CD7-targeting chimeric antigen receptors and uses thereof

Abstract

The present invention provides modified immune cells that express a chimeric antigen receptor (CAR) comprising an antigen-binding region comprising an anti-CD7 antibody, and in which expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene is suppressed or silenced. The present invention also provides uses of the modified immune cells in treating diseases associated with CD7 expression.

Description

[Technical Field]

[0001] The present invention is in the field of immunotherapy. More specifically, the present invention relates to chimeric antigen receptors that target CD7 and the use of chimeric antigen receptors in the treatment of disease. [Background technology]

[0002] In recent years, cancer immunotherapy technology has developed rapidly, and chimeric antigen receptor T cell (CAR-T) immunotherapy, as a new type of adoptive immunotherapy technology, has shown remarkable clinical efficacy in the treatment of various solid tumors and hematological tumors.

[0003] Depending on the source of T cells used to construct CAR-T cells, they can be divided into autologous CAR-T cells and allogeneic CAR-T cells (also known as universal CAR-T). The advantage of autologous CAR-T cells is that the initial T cells are derived from the patient receiving CAR-T therapy, eliminating the risk of immune rejection. However, their drawbacks are quite clear: high costs and long preparation times. Therefore, increasing research is focusing on the development of universal CAR-T cells. Universal CAR-T cells can be prepared from T cells isolated from the peripheral blood of healthy donors, significantly shortening the patient's waiting time for treatment. Furthermore, T cells obtained from healthy donors have better vitality and function than patient-derived T cells, which can increase the infectivity rate of CAR cells and improve therapeutic efficacy.

[0004] CD7 is a cell surface protein with a molecular weight of approximately 40 kDa and is a member of the immunoglobulin superfamily. CD7 is expressed on most T cells, NK cells, myeloid cells, T-cell acute lymphoblastic leukemia / lymphoma, acute myeloblastic leukemia, and chronic myeloid leukemia. It has been reported that CD7 acts as a costimulatory signal during T cell activation through binding to its ligand K12 / SECTM1. Furthermore, disruption of CD7 molecules in mouse T cell precursors still results in normal T cell development and homeostasis, suggesting that CD7 does not significantly affect T cell development and function, making it a highly suitable therapeutic target for the treatment of T-cell acute lymphoblastic leukemia (T-ALL). In fact, CD7 has been widely investigated as a target for cytotoxic molecules to treat leukemia and lymphoma.

[0005] CD7-directed CAR-T therapy, especially universal CAR-T therapy, is still very limited. Therefore, the present invention aims to provide efficient universal CAR-T cells that target CD7 and their use in the treatment of diseases. Summary of the Invention

[0006] In a first aspect, the present invention provides modified immune cells characterized by: (1) expressing a chimeric antigen receptor comprising an antigen-binding region comprising an anti-CD7 antibody; and (2) suppressing or silencing expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene.

[0007] In one embodiment, the chimeric antigen receptor comprises a CD7 antibody, a transmembrane domain, and an intracellular signaling domain.

[0008] In one embodiment, the anti-CD7 antibody comprises CDR-L1, CDR-L2, and CDR-L3 set forth in SEQ ID NOs: 1, 2, and 3, respectively, and CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOs: 4, 5, and 6. In a preferred embodiment, the anti-CD7 antibody comprises a light chain variable region having at least 90% identity to an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 7, 10, 13, 16, and 19, and a heavy chain variable region having at least 90% identity to an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 8, 11, 14, 17, and 20. In a preferred embodiment, the amino acid sequence of the anti-CD7 antibody is selected from SEQ ID NOs: 9, 12, 15, 18, and 21.

[0009] In one embodiment, the antigen-binding region of the chimeric receptor further comprises an antibody targeting a second antigen, wherein the second antigen is selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1 lRa, 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-1a, MAGE-A1, pod protein, 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 / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe antibody targeting the second antigen is 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. More preferably, the antibody targeting the second antigen is an antibody targeting CD19. In a preferred embodiment, the anti-CD19 antibody comprises a light chain variable region having at least 90% identity to an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 52 and 55, and a heavy chain variable region having at least 90% identity to an amino acid sequence set forth in a sequence selected from SEQ ID NOs: 53 and 56. In a preferred embodiment, the amino acid sequence of the anti-CD19 antibody is selected from SEQ ID NOs: 54 and 57.

[0010] In one embodiment, the transmembrane domain is selected from the transmembrane domains of proteins such as the TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 alpha, CD4, and CD28.

[0011] In one embodiment, the intracellular signaling domain is selected from the intracellular regions of proteins such as FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling domain comprises the CD3ζ intracellular region.

[0012] In one embodiment, the chimeric antigen receptor further comprises one or more costimulatory domains selected from the intracellular domains of proteins such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof. Preferably, the costimulatory domain is the intracellular region of CD27, CD28, CD134, CD137, or CD278, or a combination thereof.

[0013] In one embodiment, the TCR / CD3 genes are selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

[0014] In one embodiment, the MHC class II-related gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof, preferably selected from RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0015] In a preferred embodiment, the modified immune cells described herein comprise endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and at least one MHC class II gene selected from RFX5, RFXAP, RFXANK, and CIITA, the expression of which is suppressed or silenced. More preferably, the modified immune cells described herein comprise endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and RFX5, the expression of which is suppressed or silenced.

[0016] In one embodiment, the engineered immune cell further expresses one or more NK inhibitory ligands, an NK inhibitory molecule comprising a transmembrane domain and a costimulatory domain.

[0017] In one embodiment, the NK inhibitory ligand is an antibody that targets an NK inhibitory receptor, which is selected from the group consisting of 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), and NK cell receptor protein 1 (NKR-P1) family members. members (e.g., NKR-P1B and NKR-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 NK inhibitory receptor is selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1. More preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1. Even more preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, LIR1, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1.

[0018] In one embodiment, the NK inhibitory ligand is a natural ligand of an NK inhibitory receptor or an NK inhibitory receptor-binding domain contained therein. 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, CTLA4, CD155, CD112, CD113, Gal-9, FGL1, etc.), and NK inhibitory receptor-binding domains contained therein. More preferably, the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1 / PD-L2, CTLA-4, CD155, CD112, CD113, Gal-9, FGL1, or an NK inhibitory receptor binding region contained therein, and even more preferably, from HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, or an NK inhibitory receptor binding region contained therein. In a preferred embodiment, the NK inhibitory ligand is selected from the HLA-E extracellular region, HLA-G extracellular region, E-cadherin extracellular region, PD-L1 extracellular region, and PD-L2 extracellular region. In a preferred embodiment, the NK inhibitory ligand is the E-cadherin extracellular region, and includes EC1 and EC2, and more preferably includes EC1, EC2, EC3, EC4, and EC5. In a preferred embodiment, the NK inhibitory ligand is the extracellular domain of PD-L1 or PD-L2. In a preferred embodiment, the NK inhibitory ligand is the extracellular domain of HLA-E. In a preferred embodiment, the NK inhibitory ligand is the extracellular domain of HLA-G.

[0019] In one embodiment, the NK inhibitory molecule further comprises a CD3ζ intracellular region as an intracellular signaling domain.

[0020] In one embodiment, the immune cells are T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells. Preferably, the engineered 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.

[0021] In one aspect, the present invention further provides pharmaceutical compositions containing modified immune cells according to the present invention as an active agent, and including various pharmaceutically acceptable excipients.

[0022] In one aspect, the present invention further provides a method of treating a subject having a disease associated with CD7 expression, comprising administering to the subject an effective amount of an engineered immune cell or pharmaceutical composition of the present invention. Thus, the present invention further covers the use of the engineered immune cell in the preparation of a medicament for treating a disease associated with CD7 expression.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Chimeric Antigen Receptor

[0024] In a first aspect, the present invention provides modified immune cells characterized by: (1) expressing a chimeric antigen receptor comprising an antigen-binding region comprising an anti-CD7 antibody; and (2) suppressing or silencing expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene.

[0025] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid peptide that generally comprises an antigen-binding region (e.g., an antibody or antigen-binding portion thereof), a transmembrane domain, an optional costimulatory domain, and an intracellular signaling domain, with each domain connected by a linker. CARs utilize the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets without MHC restriction. Non-MHC-restricted antigen recognition confers on CAR-expressing immune cells antigen recognition capabilities unrelated to antigen processing, thereby bypassing a major mechanism of tumor evasion.

