CN115232797A

CN115232797A - Engineered immune cells and uses thereof

Info

Publication number: CN115232797A
Application number: CN202111354467.2A
Authority: CN
Inventors: 邢芸; 李国坤; 任江涛; 贺小宏; 王延宾; 韩露
Original assignee: Nanjing Bioheng Biotech Co Ltd
Current assignee: Nanjing Bioheng Biotech Co Ltd
Priority date: 2021-04-23
Filing date: 2021-11-16
Publication date: 2022-10-25
Also published as: WO2022223049A1

Abstract

The present invention relates to an engineered immune cell expressing (i) a cell surface molecule specifically recognizing 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 the group consisting of XCL2 and XCL1. The invention also provides the use of the engineered immune cells in the treatment of cancer, infection or autoimmune disease. Compared with the traditional engineered immune cell, the engineered immune cell has obviously improved tumor killing activity.

Description

Engineered immune cells and uses thereof

Technical Field

The present invention is in the field of immunotherapy. More specifically, the present invention relates to an 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 the group consisting of XCL2XCL1. More preferably, the cell surface molecule specifically recognizing the ligand is a chimeric antigen receptor.

Background

The tumor immunotherapy mainly eliminates tumor cells by regulating the human immune system and the tumor microenvironment and finally relying on autoimmunity. The immune system is a unified entity, and innate immunity plays an important role in tumor immunity.

Some antigen presenting cells, such as dendritic cells and macrophages, are the connecting bridge for innate and acquired immunity. The antigen presenting cell can identify and present tumor antigens to an acquired immune system, activate tumor specific T cells and further eliminate tumors. Thus, increasing 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. Successful control of tumors by CAR cells generally requires the following processes: activation of the immune system, activation and expansion of CAR cells, infiltration of activated CAR cells into tumor tissue and killing of tumor cells. However, there is a general problem with current CAR cell therapy, namely that the tumor microenvironment has an inhibitory effect on CAR cells, such that CAR cells cannot infiltrate the tumor tissue. Therefore, how to reduce the inhibitory effect of the tumor microenvironment on CAR cells, improve survival time of CAR cells, or recruit other immune cells to act synergistically with CAR cells is very important for improving the therapeutic effect of CAR cells.

Therefore, there is a need for an improved immunotherapy approach that can increase the tumor antigen presentation efficiency, induce adoptive immune response in the body, solve the problem of tumor heterogeneity and the inhibitory effect of tumor microenvironment, and thus increase the efficacy of CAR cell therapy.

Disclosure of Invention

In a first aspect, the present invention provides a novel engineered immune cell expressing (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 the group consisting of XCL2 and XCL1.

In one embodiment, the cell surface molecule specifically recognizing 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 cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell. Preferably, the T cell is a CD4+/CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a CD4-CD8-T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a regulatory T cell, a γ δ -T cell, or an α β -T cell.

In one embodiment, the cell surface molecule that specifically recognizes the ligand is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, and an endodomain comprising a costimulatory domain and/or a primary signaling domain. Wherein the ligand binding domain may be selected from IgG, fab ', F (ab ') 2, 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 specifically recognizing the ligand 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, 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-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/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, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globh, NY-BR-1, UPK2, HAVC 1, ADRB3, PANX3, NY-BR-1, and pK2 GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-L/Galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, LAP, LAM, TMPRSS2 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, PDL2, TGF β, APRIL, NKG2D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecule expressed by HIV, HCV, HBV and/or other pathogen; and/or a neoepitope or neoantigen.

In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28 and CD 278.

In one embodiment, the primary signaling domain is selected from the signaling domains of the following proteins: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the primary signalling domain is a signalling domain comprising CD3 ζ.

In one embodiment, the co-stimulatory domain is one or more intracellular regions of a protein 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 (OX 40), CD137 (4-1 BB), 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 co-stimulatory domain is an intracellular region of CD27, CD28, CD134, CD137 or CD278 or a combination thereof.

In one embodiment, the expression or activity of the exogenous cytokines (e.g., IL12, IL18, IL21, IL23, and IL 33) and/or chemokines (e.g., XCL1, XCL 2) is constitutive expression. In another embodiment, the expression or activity of the exogenous cytokine and/or chemokine is conditional expression. For example, conditional expression can be achieved by operably linking an exogenous gene to an inducible, repressible, or tissue-specific promoter.

In one embodiment, the cytokine and/or chemokine may be operably linked to a localization domain that can localize the exogenous gene of the invention to a specific cellular location for expression, e.g., the cell membrane, a specific organelle in the cytoplasm, e.g., endoplasmic reticulum, golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, leader peptides, transmembrane domains, and the like. In one embodiment, the exogenous gene cytokines and/or chemokines of the present invention are operably linked to a transmembrane domain, thereby anchoring expression at the surface of the engineered immune cell.

In a second aspect, the invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a cell surface molecule specifically recognizing 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 specifically recognizing 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 invention also provides a vector comprising the nucleic acid molecule described above. In particular, the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous Sarcoma Viruses (RSV), polyoma viruses, and adeno-associated viruses (AAV). In some embodiments, the vector further comprises elements such as an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a poly A tail (polyA), a 3'UTR, a 5' UTR, an enhancer, a terminator, an insulator, an operator, a selectable marker, a reporter gene, a targeting sequence, and/or a protein purification tag. In a specific embodiment, the vector is an in vitro transcription vector.

In one embodiment, the invention also provides a pharmaceutical composition comprising an engineered immune cell, nucleic acid molecule or vector of the invention, and one or more pharmaceutically acceptable excipients.

