Immune cell combination therapy

Abstract

A combination of an immune cell that expresses a receptor that recognizes a tumor antigen and a benzamide-based HDAC inhibitor. The invention further discloses a kit containing the immune cells and the benzamide HDAC inhibitor and a method for combined treatment of diseases such as cancer.

Description

Immune cell combination therapy Technical Field

[0001] The present invention belongs to the technical field of tumor treatment and relates to immune cell combination therapy, and specifically to a combination comprising immune cells and benzamide HDAC inhibitors, a kit and a method for treating cancer and other diseases thereof. Background Art

[0002] In recent years, adoptive cell therapy, an emerging immunotherapy, has demonstrated significant advantages in the field of cancer treatment. This therapy typically requires the modification of cells, such as through gene editing and / or transduction, to carry exogenous proteins such as chimeric antigen receptors and recombinant T cell receptors. These cells are then expanded in vitro and infused back into the patient. While these therapies have demonstrated promising efficacy against hematologic malignancies, recurrence is still possible after treatment due to factors such as antigen escape.

[0003] Therefore, there is still a need to improve existing adoptive cell therapy to reduce antigen escape, prevent recurrence, and enhance anti-tumor effects.

[0004] Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a combination therapy of immune cells and benzamide HDAC inhibitors, which has a significantly enhanced tumor killing effect compared to the use of immune cells alone.

[0006] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In order to make this application easier to understand, certain terms are defined below.

[0007] Ⅰ. Combination

[0008] In a first aspect, the present invention provides a combination comprising: an immune cell expressing a receptor that recognizes a tumor antigen; and a benzamide HDAC inhibitor.

[0009] 1. Immune cells

[0010] 1.1 Tumor antigens

[0011] The term "tumor antigen" refers to an antigen that appears or is overexpressed in the body during the occurrence and development of a tumor. The tumor antigen described in the present invention can be a solid tumor antigen or a hematologic tumor antigen. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells, such as a lineage marker. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, for example, a molecule that contains deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a tumor antigen is a tumor-associated antigen, i.e., a cell surface molecule that is overexpressed in cancer cells compared to normal cells, for example, 0.5 times overexpression, 1 times overexpression, 1.5 times overexpression, 2 times overexpression, 2.5 times overexpression, 3 times overexpression or more compared to normal cells. In some embodiments, a tumor antigen is a tumor-specific antigen, i.e., expressed only completely or as a fragment (e.g., MHC / peptide) on the cell surface of cancer cells, and is not synthesized or expressed on the surface of normal cells.

[0012] In some embodiments, the tumor antigen 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, CD138, CD151, CD171, CD179a, CD300LF, CLEC12A, CDH16, CSPG4, CS-1, CLL1, Claudin6, Claudin18.1, Claudin18.2, CEA, CEACAM6, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2,EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, 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, 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 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, mesothelin, 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 variants of tumor necrosis, and any combination thereof. Preferably,The tumor antigen is selected from the group consisting of CD7, CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, Her2, MUC1, mesothelin, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, CLL1, NKG2D ligands, and any combination thereof, more preferably CD7, CLL1, NKG2D ligands, and any combination thereof. The NKG2D ligands described in the present invention are selected from the group consisting of MICA / B, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0013] 1.2 Receptors

[0014] In some embodiments, the receptor is selected from endogenous receptors, which are endogenous molecules in immune cells that are capable of binding to tumor antigens, such as NKG2D of NK cells.

[0015] In other embodiments, the receptor is selected from an exogenous receptor, which is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor fusion protein (TFP), a T cell antigen coupler (TAC) or an immune mobilizing monoclonal T cell receptor (ImmTAC), preferably a chimeric antigen receptor or a T cell receptor, and more preferably a chimeric antigen receptor.

[0016] 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.

[0017] The term “T cell antigen coupler” or “TAC” encompasses three functional domains: (1) a tumor targeting domain, which may consist of a single-chain antibody (scFv), a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a scFv 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.

[0018] 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 region 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.

[0019] 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.

[0020] The term "chimeric antigen receptor" or "CAR" is a recombinant polypeptide construct that, when expressed in immune cells, is capable of antigen recognition based on antigen binding specificity. When it binds to the corresponding tumor antigen, it can affect the tumor cells, causing them to not grow, die, or be affected in other ways, and resulting in a reduction or elimination of the patient's tumor burden. CARs typically contain at least an antigen binding domain, a transmembrane domain, and an intracellular domain, the intracellular domain comprising at least one costimulatory domain and / or at least one primary signaling domain.

[0021] In some embodiments, the exogenous receptor is a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises at least one costimulatory domain and / or at least one primary signaling domain.

[0022] The term "antigen-binding domain" refers to any structure (such as an antibody, ligand, or receptor, etc.) that can bind to an antigen, or a functional variant thereof.

[0023] 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 fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), linear antibodies, "diabodies" with two antigen-binding sites, single-domain antibodies (sdAb) (e.g., the heavy chain variable region VH, the light chain variable region VL, nanobodies VHH, etc. of an antibody).

[0024] 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. For example, known NKG2D ligands include MICA / B, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0025] Unless the context clearly indicates otherwise, the "antigen-binding domain" of the present invention encompasses antibodies, ligands, 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 scFv or sdAbs.

[0026] 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 sequence. For example, a functional fragment of a ligand or receptor in the present invention generally refers to a fragment of the ligand or receptor that is capable of binding to the corresponding antigen, such as the extracellular region.

[0027] 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 (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes. The term "complementarity determining region" or "CDR" refers to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. For example, generally, there are three CDRs (e.g., CDR1-H, CDR2-H, and CDR3-H) in each heavy chain variable region, and three CDRs (e.g., CDR1-L, CDR2-L, and CDR3-L) in each light chain variable region. The precise amino acid sequence boundaries of the CDRs 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, and the AHo numbering scheme. The CDR sequences provided herein are determined using the Chothia numbering scheme.

