WO2022033537A1 - 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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WO2022033537A1
WO2022033537A1 PCT/CN2021/112174 CN2021112174W WO2022033537A1 WO 2022033537 A1 WO2022033537 A1 WO 2022033537A1 CN 2021112174 W CN2021112174 W CN 2021112174W WO 2022033537 A1 WO2022033537 A1 WO 2022033537A1
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cells
immune cell
engineered immune
nucleic acid
car
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PCT/CN2021/112174
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English (en)
French (fr)
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邢芸
任江涛
贺小宏
王延宾
韩露
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南京北恒生物科技有限公司
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Priority to CA3186188A priority Critical patent/CA3186188A1/en
Priority to KR1020237000648A priority patent/KR20230033702A/ko
Priority to JP2023501259A priority patent/JP2023534928A/ja
Priority to US18/020,319 priority patent/US20240009311A1/en
Priority to AU2021323846A priority patent/AU2021323846A1/en
Priority to EP21855587.8A priority patent/EP4166655A1/en
Publication of WO2022033537A1 publication Critical patent/WO2022033537A1/zh

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Definitions

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell expressing (i) a chimeric receptor, and (ii) exogenous CCL3, CCL4 and/or CCL5 genes. More preferably, the chimeric receptor is a chimeric antigen receptor or a T cell receptor.
  • Tumor immunotherapy mainly relies on autoimmunity to eliminate tumor cells by regulating the human immune system and tumor microenvironment.
  • the immune system is a unified whole, and innate immunity also plays a very important role in tumor immunity.
  • Some antigen-presenting cells such as dendritic cells and macrophages, are the bridge between innate and adaptive immunity. Antigen-presenting cells can recognize and present tumor antigens to the adaptive immune system, activate tumor-specific T cells, and then eliminate tumors. Therefore, enhancing the tumor-killing effect of the immune system by enhancing the antigen presentation process is an important research direction of tumor immunity.
  • CAR cell therapy is an important tumor cell immunotherapy.
  • the successful tumor control of CAR cells generally requires the following processes: activation of the immune system, activation and expansion of CAR cells, infiltration of tumor tissue by activated CAR cells and killing of tumor cells.
  • the tumor microenvironment has an inhibitory effect on CAR cells, so that CAR cells cannot infiltrate tumor tissue. Therefore, how to reduce the inhibitory effect of the tumor microenvironment on CAR cells, improve the survival time of CAR cells, or recruit other immune cells to synergize with CAR cells is very important to improve the therapeutic effect of CAR cells.
  • Conventional type 1 dendritic cells are a subset of dendritic cells and are the main immune cells presenting tumor antigens.
  • the results show that cDC1 can effectively present tumor-associated antigens, especially necrotic cell-associated antigens, effectively induce antigen-specific CD8+ T cell responses, and play an extremely important role in the process of tumor killing in vivo.
  • Both mouse and human studies have shown that the distribution of cDC1 in the tumor microenvironment is positively correlated with anti-tumor immune responses, and is an important evaluation parameter for tumor-related immune scores.
  • cDC1 is less distributed in mice and humans, and is almost invisible in mouse and human tumor microenvironments with low tumor immune response rates. Optimizing the role of cDC1 in tumor therapy is an important research direction to improve the effect of tumor immunotherapy.
  • Inflammatory chemokines play an important role in recruiting relevant immune cells into the tumor microenvironment, driving cellular interactions and molecular signaling cascades, etc., and determining the final outcome of the host's antitumor immune response.
  • MIP macrophage inflammatory protein
  • RANTES macrophage inflammatory protein
  • MIP-1 family-related proteins enriched at tumor sites can promote the accumulation of CD103 + cDCs at tumor sites by recruiting natural killer cells (NK) to tumor sites, and increase the production of chemokines CXCL9 and CXCL10, Effectively inhibit tumor progression.
  • NK natural killer cells
  • a new immunotherapy method is needed that can effectively differentiate or recruit cDC1 dendritic cells to improve the efficiency of tumor antigen presentation and induce adoptive immune responses in the body, thereby improving the efficacy of CAR cell therapy.
  • the present invention provides a novel engineered immune cell expressing (i) a chimeric receptor, and (ii) an exogenous CCL3, CCL4 and/or CCL5 gene.
  • the CCL3 gene is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 77 or 79, or the polypeptide encoded by the CCL3 gene has the amino acid sequence set forth in SEQ ID NO: 78 or 80 At least 90% identical.
  • the CCL4 gene is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 81 or 83, or the polypeptide encoded by the CCL4 gene has the amino acid sequence set forth in SEQ ID NO: 82 or 84 At least 90% identical.
  • the CCL5 gene is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 85 or 87, or the polypeptide encoded by the CCL5 gene has the amino acid sequence set forth in SEQ ID NO: 86 or 88 At least 90% identical.
  • the engineered immune cells further express (iii) exogenous interleukins.
  • the interleukin is IL-2, IL-7, IL-12, IL-15, IL-21, IL-17, IL-18, IL-23, IL33, or a subunit thereof , or a combination thereof, or a combination of subunits thereof, preferably IL-7.
  • the interleukin-encoding gene is the same as SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69
  • the nucleic acid sequences shown in 71, 73, and 75 have at least 90% identity, or the interleukin is identical to SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
  • the amino acid sequences shown at 64, 66, 68, 70, 72, 74, 76 are at least 90% identical.
  • the interleukin, CCL4 or CCL5 expressed by the engineered immune cells is a fusion protein or mutant that is resistant to proteolysis.
  • the chimeric receptor is a chimeric antigen receptor or a T cell receptor.
  • the chimeric receptor is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain.
  • the ligand binding domain can be selected from immunoglobulin molecules, Fab, Fab', F(ab')2, Fv fragment, scFv, disulfide-linked Fv (sdFv), heavy chain variable region of antibody (VH) or light chain variable region (VL), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies, nanobodies, and non-immunoglobulin antigen binding scaffolds
  • the chimeric receptor binds to a target selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, AFP, Folate receptor ⁇ , ERBB2(Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2 , IGF-I receptor, CAIX, LMP2, gploo, bcr-
  • the target is selected from CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUCl, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D , EGFRvIII, CS1, BCMA, mesothelin, and any combination thereof.
  • 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 intracellular signaling domain is selected from the signaling domains of the following proteins: FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain is a CD3 ⁇ -comprising signaling domain.
  • the costimulatory domain is one or more costimulatory signaling domains selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), 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, CD94, LTB, ZAP70, and combinations thereof.
  • the costimulatory domain is a costimulatory signaling domain of CD27, CD28, CD134, CD137 or CD278 or a combination thereof.
  • the expression or activity of exogenous interleukins, CCL3, CCL4 and/or CCL5 is constitutive.
  • the expression or activity of exogenous interleukin, CCL3, CCL4 and/or CCL5 is conditional expression.
  • Conditional expression is achieved, for example, by operably linking the exogenous gene to an inducible, repressible, or tissue-specific promoter.
  • the interleukin, CCL3, CCL4 and/or CCL5 can be operably linked to a localization domain that can localize the exogenous gene of the invention for expression at a specific cellular location, e.g. Cell membranes, specific organelles in the cytoplasm such as endoplasmic reticulum, Golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, guide peptides, transmembrane domains, and the like.
  • the exogenous genes interleukin, CCL3, CCL4, and/or CCL5 of the invention are operably linked to the transmembrane domain, thereby anchored for expression on the surface of engineered immune cells.
  • 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, tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells or ⁇ -T cells.
  • the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, and the like.
  • the present invention provides a nucleic acid molecule, (i) a nucleic acid sequence encoding a chimeric receptor, and (ii) a nucleic acid sequence encoding CCL3, CCL4 and/or CCL5.
  • the chimeric receptor is a chimeric antigen receptor or a T cell receptor, more preferably a chimeric antigen receptor.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding an interleukin.
  • the interleukin is IL-2, IL-7, IL-12, IL-15, IL-21, IL-17, IL-18, IL-23, IL33, or a subunit thereof, or a subunit thereof A combination, or a combination of subunits thereof, more preferably IL-7, a subunit thereof, or a combination thereof.
  • the nucleic acid is DNA or RNA.
  • the present invention also provides a vector comprising the above-described nucleic acid molecule.
  • the vector is selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV).
  • the vector further comprises an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a polyadenylation tail (polyA), 3'UTR, 5'UTR, enhancer elements such as promoters, terminators, insulators, operons, selectable markers, reporter genes, targeting sequences and/or protein purification tags.
  • the vector is an in vitro transcribed vector.
  • the present invention also provides a kit comprising the engineered immune cells, nucleic acid molecules or vectors of the present invention.
  • the present invention also provides a pharmaceutical composition comprising the engineered immune cells, nucleic acid molecules or vectors of the present invention, and a plurality of pharmaceutically acceptable excipients.
  • the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell according to the present invention, Nucleic acid molecules, vectors or pharmaceutical compositions.
  • 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, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, chorionic villus Membrane cancer, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, Hepatoma, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (eg, small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous lung cancer), melanoma, myeloma, neuroblastoma, oral cavity Cancers (e.g.
  • lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, such as B-cell lymphomas (including low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL NHL, high-grade lymphoblastic NHL, high-grade small non-cleaving cell NHL, bulky NHL), mantle cell lymphoma, AIDS-related lymph
  • the infection includes, but is 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 advantage of the engineered immune cells of the present invention is that the co-expressed CCL3, CCL4 and/or CCL5 and optional interleukins such as IL7 can effectively recruit DC cells to the tumor site, increasing the proliferation and survival of the engineered immune cells Therefore, on the one hand, the inhibitory effect of the tumor microenvironment on engineered immune cells is reduced, and the tumor killing ability of engineered immune cells is improved; on the other hand, the recruited DC cells can activate the adoptive immune recognition of the body's own T cells. Immune cells form a synergistic effect that ultimately enhances tumor suppression.
  • Figure 1 CD19 expression rate of Panc02-mCD19 cells.
  • Figure 2 CAR expression levels of CAR-T cells determined by flow cytometry.
  • Figure 3 IL-7 expression levels of CAR-T cells determined by ELISA.
  • Figure 4 CCL4 expression levels of CAR-T cells determined by ELISA.
  • Figure 5 CCL5 expression levels of CAR-T cells determined by ELISA.
  • FIG. 6 IFN- ⁇ release levels after CAR-T cells were co-cultured with target cells and non-target cells, respectively.
  • Figure 7 Changes in body weight of mice after treatment of mouse pancreatic cancer with CAR-T cells expressing CCL4 or CCL5.
  • Figure 8 Tumor growth curve in mice after treatment of mouse pancreatic cancer with CAR-T cells expressing CCL4 or CCL5.
  • Figure 9 Changes in body weight of mice after treatment of mouse pancreatic cancer with CCL3-expressing CAR-T cells.
  • FIG. 1 Tumor growth curve in mice after treatment of mouse pancreatic cancer with CCL3-expressing CAR-T cells.