In a third aspect, the invention also provides a method of treating a subject having cancer, an infection or an autoimmune disease, comprising administering to the subject an effective amount of an immune cell, a nucleic acid molecule, a vector or a pharmaceutical composition according to the invention.

In one embodiment, the cancer is a solid tumor or a hematological tumor. More specifically, the cancer is selected from: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, cancer of the biliary tract, cancer of the bladder, bone cancer, cancer of the brain and CNS, breast cancer, cancer of the peritoneum, cancer of the cervix, choriocarcinoma, colon and rectum cancer, cancer of the connective tissue, cancer of the digestive system, endometrial cancer, esophageal cancer, cancer of the eye, cancer of the head and neck, stomach cancer, glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumor, kidney cancer, larynx cancer, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumor of the urinary system vulvar and other carcinomas and sarcomas, as well as B-cell lymphomas, mantle cell lymphomas, AIDS-related lymphomas, waldenstrom macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumors, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmacytic lymphoma, preleukemia, plasmacytoid dendritic cell tumors, malignant lymphomas, malignant lymphoproliferative disorders, malignant lymphomas, and other cancers and sarcomas, as well as B-cell lymphomas, mantle cell lymphomas, AIDS-related lymphomas, and other cancers, and lymphomas, 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 I diabetes, celiac disease, graves 'disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, addison's disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus, among others.

The engineered immune cell has the advantages that the co-expressed cell factor and/or chemotactic factor can effectively promote the differentiation or recruitment of DC cells at a tumor part, increase the number of the DC cells and increase the proliferation and survival time of the engineered immune cell, so that the inhibition effect of a tumor microenvironment on the engineered immune cell is reduced, the tumor killing capacity of the engineered immune cell is improved, the increased DC cells can activate the adoptive immune recognition of the T cells of an organism to form a synergistic effect with the engineered immune cell, and the inhibition on the tumor is finally enhanced.

Drawings

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

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

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

FIG. 4 is a schematic view of: IFN- γ release levels after co-culture of CAR-T cells with target and non-target cells, respectively.

FIG. 5 is a schematic view of: killing effect of CAR-T cells on target cells at different effective target ratios.

FIG. 6: weight change profile of mice after treatment of mouse pancreatic cancer with CAR-T cells.

FIG. 7: tumor growth curves in mice after treatment of mouse pancreatic cancer with CAR-T cells.

FIG. 8: IFN- γ release levels after coculture of CAR-T cells expressing IL12, IL23 or a combination thereof with XCL1 with target and non-target cells, respectively.

FIG. 9: IFN- γ release levels after coculture of CAR-T cells expressing IL18, IL33 or a combination thereof with XCL1 with target and non-target cells, respectively.

FIG. 10: tumor suppression effect of CAR-T cells expressing IL12, IL23 or a combination thereof with XCL1 in vivo.

FIG. 11: tumor-inhibiting effect of CAR-T cells expressing IL18, IL33 or a combination thereof with XCL1 in vivo.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 that is capable of specifically binding to a target molecule (e.g., a ligand). Such surface molecules generally comprise a ligand binding domain capable of specific binding to a ligand, a transmembrane domain anchoring the surface molecule to the cell surface, and an intracellular domain responsible for signal transmission. 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 responds to antigen presentation and participates in T cell activation. Stimulation of TCRs is triggered by major histocompatibility complex Molecules (MHC) on antigen presenting cells that present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling. TCRs are composed of six peptide chains that form heterodimers, which are generally classified into α β type and γ δ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to specific antigens and MHC molecules. The variable region of the TCR may comprise or be operably linked to a ligand-binding domain, wherein the ligand-binding domain is defined as follows.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes a ligand binding domain (e.g., 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 domain linked by a linker. CARs are able to redirect the specificity and reactivity of T cells and other immune cells to a selected target in a non-MHC-restricted manner using the antigen binding properties of monoclonal antibodies. non-MHC-restricted antigen recognition gives CAR cells the ability to recognize antigens independent of antigen processing, thus bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does 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 a TCR, typically consisting of TCR subunits and an antigen-binding region linked thereto, and expressed on the cell surface. Wherein the TCR subunit comprises at least a portion of a TCR extracellular domain, a transmembrane domain, a TCR intracellular signaling domain.

As used herein, the term "T cell antigen coupler" or "TAC" includes three functional domains: 1 a tumor targeting domain comprising a single chain antibody, designed ankyrin repeat protein (DARPin), or other targeting group; 2 an extracellular domain, a single chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3 transmembrane region and the intracellular region of the CD4 co-receptor, wherein the intracellular region is linked to the 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 to a ligand (e.g., an antigen). The ligand binding domain may be an antibody structure including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies and functional fragments thereof. For example, ligand binding domains include, but are not limited to, igG, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin, DARPIN, and the like, preferably selected from Fab, scFv, sdAb, and nanobody. 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 natural specific binding polypeptide or natural receptor structure of a particular protein, such as PD1, PDL2, TGF β, APRIL and NKG2D.

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

"Single-chain antibody" or "scFv" is an antibody in which an antibody variable region (VH) and a light chain variable region (VL) are linked via a linker. The optimal length and/or amino acid composition of the linker may be selected. The length of the linker will significantly affect the variable region folding and interaction profiles of the scFv. In fact, if shorter linkers are used (e.g., between 5-10 amino acids), intra-strand folding may be prevented. For selection of the size and composition of the linker, see, e.g., hollinger et al, 1993proc Natl acad.sci.u.s.a.90; U.S. patent application publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT publication nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference in their entirety. The scFv may comprise VH and VL linked in any order, for example VH-linker-VL or VL-linker-VH.

"Single domain antibody" or "sdAb" refers to an antibody that naturally lacks a light chain, which comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also referred to as "heavy chain antibodies".