[0028] 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 the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise indicated, the scFv herein may have the VL and VH variable regions in any order, for example, the scFv may comprise VL-linker-VH or VH-linker-VL from N-terminus to C-terminus. The term "linker" refers to a molecular sequence 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. 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 GS-rich sequence) or a non-peptide chemical linker, such as the amino acid sequence shown in SEQ ID NO: 42 or 43.

[0029] 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.

[0030] The choice of antigen binding domain depends on the cell surface marker on the target cell associated with the particular disease state to be recognized, such as a "tumor antigen" as defined above.

[0031] In some embodiments, the receptor for recognizing a tumor antigen described in the present invention comprises a CD7 binding domain, which is an antibody, ligand, or functional fragment thereof that targets CD7. CD7 is a cell surface glycoprotein with a molecular weight of approximately 40 kDa and is a member of the immunoglobulin superfamily. CD7 is expressed in most T cells, NK cells, myeloid cells, T cell acute lymphoblastic leukemia / lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. It is reported that the CD7 molecule acts as a co-stimulatory signal during T cell activation by binding to its ligand K12 / SECTM1. In addition, it is reported that the destruction of the CD7 molecule in mouse T progenitor cells still results in normal T cell development and homeostasis, indicating that CD7 does not seem to have a critical impact on the development and function of T cells, making it a very suitable therapeutic target for the treatment of T cell acute lymphoblastic leukemia (T-ALL).

[0032] In some embodiments, the CD7 binding domain is selected from an antibody targeting CD7. Anti-CD7 antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting CD7 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 8, 11, 14, 17 or 20, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 7, 10, 13, 16 or 19. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:4, CDR2-H as shown in SEQ ID NO:5, and CDR3-H as shown in SEQ ID NO:6; the light chain variable region comprises CDR1-L as shown in SEQ ID NO:1, CDR2-L as shown in SEQ ID NO:2, and CDR3-L as shown in SEQ ID NO:3.

[0033] In some embodiments, the antibody targeting CD7 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: 8, 11, 14, 17 or 20, 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: 7, 10, 13, 16 or 19. Preferably, the antibody targeting CD7 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 8, 11, 14, 17 or 20 and a light chain variable region as set forth in SEQ ID NO: 7, 10, 13, 16 or 19.

[0034] In some embodiments, the antibody targeting CD7 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, 12, 15, 18 or 21. Preferably, the antibody targeting CD7 is as shown in SEQ ID NO: 9, 12, 15, 18 or 21.

[0035] In some embodiments, the receptor for recognizing a tumor antigen described in the present invention comprises a CLL1 binding domain, which is an antibody, ligand, or a functional fragment thereof that targets CLL1. CLL1, i.e., C-type lectin-like 1, also known as MICL, CLEC12A, CLEC-1, DCAL-2, and CD371, is a glycoprotein receptor and a member of the C-type lectin-like receptor family involved in immune regulation. CLL1 is expressed in hematopoietic cells, primarily in innate immune cells and bone marrow progenitor cells including monocytes, dendritic cells, and granulocytes. CLL1 is also expressed in acute myeloid leukemia (AML) blasts and leukemia stem cells (e.g., CD34+ / CD38- stem cells). Expression of CLL1 may also be associated with other myeloid leukemias, such as acute monocytic leukemia, acute promyelocytic leukemia, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS).

[0036] In some embodiments, the CLL1 binding domain is selected from an antibody targeting CLL1. Any anti-CLL1 antibody known in the art can be used in the present invention. In some embodiments, the antibody targeting CLL1 is selected from a single-domain antibody, wherein the CDR1, CDR2, and CDR3 contained in the single-domain antibody are the same as the CDR1, CDR2, and CDR3 contained in SEQ ID NO: 25. In some embodiments, the CDR1 is as shown in SEQ ID NO: 22, the CDR2 is as shown in SEQ ID NO: 23, and the CDR3 is as shown in SEQ ID NO: 24.

[0037] In some embodiments, the antibody targeting CLL1 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: 25. Preferably, the antibody targeting CLL1 of the present invention has the amino acid sequence shown in SEQ ID NO: 25.

[0038] In some embodiments, the receptor described in the present invention comprises an NKG2D ligand binding domain, which is an antibody, NKG2D, or a functional fragment thereof that targets an NKG2D ligand. NKG2D is a type II transmembrane glycoprotein that is expressed on the cell surface in the form of a homodimer formed by disulfide bonds. The extracellular region contains a C-type lectin-like region and is therefore commonly referred to as a C-type lectin-like receptor homodimer. Almost all NK cells (including humans and mice) express NKG2D on their surfaces. Human CD8+ T cells naturally express NKG2D, and some subsets of NKT cells, γδT cells, and CD4+ T cells also express NKG2D. Human NKG2D is highly conserved, and its ligands mainly include the histocompatibility complex class I chain-related protein A / B (MICA / B) and human cytomegalovirus UL16 binding protein (ULBPs, ULBP-1~6). It is generally believed that the expression of NKG2D ligands is an indicator of cellular "stress," such as during viral infection and malignant transformation, but rarely appears on the surface of healthy cells. NKG2D ligands are expressed to varying degrees on tumor cells derived from almost all human tissues. MICA is expressed in many epithelial cancer cells, malignant melanoma cells, and various leukemia cell types. Furthermore, MICA and MICB are expressed in primary hepatocellular carcinoma and are associated with the sensitivity of liver cancer cells to NK cells. ULBP molecules are expressed in tumor cells derived from primary leukemia, malignant melanoma, and glioma. Because NKG2D can recognize corresponding ligands on tumor cells, and the expression of NKG2D ligands is a specific change in tumor cells during tumor development, NKG2D ligands provide a more precise target for tumor immunotherapy and offer insights into the development and application of new immunotherapeutic approaches.

