WO2025035309A1 - Engineered immune cell and use thereof - Google Patents

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

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 inhibited or silenced, a pharmaceutical composition comprising the engineered immune cells, and the use thereof in treating cancer, infection or autoimmune diseases.

Background Art

In recent years, cell therapy, as an emerging tumor treatment method, has shown very significant clinical efficacy. Cell therapy mainly involves the in vitro expansion of immune cells, and then infusing them back into tumor patients to achieve the purpose of treatment. However, this therapy still faces problems such as rapid cell exhaustion and insufficient persistence in the patient's body, resulting in reduced efficacy. Therefore, how to improve the effect of cell therapy is one of the long-term challenges facing this field.

Summary of the invention

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.

Ⅰ Engineered Immune Cells

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

The CD276 molecule, also known as B7-H3 (B7homolog 3protein), is an important immune checkpoint molecule of the B7-CD28 family. CD276 belongs to a type I transmembrane protein with a molecular weight of 45-66kDa. The CD276 molecule has a similar molecular structure to PD-L1 (B7-H1). In the human body, CD276 mainly exists in the form of 4IgB7-H3, and some of it exists in the form of 2IgB7-H3. Many malignant tumors express CD276, and CD276 is correlated with tumor growth, metastasis, and poor prognosis. In addition, 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 the expression of CD276 has multiple locations, including cell membrane, cytoplasm, nucleus, exosomes, etc., suggesting that it may have diverse functions.

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 mobilization monoclonal T cell receptor, preferably a chimeric antigen receptor or a T cell receptor.

1.1 Functional xenobiotic receptors

In some embodiments, the engineered immune cells of the present invention may express functional exogenous receptors. The functional exogenous receptors described in the present invention include chimeric antigen receptors (CAR), T cell receptors (TCR), T cell receptor fusion proteins (TFP), T cell antigen couplers (TAC) or immune mobilization monoclonal T cell receptors (ImmTAC), etc., preferably chimeric antigen receptors or T cell receptors, more preferably chimeric antigen receptors.

The term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide, which 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, a co-stimulatory domain, and each domain is connected by a linker. CAR can redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner. 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 a primary signaling domain, optionally, one or more co-stimulatory domains. In some embodiments, the chimeric antigen receptor also includes one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc.

The term "T cell receptor" or "TCR" refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. The stimulation of TCR is triggered by the major histocompatibility complex molecule (MHC) on the antigen presenting cell, which presents the antigen peptide to the T cell and binds to the TCR complex to induce a series of intracellular signal transductions. 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 of specificity. The variable region of 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.

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

The term "T cell receptor fusion protein" or "TFP" refers to a recombinant polypeptide derived from various components of TCR, which is usually composed of a TCR subunit and an antigen binding domain connected thereto and expressed on the cell surface. Among them, the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

The term “immune mobilizing monoclonal T cell receptor” or “ImmTAC” is a combination of an engineered T cell receptor (TCR) and The anti-CD3 scFv is composed of a modified TCR that 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 between the scFv antibody fragment and CD3.

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 of the antigen.

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 co-stimulatory domains.

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 the antigen binding domain depends on the cell surface markers on the target cells associated with a specific disease state to be recognized, such as tumor antigens.

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, CD44v 6. CD44v7/8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, C D137, 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、ETV6-AML、EMR2、EGP2、EGP40、FAP、FAR、FBP、FLT3、FOSL1、FCRL5、FCAR、Flt3、Flt4、Frizzled、GD2 , GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HA VCR1, HPV E6, E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL 1. IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, M SLN, 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, R AGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1/CD248, TEM7 R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNF R2, 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 reverse transcriptase/hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variant of tumor necrosis area, 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 α, 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.

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 the 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 to 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 (sdAb) (e.g., antibody heavy chain variable region VH, light chain variable region VL, nanobody VHH, etc.).

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.

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 complete antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, diabodies, sdAb and functional fragments of ligands or receptors, preferably selected from complete antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, diabodies and sdAb, more preferably scFv and/or sdAb.

