WO2023051361A1 - Engineered immune cell and use thereof - Google Patents

Abstract

Provided is an engineered immune cell that expresses a cell surface molecule that specifically recognizes an antigen and one or more exogenous chemokines. The exogenous chemokines are selected from CXCL10 and CXCL11. Also provided is a use of the engineered immune cell in the treatment of cancer, infections or autoimmune diseases.

Description

Engineered immune cells and uses thereof

Cross References to Related Applications

This disclosure claims the priority of the Chinese patent application with application number CN202111149283.2 and titled "Engineered Immune Cells and Their Uses" filed with the China Patent Office on September 29, 2021, the entire contents of which are incorporated by reference in this disclosure middle.

technical field

The invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered Th9 cell expressing a chimeric receptor and optionally a cytokine that specifically recognizes Claudin18.2. More preferably, the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor.

Background technique

In recent years, adoptive cell therapy, as an emerging immunotherapy, has shown great advantages in the field of tumor treatment. Currently, while most adoptive cell therapies use CD8+ T cells, there are

Th1, 2, 9, 17, 22, Tfh.

Or CD4+ T cells.

This therapy usually requires the modification of cells first, such as gene editing and/or transduction to modify cells to carry exogenous proteins such as chimeric antigen receptors and recombinant T cell receptors, and then expand them in vitro , and returned to the patient. At present, these therapies have shown good efficacy against hematological malignancies, but their efficacy is not satisfactory for solid tumors. One of the reasons is that the engineered cells cannot effectively reach the tumor due to the immunosuppressive tumor microenvironment. tumor site.

Th9 cells are a recently discovered CD4+ subpopulation effector T cells, which are induced by resting T cells under the co-culture conditions of transforming growth factor β (TGF-β) and interleukin (IL) 4, and can also be produced by TGF-β alone induces Th2 cells to differentiate, and PU.1 and interferon regulatory factor 4 genes are considered to be specific transcription factors for Th9 cell differentiation. Experiments in mice have shown that Th9 cells can secrete cytokines IL-9 and IL-10 in large quantities. However, unlike mice, human Th9 cells do not secrete IL-10. Th9 cells play an important role in allergic diseases, anti-parasitic infections and autoimmune diseases. The discovery of Th9 cell subsets has found a new breakthrough for the study of etiology and clinical diagnosis and treatment of these diseases.

Therefore, there is still a need for improved cell therapies that can resist the suppressive effect of the tumor microenvironment while recruiting other immune effector cells to the tumor site to enhance the antitumor effect.

Contents of the invention

In a first aspect, the present invention provides a novel engineered Th9 cell, which expresses cell surface molecules that specifically recognize antigens and one or more exogenous cytokines, wherein the cytokines are selected from XCL1, XCL2, IL7, TSLP.

In one embodiment, the present invention provides a novel engineered Th9 cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous TSLP.

In one embodiment, the present invention provides a novel engineered Th9 cell that expresses cell surface molecules that specifically recognize antigens and one or more exogenous cytokines, wherein the cytokines are selected from XCL1, XCL2 and IL7.

In one embodiment, the cell surface molecule that specifically recognizes an antigen comprises an antigen binding region and is a chimeric antigen receptor, a T cell 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, Fd', Fv, scFv, sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies , anticalin and DARPIN. Preferably, the antigen binding region is selected from scFv, Fab, single domain antibodies and nanobodies.

In one embodiment, the cell surface molecule that specifically recognizes the antigen binds to one or more targets selected from: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD179a , DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII , tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelial PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2 (Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2, c-Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl Gene-GD2, Folate receptor β, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1 , MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutants, hTERT, sarcoma translocation breakpoints, 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, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, produced by HIV, HCV, HBV, and/or Molecules expressed by other pathogens; and/or neo-epitopes 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 co-stimulatory domain and/or primary signaling domain.

In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8α, CD4, CD28 and CD278.

