Chimeric antigen receptor targeting CD7 and application thereof

Abstract

The invention provides an engineered immune cell characterized by: (1) expressing a chimeric antigen receptor comprising an antigen binding region, the antigen binding region comprising an anti-CD7 antibody; and (2) expression of endogenous CD7, at least one TCR / CD3 gene and at least one MHC-II related gene is inhibited or silenced. The invention also provides the use of the engineered immune cell in the treatment of diseases associated with CD7 expression.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to a chimeric antigen receptor targeting CD7 and its use in treating diseases. Background Art

[0002] In recent years, cancer immunotherapy technology has developed rapidly, especially chimeric antigen receptor T cell (CAR-T)-related immunotherapy, which, as a new adoptive immunotherapy technology, has shown very significant clinical efficacy in the treatment of various solid tumors and blood 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 called universal CAR-T). The advantage of autologous CAR-T cells is that there is no risk of immune rejection because the starting T cells come from the patient receiving CAR-T therapy; but its disadvantages are also very obvious: high cost and long preparation cycle. Therefore, more and more research is focused on the development of universal CAR-T cells. Universal CAR-T can be prepared using T cells isolated from the peripheral blood of healthy donors, greatly shortening the time patients have to wait for treatment. In addition, the vitality and function of T cells obtained from healthy donors are also better than those from patient-derived T cells, which can increase the infection rate of CAR cells and improve the treatment effect.

[0004] CD7 is a cell surface glycoprotein with a molecular weight of approximately 40 kDa and is a member of the immunoglobulin superfamily. CD7 is expressed in most T cells, NK cells, myeloid cells, T cell acute lymphoblastic leukemia / lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. It has been reported that the CD7 molecule acts as a co-stimulatory signal during T cell activation by binding to its ligand K12 / SECTM1. Moreover, disruption of the CD7 molecule in mouse T progenitor cells still results in normal T cell development and homeostasis, indicating that CD7 does not seem to have a critical effect on T cell development and function, making it a very suitable therapeutic target for the treatment of T cell acute lymphoblastic leukemia (T-ALL). In fact, CD7 has been widely studied as a target for cytotoxic molecules for the treatment of leukemia and lymphoma.

[0005] Currently, CAR-T therapies targeting CD7, especially universal CAR-T therapies, are still very limited. Therefore, the present invention aims to provide a highly efficient universal CAR-T cell targeting CD7, and its use in treating diseases. Summary of the invention

[0006] In a first aspect, the present invention provides an engineered immune cell characterized in that: (1) a chimeric antigen receptor is expressed comprising an antigen binding region, wherein the antigen binding region comprises an anti-CD7 antibody; and (2) the expression of endogenous CD7, at least one TCR / CD3 gene and at least one MHC-II class-related gene is inhibited or silenced.

[0007] In one embodiment, the chimeric antigen receptor comprises an anti-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 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and CDR-H1, CDR-H2 and CDR-H3 as shown 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 10, 13, 16 and 19 and a heavy chain variable region having at least 90% identity with an amino acid sequence selected from the group consisting of 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 the group consisting of SEQ ID NOs: 9, 12, 15, 18 and 21.

[0009] In one embodiment, the antigen binding region of the chimeric antigen 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, 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-la, MAGE-A1, legumin, 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 breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, 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 with an amino acid sequence selected from SEQ ID NOs: 52 and 55 and a heavy chain variable region having at least 90% identity with an amino acid 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 NO: 54 and 57.

[0010] In one embodiment, the transmembrane domain is selected from the transmembrane domain of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the transmembrane domain of CD8 α, CD4 and CD28.

[0011] In one embodiment, the intracellular signaling domain is selected from the intracellular region of the following proteins: 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 co-stimulatory domains selected from the intracellular regions of the following proteins: 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 co-stimulatory domain is the intracellular region of CD27, CD28, CD134, CD137 or CD278, or a combination thereof.

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

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

[0015] In a preferred embodiment, the engineered immune cells of the present invention include endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and at least one MHC-II class gene selected from RFX5, RFXAP, RFXANK and CIITA, and the expression is inhibited or silenced. More preferably, the engineered immune cells of the present invention include endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and the expression of RFX5 is inhibited or silenced.

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

[0017] In one embodiment, the NK inhibitory ligand is an antibody targeting an NK inhibitory receptor selected from the group consisting of a NKG2 / CD94 component (e.g., NKG2A, NKG2B, CD94); a killer cell Ig-like receptor (KIR) family member (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3); a leukocyte Ig-like receptor (LIR) family member (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8); a NK cell receptor protein 1 (NKR-P1 ) family 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 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. Still 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 a NK inhibitory receptor or a NK inhibitory receptor binding region contained therein. Preferably, the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (eg, PD-L1 / PD-L2, CTLA4, CD155, CD112, CD113, Gal-9, FGL1, etc.), and the NK inhibitory receptor binding region 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 the NK inhibitory receptor binding region contained therein; more preferably selected from HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, or the NK inhibitory receptor binding region contained therein. In a preferred embodiment, the NK inhibitory ligand is selected from the extracellular region of HLA-E, the extracellular region of HLA-G, the extracellular region of E-cadherin, the extracellular region of PD-L1, and the extracellular region of PD-L2. In a preferred embodiment, the NK inhibitory ligand is an E-cadherin extracellular region, which comprises EC1 and EC2, more preferably EC1, EC2, EC3, EC4, and EC5. In a preferred embodiment, the NK inhibitory ligand is the extracellular region of PD-L1 or PD-L2. In a preferred embodiment, the NK inhibitory ligand is the extracellular region of HLA-E. In a preferred embodiment, the NK inhibitory ligand is the extracellular region 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 engineered immune cell is a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell. Preferably, the engineered immune cell is a T cell, such as a CD4+ / CD8+T cell, a CD4+ helper T cell (e.g., a Th1 and Th2 cell), a CD8+T cell (e.g., a cytotoxic T cell), a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell, an αβ-T cell.

[0021] In one aspect, the present invention also provides a pharmaceutical composition comprising the engineered immune cells of the present invention as an active agent, and one or more pharmaceutically acceptable excipients.

[0022] In one aspect, the present invention also provides a method for treating a subject with a disease associated with CD7 expression, comprising administering to the subject an effective amount of the engineered immune cell or pharmaceutical composition according to the present invention. Therefore, the present invention also encompasses the use of engineered immune cells in the preparation of a medicament for treating a disease associated with CD7 expression. DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] Chimeric Antigen Receptor

[0026] In a first aspect, the present invention provides an engineered immune cell characterized in that: (1) a chimeric antigen receptor is expressed comprising an antigen binding region, wherein the antigen binding region comprises an anti-CD7 antibody; and (2) the expression of endogenous CD7, at least one TCR / CD3 gene and at least one MHC-II class-related gene is inhibited or silenced.

[0027] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide, which generally includes an antigen binding region (e.g., an antibody or an antigen binding portion thereof), a transmembrane domain, an optional co-stimulatory domain, and an intracellular signaling domain, each domain being connected by a linker. CAR is able to redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner using the antigen binding properties of antibodies. Non-MHC restricted antigen recognition gives CAR-expressing immune cells the ability to recognize antigens independent of antigen processing, thereby bypassing the main mechanism of tumor escape.

