CN116103239A - Engineered immune cells and uses thereof - Google Patents

Abstract

The present invention relates to an engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance expression of IL9, and optionally a chemokine and/or a cell surface molecule that specifically recognizes an antigen. The invention also provides the use of the engineered immune cells in the treatment of cancer, infection or autoimmune disease. Compared with the traditional engineering immune cell, the engineering immune cell has obviously improved tumor killing activity.

Description

Engineered immune cells and uses thereof

Technical Field

The present invention belongs to the field of immunotherapy. More specifically, the invention relates to an engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance expression of IL 9.

Background

In recent years, adoptive cell therapy has shown great advantage in the field of tumor therapy as an emerging immunotherapy. Such therapies typically require modification of the cells, for example, by techniques such as gene editing and/or transduction, to carry exogenous proteins such as chimeric antigen receptors, recombinant T cell receptors, etc., followed by expansion in vitro and return to the patient. At present, these therapies have shown good efficacy against hematological tumors, but their efficacy is not satisfactory for solid tumors, one of which is that the engineered cells cannot reach the tumor site effectively due to the immunosuppressive tumor microenvironment.

Thus, there remains a need for improved cell therapies to counteract the inhibitory effects of the tumor microenvironment to potentiate the anti-tumor effect.

Disclosure of Invention

In a first aspect, the invention provides a novel engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance expression of IL 9. Preferably, the exogenous IL9 is wild-type IL9 or a variant thereof, which variant has the same or similar function as wild-type IL 9. More preferably, the IL9 hybridizes to SEQ ID NO:21 or 23 has at least 90% identity.

In one embodiment, the modified promoter is selected from the group consisting of a constitutive promoter and an inducible promoter. For example, expression of IL9 can be enhanced by replacing the endogenous promoter with a constitutive or inducible promoter, or inserting it into the promoter region of the endogenous IL9 gene, thereby operably linking the modified promoter to the endogenous IL9 gene. Preferably, the constitutive promoters include, but are not limited to, CMV promoter, EF1a promoter, SV40 promoter, PGK1 promoter, ubc promoter, β -actin promoter, CAG promoter; the inducible promoters include, but are not limited to, tetracycline Responsive Element (TRE) promoters, estrogen Responsive Element (ERE) promoters.

In one embodiment, the engineered immune cell further expresses an exogenous chemokine selected from XCL1 and XCL2. Preferably, the XCL1 is identical to SEQ ID NO:25 or 27 has at least 90% identity to the amino acid sequence set forth in seq id no; XCL2 and SEQ ID NO:29 has at least 90% identity.

In one embodiment, the engineered immune cell further expresses a cell surface molecule comprising an antigen binding region that specifically recognizes an antigen, selected from the group consisting of a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler, preferably a chimeric antigen receptor.

In one embodiment, the antigen binding region may be selected from IgG, fab, 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 scFv, fab, single domain antibodies, and nanobodies.

In one embodiment, the cell surface molecule that specifically recognizes 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, CD 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, B H3, 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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20 LY6K, OR E2, 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 breakpoint, ML-IAP, 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, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF beta, APRIL, NKG2D, NKG D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecules expressed by HIV, HCV, HBV and/or other pathogens; and/or neoepitopes or neoantigens.

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 domain comprising a costimulatory domain and/or a primary signaling domain.

In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, cd3ζ subunit, cd3ε subunit, cd3γ subunit, cd3δ subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the group consisting of the transmembrane domains of CD8 a, CD4, CD28 and CD 278.

In one embodiment, the primary signaling domain is an intracellular region selected from the group consisting of: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the primary signaling domain comprises a cd3ζ intracellular region.

In one embodiment, the co-stimulatory domain comprises one or more intracellular regions 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, SLP, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof. Preferably, the co-stimulatory domain is selected from the group consisting of the intracellular region of CD27, CD28, CD134, CD137, DAP10, DAP12 or CD278 or a combination thereof.

In one embodiment, the immune cell is selected from T cells, B cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells. Preferably, the T cell is a cd4+cd8+ T cell, a cd4+ T cell, a cd8+ T cell, a CD4-CD8-T cell, a tumor infiltrating cell, a memory T cell, a regulatory T cell, a naive T cell, a γδ -T cell or an αβ -T cell.

