WO2023016284A1 - 工程化免疫细胞及其用途 - Google Patents

工程化免疫细胞及其用途 Download PDF

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WO2023016284A1
WO2023016284A1 PCT/CN2022/109101 CN2022109101W WO2023016284A1 WO 2023016284 A1 WO2023016284 A1 WO 2023016284A1 CN 2022109101 W CN2022109101 W CN 2022109101W WO 2023016284 A1 WO2023016284 A1 WO 2023016284A1
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cells
immune cell
engineered immune
antigen
nucleic acid
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French (fr)
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邢芸
任江涛
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南京北恒生物科技有限公司
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    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
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    • A61K2239/29—Multispecific CARs
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas

Definitions

  • the invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and an exogenous CCL2 gene. More preferably, the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor.
  • Lymphocytes generally sense chemokines secreted by other cells through chemokine receptors, and then are directed to home to target sites. Despite the large number of chemokines and their receptors, generally only a limited number of chemokine receptors are expressed per lymphocyte. Therefore, when certain tumor cells secrete a certain chemokine in large quantities and lymphocytes (such as engineered CAR-T cells) do not express the receptors paired with the chemokine, the lymphocytes cannot be effectively transported to the tumor The microenvironment affects the therapeutic effect.
  • the present invention provides a novel engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous CCL2.
  • the engineered immune cells of the present invention further express exogenous IL7.
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a T cell receptor, preferably a chimeric antigen receptor.
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor comprising an antigen binding region, a transmembrane domain and an intracellular signaling domain comprising costimulatory domain and/or primary signaling domain.
  • the antigen binding region can be selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, single domain antibody, nanobody, diabody, anticalin and DARPIN.
  • the antigen binding region is selected from scFv, Fab, single domain antibodies and nanobodies.
  • 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,
  • the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain is selected from the transmembrane domains of CD8 ⁇ , CD4, CD28 and CD278.
  • the primary signaling domain is an intracellular region of a protein selected from the group consisting of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • said primary signaling domain comprises a CD3 ⁇ intracellular region.
  • the co-stimulatory domain comprises one or more intracellular regions of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, 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.
  • the co-stimulatory domain is selected from the intracellular region of CD27, CD28, CD134, CD137, DAP10, DAP12 or CD278 or a combination thereof.
  • the immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells.
  • 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, ⁇ -T cells or ⁇ -T cells.
  • the expression or activity of exogenous CCL2 and/or IL7 is constitutive.
  • the exogenous expression or activity of CCL2 and/or IL7 is conditional.
  • conditional expression is achieved by operably linking the exogenous gene to an inducible, repressible or tissue-specific promoter.
  • CCL2 and/or IL7 can be operably linked to a localization domain, which can localize and express the exogenous gene of the present invention on a specific cell location, such as a cell membrane.
  • the exogenous gene of the present invention such as CCL2 and/or IL7, is operably linked to the transmembrane domain, so as to be expressed on the surface of the engineered immune cell.
  • 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 CCL2.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding IL7.
  • the cell surface molecule that specifically recognizes an antigen is a chimeric antigen receptor or a T cell receptor, more preferably a chimeric antigen receptor.
  • the nucleic acid is DNA or RNA.
  • the present invention also provides a vector comprising the above-mentioned nucleic acid molecule.
  • the vector is selected from plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV).
  • RSV Rous sarcoma virus
  • AAV adeno-associated virus
  • 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.
  • said vector is an in vitro transcribed vector.
  • 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.
  • 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.
  • the cancer is a solid tumor or a hematological tumor. More specifically, the cancer is selected from the group consisting of: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatoma, Intraepithelial neoplasms, kidney cancer, laryngeal cancer, liver tumors, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma,
  • the infections include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • 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 Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.
  • 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 The expression level of CCL2 in CAR-T cells determined by ELISA.
  • FIG. 4 IFN- ⁇ release levels after CAR-T cells were co-cultured with target cells and non-target cells.
  • 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.
  • the present invention provides a novel engineered immune cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous CCL2.
  • 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).
  • a target molecule eg, an antigen
  • Such surface molecules typically comprise an antigen-binding region capable of specific 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 receptors or chimeric antigen receptors.
  • T cell receptor refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling.
  • MHC major histocompatibility complex molecules
  • TCR is composed of six peptide chains that form heterodimers, which are generally divided into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specific antigens and MHC molecules.
  • the variable region of the TCR may comprise or be operably linked to an antigen binding region, wherein the definition of the antigen binding region is as follows.
  • chimeric antigen receptor 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 Signal transduction domains (comprising co-stimulatory domains and/or primary signal transduction 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.
  • TCR T cell receptor
  • 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.
  • 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.
  • the antigen binding domain may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody.
  • the antigen binding region may also be a specific binding polypeptide or receptor structure of a specific protein, such as PD1, PDL1, PDL2, TGF ⁇ , 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.
  • the amino acid modification is preferably a conservative modification.
  • 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), beta-branched side chains (e.g.
  • 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, tyros
  • threonine valine, isoleucine
  • 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.
  • 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.
  • 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.
  • sequence identity can be determined 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 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, EGFR
  • the CAR of the invention can be designed to include an antigen-binding region specific for that antigen.
  • 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.
  • a CD19 antibody can be used as the antigen binding region of the present invention.
  • the antigen binding region is an antibody targeting CD19, which has the same CDRs as the antibody shown in SEQ ID NO: 1 or 2.
  • 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. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to the target antigen.
  • Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof.
  • the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues such as leucine and valine.
  • 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 CD8 ⁇ , 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.
  • the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antigen binding region and the transmembrane domain.
  • 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.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence.
  • 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.
  • CD8 ⁇ 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.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function, which includes a co-stimulatory domain and/or a primary signaling domain.
  • the intracellular signaling domain is responsible for intracellular signal transmission following antigen binding at the antigen binding region, resulting in activation of immune cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed.
  • the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.
  • 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.
  • the primary signaling domain can contain many immunoreceptor tyrosine-based activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM).
  • Non-limiting examples of primary signaling domains of the invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain of the CAR of the present invention may comprise a CD3 ⁇ 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.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids with a propensity to form a single ⁇ -helix.
  • At the end of the signal peptide there is usually a stretch of amino acids that is recognized and cleaved by the signal peptidase.
  • the signal peptidase can cleave during translocation or after completion to generate a free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • 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, CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • 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 CD8 ⁇ 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.
  • the CAR of the present invention may also include a switch structure to regulate the expression time of the CAR.