[0028] In one embodiment, the CAR expressed by the engineered immune cells provided by the present invention comprises an antibody or antigen-binding fragment thereof targeting CD7, a transmembrane domain, and an intracellular signaling domain.

[0029] As used herein, the term "antibody" has the broadest meaning understood by those skilled in the art, and includes monoclonal antibodies (including complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit desired biological activity. The antibodies of the present invention may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). The antibodies of the present invention also include recombinant antibodies, human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, and antigen-binding portions thereof.

[0030] As used herein, "antibody fragment" or "antigen binding portion" refers to a portion of an intact antibody, generally comprising the antigen binding site of the intact antibody and thus 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 bond-linked Fv (sdFv), antibody heavy chain variable region (VH) or light chain variable region (VL), linear antibodies, "diabodies" with two antigen binding sites, single domain antibodies, nanobodies, natural ligands of the antigen or functional fragments thereof, etc. Therefore, the "antibody" of the present invention encompasses antibody fragments or antigen binding portions of antibodies as defined above.

[0031] In one embodiment, the anti-CD7 antibody of the present invention is an anti-CD7 scFv. "Single-chain antibody" and "scFv" are used interchangeably herein and refer to an antibody formed by connecting an antibody heavy chain variable region (VH) and a light chain variable region (VL) via a linker. The optimal length and / or amino acid composition of the linker can be determined as needed. The length of the linker will significantly affect the folding and interaction of the variable region of the scFv. In fact, if a shorter linker (e.g., between 5-10 amino acids) is used, intrachain folding can be prevented. Regarding the selection of the size and composition of the linker, see, for example, 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. WO2006 / 020258 and WO2007 / 024715, the entirety of which is incorporated herein by reference. The scFv may comprise VH and VL linked in any order, such as VH-linker-VL or VL-linker-VH.

[0032] In one embodiment, the CAR of the present invention comprises an antibody targeting CD7, which comprises CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 1, 2, and 3, respectively, and CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NOs: 4, 5 and 6. Preferably, the antibody targeting CD7 of the present 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 with an amino acid sequence selected from the group consisting of 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 with an amino acid sequence selected from the group consisting of 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 shown in SEQ ID NO: 9, 12, 15, 18 or 21.

[0033] In one embodiment, in addition to an antibody targeting CD7, the antigen binding region of the chimeric antigen receptor of the present invention 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, 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-la, MAGE-A1, legumin, 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 breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut 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 α, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin and any combination thereof. Antibodies targeting the above tumor antigens known in the art can be used in the present invention. .

[0034] In a preferred embodiment, the antibody targeting the second antigen is an antibody targeting CD19, which 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 shown 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 shown 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, and the amino acid sequence of the antibody targeting CD19 is as shown in SEQ ID NO: 54 or 57.

[0035] The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of the parent but contains at least one amino acid modification (i.e., substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

[0036] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. The modifications can be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0037] 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 with the parent amino acid sequence and retains the biological activity, such as binding activity, of the parent amino acid.

[0038] As used herein, the term "sequence identity" refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity. Those skilled in the art will recognize that some 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.

[0039] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables chimeric antigen receptors to be expressed on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells), and guides immune cells to respond to target cells. The transmembrane domain can be natural or synthetic, or it can be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor is bound to the target antigen, the transmembrane domain can carry out signal transduction. Particularly suitable transmembrane domains in the present invention can be derived from, for example, TCR alpha chains, TCR beta chains, TCR gamma chains, TCR delta chains, CD3 zeta subunits, CD3 epsilon subunits, CD3 gamma subunits, CD3 delta subunits, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, the transmembrane domain can be synthetic and can mainly include hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8α chain or CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:22 or 24, 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 with the nucleotide sequence shown in SEQ ID NO:23 or 25.

[0040] In one embodiment, the chimeric antigen receptor of the present invention may also include a hinge region between the antigen binding region and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antigen binding region. The hinge region may include 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 natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or 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 fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of a CD8α chain, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably a hinge from CD8α, CD28 or IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:38, 40 or 42, 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 with the nucleotide sequence shown in SEQ ID NO:39, 41 or 43.

[0041] As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs cells to perform a specified function. The intracellular signaling domain is responsible for the primary signaling within the cell after the antigen binding region binds to the antigen, resulting in the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or an auxiliary activity, including the secretion of cytokines.

[0042] In one embodiment, the intracellular signaling domain contained in the chimeric antigen receptor of the present invention can be the cytoplasmic sequences of T cell receptors and co-receptors, which work together to initiate primary signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions. The intracellular signaling domain can contain many immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of the intracellular signaling domain 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 the CAR of the present invention may comprise a CD3ζ intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 30 or 32, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 31 or 33.

[0043] In one embodiment, the chimeric antigen receptor of the present invention further comprises one or more costimulatory domains. The costimulatory domain can be an intracellular functional signaling domain from a costimulatory molecule, which comprises the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. "Costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of a T cell. Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting examples of costimulatory domains of the present invention include, but are not limited to, intracellular regions of the following proteins: 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.

[0044] In a preferred embodiment, the costimulatory domain comprises one or more intracellular regions selected from the following proteins: DAP10, DAP12, CD27, CD28, CD134, 4-1BB or 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.

[0045] 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 with the amino acid sequence shown 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 with the nucleotide sequence shown 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 with the amino acid sequence shown 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 with the nucleotide sequence shown in SEQ ID NO: 27.

[0046] In one embodiment, the CAR of the present invention may also include a signal peptide so that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface. The core of the signal peptide may contain a long hydrophobic amino acid segment that has a tendency to form a single α-helix. At the end of the signal peptide, there is usually an amino acid segment that is recognized and cut by a signal peptidase. The signal peptidase can be cut during or after the shift to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in 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 useful in the present invention is derived from B2M or CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown 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% or 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:35 or 37.

[0047] In one embodiment, the CAR of the present invention may also include a switch structure to regulate the expression time of CAR. For example, the switch structure may be in the form of a dimerization domain, which causes conformational changes by binding to its corresponding ligand, exposing the extracellular antigen binding region, so that it binds to the targeted antigen, thereby activating the signal transduction pathway. Alternatively, the switch structure may be used to connect the antigen binding region and the signal transduction domain respectively, and only when the switch structure binds to each other (for example, in the presence of an inducing compound), the antigen binding region and the signal transduction domain can be connected together through a dimer, thereby activating the signal pathway. The switch structure may also be in the form of a masked peptide. The masking peptide can shield the extracellular antigen binding region, preventing it from binding to the targeted antigen, and when the masking peptide is cut by, for example, a protease, the extracellular antigen binding region can be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art can be used in the present invention.

[0048] In one embodiment, the CAR of the present invention may also include a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance to remove CAR cells when needed (e.g., when serious toxic side effects occur). For example, the suicide gene can be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when necessary, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes. The suicide gene may also be herpes simplex virus thymidine kinase (HSV-TK), which can cause cells to die under induced treatment with ganciclovir. The suicide gene may also be iCaspase-9, which can be induced by chemical induction drugs such as AP1903, AP20187, etc. to dimerize iCaspase-9, thereby activating downstream Caspase3 molecules, leading to apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

[0049] Inhibition or silencing of endogenous gene expression

[0050] In one embodiment, the expression of endogenous CD7, at least one TCR / CD3 gene and at least one MHC class II related gene of the engineered immune cells provided by the present invention is inhibited or silenced.