  • chimeric receptor refers to a molecule expressed on the surface of a cell capable of specifically binding to a target molecule (eg, a ligand). Such molecules generally comprise a ligand binding domain capable of specifically binding a ligand, a transmembrane domain that anchors the surface molecule to the cell surface, and an intracellular domain responsible for signaling. Examples of common such chimeric receptors include, for example, T cell receptors or chimeric antigen receptors.
  • T cell receptor refers to a membrane protein complex that is involved in T cell activation in response to antigen presentation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHCs) on antigen-presenting cells that present antigenic peptides to T cells and bind to the TCR complex to induce a cascade of intracellular signaling.
  • MHCs major histocompatibility complex molecules
  • TCR consists of six peptide chains that form heterodimers respectively, which are generally classified into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, where the variable region is responsible for binding specificity to a specific antigen and MHC molecule.
  • the variable region of the TCR may comprise or be operably linked to a ligand binding domain, wherein the ligand binding domain is defined below.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes a ligand binding domain (eg, an antibody or antibody fragment), a transmembrane domain, a co- The stimulatory domain and the intracellular signaling domain are linked by linkers.
  • CARs can exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in an MHC-non-restricted manner.
  • Non-MHC-restricted antigen recognition gives CAR cells the ability to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape.
  • CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).
  • ligand binding domain refers to any structure or functional variant thereof that can bind a ligand (eg, an antigen).
  • the ligand binding domain can be an antibody structure including, but not limited to, monoclonal, polyclonal, recombinant, human, humanized, murine, chimeric, and functional fragments thereof.
  • ligand binding domains include, but are not limited to, immunoglobulin molecules, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-linked Fv (sdFv), heavy chain variable of an antibody Region (VH) or light chain variable region (VL), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies, Nanobodies, and non-immunoglobulin antigen binding scaffolds such as recombinant fibronectin Domain, DARPIN, affimer, affilin, adnectin, affitin, obodies, repeat body, fynomer, alpha body, avimer, atrimer, centyrin, pronectin, anticalin, kunitz type domain, Armadillo repeat protein, etc.
  • immunoglobulin molecules Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-linked F
  • the ligand binding domain is selected from Fabs, scFvs, single domain antibodies, Nanobodies, or functional fragments thereof.
  • the ligand binding domains may be monovalent or bivalent, and may be monospecific, bispecific or multispecific antibodies comprising one or more ligand binding domains.
  • Fab refers to either of two identical fragments produced upon cleavage of an immunoglobulin molecule by papain, consisting of an intact light chain and an N-terminal portion of a heavy chain linked by disulfide bonds, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared to intact IgG, Fab has no Fc fragment, has higher mobility and tissue penetration, and can bind antigen monovalently without mediating antibody effects.
  • a “single chain antibody” or “scFv” is an antibody composed of an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker significantly affects the variable region folding and interaction of the scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids).
  • linker size and composition see, eg, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448; US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication Nos.
  • the scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.
  • Single-domain antibody refers to an antibody that is naturally deficient in its light chain, and which contains only one variable heavy chain region (VHH) and two conventional CH2 and CH3 regions, also referred to as a “heavy chain” antibody.
  • VHH variable heavy chain region
  • CH2 and CH3 regions also referred to as a “heavy chain” antibody
  • Nemobody or “Nb” refers to a single cloned and expressed VHH structure, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind to the target antigen. .
  • the term "functional variant” or “functional fragment” refers to a variant that substantially comprises the amino acid sequence of a parent but contains at least one amino acid modification (ie, substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variants retain the biological activity of the parent amino acid sequence.
  • the amino acid modification is preferably a conservative modification.
  • conservative modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues having similar side chains.
  • Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, 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 eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • threonine valine, isoleucine
  • aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • a “functional variant” or “functional fragment” is 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 refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical.
  • 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 ClustalW.
  • the ligand binding domains of the invention bind to one or more targets selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF
  • the target is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2(Her2/neu), MUCl, EGFR, CAIX, WT1, NY- ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D and any combination thereof.
  • the CAR of the present invention can be designed to include a ligand binding domain specific for that antigen.
  • an antibody to CD19 can be used as the ligand binding domain of the invention.
  • the chimeric antigen receptor of the invention targets CD19, CD22, or a combination thereof.
  • the chimeric antigen receptor of the present invention comprises an anti-CD19 antibody comprising the amino acid sequence set forth in positions 1-107 of SEQ ID NO: 1 or positions 1-107 of SEQ ID NO: 25
  • a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity and with SEQ ID NO: 1 positions 123-242 or Heavy chain variable having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 25 region sequence.
  • the chimeric antigen receptor of the present invention comprises an anti-CD22 antibody comprising at least 70%, preferably at least 80%, more
  • the heavy chain variable region sequence of at least 90%, 95%, 97%, 99% or 100% sequence identity has at least 70%, preferably at least 80%, more preferably light chain variable region sequences of at least 90%, 95%, 97%, 99% or 100% sequence identity.
  • transmembrane domain refers to a polypeptide that enables expression of a chimeric antigen receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells structure.
  • immune cells eg, lymphocytes, NK cells, or NKT cells
  • the transmembrane domain 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 useful 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 their functional fragments.
  • the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from the CD8 alpha chain or CD28, which is at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 3, 5 or 30 , 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleotide sequence shown in SEQ ID NO: 4, 6 or 29 , 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the present invention may further comprise a hinge region between the ligand binding domain and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand binding domain. Specifically, the hinge region serves to provide greater flexibility and accessibility to the ligand binding domain.
  • 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 can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, 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 a fully synthetic hinge sequence.
  • the hinge region comprises a hinge region portion of a CD8 ⁇ chain, FcyRIII ⁇ receptor, IgG4 or IgG1, more preferably a hinge from CD8 ⁇ , CD28 or IgG4, which is identical to SEQ ID NO: 19, 21, 23 or
  • the amino acid sequence shown in 36 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence and SEQ ID NO: 20, 22
  • the nucleotide sequences shown in , 24 or 35 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs cells to perform specified functions.
  • the intracellular signaling domain is responsible for primary intracellular signaling following binding of the ligand binding domain to an antigen, resulting in the 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 cells in which the CAR is expressed.
  • the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.
  • the chimeric antigen receptors of the invention comprise intracellular signaling domains that may be cytoplasmic sequences of T cell receptors and co-receptors that act together upon antigen receptor binding to initiate primary signaling conduction, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same or similar function.
  • the intracellular signaling domain can contain a number of immunoreceptor tyrosine-based activation motifs (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • intracellular 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 signaling domain of the CAR of the present invention may comprise a CD3 ⁇ signaling domain having at least 70% of the amino acid sequence shown in SEQ ID NO: 11, 13 or 34, preferably at least at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70% with the nucleotide sequence shown in SEQ ID NO: 12, 14 or 33 , preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptors of the invention comprise one or more costimulatory domains.
  • a costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory 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 costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), 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, CD94, LTB and ZAP70.
  • costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10
  • the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28, CD27, OX40 or a combination thereof.
  • the costimulatory domain comprised by the CAR of the present invention 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: 7 or 9 or 99% or 100% sequence identity, or the coding sequence of the costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleotide sequence shown in SEQ ID NO: 8 or 10 , 95%, 97% or 99% or 100% sequence identity.
  • the CAR of the present invention may further 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 segment of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptidases At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases.
  • the signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • Signal peptides useful in the present invention are well known to those skilled in the art, eg, signal peptides derived from CD8 ⁇ , IgG1, GM-CSFR ⁇ , B2M, and the like.
  • the signal peptide useful in the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95% with the amino acid sequence shown in SEQ ID NO: 16, 18 or 39 , 97% or 99% or 100% sequence identity.
  • the CAR of the present invention may further comprise a switch structure to regulate the expression time of the CAR.
  • the switch structure can be in the form of a dimerization domain that induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain for binding to the targeted antigen, thereby activating a signaling pathway.
  • switch domains can be used to connect the binding and signaling domains, respectively, which can dimerize only when the switch domains bind to each other (eg, in the presence of an inducing compound) linked together, thereby activating signaling pathways.
  • the switch structure can also be in the form of a masked peptide.
  • the masking peptide can mask the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure.
  • Various switch structures known to those skilled in the art can be used in the present invention.
  • the CAR of the present invention may further comprise a suicide gene, i.e., causing it to express a cell death signal that can be induced by a foreign substance, to clear the CAR cells when needed (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 desired, 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, which can be induced to dimerize by chemical inducing drugs such as AP1903, AP20187, etc., thereby activating the downstream Caspase3 molecule, leading to apoptosis.
  • chemical inducing drugs such as AP1903, AP20187, etc.
  • Various suicide genes known to those skilled in the art can be used in the present invention.
  • Chemokines can be divided into four subgroups: CXC, CC, C and CX3C according to the arrangement of their N-terminal cysteines, among which CC chemokines contain two adjacent cysteine-cysteine Acid (cys-cys or CC) residues.
  • Examples of CC chemokines are those that bind to CCR1 and CCR5, including MIP-1 ⁇ , MIP-1 ⁇ , CCL5, MCP-1, MCP-2, MCP-3, I-309, and the like.
  • Macrophage inflammatory protein has two main forms: ⁇ (MIP-1 ⁇ , also known as CCL3) and ⁇ (MIP-1 ⁇ , also known as CCL4), the first to isolate macrophages activated by free lipopolysaccharide. Phage culture medium. The two have similar sequences and activities, mainly produced by macrophages, monocytes and dendritic cells, etc., and both participate in inflammation and infection by binding to the chemokine receptor 5 (CCR5) on the cell surface. immune response. Studies have shown that CCL3 and CCL4 are involved in the host defense response of many infectious diseases such as tuberculosis, and mediate the directional migration of immune cells.
  • CCR5 chemokine receptor 5
  • CCL5 also known as RANTES, is a small molecule protein with a molecular weight of 8000.
  • the gene of human CCL5 protein is located at 17q11-21.
  • CLL5 exists in various forms such as monomer, dimer and multimer.
  • CCL5 is mainly expressed in macrophages, activated T cells, fibroblasts, glial cells, epithelial cells, endothelial cells, platelets and certain tumor cells. Sex granulocytes, T lymphocytes, natural killer cells, etc. have chemotactic or stimulatory effects.
  • CCL3, CCL4 and CCL5 all bind to CCR5, thereby recruiting immune cells such as immature myeloid dendritic cells, monocytes, macrophages, Th1, Treg, NK and plasmacytoid dendritic cells for the directed migration to inflammatory site or tumor site.
  • immune cells such as immature myeloid dendritic cells, monocytes, macrophages, Th1, Treg, NK and plasmacytoid dendritic cells for the directed migration to inflammatory site or tumor site.
