CN115725504A

CN115725504A - Engineered immune cells and uses thereof

Info

Publication number: CN115725504A
Application number: CN202110985593.1A
Authority: CN
Inventors: 邢芸; 任江涛
Original assignee: Nanjing Bioheng Biotech Co Ltd
Current assignee: Nanjing Bioheng Biotech Co Ltd
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2023-03-03
Also published as: WO2023025009A1

Abstract

The present invention relates to an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous CCL20. 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 of the invention 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 invention relates to an engineered immune cell that expresses a cell surface molecule that specifically recognizes an antigen and an exogenous CCL20 gene. More preferably, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor.

Background

In recent years, adoptive cell therapy, an emerging immunotherapy, has shown great advantages in the field of tumor treatment. Such therapies typically require that cells be first engineered, for example by gene editing and/or transduction techniques, to carry exogenous proteins such as chimeric antigen receptors, recombinant T cell receptors, etc., and then expanded in vitro and returned to the patient. Currently, these therapies have shown good efficacy against hematological tumors, but their efficacy has not been satisfactory for solid tumors, one of the reasons for which the efficient transport of engineered cells to the tumor site (i.e., the immunosuppressive tumor microenvironment) is not possible.

Lymphocytes are generally directed to home to a target site by sensing chemokines secreted by other cells through chemokine receptors. Although the number of chemokines and their receptors is large, generally only a limited number of chemokine receptors are expressed per lymphocyte. Thus, when certain tumor cells secrete a chemokine in large quantities and lymphocytes (e.g., engineered CAR-T cells) do not express the receptor paired with the chemokine, the lymphocytes cannot be effectively transported to the tumor microenvironment, thereby affecting the therapeutic effect.

Thus, there remains a need for improved cell therapies to alter the immunosuppressive microenvironment while recruiting other immune effector cells to the tumor site, enhancing the anti-tumor effect.

Disclosure of Invention

In a first aspect, the invention provides a novel engineered immune cell that expresses a cell surface molecule that specifically recognizes an antigen and exogenous CCL20.

In one embodiment, the engineered immune cells of the invention further express exogenous IL7.

In one embodiment, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor or a T cell receptor, preferably a chimeric antigen receptor.

In one embodiment, the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor comprising an antigen binding region, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain. Wherein the antigen-antigen binding region 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 antigen-antigen binding region is selected from the group consisting of scFv, fab, single domain antibody and nanobody.

In one embodiment, the cell surface molecule specifically recognizing an antigen 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, CD179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1 DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewisY, 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, globoH, NY-BR-1, UPK2, HAVR 1, ADRB3, PANX3, pNX 3 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, tie 2, 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 ∈ 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 an intracellular region of a protein selected from the group consisting of: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the primary signaling domain comprises a CD3 ζ intracellular region.

In one embodiment, the co-stimulatory domain comprises one or more intracellular regions of a protein selected from the group consisting of: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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 selected from the intracellular domains of CD27, CD28, CD134, CD137, DAP10, DAP12 or CD278 or a combination thereof.

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 γ δ -T cell, or an α β -T cell.

In one embodiment, the expression or activity of exogenous CCL20 and/or IL7 is constitutive expression. In another embodiment, the expression or activity of exogenous CCL20 and/or IL7 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, CCL20 and/or IL7 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., a cell membrane. In one embodiment, an exogenous gene of the invention, such as CCL20 and/or IL7, is operably linked to a transmembrane domain, thereby anchoring expression at the surface of an engineered immune cell.

In a second aspect, the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding CCL20. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding IL7. Preferably, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor 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 an immune cell, 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: <xnotran> , , , , , , , , CNS , , , , , , , , , , , , , (GBM), , , , , , , , , , , , , , , , , , , , , , , , , , , , , B , B (B-LBL), , AIDS , Waldenstrom , (CLL), (ALL), B (B-ALL), T (T-ALL), B , , , B , , (CML), , MALT , , , , , , </xnotran> Pre-leukemic, plasmacytoid dendritic cell tumors, and post-transplant lymphoproliferative disorders (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.

Drawings

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

FIG. 2: expression level of IL7 by CAR-T cells determined by ELISA.

