Engineered immune cell and use thereof

Abstract

Provided is an engineered immune cell, wherein CD276 molecule expression is inhibited or silenced. Also provided are a pharmaceutical composition comprising the engineered immune cell, and a use thereof in the treatment of cancer, infection or autoimmune diseases.

Description

Engineered immune cells and their uses Technical Field

[0001] The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to engineered immune cells in which the expression of CD276 molecules is suppressed or silenced, pharmaceutical compositions comprising the engineered immune cells, and their use in treating cancer, infection or autoimmune diseases. Background Art

[0002] In recent years, cell therapy, an emerging cancer treatment, has demonstrated remarkable efficacy in clinical practice. Cell therapy primarily involves expanding immune cells ex vivo and then infusing them back into cancer patients for therapeutic purposes. However, this approach still faces challenges such as rapid cell exhaustion and insufficient persistence in the patient's body, leading to reduced efficacy. Therefore, improving the effectiveness of cell therapy remains a long-standing challenge in this field.

[0003] Summary of the Invention

[0004] 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.

[0005] Ⅰ Engineered immune cells

[0006] In a first aspect, the present invention provides an engineered immune cell, wherein the expression of the CD276 molecule is inhibited or silenced.

[0007] The CD276 molecule, also known as B7-H3 (B7 homolog 3 protein), is an important immune checkpoint molecule in the B7-CD28 family. CD276 is a type I transmembrane protein with a molecular weight of 45-66 kDa. It shares a similar molecular structure with PD-L1 (B7-H1). In the human body, CD276 primarily exists as 4IgB7-H3, with some existing as 2IgB7-H3. CD276 is expressed in various malignant tumors and correlates with tumor growth, metastasis, and poor prognosis. Furthermore, CD276 is also expressed in immune cells such as monocytes, dendritic cells, myeloid-derived suppressor cells, neutrophils, macrophages, B cells, and activated T cells. Studies have shown that CD276 is expressed in various locations, including the cell membrane, cytoplasm, nucleus, and exosomes, suggesting that it may have diverse functions.

[0008] In some embodiments, the engineered immune cells further express a functional exogenous receptor that specifically recognizes an antigen. Preferably, the functional exogenous receptor that specifically recognizes an antigen is selected from a chimeric antigen receptor, a T cell receptor, a T cell receptor fusion protein, a T cell antigen coupler, and an immune mobilizing monoclonal T cell receptor, preferably a chimeric antigen receptor or a T cell receptor.

[0009] 1.1 Functional exogenous receptors

[0010] In some embodiments, the engineered immune cells of the present invention can express functional exogenous receptors. The functional exogenous receptors described in the present invention include chimeric antigen receptors (CARs), T cell receptors (TCRs), T cell receptor fusion proteins (TFPs), T cell antigen couplers (TACs), or immune mobilization monoclonal T cell receptors (ImmTACs), etc., preferably chimeric antigen receptors or T cell receptors, more preferably chimeric antigen receptors.

[0011] The term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen (e.g., tumor antigen) binding domain (e.g., ligand / receptor of an antibody or antigen), a transmembrane domain, and a primary signaling domain, optionally further comprising a costimulatory domain, with each domain connected by a linker. CAR is capable of redirecting the specificity and reactivity of T cells and other immune cells to a selected target in a non-MHC restricted manner. In some embodiments, the functional exogenous receptor of the present invention is a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a primary signaling domain, optionally further comprising one or more costimulatory domains. In some embodiments, the chimeric antigen receptor further comprises one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, and the like.

[0012] The term "T cell receptor" or "TCR" refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling. The TCR consists of six peptide chains that form heterodimers, which are generally divided into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to a specific antigen and MHC molecule. The variable region of the TCR may include an antigen binding domain or be operably connected to an antigen binding domain, wherein the definition of the antigen binding domain is as described below.

[0013] The term "T cell antigen coupler" or "TAC" includes three functional domains: (1) a tumor targeting domain, which can be composed of a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, which is linked to the protein kinase LCK and catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.

[0014] The term "T cell receptor fusion protein" or "TFP" refers to a recombinant polypeptide derived from various components of a TCR, typically consisting of a TCR subunit and an antigen-binding domain attached thereto, and expressed on the cell surface. The TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

[0015] The term "immune mobilizing monoclonal T cell receptor" or "ImmTAC" is composed of an engineered T cell receptor (TCR) and an anti-CD3 scFv, wherein the engineered TCR can specifically recognize and bind to the HLA-peptide complex on the surface of tumor cells with significantly improved affinity, and promote T cell-mediated effector function through the interaction of the scFv antibody fragment with CD3.

[0016] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes an antigen (e.g., a tumor antigen). In some embodiments, the extracellular domain comprises an antibody that specifically binds to an antigen or a ligand or receptor for the antigen.

[0017] In some embodiments, the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain and / or a primary signaling domain, and optionally, one or more costimulatory domains.

[0018] The term "antigen binding domain" refers to any structure (such as an antibody, ligand, or receptor, etc.) or a functional variant thereof that can bind to an antigen. The choice of antigen binding domain depends on the cell surface markers on the target cells associated with the specific disease state to be recognized, such as tumor antigens.

[0019] In some embodiments, the antigen is selected from the group consisting of ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, CD276, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD 138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin6, Claudin18.1, Claudin18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene Because, ETV6-AML, EMR2, EGP2, EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp1 00, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, muthsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2, VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor β, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variant of tumor necrosis, and any combination thereof. Preferably, the antigen is selected from CD276, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA and GPRC5D, more preferably selected from CD276, CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, CD7 and BCMA.