In one embodiment, the primary signaling domain is an intracellular region of a protein selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, said 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(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7-H3) , CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof. Preferably, the co-stimulatory domain is selected from the intracellular region of CD27, CD28, CD134, CD137, DAP10, DAP12 or CD278 or a combination thereof.

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

In one embodiment, the expression or activity of the exogenous chemokine and/or IL7 is constitutive. In another embodiment, the expression or activity of the exogenous chemokine and/or IL7 is conditional. For example, conditional expression is achieved by operably linking the exogenous gene to an inducible, repressible or tissue-specific promoter.

In one embodiment, the chemokine and/or IL7 can be operably linked to a localization domain, which can localize the expression of the exogenous gene of the present invention to a specific cellular location, such as a cell membrane. In one embodiment, exogenous genes of the present invention, such as chemokines and/or IL7, are operably linked to transmembrane domains, thereby anchoring expression on the surface of engineered immune cells.

In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding a chemokine, preferably further comprising a nucleic acid sequence encoding IL7. Preferably, the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor, a T cell receptor, a T cell fusion protein or a T cell antigen coupler, more preferably a chimeric antigen receptor.

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

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

In a third aspect, the present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the immune cells according to the present invention, Nucleic acid molecule, vector or pharmaceutical composition.

In the fourth aspect, the present invention also provides a combination therapy comprising engineered immune cells expressing cell surface molecules that specifically recognize antigens and one or more exogenous chemokines, the exogenous chemokines The factor is selected from CXCL10 and CXCL11. In one embodiment, the combination therapy comprises: (1) engineered immune cells expressing exogenous chemokines and exogenous IL7; (2) engineered immune cells expressing IL7 and exogenous chemokines or (3) engineered immune cells and exogenous IL7 and chemokines; wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, wherein the chemokines are selected from CXCL10 and CXCL11.

Description of drawings

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

Figure 2: The expression level of IL7 in CAR-T cells determined by ELISA.

Figure 3: Expression levels of CXCL10 and CXCL11 in CAR-T cells determined by ELISA.

Figure 4: IFN-γ release levels after co-culture of CAR-T cells expressing CXCL10 (A) or CXCL11 (B) with target and non-target cells, respectively.

Figure 5: The body weight change curve of mice after pancreatic cancer was treated with CAR-T cells.

Figure 6: Tumor growth curves of mice treated with CAR-T cells for pancreatic cancer.

Detailed description of the invention

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

Cell surface molecules that specifically recognize antigens

In a first aspect, the present invention provides a novel engineered immune cell, a cell surface molecule that specifically recognizes an antigen and one or more exogenous chemokines selected from From CXCL10 and CXCL11.

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 (eg, an antigen). Such surface molecules generally 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 signal transmission. Common examples of such surface molecules include eg T cell receptor (TCR), chimeric antigen receptor (CAR), T cell fusion protein (TFP) or T cell antigen coupler (TAC).

As used herein, the term "T cell receptor" or "TCR" is a characteristic marker on the surface of T cells that binds to CD3 in a non-covalent bond to form a complex. Antigen presenting cells present antigenic peptides to T cells through major histocompatibility complex molecules (MHC) and bind to TCR complexes to induce a series of intracellular signaling. TCR is composed of six peptide chains that form heterodimers, which are generally divided into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding 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 (such as the antigen-binding portion of an antibody), a transmembrane domain, and an intracellular Domains (comprising co-stimulatory domains and/or primary signaling domains), each domain is connected by a linker. CARs are able to exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to a target of choice in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR cells the ability to recognize antigens independently of antigen processing, thus bypassing major mechanisms of tumor escape. Furthermore, CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR) when expressed in T cells.

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

As used herein, the term "T cell antigen coupler" or "TAC" includes three functional domains: 1 tumor targeting domain, including single-chain antibody, designed ankyrin repeat protein (DARPin) or Other targeting groups; 2 extracellular region domain, single-chain antibody binding to CD3, thus bringing TAC receptor and TCR receptor closer; 3 transmembrane region and intracellular region of CD4 co-receptor, wherein, intracellular The domain-linked protein kinase, LCK, catalyzes the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) of the TCR complex as an initial step in T cell activation.