  • the engineered immune cells of the invention express (i) a chimeric receptor, and (ii) an exogenous CCL3, CCL4 and/or CCL5 gene. More preferably, the engineered immune cells of the present invention express (i) a chimeric antigen receptor, and (ii) an exogenous CCL4 and/or CCL5 gene.
  • CCL3 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: 78 or 80 % sequence identity, or the coding sequence of CCL3 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: 77 or 79 % sequence identity.
  • CCL4 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: 82 or 84 % sequence identity, or the coding sequence of CCL4 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: 81 or 83 % sequence identity.
  • CCL5 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: 86 or 88 % sequence identity, or the coding sequence of CCL5 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: 85 or 87 % sequence identity.
  • Interleukins are a class of cytokines that are produced by leukocytes and function between leukocytes. They play an important role in transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and in inflammatory responses. In general, the biological effects of interleukins are achieved through their binding to the corresponding receptors, for example, the biological properties of IL-7 are achieved through the binding of IL-7 to its receptor IL-7R.
  • interleukins useful in the present invention include, but are not limited to, IL-2, IL-7, IL-12, IL-15, IL-21, IL-17, IL-18, IL-23, IL33, or a subunit thereof, or a combination thereof, or a combination of subunits thereof, more preferably IL-7 or a subunit thereof.
  • IL-2 is mainly produced by T cells and acts in an autocrine and paracrine manner. IL-2 can not only maintain the growth of T cells and promote the production of cytokines, but also induce CD8+ T cells and CD4+ T cells to exert cytotoxic effects.
  • IL-2 can also stimulate NK cell proliferation, enhance NK killing activity, promote NK cells to produce IFN ⁇ , TNF ⁇ , TGF ⁇ and other factors, and activate macrophages to enhance the antigen presentation ability and target cell killing ability of macrophages.
  • IL-7 is mainly produced by bone marrow and thymic stromal cells, and its main functions involve the following aspects: promoting the growth of precursor B cells; inhibiting the apoptosis of peripheral T cells, inducing cell proliferation and continuous survival; The development and function of phages induce macrophages to secrete a variety of cytokines.
  • IL-12 mainly acts on T cells and NK cells.
  • IL-12 can stimulate the proliferation of activated T cells, promote the differentiation of Th0 cells to Th1 cells; induce the cytotoxic activity of CTL and NK cells and promote their secretion of cytokines such as IFN ⁇ , TNF ⁇ , GMCSF; promote NK cells and IL-1.
  • cytokines such as IFN ⁇ , TNF ⁇ , GMCSF
  • IL-15 can be produced by a variety of cells, including activated macrophages, epidermal cells, and present cells. Since the molecular structure of IL-15 is similar to that of IL-2, the ⁇ chain and ⁇ chain of IL-2R can be used to bind to target cells and exert biological activities similar to IL-2, such as stimulating the proliferation of T cells and NK cells.
  • IL-21 is produced by activated CD4+ T cells, NKT cells, Tfh cells and Th17 cells, and has high homology with IL-2 and IL-15. IL-21 has a wide range of immunoregulatory functions, and activation of it can enhance the proliferation of activated CD8+ T cells, enhance the cytotoxic activity of NK cells, and promote the proliferation and differentiation of B cells.
  • IL-17 secreted by Th17 cells, is a pro-inflammatory molecule that suppresses regulatory T cells and recruits and activates neutrophils and macrophages.
  • IL-18 is also a pro-inflammatory molecule, secreted by T cells and NK cells, and activated macrophages also secrete large amounts of IL-18.
  • IL-18 plays an important role in innate and adoptive immunity.
  • IL-23 is also secreted by T cells and NK cells, and IL-23 can promote Th17 cell differentiation.
  • Dendritic cells, monocytes, and macrophages also express low levels of IL-23 receptors, which can be activated by IL-23.
  • the sequence of IL-33 is similar to that of IL-18. It binds to the receptor ST2 through the IL-1 receptor accessory protein IL-RAcP, and the three form a heterodimer, which activates signaling pathways such as NF- ⁇ B and MAPK, and strongly induces pre- The production of inflammatory factors and chemokines, thereby exerting biological effects.
  • IL-33 is expressed in a variety of cells, including epithelial cells, fibroblasts, macrophages, dendritic cells, etc. Studies have shown that IL-33 can stimulate immature dendritic cells to produce Treg, promote Th1 cells to secrete IFN- ⁇ , and increase the expression of CD69, thereby activating NK, NKT cells and CD8+T cells, and promoting anti-tumor immunity. Studies have shown that these interleukins alone or in combination (eg, IL-33 and IL-12, IL-7+IL-12, IL-7+IL15, etc.) can exert effective anti-tumor effects.
  • interleukins alone or in combination (eg, IL-33 and IL-12, IL-7+IL-12, IL-7+IL15, etc.) can exert effective anti-tumor effects.
  • the interleukin used in the present invention is the The amino acid sequences shown in 74 and 76 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or the coding sequence thereof and SEQ ID NO: 41 , 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 have at least 70%, preferably at least 80%, more Preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • exogenous genes in the present invention such as interleukin, CCL3, CCL4, CCL5, can be constitutive expression or conditional expression.
  • the expression of exogenous interleukins, CCL3, CCL4, CCL5 is conditional.
  • the exogenous gene of the present invention can be operably linked to 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, a tumor acidic microenvironment, a tumor hypoxic microenvironment, and the like.
  • Such inducers include, for example, ciccycline, tetracycline, or analogs thereof, and analogs of tetracycline include, for example, chlortetracycline, oxytetracycline, desmethylchlorotetracycline, methycycline, doxycycline, and minocycline White.
  • Inducible promoters include, for example, Lac operon sequences, tetracycline operon sequences, galactose operon sequences, or doxycycline operon sequences, and the like.
  • the promoter is a repressible promoter, ie, in the presence of a repressor specific for the repressible promoter, the expression of the foreign gene in the cell is inhibited or not expressed.
  • Repressible promoters include, for example, Lac repressible elements or tetracycline repressible elements.
  • Inducible/repressible expression systems well known to those skilled in the art can be used in the present invention, including but not limited to Tet-on system, Tet-off system, Cre/loxP system and the like.
  • interleukins, CCL3, CCL4, CCL5 can be operably linked to localization domains that can localize the exogenous genes of the invention for expression at specific cellular locations, such as cell membranes, Specific organelles in the cytoplasm such as endoplasmic reticulum, Golgi apparatus, nucleus, etc. Localization domains include, but are not limited to, nuclear localization signals, guide peptides, transmembrane domains, and the like.
  • the exogenous genes interleukin, CCL3, CCL4, CCL5 of the invention are operably linked to the transmembrane domain so as to be anchored for expression on the surface of engineered immune cells.
  • the exogenous genes in the present invention eg, interleukin, CCL3, CCL4, CCL5 proteins
  • the present invention also provides a nucleic acid molecule comprising (i) a nucleic acid sequence encoding a chimeric receptor, and (ii) a nucleic acid sequence encoding CCL3, CCL4 and/or CCL5.
  • the nucleic acid molecule further comprises (iii) a nucleic acid sequence encoding an interleukin.
  • the interleukins include but are not limited to IL-2, IL-7, IL-12, IL-15, IL-21, IL-17, IL-18, IL-23, IL33, or subunits thereof , or a combination thereof, or a combination of subunits thereof, more preferably IL-7 or a subunit thereof.
  • the chimeric receptor is a T cell receptor or a chimeric antigen receptor, preferably a chimeric antigen receptor.
  • Chimeric antigen receptors are defined as above.
  • nucleic acid molecule includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in linear or circular form in single- and/or double-stranded form form, or their mixtures (including hybrid molecules).
  • nucleic acids according to the present invention include DNA (eg dsDNA, ssDNA, cDNA), RNA (eg dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (eg PNA).
  • the nucleic acid is DNA or RNA, more preferably mRNA.
  • Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNA)). Nucleic acids of the invention may also contain one or more modified bases, such as, for example, tritylated bases and uncommon bases such as inosine. Other modifications are also contemplated, including chemical, enzymatic, or metabolic modifications, so long as the multi-chain CARs of the invention can be expressed from polynucleotides. Nucleic acids can be provided in isolated form. In one embodiment, nucleic acids may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.
  • transcriptional control elements including promoters, enhancers, operators, repressors, and transcription termination signals
  • ribosome binding sites introns, and the like.
  • the nucleic acid sequences of the invention can be codon optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacterial, yeast, or insect cells. Codon optimization refers to the replacement of codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement code for the same amino acid. Therefore, the choice of optimal codons depends on the codon usage preferences of the host genome.
  • the present invention also provides a vector comprising the nucleic acid according to the present invention.
  • the nucleic acid sequence encoding the chimeric receptor optionally the nucleic acid sequence encoding IL-7, the nucleic acid encoding CCL3, the nucleic acid encoding CCL4, and/or the nucleic acid sequence encoding CCL5 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, in which the nucleic acid molecule 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 specific targeting sequence at the site.
  • An “expression vector” is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric antigen receptor polypeptides of the invention, in suitable host cells and the translation of their mRNAs. Suitable carriers for use in the present invention are known in the art and many are commercially available.
  • the vectors of the present invention include, but are not limited to, plasmids, viruses (eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)), and the like ), phages, phagemids, cosmids, and artificial chromosomes (including BAC and YAC).
  • viruses eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)
  • RSV Rous sarcoma virus
  • AAV adeno-associated virus
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector .
  • Engineered vectors typically also contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (eg, a multiple cloning site, MCS).
  • the vector may additionally contain a promoter , polyadenylation tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, 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 invention express chimeric receptors (eg, chimeric antigen receptors), CCL3, CCL4, and/or CCL5 genes, and optionally, interleukins, such as IL-7.
  • 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 derived from stem cells such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, Immune cells such as bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, e.g., 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 sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic 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.
  • immune cells are engineered to express chimeric antigen receptors and exogenous CCL3, CCL4 and/or CCL5 genes, and optionally an interleukin such as IL-7.
  • RNA transfection is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell.
  • RNA transfection the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell. The term is mainly used for non-viral methods in eukaryotic cells.
  • transfection is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes and deposit their cargo inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells).
  • transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means.
  • the cells or bacteria must be in a competent state.
  • techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation.
  • the present invention also provides a method of preparing an engineered immune cell, comprising introducing into said immune cell: (a) a first nucleic acid sequence encoding a chimeric receptor or a chimeric receptor encoded therein; b) encoding A second nucleic acid sequence of CCL3, CCL4 and/or CCL5 or the CCL3, CCL4 and/or CCL5 protein it encodes; and optionally (c) a third nucleic acid sequence encoding an interleukin or an interleukin it encodes.
  • the components (a), (b) and (c) described above may be sequentially introduced into the immune cells in any order.
  • the components (a), (b) and (c) described above can be introduced into immune cells simultaneously, eg, by cloning (a), (b) and (c) in one or more vectors.
  • nucleic acid or vector After the nucleic acid or vector is introduced into immune cells, those skilled in the art can expand and activate the resulting immune cells by conventional techniques.