[0020] In some embodiments, the antigen binding domain in the present invention is selected from an antibody. The term "antibody" has the broadest meaning understood by those skilled in the art, and includes complete antibodies such as monoclonal 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, which can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). The term "antibody fragment" refers to at least a portion of a complete antibody or a variant thereof, and refers to a binding domain (e.g., an antigen variable region of a complete antibody) sufficient to confer recognition and specific binding to an antigen on an antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fd', Fv fragments, scFv, disulfide-linked Fv (sdFv), linear antibodies, "diabodies" with two antigen-binding sites, single-domain antibodies (sdAbs) (e.g., the heavy chain variable region VH, the light chain variable region VL, nanobodies VHH, etc. of an antibody).

[0021] In other embodiments, the antigen-binding domain of the present invention is selected from ligands, receptors, and functional fragments thereof (i.e., functional fragments with antigen-binding ability, such as extracellular domains). The term "ligand or receptor" refers to any molecule or atom that can interact with the corresponding antigen (as a receptor or ligand) after binding. The ligand or receptor can be a naturally occurring molecule, such as an organic or inorganic molecule, or a synthetic molecule.

[0022] Unless the context clearly indicates otherwise, the "antigen-binding domain" of the present invention encompasses antibodies, ligands, receptors, and functional fragments thereof as described above. Therefore, the antigen-binding domain described in the present invention is selected from the group consisting of intact antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, diabodies, sdAbs, and functional fragments of ligands or receptors, preferably selected from the group consisting of intact antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, diabodies, and sdAbs, and more preferably scFv and / or sdAbs.

[0023] The term "heavy chain" refers to the larger of the two types of polypeptide chains found in naturally occurring conformations in antibody molecules and generally determines the class to which the antibody belongs. The term "light chain" refers to the smaller of the two types of polypeptide chains found in naturally occurring conformations in antibody molecules. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0024] The term "complementarity determining region" or "CDR" refers to the amino acid sequence in the antibody variable region that imparts antigen specificity and binding affinity. For example, in general, there are three CDRs (e.g., CDR1-H, CDR2-H, and CDR3-H) in each heavy chain variable region, and three CDRs (CDR1-L, CDR2-L, and CDR3-L) in each light chain variable region. The precise amino acid sequence boundaries of CDRs can be determined using any of many well-known schemes, including: Kabat numbering scheme, Chothia numbering scheme, IMGT numbering scheme, AHo numbering scheme, AbM numbering scheme. The precise amino acid sequence of a given CDR or FR may be different due to the different numbering schemes selected. It should be understood that "CDR" or "FR" of a given antibody or its region (e.g., its variable region) encompasses CDRs or FRs defined by any of the above-mentioned schemes or other known schemes. In the case where a specified CDR or FR contains a given amino acid sequence, it should be understood that such CDR or FR can also have the sequence of the corresponding CDR or FR defined by any of the above-mentioned schemes or other known schemes. The numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.

[0025] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one light chain variable region and at least one heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are adjacent (e.g., connected via a linker) and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise indicated, the scFv herein may have the VL and VH described in any order, for example, scFv may comprise VL-linker-VH from N-terminus to C-terminus, or may comprise VH-linker-VL. The term "linker" refers to a molecular sequence connecting two molecules or two sequences on the same molecule. In some embodiments, the linker is a peptide linker. Preferably, the linker does not adversely affect the expression, secretion or biological activity of the polypeptide. In addition, the linker is preferably not antigenic and does not elicit an immune response. In some embodiments, the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a sequence rich in GS) or a non-peptide chemical linker.

[0026] The term "single-domain antibody" or "sdAb" refers to an antibody consisting of a single variable region with three CDRs that can bind to an antigen alone without being paired with a corresponding CDR-containing polypeptide. A single-domain antibody comprises a VHH fragment derived solely from or derived from a camelid heavy chain antibody and optionally fused to a heavy chain constant region.

[0027] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes CD276, such as an antibody targeting CD276. Antibodies targeting CD276 known in the art can be used in the present invention. In some embodiments, the antibody targeting CD276 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H included in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H included in SEQ ID NO: 7, 16, 25 or 34; wherein the CDR1-L, CDR2-L and CDR3-L included in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L included in SEQ ID NO: 8, 17, 26 or 35. In some embodiments, the heavy chain variable region comprises a CDR1-H as shown in SEQ ID NO: 1, 10, 19 or 28, a CDR2-H as shown in SEQ ID NO: 2, 11, 20 or 29, and a CDR3-H as shown in SEQ ID NO: 3, 12, 21 or 30, and the light chain variable region comprises a CDR1-L as shown in SEQ ID NO: 4, 13, 22 or 31, a CDR2-L as shown in SEQ ID NO: 5, 14, 23 or 32, and a CDR3-L as shown in SEQ ID NO: 6, 15, 24 or 33.

[0028] In some embodiments, the antibody targeting CD276 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7, 16, 25 or 34, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8, 17, 26 or 35. Preferably, the antibody targeting CD276 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 7, 16, 25 or 34 and a light chain variable region as set forth in SEQ ID NO: 8, 17, 26 or 35.

[0029] In some embodiments, the antibody targeting CD276 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9, 18, 27 or 36. Preferably, the antibody targeting CD276 is as shown in SEQ ID NO: 9, 18, 27 or 36.

[0030] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes MSLN, such as an antibody targeting MSLN. Antibodies targeting MSLN known in the art can be used in the present invention. In some embodiments, the antibody targeting MSLN comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 43; wherein the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 44. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:37, CDR2-H as shown in SEQ ID NO:38, and CDR3-H as shown in SEQ ID NO:39, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO:40, CDR2-L as shown in SEQ ID NO:41, and CDR3-L as shown in SEQ ID NO:42.