"Nanobody" or "Nb" refers to a VHH structure that is cloned and expressed individually, has structural stability comparable to that of an original heavy chain antibody and binding activity to an antigen, and is the smallest unit currently known to bind to a 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 (i.e., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to an amino acid modification that does 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 those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Conservative modifications may be selected, for example, 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" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a parent amino acid sequence and retains the biological activity, e.g., binding activity, of the parent amino acid.

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of the exact same sequence have 100% identity. One 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 to be identified that is associated with a particular disease state, e.g., a tumor-specific antigen or tumor-associated antigen. Thus, in one embodiment, the ligand binding domain of the invention binds 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, 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-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/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, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globh, NY-BR-1, UPK2, HAVC 1, ADRB3, PANX3, NY-BR-1, and pK2 GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-L/Galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, LAP, and, TMPRSS2 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, PDL2, TGF β, APRIL, NKG2D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecule expressed by HIV, HCV, HBV and/or other pathogen; and/or a neoepitope or neoantigen. Depending on the antigen to be targeted, the CAR of the invention may be designed to include a ligand binding domain specific for that antigen. For example, if CD19 is the antigen to be targeted, a CD19 antibody may be used as the ligand binding domain of the invention. In one embodiment, the CAR of the invention comprises a CD19 scFv comprising an amino acid sequence that is identical to SEQ ID No. 2, positions 1-107 or SEQ ID NO:14, a light chain variable region sequence having at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in positions 1-107 of SEQ ID NO:2 from 123 to 242 or SEQ ID NO: a heavy chain variable region sequence comprising at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence depicted at positions 123-238 of position 14.

In a preferred embodiment, the cell surface molecule specifically recognizing a ligand of the present invention is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory domain and/or a primary signaling domain.

As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables a chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and to direct the cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to a target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 epsilon subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. 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 a CD8 alpha chain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 4 or 16, or the coding sequence of the CD8 alpha transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID No. 3 or 15.

In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located 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 connect a transmembrane domain to a ligand binding domain. In particular, 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 may be derived in whole or in part from a native molecule, such as from the extracellular region of CD8, CD4 or CD28, or 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, said hinge region comprises a portion of the CD8a chain, fc γ RIII a receptor, igG4 or IgG1 hinge region, more preferably a CD8a hinge, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 12 or 22, or the coding sequence of the CD8a hinge has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID No. 11 or 21.

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

In one embodiment, the chimeric antigen receptor of the invention comprises a primary signaling domain that can be cytoplasmic sequences of T cell receptors and co-receptors that work together to initiate primary signaling upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar function. The primary signaling domain may comprise 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 FcR γ, fcR β, CD3 γ, CD3 δ, CD3 e, CD3 ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signalling domain of a CAR of the invention may comprise a CD3 zeta signalling domain which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 8 or 20 or whose coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID No. 7 or 19.

In one embodiment, the chimeric antigen receptor of the present invention comprises one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule, comprising the entire intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of co-stimulatory domains of the invention include, but are not limited to, co-stimulatory signaling domains derived from: 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 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70. Preferably, the co-stimulatory domain of a CAR of the invention is from 4-1BB, CD28, CD27, OX40 or a combination thereof. In one embodiment, the CAR of the invention comprises a co-stimulatory domain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence depicted in SEQ ID No. 6 or 18, or the coding sequence of the co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence depicted in SEQ ID No. 5 or 17.

In one embodiment, the CAR of the invention may further comprise a signal peptide such that when it is expressed in a cell, for example a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long hydrophobic amino acid segment that has a tendency to form a single alpha-helix. At the end of the signal peptide there is usually a stretch of amino acids which is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during translocation or after completion to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, such as those derived from CD8 α, igG1, GM-CSFR α, and the like. In one embodiment, the signal peptide useful in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 10 or 30, 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 to the amino acid sequence shown in SEQ ID NO. 9 or 29.

In one embodiment, the CAR of the invention may further comprise a switch structure to regulate the expression time of the CAR. For example, the switch structure may be in the form of a dimerization domain that causes a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain to allow binding to the targeted antigen, thereby activating the signaling pathway. Alternatively, a switch domain may be used to connect the binding domain and the signaling domain, respectively, such that the binding domain and the signaling domain are connected together through a dimer only when the switch domains are bound to each other (e.g., in the presence of an inducing compound) to activate the signaling pathway. The switch structure may also be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain, preventing its binding to the antigen to be targeted, and when the masking peptide is cleaved, for example by a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure. Various switch configurations known to those skilled in the art may be used with the present invention.

In one embodiment, the CAR of the invention may also comprise a suicide gene, i.e., one that causes it to express a cell death signal that can be induced by a foreign substance, in order to clear the CAR cells when needed (e.g., when severe toxic side effects are produced). For example, the suicide gene may be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when desired, the CAR cells can be eliminated by adding antibodies or agents that target these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir therapy. The suicide gene can also be iCaspase-9, and iCaspase-9 can be induced to dimerize through chemical induction drugs such as AP1903, AP20187 and the like, so that downstream Caspase3 molecules are activated, and apoptosis is caused. 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 co-stimulatory domain and a CD3 zeta intracellular signaling domain, (b) a CD27 co-stimulatory domain and a CD3 zeta intracellular signaling domain, (c) a CD28 co-stimulatory domain and a CD3 zeta intracellular signaling domain, (d) an OX40 co-stimulatory domain and a CD3 zeta intracellular signaling domain, (e) a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain, (f) an OX40 co-stimulatory domain, a 4-1BB co-stimulatory domain and a CD3 zeta intracellular signaling domain, or (g) a CD28 co-stimulatory domain, an OX40 co-stimulatory domain and a CD3 zeta intracellular signaling domain.

