical compositions according to the present invention can be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or liquid extracts, or in any form particularly suited to the required administration method. Known processes for producing pharmaceuticals of the present invention include, for example, conventional mixing, dissolving, granulating, dragee-making, grinding, emulsifying, encapsulating, embedding, or lyophilizing processes. The pharmaceutical compositions containing the immune cells described herein are usually provided in the form of a solution, preferably containing a pharmaceutically acceptable buffer.

[0123] The pharmaceutical composition according to the present invention can also be administered in combination with one or more other drugs suitable for the treatment and / or prevention of diseases. Preferred examples of drugs suitable for combination include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, and the like. E), vincristine, and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase; radionuclides such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and -213, actinium-225, and astatine-213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as platelet factor 4 and melanoma growth stimulating protein; known anticancer agents such as antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. The pharmaceutical compositions of the present invention can also be used in combination with one or more other therapeutic methods, such as chemotherapy and radiotherapy.

[0124] Therapeutic applications The present invention further provides a method for treating a subject suffering from cancer, an infectious disease, or an autoimmune disease. The method comprises administering to the subject an effective amount of an NK inhibitory molecule, a nucleic acid molecule, a vector, a genetically modified immune cell, or a pharmaceutical composition according to the present invention. Thus, the present invention encompasses the use of an NK inhibitory molecule, a nucleic acid molecule, a vector, or a genetically modified immune cell in the manufacture of a medicament for treating cancer, an infectious disease, or an autoimmune disease.

[0125] In one embodiment, an effective amount of the immune cells and / or pharmaceutical compositions of the present invention is administered directly to a subject.

[0126] In another embodiment, the therapeutic method of the present invention is an ex vivo treatment. Specifically, this method comprises the following steps: (a) providing a sample containing immune cells; (b) inhibiting or silencing the expression of at least one TCR / CD3 gene and at least one MHC-related gene in the immune cells in vitro, and then introducing the NK inhibitory molecule of the present invention and optional chimeric antigen receptor into the immune cells to obtain modified immune cells; and (c) administering the modified immune cells to a subject. Preferably, the immune cells provided in step (a) are selected from B cells, macrophages, dendritic cells, monocytes, T cells, NK cells, or NKT cells. The immune cells can be obtained from a sample (particularly a blood sample) of the subject by conventional methods known in the art. Other immune cells capable of expressing the chimeric antigen receptor and NK inhibitory molecule of the present invention and performing the desired biological effector functions described herein can also be used. Furthermore, the immune cells are generally selected to be compatible with the subject's immune system, i.e., preferably, the immune cells do not induce an immunogenic response. For example, "universal recipient cells" can be used, i.e., lymphocytes that perform the desired biological effector function, are universally compatible, and can be grown and expanded in vitro. The use of such cells eliminates the need to obtain and / or provide the subject's own lymphocytes. The ex vivo transfer in step (c) can be performed by electroporating the nucleic acid or vector described herein into immune cells, or by infecting immune cells with a viral vector. The viral vector can be a lentiviral vector, adenoviral vector, adeno-associated viral vector, or retroviral vector, as described above. Other methods include the use of transfection reagents (e.g., liposomes) or transient RNA transfection.

[0127] In one embodiment, the immune cells are autologous or allogeneic cells, preferably B cells, T cells, macrophages, dendritic cells, monocytes, or NK cells, NKT cells, more preferably T cells, NK cells or NKT cells.

[0128] As used herein, the term "autologous" refers to material derived from an individual that is later reintroduced into that same individual.

[0129] As used herein, the term "allogeneic" refers to any material that is derived from a different animal or patient of the same species as the individual to whom the material is introduced.Two or more individuals are considered allogeneic to each other if the genes at one or more loci are different.In some cases, the genetic differences between allogeneic materials from individuals of the same species are sufficient to cause antigenic interactions.

[0130] As used herein, the term "subject" refers to a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal can be advantageously used as a subject in an animal model of cancer. Preferably, the subject is a human.

[0131] In one embodiment, the cancer is a cancer associated with the expression of a target that binds to the ligand-binding domain. For example, the cancer may be brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, osteosarcoma, brain cancer and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma, etc.), or tumors of the liver (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma, etc.). Cancers of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system malignancies, vulvar cancer and other cancers and sarcomas, and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma) Lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, and large nodule NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic Diseases that can be treated with the genetically modified immune cells or pharmaceutical compositions of the present invention include, but are not limited to, leukemia (T-ALL), B-cell prolymphocytic leukemia, plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myeloid leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell neoplasm, and post-transplant lymphoproliferative disorder (PTLD), as well as other diseases associated with target expression. Preferably, diseases that can be treated with the genetically modified immune cells or pharmaceutical compositions of the present invention include leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, etc.

[0132] In one embodiment, the infectious disease includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0133] In one embodiment, the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vascular inflammation, pernicious anemia, and systemic lupus erythematosus.

[0134] In one embodiment, the method further comprises administering to the subject one or more additional chemotherapeutic agents, biologic agents, drugs or treatments, in this embodiment, the chemotherapeutic agents, biologic agents, drugs or treatments are selected from radiation therapy, surgery, antibodies and / or small molecules, and any combination thereof.

[0135] The present invention will be described in detail below by way of examples with reference to the accompanying drawings. Those skilled in the art will appreciate that the accompanying drawings and examples are merely illustrative and do not limit the present invention. The embodiments and features of the embodiments of the present application can be combined with each other unless there is a contradiction.

[0136] The T cells used in all examples of the present invention were primary human CD4+CD8+ T cells isolated from healthy donors using leukapheresis with Ficoll-Paque™ PREMIUM (GE Healthcare, Lot No. 17-5442-02).