As used herein, "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 antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, murine antibody, chimeric antibody and functional fragments thereof. For example, antigen binding domains include, but are not limited to, IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, single domain antibodies, nanobodies, diabodies, anticalins, DARPIN etc., preferably selected from Fab, scFv, sdAb and Nanobodies. In the present invention, the antigen binding domain 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, PDL1, PDL2, TGFβ, APRIL and NKG2D.

The term "functional variant" or "functional fragment" refers to a variant comprising essentially the amino acid sequence of a parent but containing at least one amino acid modification (i.e. substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the Such variants retain the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment comprising the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include 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), non-polar side chains (e.g. alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, on the basis of 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% of the parent amino acid sequence. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.

As used herein, the term "sequence identity" means the degree to which two (nucleotide or amino acid) sequences in an alignment have the same residue at the same position, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies of the exact same sequence have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and Clustal W.

The choice of the antigen binding region depends on the cell surface markers to be recognized on the target cells associated with the particular disease state, such as tumor-specific or tumor-associated antigens. 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, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4 , DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR , GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2, c- Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl- GD2, Folate receptor β, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD -CT-2, Fos-related antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML- IAP, TMPRSS2ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4 , SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2 , LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, expressed by HIV, HCV, HBV, and/or other pathogens and/or neo-epitopes or neoantigens. Depending on the antigen to be targeted, the CAR of the invention can be designed to include an antigen-binding region specific for that antigen. Preferably, the target is selected from 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, a CD19 antibody can be used as the antigen binding region of the present invention.

As used herein, the term "transmembrane domain" refers to a polypeptide capable of expressing a chimeric antigen receptor on the surface of an immune cell (such as a lymphocyte, NK cell or NKT cell) and directing a cellular response of the immune cell against a target cell structure. Transmembrane domains can be natural or synthetic and can 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, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and may comprise predominantly 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% of the amino acid sequence shown in SEQ ID NO:3 or 100% sequence identity; or derived from CD8α, it has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% with the amino acid sequence shown in SEQ ID NO: 4 or 5 or 100% sequence identity.

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 link a transmembrane domain to an antigen binding region. Specifically, the hinge region is used 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 in whole or in part from the extracellular region of CD8, FcyRIIIa receptor, IgG4, IgGl, 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 an entirely synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a CD28 hinge, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence shown in SEQ ID NO:15. % or 100% sequence identity; or comprising a CD8α hinge, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity; or comprising an IgG4 hinge having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% to the amino acid sequence shown in SEQ ID NO: 18 % or 100% sequence identity.

As used herein, the term "intracellular domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a given function, which includes costimulatory domains and/or primary signaling domains. The intracellular domain is responsible for intracellular signaling following antigen binding by the antigen binding region, resulting in activation of immune cells and immune responses.

In one embodiment, the chimeric antigen receptors of the invention comprise a primary signaling domain, which may be the cytoplasmic sequence of the T cell receptor and co-receptor that function together to elicit primary signaling following antigen receptor binding , and any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. Primary signaling domains can contain many immunoreceptor tyrosine activation 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 CD66d. In a preferred embodiment, the primary signaling domain of the CAR of the present invention may comprise a CD3ζ intracellular region, and the signaling domain has at least 70% of the amino acid sequence shown in SEQ ID NO: 9, 10 or 11, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

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

In one embodiment, the CAR of the invention may also comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, and the like. In one embodiment, the signal peptide that can be used in the present invention is a B2M signal peptide, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO:12. % or 99% or 100% sequence identity; or CD8α signal peptide, it has at least 70%, preferably at least 80%, more preferably at least 90%, 95% with the amino acid sequence shown in SEQ ID NO: 13 or 14 , 97% or 99% or 100% sequence identity.

exogenous gene

In addition to cell surface molecules that specifically recognize antigens, the engineered immune cells of the present invention also express one or more exogenous chemokines selected from CXCL10 and CXCL11.

