Engineered immune cell and use thereof

Abstract

Provided is an engineered immune cell, wherein the expression of a CD96 molecule is inhibited or silenced, and the engineered immune cell expresses a functional exogenous receptor which specifically recognizes an antigen. Also provided are a pharmaceutical composition containing the engineered immune cell, and use thereof in the treatment of cancer, infections, 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 CD96 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] CD96, also known as TACTILE (T-Cell Activation, Increased Late Expression), is a member of the immunoglobulin superfamily of receptors. CD96 is a type I transmembrane protein with a molecular weight of approximately 64 kDa. It consists of three extracellular Ig-like domains, a transmembrane domain, and an intracellular domain consisting of 45 amino acids. Exon 4 of the human CD96 mRNA undergoes alternative splicing, resulting in two isoforms differing by the presence of a second extracellular Ig-like domain. The less commonly expressed CD96V1 has a V-like second extracellular Ig-like domain, while the more widely expressed CD96V2 has an I or C-like domain. The intracellular domain of human CD96 consists of a basic / proline-rich motif and a single ITIM-like domain, as well as a YXXM motif. The primary ligand for human CD96 is CD155, although mouse CD96 can also bind to CD111.

[0004] The CD96 molecule is primarily expressed on αβT cells, γδT cells, NK cells, and NKT cells, with high expression on late-activated T cells and CD8+ tumor TILs. CD96 expression is also found in diseases such as acute myeloid leukemia stem cells, acute lymphoblastic leukemia cells, and myelodysplastic syndrome. Its ligand, CD155, is expressed on antigen-presenting cells such as DCs and T cells, as well as on the surface of various tumor cells.

[0005] CD96 functions as an immunosuppressive receptor in mouse NK cells. NK cells from CD96-knockout mice secrete more IFN-γ in response to stimuli such as LPS, IL-12, and IL-18. Simultaneous blockade of the CD96, PD-1, and CTLA4 pathways significantly increases cytokine secretion and prolongs survival in tumor-bearing mice compared to blocking only the PD-1 and CTLA4 pathways. CD96 also exerts an inhibitory function in mouse T cells. Blockade of CD96 enhances the secretory function of CD8+ T cells and inhibits tumor growth in various tumor-bearing mice. Systematic studies of CD96 in regulating human T and NK cell function are lacking, and its role remains inconclusive. Studies have shown that activation of CD96 enhances the cytotoxicity of human NK cells, suggesting that CD96 plays an activation signaling role in NK cells. Blockade of CD96 does not affect NK cell cytotoxicity against ovarian cancer cells and has no significant effect on NK cell secretory function. Recent studies suggest that CD96 may play an activation signaling role in human and mouse CD8+ T cells. The role of CD96 in human T and NK cells is currently unknown. There are also no reports on its role in CAR-T cells.

[0006] Summary of the Invention

[0007] 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.

[0008] Ⅰ Engineered immune cells

[0009] In a first aspect, the present invention provides an engineered immune cell in which expression of the CD96 molecule is suppressed or silenced, and the engineered immune cell expresses a functional exogenous receptor that specifically recognizes an antigen. Compared to engineered immune cells in which expression of the CD96 molecule is not suppressed or silenced, the engineered immune cell provided by the present invention has enhanced tumor clearance and in vivo expansion.

[0010] 1.1 Functional exogenous receptors

[0011] 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.

[0012] 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 mobilizing monoclonal T cell receptors (ImmTACs), etc., preferably chimeric antigen receptors or T cell receptors, more preferably chimeric antigen receptors.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In some embodiments, the antigen is selected from the group consisting of: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7-H3, 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 B7-H3, 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 B7-H3, CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, CD7 and BCMA.

[0021] 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).

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes Claudin18.2, such as an antibody targeting Claudin18.2. Antibodies targeting Claudin18.2 known in the art can be used in the present invention. In some embodiments, the antibody targeting Claudin18.2 comprises a VHH, wherein the CDR1, CDR2, and CDR3 comprised by the VHH are identical to the CDR1, CDR2, and CDR3 comprised by one of SEQ ID NOs: 4-8. In some embodiments, the CDR1 comprised by the VHH is as shown in SEQ ID NO: 1, the CDR2 is as shown in SEQ ID NO: 2, and the CDR3 is as shown in SEQ ID NO: 3.

[0029] In some embodiments, the VHH 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 one of SEQ ID NOs: 4-8. Preferably, the antibody targeting Claudin18.2 of the present invention comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-8.