In one embodiment, the expression of IL9 and/or a chemokine is secretory or anchored. For example, IL9 and/or chemokines can be operably linked to a localization domain (e.g., a transmembrane domain) that can localize an exogenous gene of the invention to a particular cellular location for expression, e.g., a cell membrane.

In a second aspect, the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding an exogenous IL9 or a nucleic acid sequence encoding an endogenous IL9 operably linked to a modified promoter, preferably further comprising a nucleic acid sequence encoding an exogenous chemokine selected from XCL1 and XCL2. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, which is a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler, more preferably a chimeric antigen receptor.

The invention also provides a vector comprising the nucleic acid molecule. In particular, the vector is selected from the group consisting of a plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, rous Sarcoma Virus (RSV), polyoma virus, and adeno-associated virus (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 operon, 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 transcribed 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 suffering from cancer, an infection or an autoimmune disease, comprising administering to the subject an effective amount of an immune cell, nucleic acid molecule, vector or pharmaceutical composition according to the invention.

In a fourth aspect, the invention also provides a combination therapy comprising an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and an exogenous IL9. In one embodiment, the combination therapy comprises: (1) An engineered immune cell expressing exogenous IL9 and an exogenous chemokine; (2) Engineered immune cells expressing exogenous chemokines and exogenous IL9; or (3) engineering immune cells and exogenous IL9 and chemokines; wherein the engineered immune cell expresses a cell surface molecule that specifically recognizes an antigen and the chemokine is selected from XCL1 and XCL2.

Drawings

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

Fig. 2: expression level of IL9 of CAR-T cells as determined by ELISA.

Fig. 3: IFN-gamma release levels after co-culture of CAR-T cells with target cells and non-target cells, respectively.

Fig. 4: body weight change curve of mice after treatment of pancreatic cancer with CAR-T cells.

Fig. 5: tumor growth curve of mice after treatment of 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.

IL9 and chemokines

In a first aspect, the invention provides a novel engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance expression of IL 9.

IL9 is an interleukin that activates the JAK-STAT signaling pathway by binding to the receptor IL9R, thereby playing an important role in the activation and regulation of immune cells, proliferation and differentiation, and in inflammatory responses. IL9 is mainly produced by mast cells, th2 cells, th9 cells, th17 cells, treg cells, NKT cells, innate lymphocytes (innate lymphoid cells, ILCs), and the like. The relationship between IL9 and other interleukins is not clear, but is clearly different from IL2 in that it cannot induce cytotoxic activities such as CTL and LAK, but can maintain long-term growth of non-antigen-dependent Th cells.

In the present invention, the expression of IL9 is enhanced by introducing exogenous IL9 into immune cells or by operably linking endogenous IL9 to a modified promoter. For example, any genomic editing method may be used to modify the promoter/enhancer region of the IL9 locus, including replacing the endogenous promoter with a constitutive or inducible promoter, or inserting a constitutive or inducible promoter into the promoter region of the IL9 locus, to enhance endogenous expression of IL9 in an immune responsive cell. In certain embodiments, a constitutive promoter is located at the IL9 locus to drive gene expression of an endogenous IL9 gene. Suitable constitutive promoters include, but are not limited to, CMV promoter, EF1a promoter, SV40 promoter, PGK1 promoter, ubc promoter, beta-actin promoter and CAG promoter. In certain embodiments, an inducible promoter is located at the IL9 locus to drive gene expression of the endogenous IL9 gene. Examples of inducible promoters include, but are not limited to, tetracycline Responsive Element (TRE) promoters and Estrogen Responsive Element (ERE) promoters. Furthermore, it is also possible to place the enhancer element in a region other than the promoter region.

In one embodiment, the exogenous IL9 is wild-type (e.g., from a human or mouse) or a variant thereof that has the same or similar function as wild-type IL 9. More preferably, the IL9 hybridizes to SEQ ID NO:21 or 23, or a coding sequence thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:20 or 22, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