[0137] Example 1: Construction of UNKi-T immune cells that express NK inhibitory molecules and knock out TCR / HLA-I / HLA-II

[0138] The coding sequences for the B2m signal peptide (SEQ ID NO:21), NK inhibitory ligand, CD28 hinge region (SEQ ID NO:27), and CD28 transmembrane region (SEQ ID NO:11) were synthesized and cloned sequentially into pGEM-T Easy vector (Promega, lot number A1360), where the NK inhibitory ligand is the extracellular region of E-cadherin (SEQ ID NO:41, corresponding to the ECaD0 plasmid), a fusion molecule of B2M and the extracellular region of HLA-E (comprising the presenting peptide SEQ ID NO:46, B2M SEQ ID NO:37, and the HLA-E extracellular region variant SEQ ID NO:33, wherein the nucleic acid sequence of B2M is the synonymous variant SEQ ID NO:38, corresponding to the E0 plasmid), or a fusion molecule of B2M and the extracellular region of HLA-G (comprising B2M SEQ ID NO:37, and the HLA-G extracellular region SEQ ID NO:35, wherein the nucleic acid sequence of B2M is the synonymous variant SEQ ID NO:38, corresponding to the G0 plasmid). The ECad0, EO, and GO plasmids were further enriched with the CD28 costimulatory domain (SEQ ID NO: 13), resulting in ECad28, E28, and G28 plasmids, respectively. Correct insertion of the target sequence into the plasmids was confirmed by sequencing.

[0139] The coding sequences for the B2m signal peptide (SEQ ID NO: 21), anti-NKG2A-scFv (including SEQ ID NOs: 5 and 7), IgG4 hinge region (SEQ ID NO: 29), CD28 transmembrane region (SEQ ID NO: 11), and CD28 costimulatory domain (SEQ ID NO: 13) were synthesized and sequentially cloned into the pGEM-T Easy vector (Promega, lot number A1360) to obtain the A28 plasmid. The correct insertion of the target sequence into the plasmid was confirmed by sequencing.

[0140] The above plasmids were diluted in 3 ml of Opti-MEM (Gibco, Lot No. 31985-070) into a sterile tube, followed by the addition of the packaging vector psPAX2 (Addgene, Lot No. 12260) and envelope vector pMD2.G (Addgene, Lot No. 12259) at a plasmid:viral packaging vector:viral envelope vector ratio of 4:2:1. Then, 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche, Lot No. 06366236001) was added, immediately mixed to homogeneity, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to the flask of 293T cells. Virus was collected at 24 and 48 hours, pooled, and ultracentrifuged (25,000 g, 4°C, 2.5 hours) to obtain concentrated lentivirus.

[0141] T cells were activated with DynaBeads CD3 / CD28CTS™ (Gibco, lot number 40203D) and cultured for 1 day at 37°C, 5% CO2. Concentrated lentivirus was then added and cultured for 3 days. T cells expressing NK inhibitory molecules were obtained.

[0142] We then used the CRISPR system to knock out TCR / CD3 components (specifically, the TRAC gene) and MHC-related genes (specifically, B2M and RFX5) in T cells expressing NK inhibitory molecules. Specifically, activated NKi-T cells were electrotransfected with 10 μg Cas9 protein and 10 μg sgRNA (3.3 μg TRAC sgRNA (SEQ ID NO: 43) + 3.3 μg B2m sgRNA (SEQ ID NO: 44) + 3.3 μg RFX5 sgRNA (SEQ ID NO: 45)) using a BTX Agile Pulse Max electroporation machine (Harvard Apparatus BTX) at 400 V for 0.7 ms. Immediately after electrotransfection, NKi-T cells were placed in 1 ml of prewarmed medium and cultured at 37°C under 5% CO2 in the presence of IL-2 (300 IU / ml). Triple knockout UNKi-T cells were obtained. Wild-type T cells with TCR / B2M / RFC5 knockout using the CRISPR system (i.e., Mock T cells) and wild-type T cells without gene knockout (i.e., NT cells) were used as controls.

[0143] The structures of the NK inhibitory molecules contained in the UNKi-T cells prepared in this example are shown in Table 1.

[0144] [Table 1]

[0145] After 11 days, the expression efficiency of TCR / HLA-I / HLA-II in UNKi-T cells, Mock T cells, and NT cells was examined by flow cytometry using FITC Mouse Anti-Human CD3 (BD ​​Pharmingen, lot no. 555916), PE Mouse Anti-Human HLA-I (R&D lot no. FAB7098P), and APC Anti-Human DR, DP, DQ (Biolegend, lot no. 361714) antibodies. The results are shown in Table 2.

[0146] [Table 2]

[0147] Table 2 shows that the expression of TCR / B2M / RFX5 in UNKi-T cells and Mock T cells prepared according to the present invention was effectively inhibited or silenced.

[0148] Using a flow cytometer, HLA-E expression in UNKi-T cells and Mock T cells was detected with PE mouse anti-human HLA-E (biolegend, lot no. 342604) (Figure 1), HLA-G expression in UNKi-T cells and Mock T cells was detected with PE mouse anti-human HLA-G (biolegend, lot no. 335906) (Figure 2), E-cadherin expression in UNKi-T cells and Mock T cells was detected with 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) (Figure 3), and Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, F(ab′) fragment specific Anti-NKG2A scFv expression in UNKi- and Mock T cells was measured using the antibody (Jackson ImmunoResearch, Lot No. 109-065-097) and APC Streptavidin (BD, Lot No. 554067) (Figure 4).

[0149] 1 to 4 show that all of the NK inhibitory molecules are effectively expressed in the UNKi-T cells prepared according to the present invention.

[0150] Example 2. Inhibitory effect of UNKi-T cells on killing of NK cells

[0151] The effector cells used in this example were NK92 cells. Because the NK92 cell line does not express KLRG1, an E-cadherin receptor, NK92 cells overexpressing KLRG1 were first prepared.

[0152] A nucleic acid sequence encoding KLRG1 (SEQ ID NO: 54) was synthesized and cloned into the pGEM-T Easy vector (Promega, Lot No. A1360). The correct insertion of the target sequence was confirmed by sequencing. The vector was digested with SpeI enzyme, purified, and recovered to obtain a linearized vector. Following the manufacturer's recommendations, the linearized vector was used as a template to prepare mRNA using the mMESSAGE mMACHINE® T7 Ultra Kit (Invitrogen, Lot No. AM1345). Purified mRNA was obtained using the Fastpure cell / Tissue total RNA isolation kit (Vazyme, Lot No. RC101-01). NK92 cells were electrotransfected with 20 μg of the purified mRNA prepared above at 200 V for 2 ms using a BTX Agile Pulse Max electroporation machine (Harvard Apparatus BTX) to obtain NK92-KLRG1 cells. After 16 hours, KLRG1 expression was measured, and the results are shown in Figure 5. NK92 cells not transfected with KLRG1 served as a control.