Chemokines are a class of small molecule cytokine family proteins that can induce directional migration of cells. Studies have shown that chemokines play an important role in various biological activities such as immune response, cell development, and inflammatory response. According to the arrangement of amino-terminal cysteine, chemokines can be divided into four subfamilies: CXC, CC, XC, and CX3C, among which the CXC subfamily includes 17 members from CXCL1 to CXCL17, and the CC subfamily includes CCL1 to CCL28. 28 members, the XC subfamily includes XCL1 and XCL2, and the CX3C subfamily includes CX3CL1.

Both CXCL10 and CXCL11 belong to the CXC subfamily, and both play biological roles by binding to CXCR3. All of them are capable of chemoattracting lymphocytes and inhibiting angiogenesis. In one embodiment, the chemokine used in the present invention may be a wild type, a variant thereof or a functional fragment thereof, and the variant or functional fragment has the same or similar biological function as the wild type. Specifically, CXCL10 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: 25 or 27, or The coding sequence of CXCL10 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 24 or 26, and Comparable activity to wild-type CXCL10. CXCL11 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: 29 or 31, or the coding of CXCL11 The sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 28 or 30, and has the same sequence identity as the wild type CXCL11 has comparable activity.

In one embodiment, the engineered immune cells of the present invention can further express exogenous IL7. The IL7 used in the present invention may be wild-type IL7, its variants or functional fragments thereof, and the variants or functional fragments have the same or similar biological functions as wild-type IL7. Specifically, 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 shown in SEQ ID NO: 21 or 23, or The coding sequence of IL7 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 22 or 24.

The exogenous genes in the present invention, such as CXCL10, CXCL11 and/or IL7, can be expressed constitutively or conditionally. In one embodiment, the expression of exogenous CXCL10, CXCL11 and/or IL7 is conditional expression. For example, according to needs, the exogenous gene of the present invention can be operably linked with an inducible, repressible or tissue-specific promoter, thereby regulating the expression of the introduced exogenous gene at a specific time or in a specific tissue or cell type level. In one embodiment, the promoter is an inducible promoter, ie a promoter that initiates transcription only in the presence of specific environmental conditions, developmental conditions or inducers. In another embodiment, the promoter is a repressible promoter, ie, in the presence of a repressor specific for the repressible promoter, expression of the foreign gene in the cell is suppressed or not expressed.

In one embodiment, the exogenous gene of the present invention, such as CXCL10, CXCL11 and/or IL7, is secreted expression. In another embodiment, the exogenous gene is anchored expression, for example, it is operably linked with a localization domain, and the localization domain can localize the expression of the exogenous gene of the present invention on a specific cell location, For example 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 CXCL10, CXCL11 and/or IL7 of the present invention are operably linked to the transmembrane domain, so as to be expressed on the surface of engineered immune cells.

Nucleic Acids and Vectors

The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding one or more exogenous chemokines such as CXCL10 and/or CXCL11. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding IL7.

In one embodiment, the cell surface molecule that specifically recognizes an antigen is a T cell receptor or a chimeric antigen receptor, preferably a chimeric antigen receptor. Chimeric antigen receptors are defined 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 single- and/or double-stranded form, linear or circular shape, or their mixtures (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 present invention also provides a vector comprising the nucleic acid according to the present invention. Wherein, the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen, the nucleic acid sequence encoding the exogenous chemokine, and optionally the nucleic acid sequence encoding IL7 may be located in one or more vectors. When located in a vector, the nucleic acid sequences can be operably linked by a 2A peptide.