FIG. 3: expression level of CCL20 of CAR-T cells determined by ELISA.

FIG. 4: (iii) 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: weight change profile of mice after treatment of mouse pancreatic cancer with CAR-T cells.

FIG. 6: tumor growth curves in mice after treatment of mouse pancreatic cancer with CAR-T cells.

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 antigens

As used herein, the term "cell surface molecule that specifically recognizes an antigen" refers to a molecule expressed on the surface of a cell that is capable of specifically binding to a target molecule (e.g., an antigen). Such surface molecules typically comprise an antigen-binding region capable of specifically binding to an antigen, a transmembrane domain that anchors the surface molecule to the cell surface, and an intracellular domain responsible for signaling. Examples of common such surface molecules include, for example, T cell receptors or chimeric antigen receptors.

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 can comprise or be operably linked to an antigen-binding region, wherein the antigen-binding region is defined as follows.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide generally comprising an antigen binding region (e.g., an antigen-binding portion of an antibody), a transmembrane domain, and an intracellular signaling domain (comprising a costimulatory domain and/or a primary signaling domain), each linked by a linker. CARs are able to redirect the specificity and reactivity of T cells and other immune cells to selected targets 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, "antigen binding region" refers to any structure or functional variant thereof that can bind to an antigen. The antigen binding region can 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, antigen binding regions 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 antigen binding region may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody. In another embodiment, the antigen binding region may also be a specific binding polypeptide or receptor structure for a particular protein, such as PD1, PDL2, TGF β, APRIL and NKG2D.

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 often expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity. 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 antigen-binding region 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 a tumor-associated antigen. Thus, in one embodiment, the antigen binding region 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, CD179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1 DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewisY, 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, tie 2, 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. Depending on the antigen to be targeted, the CAR of the invention may be designed to include an antigen binding region specific for that antigen. Preferably, the target is selected from the group consisting of CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, folate receptor alpha, CEA, PSCA, PSMA, her2, EGFR, IL13Ra2, GD2, NKG2D, claudin18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, mesothelin, and any combination thereof. For example, if CD19 is the antigen to be targeted, CD19 antibodies may be used as the antigen binding region of the invention. In one embodiment, the antigen binding region is a CD19 targeting antibody having the same CDRs as the antibody set forth in SEQ ID NO. 1 or 2.

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 CD28 having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 3; or from CD8 alpha, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 4 or 5.

In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antigen binding region 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 an antigen binding region. In particular, the hinge region serves to provide greater flexibility and accessibility to the antigen binding region. 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 in whole or in part from the extracellular region of CD8, fc γ RIII α receptor, igG4, igG1, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a CD28 hinge having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 15; or comprises a CD8 a hinge 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 as set forth in SEQ ID NO 16 or 17; or comprises an IgG4 hinge 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. 18.

As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs a cell to a specified function, which comprises a costimulatory domain and/or a primary signaling domain. The intracellular signaling domain is responsible for intracellular signaling after the antigen binding region binds the antigen, resulting in activation of the immune cell and immune response. In other words, the intracellular 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, which may be the cytoplasmic sequences of the T cell receptor and co-receptor that work together to trigger primary signaling after 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 primary signaling domain of a CAR of the invention may comprise a CD3 ζ intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NOs 9, 10 or 11.

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, intracellular regions derived from: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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 the CAR of the invention is from 4-1BB, CD28, CD27, OX40, ICOS, DAP10, DAP12 or a combination thereof. In one embodiment, the CAR of the invention comprises a CD28 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 set forth in SEQ ID No. 6; and/or comprises a 4-1BB 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 7 or 8.

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 that is recognized and cleaved by the signal peptidase. Signal peptidases can cleave during translocation or after completion to produce free signal peptide and 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 B2M, CD8 α, igG1, GM-CSFR α, and the like. In one embodiment, the signal peptide useful in the present invention is a B2M signal peptide having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12; or a CD8 alpha signal peptide 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 13 or 14.

