WO2021169977A1 - 新型嵌合抗原受体及其用途 - Google Patents

新型嵌合抗原受体及其用途 Download PDF

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WO2021169977A1
WO2021169977A1 PCT/CN2021/077580 CN2021077580W WO2021169977A1 WO 2021169977 A1 WO2021169977 A1 WO 2021169977A1 CN 2021077580 W CN2021077580 W CN 2021077580W WO 2021169977 A1 WO2021169977 A1 WO 2021169977A1
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cancer
cells
cell
domain
chimeric antigen
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PCT/CN2021/077580
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French (fr)
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周亚丽
吴长顺
姜小燕
陈功
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南京北恒生物科技有限公司
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Priority to CA3169610A priority Critical patent/CA3169610A1/en
Priority to EP21760732.4A priority patent/EP4083073A4/en
Priority to KR1020227032920A priority patent/KR20220145374A/ko
Priority to JP2022549898A priority patent/JP7462777B2/ja
Priority to AU2021225890A priority patent/AU2021225890A1/en
Publication of WO2021169977A1 publication Critical patent/WO2021169977A1/zh

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Definitions

  • the present invention relates to the field of cellular immunotherapy, and in particular to a novel chimeric antigen receptor containing a cytokine receptor universal ⁇ chain (also called ⁇ c chain) or its intracellular region and its use.
  • a novel chimeric antigen receptor containing a cytokine receptor universal ⁇ chain also called ⁇ c chain
  • CAR-T cell immunotherapy is to genetically modify T cells in vitro so that they can recognize tumor antigens, and after being amplified to a certain number, they are returned to the patient to kill cancer cells, thereby achieving the purpose of treating tumors.
  • the intracellular signaling domain of the first-generation CAR only contains the primary signaling domain, such as CD3 ⁇ , so CAR-carrying cells (such as CAR-T cells) have poor activity and short survival time in vivo.
  • the second-generation CAR introduces a costimulatory domain, such as CD28 or 4-1BB, so that cells can continue to proliferate and enhance anti-tumor activity.
  • the third-generation CAR contains two costimulatory domains (such as CD28+4-1BB), and the fourth-generation CAR adds cytokines or costimulatory ligands to further enhance T cell responses, or adds suicide genes when needed. Make CAR-T cells self-destruct.
  • the second-generation CAR structure is still mostly used in clinical research.
  • CAR-T cell therapy still has some problems in clinical application. For example, there are a large number of tumor recurrences in the treatment of hematoma, and the response rate in the treatment of solid tumors is not high. These may be caused by the complex tumor microenvironment, CAR -Caused by factors such as T cell exhaustion.
  • the present invention provides a novel chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, and an additional signaling domain, wherein the additional The signal transduction region is composed of the ⁇ c chain or its intracellular region.
  • the amino acid sequence of the ⁇ c chain is shown in SEQ ID NO: 14; the amino acid sequence of its intracellular region is shown in SEQ ID NO: 16.
  • the costimulatory domain, intracellular signal transduction domain, and additional signal transduction region are arranged in order from the closest to the farthest distance from the cell membrane.
  • the antigen binding region is selected from scFv, Fab, single domain antibody, nanobody, antigen binding ligand, recombinant fibronectin domain, anticalin and DARPIN.
  • the antigen binding region is selected from scFv, Fab, single domain antibody and Nanobody.
  • the antigen binding region is selected from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, and chimeric antibodies.
  • the target bound by the antigen binding region is selected from: 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-1 Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24 , PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Claudin 18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GMl
  • the target is selected from 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, Claudin 18.2, NKG2D and any combination of them.
  • 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 signaling domain comprising CD3 ⁇ .
  • the costimulatory domain is one or more costimulatory signaling domains selected 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, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.
  • the costimulatory domain is a costimulatory signal transduction domain of CD27, CD28, CD134, CD137 or CD278.
  • the present invention also provides a nucleic acid comprising a sequence encoding the chimeric antigen receptor of the present invention, a vector comprising the nucleic acid, and an immune cell comprising the nucleic acid or the vector.
  • the invention provides a nucleic acid comprising a sequence encoding the chimeric antigen receptor of the invention.
  • the nucleic acid is DNA or RNA, more preferably mRNA.
  • the present invention provides a vector comprising the aforementioned nucleic acid.
  • the vector is selected from linear nucleic acid molecules, plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV), bacteriophages, bacteriophages Granules, cosmids or artificial chromosomes.
  • the vector also includes an origin for autonomous replication in immune cells, a selection marker, a restriction enzyme cleavage site, a promoter, a polyadenylic acid tail (polyA), 3'UTR, 5'UTR, enhanced Element, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag.
  • the vector is an in vitro transcribed vector.
  • the present invention provides an immune cell comprising the nucleic acid or vector of the present invention, which is capable of expressing the chimeric antigen receptor of the present invention.
  • the immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells.
  • the T cells are CD4+/CD8+ double positive T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells or ⁇ -T cells.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor of the present invention or its encoding nucleic acid, vector or immune cell containing them as defined above, and one or more pharmaceutically acceptable Of excipients.
  • the present invention provides a method for treating a subject suffering from cancer, comprising administering to the subject an effective amount of the chimeric antigen receptor, immune cell or drug combination according to the present invention Things.
  • the cancer is selected from: blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma , Colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatocellular tumor, intraepithelial tumor, Kidney cancer, laryngeal cancer, leukemia, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, Respiratory system cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testi
  • the disease that can be treated with the chimeric antigen receptor, nucleic acid, vector, immune cell or pharmaceutical composition of the present invention is selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer Wait.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide.
  • the basic structure of the hybrid polypeptide includes an antigen-binding region (for example, the antigen-binding portion of an antibody), a transmembrane domain, Co-stimulatory domain and intracellular signaling domain.
  • CAR can use the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner.
  • Non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens unrelated to antigen processing, thus bypassing the main mechanism of tumor escape.
  • the CAR when expressed in T cells, the CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).
  • TCR T cell receptor
  • the novel chimeric antigen receptor of the present invention includes, in addition to the basic structures of the antigen binding domain, transmembrane domain, costimulatory domain and intracellular signal transduction domain, it also includes an additional ⁇ c chain or its intracellular region. Signal conduction area.
  • antigen binding region refers to any structure or functional variant thereof that can bind to an antigen.
  • the antigen binding region can be an antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and functional fragments thereof.
  • the antigen binding region includes but is not limited to Fab, single chain antibody (Single Chain Antibody Fragment, scFv), single domain antibody (Single Domain Antibody, sdAb), Nanobody (Nanobody, Nb), antigen binding ligand, recombinant fiber
  • the zonulin domain, anticalin and DARPIN, etc. are preferably selected from Fab, scFv, sdAb and Nanobody.