The term "heavy chain" refers to the larger of the two types of polypeptide chains that occur 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 that occur in naturally occurring conformations in antibody molecules. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

The term "complementarity determining region" or "CDR" refers to amino acid sequences within an antibody variable region that confer 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, and it should be understood that the "CDR" or "FR" of a given antibody or its region (such as its variable region) covers the CDR or FR defined by any of the above schemes or other known schemes, and 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 schemes or other known schemes. The numbering scheme used to define the boundaries of CDR and FR in this article is the Chothia scheme.

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 joint), and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, the scFv herein may have the VL and VH in any order, for example, the scFv may include VL-joint-VH from the N-terminus to the C-terminus, or may include VH-joint-VL. The term "joint" refers to a molecular sequence connecting two molecules or two sequences on the same molecule. In some embodiments, the joint is a peptide joint. Preferably, the joint does not adversely affect the expression, secretion or biological activity of the polypeptide. In addition, the joint is preferably not antigenic and does not induce an immune response. In some embodiments, the joint may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a sequence rich in GS) or a non-peptide chemical joint.

The term "single domain antibody" or "sdAb" refers to an antibody consisting of a single variable region with three CDRs, which can bind to an antigen alone without being paired with a corresponding CDR-containing polypeptide. A single domain antibody comprises a VHH fragment derived from or derived only from a camelid heavy chain antibody and can be fused to a heavy chain constant region as desired.

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 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. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 1, 10, 19 or 28, CDR2-H as shown in SEQ ID NO: 2, 11, 20 or 29, and CDR3-H as shown in SEQ ID NO: 3, 12, 21 or 30, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 4, 13, 22 or 31, CDR2-L as shown in SEQ ID NO: 5, 14, 23 or 32, and CDR3-L as shown in SEQ ID NO: 6, 15, 24 or 33.

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 in the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 7, 16, 25 or 34 and a light chain variable region as shown in SEQ ID NO: 8, 17, 26 or 35.

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.

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 the same as 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 the same as 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.

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 in the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 43 and a light chain variable region as shown in SEQ ID NO: 44.

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.

The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of a 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 parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

The term "conservative modification" refers to amino acid modifications that do not significantly affect or change 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 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 amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including 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.

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.

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 can carry out signal transduction. Particularly suitable transmembrane domains 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.

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 may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from 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. 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 hinges. 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 with the amino acid sequence shown 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 58.

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. "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 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, 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 with 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 with the amino acid sequence shown in SEQ ID NO: 60 or 61.

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 may 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, with SEQ ID NO: 62 or 63. The amino acid sequences shown have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the intracellular region of CD3ζ.

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%, and more preferably at least 90%, 95%, 97%, or 99%, or 100% sequence identity with 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%, and 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.

In some embodiments, 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 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 masked peptide. The masking peptide may shield the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cut 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.

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 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.

1.2 Immune cells

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, the 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, a 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, etc., 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 an infection site, 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 to obtain T cells from the blood of the subject.

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. 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. Transfection of animal cells generally involves opening a transient hole or "hole" in the cell membrane to allow the uptake 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.

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, no artificial intervention method (gene editing or non-gene editing) is used to change any endogenous HLA-I class genes and/or HLA-II class genes. The expression level of native HLA-I class genes and/or HLA-II class genes.

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.

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, CASP1 is inhibited or silenced. 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.

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 breaks can also be mediated by, for example, Cas enzymes in meganucleases, zinc finger nucleases, TALE nucleases or CRISPR systems to silence genes.

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 sgRNA, a nucleic acid encoding sgRNA, or a vector; the Cas enzyme is introduced into immune cells in the form of a nucleic acid encoding the Cas enzyme or a vector. More preferably, the sgRNA is selected from one or more of SEQ ID NO: 46-51.

Ⅱ Pharmaceutical Composition

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 covers the use of the engineered immune cells in the preparation of a pharmaceutical composition or a drug.

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, coating agents, adsorbents, antiadhesives, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, 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 desired pharmaceutical composition of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water. The choice of a suitable excipient generally depends on, among other things, the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

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.