[0153] As can be seen from Figure 5, NK92-KLRG1 cells could efficiently express KLRG1.

[0154] The inhibitory effect of UNKi-T cells prepared according to the present invention on NK cell killing was examined according to the following method. UNKi-T cells prepared according to the present invention and Mock-T cells were labeled with Far-Red (Invitrogen, Lot No. C34564). Labeled UNKi-T cells and Mock-T cells were plated in a 96-well plate at a concentration of 1 x 10 cells / well and cocultured with NK92 cells (UNKi-T cells and Mock-T cells expressing HLA-E, HLA-G, or NKG2A scFv) or NK92-KLRG1 cells (UNKi-T cells expressing E-cadherin) at an effector-target ratio of 2:1. After 16 to 18 hours, the proportion of T cells in the culture was measured using a flow cytometer, and the killing rate of NK cells against T cells was calculated. The results are shown in Figure 6.

[0155] As can be seen from Figure 6, compared with mock T cells that do not express 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. Furthermore, compared with T cells expressing only an inhibitory ligand and a transmembrane domain, the addition of a costimulatory domain can further significantly enhance the inhibition of NK cell killing by T cells (see G0 vs. G28, E0 vs. E28, and ECaD0 vs. ECaD28). Therefore, NK inhibitory molecules containing an inhibitory ligand, a transmembrane domain, and a costimulatory domain prepared according to the present invention significantly reduce the killing effect of NK cells on UNKi-T cells, effectively reducing the risk of HvGD.

[0156] Example 3. Killing effect of UNKi-T cells on NK cells E28z-UNKi-T and A28z-UNKi-T cells prepared according to the method described in Example 1 differ from E28-UNKi-T and A28-UNKi-T cells only in that they further contain the CD3ζ intracellular signaling domain (SEQ ID NO: 17).

[0157] (1) Detection of NK inhibitory molecule expression Anti-NKG2A scFv expression in A28z-UNKi-T cells and mock T cells 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) (Figure 7). HLA-E expression in E28z-UNKi-T cells and mock T cells was detected using PE mouse anti-human HLA-E (biolegend, lot no. 342604) using a flow cytometer (Figure 8).

[0158] The above results demonstrate that NK inhibitory molecules can be effectively expressed in E28z-UNKi-T cells and A28z-UNKi-T cells prepared according to the present invention.

[0159] (2) Detection of CD107a expression Cytotoxic T lymphocytes (CTL cells) contain high concentrations of cytotoxic granules in the cytosol, which exist in the form of vesicles. Lysosome-associated membrane protein I (CD107a) is a major component of these vesicle membrane proteins. When CTL cells kill target cells, the cytotoxic granules reach the plasma membrane and fuse with it (transporting CD107a molecules to the plasma membrane surface), triggering the release of the granule contents and resulting in the death of the target cell. Therefore, CD107a molecules are a sensitive marker of CTL cell degranulation and can reflect the killing activity of the cells.

[0160] Target cells (NK92 cells) were seeded at a concentration of 1 x 10 cells / well in a 96-well plate. Mock T cells, E28z-UNKi T cells, and A28z-UNKi T cells were added to each well at a 1:1 ratio. At the same time, 10 μl PE-anti-human CD107a (BD Pharmingen, Lot No. 555801) was added and co-cultured at 37°C and 5% CO2. After 1 hour, Goigstop (BD Pharmingen, Lot No. 51-2092KZ) was added and incubated for an additional 2.5 hours. Subsequently, 5 μl APC-anti-human CD8 (BD Pharmingen, Lot No. 555369) and 5 μl FITC-anti-human CD4 (BD Pharmingen, Lot No. 561005) were added to each well. After 30 minutes of incubation at 37°C, CD107a expression was analyzed using a flow cytometer. The results are shown in Figure 9A (CD4+ T cell toxicity) and Figure 9B (CD8+ T cell toxicity).

[0161] The above results indicate that mock T cells, which do not express NK inhibitory molecules, hardly kill target cells. Conversely, after co-culture of E28z-UNKi-T cells and A28z-UNKi-T cells prepared according to the present invention with target cells, the expression rate of CD107a significantly increased, and the UNKi-T cells according to the present invention have a significant killing effect on NK cells.

[0162] (3) Detection of IFN-γ secretion Target cells (NK92 cells) were seeded at 1 x 105 cells / well in a 96-well plate, and Mock T cells, E28z-UNKi-T cells, and A28z-UNKi-T cells were added to each well at a 1:1 ratio. They were co-cultured at 37°C and 5% CO2, and the cell co-culture supernatant was collected after 18 to 24 hours.

[0163] Purified anti-human IFN-γ antibody (Biolegend, lot no. 506502) was used to capture the IFN-γ signaling signal in a 96-well plate and incubated overnight at 4°C. The antibody solution was then removed. 250 μL of 2% BSA (Sigma, lot no. V900933-1kg) in PBST (1X PBS containing 0.1% Tween) was added and incubated at 37°C for 2 hours. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or standard solution was added to each well and incubated at 37°C for 1 hour. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). Next, 50 μL of the detection antibody, Anti-Interferon gamma (MD-1) (Biotin) (Abcam, lot number ab25017), was added to each well and incubated at 37°C for 1 hour. Afterwards, the plate was washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). HRP Streptavidin (Biolegend, lot number 405210) was added and incubated at 37°C for 30 minutes. The supernatant was discarded, and the plate was washed five times with 250 μL of PBST (1X PBS containing 0.1% Tween). 50 μL of TMB substrate solution was added to each well. After 30 minutes of incubation at room temperature in the dark, the reaction was stopped by adding 50 μL of 1 mol / L H2SO4 to each well. Within 30 minutes of stopping the reaction, the absorbance at 450 nm was measured using a microplate reader, and the cytokine content was calculated using a standard curve (drawn based on the standard readings and concentrations). The results are shown in Figure 10.