  • the antigen binding region can be monovalent or bivalent, and can be a monospecific, bispecific or multispecific antibody.
  • the antigen binding region can also be a specific binding polypeptide or receptor structure of a specific protein, such as PD1, PDL1, PDL2, TGF ⁇ , APRIL, and NKG2D.
  • Fab refers to any of the two identical fragments produced by papain cleavage of immunoglobulin molecules, consisting of a complete light chain and a heavy chain N-terminal part connected by disulfide bonds, wherein the heavy chain N-terminal part includes Heavy chain variable region and CH1. Compared with intact IgG, Fab has no Fc fragment, has higher fluidity and tissue penetration, and can bind to antigen monovalently without mediating antibody effects.
  • a “single chain antibody” or “scFv” is an antibody in which the variable region of the heavy chain (VH) of the antibody and the variable region of the light chain (VL) are connected by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker will significantly affect the folding and interaction of the variable region of scFv. In fact, if a shorter linker (for example, between 5-10 amino acids) is used, intra-chain folding can be prevented.
  • a shorter linker for example, between 5-10 amino acids
  • intra-chain folding can be prevented.
  • the size and composition of the linker see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90: 6444-6448; U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication Nos. WO2006/020258 and WO2007/024715, the entire contents
  • Single domain antibody or “sdAb” refers to an antibody that naturally lacks the light chain.
  • the antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as the “heavy chain”.
  • VHH heavy chain variable region
  • CH2 and CH3 regions also known as the "heavy chain”.
  • Nemobody or “Nb” refers to a separately cloned and expressed VHH structure, which has structural stability and antigen binding activity equivalent to that of the original heavy chain antibody, and is the smallest unit currently known to bind the target antigen .
  • the term "functional variant” or “functional fragment” refers to a variant that essentially contains the amino acid sequence of the 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 variant retains the biological activity of the parent amino acid sequence.
  • the amino acid modification is preferably a conservative modification.
  • conservative modification refers to an amino acid modification that does not significantly affect or change 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 receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain.
  • Amino acid residue families with similar side chains have been defined in the art, including basic side chains (such as lysine, arginine, histidine), acidic side chains (such as 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), ⁇ -branched side chains (e.g.
  • basic side chains such as lysine, arginine, histidine
  • acidic side chains such as aspartic acid, glutamic acid
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • non-polar side chains e
  • amino acids involved threonine, valine, isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine.
  • Conservative modifications can be selected, for example, based on polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity in the amphipathic properties of the residues involved.
  • a “functional variant” or “functional fragment” has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.
  • sequence identity refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is usually expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Therefore, two copies with exactly the same sequence have 100% identity.
  • Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.
  • the antigen binding region of the present invention binds to one or more targets selected from: 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-1 Ra, PSCA, PRSS21, VEGFR2 LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAI
  • the target is selected from: 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, Claudin 18.2, NKG2D and any combination thereof.
  • the CAR of the present invention can be designed to include an antigen binding region specific for the antigen.
  • a CD19 antibody can be used as the antigen binding region of the present invention.
  • transmembrane domain refers to a polypeptide that enables chimeric antigen receptors to be expressed on the surface of immune cells (such as lymphocytes, NK cells, or NKT cells), and guides immune cells to respond to target cells. structure.
  • the transmembrane domain can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. When the chimeric receptor polypeptide binds to the target antigen, the transmembrane domain is capable of signal transduction.
  • Transmembrane domains particularly suitable for use in the present invention can 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 mainly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from a human CD8 ⁇ chain, which has at least 70%, preferably at least 80%, of the amino acid sequence shown in SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 3 , More preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antigen binding region and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen binding region.
  • the hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region may be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from the constant region of an antibody.
  • 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 the hinge region of CD8 ⁇ chain, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably CD8 ⁇ hinge, which is the same as the amino acid sequence shown in SEQ ID NO: 12 or the same as SEQ ID NO:
  • the nucleotide sequence shown in 11 has 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 the cell to perform a specified function.
  • the intracellular signal transduction domain is responsible for the primary signal transmission in the cell after the antigen binding region binds to the antigen, which leads to the activation of immune cells and immune response.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of immune cells in which CAR is expressed.
  • the effector function of T cells may be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • the intracellular signaling domain contained in the chimeric antigen receptor of the present invention may be the cytoplasmic sequence of the T cell receptor and the co-receptor, which act together to trigger the primary signal after the antigen receptor is bound. Conduction, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions.
  • the intracellular signal transduction domain can contain many immunoreceptor tyrosine activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM).
  • Non-limiting examples of intracellular signaling domains of the present invention include, but are not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the signal transduction domain of the CAR of the present invention may include the CD3 ⁇ signal transduction domain, which is in accordance with the amino acid sequence shown in SEQ ID NO: 8 or the nucleus shown in SEQ ID NO: 7
  • the nucleotide sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor of the invention comprises one or more costimulatory domains.
  • the costimulatory domain may be an intracellular functional signaling domain derived from a costimulatory molecule, which includes the entire intracellular part of the costimulatory molecule, or a functional fragment thereof.
  • a "costimulatory molecule” refers to a homologous binding partner that specifically binds to a costimulatory ligand on T cells, thereby mediating a costimulatory response (for example, proliferation) of T cells.
  • Co-stimulatory molecules include, but are not limited to, Class 1 MHC molecules, BTLA and Toll ligand receptors.
  • Non-limiting examples of costimulatory domains of the present 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, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.
  • costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7
  • the costimulatory domain of the CAR of the present invention is a 4-1BB and/or CD28 fragment, and more preferably has at least 70% of the amino acid sequence shown in SEQ ID NO: 6 or the nucleotide sequence shown in SEQ ID NO: 5 %, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor of the present invention in addition to the costimulatory domain and the intracellular signal transduction domain for signal transduction, also contains at least one additional signal transduction region consisting of a ⁇ c chain Or its intracellular region.
  • the intracellular region (that is, the structure for signal transduction) of the chimeric antigen receptor of the present invention is composed of three types of signal transduction: the costimulatory domain, the intracellular signal transduction domain, and the ⁇ c chain or its intracellular region. Structure and composition.
  • the chimeric antigen receptor of the present invention does not contain the fourth signal transduction structure, such as the signal transduction regions of other cytokines, such as IL-2Ra, IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra, etc. Inner zone.
  • the fourth signal transduction structure such as the signal transduction regions of other cytokines, such as IL-2Ra, IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra, etc. Inner zone.
  • ⁇ c chain refers to the ⁇ chain shared by the receptors of cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.
  • the ⁇ c chain was initially identified in the IL-2 receptor, and it was later discovered that it is also involved in the composition of receptors such as IL-4, IL-7, IL-9, IL-15, and IL-21, so it is also called the universal ⁇ chain .