[0051] CD7 is expressed not only in tumor cells, such as T-cell acute lymphoblastic leukemia / lymphoma, acute myeloid leukemia, and chronic myeloid leukemia, but also in most normal T cells and NK cells. Therefore, in order to avoid mutual recognition and killing between CAR cells targeting CD7, it is necessary to inhibit or silence the expression of the endogenous CD7 gene of CAR cells.

[0052] The T cell receptor (TCR) is a characteristic marker on the surface of all T cells. It binds to CD3 with non-covalent bonds to form a TCR / CD3 complex, and binds to the specific MHC-antigen peptide complex on the surface of antigen-presenting cells to generate specific antigen stimulation signals, activate T cells, and play a killing role. Therefore, inhibiting or silencing the expression of endogenous TCR / CD3 genes in CAR-T cells can avoid their attack on normal cells or tissues in the patient's body, thereby avoiding or reducing the risk of graft-versus-host disease (GvHD). TCR is a heterodimer composed of two different peptide chains, which is usually divided into two categories: α / β type and γ / δ type, of which more than 95% of peripheral T lymphocytes express TCRα / β. The TCRα chain is encoded by the TRAC gene, and the β chain is encoded by the TRBC gene. Each peptide chain of TCR includes 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 have the ability to transmit antigen stimulation signals. TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity exists in the V region; the V region has three hypervariable regions CDR1, CDR2, and CDR3, among which CDR3 has the largest variation, which directly determines the antigen binding specificity of TCR. When TCR recognizes the MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the MHC molecule, while CDR3 directly binds to the antigen peptide. CD3 includes four subunits: γ, δ, ε, and ζ, usually in the form of dimers εγ, εδ, and ζζ. These four subunits all contain a conserved immunoreceptor tyrosine-based activation motif (ITAM), in which two tyrosine residues are phosphorylated by tyrosine protein kinases and transmit activation signals to T cells. Therefore, in one embodiment, at least one TCR / CD3 gene is selected from: TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0053] By detecting the composition of peripheral blood lymphocytes in patients, the inventors found that CD4+CD7-T cells accounted for about 20% of the total CD3+T cells ( Figure 1 ). Since CAR cells targeting CD7 cannot recognize the CD4+CD7-T group, they cannot effectively kill this group. Instead, CD4+CD7-T cells will kill exogenous CAR cells by recognizing MHC-II molecules. Therefore, in order to prevent CD4+CD7-T cells in the patient from killing exogenous CAR-T cells, the inventors considered inhibiting or silencing the expression of endogenous MHC-II related genes in CAR-T cells.

[0054] In the present invention, MHC class II-related genes include MHC class II genes themselves, as well as genes that interact with MHC class II genes or regulate their expression.

[0055] The major histocompatibility complex (MHC) was originally characterized as a protein that plays a major role in transplantation reactions. It is expressed on the surface of all higher vertebrates and is called H-2 in mice and HLA in human cells. There are two main types of MHC: class I and class II. Class I MHC proteins are heterodimers of two proteins: one is the transmembrane protein α chain encoded by the MHC I gene, and the other is the β2 microglobulin chain of the extracellular protein encoded by a gene not located in the MHC gene cluster. The α chain includes three domains, and foreign peptides bind to two domains α1 and α2 that are also the most variable 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). Furthermore, class I MHC proteins tend to be expressed in almost all nucleated cells and platelets (and red blood cells in mice), whereas class II MHC proteins are more selectively expressed. Typically, class II MHC proteins are expressed on B cells, some macrophages and monocytes, activated T cells, Langerhans cells, and dendritic cells.

[0056] The class II HLA cluster of humans contains three major loci, DP, DQ and DR. Class II molecules are heterodimers consisting of an α chain and a β chain, both anchored in the membrane, wherein the α chain is about 33-35 kDa and contains 5 exons. Exon 1 encodes a leader peptide, exons 2 and 3 encode two extracellular domains, exon 4 encodes a transmembrane domain, and exon 5 encodes a cytoplasmic tail. Therefore, in one embodiment, the MHC-II class-related genes are selected from: HLA-DPA, HLA-DQ and HLA-DRA.

[0057] The expression of MHC-II class genes also depends on a variety of important positive regulatory proteins, such as RFX complex, CIITA, etc. The RFX complex is composed of three subunits: RFXANK (also known as RFXB), RFX5 and RFX auxiliary protein (also known as RFXAP). The RFX complex promotes the expression of class II MHC molecules by promoting the binding of other transcription factors to the promoter of class II MHC molecules and enhancing the specificity of promoter binding. CIITA is the main control factor for class II MHC expression. CIITA includes an N-terminal rich in acidic amino acids, a PST region rich in Pro, Ser, and Thr, a middle GTP binding region, and a C-terminal rich in Leu repeat sequences (LRR), wherein the N-terminal acidic region and the PST region are transcriptional activation regions. Therefore, in one embodiment, MHC-II class related genes are selected from: RFX5, RFXAP, RFXANK and CIITA.

[0058] Therefore, in one embodiment, the MHC-II class related gene is selected from: HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA, and more preferably RFX5.

[0059] In one embodiment, the endogenous MHC-I class genes (eg, HLA-A, HLA-B, HLA-C, B2M, etc.) in the CAR-T cells are functional. In another embodiment, the expression of the endogenous MHC-I class genes in the CAR-T cells is also inhibited or silenced.

[0060] In one embodiment, the engineered immune cells expressing chimeric antigen receptors targeting CD7 described in the present invention include endogenous CD7, at least one TCR / CD3 gene, and at least one MHC-II class-related gene whose expression is inhibited or silenced, wherein the at least one TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof; the at least one MHC-II class-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 chimeric antigen receptors targeting CD7 described in the present invention include endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and at least one MHC-II class gene selected from RFX5, RFXAP, RFXANK, and CIITA whose expression is inhibited or silenced. More preferably, the engineered immune cells expressing the chimeric antigen receptor targeting CD7 of the present invention include endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and the expression of RFX5 is inhibited or silenced. In one embodiment, the expression of MHC-I class genes, such as HLA-A, HLA-B, HLA-C and B2M in the engineered immune cells is functional.

[0061] In one embodiment, in addition to CD7, MHC class II related genes and TCR / CD3 genes, the engineered immune cells of the present invention may also include at least one gene selected from the group consisting of CD52, GR, dCK and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10B. 0A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, I L10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0062] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to, for example, DNA fragmentation mediated by large-range nucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems, or gene inactivation by antisense oligonucleotides, RNAi, shRNA and other technologies.

[0063] NK inhibitory molecules

[0064] The inventors also found that a large proportion (about 20%) of CD7-NK cells ( Figure 1 ), which cannot be targeted and killed by CD7 CAR. Moreover, when the expression of MHC-II gene is inhibited or silenced, these cells may kill CAR cells.

[0065] Therefore, in one embodiment, in order to inhibit the killing of CAR-T cells by NK cells in the patient, the engineered immune cells further express NK inhibitory molecules, which include one or more NK inhibitory ligands, transmembrane domains and co-stimulatory domains. For example, the NK inhibitory molecule includes one or two NK inhibitory ligands, transmembrane domains and co-stimulatory domains.

[0066] The definitions of the transmembrane domain and the costimulatory domain contained in the NK inhibitory molecule are the same as those of the transmembrane domain and the costimulatory domain contained in the above-mentioned chimeric antigen receptor.