[0030] 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, or it can 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 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: 9, 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: 10 or 11.

[0031] 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: 12. 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: 13 or 14. 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: 15.

[0032] 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: 16. 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: 17 or 18.

[0033] 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: 19 or 20.

[0034] 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: 21. 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:22 or 23.

[0035] 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 can shield the extracellular binding domain, preventing it from binding to the targeted antigen. When the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art can be used in the present invention.

[0036] 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.

[0037] 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.

[0038] As used herein, 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.

[0039] 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.

[0040] 1.2 Immune cells

[0041] As used herein, the term "immune cell" refers to any cell with one or more effector functions (e.g., cytotoxicity / 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.

[0042] 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.

[0043] 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).

[0044] 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.

[0045] 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.

[0046] 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 breakage can also be mediated by, for example, a large range of nucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems, thereby silencing genes. For example, in some embodiments, the present invention achieves inhibition or silencing of CD96 expression by introducing Cas enzymes and sgRNA targeting CD96 into immune cells. Preferably, the sgRNA is introduced into immune cells in the form of sgRNA, nucleic acid encoding sgRNA or a vector; the Cas enzyme is introduced into immune cells in the form of a nucleic acid encoding Cas enzyme or a vector. More preferably, the sgRNA is selected from one or more of SEQ ID NO: 24-26.

[0047] Ⅱ Pharmaceutical Composition

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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, engineered immune cells as described herein are typically provided in solution form and preferably comprise a pharmaceutically acceptable buffer.

[0052] 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. Furthermore, the pharmaceutical compositions of the present invention may also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0053] III. Treatment

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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.

[0058] 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.

[0059] 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 accompanying drawings and embodiments of the present invention are for illustrative purposes only and do not constitute any limitation to the present invention. The embodiments of the present invention and the features therein may be combined with each other unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1: CD96 expression during T cell activation.

[0061] Figure 2: CAR molecule expression levels on CAR-T cells with and without CD96 knockout.

[0062] Figure 3: Expansion of T cells with and without CD96 knockout.

[0063] Figure 4: Knockout efficiency of CD96 knockout in T cells.

[0064] Figure 5: Expression levels of CD45RO and CD62L molecules on the surface of T cells with and without CD96 knockout.

[0065] Figure 6: In vitro killing effect of CAR-T cells with and without CD96 knockout on target cells.

[0066] Figure 7: Release levels of cytokines IL-2, IFN-γ, and TNF-α after co-culture of CAR-T cells with and without CD96 knockout and target cells.

[0067] Figure 8: CD107a positivity rate after co-culture of CAR-T cells with and without CD96 knockout and target cells.

[0068] Figure 9: Tumor inhibitory effect of CAR-T cells with and without CD96 knockout.

[0069] Figure 10: Changes in the number of T cells in the peripheral blood of mice after infusion of CAR-T with and without CD96 knockout. DETAILED DESCRIPTION

[0070] Example 1 Expression of CD96 during T cell activation

[0071] T cells were activated using DynaBeads CD3 / CD28 CTS™ (Gibco, Cat. No. 40203D) at 37°C and 5% CO2, and CD96 expression in T cell subsets was measured by flow cytometry at different time points. As shown in Figure 1, with T cell activation, CD96 abundance in T cells initially increased slightly, then decreased, and then continued to increase. No significant differences were found between CD4-positive and CD8-positive T cell subsets.

[0072] Example 2 Preparation of CD96-knockout anti-CLDN18.2 CAR-T cells and verification of their function

[0073] 2.1 Preparation of CLDN18.2-targeted CAR-T cells

[0074] Sequences encoding the following proteins were synthesized and cloned into the MSCV retroviral vector: CD8α signal peptide (SEQ ID NO: 22), anti-CLDN18.2 (i.e., Claudin18.2) antibody 150V5 (SEQ ID NO: 4), CD8a hinge region (SEQ ID NO: 13), CD8α transmembrane region (SEQ ID NO: 10), 4-1BB intracellular region (SEQ ID NO: 17), CD3ζ intracellular region (SEQ ID NO: 19), and the correct insertion of the target sequence was confirmed by sequencing.

[0075] Retrovirus was produced using the 293GP packaging cell line. Briefly, 31.5 μg of MSCV vector plasmid and 10.5 μg of VSVG envelope plasmid were co-transfected into 293GP cells grown to 70% confluency in a T175 flask using X-treme GENE HP DNA Transfection Reagent (Roche, Cat. No. 06366236001). The cells were cultured at 32°C and 5% CO2. Viral supernatants were collected 72, 96, 120, and 144 hours after transfection, separated by centrifugation from cell debris, and frozen at -80°C until use.