The C-type chemokine family, also known as lymphokines, consists of two members XCL1 and XCL2, mainly produced by cd8+ T cells and natural killer cells. XCL1 has unique sequence features and two mutually convertible protein spatial conformations, making XCL1 distinct from other chemokines and perform unique functions. XCL 1-specific receptor XCR1 is a G protein-coupled receptor family member, and the interaction of both plays an important role not only in negative selection of thymus and establishment of autoimmune tolerance, but also in initiating cross antigen presentation and mediating cytotoxic immune responses. XCL1 not only regulates the balance of the immune system and maintains intestinal immune homeostasis, but is also associated with a variety of diseases such as autoimmune diseases, nephritis, tuberculosis, and infection with human immunodeficiency virus. XCL2 has 97% identity to the nucleic acid sequence of XCL1 and the amino acid sequence differs by only two residues. XCL2 has been found to be very similar in expression profile, structure and function to XCL1, e.g. like XCL1 XCL2 also has two interconvertible protein spatial conformations, haplotype and dimer, wherein the haplotype conformation binds and activates XCR1 and the dimer conformation has a higher affinity for hairpin structures in glycosaminoglycans (GAGs). The receptor XCR1 of XCL1 and XCL2 is selectively expressed on DC (cDC 1) cells with antigen presenting ability, and studies have found that the introduction of XCL1 can effectively improve the therapeutic effects of anti-tumor immunotherapy and targeted vaccines.

In one embodiment, XCL1 and/or XCL2 used in the present invention is wild-type (e.g. from human or murine) or a variant thereof, said variant having the same or similar function as the wild-type. Preferably XCL1 and SEQ ID NO:25 or 27, or the coding sequence of XCL1 has at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:24 or 26, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. XCL2 and SEQ ID NO:29, or the coding sequence of XCL2 has at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:28, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In one embodiment, the expression of exogenous genes (e.g., IL9 and/or chemokines) in the present invention is secretory expression. In another embodiment, the exogenous gene is expressed anchored, e.g., operably linked to a localization domain that can localize the exogenous gene of the invention to a particular cellular location, e.g., cell membrane, 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 on the surface of an engineered immune cell.

Cell surface molecules specifically recognizing antigens

In another aspect, the engineered immune cells of the invention further express 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 (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" is a characteristic marker on the surface of a T cell that associates non-covalently with CD3 to form a complex. Antigen presenting cells present antigen peptides to T cells via major histocompatibility complex Molecules (MHC) and bind to TCR complexes to induce a range of intracellular signaling. TCRs consist of six peptide chains, each forming a heterodimer, which are generally classified into the αβ and γδ types. Each peptide chain comprises a constant region and a variable region, wherein the variable region is responsible for binding to specific antigen and MHC molecules.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen binding region (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 of which are 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 antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed within T cells, the CAR advantageously does not dimerize with the alpha and beta chains of endogenous T Cell Receptors (TCRs).

As used herein, the term "T cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from the components of a TCR, typically consisting of TCR subunits and antigen-binding regions linked thereto, 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 tumor targeting domain, comprising a single chain antibody, a designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting group; 2 extracellular domain, a single chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3 and an intracellular region of a CD4 co-receptor, wherein the intracellular region is linked to the protein kinase LCK, which 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, "antigen binding region" refers to any structure or functional variant thereof that can bind to an antigen. The antigen binding region 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, antigen binding regions include, but are not limited to IgG, fab, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies, anticalin, DARPIN, and the like, preferably selected from Fab, scFv, sdAb and nanobodies. 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 of a specific protein, such as PD1, PDL2, tgfβ, 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 that 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 may be introduced into the chimeric antigen receptor of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications may be chosen, for example, based on polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the similarity of 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 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 identical residues at identical positions 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 with identical sequences have 100% identity. Those skilled in the art will recognize that some algorithms may 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, e.g., a tumor specific antigen or tumor associated antigen, associated with the particular disease state. 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, CD 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, B H3, 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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20 LY6K, OR E2, 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 breakpoint, ML-IAP, 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, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF beta, APRIL, NKG2D, NKG D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecules expressed by HIV, HCV, HBV and/or other pathogens; and/or neoepitopes or neoantigens. Depending on the antigen to be targeted, the CARs 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 α, CEA, PSCA, PSMA, her2, EGFR, IL13Ra2, GD2, NKG2D, claudin18.2, ROR1, egfrvlll, 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.

