CN115786268A - Engineered immune cells and uses thereof - Google Patents

Abstract

The present invention relates to an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen, exogenous IL7 and CXCL9. The invention also provides the use of the engineered immune cells in the treatment of cancer, infection or autoimmune disease. Compared with the traditional engineered immune cell, the engineered immune cell has obviously improved tumor killing activity.

Description

Engineered immune cells and uses thereof

Technical Field

The present invention is in the field of immunotherapy. More specifically, the invention relates to an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen, exogenous IL7 and CXCL9. 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 therapy. 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 being the inability to effectively transport the engineered cells to the tumor site (i.e., the immunosuppressive tumor microenvironment).

Thus, there remains a need for improved cell therapies to counteract the inhibitory effects of the tumor microenvironment, while recruiting other immune effector cells to the tumor site to potentiate 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 IL7 and CXCL9.

In one embodiment, the cell surface molecule specifically recognizing an antigen is a chimeric antigen receptor, a T cell fusion protein or a T cell antigen coupler, 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 domain comprising a costimulatory 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: <xnotran> CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40 3245 zxft 3245 44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2 3732 zxft 3732 40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa , TIM3, TSHR, CD19, BAFF-3963 zxft 3963-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn , PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, , PSCA, PRSS21, VEGFR2, lewisY, PDGFR- β, SSEA-4, AFP, folate α, erbB2 (Her 2/neu), erbB3, erbB4, MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, c-Met, prostase, PAP, ELF2M, ephrin B2, IGF-I , CAIX, LMP2, gpl00, bcr-abl, , ephA2, fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o- -GD2, folate β, TEM7 4325 zxft 4325 6, GPRC5 3536 zxft 3536 61, ALK, , PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6 3926 zxft 3926 51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, , HPV E6, E7, ETV6-AML, 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos 1, p53, p53 , PSA, , PCTA-l/Galectin 8, melanA/MARTl, ras , hTERT, , ML-IAP, </xnotran> 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, NKG D ligand, and/or a biotinylated molecule specific for antigen, biotinylated molecule, molecule expressed by HIV, HBV, HCV and/or other pathogens; 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 δ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28 and CD 278.

In one embodiment, the primary signaling domain is an intracellular region of a protein selected from the group consisting of: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 epsilon, CD3 δ, 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, NKG2 8978 zx8978, 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 CXCL9 and/or IL7 is constitutive expression. In another embodiment, the exogenous CXCL9 and/or IL7 expression or activity 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, CXCL9 and/or IL7 can 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, exogenous genes of the invention, e.g., CXCL9 and/or IL7, are 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, a nucleic acid sequence encoding CXCL9 and a nucleic acid sequence encoding IL7. Preferably, the cell surface molecule specifically recognizing an antigen 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 described above. In particular, the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous Sarcoma Viruses (RSV), polyoma viruses, and adeno-associated viruses (AAV). In some embodiments, the vector further comprises elements such as an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a poly A tail (polyA), a 3'UTR, a 5' UTR, an enhancer, a terminator, an insulator, an operator, a selectable marker, a reporter gene, a targeting sequence, and/or a protein purification tag. In a specific embodiment, the vector is an in vitro transcription vector.

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

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

In a fourth aspect, the invention also provides a combination therapy comprising: (1) An engineered immune cell expressing CXCL9 and a composition comprising exogenous IL 7; (2) Engineered immune cells expressing IL7 and compositions comprising exogenous CXCL 9; or (3) an engineered immune cell that expresses a cell surface molecule that specifically recognizes an antigen, and a composition comprising exogenous IL7 and CXCL9.

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 CXCL9 by CAR-T cells determined by ELISA.

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

FIG. 5: weight change profile of mice after treatment of pancreatic cancer in mice 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

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 IL7 and CXCL9.

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 of the T cell surface that binds to CD3 in a non-covalent bond to form a complex. Antigen presenting cells present antigenic peptides to T cells via major histocompatibility complex Molecules (MHC) 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.

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 domain (comprising a costimulatory domain and/or a primary signaling domain) connected 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, the term "T cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from components of a TCR, typically consisting of TCR subunits and antigen-binding regions linked thereto, and expressed on the cell surface. Wherein the TCR subunit comprises at least a portion of a TCR extracellular domain, a transmembrane domain, a TCR intracellular signaling domain.