In one embodiment, the CAR of the invention may further comprise a switch structure to regulate the time of expression of the CAR. For example, the switch structure may be in the form of a dimerization domain that causes a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain to allow binding to the targeted antigen, thereby activating a 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 via a dimer only when the switch domains are bound to each other (e.g., in the presence of an inducing compound), thereby activating 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 may also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by treatment with ganciclovir. The suicide gene can also be iCaspase-9, and the iCaspase-9 can be induced to dimerize by 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.

Exogenous gene

In addition to cell surface molecules that specifically recognize antigens, the engineered immune cells of the invention also express exogenous CCL20.

Chemotactic cytokines are classified into four subfamilies of CXC, CC, XC and CX3C according to the arrangement of cysteine at the amino terminal. CCL20 is a member of the CC subfamily, also known as macrophage inflammatory protein 3 α (MIP-3 α). CCL20 has a strong chemotactic effect on lymphocytes and dendritic cells. CCL20 plays a role in autoimmune diseases (e.g., rheumatoid arthritis, psoriasis, etc.) and malignant tumors (e.g., cancers of the liver, colon, breast, pancreas, stomach, etc.) by binding to the ligand CCR 6.

In one embodiment, CCL20 used in the invention is substantially identical to SEQ ID NO:25 or 27, or the coding sequence of CCL20 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:24 or 26, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In a preferred embodiment, the engineered immune cells of the invention further express IL7. In one embodiment, IL7 used in the present invention is substantially identical to SEQ ID NO:21 or 23, or the coding sequence for IL7 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:22 or 24, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

The expression of exogenous genes in the present invention, such as CCL20 and/or IL7, may be constitutive expression or conditional expression.

In one embodiment, the expression of exogenous CCL20 and/or IL7 is a conditional expression. For example, the exogenous gene of the present invention may be operably linked to an inducible, repressible or tissue-specific promoter, as desired, to regulate the expression level of the introduced exogenous gene at a particular time or in a particular 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, tetracycline or analogs thereof including, for example, chlortetracycline, oxytetracycline, demethylclocycline, 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 another embodiment, the promoter is a repressible promoter, i.e., expression of the foreign gene in the cell is inhibited or not expressed in the presence of a repressor specific for the repressible promoter. Repressible promoters include, for example, lac repressible elements or tetracycline repressible elements. Inducible/repressible expression systems well known to those skilled in the art can be used in the present invention, including but not limited to the Tet-on system, tet-off system, cre/loxP system, and the like.

In one embodiment, CCL20 and/or IL7 may be operably linked to a localization domain that can localize the exogenous gene of the invention for expression at a specific cellular location, such as a cell membrane or the like. Localization domains include, but are not limited to, nuclear localization signals, leader peptides, transmembrane domains, and the like. In one embodiment, the exogenous genes CCL20 and/or IL7 of the invention are operably linked to a transmembrane domain, thereby anchoring expression at the surface of an engineered immune cell.

In one embodiment, the exogenous gene of the invention, e.g., CCL20 and/or IL7 protein, can be wild-type or a fusion protein or mutant with specific properties (e.g., resistance to proteolysis).

Nucleic acids and vectors

The invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding CCL20. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding IL7.

In one embodiment, the cell surface molecule specifically recognizing an antigen is a T cell receptor or a chimeric antigen receptor, preferably a chimeric antigen receptor. The chimeric antigen receptor is defined as above.

As used herein, the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in linear or circular form, in single-and/or double-stranded form, 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.

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 an antigen, the nucleic acid sequence encoding CCL20 and optionally the nucleic acid sequence encoding IL7 may be located in one or more vectors.

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 for delivering an isolated nucleic acid to the interior of a cell, for example, by homologous recombination or by using a hybrid recombinase that targets sequences at a site specifically. 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 for use in the present invention are known in the art and many are commercially available. In one embodiment, vectors of the invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, oncolytic viruses, lentiviruses, adenoviruses, vaccinia viruses, rous sarcoma viruses, polyoma viruses, and adeno-associated viruses (AAV), etc.), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BAC and YACs). The vector itself is usually a nucleotide sequence, usually a DNA sequence comprising an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors typically also contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally comprise elements such as a promoter, poly A tail (polyA), 3' UTR, enhancer, terminator, insulator, operator, selectable marker, reporter gene, targeting sequence and/or protein purification tag. In a specific embodiment, the vector is an in vitro transcription 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 an antigen and an exogenous CCL20 gene, and optionally an exogenous IL7 gene.