[0026] In one embodiment, the CAR expressed by the engineered immune cells provided herein comprises an antibody or antigen-binding fragment thereof that targets CD7, a transmembrane domain, and an intracellular signaling domain.

[0027] As used herein, the term "antibody" has the broadest meaning as understood by those skilled in the art and includes monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides having one or more CDR sequences capable of exhibiting a desired biological activity. Antibodies according to the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). Antibodies of the present invention also include recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and antigen-binding portions thereof.

[0028] As used herein, the term "antibody fragment" or "antigen-binding portion" refers to a portion of an intact antibody, generally comprising the antigen-binding site of the intact antibody, thereby retaining the ability to bind to an antigen. Examples of antibody fragments in the present invention include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-bonded Fv (sdFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, linear antibodies, "double bodies" having two antigen-binding sites, single-domain antibodies, nanobodies, natural ligands of the antigen, or functional fragments thereof. Therefore, the "antibody" of the present invention covers antibody fragments or antigen-binding portions of the antibodies defined above.

[0029] In one embodiment, the anti-CD7 antibody of the present invention is an anti-CD7 scFv. The terms "single-chain antibody" and "scFv" are used interchangeably herein and refer to an antibody in which an antibody heavy chain variable region (VH) and a light chain variable region (VL) are linked by a linker. The optimal length and / or amino acid composition of the linker may be specified as needed. The length of the linker clearly affects the folding and interaction conditions of the variable regions of the scFv. In fact, the use of a short linker (e.g., 5-10 amino acids) can prevent intrachain folding. For selection of linker size and composition, see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO 2006 / 020258 and WO 2007 / 024715, the entire contents of which are incorporated herein by reference. An scFv can comprise a VH and a VL linked in any order, e.g., VH-linker-VL or VL-linker-VH.

[0030] In one embodiment, a CAR of the invention comprises an antibody that targets CD7, comprising CDR-L1, CDR-L2, and CDR-L3 set forth in SEQ ID NOs: 1, 2, and 3, respectively, and CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOs: 4, 5, and 6. Preferably, the CD7-targeting antibody of the invention comprises a light chain variable region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to an amino acid sequence set forth in one selected from SEQ ID NOs: 7, 10, 13, 16, and 19, and a heavy chain variable region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to an amino acid sequence set forth in one selected from SEQ ID NOs: 8, 11, 14, 17, and 20. More preferably, the chimeric antigen receptor of the present invention comprises an anti-CD7 antibody, the amino acid sequence of which is set forth in SEQ ID NO: 9, 12, 15, 18, or 21.

[0031] In one embodiment, the antigen-binding region comprised in the chimeric antigen receptor of the present invention further comprises an antibody targeting a second antigen in addition to an antibody targeting CD7, wherein the second antigen is selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1. lRa, 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, 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, pod protein, 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 / 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-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut The tumor antigen is 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. Preferably, the tumor antigen is selected from 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, and any combination thereof. Any antibody targeting the above tumor antigens known in the art can be used in the present invention.

[0032] In a preferred embodiment, the antibody targeting the second antigen is an antibody targeting CD19, and comprises a light chain variable region sequence having 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: 52 or 55, and a heavy chain variable region sequence having 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: 53 or 56. More preferably, the chimeric antigen receptor of the present invention comprises an antibody targeting CD7 and an antibody targeting CD19, wherein the amino acid sequence of the antibody targeting CD19 is set forth in SEQ ID NO: 54 or 57.

[0033] The term "functional variant" or "functional fragment" means a variant that essentially comprises a parent amino acid sequence, but that contains at least one amino acid modification (substitution, deletion, insertion, etc.) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

[0034] 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 are 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, formadzinone, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications may be selected based, for example, on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0035] Thus, a "functional variant" or "functional fragment" has at least 75%, 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 to the parent amino acid sequence and retains the biological activity of the parent amino acid, e.g., binding activity.

[0036] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences share the same residues at identical positions in a comparison, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with the exact same sequence share 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as 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.

[0037] As used herein, the term "transmembrane domain" refers to a polypeptide structure that allows a chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, an NK cell, or an NKT cell) and guides the immune cell's cellular response to a target cell. The transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. When the chimeric antigen receptor binds to a target antigen, the transmembrane domain is capable of signal transduction. This term particularly applies to transmembrane domains of the present invention, and may be derived from, 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 functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and comprise predominantly hydrophobic residues, such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8 alpha chain or CD28 and 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: 22 or 24, or the nucleotide sequence whose coding sequence 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: 23 or 25.

[0038] In one embodiment, the chimeric antigen receptor of the present invention may comprise a hinge region located between the antigen-binding region 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 antigen-binding region. Specifically, the hinge region provides greater flexibility and accessibility to the antigen-binding region. The hinge region may comprise 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 natural 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 completely synthetic hinge sequence. In preferred embodiments, the hinge region comprises a portion of the hinge region of the CD8α chain, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably a hinge from CD8α, CD28 or IgG4, 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: 38, 40 or 42, or the nucleotide sequence whose 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: 39, 41 or 43.

[0039] As used herein, the term "intracellular signaling domain" refers to a protein portion that transduces an effector function signal and directs a cell to perform a predetermined function. The intracellular signaling domain is responsible for the primary intracellular signaling when the antigen-binding region binds to an antigen, thereby leading to the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for at least one of the normal effector functions of immune cells expressing a CAR. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion.

[0040] In one embodiment, the intracellular signaling domain contained in the chimeric antigen receptor of the present invention may be any synthetic sequence having the same or similar function as the cytoplasmic sequences of T cell receptors and coreceptors, which function together to induce primary signal transduction after antigen receptor binding, or any derivatives or variants of these sequences. The intracellular signaling domain may contain any of a number of immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the present invention include, but are not limited to, the intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of a CAR of the invention may comprise the CD3ζ intracellular region, 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: 30 or 32, 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: 31 or 33.

[0041] In one embodiment, the chimeric antigen receptor of the present invention further comprises one or more costimulatory domains. The costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, including the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. The term "costimulatory molecule" refers to a cognate binding gametophyte that mediates a costimulatory response (e.g., proliferation) of T cells by specifically binding to a costimulatory ligand in T cells. Examples of costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of costimulatory domains of the present invention include, but are not limited to, the intracellular regions of proteins such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM1), 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, CD94, LTB, and ZAP70, and combinations thereof.

[0042] In a preferred embodiment, the costimulatory domain comprises the intracellular region of one or more proteins selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB, and CD278. For example, in one embodiment, the costimulatory domain comprises the intracellular region of 4-1BB. In one embodiment, the costimulatory domain comprises the intracellular region of CD28. In one embodiment, the costimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of CD28.

[0043] In one embodiment, the intracellular region of 4-1BB 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: 28, 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: 29. In one embodiment, the intracellular region of CD28 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: 26, 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: 27.

[0044] In one embodiment, the CAR of the present invention may contain a signal peptide so that, when expressed in a cell, e.g., a T cell, the nascent protein is introduced into the endoplasmic reticulum and then to the cell surface. The core of the signal peptide may 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 cleaves the signal peptide during or after translocation, generating a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides applicable to the present invention are well known to those skilled in the art, such as signal peptides derived from CD8α, IgG1, GM-CSFRα, B2M, etc. In one embodiment, the signal peptide applicable to the present invention is derived from B2M or CD8α and 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: 34 or 36, or its coding sequence 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: 35 or 37.

[0045] In one embodiment, the CAR of the present invention may further comprise a switch structure to control the expression time of the CAR. For example, the switch structure may be in the form of a dimerization domain, which undergoes a conformational change upon binding to its corresponding ligand, exposing the extracellular antigen-binding region, thereby binding to the targeted antigen and activating the signaling pathway. Alternatively, the antigen-binding region and the signaling domain may be linked by a switch domain, such that only when the switch domains are bound to each other (e.g., in the presence of an inducing compound) do the antigen-binding region and the signaling domain dimerize to activate the signaling pathway. The switch structure may also be in the form of a masking peptide. The masking peptide can mask the extracellular antigen-binding region and prevent binding to the targeted antigen. After the masking peptide is cleaved, for example, by a protease, the extracellular antigen-binding region is exposed, resulting in a "normal" CAR structure. Various switch structures known to those skilled in the art can all be applied to the present invention.

