Engineered immune cell and use thereof

Abstract

The present invention relates to an engineered immune cell, wherein the expression of FKBP12 is inhibited or silenced, and an exogenous chimeric antigen receptor and / or immunosuppressive molecule are expressed. The present invention also provides a composition comprising the engineered immune cell and use thereof in the treatment of cancers, infections, or autoimmune diseases.

Description

Engineered immune cells and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT International Application No. PCT / CN2024 / 101699, entitled “Engineered immune cells and uses thereof” and filed on June 26, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of cell therapy. More specifically, the present application relates to an engineered immune cell and uses thereof. BACKGROUND

[0004] As a new emerging immunotherapy, adoptive cell therapy has developed very rapidly in recent years. In particular, chimeric antigen receptor therapy (CAR-T therapy) has multiple products on the market and has shown excellent therapeutic effect in clinical trials. However, the significant efficacy of adoptive cell therapy is accompanied by a variety of adverse reactions, including graft rejection, cytokine release syndrome, nervous system toxicity, tumor lysis syndrome, cytopenia, infection, hypogammaglobulinemia, and hepatitis B virus activation, etc.

[0005] Therefore, it is still necessary to improve the existing adoptive cell therapy to reduce the occurrence of adverse reactions and achieve better therapeutic effect. SUMMARY

[0006] Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the application belongs.

[0007] In a first aspect, the present application provides an engineered immune cell, wherein the expression of FKBP12 is inhibited or silenced, and the engineered immune cell expresses a chimeric antigen receptor and / or an immunosuppressive molecule; the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and a primary signaling domain; the immunosuppressive molecule comprises an immunosuppressive protein binding domain and a transmembrane domain and does not comprise a primary signaling domain.

[0008] FKBP12 (FK506 binding protein 12) is a small molecular weight protein widely present in mammalian cells, belonging to the immunophilin family. The structure of FKBP12 consists of a typical immunophilin folding domain, which enables it to have the activity of a peptidyl prolyl cis-trans isomerase (PPIase), capable of catalyzing the cis-trans isomerization of proline residues in proteins. This property enables FKBP12 to play an important role in protein folding and functional regulation. FKBP12 can bind to FK506 to form a complex, inhibiting calcineurin, thereby inhibiting the activation of immune cells, achieving an immunosuppressive effect.

[0009] Chimeric antigen receptor

[0010] 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., an antibody or a ligand for an antigen), a transmembrane domain, a primary signaling domain, and optionally a costimulatory domain, each of which is connected by a linker. CARs are 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 chimeric antigen receptors of the present application comprise 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 receptors further comprise one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc.

[0011] In some embodiments, the "binding domain" in the present application is selected from an antibody. The term "antibody" has the broadest meaning understood by one of skill in the art and includes intact antibodies, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), etc., and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that are capable of exhibiting the desired biological activity, which can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). The term "antibody fragment" refers to at least a portion of an intact antibody or variant thereof and refers to a binding domain sufficient to confer on the antibody fragment the ability to recognize and specifically bind to a target (e.g., an antigen), such as the antigen variable region of an intact antibody. Examples of antibody fragments include, but are not limited to: Fab, Fab', F(ab')2, Fd fragments, Fd', Fv fragments, scFv, disulfide-linked Fv (sdFv), linear antibodies, "diabodies" with two antigen binding sites, single domain antibodies (sdAb) (e.g., a heavy chain variable region VH of an antibody, a light chain variable region VL, a nanobody VHH, etc.).

[0012] In some embodiments, the "binding domain" in the present application is selected from a ligand, a receptor and a functional fragment thereof (i.e. a functional fragment having the ability to bind to an antigen or an immunoinhibitory protein, such as an extracellular region) that binds to an antigen or an immunoinhibitory protein. The term "ligand or receptor" refers to any molecule or atom that is capable of producing an interaction upon binding to an antigen or an immunoinhibitory protein. The ligand or receptor can be a naturally occurring molecule, such as an organic or inorganic molecule, or a synthetic molecule. For example, known ligands of NKG2A include HLA-E, known ligands of TIM3 include Galectin9, HMGB1 and CEACAM1, known ligands of LAG3 include Galectin3, LSECtin, FGL1 and some MHC class II molecules, known ligands of TIM3 include CD112, CD113, CD155 and Nectin4, known ligands of CTLA4 include CD80 and CD86, known ligands of PD1 include PDL1 and PDL2, known ligands of SIRPa include CD47, and known ligands of LILRB1 include HLA-G.

[0013] Unless the context clearly indicates otherwise, the "binding domain" of the present application encompasses antibodies, ligands and functional fragments thereof as described above. Thus, the binding domain described in the present application is selected from the group consisting of a whole antibody, a Fab, a Fab', a F(ab')2, a Fd fragment, a Fd', a Fv fragment, a scFv, a sdFv, a linear antibody, a diabody, a sdAb, a functional fragment of a ligand or a receptor.

[0014] The term "functional variant" or "functional fragment" refers to a variant or fragment that contains at least one amino acid modification (i.e. substitution, deletion or insertion) compared to the parent amino acid sequence but retains the biological activity of the parent amino acid. For example, a functional fragment of a ligand or a receptor in the present application generally refers to a fragment of the ligand or the receptor that is capable of binding to the corresponding antigen or immunoinhibitory protein, such as an extracellular region.

[0015] The term "heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule 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 present in the naturally occurring conformation of an antibody molecule. Kappa (K) and lambda (l) light chains refer to the two major antibody light chain isotypes.

[0016] The term "complementarity determining region" or "CDR" refers to the amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity. For example, generally, there are three CDRs in each heavy chain variable region (CDR1-H, CDR2-H, and CDR3-H), and three CDRs in each light chain variable region (CDR1-L, CDR2-L, and CDR3-L). The precise amino acid sequence boundaries of a CDR can be determined using any of a number of well-known schemes, including: the Kabat numbering scheme, the Chothia numbering scheme, the IMGT numbering scheme, the AHo numbering scheme, the AbM numbering scheme. The precise amino acid sequence of a given CDR or FR can differ depending on the numbering scheme chosen, it is understood that "CDRs" or "FRs" of a given antibody or region thereof, such as a variable region thereof, encompass the CDRs or FRs defined by any of the above schemes or other known schemes, and where a given CDR or FR contains a given amino acid sequence, it is 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 herein to define the boundaries of CDRs and FRs is the Chothia scheme.

[0017] The term "single chain antibody" or "scFv" refers to a fusion protein comprising at least one light chain variable region (VL) and at least one heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are contiguous (e.g., connected via a linker), and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFvs herein can have the VL and VH in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and can comprise VL-linker-VH or VH-linker-VL from N- to C-terminus.

[0018] The term "linker" refers to a sequence of molecules that connects 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. Furthermore, the linker is preferably not antigenic and does not elicit an immune response. In some embodiments, the linker can be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a GS-rich sequence), or a non-peptide chemical linker, such as (G4S)n, (EAAAK)n, where n can be 1, 2, 3, 4, 5, or 6. In one embodiment, the linker described in the present application has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51 or 52.