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 cell may be a T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell, or an immune cell obtained from a stem cell source such as iPSC, ESC or the like. Preferably, the immune cell is a T cell. The T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, 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+ helper T cells (e.g., th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), CD4-CD8-T cells, tumor infiltrating cells, memory T cells, naive 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 one embodiment, the immune cell of the invention 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, 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, TALEN nucleases or Cas enzymes in CRISPR systems.

Pharmaceutical composition

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 active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Edited by Gennaro AR,19th ed. Pennsylvania mack Publishing company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity 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 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.

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, the method of treatment comprises administering to a subject an effective amount of an immune cell and/or pharmaceutical composition of the invention.

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 bound by an antigen binding region. For example, the cancer includes, but is not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, kidney cancer, larynx cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer and squamous lung cancer), lymphoma (including hodgkin lymphoma and non-hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, and prostate cancer retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urological malignancies, vulval cancer and other carcinomas and sarcomas, and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cracked cytolytic NHL, large lump disease NHL), B lymphoblastic lymphoma (B-LBL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic Lymphocytic Leukemia (CLL); large lump disease NHL, 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 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, plasmablatic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); and other diseases associated with target expression. Preferably, the diseases that can be treated with the engineered immune cells or 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 drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the 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

1.1 construction of retroviral plasmids

MSCV-mCD19-CAR plasmid was constructed comprising the coding sequences for CD19-scFv (SEQ ID NO: 2), CD8 a hinge region (SEQ ID NO: 17), CD8 a transmembrane region (SEQ ID NO: 5), 41BB costimulatory domain (SEQ ID NO: 7) and CD3 ζ intracellular region (SEQ ID NO: 11).

MSCV-mCD19-CAR-IL7 plasmid was constructed, which further comprises the coding sequence of T2A (SEQ ID NO: 19) and IL7 (SEQ ID NO: 23) on the basis of MSCV-mCD19-CAR plasmid.

MSCV-mCD19-CAR-CCL20 plasmid was constructed, which further comprises the coding sequence of T2A (SEQ ID NO: 19) and CCL20 (SEQ ID NO: 25) on the basis of MSCV-mCD19-CAR plasmid.

1.2. Preparation of retrovirus

In T175 flasks at 30X 10 ⁶ Density of cells/flask 293T cells were inoculated into 30ml DMEM medium containing 10% fetal bovine serum at 37 ℃ 5% ₂ The cells were cultured overnight in an incubator for virus packaging.

To a sterile tube were added 3ml of Opti-MEM (Gibco, cat # 31985-070), 45. Mu.g of the prepared retroviral plasmid and 15. Mu.g of the packaging vector pCL-Eco (Shanghai Hao Wu Biotech Co., ltd., cat # P3029). 120 μ l of X-trememe GENE HP DNA transfection reagent (Roche, cat. No. 06366236001) was then added, mixed immediately and incubated at room temperature for 15min. The plasmid/vector/transfection reagent mixture was then added dropwise to a pre-prepared 293T cell culture flask at 37 ℃ 5% ₂ Culturing overnight under the condition. The culture was collected 72 hours after transfection, and centrifuged (2000 g,4 ℃,10 minutes) to obtain a retrovirus supernatant.

1.3 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 with RetroNectin overnight, then 500 μ L of retroviral supernatant was added and complete medium 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 changed and the cell density was adjusted to 1X 10 ⁶ Individual cells/mL. Three days after infection, cells were collected for subsequent analysis. The collected cells are mCD19-CAR cells, mCD19-CAR + CCL20 cells and mCD19-CAR + IL7+ CCL20 cells.

Example 2 detection of expression of CAR-T cells

2.1 expression levels of cell surface CAR

2X 10 of the product prepared in example 1 was 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 CAR positive efficiency in mCD19-CAR cells, mCD19-CAR + CCL20 cells and mCD19-CAR + IL7+ CCL20 cells was greater than 60% compared to untreated NT cells, indicating that these cells all efficiently expressed CAR.