[0067] In one embodiment, the NK inhibitory ligand is an antibody targeting an NK inhibitory receptor selected from the group consisting of a NKG2 / CD94 component (e.g., NKG2A, NKG2B, CD94); a killer cell Ig-like receptor (KIR) family member (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3); a leukocyte Ig-like receptor (LIR) family member (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8); a NK cell receptor protein 1 (NKR-P1 ) family 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 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. Still more preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, LIR1, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1 and KLRG1.

[0068] In one embodiment, the NK inhibitory ligand is an antibody targeting a NK inhibitory receptor, which antibody is a whole antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody, sdAb or nanobody.

[0069] 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 58, 61 or 76. More preferably, the amino acid sequence of the antibody targeting NKG2A is shown in SEQ ID NO: 60, 63 or 78. Other antibodies targeting NKG2A known in the art can also be used in the present invention, such as Z270 (available from Immunotech, France), Z199 (available from Beckman Coulter, USA), 20D5 (available from BD Biosciences Pharmingen, USA), P25 (available from Moretta et al, Univ. Genova, Italy), etc.

[0070] In a preferred embodiment, the NK inhibitory ligand is an antibody targeting KIR, such as KIR2DL1, KIR2DL2 / 3 and KIR3DL1. More preferably, the antibody targeting KIR 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 66 or 80. More preferably, the amino acid sequence of the antibody targeting KIR is shown in SEQ ID NO: 67 or 81. Other KIR-targeting antibodies known in the art can also be used in the present invention, such as GL183 (targeting KIR2DL2 / L3, available from Immunotech, France and Beckton Dickinson, USA), EB6 (targeting KIR2DL1, available from Immunotech, France and Beckton Dickinson, USA), AZ138 (targeting KIR3DL1, available from Moretta et al, Univ. Genova, Italy), Q66 (targeting KIR3DL2, available from Immunotech, France), Z27 (targeting KIR3DL1, available from Immunotech, France and Beckton Dickinson, USA), etc.

[0071] 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 shown 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 shown in SEQ ID NO: 69 or 72. More preferably, the amino acid sequence of the antibody targeting LIR1 is shown in SEQ ID NO: 70 or 73.

[0072] In one embodiment, the NK inhibitory ligand is a natural ligand of a NK inhibitory receptor or a NK inhibitory receptor binding region contained therein. Preferably, the NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, Cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (eg, PD-L1 / PD-L2, CD155, CD112, CD113, Gal-9, FGL1, etc.), and the NK inhibitory receptor binding region 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 NK inhibitory receptor binding regions contained therein; more preferably selected from HLA-E, HLA-F, HLA-G, cadherin, or NK inhibitory receptor binding regions contained therein. More preferably, the NK inhibitory ligand is selected from the extracellular region of HLA-E, the extracellular region of HLA-G, the extracellular region of E-Cadherin, the extracellular region of PD-L1, and the extracellular region of PD-L2.

[0073] In a preferred embodiment, the NK inhibitory ligand is an E-cadherin extracellular region, which comprises domains EC1 and EC2, more preferably comprises 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 with the amino acid sequence shown in SEQ ID NO: 44 (comprising domains EC1 and EC2) or SEQ ID NO: 48 (comprising domains EC1, EC2, EC3, EC4 and EC5).

[0074] In one embodiment, the NK inhibitory ligand is the extracellular region of PD-L1 or PD-L2. Preferably, the PD-L1 extracellular region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 45, and the PD-L2 extracellular region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 46.

[0075] In one embodiment, the NK inhibitory ligand is an HLA-E extracellular region or an HLA-G extracellular region. Preferably, the HLA-E extracellular region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 50 (wild type) or SEQ ID NO: 51 (mutant containing Y84C mutation), and the HLA-G extracellular region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 49. It is known in the art that non-classical HLA-I class molecules (such as HLA-E, HLA-G) need to form a complex with B2M to exert their function. Therefore, when B2M needs to be knocked out, in order to enable non-classical HLA-I class molecules to normally exert their inhibitory function, it is necessary to introduce a B2M with a synonymous mutation in the DNA sequence to avoid being knocked out by gene editing tools. In other words, in cells in which B2M is knocked out, the NK inhibitory ligand is a fusion molecule of B2M and the extracellular region of HLA-E or HLA-G.

[0076] 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.

[0077] In one embodiment, the NK inhibitory molecule further comprises an intracellular signaling domain. In one embodiment, the intracellular signaling domain is selected from the intracellular region of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. Preferably, the intracellular signaling domain comprises the intracellular region of CD3ζ.

[0078] In one embodiment, the NK inhibitory molecule may further 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 having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 44. Those skilled in the art may also select other suitable signal peptides as needed.

[0079] Engineered immune cells

[0080] The present invention also provides an engineered immune cell, characterized in that: (1) a chimeric antigen receptor comprising an antigen binding region is expressed, wherein the antigen binding region comprises an anti-CD7 antibody and an optional second antigen region; (2) the expression of endogenous CD7, at least one TCR / CD3 gene, and at least one MHC-II class-related gene is inhibited or silenced. In one embodiment, the engineered immune cell of the present invention further expresses a NK inhibitory molecule, wherein the NK inhibitory molecule comprises one or more NK inhibitory ligands, a transmembrane domain, and a co-stimulatory domain.

[0081] As used herein, the term "immune cell" refers to any cell of the immune system with one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and / or NKT cells, or immune cells derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Preferably, immune cells are T cells. T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines cultured in vitro such as Jurkat, SupT1, etc., or T cells obtained from subjects. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissues from infection sites, ascites, pleural effusion, spleen tissue and tumors. T cells can also be concentrated or purified. T cells can be in any developmental stage, including but not limited to, CD4+ / CD8+T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+T cells (such as, cytotoxic T cells), tumor infiltrating cells, memory T cells, immature T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, immune cells are human T cells. Various techniques known to those skilled in the art can be used, such as Ficoll separation of the blood of the subject to obtain T cells. In the present invention, immune cells are engineered to express chimeric antigen receptors and suppress or silence the expression of endogenous CD7, at least one TCR / CD3 gene and at least one MHC-II class related gene.

[0082] The nucleic acid sequence encoding the chimeric antigen receptor polypeptide and the encoding nucleic acid sequence of the optional NK inhibitory molecule can be introduced into immune cells by conventional methods known in the art (such as by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, which is the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of viral-mediated nucleic acid molecules or polynucleotides. The transfection of animal cells generally involves opening a transient hole or "hole" in the cell membrane to allow the intake of materials. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, also into non-animal eukaryotic cells (including plant cells). Therefore, transformation is the genetic change of bacteria or non-animal eukaryotic cells, which is produced by direct uptake from its surroundings through the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be achieved by artificial means. In order for transformation to occur, the cell or bacterium must be in a state of competence. For prokaryotic transformation, techniques may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection and electroporation.

[0083] After the nucleic acid or vector is introduced into the immune cells, those skilled in the art can amplify and activate the obtained immune cells by conventional techniques.

[0084] Pharmaceutical composition

[0085] The present invention also provides a pharmaceutical composition comprising the engineered immune cells of the present invention as an active agent, and one or more pharmaceutically acceptable excipients.

