CN115232797A - Engineered immune cells and their uses - Google Patents

Abstract

The present invention relates to an engineered immune cell that expresses (i) a cell surface molecule that specifically recognizes a ligand, (ii) one or more exogenous cytokines selected from IL12, IL18, IL-21, IL23 and IL33, and (iii) one or more exogenous chemokines selected from the group consisting of XCL2 and XCL1. The present invention also provides the use of the engineered immune cells in the treatment of cancer, infection or autoimmune disease. Compared with traditional engineered immune cells, the engineered immune cells of the present invention have significantly improved tumor-killing activity.

Description

Engineered immune cells and their uses

technical field

The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell that expresses (i) cell surface molecules that specifically recognize ligands, (ii) exogenous cytokines selected from IL12, IL18, IL-21, IL23 and IL33, and (iii) an exogenous chemokine selected from XCL2XCL1. More preferably, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor.

Background technique

Tumor immunotherapy mainly relies on autoimmunity to eliminate tumor cells by regulating the human immune system and tumor microenvironment. The immune system is a unified whole, and innate immunity also plays a very important role in tumor immunity.

Some antigen-presenting cells, such as dendritic cells and macrophages, are the bridge between innate and adaptive immunity. Antigen-presenting cells can recognize and present tumor antigens to the adaptive immune system, activate tumor-specific T cells, and then eliminate tumors. Therefore, enhancing the tumor-killing effect of the immune system by enhancing the antigen presentation process is an important research direction of tumor immunity.

CAR cell therapy is an important tumor cell immunotherapy. The successful tumor control of CAR cells generally requires the following processes: activation of the immune system, activation and expansion of CAR cells, infiltration of tumor tissue by activated CAR cells and killing of tumor cells. However, there is a common problem in current CAR cell therapy, that is, the tumor microenvironment has an inhibitory effect on CAR cells, so that CAR cells cannot infiltrate tumor tissue. Therefore, how to reduce the inhibitory effect of the tumor microenvironment on CAR cells, improve the survival time of CAR cells, or recruit other immune cells to synergize with CAR cells is very important to improve the therapeutic effect of CAR cells.

Therefore, an improved immunotherapy method is needed, which can improve the efficiency of tumor antigen presentation, induce the adoptive immune response of the body, solve the problem of tumor heterogeneity and the inhibitory effect of the tumor microenvironment, so as to improve the efficacy of CAR cell therapy.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a novel engineered immune cell that expresses (i) a cell surface molecule that specifically recognizes a ligand, (ii) an exogenous cytokine selected from the group consisting of IL12, IL18, IL-21, IL23 and IL33, and (iii) an exogenous chemokine selected from XCL2 and XCL1.

In one embodiment, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler or a T cell receptor, preferably a chimeric antigen receptor.

In one embodiment, the cytokine or chemokine protein is a fusion protein or mutant that is resistant to proteolysis.

In one embodiment, the immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells. Preferably, the T cells are CD4+/CD8+ T cells, CD4+ helper T cells, CD8+ T cells, CD4-CD8- T cells, tumor infiltrating cells, memory T cells, naive T cells, regulatory T cells, γδ -T cells or αβ-T cells.

In one embodiment, the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain and an intracellular domain comprising a common Stimulatory domains and/or primary signaling domains. Wherein, the ligand binding domain can be selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin and Darpin. Preferably, the ligand binding domain is selected from the group consisting of scFv, Fab, single domain antibodies and Nanobodies.

In one embodiment, the cell surface molecule that specifically recognizes the ligand binds to a target selected from CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23 , CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, IL-22Ra, IL-2, mesothelin, PSCA , PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2, c-Met , Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2 , Folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY -ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, Podin, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD- CT-2, Fos-associated antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP , TMPRS S2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, expressed by HIV, HCV, HBV, and/or other pathogens molecules; and/or neoepitopes or neoantigens.

In one embodiment, 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, CD45, CD4, CD5, CD8α, 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.

In one embodiment, 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 is a CD3ζ-comprising signaling domain.

In one embodiment, the costimulatory domain is one or more intracellular 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 combinations thereof. Preferably, the costimulatory domain is the intracellular region of CD27, CD28, CD134, CD137 or CD278 or a combination thereof.

In one embodiment, the expression or activity of exogenous cytokines (eg, IL12, IL18, IL21, IL23, and IL33) and/or chemokines (eg, XCL1, XCL2) is constitutive. In another embodiment, the expression or activity of the exogenous cytokine and/or chemokine is conditional expression. Conditional expression is achieved, for example, by operably linking the exogenous gene to an inducible, repressible, or tissue-specific promoter.

In one embodiment, cytokines and/or chemokines can be operably linked to a localization domain that can localize the exogenous gene of the invention for expression at a specific cellular location, eg, cell membrane, cytoplasm Specific organelles such as endoplasmic reticulum, Golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, guide peptides, transmembrane domains, and the like. In one embodiment, the exogenous genetic cytokines and/or chemokines of the invention are operably linked to the transmembrane domain so as to be anchored for expression on the surface of engineered immune cells.

In a second aspect, the present invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes a ligand, (ii) a nucleic acid sequence encoding a cytokine selected from the group consisting of IL21, IL12 and IL23, and (iii) a nucleic acid sequence encoding a chemokine selected from the group consisting of XCL1 and XCL2. Preferably, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler or a T cell receptor, more preferably a chimeric antigen receptor. Preferably, the nucleic acid is DNA or RNA.

The present invention also provides a vector comprising the above-described nucleic acid molecule. Specifically, the vector is selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV). In some embodiments, the vector further comprises an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a polyadenylation tail (polyA), 3'UTR, 5'UTR, enhancer elements such as promoters, terminators, insulators, operons, selectable markers, reporter genes, targeting sequences and/or protein purification tags. In a specific embodiment, the vector is an in vitro transcribed vector.

In one embodiment, the present invention also provides a pharmaceutical composition comprising the engineered immune cells, nucleic acid molecules or vectors of the present invention, and one or more pharmaceutically acceptable excipients.

In a third aspect, the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell according to the present invention, Nucleic acid molecules, vectors or pharmaceutical compositions.

In one embodiment, 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 tumor, kidney cancer, throat cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectum cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system, vulvar cancer and other cancers and sarcomas, and B cells Lymphoma, mantle cell lymphoma, AIDS-related lymphoma, 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 tumor, 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 tree Stem cell tumor, and post-transplant lymphoproliferative disorder (PTLD).

In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

In one embodiment, the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.

The advantage of the engineered immune cells of the present invention is that the co-expressed cytokines and/or chemokines can effectively promote the differentiation or recruitment of DC cells at the tumor site, increase the number of DC cells, increase the proliferation of engineered immune cells and On the one hand, the inhibitory effect of the tumor microenvironment on engineered immune cells can be reduced, and the tumor killing ability of engineered immune cells can be improved. On the other hand, the increased DC cells can activate the adoptive immune recognition of the body's own T cells. The immune cells form a synergistic effect, which ultimately enhances tumor suppression.

Description of drawings

Figure 1: CAR expression levels of CAR-T cells determined by flow cytometry.

Figure 2: XCL1 expression levels of CAR-T cells determined by ELISA.

Figure 3: IL-21 expression levels of CAR-T cells determined by ELISA.

Figure 4: IFN-γ release levels after CAR-T cells were co-cultured with target cells and non-target cells, respectively.

Figure 5: The killing effect of CAR-T cells on target cells under different effector-target ratios.

Figure 6: Changes in body weight of mice after treating pancreatic cancer with CAR-T cells.

Figure 7: Tumor growth curve in mice after treatment of mouse pancreatic cancer with CAR-T cells.

Figure 8: IFN-γ release levels after CAR-T cells expressing IL12, IL23 or their combination with XCL1 were co-cultured with target cells and non-target cells, respectively.

Figure 9: IFN-γ release levels after CAR-T cells expressing IL18, IL33 or their combination with XCL1 were co-cultured with target cells and non-target cells, respectively.

Figure 10: Tumor inhibitory effect of CAR-T cells expressing IL12, IL23 or their combination with XCL1 in vivo.

Figure 11: Tumor inhibitory effect of CAR-T cells expressing IL18, IL33 or their combination with XCL1 in vivo.

Detailed description of the invention

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Cell surface molecules that specifically recognize ligands

As used herein, the term "cell surface molecule that specifically recognizes a ligand" refers to a molecule expressed on the surface of a cell capable of specifically binding to a target molecule (eg, a ligand). Such surface molecules generally comprise a ligand binding domain capable of specifically binding the ligand, a transmembrane domain that anchors the surface molecule to the cell surface, and an intracellular domain responsible for signaling. Examples of common such surface molecules include, for example, T cell receptors (TCRs), chimeric antigen receptors (CARs), T cell fusion proteins (TFPs), or T cell antigen couplers (TACs).

As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex that is involved in T cell activation in response to antigen presentation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHCs) on antigen-presenting cells that present antigenic peptides to T cells and bind to the TCR complex to induce a cascade of intracellular signaling. TCR consists of six peptide chains that form heterodimers respectively, which are generally classified into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specificity to a specific antigen and MHC molecule. The variable region of the TCR may comprise or be operably linked to a ligand binding domain, wherein the ligand binding domain is defined below.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes a ligand-binding domain (eg, an antigen-binding portion of an antibody), a transmembrane domain, and An intracellular domain comprising a costimulatory domain and/or a primary signaling domain, each of which is connected by a linker. CARs can exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in an MHC-non-restricted manner. Non-MHC-restricted antigen recognition gives CAR cells the ability to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed within T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

As used herein, the term "T cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from components of the TCR, typically consisting of a TCR subunit and an antigen-binding region linked thereto, and expressed on the cell surface. Wherein, the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

As used herein, the term "T cell antigen coupler" or "TAC" includes three functional domains: 1 Tumor targeting domains, including single chain antibodies, designed ankyrin repeat proteins (DARPins) or Other targeting groups; 2 extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor close to the TCR receptor; 3 transmembrane region and intracellular region of the CD4 co-receptor, where the intracellular Domain-linked protein kinase LCK, catalyzes the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) of the TCR complex as an initial step in T cell activation.