Exogenous gene

In addition to the cell surface molecules specifically recognizing the antigen, the engineered immune cells of the invention express one or more exogenous cytokines selected from the group consisting of IL12, IL18, IL21, IL23 and IL33 and one or more exogenous chemokines selected from the group consisting of XCL1 and XCL2.

The cytokines used in the present invention are mainly interleukins selected from the group consisting of IL-21, IL12, IL18, IL33 and IL23. Interleukins are produced by and function among leukocytes, playing important roles in, for example, transmitting messages, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and in inflammatory responses. In general, the biological effects of interleukins are achieved by their binding to the corresponding receptors, for example the biological properties of IL-21 are achieved by the binding of IL-21 to its receptor IL-21R. At present, at least 38 interleukins, named IL1 to IL38, are found, and their functions are complex. Based on structural homology, interleukins can be classified into several protein families, such as the IL1 family (including IL1 α, IL1 β, IL18, IL37, IL38, IL33, etc.), the IL2 family (including IL2, IL4, IL13, IL15, IL21, etc.), the IL6 family (including IL6, IL12, IL23, IL27, IL35, etc.), the IL10 family (including IL10, IL19, IL20, IL22, IL26, etc.), the IL17 family (including IL17 and IL 25), 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, said variant having the same or similar, even better function as the wild type.

The amino acid sequence of the wild-type hIL-21 propeptide is shown in SEQ ID NO:32, the amino acid sequence of the mature peptide is shown as SEQ ID NO:34 (consisting of amino acids 30 to 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 thereof can enhance proliferation of activated CD8+ T cells, enhance cytotoxic activity of NK cells, and promote proliferation and differentiation of B cells.

In one embodiment, the invention uses IL-21 is wild type IL-21 or IL-21 variants, such as at least retained all or most IL-21 activity, even with improved activity of IL-21 variants.

In one embodiment, the IL-21 variant used in the present invention may be, for example, a variant of IL-21 produced by deletion and/or substitution of SEQ ID NO:34, amino acids 65-96, such as: (1) deletion and/or substitution of SEQ ID NO:34, amino acids 83-86, amino acids 83-88, amino acids 83-90, amino acids 82-88, amino acids 77-92, amino acids 71-92, amino acids 65-92, and amino acids 77-96; (2) A variant sequence comprising an additional N-terminal Met based on (1); (3) Variant sequences obtained by conservative modifications of up to 10 amino acids in the sequences of (1) and (2). See, for example, WO2006111524, the entire contents of which are incorporated herein by reference.

In one embodiment, the IL-21 gene used in the present invention encodes a polypeptide that hybridizes to the complement of SEQ ID NO: 32. 34 or 36, or the IL-21 gene has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31. 33 or 35, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

IL12 is a heterodimeric cytokine, encoded by 2 independent genes, IL-12A (p 35) and IL-12B (p 40). IL12 is produced by dendritic cells, macrophages, neutrophils and other antigen presenting cells and promotes proliferation of T helper 1 (Th 1); inducing NK cells and T cells to generate gamma interferon and improving the cytotoxicity of the NK cells; promoting the formation of cytotoxic T cells, and the like.

In one embodiment, the invention uses IL12 is wild type IL12 or IL12 variants, such as at least retained all or most IL12 activity, even with improved activity of IL12 variants. In one embodiment, the IL12 used in the present invention is substantially identical to SEQ ID NO:40 or 42, or a gene encoding IL-12 and SEQ ID NO:39 or 41 have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100%.

IL23 is also a heterodimeric cytokine consisting of two subunits, p40 and p 19. IL23 is produced primarily by activated dendritic cells, macrophages and monocytes, among others. 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 invention uses IL-23 is wild type IL-23 or IL-23 variants, such as at least retained all or most IL-23 activity, even with improved activity of IL-23 variants. In one embodiment, IL-23 used in the present invention is substantially identical to the sequence of SEQ ID NO:48 or 50, or the gene encoding IL-23 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:47 or 49, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity. IL18 is produced primarily by macrophages, and may be produced by a variety of immune and non-immune cells such as monocytes, dendritic cells, epithelial cells, fibroblasts, and the like. IL18 mediates MyD 88-NF-. Kappa.B signaling pathways by binding to the heterodimeric receptor IL18 R.alpha.R.beta.and is capable of strongly inducing secretion of IFN.gamma.. Since IL18 induces the proliferation and enhances the activity of immune cells, its role in anti-tumor, anti-infection and immunomodulation has been extensively studied in recent years.

In one embodiment, the IL18 used in the present invention is a wild-type IL18 or IL18 variant, e.g. an IL18 variant which retains at least all or most of the IL18 activity, even with improved activity. In one embodiment, IL18 used in the present invention is substantially identical to SEQ ID NO:44 or 46, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% 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% sequence identity with the amino acid sequence set forth in SEQ ID NO:43 or 45, have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100%.

IL33 is a bifunctional protein, which can be used as a molecule positioned in the nucleus to play the role of a transcription factor, can be secreted out of the cell to play the role of a cell factor, and is closely related to a plurality of diseases such as allergy, autoimmune diseases, cardiovascular diseases, infection, tumor and the like. IL33 is expressed in a variety of cells, including epithelial cells, fibroblasts, macrophages, endothelial cells, mast cells, dendritic cells, and osteoblasts, among others. These cells release IL33 upon injury, and IL33 may produce different inflammatory responses depending on the cell type.