[0046] In one embodiment, the CAR of the present invention may contain a suicide gene, i.e., a cell death signal induced by an exogenous substance is expressed to eliminate CAR cells when needed (e.g., when serious side effects occur). For example, the suicide gene may be in the form of an inserted epitope, such as the CD20 epitope or RQR8, and CAR cells can be eliminated when needed by adding antibodies or reagents targeting these epitopes. The suicide gene may be herpes simplex virus thymidine kinase (HSV-TK), which can be induced to die by treating cells with ganciclovir. The suicide gene may be iCaspase-9, which can be induced to dimerize using 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. Repression or silencing of endogenous gene expression

[0047] In one embodiment, the expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-associated gene of the modified immune cells provided herein is suppressed or silenced.

[0048] CD7 is expressed not only in tumor cells such as T-cell acute lymphoblastic leukemia / lymphoma, acute myeloblastic leukemia, and chronic myeloid leukemia, but also in most normal T cells and NK cells. Therefore, to avoid cross-recognition and killing between CD7-targeting CAR cells, it is necessary to suppress or silence the expression of the endogenous CD7 gene on CAR cells.

[0049] T cell surface receptors (TCRs) are characteristic features of all T cells. They bind noncovalently to CD3 to form the TCR / CD3 complex, and upon binding to specific MHC-antigen peptide complexes on the surface of antigen-presenting cells, they generate specific antigen-stimulating signals, activating and killing T cells. Therefore, suppressing or silencing the expression of the endogenous TCR / CD3 gene in CAR T cells can prevent them from attacking normal cells or tissues in the patient's body and also reduce the risk of graft-versus-host disease (GvHD). TCRs are heterodimers consisting of two distinct peptide chains and are generally divided into two types: α / β and γ / δ. More than 95% of peripheral T lymphocytes express both TCRα and β. The TCRα chain is encoded by the TRAC gene, and the β chain is encoded by the TRBC gene. Each peptide chain of a TCR 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 resides in the V region. The V region contains three hypervariable regions, CDR1, CDR2, and CDR3, of which CDR3 is the most mutated and therefore determines the antigen-binding specificity of the TCR. When a TCR recognizes an MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the MHC molecule, while CDR3 binds directly to the antigen peptide. CD3 contains four subunits: gamma, delta, epsilon, and zeta, and typically exists in the form of dimers: epsilon gamma, epsilon delta, and zeta zeta. All four subunits contain a conserved immunoreceptor tyrosine-based activation motif (ITAM), two of which tyrosine residues transduce activation signals to T cells after phosphorylation by tyrosine protein kinases. Thus, in one embodiment, the at least one TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0050] By examining the composition of peripheral blood lymphocytes in patients, the inventors discovered that CD4+CD7- T cells account for approximately 20% of the total number of CD3+ T cells (Figure 1). CAR cells targeting CD7 cannot recognize CD4+CD7- T cells and therefore cannot effectively kill this group. Conversely, CD4+CD7- T cells further kill exogenous CAR cells by recognizing MHC class II molecules. Therefore, to avoid the killing of exogenous CAR-T cells by CD4+CD7- T cells in patients, the inventors considered suppressing or silencing the expression of endogenous MHC class II-related genes in CAR-T cells.

[0051] In the present invention, MHC class II-related genes include MHC class II genes themselves, and genes that interact with or control the expression of MHC class II genes.

[0052] The major histocompatibility complex (MHC) was first characterized as a protein that plays a major 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: the transmembrane α chain encoded by the MHC I gene and the extracellular β2-microglobulin chain encoded by a gene not located within the MHC gene cluster. The α chain contains three domains, and foreign peptides are bound to the two most variable domains, α1 and α2, at the N-terminus. 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 (e.g., CD8+ T cells), while class II MHC presents antigens to helper T cells (e.g., CD4+ T cells). Also, class I MHC proteins tend to be expressed on almost all nucleated cells and platelets (and mouse red blood cells), while class II MHC proteins are more selectively expressed. Class II MHC proteins are typically expressed on B cells, some macrophages and monocytes, activated T cells, Langerhans cells, and dendritic cells.

[0053] The human class II HLA cluster contains three major loci: DP, DQ, and DR. Class II molecules are heterodimers consisting of an α chain and a β chain, both of which are membrane-anchored, with the α chain being approximately 33-35 kDa and containing five exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode the two extracellular domains, exon 4 encodes the transmembrane domain, and exon 5 encodes the cytoplasmic tail. Thus, in one embodiment, the MHC class II-associated gene is selected from HLA-DPA, HLA-DQ, and HLA-DRA.

[0054] Expression of MHC class II genes is also determined by several important positive regulatory proteins, such as the RFX complex and CIITA. The RFX complex is composed of three subunits: RFXANK (also known as RFXB), RFX5, and RFX helper protein (also known as RFXAP). The RFX complex promotes the expression of MHC class II molecules by promoting the binding of other transcription factors to the promoter of MHC class II molecules and enhancing the specificity of promoter binding. CIITA is a master regulator of MHC class II expression. CIITA contains 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-rich repeats (LRRs), where the N-terminal acidic region and the PST region are transcriptional activation regions. Therefore, in one embodiment, the MHC class II-related gene is selected from RFX5, RFXAP, RFXANK, and CIITA.

[0055] Thus, in one embodiment, the MHC class II-related gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK and CIITA, preferably RFX5, RFXAP, RFXANK and CIITA, more preferably RFX5.

[0056] In one embodiment, the endogenous MHC-I class genes (e.g., HLA-A, HLA-B, HLA-C, B2M, etc.) in the CAR-T cells are functional. In another embodiment, the expression of endogenous MHC-I class genes in the CAR-T cells is also suppressed or silenced.

[0057] In one embodiment, the engineered immune cells expressing a CD7-targeting chimeric antigen receptor described in the present invention comprise endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene, the expression of which is suppressed or silenced, wherein the at least one TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and a combination thereof, and the at least one MHC class II-related gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK, and CIITA. In a preferred embodiment, the engineered immune cells expressing a CD7-targeting chimeric antigen receptor described in the present invention comprise endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and at least one MHC class II gene selected from RFX5, RFXAP, RFXANK, and CIITA, the expression of which is suppressed or silenced. More preferably, the modified immune cells expressing a CD7-targeting chimeric antigen receptor described in the present invention contain endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and RFX5, the expression of which is suppressed or silenced. In one embodiment, the expression of MHC-I class genes, such as HLA-A, HLA-B, HLA-C, and B2M, in the modified immune cells is functional.

[0058] In one embodiment, in addition to CD7, MHC class II-associated genes, and TCR / CD3 genes, the modified immune cells of the present invention may also contain other genes, such as CD52, GR, dCK, and other 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, TGF-β ... The gene may include at least one gene selected from immune checkpoint genes such as BRII, 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, and the expression of the gene may be suppressed or silenced.

[0059] Methods for suppressing gene expression or silencing genes are well known to those skilled in the art and include, but are not limited to, mediating DNA cleavage by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes of the CRISPR system, or inactivating genes by techniques such as antisense oligonucleotides, RNAi, shRNA, etc. NK inhibitory molecules

[0060] The inventors also discovered that there is a large proportion (approximately 20%) of CD7-NK cells in patients that cannot be targeted and killed by CD7CAR (Figure 1), and this portion of cells has the potential to kill CAR cells if MHC-II gene expression is suppressed or silenced.

[0061] Thus, in one embodiment, to inhibit killing of CAR-T cells by NK cells in a patient, the engineered immune cells further express an NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain, for example, the NK inhibitory molecule comprises one or two NK inhibitory ligands, a transmembrane domain, and a costimulatory domain.

[0062] The definitions of the transmembrane domain and costimulatory domain contained in the NK inhibitory molecule are the same as those of the transmembrane domain and costimulatory domain contained in the chimeric antigen receptor.

[0063] In one embodiment, the NK inhibitory ligand is an antibody that targets an NK inhibitory receptor, which is selected from the group consisting of 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), and NK cell receptor protein 1 (NKR-P1) family members. members (e.g., NKR-P1B and NKR-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, said NK inhibitory receptor is preferably selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1. More preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1. Even more preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, LIR1, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, and KLRG1.