[0086] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which 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, coatings, adsorbents, antiadhesives, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension regulators. It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. In general, the choice of suitable excipients depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

[0087] The pharmaceutical composition according to the present invention can be applied to a variety of routes. Typically, administration is completed parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

[0088] The pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts, or in the form particularly suitable for the desired method of administration. The process known to the present invention for producing drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions comprising, for example, immune cells as described herein are generally provided in solution form, and preferably include a pharmaceutically acceptable buffer.

[0089] The pharmaceutical composition according to the present invention can also be used in combination with one or more other medicaments suitable for treating and / or preventing the disease to be treated. Preferred examples of the medicament suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreateglucuronate, auristatin E, E), vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial 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, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides. In addition, the pharmaceutical composition of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0090] Therapeutic applications

[0091] The present invention also provides a method for treating a subject suffering from a disease associated with CD7 expression, comprising administering to the subject an effective amount of the immune cell or pharmaceutical composition according to the present invention. Therefore, the present invention also encompasses the use of the engineered immune cell and pharmaceutical composition in the preparation of a medicament for treating a disease associated with CD7 expression.

[0092] In one embodiment, an effective amount of the immune cells and / or pharmaceutical compositions of the invention are administered directly to a subject.

[0093] In another embodiment, the treatment method of the present invention is ex vivo treatment. Specifically, the method comprises the following steps: (a) providing a sample, the sample comprising immune cells; (b) introducing the chimeric antigen receptor of the present invention and exogenous genes (e.g., NK inhibitory molecules) into the immune cells in vitro, and inhibiting or silencing the expression of specific genes in the immune cells (e.g., endogenous CD7, TCR / CD3 genes, and MHC-II class-related genes), obtaining modified immune cells, (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 conventional methods known in the art. However, other immune cells capable of expressing the chimeric antigen receptor of the present invention and exerting the desired biological effector function as described herein may also be used. In addition, the immune cells generally selected are compatible with the subject's immune system, i.e., preferably, the immune cells do not induce an immunogenic response. For example, "universal recipient cells" can be used, i.e., lymphocytes that are generally compatible and can grow and expand in vitro and that exert the desired biological effector function. The use of such cells will not require the acquisition and / or provision of the subject's own lymphocytes. The ex vivo introduction of step (c) can be implemented by introducing the nucleic acid or vector described herein into immune cells via electroporation or by infecting immune cells with a viral vector, the viral vector being a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector as described above. Other conceivable methods include the use of a transfection agent (such as liposomes) or transient RNA transfection.

[0094] In one embodiment, the immune cell is an autologous or allogeneic cell, preferably a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell and / or a NKT cell, more preferably a T cell, a NK cell or a NKT cell.

[0095] As used herein, the term "autologous" refers to any material derived from an individual that will later be reintroduced into that same individual.

[0096] As used herein, the term "allogeneic" refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may differ genetically enough to allow antigenic interactions to occur.

[0097] As used herein, the term "subject" is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.

[0098] In one embodiment, diseases associated with CD7 expression include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) and solid tumors. Hematological tumors are cancers of the blood or bone marrow and include, but are not limited to, acute leukemias (such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia and myeloblastic, promyelocytic, myelo-monocytic, monocytic and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, T-lymphoblastic lymphoma (T-LBL), early pre-T lymphoblastic leukemia (ETP-ALL), extranodal NK / T-cell lymphoma, small lymphocytic lymphoma (SLL) and myelodysplasia. Solid tumor is the abnormal mass of the tissue that does not usually comprise cyst or liquid zone, and it can be benign or malignant.Dissimilar solid tumors are named with the cell type that forms them (such as sarcoma, cancer and lymphoma).The example of solid tumor includes but is not limited to fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, pancreatic cancer, ovarian cancer, peritoneum, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, gastric 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 ureteral cancer.

[0099] 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 precursor T-lymphoblastic leukemia (ETP-ALL) and extranodal NK / T-cell lymphoma.

[0100] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples. It should be noted that those skilled in the art should understand that the accompanying drawings and embodiments of the present invention are only for illustrative purposes and do not constitute any limitation to the present invention. The embodiments in this application and the features in the embodiments can be combined with each other without contradiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 : Shows the ratio of CD7+ cells and CD7- cells in the patient's peripheral blood lymphocytes.

[0102] Figure 2 : Shows the expression level of scFv on CAR7-dKO T cells and CAR7-tKO T cells.

[0103] Figure 3 : Shows the killing ability of CAR7-dKO T cells and CAR7-tKO T cells on target cells.

[0104] Figure 4 : Shows the cytokine release levels after co-culture of CAR7-dKO T cells and CAR7-tKO T cells with target cells.

[0105] Figure 5 : Shows the inhibitory effect of NK inhibitory molecules on NK cell killing. A: NK inhibitory molecules include anti-KIR scFv; B: NK inhibitory molecules include anti-LIR scFv; C: NK inhibitory molecules include anti-NKG2A scFv, HLA-G extracellular region, HLA-E extracellular region or E-Cad extracellular region.

[0106] Figure 6 : The killing effect of CD4+T cells (A) and CD8+T cells (B) expressing NK inhibitory molecules including CD3ζ on NK cells.

[0107] Figure 7 : Shows the expression level of scFv on CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0108] Figure 8 : Shows the expression levels of NK inhibitory molecules in CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0109] Fig. 9 : Shows the killing ability of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells on target cells.

[0110] Fig.10: Shows the cytokine release levels of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells after co-culture with target cells.

[0111] Fig.11 : Shown are scFv expression levels on CAR7-E T cells, CAR7-PDL1 T cells, and CAR7-EPDL1 T cells.

[0112] Fig.12 : Shows the expression level of HLA-E in CAR7-E T cells and the expression level of PDL1 in CAR7-PDL1 T cells.

[0113] Fig.13 : Shows the expression levels of HLA-E and PDL1 in CAR7-EPDL1 T cells.

[0114] Fig.14 : Shows the killing ability of CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells on target cells.

[0115] Fig.15 : Shows the cytokine release levels of CAR7-E T cells, CAR7-PDL1 T cells, and CAR7-EPDL1 T cells after co-culture with target cells.

[0116] Fig.16 : Shows the expression levels of CD7 scFv and CD19 scFv in CAR7-19 T cells.

[0117] Fig.17 : Shows the killing ability of CAR7-19 T cells against two target cells.

[0118] Fig.18 : Shows the cytokine release levels after co-culture of CAR7-19 T cells with two target cells. DETAILED DESCRIPTION

[0119] Example 1. Preparation of universal anti-CD7 CAR T cells

[0120] The sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Catalog No.: PPL00157-4a) in sequence: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 28), CD3ζ intracellular signaling domain (SEQ ID NO: 30), and the correct insertion of the target sequence was confirmed by sequencing.

[0121] After adding 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) to dilute the above plasmid in a sterile tube, add packaging vector psPAX2 (Addgene, Catalog No. 12260) and envelope vector pMD2.G (Addgene, Catalog No. 12259) according to the ratio of plasmid: viral packaging vector: viral envelope vector = 4:2:1. Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, Catalog No. 06366236001), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid / vector / transfection reagent mixture dropwise to the culture flask of 293T cells. Collect the virus at 24 hours and 48 hours, combine them, and ultracentrifuge (25000g, 4°C, 2.5 hours) to obtain concentrated lentivirus.