In one embodiment, the IL33 used in the present invention is a wild-type IL33 or IL33 variant, e.g. an IL33 variant which at least retains all or most of the activity of IL33, even with improved activity. For example, it has been found that the full length of IL33 can be cleaved to the mature form of IL33 _95-270 (consisting of amino acids 95 to 270 of full-length IL 33), IL33 _99-270 (consisting of amino acids 99 to 270 of full-length IL 33) and IL33 _109-270 (consisting of amino acids 109-270 of full-length IL 33), these fragments are released extracellularly by injured or necrotic cells and are 10-fold more biologically active than full-length IL33 (see Lefrancais, E et al, IL-33 is processed in biological activity by neutral phenaphil enzyme and cathepsin G.Proc.Natl Acad.Sci.USA 109, 1673-1678). In one embodiment, IL33 used in the present invention is substantially identical to SEQ ID NO: 52. 54, 55, 56 or 57, or a gene encoding IL33 and SEQ ID NO: 51. 53, 58, 59 or 60 or a pharmaceutically acceptable salt thereofThe columns have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100%.

The C-type chemokine family, also known as lymphokines, includes two members XCL1 and XCL2, which are produced primarily by CD8+ T cells and natural killer cells. XCL1 has unique sequence characteristics and two interconvertible protein spatial conformations, which distinguishes XCL1 from other chemokines and performs unique functions. XCL 1-specific receptor XCR1 is a member of the G protein-coupled receptor family, and the interaction between them not only plays an important role in negative selection of the thymus and establishment of autoimmune tolerance, but also initiates cross-antigen presentation and mediates cytotoxic immune responses. XCL1 not only regulates the immune system balance and maintains intestinal immune homeostasis, but also is associated with various diseases such as autoimmune diseases, nephritis, tuberculosis, and hiv infection. XCL2 has 97% identity to the nucleic acid sequence of XCL1, wherein the two amino acid residues at positions 7 and 8 are different: asp and Lys are contained in XCL1, and His and Arg are contained in XCL2. It has been found that XCL2 is very similar to XCL1 in expression profile, structure and function, for example, as in XCL1, XCL2 also has two interconvertible protein spatial conformations, a monomeric conformation that binds and activates XCR1 and a dimeric conformation that has a higher affinity for hairpin structures in glycosaminoglycans (GAGs). The receptors XCR1 of XCL1 and XCL2 are selectively expressed on DC (cDC 1) cells with antigen presenting capability, and research shows that the introduction of XCL1 can effectively improve the curative effect of antitumor immunotherapy and targeted vaccine.

In one embodiment, XCL1 as used herein is substantially identical to SEQ ID NO:24 or 26, or the coding sequence of XCL1 shares at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:23 or 25, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

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

Expression of foreign genes

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

In one embodiment, the expression of the exogenous cytokine and/or chemokine is a conditional expression. For example, the foreign gene of the present invention may be operably linked to an inducible or tissue-specific promoter, as necessary, to regulate the expression level of the introduced foreign gene at a specific time or in a specific tissue, cell type. In one embodiment, the promoter is an inducible promoter, i.e., a promoter that initiates transcription only in the presence of a particular environmental condition, developmental condition, or inducer. Such environmental conditions include, for example, tumor acidic microenvironments, tumor hypoxic microenvironments, and the like. Such inducers include, for example, cermetin, tetracycline or analogs thereof, including, for example, chlortetracycline, oxytetracycline, demethylchlortetracycline, methacycline, doxycycline and minocycline. Inducible promoters include, for example, lac operator sequences, tetracycline operator sequences, galactose operator sequences, or doxycycline operator sequences, among others. In one embodiment, the expression of the cytokine and/or chemokine is secretory or anchored, e.g., may be operably linked to a localization domain that can localize the exogenous gene of the invention to a specific cellular location, e.g., the cell membrane, a specific organelle in the cytoplasm, e.g., endoplasmic reticulum, golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, leader peptides, transmembrane domains, and the like. In one embodiment, the exogenous gene of the invention is operably linked to a transmembrane domain, thereby anchoring expression at the surface of an engineered immune cell.

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

Nucleic acids

The 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 the group consisting of IL12, IL18, IL-21, IL23 and IL33, and (iii) a nucleic acid sequence encoding a chemokine selected from the group consisting of 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. The definition of chimeric antigen receptor is as described 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, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

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

The nucleic acid sequences of the invention may be codon optimized for optimal expression in a desired host cell (e.g., an immune cell); 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, with the codons before and after the replacement encoding the same amino acid. Thus, the choice of optimal codons depends on the codon usage bias of the host genome.

Carrier

The invention also provides a vector comprising a nucleic acid according to the invention. Wherein the nucleic acid sequence encoding a cell surface molecule specifically recognizing the ligand, the nucleic acid sequence encoding a cytokine selected from the group consisting of IL12, IL18, IL-21, IL23, IL33, the nucleic acid sequence encoding a chemokine selected from the group consisting of XCL1 and XCL2 may be located in one or more vectors. When located in the same vector, these nucleic acid sequences may be linked by, for example, a 2A peptide.

As used herein, the term "vector" is a vector nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into a host cell where it can be replicated and/or expressed, for example.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium through which an isolated nucleic acid is delivered to the interior of a cell, for example, by homologous recombination or by using a hybrid recombinase that targets sequences at specific sites. An "expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding the chimeric antigen receptor polypeptides of the invention) in a suitable host cell and the translation of their mRNA. Suitable carriers useful in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the invention include, but are not limited to, plasmids, viruses (e.g., retrovirus, lentivirus, adenovirus, vaccinia virus, rous sarcoma virus (RSV, polyoma virus and adeno-associated virus (AAV), etc.), bacteriophage, phagemid, cosmid, and artificial chromosomes (including BAC and YAC). The vectors themselves are typically nucleotide sequences, typically DNA sequences comprising an insert (transgene) and a larger sequence as the "backbone" of the vector.