[0031] In some embodiments, the antibody targeting MSLN comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 43, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 44. Preferably, the antibody targeting MSLN of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 43 and a light chain variable region as set forth in SEQ ID NO: 44.

[0032] In some embodiments, the antibody targeting MSLN is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 45. Preferably, the antibody targeting MSLN is as shown in SEQ ID NO: 45.

[0033] 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.

[0034] The term "conservative modification" refers to amino acid modifications that do 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. Modifications can be introduced into the chimeric antigen receptors 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 by an amino acid residue with a similar side chain. Families of amino acid residues with 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), non-polar 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 may be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0035] Thus, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the parent amino acid sequence and retains the biological activity, e.g., binding activity, of the parent amino acid.

[0036] The term "transmembrane domain" refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the cell surface and anchors the antigen binding domain to the cell membrane. The transmembrane domain can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. When the target binding domain binds to the target, the transmembrane domain is capable of signal transduction. Particularly suitable transmembrane domains for use in the present invention can be derived from TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, etc. In some embodiments, the transmembrane domain is derived from the following molecules: CD8α, CD4, CD28 or 4-1BB, or the transmembrane domain can be synthetic and can mainly contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from 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: 52, or the transmembrane domain is derived from 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: 53 or 54.

[0037] In some embodiments, the chimeric antigen receptor further comprises a hinge region between the antigen binding domain and the transmembrane domain. The term "hinge region" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antigen binding domain. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antigen binding domain. The hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from natural molecules, such as 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 can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it can be a fully synthetic hinge sequence. Preferably, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably selected from CD8α, CD28 or IgG4 hinge. In some embodiments, the hinge region is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the hinge region is from CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56 or 57. In some embodiments, the hinge region is from IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58.

[0038] The term "costimulatory domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function, which is an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular region of the costimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (e.g., proliferation and survival). The costimulatory signaling domain of any costimulatory molecule is suitable for use in the chimeric antigen receptor described herein. The costimulatory domain of the present invention includes but is not limited to the intracellular region derived from the following protein: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, 4-1BB, CD270, CD272, B7-H3, ICOS, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, ZAP70 and any combination thereof. Preferably, the costimulatory domain is 4-1BB and / or CD28. In some embodiments, the costimulatory domain is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 59. In other embodiments, the costimulatory domain is from 4-1BB, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 60 or 61.

[0039] The term "primary signaling domain" refers to a protein structure that works together to initiate primary signaling after antigen-receptor binding, which is generally an intracellular sequence of a T cell receptor and a co-receptor. The primary signaling domain generally comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The primary signaling domain in the present invention can be derived from the intracellular region of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2 and CD66d, etc. Preferably, the CAR of the present invention comprises a CD3ζ intracellular region, for example, a CD3ζ intracellular region having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% to the amino acid sequence shown in SEQ ID NO: 62 or 63.

[0040] In some embodiments, the chimeric antigen receptor of the present invention further comprises a signal peptide so that when it is expressed in a cell (e.g., a T cell), the nascent protein is directed to the endoplasmic reticulum and subsequently 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 cut during or after the translocation 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 B2M, CD8α, IgG1, GM-CSFRα, etc. In some embodiments, the signal peptide of the present invention is from B2M, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:64. In some embodiments, the signal peptide of the present invention is derived from 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:65 or 66.

[0041] In some embodiments, the CAR of the present invention may further comprise 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 a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain so that it binds to the targeted antigen, thereby activating the signal transduction pathway. Alternatively, the switch domain may be used to connect the binding domain and the signal transduction domain respectively, and only when the switch domains bind to each other (for example, in the presence of an inducing compound) can the binding domain and the signal transduction domain be connected together through a dimer, thereby activating the signal pathway. The switch structure may also be in the form of a masking peptide. The masking peptide may shield the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, for example, a protease, the extracellular binding domain may be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art may be used in the present invention.

[0042] In some embodiments, 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, so as to remove the CAR cells when needed (e.g., when serious toxic side effects are produced). For example, the suicide gene can be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when needed, the CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which can cause cells to die under the induction of ganciclovir treatment. The suicide gene can 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 cell apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

[0043] 1.2 Immune cells

[0044] As used herein, the term "immune cell" refers to any cell with one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC) of the immune system. For example, an immune cell can be a T cell, a macrophage, a neutrophil, a dendritic cell, a monocyte, a NK cell or a NKT cell, or an immune cell derived from a stem cell (e.g., an adult stem cell, an embryonic stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell). Preferably, the immune cell is a T cell. The T cell can be any T cell, such as a T cell cultured in vitro, such as a primary T cell, or a T cell from a T cell line cultured in vitro such as Jurkat, SupT1, or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. 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. Multiple techniques well known to those skilled in the art can be used, such as Ficoll separation to obtain T cells from the blood of the experimenter.

[0045] When the engineered immune cells of the present invention express functional exogenous receptors (such as CAR or TCR), the functional exogenous receptors can be introduced into immune cells using 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. One 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. Transfection of animal cells generally involves opening a transient pore or "hole" in the cell membrane to allow the uptake of materials. Calcium phosphate can be used, 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 for transfection. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, also into non-animal eukaryotic cells (including plant cells). Therefore, transformation is the genetic alteration of bacteria or non-animal eukaryotic cells, which is produced by direct uptake from their surroundings by 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 competent state. For prokaryotic transformation, techniques may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation.

[0046] In some embodiments, the expression of endogenous HLA-I class genes and / or HLA-II class genes in the engineered immune cells of the present invention is not modified. That is, the expression level of any endogenous HLA-I class gene and / or HLA-II class gene is not altered by any artificial intervention method (gene editing or non-gene editing).