As used herein, the term "T cell antigen coupler" or "TAC" includes three functional domains: 1 a tumor targeting domain comprising a single chain antibody, designed ankyrin repeat protein (DARPin), or other targeting group; 2 an extracellular domain, a single chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3 transmembrane region and the intracellular region of the CD4 co-receptor, wherein the intracellular region is linked to the protein kinase LCK, catalyzes the phosphorylation of the Immunoreceptor Tyrosine Activation Motif (ITAM) of the TCR complex as an initial step in T cell activation.

As used herein, "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 can 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. Such 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 having 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 selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

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

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of 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: <xnotran> CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40 3245 zxft 3245 44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2 3732 zxft 3732 40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa , TIM3, TSHR, CD19, BAFF-3963 zxft 3963-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn , PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, , PSCA, PRSS21, VEGFR2, lewisY, PDGFR- β, SSEA-4, AFP, folate α, erbB2 (Her 2/neu), erbB3, erbB4, MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, c-Met, prostase, PAP, ELF2M, ephrin B2, IGF-I , CAIX, LMP2, gpl00, bcr-abl, , ephA2, fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o- -GD2, folate β, TEM7 4325 zxft 4325 6, GPRC5 3536 zxft 3536 61, ALK, , PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6 3926 zxft 3926 51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, , HPV E6, E7, ETV6-AML, 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos 1, p53, p53 , PSA, , PCTA-l/Galectin 8, melanA/MARTl, ras , hTERT, , ML-IAP, </xnotran> 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, NKG D ligand, and/or a biotinylated molecule specific for antigen, biotinylated molecule, molecule expressed by HIV, HBV, HCV and/or other pathogens; 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.

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. 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 δ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, 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 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 depicted in SEQ ID NO 18.

As used herein, the term "intracellular 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 domain is responsible for intracellular signaling after binding of the antigen at the antigen binding region, resulting in activation of the immune cell and immune response.

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 activation motifs. 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 δ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the primary signalling 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 the amino acid sequence set forth in SEQ ID No. 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, which comprises 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, NKG2 8978 zx8978, PD-1, LIGHT, TRIFT 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. Signal peptides useful in the present invention are well known to those skilled in the art, such as those derived from B2M, CD 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 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.

Exogenous gene

In addition to cell surface molecules that specifically recognize antigens, the engineered immune cells of the invention also express exogenous IL7 and CXCL9.

CXCL9 is one of the members of the CXC subfamily, is expressed mainly on T cells and NK cells, and plays an important role in autoimmune diseases, tumor therapy, and allogeneic transplantation in the body. CXCL9 has been reported to promote the polarization of effector Th1 and Th17 cells, thereby enhancing immune responses and anti-tumor effects.

In one embodiment, CXCL9 for use in the invention can be wild-type CXCL9, a variant or functional fragment thereof having the same or similar biological function as wild-type CXCL9, or a functional fragment thereof. Specifically, CXCL9 is identical to SEQ ID NO:25 or 27 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence for CXCL9 shares sequence identity with 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 and comparable activity to wild-type CXCL9.

In one embodiment, the IL7 used in the present invention may be a wild-type IL7, a variant or functional fragment thereof having the same or similar biological function as wild-type IL7. Specifically, the peptide has a sequence similar 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 CXCL9 and/or IL7, may be constitutive expression or conditional expression. In one embodiment, expression of exogenous CXCL9 and/or IL7 is conditional. 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. 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.

In one embodiment, CXCL9 and/or IL7 can be operably linked to a localization domain that can localize the exogenous gene of the invention for expression at 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 genes CXCL9 and/or IL7 of the invention are operably linked to a transmembrane domain, thereby anchoring surface expression in engineered immune cells.

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, a nucleic acid sequence encoding CXCL9 and 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 definition of chimeric antigen receptor is as described above.

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

The invention also provides a vector comprising a nucleic acid according to the invention. Wherein the nucleic acid sequence encoding a cell surface molecule specifically recognizing the antigen, the nucleic acid sequence encoding CXCL9 and the nucleic acid sequence encoding IL7 can 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 used as a vehicle for transferring (foreign) genetic material into a host cell where it can, for example, be replicated and/or expressed.

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, exogenous IL7 and CXCL9.

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 isolation.