2.2Expression level of IL7

The supernatant of CAR-T cells was collected and the level of IL7 secretion in the cells was detected using the Mouse IL-7DuoSet ELISA kit (R & D Systems, cat # DY 407) according to the manufacturer's recommendations and the results are shown in FIG. 2. It can be seen that mCD19-CAR + IL7+ CCL20 cells can efficiently express IL7.

2.3Expression level of CCL20

The supernatant of CAR-T cells was collected and the level of CCL20 secretion in the cells was determined using the Mouse CCL20 DuoSet ELISA kit (R & D Systems, cat # DY 479) according to the manufacturer's recommendations and the results are shown in figure 3. It can be seen that both mCD19-CAR + CCL20 cells and mCD19-CAR + IL7+ CCL20 cells efficiently expressed CCL20.

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

In 96-hole round bottom plate with 2X 10 ⁵ NT cells, CD19-CAR cells, mCD19-CAR + CCL20 cells and mCD19-CAR + IL7+ CCL20 cells were added at a concentration of 100. Mu.l per cell, respectively. Then 1X 10 in each well ⁴ Each Panc02-mCD19 target cell or Panc02 non-target cell was added at a concentration of 100. Mu.l. After incubation at 37 ℃ for 24h, culture supernatants were collected. According to the manufacturer's recommendations, use the Mouse IFN-gamma DuoSet ELISA kit (R)&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 non-target cells Panc02, but only significantly elevated IFN- γ levels were detected after co-culture with target cells Panc02-CD19, and that the NT cells did not express IFN- γ, indicating that killing of CAR-T cells in this example was specific. In addition, the level of IFN-gamma of the mCD19-CAR + CCL20 cell is obviously higher than that of the CD19-CAR cell, and the additional expression of the CCL20 gene can obviously improve the killing activity of the CAR-T cell. In addition, the level of IFN- γ was also significantly higher for CAR-T cells expressing IL7+ CCL20 combination than CAR-T cells expressing CCL20 alone, suggesting that the addition of IL7 could further improve the killing activity of CAR-T cells.

Example 4 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 And the Panc02-mCD19 pancreatic cancer cells. Mice inoculated with pancreatic cancer cells were randomly divided into 3 groups of 5 mice each. When the tumor volume grows to 100mm ³ In time, 1X 10 injections were given via tail vein to each group of mice ⁶ N isT cells, mCD19-CAR cells or mCD19-CAR + IL7+ CCL20 cells. Mice were monitored for changes in body weight and tumor volume until the end of the experiment.

The body weight changes of the mice are shown in fig. 5. It can be seen that there was no significant difference in body weight of mice in each group compared to the control group after CAR-T cell administration, indicating that CAR-T cell administration did not have a significant toxic side effect on the mice.

The change in tumor volume in mice is shown in figure 6. It can be seen that the anti-tumor effect of the mCD19-CAR + IL7+ CCL20 cell is significantly better than that of the conventional CAR-T cell, and the additional expression of IL7+ CCL20 can generate a synergistic effect with the CAR-T cell to enhance the anti-tumor effect of the CAR-T cell.

The above results indicate that co-expressing CCL20 or its combination with IL7 is effective in enhancing the inhibitory effect of CAR-expressing engineered immune cells on tumor cells.

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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<211> 113

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> intracellular domain of CD3 ζ