As used herein, "ligand binding domain" refers to any structure or functional variant thereof that can bind a ligand (eg, an antigen). The ligand binding domain can be an antibody structure including, but not limited to, monoclonal, polyclonal, recombinant, human, humanized, murine, chimeric, and functional fragments thereof. For example, ligand binding domains include, but are not limited to, IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies, Anticalin, DARPIN, etc., are preferably selected from Fab, scFv, sdAb and Nanobodies. In the present invention, the ligand binding domain may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody. In another embodiment, the ligand binding domain may also be a native specific binding polypeptide or native receptor structure of a particular protein, such as PDl, PDLl, PDL2, TGF[beta], APRIL and NKG2D.

"Fab" refers to either of two identical fragments produced upon cleavage of an immunoglobulin molecule by papain, consisting of an intact light chain and an N-terminal portion of a heavy chain linked by disulfide bonds, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared to intact IgG, Fab has no Fc fragment, has higher mobility and tissue penetration, and can bind antigen monovalently without mediating antibody effects.

A "single chain antibody" or "scFv" is an antibody composed of an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker. The optimal length and/or amino acid composition of the linker can be selected. The length of the linker significantly affects the variable region folding and interaction of scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids). For selection of linker size and composition, see, eg, Hollinger et al., 1993 ProcNatl Acad. Sci. U.S.A. 90:6444-6448; US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication No. WO2006 /020258 and WO2007/024715, the entire contents of which are incorporated herein by reference. The scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.

"Single-domain antibody" or "sdAb" refers to an antibody that is naturally deficient in its light chain, and which contains only one variable heavy chain region (VHH) and two conventional CH2 and CH3 regions, also referred to as a "heavy chain" Antibody".

"Nanobody" or "Nb" refers to a single cloned and expressed VHH structure, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind to the target antigen. .

The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of a parent but contains at least one amino acid modification (ie, substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variants retain the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

Thus, a "functional variant" or "functional fragment" is at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, 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.

The choice of ligand binding domain depends on the cell surface marker on the target cell associated with the particular disease state to be identified, eg, tumor-specific antigen or tumor-associated antigen. Thus, in one embodiment, the ligand binding domains of the invention bind to one or more targets selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD179a , DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII , tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, IL-22Ra, IL-2, mesothelial protein, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2, c-Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl Base-GD2, Folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, podin, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1 , MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TM PRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, expressed by HIV, HCV, HBV, and/or other pathogens molecules; and/or neoepitopes or neoantigens. Depending on the antigen to be targeted, the CAR of the present invention can be designed to include a ligand binding domain specific for that antigen. For example, if CD19 is the antigen to be targeted, an antibody to CD19 can be used as the ligand binding domain of the invention. In one embodiment, the CAR of the present invention comprises a CD19 scFv comprising at least 90%, 95%, A light chain variable region sequence of 97% or 99% or 100% sequence identity and having at least 90% of the amino acid sequence shown in SEQ ID NO: 2, positions 123-242 or SEQ ID NO: 14, positions 123-238 , 95%, 97% or 99% or 100% sequence identity of heavy chain variable region sequences.

In a preferred embodiment, the cell surface molecule of the invention that specifically recognizes a ligand is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain and an intracellular domain, wherein the intracellular structure Domains comprise costimulatory domains and/or primary signaling domains.

As used herein, the term "transmembrane domain" refers to a polypeptide that enables expression of a chimeric antigen receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells structure. 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 chimeric antigen receptor binds to the target antigen. Transmembrane domains particularly useful in the present invention may be derived from, for example, 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, CD154 and their functional fragments. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8α chain which is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence of the CD8α transmembrane domain and the nucleotide sequence shown in SEQ ID NO: 3 or 15 have at least 70%, preferably at least 80%, more preferably at least 90% %, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptors of the present invention may further comprise a hinge region between the ligand binding domain and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand binding domain. Specifically, the hinge region serves to provide greater flexibility and accessibility to the ligand binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of a CD8α chain, FcγRIIIα receptor, IgG4 or IgG1, more preferably a CD8α hinge, which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 12 or 22 , preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence of the CD8α hinge and the nucleotide sequence shown in SEQ ID NO: 11 or 21 have At least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

As used herein, the term "intracellular domain" refers to the intracellular portion of a cell surface molecule that specifically recognizes a ligand, including costimulatory domains and/or primary signaling domains. The term "primary signaling domain" refers to the portion of a protein that transduces effector function signals and directs cells to perform specified functions. The primary signaling domain is responsible for the intracellular primary signaling following binding of the ligand binding domain to the antigen, resulting in the activation of immune cells and immune responses. In other words, the primary signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.

In one embodiment, the chimeric antigen receptors of the invention comprise primary signaling domains that may be cytoplasmic sequences of T cell receptors and co-receptors that act together upon antigen receptor binding to initiate primary signaling , as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar function. Primary signaling domains can contain a number of immunoreceptor tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of primary signaling domains of the invention include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR of the present invention may comprise a CD3ζ signaling domain having at least 70%, preferably at least 80% of the amino acid sequence shown in SEQ ID NO: 8 or 20. , more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, with the nucleotide sequence shown in SEQ ID NO: 7 or 19 %, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptors of the invention comprise one or more costimulatory domains. A costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors. Non-limiting examples of costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: 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 and ZAP70. Preferably, the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40 or a combination thereof. In one embodiment, the costimulatory domain comprised by the CAR of the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 6 or 18 or 99% or 100% sequence identity, or the coding sequence of the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleotide sequence shown in SEQ ID NO: 5 or 17 , 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the CAR of the present invention may further comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long segment of hydrophobic amino acids that has a tendency to form a single alpha-helix. At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases. The signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases. Signal peptides useful in the present invention are well known to those skilled in the art, eg, signal peptides derived from CD8α, IgG1, GM-CSFRα, and the like. In one embodiment, the signal peptide useful in the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence set forth in SEQ ID NO: 10 or 30 or 100% sequence identity, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the CAR of the present invention may further comprise a switch structure to regulate the expression time of the CAR. For example, the switch structure can be in the form of a dimerization domain that induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain for binding to the targeted antigen, thereby activating a signaling pathway. Alternatively, switch domains can be used to connect the binding and signaling domains, respectively, which can dimerize only when the switch domains bind to each other (eg, in the presence of an inducing compound) linked together, thereby activating signaling pathways. The switch structure can also be in the form of a masked 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, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art can be used in the present invention.

In one embodiment, the CAR of the present invention may further comprise a suicide gene, i.e., causing it to express a cell death signal that can be induced by a foreign substance, to clear the CAR cells when needed (eg, when severe toxic side effects occur). For example, suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when desired, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir treatment. The suicide gene can also be iCaspase-9, which can be induced to dimerize by chemical inducing drugs 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 invention.

In an exemplary embodiment, the chimeric antigen receptor comprises: (a) a 4-1BB costimulatory domain and a CD3ζ intracellular signaling domain, (b) a CD27 costimulatory domain and a CD3ζ intracellular signaling domain Transduction domain, (c) CD28 costimulatory domain and CD3ζ intracellular signaling domain, (d) OX40 costimulatory domain and CD3ζ intracellular signaling domain, (e) CD28 costimulatory domain, 4-1BB Costimulatory domain and CD3ζ intracellular signaling domain, (f) OX40 costimulatory domain, 4-1BB costimulatory domain and CD3ζ intracellular signaling domain, or (g) CD28 costimulatory domain, OX40 costimulatory domain Stimulatory domain and CD3ζ intracellular signaling domain.

exogenous gene

In addition to cell surface molecules that specifically recognize antigens, the engineered immune cells of the present invention also express one or more exogenous cytokines and one or more exogenous chemokines selected from the group consisting of IL12, IL18, IL21, IL23 and IL33, the chemokine is selected from XCL1 and XCL2.

The cytokines used in the present invention are mainly interleukins, which are selected from IL-21, IL12, IL18, IL33 and IL23. Interleukins are produced by leukocytes and function among leukocytes, such as transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and playing an important role in inflammatory responses. In general, the biological effects of interleukins are achieved through their binding to the corresponding receptors, for example, the biological properties of IL-21 are achieved through the binding of IL-21 to its receptor IL-21R. At least 38 interleukins have been found, named IL1 to IL38, and their functions are complex. According to structural homology, interleukins can be divided into several protein families, such as IL1 family (including IL1α, IL1β, IL18, IL37, IL38, IL33, etc.), IL2 family (including IL2, IL4, IL13, IL15, IL21, etc.) ), IL6 family (including IL6, IL12, IL23, IL27, IL35, etc.), IL10 family (including IL10, IL19, IL20, IL22, IL26, etc.), IL17 family (including IL17 and IL25) and other interleukins (including IL5, IL7 , IL9, IL11, IL14, IL16, IL31, IL32, etc.). In one embodiment, the cytokine used in the present invention is a wild-type or a variant thereof that has the same or similar, or even better, function than the wild-type.

The amino acid sequence of the propeptide of wild-type hIL-21 is shown in SEQ ID NO: 32, and the amino acid sequence of the mature peptide is shown in SEQ ID NO: 34 (consisting of amino acids 30-162 of SEQ ID NO: 32).

IL-21 is produced by activated CD4+ T cells, NKT cells, Tfh cells and Th17 cells, and has high homology with IL-2 and IL-15. IL-21 has a wide range of immunoregulatory functions, and activation of it can enhance the proliferation of activated CD8+ T cells, enhance the cytotoxic activity of NK cells, and promote the proliferation and differentiation of B cells.

In one embodiment, the IL-21 used in the present invention is wild-type IL-21 or an IL-21 variant, eg, an IL-21 variant that retains at least all or most of the IL-21 activity, even with improved activity.

In one embodiment, the IL-21 variant used in the present invention may be, for example, a variant sequence obtained by deleting and/or substituting one or more amino acids in amino acids 65-96 of SEQ ID NO: 34, for example: (1) Deletion and/or substitution of amino acids 83-86, amino acids 83-88, amino acids 83-90, amino acids 82-88, amino acids 77-92, and amino acids of SEQ ID NO: 34 Variant sequences obtained from amino acids 71-92, amino acids 65-92, and amino acids 77-96; (2) a variant sequence containing additional N-terminal Met on the basis of (1); (3) in Variant sequences obtained by conservatively modifying up to 10 amino acids in the sequences of (1) and (2). See, eg, WO2006111524, the entire contents of which are incorporated herein by reference.

In one embodiment, the polypeptide encoded by the IL-21 gene used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 32, 34 or 36 , 97% or 99% or 100% sequence identity, or the IL-21 gene has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleic acid sequence shown in SEQ ID NO: 31, 33 or 35, 95%, 97% or 99% or 100% sequence identity.