[0076] T cells were activated with an appropriate amount of CD3 / CD28 magnetic beads at 37°C and 5% CO2, marked as Day 0. On Day 2 (Day 2), Cas9 protein and sgRNA targeting the CD96 gene (one of SEQ ID NOs: 24-26) were co-transfected to knock out the CD96 molecule. After overnight culture, the electroporated T cells were added with an appropriate amount of retroviral supernatant on Day 3 and centrifuged in a Retronectin-coated culture plate for transduction. Culture was continued from Day 11 to Day 14 to obtain CD96-knockout anti-CLDN18.2 CAR-T cells (150V5CD96KO). Unmodified wild-type T cells (NT / NT CD96WT), wild-type T cells after CD96 knockout (NT CD96KO), and anti-CLDN18.2 CAR-T cells without CD96 knockout (150V5CD96WT) were used as controls. As shown in Figure 2, the CAR-T cell positivity rate was approximately 50%, and knocking out CD96 did not affect CAR expression. Furthermore, as shown in Figure 3, knocking out CD96 had no significant adverse effect on the expansion of T cells, especially CAR-T cells.

[0077] 2.2 Detection of CD96 knockout efficiency

[0078] Under culture conditions of 37°C and 5% CO2, flow cytometry was used to detect the expression of CD96 molecules on the surface of T cells in each group on days 5, 7, and 9 after activation. As shown in Figure 4, the expression of CD96 molecules on the surface of T cells was effectively inhibited.

[0079] 2.3 Detection of CAR-T cell memory phenotype

[0080] On Day 11 after activation, flow cytometry was used to analyze the expression of CD45RO and CD62L molecules on the surface of T cells in each group under 37°C and 5% CO2 conditions to determine their memory cell phenotype. As shown in Figure 5, knocking out CD96 had no significant effect on the memory phenotype of T cells.

[0081] 2.4 Detection of the in vitro killing effect of CAR-T cells on target cells

[0082] Target cells (NUGC4 cells) or non-target cells (Raji cells) expressing luciferase gene were cultured at a rate of 1×10 4NT CD96WT, NT CD96KO, 150V5CD96WT, and 150V5CD96KO were then added to the 96-well plates for co-culture at effector-to-target ratios (i.e., effector T cell to target cell ratios) of 16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, and 1:16. Fluorescence was measured 18 hours later using a microplate reader. The killing efficiency was calculated as (target cell mean fluorescence - sample mean fluorescence) / target cell mean fluorescence × 100%. The killing effect is shown in Figure 6.

[0083] It can be seen that at different effector-target ratios, each CAR-T cell of the present invention showed a strong killing effect on target cells (NUGC4 cells), but had no obvious killing effect on non-target cells (Raji cells), indicating that each CAR-T cell only showed specific killing of cells expressing the target molecule CLDN18.2. More importantly, knocking out the CD96 molecule had no adverse effect on the specific killing function of CAR-T cells in vitro.

[0084] 2.5 Detection of cytokine release levels of CAR-T cells

[0085] Target cells (NUGC4 cells) or non-target cells (Raji cells) were cultured at a rate of 1×10 5 T cells from each group were seeded into 96-well plates at a density of 1:1 / well. T cells from each group were then added to the 96-well plates for co-culture at an effector-target ratio of 1:1. The co-culture supernatant was collected 24 hours later. The levels of IL-2, IFN-γ, and TNF-α in the co-culture supernatant were measured using the Human IL-2 DuoSet ELISA Kit (R&D systems, Catalog No. DY202), Human IFN-gamma DuoSet ELISA Kit (R&D systems, Catalog No. DY285), and Human TNF-alpha DuoSet ELISA Kit (R&D systems, Catalog No. DY210), respectively. The results are shown in Figure 7.

[0086] It can be seen that compared with NT cells, after co-culture of the CAR-T cells of the present invention with target cells, the release levels of cytokines IL-2, IFN-γ and TNF-α were significantly increased, and they showed specificity for the target molecule CLDN18.2, and knocking out CD96 had no adverse effect on the specific cytokine release ability of CAR-T cells.