[0064] In one embodiment, the NK inhibitory ligand is an antibody that targets an NK inhibitory receptor, and said antibody is an intact antibody, a Fab, a Fab', a F(ab')2, an Fv fragment, an scFv antibody fragment, a linear antibody, an sdAb, or a nanobody.

[0065] In a preferred embodiment, the NK inhibitory ligand is an antibody targeting NKG2A. More preferably, the antibody targeting NKG2A comprises a light chain variable region sequence having 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, 62, or 77, and a heavy chain variable region sequence having 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, 61, or 76. More preferably, the amino acid sequence of the antibody targeting NKG2A is set forth in SEQ ID NO: 60, 63, or 78. Other antibodies that target NKG2A known in the art, such as 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), are also applicable to the present invention.

[0066] In a preferred embodiment, the NK inhibitory ligand is an antibody targeting a KIR, such as KIR2DL1, KIR2DL2 / 3, and KIR3DL1. More preferably, the KIR-targeting antibody comprises a light chain variable region sequence having 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: 65 or 79, and a heavy chain variable region sequence having 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: 66 or 80. More preferably, the amino acid sequence of the KIR-targeting antibody is set forth in SEQ ID NO: 67 or 81. Other antibodies that target KIR known in the art, such as GL183 (targeting KIR2DL2 / L3 and available from Immunotech, France and Beckton Dickinson, USA), EB6 (targeting KIR2DL1 and available from Immunotech, France and Beckton Dickinson, USA), AZ138 (targeting KIR3DL1 and available from Moretta et al., Univ. Genova, Italy), Q66 (targeting KIR3DL2 and available from Immunotech, France), and Z27 (targeting KIR3DL1 and available from Immunotech, France and Beckton Dickinson, USA), are also applicable to the present invention.

[0067] In a preferred embodiment, the NK inhibitory ligand is an antibody targeting LIR1. More preferably, the antibody targeting LIR1 comprises a light chain variable region sequence having 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: 68 or 71, and a heavy chain variable region sequence having 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: 69 or 72. More preferably, the amino acid sequence of the antibody targeting LIR1 is set forth in SEQ ID NO: 70 or 73.

[0068] In one embodiment, the NK inhibitory ligand is a natural ligand of an NK inhibitory receptor or an NK inhibitory receptor-binding domain contained therein. 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 NK inhibitory receptor-binding domains contained therein. More preferably, the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1 / PD-L2, CTLA-4, CD155, CD112, CD113, Gal-9, FGL1, or an NK inhibitory receptor binding region contained therein, even more preferably from HLA-E, HLA-F, HLA-G, cadherin, or an NK inhibitory receptor binding region contained therein, and more preferably from the extracellular region of HLA-E, HLA-G, E-cadherin, PD-L1, and PD-L2.

[0069] In a preferred embodiment, the NK inhibitory ligand is an E-cadherin extracellular region, comprising domains EC1 and EC2, more preferably comprising domains EC1, EC2, EC3, EC4, and EC5. Preferably, the E-cadherin extracellular 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: 44 (comprising domains EC1 and EC2) or SEQ ID NO: 48 (comprising domains EC1, EC2, EC3, EC4, and EC5).

[0070] In one embodiment, the NK inhibitory ligand is the extracellular domain of PD-L1 or PD-L2. Preferably, the PD-L1 extracellular domain 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: 45, and the PD-L2 extracellular domain 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: 46.

[0071] In one embodiment, the NK inhibitory ligand is an HLA-E extracellular domain or an HLA-G extracellular domain. Preferably, the HLA-E extracellular domain 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: 50 (wild type) or SEQ ID NO: 51 (including the Y84C mutation variant), and the HLA-G extracellular domain 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: 49. As is known in the art, non-classical HLA class I molecules (e.g., HLA-E, HLA-G) can perform their functions as long as they form a complex with B2M. Therefore, if B2M needs to be knocked out, a synonymous mutation in the DNA sequence of B2M must be introduced to prevent knockout by gene editing tools so that non-classical HLA class I molecules can normally perform their inhibitory functions. In other words, in cells where B2M is knocked out, the NK inhibitory ligand is a fusion molecule of B2M and the extracellular domain of HLA-E or HLA-G.

[0072] In another embodiment, the NK inhibitory molecule comprises at least two NK inhibitory ligands, and the NK inhibitory ligands are selected from anti-NKG2A scFv, anti-KIR scFv, anti-LIR1 scFv, HLA-E extracellular region, HLA-G extracellular region, E-cadherin extracellular region, PD-L1 extracellular region, and PD-L2 extracellular region, more preferably PD-L1 extracellular region and HLA-E extracellular region.

[0073] In one embodiment, the NK inhibitory molecule further comprises an intracellular signaling domain selected from the intracellular regions of proteins such as FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling domain comprises the intracellular region of CD3ζ.

[0074] In one embodiment, the NK inhibitory molecule may comprise a signal peptide, such as a signal peptide from PDL1 or B2M. In one embodiment, the NK inhibitory molecule comprises a PD-L1 signal peptide, which has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44. Those skilled in the art can select other suitable signal peptides according to their needs. Engineered immune cells

[0075] The present invention further provides modified immune cells that (1) express a chimeric antigen receptor comprising an antigen-binding region comprising an anti-CD7 antibody and an optional second antigen region, and (2) have suppressed or silenced expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene. In one embodiment, the modified immune cells of the present invention further express one or more NK inhibitory ligands, an NK inhibitory molecule comprising a transmembrane domain, and a costimulatory domain.

[0076] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., killing of cytotoxic cells, secretion of cytokines, induction of ADCC and / or CDC). For example, the immune cell may be a T cell, macrophage, dendritic cell, monocyte, NK cell, and / or NKT cell, or may be an immune cell derived from a stem cell, such as an adult stem cell, embryonic stem cell, umbilical cord blood stem cell, progenitor cell, bone marrow stem cell, induced pluripotent stem cell, totipotent stem cell, or hematopoietic stem cell. Preferably, the immune cell is a T cell. The T cell may be any T cell, for example, an ex vivo cultured T cell such as a primary T cell, or a T cell from an in vitro cultured T cell line such as Jurkat, SupT1, or the like, 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 multiple sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. T cells may be enriched or purified. T cells may be at any stage of development, including, but 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, and αβ-T cells. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained by separating a subject's blood using various techniques known to those skilled in the art, such as Ficoll. In the present invention, immune cells are modified to express a chimeric antigen receptor and suppress or silence the expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene.

[0077] Nucleic acid sequences encoding the chimeric antigen receptor polypeptide and any NK inhibitory molecule are introduced into immune cells using common methods known in the art (e.g., 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 approaches in eukaryotic cells. The term "transduction" generally describes virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells generally involves transiently opening pores or "holes" in the cell membrane to allow material uptake. Transfection can be achieved using calcium phosphate, electroporation, cell extrusion, or by mixing cationic lipids with materials to generate lipid bodies that fuse with the cell membrane and deposit these carriers internally. Exemplary techniques used to transfect 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 intended to describe the transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, as well as non-viral vectors in non-animal eukaryotic cells (including plant cells). Thus, transformation is the genetic alteration of bacteria or non-animal eukaryotic cells, resulting from the direct uptake and subsequent integration of exogenous genetic material (nucleic acid molecules) from their surroundings through the cell membrane. Transformation may also be achieved by artificial means. For transformation to occur, the cell or bacterium must be in a competent state. For prokaryotic transformation, techniques include heat shock-mediated uptake, fusion of whole cells with bacterial protoplasts, microinjection, and electroporation.

[0078] Once the nucleic acid or vector has been introduced into immune cells, one skilled in the art can expand and activate the obtained immune cells using common techniques. Pharmaceutical Composition

[0079] The present invention further provides pharmaceutical compositions containing modified immune cells according to the present invention as an active agent, and including various pharmaceutically acceptable excipients.

[0080] 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., achieves the desired therapeutic effect without any undesired local or systemic effects) and is well known in the art (see, e.g., 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, coating agents, adsorbents, anti-adherents, 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. Those skilled in the art will know how to select appropriate excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients in pharmaceutical compositions for use in the present invention are saline, buffered saline, dextrose, and water. Generally, the selection of an appropriate excipient will depend on the particular active agent used, the disease being treated, and the desired dosage form of the pharmaceutical composition.