[0039] In one specific embodiment, the NKG2D or a functional fragment thereof is contained in an endogenous receptor of the immune cell of the present invention. In another specific embodiment, the antibody targeting the NKG2D ligand, NKG2D or a functional fragment thereof is contained in an exogenous receptor of the immune cell of the present invention.

[0040] The term "transmembrane domain" or "transmembrane region" refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of immune cells (e.g., T cells, NK cells, or NKT cells) and guides immune cells to produce a cellular response against target cells. The transmembrane domain can be natural or synthetic, or it can be derived from any membrane-bound protein or transmembrane protein. When the chimeric receptor polypeptide binds to the target antigen, the transmembrane domain can perform signal transduction. The antigen binding domain and the transmembrane domain can be connected by a hinge region or a linker. In some embodiments, the transmembrane domain is selected from the transmembrane domain of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8α, CD4, CD28 and CD278. Preferably, the transmembrane domain is derived from the CD8α chain and has at least 80%, 85%, 90%, 95% or 99% identity or 100% identity with the amino acid sequence shown in SEQ ID NO: 27 or 28 (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity or 100% identity).

[0041] The term "hinge region" refers to the segment between the antigen binding domain and the transmembrane domain. In some embodiments, the hinge region can be used to provide flexibility to the antigen binding domain (e.g., scFv). In some embodiments, for example, when the antibody for detecting scFv is ineffective or unavailable, the hinge region can be used to detect the expression of CAR on the cell surface. In some cases, the hinge region is derived from an immunoglobulin molecule and needs to be optimized according to the position of the epitope on the target, such as the hinge region of an IgG4 molecule. In some cases, the hinge does not belong to an immunoglobulin molecule, but instead belongs to another molecule, such as the hinge region of a CD8α molecule. The CD8α hinge region may contain cysteine ​​and proline residues, which can play a role in the interaction between CD8 co-receptors and MHC molecules. In some embodiments, cysteine ​​and proline residues can affect the performance of CAR and can therefore be engineered to affect the performance of CAR. In some embodiments, the chimeric antigen receptor further comprises a hinge region present between the antigen binding domain and the transmembrane domain. In some embodiments, the hinge region is derived from the hinge region of the following proteins: CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1. Alternatively, the amino acid sequence of the hinge region is at least 80%, 85%, 90%, 95% or 99% identical or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 35-38 (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or 100% identical). In some embodiments, the hinge region consists of the amino acid sequence shown in any one of SEQ ID NOs: 35-38.

[0042] The term "intracellular domain" or "intracellular region" refers to a portion of a protein that transduces immune effector function signals and directs cells to perform specific functions. The intracellular region can be the full-length sequence of the signal intracellular region, but in many cases it is not necessary to use the full-length sequence, and a truncated portion thereof that has the function of transducing immune effector function signals can be used to replace the full-length sequence. Therefore, the intracellular region can be a full-length or truncated portion of the intracellular portion of the signal molecule that is sufficient to transduce the immune effector function signal. In one aspect, the intracellular domain comprises at least one costimulatory domain and / or at least one primary signaling domain. The term "primary signaling domain" can be an intracellular functional signaling domain from a molecule responsible for primary stimulation or antigen-dependent stimulation, which can include an immunoreceptor tyrosine-based activation motif (ITAM). The term "costimulatory domain" can be an intracellular functional signaling domain from a costimulatory molecule, which can include the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. The term "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell.

[0043] In some embodiments, the primary signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the primary signaling domain comprises the signaling domain of CD3ζ. More preferably, the primary signaling domain is at least 80%, 85%, 90%, 95% or 99% identical or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 32-34 (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or 100% identical).

[0044] In some embodiments, the costimulatory domain is one or more costimulatory signaling domains selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and any combination thereof. Preferably, the costimulatory domain is a costimulatory signaling domain of CD27, CD28, CD134, CD137 or CD278 or a combination thereof. Preferably, the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40 and any combination thereof. More preferably, the costimulatory domain has at least 80%, 85%, 90%, 95% or 99% identity or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 29-31 (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity or 100% identity).

[0045] In some embodiments, the chimeric antigen receptor comprises: (a) a 4-1BB costimulatory domain and a CD3ζ primary signaling domain, (b) a CD27 costimulatory domain and a CD3ζ primary signaling domain, (c) a CD28 costimulatory domain and a CD3ζ primary signaling domain, (d) an OX40 costimulatory domain and a CD3ζ primary signaling domain, (e) a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain, (f) an OX40 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ primary signaling domain, or (g) a CD28 costimulatory domain, an OX40 costimulatory domain, and a CD3ζ primary signaling domain.

[0046] The term "signal peptide" can allow the nascent protein to be directed to the endoplasmic reticulum and subsequently to the cell surface when the chimeric antigen receptor is expressed in a cell, such as a T cell. The core of the signal peptide can 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. In some embodiments, the chimeric antigen receptor also comprises a signal peptide, and the signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from CD8α, IgG1, GM-CSFRα, etc. In some embodiments, the signal peptide of the present invention has at least 80%, 85%, 90%, 95% or 99% identity or 100% identity to the amino acid sequence of any one of SEQ ID NOs:39-41 (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity or 100% identity).

[0047] The term "identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment can be achieved in a variety of ways within the skill of the art using publicly available calculator software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software, to determine the percentage of amino acid sequence identity. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full-length sequences being compared.

[0048] The term "variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant. The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence, and capable of having one or more activities of the reference amino acid sequence.

[0049] 1.3 Cells

[0050] The term "immune cell" or "immune effector cell" refers to a cell that participates in an immune response (e.g., promotes an immune effector response). Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, monocytes, macrophages, neutrophils, dendritic cells, etc.