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

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a sequence at a specific targeting site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding chimeric antigen receptor polypeptides of the invention, and the translation of their mRNA in a suitable host cell. Suitable vectors 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 (such as retroviruses, oncolytic viruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus, polyoma virus, and adeno-associated virus (AAV ), etc.), phage, phagemid, cosmid and artificial chromosome (including BAC and YAC). The vector itself is usually a sequence of nucleotides, usually a DNA sequence containing the insert (transgene) and a larger sequence that acts as the "backbone" of the vector. The engineered vector usually also contains 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 (such as a multiple cloning site, MCS). The vector may additionally comprise elements such as a promoter, a polyA tail (polyA), a 3' 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, said vector is an in vitro transcribed vector.

engineered immune cells

The present invention also provides an engineered immune cell comprising the nucleic acid or vector of the present invention. In other words, the engineered immune cells of the present invention express cell surface molecules that specifically recognize antigens and one or more exogenous chemokines, such as CXCL10 and/or CXCL11. In one embodiment, the engineered immune cells of the present invention further express exogenous IL7.

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, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells obtained from stem cell sources such as iPSCs, ESCs, hematopoietic stem cells, etc. Preferably, the immune cells are T cells. The T cells may be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be enriched or purified. T cells can be at any developmental stage, including, but not limited to, CD4+CD8+ T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), CD4-CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.

In one embodiment, the immune cells of the present invention further comprise suppressed or silenced expression of at least one endogenous gene selected from the group consisting of: CD52, GR, TCRα, TCRβ, CD3γ, CD3δ, CD3ε , CD247ζ, HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More specifically, the expression of at least TCR components (including TCRα, TCRβ genes) or CD3 components (including CD3γ, CD3δ, CD3ε, CD247ζ) in immune cells is inhibited or silenced. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD). The method of suppressing or silencing a gene is known in the art, for example, DNA fragmentation is mediated by meganuclease, zinc finger nuclease, TALEN nuclease or Cas enzyme in the CRISPR system, thereby knocking out the gene; or by shRNA, RNAi and other ways to inhibit gene expression.

Pharmaceutical Compositions and Combination Therapies

The present invention also provides a pharmaceutical composition, which comprises the engineered immune cell, nucleic acid molecule or carrier described in the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the nucleic acid molecules, vectors or engineered immune cells in the preparation of pharmaceutical compositions.

As used herein, the term "pharmaceutically acceptable excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse desired local or systemic effect), which are well known in the art (see, for example, 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, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . The selection of suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in pharmaceutical compositions of the 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 compositions according to the present invention are suitable for various routes of administration. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

The pharmaceutical composition according to the invention can also be administered in combination with one or more other agents suitable for the treatment and/or prophylaxis of the disease to be treated.

The present invention also provides a combination therapy comprising engineered immune cells expressing cell surface molecules that specifically recognize antigens and one or more exogenous chemokines selected from the group consisting of CXCL10 and CXCL11. In one embodiment, the combination therapy comprises: (1) engineered immune cells expressing exogenous chemokines and exogenous IL7; (2) engineered immune cells expressing IL7 and exogenous chemokines or (3) engineered immune cells and exogenous IL7 and chemokines; wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, wherein the chemokines are selected from CXCL10 and CXCL11.

therapeutic application

The present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the nucleic acid molecule, vector, engineered immune cell according to the present invention or pharmaceutical compositions. Therefore, the present invention also covers the use of the nucleic acid molecules, vectors, and engineered immune cells in the preparation of drugs for treating cancer, infection, or autoimmune diseases.

In one embodiment, the method of treatment comprises administering to a subject an effective amount of an immune cell and/or a pharmaceutical composition of the present 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 cells or NKT cells.

As used herein, the term "autologous" refers to any material derived from an individual that will later be reintroduced into that same individual. As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species or a different patient as the individual into whom the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci differ. In some cases, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.

As used herein, the term "subject" is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cows, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.