  • the switch structure can be in the form of a dimerization domain, which induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain to allow it to bind to the targeted antigen, thereby activating the signaling pathway.
  • a switch domain can be used to link the binding and signaling domains separately, and the binding and signaling domains can pass through the dimer only when the switch domains are associated with each other (e.g. in the presence of an inducing compound). Link together to activate the signaling pathway.
  • the switch structure can also be in the form of a masking peptide.
  • the masking peptide can mask the extracellular binding domain and prevent it from binding to the targeted antigen.
  • the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it an "ordinary" CAR structure.
  • Various switch configurations known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may also contain a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance, so as to eliminate CAR cells when necessary (eg, when severe toxic side effects occur).
  • suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when necessary, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes.
  • the suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir treatment.
  • HSV-TK herpes simplex virus thymidine kinase
  • the suicide gene can also be iCaspase-9, and the dimerization of iCaspase-9 can be induced by chemically inducing drugs such as AP1903 and AP20187, thereby activating the downstream Caspase3 molecule and leading to cell apoptosis.
  • chemically inducing drugs such as AP1903 and AP20187
  • the engineered immune cells of the present invention also express exogenous CCL2.
  • Chemotactic cytokines can be divided into four subfamilies: CXC, CC, XC and CX3C according to the arrangement of the amino-terminal cysteine, and CCL2 is a member of the CC subfamily.
  • CCL2 has chemotactic activity on monocytes and basophils, and is one of the key chemokines regulating monocyte/macrophage migration and infiltration.
  • CCL2 is involved in various diseases, such as cancer (such as breast cancer, colorectal cancer, prostate cancer, melanoma, gastric cancer and ovarian cancer, etc.), bone remodeling and bone-related tumors, autoimmune diseases, neurological diseases, bacteria and viruses important role in the development of infection.
  • the CCL2 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 25 or 27 % sequence identity, or the coding sequence of CCL2 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the nucleotide sequence shown in SEQ ID NO: 24 or 26 % sequence identity.
  • the engineered immune cells of the present invention further express IL7.
  • the IL7 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 21 or 23 % sequence identity, 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% with the nucleic acid sequence shown in SEQ ID NO: 22 or 24 % sequence identity.
  • exogenous genes in the present invention can be expressed constitutively or conditionally.
  • the expression of exogenous CCL2 and/or IL7 is conditional.
  • 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.
  • the promoter is an inducible promoter, ie a promoter that initiates transcription only in the presence of specific environmental conditions, developmental conditions or inducers.
  • environmental conditions include, for example, an acidic tumor microenvironment, a hypoxic tumor microenvironment, and the like.
  • Such inducers include, for example, cyclocycline, tetracycline, or analogs thereof.
  • Analogs of tetracycline include, for example, chlortetracycline, oxytetracycline, desmethylchlortetracycline, methacycline, doxycycline, and minocycline.
  • Inducible promoters include, for example, a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, and the like.
  • 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.
  • Repressible promoters include, for example, a Lac repressible element or a tetracycline repressible element.
  • Inducible/repressible expression systems well known to those skilled in the art can be used in the present invention, including but not limited to Tet-on system, Tet-off system, Cre/loxP system, etc.
  • CCL2 and/or IL7 can be operably linked to a localization domain, and the localization domain can localize and express the exogenous gene of the present invention on a specific cell location, such as the cell membrane.
  • Localization domains include, but are not limited to, nuclear localization signals, leader peptides, transmembrane domains, and the like.
  • the exogenous gene CCL2 and/or IL7 of the present invention is operably linked to the transmembrane domain, so as to be expressed on the surface of the engineered immune cells.
  • the exogenous gene in the present invention can be a wild type or a fusion protein or a mutant with specific properties (eg resistance to protease hydrolysis).
  • the present invention also provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen and a nucleic acid sequence encoding CCL2.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding IL7.
  • 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.
  • 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).
  • 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).
  • 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.
  • the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen, the nucleic acid sequence encoding CCL2, and optionally the nucleic acid sequence encoding IL7 may be located in one or more vectors.
  • 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.
  • 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.
  • vectors of the present invention include, but are not limited to, plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc. ), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs).
  • viruses such as retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc.
  • phages phagemids
  • cosmids include BACs and YACs.
  • the vector itself is usually a sequence of nucleotides, usually a DNA sequence containing an 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 required), a selectable marker and a restriction enzyme cleavage site (such as a multiple cloning site, MCS).
  • the vector may additionally contain a promoter , polyadenylation tail (polyA), 3' UTR, enhancer, terminator, insulator, operator, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements.
  • the vector is an in vitro transcribed vector.
  • the present invention also provides an engineered immune cell comprising the nucleic acid or vector of the present invention.
  • the engineered immune cells of the present invention express cell surface molecules that specifically recognize antigens and exogenous CCL2 gene, and optionally exogenous IL7 gene.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the 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 cord blood.
  • 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.
  • 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, ⁇ -T cells, ⁇ -T cells, etc.
  • 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.
  • the immune cells of the invention further comprise at least one inactivated gene selected from the group consisting of: CD52, GR, TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ , HLA-I, HLA-II, B2M , immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More specifically, at least TCR components (including TCR ⁇ , TCR ⁇ genes) or CD3 components (including CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ ) in immune cells are inactivated. This inactivation renders the TCR-CD3 complex nonfunctional in the cell. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD).
  • GvHD graft-versus-host disease
  • DNA fragmentation is mediated by meganuclease, zinc finger nuclease, TALEN nuclease or Cas enzyme in the CRISPR system, thereby inactivating the gene.
  • 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 or medicaments.
  • 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 .
  • suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical compositions of the present invention.
  • excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • 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 present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, especially ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form especially adapted to the desired method of administration.
  • Processes known in the present invention for the production of medicaments may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution, and preferably comprise a pharmaceutically acceptable buffer.
  • 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.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, Auristatin E, vincristine, and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth
  • 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 cell or the pharmaceutical composition according to the present invention. Therefore, the present invention also covers the use of the engineered immune cells in the preparation of medicaments for treating cancer, infection or autoimmune diseases.
  • 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.
  • 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.
  • autologous refers to any material derived from an individual that will later be reintroduced into that same individual.
  • 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.
  • 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.
  • the cancer is a cancer associated with expression of a target bound by the antigen binding region.
  • 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-
  • 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 and the like.
  • the infections include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • 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 Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.
  • the method further comprises administering to the subject one or more additional chemotherapeutic agents, biologics, drugs or treatments.
  • the chemotherapeutic agent, biologic, drug or treatment is selected from radiation therapy, surgery, antibody agents and/or small molecules and any combination thereof.