[0087] 2.6 Detection of CRA-T cell degranulation function

[0088] Target cells (NUGC4 cells) or non-target cells (Raji cells) were cultured at a rate of 1×10 5T cells from each group were seeded into a 96-well plate at a density of 1:1 / well. T cells from each group were then added to the 96-well plate for co-culture at an effector-target ratio of 1:1. The protein transport inhibitor Monensin (BD GolgiStop, BD Biosciences) and the flow cytometry antibody CD107a-PE (Biolegend, Catalog No. 328608) were also added. After 4 hours of co-culture, conventional flow cytometry staining was performed, and the CD107a positivity rate was calculated. The results are shown in Figure 8.

[0089] It can be seen that compared with NT cells, the expression of CD107a of each CAR-T cell of the present invention was significantly increased after co-culture with target cells, and showed specificity for the target molecule CLDN18.2, and knockout of CD96 had no adverse effect on the specific degranulation ability of CAR-T cells.

[0090] Example 3 Verification of in vivo expansion and tumor suppression effect of CD96 knockout anti-CLDN18.2 CAR-T cells

[0091] A gastric cancer CDX subcutaneous xenograft tumor model was established using severely immunodeficient NPI mice (Admore Biotechnology). 7-week-old healthy female NPI mice were selected and 4×10 6 NUGC4 cells were randomly divided into three groups on Day 20, namely NT group, 150V5CD96WT group, and 150V5CD96KO group. 3×10 6 NT cells or corresponding CAR-T cells were used to monitor the changes in tumor burden in mice. The results are shown in Figure 9.

[0092] As can be seen, compared with the NT group, each CAR-T cell of the present invention exhibited a significant tumor-suppressing effect. Group analysis of the tumor growth curves of mice in different groups showed that the tumors in the 150V5CD96KO group were rapidly cleared, while some mice in the 150V5CD96WT group experienced tumor volume reduction and recurrence, while the 150V5CD96KO group did not experience recurrence, indicating that CAR-T cells with CD96 knockout have a stronger tumor-clearing effect.

[0093] On the 19th and 39th days after CAR-T infusion, the number of T cells in the peripheral blood of mice was detected by flow cytometry. The results are shown in Figure 10.

[0094] It can be seen that knocking out CD96 in CAR-T cells can significantly increase the number of CAR-T cells in the peripheral blood of mice. On the 19th day after infusion, both CD4-positive T cells and CD8-positive T cell subsets increased significantly. After the 39th day, the CD4-positive T cell subset was the main one, indicating that CAR-T cells with CD96 knockout have stronger in vivo expansion ability.

[0095] The above results show that CAR-T cells with CD96 knockout have stronger tumor clearance effect and in vivo expansion ability, and have no obvious adverse effects on the cytokine release and degranulation ability of CAR-T cells.

[0096] 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 CD96 molecule is inhibited or silenced, and the engineered immune cell expresses a functional exogenous receptor that specifically recognizes an antigen.

2. The engineered immune cell according to claim 1, 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.

3. The engineered immune cell of claim 2, 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.

4. The engineered immune cell according to claim 3, wherein the antigen specifically recognized by the functional exogenous receptor is selected from: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7-H3, 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. , 5. The engineered immune cell of claim 3 or 4, 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.

6. The engineered immune cell according to any one of claims 3 to 5, wherein the antigen binding domain is selected from an antibody targeting Claudin18.

2.

7. The engineered immune cell according to any one of claims 6, wherein the antibody targeting Claudin18.2 comprises a VHH, wherein the CDR1, CDR2 and CDR3 comprised by the VHH are identical to the CDR1, CDR2 and CDR3 comprised by one of SEQ ID NOs: 4-8.

8. The engineered immune cell according to claim 7, wherein the antibody targeting Claudin18.2 comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 4-8.

9. The engineered immune cell according to any one of claims 3-8, 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.

10. The engineered immune cell according to any one of claims 3-9, 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.

11. The engineered immune cell of any one of claims 3-10, 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.

12. The engineered immune cell according to any one of claims 1-11, 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.

13. The engineered immune cell according to claim 12, 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.

14. The engineered immune cell according to any one of claims 1 to 13, 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.

15. The engineered immune cell according to any one of claims 1-14, 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.

16. The engineered immune cell of claim 15, 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.

17. The engineered immune cell according to any one of claims 1-16, 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.

18. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1-16 and one or more pharmaceutically acceptable excipients.

19. Use of the engineered immune cell according to any one of claims 1 to 16 in the preparation of a medicament for treating cancer, infection or autoimmune disease.

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