[0047] In some embodiments, the expression of at least one endogenous HLA-I class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene and at least one endogenous HLA-I class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-I class and HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene, at least one endogenous HLA-I class gene, and at least one endogenous HLA-II class gene of the engineered immune cells of the present invention is suppressed or silenced. Preferably, the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C, and B2M. Preferably, the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA. Preferably, the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε and CD3ζ.

[0048] In some embodiments, the expression of one or more endogenous genes 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, TNFRSF10A, CASP8, and CASP1 is suppressed or silenced in the engineered immune cells of the present invention. 0. CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, I L10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0049] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art. For example, antisense RNA, RNA bait, RNA aptamer, siRNA, shRNA, miRNA, trans dominant negative protein (TNP), chimeric / fusion protein, chemokine ligand, anti-infective cell protein, intracellular antibody (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors can be used to inhibit gene expression. In addition, DNA fragmentation can also be mediated by, for example, a large range of nucleases, zinc finger nucleases, TALE nucleases or the Cas enzyme in the CRISPR system to silence genes.

[0050] For example, in some embodiments, the present invention achieves inhibition or silencing of CD276 expression by introducing a Cas enzyme and an sgRNA targeting CD276 into immune cells. Preferably, the sgRNA is introduced into immune cells in the form of an sgRNA, a nucleic acid encoding an sgRNA, or a vector; and the Cas enzyme is introduced into immune cells in the form of a nucleic acid encoding a Cas enzyme or a vector. More preferably, the sgRNA is selected from one or more of SEQ ID NOs: 46-51.

[0051] Ⅱ Pharmaceutical Composition

[0052] In a second aspect, the present invention provides a pharmaceutical composition comprising the engineered immune cells of the present invention as an active agent and one or more pharmaceutically acceptable excipients. Therefore, the present invention also encompasses the use of the engineered immune cells in the preparation of a pharmaceutical composition or medicament.

[0053] 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), and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavorings, colorants, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coatings, isotonic agents, absorption delaying agents, stabilizers, and tension modifiers. It is known to those skilled in the art to select suitable excipients to prepare the pharmaceutical compositions desired in the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. The choice of suitable excipients will generally depend on, inter alia, the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

[0054] The pharmaceutical composition according to the present invention can be applied in a variety of ways. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0055] 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 a form particularly suitable for the desired method of administration. The processes known in 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 typically provided in solution form and preferably comprise a pharmaceutically acceptable buffer.

[0056] The pharmaceutical composition according to the present invention can also be used in combination with one or more other medicaments (biological agents such as antibody reagents, and / or small molecules) or therapeutic methods (such as surgery, chemotherapy or radiotherapy) that are applicable to the treatment and / or prevention of the disease to be treated. Preferred examples of the medicament suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium porphyrin II (sorfimer sodium photofrin II), temozolomide, topotecan, trimetreate glucuronate (trimetreate glucuronate), auristatin E (auristatin E), vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes and RNA enzymes; radionuclides such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as platelet factor 4 and melanoma growth stimulating protein; antibodies or fragments thereof such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides. In addition, the pharmaceutical compositions of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0057] III. Treatment

[0058] In a third aspect, the present invention provides a method for preventing or treating cancer, infection or autoimmune disease, comprising administering to a subject the engineered immune cell or pharmaceutical composition described above.

[0059] The present invention also provides use of the engineered immune cells or pharmaceutical compositions described above in the preparation of drugs for cancer, infection or autoimmune diseases.

[0060] In some embodiments, the cancer includes, but is not limited to, brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumors, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), Lung cancer (including adenomatous, adenomatous, and squamous lung cancer), lymphoma (including Hodgkin and non-Hodgkin lymphomas), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancers of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignancies of the urinary system, vulvar cancer, and other carcinomas and sarcomas, and B-cell lymphoma (including low-grade / follicular Non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large bulky disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL) , T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myeloid leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell neoplasm, and post-transplant lymphoproliferative disorder (PTLD).

[0061] In some embodiments, the infection includes but is not limited to infections caused by viruses, bacteria, fungi and parasites. Preferably, the diseases that can be treated with the engineered immune cells, cell populations or pharmaceutical compositions of the present invention are selected from the group consisting of hepatitis C virus (HCV), hepatitis B virus (HBV), immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) infections.

[0062] In some embodiments, the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus. Preferably, the diseases that can be treated with the engineered immune cells, cell populations or pharmaceutical compositions of the present invention are selected from the group consisting of multiple sclerosis, systemic lupus erythematosus and the like. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are for illustrative purposes only and do not constitute any limitation to the present invention. In the absence of contradiction, the embodiments of the present invention and the features in the embodiments may be combined with each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1: CAR molecule expression levels on CAR-T cells prepared based on different anti-CD276 antibodies.

[0064] Figure 2: Expansion fold of CAR-T cells prepared based on different anti-CD276 antibodies.

[0065] Figure 3: In vitro killing effect of CAR-T cells prepared based on different anti-CD276 antibodies on target cells.

[0066] Figure 4: Cytokine release levels of CAR-T cells prepared based on different anti-CD276 antibodies.

[0067] Figure 5: Knockout effect of CD276 molecules in CAR-T cells prepared based on BH329V5.

[0068] Figure 6: CAR molecule expression levels on CAR-T cells prepared based on BH329V5.

[0069] Figure 7: In vitro killing effect of CAR-T cells prepared based on BH329V5 on target cells.

[0070] Figure 8: Cytokine release levels of CAR-T cells prepared based on BH329V5.