IL12 is a heterodimeric cytokine encoded by two independent genes IL-12A (p35) and IL-12B (p40). IL12 is produced by dendritic cells, macrophages, neutrophils and other antigen-presenting cells, and can promote the proliferation of T helper 1 (Th1) cells; induce NK cells and T cells to produce gamma interferon, and improve NK cells Toxicity; promote the formation of cytotoxic T cells, etc.

In one embodiment, the IL12 used in the present invention is wild-type IL12 or an IL12 variant, eg, an IL12 variant that retains at least all or most of the IL12 activity, even with improved activity. In one embodiment, the IL12 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 40 or 42 % sequence identity, or the gene encoding IL-12 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% with the nucleic acid sequence shown in SEQ ID NO: 39 or 41 or 100% sequence identity.

IL23 is also a heterodimeric cytokine composed of two subunits, p40 and p19. IL23 is mainly produced by activated dendritic cells, macrophages and monocytes. IL23 has been reported to play an important role in experimental autoimmune encephalomyelitis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, tumor development, and infection.

In one embodiment, the IL-23 used in the present invention is wild-type IL-23 or an IL-23 variant, eg, an IL-23 variant that retains at least all or most of the IL-23 activity, even with improved activity. In one embodiment, the IL-23 used in the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence shown in SEQ ID NO: 48 or 50 or 100% sequence identity, or the gene encoding IL-23 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or the nucleic acid sequence shown in SEQ ID NO: 47 or 49 99% or 100% sequence identity. IL18 is mainly produced by macrophages, but can also be produced by various immune and non-immune cells such as monocytes, dendritic cells, epithelial cells, and fibroblasts. IL18 mediates the MyD88-NFκB signaling pathway by binding to the heterodimeric receptor IL18Rα/Rβ and can strongly induce the secretion of IFNγ. Since IL18 can induce the proliferation and enhance the activity of immune cells, its role in anti-tumor, anti-infection and immune regulation has been widely studied in recent years.

In one embodiment, the IL18 used in the present invention is wild-type IL18 or an IL18 variant, eg, an IL18 variant that retains at least all or most of the IL18 activity, even with improved activity. In one embodiment, the IL18 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 44 or 46 % sequence identity, or the gene encoding IL18 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleic acid sequence shown in SEQ ID NO: 43 or 45 % sequence identity.

IL33 is a bifunctional protein that can act as a transcription factor as a molecule located in the nucleus, and can be secreted to the outside of the cell to act as a cytokine. It is associated with many diseases such as allergies, autoimmune diseases, cardiovascular Infections and tumors are closely related. IL33 is expressed in a variety of cells, including epithelial cells, fibroblasts, macrophages, endothelial cells, mast cells, dendritic cells, and osteoblasts. These cells release IL33 when they are injured, and IL33 can produce different inflammatory responses depending on the cell type.

In one embodiment, the IL33 used in the present invention is wild-type IL33 or an IL33 variant, eg, an IL33 variant that retains at least all or most of the IL33 activity, even with improved activity. For example, it was found that full-length IL33 can be cleaved into mature forms _{IL33 95-270} (consisting of amino acids 95-270 of full-length IL33), _{IL33 99-270} (consisting of amino acids 99-270 of full-length IL33) composition) and _{IL33 109-270} (consisting of amino acids 109-270 of full-length IL33), these fragments are both released extracellularly from damaged or necrotic cells and are 10-fold more biologically active than full-length IL33 (see Lefrancais, E. et al. IL-33 is processed into mature bioactive forms by neutrophilelastase and cathepsin G. Proc. Natl Acad. Sci. USA 109, 1673-1678). In one embodiment, the IL33 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 52, 54, 55, 56 or 57 % or 99% or 100% sequence identity, or the gene encoding IL33 has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleic acid sequence shown in SEQ ID NO: 51, 53, 58, 59 or 60 %, 95%, 97% or 99% or 100% sequence identity.

The C-type chemokine family, also known as lymphoid chemokines, includes two members, XCL1 and XCL2, which are mainly produced by CD8+ T cells and natural killer cells. XCL1 has unique sequence features and two interchangeable protein spatial conformations, which make XCL1 different from other chemokines and play unique functions. The XCL1-specific receptor XCR1 is a member of the G protein-coupled receptor family. The interaction between the two not only plays an important role in the negative selection of the thymus and the establishment of autoimmune tolerance, but also can initiate cross-antigen presentation and mediate cytotoxic immune responses. . XCL1 not only regulates immune system balance and maintains intestinal immune homeostasis, but is also associated with various diseases, such as autoimmune diseases, nephritis, tuberculosis and human immunodeficiency virus infection. The nucleic acid sequences of XCL2 and XCL1 are 97% identical, with two amino acid residues different at positions 7 and 8: Asp and Lys in XCL1, and His and Arg in XCL2. Studies have found that XCL2 and XCL1 are very similar in expression profile, structure and function. For example, like XCL1, XCL2 also has two interconvertible protein spatial conformations, haplotype and dimer, in which the monomeric conformation binds and interacts with each other. Activates XCR1, and the dimerized conformation has a higher affinity for the hairpin structure in Glycosaminoglycans (GAGs). The receptors of XCL1 and XCL2, XCR1, are selectively expressed on DC (cDC1) cells with antigen-presenting ability. Some studies have found that the introduction of XCL1 can effectively improve the efficacy of anti-tumor immunotherapy and targeted vaccines.

In one embodiment, XCL1 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 24 or 26 % sequence identity, or the coding sequence of XCL1 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleic acid sequence shown in SEQ ID NO: 23 or 25 % sequence identity.

In one embodiment, the XCL2 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 38 Sequence identity, or the coding sequence of XCL2 is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% identical to the nucleic acid sequence set forth in SEQ ID NO:37 sex.

Expression of foreign genes

The expression of exogenous genes, eg cytokines and/or chemokines in the present invention may be constitutive or conditional.

In one embodiment, the expression of exogenous cytokines and/or chemokines is conditional expression. For example, if desired, the exogenous gene of the present invention can be operably linked to an inducible or tissue-specific promoter to regulate the expression level of the introduced exogenous gene at a specific time or in a specific tissue or cell type. In one embodiment, the promoter is an inducible promoter, ie, a promoter that initiates transcription only in the presence of specific environmental conditions, developmental conditions, or inducers. Such environmental conditions include, for example, a tumor acidic microenvironment, a tumor hypoxic microenvironment, and the like. Such inducers include, for example, ciccycline, tetracycline, or analogs thereof, and analogs of tetracycline include, for example, chlortetracycline, oxytetracycline, desmethylchlorotetracycline, methycycline, doxycycline, and minocycline white. Inducible promoters include, for example, Lac operon sequences, tetracycline operon sequences, galactose operon sequences, or doxycycline operon sequences, and the like. In one embodiment, the expression of cytokines and/or chemokines is secreted or anchored, eg, can be operably linked to a localization domain that can localize an exogenous gene of the invention Expressed at specific cellular locations, such as cell membranes, specific organelles in the cytoplasm such as endoplasmic reticulum, Golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, guide peptides, transmembrane domains, and the like. In one embodiment, the exogenous gene of the present invention is operably linked to the transmembrane domain, thereby anchored for expression on the surface of engineered immune cells.

In one embodiment, the exogenous gene in the present invention, eg, IL12, IL18, IL-21, IL23, IL33, XCL2 or XCL1 protein may be wild type or a fusion protein with specific properties (eg resistance to proteolysis) or mutant.

nucleic acid

The present invention also provides a nucleic acid molecule comprising (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes a ligand, (ii) a nucleic acid sequence encoding a cytokine (selected from IL12, IL18, IL-21, IL23 and IL33). a nucleic acid sequence, and (iii) a nucleic acid sequence encoding a chemokine (selected from XCL2 and XCL1).

In one embodiment, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler, or a T cell receptor, preferably a chimeric antigen receptor. Chimeric antigen receptors are defined as above.

As used herein, the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in linear or circular form in single- and/or double-stranded form form, or their mixtures (including hybrid molecules). Thus, nucleic acids according to the present invention include DNA (eg dsDNA, ssDNA, cDNA), RNA (eg dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (eg PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNA)). Nucleic acids of the invention may also contain one or more modified bases, such as, for example, tritylated bases and uncommon bases such as inosine. Other modifications are also contemplated, including chemical, enzymatic, or metabolic modifications, so long as the multi-chain CARs of the invention can be expressed from polynucleotides. Nucleic acids can be provided in isolated form. In one embodiment, nucleic acids may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.

The nucleic acid sequences of the invention can be codon optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacterial, yeast, or insect cells. Codon optimization refers to the replacement of codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement code for the same amino acid. Therefore, the choice of optimal codons depends on the codon usage preferences of the host genome.

carrier

The present invention also provides a vector comprising the nucleic acid according to the present invention. Wherein, the nucleic acid sequences encoding cell surface molecules that specifically recognize ligands, the nucleic acid sequences encoding cytokines (selected from IL12, IL18, IL-21, IL23, and IL33), the nucleic acid sequences encoding chemokines (selected from XCL1 and XCL2) Nucleic acid sequences can be located in one or more vectors. When located in the same vector, these nucleic acid sequences can be linked by, for example, the 2A peptide.

As used herein, the term "vector" is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell, in which the nucleic acid molecule can eg be replicated and/or expressed.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a specific targeting sequence at the site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric antigen receptor polypeptides of the invention, in suitable host cells and the translation of their mRNAs. Suitable carriers for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)), and the like ), phages, phagemids, cosmids, and artificial chromosomes (including BAC and YAC). The vector itself is usually a nucleotide sequence, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector .Engineered vectors typically also contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (eg, a multiple cloning site, MCS). The vector may additionally contain a promoter , polyadenylation tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements. In a specific embodiment , the vector is an in vitro transcribed vector.

engineered immune cells

The present invention also provides an engineered immune cell comprising the nucleic acid or vector of the present invention. In other words, the engineered immune cells of the present invention express a cell surface molecule that specifically recognizes a ligand, one or more exogenous cytokines (selected from IL12, IL18, IL-21, IL23, IL33), and a or a plurality of exogenous chemokines selected from the XCL1 and XCL2 genes.

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells obtained from stem cell sources such as cord blood. Preferably, the immune cells are T cells. The T cells can be any T cells, such as T cells cultured in vitro, eg, primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4-CD8- T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells) ), tumor infiltrating cells, memory T cells, naive T cells, regulatory T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation. In the present invention, immune cells are engineered to express chimeric antigen receptors as well as exogenous cytokines, and chemokines.