[0081] The pharmaceutical compositions of the present invention are suitable for administration by several routes. Generally, administration is accomplished parenterally. Parenteral administration methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0082] The pharmaceutical compositions of the present invention can be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, including, inter alia, ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, liquid extracts, or in a form specifically adapted for the desired administration method. The process for producing a drug known in the present invention may include, for example, routine mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, encapsulating, or lyophilizing processes. For example, pharmaceutical compositions containing immune cells as described herein are generally provided in the form of a solution and preferably contain a pharmaceutically acceptable excipient.

[0083] The pharmaceutical composition of the present invention can be administered in combination with one or more other drugs used in the treatment and / or prevention of the disease to be treated. Preferred examples of drugs to be used in combination include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium porphyrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, and the like. E), known anticancer drugs such as vincristine and adriamycin, peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes and RNA enzymes, radionuclides such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and -213, actinium-225 and astatine-213, prodrugs of antibody-directed enzyme prodrugs, immunostimulants such as platelet factor 4 and melanoma growth stimulating protein, antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heteroprotein domains, homoprotein domains, viral / bacterial protein domains, and viral / bacterial peptides. The pharmaceutical compositions of the present invention may also be used in combination with one or more other types of treatment, such as chemotherapy, radiation therapy, etc. Therapeutic applications

[0084] The present invention further provides a method for treating a subject having a disease associated with CD7 expression, comprising administering to the subject an effective amount of the immune cells or pharmaceutical composition described in the present invention. Thus, the present invention further covers the use of the modified immune cells and pharmaceutical compositions in the preparation of a medicament for treating a disease associated with CD7 expression.

[0085] In one embodiment, an effective amount of the immune cells and / or pharmaceutical compositions of the present invention is directly administered to a subject.

[0086] In another embodiment, the therapeutic method of the present invention is an ex vivo treatment. Specifically, this method comprises the steps of: (a) providing a sample containing immune cells; (b) introducing the chimeric antigen receptor of the present invention and an exogenous gene (e.g., an NK inhibitory molecule) into the immune cells ex vivo to suppress or silence the expression of specific genes (e.g., endogenous CD7, TCR / CD3 genes, and MHC class II-related genes) in the immune cells, thereby obtaining modified immune cells; and (c) administering the modified immune cells to a subject in need thereof. Preferably, the immune cells provided in step (a) are selected from macrophages, dendritic cells, monocytes, T cells, NK cells, and / or NKT cells, and the immune cells can be obtained from a sample (particularly a blood sample) of a subject by a general method known in the art. However, other immune cells that express the chimeric antigen receptor of the present invention and can exert the desired biological effect function described herein may also be used. Additionally, the selected immune cells are generally compatible with the subject's immune system, i.e., preferably, the immune cells do not induce an immunogenic response. For example, "universal recipient cells," i.e., universally compatible ex vivo cultured and expanded lymphocytes that perform the desired biological effect function, may be used. The use of such cells obviates the need to obtain and / or provide the subject's own lymphocytes. The ex vivo introduction in step (c) can be carried out by introducing the nucleic acid or vector described herein into immune cells via electroporation, or by infecting immune cells with a viral vector, such as the lentiviral, adenoviral, adeno-associated viral, or retroviral vectors described above. Other conceivable methods include the use of transfection agents (e.g., lipid bodies) or transient RNA transfection.

[0087] In one embodiment, the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells and / or NKT cells, more preferably T cells, NK cells or NKT cells.

[0088] As used herein, the term "autologous" means any material that originates from an individual and is later reintroduced into that same individual.

[0089] As used herein, the term "allogeneic" means that any material is derived from a different animal or patient of the same species as the individual into whom the material was introduced. Two or more individuals are considered allogeneic to one another if they differ in genes at one or more loci. In some cases, differences in the genes of allogeneic material from each individual of the same species make antigenic interactions more likely.

[0090] 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. Non-human mammals are advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.

[0091] In one embodiment, disorders associated with CD7 expression include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) and solid tumors. Hematological tumors are cancers of the blood or bone marrow, including acute leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia, and myeloblastic, promyelocytic, granulomonocytic, monocytic, and erythroleukemia), chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphoblastic leukemia), polycythemia vera, lymphomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma (painless hyperhidrosis), and leukemias (painless hyperhidrosis). Solid tumors include, but are not limited to, leukemia (large-scale forms), multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt's lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, T-lymphoblastic lymphoma (T-LBL), early pro-T lymphoblastic leukemia (ETP-ALL), extranodal NK / T-cell lymphoma, small lymphocytic lymphoma (SLL), and myelodysplasia. Solid tumors are abnormal masses that usually do not contain cystic or fluid areas of tissue and may be benign or malignant. Different types of solid tumors are named according to the cell type that formed them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, pancreatic cancer, ovarian cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, melanoma, kidney cancer, throat cancer, soft tissue cancer, stomach cancer, testicular cancer, colon cancer, esophageal cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, thyroid cancer, and ureter cancer.

[0092] In one embodiment, the disease associated with CD7 expression is preferably selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), T-lymphoblastic lymphoma (T-LBL), early pro-T lymphoblastic leukemia (ETP-ALL), and extranodal NK / T-cell lymphoma.

[0093] The present invention will be described in detail below with reference to the drawings and examples. It should be noted that those skilled in the art should understand that the drawings and examples of the present invention are merely illustrative and do not constitute any limitations on the present invention. Where not inconsistent, the features of the embodiments and examples in this application can be combined with each other. [Brief explanation of the drawings]

[0094] [Figure 1] The ratio of CD7+ cells to CD7- cells in the peripheral blood lymphocytes of patients is shown. [Figure 2] The expression levels of scFv in CAR7-dKO and CAR7-tKO T cells are shown. [Figure 3] 1 shows the target cell killing ability of CAR7-dKO T cells and CAR7-tKO T cells. [Figure 4] Cytokine release levels after co-culture of CAR7-dKO and CAR7-tKO T cells with target cells are shown. [Figure 5] Figure 1 shows the inhibitory effect of NK inhibitory molecules on NK cell killing. A: NK inhibitory molecules contain anti-KIR scFv. B: NK inhibitory molecules contain anti-LIR scFv. C: NK inhibitory molecules contain anti-NKG2A scFv, HLA-G extracellular region, HLA-E extracellular region, or E-Cad extracellular region. [Figure 6] This shows the killing effect of NK cells by CD4+ T cells (A) and CD8+ T cells (B) containing the NK inhibitory molecule CD3ζ. [Figure 7] 1 shows the expression levels of scFv in CAR7-NKi-dKO and CAR7-NKi-tKO T cells. [Figure 8] 1 shows the expression levels of NK inhibitory molecules in CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells. [Figure 9] 1 shows the target cell-killing ability of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells. [Figure 10]Cytokine release levels after co-culture of CAR7-NKi-dKO and CAR7-NKi-tKO T cells with target cells are shown. [Figure 11] ScFv expression levels in CAR7-E T cells, CAR7-PDL1 T cells, and CAR7-EPDL1 T cells are shown. [Figure 12] The expression levels of HLA-E in CAR7-E T cells and PDL1 in CAR7-PDL1 T cells are shown. [Figure 13] Shown are the expression levels of HLA-E and PDL1 in CAR7-EPDL1 T cells. [Figure 14] Shows the target cell killing ability of CAR7-E T cells, CAR7-PDL1 T cells, and CAR7-EPDL1 T cells. [Figure 15] Cytokine release levels after co-culture of CAR7-E T cells, CAR7-PDL1 T cells, and CAR7-EPDL1 T cells with target cells are shown. [Figure 16] 1 shows the expression levels of CD7 scFv and CD19 scFv in CAR7-19 T cells. [Figure 17] Shows the killing ability of CAR7-19 T cells against two types of target cells. [Figure 18] Cytokine release levels after co-culture of CAR7-19 T cells with two types of target cells are shown. DETAILED DESCRIPTION OF THE INVENTION

[0095] Example 1. Preparation of anti-CD7 universal CAR T cells Sequences encoding proteins such as the CD8α signal peptide (SEQ ID NO: 36), anti-CD7scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 28), and CD3ζ intracellular signaling domain (SEQ ID NO: 30) were synthesized and cloned sequentially into the pLVX vector (Public Protein / Plasmid Library (PPL), product number: PPL00157-4a), and correct insertion of the target sequences was confirmed by sequencing.