  • IL-2R ⁇ the IL-2 receptor
  • ⁇ chain also called IL-2R ⁇
  • ⁇ c chain also called IL-2R ⁇ or IL-2Rg
  • the IL-2 receptor comprising all three chains has the highest affinity for IL-2 cytokine.
  • the three chains of IL-2 receptor are anchored on the cell membrane, and the biochemical signal is transmitted to the cell through the binding with IL-2.
  • the specific components of cytokine receptors such as IL-2R ⁇ , IL-4R ⁇ , IL-7R ⁇ , IL-9R ⁇ , IL-21R, etc., are responsible for binding to JAK1, while the ⁇ c chain is responsible for binding to JAK1.
  • cytokine receptors When these cytokine receptors bind to cytokines, three main signal pathways are activated, including MAP kinase, PI3 kinase and JAK-STAT pathway, which in turn regulate the survival and proliferation of T cells and NK cells.
  • ⁇ c chain is a glycoprotein with a molecular weight of 64kD, composed of 347 amino acids, including an extracellular region of 232 amino acids, a transmembrane region of 29 amino acids, and an intracellular region of 86 amino acids.
  • the intracellular region contains the Src homologous region, which is essential to promote cell growth and IL-2 mediated expression of c-myc, c-fos, c-jun and other genes.
  • the ⁇ c chain that can be used in the present invention has at least 70%, preferably at least 80%, more preferably at least the amino acid sequence shown in SEQ ID NO: 14 or the nucleotide sequence shown in SEQ ID NO: 13 90%, 95%, 97% or 99% or 100% sequence identity.
  • the ⁇ c chain intracellular region that can be used in the present invention has at least 70%, preferably at least 80%, with the amino acid sequence shown in SEQ ID NO: 16 or the nucleotide sequence shown in SEQ ID NO: 15, More preferred is at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the ⁇ c chain of the present invention is composed of SEQ ID NO: 14, and its intracellular region is composed of SEQ ID NO: 16.
  • the CAR of the present invention may also contain a signal peptide so that when it is expressed in a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids, which has a tendency to form a single ⁇ -helix.
  • At the end of the signal peptide there is usually an amino acid segment that is recognized and cleaved by signal peptidase.
  • Signal peptidase can cleave during or after translocation to produce free signal peptide and mature protein. Then, the free signal peptide is digested by a specific protease.
  • the signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the chimeric antigen receptor of the present invention includes a CD8 ⁇ transmembrane domain, a 4-1BB costimulatory domain, a CD3 ⁇ signaling domain, and a ⁇ c chain or its intracellular region. More preferably, the chimeric antigen receptor further comprises a CD8 ⁇ signal peptide, a CD8 ⁇ hinge region and/or a CD28 costimulatory domain.
  • the costimulatory domain, intracellular signal transduction domain and additional signal transduction domain are arranged in order from the closest to the farthest distance from the cell membrane, that is, the co-stimulatory domain, the intracellular signal transduction domain and the additional signal transduction region
  • the stimulus domain is closest to the cell membrane, and the additional signal transduction zone is the farthest from the cell membrane.
  • the present invention also provides a nucleic acid comprising a sequence encoding the chimeric antigen receptor of the present invention.
  • nucleic acid includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each of which is linear or circular in single-stranded and/or double-stranded form , Or their mixtures (including hybrid molecules). Therefore, the nucleic acid according to the present invention includes DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.
  • Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as those found in peptide nucleic acids (PNA)).
  • the nucleic acid of the present invention may also contain one or more modified bases, such as, for example, trityl bases and unusual bases (such as inosine). Other modifications are also conceivable, including chemical, enzymatic or metabolic modifications, as long as the multi-chain CAR of the present invention can be expressed from polynucleotides.
  • the nucleic acid can be provided in an isolated form.
  • the nucleic acid may also include regulatory sequences, such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.
  • the nucleic acid sequence of the present invention can be codon-optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacteria, yeast, or insect cells.
  • Codon optimization refers to the replacement of codons that are generally rare in the highly expressed genes of a given species in the target sequence with codons that are generally common in the highly expressed genes of such species, and the codons before and after the replacement Code the same amino acid. Therefore, the choice of the best codon depends on the codon usage preference of the host genome.
  • the present invention also provides a vector comprising one or more nucleic acids as described in the present invention.
  • vector is a nucleic acid molecule used as a vehicle for transferring (exogenous) genetic material into a host cell, where the nucleic acid molecule can be replicated and/or expressed, for example.
  • Targeting vector is a medium that delivers an isolated nucleic acid to the inside of a cell by, for example, homologous recombination or using a hybrid recombinase that specifically targets the 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 polypeptide of the present invention) in a suitable host cell and the translation of their mRNA. Suitable vectors that can be used in the present invention are known in the art, and many are commercially available.
  • the vector of the present invention includes, but is not limited to, linear nucleic acid molecules (e.g.
  • DNA or RNA DNA or RNA
  • plasmids viruses
  • viruses e.g. retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV, multiple Oncovirus and adeno-associated virus (AAV), etc.
  • phage phagemid
  • cosmid and artificial chromosome including BAC and YAC
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgene) And the larger sequence as the "backbone" of the vector.
  • the engineered vector usually also contains a starting point for autonomous replication in the host cell (if stable expression of the polynucleotide is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site) , MCS).
  • the vector may additionally include a promoter, polyadenylic acid tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification Elements such as tags, etc.
  • the vector is an in vitro transcribed vector.
  • the present invention provides engineered immune cells, which comprise chimeric antigen receptors or nucleic acid encoding them.
  • 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 may be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells.
  • the immune cells are T cells.
  • the T cell may be any T cell, such as a T cell cultured in vitro, such as a primary T cell, or a T cell derived from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or a T cell obtained from a subject.
  • T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be any type of T cells and can be at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxicity) T cells), tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • Various techniques known to those skilled in the art, such as Ficoll isolation can be used to obtain T cells from the blood of the subject.
  • immune cells are engineered to express chimeric antigen receptor polypeptides.
  • the nucleic acid sequence encoding the chimeric antigen receptor polypeptide can be introduced into immune cells by conventional methods known in the art (such as by transduction, transfection, transformation, etc.) to express the chimeric antigen receptor polypeptide of the present invention.
  • Transfection is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells.
  • RNA transfection the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells.
  • the term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells usually involves opening transient holes 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 produce liposomes that fuse with cell membranes and deposit their cargoes 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 and non-animal eukaryotic cells (including plant cells). Therefore, transformation is a genetic modification of bacteria or non-animal eukaryotic cells, 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. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation.
  • the immune cell of the present invention further comprises at least one inactivating gene selected from the group consisting of CD52, GR, TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ , HLA-I, HLA-II genes , Immune checkpoint genes such as PD1 and CTLA-4. More specifically, at least TCR ⁇ or TCR ⁇ genes in immune cells are inactivated. This inactivation renders the TCR non-functional in the cell. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD).