  • the engineered immune cells of the present invention may further comprise suppressed or silenced expression of at least one gene selected from the group consisting of CD52, GR, dCK, TCR/CD3 genes (eg, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6 , CASP7, FADD, FA
  • RNA silencing genes are well known to those skilled in the art.
  • antisense RNA RNA decoys, RNA aptamers, siRNA, shRNA/miRNA, trans dominant negative protein (TNP), chimeric/fusion proteins, chemokine ligands, anti-infective cellular proteins, cellular Intrabodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors to inhibit gene expression.
  • gene silencing can also be achieved by mediating DNA fragmentation, eg, by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.
  • the present invention provides a kit comprising the engineered immune cells, nucleic acid molecules or vectors of the present invention.
  • kit of the present invention further comprises instructions.
  • the present invention also provides a pharmaceutical composition comprising the engineered immune cells, nucleic acid molecules or carriers of the present invention as active agents, and a variety of pharmaceutically acceptable excipients. Therefore, the present invention also encompasses the use of the nucleic acid molecule, vector or engineered immune cell in the preparation of a pharmaceutical composition or medicament.
  • the term "pharmaceutically acceptable excipient” means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention.
  • excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends, among other things, 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 may be suitable for administration by various routes. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular 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 particularly suitable for the desired method of administration.
  • Processes known in the present invention for the manufacture of pharmaceuticals 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.
  • compositions according to the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • 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, trimetate 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 210 and 213, act
  • the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the present invention. Accordingly, the present invention also encompasses the use of the nucleic acid molecules, vectors, engineered immune cells and pharmaceutical compositions in the manufacture of medicaments for the treatment of cancer, infection or autoimmune diseases.
  • an effective amount of an immune cell and/or pharmaceutical composition of the invention is administered directly to a subject.
  • the method of treatment of the present invention is an ex vivo treatment.
  • the method comprises the steps of: (a) providing a sample comprising immune cells; (b) in vitro combining a chimeric receptor of the invention (eg, a chimeric antigen receptor) and an exogenous to-be-expressed The genes are introduced into the immune cells, modified immune cells are obtained, and (c) the modified immune cells are administered to a subject in need thereof.
  • the immune cells provided in step (a) are selected from macrophages, dendritic cells, monocytes, T cells, NK cells and/or NKT cells; Conventional methods are obtained from a subject's sample, particularly a blood sample.
  • immune cells capable of expressing the chimeric antigen receptors and exogenous genes of the invention and performing the desired biological effector functions as described herein may also be used.
  • the immune cells are typically selected to be compatible with the subject's immune system, ie preferably the immune cells do not elicit an immunogenic response.
  • "universal recipient cells,” ie, universally compatible lymphocytes that can be grown and expanded in vitro, can be used that perform the desired biological effector function. The use of such cells would not require obtaining and/or providing the subject's own lymphocytes.
  • step (c) can be carried out by introducing a nucleic acid or vector as described herein into immune cells via electroporation or by infecting immune cells with a viral vector such as a lentiviral vector, adenovirus Viral vector, adeno-associated viral vector or retroviral vector.
  • a viral vector such as a lentiviral vector, adenovirus Viral vector, adeno-associated viral vector or retroviral vector.
  • transfection reagents such as liposomes
  • transient RNA transfection transient RNA transfection.
  • 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 or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. 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.
  • the mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples.
  • Mammals other than humans can advantageously be used as subjects representing animal models of cancer.
  • the subject is a human.
  • the cancer is a cancer associated with expression of a ligand binding domain-bound target.
  • cancers include, but are not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous lung cancer), lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma
  • the diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, and the like.
  • the infection includes, but is 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, biological agents, 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.
  • the total cDNA sequence of mouse spleen was obtained by reverse transcription PCR, and then the mouse mCD19 sequence containing XbaI and SalI restriction sites was obtained by PCR. Then, the mCD19 gene was recombined into the pLVX vector (PPL, Cat No.: PPL00157-4a) to obtain the pLV-mCD19 plasmid.
  • 293T cells were seeded in 30 ml of DMEM medium containing 10 % fetal bovine serum at a density of 30 x 106 cells/flask, and cultured overnight in a 37°C, 5% CO2 incubator.
  • the plasmid/vector/transfection reagent mixture was then added dropwise to a pre-prepared culture flask of 293T cells and incubated overnight at 37°C, 5% CO2 . Cultures were collected 24 hours and 48 hours after transfection, pooled and ultracentrifuged (25000g, 4°C, 2.5h) to obtain concentrated pLV-mCD19 lentivirus, which was stored at -80°C.
  • RPMI-1640 medium Gibco, Cat. No. C12430500BT
  • fetal bovine serum 1 ⁇ 10 6 mouse pancreatic cancer cells Panc02 (from China Pharmaceutical University Experiment) Chamber gift
  • 200 ⁇ l of pLV-mCD19 lentivirus were incubated at 37° C., 5% CO 2 for 48 h.
  • the cells were then digested into a single cell suspension with 0.25% trypsin, and the cells were diluted and transferred to a 96-well plate for continued culture until monoclonal cells appeared.
  • Monoclonal cells were picked, trypsinized again into single cells with 0.25%, and resuspended in 200 ⁇ l opti-MEM medium.
  • APC anti-mouse CD19 antibody Biolegend, Cat. No. 115512
  • the infection efficiency was detected by flow cytometry, and CD19-positive clones were screened. After the positive clones were passaged 3-4 times, the expression level of CD19 was detected by flow cytometry. Panc02 cells that were not infected with the virus were used as controls.
  • the artificially synthesized CD19-scFv (SEQ ID NO: 26), CD8a hinge and transmembrane regions (SEQ ID NO: 35 and 29), 41bb intracellular domain (SEQ ID NO: 31) and CD3 ⁇ intracellular domain (SEQ ID NO: 31) were synthesized in sequence.
  • the coding sequence fragments of T2A (SEQ ID NO: 37) and IL-7 (SEQ ID NO: 43) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends.
  • the fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-IL-7 plasmid.
  • the coding sequence fragments of T2A (SEQ ID NO: 37) and CCL4 (SEQ ID NO: 83) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends.
  • the fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-CCL4 plasmid.
  • the coding sequence fragments of T2A (SEQ ID NO: 37) and CCL5 (SEQ ID NO: 87) were artificially synthesized in turn, and EcoRI/SalI restriction sites were added at both ends.
  • the fragment was cloned into the MSCV-mCD19-CAR vector to obtain the MSCV-mCD19-CAR-CCL5 plasmid.
  • 293T cells were seeded in 30 ml of DMEM medium containing 10 % fetal bovine serum at a density of 30 x 106 cells/flask, and cultured overnight in a 37°C, 5% CO2 incubator with in virus packaging.
  • 06366236001 mix immediately, and incubate at room temperature for 15 min.
  • the plasmid/vector/transfection reagent mixture was then added dropwise to a pre-prepared culture flask of 293T cells and incubated overnight at 37°C under 5% CO2 conditions. Cultures were harvested 72 hours after transfection and centrifuged (2000 g, 4°C, 10 min) to obtain retroviral supernatant.
  • T lymphocytes were isolated from mouse spleen and T cells were activated with DynaBeads CD3/CD28CTS TM (Gibco, Cat. No. 40203D), and then cultured for 1 day at 37°C and 5% CO 2 .
  • Activated T cells were seeded into 24-well plates pre-coated with RetroNectin overnight at a density of 3 ⁇ 10 6 cells/mL per well, and then 500 ⁇ L of complete medium (NT, control), MSCV-mCD19-CAR, and Virus, MSCV-mCD19-CAR-CCL4 virus, MSCV-mCD19-CAR-CCL5 virus, MSCV-mCD19-CAR-IL-7 virus + MSCV-mCD19-CAR-CCL4 virus, or MSCV-mCD19-CAR-IL-7 Virus+MSCV-mCD19-CAR-CCL5 virus and supplemented with complete medium to 2mL.
  • complete medium NT, control
  • MSCV-mCD19-CAR and Virus
  • MSCV-mCD19-CAR-CCL4 virus MSCV-mCD19-CAR-CCL5 virus
  • MSCV-mCD19-CAR-IL-7 virus + MSCV-mCD19-CAR-CCL5 virus or
  • the 24-well plate was placed in a centrifuge for centrifugation, and centrifuged at 32°C and 2000g for 2h. Immediately thereafter, the 24-well plate was placed in a 37°C, CO 2 incubator for static culture. Change fresh medium the next day and adjust the cell density to 1 x 10 6 cells/mL. Three days after infection, cells were collected for subsequent analysis. The collected cells are NT cells, mCD19-CAR cells, mCD19-CAR+CCL4 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+IL7+CCL4 cells, and mCD19-CAR+IL7+CCL5 cells.
  • CAR positivity in mCD19-CAR cells, mCD19-CAR+CCL4 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+IL7+CCL4 cells, and mCD19-CAR+IL7+CCL5 cells compared to controls The efficiencies were all around 60%, indicating that these cells could effectively express CAR.
  • the supernatant of the CAR-T cells prepared in Example 2 was collected, and the Mouse IL-7 DuoSet ELISA kit (R&D Systems, Cat. No. DY407) was used to detect the level of IL-7 secretion in the cells according to the manufacturer's recommendations. The results are shown in the figure. 3 shown.
  • both CAR T cells transfected with MSCV-mCD19-CAR-IL-7 virus can efficiently express IL-7.
  • the supernatant of the CAR-T cells prepared in Example 2 was collected, and the level of CCL4 secretion in the cells was detected with the Mouse CCL4 DuoSet ELISA kit (R&D Systems, Cat. No. DY451) according to the manufacturer's recommendations. The results are shown in Figure 4 .
  • the supernatant of the CAR-T cells prepared in Example 2 was collected, and the level of CCL5 secretion in the cells was detected with the Mouse CCL5 DuoSet ELISA kit (R&D Systems, Cat. No. DY478) according to the manufacturer's recommendations. The results are shown in Figure 5 .
  • NT cells mCD19 -CAR cells, mCD19-CAR+IL-7 cells, mCD19-CAR+CCL4 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+IL7+CCL4 cells, and mCD19-CAR+IL7+CCL5 cells.
  • Target Panc02-mCD19 cells or non-target Panc02 cells were then added to each well at a concentration of 1 x 104 cells/100 ⁇ l, respectively. After 24 h incubation at 37°C, the culture supernatant was collected. The expression levels of IFN- ⁇ in culture supernatants were detected using the Mouse IFN-gamma DuoSet ELISA kit (R&D, Cat. No. DY485) according to the manufacturer's recommendations.
  • Panc02-mCD19 pancreatic cancer cells prepared in Example 1 were subcutaneously inoculated into the axilla of the left forelimb of healthy C57BL/6 mice.
  • mice inoculated with pancreatic cancer cells were randomly divided into 6 groups of 5 mice each.