[0019] The term "single domain antibody" or "sdAb" refers to a single antigen binding polypeptide having three complementarity determining regions (CDRs), including full length antibodies (e.g., HCAb) as well as antigen binding fragments thereof (e.g., VH, VL, VHH). In some cases, the single domain antibody is selected from or engineered from a camelid- or shark-derived HCAb, and the heavy chain variable domain thereof is referred to herein as "VHH". A VHH has the following basic structure from N- to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0020] In some embodiments, the antigen bound by the antigen binding domain is selected from the group consisting of: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, 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, Claudin 6, Claudin 18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2 ETS fusion gene, ETV6-AML, EMR2, EGP2, FAP, FcRH5, FCRL5, FCRH2, FCRH1, FCRH4, FCRH5, FCRH6, FCRH7, Fc-epsilon Receptor 1, Fc-gamma Receptor 1, Fc-gamma Receptor 2, Fc-gamma Receptor 3, Fc-gamma Receptor 4, Fc-gamma Receptor 5, Fc-gamma Receptor 6, Fc-gamma Receptor 7, Fc-gamma Receptor 8, Fc-gamma Receptor 9, Fc-gamma Receptor 10, Fc-gamma Receptor 11, Fc-gamma Receptor 12, Fc-gamma Receptor 13, Fc-gamma Receptor 14, Fc-gamma Receptor 15, Fc-gamma Receptor 16, Fc-gamma Receptor 17, Fc-gamma Receptor 18, Fc-gamma Receptor 19, Fc-gamma Receptor 20, Fc-gamma Receptor 21, Fc-gamma Receptor 22, Fc-gamma Receptor 23, Fc-gamma Receptor 24, Fc-gamma Receptor 25, Fc-gamma Receptor 26, Fc-gamma Receptor 27, Fc-gamma Receptor 28, Fc-gamma Receptor 29, Fc-gamma Receptor 30, Fc-gamma Receptor 31,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, HAVCR1, HPV E6, E7, HVEM, HIV-1 Gag, HLA-A1, HLA-A2, IL6R, IL-11 Ra, IL-13 Ra, 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-beta, PCTA-1 / Galectin 8, p53, p53 mutants, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutants, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp 17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCR alpha, TCR beta, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2 VEGFR, WT1, XAGE1, 5T4, 8H9, alpha v beta 6 integrin, CA9, folate receptor alpha, ephrinB2, 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 carboxyl esterase, cyclin B1, fibronectin, tenascin, carcinoembryonic variant of the tumor necrosis zone, and any combination thereof. Preferably,The antigen is selected from the group consisting of CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, and any combination thereof, more preferably selected from the group consisting of CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, CD7, BCMA, and any combination thereof, for example from the group consisting of CD19 and BCMA.

[0021] In some embodiments, the antigen binding domain comprises a CD19 binding domain, e.g., an antibody targeting CD19. Any of the antibodies targeting CD19 known in the art can be used in the present application. In some embodiments, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises CDR1-H, CDR2-H, and CDR3-H identical to CDR1-H, CDR2-H, and CDR3-H comprised by SEQ ID NO: 47, 55, or 64; wherein the light chain variable region comprises CDR1-L, CDR2-L, and CDR3-L identical to CDR1-L, CDR2-L, and CDR3-L comprised by SEQ ID NO: 48, 56, or 65. In some embodiments, the heavy chain variable region comprises CDR1-H as set forth in SEQ ID NO: 41, CDR2-H as set forth in SEQ ID NO: 42, CDR3-H as set forth in SEQ ID NO: 43, and the light chain variable region comprises CDR1-L as set forth in SEQ ID NO: 44, CDR2-L as set forth in SEQ ID NO: 45, CDR3-L as set forth in SEQ ID NO: 46. In other embodiments, the heavy chain variable region comprises CDR1-H as set forth in SEQ ID NO: 58, CDR2-H as set forth in SEQ ID NO: 59, CDR3-H as set forth in SEQ ID NO: 60, and the light chain variable region comprises CDR1-L as set forth in SEQ ID NO: 61, CDR2-L as set forth in SEQ ID NO: 62, CDR3-L as set forth in SEQ ID NO: 63.

[0022] In some embodiments, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region that 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: 47, 55, or 64, and a light chain variable region that 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: 48, 56, or 65. Preferably, the antibody targeting CD19 in the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 47 and a light chain variable region as set forth in SEQ ID NO: 48, or comprises a heavy chain variable region as set forth in SEQ ID NO: 55 and a light chain variable region as set forth in SEQ ID NO: 56.

[0023] In some embodiments, the antibody targeting CD19 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: 49, 57, or 66. Preferably, the antibody targeting CD19 is as set forth in SEQ ID NO: 49, 57, or 66.

[0024] In some embodiments, the antigen binding domain comprises a BCMA binding domain, e.g., an antibody targeting BCMA. Any of the antibodies targeting BCMA known in the art can be used in the present application. In some embodiments, the antibody targeting BCMA comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1-H, CDR2-H, and CDR3-H identical to those comprised in any one of SEQ ID NOs: 74-97. In some embodiments, the heavy chain variable region comprises CDR1-H as set forth in SEQ ID NO: 67, 70, or 73, CDR2-H as set forth in SEQ ID NO: 68 or 71, and CDR3-H as set forth in SEQ ID NO: 69 or 72. In other embodiments, the heavy chain variable region comprises CDR1-H as set forth in SEQ ID NO: 58, CDR2-H as set forth in SEQ ID NO: 59, and CDR3-H as set forth in SEQ ID NO: 60, and the light chain variable region comprises CDR1-L as set forth in SEQ ID NO: 61, CDR2-L as set forth in SEQ ID NO: 62, and CDR3-L as set forth in SEQ ID NO: 63.

[0025] In some embodiments, the antibody targeting BCMA comprises a heavy chain variable region that 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: 74-97. Preferably, the antibody targeting BCMA in the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 74-97.

[0026] In some embodiments, the antigen binding domain comprises a BCMA binding domain and a CD19 binding domain as previously described. The BCMA binding domain and the CD19 binding domain are connected by a linker.

[0027] In some embodiments, the chimeric antigen receptor further comprises a signal peptide and / or a hinge region. In some embodiments, the binding domain and the transmembrane domain are further connected by a hinge region. The term "hinge region" generally refers to any oligo- or polypeptide that functions to link the transmembrane domain to the antibody. In particular, the hinge region serves to provide greater flexibility and accessibility to the antibody. 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 a natural molecule, such as the extracellular region of CD8, CD4, or CD28, in whole or in part, or from an antibody constant region, in whole or in part. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a completely synthetic hinge sequence. Preferably, the hinge region comprises a hinge region portion of CD8a, CD28, FcyRIIIa receptor, IgG4, or IgGl, more preferably CD8a, 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%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the hinge region is from CD8a, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or 31. 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%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32.