[0051] In some embodiments, the immune cells described herein express endogenous receptors that recognize tumor antigens, for example, NK cells express NKG2D that recognize NKG2D ligands (MICA / B and ULBPs).

[0052] In some embodiments, the immune cells described herein express an exogenous receptor as defined above that recognizes a tumor antigen, for example, the immune cells express a chimeric antigen receptor that recognizes CD7 or CLL1.

[0053] In some embodiments, the immune cell is a primary cell or derived from a stem cell. Immune cells can be obtained from a variety of sources, such as from a subject (e.g., from a subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, tumor, etc.), or from a cell line cultured in vitro (e.g., Jurkat, SupT1, NK92, etc.), or differentiated from stem cells, such as embryonic stem cells, adult stem cells (e.g., 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 iPSC, etc.).

[0054] In some embodiments, the immune cells are selected from T cells, NK cells, NKT cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, or any combination thereof. Preferably, the T cells are CD4+ / CD8+ T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, or αβ-T cells, or any combination thereof.

[0055] In some embodiments, T cells can be derived from primary T cells, T cell lines, etc. The primary T cells can be derived from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors of a subject (such as a human, mouse or rat, cat, dog, cow, horse, sheep, goat, or other livestock); and the T cell line is selected from Jurkat, H9, SupT1, etc.

[0056] In some embodiments, the NK cells can be derived from primary NK cells, stem cell-derived NK cells, NK cell lines, etc. Wherein, the primary NK cells can be derived from a subject (such as a human, mouse or rat, cat, dog, cow, horse, sheep, goat or other livestock, etc.), and are selected from peripheral blood NK cells, umbilical cord blood NK cells, spleen NK cells, etc.; the stem cell-derived NK cells can be selected from NK cells derived from pluripotent stem cells (IPSC), NK cells derived from embryonic stem cells (ESC), NK cells derived from hematopoietic stem cells (HSPC), etc.; the NK cell line is selected from NK92, NK92.26.5, NK92.MI, NK92Ci, NK92Fc, NK3.3, NKL, NKG, NK-YT, NK-YTS, KHYG-1, HATAK cells, etc.

[0057] The term "subject" can be a mammal in need of treatment, such as a human or veterinary patient (e.g., a rodent such as a mouse or rat, cat, dog, cow, horse, sheep, goat or other livestock). In some embodiments, a "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, etc. The subject may be suspected of having a disease characterized by cell proliferation or have a risk of developing a disease characterized by cell proliferation, be diagnosed as having a disease characterized by cell proliferation, or be a control subject confirmed not to have a disease characterized by cell proliferation. Diagnostic methods for diseases characterized by cell proliferation and the clinical division of such diagnosis are known to those skilled in the art.

[0058] In some embodiments, the expression of endogenous HLA-I class genes and / or HLA-II class genes in the 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).

[0059] In some embodiments, the expression of at least one endogenous HLA-I class gene of the 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 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 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 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 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 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.

[0060] 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, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL 10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0061] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to, for example, DNA or RNA breakage mediated by large-range nucleases, zinc finger nucleases, TALENs, CRISPR / Cas systems, base editors, or gene inactivation by antisense oligonucleotides, RNAi, shRNA, transposons, mutations, and the like.

[0062] In some embodiments, the immune cells are autologous immune cells or allogeneic immune cells. The term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual. The term "xenogeneic" refers to a transplant derived from an animal of a different species. The term "allogeneic" refers to any material derived from a different animal of the same species as the individual that introduced the material. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some cases, allogeneic material from individuals of the same species can be sufficiently different genetically to interact antigenically.

[0063] The term "vector" is used as a medium nucleic acid molecule for transferring (exogenous) genetic material into a host cell, in which the nucleic acid molecule can, for example, be replicated and / or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a sequence at a specific targeting site. An "expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding chimeric antigen receptor polypeptides of the present invention) in a suitable host cell and for the translation of their mRNA. Suitable vectors that can be used for the present invention are known in the art, and many are commercially available. In some embodiments, the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses (RSV, polyoma viruses, and adeno-associated viruses (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors also typically contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is required), a selection marker, and a restriction enzyme cleavage site (such as a multiple cloning site MCS). The vector may additionally contain elements such as a promoter, a polyadenylic acid tail (polyA), a 3'UTR, an enhancer, a terminator, an insulator, an operator, a selection marker, a reporter gene, a targeting sequence, and / or a protein purification tag. In a specific embodiment, the vector is an in vitro transcribed vector.

[0064] Nucleic acid sequences encoding exogenous receptor polypeptides can be introduced into immune cells using conventional methods known in the art (e.g., by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. An example is RNA transfection, which is the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. This term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the transfer of nucleic acid molecules or polynucleotides mediated by viruses. Transfection of animal cells generally involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside. 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.

[0065] 2. Chidamide

[0066] Histone deacetylases (HDACs) are a class of proteases that play a crucial role in chromatin structural modification and gene expression regulation, modulating the expression of tumor suppressor genes and enhancing the activity of transcription factors. HDAC inhibitors, a new class of therapeutic agents, can be subdivided into structural classes: hydroxamic acids, cyclic peptides, carboxylic acids, benzamides, and electrophilic ketones. While HDAC inhibitors vary in structure, they generally contain three components: (1) an enzyme surface recognition region, which closely contacts residues at the edge of the enzyme pocket and includes various aromatic rings, fused rings, and heteroaromatic rings; (2) a linker region, composed of hydrophobic structural segments of a defined length, which effectively contacts the narrow pocket and includes linear aliphatic chains, trans-phenylpropene, and heteroaromatic rings; and (3) a zinc binding group (ZBG), which directly interacts with the zinc ion in the active site and forms hydrogen bonds with histidine and tyrosine residues. These groups include carboxyl groups, sulfhydryl groups, hydroxamic acid groups, benzamide groups, and trifluoromethylketone groups.