  • MSCV-mCD19-CAR plasmid which contains CD19-scFv (SEQ ID NO: 2), CD8 ⁇ hinge region (SEQ ID NO: 17), CD8 ⁇ transmembrane region (SEQ ID NO: 5), 41BB co-stimulatory domain ( SEQ ID NO: 7) and the coding sequence of CD3 ⁇ 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-CCL2 plasmid was constructed, which further included the coding sequences of T2A (SEQ ID NO: 19) and CCL2 (SEQ ID NO: 25) on the basis of the MSCV-mCD19-CAR plasmid.
  • Opti-MEM Gibco, Cat. No. 31985-070
  • 45 ⁇ g of prepared retroviral plasmid 15 ⁇ g of packaging vector pCL-Eco (Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029)
  • packaging vector pCL-Eco Shanghai Hewu Biotechnology Co., Ltd., Cat. No. P3029
  • 120 ⁇ l of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001)
  • the plasmid/vector/transfection reagent mixture was added dropwise into the pre-prepared 293T cell culture flask, and cultured overnight at 37°C and 5% CO 2 . Cultures were harvested 72 hours after transfection and centrifuged (2000g, 4°C, 10 minutes) to obtain retroviral supernatants.
  • T lymphocytes were isolated from mouse spleen, and T cells were activated with DynaBeads CD3/CD28 CTS TM (Gibco, Cat. No. 40203D), and then cultured at 37°C and 5% CO 2 for 1 day.
  • Activated T cells were inoculated into 24-well plates pre-coated overnight with RetroNectin at a density of 3 ⁇ 106 cells/mL per well, then 500 ⁇ L of retrovirus supernatant was added, and complete medium was supplemented to 2 mL.
  • the 24-well plate Place the 24-well plate in a centrifuge for centrifugal infection, and centrifuge at 2000g for 2h at 32°C. Then, the 24-well plate was immediately placed in a 37°C, CO 2 incubator for static culture. Replace the fresh medium the next day, and adjust the cell density to 1 ⁇ 106 cells/mL. Three days after infection, cells were harvested for subsequent analysis. The collected cells are mCD19-CAR cells, mCD19-CAR+CCL2 cells and mCD19-CAR+IL7+CCL2 cells.
  • CAR-T cells 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 compared with untreated NT cells, the CAR-positive efficiency in mCD19-CAR cells, mCD19-CAR+CCL2 cells and mCD19-CAR+IL7+CCL2 cells was all greater than 60%, indicating that these cells can all be effective Express CAR.
  • H&L Goat Anti-Rat IgG
  • APC Streptavidin BD Pharmingen, Cat. No. 554067
  • the IFN- ⁇ level of CAR-T cells expressing the combination of IL7+CCL2 was also significantly higher than that of CAR-T cells expressing only CCL2, indicating that the addition of IL7 can further improve the killing activity of CAR-T cells.
  • 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 4 groups, 5 mice in each group.
  • When the tumor volume grew to 100 mm 3 , 5 ⁇ 10 6 NT cells, mCD19-CAR cells, mCD19-CAR+CCL2 cells or mCD19-CAR+IL7+CCL2 cells were injected into the mice of each group through the tail vein. Monitor the mice for body weight and tumor volume changes until the end of the experiment.
  • mice The body weight changes of the mice are shown in Figure 5. It can be seen that after administration of CAR-T cells, the weight of the mice in each group was not significantly different from that of the control group, and during the observation period, when the mouse tumor did not exceed 1500mm 3 , the mice were active and had normal coat color. This shows that the administration of CAR-T cells will not have obvious toxic side effects in mice.
  • mice The tumor volume changes in mice are shown in Figure 6. It can be seen that the anti-tumor effect of mCD19-CAR+CCL2 cells is better than that of conventional CAR-T cells, indicating that the additionally expressed CCL2 can synergize with CAR-T cells and enhance the anti-tumor effect of the latter. In addition, the inventors also unexpectedly found that further expression of IL7 can significantly increase the promoting effect of CCL2 on CAR-T cells, thereby more significantly inhibiting tumor growth.