  • the tumor volume grew to 100mm3 , 1 ⁇ 106 NT cells, mCD19 -CAR cells, mCD19-CAR+CCL4 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+CCL5 cells, mCD19-CAR+IL7+CCL4 cells and mCD19-CAR+IL7+CCL5 cells.
  • mice were monitored for changes in body weight and tumor volume until the end of the experiment.
  • CAR-T cells expressing CCL3 were prepared according to the method described in Example 2, wherein the coding sequence fragments of T2A (SEQ ID NO: 37) and CCL3 (SEQ ID NO: 79) were cloned into the MSCV-mCD19-CAR vector to obtain MSCV-mCD19-CAR-CCL3 plasmid and packaged as retrovirus. Activated T cells were transfected with this virus to obtain mCD19-CAR+CCL3 cells. Activated T cells were simultaneously transfected with MSCV-mCD19-CAR-CCL3 virus and MSCV-mCD19-CAR-IL-7 virus to obtain mCD19-CAR+IL7+CCL3 cells.

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Abstract

提供了一种工程化免疫细胞，其表达(i)嵌合受体，和(ii)外源性的CCL3、CCL4和/或CCL5，具有提高的肿瘤杀伤活性，可用于治疗癌症、感染或自身免疫性疾病。

Description

工程化免疫细胞及其用途 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(i)嵌合受体，和(ii)外源性的CCL3、CCL4和/或CCL5基因。更优选地，所述嵌合受体是嵌合抗原受体或T细胞受体。
背景技术
肿瘤免疫治疗主要是通过调节人体免疫系统和肿瘤微环境，最终依靠自身免疫来清除肿瘤细胞。免疫系统是一个统一的整体，固有免疫在肿瘤免疫中也起到十分重要的作用。
一些抗原递呈细胞，如树突状细胞及巨噬细胞是固有免疫和获得性免疫的连接桥梁。抗原递呈细胞可以对肿瘤抗原进行识别并递呈给获得性免疫系统，激活肿瘤特异性T细胞，进而对肿瘤进行清除。因而通过增强抗原递呈过程来增加免疫系统杀伤肿瘤的效果是肿瘤免疫的重要研究方向。
CAR细胞治疗是重要的肿瘤细胞免疫疗法。CAR细胞成功控制肿瘤一般而言需要经过以下几个过程：免疫系统激活、CAR细胞的活化和扩增、活化的CAR细胞浸润肿瘤组织并杀死肿瘤细胞。然而，目前CAR细胞疗法普遍存在一个问题，即肿瘤微环境对CAR细胞有抑制作用，使得CAR细胞无法浸润肿瘤组织。因此，如何降低肿瘤微环境对CAR细胞的抑制作用，提高CAR细胞的存活时间，或者招募其他免疫细胞与CAR细胞协同作用，对于提高CAR细胞的治疗效果非常重要。
传统1型树突状细胞(conventional DC1，cDC1)是树突状细胞的一类亚群，是呈递肿瘤抗原的主要免疫细胞。研究结果显示，cDC1可以有效递呈肿瘤相关抗原，尤其是坏死细胞相关抗原，有效诱导抗原特异性CD8+T细胞应答，在体内肿瘤杀伤过程中发挥极其重要的作用。在小鼠及人的研究中均显示，cDC1在肿瘤微环境中的分布与抗肿瘤免疫应答呈正相关，是一个重要的肿瘤相关免疫评分的评价参数。cDC1在小鼠及人体内分布较少，在肿瘤免疫应答率低的小鼠及人肿瘤微环境中几乎不可 见。优化cDC1在肿瘤治疗中的作用是提高肿瘤免疫治疗效果的一个重要研究方向。
炎症趋化因子在招募相关免疫细胞进入肿瘤微环境、驱动细胞相互作用和分子信号级联反应等方面起着重要作用，决定着宿主抗肿瘤免疫反应的最终结果。研究表明，巨噬细胞炎性蛋白(MIP)-1家族及相关蛋白，由CCL3(MIP-1a)、CCL4(MIP-1b)和CCL5(RANTES)组成，可通过直接招募抗原呈递细胞(例如招募树突状细胞到肿瘤部位)而成为某些肿瘤免疫细胞浸润的主要决定因素。另外据报道，肿瘤部位富集的MIP-1家族相关蛋白可以通过募集自然杀伤细胞(NK)至肿瘤部位，进而促进CD103 +cDC在肿瘤部位的积累，并增加趋化因子CXCL9和CXCL10的产生，有效抑制肿瘤进展。
因此，需要一种新的免疫治疗手段，可以有效分化或招募cDC1树突状细胞，以提高肿瘤抗原呈递效率，诱导机体过继性免疫反应，从而提高CAR细胞治疗的疗效。
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(i)嵌合受体，和(ii)外源性的CCL3、CCL4和/或CCL5基因。
在一个实施方案中，CCL3基因与SEQ ID NO：77或79所示的核酸序列具有至少90％同一性，或者所述CCL3基因编码的多肽与SEQ ID NO：78或80所示的氨基酸序列具有至少90％同一性。
在一个实施方案中，CCL4基因与SEQ ID NO：81或83所示的核酸序列具有至少90％同一性，或者所述CCL4基因编码的多肽与SEQ ID NO：82或84所示的氨基酸序列具有至少90％同一性。
在一个实施方案中，CCL5基因与SEQ ID NO：85或87所示的核酸序列具有至少90％同一性，或者所述CCL5基因编码的多肽与SEQ ID NO：86或88所示的氨基酸序列具有至少90％同一性。
在一个实施方案中，所述工程化免疫细胞进一步表达(iii)外源性的白细胞介素。
在一个实施方案中，所述白细胞介素是IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、IL33、或其亚基、或其组合、或其亚基的组合，优选是IL-7。
在一个实施方案中，其中所述白细胞介素的编码基因与SEQ ID NO：41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75所示的核酸 序列具有至少90％同一性，或者所述白细胞介素与SEQ ID NO：42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76所示的氨基酸序列具有至少90％同一性。
在一个实施方案中，所述工程化免疫细胞表达的白细胞介素、CCL4或CCL5是可以抵抗蛋白酶水解的融合蛋白或者突变体。
在一个实施方案中，所述嵌合受体是嵌合抗原受体或T细胞受体。优选地，所述嵌合受体是嵌合抗原受体，其包含配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。其中，配体结合结构域可以选自免疫球蛋白分子、Fab、Fab'、F(ab')2、Fv片段、scFv、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、由VH和CH1结构域组成的Fd片段、线性抗体、单结构域抗体、纳米抗体，以及非免疫球蛋白抗原结合支架
在一个实施方案中，所述嵌合受体与选自以下的靶标结合：TSHR、CD19、CD123、CD22、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、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、豆荚蛋白、HPV E6、E7、MAGE Al、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、前列腺特异性蛋白、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、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、Cyclin Bl、MYCN、RhoC、TRP-2、CYP1B1、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和它们的任意组合。优选地，所述靶标选自CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、MUC1、AFP、Folate受体α、CEA、PSCA、PSMA、Her2、EGFR、IL13Ra2、GD2、NKG2D、EGFRvIII、CS1、BCMA、间皮素和它们的任意组合。
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，所述胞内信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。优选地，所述胞内信号传导结构域是包含CD3ζ的信号传导结构域。
在一个实施方案中，所述共刺激结构域是一个或多个选自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、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、CD94、LTB、ZAP70以及它们的组合。优选地，所述共刺激结构域是CD27、CD28、CD134、CD137或CD278的共刺激信号传导结构域或它们的组合。
在一个实施方案中，外源性的白细胞介素、CCL3、CCL4和/或CCL5的表达或活性是组成型表达。在另一个实施方案中，外源性的白细胞介素、CCL3、CCL4和/或CCL5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，白细胞介素、CCL3、CCL4和/或CCL5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、细胞质中的特定细胞器例如内质网、高尔基体、细胞核等。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白细胞介素、CCL3、CCL4和/或CCL5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，所述免疫细胞是选自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、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，免疫细胞衍生自干细胞，例如成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等。
在第二个方面，本发明提供一种核酸分子，(i)编码嵌合受体的核酸序列，和(ii)编码CCL3、CCL4和/或CCL5的核酸序列。优选地，所述嵌合受体是嵌合抗原受体或T细胞受体，更优选嵌合抗原受体。
在一个实施方案中，核酸分子进一步包含编码白细胞介素的核酸序列。优选地，所述白细胞介素是IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、IL33、或其亚基、或其组合、或其亚基的组合，更优选是IL-7、其亚基或其组合。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，本发明还提供一种试剂盒，其包含本发明所述的工程化免疫细胞、核酸分子或载体。
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、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)、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、间皮瘤、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、Waldenstrom巨球蛋白血症、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤，例如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)、套细胞淋巴瘤、AIDS相关淋巴瘤、伯基特氏淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、MALT淋巴瘤、边缘区淋巴瘤、浆母细胞性淋巴瘤、浆细胞样树突状细胞瘤等)、白血病(包括急性白血病，例如急性淋巴细胞白血病、急性髓细胞白血病、急性非淋巴细胞白血病诸如急性粒细胞白血病(包括未分化型和部分分化型)、急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、红白血病、急性巨核细胞白血病；慢性白血病，例如慢性髓细胞白血病、慢性淋巴细胞白血病、慢性单核细胞白血病；和其他特殊类型的白血病例如毛细胞白血病、幼淋巴细胞白血病、浆细胞白血病、成人T细胞白血病、嗜酸性粒细胞白血病、嗜碱性粒细胞白血病等)、母细胞性浆细胞样树突状细胞瘤、恶性淋巴组织增生疾病、骨髓发育不良、多发性骨髓瘤、骨髓增生异常、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。
本发明的工程化免疫细胞的优势之处在于，共表达的CCL3、CCL4和/或CCL5以及任选的白细胞介素例如IL7能够有效募集DC细胞至肿瘤部位，增加工程化免疫细胞的增殖及存活时间，从而一方面降低肿瘤微环境对工程化免疫细胞的抑制作用， 提高工程化免疫细胞的肿瘤杀伤能力，另一方面募集的DC细胞能够激活机体自身T细胞的过继性免疫识别，与工程化免疫细胞形成协同效应，最终增强对肿瘤的抑制。
附图说明
图1：Panc02-mCD19细胞的CD19表达率。
图2：通过流式细胞术测定的CAR-T细胞的CAR表达水平。
图3：通过ELISA测定的CAR-T细胞的IL-7表达水平。
图4：通过ELISA测定的CAR-T细胞的CCL4表达水平。
图5：通过ELISA测定的CAR-T细胞的CCL5表达水平。
图6：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7：用表达CCL4或CCL5的CAR-T细胞治疗小鼠胰腺癌后，小鼠的体重变化曲线。
图8：用表达CCL4或CCL5的CAR-T细胞治疗小鼠胰腺癌后，小鼠的肿瘤生长曲线。
图9：用表达CCL3的CAR-T细胞治疗小鼠胰腺癌后，小鼠的体重变化曲线。