As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric antigen receptor on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and directs 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. Particularly suitable transmembrane domains for use in the present invention may be derived from, for example, a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a cd3ζ subunit, a cd3ε subunit, a cd3γ subunit, a cd3δ subunit, CD45, CD4, CD5, CD8 alpha, 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 predominantly comprise hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 3; or from CD 8. 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 invention can further comprise a hinge region 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 natural molecule, such as from the extracellular region of CD8, fcyriii alpha receptor, igG4, igG1, CD4 or CD28, or from the 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 shown in SEQ ID NO. 15; or comprises a CD8 alpha 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. 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 domain" refers to a portion of a protein that transduces an effector function signal and directs a cell to perform a specified function, comprising a co-stimulatory domain and/or a primary signaling domain. Intracellular domains are responsible for intracellular signaling after antigen binding by antigen binding regions, leading to activation of immune cells and immune responses.

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 function together to elicit primary signaling upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar functions. The primary signaling domain may comprise a number of immunoreceptor tyrosine-activating motifs. Non-limiting examples of primary signaling domains of the invention include, but are not limited to, those derived from fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66 d. In a preferred embodiment, the primary signaling domain of a CAR of the invention may comprise a cd3ζ intracellular domain having 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 depicted in SEQ ID No. 9, 10 or 11.

In one embodiment, the chimeric antigen receptor of the 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 whole intracellular portion of said 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 costimulatory domains of the present invention include, but are not limited to, intracellular regions derived from the following proteins: 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, SLP, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the co-stimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40, ICOS, DAP10, DAP12, or a combination thereof. In one embodiment, a 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 set forth in SEQ ID NO. 7 or 8.

In one embodiment, the CAR of the invention may further comprise a signal peptide such that when expressed in a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. Signal peptides useful in the present invention are well known to those skilled in the art, e.g., those derived from B2M, CD alpha, igG1, GM-CSFR alpha, etc. 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 shown 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.

Nucleic acids and vectors

The invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding an exogenous IL9 or a nucleic acid sequence encoding an endogenous IL9 operably linked to a modified promoter. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding one or more chemokines selected from XCL1 and XCL2. Preferably, the nucleic acid molecule further comprises a cell surface molecule that specifically recognizes an antigen, such as a chimeric antigen receptor, a T cell fusion protein, and a T cell antigen coupler 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, linear or circular, each in single-and/or double-stranded form, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the invention include DNA (e.g., dsDNA, ssDNA, cDNA), RNA (e.g., dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (e.g., PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

The invention also provides a vector comprising a nucleic acid molecule according to the invention. Wherein the nucleic acid sequence encoding the exogenous IL9 or the nucleic acid sequence encoding the endogenous IL9 operably linked to the modified promoter, the nucleic acid sequence encoding the chemokine, and the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen may be located in one or more vectors. When in a vector, the nucleic acid sequences may be operably linked by a 2A peptide.

As used herein, the term "vector" is a vector nucleic acid molecule that serves to transfer (exogenous) genetic material into a host cell in which the nucleic acid molecule may, for example, replicate and/or express.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or the use of hybrid recombinases that specifically target sequences at a site. An "expression vector" is a vector for transcription of heterologous nucleic acid sequences (e.g., those encoding chimeric antigen receptor polypeptides of the invention) in a suitable host cell, as well as translation of their mRNA. Suitable carriers useful 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, polyomaviruses, adeno-associated viruses (AAV), and the like), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is typically a nucleotide sequence, typically a DNA sequence comprising an insert (transgene) and a larger sequence that acts as the "backbone" of the vector. The engineered vector typically also comprises 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 promoters, poly A tails (polyA), 3' UTRs, enhancers, terminators, insulators, operators, selectable markers, reporter genes, targeting sequences and/or protein purification tags. In a specific embodiment, the vector is an in vitro transcribed vector.

Engineered immune cells

The invention also provides an engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance expression of IL 9. Preferably, the engineered immune cell further comprises an exogenous chemokine selected from XCL1 and XCL2. Preferably, the engineered immune cell further comprises a cell surface molecule that specifically recognizes an antigen, such as a chimeric antigen receptor, a T cell fusion protein, and a T cell antigen coupler as described above.

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 ipscs, ESCs, hematopoietic stem cells, and the like. Preferably, the immune cells are T cells. 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 an infected site, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. T cells may be at any stage of development, 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, regulatory T cells, naive T cells, γδ -T cells, αβ -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 isolation.