Engineered immune cells

The invention also provides an engineered immune cell comprising a nucleic acid or vector of the invention. In other words, the engineered immune cells of the invention express a cell surface molecule that specifically recognizes the ligand, one or more exogenous cytokines selected from IL12, IL18, IL-21, IL23, IL33, and one or more exogenous chemokines selected from 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 (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cells may be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells obtained from stem cell sources such as cellular umbilical cord blood. Preferably, the immune cell is a T cell. The T cell may be any T cell, such as a T cell cultured in vitro, e.g., a primary T cell, or a T cell derived from a T cell line cultured in vitro, e.g., jurkat, supT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. The T cells may be at any developmental stage, including, but not limited to, CD4+/CD8+ T cells, CD4-CD8-T cells, CD4+ helper T cells (e.g., th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, regulatory T cells, γ δ -T cells, α β -T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled 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 (e.g., by transduction, transfection, transformation, and the like). "transfection" is the process of introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. One example is RNA transfection, the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. The term is used primarily for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection may be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of a nucleic acid molecule or polynucleotide (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). The transformation may be achieved by artificial means. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques may include heat shock mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation.

In yet another embodiment, the immune cell of the invention further comprises at least one inactivated gene selected from the group consisting of: CD52, GR, TCR alpha, TCR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD247 zeta, HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More particularly, at least the TCR component (including TCR α, TCR β genes) or the CD3 component (including CD3 γ, CD3 δ, CD3 ε, CD247 ζ) in the immune cell is 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, for example, by mediating DNA cleavage by meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems, thereby inactivating the gene.

Pharmaceutical compositions and combination therapies

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

As used herein, the term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesirable local or systemic effects) and are well known in the art (see, e.g., remington's pharmaceutical sciences. Edied 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, anti-adherents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonizing agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients to prepare the desired pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical composition according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Methods of parenteral delivery 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 in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or extracts of fluid extracts, or in a form which is particularly suitable for the desired method of administration. Processes known in the art for the manufacture of medicaments may comprise, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as described herein are typically provided in solution form and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical compositions according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the diseases to be treated. Preferred examples of the pharmaceutical agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rebeccin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium II (sorfimer sodium phosphate II), temozolomide, topotecan, glucuronic acid trimetrexate (trimetrenate glucoside), oritavastin E (auristatin E), vincristine and adriamycin; 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, and the like; antibodies or fragments thereof, such as anti-CD 3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains, and viral/bacterial peptides. In addition, the pharmaceutical compositions of the present invention may also be used in combination with one or more other therapeutic methods, such as chemotherapy, radiation therapy.

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

Therapeutic applications

The invention also provides a method of treating a subject having cancer, an infection or an autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the invention. Thus, the invention also encompasses the use of said engineered immune cells in the preparation 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 cell is an autologous or allogeneic cell, preferably a T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell, more preferably a T cell, NK cell or NKT cell.

As used herein, the term "autologous" means that any material derived from an individual will be reintroduced into the same individual at a later time.

As used herein, the term "allogeneic" refers to any material derived from a different animal or patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered allogeneic to each other. In some cases, genetic differences in allogenic material from individuals of the same species may be sufficient for antigen interactions to occur.

As used herein, the term "subject" is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously 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 a target to which the ligand binding domain binds, such as a hematologic tumor or a solid tumor. For example, the cancer includes, but is not limited to: <xnotran> , , , , , , , , CNS , , , , , , , , , , , , ( ), (GBM), , , , , , , ( , , ), ( ), , , , ( , , ), , , , , , , , , , , , , , , , , B ( / (NHL), (SL) NHL, / NHL, NHL, NHL, NHL, NHL, NHL), , AIDS , Waldenstrom , (CLL), (ALL), B (B-ALL), </xnotran> T cell acute lymphocytic leukemia (T-ALL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmacytoma, 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 the pharmaceutical compositions of the invention are selected from: leukemia, lymphoma, multiple myeloma, cerebral glioma, pancreatic cancer, gastric cancer, and the like.

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

The invention will be described in detail below with reference to the drawings and examples. It should be noted that the appended drawings and their examples are provided for illustrative purposes only and are not intended to limit the present invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Detailed Description

Example 1 preparation of CAR-T cells

1. Construction of retroviral plasmids

The sequence fragments of the coding sequence of mCD19-scFv (SEQ ID NO: 13), mCD8a hinge region (SEQ ID NO: 21), mCD8a transmembrane region (SEQ ID NO: 15), mouse 41bb intracellular domain (SEQ ID NO: 17) and mouse CD3 zeta intracellular domain (SEQ ID NO: 19) which are connected in sequence are artificially synthesized, and XhoI/EcoRI enzyme cutting sites are added at two ends. The fragment was cloned into MSCV vector to obtain MSCV-mCD19-CAR plasmid.

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

A fragment of the coding sequence connecting T2A and mouse XCL1 (SEQ ID NO: 25) in sequence was synthesized artificially and EcoRI/SalI cleavage sites were added to both ends. Cloning the fragment into MSCV-mCD19-CAR vector to obtain MSCV-mCD19-CAR-XCL1 plasmid.

2. Preparation of retrovirus

In T175 flasks, 30X 10 ⁶ Density of individual cells/flask 293T cells were inoculated into 30ml of DMEM medium containing 10% fetal bovine serum and cultured overnight in a 5% CO2 incubator at 37 ℃ for virus packaging.