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Arg

<210> 11

<211> 109

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCD3 ζ intracellular domain

<400> 11

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> 12

<211> 20

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> B2M Signal peptide

<400> 12

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala

20

<210> 13

<211> 21

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> CD8 alpha signal peptide

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 14

<211> 24

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCD8 alpha signal peptide

<400> 14

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> 15

<211> 39

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> CD28 hinge region

<400> 15

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 16

<211> 45

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> CD8 a hinge region

<400> 16

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

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

20 25 30

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

35 40 45

<210> 17

<211> 45

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCD8 α hinge region

<400> 17

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> 18

<211> 12

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> IgG4 hinge region

<400> 18

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 19

<211> 18

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> T2A

<400> 19

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

1 5 10 15

Gly Pro

<210> 20

<211> 402

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hIL-7

<400> 20

atgttccatg tttcttttag gtatatcttt ggacttcctc ccctgatcct tgttctgttg 60

ccagtagcat catctgattg tgatattgaa ggtaaagatg gcaaacaata tgagagtgtt 120

ctaatggtca gcatcgatca attattggac agcatgaaag aaattggtag caattgcctg 180

aataatgaat ttaacttttt taaaagacat atctgtgatg ctaataaggt taaaggaaga 240

aaaccagctg ccctgggtga agcccaacca acaaagagtt tggaagaaaa taaatcttta 300

aaggaacaga aaaaactgaa tgacttgtgt ttcctaaaga gactattaca agagataaaa 360

acttgttgga ataaaatttt gatgggcact aaagaacact ga 402

<210> 21

<211> 133

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hIL-7

<400> 21

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

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

20 25 30

Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Val Lys Gly Arg

65 70 75 80

Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu

85 90 95

Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu

100 105 110

Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met

115 120 125

Gly Thr Lys Glu His

130

<210> 22

<211> 465

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mIL-7

<400> 22

atgttccatg tttcttttag atatatcttt ggaattcctc cactgatcct tgttctgctg 60

cctgtcacat catctgagtg ccacattaaa gacaaagaag gtaaagcata tgagagtgta 120

ctgatgatca gcatcgatga attggacaaa atgacaggaa ctgatagtaa ttgcccgaat 180

aatgaaccaa acttttttag aaaacatgta tgtgatgata caaaggaagc tgcttttcta 240

aatcgtgctg ctcgcaagtt gaagcaattt cttaaaatga atatcagtga agaattcaat 300

gtccacttac taacagtatc acaaggcaca caaacactgg tgaactgcac aagtaaggaa 360

gaaaaaaacg taaaggaaca gaaaaagaat gatgcatgtt tcctaaagag actactgaga 420

gaaataaaaa cttgttggaa taaaattttg aagggcagta tataa 465

<210> 23

<211> 154

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mIL-7

<400> 23

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Ile Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Thr Ser Ser Glu Cys His Ile Lys Asp Lys