[0071] Figure 9: Tumor inhibitory effect of CAR-T cells prepared based on BH329V5.

[0072] Figure 10: CAR molecule expression levels on CAR-T cells prepared based on BH28-3V5.

[0073] Figure 11: Knockout effect of CD276 molecules in CAR-T cells prepared based on BH28-3V5.

[0074] Figure 12: In vitro killing effect of CAR-T cells prepared based on BH28-3V5 on target cells.

[0075] Figure 13: Cytokine release levels of CAR-T cells prepared based on BH28-3V5.

[0076] Figure 14: Tumor inhibitory effect of CAR-T cells prepared based on BH28-3V5.

[0077] Figure 15: Expansion of CAR-T cells prepared based on BH28-3V5 in mice. DETAILED DESCRIPTION

[0078] Example 1. Preparation of CD276-knockout CAR-T cells based on different anti-CD276 antibodies and verification of their functions

[0079] 1.1 Preparation of CAR-T cells

[0080] Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Catalog No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 65), anti-CD276 single-chain antibody (SEQ ID NO: 9, 18, or 27), CD8α hinge region (SEQ ID NO: 56), CD8α transmembrane region (SEQ ID NO: 53), 4-1BB intracellular region (SEQ ID NO: 60), and CD3ζ intracellular region (SEQ ID NO: 62). Correct insertion of the target sequences was confirmed by sequencing. The amino acid sequence of anti-CD276 antibody 376.96 is shown in SEQ ID NO: 9; the amino acid sequence of anti-CD276 antibody 8H9 is shown in SEQ ID NO: 18; and the amino acid sequence of anti-CD276 antibody MGA271 is shown in SEQ ID NO: 27.

[0081] After diluting the plasmid in 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) in a sterile tube, add the packaging vector psPAX2 (Addgene, Catalog No. 12260) and the envelope vector pMD2.G (Addgene, Catalog No. 12259) at a ratio of 4:2:1 plasmid:viral packaging vector:viral envelope vector. Next, add 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche, Catalog No. 06366236001), mix immediately, and incubate at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture is then added dropwise to the 293T cell culture flask. Viruses are collected at 24 and 48 hours, pooled, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours).

[0082] T cells were activated with DynaBeads CD3 / CD28CTSTM (Gibco, Cat. No. 40203D) and cultured at 37°C and 5% CO2 for 1 day. Then, concentrated lentivirus was added and cultured for 1 day. sgRNA (one of SEQ ID NO: 46-51) was used to electroporate the CD276 molecule to knock out the CD276 molecule, and each CAR-T cell with CD276 knocked out was obtained. Depending on the antibody, they were numbered KO-376.96, KO-8H9, and KO-MGA271, respectively. Unmodified wild-type T cells (NT) and CAR-T cells with no CD276 molecules knocked out (WT-376.96, WT-8H9, WT-MGA271) were used as controls.

[0083] 1.2 Detection of CD276 and CAR molecule expression levels on CAR-T cells

[0084] After 7 days of culture at 37°C and 5% CO2, T cells were stained with APC anti-human CD276 (B7-H3) antibody (Biolegend, Catalog No. 351005), and the expression level of CD276 molecules on CAR-T cells was measured by flow cytometry. T cells were also stained with FITC-Rabbit anti-mouse IgG, F(ab')specific (Jackson Immunoresearch, Catalog No. 315-095-006), and the expression level of CAR molecules on CAR-T cells was measured by flow cytometry. The results are shown in Figure 1.

[0085] It can be seen that the expression level of CD276 in the knocked-out CAR-T cells is reduced, while the CD276 single-chain antibody can be effectively expressed, indicating that knocking out the CD276 molecule has no adverse effect on the expression of the chimeric antigen receptor molecule.

[0086] In addition, from the amplification data of each CAR-T (Figure 2), it can be seen that compared with the wild-type CAR-T that retains CD276, the CD276 knockout CAR-T has better amplification ability.

[0087] 1.3 Detection of the in vitro killing effect of CAR-T cells on target cells

[0088] 1×10 4 Each target cell expressing the luciferase gene (NUGC4 cells, DLD1 cells, Huh7 cells) was plated into a 96-well plate at a concentration of 10 cells / well. NT cells and each CAR-T cell were then plated into a 96-well plate for co-culture at effector-target ratios (i.e., the ratio of effector T cells to target cells) of 16:1, 8:1, 4:1, and 2:1. After 16-18 hours, the fluorescence value was measured using a microplate reader. The killing efficiency was calculated according to the formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, and the results are shown in Figure 3.

[0089] It can be seen that at various effector-target ratios, the CAR-T cells of the present invention showed a strong killing effect on target cells (DLD1 cells, Huh7 cells, NUGC4 cells). More importantly, knocking out the CD276 molecule had no adverse effect on the specific in vitro killing function of CAR-T cells.

[0090] 1.4 Detection of cytokine release levels of CAR-T cells

[0091] 1×10 5 Target cells (DLD1 cells, HCT116 cells, NUGC4 cells, MDA-MB-231 cells, Huh7 cells) and non-target cells (Nalm6 cells) were plated in a 96-well plate at a concentration of 1 cell / well. CAR-T cells and NT cells (negative control) were added at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours of co-culture.

[0092] According to the manufacturer's recommendations, the Human IL-2 DuoSet ELISA Kit (R&D systems, Catalog No. DY202) and Human IFN-gamma DuoSet ELISA Kit (R&D systems, Catalog No. DY285) were used to detect the levels of IL2 and IFN-γ in the co-culture supernatant, respectively. The results are shown in Figure 4.