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.

The pharmaceutical composition according to the present invention can also be used in combination with one or more other agents (biological agents such as antibody reagents, and/or small molecules) or treatment methods (such as surgery, chemotherapy or radiotherapy) suitable for treating and/or preventing the disease to be treated. Preferred examples of agents suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, tadalafil, tadalafil, succinimidyl ... The invention relates to a pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises a 5-hydroxy-1,4-dimethoate 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1,4-dopamine 1

III. Treatment

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 as described above.

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.

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 adenocarcinoma, adenocarcinoma, and squamous lung cancer), lymphoma (including Hodgkin's and non-Hodgkin's lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, colorectal 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 mass disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic 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).

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: hepatitis C virus (HCV), hepatitis B virus (HBV), immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) infections, etc.

In some embodiments, the autoimmune disease includes but is 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, etc. Preferably, the disease that can be treated with the engineered immune cell, cell population or pharmaceutical composition of the present invention is selected from: multiple sclerosis, systemic lupus erythematosus, etc. The present invention will be described in detail 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 only for illustrative purposes 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 can be combined with each other.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2: Expansion folds of CAR-T cells prepared based on different anti-CD276 antibodies.

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

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

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

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

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

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

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

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

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

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

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

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

Figure 15: Expansion of CAR-T cells prepared based on BH28-3V5 in mice.

DETAILED DESCRIPTION

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

1.1 Preparation of CAR-T cells

Sequences encoding the following proteins were synthesized and cloned into 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), CD3ζ intracellular region (SEQ ID NO: 62), and the correct insertion of the target sequence was confirmed by sequencing. Among them, 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; the amino acid of anti-CD276 antibody MGA271 is shown in SEQ ID NO: 27.

After adding 3 ml Opti-MEM (Gibco, Catalog No. 31985-070) to a sterile tube to dilute the above plasmid, 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 120 μl 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℃, 2.5 hours) to obtain concentrated lentivirus.

T cells were activated with DynaBeads CD3/CD28CTSTM (Gibco, Cat. No. 40203D) and cultured at 37°C and 5% CO ₂ for 1 day. Then, concentrated lentivirus was added and cultured for 1 day. The CD276 molecule was knocked out by electroporation using sgRNA (one of SEQ ID NO: 46-51), and each CAR-T cell with CD276 knocked out was obtained, which was numbered KO-376.96, KO-8H9, and KO-MGA271 according to the different antibodies. Unmodified wild-type T cells (NT) and CAR-T cells (WT-376.96, WT-8H9, WT-MGA271) without knocking out CD276 molecules were used as controls.

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

After culturing for 7 days at 37°C and 5% CO ₂ , the T cells were stained with the antibody APC anti-human CD276 (B7-H3) antibody (Biolegend, Catalog No. 351005), and the expression level of CD276 molecules on CAR-T cells was detected by flow cytometry. T cells were 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 detected by flow cytometry. The relevant results are shown in Figure 1.

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.

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.

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

Each target cell expressing the luciferase gene (NUGC4 cells, DLD1 cells, Huh7 cells) was plated into a 96-well plate at a concentration of 1×10 ⁴ cells/well, and then NT cells and each CAR-T cell 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 16:1, 8:1, 4:1, and 2:1. After 16-18 hours, the fluorescence value was measured using an ELISA reader. According to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, the killing efficiency was calculated, and the results are shown in Figure 3.

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.

1.4 Detection of cytokine release levels of CAR-T cells

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 1×10 ⁵ cells/well, and CAR-T cells and NT cells (negative control) were added at a ratio of 1:1. After 18-24 hours of co-culture, the cell co-culture supernatant was collected.

According to the manufacturer’s recommendations, 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.

It can be seen that compared with NT cells, after each CAR-T cell of the present invention was co-cultured with each target cell, the release levels of cytokines IL2 and IFN-γ 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.