[0028] In some embodiments, the chimeric antigen receptors of the application further comprise a signal peptide, such 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 can contain a long hydrophobic stretch of amino acids with a propensity to form a single a-helix. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by specific proteases. Signal peptides useful in the application are well known to those skilled in the art, e.g., signal peptides derived from B2M, CD8a, IgGl, GM-CSFRa, etc. In some embodiments, the signal peptide useful in the application 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 set forth in SEQ ID NO: 38. In some embodiments, the signal peptide useful in the application is from CD8a, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39 or 40.

[0029] In some embodiments, the CAR of the application can further comprise a switch structure to regulate the expression timing of the CAR. For example, the switch structure can be in the form of a dimerization domain that causes a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain for binding to the targeted antigen, thereby activating the signaling pathway. Alternatively, a switch domain can also be used to link the binding domain and the signaling domain separately, and only when the switch domains bind to each other (e.g., in the presence of an inducing compound), the binding domain and the signaling domain can be linked together through the dimer, thereby activating the signaling pathway. The switch structure can also be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, e.g., 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 application.

[0030] In some embodiments, the CAR of the present application can also comprise a suicide gene, i.e., one that expresses a cell death signal inducible by an exogenous agent to eliminate the CAR cells when needed, e.g., when severe 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., which can be used to eliminate the CAR cells when needed by adding an antibody or agent that targets these epitopes. The suicide gene can also be a herpes simplex virus thymidine kinase (HSV-TK), which can cause the cell to die upon induction by ganciclovir treatment. The suicide gene can also be iCaspase-9, which can be induced to dimerize by chemical inducers such as AP1903, AP20187, etc., thereby activating the downstream Caspase3 molecule, leading to apoptosis. Various suicide genes known to those skilled in the art can be used in the present application.

[0031] The term "transmembrane domain" refers to a polypeptide structure that is capable of expressing a protein molecule on the surface of a cell and anchoring a target binding domain to the cell membrane. The transmembrane domain can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the target binding domain binds to a target. Particularly suitable transmembrane domains for use in the present application can be derived from, for example, TCRa chain, TCRP chain, TCRy chain, TCR5 chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD28, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD112, CD113, CD134, CD137, CD154, CD155, KIRDS2, OX40, CD2, CD27, CD18, ICOS, 4-1BB, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, BCMA, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, LSECtin, PDL1, PDL2, CEACAM1, MHC class I molecule, MHC class II molecule, CRT AM, Ly9, CD160, PSGL1, CD100, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. In some embodiments, the transmembrane domain is derived from a molecule that is CD8a, CD4, CD28, or 4-1BB, or the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD28 having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26, or the transmembrane domain is derived from CD8a having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27 or 28.In other embodiments, the transmembrane domain comprised by the immunosuppression molecule in the present application is derived from a ligand or receptor targeting an immunosuppression protein, such as HLA-E, CEACAM1, LSECtin, CD47, CD112, CD113, CD155, Nectin4, CD80, CD86, PDL1, PDL2, and some MHC class II molecules, etc. Preferably, the transmembrane domain 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 set forth in any one of SEQ ID NO: 12, 24, 25. More preferably, the transmembrane domain is linked, preferably directly linked, to the extracellular domain of the corresponding ligand or receptor.

[0032] The term "co-stimulatory domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as effector function, which is an intracellular functional signaling domain from a co-stimulatory molecule, comprises the entire intracellular region of the co-stimulatory molecule, or a functional fragment thereof. A "co-stimulatory molecule" refers to a cognate binding partner that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response (e.g., proliferation and survival). The co-stimulatory signaling domain of any co-stimulatory molecule is suitable for use in the chimeric antigen receptors or immunosuppression molecules described herein. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Co-stimulatory domains of the present application include, but are not limited to, the intracellular region derived from 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 combinations thereof. Preferably, the co-stimulatory domain of the CARs of the present application is 4-1BB and / or CD28. In some embodiments, the co-stimulatory domain is 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 set forth in SEQ ID NO: 33. In some embodiments, the co-stimulatory domain is from 4-1BB, 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 set forth in SEQ ID NO: 34 or 35.

[0033] The term "primary signaling domain" refers to protein structures that act together to initiate primary signaling following antigen-receptor engagement, which are generally intracellular sequences of T cell receptors and co-receptors. Primary signaling domains generally comprise one or more Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Primary signaling domains in the present application include, but are not limited to, intracellular regions derived from FcRy, FcRP, CD3y, CD3d, CD3s, CD3z, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2, and CD66d. In some embodiments, the immunosuppressive molecules of the present application do not comprise a CD3z intracellular region, e.g., a CD3z intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36 or 37.

[0034] The term "sequence identity" indicates the degree of identity between two (nucleotide or amino acid) sequences in an alignment, and is generally expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity. Those skilled in the art know that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197), and ClustalW.

[0035] Immunosuppressive molecules

[0036] The term "immune inhibitory molecule" refers to a molecule that is capable of binding to an immune inhibitory protein (e.g., PD1, NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPa, KIR2DL1 / 2 / 3, LILRB1, etc.) and thereby inhibits the immune rejection of foreign cells in a subject, e.g., reduces the killing function of immune cells (e.g., T cells, NK cells, etc.) in a subject or inhibits the over-proliferation of immune cells. The term "immune inhibitory protein" refers to a ligand or receptor that exerts an inhibitory effect on the immune response, which plays an important role in maintaining self-tolerance, modulating the duration and amplitude of the immune response, and thus avoiding damage and destruction of normal tissues by the immune system. The immune inhibitory protein in the present application is selected from PD1, NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPa, KIR2DL1 / 2 / 3, LILRB1, or a combination thereof. The immune inhibitory molecule described in the present application comprises an immune inhibitory protein binding domain and a transmembrane domain and does not comprise a primary signaling domain, wherein the immune inhibitory protein is selected from PD1, NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPa, KIR2DL1 / 2 / 3, LILRB1, or a combination thereof.

[0037] In some embodiments, the "immune inhibitory protein binding domain" in the present application is selected from a ligand, a receptor, and functional fragments thereof (i.e., functional fragments having the ability to bind to an immune inhibitory protein, such as an extracellular region) that binds to an immune inhibitory protein. In some embodiments, the immune inhibitory molecule further comprises a costimulatory domain. In this case, the three domains of the immune inhibitory protein binding domain, the transmembrane domain, and the costimulatory domain comprised by the immune inhibitory molecule do not come from the same molecule at the same time. For example, two domains come from the same molecule, and the other domain comes from a different molecule, or all three domains come from different molecules. In other words, the immune inhibitory molecule of the present application is not a full-length ligand or receptor itself. In other embodiments, the immune inhibitory molecule does not comprise a costimulatory domain.

[0038] In some embodiments, the immune inhibitory molecule further comprises a signal peptide and / or a hinge region.

[0039] The definitions of the binding domain, antibody, ligand or receptor, functional variant, and the transmembrane domain, costimulatory domain, primary signaling domain, hinge region, signal peptide, etc. used when describing the structure of the immune inhibitory molecule in the present application are described in the section "Chimeric antigen receptor" above.