[0093] It can be seen that compared with NT cells, the release levels of cytokines IL2 and IFN-γ of each CAR-T cell of the present invention after co-culture with each target cell were significantly increased, and this cytokine release was specific. Similarly, knocking out the CD276 molecule had no adverse effect on the specific cytokine release ability of CAR-T cells.

[0094] Example 2. Preparation of CD276-knockout CAR-T cells based on BH329V5 antibody and verification of their function

[0095] 2.1 Preparation of CAR-T cells

[0096] Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Catalog No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 65), anti-CD276 single-chain antibody BH329V5 (SEQ ID NO: 45), CD8α hinge region (SEQ ID NO: 56), CD8α transmembrane region (SEQ ID NO: 53), 4-1BB intracellular region (SEQ ID NO: 60) or CD28 intracellular region (SEQ ID NO: 59), CD3ζ intracellular region (SEQ ID NO: 62), and the correct insertion of the target sequence was confirmed by sequencing.

[0097] After diluting the plasmid in 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) in a sterile tube, add the packaging vector psPAX2 (Addgene, Catalog No. 12260) and the envelope vector pMD2.G (Addgene, Catalog No. 12259) at a ratio of 4:2:1 plasmid:viral packaging vector:viral envelope vector. Next, add 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche, Catalog No. 06366236001), mix immediately, and incubate at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture is then added dropwise to the 293T cell culture flask. Viruses are harvested at 24 and 48 hours, pooled, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours).

[0098] T cells were activated with DynaBeads CD3 / CD28CTSTM (Gibco, Cat. No. 40203D) and cultured at 37 ° C and 5% CO2 for 1 day. Then, concentrated lentivirus was added and cultured for 1 day. sgRNA (one of SEQ ID NO: 46-51) was used to electroporate the CD276 molecule to knock out the CD276 molecule, and each CAR-T cell with CD276 knocked out was obtained, numbered as KO BH329V5 (containing the 4-1BB intracellular region) and KO BH329V5-828z (containing the CD28 intracellular region). Unmodified wild-type T cells (NT) and CAR-T cells with no CD276 molecules knocked out (WT BH329V5, WT BH329V5-828z) were used as controls.

[0099] 2.2 Detection of CD276 knockout effect and CAR molecule expression level in CAR-T cells

[0100] After 7 days of culture at 37°C and 5% CO₂, T cells from each group were sampled and sequenced to detect CD276 knockout. The relevant data are as follows. The sequencing data (Figure 5) demonstrate that the sgRNA selected in this invention can effectively knockout CD276 in T cells.

[0101] On day 9 of culture, T cells were stained with FITC-Rabbit anti-mouse IgG, F(ab')-specific (Jackson ImmunoResearch, Cat. No. 315-095-006), and the expression level of the CAR molecule on the CAR-T cells was measured by flow cytometry. The results are shown in Figure 6. As can be seen, the CD276 single-chain antibody was effectively expressed, indicating that knocking out the CD276 molecule had no adverse effect on the expression of the chimeric antigen receptor molecule.

[0102] 2.3 Detection of the in vitro killing effect of CAR-T cells on target cells

[0103] 1×10 4 Each target cell expressing the luciferase gene (NUGC4 cells, DLD1 cells, Huh7 cells) or non-target cells (Nalm6 cells) was plated into a 96-well plate at a concentration of 10 cells / well. Then, NT cells and each CAR-T cell were plated into a 96-well plate at an effector-target ratio (i.e., the ratio of effector T cells to target cells) of 16:1, 8:1, 4:1, 2:1, and 1:1 for co-culture. After 16-18 hours, the fluorescence value was measured using a microplate reader. The killing efficiency was calculated according to the formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, and the results are shown in Figure 7.

[0104] It can be seen that under various effect-target ratios, each CAR-T cell of the present invention showed a strong killing effect on target cells (NUGC4 cells, DLD1 cells, Huh7 cells), while the killing of non-target cells (Nalm6 cells) was weaker, indicating that each CAR-T cell only showed specific killing of cells expressing CD276. More importantly, knocking out the CD276 molecule had no significant adverse effect on the specific in vitro killing ability of CAR-T cells.

[0105] 2.4 Detection of cytokine release levels of CAR-T cells

[0106] 1×10 5 Target cells (DLD1 cells, HCT116 cells, NUGC4 cells, MDA-MB-231 cells, Huh7 cells) and non-target cells (Nalm6 cells) were plated in a 96-well plate at a concentration of 1 cell / well. CAR-T cells and NT cells (negative control) were added at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours of co-culture.

[0107] According to the manufacturer's recommendations, the Human IL-2 DuoSet ELISA Kit (R&D systems, Catalog No. DY202) and Human IFN-gamma DuoSet ELISA Kit (R&D systems, Catalog No. DY285) were used to detect the levels of IL2 and IFN-γ in the co-culture supernatant, respectively. The results are shown in Figure 8.

[0108] It can be seen that compared with NT cells, the release levels of cytokines IL2 and IFN-γ of each CAR-T cell of the present invention after co-culture with each target cell were significantly increased, and this cytokine release was specific. Similarly, knocking out the CD276 molecule had no adverse effect on the specific cytokine release ability of CAR-T cells, on the contrary, in most cases it promoted it.

[0109] 2.5 Verification of the tumor suppression effect of CAR-T cells

[0110] Twenty-five healthy female NPI mice of approximately 7 weeks old were divided into five groups: NT group (negative control), WT BH329V5 group, WT BH329V5-828z group, KO BH329V5 group, and KO BH329V5-828z group.