[0164] From the above results, the cytokine IFN-γ release levels of the E28z-UNKi-T cells and A28z-UNKi-T cells of the present invention were much higher than those of Mock T cells, which resulted in a significantly higher killing effect on NK92 target cells.

[0165] Example 4. UNKi-T cells targeting KIR or LIR1 and their inhibitory effect on NK cell killing The coding sequences for the B2m signal peptide (SEQ ID NO: 21), anti-KIR-scFv (including SEQ ID NOs: 55 and 56) or anti-LIR1 scFv (including SEQ ID NOs: 57 and 58, or including SEQ ID NOs: 59 and 60), IgG4 hinge region (SEQ ID NO: 29), CD8α transmembrane region (SEQ ID NO: 9), and CD28 costimulatory domain (SEQ ID NO: 13) were synthesized and sequentially cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), lot number: PPL00157-4a) to obtain KIRG4, LIRG4-1, and LIRG4-2 plasmids. The correct insertion of the target sequences into the plasmids was confirmed by sequencing.

[0166] UNKi-T cells were prepared according to the knockout and infection methods described in Example 1, and the CD3 / HLA-I / HLA-II expression efficiency in UNKi-T cells, mock T cells, and NT cells was examined by flow cytometry using FITC Mouse Anti-Human CD3 (BD ​​Pharmingen, lot no. 555916), PE Mouse Anti-Human HLA-I (R&D lot no. FAB7098P), and APC Anti-Human DR, DP, DQ (Biolegend, lot no. 361714) antibodies. The results are shown in Table 3.

[0167] [Table 3]

[0168] Table 3 shows that the expression of CD3 / HLA-I / HLA-II in UNKi-T cells and mock T cells prepared according to the present invention was effectively inhibited or silenced.

[0169] The scFv expression in LIRG4-UNKi-T cells 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). The results are shown in Figure 11. The expression of scFv in LIRG4-UNKi-1 and LIRG4-UNKi-2 T cells was detected using recombinant human LILRB1 protein (Sino Biological, Lot No. 16014-H02H) as the primary antibody and APC anti-human IgG Fc (Biolegend, Lot No. 409306) as the secondary antibody. The results are shown in Figure 12. Figures 11 and 12 demonstrate that the scFvs in the UNKi-T cells prepared according to the present invention were both effectively expressed.

[0170] UNKi-T cells and NK92 cells were co-cultured according to the method of Example 2, and the inhibitory effect of UNKi-T cells on NK cell killing was examined. The results are shown in Figure 13. The above results demonstrate that, compared with mock T cells, UNKi-T cells targeting KIR (Figure 13A) or LIR1 (Figure 13B) prepared in this example can significantly reduce the killing effect of NK cells on T cells.

[0171] Example 5. T cells targeting SIGLC7, SIGLEC9, or KLRG1 and their inhibitory effects on NK cell killing The coding sequences for the 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), CD8α transmembrane region (SEQ ID NO: 9), and CD28 costimulatory domain (SEQ ID NO: 13) were synthesized and sequentially cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), lot number: PPL00157-4a) to obtain the SC7G4, SC7 / SC9G4, and K1G4 plasmids. The correct insertion of the target sequences into the plasmids was confirmed by sequencing.

[0172] The above-mentioned plasmids were transferred into T cells according to the method of Example 1, and the B2M gene was knocked out to obtain NKi-T cells expressing NK inhibitory molecules and in which B2M was knocked out (i.e., SC7G4-T cells, SC7 / SC9G4-T cells, and K1G4-T cells). T cells in which B2M was only knocked out were used as negative controls (NT).

[0173] The scFv expression in SC7G4, SC7 / SC9G4, and K1G4 T cells was measured using 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 shown in Figure 14. These results demonstrate that the scFvs of the NKi-T cells prepared according to the present invention were all effectively expressed.

[0174] According to the method described in Example 2, the above NKi-T cells were co-cultured with NK92-KLRG1 cells to examine their inhibitory effect on NK cell killing. The results are shown in Figure 15. The above results demonstrate that, compared with NT, T cells targeting SIGLEC7, SIGLEC9, or KLRG1 prepared according to this example can significantly reduce the killing effect of NK cells on T cells.

[0175] Example 6. Targeting PD-1 to T cells and their inhibitory effects on NK cell proliferation The coding sequences for the PDL1 signal peptide (sequence number 121), PDL1 extracellular domain (sequence number 70), PDL1 transmembrane domain (sequence number 120), and CD28 costimulatory domain (sequence number 13) were synthesized and sequentially cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), lot number: PPL00157-4a) to obtain the PDL1 plasmid. Sequencing confirmed that the target sequence had been correctly inserted into the plasmid.

[0176] The above plasmid was transferred into T cells according to the method described in Example 1 to obtain NKi-T cells expressing NK inhibitory molecules, i.e., PDL1-T cells. Expression was examined using an anti-PD-L1 antibody (manufacturer: Soleivo, lot number: 10084-R312-A). The results are shown in Figure 16. The above results demonstrate that PDL1 is effectively expressed. Untreated T cells were used as a negative control (NT).

[0177] The above NKi-T cells were cultured and treated with mitomycin C. Two allogeneic PBMCs (donor 1 and donor 2) were labeled with Far-Red and co-cultured at a T cell:PBMC ratio of 1:2. Half-medium changes were performed every 2–3 days. After 8 days, cells were counted and stained with PE anti-human CD3 (manufacturer: biolegend, lot number: 317308) and FITC anti-human CD56 (manufacturer: biolegend, lot number: 362546). NK cell population ratios were then determined by flow cytometry. The number of NK cells was calculated by multiplying the total number of cells by the NK cell population ratio. The results are shown in Figure 17. These results demonstrate that NKi-T cells targeting PD-1 can significantly inhibit NK cell proliferation.

[0178] Example 7. Inhibitory effects of KIR-targeted B cells and Huh7 cells on NK cell killing

[0179] The KIRG4 plasmid prepared in Example 4 was transferred into B cells and Huh7 cells (liver cancer cells) according to the method of Example 1, and the B2M gene was knocked out to obtain NKi-B cells and NKi-Huh7 cells expressing NK inhibitory molecules and in which B2M was knocked out. B cells and Huh7 cells (NC cells) in which only the B2M gene was knocked out were used as negative controls.