[0096] The above plasmid was diluted with 3 ml of Opti-MEM (Gibco, product number 31985-070) in a sterile tube, followed by the packaging vector psPAX2 (Addgene, product number 12260) and the envelope vector pMD2.G (Addgene, product number 12259) in a ratio of 4:2:1 (plasmid:viral packaging vector:viral envelope vector). 120 μl of X-treme GENE HP DNA transfection reagent (Roche, product number 06366236001) was then added, mixed thoroughly, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to a 293T cell culture flask. After 24 and 48 hours, the virus was collected, combined, and ultracentrifuged (25,000 g, 4°C, 2.5 hours) to obtain concentrated lentivirus.

[0097] DynaBeads CD3 / CD28 CTS TM Wild-type T cells were activated using a 500-kDa ELISA kit (Gibco, product number 40203D) and subsequently cultured at 37°C and 5% CO2 for one day. The TCR / CD3 component (specifically, the TRAC gene), CD7 gene, and any MHC class II-related gene (specifically, RFX5) were then knocked out in the wild-type T cells using the CRISPR system to obtain TCR / CD7 double knockout dKO-T cells and TCR / CD7 / RFX5 triple knockout tKO-T cells. Wild-type T cells with no gene knockout (i.e., NT cells) served as controls.

[0098] The gene editing efficiency of TCR / CD7 / RFX5 in T cells was detected by flow cytometry using FITC Mouse Anti-Human CD3 (BD Pharmingen, product number 555916), PE mouse anti-human CD7 (Biolegend, product number 395604), and APC anti-human DR, DP, DQ (Biolegend, product number 361714) antibodies. The results are shown in Table 1.

[0099] Table 1. Gene expression efficiency in T cells JPEG0007725031000001.jpg46170

[0100] As can be seen from Table 1, the expression of relevant genes in the dKO-T cells and tKO-T cells prepared in the present invention is effectively suppressed or silenced.

[0101] The concentrated lentivirus was transfected into dKO and tKO T cells to obtain CAR7-dKO and CAR7-tKO T cells. Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab')2 Fragment Specific (min x Hu, Bov, Hrs, Sr, Prot) (Jackson Immunoresearch, product number 109-065-097) was used as the primary antibody, and APC Streptavidin (BD Pharmingen, product number 554067) or PE Streptavidin (BD Pharmingen, product number 554061) was used as the secondary antibody. The expression levels of scFv in CAR7-dKO and CAR7-tKO T cells were detected by flow cytometry. The results are shown in Figure 2.

[0102] As can be seen from this, all of the scFvs in the CAR T cells prepared in the present invention can be effectively expressed. Example 2: Target cell killing effect and cytokine release by CAR T cells 2.1 Killing effect of CAR-T cells on target cells

[0103] To detect the killing ability of CAR-T cells on target cells, we first 4 Jurkat target cells carrying a fluorescein gene were placed in a 96-well plate. CAR T cells and NT cells were then cultured together in the 96-well plate at effector:target ratios (i.e., the ratio of effector T cells to target cells) of 0.5:1, 0.25:1, and 0.125:1. After 16–18 hours, fluorescence was measured using a microplate reader. Killing efficiency was calculated using the formula: (mean target cell fluorescence – mean sample fluorescence) / mean target cell fluorescence × 100%. The results are shown in Figure 3.

[0104] As can be seen, compared with NT, both CAR7-dKO T cells and CAR7-tKO T cells have specific killing ability against target cells, and CAR7-tKO T cells have higher killing ability than CAR7-dKO T cells. 2.2 Cytokine release from CAR-T cells

[0105] When T cells kill target cells, the target cells are reduced in number and also release cytokines. The level of cytokine IFNγ released when the CAR T cells of the present invention kill target cells is measured by sandwich method (ELISA) according to the following steps. (1) Collection of cell co-culture supernatant

[0106] 1x10 5 Jurkat target cells were placed in a 96-well plate at 100 μg / well. CAR T and NT cells (negative control) were then co-cultured with the target cells at a ratio of 0.125:1. After 18-24 hours, the cell co-culture supernatant was collected. (2) Detection of IFNγ secretion in the supernatant by ELISA

[0107] A 96-well plate was coated with the capture antibody Purified Anti-Human IFN-γ Antibody (Biolegend, Product No. 506502) and incubated overnight at 4°C. The antibody solution was then removed and 250 μL of 2% BSA (Sigma, Product No. V900933-1kg) in PBST (1X PBS containing 0.1% Tween) was added and incubated for 2 hours at 37°C. 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 for 1 hour at 37°C. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). Then, 50 μL of detection antibody, Anti-Interferon gamma antibody [MD-1] (Biotin) (Abcam, product 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, product number 405210) was added again 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. The reaction was allowed to proceed at room temperature in the dark for 30 minutes, after which 50 μL of 1 mol / L H2SO4 was added to each well to stop the reaction. During the 30 minutes that the reaction was stopped, the absorbance at 450 nm was detected using a microplate reader, and the cytokine content was calculated based on a standard curve (drawn based on the readings and concentrations of the standards). The results are shown in Figure 4.

[0108] As can be seen, the cytokine release from target cells by CAR7-dKO T cells and CAR7-tKO T cells was higher than that of the control NT group, and CAR7-tKO T cells released a higher level than CAR7-dKO T cells.

[0109] As can be seen from the above results, the target cell killing ability and cytokine release levels of CAR7-tKO T cells were both higher than those of CAR7-dKO T cells, demonstrating that knockout of the MHC class II gene enhanced the killing activity of CAR-T cells. This was unexpected, as previous reports have suggested that MHC class II gene expression is associated with immune rejection, but there have been no reports on the relationship between MHC class II gene expression and the activity of CAR-T cells themselves. Example 3. Inhibitory effect of NK inhibitory molecules on NK cell killing

[0110] Coding sequences for proteins such as B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, CD28 hinge region (SEQ ID NO: 40), and CD28 transmembrane region (SEQ ID NO: 24) were synthesized and inserted into the pLVX vector (Public Protein / Plasmid). The NK inhibitory ligand was cloned sequentially into the PPL Library (Product No. PPL00157-4a), where the NK inhibitory ligand was the extracellular domain of E-cadherin (SEQ ID NO: 47, corresponding to the ECaD0 plasmid), a fusion molecule of B2M and the extracellular domain of HLA-E (comprising the presenting peptide (SEQ ID NO: 75), B2M (SEQ ID NO: 74), and an HLA-E extracellular domain variant (SEQ ID NO: 51), with a synonymous mutation in the B2M coding sequence (SEQ ID NO: 82), corresponding to the E0 plasmid), or a fusion molecule of B2M and the extracellular domain of HLA-G (comprising B2M (SEQ ID NO: 74) and the HLA-G extracellular domain (SEQ ID NO: 49), with a synonymous mutation in the B2M coding sequence (SEQ ID NO: 82), corresponding to the G0 plasmid). The ECaD0, E0, and G0 plasmids further contained the CD28 costimulatory domain (SEQ ID NO: 26), yielding the ECaD28, E28, and G28 plasmids, respectively. Correct insertion of the target sequence was confirmed by sequencing.

[0111] The coding sequences for proteins such as the B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, IgG4 hinge region (SEQ ID NO: 42), CD8α transmembrane region (SEQ ID NO: 22), and CD28 costimulatory domain (SEQ ID NO: 26) were synthesized and sequentially cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Cat. No. PPL00157-4a). The NK inhibitory ligand was an anti-NKG2A scFv (SEQ ID NO: 63, corresponding to the A28 plasmid), an anti-KIR scFv (SEQ ID NO: 67, corresponding to the KIRG4 plasmid), or an anti-LIR1 scFv (SEQ ID NO: 70, corresponding to the LIR1-1 plasmid). Correct insertion of the target sequences was confirmed by sequencing.

[0112] The coding sequences for proteins such as CD8α signal peptide (SEQ ID NO: 36), anti-LIR1 scFv (SEQ ID NO: 73), CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), and 4-1BB costimulatory domain (SEQ ID NO: 28) were synthesized and cloned sequentially into the pLVX vector (Public Protein / Plasmid Library (PPL), product number: PPL00157-4a) to obtain the LIR1-2 plasmid. Correct insertion of the target sequence was confirmed by sequencing.