[0111] On day 0 (D0), each mouse was injected subcutaneously with 4 × 10 6 NUGC4 cells. After 26 days (D26), 10×10 6The changes in tumor burden in mice were assessed weekly, and the results are shown in Figure 9.

[0112] It can be seen that compared with the NT group, each CAR-T cell of the present invention showed a significant tumor inhibition effect, especially the tumor inhibition effect of the WT BH329V5 group, the KO BH329V5 group and the KO BH329V5-828z group was further reduced compared with the WT BH329V5-828z group, indicating that the CD276 knockout CAR-T cells showed a better in vivo tumor inhibition effect than the non-knockout CAR-T cells, and this effect was present in different intracellular domain combinations. At the same time, compared with the NT group, the survival time of mice in each experimental group of the present invention was significantly prolonged.

[0113] Example 3. Preparation of CD276-knockout MSLN-targeted CAR-T cells and verification of their function

[0114] 3.1 Preparation of CAR-T cells

[0115] Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), Catalog No. PPL00157-4a): CD8α signal peptide (SEQ ID NO: 65), anti-MSLN single-chain antibody BH28-3V5 (SEQ ID NO: 45), CD8α hinge region (SEQ ID NO: 56), CD8α transmembrane region (SEQ ID NO: 53), 4-1BB intracellular region (SEQ ID NO: 60), and CD3ζ intracellular region (SEQ ID NO: 62). The correct insertion of the target sequences was confirmed by sequencing.

[0116] After diluting the plasmid in 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) in a sterile tube, add the packaging vector psPAX2 (Addgene, Catalog No. 12260) and the envelope vector pMD2.G (Addgene, Catalog No. 12259) at a ratio of 4:2:1 for plasmid:viral packaging vector:viral envelope vector. Next, add 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche, Catalog No. 06366236001), mix immediately, and incubate at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture is then added dropwise to the 293T cell culture flask. Viruses are collected at 24 and 48 hours, combined, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours).

[0117] T cells were activated with DynaBeads CD3 / CD28CTSTM (Gibco, Cat. No. 40203D) and cultured at 37 ° C and 5% CO2 for 1 day. Then, concentrated lentivirus was added and cultured for another day. CD276 molecules were knocked out by electroporation using sgRNA (one of SEQ ID NO: 46-51) to obtain MSLN-targeted CAR-T cells KO BH28-3V5 with CD276 knocked out. Unmodified wild-type T cells (NT) and MSLN-targeted CAR-T cells (WT BH28-3V5) with no CD276 knockout were used as controls.

[0118] 3.2 Detection of CD276 and CAR molecule expression levels on CAR-T cells

[0119] After 7 days of culture at 37°C and 5% CO₂, T cells were stained with FITC-Rabbit anti-mouse IgG, F(ab')-specific (Jackson ImmunoResearch, Catalog No. 315-095-006), and the expression levels of CAR molecules on CAR-T cells were measured by flow cytometry. The results are shown in Figure 10. As can be seen, CD276 single-chain antibody was effectively expressed, indicating that knocking out CD276 molecules had no adverse effect on the expression of chimeric antigen receptor molecules.

[0120] After 7 days of culture at 37°C and 5% CO₂, T cells from each group were sampled and sequenced to detect CD276 knockout. The relevant data are as follows. The sequencing data (Figure 11) demonstrate that the sgRNA selected in this invention can effectively knockout CD276 in T cells.

[0121] 3.3 Detection of the in vitro killing effect of CAR-T cells on target cells

[0122] 1×10 4 Each target cell (Hela cell, NUGC4 cell) or non-target cell (Huh7 cell) expressing the luciferase gene was plated into a 96-well plate at a concentration of 10 cells / well. Then, NT cells and each CAR-T cell were plated into a 96-well plate at an effector-target ratio (i.e., the ratio of effector T cells to target cells) of 8:1, 4:1, 2:1, 1:1, 1:2, and 1:4 for co-culture. After 16-18 hours, the fluorescence value was measured using a microplate reader. The killing efficiency was calculated according to the formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, and the results are shown in Figure 12.

[0123] It can be seen that under various effector-target ratios, each CAR-T cell of the present invention showed a strong killing effect on target cells (Hela cells, NUGC4 cells), while the killing of non-target cells (Huh7 cells) was weaker, indicating that each CAR-T cell only showed specific killing of cells expressing MSLN. More importantly, knocking out the CD276 molecule had no adverse effect on the specific in vitro killing function of CAR-T cells.

[0124] 3.4 Detection of cytokine release levels of CAR-T cells

[0125] 1×10 5 Target cells (Hela cells, NUGC4 cells, HCT116 cells) and non-target cells (Huh7 cells) were plated in a 96-well plate at a concentration of 1 cell / well. CAR-T cells and NT cells (negative control) were added at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours of co-culture.

[0126] According to the manufacturer's recommendations, the Human IL-2 DuoSet ELISA Kit (R&D systems, Catalog No. DY202) and Human IFN-gamma DuoSet ELISA Kit (R&D systems, Catalog No. DY285) were used to detect the levels of IL2 and IFN-γ in the co-culture supernatant, respectively. The results are shown in Figure 13.

[0127] It can be seen that compared with NT cells, the release levels of cytokines IL2 and IFN-γ of each CAR-T cell of the present invention after co-culture with each target cell were significantly increased, and this cytokine release was specific. Similarly, knocking out the CD276 molecule had no adverse effect on the specific cytokine release ability of CAR-T cells.

[0128] Example 4. Verification of the in vivo tumor suppression effect of CAR-T cells targeting MSLN with CD276 knockout

[0129] Fifteen healthy female NPI mice of approximately 7 weeks old were divided into three groups: NT group (negative control), WT BH28-3V5 group, and KO BH28-3V5 group.