[0067] Zn-containing benzamide HDAC inhibitors 2+Benzamides, with a chelating group of benzamide, are generally less active than their hydroxamic acid counterparts. Benzamides are selective inhibitors, primarily inhibiting class I HDACs (including HDAC isoforms 1, 2, and 3) and some class IIa HDACs, with no inhibitory effect on class IIb HDACs. These compounds have attracted attention for their favorable human pharmacokinetic and pharmacodynamic properties, as well as their excellent in vitro and in vivo antitumor activity. Implanted benzamide HDAC inhibitors include CI-994, MS-275, MGCD0103, and chidamide.

[0068] Among them, Chidamide (trade name: Epidaza) was approved for global marketing in January 2015. Its indication is relapsed and refractory peripheral T-cell lymphoma. It is the world's first benzamide-type selective oral inhibitor of histone deacetylase approved for marketing. As a structural analogue of MS-275 (artificial benzamide derivative), Chidamide can target HDAC1, 2, 3, and 10. Its indications include (1) for patients with relapsed or refractory peripheral T-cell lymphoma (PTCL) who have received at least one systemic chemotherapy; (2) in combination with aromatase inhibitors for hormone receptor-positive, human epidermal growth factor receptor-2-negative, postmenopausal, and locally advanced or metastatic breast cancer that has relapsed or progressed after endocrine therapy. Chidamide has a stable structure, a long half-life, and relatively low cytotoxicity. It is an emerging anti-tumor drug.

[0069] The benzamide HDAC inhibitors described in the present invention are selected from CI-994, MS-275, MGCD0103, chidamide, and derivatives thereof. In some embodiments, the derivatives are selected from pharmaceutically acceptable salts, enantiomers, and crystalline forms of CI-994, MS-275, MGCD0103, or chidamide.

[0070] In some embodiments, the HDAC inhibitor is selected from chidamide and its derivatives; the derivatives of chidamide are selected from pharmaceutically acceptable salts, enantiomers, crystalline form A or crystalline form B thereof. The pharmaceutically acceptable salt of chidamide refers to the product of chidamide and at least one of the following acid addition salts, including but not limited to acetate, aspartate, benzoate, benzenesulfonate, bicarbonate, carbonate, bisulfate, sulfate, borate, camphorsulfonate, citrate, edisylate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, benzoate, hydrochloride, hydrobromide, iodate, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, hydrochloride, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, monohydrogen phosphate, dihydrogen phosphate, saccharate, stearate, succinate, tartrate, toluenesulfonate, trifluoroacetate. For crystal forms A and B of chidamide, see CN201210489178.8. For the enantiomers of Chidamide, see CN201410136761.X.

[0071] II. Reagent Kit

[0072] In a second aspect, the present invention provides a kit comprising the immune cells described above and a benzamide HDAC inhibitor.

[0073] The immune cells and benzamide HDAC inhibitors described in the present invention can be prepared separately or simultaneously into the same or different formulations, such as solid, liquid, gaseous or lyophilized formulations, and can particularly be 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 the formulations may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. The formulations comprising the immune cells described in the present invention are typically provided in the form of liquid formulations or lyophilized formulations.

[0074] In some embodiments, a pharmaceutically acceptable carrier may also be added to the formulation. In some embodiments, the pharmaceutically acceptable carrier may include a buffer (e.g., neutral buffered saline, sulfate buffered saline, etc.), a carbohydrate (glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid such as glycine; antioxidant), a chelating agent (e.g., EDTA or glutathione), an adjuvant (e.g., aluminum hydroxide), a preservative, or any combination thereof. The term "pharmaceutically acceptable carrier" refers to a carrier matrix or vehicle that does not interfere with the effectiveness of the biological activity of the active ingredient and does not produce toxicity to the host or subject.

[0075] III. Application and Treatment Methods

[0076] In a third aspect, the present invention also provides the use of the above combination or kit in the preparation of a drug for preventing or treating cancer, infection or autoimmune disease, and a method for treating a disease using the above combination or preparation.

[0077] In the present invention, the immune cells and the benzamide HDAC inhibitor are administered in no particular order; the benzamide HDAC inhibitor can be administered first and then the immune effector cells; they can also be administered simultaneously; the immune cells can also be administered first and then the benzamide HDAC inhibitor. In some embodiments, the immune cell therapy is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, before the administration of the benzamide HDAC inhibitor. In some embodiments, the immune cell therapy is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 day, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, after administration of the benzamide HDAC inhibitor.

[0078] The term "treat" refers to reducing or improving the progression, severity and / or duration of a proliferative disorder, or improving one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder, caused by the administration of one or more therapies (e.g., one or more therapeutic agents, such as the immune cells and benzamide HDAC inhibitors of the present invention). In some embodiments, the term "treat" refers to improving at least one measurable physical parameter of a proliferative disorder, such as the growth of a tumor, which is not necessarily discernible to the patient. In some embodiments, the term "treat" refers to physically, for example, by stabilizing a discernible symptom, or physiologically, for example, by stabilizing a physical parameter, or both, inhibiting the progression of a proliferative disorder. In some embodiments, the term "treat" refers to reducing or stabilizing tumor size or cancer cell count.

[0079] The term "prophylaxis" means preventative or protective treatment against a disease or disease state.

[0080] In some embodiments, the cancer is a solid tumor or a hematological tumor. More specifically, the cancer is selected from the group consisting of: 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, glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system, vulvar cancer and other cancers and sarcomas, with and B-cell lymphoma, 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's 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).

[0081] In some embodiments, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0082] 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.

[0083] The combination or formulation provided by the present invention can be administered in a manner suitable for the disease to be treated (or prevented), and the amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, and the results of clinical trials.