[0122] Using DynaBeads CD3 / CD28 CTS TM (Gibco, Cat. No. 40203D) was used to activate wild-type T cells and cultured for 1 day at 37°C and 5% CO2. The CRISPR system was then used to knock out the TCR / CD3 components (specifically the TRAC gene), CD7 gene, and optional MHC-II class-related genes (specifically RFX5) in wild-type T cells to obtain TCR / CD7 double knockout dKO-T cells and TCR / CD7 / RFX5 triple knockout tKO-T cells. Wild-type T cells (i.e., NT cells) without knockout genes were used as controls.

[0123] The gene editing efficiency of TCR / CD7 / RFX5 in T cells was detected by flow cytometry using FITC Mouse Anti-Human CD3 (BD ​​Pharmingen, Catalog No. 555916) antibody, PE mouse anti-human CD7 (biolegend Catalog No. 395604) and APC anti-human DR, DP, DQ (biolegend, Catalog No. 361714) antibodies. The results are shown in Table 1.

[0124] Table 1. Gene expression efficiency in T cells

[0125] name TCR / CD3 CD7 RFX5 / MHC-II dKO-T 2.2％ 5.3％ 92％ tKO-T 3％ 8.9％ 11.2％ NT 98％ 94％ 92％

[0126] It can be seen from Table 1 that the expression of related genes in the dKO-T cells and tKO-T cells prepared by the present invention is effectively inhibited or silenced.

[0127] The concentrated lentivirus was added to dKO-T cells and tKO-T cells to obtain CAR7-dKO T cells and CAR7-tKOT cells. Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab')2 Fragment Specific (min XHu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat. no. 109-065-097) was used as the primary antibody, and APC Streptavidin (BD Pharmingen, cat. no. 554067) or PE Streptavidin (BD Pharmingen, cat. no. 554061) was used as the secondary antibody. The expression level of scFv on CAR7-dKO T cells and CAR7-tKOT cells was detected by flow cytometry. The results are shown in Figure 2. Figure 2 shown.

[0128] It can be seen that the scFv in the CAR T cells prepared by the present invention can be effectively expressed.

[0129] Example 2: Killing effect of CAR T cells on target cells and cytokine release

[0130] 2.1 CAR-T cell killing effect on target cells

[0131] In order to detect the killing ability of CAR-T cells on target cells, 1x10 4 / well Jurkat target cells carrying the fluorescein gene were plated into a 96-well plate, and then CAR T cells and NT cells were plated into a 96-well plate for co-culture at an effector-target ratio (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, the fluorescence value was measured using an ELISA reader. The killing efficiency was calculated according to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, and the results are shown in the figure. Figure 3 shown.

[0132] It can be seen that compared with NT, both CAR7-dKO T cells and CAR7-tKO T cells have specific killing of target cells, and the killing ability of CAR7-tKO T cells is higher than that of CAR7-dKO T cells.

[0133] 2.2 Cytokine release of CAR-T cells

[0134] When T cells kill target cells, the number of target cells decreases and cytokines are released. According to the following steps, enzyme-linked immunosorbent assay (ELISA) is used to measure the release level of cytokine IFNγ when the CAR T cells of the present invention kill target cells.

[0135] (1) Collecting cell co-culture supernatant

[0136] 1x10 5 Jurkat target cells were plated in a 96-well plate at 1:1 ratio. CAR T cells and NT cells (negative control) were then co-cultured with the target cells at a ratio of 0.125:1. The cell co-culture supernatant was collected after 18-24 hours.

[0137] (2) ELISA to detect IFNγ secretion in the supernatant

[0138] The 96-well plate was coated with the capture antibody Purified anti-human IFN-γAntibody (Biolegend, Catalog No. 506502) and incubated overnight at 4°C, then the antibody solution was removed, and 250 μL of PBST (1XPBS containing 0.1% Tween) solution containing 2% BSA (sigma, Catalog No. V900933-1kg) was added and incubated at 37°C for 2 hours. The plate was then washed 3 times with 250 μL PBST (1XPBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or standard was added to each well and incubated at 37°C for 1 hour, and then the plate was washed 3 times with 250 μL PBST (1XPBS containing 0.1% Tween). Then add 50 μL of detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, catalog number ab25017) to each well, incubate at 37°C for 1 hour, and wash the plate 3 times with 250 μL PBST (1XPBS containing 0.1% Tween). Then add HRP Streptavidin (Biolegend, catalog number 405210), incubate at 37°C for 30 minutes, discard the supernatant, add 250 μL PBST (1XPBS containing 0.1% Tween), and wash 5 times. Add 50 μL TMB substrate solution to each well. Allow the reaction to occur in the dark at room temperature for 30 minutes, then add 50 μL 1 mol / L H 2 SO 4 Within 30 minutes of stopping the reaction, use an enzyme reader to detect the absorbance at 450nm, and calculate the cytokine content according to the standard curve (drawn according to the reading value and concentration of the standard). The results are as follows: Figure 4 shown.

[0139] It can be seen that the factor release of target cells by CAR7-dKO T cells and CAR7-tKO T cells was significantly higher than that of the control NT group, and the release level of CAR7-tKO T cells was higher than that of CAR7-dKO T cells.

[0140] From the above results, it can be seen that the killing ability of CAR7-tKO T cells on target cells and the release level of cytokines are higher than those of CAR7-dKO T cells, indicating that the knockout of MHC-II class genes enhances the killing activity of CAR-T cells. This is unexpected, because existing reports generally believe that the expression of MHC-II class genes is related to immune rejection, and there has been no report on the relationship between MHC-II class genes and the activity of CAR-T cells themselves.

[0141] Example 3. Inhibitory effect of NK inhibitory molecules on NK cell killing

[0142] The coding sequences of the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Catalog No.: PPL00157-4a) in sequence: B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), wherein the NK inhibitory ligand is the extracellular region of E-cadherin (SEQ ID NO: 47, corresponding to ECad0 plasmid), a fusion molecule of B2M and HLA-E extracellular region (comprising presenting peptide SEQ ID NO: 75, B2M SEQ ID NO: 74 and HLA-E extracellular region mutant SEQ ID NO: 51, wherein the coding sequence of B2M SEQ ID NO: 82 comprises a synonymous mutation, corresponding to E0 plasmid) or a fusion molecule of B2M and HLA-G extracellular region (comprising B2M SEQ ID NO: 74 and HLA-G extracellular region SEQ ID NO: 51). NO: 49, wherein the coding sequence of B2M SEQ ID NO: 82 contains a synonymous mutation, corresponding to the G0 plasmid). The CD28 co-stimulatory domain (SEQ ID NO: 26) is further included in the ECad0, E0 and G0 plasmids to obtain ECad28, E28 and G28 plasmids, respectively. The correct insertion of the target sequence in the plasmid is confirmed by sequencing.