20 25 30

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

35 40 45

Asp Lys Met Thr Gly Thr Asp Ser Asn Cys Pro Asn Asn Glu Pro Asn

50 55 60

Phe Phe Arg Lys His Val Cys Asp Asp Thr Lys Glu Ala Ala Phe Leu

65 70 75 80

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

85 90 95

Glu Glu Phe Asn Val His Leu Leu Thr Val Ser Gln Gly Thr Gln Thr

100 105 110

Leu Val Asn Cys Thr Ser Lys Glu Glu Lys Asn Val Lys Glu Gln Lys

115 120 125

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

130 135 140

Cys Trp Asn Lys Ile Leu Lys Gly Ser Ile

145 150

<210> 24

<211> 294

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCCL20

<400> 24

atggcctgcg gtggcaagcg tctgctcttc cttgctttgg catgggtact gctggctcac 60

ctctgcagcc aggcagaagc agcaagcaac tacgactgtt gcctctcgta catacagacg 120

cctcttcctt ccagagctat tgtgggtttc acaagacaga tggccgatga agcttgtgac 180

attaatgcta tcatctttca cacgaagaaa agaaaatctg tgtgcgctga tccaaagcag 240

aactgggtga aaagggctgt gaacctcctc agcctaagag tcaagaagat gtaa 294

<210> 25

<211> 97

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCCL20

<400> 25

Met Ala Cys Gly Gly Lys Arg Leu Leu Phe Leu Ala Leu Ala Trp Val

1 5 10 15

Leu Leu Ala His Leu Cys Ser Gln Ala Glu Ala Ala Ser Asn Tyr Asp

20 25 30

Cys Cys Leu Ser Tyr Ile Gln Thr Pro Leu Pro Ser Arg Ala Ile Val

35 40 45

Gly Phe Thr Arg Gln Met Ala Asp Glu Ala Cys Asp Ile Asn Ala Ile

50 55 60

Ile Phe His Thr Lys Lys Arg Lys Ser Val Cys Ala Asp Pro Lys Gln

65 70 75 80

Asn Trp Val Lys Arg Ala Val Asn Leu Leu Ser Leu Arg Val Lys Lys

85 90 95

Met

<210> 26

<211> 291

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hCCL20

<400> 26

atgtgctgta ccaagagttt gctcctggct gctttgatgt cagtgctgct actccacctc 60

tgcggcgaat cagaagcagc aagcaacttt gactgctgtc ttggatacac agaccgtatt 120

cttcatccta aatttattgt gggcttcaca cggcagctgg ccaatgaagg ctgtgacatc 180

aatgctatca tctttcacac aaagaaaaag ttgtctgtgt gcgcaaatcc aaaacagact 240

tgggtgaaat atattgtgcg tctcctcagt aaaaaagtca agaacatgta a 291

<210> 27

<211> 96

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCCL20

<400> 27

Met Cys Cys Thr Lys Ser Leu Leu Leu Ala Ala Leu Met Ser Val Leu

1 5 10 15

Leu Leu His Leu Cys Gly Glu Ser Glu Ala Ala Ser Asn Phe Asp Cys

20 25 30

Cys Leu Gly Tyr Thr Asp Arg Ile Leu His Pro Lys Phe Ile Val Gly

35 40 45

Phe Thr Arg Gln Leu Ala Asn Glu Gly Cys Asp Ile Asn Ala Ile Ile

50 55 60

Phe His Thr Lys Lys Lys Leu Ser Val Cys Ala Asn Pro Lys Gln Thr

65 70 75 80

Trp Val Lys Tyr Ile Val Arg Leu Leu Ser Lys Lys Val Lys Asn Met

85 90 95

Claims

1. An engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous CCL20.

2. The engineered immune cell of claim 1, wherein the engineered immune cell further expresses exogenous IL7.

3. The engineered immune cell of claim 1, wherein the CCL20 is identical to the amino acid sequence of SEQ ID NO:25 or 27, or a coding sequence thereof which shares at least 90% identity with the amino acid sequence set forth in SEQ ID NO:24 or 26 have at least 90% identity.

4. The engineered immune cell of claim 2, wherein the IL7 is identical to the amino acid sequence of SEQ ID NO:21 or 23, or a coding sequence thereof which shares at least 90% identity with the amino acid sequence set forth in SEQ ID NO:22 or 24 have at least 90% identity.

5. The engineered immune cell of any one of claims 1-4, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a T cell receptor.

6. The engineered immune cell of claim 5, wherein the chimeric antigen receptor comprises an antigen binding region, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain.

7. The engineered immune cell of claim 6, wherein the antigen binding region 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 antigen.

8. The engineered immune cell of claim 7, wherein the antigen-antigen binding region 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, CD179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1 DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewisY, 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, globoH, NY-BR-1, UPK2, HAVR 1, ADRB3, PANX3, pNX 3 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, tie 2, 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.

9. The engineered immune cell of claim 6, 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 ∈ 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.

10. The engineered immune cell of any one of claims 6-10, wherein the primary signaling domain is selected from the intracellular regions of: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

11. The engineered immune cell of claim 6, wherein the co-stimulatory domain comprises one or more intracellular regions of a protein selected from the group consisting of: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, 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.

12. The engineered immune cell of any one of claims 1-11, wherein the immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.

13. The engineered immune cell of claim 12, 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 γ δ -T cell, or an α β -T cell.

14. The engineered immune cell of any one of claims 1-13, wherein the expression of CCL20 and/or IL7 is conditional expression or constitutive expression.

15. The engineered immune cell of any one of claims 1-14, wherein the CCL20 and/or IL7 is operably linked to a localization domain.

16. A nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding CCL20.

17. The nucleic acid molecule of claim 16, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor.

18. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding IL7.

19. A vector comprising the nucleic acid molecule of any one of claims 16-18.

20. The vector of claim 18, 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).

21. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 1-15, a nucleic acid molecule according to any one of claims 16-18 or a vector according to any one of claims 19-20, and one or more pharmaceutically acceptable excipients.

22. A method of treating a subject having cancer, an infection, or an autoimmune disease, comprising administering to the subject the engineered immune cell of any one of claims 1-15 or the pharmaceutical composition of claim 21.