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Abstract

本发明涉及一种工程化免疫细胞，其表达(i)特异性识别抗原的细胞表面分子，和(ii)外源性的CCL2。本发明还提供了该工程化免疫细胞在治疗癌症、感染或自身免疫性疾病中的用途。与传统的工程化免疫细胞细胞相比，本发明的工程化免疫细胞具有显著提高的肿瘤杀伤活性。

Description

工程化免疫细胞及其用途
相关申请的交叉引用
本发明要求于2021年08月11日提交中国专利局的申请号为CN202110917589.1、名称为“工程化免疫细胞及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及一种工程化免疫细胞，其表达特异性识别抗原的细胞表面分子和外源性的CCL2基因。更优选地，所述特异性识别抗原的细胞表面分子是嵌合抗原受体。
背景技术
近年来，过继细胞疗法作为一种新兴的免疫疗法，已经在肿瘤治疗领域展现出巨大的优势。这种疗法通常需要先将细胞进行改造，例如通过基因编辑和/或转导等技术将细胞改造为携带嵌合抗原受体、重组T细胞受体等外源性蛋白，然后在体外进行扩增，并回输给患者。目前，这些疗法针对血液肿瘤已经显示出良好的疗效，但对于实体瘤而言，其功效尚不能令人满意，其中原因之一就是无法将改造后的细胞有效运输至肿瘤部位(即，免疫抑制性的肿瘤微环境)。
淋巴细胞一般通过趋化因子受体来感知其他细胞分泌的趋化因子，进而被定向归巢至靶标位点。尽管趋化因子及其受体的数量众多，但一般每个淋巴细胞仅表达有限数量的趋化因子受体。因此，当某些肿瘤细胞大量分泌某一种趋化因子而淋巴细胞(例如改造后的CAR-T细胞)不表达与该趋化因子配对的受体时，该淋巴细胞无法有效被运输到肿瘤微环境，进而影响治疗效果。
因此，仍然需要改进的细胞疗法，以改变免疫抑制微环境，同时招募其他免疫效应细胞至肿瘤部位，加强抗肿瘤效果。
发明内容
在第一个方面，本发明提供一种新的工程化免疫细胞，其表达特异性识别抗原的细胞表面分子和外源性的CCL2。
在一个实施方案中，本发明的工程化免疫细胞进一步表达外源性的IL7。
在一个实施方案重，所述特异性识别抗原的细胞表面分子是嵌合抗原受体或T细胞受体，优选是嵌合抗原受体。
在一个实施方案中，所述特异性识别抗原的细胞表面分子是嵌合抗原受体，其包含抗原结合区、跨膜结构域和胞内信号传导结构域，所述胞内信号传导结构域包含共刺激结构域和/或初级信号传导结构域。其中，抗原抗原结合区可以选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双 体、anticalin和DARPIN。优选地，所述抗原抗原结合区选自scFv、Fab、单结构域抗体和纳米抗体。
在一个实施方案中，所述特异性识别抗原的细胞表面分子与选自以下的一个或多个靶标结合：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轻链、TIM3、TSHR、CD19、BAFF-R、CLL-1、EGFRvIII、tEGFR、GD2、GD3、BCMA、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、IL-llRa、IL-22Ra、IL-2、间皮素、PSCA、PRSS21、VEGFR2、LewisY、PDGFR-β、SSEA-4、AFP、Folate受体α、ErbB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、NCAM、Claudin18.2、c-Met、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gpl00、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM7R、CLDN6、GPRC5D、CXORF61、ALK、多聚唾液酸、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、豆荚蛋白、HPV E6、E7、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PSA、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、TMPRSS2ETS融合基因、NA17、PAX3、雄激素受体、孕酮受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2D配体，和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。
在一个实施方案中，所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。优选地，跨膜结构域选自CD8α、CD4、CD28和CD278的跨膜结构域。
在一个实施方案中，所述初级信号传导结构域是选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。优选地，所述初级信号传导结构域包含CD3ζ胞内区。
在一个实施方案中，所述共刺激结构域包含一个或多个选自以下蛋白的胞内区： 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以及它们的组合。优选地，所述共刺激结构域选自CD27、CD28、CD134、CD137、DAP10、DAP12或CD278的胞内区或它们的组合。
在一个实施方案中，所述免疫细胞选自T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。优选地，所述T细胞是CD4+CD8+T细胞、CD4+辅助T细胞、CD8+T细胞、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。
在一个实施方案中，外源性的CCL2和/或IL7的表达或活性是组成型表达。在另一个实施方案中，外源性的CCL2和/或IL7的表达或活性是条件型表达。例如，通过将外源性基因与诱导型、阻遏型或组织特异性启动子可操作连接从而实现条件型表达。
在一个实施方案中，CCL2和/或IL7可以与定位结构域可操作连接，所述定位结构域可以将本发明的外源性基因定位在特定的细胞位置上表达，例如细胞膜。在一个实施方案中，本发明的外源性基因例如CCL2和/或IL7与跨膜结构域可操作连接，从而锚定在工程化免疫细胞的表面表达。
在第二个方面，本发明提供一种核酸分子，其包含编码特异性识别抗原的细胞表面分子的核酸序列和编码CCL2的核酸序列。在一个实施方案中，所述核酸分子进一步包含编码IL7的核酸序列。优选地，所述特异性识别抗原的细胞表面分子是嵌合抗原受体或T细胞受体，更优选嵌合抗原受体。优选地，所述核酸是DNA或RNA。
本发明还提供包含上述核酸分子的载体。具体地，所述载体选自质粒、逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV)、多瘤病毒和腺相关病毒(AAV)。在一些实施方案中，该载体还包含在免疫细胞中自主复制的起点、选择标记、限制酶切割位点、启动子、多聚腺苷酸尾(polyA)、3’UTR、5’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
在一个实施方案中，本发明还提供一种药物组合物，其包含本发明所述的工程化免疫细胞、核酸分子或载体，和一种或多种药学上可接受的赋型剂。
在第三个方面，本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的免疫细胞、核酸分子、载体或药物组合物。
在一个实施方案中，所述癌症是实体瘤或血液肿瘤。更具体地，所述癌症选自：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、 消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌、淋巴瘤、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤、B淋巴母细胞淋巴瘤(B-LBL)、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)。
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。
附图说明
图1：通过流式细胞术测定的CAR-T细胞的CAR表达水平。
图2：通过ELISA测定的CAR-T细胞的IL7的表达水平。
图3：通过ELISA测定的CAR-T细胞的CCL2的表达水平。
图4：CAR-T细胞分别与靶细胞和非靶细胞共培养后的IFN-γ释放水平。
图5：用CAR-T细胞治疗小鼠胰腺癌后，小鼠的体重变化曲线。
图6：用CAR-T细胞治疗小鼠胰腺癌后，小鼠的肿瘤生长曲线。
发明详述
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
特异性识别抗原的细胞表面分子