  • GvHD graft-versus-host disease
  • DNA fragmentation is mediated by meganuclease, zinc finger nuclease, TALE nuclease, or Cas enzyme in the CRISPR system, thereby inactivating the gene.
  • the present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor, nucleic acid, carrier or engineered immune cell of the present invention as an active agent, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the chimeric antigen receptor, nucleic acid, vector or engineered immune cell in the preparation of pharmaceutical compositions or medicines.
  • the term "pharmaceutically acceptable excipient” refers to pharmacologically and/or physiologically compatible with the subject and the active ingredient (that is, capable of eliciting the desired therapeutic effect without causing any undesirable effects).
  • the carriers and/or excipients for the desired local or systemic effects are well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical composition of the present invention.
  • Exemplary excipients used in the pharmaceutical composition of the present invention include saline, buffered saline, dextrose, and water.
  • suitable excipients depends inter alia on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.
  • composition according to the present invention can be applied to various routes of administration. Usually, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.
  • the pharmaceutical composition according to the present invention can also be prepared into various forms, such as solid, liquid, gaseous or lyophilized form, especially ointment, cream, transdermal patch, gel, powder, tablet, solution, gas In the form of spray, granule, pill, suspension, emulsion, capsule, syrup, elixir, extract, tincture, or liquid extract extract, or a form particularly suitable for the desired method of administration.
  • the processes known in the present invention for the production of drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or freeze-drying processes.
  • Pharmaceutical compositions containing immune cells such as those described herein are usually provided in the form of a solution, and preferably contain a pharmaceutically acceptable buffer.
  • the pharmaceutical composition according to the present invention can 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 the combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , Gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, Austria Auristatin E (auristatin E), vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth
  • the present invention also provides a method for preparing engineered immune cells, which includes introducing the chimeric antigen receptor of the present invention or its encoding nucleic acid sequence into immune cells, so that the immune cells express the chimeric antigen receptor of the present invention.
  • the immune cells are human immune cells, more preferably human T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells.
  • nucleic acids or vectors into immune cells and expressing them are known in the art.
  • the nucleic acid or vector can be introduced into immune cells by physical methods, such as calcium phosphate precipitation method, lipofection method, particle bombardment method, microinjection method, electroporation method, etc.
  • chemical methods can also be used, such as through colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids
  • the body introduces the nucleic acid or vector.
  • biological methods can also be used to introduce nucleic acids or vectors.
  • viral vectors especially retroviral vectors
  • retroviral vectors have become the most common method for inserting genes into mammalian, such as human cells.
  • Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus.
  • nucleic acid or vector After the nucleic acid or vector is introduced into the immune cells, those skilled in the art can amplify and activate the obtained immune cells by conventional techniques.
  • the present invention also provides a method for treating a subject suffering from cancer, which comprises administering to the subject an effective amount of the immune cell or the pharmaceutical composition of the present invention.
  • an effective amount of the immune cells and/or pharmaceutical composition of the present invention is directly administered to the subject.
  • the treatment method of the present invention is ex vivo treatment.
  • the method includes the following steps: (a) providing a sample of the subject, the sample containing immune cells; (b) introducing the chimeric antigen receptor of the present invention into the immune cells in vitro to obtain modified Immune cells, (c) administering the modified immune cells to a subject in need thereof.
  • the immune cells provided in step (a) are selected from T cells, NK cells and/or NKT cells; and the immune cells can be obtained from a sample of a subject (especially a blood sample) by conventional methods known in the art. ).
  • other immune cells capable of expressing the chimeric antigen receptor of the present invention and exerting the desired biological effect function as described herein can also be used.
  • step (c) can be carried out by introducing the nucleic acid or vector described herein into immune cells via electroporation or by infecting immune cells with a viral vector, the viral vector being the aforementioned lentiviral vector, adenoma Viral vector, adeno-associated virus vector or retroviral vector.
  • transfection reagents such as liposomes
  • 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 a different patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some cases, the genetic differences of allogeneic materials from individual individuals of the same species may be sufficient for antigenic interaction to occur.
  • the term "subject" is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer.
  • the subject is a human.
  • the disease is cancer associated with the expression of the target bound by the antigen binding region.
  • the cancer includes, but is not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , Choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, glandular lung cancer, and squamous lung cancer), lymphoma (including Hodgkin's lymphoma and Non-Hodgkin's lymphoma),
  • the disease that can be treated with the engineered immune cell or pharmaceutical composition of the present invention is selected from the group consisting of leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer and the like.
  • the method further comprises administering one or more additional chemotherapeutic agents, biological agents, drugs, or treatments to the subject.
  • the chemotherapeutic agent, biological agent, drug or treatment is selected from radiotherapy, surgery, antibody agents and/or small molecules and any combination thereof.
  • Figure 1 CAR expression level of CAR-T cells measured by flow cytometry.
  • Figure 2 The killing effect of CAR-T cells on target cells. Two-way ANOVA was used for analysis, and T test was used for statistical analysis. * Indicates that the P value is less than 0.05, reaching a significant level.
  • Figure 3 The release levels of IL-2 (A) and IFN ⁇ (B) after CAR-T cells were co-cultured with target cells and non-target cells.
  • FIG. 5 On day D21, the expansion levels of CD3+ (A), CD8+ (B) and CD4+ (C) T cells in mice.
  • Figure 7 The killing effect of CAR-T cells on target cells. Two-way ANOVA was used for analysis, and T test was used for statistical analysis. * Indicates that the P value is less than 0.05, reaching a significant level.
  • SEQ ID NO describe SEQ ID NO: 1 The nucleotide sequence of CD19-scFv
  • the T cells used in all the examples of the present invention are primary human CD4+CD8+T cells isolated from healthy donors by Ficoll-PaqueTM PREMIUM (GE Healthcare, article number 17-5442-02) using leukocyte separation.
  • Nalm6 tumor cells were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.
  • CD8 ⁇ signal peptide SEQ ID NO: 9
  • anti-CD19scFv SEQ ID NO: 1
  • CD8 ⁇ hinge region SEQ ID NO: 11
  • CD8 ⁇ transmembrane region SEQ ID NO: 3
  • 4-1BB costimulatory domain SEQ ID NO: 5
  • CD3 ⁇ intracellular signaling domain SEQ ID NO: 7
  • the only difference between the bbzg-CAR plasmid and the bbz-CAR plasmid is that it also includes a gamma chain intracellular region (SEQ ID NO: 15) connected to the CD3 ⁇ intracellular signal transduction domain.
  • SEQ ID NO: 15 a gamma chain intracellular region connected to the CD3 ⁇ intracellular signal transduction domain.