In one embodiment, the immune cells of the invention further comprise the inhibition or silencing of the expression of at least one endogenous gene selected from the group consisting of: CD52, GR, TCRα, TCRβ, 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, expression of at least a TCR component (including TCR α, TCR β genes) or a CD3 component (including CD3 γ, CD3 δ, CD3 epsilon, CD247 ζ) in an immune cell is inhibited or silenced. This strategy is particularly useful for avoiding graft versus host disease (GvHD). Methods of inhibiting or silencing a gene are known in the art, such as by DNA cleavage mediated by a meganuclease, zinc finger nuclease, TALEN nuclease, or Cas enzyme in a CRISPR system, thereby knocking out the gene; or inhibiting gene expression by shRNA, RNAi and the like.

Pharmaceutical composition and combination therapy

The invention also provides a pharmaceutical composition comprising an 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 in the preparation of a pharmaceutical composition.

As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesired local or systemic effects), as is well known in the art (see, e.g., remington's Pharmaceutical sciences.edition 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-adherent agents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonicity agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients is known to those skilled in the art to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the 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 a variety of routes. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

The pharmaceutical composition 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 disease to be treated.

The invention also provides a combination therapy comprising an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous IL9. In another embodiment, the combination therapy comprises: (1) An engineered immune cell expressing exogenous IL9 and an exogenous chemokine; (2) Engineered immune cells expressing exogenous chemokines and exogenous IL9; or (3) engineering immune cells and exogenous IL9 and chemokines; wherein the engineered immune cell expresses a cell surface molecule that specifically recognizes an antigen and the chemokine is selected from XCL1 and XCL2.

Therapeutic application

The invention also provides a method of treating a subject suffering from cancer, an infection or an autoimmune disease comprising administering to the subject an effective amount of a nucleic acid molecule, vector, engineered immune cell or pharmaceutical composition according to the invention. Thus, the invention also covers the use of said nucleic acid molecules, vectors, engineered immune cells for the preparation of a medicament for the treatment of cancer, infections or autoimmune diseases.

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 cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, more preferably T cells, NK cells or NKT cells.

As used herein, the term "autologous" refers to any material derived from an individual that will be later reintroduced into that same individual. As used herein, the term "allogeneic" refers to any material derived from a different animal or different patient of the same species as the individual into which the material was introduced. Two or more bodies are considered to be allogeneic to each other when the genes at one or more loci are different. In some cases, genetic variation of allogeneic 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 may 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, such as a hematological tumor or a solid tumor. For example, the cancers include, but are not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, renal cancer, laryngeal cancer, liver tumors, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenoid lung cancer and squamous lung cancer), lymphomas (including Hodgkin's lymphoma and non-Hodgkin's lymphoma), melanomas, myelomas, neuroblastomas, oral cancer (e.g., lip, tongue, mouth and throat), ovarian cancer, pancreatic cancer, prostate cancer retinoblastoma, rhabdomyosarcoma, rectal cancer, cancers of the respiratory system, salivary gland carcinoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system, vulvar cancer, and other carcinomas and sarcomas, as well as B-cell lymphomas (including low-grade/follicular non-hodgkin lymphoma (NHL), small Lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, higher immunoblastic NHL, higher lymphoblastic NHL, higher small-grade non-cracked cellular NHL, large-tumor-mass NHL), B-lymphoblastic lymphoma (B-LBL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic Lymphocytic Leukemia (CLL) Acute Lymphoblastic Leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, lymphoblastic plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastoid lymphoma, pre-leukemia, plasmacytoid dendritic cell tumor, post-transplant lymphoproliferative disorder (PTLD); and other diseases associated with target expression. Preferably, the disease that can be treated with the engineered immune cells or pharmaceutical compositions of the invention is selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, liver cancer, breast cancer, esophageal cancer, thyroid cancer, prostate cancer, bone cancer, lung cancer, etc.

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, systemic lupus erythematosus, and the like.

In one embodiment, the method further comprises administering one or more additional chemotherapeutic agents, biological agents, drugs, or treatments to the subject. 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 accompanying drawings in combination with examples. It should be noted that, those skilled in the art should understand that the drawings and the embodiments of the present invention are only for illustrative purposes and should not limit the present invention in any way. The embodiments and features of the embodiments in this application may be combined with each other without contradiction.

Detailed Description

Example 1 preparation of CAR-T cells

MSCV-mCD19-CAR plasmids were constructed comprising the coding sequences of CD19-scFv (SEQ ID NO: 2), CD 8. Alpha. Hinge region (SEQ ID NO: 17), CD 8. Alpha. Transmembrane region (SEQ ID NO: 5), 41BB costimulatory domain (SEQ ID NO: 8) and CD3 zeta intracellular region (SEQ ID NO: 11).