In one embodiment, the immune cell of the invention further comprises suppressed or silenced expression of at least one endogenous gene selected from the group consisting of: CD52, GR, TCR alpha, TCR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD247 delta, 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 the 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, e.g., by mediating DNA cleavage by meganucleases, zinc finger nucleases, TALEN nucleases or Cas enzymes in CRISPR systems, thereby knocking out the gene; or by shRNA, RNAi, or the like.

Pharmaceutical compositions and combination therapies

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

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) and 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, isotonizing agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients to prepare the desired pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the selection 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 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.

The present invention also provides a combination therapy comprising: (1) An engineered immune cell expressing CXCL9 and a composition comprising exogenous IL 7; (2) Engineered immune cells expressing IL7 and compositions comprising exogenous CXCL 9; or (3) an engineered immune cell that expresses a cell surface molecule that specifically recognizes an antigen, and a composition comprising exogenous IL7 and CXCL9.

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 a nucleic acid molecule, vector, engineered immune cell, or pharmaceutical composition according to the invention. Thus, the invention also encompasses the use of said nucleic acid molecule, vector, engineered immune cell for 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 to which an antigen binding region binds, such as a hematologic tumor or a solid tumor. For example, the cancer includes, but is not limited to: <xnotran> , , , , , , , , CNS , , , , , , , , , , , , ( ), (GBM), , , , , , , ( , , ), ( ), , , , ( , , ), , , , , , , , , , , , , , , , , B ( / (NHL), (SL) NHL, / NHL, NHL, NHL, NHL, NHL, NHL), B (B-LBL), , AIDS , Waldenstrom , (CLL), </xnotran> 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 disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmacytic lymphoma, pre-leukemic, 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, 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, and systemic lupus erythematosus, among others.

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 accompanying 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-CXCL9 plasmid was constructed, which further comprises the coding sequence of T2A (SEQ ID NO: 19) and CXCL9 (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 Individual cells/flask 293T cells were seeded in 30ml DMEM medium containing 10% foetal calf serum at 37 ℃ 5% ₂ The cells were cultured overnight in an incubator for virus packaging.

To a sterile tube, 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 cereal noon Biotech Co., ltd., cat # P3029) were added. Then 120 mu l X-treme GENE HP DNA transfection reagent (Roche, cat # 06366236001) was addedI.e., mixed well 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 Incubated under conditions overnight. 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 overnight with RetroNectin, 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 centrifugation infection 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 + CXCL9 cells, mCD19-CAR + IL7 cells and mCD19-CAR + IL7+ CXCL9 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 were taken out ⁵ Individual CAR-T cells, treated with Goat Anti-Rat IgG (H)&L) Biotin (BioVision, cat # 6910-250) as a primary antibody and APC Streptavidin (BD Pharmingen, cat # 554067) as a secondary antibody, the expression level of CAR on CAR T cells was examined by flow cytometry, and the results are shown in FIG. 1. It can be seen that CAR was efficiently expressed in all CAR-T cells compared to untreated NT cells.

2.2 Expression level of IL7

The supernatant of CAR-T cells was collected and the level of IL7 secretion in the cells was determined 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 both mCD19-CAR + IL7 cells and mCD19-CAR + IL7+ CXCL9 cells can efficiently express IL7.

2.3 Expression level of CXCL9

The supernatant of CAR-T cells was collected and the level of CXCL9 secretion in the cells was measured using the Mouse CXCL9 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 + CXCL9 cells and mCD19-CAR + IL7+ CXCL9 cells can efficiently express CXCL9.

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 + IL7 cells, mCD19-CAR + CXCL9 cells and mCD19-CAR + IL7+ CXCL9 cells were added at a concentration of 100. Mu.l each. Then 1X 10 in each well ⁴ At a concentration of 100. Mu.l of each cell, either Panc02-mCD19 target cells or Panc02 non-target cells were added. 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 IFN- γ release 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 the killing of CAR-T cells in this example is specific. In addition, the level of IFN- γ was significantly higher for mCD19-CAR + CXCL9 cells and mCD19-CAR + IL7 cells than for CD19-CAR cells, indicating that both the additionally expressed IL7 and CXCL9 genes alone significantly increased the killing activity of CAR-T cells. Furthermore, the inventors have also unexpectedly found that CAR-T cells expressing the IL7+ CXCL9 combination have significantly higher IFN- γ levels than CAR-T cells expressing either CXCL9 or IL7 alone, indicating that IL7 and CXCL9 can produce a synergistic effect, further enhancing the killing activity of CAR-T cells.