[0113] The above plasmid was diluted with 3 ml of Opti-MEM (Gibco, product number 31985-070) in a sterile tube, followed by the packaging vector psPAX2 (Addgene, product number 12260) and the envelope vector pMD2.G (Addgene, product number 12259) in a ratio of 4:2:1 (plasmid:viral packaging vector:viral envelope vector). 120 μl of X-treme GENE HP DNA transfection reagent (Roche, product number 06366236001) was then added, mixed thoroughly, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to a 293T cell culture flask. After 24 and 48 hours, the virus was collected, combined, and ultracentrifuged (25,000 g, 4°C, 2.5 hours) to obtain concentrated lentivirus.

[0114] T cells were activated using DynaBeads CD3 / CD28 CTS™ (Gibco, product number 40203D) and cultured at 37°C and 5% CO for 1 day. Concentrated lentivirus was then added and cultured for 3 days to obtain T cells expressing NK inhibitory molecules, i.e., UNKi T cells.

[0115] Then, the CRISPR system was used to knock out TCR / CD3 components (specifically, the TRAC gene) and MHC-related genes (specifically, B2M and RFX5) in wild-type T cells (Mock T cells, used as a control) and the UNKi-T cells, and flow cytometry was used to confirm that each gene was effectively knocked out.

[0116] The inhibitory effect of the UNKi-T cells prepared in the present invention on the killing activity of NK cells was then detected according to the following method: Far-Red (Invitrogen, product number C34564) was used to mark the UNKi-T cells prepared in the present invention and Mock-T cells. 4Marked UNKi-T cells and mock T cells were placed in a 96-well plate at a concentration of 100 cells / well and cocultured with NK92 cells (UNKi-T cells and mock T cells expressing the HLA-E extracellular domain, HLA-G extracellular domain, anti-NKG2A scFv, anti-KIR scFv, or anti-LIR1 scFv) or NK92-KLRG1 cells (UNKi-T cells expressing the E-cadherin extracellular domain, prepared by transfecting NK92 cells with the KLRG1 gene) at a 2:1 effector:target ratio. After 16-18 hours, the proportion of T cells in the culture was determined using flow cytometry, and the killing effect of NK cells on T cells was calculated. The results are shown in Figure 5.

[0117] As can be seen from Figure 5, compared with mock T cells that do not express NK inhibitory molecules, UNKi-T cells expressing inhibitory ligands such as anti-KIR scFv, anti-LIR1 scFv, anti-NKG2A scFv, HLA-G extracellular domain, HLA-E extracellular domain, and E-cadherin extracellular domain significantly reduced the killing of T cells by NK cells. Furthermore, compared with T cells expressing only an inhibitory ligand and a transmembrane domain (i.e., without a costimulatory domain) (G0, E0, Ecad0), the addition of a costimulatory domain significantly increased the suppression of NK cell killing by T cells (G28, E28, Ecad28). Therefore, the NK inhibitory molecules described in this invention, which contain an inhibitory ligand, a transmembrane domain, and a costimulatory domain, significantly reduced the killing of UNKi-T cells by NK cells, effectively reducing the risk of HvGD.

[0118] In some cases, it is necessary not only to inhibit NK cells from killing CAR-T cells, but also to have T cells kill NK cells. Therefore, the inventors further added the CD3ζ intracellular signaling domain (SEQ ID NO: 30) to the E28 plasmid and A28 plasmid cells, packaged it as a lentivirus according to the above method, and infected T cells in which the TCR / CD3 component (specifically, the TRAC gene) and MHC-related genes (specifically, B2M and RFX5) were effectively knocked out to obtain E28z-UNKi-T cell nuclei and A28z-UNKi-T cells.

[0119] The killing of NK cells by UNKi-T cells was detected by the following method: 1 x 10 5 Target cells (NK92 cells) were plated at a concentration of 10 cells / well into a 96-well plate. Mock T cells, E28z-UNKi-T cells, and A28z-UNKi-T cells were then added at a 1:1 ratio to each well. At the same time, 10 μl of PE-anti-human CD107a (BD Pharmingen, product number 555801) was added and co-cultured at 37°C, 5% CO. After 1 hour, Goigstop (BD Pharmingen, product number 51-2092KZ) was added and the cells were incubated for a further 2.5 hours. Then, 5 μl of APC-anti-human CD8 (BD Pharmingen, product number: 555369) and 5 μl of FITC-anti-human CD4 (BD Pharmingen, product number: 561005) were added to each well and incubated at 37°C for 30 minutes. CD107a expression was detected by flow cytometry, and the results are shown in Figure 6A (CD4+ T cell toxicity) and Figure 6B (CD8+ T cell toxicity).

[0120] As can be seen from this, mock T cells that do not express NK inhibitory molecules have almost no killing effect on target cells. In contrast, after co-culture of target cells with E28z-UNKi-T cells and A28z-UNKi-T cells prepared in the present invention, the expression rate of CD107a is significantly increased, indicating that the UNKi-T cells of the present invention can significantly kill NK cells. Example 4. Preparation of universal CAR T cells expressing NK inhibitory molecules

[0121] Sequences encoding proteins such as CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), CD3ζ intracellular signaling domain (SEQ ID NO: 30), F2A, E-cadherin extracellular region (SEQ ID NO: 48), CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), and CD28 intracellular region (SEQ ID NO: 26) are synthesized and cloned into an MSCV vector, and correct insertion of the target sequence is confirmed by sequencing.

[0122] Dilute the plasmid into a sterile tube with 3 ml of Opti-MEM (Gibco, product number 31985-070), then add the packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., product number P3029) at a plasmid:viral packaging vector ratio of 3:1. Then, add 120 μl of X-treme GENE HP DNA transfection reagent (Roche, product number 06366236001), mix thoroughly, and incubate at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to a 293GP cell culture flask. After 72 and 96 hours, the virus was collected, combined, and centrifuged (2000 rpm, 4°C, 10 minutes) to defragment the virus, obtaining the retroviral supernatant.

[0123] Based on the knockout strategy described in Example 1, TCR / CD7 double knockout dKO T cells and TCR / CD7 / RFX5 triple knockout tKO T cells were prepared. Wild-type T cells (i.e., NT cells) without gene knockout served as controls.

[0124] A 24-well plate was coated with retronectin and incubated overnight at 4°C. The solution was then removed and 300 μL of 5% FBS (Gibco, product number) in PBS was added and allowed to stand at room temperature for 30 minutes. The supernatant was then removed and the plate was washed twice with 1 mL of PBS. 2 mL of retroviral supernatant and 0.5 M dKO-T cells or tKO-T cells were added to each well. The plate was then centrifuged at 2000 g and 32°C for 2 hours and cultured in a carbon dioxide incubator to obtain CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0125] After 7 days of culture, the expression levels of CD7 scFv in CAR7-NKi-dKO and CAR7-NKi-tKO T cells were detected by flow cytometry using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (min x Hu, Bov, Hrs, Sr, Prot) (Jackson Immunoresearch, product number 115-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, product number 554067) or PE Streptavidin (BD Pharmingen, product number 554061) as the secondary antibody. The results are shown in Figure 7. E-cadherin expression in CAR T cells was detected using an E-cadherin monoclonal antibody (Invitrogen product number 13-5700) and a goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, product number A-11001). The results are shown in Figure 8.

[0126] As can be seen from this, both the anti-CD7 scFv and the NKi inhibitory molecule can be effectively expressed in the CAR T cells prepared in the present invention.

[0127] The killing effects of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells on Jurkat target cells were determined according to the method described in Section 2.1 of Example 2, and the results are shown in Figure 9. As can be seen, both types of CAR-T cells were able to significantly kill target cells at each effector:target ratio, and at an effector:target ratio of 0.125:1, CAR7-NKi-tKO T cells had a superior killing effect to CAR7-NKi-dKO T cells.