[0143] The coding sequences of the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), catalog number: PPL00157-4a) in sequence: B2m signal peptide (SEQ ID NO: 34), NK inhibitory ligand, IgG4 hinge region (SEQ ID NO: 42), CD8α transmembrane region (SEQ ID NO: 22), CD28 co-stimulatory domain (SEQ ID NO: 26), wherein the NK inhibitory ligand is anti-NKG2A scFv (SEQ ID NO: 63, corresponding to A28 plasmid), anti-KIR scFv (SEQ ID NO: 67, corresponding to KIRG4 plasmid) or anti-LIR1 scFv (SEQ ID NO: 70, corresponding to LIR1-1 plasmid). The correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0144] The coding sequences of the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Catalog No.: PPL00157-4a) in sequence: CD8α signal peptide (SEQ ID NO: 36), anti-LIR1 scFv (SEQ ID NO: 73), CD28 hinge region (SEQ ID NO: 40), CD28 transmembrane region (SEQ ID NO: 24), 4-1BB co-stimulatory domain (SEQ ID NO: 28) to obtain LIR1-2 plasmid. The correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0145] After adding 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) to dilute the above plasmid in a sterile tube, add packaging vector psPAX2 (Addgene, Catalog No. 12260) and envelope vector pMD2.G (Addgene, Catalog No. 12259) according to the ratio of plasmid: viral packaging vector: viral envelope vector = 4:2:1. Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, Catalog No. 06366236001), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid / vector / transfection reagent mixture dropwise to the culture flask of 293T cells. Collect the virus at 24 hours and 48 hours, combine them, and ultracentrifuge (25000g, 4°C, 2.5 hours) to obtain concentrated lentivirus.

[0146] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, concentrated lentivirus was added and cultured for 3 days to obtain T cells expressing NK inhibitory molecules, namely UNKi-T cells.

[0147] Then, the CRISPR system was used to knock out the 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.

[0148] Then, the inhibitory effect of the UNKi-T cells prepared by the present invention on the killing effect of NK cells was detected according to the following method: the UNKi-T cells and Mock-T cells prepared by the present invention were labeled with Far-Red (invitrogen, catalog number C34564). 4The labeled UNKi-T cells and Mock T cells were plated into 96-well plates at a concentration of 10 cells / well, and NK92 cells (UNKi-T cells and Mock T cells expressing the HLA-E extracellular region, HLA-G extracellular region, anti-NKG2A scFv, anti-KIR scFv or anti-LIR1 scFv) or NK92-KLRG1 cells (UNKi-T cells expressing the E-cadherin extracellular region, prepared by introducing the KLRG1 gene into NK92 cells) were added at a 2:1 effector-target ratio for co-culture. After 16-18 hours, the proportion of T cells in the culture was detected by flow cytometry, and the killing effect of NK cells on T cells was calculated. The results are shown in the figure. Figure 5 shown.

[0149] from Figure 5 It can be seen that compared with Mock T cells that do not express NK inhibitory molecules, UNKi-T cells expressing inhibitory ligands such as anti-KIR scFv, anti-LIR1 scFv, anti-NKG2A scFv, HLA-G extracellular region, HLA-E extracellular region, and E-cadherin extracellular region can significantly reduce the killing effect of NK cells on T cells. Moreover, compared with T cells (G0, E0, Ecad0) that only express inhibitory ligands and transmembrane domains (i.e., not including costimulatory domains), the addition of costimulatory domains can further significantly enhance the inhibition of T cells on NK cell killing (G28, E28, ECad28). Therefore, the NK inhibitory molecules comprising inhibitory ligands, transmembrane domains and costimulatory domains of the present invention can significantly reduce the killing effect of NK cells on UNKi-T cells, so as to effectively reduce the risk of HvGD.

[0150] In addition, in some cases, it is not only necessary to inhibit the killing of CAR-T cells by NK cells, but even further to require T cells to kill NK cells. Therefore, the inventors further included the CD3ζ intracellular signaling domain (SEQ ID NO: 30) on the basis of E28 plasmid and A28 plasmid cells, and packaged it into a lentivirus according to the above method, and infected T cells in which TCR / CD3 components (specifically TRAC gene) and MHC-related genes (specifically B2M and RFX5) were effectively knocked out to obtain E28z-UNKi-T cells and A28z-UNKi-T cells.

[0151] The killing of NK cells by UNKi-T cells was detected by the following method: 1x10 5The target cells (NK92 cells) were plated in a 96-well plate at a concentration of cells / well, and then Mock T cells, E28z-UNKi-T cells and A28z-UNKi-T cells were added to each well at a ratio of 1:1, and 10μl PE-anti-human CD107a (BD Pharmingen, Catalog No. 555801) was added at the same time, and co-cultured at 37°C, 5% CO2. After 1 hour, Goigstop (BD Pharmingen, Catalog No. 51-2092KZ) was added and incubated for 2.5 hours. Then 5μl APC-anti human CD8 (BD Pharmingen, Catalog No.: 555369) and 5μl FITC-anti human CD4 (BD Pharmingen, Catalog No.: 561005) were added to each well. After incubation at 37°C for 30 minutes, the expression of CD107a was detected by flow cytometry. The results are as follows: Figure 6 A (CD4+ T cell cytotoxicity) and Figure 6 B (CD8+ T cell toxicity).

[0152] It can be seen that the Mock T cells that do not express NK inhibitory molecules have almost no killing effect on target cells. In contrast, after the E28z-UNKi-T cells and A28z-UNKi-T cells prepared by the present invention were co-cultured with target cells, the expression rate of CD107a was significantly increased, indicating that the UNKi-T cells of the present invention can significantly kill NK cells.

[0153] Example 4. Preparation of universal CAR T cells expressing NK inhibitory molecules

[0154] Sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), 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), and the correct insertion of the target sequence was confirmed by sequencing.

[0155] After adding 3 ml of Opti-MEM (Gibco, catalog number 31985-070) to a sterile tube to dilute the above plasmid, add the packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., catalog number P3029) according to the ratio of plasmid: viral packaging vector = 3:1. Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid / vector / transfection reagent mixture dropwise to the culture bottle of 293GP cells. Collect the virus at 72 hours and 96 hours, combine them, and centrifuge (2000rpm, 4°C, 10min) to remove fragments to obtain the retroviral supernatant.

[0156] TCR / CD7 double knockout dKO T cells and TCR / CD7 / RFX5 triple knockout tKO T cells were prepared by referring to the knockout method in Example 1. Wild-type T cells (ie, NT cells) without gene knockout were used as controls.

[0157] Retronectin was used to coat a 24-well plate and incubated overnight at 4°C, then the solution was removed, 300 μL of PBS solution containing 5% FBS (Gibco, catalog number) was added, and the plate was placed at room temperature for 30 min. 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, centrifuged at 2000 g, 32°C for 2 h, and then placed in a carbon dioxide incubator for culture to obtain CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells.

[0158] After 7 days of culture, Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat. no. 115-065-072) was used as the primary antibody, and APC Streptavidin (BD Pharmingen, cat. no. 554067) or PE Streptavidin (BD Pharmingen, cat. no. 554061) was used as the secondary antibody. The expression level of CD7 scFv in CAR7-NKi-dKOT cells and CAR7-NKi-tKO T cells was detected by flow cytometry. The results are shown in Figure 7E-cadherin monoclonal antibody (Invitrogen Catalog No. 13-5700) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, Catalog No. A-11001) were used to detect E-Cadherin expression in CAR T cells. The results are shown in Figure 8 shown.

[0159] It can be seen that both anti-CD7 scFv and NKi inhibitory molecules can be effectively expressed in the CAR T cells prepared by the present invention.