A MSCV-mCD19-CAR-IL19 plasmid was constructed which further comprises the coding sequences of T2A (SEQ ID NO: 19) and IL19 (SEQ ID NO: 23) on the basis of the MSCV-mCD19-CAR plasmid.

A MSCV-mCD19-CAR-TSLP plasmid was constructed which further contained the coding sequences of T2A (SEQ ID NO: 19) and XCL1 (SEQ ID NO: 25) on the basis of the MSCV-mCD19-CAR plasmid.

Packaging the above plasmid into retrovirus and further transfecting activated T cells to obtain mCD19-CAR cells of conventional structure, and CAR-T cells expressing IL9 or IL9+XCL1, i.e., mCD19-CAR+IL9 cells, mCD19-CAR+IL9+XCL1 cells (obtained by co-transfection with retrovirus packaged with MSCV-mCD19-CAR-IL9 plasmid and retrovirus packaged with MSCV-mCD19-CAR-XCL1 plasmid).

Example 2 detection of expression of CAR-T cells

2.1 expression level of cell surface CAR

The 2X 10 preparation from example 1 was removed ⁵ Individual CAR-T cells were treated with gold Anti-ray IgG (H &L) Biotin (BioVision, cat. No. 6910-250) as primary antibody, APC strepitavidin (BD Pharmingen, cat. No. 554067) as secondary antibody, and the expression level of CAR on CAR T cells was examined by flow cytometry, the results are shown in FIG. 1. It can be seen that CARs in all CAR-T cells are efficiently expressed compared to untreated NT cells.

2.2Expression level of IL9

Supernatant from CAR-T cells was collected and assayed for secretion of IL9 in the cells using the Mouse IL9 DuoSet ELISA kit (R & D Systems, cat# DY 409) according to the manufacturer's recommendations, and the results are shown in figure 2. It can be seen that both mCD19-car+il9 cells and mCD19-car+il9+xcl1 cells can efficiently express IL9.

EXAMPLE 3 detection of IFN- γ secretion levels by CAR-T cells

In a 96-well round bottom plate with a ratio of 2×10 ⁵ NT cells, CD19-CAR cells, mCD19-CAR+IL9 cells and mCD19-CAR+IL9+XCL1 cells were added at a concentration of 100. Mu.l each. Then at 1X 10 in each well ⁴ Panc02-mCD19 target cells or Panc02 non-target cells were added at a concentration of 100. Mu.l of each cell, respectively. After incubation at 37℃for 24h, the culture supernatant was collected. According to the manufacturer's recommendations, using the Mouse IFN-gamma DuoSet ELISA kit (R&D, product number DY 485) the expression level of IFN-y in the culture supernatant was examined. The detection results are shown in FIG. 3.

It can be seen that no release of IFN-gamma was detected in non-target cells Panc02, whereas only significantly elevated IFN-gamma levels were detected after co-culture with target cells Panc02-CD19, and that NT cells did not express IFN-gamma, indicating that killing of CAR-T cells in this example was specific. In addition, the IFN-gamma level of mCD19-CAR+IL9 cells is significantly higher than that of mCD19-CAR cells, indicating that IL9 gene alone can significantly increase the killing activity of CAR-T cells. Furthermore, the inventors have unexpectedly found that the IFN- γ levels of CAR-T cells expressing the IL9+ XCL1 combination are significantly higher than CAR-T cells expressing IL9 alone, suggesting that IL9 and XCL1 may produce a synergistic effect, further enhancing the killing activity of CAR-T cells.

Example 4 validation of tumor suppression effect of CAR-T cells

The left forelimb armpit part of the healthy C57BL/6 mice was inoculated subcutaneously with 5X 10 ⁵ And (3) Panc02-mCD19 pancreatic cancer cells. Mice vaccinated with pancreatic cancer cells were randomly divided into 4 groups of 6 mice each. Until the tumor volume grows to 100mm ³ At this time, mice of each group were injected via tail vein with 1×10 ⁶ NT cells, mCD19-CAR cells, mCD19-car+il9 cells or mCD19-car+il9+xcl1 cells. The mice were monitored for body weight and tumor volume changes until the end of the experiment.