[0180] The expression of scFv in NKi-B cells and NKi-Huh7 cells 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). The results are shown in Figure 18. These results demonstrate that NK inhibitory molecules are effectively expressed in B cells and Huh7 cells.

[0181] The cells prepared according to the method of Example 2 were co-cultured with NK cells to examine the inhibitory effect of the cells on the killing effect of NK cells. The results are shown in Figure 19. The results show that, compared with NC cells, the NKi-B cells and NKi-Huh7 cells prepared according to this example can significantly reduce the killing effect of NK cells.

[0182] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will appreciate that the present invention may have various changes and modifications. Those skilled in the art will understand that any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are within the scope of protection of the present invention. [Sequence List Free Text]

[0183] SEQ ID NO: 1: mNKG2A scFv-VH SEQ ID NO: 2: mNKG2A scFv-VH SEQ ID NO: 3: mNKG2A scFv-VL SEQ ID NO: 4: mNKG2A scFv-VL SEQ ID NO: 5: hNKG2A scFv-VH SEQ ID NO: 6: hNKG2A scFv-VH SEQ ID NO: 7: hNKG2A scFv-VL SEQ ID NO: 8: hNKG2A scFv-VL SEQ ID NO: 9: CD8α transmembrane domain SEQ ID NO: 10: CD8α transmembrane domain SEQ ID NO: 11: CD28 transmembrane domain SEQ ID NO: 12: CD28 transmembrane domain SEQ ID NO: 13: CD28 costimulatory domain SEQ ID NO: 14: CD28 costimulatory domain SEQ ID NO: 15: 4-1BB costimulatory domain SEQ ID NO: 16: 4-1BB costimulatory domain SEQ ID NO: 17: CD3 zeta signaling domain SEQ ID NO: 18: CD3 zeta signaling domain SEQ ID NO: 19: CD3 zeta signaling domain mutant SEQ ID NO: 20: CD3 zeta signaling domain mutant SEQ ID NO: 21: B2M signal peptide SEQ ID NO: 22: B2M signal peptide SEQ ID NO: 23: CD8α signal peptide SEQ ID NO: 24: CD8α signal peptide SEQ ID NO: 25: CD8α hinge region SEQ ID NO: 26: CD8α hinge region SEQ ID NO: 27: CD28 hinge region SEQ ID NO: 28: CD28 hinge region SEQ ID NO: 29: IgG4 hinge region SEQ ID NO: 30: IgG4 hinge region SEQ ID NO: 31: HLA-E extracellular domain SEQ ID NO: 32: HLA-E extracellular domain SEQ ID NO: 33: HLA-E extracellular domain mutant SEQ ID NO: 34: HLA-E extracellular domain mutant SEQ ID NO: 35: HLA-G extracellular domain SEQ ID NO: 36: HLA-G extracellular domain SEQ ID NO: 37: B2M SEQ ID NO: 38: Synonymous mutation B2M SEQ ID NO: 39: E-cadherin EC1-EC5 SEQ ID NO: 40: E-cadherin EC1-EC5 SEQ ID NO: 41: E-cadherin EC1-EC2 SEQ ID NO: 42: E-cadherin EC1-EC2 SEQ ID NO: 43: TRAC sgRNA SEQ ID NO: 44: B2M sgRNA SEQ ID NO: 45: RFX5 sgRNA SEQ ID NO: 46: Presenting peptide SEQ ID NO: 47: Presenting peptide SEQ ID NO: 48: Presenting peptide SEQ ID NO: 49: Presenting peptide SEQ ID NO: 50: Presenting peptide SEQ ID NO: 51: Presenting peptide SEQ ID NO: 52: Presenting peptide SEQ ID NO: 53: Presenting peptide SEQ ID NO: 54: KLRG1 SEQ ID NO: 55: KIR scFv VL SEQ ID NO: 56: KIR scFv VH SEQ ID NO: 57: KIR scFv SEQ ID NO: 58: KIR scFv VL SEQ ID NO: 59: KIR scFv VH SEQ ID NO: 60: KIR scFv SEQ ID NO: 61: LIR1 scFv-1 VL SEQ ID NO: 62: LIR1 scFv-1 VH SEQ ID NO: 63: LIR1 scFv-2 SEQ ID NO: 64: LIR1 scFv-2 VL SEQ ID NO: 65: LIR1 scFv-2 VH SEQ ID NO: 66: LIR1 scFv-2 SEQ ID NO: 67: NKG2A scFv VH SEQ ID NO: 68: NKG2A scFv VL SEQ ID NO: 69: NKG2A scFv SEQ ID NO: 70: PDL1 extracellular domain SEQ ID NO: 71: PDL2 extracellular domain SEQ ID NO: 72: NKG2A scFv CDR-L1 SEQ ID NO: 73: NKG2A scFv CDR-L2 SEQ ID NO: 74: NKG2A scFv CDR-L3 SEQ ID NO: 75: NKG2A scFv CDR-H1 SEQ ID NO: 76: NKG2A scFv CDR-H2 SEQ ID NO: 77: NKG2A scFv CDR-H3 SEQ ID NO: 78: NKG2A scFv CDR-L1 SEQ ID NO: 79: NKG2A scFv CDR-L2 SEQ ID NO: 80: NKG2A scFv CDR-L3 SEQ ID NO: 81: NKG2A scFv CDR-H1 SEQ ID NO: 82: NKG2A scFv CDR-H2 SEQ ID NO: 83: NKG2A scFv CDR-H3 SEQ ID NO: 84: KIR scFv CDR-L1 SEQ ID NO: 85: KIR scFv CDR-L2 SEQ ID NO: 86: KIR scFv CDR-L3 SEQ ID NO: 87: KIR scFv CDR-H1 SEQ ID NO: 