[0040] In some embodiments, the immunosuppressive molecule of the present application comprises a PD1 binding molecule comprising a PD1 binding domain that is an antibody, a ligand or a functional fragment thereof targeting PD1. PD1, also known as CD279, is a member of the immunoglobulin superfamily, is a type I transmembrane protein mainly expressed on activated T cells, NK cells, B cells, macrophages, dendritic cells and monocytes, comprising an extracellular immunoglobulin variable domain, a transmembrane domain and a cytoplasmic tail domain, which contains two tyrosine motifs: an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Studies have shown that ITSM is essential for the function of PD1 to suppress the activity of T cells. The ligands of PD1 include PDL1 (B7-H1, CD274) and PDL2 (B7-DC, CD273), which belong to the B7 family of proteins. PD1 exerts immune negative regulation by interacting with its ligands PDL1 and PDL2. When PD1 interacts with the ligand, its intracellular ITSM is phosphorylated and recruits the corresponding phosphatases SHP-1 and SHP-2, resulting in the dephosphorylation of downstream signaling molecules and thus down-regulating the level of immune cell response. This negative regulation mechanism of the immune system is a key molecular basis for maintaining immune tolerance of the body. A large number of studies have shown that the overexpression of immune negative regulatory molecules such as PD1 and their interaction with receptors PD-L1 / PD-L2 induce the body's immune suppression state, which plays an important role in the pathogenesis of cancer and chronic infectious diseases such as HIV, HCV and HBV.

[0041] In some embodiments, the PD1 binding domain is an antibody targeting PD1. Any anti-PD1 antibody known in the art can be used in the present application. In some embodiments, the antibody targeting PD1 comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising CDR1-H, CDR2-H and CDR3-H identical to those contained in SEQ ID NO: 19, and the light chain variable region comprising CDR1-L, CDR2-L and CDR3-L identical to those contained in SEQ ID NO: 20. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 13, CDR2-H as shown in SEQ ID NO: 14, CDR3-H as shown in SEQ ID NO: 15, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 16, CDR2-L as shown in SEQ ID NO: 17, CDR3-L as shown in SEQ ID NO: 18.

[0042] In some embodiments, the antibody targeting PD1 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: 19, 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: 20. Preferably, the antibody targeting PD1 in the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 19 and a light chain variable region as set forth in SEQ ID NO: 20.

[0043] In some embodiments, the antibody targeting PD1 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: 21. Preferably, the antibody targeting PD1 is as set forth in SEQ ID NO: 21.

[0044] In other embodiments, the PD1 binding molecule of the present application comprises a PD1 binding domain that is a ligand binding to PD1, such as PDL1, PDL2, or a functional fragment thereof (i.e., a fragment having the function of binding to PD1, such as the extracellular region of PDL1 or PDL2). Preferably, the ligand binding to PD1 comprises an amino acid sequence that 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: 24 or 25. More preferably, the ligand binding to PD1 comprises an amino acid sequence as set forth in SEQ ID NO: 24 or 25. In this embodiment, the transmembrane region of the PD1 binding molecule is preferably the transmembrane region of PDL1 or PDL2, and optionally, the PD1 binding molecule comprises the intracellular region of PDL1 or PDL2.

[0045] In some embodiments, the immunosuppressive molecule of the present application comprises a PD1 binding molecule comprising a NKG2A binding domain, which is an antibody, ligand or functional fragment thereof targeting NKG2A. NKG2A, also known as KLRC1 or CD159A, is a type II transmembrane protein expressed mainly in cytotoxic lymphocytes (such as CD8+ T cells and NK cells) and belongs to the C-type lectin superfamily, containing a cytoplasmic domain, a transmembrane domain and an extracellular lectin-like domain, with two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its intracellular part, which are involved in inhibitory signal transduction. NKG2A can form a heterodimeric receptor with CD94, and NKG2A / CD94 transmits inhibitory signals to NK cells and CD8+ T cells, etc. The natural ligand of NKG2A is HLA-E, and the interaction between NKG2A and HLA-E helps tumor immune escape. It has been proved that disrupting the interaction between NKG2A and HLA-E can effectively enhance the anti-tumor immune response. In addition, more and more research results show that NKG2A also plays an important role in other immune-related diseases, including viral infection, autoimmune disease, inflammatory disease, parasitic infection, etc.

[0046] In some embodiments, the NKG2A binding domain is an antibody targeting NKG2A. Any anti-NKG2A antibody known in the art can be used in the present application. In some embodiments, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises CDR1-H, CDR2-H and CDR3-H identical to those comprised in SEQ ID NO: 7, and the light chain variable region comprises CDR1-L, CDR2-L and CDR3-L identical to those comprised in SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 1, CDR2-H as shown in SEQ ID NO: 2, CDR3-H as shown in SEQ ID NO: 3, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 4, CDR2-L as shown in SEQ ID NO: 5, CDR3-L as shown in SEQ ID NO: 6.

[0047] In some embodiments, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region that 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, and a light chain variable region that 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. Preferably, the antibody targeting NKG2A in the present application comprises a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 8.

[0048] In some embodiments, the antibody targeting NKG2A 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. Preferably, the antibody targeting NKG2A is as set forth in SEQ ID NO: 9.

[0049] In other embodiments, the immunosuppressive molecule of the present application comprises a NKG2A binding domain that is a ligand binding to NKG2A, such as HLA-E or a functional fragment thereof (i.e., a fragment having NKG2A binding function, such as the extracellular region of HLA-E). Preferably, the ligand binding to NKG2A comprises an amino acid that 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: 10 or 11. More preferably, the ligand binding to NKG2A comprises an amino acid sequence as set forth in SEQ ID NO: 10 or 11. In this embodiment, the transmembrane region of the immunosuppressive molecule can be the transmembrane region of HLA-E. But typically, the immunosuppressive molecule in this embodiment does not comprise the intracellular region of HLA-E. In other words, the immunosuppressive molecule is not full-length HLA-E.

[0050] Immune cells

[0051] The term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, an immune cell can be a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell. An immune cell can be obtained from a variety of sources, for example, it can be obtained from a subject (e.g., isolated from a subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, a tumor, etc.), or from an in vitro cultured cell line (e.g., Jurkat, SupTl, NK92, etc.), or differentiated from a stem cell (e.g., derived from umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSCs, etc.). Preferably, the immune cell is a T cell or an NK cell, more preferably a T cell. The T cell can be concentrated or purified. The T cell can be at any stage of development, including but not limited to: CD4+CD8+T cells, CD4+T cells (e.g., Thl and Th2 cells), CD8+T cells (e.g., cytotoxic T cells), CD4-CD8- T cells, tumor infiltrating cells, memory T cells, naive T cells, gamma delta-T cells, alpha beta-T cells, etc. Preferably, the immune cell is a human T cell, which can be obtained using a variety of techniques known to one of skill in the art, such as isolating T cells from a subject's blood using Ficoll.