The body weight change of the mice is shown in fig. 4. As can be seen, there was no significant difference in body weight of the mice of each group compared to the control group after administration of CAR-T cells, indicating that administration of CAR-T cells did not have significant toxic side effects on mice.

The tumor volume change in mice is shown in figure 5. It can be seen that the additional expression of IL9 can significantly enhance the tumor-inhibiting effect of CAR-T cells compared to conventional ACR-T cells. The inventors have also unexpectedly found that the in vivo tumor-inhibiting effect of CAR-T cells expressing the IL9+ XCL1 combination also has significant advantages over CAR-T cells expressing IL9 alone, indicating that IL9 can act synergistically with chemokine production.

The results show that enhancing the expression level of IL9 and chemokines thereof can effectively enhance the inhibition effect of the engineering immune cells expressing the CAR on tumor cells.

It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the present invention, but various modifications and variations of the present invention will be apparent to those skilled in the art. It will be understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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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 alpha 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 alpha 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> 435

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hIL9

<400> 20

atgcttctgg ccatggtcct tacctctgcc ctgctcctgt gctccgtggc aggccagggg 60

tgtccaacct tggcggggat cctggacatc aacttcctca tcaacaagat gcaggaagat 120

ccagcttcca agtgccactg cagtgctaat gtgaccagtt gtctctgttt gggcattccc 180

tctgacaact gcaccagacc atgcttcagt gagagactgt ctcagatgac caataccacc 240

atgcaaacaa gatacccact gattttcagt cgggtgaaaa aatcagttga agtactaaag 300

aacaacaagt gtccatattt ttcctgtgaa cagccatgca accaaaccac ggcaggcaac 360

gcgctgacat ttctgaagag tcttctggaa attttccaga aagaaaagat gagagggatg 420

agaggcaaga tatga 435

<210> 21

<211> 144

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hIL9

<400> 21

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

1 5 10 15

Ala Gly Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe

20 25 30

Leu Ile Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser

35 40 45

Ala Asn Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys

50 55 60

Thr Arg Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr

65 70 75 80

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

85 90 95

Glu Val Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro

100 105 110

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

115 120 125

Leu Glu Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile

130 135 140

<210> 22

<211> 435

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mIL9

<400> 22

atgttggtga catacatcct tgcctctgtt ttgctcttca gttctgtgct gggccagaga 60

tgcagcacca catggggcat cagagacacc aattacctta ttgaaaatct gaaggatgat 120

ccaccgtcaa aatgcagctg cagcggcaac gtgaccagct gcttgtgtct ctccgtccca 180

actgatgatt gtaccacacc gtgctacagg gagggactgt tacagctgac caatgccaca 240

cagaaatcaa gactcttgcc tgttttccat cgggtgaaaa ggatagttga agtcctaaag 300

aacatcacgt gtccgtcctt ttcctgcgaa aagccatgca accagaccat ggcaggcaac 360

acactgtcat ttctgaagag tctcctgggg acgttccaga agacagagat gcaaaggcag 420

aaaagccgac catga 435

<210> 23

<211> 144

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mIL9

<400> 23

Met Leu Val Thr Tyr Ile Leu Ala Ser Val Leu Leu Phe Ser Ser Val

1 5 10 15

Leu Gly Gln Arg Lys Ser Thr Thr Trp Gly Ile Arg Asp Thr Asn Tyr

20 25 30

Leu Ile Glu Asn Leu Lys Asp Asp Pro Pro Ser Lys Cys Ser Cys Ser

35 40 45

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

50 55 60

Thr Thr Pro Cys Tyr Arg Glu Gly Leu Leu Gln Leu Thr Asn Ala Thr

65 70 75 80

Gln Lys Ser Arg Leu Leu Pro Val Phe His Arg Val Lys Arg Ile Val

85 90 95

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

100 105 110

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

115 120 125

Leu Gly Thr Phe Gln Lys Thr Glu Met Gln Arg Gln Lys Ser Arg Pro

130 135 140

<210> 24

<211> 345

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mXCL1

<400> 24

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> 25

<211> 114

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mXCL1

<400> 25

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> 26

<211> 345

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hXCL1

<400> 26

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> 27

<211> 114

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hXCL1

<400> 27

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> 28

<211> 345

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hXCL2

<400> 28

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> 29

<211> 114

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hXCL2

<400> 29

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

Claims (26)

1. An engineered immune cell comprising exogenous IL9 or a modified promoter operably linked to endogenous IL9 to enhance expression of IL 9.