图10.用表达CCL3的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。
嵌合受体
如本文所用，术语“嵌合受体”是指在细胞表面表达的能够与靶分子(例如配体)特异性结合的分子。此类分子一般包含能够与配体特异性结合的配体结合结构域、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。常见的此类嵌合受体的实例包括例如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的可变区可以包含配体结合结构域或与配体结合结构域可操作连接，其中配体结合结构域的定义如下所述。
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配体结合结构域(例如抗体或抗体片段)、跨膜结构域、共刺激结构域和细胞内信号传导结构域，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，“配体结合结构域”是指可以与配体(例如抗原)结合的任何结构或其功能性变体。配体结合结构域可以是抗体结构，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。配体结合结构域的实例包括但不限于免疫球蛋白分子、Fab、Fab'、F(ab')2、Fv片段、scFv、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、由VH和CH1结构域组成的Fd片段、线性抗体、单结构域抗体、纳米抗体、以及非免疫球蛋白抗原结合支架，例如重组纤连蛋白结构域、DARPIN、亲合体、affilin、adnectin、affitin、obodies、repebody、fynomer、alpha body、avimer、atrimer、centyrin、pronectin、anticalin、kunitz型结构域、Armadillo重复蛋白等。优选地，配体结合结构域选自Fab、scFv、单结构域抗体、纳米抗体，或其功能性片段。在本发明中，配体结合结构域可以是单价或二价，且可以是包含一个或多个配体结合结构域的单特异性、双特异性或多特异性的抗体。
“Fab”是指免疫球蛋白分子被木瓜蛋白酶裂解后产生的两个相同片段中的任一个，由通过二硫键连接的完整轻链和重链N端部分组成，其中重链N端部分包括重链可变区和CH1。与完整的IgG相比，Fab没有Fc片段，流动性和组织穿透能力较高，并且无需介导抗体效应即可单价结合抗原。
“单链抗体”或“scFv”是由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。可以选择接头的最佳长度和/或氨基酸组成。接头的长度会明 显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人,1993Proc Natl Acad.Sci.U.S.A.90:6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。
“单结构域抗体”或“sdAb”是指一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，也称为“重链抗体”。
“纳米抗体”或“Nb”是指单独克隆并表达出来的VHH结构，其具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是目前已知的可结合目标抗原的最小单位。
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。
配体结合结构域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，本发明的配体结合结构域与选自以下的一个或多个靶标结合：TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gplOO、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、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、豆荚蛋白、HPV E6、E7、MAGE Al、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、前列腺特异性蛋白、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、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、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和它们的任意组合。优选地，所述靶标选自：CD19、CD20、CD22、BAFF-R、CD33、 EGFRvIII、BCMA、GPRC5D、PSMA、ROR1、FAP、ERBB2(Her2/neu)、MUC1、EGFR、CAIX、WT1、NY-ESO-1、CD79a、CD79b、GPC3、Claudin18.2、NKG2D和它们的任意组合。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配体结合结构域。例如，如果CD19是待靶向的抗原，则CD19抗体可用作本发明的配体结合结构域。
在一个实施方案中，本发明的嵌合抗原受体靶向CD19、CD22或其组合。在一个优选的实施方案中，本发明的嵌合抗原受体包含抗CD19抗体，其包含与SEQ ID NO：1第1-107位或SEQ ID NO：25第1-107位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％、99％或100％序列同一性的轻链可变区序列和与SEQ ID NO：1第123-242位或SEQ ID NO：25第123-238位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％、99％或100％序列同一性的重链可变区序列。在一个优选的实施方案中，本发明的嵌合抗原受体包含抗CD22抗体，其包含与SEQ ID NO：27第1-124位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％、99％或100％序列同一性的重链可变区序列和与SEQ ID NO：27第143-249位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％、99％或100％序列同一性的轻链可变区序列。
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CD8α链或CD28，其与SEQ ID NO:3、5或30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，或其编码序列与SEQ ID NO:4、6或29所示的核苷酸序列具有至少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配体结合结构域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配体结合结构域的任何寡肽或多肽。具体地，铰链区用来为配体结合结构域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、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CD8α链、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选来自CD8α、CD28或IgG4的铰链，其与SEQ ID NO:19、21、23或36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，或者其编码序列与SEQ ID NO:20、22、24或35所示的核苷酸序列具有至少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。胞内信号传导结构域负责在配体结合结构域结合抗原以后的细胞内初级信号传递，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的胞内信号传导结构域可以是T细胞受体和共受体的细胞质序列，其在抗原受体结合以后一同起作用以引发初级信号传导，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。胞内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)。本发明的胞内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的信号传导结构域可以包含CD3ζ信号传导结构域，该信号传导结构域与SEQ ID NO:11、13或34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，或其编码序列与SEQ ID NO:12、14或33所示的核苷酸序列具 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共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、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、CD94、LTB以及ZAP70。优选地，本发明CAR的共刺激结构域来自4-1BB、CD28、CD27、OX40或其组合。在一个实施方案中，本发明的CAR包含的共刺激结构域与SEQ ID NO:7或9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，或该共刺激结构域的编码序列与SEQ ID NO:8或10所示的核苷酸序列具有至少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CD8α、IgG1、GM-CSFRα、B2M等的信号肽。在一个实施方案中，可用于本发明的信号肽与SEQ ID NO:15、17或40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，或该信号肽的编码序列与SEQ ID NO:16、18或39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。
CC趋化因子
趋化因子根据其N端半胱氨酸的排列方式，可分为CXC、CC、C和CX3C四个亚族，其中CC趋化因子因含有两个相邻的半胱氨酸-半胱氨酸(cys-cys或C-C)残基而得名。CC趋化因子的实例是与CCR1和CCR5结合的那些，包括MIP-1α、MIP-1β、CCL5、MCP-1、MCP-2、MCP-3、I-309等。
巨噬细胞炎性蛋白(MIP)具有两种主要的形式：α(MIP-1α，也称为CCL3)和β(MIP-1β，也称为CCL4),最先分离自由脂多醣所活化的巨噬细胞培养液。这两者具有相似的序列和活性，主要由巨噬细胞、单核细胞和树突状细胞等产生，均通过与细胞表面的趋化因子受体5(CCR5)的结合来参与对炎症和感染的免疫应答。已有研究表明，CCL3和CCL4参与肺结核等许多传染病的宿主防御反应，介导免疫细胞的定向迁移。
CCL5也称为RANTES，是一种分子量为8000的小分子蛋白,人CCL5蛋白的基 因定位于17q11-21。CLL5以单体、二聚体、多聚体等多种形式存在。CCL5主要表达于巨噬细胞、活化的T细胞、成纤维细胞、胶质细胞、上皮细胞、内皮细胞、血小板和特定的肿瘤细胞,对多种白细胞如单核细胞、嗜酸性粒细胞、嗜碱性粒细胞、T淋巴细胞、自然杀伤细胞等具有趋化或刺激作用。
CCL3、CCL4和CCL5均与CCR5结合，从而募集免疫细胞，例如未成熟的骨髓树突细胞、单核细胞、巨噬细胞、Th1、Treg、NK和浆细胞样树突细胞等定向迁移至炎性部位或肿瘤部位。
在一个实施方案中，本发明的工程化免疫细胞表达(i)嵌合受体,和(ii)外源性的CCL3、CCL4和/或CCL5基因。更优选地，本发明的工程化免疫细胞表达(i)嵌合抗原受体,和(ii)外源性的CCL4和/或CCL5基因。
在一个实施方案中，本发明使用的CCL3与SEQ ID NO：78或80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CCL3的编码序列与SEQ ID NO：77或79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。
在一个实施方案中，本发明使用的CCL4与SEQ ID NO：82或84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CCL4的编码序列与SEQ ID NO：81或83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。
在一个实施方案中，本发明使用的CCL5与SEQ ID NO：86或88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CCL5的编码序列与SEQ ID NO：85或87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。
白细胞介素
白细胞介素是由白细胞产生，且在白细胞间发挥功能的一类细胞因子，在传递信息，激活与调节免疫细胞，介导T、B细胞活化、增殖与分化及在炎症反应中起重要作用。一般而言，白细胞介素的生物学效应通过其与相应受体的结合来实现，例如 IL-7的生物学特性通过IL-7与其受体IL-7R的结合来实现。