In one embodiment, the cancer is a cancer associated with expression of a target bound by the 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 cancers, breast cancer, peritoneal cancer, cervical cancer , choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatoma, intraepithelial neoplasia, renal cancer, laryngeal cancer, liver tumors, lung cancer (such as small cell lung cancer, non-small cell lung cancer, adenoid lung cancer, and squamous lung cancer), lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (eg, lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer Carcinoma, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system malignancies, vulvar and other carcinomas and sarcomas, and B cell Lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cellular NHL, large-bulk disease NHL), B-lymphoblastic lymphoma (B-LBL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, Chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic Plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hair cell Leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); Diseases related to target expression. Preferably, the diseases that can be treated with the engineered immune cells or the pharmaceutical composition of the present 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 infections include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites.

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

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

The present invention will be described in detail below with reference to the accompanying drawings and 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 the purpose of illustration, and shall not constitute any limitation to the present invention. In the case of no contradiction, the embodiments in the present application and the features in the embodiments can be combined with each other.

Detailed ways

Example 1. Preparation of CAR-T cells

Construction of MSCV-mCD19-CAR plasmid, which contains CD19-scFv (SEQ ID NO: 2), CD8α hinge region (SEQ ID NO: 17), CD8α transmembrane region (SEQ ID NO: 5), 41BB co-stimulatory domain ( SEQ ID NO: 8) and the coding sequence of CD3ζ intracellular region (SEQ ID NO: 11).

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

The MSCV-mCD19-CAR-CXCL10 plasmid was constructed, which further included the coding sequences of T2A (SEQ ID NO: 19) and CXCL10 (SEQ ID NO: 25) on the basis of the MSCV-mCD19-CAR plasmid.

The MSCV-mCD19-CAR-CXCL11 plasmid was constructed, which further included the coding sequences of T2A (SEQ ID NO: 19) and CXCL11 (SEQ ID NO: 29) on the basis of the MSCV-mCD19-CAR plasmid.

The above plasmids were packaged into retroviruses and further transfected into activated T cells to obtain mCD19-CAR cells with traditional structure and CAR-T cells expressing IL7, CXCL10, CXCL11 and their combinations, namely, mCD19-CAR+IL7 Cells, mCD19-CAR+CXCL10 cells, mCD19-CAR+CXCL11 cells, mCD19-CAR+IL7+CXCL10 cells (using retrovirus packaged with MSCV-mCD19-CAR-IL7 plasmid and packaged with MSCV-mCD19-CAR-CXCL10 plasmid retrovirus co-transfection) and mCD19-CAR+IL7+CXCL11 cells (retrovirus packaged with MSCV-mCD19-CAR-IL7 plasmid and retrovirus packaged with MSCV-mCD19-CAR-CXCL11 plasmid obtained by co-transfection).

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

2.1 Expression level of cell surface CAR

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

2.2 Expression level of IL7

The supernatant of CAR-T cells was collected, and the secretion level of IL7 in the cells was detected with the Mouse IL-7 DuoSet ELISA kit (R&D Systems, Cat. No. DY407) according to the manufacturer's recommendation. The results are shown in Figure 2. It can be seen that both mCD19-CAR+IL7+CXCL10 cells and mCD19-CAR+IL7+CXCL11 cells can effectively express IL7.

2.3 Expression levels of CXCL10 and CXCL11

The supernatant of CAR-T cells was collected, and according to the manufacturer's recommendations, the secretion level of CXCL10 in the cells was detected with the Mouse CXCL10 DuoSet ELISA kit (R&D Systems, Cat. No. DY466), and the Mouse CXCL11 DuoSet ELISA kit (R&D Systems, Cat. No. DY572) to detect the secretion level of CXCL11 in the cells, and the results are shown in Figure 3. It can be seen that both mCD19-CAR+CXCL10 cells and mCD19-CAR+IL7+CXCL10 cells can effectively express CXCL10, while both mCD19-CAR+CXCL11 cells and mCD19-CAR+IL7+CXCL11 cells can effectively express CXCL11.