Example 2. Preparation of CD276 knockout CAR-T cells based on BH329V5 antibody and verification of its function

2.1 Preparation of CAR-T cells

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.

After adding 3 ml Opti-MEM (Gibco, Catalog No. 31985-070) to a sterile tube to dilute the above plasmid, 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℃, 2.5 hours) to obtain concentrated lentivirus.

T cells were activated with DynaBeads CD3/CD28CTSTM (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 1 day. Then, concentrated lentivirus was added and culture was continued for 1 day. The CD276 molecule was knocked out by electroporation using sgRNA (one of SEQ ID NO:46-51), 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 without CD276 knockout (WT BH329V5, WT BH329V5-828z) were used as controls.

2.2 Detection of the knockout effect of CD276 molecules and the expression level of CAR molecules in CAR-T cells

After culturing for 7 days at 37°C and 5% _CO2 , samples of T cells in each group were taken and sent for sequencing to detect the knockout of CD276 molecules. The relevant data are as follows. It can be seen from the sequencing data (Figure 5) that the sgRNA selected in the present invention can effectively knock out the CD276 molecules of T cells.

On the 9th day of culture, FITC-Rabbit anti-mouse IgG, F(ab')specific (jackson immunoresearch, catalog number 315-095-006) was used to stain T cells, and the expression level of CAR molecules on CAR-T cells was detected by flow cytometry. The relevant results are shown in Figure 6. It can be seen that CD276 single-chain antibody can be effectively expressed. This shows that knocking out CD276 molecules has no adverse effect on the expression of chimeric antigen receptor molecules.

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

Each target cell (NUGC4 cell, DLD1 cell, Huh7 cell) or non-target cell (Nalm6 cell) expressing luciferase gene was plated into a 96-well plate at a concentration of 1×10 ⁴ cells/well, and then NT cells and each CAR-T cell 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 16:1, 8:1, 4:1, 2:1, and 1:1, and the fluorescence value was measured by a microplate reader after 16-18 hours. 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 Figure 7.

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

2.4 Detection of cytokine release levels of CAR-T cells

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 1×10 ⁵ cells/well, and CAR-T cells and NT cells (negative control) were added at a ratio of 1:1. After 18-24 hours of co-culture, the cell co-culture supernatant was collected.

According to the manufacturer’s recommendations, 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.

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

2.5 Verification of the tumor suppression effect of CAR-T cells

Twenty-five healthy female NPI mice of approximately 7 weeks old were divided into 5 groups: NT group (negative control), WT BH329V5 group, WT BH329V5-828z group, KO BH329V5 group, and KO BH329V5-828z group.

On day 0 (D0), 4×10 ⁶ NUGC4 cells were injected subcutaneously into each mouse. 26 days later (D26), 10×10 ⁶ NT cells or corresponding CAR-T cells were injected into the tail vein of each mouse according to the grouping. The changes in tumor burden of mice were evaluated weekly, and the results are shown in Figure 9.

It can be seen that compared with the NT group, each CAR-T cell of the present invention showed significant tumor inhibition effect, especially the tumor inhibition effect of the WT BH329V5 group, KO BH329V5 group and KO BH329V5-828z group was further reduced compared with the WT BH329V5-828z group, indicating that CD276 knockout CAR-T cells showed a better in vivo tumor inhibition effect than non-knockout CAR-T cells, and this effect existed 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.

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

3.1 Preparation of CAR-T cells

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), CD3ζ intracellular region (SEQ ID NO: 62), and the correct insertion of the target sequence was confirmed by sequencing.

After adding 3 ml Opti-MEM (Gibco, Catalog No. 31985-070) to a sterile tube to dilute the above plasmid, 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 120 μl 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.

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 1 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 without CD276 knockout (WT BH28-3V5) were used as controls.

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

After culturing for 7 days at 37°C and 5% CO ₂ , T cells were stained with FITC-Rabbit anti-mouse IgG, F(ab')specific (jackson immunoresearch, catalog number 315-095-006), and the expression level of CAR molecules on CAR-T cells was detected by flow cytometry. The relevant results are shown in Figure 10. It can be seen that CD276 single-chain antibody can be effectively expressed. This shows that knocking out the CD276 molecule has no adverse effect on the expression of chimeric antigen receptor molecules.