在第一个方面，本发明提供一种新的工程化免疫细胞，其表达特异性识别抗原的细胞表面分子和外源性的CCL2。
如本文所用，术语“特异性识别抗原的细胞表面分子”是指在细胞表面表达的能够与靶分子(例如抗原)特异性结合的分子。此类表面分子一般包含能够与抗原特异性结合的抗原结合区、将表面分子锚定在细胞表面的跨膜结构域，以及负责信号传递 的胞内结构域。常见的此类表面分子的实例包括例如T细胞受体或嵌合抗原受体。
如本文所用，术语“T细胞受体”或“TCR”是指响应于抗原呈递并参与T细胞活化的膜蛋白复合体。TCR的刺激由抗原呈递细胞上的主要组织相容性复合体分子(MHC)触发，所述抗原呈递细胞将抗原肽呈递至T细胞并且结合至TCR复合体以诱发一系列胞内信号传导。TCR由分别形成异二聚体的六条肽链组成，其一般分为αβ型和γδ型。每条肽链包括恒定区和可变区，其中可变区负责结合特异性的特定的抗原和MHC分子。TCR的可变区可以包含抗原结合区或与抗原结合区可操作连接，其中抗原结合区的定义如下所述。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括抗原结合区(例如抗体的抗原结合部分)、跨膜结构域和胞内信号传导结构域(包含共刺激结构域和/或初级信号传导结构域)，各个结构域之间通过接头连接。CAR能够利用单克隆抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。非MHC限制性的抗原识别给予CAR细胞与抗原处理无关的识别抗原的能力，因此绕过了肿瘤逃逸的主要机制。此外，当在T细胞内表达时，CAR有利地不与内源性T细胞受体(TCR)的α链和β链二聚化。
如本文所用，“抗原结合区”是指可以与抗原结合的任何结构或其功能性变体。抗原结合区可以是抗体结构，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体、嵌合抗体及其功能性片段。例如，抗原结合区包括但不限于IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双体、anticalin、DARPIN等，优选选自Fab、scFv、sdAb和纳米抗体。在本发明中，抗原结合区可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。在另一个实施方案中，抗原结合区也可以是特定蛋白的特异性结合多肽或受体结构，所述特定蛋白是例如PD1、PDL1、PDL2、TGFβ、APRIL和NKG2D。
术语“功能性变体”或“功能性片段”是指基本上包含亲本的氨基酸序列但与该亲本氨基酸序列相比含有至少一个氨基酸修饰(即取代、缺失或插入)的变体，条件是所述变体保留亲本氨基酸序列的生物活性。在一个实施方案中，所述氨基酸修饰优选是保守型修饰。
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守修饰包括氨基酸取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨 酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
因此，“功能性变体”或“功能性片段”与亲本氨基酸序列具有至少75％，优选至少76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性，并且保留亲本氨基酸的生物活性，例如结合活性。
如本文所用，术语“序列同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
抗原结合区的选择取决于待识别的与具体疾病状态相关的靶细胞上的细胞表面标记，例如肿瘤特异性抗原或肿瘤相关抗原。因此，在一个实施方案中，本发明的抗原结合区与选自以下的一个或多个靶标结合：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轻链、TIM3、TSHR、CD19、BAFF-R、CLL-1、EGFRvIII、tEGFR、GD2、GD3、BCMA、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、IL-llRa、IL-22Ra、IL-2、间皮素、PSCA、PRSS21、VEGFR2、LewisY、PDGFR-β、SSEA-4、AFP、Folate受体α、ErbB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、NCAM、Claudin18.2、c-Met、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gpl00、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM7R、CLDN6、GPRC5D、CXORF61、ALK、多聚唾液酸、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、豆荚蛋白、HPV E6、E7、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PSA、存活蛋白和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、TMPRSS2ETS融合基因、NA17、PAX3、雄激素受体、孕酮受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯 酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2D配体，和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。根据待靶向的抗原，本发明的CAR可以被设计为包括对该抗原具有特异性的抗原结合区。优选地，所述靶标选自CD7、CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、AFP、Folate受体α、CEA、PSCA、PSMA、Her2、EGFR、IL13Ra2、GD2、NKG2D、Claudin18.2、ROR1、EGFRvIII、CS1、BCMA、GPRC5D、间皮素和它们的任意组合。例如，如果CD19是待靶向的抗原，则CD19抗体可用作本发明的抗原结合区。在一个实施方案中，所述抗原结合区是靶向CD19的抗体，其具有与SEQ ID NO:1或2所示的抗体相同的CDR。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154及其功能性片段。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD28，其与SEQ ID NO:3所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；或源自CD8α，其与SEQ ID NO:4或5所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体还可以包含位于抗原结合区和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、FcγRIIIα受体、IgG4、IgG1、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD28铰链，其与SEQ ID NO:15所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；或包含CD8α铰链，其与SEQ ID NO:16或17所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；或包含IgG4铰链，其与SEQ ID NO:18所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分，其包含共刺激结构域和/或初级信号传导结构域。胞内信号传导结构域负责在抗原结合区结合抗原以后的细胞内的信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应子功能的至少一种。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