[0084] The administration of the combination or formulation of the present invention can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The combination or formulation provided by the present invention can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. Preferably, the oncolytic virus is administered by intratumoral injection or intravenous injection; the engineered immune cells are administered by intravenous injection.

[0085] The combinations or formulations provided herein can also be used in combination with surgery, chemotherapy, radiotherapy, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoadsorbents (such as CAMPATH, anti-CD3 antibodies or other antibody therapies), cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccines for the treatment of diseases.

[0086] In some embodiments, the combination or formulation provided by the present invention can be specifically used in combination with the following chemotherapeutic drugs: alkylating agents (such as uramustine, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphamide, temozolomide, thiotepa, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, oxaliplatin, dactinomycin, hexamethylmelamine, cisplatin, etc.), antimetabolites (such as folic acid antagonists, pyrimidines analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine), drugs that inhibit the calcium-dependent phosphatase calcineurin or p70S6 kinase FK506 or p70S6 kinase, mTOR inhibitors (e.g., temsirolimus, defotiolimus, everolimus, rapamycin, semamod, temsirolimus, inner salts, XL765), immunomodulators (e.g., atezolizumab, pegfilgrastim, lenalidomide, thalidomide, IRX-2 (including leukopenia),

[00135] The present invention also includes but is not limited to: a mixture of human cytokines such as interleukin 1, interleukin 2, and interferon gamma, etc.), anthracyclines (e.g., doxorubicin, bleomycin, daunorubicin, mitoxantrone, epirubicin, idarubicin, mitomycin C, geldanamycin, herbimycin, griseomycin, desacetyl griseomycin, etc.), vinca alkaloids (e.g., tartaric acid, vinorelbine, vincristine, vindesine, vinblastine, vinorelbine, etc.), proteasome inhibitors (e.g., bortezomib, carfilzomib, marezomib, ixazomib, citrate, delanzomib, etc.), GITR agonists (e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies)), protein tyrosine phosphatase inhibitors (e.g., SHP-1 inhibitors, SHP-2 inhibitors), CDK4 kinase inhibitors (e.g., palbociclib), BTK inhibitors (e.g., ibrutinib), MKN kinase inhibitors (e.g., crizotinib, ensartinib, etc.), DGK kinase inhibitors, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1: CD7 expression levels in KG1 and HL60 cells measured by flow cytometry after 48 h of CHI treatment.

[0088] Figure 2: MICA / B expression levels in KG1 and HL60 cells measured by flow cytometry after 48 h of CHI treatment.

[0089] Figure 3: ULBP1 expression levels in KG1 and HL60 cells measured by flow cytometry after 48 h of CHI treatment.

[0090] FIG4 : ULBP3 expression levels in KG1 and HL60 cells measured by flow cytometry after 48 h of CHI treatment.

[0091] FIG5 : CLL1 expression levels in KG1 cells measured by flow cytometry after 48 h of CHI treatment.

[0092] Figure 6: Expression levels of CD7-CAR T cell scFv determined by flow cytometry.

[0093] Figure 7: Expression levels of CLL1-CAR T cell VHHs determined by flow cytometry.

[0094] Figure 8: Cytotoxic effect of CD7-CAR T cells on CHI-treated and untreated HL60 cells.

[0095] Figure 9: Killing effect of CLL1-CAR T cells on CHI-treated and untreated KG1 cells.

[0096] Figure 10: Cytotoxic effect of NK92 cells on CHI-treated and untreated KG1 and HL60 cells.

[0097] Figure 11: IL-2 and IFNγ release levels of CLL1-CAR T cells after co-culture with CHI-treated and untreated KG1 cells, respectively.

[0098] Two-way ANOVA and T test were used for statistical analysis. * indicates a P value less than 0.05, reaching the significant level. DETAILED DESCRIPTION

[0099] The present invention will be further described below in conjunction with specific examples and accompanying drawings. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, where specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0100] Example 1: Effects of Chidamide Treatment on KG1 and HL60 Cells on Their Surface Antigens

[0101] 1.1 Preparation of Chidamide (CHI) Solution

[0102] Chidamide powder (Bibco Pharmaceuticals, Catalog No. BD449968) was pre-dissolved in DMSO (BBI, Catalog No. 67-68-5), and then dissolved in DPBS (hyclone, Catalog No. SH30028.02) to 100 μM as a stock solution. When used, it was further diluted with RPMI1640 medium (Gibco, Catalog No. C22400500BT) to a 1 μM working solution.

[0103] 1.2 Chidamide (CHI) treatment of KG1 and HL60 cells in vitro

[0104] KG1 and HL60 cell lines were revived using RPMI1640 medium + 20% FBS (Gibco, Cat. No. 10099-141C) at 37°C and 5% CO2 for one week. After the cells recovered, they were plated. First, the KG1 and HL60 cells were counted and the cell density was adjusted to 1×10 based on the results. 6 cells / mL and then plated into 12-well plates. The cells were divided into two groups: the experimental group was cultured in RPMI1640 medium with 1 μM CHI and 20% FBS; the control group was cultured in RPMI1640 medium with 0 μM CHI and DMSO (maintained at the same DMSO concentration as the experimental group). Two wells of cells were plated in each group.

[0105] After 48 hours of drug treatment, PE anti-human CD371 (Biolegend, Catalog No. 353604), PE anti-human CD7 (Biolegend, Catalog No. 343106), PE anti-human MICA / B (Biolegend, Catalog No. 320906), PE anti-human ULBP1 (R&D, Catalog No. FAB1380P), and PE anti-human ULBP3 (R&D, Catalog No. FAB1517P) were used to detect the expression levels of their corresponding antigens on KG1 cells and HL60 cells by flow cytometry. The results are shown in Figures 1-5.