[0052] In some embodiments, the immune cell is derived from a stem cell.

[0053] The term "stem cell" refers to a primitive cell with self-replicating, multi-directional differentiation, and homing potential, which is the origin cell of the body and is the progenitor cell that forms various tissues and organs of the human body. During the process of cell differentiation, due to the uneven distribution of regulatory differentiation proteins in the cytoplasm, one daughter cell irreversibly goes to the terminal differentiation of a functionally specific differentiated cell, until it completely loses the ability to divide and eventually dies of aging. In order to make up for this deficiency, the body retains a part of undifferentiated primitive cells, which retain the characteristics of the parent. This part of the retained undifferentiated primitive cells, called stem cells, can generate differentiated cells by division according to the developmental pathway if needed. The stem cells described in the present application can be embryonic stem cells, adult stem cells (e.g., umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells, etc.), and pluripotent stem cells (e.g., induced pluripotent stem cells iPSCs, etc.).

[0054] In some embodiments, the expression of the endogenous HLA-Class I genes and / or HLA-Class II genes of the engineered immune cells of the application is not modified. That is, the expression level of any one of the endogenous HLA-Class I genes and / or HLA-Class II genes is not altered by any means of artificial intervention (gene editing or non-gene editing).

[0055] In some embodiments, the expression of at least one endogenous HLA-Class I gene of the engineered immune cells of the application is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-Class II gene of the engineered immune cells of the application is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene of the engineered immune cells of the application is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene and at least one endogenous HLA-Class I gene of the engineered immune cells of the application is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-Class I and HLA-Class II gene of the engineered immune cells of the application is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene, at least one endogenous HLA-Class I gene and at least one endogenous HLA-Class II gene of the engineered immune cells of the application is suppressed or silenced. Preferably, the HLA-Class I gene is selected from the group consisting of HLA-A, HLA-B, HLA-C and B2M. Preferably, the HLA-Class II gene is selected from the group consisting of HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK and CIITA, preferably from RFX5, RFXAP, RFXANK and CIITA. Preferably, the TCR / CD3 gene is selected from the group consisting of TRAC, TRBC, CD3y, CD35, CD3s and CD3z.

[0056] In some embodiments, the engineered immune cell of the application has one or more of the expression of the 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, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3 is inhibited or silenced.

[0057] Methods of inhibiting gene expression or silencing a gene are well known to those skilled in the art and include, but are not limited to, inactivation of the gene by, for example, meganucleases, zinc-finger nucleases, TALENs, CRISPR / Cas systems, base editors mediated DNA or RNA break, or by antisense oligonucleotides, RNAi, shRNA, transposons, mutations, and the like.

[0058] In some embodiments, the engineered immune cell of the application is an allogeneic cell. As used herein, the term “allogeneic” refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to one another when they differ in their genes at one or more loci. In some cases, allogeneic material from various individuals of the same species can differ in their genes enough to have an antigenic interaction.

[0059] Pharmaceutical composition

[0060] The present application also provides a pharmaceutical composition comprising the engineered immune cell of the application as an active agent, and one or more pharmaceutically acceptable excipients. Accordingly, the present application also encompasses the use of the engineered immune cell for the preparation of a pharmaceutical composition or a medicament.

[0061] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible, in pharmacology and / or physiology, with the subject and the active ingredient (i.e., capable of producing the desired therapeutic effect without causing any undesirable local or systemic effects) and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, encapsulating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of a suitable excipient to prepare the desired pharmaceutical composition of the present application is known to one skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present application include saline, buffered saline, dextrose, and water. Generally, the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0062] The pharmaceutical compositions according to the present application can be suitable for administration by a variety of routes. 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.

[0063] The pharmaceutical compositions according to the present application 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 fluidextracts, or in a form particularly suitable for the desired method of administration. Processes known to the present application for the production of medicaments can include, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions comprising, for example, the immune cells described herein are typically provided in solution form, and preferably comprise a pharmaceutically acceptable buffer.

[0064] The pharmaceutical composition according to the present application can also be administered in combination with one or more other agents (biological agents such as antibody reagents, and / or small molecules) or therapeutic methods (e.g., surgery, chemotherapy, or radiation therapy) that are useful in the treatment and / or prevention of 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, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225, and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulatory protein, and the like; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. In addition, the pharmaceutical composition of the present application can also be used in combination with other therapeutic method(s), e.g., chemotherapy, radiation therapy.

[0065] Uses, methods of treatment

[0066] The present application also provides uses of the pharmaceutical composition or engineered immune cells as described above in the manufacture of a medicament for the treatment of cancer, infection, or autoimmune disease, and methods for treating a subject having cancer, infection, or autoimmune disease.

[0067] In some embodiments, the cancer is a cancer associated with expression of a target that binds to a functional exogenous receptor. For example, 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, cancer of the peritoneum, cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), hepatic carcinoma, hepatoma, intra-epithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (e.g., small cell lung carcinoma, non-small cell lung carcinoma, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoma (including Hodgkin's and non-Hodgkin's lymphoma), melanoma, myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland carcinoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, cancer of the vulva, as well as other carcinomas and sarcomas, and B-cell lymphoma (including low grade / follicular non-Hodgkin's 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, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's 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's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative conditions, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, plasmablastic lymphoma, pre-leukemia, plasmacytoid dendritic cell neoplasm, and post-transplant lymphocyte

[0068] In some embodiments, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0069] 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, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, systemic lupus erythematosus, and the like.

[0070] The present application will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and examples of the present application are only for the purpose of illustration and should not be construed as any limitation to the present application. The embodiments of the present application and the features in the embodiments can be combined with each other without contradiction. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1: scFv expression level in CAR19 T and CAR19-FK KO T cells.

[0072] Figure 2: FKBP12 knockout effect of CAR19-FK KO T cells.

[0073] Figure 3: In vitro killing effect of CAR19 T and CAR19-FK KO T cells on target cells.

[0074] Figure 4: Cytokine release level after co-incubation of CAR19 T and CAR19-FK KO T cells with target cells.

[0075] Figure 5: scFv expression level in CAR19PA T and CAR19PA-FK KO T cells.

[0076] Figure 6: PDL1 expression level in CAR19PA T and CAR19PA-FK KO T cells.

[0077] Figure 7: FKBP12 knockout effect of CAR19PA-FK KO T cells.

[0078] Figure 8: In vitro killing effect of CAR19PA T and CAR19PA-FK KO T cells on target cells.

[0079] Figure 9: Cytokine release level after co-incubation of CAR19PA T and CAR19PA-FK KO T cells with target cells.

[0080] Figure 10: scFv and VHH expression level in CAR19 / B_P T and CAR19 / B_P-FK KO T cells.

[0081] Figure 11: PDL1 expression level in CAR19 / B_P T and CAR19 / B_P-FK KO T cells.

[0082] Figure 12: FKBP12 knockout effect of CAR19 / B_P-FK KO T cells.

[0083] Figure 13: In vitro killing effect of CAR19 / B_P T and CAR19 / B_P-FK KO T cells on target cells.