在一个实施方案中，本发明的嵌合抗原受体包含初级信号传导结构域，其可以是在抗原受体结合以后一同起作用以引发初级信号传导的T细胞受体和共受体的细胞质序列，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。初级信号传导结构域可以包含许多免疫受体酪氨酸激活基序(Immunoreceptor Tyrosine-based Activation Motifs,ITAM)。本发明的初级信号传导结构域的非限制性施例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在优选的实施方式中，本发明CAR的初级信号传导结构域可以包含CD3ζ胞内区，该信号传导结构域与SEQ ID NO:9、10或11所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体包含一个或多个共刺激结构域。共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的胞内区：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以及ZAP70。优选地，本发明CAR的共刺激结构域来自4-1BB、CD28、CD27、OX40、ICOS、DAP10、DAP12或其组合。在一个实施方案中，本发明的CAR包含CD28共刺激结构域，其与SEQ ID NO:6所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；和/或包含4-1BB共刺激结构域，其与SEQ ID NO:7或8所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的CAR还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割， 以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自B2M、CD8α、IgG1、GM-CSFRα等的信号肽。在一个实施方案中，可用于本发明的信号肽是B2M信号肽，其与SEQ ID NO:12所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性；或是CD8α信号肽，其与SEQ ID NO:13或14所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的CAR还可以包含开关结构，以调控CAR的表达时间。例如，开关结构可以是二聚化结构域的形式，通过与其相应配体的结合引起构象变化，暴露胞外结合结构域，使其与被靶向抗原结合，从而激活信号传导通路。或者，也可以使用开关结构域分别连接结合结构域和信号传导结构域，仅当开关结构域互相结合(例如在诱导化合物的存在下)时，结合结构域和信号传导结构域才能通过二聚体连接在一起，从而激活信号通路。开关结构还可以是掩蔽肽的形式。掩蔽肽可以遮蔽胞外结合结构域，阻止其与被靶向抗原的结合，当通过例如蛋白酶切割掩蔽肽后，就可以暴露胞外结合结构域，使其成为一个“普通”的CAR结构。本领域技术人员知晓的各种开关结构均可用于本发明。
在一个实施方案中，本发明的CAR还可以包含自杀基因，即，使其表达一个可通过外源物质诱导的细胞死亡信号，以在需要时(例如产生严重的毒副作用时)清除CAR细胞。例如，自杀基因可以是插入的表位的形式，例如CD20表位、RQR8等，当需要时，可以通过加入靶向这些表位的抗体或试剂来消除CAR细胞。自杀基因也可以是单纯疱疹病毒胸苷激酶(HSV-TK)，该基因可使细胞在接受更昔洛韦治疗诱导下死亡。自杀基因还可以是iCaspase-9，可以通过化学诱导药物如AP1903、AP20187等诱导iCaspase-9发生二聚化，从而激活下游的Caspase3分子，导致细胞凋亡。本领域技术人员知晓的各种自杀基因均可用于本发明。
外源性基因
除了特异性识别抗原的细胞表面分子，本发明的工程化免疫细胞还表达外源性的CCL2。
趋化性细胞因子根据其氨基端半胱氨酸的排列方式，可分为CXC、CC、XC和CX3C四个亚族，CCL2是CC亚族的成员之一。CCL2对单核细胞和嗜碱性粒细胞具有趋化活性，是调节单核细胞/巨噬细胞迁移和浸润的关键趋化因子之一。CCL2在多种疾病，例如癌症(例如乳腺癌、结直肠癌、前列腺癌、黑色素瘤、胃癌和卵巢癌等)、骨重塑和骨相关肿瘤、自身免疫性疾病、神经系统疾病、细菌与病毒感染的发展进程中发挥重要作用。
在一个实施方案中，本发明使用的CCL2与SEQ ID NO：25或27所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或CCL2的编码序列与SEQ ID NO：24或26所示的核酸序列具有至 少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个优选的实施方案中，本发明的工程化免疫细胞还进一步表达IL7。在一个实施方案中，本发明使用的IL7与SEQ ID NO：21或23所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或IL7的编码序列与SEQ ID NO：22或24所示的核酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
本发明中的外源性基因，例如CCL2和/或IL7的表达可以是组成型表达或条件型表达。
在一个实施方案中，外源性CCL2和/或IL7的表达是条件型表达。例如，根据需要，可以将本发明的外源基因与诱导型、阻遏型或组织特异性启动子可操作连接，从而在特定的时间或特定的组织、细胞类型内调控引入的外源基因的表达水平。在一个实施方案中中，启动子是诱导型启动子，即，仅在特定环境条件、发育条件或诱导物存在下启动转录的启动子。此类环境条件包括例如肿瘤酸性微环境、肿瘤低氧微环境等。此类诱导物包括例如西环素、四环素或其类似物，四环素的类似物包括例如金霉素、土霉素、去甲基氯四环素、甲烯土霉素、多西环素和米诺环素。诱导型启动子包括例如Lac操纵子序列、四环素操纵子序列、半乳糖操纵子序列或多西环素操纵子序列等。在另一个实施方案中，启动子是阻遏型启动子，即，在存在对阻遏型启动子具有特异性的阻遏物的情况下，外源基因在细胞中的表达被抑制或不表达。阻遏型启动子包括例如Lac阻遏型元件或四环素阻遏型元件。本领域技术人源熟知的诱导型/阻遏型表达系统可用于本发明，包括但不限于Tet-on系统、Tet-off系统、Cre/loxP系统等。
在一个实施方案中，CCL2和/或IL7可以与定位结构域可操作连接，所述定位结构域可以将本发明的外源性基因定位在特定的细胞位置上表达，例如细胞膜等。定位结构域包括但不限于核定位信号、引导肽、跨膜结构域等。在一个实施方案中，本发明的外源性基因CCL2和/或IL7与跨膜结构域可操作连接，从而锚定在工程化免疫细胞的表面表达。
在一个实施方案中，本发明中的外源性基因，例如CCL2和/或IL7蛋白可以是野生型或具有特定性能(例如抵抗蛋白酶水解)的融合蛋白或者突变体。
核酸和载体
本发明还提供一种核酸分子，其包含编码特异性识别抗原的细胞表面分子的核酸序列和编码CCL2的核酸序列。在一个实施方案中，所述核酸分子进一步包含编码IL7的核酸序列。
在一个实施方案中，所述特异性识别抗原的细胞表面分子是T细胞受体或嵌合抗原受体，优选嵌合抗原受体。嵌合抗原受体的定义如上所述。
如本文所用，术语“核酸分子”包括核糖核苷酸和脱氧核糖核苷酸的序列，如经修饰的或未经修饰的RNA或DNA，各自为单链和/或双链形式的线性或环状，或它 们的混合物(包括杂合分子)。因此，根据本发明的核酸包括DNA(比如dsDNA、ssDNA、cDNA)、RNA(比如dsRNA、ssRNA、mRNA、ivtRNA)，它们的组合或衍生物(比如PNA)。优选地，所述核酸是DNA或RNA，更优选mRNA。
本发明还提供一种载体，包含如本发明所述的核酸。其中，编码特异性识别抗原的细胞表面分子的核酸序列、编码CCL2的核酸序列和任选的编码IL7的核酸序列可以位于一个或多个载体中。
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。
载体一般包括靶向载体和表达载体。“靶向载体”是通过例如同源重组或使用特异性靶向位点处序列的杂合重组酶将分离的核酸递送至细胞内部的介质。“表达载体”是用于异源核酸序列(例如编码本发明的嵌合抗原受体多肽的那些序列)在合适的宿主细胞中的转录以及它们的mRNA的翻译的载体。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一个实施方案中，本发明的载体包括但不限于质粒、病毒(例如逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV、多瘤病毒和腺相关病毒(AAV)等)、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核苷酸序列，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。工程化载体通常还包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
工程化免疫细胞
本发明还提供一种工程化免疫细胞，其包含本发明的核酸或载体。换言之，本发明的工程化免疫细胞表达特异性识别抗原的细胞表面分子和外源性CCL2基因，以及任选的外源性的IL7基因。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，或者是从细胞脐带血等干细胞来源获得的免疫细胞。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+CD8+T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、CD4-CD8-T细胞、肿瘤浸润细胞、 记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。