Nucleic acid sequences encoding chimeric antigen receptor polypeptides, as well as cytokines and chemokines, can be introduced into immune cells using conventional methods known in the art (eg, by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell. An example is RNA transfection, the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes 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. perforation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells). Thus, transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means. For transformation to occur, the cells or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation.

In yet another embodiment, the immune cells of the present invention further comprise at least one inactivated gene selected from the group consisting of CD52, GR, TCRα, TCRβ, CD3γ, CD3δ, CD3ε, CD247ζ, HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More particularly, at least TCR components (including TCRα, TCRβ genes) or CD3 components (including CD3γ, CD3δ, CD3ε, CD247ζ) in immune cells are inactivated. This inactivation renders the TCR-CD3 complex non-functional in the cell. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD). Methods of inactivating a gene are known in the art, eg, by mediated DNA fragmentation by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, thereby inactivating the gene.

Pharmaceutical Compositions and Combination Therapies

The present invention also provides a pharmaceutical composition comprising the engineered immune cell, nucleic acid molecule or carrier of the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also encompasses the use of the nucleic acid molecule, vector or engineered immune cell in the preparation of a pharmaceutical composition or medicament.

As used herein, the term "pharmaceutically acceptable excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water. In general, the selection of suitable excipients depends, among other things, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical compositions according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration. Processes known in the present invention for the manufacture of pharmaceuticals may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical compositions according to the present invention may also be administered in combination with one or more other agents suitable for 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, trimetateglucuronate, orris Statins E (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-stimulating protein, etc.; antibodies or their Fragments, 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 invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

The present invention also provides a combination therapy comprising (1) an engineered immune cell and a chemokine expressing an exogenous cytokine; (2) an engineered immune cell and a cytokine expressing an exogenous chemokine; or (3) engineered immune cells, cytokines and chemokines, wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, and the cytokines are selected from the group consisting of IL12, IL18, IL-21, IL23 and IL33, The chemokine is selected from XCL1 and XCL2.

therapeutic application

The present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the present invention. Accordingly, the present invention also encompasses the use of the engineered immune cells in the manufacture of a medicament for the treatment of cancer, infection or autoimmune disease.

In one embodiment, an effective amount of an immune cell and/or pharmaceutical composition of the invention is administered directly to a subject.

In one embodiment, the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, more preferably T cells, NK cells cells or NKT cells.

As used herein, the term "autologous" refers to any material derived from an individual to be later reintroduced into that same individual.

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 each other when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.

As used herein, the term "subject" is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects representing animal models of cancer. Preferably, the subject is a human.

In one embodiment, the cancer is a cancer associated with expression of the target bound by the ligand binding domain, eg, a hematological tumor or a solid tumor. For example, such cancers include, but are not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous lung cancer), lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectum cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system, vulvar cancer and other cancers and sarcomas, and B cells Lymphomas (including low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade Lymphoblastic NHL, high-grade small non-cleaving cell NHL, bulky NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myeloid leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); and other diseases associated with target expression. Preferably, the diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, and the like.

In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

In one embodiment, the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.

In one embodiment, the method further comprises administering to the subject one or more additional chemotherapeutic agents, biological agents, drugs or treatments. In this embodiment, the chemotherapeutic agent, biologic, drug or treatment is selected from radiation therapy, surgery, antibody agents and/or small molecules and any combination thereof.

The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with examples. It should be noted that those skilled in the art should understand that the accompanying drawings and the embodiments of the present invention are only for the purpose of illustration and do not constitute any limitation to the present invention. The embodiments in the present application and the features in the embodiments may be combined with each other where there is no contradiction.

Detailed ways

Example 1. Preparation of CAR-T cells

1. Construction of Retroviral Plasmids

Artificially synthesized mCD19-scFv (SEQ ID NO: 13), mCD8a hinge region (SEQ ID NO: 21), mCD8a transmembrane region (SEQ ID NO: 15), murine 41bb intracellular domain (SEQ ID NO: 17) ) and the coding sequence fragment of the murine CD3ζ intracellular domain (SEQ ID NO: 19), and added XhoI/EcoRI restriction sites at both ends. The fragment was cloned into the MSCV vector to obtain the MSCV-mCD19-CAR plasmid.

The coding sequence fragments of T2A and murine IL-21 (SEQ ID NO: 35) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-IL-21 plasmid.

The coding sequence fragments of T2A and murine XCL1 (SEQ ID NO: 25) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends. The fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-XCL1 plasmid.

2. Preparation of Retrovirus

In a T175 flask, 293T cells were seeded in 30 ml of DMEM medium containing ¹⁰ % fetal bovine serum at a density of 30 x 106 cells/flask, and cultured overnight in a 37°C, 5% CO2 incubator for Virus packaging.

Add 3 ml Opti-MEM (Gibco, Cat. No. 31985-070), 45 μg retroviral plasmid (MSCV-mCD19-CAR plasmid, MSCV-mCD19-CAR-IL-21 plasmid or MSCV-mCD19-CAR-XCL1) to a sterile tube plasmid) and 15 μg of the packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029). Then, 120 μl of X-tremeGENE HP DNA Transfection Reagent (Roche, Cat. No. 06366236001) was added, mixed immediately, and incubated at room temperature for 15 min. The plasmid/vector/transfection reagent mixture was then added dropwise to a pre-prepared culture flask of 293T cells and incubated overnight at 37°C under 5% CO2 conditions. Cultures were harvested 72 hours after transfection and centrifuged (2000 g, 4°C, 10 min) to obtain retroviral supernatant.

3. Preparation of CAR-T cells

T lymphocytes were isolated from mouse spleen and T cells were activated with DynaBeads CD3/CD28 CTS ^™ (Gibco, Cat. No. 40203D) and then cultured at 37°C and 5% CO ₂ for 1 day.

Activated T cells were seeded into 24-well plates pre-coated with RetroNectin overnight at a density of 3 × 10 ⁶ cells/mL per well, and then 500 μL of complete medium (NT, control), MSCV-mCD19-CAR, and 500 μL of complete medium (NT, control) were added, respectively. virus, MSCV-mCD19-CAR-IL-21 virus, or MSCV-mCD19-CAR-IL-21 virus + MSCV-mCD19-CAR-XCL1 virus, and supplemented with complete medium to 2 mL.

The 24-well plate was placed in a centrifuge for centrifugation, and centrifuged at 32°C and 2000g for 2h. Then, the 24-well plate was immediately placed in a 37°C, CO2 incubator for static culture. Change fresh medium the next day and adjust the cell density to 1 x 10 ⁶ cells/mL. Three days after infection, cells were collected for subsequent analysis. The collected cells are NT cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells, and mCD19-CAR-IL-21-XCL1 cells.

Example 2. Detection of the expression of CAR-T cells

1. Expression level of cell surface CAR

Take out 2×10 ⁵ CAR-T cells prepared in Example 1, use Goat Anti-Rat IgG (H&L) Biotin (BioVision, Cat. No. 6910-250) as the primary antibody, and APC Streptavidin (BD Pharmingen, Cat. No. 554067) as the secondary antibody , the expression level of CAR on CAR T cells was detected by flow cytometry, and the results are shown in Figure 1.

It can be seen that the CAR positive efficiency in mCD19-CAR, mCD19-CAR-IL-21, and mCD19-CAR-IL-21-XCL1 cells were all greater than 50% compared with the control, indicating that these cells can effectively express CAR.

2. Expression level of XCL1

The supernatant of the CAR-T cells prepared in Example 1 was collected, and the MouseXCL1DuoSet ELISA kit (R&D Systems, Cat. No. DY486) was used to detect the level of XCL1 secretion in the cells according to the manufacturer's recommendations. The results are shown in Figure 2.

It can be seen that CAR T cells containing mCD19-CAR-XCL1 can efficiently secrete XCL1.

3. The expression level of IL-21

Collect the supernatant of the CAR-T cells prepared in Example 1, and use Mouse IL-21DuoSet ELISA kit (R&D Systems, Cat. No. DY594) to detect the level of IL-21 secretion in the cells according to the manufacturer's recommendations. The results are shown in the figure 3 shown.

It can be seen that both CAR T cells containing mCD19-CAR-IL-21 can efficiently express IL-21.

Example 3. Detection of IFN-γ secretion level of CAR-T cells

NT cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells, and mCD19-CAR-IL-21-XCL1 cells were added at a concentration of ² x 105 cells/100 μl in a 96-well round bottom plate, respectively. Target cells Panc02-mCD19 cells or non-target cells Panc02 cells were then added to each well at a concentration of 1×10 ⁴ cells/100 μl, respectively. After 24 h incubation at 37°C, the culture supernatant was collected. The expression levels of IFN-γ in culture supernatants were detected using the Mouse IFN-gamma DuoSet ELISA kit (R&D, Cat. No. DY485) according to the manufacturer's recommendations.

The detection results are shown in Figure 4. It can be seen that the release of IFN-γ was not detected in the non-target cells Panc02, while a significantly elevated level of IFN-γ release was detected in the target cells Panc02-CD19, indicating that the CART cells in this example were not effective against the target cells. Cells have specific killing activity.

Example 4. In vitro tumor killing effect verification of CAR-T cells

In a 96-well plate, 100 μl containing 1×10 ⁴ Panc02-mCD19 cells were added to each well as target cells, and 100 μl NT was added at the ratio of 10:1, 5:1, 2.5:1, and 1.25:1. cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells, or mCD19-CAR-IL-21-XCL1 cells were used as effector cells. The 96-well plate was incubated at 37°C overnight, and then 50 μl of 5× D-Luciferin solution (XenoLight, Cat#12279) was added to each well, and the fluorescence value was immediately measured by a microplate reader. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of samples)/mean fluorescence of target cells×100%, the killing efficiency was calculated, and the results are shown in FIG. 5 .

It can be seen that the additional expression of IL-21 slightly increased the killing ability of CAR-T cells against target cells compared with mCD19-CAR cells alone. Unexpectedly, the inventors found that compared with CAR-T cells expressing only IL-21, the combination of expressing XCL1+IL-21 can further significantly improve the killing ability of CAR-T cells to target cells, especially in low-efficiency targets. than (eg 1.25:1) case.

Example 5. Validation of tumor suppressor effect of CAR-T cells

5×10 ⁵ Panc02-mCD19 pancreatic cancer cells overexpressing CD19 were subcutaneously inoculated into the axilla of the left forelimb of healthy C57BL/6 mice.