  • the 4-1BB costimulatory domain, the CD3 ⁇ intracellular signal transduction domain and the ⁇ chain intracellular region are arranged in order from the closest to the farthest distance from the cell membrane.
  • Opti-MEM After adding 3ml Opti-MEM (Gibco, article number 31985-070) to the sterile tube to dilute the above plasmid, add the packaging vector psPAX2 (Addgene, Product number 12260) and the envelope vector pMD2.G (Addgene, product number 12259). Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, catalog number 0636236601), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid/vector/transfection reagent mixture dropwise to the 293T cell culture flask . The virus was collected at 24 hours and 48 hours, and after combining them, ultracentrifugation (25000 g, 4°C, 2.5 hours) was used to obtain concentrated lentivirus.
  • T cells were activated with DynaBeads CD3/CD28CTSTM (Gibco, catalog number 40203D), and cultured at 37°C and 5% CO 2 for 1 day. Then, the concentrated lentivirus was added, and after continuous culture for 3 days, traditional con-CAR T cells targeting CD19 (used as a control) and the bbzg-CAR T cells of the present invention were obtained.
  • the bbzg-CAR T cell of the present invention can effectively express scFv, and its expression level is slightly higher than that of the traditional bbz-CAR T cell, indicating that the addition of the ⁇ chain intracellular region will not affect the surface expression of the CAR structure.
  • Example 2 The killing effect of CAR T cells on target cells and the release of cytokines
  • T cells kill target cells the number of target cells will decrease.
  • target cells that can express luciferase the number of target cells decreases, and the secreted luciferase also decreases.
  • Luciferase can catalyze the conversion of luciferin to oxidized luciferin, and during this oxidation process, bioluminescence will be produced, and the intensity of this luminescence will depend on the level of luciferase expressed by the target cell. Therefore, the detected fluorescence intensity can reflect the killing ability of T cells to target cells.
  • Nalm6 target cells carrying the fluorescein gene were first spread into a 96-well plate at 1 ⁇ 10 4 /well, and then an effective target ratio of 32:1 (ie effector T cells and Target cell ratio) Spread bbzg-CAR T cells, Con-CAR T cells (positive control) and untransfected T cells (negative control) into a 96-well plate for co-cultivation, and use a microplate reader after 16-18 hours Determine the fluorescence value. According to the calculation formula: (target cell fluorescence average value-sample fluorescence average value)/target cell fluorescence average value ⁇ 100%, the killing efficiency is calculated, and the result is shown in FIG. 2.
  • the bbzg-CAR T cells of the present invention have a significantly higher killing effect on target cells than traditional bbz-CAR T cells.
  • ELISA enzyme-linked immunosorbent assay
  • the reaction was allowed to occur in the dark at room temperature for 30 minutes, and then 50 ⁇ L of 1 mol/L H 2 SO 4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, use a microplate reader to detect the absorbance at 450 nm, and calculate the cytokine content according to the standard curve (drawn according to the reading and concentration of the standard). The result is shown in Figure 3.
  • the release of IFN ⁇ was not detected in the non-target cells 293F, indicating that the killing of bbz-CAR T cells and bbzg-CAR T cells is specific.
  • the IL2 release level of bbzg-CAR T cells is significantly lower than that of traditional CAR T cells, but the release level of IFN- ⁇ is significantly higher than that of traditional CAR T cells.
  • the cytokine release of the bbzg-CAR T cells of the present invention is comparable to that of traditional CAR-T cells.
  • mice Twenty 8-week-old healthy female NCG mice were divided into four groups: PBS group, NT group (negative control), bbz-CART group (positive control) and bbzg-CAR T group.
  • PBS group On day 0 (D0), 1 ⁇ 10 6 Nalm6 cells were injected into the tail vein of each mouse.
  • D7 Seven days later (D7), PBS solution or 2 ⁇ 10 6 NT cells, con-CAR T cells or bbzg-CAR T cells were injected into the tail vein of each mouse according to the grouping situation. The survival rate and tumor burden of the mice were evaluated weekly.
  • the in vivo optical imaging technology of living animals was used to evaluate the changes in tumor burden of each group of mice.
  • the tumor burden of mice was detected on D7, D14, D21, D28, D35, D42, D49 and expressed in Photons/s. The results are shown in Figure 4.
  • mice in the PBS and NT groups progressed rapidly, reaching the highest value at D21 (and then died).
  • the tumor burden decreased rapidly, but gradually rebounded on D28 or D35.
  • mice in the bbzg-CAR T group not only decreased their tumor burden rapidly after treatment, but also maintained a low level until D49 without recurrence. This indicates that the bbzg-CAR T cell of the present invention can effectively inhibit tumor growth, and the effect is significantly better than that of the traditional bbz-CAR T cell.
  • the inventor also monitored the expansion of T cells in the two groups of mice treated with bbz-CART cells and bbzg-CAR T cells on the 21st day. Specifically, blood was taken from the submandibular vein of the mouse on D21, and Trucount FACS analysis (hCD3, hCD8, hCD4 expression levels) was performed, and the results are shown in FIG. 5.
  • T cell expansion was detected in both the bbz-CAR T group and the bbzg-CAR T group.
  • the expansion of CD4+ T cells in the two groups was similar, the expansion of CD3+ and CD8+ T cells in the bbzg-CAR group T was significantly higher than that in the bbz-CAR T group. Therefore, although the tumor burden on the 21st day of the two groups was similar ( Figure 4), the bbzg-CAR T group had significantly more T cells proliferated, making it possible to keep the tumor burden at a low level. On the contrary, due to less expansion of T cells and continuous depletion, the tumor burden of the bbz-CAR T group mice rebounded afterwards.
  • mice in each group were compared to the survival percentage of mice in each group as of the end of the experiment (ie, 105 days after inoculation with tumor cells Nalm6) ( Figure 6).
  • Figure 6 the survival percentage of mice in each group as of the end of the experiment.
  • all the mice in the PBS and NT groups died, and only one (20%) of the mice treated with bbz-CAR T cells survived, while the mice treated with bbzg-CAR T cells still survived 60%.
  • the bbzg-CAR T cells of the present invention can effectively inhibit tumors and improve the survival rate.
  • the bbzg-CAR T cells of the present invention can greatly promote the expansion of T cells due to the introduction of the cytokine receptor universal gamma chain, thereby improving the continuous killing of tumor cells. Effect, improve the tumor suppression effect in the body and increase the survival of mice.
  • the ⁇ c chain intracellular region (SEQ ID NO: 65) was inserted between the 4-1BB costimulatory domain and the CD3 ⁇ primary signaling domain of the bbz-CAR plasmid to obtain the bbgz-CAR plasmid, which is similar to the bbzg-CAR plasmid. The only difference lies in the position of the intracellular region of the ⁇ c chain. According to the method of Example 1, bbgz-CAR T cells were prepared.