在一个实施方案中，本发明的免疫细胞还包含至少一种失活基因，其选自以下：CD52、GR、TCRα、TCRβ、CD3γ、CD3δ、CD3ε、CD247ζ、HLA-I、HLA-II、B2M、免疫检查点基因如PD1、CTLA-4、LAG3和TIM3。更特别地，免疫细胞中的至少TCR组分(包括TCRα、TCRβ基因)或CD3组分(包括CD3γ、CD3δ、CD3ε、CD247ζ)被失活。这种失活使得TCR-CD3复合物在细胞中没有功能。该策略对于避免移植物抗宿主病(GvHD)特别有用。使基因失活的方法是本领域已知的，例如通过大范围核酸酶、锌指核酸酶、TALEN核酸酶或CRISPR系统中的Cas酶介导DNA断裂，从而使该基因失活。
药物组合物
本发明还提供一种药物组合物，其包含本发明所述的工程化免疫细胞、核酸分子或载体作为活性剂，和一种或多种药学上可接受的赋型剂。因此，本发明还涵盖所述核酸分子、载体或工程化免疫细胞在制备药物组合物或药物中的用途。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
治疗应用
本发明还提供一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用有效量的根据本发明所述的免疫细胞或药物组合物。因此，本发明还涵盖所述工程化免疫细胞在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。
在一个实施方案中，所述治疗方法包括向受试者施用有效量的本发明的免疫细胞和/或药物组合物。
在一个实施方案中，所述免疫细胞是自体或同种异体的细胞，优选T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，更优选T细胞、NK细胞或NKT细胞。
如本文所用，术语“自体”是指来源于个体的任何材料稍后将被再引入该相同个体中。如本文所用，术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些情况下，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。
如本文所用，术语“受试者”是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。
在一个实施方案中，所述癌症是与抗原结合区结合的靶标表达有关的癌症。例如，所述癌症包括但不限于：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌(包括胃肠癌)、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌(例如小细胞肺癌、非小细胞肺癌、腺状肺癌和鳞状肺癌)、淋巴瘤(包 括霍奇金淋巴瘤和非霍奇金淋巴瘤)、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中间级/滤泡性NHL、中间级扩散性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小型非裂化细胞性NHL、大肿块病NHL)、B淋巴母细胞淋巴瘤(B-LBL)、套细胞淋巴瘤、AIDS相关淋巴瘤、以及Waldenstrom巨球蛋白血症、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(B-ALL)、T细胞急性淋巴细胞白血病(T-ALL)、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、慢性骨髓性白血病(CML)、恶性淋巴组织增生疾病、MALT淋巴瘤、毛细胞白血病、边缘区淋巴瘤、多发性骨髓瘤、骨髓发育不良、浆母细胞性淋巴瘤、白血病前期、浆细胞样树突状细胞瘤、以及移植后淋巴细胞增生性紊乱(PTLD)；以及其他与靶标表达有关的疾病。优选地，可以用本发明的工程化免疫细胞或药物组合物治疗的疾病选自：白血病、淋巴瘤、多发性骨髓瘤、脑神经胶质瘤、胰腺癌、胃癌等。
在一个实施方案中，所述感染包括但不限于由病毒、细菌、真菌和寄生虫引起的感染。
在一个实施方案中，所述自身免疫性疾病包括但不限于I型糖尿病、腹腔疾病、格雷夫斯病、炎症性肠病、多发性硬化症、银屑病、类风湿性关节炎、艾迪生病、干燥综合征、桥本甲状腺炎、重症肌无力、血管炎、恶性贫血与系统性红斑狼疮等。
在一个实施方案中，所述方法还进一步包括向所述受试者施用一种或多种额外的化疗剂、生物制剂、药物或治疗。在该实施方案中，化疗剂、生物制剂、药物或治疗选自放射疗法、手术、抗体试剂和/或小分子和它们的任意组合。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
具体实施方式
实施例1.制备CAR-T细胞
1.1构建逆转录病毒质粒
构建MSCV-mCD19-CAR质粒，其包含CD19-scFv(SEQ ID NO：2)、CD8α铰链区(SEQ ID NO：17)、CD8α跨膜区(SEQ ID NO：5)、41BB共刺激结构域(SEQ ID NO：7)和CD3ζ胞内区(SEQ ID NO：11)的编码序列。
构建MSCV-mCD19-CAR-IL7质粒，其是在MSCV-mCD19-CAR质粒的基础上进一步包含T2A(SEQ ID NO：19)和IL7(SEQ ID NO：23)的编码序列。
构建MSCV-mCD19-CAR-CCL2质粒，其是在MSCV-mCD19-CAR质粒的基础上进一步包含T2A(SEQ ID NO：19)和CCL2(SEQ ID NO：25)的编码序列。
1.2.制备逆转录病毒
在T175培养瓶中，以30×10 6个细胞/瓶的密度将293T细胞接种于30ml含有10％胎牛血清的DMEM培养基中，于37℃、5％CO 2培养箱中培养过夜，用于病毒包装。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)、45μg制备的逆转录病毒质粒和15μg包装载体pCL-Eco(上海禾午生物科技有限公司，货号P3029)。然后加入120μl X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，室温孵育15min。然后将该质粒/载体/转染试剂混合物逐滴加入到预先准备好的293T细胞的培养瓶中，于37℃，5％CO 2条件下培养过夜。在转染后72小时收集培养物，离心(2000g，4℃，10分钟)，获得逆转录病毒上清液。
1.3制备CAR-T细胞
从小鼠脾脏分离T淋巴细胞，并用DynaBeads CD3/CD28 CTS TM(Gibco,货号40203D)激活T细胞，然后在37℃和5％CO 2下培养1天。
以每孔3×10 6个细胞/mL的密度将激活的T细胞接种至预先用RetroNectin包被过夜的24孔板中，然后加入500μL逆转录病毒上清液，并补充完全培养基至2mL。
将24孔板放于离心机进行离心感染，于32℃，2000g离心2h。然后，立刻将24孔板放置于37℃、CO 2培养箱静置培养。第二天更换新鲜培养基，并调整细胞密度为1×10 6个细胞/mL。感染三天后，收集细胞用于后续分析。收集的细胞即为mCD19-CAR细胞、mCD19-CAR+CCL2细胞和mCD19-CAR+IL7+CCL2细胞。
实施例2.检测CAR-T细胞的表达
2.1细胞表面CAR的表达水平
取出实施例1制备的2×10 5个CAR-T细胞，用Goat Anti-Rat IgG(H&L)Biotin(BioVision,货号6910-250)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)作为二抗，通过流式细胞术检测CAR T细胞上的CAR的表达水平，结果如图1所示。可以看出，与未经处理的NT细胞相比，mCD19-CAR细胞、mCD19-CAR+CCL2细胞和mCD19-CAR+IL7+CCL2细胞中的CAR阳性效率均大于60％，表明这些细胞均可有效表达CAR。
2.2  IL7的表达水平
收集CAR-T细胞的上清液，根据制造商的建议，用Mouse IL-7 DuoSet ELISA kit试剂盒(R&D Systems,货号DY407)检测细胞中的IL7的分泌水平，结果如图2所示。可以看出，mCD19-CAR+IL7+CCL2细胞可有效表达IL7。
2.3  CCL2的表达水平
收集CAR-T细胞的上清液，根据制造商的建议，用Mouse CCL2 DuoSet ELISA kit试剂盒(R&D Systems,货号DY479)检测细胞中的CCL2的分泌水平，结果如图3所示。可以看出，mCD19-CAR+CCL2细胞和mCD19-CAR+IL7+CCL2细胞均可有效 表达CCL2。