在一个实施方案中，可用于本发明的白细胞介素包括但不限于IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、IL33、或其亚基、或其组合、或其亚基的组合，更优选IL-7或其亚基。IL-2主要由T细胞产生，通过自分泌和旁分泌的方式发挥作用。IL-2不仅能维持T细胞生长，促进细胞因子的产生，而且能够诱导CD8+T细胞和CD4+T细胞发挥细胞毒性作用。此外，IL-2还能够刺激NK细胞增殖，增强NK杀伤活性，促进NK细胞产生IFNγ、TNFβ、TGFβ等因子，以及激活巨噬细胞，增强巨噬细胞的抗原呈递能力和靶细胞杀伤能力。IL-7主要由骨髓及胸腺基质细胞产生，其主要功能涉及以下几个方面：促进前体B细胞生长；抑制外周T细胞凋亡，诱导细胞增殖、持续存活；以及影响树突状细胞、巨噬细胞的发育和功能，诱导巨噬细胞分泌多种细胞因子。IL-12主要作用于T细胞和NK细胞。具体而言，IL-12可以刺激活化型T细胞增殖，促进Th0细胞向Th1细胞分化；诱导CTL和NK细胞的细胞毒性活性并促进其分泌IFNγ、TNFα、GMCSF等细胞因子；促进NK细胞和IL-2Rα、TNF受体以及CD56的表达，增强对肿瘤细胞的ADCC效应。IL-15可由活化的巨噬细胞、表皮细胞和呈现为细胞等多种细胞产生。由于IL-15的分子结构与IL-2比较相似，可以利用IL-2R的β链和γ链与靶细胞结合，发挥与IL-2类似的生物学活性，例如刺激T细胞和NK细胞的增殖，诱导B细胞增殖和分化等。IL-21由活化的CD4+T细胞、NKT细胞、Tfh细胞和Th17细胞产生，与IL-2、IL-15均具有较高的同源性。IL-21具有广泛的免疫调节功能，激活它可以增强活化型CD8+T细胞的增殖，增强NK细胞的细胞毒性活性，促进B细胞的增殖与分化。IL-17由Th17细胞分泌，为促炎性分子，能够抑制调节性T细胞，并且招募和激活中性粒细胞和巨噬细胞。IL-18同样为促炎性分子，由T细胞和NK细胞分泌，激活的巨噬细胞也会分泌大量IL-18。有报道显示，IL-18在固有免疫和过继性免疫过程中发挥重要作用。IL-23同样由T细胞和NK细胞分泌，IL-23可以促进Th17细胞分化。树突状细胞、单核细胞、巨噬细胞同样表达低水平IL-23受体，可被IL-23激活。IL-33的序列与IL-18相似，其通过IL-1受体辅助蛋白IL-RAcP与受体ST2结合，三者形成异二聚体，激活NF-κB、MAPK等信号通路，强烈诱导前炎性因子和趋化因子的产生，从而发挥生物学效应。IL-33在多种细胞中表达，包括上皮细胞、纤维母细胞、巨噬细胞、树突状细胞等。研究表明，IL-33可以刺激未成熟的树突状细胞产生Treg，促进Th1细胞分泌IFN- γ，增加CD69的表达，从而激活NK、NKT细胞和CD8+T细胞，促进抗肿瘤免疫。研究表明，这些白细胞介素单独或组合(例如IL-33和IL-12、IL-7+IL-12、IL-7+IL15等)能够发挥有效的抗肿瘤作用。
在一个实施方案中，本发明使用的白细胞介素与SEQ ID NO：42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76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，或其编码序列与SEQ ID NO：41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75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。
外源基因的表达
本发明中的外源性基因，例如白细胞介素、CCL3、CCL4、CCL5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，外源性的白细胞介素、CCL3、CCL4、CCL5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，白细胞介素、CCL3、CCL4、CCL5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、细胞质中的特定细胞器例如内质网、高尔基体、细胞核等。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 源性基因白细胞介素、CCL3、CCL4、CCL5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，例如白细胞介素、CCL3、CCL4、CCL5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
本发明还提供一种核酸分子，其包含(i)编码嵌合受体的核酸序列，和(ii)编码CCL3、CCL4和/或CCL5的核酸序列。
在一个实施方案中，核酸分子进一步包含(iii)编码白细胞介素的核酸序列。优选地，所述白细胞介素包括但不限于IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、IL33、或其亚基、或其组合、或其亚基的组合，更优选IL-7或其亚基。
在一个实施方案中，所述嵌合受体是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。
核酸可以包含常规的磷酸二酯键或非常规的键(如酰胺键，比如在肽核酸(PNA)中发现的)。本发明的核酸还可含有一种或多种经修饰的碱基，比如，例如三苯甲基化的碱基和不常见的碱基(比如肌苷)。也可以想到其它修饰，包括化学、酶促或代谢修饰，只要本发明的多链CAR可以从多核苷酸表达即可。核酸可以以分离的形式提供。在一个实施方案中，核酸也可以包括调节序列，比如转录控制元件(包括启动子、增强子、操纵子、抑制子和转录终止信号)、核糖体结合位点、内含子等。
可以对本发明的核酸序列进行密码子优化以在所需的宿主细胞(如，免疫细胞)中进行最佳表达；或者用于在细菌、酵母菌或昆虫细胞中表达。密码子优化是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。
载体
本发明还提供一种载体，包含如本发明所述的核酸。其中，编码嵌合受体的核酸序列、任选的编码IL-7的核酸序列、编码CCL3的核酸、CCL4的核酸和/或编码CCL5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。
工程化免疫细胞及其制备方法
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嵌合受体(例如嵌合抗原受体)、CCL3、CCL4和/或CCL5基因，以及任选的白细胞介素，例如IL-7。
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，或者是衍生自干细胞，例如成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等的免疫细胞。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)、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。在本发明中，免疫细胞被工程化以表达嵌合抗原受体以及外源性的CCL3、CCL4和/或CCL5基因，以及任选的白细胞介素，例如IL-7。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以将编码嵌合受体(例如嵌合抗原受体)多肽的核酸序列以及CCL3、CCL4和/或CCL5基因，以及任选的白细胞介素基因，例如IL-7引入免疫细胞。“转染”是将核酸分子或多核苷酸(包括载体)引入靶细胞的过程。一个例子是RNA转染，即将RNA(比如体外转录的RNA，ivtRNA)引入宿主细胞的过程。该术语主要用于真核细胞中的非病毒方法。术语“转导”通常用于描述病毒介导的核酸分子或多核苷酸的转移。动物细胞的转染通常涉及在细胞膜中打开瞬时的孔或“洞”，以允许摄取材料。可以使用磷酸钙、通过电穿孔、通过细胞挤压或通过将阳离子脂质与材料混合以产生与细胞膜融合并将它们的运载物沉积入内部的脂质体，进行转染。用于转染真核宿主细胞的示例性技术包括脂质囊泡介导的摄取、热休克介导的摄取、磷酸钙介导的转染(磷酸钙/DNA共沉淀)、显微注射和电穿孔。术语“转化”用于描述核酸分子或多核苷酸(包括载体)向细菌中、也向非动物真核细胞(包括植物细胞)中的非病毒转移。因此，转化是细菌或非动物真核细胞的基因改变，其通过细胞膜从其周围直接摄取并随后并入外源遗传材料(核酸分子)而产生。转化可以通过人工手段实现。为了发生转化，细胞或细菌必须处于感受态的状态。对于原核转化，技术可包括热休克介导的摄取、与完整细胞的细菌原生质体融合、显微注射和电穿孔。
因此，本发明还提供一种制备工程化免疫细胞的方法，包括将以下引入所述免疫细胞：(a)编码嵌合受体的第一核酸序列或其编码的嵌合受体；b)编码CCL3、CCL4和/或CCL5的第二核酸序列或其编码的CCL3、CCL4和/或CCL5蛋白；和任选的(c) 编码白细胞介素的第三核酸序列或其编码的白细胞介素。
在一个实施方案中，可以将上述组分(a)、(b)和(c)以任何顺序先后依次引入免疫细胞。在另一个实施方案中，可以将上述组分(a)、(b)和(c)同时引入免疫细胞，例如将(a)、(b)和(c)克隆在一个或多个载体中。
将核酸或载体引入免疫细胞后，本领域技术人员可以通过常规技术对所得免疫细胞进行扩增和活化。
在一个实施方案中，本发明的工程化免疫细胞还可以包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK、TCR/CD3基因(例如TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ)、MHC相关基因(HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA)和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的。例如，可以使用反义RNA、RNA诱饵、RNA适体、siRNA、shRNA/miRNA、反式显性阴性蛋白(TNP)、嵌合/融合蛋白、趋化因子配体、抗感染性细胞蛋白、细胞内抗体(sFv)、核苷类似物(NRTI)、非核苷类似物(NNRTI)、整合酶抑制剂(寡核苷酸、二核苷酸和化学剂)和蛋白酶抑制剂来抑制基因的表达。另外，也可以通过例如大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂，从而使基因沉默。
试剂盒和药物组合物
本发明该提供一种试剂盒，其包含本发明的工程化免疫细胞、核酸分子或载体。
在一个优选的实施方案中，本发明的试剂盒还包含说明书。
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，本发明还涵盖所述核酸分子、载体、工程化免疫细胞以及药物组合物在制备治疗癌症、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，直接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。
在另一个实施方案中，本发明的治疗方法是离体治疗。具体地，该方法包括以下步骤：(a)提供样品，所述样品包含免疫细胞；(b)在体外将本发明的嵌合受体(例如嵌合抗原受体)以及待表达的外源性基因引入所述免疫细胞，获得经修饰的免疫细胞，(c)向有此需要的受试者施用所述经修饰的免疫细胞。优选地，步骤(a)中提供的免疫细胞选自巨噬细胞、树突状细胞、单核细胞、T细胞、NK细胞和/或NKT细胞；并且所述免疫细胞可以通过本领域已知的常规方法从受试者的样品(特别是血液样品)中获得。然而，也可以使用能够表达本发明的嵌合抗原受体和外源性基因并发挥如本文所述的所需生物效应功能的其它免疫细胞。此外，通常选择的免疫细胞与受试者的免疫系统相容，即优选所述免疫细胞不引发免疫原性响应。例如，可以使用“通用接受体细胞”，即发挥所需生物效应功能的普遍相容的可在体外生长和扩增的淋巴细胞。使用此类细胞将不需要获得和/或提供受试者自身淋巴细胞。步骤(c)的离体引入可以通过经由电穿孔将本文所述的核酸或载体引入免疫细胞或通过用病毒载体感染免疫细胞来实施，所述病毒载体为如前所述的慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体。其它可想到的方法包括使用转染试剂(比如脂质体)或瞬时RNA转染。
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，所述癌症是与配体结合结构域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)、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 构建小鼠胰腺癌细胞系Panc02-mCD19
1.制备pLV-mCD19质粒
以小鼠脾脏总mRNA为模板，通过反转录PCR得到小鼠脾脏总cDNA序列后，再通过PCR获得含有XbaI及SalI酶切位点的小鼠mCD19序列。然后，将mCD19基因重组至pLVX载体(PPL，Cat No.:PPL00157-4a)中，得到pLV-mCD19质粒。
2.慢病毒包装
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。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)、34μg pLV-mCD19质粒、8.5μg pMD2.G载体(Addgene，货号12259)和17μg psPAX2载体(Addgene，货号12260)。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24小时和48小时收集培养物，合并后超速离心(25000g，4℃，2.5h)，获得浓缩的pLV-mCD19慢病毒，将其保存于-80℃。
3.筛选Panc02-mCD19细胞系
在6孔细胞培养板的每个孔中，加入含10％胎牛血清的RPMI-1640培养基(Gibco，货号C12430500BT)、1×10 6个小鼠胰腺癌细胞Panc02(来自中国药科大学实验室馈赠)和200μl pLV-mCD19慢病毒，于37℃、5％CO 2条件下孵育48h。然后用0.25％胰酶将细胞消化成单细胞悬液，并将细胞稀释后转移至96孔板中继续培养，直至出现单克隆细胞。挑选单克隆细胞，再次用0.25％胰酶消化成单细胞，并用200μl opti-MEM培养基重悬细胞。用APC anti-mouse CD19抗体(Biolegend，货号115512)，通过流式细胞术检测感染效率，并筛选出CD19阳性克隆。将阳性克隆传代3-4次后，再用流式细胞术检测CD19表达水平。未经病毒感染的Panc02细胞用作对照。
结果如图1所示。最后筛选的Panc02-mCD19细胞系中，CD19表达率为100％。
实施例2.制备CAR-T细胞
1.构建逆转录病毒质粒
人工合成依次连接的CD19-scFv(SEQ ID NO：26)、CD8a铰链区及跨膜区(SEQ ID NO：35和29)、41bb胞内域(SEQ ID NO：31)和CD3ζ胞内域(SEQ ID NO：33)的编码序列片段，并在两端加入XhoI/EcoRI酶切位点。将片段克隆入MSCV载体，获得MSCV-mCD19-CAR质粒。