[0106] It can be seen that CHI treatment for 48 h can significantly increase the expression levels of corresponding CD7, MICA / B, ULBP1, ULBP3 and CLL1 on KG1 and HL60 cells.

[0107] Example 2: Construction of CAR T cells

[0108] The following coding sequences were synthesized and cloned into the pGEM-T Easy vector in sequence: CD8α signal peptide (SEQ ID NO: 40), anti-CD7scFv (SEQ ID NO: 21), CD8α hinge region (SEQ ID NO: 36), CD8α transmembrane region (SEQ ID NO: 27), 4-1BB co-stimulatory domain (SEQ ID NO: 30), CD3ζ intracellular signaling domain (SEQ ID NO: 33) to obtain a traditional CD7-CAR plasmid, and the correct insertion of the target sequence was confirmed by sequencing.

[0109] The following coding sequences were synthesized and cloned into the pGEM-T Easy vector in sequence: CD8α signal peptide (SEQ ID NO: 40), anti-CLL1 VHH (SEQ ID NO: 25), CD8α hinge region (SEQ ID NO: 36), CD8α transmembrane region (SEQ ID NO: 27), 4-1BB co-stimulatory domain (SEQ ID NO: 30), CD3ζ intracellular signaling domain (SEQ ID NO: 33) to obtain the traditional CLL1-CAR plasmid, and the correct insertion of the target sequence was confirmed by sequencing.

[0110] After diluting the CD7-CAR and CLL1-CAR plasmids separately in 3 ml of Opti-MEM (Gibco, Catalog No. 31985-070) in a sterile tube, the packaging vector psPAX2 (Addgene, Catalog No. 12260) and the envelope vector pMD2.G (Addgene, Catalog No. 12259) were added at a ratio of 4:2:1 between plasmid:viral packaging vector:viral envelope vector. Next, 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche, Catalog No. 06366236001) was added, mixed immediately, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to the 293T cell culture flask. Viruses were collected at 24 and 48 hours, pooled, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours).

[0111] T cells were activated with DynaBeads CD3 / CD28 CTS™ (Gibco, Cat. No. 40203D) and cultured for 2 days at 37°C and 5% CO2. Then, concentrated lentivirus was added at an MOI of 3 to obtain CAR T cells targeting CD7 and CLL1, respectively.

[0112] After culturing for 7 days at 37°C and 5% CO2, the expression level of scFv on CD7-CAR T cells was detected by flow cytometry using Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab') Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (Jackson Immunoresearch, Catalog No. 109-065-097) as the primary antibody and APC Streptavidin (BD Pharmingen, Catalog No. 554067) or PE Streptavidin (BD Pharmingen, Catalog No. 554061) as the secondary antibody. The results are shown in Figure 6.

[0113] Anti-Camelid VHH cocktail (AF 488) (GenScript, Catalog No. A02021-200) was used as the primary antibody, and the expression level of VHH on CLL1-CAR T cells was detected by flow cytometry. The results are shown in Figure 7 (NT is unmodified wild-type T cells).

[0114] It can be seen that the scFv of the CD7-CAR T cells and the VHH of the CLL1-CAR T cells of the present invention can be effectively expressed.

[0115] Example 3: Cytotoxicity and cytokine release of CAR T cells against KG1 or HL60 cells pretreated with CHI

[0116] 3.1 Cytotoxicity of CAR T cells against KG1 or HL60 cells treated with CHI for 48 h

[0117] When T cells kill target cells, the number of target cells decreases. When T cells are co-cultured with fluorescently labeled target cells, the fluorescence intensity decreases as the number of target cells decreases. Therefore, the detected fluorescence intensity can reflect the T cell's ability to kill target cells.

[0118] To investigate whether pretreatment of KG1 or HL60 cells with CHI would affect the killing ability of CAR T cells against tumor cells, KG1 or HL60 cells were first treated with CHI for 48 h, with the 0 μM group serving as the control. The supernatant was then discarded, and KG1 or HL60 cells were treated with Far-red dye (Invitrogen, Cat. No. C34564A) to label them with APC fluorescence. 4CHI-treated and untreated KG1 or HL60 cells were plated into 96-well plates. CLL1-CAR T or CD7-CAR T cells were then plated into 96-well plates for co-culture at a 4:1 effector-target ratio (i.e., the ratio of effector T cells to target cells). The culture medium was RPMI1640 without CHI + X-vivo15 (1:1). After 24 hours, the fluorescence value was measured by flow cytometry. The killing efficiency was calculated according to the formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean × 100%. The results are shown in Figures 8 and 9.

[0119] It can be seen that compared with the control group, the killing effect of CD7-CAR T cells and CLL1-CAR T cells on KG1 or HL60 cells pretreated with CHI was significantly higher than that on KG1 cells not treated with CHI.

[0120] Following the same plating and treatment methods as described above, NK92 cells were co-cultured with KG1 and HL60 cell lines, and their killing efficiency was determined. The results are shown in FIG10 .

[0121] It can be seen that the killing effect of NK cells on KG1 and HL60 cells pretreated with CHI is significantly higher than that on KG1 and HL60 cells not treated with CHI.

[0122] 3.2 Cytokine Release by CAR T Cells

[0123] When T cells kill target cells, the number of target cells decreases and cytokines such as IL-2 and IFN-γ are released. Follow the steps below to measure the levels of IL-2 and IFN-γ released by CAR-T cells during target cell killing using an enzyme-linked immunosorbent assay (ELISA).

[0124] (1) Collect cell co-culture supernatant

[0125] 5×10 4 Target cells (CHI-treated and untreated KG1) were plated in a 96-well plate at a ratio of 1:1. CLL1-CAR T cells were co-cultured with target cells at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours.