  • the killing effect of CAR-T cells on target cells was detected according to the method described in Example 2, and the results are shown in FIG. 7. It can be seen that the killing effect of bbgz-CAR T cells on target cells is equivalent to that of traditional bbz-CAR T cells, but both are significantly lower than the killing effect of bbzg-CAR T cells. This indicates that the position of the additional signal transduction region (ie, the ⁇ c chain or its intracellular region) in the CAR structure has an important impact on the killing activity of CAR T cells.

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Abstract

本发明提供一种新型的嵌合抗原受体，其包含抗原结合区、跨膜结构域、共刺激结构域、胞内信号传导结构域和额外信号传导区，其中所述额外信号传导区由γc链或其胞内区组成。本发明还提供包含本发明的新型嵌合抗原受体的工程化免疫细胞及其药物组合物，以及所述工程化免疫细胞/药物组合物在治疗癌症中的用途。

Description

新型嵌合抗原受体及其用途 技术领域
本发明涉及细胞免疫治疗领域，尤其涉及包含细胞因子受体通用γ链(也称为γc链)或其胞内区的新型嵌合抗原受体及其用途。
背景技术
近几年，癌症免疫治疗技术发展迅速，尤其是嵌合抗原受体T细胞(CAR-T)相关的免疫疗法在血液瘤的治疗上获得了优异的临床效果。CAR-T细胞免疫疗法是将T细胞在体外进行基因改造，使其能够识别肿瘤抗原，在扩增到一定数量后回输至病人体内，进行癌细胞杀伤，从而达到治疗肿瘤的目的。
目前，随着技术的发展，已经出现了四代不同的CAR结构。第一代CAR的胞内信号传导结构域仅包含初级信号传导结构域，例如CD3ζ，因此携带CAR的细胞(例如CAR-T细胞)活性差，体内存活时间短。第二代CAR引入了共刺激结构域，例如CD28或4-1BB，使得细胞能够持续增殖，增强抗肿瘤活性。第三代CAR则包含两个共刺激结构域(例如CD28+4-1BB)，第四代CAR则加入了细胞因子或共刺激配体以进一步增强T细胞应答，或加入自杀基因以在需要时使CAR-T细胞自我毁灭。现在临床研究中大多使用的仍然是第二代CAR结构。
然而，CAR-T细胞疗法在临床应用中仍然存在一些问题，例如在血液瘤治疗中存在大量肿瘤复发现象，在实体瘤治疗中应答率不高等等，这些可能是由复杂的肿瘤微环境、CAR-T细胞耗竭等因素造成。
因此，仍然需要对现有的CART细胞疗法进行改进，以促进CAR-T细胞在体内的增殖，抵抗肿瘤微环境的免疫抑制作用，进而提高CAR-T细胞疗法对肿瘤的整体治疗效果。
发明概述
在第一个方面，本发明提供一种新型嵌合抗原受体，其包含抗原结合区、跨膜结构域、共刺激结构域、胞内信号传导结构域和额外信号传导区，其中所述额外信号传导区由γc链或其胞内区组成。在一个优选的实施方案中，γc链的氨基酸序列如SEQ ID NO： 14所示；其胞内区的氨基酸序列如SEQ ID NO：16所示。
在一个实施方案中，所述共刺激结构域、胞内信号传导结构域和额外信号传导区按照与细胞膜的距离从近到远依次排列。
在一个实施方案中，所述抗原结合区选自scFv、Fab、单结构域抗体、纳米抗体、抗原结合配体、重组纤连蛋白结构域、anticalin和DARPIN。优选地，所述抗原结合区选自scFv、Fab、单结构域抗体和纳米抗体。
在一个实施方案中，所述抗原结合区选自单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体和嵌合抗体。
在一个实施方案中，所述抗原结合区结合的靶标选自：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、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、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、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。
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、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。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。
在第二个方面，本发明还提供包含编码本发明的嵌合抗原受体的序列的核酸、包含所述核酸的载体、以及包含所述核酸或载体的免疫细胞。
在一个实施方案中，本发明提供一种核酸，其包含编码本发明的嵌合抗原受体的序列。优选地，所述核酸是DNA或RNA，更优选mRNA。
在一个实施方案中，本发明提供包含上述核酸的载体。具体地，所述载体选自线性核酸分子、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)、噬菌体、噬菌粒、粘粒或人工染色体。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，本发明提供包含本发明的核酸或载体的免疫细胞，其能够表达本发明的嵌合抗原受体。在一个具体的实施方案中，所述免疫细胞选自T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞或NKT细胞。优选地，所述T细胞是CD4+/CD8+双阳性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。
在第三个方面，本发明提供一种药物组合物，包含如上定义的本发明的嵌合抗原受体或其编码核酸、载体或包含它们的免疫细胞，和一种或多种药学上可接受的赋型剂。
在第四个方面，本发明提供一种治疗患有癌症的受试者的方法，包括向所述受试者 施用有效量的根据本发明所述的嵌合抗原受体、免疫细胞或药物组合物。
在一个实施方案中，所述癌症选自：胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、白血病、肝肿瘤、肺癌、淋巴瘤、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤、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)。优选地，可以用本发明的嵌合抗原受体、核酸、载体、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。
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。
嵌合抗原受体
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，该杂合多肽的基础结构包括抗原结合区(例如抗体的抗原结合部分)、跨膜结构域、共刺激结构域和细胞内信号传导结构域。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表达CAR的T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。本发明的新型嵌合抗原受体除了包含抗原结合区、跨膜结构域、共刺激结构域和细胞内信号传导结构域这些基础结构之外，还包括由γc链或其 胞内区组成的额外信号传导区。
如本文所用，“抗原结合区”是指可以与抗原结合的任何结构或其功能性变体。抗原结合区可以是抗体结构，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、嵌合抗体及其功能性片段。例如，抗原结合区包括但不限于Fab、单链抗体(Single Chain Antibody Fragment,scFv)、单结构域抗体(Single Domain Antibody,sdAb)、纳米抗体(Nanobody，Nb)、抗原结合配体、重组纤连蛋白结构域、anticalin和DARPIN等，优选选自Fab、scFv、sdAb和纳米抗体。在本发明中，抗原结合区可以是单价或二价，且可以是单特异性、双特异性或多特异性的抗体。在另一个实施方案中，抗原结合区也可以是特定蛋白的特异性结合多肽或受体结构，所述特定蛋白是例如PD1、PDL1、PDL2、TGFβ、APRIL和NKG2D。