[0084] Figure 14: Cytokine release levels after co-incubation of CAR19 / B_P T and CAR19 / B_P-FK KO T cells with target cells.

[0085] Figure 15: scFv expression levels in CARB_P T and CARB_P-FK KO T cells.

[0086] Figure 16: PDL1 expression levels in CARB_P T and CARB_P-FK KO T cells.

[0087] Figure 17: FKBP12 knockout effect of CARB_P-FK KO T cells.

[0088] Figure 18: Cytokine release levels after co-incubation of CARB_P T and CARB_P-FK KO T cells with target cells. DETAILED DESCRIPTION

[0089] Example 1: Construction of anti-CD19 CAR-T cells with FKBP12 knockout and verification of their functions

[0090] 1.1 Construction of anti-CD19 CAR-T cells with FKBP12 knockout

[0091] The following coding sequences were synthesized and cloned into pGEM-T Easy vector (Promega) in turn: anti-CD19 scFv (SEQ ID NO: 49), CD8a hinge region (SEQ ID NO: 29), CD8a transmembrane region (SEQ ID NO: 27), 4-1BB costimulatory domain (SEQ ID NO: 34), CD3 zeta intracellular region (SEQ ID NO: 36), to obtain CAR19 plasmid, and the correct insertion of target sequences in the plasmid was confirmed by sequencing.

[0092] In a sterile tube, add 3ml Opti-MEM (Gibco) to dilute the CAR19 plasmid, then add the packaging vector psPAX2 (Addgene) and the envelope vector pMD2.G (Addgene) according to the ratio of plasmid: virus packaging vector: virus envelope vector = 4:2:1. Then, add 120μl X-treme GENE HP DNA transfection reagent (Roche), mix immediately, incubate at room temperature for 15min, then add the plasmid / vector / transfection reagent mixture dropwise to the culture bottle of 293T cells. Collect the virus at 24h and 48h, combine them, and obtain concentrated lentivirus by ultracentrifugation (25000g, 4℃, 2.5h).

[0093] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, Cat No.40203D) and cultured at 37℃ and 5%CO2 for 1 day. Then, add the concentrated lentivirus and continue to culture for 1 day. sgRNA (SEQ ID NO:53) was used for electroporation to knock out FKBP12, and the anti-CD19 CAR-T cells with FKBP12 knockout were numbered as CAR19-FK KO T. Unmodified wild-type T cells (NT) and anti-CD19 CAR-T cells without FKBP12 knockout (CAR19 T) were used as controls.

[0094] 1.2 Detection of scFv expression level in CAR-T cells and knockout effect of FKBP12

[0095] Flow cytometry was used to detect the expression of anti-CD19 scFv in CAR19 T and CAR19-FK KO T cells using FITC goat anti-human Fab (Thermo Fisher). As can be seen, the scFv in the CAR-T cells prepared in this embodiment can be effectively expressed (Figure 1).

[0096] After 7 days of culture at 37℃ and 5%CO2, sample the T cells in each group and send for sequencing to detect the knockout of FKBP12. From the sequencing data (Figure 2), it can be seen that FKBP12 in CAR19-FK KO T cells is effectively knocked out.

[0097] 1.3 Detection of in vitro killing effect of CAR-T cells on target cells

[0098] 1×10 4The target cells (Raji cells) expressing luciferase gene were plated in 96-well plates at a concentration of 1 cell / well, and then the NT cells and each CAR-T cell were plated in 96-well plates at an effector-to-target ratio (i.e. the ratio of effector T cells to target cells) of 0.625:1, 1.25:1, 2.5:1, 5:1, and 10:1, respectively, for co-culture. After 48 hours, the Raji group fluorescence value was determined using a microplate reader. According to the calculation formula: (target cell fluorescence mean value-sample fluorescence mean value) / target cell fluorescence mean value x 100%, the killing efficiency was calculated, and the results are shown in Figure 3.

[0099] As can be seen, under various effector-to-target ratios, each CAR-T cell of the application showed strong killing effect on target cells (Nalm6 cells and Raji cells), and knocking out FKBP12 had no significant adverse effect on the in vitro killing ability of CAR-T cells.

[0100] 1.4 Detection of cytokine release level of CAR-T cells

[0101] CAR-T cells were treated with FK506 at a final concentration of 100 nM 48 h in advance, and then centrifuged and replaced with plating. On the plating day, 1 x 10 5 Target cells (Raji cells) were plated in 96-well plates at a concentration of 1 cell / well, and each CAR-T cell and NT cell was added at a ratio of 1:1, and FK506 was added at a final concentration of 100 nM at the same time for co-culture for 18-24 hours, and then the co-culture supernatant was collected.

[0102] According to the manufacturer's recommendations, Human IL-2 DuoSet ELISA Kit (R&D systems, Cat. No. DY202) and Human IFN-gamma DuoSet ELISA Kit (R&D systems, Cat. No. DY285) were used to detect the content of IL2 and IFN-gamma in the co-culture supernatant, and the results are shown in Figure 4.

[0103] As can be seen, after the addition of FK506, the release levels of IL2 and IFN-gamma were significantly reduced after the co-culture of CAR19 T cells without knocking out FKBP12 and each target cell; while the release levels of IL2 and IFN-gamma were restored after the co-culture of CAR19 T cells with knocking out FKBP12 and each target cell, indicating that after the addition of FK506, CAR-T cells with knocking out FKBP12 can effectively resist the functional inhibition of FK506 on CAR-T. In addition, knocking out FKBP12 has no adverse effect on the specific cytokine release ability of CAR-T cells, on the contrary, it is a promoting effect in most cases.

[0104] Example 2: Construction of anti-CD19 CAR-T cells with knock-out of FKBP12 and expression of immunosuppressive molecules and verification of their functions

[0105] 2.1 Construction of anti-CD19 CAR-T cells with knock-out of FKBP12

[0106] In the CAR19 plasmid prepared in 1.1, further include PD1 binding molecule and NKG2A binding molecule (connected by 2A peptide), the structure of PD1 binding molecule is as follows: PD-L1 signal peptide (SEQ ID NO: 54), PD-L1 extracellular domain (SEQ ID NO: 22), PD-L1 transmembrane domain (SEQ ID NO: 24), the structure of NKG2A binding molecule is as follows: B2M signal peptide (SEQ ID NO: 38), NKG2A binding domain (SEQ ID NO: 9), IgG4 hinge region (SEQ ID NO: 32), CD28 transmembrane region (SEQ ID NO: 26), CD28 costimulatory domain (SEQ ID NO: 33), obtain CAR19-PA plasmid, and confirm the correct insertion of target sequence in the plasmid by sequencing.

[0107] According to the method in Example 1.1, the CAR19-PA plasmid is transfected into T cells to obtain anti-CD19 CAR-T cells with knock-out of FKBP12 and expression of PD1 binding molecule and NKG2A binding molecule, numbered as CAR19PA-FK KO T. Unmodified wild type T cells (NT), anti-CD19 CAR-T cells without knock-out of FKBP12 and expression of PD1 binding molecule and NKG2A binding molecule (CAR19PA T) are used as controls.