[0128] Cytokine release levels were detected after co-culture of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells with Jurkat target cells according to the method described in Section 2.2 of Example 2, and the results are shown in Figure 10. As can be seen, the cytokine release levels of the CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells of the present invention were both significantly higher than those of the control NT cells, and the release level of the CAR7-NKi-tKO T cell group was significantly higher than that of the CAR7-NKi-dKO T cell group. Example 5. Preparation of universal CAR T cells expressing NK inhibitory molecules and verification of their function

[0129] Sequences encoding proteins such as CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD28 hinge region (SEQ ID NO: 40), CD8α transmembrane region (SEQ ID NO: 22), CD28 intracellular region (SEQ ID NO: 26), CD3ζ intracellular signaling domain (SEQ ID NO: 32), F2A, PD-L1 signal peptide (SEQ ID NO: 44), PD-L1 extracellular region (SEQ ID NO: 45), CD28 transmembrane region (SEQ ID NO: 24), and 4-1BB intracellular region (SEQ ID NO: 28) were synthesized and cloned into the MSCV vector, followed by sequencing to confirm correct insertion of the target sequences (plasmid name: CAR7-PDL1).

[0130] Sequences encoding proteins such as CD8α signal peptide (sequence number 36), anti-CD7 scFv (sequence number 21), CD8α hinge region (sequence number 38), CD28 transmembrane region (sequence number 24), 4-1BB intracellular region (sequence number 28), CD3ζ intracellular signaling domain (sequence number 32), F2A, B2M signal peptide (sequence number 34), HLA-E extracellular region (sequence number 50), CD28 transmembrane region (sequence number 24), and CD28 intracellular region (sequence number 26) were synthesized and cloned into an MSCV vector, and correct insertion of the target sequence was confirmed by sequencing (plasmid name: CAR7-E).

[0131] Sequences encoding proteins such as CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), CD3ζ intracellular signaling domain (SEQ ID NO: 32), F2A, B2M signal peptide (SEQ ID NO: 34), HLA-E extracellular region (SEQ ID NO: 50), connecting peptide (SEQ ID NO: 64), PD-L1 extracellular region (SEQ ID NO: 45), CD28 transmembrane region (SEQ ID NO: 24), and CD28 intracellular region (SEQ ID NO: 26) were synthesized and cloned into the MSCV vector, followed by sequencing to confirm correct insertion of the target sequence (plasmid name: CAR7-EPDL1).

[0132] The above-mentioned plasmids are packaged as retroviruses according to the method described in Example 3, and tKO-T cells are infected with them to obtain CAR7-E T cells, CAR7-PDL1 T cells, and CAR7-EPDL1 T cells, respectively.

[0133] After 7 days of culture, the expression levels of scFvs in the three types of cells were detected by flow cytometry using Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab')2 Fragment Specific (min X Hu, Bov, Hrs, Sr, Prot) (Jackson Immunoresearch, Product No. 109-065-097) as the primary antibody and APC Streptavidin (BD Pharmingen, Product No. 554067) or PE Streptavidin (BD Pharmingen, Product No. 554061) as the secondary antibody. The results are shown in Figure 11. The expression of HLA-E and PDL1 in the CAR T cells was detected using PE mouse anti-human HLA-E (Biolegend, Product No. 342604) and PE anti-human PDL1 (Biolegend, Product No. 329706), respectively. The results are shown in Figures 12 and 13.

[0134] As can be seen from this, both the anti-CD7 scFv and NK inhibitory molecules (HLA-E and PD-L1) can be effectively expressed in the CAR T cells prepared in the present invention.

[0135] The killing effects of the above three types of CAR T cells on Jurkat target cells were detected according to the method described in 2.1 of Example 2, and the results are shown in Figure 14. As can be seen, all three types of CAR-T cells can significantly kill target cells.

[0136] The cytokine release levels after co-culture of each of the three types of CAR-T cells with Jurkat target cells were detected according to the method described in Section 2.2 of Example 2, and the results are shown in Figure 15. Compared with the NT group, the cytokine release levels of all three types of CAR-T cells were significantly higher. Example 6. Preparation of dual-targeted CAR-T cells and verification of their function

[0137] Sequences encoding proteins such as the CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 20), connecting peptide (SEQ ID NO: 64), anti-CD19 scFv (SEQ ID NO: 54), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), and CD3ζ intracellular signaling domain (SEQ ID NO: 32) were synthesized and cloned sequentially into the pLVX vector (Public Protein / Plasmid Library (PPL), product number: PPL00157-4a), and correct insertion of the target sequence was confirmed by sequencing.

[0138] The above-mentioned plasmid vector was packaged as a lentivirus according to the method of Example 1, and tkO-T cells were infected to obtain CAR7-19 T cells. Unmodified wild-type T cells were used as a negative control (NT).

[0139] The scFv expression level of CAR7-19 T cells was detected by flow cytometry using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (min × Hu, Bov, Hrs, Sr, Prot) (Jackson Immunoresearch, product number 115-065-072) or Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab')2 Fragment Specific (min × Hu, Bov, Hrs, Sr, Prot) (Jackson Immunoresearch, product number 109-065-097) as the primary antibody and FITC Streptavidin (BD Pharmingen, product number 554060) or PE Streptavidin (BD Pharmingen, product number 554061) as the secondary antibody. The results are shown in Figure 16.

[0140] As can be seen from this, both CD7 scFv and CD19 scFv can be effectively expressed in the CAR T cells prepared in the present invention.

[0141] The killing ability and cytokine release level of CAR7-19 T cells were detected according to the method in Example 2, and the results are shown in Figure 17 and Figure 18, respectively. As can be seen, CAR7-19 T cells have specific killing ability and significantly increased cytokine release against both Nalm6 and Jurkat target cells.

[0142] It should be understood that the above is only a preferred embodiment of the present invention, and does not limit the present invention, and those skilled in the art can make various modifications and changes to the present invention. As understood by those skilled in the art, any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. An engineered immune cell, (1) expressing a chimeric antigen receptor comprising an antigen-binding region comprising an anti-CD7 antibody; (2) having reduced or silenced expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC class II-related gene; The modified immune cell further expresses an NK inhibitory molecule comprising one or more NK inhibitory ligands, a transmembrane domain, and a costimulatory domain.

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

3. The modified immune cell of claim 2, wherein the anti-CD7 antibody comprises CDR-L1, CDR-L2, and CDR-L3 represented by SEQ ID NOs: 1, 2, and 3, respectively, and CDR-H1, CDR-H2, and CDR-H3 represented by SEQ ID NOs: 4, 5, and 6, respectively.

4. The antigen-binding region of the chimeric antigen receptor further comprises an antibody or functional fragment thereof that targets a second antigen, wherein the second antigen is selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, and IL-1. lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB 2 (Her2 / neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, 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, pod protein, 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 / 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-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut4. The modified immune cell of claim 2 or 3, characterized in that the target polypeptide is 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.

5. The modified immune cell of claim 2 , wherein the chimeric antigen receptor comprises an anti-CD7 antibody and an anti-CD19 antibody.

6. 6. The modified immune cell of any one of claims 2 to 5, wherein the transmembrane domain is selected from the transmembrane domains of proteins such as TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

7. 7. The modified immune cell of any one of claims 2 to 6, wherein the intracellular signaling domain is selected from the intracellular regions of proteins such as FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

8. The chimeric antigen receptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BT 8. The modified immune cell of any one of claims 1 to 7, further comprising one or more costimulatory domains selected from the intracellular regions of proteins such as CD47 (B7-H3), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.

9. 2. The modified immune cell of claim 1, wherein the TCR / CD3 genes are selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

10. The modified immune cell of claim 1, wherein the MHC class II-related gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

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

12. 2. The modified immune cell of claim 1, wherein the NK inhibitory ligand is an antibody or a functional fragment thereof that targets an NK inhibitory receptor, and the NK inhibitory receptor is selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4, and KLRG1.

13. The modified immune cell of claim 1, wherein the NK inhibitory ligand is HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1 / PD-L2, CTLA-4, CD155, CD112, CD113, Gal-9, FGL1, or an NK inhibitory receptor binding domain contained therein.

14. The modified immune cell of claim 1, wherein the NK inhibitory molecule further comprises a CD3ζ intracellular region as an intracellular signaling domain.

15. The modified immune cell according to any one of claims 1 to 14, wherein the modified immune cell is a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell, or a NKT cell.

16. 16. A pharmaceutical composition comprising the modified immune cells of any one of claims 1 to 15 and various pharmaceutically acceptable excipients.

17. 17. Use of the modified immune cells of any one of claims 1 to 15 or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating a disease related to CD7 expression.

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