“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。
“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。
抗原结合区的选择取决于待识别的与具体疾病状态相关的靶细胞上的细胞表面标记，例如肿瘤特异性抗原或肿瘤相关抗原。因此，在一个实施方案中，本发明的抗原结合区与选自以下的一个或多个靶标结合：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-TES1、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抗原结合区。例如，如果CD19是待靶向的抗原，则CD19抗体可用作本发明的抗原结合区。
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。当嵌合受体多肽与靶抗原结合时，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人CD8α链，其与SEQ ID NO:4所示的氨基酸序列或与SEQ ID NO：3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抗原结合区和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、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CD8α铰链，其与SEQ ID NO:12所示的氨基酸序列或与SEQ ID NO：11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抗原结合区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的信号转导结构域可以包含CD3ζ信号传导结构域，该信号传导结构域与SEQ ID NO:8所示的氨基酸序列或SEQ ID NO：7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、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。优选地，本发明CAR的共刺激结构域是4-1BB 和/或CD28片段，更优选与SEQ ID NO:6所示的氨基酸序列或SEQ ID NO：5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。
在一个方面，除了共刺激结构域和细胞内信号传导结构域用作信号传导之外，本发明的嵌合抗原受体还包含至少一个额外的信号传导区，所述额外信号传导区由γc链或其胞内区组成。换言之，本发明的嵌合抗原受体的胞内区(即，用于信号传导的结构)由共刺激结构域、胞内信号传导结构域，和γc链或其胞内区这三种信号传导结构组成。这意味着本发明的嵌合抗原受体不包含第四种信号传导结构，例如其他细胞因子的信号传导区，如IL-2Ra、IL2Ra、IL2Rb、IL4Ra、IL7Ra、IL9Ra、IL15Ra、IL21Ra等的胞内区。
如本文所用，术语“γc链”是指细胞因子IL-2、IL-4、IL-7、IL-9、IL-15、IL-21的受体共有的γ链。γc链最初在IL-2受体中被鉴定，后来发现它也参与IL-4、IL-7、IL-9、IL-15、IL-21等受体的组成，因此也称为通用γ链。例如，IL-2受体有三种形式，由α链(也称为IL-2Rα)、β链(也称为IL-2Rβ)和γc链(也称为IL-2Rγ或IL-2Rg)的不同组合形成，其中与其他组合相比，包含全部三条链的IL-2受体与IL-2细胞因子具有最高的亲和力。IL-2受体的三条链锚定在细胞膜上，通过与IL-2的结合将生化信号传递到细胞内。在这些γ链依赖性的细胞因子中，细胞因子受体特有的组分，例如IL-2Rβ、IL-4Rα、IL-7Rα、IL-9Rα、IL-21R等负责与JAK1结合，而γc链则与JAK3结合。当这些细胞因子受体与细胞因子结合时，三个主要的信号通路被激活，包括MAP激酶、PI3激酶和JAK-STAT通路，进而调控T细胞和NK细胞的存活以及增殖。
γc链是分子量为64kD的糖蛋白，由347个氨基酸组成，其中包括232个氨基酸的胞外区，29个氨基酸的跨膜区和86个氨基酸的胞内区。胞内区中含有Src同源区，对促进细胞的生长以及IL-2介导的c-myc、c-fos、c-jun等基因的表达至关重要。在一个实施方案中，可用于本发明的γc链与SEQ ID NO:14所示的氨基酸序列或SEQ ID NO：13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，可用于本发明的γc链胞内区与SEQ ID NO:16所示的氨基酸序列或SEQ ID NO：15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。优选地，本发明的γc链由SEQ ID NO：14组成，其胞内区由SEQ ID NO：16组成。
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等的信号肽。
在一个优选的实施方案中，本发明的嵌合抗原受体包括CD8α跨膜结构域、4-1BB共刺激结构域、CD3ζ信号传导结构域和γc链或其胞内区。更优选地，所述嵌合抗原受体进一步包含CD8α信号肽、CD8α铰链区和/或CD28共刺激结构域。
还在一个优选的实施方案中，在本发明的嵌合抗原受体中，共刺激结构域、胞内信号传导结构域和额外信号传导区按照与细胞膜的距离从近到远依次排列，即共刺激结构域离细胞膜最近，而额外信号传导区离细胞膜最远。
核酸
本发明还提供一种核酸，其包含编码本发明的嵌合抗原受体的序列。
如本文所用，术语“核酸”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一种或多种核酸。
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线性核酸分子(例如DNA或RNA)、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
本发明提供工程化免疫细胞，其包含嵌合抗原受体或其编码核酸。
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。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细胞可以是任何类型的T细胞并且可以处于任何发育阶段，包括但不限于，CD4+/CD8+双阳性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多肽。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以将编码嵌合抗原受体多肽的核酸序列引入免疫细胞，使其表达本发明的嵌合抗原受体多肽。“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。
还在一个实施方案中，本发明的免疫细胞还包含至少一种失活基因，其选自以下：CD52、GR、TCRα、TCRβ、CD3γ、CD3δ、CD3ε、CD247ζ、HLA-I、HLA-II基因、免疫检查点基因如PD1和CTLA-4。更特别地，免疫细胞中的至少TCRα或TCRβ基因被失活。这种失活使得TCR在细胞中没有功能。该策略对于避免移植物抗宿主病(GvHD)特别有用。使基因失活的方法是本领域已知的，例如通过大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂，从而使该基因失活。
药物组合物
本发明还提供一种药物组合物，其包含本发明所述的嵌合抗原受体、核酸、载体或工程化免疫细胞作为活性剂，和一种或多种药学上可接受的赋型剂。因此，本发明还涵盖所述嵌合抗原受体、核酸、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IL-2，趋化因子比如IL-8、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页可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
制备工程化免疫细胞的方法