[0108] 2.2 Detection of scFv expression level and FKBP12 knock-out level in each CAR-T cell

[0109] Using flow cytometry, FITC goat anti-human Fab (Thermo Fisher) is used to detect the expression of anti-CD19 scFv in CAR19PA T and CAR19PA-FK KO T cells. As can be seen, the scFv in the CAR-T cells prepared in this embodiment can be effectively expressed (Figure 5).

[0110] Using flow cytometry, PD1 protein (ACRO) is used to detect the expression of PDL1 in CAR19PA T and CAR19PA-FK KO T cells. As can be seen, PDL1 in CAR19PA T and CAR19PA-FK KO T cells can be effectively expressed (Figure 6).

[0111] After culturing for 7 days at 37°C and 5% CO2, the T cells in each group were sampled and sent for sequencing to detect the knockout of FKBP12. As can be seen from the sequencing data (Figure 7), FKBP12 in CAR19PA-FK KO T cells was effectively knocked out.

[0112] 2.3 Detection of in vitro killing effect of CAR-T cells on target cells

[0113] The in vitro killing effect of CAR-T cells on target cells was detected according to the method in Example 1.3, and the results are shown in Figure 8.

[0114] As can be seen, under various effector-to-target ratios, each CAR-T cell of the application showed strong killing effect on target cells (Raji cells), and knocking out FKBP12 had no significant adverse effect on the in vitro killing ability of CAR-T cells.

[0115] 2.4 Detection of cytokine release level of CAR-T cells

[0116] The cytokine release level after co-culturing CAR-T cells with target cells (Raji cells) was detected according to the method in Example 1.4, and the results are shown in Figure 9.

[0117] As can be seen, after adding FK506, the release levels of IL2 and IFN-γ after co-culturing CAR19PA T cells without knocking out FKBP12 with each target cell were significantly reduced; while the release levels of IL2 and IFN-γ after co-culturing CAR19PA T cells with each target cell were restored after knocking out FKBP12, indicating that after adding FK506, knocking out FKBP12 can effectively resist the functional inhibition of FK506 on CAR19PA T cells. In addition, knocking out FKBP12 had no adverse effect on the specific cytokine release ability of CAR19PA T cells.

[0118] Example 3: Construction of anti-CD19 / BCMA CAR-T cells knocking out FKBP12 and expressing immunosuppressive molecules and verification of their functions

[0119] 3.1 Construction of anti-CD19 / BCMA CAR-T cells knocking out FKBP12

[0120] The following coding sequence was synthesized and cloned into pGEM-T Easy vector (Promega) in turn: anti-BCMA VHH (SEQ ID NO: 75), linker (SEQ ID NO: 51), anti-CD19 scFv (SEQ ID NO: 66), CD8a hinge region (SEQ ID NO: 29), CD8a transmembrane region (SEQ ID NO: 27), 4-1BB costimulatory domain (SEQ ID NO: 34), CD3 zeta intracellular region (SEQ ID NO: 36), to obtain CAR19 / B plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0121] The PD1 binding molecule (connected by 2A peptide) was further contained in the CAR19 / B plasmid prepared in 3.1, and the structure of the PD1 binding molecule was as follows: PD-L1 signal peptide (SEQ ID NO: 54), PD-L1 extracellular domain (SEQ ID NO: 22), PD-L1 transmembrane domain (SEQ ID NO: 24), CD28 costimulatory domain (SEQ ID NO: 33), to obtain CAR19 / B-P plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0122] The CAR19 / B-P plasmid was transformed into T cells according to the method in Example 1.1, to obtain anti-CD19 / BCMA CAR-T cells knocking out FKBP12 and expressing PD1 binding molecules, numbered as CAR19 / B_P-FK KO T. Unmodified wild-type T cells (NT) and anti-CD19 / BCMA CAR-T cells not knocking out FKBP12 and expressing PD1 binding molecules (CAR19 / B_P T) were used as controls.

[0123] 3.2 Detection of scFv expression level and FKBP12 knockout level in each CAR-T cell

[0124] FITC goat anti-human Fab (Thermo Fisher) and MonoRab TM Rabbit Anti-Camelid VHH Cocktail[iFluor 488] (KPL, Cat No: A02021) was used to detect the expression of anti-CD19 scFv and anti-BCMA VHH in CAR19 / B_P T and CAR19 / B_P-FK KO T cells, respectively. As can be seen, the scFv and VHH in the CAR-T cells prepared in this example can be effectively expressed (Figure 10).

[0125] The expression of PDL1 in CAR19 / B_P T and CAR19 / B_P-FK KO T cells was detected using a flow cytometer with PD1 protein (ACRO). As can be seen, PDL1 in CAR19 / B_P T and CAR19 / B_P-FK KO T cells can be effectively expressed (Figure 11).

[0126] After 7 days of culture at 37°C and 5% CO2, the T cells in each group were sampled and sequenced to detect the knockout of FKBP12. As can be seen from the sequencing data (Figure 12), FKBP12 in CAR19 / B_P-FK KO T cells was effectively knocked out.

[0127] 3.3 Detection of in vitro killing effect of CAR-T cells on target cells

[0128] The in vitro killing effect of CAR-T cells on target cells (Nalm6, Raji and RPMI8226 cells) was detected according to the method in Example 1.3, and the results are shown in Figure 13.

[0129] As can be seen, under various effector-to-target ratios, each CAR-T cell of the application showed strong killing effect on target cells (Nalm6, Raji and RPMI8226 cells), and knocking out FKBP12 had no significant adverse effect on the in vitro killing ability of CAR-T cells.

[0130] 3.4 Detection of cytokine release level of CAR-T cells

[0131] The cytokine release level after co-culture of CAR-T cells with target cells (Raji cells and RPMI8226 cells) was detected according to the method in Example 1.4, and the results are shown in Figure 14.

[0132] As can be seen, after co-culture of CAR19 / B_P T cells without knocking out FKBP12 with each target cell, the release levels of IL2 and IFN-γ were significantly reduced after the addition of FK506; while after co-culture of CAR19 / B_P-FK KO cells with each target cell, the release levels of IL2 and IFN-γ were restored, indicating that after the addition of FK506, knocking out FKBP12 can effectively resist the functional inhibition of FK506 on CAR19 / B_P T cells. In addition, knocking out FKBP12 had no adverse effect on the specific cytokine release ability of CAR19 / B_P T.