Mice inoculated with pancreatic cancer cells were randomly divided into 5 groups of 7 mice each. When the tumor volume grew to 100mm, each mouse was injected with PBS, ⁵ x ¹⁰ NT cells, mCD19-CAR cells, mCD19-CAR-IL-21 cells or mCD19-CAR-IL-21- XCL1 cells.

The mice were monitored for changes in body weight and tumor volume until the end of the experiment.

Changes in body weight of mice are shown in Figure 6. It can be seen that after the administration of CAR-T cells, the body weight of the mice in each group was not significantly different from that of the control group, and during the observation period, when the mouse tumor did not exceed 1500 mm, the mice ^were active and had normal coat color. This shows that administration of CAR-T cells will not have obvious toxic and side effects on mice.

Changes in tumor volume in mice are shown in Figure 7. It can be seen that the tumor suppressor effect of mCD19-CAR-IL-21 cells expressing only IL-21 is comparable to that of conventional mCD19-CAR cells, indicating that IL-21 alone cannot improve the tumor suppressor effect of CAR-T cells. Unexpectedly, the inventors found that the combination of IL-21+XCL1 significantly improved the tumor suppressor effect of CAR-T cells, and maintained the tumor volume at a low level without recurrence until the end of the experiment.

The above results indicate that the combination of co-expressing IL-21+XCL1 can significantly enhance the inhibitory effect of engineered immune cells on target pancreatic cancer cells compared with CAR-T cells expressing IL-21 alone.

Example 6. Validation of the activity of CAR-T cells expressing cytokines and chemokines

According to the method of Example 1, co-expressing individual cytokines IL12 (SEQ ID NO:42), IL23 (SEQ ID NO:50), IL18 (SEQ ID NO:46), IL33 (SEQ ID NO:54), and the same were prepared CAR-T cells combined with XCL1. CAR-T cells co-expressing IL15(SEQ ID NO:61)+XCL1 and IL17(SEQ ID NO:62)+XCL1 were also prepared as controls.

According to the method of Example 3, the secretion level of IFN-γ of the above-mentioned CAR-T cells was detected. After co-culture with the target cells Panc02-mCD19, the IFN-γ secretion level of CAR-T cells expressing IL12+XCL1 and IL23+XCL1 was comparable to that of CAR-T cells expressing only IL12 or IL23 (see Figure 8), while the The IFN-γ secretion level of IL18+XCL1 and IL33+XCL1 CAR-T cells was significantly higher than that of CAR-T cells expressing only IL18 or IL33 (see Figure 9).

According to the method of Example 5, the tumor-inhibitory effect of the above-mentioned CAR-T cells in vivo was detected. Although CAR-T cells expressing IL12 or IL23 alone showed very excellent tumor suppressive effect, CAR-T cells expressing IL12+XCL1 or IL23+XCL1 combination still had significantly better tumor suppressive effect than CAR expressing IL12 or IL23 alone -T cells (see Figure 10). In addition, CAR-T cells expressing IL18+XCL1 and IL33+XCL1 also showed significant tumor suppressive effects, while CAR-T cells expressing IL15+XCL1 and IL17+XCL1 had a similar tumor suppressive effect to traditional mCD19- CAR T cells were comparable or slightly worse (see Figure 11). This also shows that not any combination of interleukin and XCL1 can achieve a significant inhibitory effect on tumors, which may be related to the complex function of interleukin.

It should be noted that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. It is understood by those skilled in the art that any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Nanjing Beiheng Biotechnology Co., Ltd.

<120> Engineered immune cells and their uses

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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

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Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

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Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

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Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

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Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

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Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

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Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

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Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

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Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

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Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

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<213> Artificial Sequence (Artificial Sequence)

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ggcaccaagc tggaactgaa gggcggaggc ggaagtggag gcggaggatc tggcggcgga 360

ggctctgaag tgcagctgca gcagtctggc gctgaactgg tccggcctgg cactagcgtg 420

aagctgtcct gcaaggtgtc cggcgacacc atcaccttct actacatgca cttcgtgaag 480

cagaggccag gacagggcct ggaatggatc ggcagaatcg accctgagga cgagagcacc 540

aagtacagcg agaagttcaa gaacaaggcc accctgaccg ccgacaccag cagcaacacc 600

gcctacctga agctgtctag cctgacctcc gaggacaccg ccacctactt ttgcatctac 660

ggcggctact acttcgacta ctggggccag ggcgtgatgg tcaccgtgtc cagc 714

<210> 14

<211> 238

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD19 scFv

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Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Thr Ser Leu Gly

1 5 10 15

Glu Thr Val Thr Ile Gln Cys Gln Ala Ser Glu Asp Ile Tyr Ser Gly

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asp Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr

65 70 75 80

Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln

115 120 125

Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys

130 135 140

Lys Val Ser Gly Asp Thr Ile Thr Phe Tyr Tyr Met His Phe Val Lys

145 150 155 160

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Glu

165 170 175

Asp Glu Ser Thr Lys Tyr Ser Glu Lys Phe Lys Asn Lys Ala Thr Leu

180 185 190

Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Lys Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ile Tyr Gly Gly Tyr Tyr

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Phe Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser

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<210> 15

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD8α transmembrane domain

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atctgggcac ccttggccgg aatctgcgtg gcccttctgc tgtccttgat catcactctc 60

atc 63

<210> 16

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD8α transmembrane domain

<400> 16

Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu

1 5 10 15

Ile Ile Thr Leu Ile

20

<210> 17

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> m4-1BB costimulatory domain

<400> 17

aggaaaaaat tcccccacat attcaagcaa ccatttaaga agaccactgg agcagctcaa 60

gaggaagatg cttgtagctg ccgatgtcca caggaagaag aaggaggagg aggaggctat 120

gagctg 126

<210> 18

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> m4-1BB costimulatory domain

<400> 18

Arg Lys Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys Lys Thr Thr

1 5 10 15

Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys Pro Gln Glu

20 25 30

Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

35 40

<210> 19

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD3ζ intracellular signaling domain

<400> 19

agcaggagtg cagagactgc tgccaacctg caggacccca accagctcta caatgagctc 60

aatctagggc gaagagagga atatgacgtc ttggagaaga agcgggctcg ggatccagag 120

atgggaggca aacagcagag gaggaggaac ccccaggaag gcgtatacaa tgcactgcag 180

aaagacaaga tggcagaagc ctacagtgag atcggcacaa aaggcgagag gcggagaggc 240

aaggggcacg atggccttta ccagggtctc agcactgcca ccaaggacac ctatgatgcc 300

ctgcatatgc agaccctggc ccctcgc 327

<210> 20

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD3ζ intracellular signaling domain

<400> 20

Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp Pro Asn Gln Leu

1 5 10 15

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Glu

20 25 30

Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys Gln Gln Arg Arg

35 40 45

Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln Lys Asp Lys Met

50 55 60

Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu Arg Arg Arg Gly

65 70 75 80

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

85 90 95

Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro Arg

100 105

<210> 21

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD8α hinge region

<400> 21

actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60

ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120

gcctgtgata tttac 135

<210> 22

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD8α hinge region

<400> 22

Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr

1 5 10 15

Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser

20 25 30

Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr

35 40 45

<210> 23

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hXCL-1

<400> 23

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggtgtaggga gtgaagtctc agataagagg acctgtgtga gcctcactac ccagcgactg 120

ccggttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180

attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacgtgggt gagagacgtg 240

gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300

ggaacccagc aatcgaccaa tacagctgtg accctgactg gctag 345

<210> 24

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hXCL-1

<400> 24

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser Asp Lys Arg Thr Cys

20 25 30

Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr

35 40 45

Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg

50 55 60

Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val

65 70 75 80

Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln

85 90 95

Thr Lys Pro Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu

100 105 110

Thr Gly

<210> 25

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mXCL-1

<400> 25

atgagacttc tcctcctgac tttcctggga gtctgctgcc tcaccccatg ggttgtggaa 60

ggtgtgggga ctgaagtcct agaagagagt agctgtgtga acttacaaac ccagcggctg 120

ccagttcaaa aaatcaagac ctatatcatc tgggaggggg ccatgagagc tgtaattttt 180

gtcaccaaac gaggactaaa aatttgtgct gatccagaag ccaaatgggt gaaagcagcg 240

atcaagactg tggatggcag ggccagtacc agaaagaaca tggctgaaac tgttcccaca 300

ggagcccaga ggtccaccag cacagcagta accctgactg ggtaa 345

<210> 26

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mXCL-1

<400> 26

Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro

1 5 10 15

Trp Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys

20 25 30

Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr

35 40 45

Ile Ile Trp Glu Gly Ala Met Arg Ala Val Ile Phe Val Thr Lys Arg

50 55 60

Gly Leu Lys Ile Cys Ala Asp Pro Glu Ala Lys Trp Val Lys Ala Ala

65 70 75 80

Ile Lys Thr Val Asp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu

85 90 95

Thr Val Pro Thr Gly Ala Gln Arg Ser Thr Ser Thr Ala Val Thr Leu

100 105 110

Thr Gly

<210> 27

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> T2A

<400> 27

gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

<210> 28

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> T2A

<400> 28

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 29

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD8α signal peptide

<400> 29

atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60

atcctgggga gt 72

<210> 30

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCD8α signal peptide

<400> 30

Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu

1 5 10 15

Gly Glu Ser Ile Ile Leu Gly Ser

20

<210> 31

<211> 489

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL-21 propeptide

<400> 31

atgagatcca gtcctggcaa catggagagg attgtcatct gtctgatggt catcttcttg 60

gggacactgg tccacaaatc aagctcccaa ggtcaagatc gccacatgat tagaatgcgt 120

caacttatag atattgttga tcagctgaaa aattatgtga atgacttggt ccctgaattt 180

ctgccagctc cagaagatgt agagacaaac tgtgagtggt cagctttttc ctgttttcag 240

aaggcccaac taaagtcagc aaatacagga aacaatgaaa ggataatcaa tgtatcaatt 300

aaaaagctga agaggaaacc accttccaca aatgcaggga gaagacagaa acacagacta 360

acatgccctt catgtgattc ttatgagaaa aaaccaccca aagaattcct agaaagattc 420

aaatcacttc tccaaaagat gattcatcag catctgtcct ctagaacaca cggaagtgaa 480

gattcctga 489

<210> 32

<211> 162

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL-21 propeptide

<400> 32

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln

20 25 30

Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln

35 40 45

Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro

50 55 60

Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile

85 90 95

Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala

100 105 110

Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr

115 120 125

Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu

130 135 140

Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu

145 150 155 160

Asp Ser

<210> 33

<211> 402

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL-21 mature peptide

<400> 33

caaggtcaag atcgccacat gattagaatg cgtcaactta tagatattgt tgatcagctg 60

aaaaattatg tgaatgactt ggtccctgaa tttctgccag ctccagaaga tgtagagaca 120

aactgtgagt ggtcagcttt ttcctgtttt cagaaggccc aactaaagtc agcaaataca 180

ggaaacaatg aaaggataat caatgtatca attaaaaagc tgaagaggaa accaccttcc 240

acaaatgcag ggagaagaca gaaacacaga ctaacatgcc cttcatgtga ttcttatgag 300

aaaaaaccac ccaaagaatt cctagaaaga ttcaaatcac ttctccaaaa gatgattcat 360

cagcatctgt cctctagaac acacggaagt gaagattcct ga 402

<210> 34

<211> 133

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL-21 mature peptide

<400> 34

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser

130

<210> 35

<211> 441

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL-21

<400> 35

atggagagga cccttgtctg tctggtagtc atcttcttgg ggacagtggc ccataaatca 60

agcccccaag ggccagatcg cctcctgatt agacttcgtc accttattga cattgttgaa 120

cagctgaaaa tctatgaaaa tgacttggat cctgaacttc tatcagctcc acaagatgta 180

aaggggcact gtgagcatgc agcttttgcc tgttttcaga aggccaaact caagccatca 240

aaccctggaa acaataagac attcatcatt gacctcgtgg cccagctcag gaggaggctg 300

cctgccagga ggggaggaaa gaaacagaag cacatagcta aatgcccttc ctgtgattcg 360

tatgagaaaa ggacacccaa agaattccta gaaagactaa aatggctcct tcaaaagatg 420

attcatcagc atctctccta g 441

<210> 36

<211> 146

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL-21

<400> 36

Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val

1 5 10 15

Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu

20 25 30

Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp

35 40 45

Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys

50 55 60

Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser

65 70 75 80

Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu

85 90 95

Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile

100 105 110

Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu

115 120 125

Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His

130 135 140

Leu Ser

145

<210> 637

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hXCL2

<400> 637

atgagacttc tcatcctggc cctccttggc atctgctctc tcactgcata cattgtggaa 60

ggtgtaggga gtgaagtctc acataggagg acctgtgtga gcctcactac ccagcgactg 120

ccagttagca gaatcaagac ctacaccatc acggaaggct ccttgagagc agtaattttt 180

attaccaaac gtggcctaaa agtctgtgct gatccacaag ccacgtgggt gagagacgtg 240

gtcaggagca tggacaggaa atccaacacc agaaataaca tgatccagac caagccaaca 300

ggaacccagc aatcgaccaa tacagctgtg accctgactg gctag 345

<210> 38

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hXCL2

<400> 38

Met Arg Leu Leu Ile Leu Ala Leu Leu Gly Ile Cys Ser Leu Thr Ala

1 5 10 15

Tyr Ile Val Glu Gly Val Gly Ser Glu Val Ser His Arg Arg Thr Cys

20 25 30

Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile Lys Thr Tyr

35 40 45

Thr Ile Thr Glu Gly Ser Leu Arg Ala Val Ile Phe Ile Thr Lys Arg

50 55 60

Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val Arg Asp Val

65 70 75 80

Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn Met Ile Gln

85 90 95

Thr Lys Pro Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala Val Thr Leu

100 105 110

Thr Gly

<210> 39

<211> 660

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL12

<400> 39

atgtgtccag cgcgcagcct cctccttgtg gctaccctgg tcctcctgga ccacctcagt 60

ttggccagaa acctccccgt ggccactcca gacccaggaa tgttcccatg ccttcaccac 120

tcccaaaacc tgctgagggc cgtcagcaac atgctccaga aggccagaca aactctagaa 180

ttttaccctt gcacttctga agagattgat catgaagata tcacaaaaga taaaaccagc 240

acagtggagg cctgtttacc attggaatta accaagaatg agagttgcct aaattccaga 300

gagacctctt tcataactaa tgggagttgc ctggcctcca gaaagacctc ttttatgatg 360

gccctgtgcc ttagtagtat ttatgaagac ttgaagatgt accaggtgga gttcaagacc 420

atgaatgcaa agcttctgat ggatcctaag aggcagatct ttctagatca aaacatgctg 480

gcagttattg atgagctgat gcaggccctg aatttcaaca gtgagactgt gccacaaaaa 540

tcctcccttg aagaaccgga ttttttataaa actaaaatca agctctgcat acttcttcat 600

gctttcagaa ttcgggcagt gactattgac agagtgacga gctatctgaa tgcttcctaa 660

<210> 40

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL12

<400> 40

Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu

1 5 10 15

Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro

20 25 30

Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val

35 40 45

Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys

50 55 60

Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser

65 70 75 80

Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys

85 90 95

Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala

100 105 110

Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr

115 120 125

Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys

130 135 140

Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu

145 150 155 160

Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr

165 170 175

Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys

180 185 190

Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr

195 200 205

Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser

210 215

<210> 41

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL12

<400> 41

atgtgtcaat cacgctacct cctctttttg gccacccttg ccctcctaaa ccacctcagt 60

ttggccaggg tcattccagt ctctggacct gccaggtgtc ttagccagtc ccgaaacctg 120

ctgaagacca cagatgacat ggtgaagacg gccagagaaa aactgaaaca ttattcctgc 180

actgctgaag acatcgatca tgaagacatc acacgggacc aaaccagcac attgaagacc 240

tgtttaccac tggaactaca caagaacgag agttgcctgg ctactagaga gacttcttcc 300

acaacaagag ggagctgcct gcccccacag aagacgtctt tgatgatgac cctgtgcctt 360

ggtagcatct atgaggactt gaagatgtac cagacagagt tccaggccat caacgcagca 420

cttcagaatc acaaccatca gcagatcatt ctagacaagg gcatgctggt ggccatcgat 480

gagctgatgc agtctctgaa tcataatggc gagactctgc gccagaaacc tcctgtggga 540

gaagcagacc cttacagagt gaaaatgaag ctctgcatcc tgcttcacgc cttcagcacc 600

cgcgtcgtga ccatcaacag ggtgatgggc tatctgagct ccgcctga 648

<210> 42

<211> 215

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL12

<400> 42

Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu

1 5 10 15

Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly Pro Ala Arg

20 25 30

Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Asp Asp Met Val

35 40 45

Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp

50 55 60

Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr

65 70 75 80

Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg

85 90 95

Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr

100 105 110

Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys

115 120 125

Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His

130 135 140

Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp

145 150 155 160

Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys

165 170 175

Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys

180 185 190

Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val

195 200 205

Met Gly Tyr Leu Ser Ser Ala

210 215

<210> 43

<211> 582

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hIL18

<400> 43

atggctgctg aaccagtaga agacaattgc atcaactttg tggcaatgaa atttattgac 60

aatacgcttt actttatagc tgaagatgat gaaaacctgg aatcagatta ctttggcaag 120

cttgaatcta aattatcagt cataagaaat ttgaatgacc aagttctctt cattgaccaa 180

ggaaatcggc ctctatttga agatatgact gattctgact gtagagataa tgcaccccgg 240

accatattta ttataagtat gtataaagat agccagccta gaggtatggc tgtaactatc 300

tctgtgaagt gtgagaaaat ttcaactctc tcctgtgaga acaaaattat ttcctttaag 360

gaaatgaatc ctcctgataa catcaaggat acaaaaagtg acatcatatt ctttcagaga 420

agtgtcccag gacatgataa taagatgcaa tttgaatctt catcatacga aggatacttt 480

ctagcttgtg aaaaagagag agaccttttt aaactcattt tgaaaaaaga ggatgaattg 540

ggggatagat ctataatgtt cactgttcaa aacgaagact ag 582

<210> 44

<211> 193

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hIL18

<400> 44

Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met

1 5 10 15

Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn

20 25 30

Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile

35 40 45

Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro

50 55 60

Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg

65 70 75 80

Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met

85 90 95

Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys

100 105 110

Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile

115 120 125

Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly

130 135 140

His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe

145 150 155 160

Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys

165 170 175

Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu

180 185 190

Asp

<210> 45

<211> 579

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL18

<400> 45

atggctgcca tgtcagaaga ctcttgcgtc aacttcaagg aaatgatgtt tattgacaac 60

acgctttact ttatacctga agaaaatgga gacctggaat cagacaactt tggccgactt 120

cactgtacaa ccgcagtaat acggaatata aatgaccaag ttctcttcgt tgacaaaaga 180

cagcctgtgt tcgaggatat gactgatatt gatcaaagtg ccagtgaacc ccagaccaga 240

ctgataatat acatgtacaa agacagtgaa gtaagaggac tggctgtgac cctctctgtg 300

aaggatagta aaatgtctac cctctcctgt aagaacaaga tcatttcctt tgaggaaatg 360

gatccacctg aaaatattga tgatatacaa agtgatctca tattctttca gaaacgtgtt 420

ccaggacaca acaagatgga gtttgaatct tcactgtatg aaggacactt tcttgcttgc 480

caaaaggaag atgatgcttt caaactcatt ctgaaaaaaa aggatgaaaa tggggataaa 540

tctgtaatgt tcactctcac taacttacat caaagttag 579

<210> 46

<211> 192

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL18

<400> 46

Met Ala Ala Met Ser Glu Asp Ser Cys Val Asn Phe Lys Glu Met Met

1 5 10 15

Phe Ile Asp Asn Thr Leu Tyr Phe Ile Pro Glu Glu Asn Gly Asp Leu

20 25 30

Glu Ser Asp Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg

35 40 45

Asn Ile Asn Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe

50 55 60

Glu Asp Met Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg

65 70 75 80

Leu Ile Ile Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val

85 90 95

Thr Leu Ser Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn

100 105 110

Lys Ile Ile Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp

115 120 125

Ile Gln Ser Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn

130 135 140

Lys Met Glu Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys

145 150 155 160

Gln Lys Glu Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu

165 170 175

Asn Gly Asp Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

180 185 190

<210> 47

<211> 570

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hIL23

<400> 47

atgctgggga gcagagctgt aatgctgctg ttgctgctgc cctggacagc tcagggcaga 60

gctgtgcctg ggggcagcag ccctgcctgg actcagtgcc agcagctttc acagaagctc 120

tgcacactgg cctggagtgc acatccacta gtgggacaca tggatctaag agaagaggga 180

gatgaagaga ctacaaatga tgttccccat atccagtgtg gagatggctg tgacccccaa 240

ggactcaggg acaacagtca gttctgcttg caaaggatcc accagggtct gattttttat 300

gagaagctgc taggatcgga tattttcaca ggggagcctt ctctgctccc tgatagccct 360

gtggcgcagc ttcatgcctc cctactgggc ctcagccaac tcctgcagcc tgagggtcac 420

cactgggaga ctcagcagat tccaagcctc agtcccagcc agccatggca gcgtctcctt 480

ctccgcttca aaatccttcg cagcctccag gcctttgtgg ctgtagccgc ccgggtcttt 540

gcccatggag cagcaaccct gagtccctaa 570

<210> 48

<211> 189

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hIL23

<400> 48

Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr

1 5 10 15

Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln

20 25 30

Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His

35 40 45

Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr

50 55 60

Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln

65 70 75 80

Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly

85 90 95

Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu

100 105 110

Pro Ser Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu

115 120 125

Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr

130 135 140

Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu

145 150 155 160

Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala

165 170 175

Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro

180 185

<210> 49

<211> 591

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL23

<400> 49

atgctggatt gcagagcagt aataatgcta tggctgttgc cctgggtcac tcagggcctg 60

gctgtgccta ggagtagcag tcctgactgg gctcagtgcc agcagctctc tcggaatctc 120

tgcatgctag cctggaacgc acatgcacca gcgggacata tgaatctact aagagaagaa 180

gaggatgaag agactaaaaa taatgtgccc cgtatccagt gtgaagatgg ttgtgaccca 240

caaggactca aggacaacag ccagttctgc ttgcaaagga tccgccaagg tctggctttt 300

tataagcacc tgcttgactc tgacatcttc aaaggggagc ctgctctact ccctgatagc 360

cccatggagc aacttcacac ctccctacta ggactcagcc aactcctcca gccagaggat 420

cacccccggg agacccaaca gatgcccagc ctgagttcta gtcagcagtg gcagcgcccc 480

cttctccgtt ccaagatcct tcgaagcctc caggcctttt tggccatagc tgcccgggtc 540

tttgcccacg gagcagcaac tctgactgag cccttagtgc caacagctta a 591

<210> 50

<211> 196

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL23

<400> 50

Met Leu Asp Cys Arg Ala Val Ile Met Leu Trp Leu Leu Pro Trp Val

1 5 10 15

Thr Gln Gly Leu Ala Val Pro Arg Ser Ser Ser Pro Asp Trp Ala Gln

20 25 30

Cys Gln Gln Leu Ser Arg Asn Leu Cys Met Leu Ala Trp Asn Ala His

35 40 45

Ala Pro Ala Gly His Met Asn Leu Leu Arg Glu Glu Glu Asp Glu Glu

50 55 60

Thr Lys Asn Asn Val Pro Arg Ile Gln Cys Glu Asp Gly Cys Asp Pro

65 70 75 80

Gln Gly Leu Lys Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln

85 90 95

Gly Leu Ala Phe Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly

100 105 110

Glu Pro Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr Ser

115 120 125

Leu Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg Glu

130 135 140

Thr Gln Gln Met Pro Ser Leu Ser Ser Ser Gln Gln Trp Gln Arg Pro

145 150 155 160

Leu Leu Arg Ser Lys Ile Leu Arg Ser Leu Gln Ala Phe Leu Ala Ile

165 170 175

Ala Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Thr Glu Pro Leu

180 185 190

Val Pro Thr Ala

195

<210> 51

<211> 813

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hIL33

<400> 51

atgaagccta aaatgaagta ttcaaccaac aaaatttcca cagcaaagtg gaagaacaca 60

gcaagcaaag ccttgtgttt caagctggga aaatcccaac agaaggccaa agaagtttgc 120

cccatgtact ttatgaagct ccgctctggc cttatgataa aaaaggaggc ctgttacttt 180

aggagagaaa ccaccaaaag gccttcactg aaaacaggta gaaagcacaa aagacatctg 240

gtactcgctg cctgtcaaca gcagtctact gtggagtgct ttgcctttgg tatatcaggg 300

gtccagaaat atactagagc acttcatgat tcaagtatca caggaatttc acctattaca 360

gagtatcttg cttctctaag cacatacaat gatcaatcca ttacttttgc tttggaggat 420

gaaagttatg agatatatgt tgaagacttg aaaaaagatg aaaagaaaga taaggtgtta 480

ctgagttact atgagtctca acacccctca aatgaatcag gtgacggtgt tgatggtaag 540

atgttaatgg taaccctgag tcctacaaaa gacttctggt tgcatgccaa caacaaggaa 600

cactctgtgg agctccataa gtgtgaaaaa ccactgccag accaggcctt ctttgtcctt 660

cataatatgc actccaactg tgtttcattt gaatgcaaga ctgatcctgg agtgtttata 720

ggtgtaaagg ataatcatct tgctctgatt aaagtagact cttctgagaa tttgtgtact 780

gaaaatatct tgtttaagct ctctgaaact tag 813

<210> 52

<211> 270

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>hIL33

<400> 52

Met Lys Pro Lys Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys

1 5 10 15

Trp Lys Asn Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser

20 25 30

Gln Gln Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg

35 40 45

Ser Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr

50 55 60

Thr Lys Arg Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His Leu

65 70 75 80

Val Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys Phe Ala Phe

85 90 95

Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser

100 105 110

Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser Thr

115 120 125

Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr Glu

130 135 140

Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val Leu

145 150 155 160

Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly

165 170 175

Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe

180 185 190

Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys Cys

195 200 205

Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His

210 215 220

Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe Ile

225 230 235 240

Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser Glu

245 250 255

Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr

260 265 270

<210> 53

<211> 801

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL33

<400> 53

atgagaccta gaatgaagta ttccaactcc aagatttccc cggcaaagtt cagcagcacc 60

gcaggcgaag ccctggtccc gccttgcaaa ataagaagat cccaacagaa gaccaaagaa 120

ttctgccatg tctactgcat gagactccgt tctggcctca ccataagaaa ggagactagt 180

tattttagga aagaacccac gaaaagatat tcactaaaat cgggtaccaa gcatgaagag 240

aacttctctg cctatccacg ggattctagg aagagatcct tgcttggcag tatccaagca 300

tttgctgcgt ctgttgacac attgagcatc caaggaactt cacttttaac acagtctcct 360

gcctccctga gtacatacaa tgaccaatct gttagttttg ttttggagaa tggatgttat 420

gtgatcaatg ttgacgactc tggaaaagac caagagcaag accaggtgct actacgctac 480

tatgagtctc cctgtcctgc aagtcaatca ggcgacggtg tggatgggaa gaagctgatg 540

gtgaacatga gtcccatcaa agacacagac atctggctgc atgccaacga caaggactac 600

tccgtggagc ttcaaagggg tgacgtctcg cctccggaac aggccttctt cgtccttcac 660

aaaaagtcct cggactttgt ttcatttgaa tgcaagaatc ttcctggcac ttacatagga 720

gtaaaagata accagctggc tctagtggag gagaaagatg agagctgcaa caatattatg 780

tttaagctct cgaaaatcta a 801

<210> 54

<211> 266

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL33

<400> 54

Met Arg Pro Arg Met Lys Tyr Ser Asn Ser Lys Ile Ser Pro Ala Lys

1 5 10 15

Phe Ser Ser Thr Ala Gly Glu Ala Leu Val Pro Pro Cys Lys Ile Arg

20 25 30

Arg Ser Gln Gln Lys Thr Lys Glu Phe Cys His Val Tyr Cys Met Arg

35 40 45

Leu Arg Ser Gly Leu Thr Ile Arg Lys Glu Thr Ser Tyr Phe Arg Lys

50 55 60

Glu Pro Thr Lys Arg Tyr Ser Leu Lys Ser Gly Thr Lys His Glu Glu

65 70 75 80

Asn Phe Ser Ala Tyr Pro Arg Asp Ser Arg Lys Arg Ser Leu Leu Gly

85 90 95

Ser Ile Gln Ala Phe Ala Ala Ser Val Asp Thr Leu Ser Ile Gln Gly

100 105 110

Thr Ser Leu Leu Thr Gln Ser Pro Ala Ser Leu Ser Thr Tyr Asn Asp

115 120 125

Gln Ser Val Ser Phe Val Leu Glu Asn Gly Cys Tyr Val Ile Asn Val

130 135 140

Asp Asp Ser Gly Lys Asp Gln Glu Gln Asp Gln Val Leu Leu Arg Tyr

145 150 155 160

Tyr Glu Ser Pro Cys Pro Ala Ser Gln Ser Gly Asp Gly Val Asp Gly

165 170 175

Lys Lys Leu Met Val Asn Met Ser Pro Ile Lys Asp Thr Asp Ile Trp

180 185 190

Leu His Ala Asn Asp Lys Asp Tyr Ser Val Glu Leu Gln Arg Gly Asp

195 200 205

Val Ser Pro Pro Glu Gln Ala Phe Phe Val Leu His Lys Lys Ser Ser

210 215 220

Asp Phe Val Ser Phe Glu Cys Lys Asn Leu Pro Gly Thr Tyr Ile Gly

225 230 235 240

Val Lys Asp Asn Gln Leu Ala Leu Val Glu Glu Lys Asp Glu Ser Cys

245 250 255

Asn Asn Ile Met Phe Lys Leu Ser Lys Ile

260 265

<210> 55

<211> 176

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL33-95-270

<400> 55

Ala Phe Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp

1 5 10 15

Ser Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu

20 25 30

Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser

35 40 45

Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys

50 55 60

Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly

65 70 75 80

Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys

85 90 95

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

100 105 110

Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn

115 120 125

Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val

130 135 140

Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser

145 150 155 160

Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr

165 170 175

<210> 56

<211> 172

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL33-99-270