[0126] (2) ELISA detection of IFN-γ secretion in the supernatant

[0127] A 96-well plate was coated with the capture antibody Purified anti-human IFN-γ Antibody (Biolegend, Catalog No. 506502) and incubated overnight at 4°C. The antibody solution was then removed and 250 μL of PBST (1X PBS containing 0.1% Tween) containing 2% BSA (Sigma, Catalog No. V900933-1kg) was added, followed by incubation at 37°C for 2 hours. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or standard was added to each well and incubated at 37°C for 1 hour. The plate was then washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). 50 μL of detection antibody, Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, Catalog No. ab25017), was then added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with 250 μL of PBST (1X PBS containing 0.1% Tween). HRP Streptavidin (Biolegend, Catalog No. 405210) was then added. After incubation at 37°C for 30 minutes, the supernatant was discarded, and 250 μL of PBST (1X PBS containing 0.1% Tween) was added and washed five times. 50 μL of TMB substrate solution was added to each well. The reaction was allowed to proceed in the dark at room temperature for 30 minutes, after which 50 μL of 1 M HSO was added to each well to stop the reaction. Within 30 minutes after stopping the reaction, the absorbance at 450 nm was measured using a microplate reader, and the cytokine content was calculated according to the standard curve (plotted according to the reading value and concentration of the standard). The results are shown in Figure 11.

[0128] It can be seen that when killing KG1 cells pretreated with CHI, the levels of IL-2 and IFN-γ released by CLL1-CAR T cells were significantly higher than when killing KG1 cells that were not treated with CHI. In general, the CHI pretreatment step in the present invention can increase the level of factor release by CLL1-CAR T cells during the process of killing target cells.

[0129] The above results indicate that the combined administration of benzamide HDAC inhibitors (such as chidamide) and immune cells targeting tumor antigens (such as CD7, MICA / B, ULBP1, ULBP3 or CLL1, etc.) can significantly enhance the tumor cell-killing activity of immune cells.

[0130] 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. A combination comprising: Immune cells expressing receptors that recognize tumor antigens and benzamide HDAC inhibitors.

2. The combination according to claim 1, wherein the receptor is selected from endogenous receptors and exogenous receptors that recognize tumor antigens, and the exogenous receptor is selected from: chimeric antigen receptors, T cell receptors, T cell receptor fusion proteins, T cell antigen couplers, immune mobilizing monoclonal T cell receptors and any combination thereof.

3. The combination according to claim 1 or 2, wherein the tumor antigen 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, CD3 0. CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79 b. CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD138, CD151, CD171, CD179a, CD300LF, CLEC12A, CD H16, CSPG4, CS-1, CLL1, Claudin6, Claudin18.1, Claudin18.2, CEA, CEACAM6, c-Met, CAIX, CXORF61 , CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2, EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130 , GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, 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, 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 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, mesothelin, 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.

4. The combination of claim 3, wherein the tumor antigen is selected from the group consisting of CD7, CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, Her2, MUC1, mesothelin, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, CLL1, NKG2D ligand, and any combination thereof.

5. The combination of claim 2, wherein the exogenous receptor is a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a costimulatory domain and / or a primary signaling domain.

6. The combination according to claim 5, wherein the antigen binding domain 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.

7. The combination according to any one of claims 1 to 6, wherein the receptor comprises a CD7 binding domain, a CLL1 binding domain, an NKG2D ligand binding domain or any combination thereof; wherein the CD7 binding domain is an antibody, a ligand or a functional fragment thereof targeting CD7; the CLL1 binding domain is an antibody, a ligand or a functional fragment thereof targeting CLL1; and the NKG2D ligand binding domain is an antibody, NKG2D or a functional fragment thereof targeting an NKG2D ligand.

8. The combination according to claim 7, wherein the antibody targeting CD7 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H comprised in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H comprised in SEQ ID NO: 8, 11, 14, 17 or 20, and the CDR1-L, CDR2-L and CDR3-L comprised in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L comprised in SEQ ID NO: 7, 10, 13, 16 or 19; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, 11, 14, 17 or 20, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, 10, 13, 16 or 19.

9. The combination according to claim 7, wherein the antibody targeting CLL1 is selected from a single-domain antibody, and the CDR1, CDR2 and CDR3 contained in the single-domain antibody are identical to the CDR1, CDR2 and CDR3 contained in SEQ ID NO: 25; preferably, the antibody targeting CLL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

25.

10. The combination of claim 5, wherein the transmembrane domain is selected from the group consisting of transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

11. The combination of claim 5, wherein the primary signaling domain is selected from the group consisting of signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

12. The combination of claim 5, wherein the costimulatory domain is one or more costimulatory signaling domains selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70.

13. The combination according to claim 5, wherein the chimeric antigen receptor further comprises a hinge region between the antigen binding domain and the transmembrane domain that is derived from the following proteins: CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1.

14. The combination according to any one of claims 1 to 13, wherein the immune cells are selected from T cells, NK cells, NKT cells, B cells, monocytes, macrophages, neutrophils, dendritic cells or any combination thereof.

15. The combination according to claim 14, wherein in the immune cells, the expression of one or more genes selected from endogenous HLA-I class genes, HLA-II class genes, and TCR / CD3 genes is inhibited or silenced.

16. The combination according to claim 15, 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; and the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and any combination thereof.

17. The combination according to claims 1-16, wherein the benzamide HDAC inhibitor is selected from CI-994, MS-275, MGCD0103, chidamide and derivatives thereof.

18. The combination according to claims 1-17, wherein the HDAC inhibitor is selected from chidamide and its derivatives; the chidamide derivatives are selected from pharmaceutically acceptable salts, enantiomers, crystalline form A or crystalline form B thereof.

19. A kit comprising the combination according to any one of claims 1 to 18.

20. Use of the combination according to any one of claims 1 to 18, or the kit according to claim 19, in the preparation of a medicament for preventing or treating cancer, infection or autoimmune disease.

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