[0160] The killing effects of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells on Jurkat target cells were detected according to the method described in 2.1 of Example 2. The results are as follows: Fig. 9 As shown in the figure, it can be seen that both CAR-T cells can significantly kill target cells at various effector-target ratios, and at an effector-target ratio of 0.125:1, the killing effect of CAR7-NKi-tKO T cells is better than that of CAR7-NKi-dKO T cells.

[0161] The cytokine release levels of CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells after co-culture with Jurkat target cells were detected according to the method described in 2.2 of Example 2. The results are as follows: Fig.10 As shown. It can be seen that the cytokine release levels of the CAR7-NKi-dKO T cells and CAR7-NKi-tKO T cells of the present invention are significantly higher than those of the control NT cells, and the release level of the CAR7-NKi-tKO T cell group is significantly higher than that of the CAR7-NKi-dKO T cell group.

[0162] Example 5. Preparation of universal CAR T cells expressing NK inhibitory molecules and verification of their function

[0163] The sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD28 hinge region (SEQ ID NO: 40), CD8α transmembrane region (SEQ ID NO: 22), CD28 intracellular region (SEQ ID NO: 26), 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), 4-1BB intracellular region (SEQ ID NO: 28), and the correct insertion of the target sequence was confirmed by sequencing (plasmid name: CAR7-PDL1).

[0164] The sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD28 transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 28), CD3ζ intracellular signaling domain (SEQ ID NO: 32), F2A, B2M signal peptide (SEQ ID NO: 34), HLA-E extracellular region (SEQ ID NO: 50), CD28 transmembrane region (SEQ ID NO: 24), CD28 intracellular region (SEQ ID NO: 26), and the correct insertion of the target sequence was confirmed by sequencing (plasmid name: CAR7-E).

[0165] The sequences encoding the following proteins were synthesized and cloned into the MSCV vector: CD8α signal peptide (SEQ ID NO: 36), anti-CD7 scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 38), CD8α transmembrane region (SEQ ID NO: 22), 4-1BB intracellular region (SEQ ID NO: 28), 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 (SEQID NO: 45), CD28 transmembrane region (SEQ ID NO: 24), CD28 intracellular region (SEQ ID NO: 26), and the correct insertion of the target sequence was confirmed by sequencing (plasmid name: CAR7-EPDL1).

[0166] The plasmid was packaged into retrovirus according to the method described in Example 3, and tKO-T cells were infected to obtain CAR7-E T cells, CAR7-PDL1 T cells and CAR7-EPDL1 T cells, respectively.

[0167] After 7 days of culture, Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab') 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat. no. 109-065-097) was used as the primary antibody, APC Streptavidin (BD Pharmingen, cat. no. 554067) or PE Streptavidin (BD Pharmingen, cat. no. 554061) was used as the secondary antibody, and the expression levels of scFv in the three cells were detected by flow cytometry. The results are shown in Fig.11 PE mouse anti-human HLA-E (biolegend, catalog number 342604) and PE anti-human PDL1 (biolegend, catalog number 329706) were used to detect the expression of HLA-E and PDL1 in CAR T cells, respectively. The results are shown in Fig.12 and Fig.13 shown.

[0168] It can be seen that the anti-CD7 scFv and NK inhibitory molecules (HLA-E and PD-L1) in the CAR T cells prepared by the present invention can be effectively expressed.

[0169] The killing effects of three CAR-T cells on Jurkat target cells were detected according to the method described in 2.1 of Example 2. The results are as follows: Fig.14 As shown in Figure 2, it can be seen that all three CAR-T cells can significantly kill target cells.

[0170] The cytokine release levels of the three CAR-T cells after co-culture with Jurkat target cells were detected according to the method described in 2.2 of Example 2. The results are as follows: Fig.15 Compared with the NT group, the levels of cytokine release of the three CAR-T cells were significantly increased.

[0171] Example 6. Preparation of dual-target CAR-T cells and verification of their functions

[0172] The sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library

[0173] (PPL, catalog number: PPL00157-4a): 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), CD3ζ intracellular signaling domain (SEQ ID NO: 32) and confirm the correct insertion of the target sequence by sequencing.

[0174] The plasmid vector was packaged into 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 negative control (NT).

[0175] Use Biotin-SP(long spacer)AffiniPure Goat Anti-Mouse IgG,F(ab') 2 Fragment Specific(min 2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat. no. 109-065-097) was used as the primary antibody, and FITC Streptavidin (BD Pharmingen, cat. no. 554060) or PE Streptavidin (BD Pharmingen, cat. no. 554061) was used as the secondary antibody. The expression level of scFv in CAR7-19 T cells was detected by flow cytometry. The results are shown in Fig.16 shown.

[0176] It can be seen that both CD7 scFv and CD19 scFv in the CAR T cells prepared by the present invention can be effectively expressed.

[0177] According to the method in Example 2, the killing function and cytokine release level of CAR7-19 T cells were detected respectively, and the results were as follows: Fig.17 and Fig.18As shown in the figure, it can be seen that CAR7-19 T cells have specific killing and significantly increased cytokine release on both Nalm6 and Jurkat target cells.

[0178] It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. It is understood by those skilled in the art that any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An engineered immune cell, Features: (1) expressing a chimeric antigen receptor comprising an antigen binding region, wherein the antigen binding region comprises an anti-CD7 antibody; (2) the expression of endogenous CD7, at least one TCR / CD3 gene and at least one MHC-II class related gene is inhibited or silenced; the engineered immune cells further express NK inhibitory molecules, wherein the NK inhibitory molecules comprise one or more NK inhibitory ligands, a transmembrane domain and a co-stimulatory domain; the NK inhibitory ligand is an antibody or a functional fragment thereof targeting an NK inhibitory receptor, wherein the NK inhibitory receptor is selected from NKG2A, LIR1, KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, PD1 and KLRG1, or the NK inhibitory ligand is HLA-E, HLA-G, cadherin, PD-L1 / PD-L2, or the NK inhibitory receptor binding regions they contain.

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

4. The engineered immune cell of any one of claims 1 to 3, wherein the antigen binding region of the chimeric antigen receptor further comprises an antibody or a functional fragment thereof targeting a second antigen, the second antigen being selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 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 breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut 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 engineered immune cell of claim 4, wherein the chimeric antigen receptor comprises an anti-CD7 antibody and an anti-CD19 antibody.

6. The engineered immune cell of claim 1 or 2, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

7. The engineered immune cell of claim 2, wherein the intracellular signaling domain is selected from the intracellular regions of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

8. The engineered immune cell of claim 1 or 2, wherein the chimeric antigen receptor further comprises one or more co-stimulatory domains selected from the intracellular regions of the following proteins: 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.

9. The engineered immune cell of claim 1, wherein the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

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

11. The engineered immune cell of any one of claims 1-10, wherein the expression of endogenous CD7, at least one TCR / CD3 gene selected from TRAC and TRBC, and at least one MHC-II class gene selected from RFX5, RFXAP, RFXANK and CIITA of the engineered immune cell is inhibited or silenced.

12. The engineered immune cell of claim 11, wherein the NK inhibitory molecule further comprises a CD3ζ intracellular region as an intracellular signaling domain.

13. The engineered immune cell of claim 1, wherein the engineered immune cell is a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell, or a NKT cell.

14. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1 to 13, and one or more pharmaceutically acceptable excipients.

15. Use of the engineered immune cell according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating a disease associated with CD7 expression, wherein the disease associated with CD7 expression is 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.

Images