To a sterile tube, 3ml of Opti-MEM (Gibco, cat # 31985-070), 45. Mu.g of a retrovirus plasmid (MSCV-mCD 19-CAR plasmid, MSCV-mCD19-CAR-IL-21 plasmid, or MSCV-mCD19-CAR-XCL1 plasmid), and 15. Mu.g of a packaging vector pCL-Eco (Haematococcus, mcSt.M., cat # P3029) were added. Then 120. Mu.l of X-trememe GENE HP DNA transfection reagent (Roche, cat # 06366236001) was added, mixed immediately and incubated at room temperature for 15min. This plasmid/vector/transfection reagent mixture was then added dropwise to a flask of 293T cells prepared in advance, and incubated overnight at 37 ℃,5% CO2. The culture was collected 72 hours after transfection, and centrifuged (2000 g,4 ℃,10 minutes) to obtain a retrovirus supernatant.

3. Preparation of CAR-T cells

T lymphocytes were isolated from mouse spleen and CTS with DynaBeads CD3/CD28 ^TM (Gibco, cat # 40203D) activates T cells, then CO at 37 ℃ and 5% ₂ The culture was performed for 1 day.

At a rate of 3X 10 per hole ⁶ Density of individual cells/mL activated T cells were seeded into 24-well plates previously coated overnight with RetroNectin, then 500 μ L complete media (NT, control), MSCV-mCD19-CAR virus, MSCV-mCD19-CAR-IL-21 virus, or MSCV-mCD19-CAR-IL-21 virus + MSCV-mCD19-CAR-XCL1 virus, respectively, were added and complete media was supplemented to 2mL.

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

Example 2 detection of expression of CAR-T cells

1. Expression levels of cell surface CARs

2X 10 of the product prepared in example 1 were taken out ⁵ Individual CAR-T cells, treated with Goat Anti-Rat IgG (H)&L) Biotin (BioVision, cat # 6910-250) as a primary antibody and APC Streptavidin (BD Pharmingen, cat # 554067) as a secondary antibody, the expression level of CAR on CAR T cells was examined by flow cytometry, and the results are shown in FIG. 1.

It can be seen that the positive efficiency of CAR in mCD19-CAR, mCD19-CAR-IL-21-XCL1 cells was all greater than 50% compared to the control, indicating that these cells all efficiently express CAR.

Expression level of XCL1

The supernatant of the CAR-T cells prepared in example 1 was collected and the level of XCL1 secretion in the cells was determined using the Mouse XCL1DuoSet ELISA kit (R & D Systems, cat # DY 486) according to the manufacturer's recommendations and the results are shown in FIG. 2.

It can be seen that CAR T cells comprising mCD19-CAR-XCL1 can effectively secrete XCL1.

Expression level of IL-21

Supernatants of the CAR-T cells prepared in example 1 were collected and IL-21 secretion levels in the cells were measured using the Mouse IL-21DuoSet ELISA kit (R & D Systems, cat # DY 594) according to the manufacturer's recommendations and the results are shown in FIG. 3.

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

Example 3 detection of IFN- γ secretion levels in CAR-T cells

In 96-hole round bottom plate with 2X 10 ⁵ NT cells, mCD19-CAR-IL-21 cells and mCD19-CAR-IL-21-XCL1 cells were added at a concentration of 100. Mu.l per cell, respectively. Then 1X 10 in each well ⁴ Target cells Panc02-mCD19 cells or non-target cells Panc02 cells were added at a concentration of 100. Mu.l each. After incubation at 37 ℃ for 24h, culture supernatants were collected. The Mouse IFN-gamma DuoSet ELISA kit (R) was used according to the manufacturer's recommendations&D, cat No. DY 485) the expression level of IFN-. Gamma.in the culture supernatant was determined.

The results of the detection are shown in FIG. 4. It can be seen that no release of IFN- γ was detected in none of the non-target cells Panc02, whereas significantly elevated levels of IFN- γ release were detected in the target cells Panc02-CD19, indicating that the CAR T cells in this example have specific killing activity against the target cells.

Example 4 validation of tumor killing Effect of CAR-T cells in vitro

In a 96-well plate, 100. Mu.l of a solution containing 1X 10 ⁴ Taking the Panc02-mCD19 cells as target cells, and carrying out cell separation according to an effective target ratio of 10: 1. 5: 1. 2.5: 1. 1.25: 1. Mu.l NT cells, mCD19-CAR-IL-21 cells or mCD19-CAR-IL-21-XCL1 cells were added as effector cells. The 96-well plates were incubated overnight at 37 ℃ and then 50. Mu.l of 5 XD-luciferase solution (XenoLight, cat # 12279) was added to each well and fluorescence was immediately measured using a microplate reader. According to the calculation formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and 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 on target cells compared to mCD19-CAR cells alone. Unexpectedly, the inventors found that expression of the XCL1+ IL-21 combination further significantly improved the killing ability of CAR-T cells against target cells relative to CAR-T cells expressing only IL-21, especially in the case of low target ratios (e.g. 1.25.

Example 5 demonstration of tumor-inhibiting Effect of CAR-T cells

5X 10 subcutaneous inoculations were made in the left forelimb axilla of healthy C57BL/6 mice ⁵ A Panc02-mCD19 pancreatic cancer cell that overexpresses CD 19.

Mice vaccinated with pancreatic cancer cells were randomly divided into 5 groups of 7 mice each. When the tumor volume grows to 100mm ³ In time, each mouse was injected via tail vein with PBS, 5X 10 ⁶ An NT cell, an mCD19-CAR-IL-21 cell, or an mCD19-CAR-IL-21-XCL1 cell.