Example 4 demonstration of tumor-inhibiting Effect of CAR-T cells

In healthy C57BL/6 miceThe axillary part of the left forelimb is inoculated with 5 multiplied by 10 under the skin 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 At the same time, 1X 10 injections were administered into each group of mice via the tail vein ⁶ Individual NT cells, mCD19-CAR + IL7 cells, mCD19-CAR + CXCL9 cells or mCD19-CAR + IL7+ CXCL9 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.

Changes in tumor volume in mice are shown in figure 6. It can be seen that expression of IL7 alone can enhance the tumor-suppressing effect of CAR-T cells, while CXCL9 alone has a tumor-suppressing effect comparable to that of conventional CAR-T cells, indicating that CXCL9 does not promote CAR-T cells as well as IL7. Furthermore, the inventors have also unexpectedly found that the in vivo anti-tumor effect of mCD19-CAR + IL7+ CXCL9 cells is significantly better than that of CAR-T cells expressing only IL7 or CXCL9, indicating that the additionally expressed IL7+ CXCL9 can produce a synergistic effect with CAR-T cells, enhancing the anti-tumor effect of the latter.

The above results indicate that the combination of co-expressing CXCL9 and 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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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> 381

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCXCL9

<400> 24

atgaagtccg ctgttctttt cctcttgggc atcatcttcc tggagcagtg tggagttcga 60

ggaaccctag tgataaggaa tgcacgatgc tcctgcatca gcaccagccg aggcacgatc 120

cactacaaat ccctcaaaga cctcaaacag tttgccccaa gccccaattg caacaaaact 180

gaaatcattg ctacactgaa gaacggagat caaacctgcc tagatccgga ctcggcaaat 240

gtgaagaagc tgatgaaaga atgggaaaag aagatcagcc aaaagaaaaa gcaaaagagg 300

gggaaaaaac atcaaaagaa catgaaaaac agaaaaccca aaacacccca aagtcgtcgt 360

cgttcaagga agactacata a 381

<210> 25

<211> 126

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCXCL9

<400> 25

Met Lys Ser Ala Val Leu Phe Leu Leu Gly Ile Ile Phe Leu Glu Gln

1 5 10 15

Cys Gly Val Arg Gly Thr Leu Val Ile Arg Asn Ala Arg Cys Ser Cys

20 25 30

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

35 40 45

Lys Gln Phe Ala Pro Ser Pro Asn Cys Asn Lys Thr Glu Ile Ile Ala

50 55 60

Thr Leu Lys Asn Gly Asp Gln Thr Cys Leu Asp Pro Asp Ser Ala Asn

65 70 75 80

Val Lys Lys Leu Met Lys Glu Trp Glu Lys Lys Ile Ser Gln Lys Lys

85 90 95

Lys Gln Lys Arg Gly Lys Lys His Gln Lys Asn Met Lys Asn Arg Lys

100 105 110

Pro Lys Thr Pro Gln Ser Arg Arg Arg Ser Arg Lys Thr Thr

115 120 125

<210> 26

<211> 378

<212> DNA

<213> Artificial sequence(Artificial Sequence)

<220>

<223> hCXCL9

<400> 26

atgaagaaaa gtggtgttct tttcctcttg ggcatcatct tgctggttct gattggagtg 60

caaggaaccc cagtagtgag aaagggtcgc tgttcctgca tcagcaccaa ccaagggact 120

atccacctac aatccttgaa agaccttaaa caatttgccc caagcccttc ctgcgagaaa 180

attgaaatca ttgctacact gaagaatgga gttcaaacat gtctaaaccc agattcagca 240

gatgtgaagg aactgattaa aaagtgggag aaacaggtca gccaaaagaa aaagcaaaag 300

aatgggaaaa aacatcaaaa aaagaaagtt ctgaaagttc gaaaatctca acgttctcgt 360

caaaagaaga ctacataa 378

<210> 27

<211> 125

<212> PRT

<213> Artificial sequence(Artificial Sequence)