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

Add NT cells, CD19-CAR cells, mCD19-CAR+CXCL10 cells, and mCD19-CAR+IL7+CXCL10 cells at a concentration of 2 × ¹⁰⁵ cells/100 μl in a 96-well round bottom plate, respectively. Then Panc02-mCD19 target cells or Panc02 non-target cells were added to each well at a concentration of 1× ¹⁰⁴ cells/100 μl. After culturing at 37° C. for 24 h, the culture supernatant was collected. The expression level of IFN-γ in the culture supernatant was detected with the Mouse IFN-gamma DuoSet ELISA Kit (R&D, Cat. No. DY485) according to the manufacturer's recommendations. The detection results are shown in Figure 4A. The IFN-γ secretion levels of mCD19-CAR+CXCL11 cells and mCD19-CAR+IL7+CXCL11 cells were detected by the same method, and the results are shown in Figure 4B.

It can be seen that the release of IFN-γ was not detected in the non-target cells Panc02, but significantly increased IFN-γ levels were only detected after co-culture with target cells Panc02-CD19, and NT cells did not express IFN-γ γ, which indicates that the killing of CAR-T cells in this example is specific. In addition, the IFN-γ levels of mCD19-CAR+CXCL10 cells and mCD19-CAR+CXCL11 cells were significantly higher than those of mCD19-CAR cells, indicating that CXCL10 or CXCL11 genes alone can significantly enhance the killing activity of CAR-T cells. In addition, the inventors also unexpectedly found that the IFN-γ level of CAR-T cells expressing the combination of IL7+CXCL10 or IL7+CXCL11 was significantly higher than that of CAR-T cells expressing only CXCL10 or CXCL11, indicating that IL7 and chemokine For example, CXCL10 or CXCL11 can produce a synergistic effect to further enhance the killing activity of CAR-T cells.

Example 4. Verification of the tumor suppressive effect of CAR-T cells

5×10 ⁵ Panc02-mCD19 pancreatic cancer cells were inoculated subcutaneously in the axilla of the left forelimb of healthy C57BL/6 mice. The mice inoculated with pancreatic cancer cells were randomly divided into 5 groups, 5 mice in each group. When the tumor volume grew to ^100mm3 , mice in each group were injected with 1× ¹⁰⁶ NT cells, mCD19-CAR cells, mCD19-CAR+IL7 cells, mCD19-CAR+IL7+CXCL10 cells or mCD19-CAR via tail vein +IL7+CXCL11 cells. Monitor the mice for body weight and tumor volume changes until the end of the experiment.

The body weight changes of the mice are shown in Figure 5. It can be seen that after administration of CAR-T cells, the body weight of the mice in each group has no significant difference compared with the control group, indicating that the administration of CAR-T cells will not have obvious toxic side effects on the mice.

The tumor volume changes in the mice are shown in Figure 6. It can be seen that expressing IL7 alone can enhance the tumor suppressive effect of CAR-T cells. The inventors also unexpectedly found that mCD19-CAR+IL7+CXCL10 cells and mCD19-CAR+IL7+CXCL11 cells have similar anti-tumor effects in vivo, both of which are significantly better than CAR-T cells expressing only IL7. The tumor burden was kept very low and there were no recurrences. This indicates that the combination of additionally expressed chemokine CXCL10 or CXCL11 and IL7 can have a synergistic effect with CAR-T cells and enhance the anti-tumor effect of the latter.

The above results indicate that the combination of co-expressed chemokines and IL7 can effectively enhance the inhibitory effect of CAR-expressing engineered immune cells on tumor cells.