After culturing for 7 days at 37°C and 5% _CO2 , samples of T cells in each group were taken and sent for sequencing to detect the knockout of CD276 molecules. The relevant data are as follows. It can be seen from the sequencing data (Figure 11) that the sgRNA selected in the present invention can effectively knock out the CD276 molecules of T cells.

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

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 1×10 ⁴ cells/well, and then NT cells and each CAR-T cell 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 8:1, 4:1, 2:1, 1:1, 1:2, and 1:4, and the fluorescence value was measured by an ELISA instrument after 16-18 hours. According to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%, the killing efficiency was calculated, and the results are shown in Figure 12.

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

3.4 Detection of cytokine release levels of CAR-T cells

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 1×10 ⁵ cells/well, and CAR-T cells and NT cells (negative control) were added at a ratio of 1:1. After 18-24 hours of co-culture, the cell co-culture supernatant was collected.

According to the manufacturer's recommendations, Human IL-2 DuoSet ELISA Kit (R&D systems, catalog number DY202) and Human IFN-gamma DuoSet ELISA Kit (R&D systems, catalog number 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.

It can be seen that compared with NT cells, after each CAR-T cell of the present invention was co-cultured with each target cell, the release levels of cytokines IL2 and IFN-γ 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.

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

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

On day 0 (D0), 4×10 ⁶ Hela cells were injected subcutaneously into each mouse. Five days later (D5), 4×10 ⁶ NT cells or corresponding CAR-T cells were injected into the tail vein of each mouse according to the grouping. The changes in tumor burden of mice were evaluated weekly, and the results are shown in Figure 14.

It can be seen that, first, compared with the NT group, each CAR-T cell of the present invention showed a significant tumor-suppressing effect. At the same time, the CAR-T cells with CD276 knockout showed a more excellent in vivo tumor-suppressing 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 quickly cleared, and the tumor volume of some mice in the WT BH28-3V5 group was reduced and then relapsed. This shows that CAR-T cells with CD276 knockout have a stronger tumor-clearing effect. Secondly, the weight of mice in each group was not much different, indicating that knocking out CD276 would 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.

In addition, we also collected peripheral blood samples from mice for analysis on days 14 and 21. The data are shown in Figure 15. At different time points, the number 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 ability.

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

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 (20)

An engineered immune cell in which the expression of the CD276 molecule is inhibited or silenced. The engineered immune cell according to claim 1, wherein the engineered immune cell also expresses a functional exogenous receptor that specifically recognizes an antigen. 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 mobilization monoclonal T cell receptor, preferably a chimeric antigen receptor or a T cell receptor. The engineered immune cell according to 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. 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, CD72, CD79a, CD79b, CD80, CD81, CD8 6. CD97, CD123, CD133, CD137, CD138, 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, ETV6-AML, EMR2, EGP2, EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt 4. Frizzled, GD2, GD3, gp100, 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, LIFR P, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LIL RA2, 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 alpha, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor beta, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase reverse transcriptase/hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variant of tumor necrosis area, and any combination thereof. The engineered immune cell according to 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. 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. 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 contained in SEQ ID NO: 8, 17, 26 or 35. , CDR2-L and CDR3-L are the same; 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:43; 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. The engineered immune cell according to 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. The engineered immune cell according to any one of claims 4 to 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. The engineered immune cell according to any one of claims 4 to 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. The engineered immune cell according to any one of claims 4 to 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. 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. 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. 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, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL 10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. The engineered immune cell according to any one of claims 1 to 15, wherein the immune cell is selected from B cells, T cells, macrophages, dendritic cells, neutrophils, monocytes, NK cells and NKT cells. The engineered immune cell of claim 16, wherein the cell is selected from 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. The engineered immune cell according to any one of claims 1 to 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. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1 to 18 and one or more pharmaceutically acceptable excipients. 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.