[0133] Example 4: Construction of anti-BCMA CAR-T cells with FKBP12 knockout and expression of immunosuppressive molecules and verification of their functions

[0134] 4.1 Construction of anti-BCMA CAR-T cells with FKBP12 knock-out

[0135] The following coding sequences were synthesized and cloned into pGEM-T Easy vector (Promega) in turn: anti-BCMA VHH (SEQ ID NO: 81), CD8a hinge region (SEQ ID NO: 29), CD8a transmembrane region (SEQ ID NO: 27), 4-1BB costimulatory domain (SEQ ID NO: 34), CD3 zeta intracellular region (SEQ ID NO: 36), to obtain CARB plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0136] The PD1 binding molecule (connected by 2A peptide) was further contained in the CARB plasmid prepared in 3.1, and the structure of the PD1 binding molecule was as follows: PD-L1 signal peptide (SEQ ID NO: 54), PD-L1 extracellular domain (SEQ ID NO: 22), PD-L1 transmembrane domain (SEQ ID NO: 24), CD28 costimulatory domain (SEQ ID NO: 33), to obtain CARB-P plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0137] The CARB-P plasmid was transformed into T cells according to the method in Example 1.1, to obtain anti-BCMA CAR-T cells with FKBP12 knock-out and expressing PD1 binding molecules, numbered as CARB_P-FK KO T. Unmodified wild-type T cells (NT) and anti-BCMA CAR-T cells without FKBP12 knock-out and expressing PD1 binding molecules (CARB_P T) were used as controls.

[0138] 4.2 Detection of the expression level of scFv and the FKBP12 knock-out level in each CAR-T cell

[0139] The expression of anti-BCMA VHH in CARB_P T and CARB_P-FK KO T cells was detected using flow cytometry with MonoRabTMRabbit Anti-Camelid VHH Cocktail[iFluor 488] (KPL, item number: A02021). As can be seen, the VHH in the CAR-T cells prepared in this example can be effectively expressed (Figure 15).

[0140] The expression of PDL1 in CARB_P T and CARB_P-FK KO T cells was detected using flow cytometry with PD1 protein (ACRO). As can be seen, PDL1 can be effectively expressed in CARB_P T and CARB_P-FK KO T cells (Figure 16).

[0141] After culturing for 7 days at 37℃ and 5% CO2, the T cells in each group were sampled and sequenced to detect the knockout of FKBP12. As can be seen from the sequencing data (Figure 17), FKBP12 in CARB_P-FK KO T cells was effectively knocked out.

[0142] 4.3 Detection of the cytokine release level of CAR-T cells

[0143] The cytokine release level of CAR-T cells after co-culturing with target cells (Raji cells and RPMI8226 cells) was detected according to the method in Example 1.4, and the results are shown in Figure 18.

[0144] As can be seen, after adding FK506, the release levels of IL2 and IFN-γ of CARB_P T cells without knocking out FKBP12 after co-culturing with each target cell were significantly reduced; and the release levels of IL2 and IFN-γ of CARB_P-FK KO cells with knocking out FKBP12 after co-culturing with each target cell were restored, indicating that after adding FK506, knocking out FKBP12 can effectively resist the functional inhibition of FK506 on CARB_P T cells. In addition, knocking out FKBP12 has no adverse effect on the specific cytokine release ability of CARB_P T cells.

[0145] In summary, knocking out FKBP12 can effectively resist the functional inhibition of FK506 on engineered immune cells expressing chimeric antigen receptors and / or immunosuppressive molecules, and has no significant adverse effect on the target cell killing effect and cytokine release level of engineered immune cells.

[0146] It should be noted that the above is only a preferred embodiment of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An engineered immune cell wherein the expression of FKBP12 is inhibited or silenced, and an exogenous chimeric antigen receptor and / or immunosuppressive molecule is expressed; the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, and a primary signal transduction domain; the immunosuppressive molecule comprises an immunosuppressive protein-binding domain and a transmembrane domain and does not contain a primary signal transduction domain.

2. The engineered immune cell according to claim 1, wherein the immunosuppressive protein is selected from PD1, NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPα, KIR2DL1 / 2 / 3, LILRB1, or a combination thereof.

3. The engineered immune cells according to claim 1 or 2, wherein the antigen specifically recognized by the chimeric antigen receptor is selected from: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, 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, Claudin 6, Claudin18.1, Claudin 18.2, 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, FB P, 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, 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 / galactoglobulin 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, UPK2VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, hepatocyte glycoside B2, tyrosinase, fucose GM1, o-acetyl-GD2, folate receptor β, polysialic acid, spermatin 17, survivin and telomerase, sarcoma translocation breakpoint, human telomere reverse transcriptase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tendinin, carcinoembryonic variants of tumor necrosis and any combination thereof.

4. The engineered immune cell according to any one of claims 1-3, wherein the antigen-binding domain or immunosuppressive protein domain is selected from intact antibodies, Fab, Fab', F(ab')2, Fd fragments, Fd', Fv fragments, scFv, sdFv, linear antibodies, dimers, sdAb, and functional fragments of ligands or receptors.

5. The engineered immune cell according to any one of claims 1-4, wherein the chimeric antigen receptor and / or immunosuppressive molecule further comprises a co-stimulatory domain.

6. The engineered immune cell according to any one of claims 1-5, wherein the co-stimulatory domain is selected from the intracellular regions 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, ZAP70, and any combination thereof.

7. The engineered immune cell according to any one of claims 1-6, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD28, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD112, CD113, CD134. , CD137, CD154, CD155, Nectin4, KIRDS2, OX40, CD2, CD27, CD18, ICOS, 4-1BB, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp 80. CD160, BCMA, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, LSECtin, PDL1, PDL2, CEACAM1, MHC class I molecules, MHC class II molecules, CRTAM, Ly9, CD160, PSGL1, CDIOO, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

8. The engineered immune cell according to any one of claims 1-7, wherein the primary signal transduction domain is selected from the intracellular regions of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2, and CD66d.

9. The engineered immune cell according to any one of claims 1-8, wherein the chimeric antigen receptor and / or immunosuppressive molecule further comprises a hinge region derived from the following proteins: CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1.

10. The engineered immune cells according to any one of claims 1-9, wherein the expression of one or more genes among endogenous HLA class I genes, HLA class II genes, and TCR / CD3 genes is suppressed or silenced in the engineered immune cells.

11. The engineered immune cells according to claim 10, wherein the HLA-I class genes are selected from HLA-A, HLA-B, HLA-C, B2M and any combination thereof; the HLA-II class genes are selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and any combination thereof; and the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and any combination thereof.

12. The engineered immune cells according to any one of claims 1-11, wherein the expression of one or more endogenous genes selected from the following is suppressed or silenced: 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.

13. The engineered immune cell according to any one of claims 1-12, wherein the immune cell is selected from B cells, T cells, macrophages, dendritic cells, monocytes, NK cells and NKT cells.

14. The engineered immune cell according to claim 13, wherein the immune 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.

15. The engineered immune cell according to claim 14, wherein the immune cell is derived from stem cells, and the stem cells are selected from embryonic stem cells, umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells and induced pluripotent stem cells.

16. A pharmaceutical composition comprising engineered immune cells as described in any one of claims 1-15, and one or more pharmaceutically acceptable excipients.

17. Use of the engineered immune cells according to any one of claims 1-15 or the pharmaceutical composition according to claim 16 in the preparation of a medicament for the prevention or treatment of cancer, infection or autoimmune diseases.

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