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

Claims (25)

1. An engineered immune cell that expresses cell surface molecules that specifically recognize antigens and one or more exogenous chemokines selected from CXCL10 and CXCL11.
2. The engineered immune cell of claim 1, wherein the engineered immune cell further expresses exogenous IL7.
3. The engineered immune cell of claim 1, wherein the CXCL10 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 25 or 27, or its coding sequence is the same as that shown in SEQ ID NO: 24 or 26 The nucleic acid sequence has at least 90% identity, and the CXCL11 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 29 or 31, or its coding sequence has at least 90% identity with the nucleic acid sequence shown in SEQ ID NO: 28 or 30 At least 90% identity.
4. The engineered immune cell of claim 2, wherein the IL7 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 21 or 23, or its coding sequence is the same as that shown in SEQ ID NO: 22 or 24 Nucleic acid sequences are at least 90% identical.
5. The engineered immune cell according to any one of claims 1-4, wherein the cell surface molecule that specifically recognizes an antigen comprises an antigen binding region, and is a chimeric antigen receptor, a T cell receptor, a T cell fusion protein or T cell antigen coupler.
6. The engineered immune cell of claim 5, wherein the chimeric antigen receptor comprises an antigen binding region, a transmembrane domain and an intracellular domain, and the intracellular domain comprises a co-stimulatory domain and/or a primary signal conduction domain.
7. The engineered immune cell of claim 5, wherein the antigen binding region is selected from the group consisting of IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, single domain Antibodies, nanobodies, diabodies, anticalins and DARPIN antigens.
8. The engineered immune cell of claim 5, 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, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1 , EGFRvIII, tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, Mesothelin, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18. 2. c-Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o -Acetyl-GD2, Folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT -1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoid translocation Dot, 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, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, produced by HIV, HCV, HBV and and/or molecules expressed by other pathogens; and/or neo-epitopes or neoantigens.
9. The engineered immune cell according to claim 6, wherein the transmembrane domain is selected from the transmembrane domain of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
10. The engineered immune cell according to any one of claims 6-9, wherein the primary signaling domain is selected from the intracellular region of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.
11. The engineered immune cell of claim 6, wherein the co-stimulatory domain comprises one or more intracellular regions selected 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(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, or ZAP70.
12. The engineered immune cell according to any one of claims 1-11, wherein the immune cell is selected from T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.
13. The engineered immune cell of claim 12, wherein the T cells are CD4+CD8+T cells, CD4+ helper T cells, CD8+T cells, CD4-CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells or αβ-T cells.
14. The engineered immune cell according to any one of claims 1-13, wherein the expression of the chemokine and/or IL7 is conditional expression or constitutive expression.
15. The engineered immune cell according to any one of claims 1-14, wherein the chemokine and/or IL7 is operably linked to the localization domain.
16. A nucleic acid molecule comprising a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding one or more chemokines, wherein the chemokines are selected from CXCL10 and CXCL11.
17. The nucleic acid molecule of claim 16, wherein said nucleic acid molecule further comprises a nucleic acid sequence encoding IL7.
18. The nucleic acid molecule of claim 16, wherein the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor, a T cell receptor, a T cell fusion protein, or a T cell antigen coupler.
19. A vector comprising the nucleic acid molecule of any one of claims 16-18.
20. The vector of claim 19, wherein the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyoma virus, and adeno-associated virus (AAV).
21. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 1-15, the nucleic acid molecule according to any one of claims 16-18 or the carrier according to any one of claims 19-20 , and one or more pharmaceutically acceptable excipients.
22. The engineered immune cell according to any one of claims 1-15, the nucleic acid molecule according to any one of claims 16-18, the carrier according to any one of claims 19-20 or the drug according to claim 21 Use of a composition for the manufacture of a medicament for treating a subject suffering from cancer, an infection or an autoimmune disease.
23. The use of claim 22, wherein the cancer is a hematological tumor or a solid tumor.
24. A combination therapy comprising engineered immune cells expressing cell surface molecules that specifically recognize antigens and one or more exogenous chemokines selected from the group consisting of CXCL10 and CXCL11.
25. A combination therapy comprising: (1) engineered immune cells expressing exogenous chemokines and exogenous IL7; (2) engineered immune cells expressing IL7 and exogenous chemokines; or (3) ) engineered immune cells and exogenous IL7 and chemokines; wherein the engineered immune cells express cell surface molecules that specifically recognize antigens, wherein the chemokines are selected from CXCL10 and CXCL11.

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