2. The engineered immune cell of claim 1, wherein the modified promoter is a constitutive promoter or an inducible promoter.

3. The engineered immune cell of claim 1, wherein the exogenous IL9 is wild-type IL9 or a variant thereof that has the same or similar function as wild-type IL 9.

4. The engineered immune cell of claim 1, wherein the IL9 is identical to SEQ ID NO:21 or 23 has at least 90% identity.

5. The engineered immune cell of claim 1, wherein the engineered immune cell further expresses an exogenous chemokine selected from XCL1 and XCL2.

6. The engineered immune cell of claim 5, wherein the XCL1 is identical to SEQ ID NO:25 or 27 has at least 90% identity to the amino acid sequence set forth in seq id no; XCL2 and SEQ ID NO:29 has at least 90% identity.

7. The engineered immune cell of claim 1, further expressing a cell surface molecule comprising an antigen binding region that specifically recognizes an antigen.

8. The engineered immune cell of claim 1, wherein the cell surface molecule is a chimeric antigen receptor, a T cell fusion protein, and a T cell antigen coupler.

9. The engineered immune cell of claim 7, wherein the antigen binding region is selected from the group consisting of IgG, fab, fab ', F (ab ') 2, fd ', fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin, and DARPIN antigen.

10. The engineered immune cell of claims 7-9, wherein the 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, CD 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, B H3, 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 GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20 LY6K, OR E2, 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 breakpoint, ML-IAP, 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, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF beta, APRIL, NKG2D, NKG D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecules expressed by HIV, HCV, HBV and/or other pathogens; and/or neoepitopes or neoantigens.

11. The engineered immune cell of any one of claims 7-10, wherein the cell surface molecule is a chimeric antigen receptor comprising an antigen binding region, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and/or a primary signaling domain.

12. The engineered immune cell of claim 11, wherein the transmembrane domain is selected from the group consisting of the transmembrane domains of: TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, cd3ζ subunit, cd3ε subunit, cd3γ subunit, cd3δ subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

13. The engineered immune cell of any one of claims 11-12, wherein the primary signaling domain is selected from the intracellular regions of: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66d.

14. The engineered immune cell of any one of claims 11-13, wherein the co-stimulatory domain comprises one or more intracellular regions 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, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, DAP12, LAT, NKG2C, SLP, PD-1, LIGHT, TRIM or ZAP70.

15. The engineered immune cell of any one of claims 1-14, wherein the immune cell is selected from a T cell, B cell, macrophage, dendritic cell, monocyte, NK cell, or NKT cell.

16. The engineered immune cell of claim 15, wherein the T cell is a cd4+cd8+ T cell, a cd4+ T cell, a cd8+ T cell, a CD4-CD8-T cell, a tumor infiltrating cell, a memory T cell, a regulatory T cell, a naive T cell, a γδ -T cell, or an αβ -T cell.

17. A nucleic acid molecule comprising a nucleic acid sequence encoding an exogenous IL9 or a nucleic acid sequence encoding an endogenous IL9 operably linked to a modified promoter.

18. The nucleic acid molecule of claim 17, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding an exogenous chemokine selected from XCL1 and XCL2.

19. The nucleic acid molecule of claim 17 or 18, further comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, the cell surface molecule being a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler.

20. A vector comprising the nucleic acid molecule of any one of claims 17-19.

21. The vector of claim 20, wherein the vector is selected from the group consisting of a plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, rous Sarcoma Virus (RSV), polyomavirus, and adeno-associated virus (AAV).

22. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-16, the nucleic acid molecule of any one of claims 17-19, or the vector of any one of claims 20-21, and one or more pharmaceutically acceptable excipients.

23. Use of an engineered immune cell of any one of claims 1-16, a nucleic acid molecule of any one of claims 17-19, a vector of any one of claims 20-21, or a pharmaceutical composition of claim 22 in the manufacture of a medicament for treating a subject having 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 an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous IL9.

26. A combination therapy comprising: (1) An engineered immune cell expressing exogenous IL9 and an exogenous chemokine; (2) Engineered immune cells expressing exogenous chemokines and exogenous IL9; or (3) engineering immune cells and exogenous IL9 and chemokines; wherein the engineered immune cell expresses a cell surface molecule that specifically recognizes an antigen and the chemokine is selected from XCL1 and XCL2.

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