<400> 56

Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser Ile Thr

1 5 10 15

Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser Thr Tyr Asn

20 25 30

Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr Glu Ile Tyr

35 40 45

Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val Leu Leu Ser

50 55 60

Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly Val Asp

65 70 75 80

Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe Trp Leu

85 90 95

His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys Cys Glu Lys

100 105 110

Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His Ser Asn

115 120 125

Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe Ile Gly Val

130 135 140

Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser Glu Asn Leu

145 150 155 160

Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr

165 170

<210> 57

<211> 162

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL33-109-270

<400> 57

His Asp Ser Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala

1 5 10 15

Ser Leu Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp

20 25 30

Glu Ser Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys

35 40 45

Asp Lys Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu

50 55 60

Ser Gly Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro

65 70 75 80

Thr Lys Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu

85 90 95

Leu His Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu

100 105 110

His Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro

115 120 125

Gly Val Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val

130 135 140

Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser

145 150 155 160

Glu Thr

<210> 58

<211> 531

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL33-95-270

<400> 58

gcctttggta tatcaggggt ccagaaatat actagagcac ttcatgattc aagtatcaca 60

ggaatttcac ctattacaga gtatcttgct tctctaagca catacaatga tcaatccatt 120

acttttgctt tggaggatga aagttatgag atatatgttg aagacttgaa aaaagatgaa 180

aagaaagata aggtgttact gagttactat gagtctcaac acccctcaaa tgaatcaggt 240

gacggtgttg atggtaagat gttaatggta accctgagtc ctacaaaaga cttctggttg 300

catgccaaca acaaggaaca ctctgtggag ctccataagt gtgaaaaacc actgccagac 360

caggccttct ttgtccttca taatatgcac tccaactgtg tttcatttga atgcaagact 420

gatcctggag tgtttatagg tgtaaaggat aatcatcttg ctctgattaa agtagactct 480

tctgagaatt tgtgtactga aaatatcttg tttaagctct ctgaaactta g 531

<210> 59

<211> 519

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL33-99-270

<400> 59

tcaggggtcc agaaatatac tagagcactt catgattcaa gtatcacagg aatttcacct 60

attacagagt atcttgcttc tctaagcaca tacaatgatc aatccattac ttttgctttg 120

gaggatgaaa gttatgagat atatgttgaa gacttgaaaa aagatgaaaa gaaagataag 180

gtgttactga gttactatga gtctcaacac ccctcaaatg aatcaggtga cggtgttgat 240

ggtaagatgt taatggtaac cctgagtcct acaaaagact tctggttgca tgccaacaac 300

aaggaacact ctgtggagct ccataagtgt gaaaaaccac tgccagacca ggccttcttt 360

gtccttcata atatgcactc caactgtgtt tcatttgaat gcaagactga tcctggagtg 420

tttataggtg taaaggataa tcatcttgct ctgattaaag tagactcttc tgagaatttg 480

tgtactgaaa atatcttgtt taagctctct gaaacttag 519

<210> 60

<211> 489

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hIL33-109-270

<400> 60

catgattcaa gtatcacagg aatttcacct attacagagt atcttgcttc tctaagcaca 60

tacaatgatc aatccattac ttttgctttg gaggatgaaa gttatgagat atatgttgaa 120

gacttgaaaa aagatgaaaa gaaagataag gtgttactga gttactatga gtctcaacac 180

ccctcaaatg aatcaggtga cggtgttgat ggtaagatgt taatggtaac cctgagtcct 240

acaaaagact tctggttgca tgccaacaac aaggaacact ctgtggagct ccataagtgt 300

gaaaaaccac tgccagacca ggccttcttt gtccttcata atatgcactc caactgtgtt 360

tcatttgaat gcaagactga tcctggagtg tttataggtg taaaggataa tcatcttgct 420

ctgattaaag tagactcttc tgagaatttg tgtactgaaa atatcttgtt taagctctct 480

gaaacttag 489

<210> 61

<211> 162

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL15

<400> 61

Met Lys Ile Leu Lys Pro Tyr Met Arg Asn Thr Ser Ile Ser Cys Tyr

1 5 10 15

Leu Cys Phe Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

20 25 30

Val Phe Ile Leu Gly Cys Val Ser Val Gly Leu Pro Lys Thr Glu Ala

35 40 45

Asn Trp Ile Asp Val Arg Tyr Asp Leu Glu Lys Ile Glu Ser Leu Ile

50 55 60

Gln Ser Ile His Ile Asp Thr Thr Leu Tyr Thr Asp Ser Asp Phe His

65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Asn Cys Phe Leu Leu Glu Leu Gln

85 90 95

Val Ile Leu His Glu Tyr Ser Asn Met Thr Leu Asn Glu Thr Val Arg

100 105 110

Asn Val Leu Tyr Leu Ala Asn Ser Thr Leu Ser Ser Asn Lys Asn Val

115 120 125

Ala Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Thr Phe

130 135 140

Thr Glu Phe Leu Gln Ser Phe Ile Arg Ile Val Gln Met Phe Ile Asn

145 150 155 160

Thr Ser

<210> 62

<211> 158

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mIL17

<400> 62

Met Ser Pro Gly Arg Ala Ser Ser Val Ser Leu Met Leu Leu Leu Leu

1 5 10 15

Leu Ser Leu Ala Ala Thr Val Lys Ala Ala Ala Ile Ile Pro Gln Ser

20 25 30

Ser Ala Cys Pro Asn Thr Glu Ala Lys Asp Phe Leu Gln Asn Val Lys

35 40 45

Val Asn Leu Lys Val Phe Asn Ser Leu Gly Ala Lys Val Ser Ser Arg

50 55 60

Arg Pro Ser Asp Tyr Leu Asn Arg Ser Thr Ser Pro Trp Thr Leu His

65 70 75 80

Arg Asn Glu Asp Pro Asp Arg Tyr Pro Ser Val Ile Trp Glu Ala Gln

85 90 95

Cys Arg His Gln Arg Cys Val Asn Ala Glu Gly Lys Leu Asp His His

100 105 110

Met Asn Ser Val Leu Ile Gln Gln Glu Ile Leu Val Leu Lys Arg Glu

115 120 125

Pro Glu Ser Cys Pro Phe Thr Phe Arg Val Glu Lys Met Leu Val Gly

130 135 140

Val Gly Cys Thr Cys Val Ala Ser Ile Val Arg Gln Ala Ala

145 150 155

Claims (25)

1. An engineered immune cell expressing (i) a cell surface molecule that specifically recognizes a ligand, (ii) one or more exogenous cytokines selected from the group consisting of IL12, IL18, IL-21, IL23 and IL33, and (iii) one or more exogenous chemokines selected from XCL2 and XCL1. 2. The engineered immune cell of claim 1, wherein the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor, a T cell fusion protein, a T cell antigen coupler, or a T cell receptor. 3. The engineered immune cell of claim 2, wherein the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, and an intracellular domain , the intracellular domain comprises a costimulatory domain and/or a primary signaling domain. 4. The engineered immune cell of any one of claims 1-3, wherein the cytokine is wild-type or a variant thereof, the variant having the same or similar function as the wild-type. 5. The engineered immune cell of claim 4, wherein the IL-21 is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 32, 34 or 36; IL12 is identical to SEQ ID NO: 40 or 42 The amino acid sequence shown is at least 90% identical, IL18 is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 44 or 46, and IL23 is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 48 or 50 Identity, IL33 is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 52, 54, 55, 56 or 57. 6. The engineered immune cell of any one of claims 1-5, wherein the XCL1 gene and SEQ ID The nucleic acid sequence shown in NO: 23 or 25 is at least 90% identical, or the polypeptide encoded by the XCL1 gene has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24 or 26, wherein the XCL2 gene is identical to The nucleic acid sequence shown in SEQ ID NO: 37 has at least 90% identity, or the polypeptide encoded by the XCL2 gene has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 38. 7. The engineered immune cell of any one of claims 1-5, wherein the immune cell is selected from T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells. 8. The engineered immune cell of claim 7, wherein the T cells are CD4+/CD8+ T cells, CD4+ helper T cells, CD8+ T cells, CD4-CD8-T cells, tumor-infiltrating cells, memory T cells , naive T cells, regulatory T cells, γδ-T cells or αβ-T cells. 9. The engineered immune cell of any one of claims 3-8, wherein the ligand binding domain is selected from the group consisting of IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv , sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies, anticalins and DARPIN. 10. The engineered immune cell of claim 3, wherein the ligand binding domain binds to a target selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, IL-22Ra, IL-2, Cortexin, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor alpha, ErbB2(Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2 , c-Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o- Acetyl-GD2, Folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP , WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, podin, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1 , MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, T MPRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, expressed by HIV, HCV, HBV and/or other pathogens molecule; and/or Neo-epitopes or neo-antigens. 11. The engineered immune cell of any one of claims 3-10, 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, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. 12. The engineered immune cell of any one of claims 3-11, wherein 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. 13. The engineered immune cell of any one of claims 3-12, 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(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 their combinations. 14. The engineered immune cell of any one of claims 1-13, wherein the immune cell further comprises at least one inactivated gene selected from the group consisting of: CD52, GR, TCRα, TCRβ, CD3γ, CD3δ, CD3ε , CD247ζ, HLA-I, HLA-II, B2M, PD1, CTLA-4, LAG3 and TIM3. 15. The engineered immune cell of any one of claims 1-14, wherein the expression of cytokines and chemokines is constitutive expression or conditional expression. 16. The engineered cell of claim 15, wherein the cytokine, chemokine is operably linked to an inducible or tissue-specific promoter so as to be conditionally expressed. 17. The engineered immune cell of any one of claims 1-16, wherein the cytokine, chemokine is operably linked to a localization domain. 18. A nucleic acid molecule comprising: (i) a nucleic acid sequence encoding a cell surface molecule that specifically recognizes a ligand, (ii) a nucleic acid sequence encoding a cytokine selected from the group consisting of IL12, IL18, IL-21 , IL23 and IL333, and (iii) a nucleic acid sequence encoding a chemokine selected from XCL1 and/or XCL2. 19. The nucleic acid molecule of claim 18, wherein the cell surface molecule that specifically recognizes a ligand is a chimeric antigen receptor. 20. A vector comprising the nucleic acid molecule of any one of claims 18-19. 21. The vector of claim 20, wherein the vector is selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus, and adeno-associated virus (AAV). 22. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-17, the nucleic acid molecule of any one of claims 18-19, or any one of claims 20-21 carrier, and one or more pharmaceutically acceptable excipients. 23. The engineered immune cell of any one of claims 1-17, the nucleic acid molecule of any one of claims 18-19, or the vector of any one of claims 20-21, or the Use of the pharmaceutical composition in the preparation of a medicament for treating cancer, infection or autoimmune disease. 24. The use of claim 23, wherein the cancer is a hematological tumor or a solid tumor. 25. A combination therapy comprising: (1) engineered immune cells and chemokines expressing exogenous cytokines; (2) engineered immune cells and cytokines expressing exogenous chemokines; or ( 3) Engineered immune cells, cytokines and chemokines, wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, and the cytokines are selected from IL12, IL18, IL-21, IL23 and IL33, and The chemokine is selected from XCL1 and XCL2.

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