人工合成依次连接T2A(SEQ ID NO：37)和IL-7(SEQ ID NO：43)的编码序列片段，并在两端加入EcoRI/SalI酶切位点。将片段克隆入MSCV-mCD19-CAR载体，获得MSCV-mCD19-CAR-IL-7质粒。
人工合成依次连接T2A(SEQ ID NO：37)和CCL4(SEQ ID NO：83)的编码序列片段，并在两端加入EcoRI/SalI酶切位点。将片段克隆入MSCV-mCD19-CAR载体，获得MSCV-mCD19-CAR-CCL4质粒。
人工合成依次连接T2A(SEQ ID NO：37)和CCL5(SEQ ID NO：87)的编码序列片段，并在两端加入EcoRI/SalI酶切位点。将片段克隆入MSCV-mCD19-CAR载体，获得MSCV-mCD19-CAR-CCL5质粒。
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逆转录病毒质粒(MSCV-mCD19-CAR质粒、MSCV-mCD19-CAR-IL-7质粒、MSCV-mCD19-CAR-CCL4质粒或MSCV-mCD19-CAR-CCL5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％CO2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。
3.制备CAR-T细胞
从小鼠脾脏分离T淋巴细胞，并用DynaBeads CD3/CD28CTS 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，然后分别加入500μL完全培养基(NT，对照)、MSCV-mCD19-CAR病毒、MSCV-mCD19-CAR-CCL4病毒、MSCV-mCD19-CAR-CCL5病毒、MSCV-mCD19-CAR-IL-7病毒+MSCV-mCD19-CAR-CCL4病毒，或MSCV-mCD19-CAR-IL-7病毒+MSCV-mCD19-CAR-CCL5病毒，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NT细胞、mCD19-CAR细胞、mCD19-CAR+CCL4细胞、mCD19-CAR+CCL5细胞、mCD19-CAR+IL7+CCL4细胞、和mCD19-CAR+IL7+CCL5细胞。
实施例3.检测CAR-T细胞的表达
1.细胞表面CAR的表达水平
取出实施例2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2所示。
可以看出，与对照相比，mCD19-CAR细胞、mCD19-CAR+CCL4细胞、 mCD19-CAR+CCL5细胞、mCD19-CAR+IL7+CCL4细胞、和mCD19-CAR+IL7+CCL5细胞中的CAR阳性效率均为60％左右，表明这些细胞均可有效表达CAR。
2.IL-7的表达水平
收集实施例2制备的CAR-T细胞的上清液，根据制造商的建议，用Mouse IL-7DuoSet ELISA kit试剂盒(R&D Systems,货号DY407)检测细胞中的IL-7分泌水平，结果如图3所示。
可以看出，用MSCV-mCD19-CAR-IL-7病毒转染的两种CAR T细胞均可有效表达IL-7。
3.CCL4的表达水平
收集实施例2制备的CAR-T细胞的上清液，根据制造商的建议，用Mouse CCL4 DuoSet ELISA kit试剂盒(R&D Systems,货号DY451)检测细胞中的CCL4分泌水平，结果如图4所示。
可以看出，用MSCV-mCD19-CAR-CCL4病毒转染的两种CAR T细胞可有效表达CCL4。
4.CCL5的表达水平
收集实施例2制备的CAR-T细胞的上清液，根据制造商的建议，用Mouse CCL5 DuoSet ELISA kit试剂盒(R&D Systems,货号DY478)检测细胞中的CCL5分泌水平，结果如图5所示。
可以看出，用MSCV-mCD19-CAR-CCL5病毒转染的两种CAR T细胞可有效表达CCL5。
实施例4.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、mCD19-CAR细胞、mCD19-CAR+IL-7细胞、mCD19-CAR+CCL4细胞、mCD19-CAR+CCL5细胞、mCD19-CAR+IL7+CCL4细胞、和mCD19-CAR+IL7+CCL5细胞。然后在各孔中以1×10 4个细胞/100μl的浓度分别加入靶标Panc02-mCD19细胞或非靶标Panc02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6所示。可以看出，在非靶细胞Panc02中均没有检测到IFN-γ的释放，且NT细胞不表达IFN-γ，表明本实施例中的CAR T细胞的杀伤都是特异性的。 并且，在杀伤靶细胞时，与仅表达CAR的T细胞相比，额外表达单独的CCL4或CCL5降低了IFN-γ的释放水平，而表达IL-7+CCL4或IL-7+CCL5则增加了IFN-γ的释放水平。
实施例5.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个实施例1制备的Panc02-mCD19胰腺癌细胞。
将接种了胰腺癌细胞的小鼠随机分为6组，每组5只。待肿瘤体积生长至100mm 3时，向每只小鼠经尾静脉注射1×10 6个实施例2制备的NT细胞、mCD19-CAR细胞、mCD19-CAR+CCL4细胞、mCD19-CAR+CCL5细胞、mCD19-CAR+IL7+CCL4细胞和mCD19-CAR+IL7+CCL5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7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8所示。可以看出，与NT细胞和常规CAR-T细胞相比，仅表达CCL4或CCL5的CAR-T细胞能增强抗肿瘤效果，表明单独的CCL4或CCL5可与CAR-T细胞产生协同作用。此外，发明人还出乎意料地发现，进一步表达IL-7可以显著增加CCL4对CAR-T细胞的促进作用，从而更显著地抑制肿瘤生长。与此相比，IL-7对于CCL5的促进作用低于对CCL4的作用，但IL-7+CCL5+CAR的组合对肿瘤的抑制效果仍然略微优于单独的CAR。
以上结果表明，共表达CCL4、CCL5或其与IL-7的组合能够有效增强表达CAR的工程化免疫细胞对靶标胰腺癌细胞的抑制效果。
实施例6.表达CCL3的CAR-T细胞的肿瘤抑制效果验证
根据实施例2所述的方法制备表达CCL3的CAR-T细胞，其中将T2A(SEQ ID NO：37)和CCL3(SEQ ID NO：79)的编码序列片段克隆入MSCV-mCD19-CAR载体，获得MSCV-mCD19-CAR-CCL3质粒并将其包装为逆转录病毒。用该病毒转染激活的T细胞，获得mCD19-CAR+CCL3细胞。用MSCV-mCD19-CAR-CCL3病毒和MSCV-mCD19-CAR-IL-7病毒同时转染激活的T细胞，获得mCD19-CAR+IL7+CCL3细胞。
根据实施例5所述的方法验证CAR-T细胞对肿瘤的体内抑制效果。图9示出了小鼠的体重变化，表明施用CAR-T细胞不会对小鼠有明显的毒副反应。图10示出了小鼠的肿瘤体积变化。可以看出，单独的CCL3可与CAR-T细胞产生协同作用，而进一步表达IL7则可以显著增加CCL3对CAR-T细胞的促进作用，从而更显著地抑制肿瘤生长。
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 (25)

  1. 一种工程化免疫细胞，其表达(i)嵌合受体，和(ii)外源性的CCL3、CCL4和/或CCL5基因。
  2. 权利要求1所述的工程化免疫细胞，其中所述CCL3基因与SEQ ID NO：77或79所示的核酸序列具有至少90％同一性，或者所述CCL3基因编码的多肽与SEQ ID NO：78或80所示的氨基酸序列具有至少90％同一性；CCL4基因与SEQ ID NO：81或83所示的核酸序列具有至少90％同一性，或者所述CCL4基因编码的多肽与SEQ ID NO：82或84所示的氨基酸序列具有至少90％同一性；CCL5基因与SEQ ID NO：85或87所示的核酸序列具有至少90％同一性，或者所述CCL5基因编码的多肽与SEQ ID NO：86或88所示的氨基酸序列具有至少90％同一性。
  3. 权利要求1-2任一项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(iii)外源性的白细胞介素。
  4. 权利要求3所述的工程化免疫细胞，其中所述白细胞介素是IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、IL-33、或其亚基、或其组合、或其亚基的组合。
  5. 权利要求4所述的工程化免疫细胞，其中所述白细胞介素的编码基因与SEQ ID NO：41、43、45、47、49、51、53、55、57、59、61、63、65、67、69、71、73、75所示的核酸序列具有至少90％同一性，或者所述白细胞介素与SEQ ID NO：42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76所示的氨基酸序列具有至少90％同一性。
  6. 权利要求1-5任一项所述的工程化免疫细胞，其中所述嵌合受体是嵌合抗原受体或T细胞受体。
  7. 权利要求6所述的工程化免疫细胞，其中所述嵌合受体是包含以下的嵌合抗原受体：配体结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。
  8. 权利要求7所述的工程化免疫细胞，其中所述配体结合结构域选自免疫球蛋白分子、Fab、Fab'、F(ab')2、Fv片段、scFv、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH)或轻链可变区(VL)、由VH和CH1结构域组成的 Fd片段、线性抗体、单结构域抗体、纳米抗体，以及非免疫球蛋白抗原结合支架。
  9. 权利要求7或8所述的工程化免疫细胞，其中所述配体结合结构域与选自以下的靶标结合：TSHR、CD19、CD123、CD22、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-l lRa、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-I受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GMl、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD 179a、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、豆荚蛋白、HPV E6、E7、MAGE Al、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、前列腺特异性蛋白、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、ERG(TMPRSS2 ETS融合基因)、NA17、PAX3、雄激素受体、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和它们的任意组合。
  10. 权利要求7-9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。
  11. 权利要求7-10任一项所述的工程化免疫细胞，其中所述胞内信号传导结构域选自以下蛋白的信号传导结构域：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  12. 权利要求7-11任一项所述的工程化免疫细胞，其中所述共刺激结构域是一个或多个选自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、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、CD94、LTB、ZAP70以及它们的组合。
  13. 权利要求1-12任一项所述的工程化免疫细胞，其中所述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、PD1、CTLA-4、LAG3和TIM3。
  14. 权利要求1-13任一项所述的工程化免疫细胞，其中所述CCL3、CCL4和/或CCL5基因的表达是条件型表达。
  15. 权利要求1-13任一项所述的工程化免疫细胞，其中所述CCL3、CCL4和/或CCL5基因与定位结构域可操作连接。
  16. 权利要求1-15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。
  17. 权利要求16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、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。
  18. 权利要求1-17所述的工程化免疫细胞，其中所述免疫细胞衍生自成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞。
  19. 一种核酸分子，其包含：(i)编码嵌合受体的核酸序列，和(ii)编码CCL3、CCL4和/或CCL5的核酸序列。
  20. 权利要求19所述的核酸分子，其中所述嵌合受体是嵌合抗原受体。
  21. 权利要求19所述的核酸分子，其进一步包含编码白细胞介素的核酸序 列。
  22. 权利要求21所述的核酸分子，其中所述白细胞介素是IL-2、IL-7、IL-12、IL-15、IL-21、IL-17、IL-18、IL-23、IL33、其亚基、或其组合、或其亚基的组合。
  23. 一种载体，其包含权利要求19-22任一项所述的核酸分子。
  24. 一种药物组合物，其包含权利要求1-18任一项所述的工程化免疫细胞、权利要求19-22任一项所述的核酸分子或权利要求23所述的载体，和一种多种药学上可接受的赋型剂。
  25. 权利要求1-18任一项所述的工程化免疫细胞、权利要求19-22任一项所述的核酸分子、权利要求23所述的载体、或权利要求24所述的药物组合物在制备治疗癌症、感染或自身免疫性疾病的药物中的用途。
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