88: KIR scFv CDR-H2 SEQ ID NO: 89: KIR scFv CDR-H3 SEQ ID NO: 90: LIR1 scFv-1 CDR-L1 SEQ ID NO: 91: LIR1 scFv-1 CDR-L2 SEQ ID NO: 92: LIR1 scFv-1 CDR-L3 SEQ ID NO: 93: LIR1 scFv-1 CDR-H1 SEQ ID NO: 94: LIR1 scFv-1 CDR-H2 SEQ ID NO: 95: LIR1 scFv-1 CDR-H3 SEQ ID NO: 96: LIR1 scFv-2 CDR-L1 SEQ ID NO: 97: LIR1 scFv-2 CDR-L2 SEQ ID NO: 98: LIR1 scFv-2 CDR-L3 SEQ ID NO: 99: LIR1 scFv-2 CDR-H1 SEQ ID NO: 100: LIR1 scFv-2 CDR-H2 SEQ ID NO: 101: LIR1 scFv-2 CDR-H3 SEQ ID NO: 102: SIGLEC7 scFv-1 CDR-L1 SEQ ID NO: 103: SIGLEC7 scFv-1 CDR-L2 SEQ ID NO: 104: SIGLEC7 scFv-1 CDR-L3 SEQ ID NO: 105: SIGLEC7 scFv-1 CDR-H1 SEQ ID NO: 106: SIGLEC7 scFv-1 CDR-H2 SEQ ID NO: 107: SIGLEC7 scFv-1 CDR-H3 SEQ ID NO: 108: SIGLEC7 scFv-1 VL SEQ ID NO: 109: SIGLEC7 scFv-1 VH SEQ ID NO: 110: SIGLEC7 scFv-1 SEQ ID NO: 111: KLRG1 scFv-1 CDR-L1 SEQ ID NO: 112: KLRG1 scFv-1 CDR-L2 SEQ ID NO: 113: KLRG1 scFv-1 CDR-L3 SEQ ID NO: 114: KLRG1 scFv-1 CDR-H1 SEQ ID NO: 115: KLRG1 scFv-1 CDR-H2 SEQ ID NO: 116: KLRG1 scFv-1 CDR-H3 SEQ ID NO: 117: KLRG1 scFv-1 VL SEQ ID NO: 118: KLRG1 scFv-1 VH SEQ ID NO: 119: KLRG1 scFv-1 SEQ ID NO: 120: PDL1 transmembrane domain SEQ ID NO: 121: PDL1 signal peptide SEQ ID NO: 122: SIGLEC7 scFv-2 CDR-L1 SEQ ID NO: 123: SIGLEC7 scFv-2 CDR-L2 SEQ ID NO: 124: SIGLEC7 scFv-2 CDR-L3 SEQ ID NO: 125: SIGLEC7 scFv-2 CDR-H1 SEQ ID NO: 126: SIGLEC7 scFv-2 CDR-H2 SEQ ID NO: 127: SIGLEC7 scFv-2 CDR-H3 SEQ ID NO: 128: SIGLEC7 scFv-2 VL SEQ ID NO: 129: SIGLEC7 scFv-2 VH SEQ ID NO: 130: SIGLEC7 scFv-2 SEQ ID NO: 131: SIGLEC7 scFv-3 CDR-L1 SEQ ID NO: 132: SIGLEC7 scFv-3 CDR-L2 SEQ ID NO: 133: SIGLEC7 scFv-3 CDR-L3 SEQ ID NO: 134: SIGLEC7 scFv-3 CDR-H1 SEQ ID NO: 135: SIGLEC7 scFv-3 CDR-H2 SEQ ID NO: 136: SIGLEC7 scFv-3 CDR-H3 SEQ ID NO: 137: SIGLEC7 scFv-3 VL SEQ ID NO: 138: SIGLEC7 scFv-3 VH SEQ ID NO: 139: SIGLEC7 scFv-3 SEQ ID NO: 140: SIGLEC7 scFv-4 CDR-L1 SEQ ID NO: 141: SIGLEC7 scFv-4 CDR-L2 SEQ ID NO: 142: SIGLEC7 scFv-4 CDR-L3 SEQ ID NO: 143: SIGLEC7 scFv-4 CDR-H1 SEQ ID NO: 144: SIGLEC7 scFv-4 CDR-H2 SEQ ID NO: 145: SIGLEC7 scFv-4 CDR-H3 SEQ ID NO: 146: SIGLEC7 scFv-4 VL SEQ ID NO: 147: SIGLEC7 scFv-4 VH SEQ ID NO: 148: SIGLEC7 scFv-4 SEQ ID NO: 149: KLRG1 scFv-2 CDR-L1 SEQ ID NO: 150: KLRG1 scFv-2 CDR-L2 SEQ ID NO: 151: KLRG1 scFv-2 CDR-L3 SEQ ID NO: 152: KLRG1 scFv-2 CDR-H1 SEQ ID NO: 153: KLRG1 scFv-2 CDR-H2 SEQ ID NO: 154: KLRG1 scFv-2 CDR-H3 SEQ ID NO: 155: KLRG1 scFv-2 VL SEQ ID NO: 156: KLRG1 scFv-2 VH SEQ ID NO: 157: KLRG1 scFv-2 SEQ ID NO: 158: KLRG1 scFv-3 CDR-L1 SEQ ID NO: 159: KLRG1 scFv-3 CDR-L2 SEQ ID NO: 160: KLRG1 scFv-3 CDR-L3 SEQ ID NO: 161: KLRG1 scFv-3 CDR-H1 SEQ ID NO: 162: KLRG1 scFv-3 CDR-H2 SEQ ID NO: 163: KLRG1 scFv-3 CDR-H3 SEQ ID NO: 164: KLRG1 scFv-3 VL SEQ ID NO: 165: KLRG1 scFv-3 VH SEQ ID NO: 166: KLRG1 scFv-3 SEQ ID NO: 167: KLRG1 scFv-4 CDR-L1 SEQ ID NO: 168: KLRG1 scFv-4 CDR-L2 SEQ ID NO: 169: KLRG1 scFv-4 CDR-L3 SEQ ID NO: 170: KLRG1 scFv-4 CDR-H1 SEQ ID NO: 171: KLRG1 scFv-4 CDR-H2 SEQ ID NO: 172: KLRG1 scFv-4 CDR-H3 SEQ ID NO: 173: KLRG1 scFv-4 VL SEQ ID NO: 174: KLRG1 scFv-4 VH SEQ ID NO: 175: KLRG1 scFv-4 SEQ ID NO: 176: SIGLEC7 / 9 scFv-1 CDR-L1 SEQ ID NO: 177: SIGLEC7 / 9 scFv-1 CDR-L2 SEQ ID NO: 178: SIGLEC7 / 9 scFv-1 CDR-L3 SEQ ID NO: 179: SIGLEC7 / 9 scFv-1 CDR-H1 SEQ ID NO: 180: SIGLEC7 / 9 scFv-1 CDR-H2 SEQ ID NO: 181: SIGLEC7 / 9 scFv-1 CDR-H3 SEQ ID NO: 182: SIGLEC7 / 9 scFv-1 VL SEQ ID NO: 183: SIGLEC7 / 9 scFv-1 VH SEQ ID NO: 184: SIGLEC7 / 9 scFv-1 SEQ ID NO: 185: SIGLEC7 / 9 scFv-1-2 VL SEQ ID NO: 186: SIGLEC7 / 9 scFv-1-2 VH SEQ ID NO: 187: SIGLEC7 / 9 scFv-1-2 SEQ ID NO: 188: SIGLEC7 / 9 scFv-2 CDR-L1 SEQ ID NO: 189: SIGLEC7 / 9 scFv-2 CDR-L2 SEQ ID NO: 190: SIGLEC7 / 9 scFv-2 CDR-L3 SEQ ID NO: 191: SIGLEC7 / 9 scFv-2 CDR-H1 SEQ ID NO: 192: SIGLEC7 / 9 scFv-2 CDR-H2 SEQ ID NO: 193: SIGLEC7 / 9 scFv-2 CDR-H3 SEQ ID NO: 194: SIGLEC7 / 9 scFv-2 VL SEQ ID NO: 195: SIGLEC7 / 9 scFv-2 VH SEQ ID NO: 196: SIGLEC7 / 9 scFv-2