<220>

<223> mCXCL9

<400> 27

Met Lys Lys Ser Gly Val Leu Phe Leu Leu Gly Ile Ile Leu Leu Val

1 5 10 15

Leu Ile Gly Val Gln Gly Thr Pro Val Val Arg Lys Gly Arg Cys Ser

20 25 30

Cys Ile Ser Thr Asn Gln Gly Thr Ile His Leu Gln Ser Leu Lys Asp

35 40 45

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

50 55 60

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

65 70 75 80

Asp Val Lys Glu Leu Ile Lys Lys Trp Glu Lys Gln Val Ser Gln Lys

85 90 95

Lys Lys Gln Lys Asn Gly Lys Lys His Gln Lys Lys Lys Val Leu Lys

100 105 110

Val Arg Lys Ser Gln Arg Ser Arg Gln Lys Lys Thr Thr

115 120 125

Claims (21)

1. An engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous IL7 and CXCL9.

2. The engineered immune cell of claim 1, wherein the CXCL9 is identical to SEQ ID NO:25 or 27, or a coding sequence thereof which is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:24 or 26 have at least 90% identity; wherein said IL7 is substantially identical to 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.

3. The engineered immune cell of any one of claims 1-2, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler.

4. The engineered immune cell of claim 3, wherein the chimeric antigen receptor comprises an antigen binding region, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and/or a primary signaling domain.

5. The engineered immune cell of claim 4, 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.

6. The engineered immune cell of claim 4, wherein the antigen-antigen binding region binds to one or more targets selected from the group consisting of: <xnotran> CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40 3245 zxft 3245 44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2 3732 zxft 3732 40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa , TIM3, TSHR, CD19, BAFF-3963 zxft 3963-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn , PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, , PSCA, PRSS21, VEGFR2, lewisY, PDGFR- β, SSEA-4, AFP, folate α, erbB2 (Her 2/neu), erbB3, erbB4, MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, c-Met, prostase, PAP, ELF2M, ephrin B2, IGF-I , CAIX, LMP2, gpl00, bcr-abl, , ephA2, fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o- -GD2, folate β, TEM7 4325 zxft 4325 6, GPRC5 3536 zxft 3536 61, ALK, , PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6 3926 zxft 3926 51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, , HPV E6, E7, ETV6-AML, 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos 1, p53, p53 , PSA, , PCTA-l/Galectin 8, melanA/MARTl, ras , hTERT, , ML-IAP, </xnotran> 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, NKG D ligand, and/or a biotinylated molecule specific for antigen, biotinylated molecule, molecule expressed by HIV, HBV, HCV and/or other pathogens; and/or a neoepitope or neoantigen.

7. The engineered immune cell of claim 4, 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 δ 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 and CD154.

8. The engineered immune cell of any one of claims 4-7, wherein the primary signaling domain is selected from the intracellular regions of: fcR γ, fcR β, CD3 γ, CD3 δ, CD3 e, CD3 δ, CD22, CD79a, CD79b, and CD66d.

9. The engineered immune cell of claim 4, 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, NKG2 8978 zx8978, PD-1, LIGHT, TRIM or ZAP70.

10. The engineered immune cell of any one of claims 1-9, wherein the immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.

11. The engineered immune cell of claim 10, 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.

12. The engineered immune cell of any one of claims 1-11, wherein the expression of CXCL9 and/or IL7 is conditional or constitutive expression.

13. The engineered immune cell of any one of claims 1-12, wherein the CXCL9 and/or IL7 is operably linked to a localization domain.

14. A nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, a nucleic acid sequence encoding CXCL9 and a nucleic acid sequence encoding IL7.

15. The nucleic acid molecule of claim 14, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler.

16. A vector comprising the nucleic acid molecule of any one of claims 14-15.

17. The vector of claim 16, 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).

18. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 1-13, a nucleic acid molecule according to any one of claims 14-15 or a vector according to any one of claims 16-17, and one or more pharmaceutically acceptable excipients.

19. Use of the engineered immune cell of any one of claims 1-13, the nucleic acid molecule of any one of claims 14-15, the vector of any one of claims 16-17, or the pharmaceutical composition of claim 18 in the manufacture of a medicament for treating a subject having cancer, an infection, or an autoimmune disease.

20. The use of claim 19, wherein the cancer is a hematological tumor or a solid tumor.

21. A combination therapy comprising: (1) An engineered immune cell expressing CXCL9 and a composition comprising exogenous IL 7; (2) Engineered immune cells expressing IL7 and compositions comprising exogenous CXCL 9; or (3) engineered immune cells and a composition comprising exogenous IL7 and CXCL 9; wherein the engineered immune cell expresses a cell surface molecule that specifically recognizes an antigen.

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