[0130] On day 0 (D0), each mouse was injected subcutaneously with 4 × 10 6 After 5 days (D5), 4×10 6 The changes in tumor burden in mice were assessed weekly, and the results are shown in Figure 14.

[0131] It can be seen that, first, compared with the NT group, each CAR-T cell of the present invention showed a significant tumor suppression effect. At the same time, the CAR-T cells with CD276 knockout showed a more excellent in vivo tumor suppression effect than the CAR-T cells without knockout. The tumor growth curves of mice in different groups were grouped and analyzed. It can be seen that the tumors of all mice in the KO BH28-3V5 group were rapidly cleared, and the tumor volume of some mice in the WT BH28-3V5 group was reduced and then relapsed. This shows that the CAR-T cells with CD276 knockout have a stronger tumor clearance effect. Secondly, the weight of mice in each group was not much different, indicating that knocking out CD276 does not enhance the toxicity of CAR-T cells to the host. Finally, compared with the NT group and the WT BH28-3V5 group, the survival time of mice in the KO BH28-3V5 group was significantly prolonged.

[0132] In addition, we collected peripheral blood samples from mice for analysis on days 14 and 21. As shown in Figure 15, at different time points, the numbers of T cells, CD4-positive T cells, and CD8-positive T cells in the KO BH28-3V5 group were higher than those in the WT group, indicating that CAR-T cells with CD276 knockout have stronger in vivo expansion capacity.

[0133] The above results show that CAR-T cells with CD276 knockout have stronger tumor killing activity and in vivo expansion ability, and can prolong the survival of animals, without obvious adverse effects on the cytokine release and safety of CAR-T cells.

[0134] 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 be modified and varied in various ways. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. An engineered immune cell, wherein the expression of CD276 molecule is inhibited or silenced.

2. The engineered immune cell according to claim 1, wherein the engineered immune cell also expresses a functional exogenous receptor that specifically recognizes an antigen.

3. The engineered immune cell according to claim 2, wherein the functional exogenous receptor that specifically recognizes an antigen is selected from a chimeric antigen receptor, a T cell receptor, a T cell receptor fusion protein, a T cell antigen coupler, and an immune mobilizing monoclonal T cell receptor, preferably a chimeric antigen receptor or a T cell receptor.

4. The engineered immune cell of claim 3, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; the intracellular signaling domain comprises a primary signaling domain and / or a co-stimulatory domain.

5. The engineered immune cell according to claim 4, wherein the antigen specifically recognized by the functional exogenous receptor is selected from: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, CD276, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24 , CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD 72. CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD138, CD151, CD171, CD179a, CD300L F, CDH16, CSPG4, CS1, Claudin6, Claudin18.1, Claudin18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXOR F61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR 2. EGP2, EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp13 0. GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2, VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor β, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variant of tumor necrosis zone, and any combination thereof. , 6. The engineered immune cell of claim 4 or 5, wherein the antigen binding domain is selected from the group consisting of a complete antibody, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, diabody, and sdAb.

7. The engineered immune cell according to any one of claims 4-6, wherein the antigen binding domain is selected from antibodies targeting CD276 and / or MSLN.

8. The engineered immune cell according to claim 7, wherein the antibody targeting CD276 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 7, 16, 25 or 34; wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 8, 17, 26 or 35; and / or, the antibody targeting MSLN comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: The CDR1-H, CDR2-H and CDR3-H contained in NO:43 are the same; wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO:

44.

9. The engineered immune cell of claim 8, wherein the antibody targeting CD276 comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region is at least 90% identical to SEQ ID NO: 7, 16, 25 or 34, and the light chain variable region is at least 90% identical to SEQ ID NO: 8, 17, 26 or 35; and / or, the antibody targeting MSLN comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region is at least 90% identical to SEQ ID NO: 43, and the light chain variable region is at least 90% identical to SEQ ID NO:

44.

10. The engineered immune cell according to any one of claims 4-9, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

11. The engineered immune cell according to any one of claims 4-10, wherein the primary signaling domain is selected from the intracellular region of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2 and CD66d.

12. The engineered immune cell of any one of claims 4-11, wherein the co-stimulatory domain is selected from the intracellular region of one or more of the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, 4-1BB, CD270, CD272, B7-H3, ICOS, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, and ZAP70.

13. The engineered immune cell according to any one of claims 1 to 12, wherein in the engineered immune cell, the expression of one or more genes among endogenous HLA-I class genes, HLA-II class genes, and TCR / CD3 genes is inhibited or silenced.

14. The engineered immune cell according to claim 13, wherein the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C, B2M and any combination thereof; the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and any combination thereof; the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and any combination thereof.

15. The engineered immune cell according to any one of claims 1 to 14, wherein the expression of one or more endogenous genes selected from the group consisting of CD52, GR, dCK, PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP1 0. CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, I L10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

16. The engineered immune cell according to any one of claims 1-15, wherein the immune cell is selected from the group consisting of B cells, T cells, macrophages, dendritic cells, neutrophils, monocytes, NK cells and NKT cells.

17. The engineered immune cell of claim 16, wherein the cell is selected from the group consisting of CD4+CD8+T cells, CD4+T cells, CD8+T cells, CD4-CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, and αβ-T cells.

18. The engineered immune cell according to any one of claims 1-17, wherein the immune cell is derived from embryonic stem cells, umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells and induced pluripotent stem cells.

19. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1-18 and one or more pharmaceutically acceptable excipients.

20. Use of the engineered immune cell according to any one of claims 1 to 18 in the preparation of a medicament for treating cancer, infection or autoimmune disease.

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