Claims

1. An NK inhibitory molecule, An NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain, wherein the NK inhibitory ligands specifically bind to an NK inhibitory receptor (NKIR) to inhibit NK cell killing of a genetically modified immune cell expressing the NK inhibitory molecule, and the NK inhibitory molecule does not comprise an intracellular signaling domain.

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

3. The NK inhibitory molecule of claim 1 or 2, wherein the NKIR is selected from an immune checkpoint receptor, 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, a carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a sialic acid-binding immunoglobulin-like lectin (SIGLEC) family member, a leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), a Ly49 family member, and a killer cell lectin-like receptor G1 (KLRG1).

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

5. The NK inhibitory molecule of claim 3 or 4, wherein the NKIR is selected from PD1, NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9, and KLRG1.

6. The NK inhibitory molecule of any one of claims 1 to 5, wherein the NK inhibitory ligand is an antibody or functional fragment thereof that targets NKIR, and the antibody or functional fragment thereof is selected from an intact antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody, sdAb, or nanobody.

7. The NK inhibitory molecule of any one of claims 1 to 5, wherein the NK inhibitory ligand is an antibody that targets PD1, NKG2A, LIR1, KIR, SIGLEC7, SIGLEC9, and / or KLRG1.

8. (i) the antibody targeting NKG2A comprises (1) CDR-L1 set forth in SEQ ID NO: 72, CDR-L2 set forth in SEQ ID NO: 73, CDR-L3 set forth in SEQ ID NO: 74, CDR-H1 set forth in SEQ ID NO: 75, CDR-H2 set forth in SEQ ID NO: 76, and CDR-H3 set forth in SEQ ID NO: 77, or (2) CDR-L1 set forth in SEQ ID NO: 78, CDR-L2 set forth in SEQ ID NO: 79, CDR-L3 set forth in SEQ ID NO: 80, CDR-H1 set forth in SEQ ID NO: 81, CDR-H2 set forth in SEQ ID NO: 82, and CDR-H3 set forth in SEQ ID NO: 83; (ii) the antibody targeting LIR1 comprises (1) CDR-L1 set forth in SEQ ID NO: 90, CDR-L2 set forth in SEQ ID NO: 91, CDR-L3 set forth in SEQ ID NO: 92, CDR-H1 set forth in SEQ ID NO: 93, CDR-H2 set forth in SEQ ID NO: 94, and CDR-H3 set forth in SEQ ID NO: 95, or (2) CDR-L1 set forth in SEQ ID NO: 96, CDR-L2 set forth in SEQ ID NO: 97, CDR-L3 set forth in SEQ ID NO: 98, CDR-H1 set forth in SEQ ID NO: 99, CDR-H2 set forth in SEQ ID NO: 100, and CDR-H3 set forth in SEQ ID NO: 101; (iii) the antibody targeting the KIR comprises CDR-L1 set forth in SEQ ID NO: 84, CDR-L2 set forth in SEQ ID NO: 85, CDR-L3 set forth in SEQ ID NO: 86, CDR-H1 set forth in SEQ ID NO: 87, CDR-H2 set forth in SEQ ID NO: 88, and CDR-H3 set forth in SEQ ID NO: 89; (iv) The antibody targeting SIGLEC7, SIGLEC9, or both comprises: (1) CDR-L1 shown in SEQ ID NO: 102, CDR-L2 shown in SEQ ID NO: 103, CDR-L3 shown in SEQ ID NO: 104, CDR-H1 shown in SEQ ID NO: 105, CDR-H2 shown in SEQ ID NO: 106, and CDR-H3 shown in SEQ ID NO: 107; (2) CDR-L1 shown in SEQ ID NO: 122, CDR-H2 shown in SEQ ID NO: 123, and CDR-H3 shown in SEQ ID NO: 124; (3) CDR-L1 shown in SEQ ID NO: 131, CDR-L2 shown in SEQ ID NO: 132, CDR-L3 shown in SEQ ID NO: 133, CDR-H1 shown in SEQ ID NO: 134, CDR-H2 shown in SEQ ID NO: 135, and CDR-H3 shown in SEQ ID NO:

136. (4) CDR-L1 set forth in SEQ ID NO: 140, CDR-L2 set forth in SEQ ID NO: 141, CDR-L3 set forth in SEQ ID NO: 142, CDR-H1 set forth in SEQ ID NO: 143, CDR-H2 set forth in SEQ ID NO: 144, and CDR-H3 set forth in SEQ ID NO: 155; (5) CDR-L1 set forth in SEQ ID NO: 176, CDR-L2 set forth in SEQ ID NO: 177, CDR-L3 set forth in SEQ ID NO: 178, CDR-H1 set forth in SEQ ID NO: 179, CDR-H2 set forth in SEQ ID NO: 180, and CDR-H3 set forth in SEQ ID NO: 181; or (6) CDR-L1 set forth in SEQ ID NO: 188, CDR-L2 set forth in SEQ ID NO: 189, CDR-L3 set forth in SEQ ID NO: 190, CDR-H1 set forth in SEQ ID NO: 191, CDR-H2 set forth in SEQ ID NO: 192, and CDR-H3 set forth in SEQ ID NO: 193; and / or (v) The antibody targeting KLRG1 comprises: (1) CDR-L1 shown in SEQ ID NO: 111, CDR-L2 shown in SEQ ID NO: 112, CDR-L3 shown in SEQ ID NO: 113, CDR-H1 shown in SEQ ID NO: 114, CDR-H2 shown in SEQ ID NO: 115, and CDR-H3 shown in SEQ ID NO: 116; (2) CDR-L1 shown in SEQ ID NO: 149, CDR-L2 shown in SEQ ID NO: 150, CDR-L3 shown in SEQ ID NO: 151, CDR-H1 shown in SEQ ID NO: 152, CDR-H2 shown in SEQ ID NO: 153, and CDR-H3 shown in SEQ ID NO: 154; (3) CDR-L1 set forth in SEQ ID NO: 158, CDR-L2 set forth in SEQ ID NO: 159, CDR-L3 set forth in SEQ ID NO: 160, CDR-H1 set forth in SEQ ID NO: 161, CDR-H2 set forth in SEQ ID NO: 162, and CDR-H3 set forth in SEQ ID NO: 163, or (4) CDR-L1 set forth in SEQ ID NO: 167, CDR-L2 set forth in SEQ ID NO: 168, CDR-L3 set forth in SEQ ID NO: 169, CDR-H1 set forth in SEQ ID NO: 170, CDR-H2 set forth in SEQ ID NO: 171, and CDR-H3 set forth in SEQ ID NO:

172. The NK inhibitory molecule of claim 7.

9. The NK inhibitory molecule according to any one of claims 1 to 5, wherein the NK inhibitory ligand is selected from PD-L1 / PD-L2, HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, CD155, CD112, CD113, Gal-9, FGL1, and NKIR-binding domains contained therein.

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

11. (i) the PD-L1 extracellular domain has the amino acid sequence shown in SEQ ID NO: 70; (ii) the PD-L2 extracellular domain has the amino acid sequence set forth in SEQ ID NO: 71; (iii) the extracellular region of HLA-E has the amino acid sequence shown in SEQ ID NO: 31 or 33; (iv) the extracellular region of HLA-G has the amino acid sequence shown in SEQ ID NO: 35; (v) the E-cadherin extracellular region has the amino acid sequence set forth in SEQ ID NO: 39 or 41; The NK inhibitory molecule of claim 10.

12. The NK inhibitory molecule of claim 1, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, 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 of claim 1, wherein the costimulatory domain is selected from the costimulatory signaling domains of proteins such as 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 combinations thereof.

14. A nucleic acid molecule encoding the NK inhibitory molecule of any one of claims 1 to 13.

15. A vector comprising the nucleic acid molecule of claim 14.

16. (1) A genetically modified immune cell characterized by expressing the NK inhibitory molecule according to any one of claims 1 to 13, and (2) inhibiting or silencing the expression of at least one MHC-related gene.

17. 17. The genetically modified immune cell of claim 16, further expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a ligand-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

18. 18. The genetically modified immune cell of claim 16 or 17, wherein the at least one MHC-associated gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

19. 19. The genetically modified immune cell of claim 18, wherein the at least one MHC-associated gene is selected from B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

20. The genetically modified immune cell of claim 19, wherein the MHC-associated gene comprises B2M, and the NK inhibitory ligand is a fusion molecule of B2M and the extracellular domain of a non-classical HLA-I molecule.

21. The genetically modified immune cell of claim 20, wherein the non-classical HLA-I molecule is HLA-E or HLA-G.

22. The genetically modified immune cell of claim 20, wherein the NK inhibitory molecule further comprises a presenting peptide selected from the group consisting of SEQ ID NOs: 46-53.

23. 23. The genetically modified immune cell of any one of claims 16 to 22, wherein expression of at least one TCR / CD3 gene of the genetically modified immune cell is inhibited or silenced, and the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

24. CD52, GR, dCK, PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM , TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD 10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3. The genetically modified immune cell of any one of claims 16 to 23, wherein expression of one or more genes selected from the group consisting of IL10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3 is inhibited or silenced.

25. The ligand binding domain is 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, CD179a, 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-llRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD-20, AFP, Folate receptor α, ERBB2 (Her2 / neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostate, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-ab1, tyrosine kinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related 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 (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut 18. The genetically modified immune cell of claim 17, which binds to a target selected from 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.

26. 26. The genetically modified immune cell of claim 25, wherein the target is selected from CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin, Claudin 18.2, ROR1, NY-ESO-1, MAGE-A4, and any combination thereof.

27. The genetically modified immune cell of any one of claims 16 to 26, wherein the genetically modified immune cell is a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell, or a NKT cell.

28. 28. The genetically modified immune cell of claim 27, wherein the genetically modified 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.

29. A pharmaceutical composition comprising the NK inhibitory molecule of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, or the genetically modified immune cell of any one of claims 16 to 28, and one or more pharmaceutically acceptable excipients.

30. Use of the NK inhibitory molecule of any one of claims 1 to 13, the nucleic acid molecule of claim 14, the vector of claim 15, or the genetically modified immune cell of any one of claims 16 to 28 in the preparation of a medicament for treating cancer, an infectious disease, or an autoimmune disease.

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