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

The body weight change of the mice is shown in fig. 6. As can be seen, the body weight of mice in each group was not significantly different from that of the control group after administration of CAR-T cells, and the tumor in the mice did not exceed 1500mm in the observation period ³ The mice were lively and had normal hair color, indicating that CAR-T cells administered did not have significant toxic side effects on the mice.

The change in tumor volume in mice is shown in figure 7. It can be seen that mCD19-CAR-IL-21 cells expressing only IL-21 had comparable tumor suppression effects to conventional mCD19-CAR cells, indicating that IL-21 alone did not improve the tumor suppression effects of CAR-T cells. Unexpectedly, the inventors found that the IL-21+ xcl1 combination significantly improved the tumor suppression effect of CAR-T cells, maintaining the tumor volume at a low level without recurrence until the end of the experiment.

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

Example 6 validation of the Activity of cytokine-and chemokine-expressing CAR-T cells

According to the method of example 1, CAR-T cells were prepared which co-expressed the cytokines IL12 (SEQ ID NO: 42), IL23 (SEQ ID NO: 50), IL18 (SEQ ID NO: 46), IL33 (SEQ ID NO: 54) alone and in combination 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.

The level of IFN- γ secretion of the CAR-T cells described above was tested according to the method of example 3. After co-culture with the target cells Panc02-mCD19, the level of IFN- γ secretion by CAR-T cells expressing IL12+ XCL1 and IL23+ XCL1 was comparable to CAR-T cells expressing only IL12 or IL23 (see fig. 8), whereas the level of IFN- γ secretion by CAR-T cells expressing IL18+ XCL1 and IL33+ XCL1 was significantly higher than that by CAR-T cells expressing only IL18 or IL33 (see fig. 9).

The above CAR-T cells were tested for anti-tumor effect in vivo according to the method of example 5. Although CAR-T cells expressing IL12 or IL23 alone showed very excellent tumor suppression effect, CAR-T cells expressing IL12+ XCL1 or IL23+ XCL1 combination still had significantly better tumor suppression effect than CAR-T cells expressing IL12 or IL23 alone (see fig. 10). In addition, CAR-T cells expressing IL18+ XCL1 and IL33+ XCL1 also showed significant tumor suppression effect, while CAR-T cells expressing IL15+ XCL1 and IL17+ XCL1 showed comparable or slightly inferior tumor suppression effect to traditional mCD19-CAR T cells (see fig. 11). This also indicates that not any combination of interleukins with XCL1 achieves a significant tumor-inhibiting effect, which may be associated with a complex function of interleukins.

It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

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<223> mCD8a 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 Co-stimulatory 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 co-stimulatory 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> mCD3 zeta 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> mCD3 zeta 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> mCD8 α 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> mCD8a 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> mCD8 alpha 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> mCD8 alpha 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 tttttataaa 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 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

1. An engineered immune cell that expresses (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 the group consisting of 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 comprising 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 having the same or similar function as wild-type.

5. The engineered immune cell of claim 4, wherein the IL-21 is identical to the amino acid sequence of SEQ ID NO: 32. 34 or 36 have at least 90% identity; IL12 is similar to SEQ ID NO:40 or 42, IL18 has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:44 or 46, and IL23 has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:48 or 50, IL33 has at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 52. 54, 55, 56 or 57 has at least 90% identity.

6. The engineered immune cell of any one of claims 1-5, wherein said XCL1 gene is homologous to SEQ ID

NO:23 or 25, or the XCL1 gene encodes a polypeptide which has at least 90% identity to the nucleic acid sequence shown in SEQ ID NO:24 or 26, wherein said XCL2 gene has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:37, or the polypeptide encoded by the XCL2 gene has at least 90% identity to the nucleic acid sequence as set forth in SEQ ID NO:38 has at least 90% identity.

7. The engineered immune cell of any one of claims 1-5, wherein the immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.

8. The engineered immune cell of claim 7, wherein the T cell is a CD4+/CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a CD4-CD8-T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a regulatory T cell, a γ δ -T cell, or an α β -T cell.

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 ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin, 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, 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-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/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, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globh, NY-BR-1, UPK2, HAVC 1, ADRB3, PANX3, NY-BR-1, and pK2 GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-L/Galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, LAP, LAM, TMPRSS2 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, PDL2, TGF β, APRIL, NKG2D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecule expressed by HIV, HCV, HBV and/or other pathogen; and/or

A neoepitope or a neoantigen.

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: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

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: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, 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 of a protein 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 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70 and combinations thereof.

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, TCR α, TCR β, CD3 γ, CD3 δ, CD3 ε, CD247 ζ, 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 the cytokine, chemokine 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: (ii) a nucleic acid sequence encoding a cell surface molecule which specifically recognizes the 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 the group consisting of 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 a plasmid, a retrovirus, a lentivirus, an adenovirus, a vaccinia virus, a Rous Sarcoma Virus (RSV), a polyoma virus, and an adeno-associated virus (AAV).

22. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 1-17, a nucleic acid molecule according to any one of claims 18-19 or a vector according to any one of claims 20-21, and one or more pharmaceutically acceptable excipients.

23. Use of an engineered immune cell according to any one of claims 1 to 17, a nucleic acid molecule according to any one of claims 18 to 19 or a vector according to any one of claims 20 to 21, or a pharmaceutical composition according to claim 22, for the manufacture of a medicament for the treatment of cancer, an infection or an 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 that express exogenous cytokines; (2) Engineered immune cells and cytokines that express exogenous chemokines; or (3) engineered immune cells, cytokines, and chemokines, wherein the engineered immune cells express a cell surface molecule that specifically recognizes an antigen, the cytokines are selected from the group consisting of IL12, IL18, IL-21, IL23, and IL33, and the chemokines are selected from the group consisting of XCL1 and XCL2.