实施例3.检测CAR-T细胞的IFN-γ分泌水平
在96孔圆底板中以2×10 5个细胞/100μl的浓度分别加入NT细胞、CD19-CAR细胞、mCD19-CAR+CCL2细胞和mCD19-CAR+IL7+CCL2细胞。然后在各孔中以1×10 4个细胞/100μl的浓度分别加入Panc02-mCD19靶细胞(表达mCD19的Panc02细胞)或Panc02非靶细胞。在37℃培养24h后，收集培养物上清液。根据制造商的建议，用Mouse IFN-gamma DuoSet ELISA试剂盒(R&D，货号DY485)检测培养物上清液中IFN-γ的表达水平。
检测结果如图4所示。可以看出，在非靶细胞Panc02中均没有检测到IFN-γ的释放，而仅在与靶细胞Panc02-CD19共培养后检测到显著升高的IFN-γ水平，且NT细胞不表达IFN-γ，这表明本实施例中的CAR-T细胞的杀伤都是特异性的。此外，mCD19-CAR+CCL2细胞的IFN-γ水平显著高于CD19-CAR细胞，表明额外表达的CCL2基因能显著提高CAR-T细胞的杀伤活性。此外，表达IL7+CCL2组合的CAR-T细胞的IFN-γ水平也显著高于仅表达CCL2的CAR-T细胞，表明IL7的加入可以进一步提高CAR-T细胞的杀伤活性。
实施例4.CAR-T细胞的肿瘤抑制效果验证
在健康C57BL/6小鼠的左前肢腋下部位，经皮下接种5×10 5个Panc02-mCD19胰腺癌细胞。将接种了胰腺癌细胞的小鼠随机分为4组，每组5只。待肿瘤体积生长至100mm 3时，向各组小鼠经尾静脉注射5×10 6个NT细胞、mCD19-CAR细胞、mCD19-CAR+CCL2细胞或mCD19-CAR+IL7+CCL2细胞。监测小鼠的体重和肿瘤体积变化，直至实验结束。
小鼠的体重变化如图5所示。可以看出，施用CAR-T细胞后，各组小鼠的体重与对照组相比没有显著差异，且在观察周期中，当小鼠肿瘤未超过1500mm 3时，小鼠行动活泼，毛色正常，这表明，施用CAR-T细胞不会对小鼠有明显的毒副反应。
小鼠的肿瘤体积变化如图6所示。可以看出，mCD19-CAR+CCL2细胞的抗肿瘤作用优于常规CAR-T细胞，表明额外表达的CCL2能与CAR-T细胞产生协同作用，增强后者的抗肿瘤效果。此外，发明人还出乎意料地发现，进一步表达IL7可以显著增加CCL2对CAR-T细胞的促进作用，从而更显著地抑制肿瘤生长。
以上结果表明，共表达CCL2或其与IL7的组合能够有效增强表达CAR的工程化免疫细胞对肿瘤细胞的抑制效果。
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (22)

  1. 一种工程化免疫细胞，其表达特异性识别抗原的细胞表面分子和外源性的CCL2。
  2. 权利要求1所述的工程化免疫细胞，其中所述工程化免疫细胞进一步表达外源性的IL7。
  3. 权利要求1所述的工程化免疫细胞，其中所述CCL2与SEQ ID NO：25或27所示的氨基酸序列具有至少90％同一性，或者其编码序列与SEQ ID NO：24或26所示的核酸序列具有至少90％同一性。
  4. 权利要求2所述的工程化免疫细胞，其中所述IL7与SEQ ID NO：21或23所示的氨基酸序列具有至少90％同一性，或者其编码序列与SEQ ID NO：22或24所示的核酸序列具有至少90％同一性。
  5. 权利要求1-4任一项所述的工程化免疫细胞，其中所述特异性识别抗原的细胞表面分子是嵌合抗原受体或T细胞受体。
  6. 权利要求5所述的工程化免疫细胞，其中所述嵌合抗原受体包含抗原结合区、跨膜结构域和胞内信号传导结构域，所述胞内信号传导结构域包含共刺激结构域和/或初级信号传导结构域。
  7. 权利要求6所述的工程化免疫细胞，其中所述抗原结合区选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体、单结构域抗体、纳米抗体、双体、anticalin和DARPIN抗原。
  8. 权利要求7所述的工程化免疫细胞，其中所述抗原抗原结合区与选自以下的一个或多个靶标结合：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轻链、TIM3、TSHR、CD19、BAFF-R、CLL-1、EGFRvIII、tEGFR、GD2、GD3、BCMA、Tn抗原、PSMA、ROR1、FLT3、FAP、TAG72、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、IL-llRa、IL-22Ra、IL-2、间皮素、PSCA、PRSS21、VEGFR2、LewisY、PDGFR-β、SSEA-4、AFP、Folate受体α、ErbB2(Her2/neu)、ErbB3、ErbB4、MUC1、MUC16、EGFR、CS1、NCAM、Claudin18.2、c-Met、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gpl00、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM7R、CLDN6、GPRC5D、CXORF61、ALK、多聚唾液酸、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、豆荚蛋白、HPV E6、E7、ETV6-AML、精子蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、PSA、存活蛋白 和端粒酶、PCTA-l/Galectin 8、MelanA/MARTl、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、TMPRSS2 ETS融合基因、NA17、PAX3、雄激素受体、孕酮受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES 1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、PD1、PDL1、PDL2、TGFβ、APRIL、NKG2D、NKG2D配体，和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。
  9. 权利要求6所述的工程化免疫细胞，其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  10. 权利要求6-10任一项所述的工程化免疫细胞，其中所述初级信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  11. 权利要求6所述的工程化免疫细胞，其中所述共刺激结构域包含一个或多个选自以下蛋白质的胞内区：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以及它们的组合。
  12. 权利要求1-11任一项所述的工程化免疫细胞，其中所述免疫细胞选自T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞、或NKT细胞。
  13. 权利要求12所述的工程化免疫细胞，其中所述T细胞是CD4+CD8+T细胞、CD4+辅助T细胞、CD8+T细胞、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞或αβ-T细胞。
  14. 权利要求1-13任一项所述的工程化免疫细胞，其中所述CCL2和/或IL7的表达是条件型表达或组成型表达。
  15. 权利要求1-14任一项所述的工程化免疫细胞，其中所述CCL2和/或IL7与定位结构域可操作连接。
  16. 一种核酸分子，其包含编码特异性识别抗原的细胞表面分子的核酸序列和编码CCL2的核酸序列。
  17. 权利要求16所述的核酸分子，其中所述特异性识别抗原的细胞表面分子是嵌合抗原受体。
  18. 权利要求16所述的核酸分子，其中所述核酸分子进一步包含编码IL7的核酸序列。
  19. 一种载体，其包含权利要求16-18任一项所述的核酸分子。
  20. 权利要求18所述的载体，其中所述载体选自质粒、逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV)、多瘤病毒和腺相关病毒(AAV)。
  21. 一种药物组合物，其包含权利要求1-15任一项所述的工程化免疫细胞、权利要求16-18任一项所述的核酸分子或权利要求19-20任一项所述的载体，和一种或多种药学上可接受的赋型剂。
  22. 一种治疗患有癌症、感染或自身免疫性疾病的受试者的方法，包括向所述受试者施用权利要求1-15任一项所述的工程化免疫细胞或权利要求21所述的药物组合物。
PCT/CN2022/109101 2021-08-11 2022-07-29 工程化免疫细胞及其用途 WO2023016284A1 (zh)

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