本发明还提供一种制备工程化免疫细胞的方法，包括将本发明的嵌合抗原受体或其编码核酸序列引入免疫细胞，以使所述免疫细胞表达本发明的嵌合抗原受体。
在一个实施方案中，所述免疫细胞是人免疫细胞，更优选人T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞。
将核酸或载体引入免疫细胞并进行表达的方法是本领域已知的。例如，可以通过物理方法，如括磷酸钙沉淀法、脂质转染法、粒子轰击法、显微注射法、电穿孔法等将核酸或载体导入免疫细胞。或者，也可以采用化学方法，如通过胶体分散系统，如大分子复合物、纳米胶囊、微球、珠粒以及基于脂质的系统，包括水包油乳液、胶束、混合胶束及脂质体引入核酸或载体。此外，还可以使用生物方法引入核酸或载体。例如，病毒载体，尤其是逆转录病毒载体等已经成为将基因插入哺乳动物，例如人细胞中的最常用方法。其它病毒载体可以来源于慢病毒、痘病毒、单纯疱疹病毒I、腺病毒及腺相关病毒等。
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。
治疗应用
本发明还提供一种治疗患有癌症的受试者的方法，包括向所述受试者施用有效量的本发明所述的免疫细胞或药物组合物。
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)提供受试者的样品，所述样品包含免疫细胞；(b)在体外将本发明的嵌合抗原受体引入所述免疫细胞，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，所述疾病是与抗原结合区结合的靶标表达有关的癌症。例如，所述癌症包括但不限于：脑神经胶质瘤、胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌(包括胃肠癌)、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、肝肿瘤、肺癌(例如小细胞肺癌、非小细胞肺癌、腺状肺癌和鳞状肺癌)、淋巴瘤(包括霍奇金淋巴瘤和非霍奇金淋巴瘤)、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌(例如唇、舌、口和咽)、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤(包括低级/滤泡性非霍奇金淋巴瘤(NHL)、小淋巴细胞性(SL)NHL、中间级/滤泡性NHL、中间级扩散性NHL、高级成免疫细胞性NHL、高级成淋巴细胞性NHL、高级小型非裂化细胞性NHL、大肿块病NHL)、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。
附图说明
图1：通过流式细胞术测定的CAR-T细胞的CAR表达水平。
图2：CAR-T细胞对靶细胞的杀伤效果。用Two-way ANOVA分析，并用T test进行统计学分析。*表示P值小于0.05，达到显著水平。
图3：CAR-T细胞分别与靶细胞和非靶细胞共培养后的IL-2(A)和IFNγ(B)释放水平。
图4：经处理小鼠体内的肿瘤负荷随时间的变化。
图5：在D21天，小鼠体内的CD3+(A)、CD8+(B)和CD4+(C)T细胞的扩增水平。
图6：经处理小鼠的存活率随时间的变化。
图7：CAR-T细胞对靶细胞的杀伤效果。用Two-way ANOVA分析，并用T test进行统计学分析。*表示P值小于0.05，达到显著水平。
具体实施方式
在以下实施例中所用的序列总结如下表1所示。
表1.本发明中所用的序列
SEQ ID NO 描述
SEQ ID NO：1 CD19-scFv的核苷酸序列
SEQ ID NO：2 CD19-scFv的氨基酸序列
SEQ ID NO：3 跨膜结构域CD8α的核苷酸序列
SEQ ID NO：4 跨膜结构域CD8α的氨基酸序列
SEQ ID NO：5 共激活结构域4-1BB的核苷酸序列
SEQ ID NO：6 共激活结构域4-1BB的氨基酸序列
SEQ ID NO：7 信号传导结构域CD3ζ的核苷酸序列
SEQ ID NO：8 信号传导结构域CD3ζ的氨基酸序列
SEQ ID NO：9 信号肽CD8α的核苷酸序列
SEQ ID NO：10 信号肽CD8α的氨基酸序列
SEQ ID NO：11 CD8α铰链区的核苷酸序列
SEQ ID NO：12 CD8α铰链区的氨基酸序列
SEQ ID NO：13 γc链的核苷酸序列
SEQ ID NO：14 γc链的氨基酸序列
SEQ ID NO：15 γc链胞内区的核苷酸序列
SEQ ID NO：16 γc链胞内区的氨基酸序列
本发明所有实施例中使用的T细胞是通过Ficoll-PaqueTM PREMIUM(GE Healthcare，货号17-5442-02)采用白细胞分离术从健康供体分离的原代人CD4+CD8+T细胞。Nalm6肿瘤细胞购买自南京集萃药康生物科技有限公司。
实施例1：构建CAR T细胞
合成以下编码序列，并将其依次克隆至pGEM-T Easy载体(Promega，货号A1360)：CD8α信号肽(SEQ ID NO：9)、抗CD19scFv(SEQ ID NO：1)、CD8α铰链区(SEQ ID NO：11)、CD8α跨膜区(SEQ ID NO：3)、4-1BB共刺激结构域(SEQ ID NO：5)、CD3ζ胞内信号传导结构域(SEQ ID NO：7)，获得传统的bbz-CAR质粒，并通过测序确认目标序列的正确插入。用同样的方法获得bbzg-CAR质粒，其与bbz-CAR质粒的唯一区别在于，还包括与CD3ζ胞内信号传导结构域连接的γ链胞内区(SEQ ID NO：15)。在bbzg-CAR质粒里，4-1BB共刺激结构域、CD3ζ胞内信号传导结构域和γ链胞内区按照离细胞膜的距离从近到远依次排列。
在无菌管中加入3ml Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒:病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene， 货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120ul X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO 2下培养1天。然后，加入浓缩的慢病毒，持续培养3天后，获得靶向CD19的传统con-CAR T细胞(用作对照)和本发明的bbzg-CAR T细胞。
在37℃和5％CO2下培养11天之后，使用Biotin-SP(long spacer)AffiniPure Goat Anti-Mouse IgG,F(ab') 2Fragment Specific(min X Hu,Bov,Hrs Sr Prot)(jackson immunoresearch，货号115-065-072)作为一抗，APC Streptavidin(BD Pharmingen，货号554067)或PE Streptavidin(BD Pharmingen，货号554061)作为二抗，通过流式细胞仪检测Fite-CAR T细胞上的scFv的表达水平，结果如图1所示(NT是未经修饰的野生型T细胞)。
可以看出，本发明的bbzg-CAR T细胞可以有效表达scFv，并且其表达水平略高于传统的bbz-CAR T细胞，表明γ链胞内区的加入不会影响CAR结构的表面表达。
实施例2：CAR T细胞对靶细胞的杀伤效果和细胞因子释放
2.1 CAR-T细胞对靶细胞的杀伤效果
当T细胞对靶细胞有杀伤时，靶细胞的数量就会减少。将T细胞和带有可表达荧光素酶的靶细胞共培养后，靶细胞数量减少的同时，分泌的荧光素酶也会随之减少。荧光素酶可以催化荧光素转化为氧化性荧光素，而在此氧化过程中，会产生生物发光，并且这种发光的强度将取决于靶细胞表达的荧光素酶的水平。因此，检测的荧光强度能够反应T细胞对靶细胞的杀伤能力。
为了检测CAR-T细胞对靶细胞的杀伤能力，首先以1x10 4/孔将携带荧光素基因的Nalm6靶细胞铺入96孔板中，然后以32:1的效靶比(即效应T细胞与靶细胞之比)将bbzg-CAR T细胞、Con-CAR T细胞(阳性对照)和未转染T细胞(阴性对照)铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图2所示。
可以看出，与NT相比，本发明的bbzg-CAR T细胞对靶细胞的杀伤效果显著高于传统bbz-CAR T细胞。
2.2 CAR-T细胞的细胞因子释放
T细胞杀伤靶细胞时，靶细胞数量减少的同时也会释放细胞因子IL2和IFN-γ等。根据以下步骤，使用酶联免疫吸附法(ELISA)来测定Fite-CARX T细胞杀伤靶细胞时细胞因子IL2和IFNγ的释放水平。
(1)收集细胞共培养上清液
以1x10 5/孔将靶细胞(Nalm6和Raji)和非靶细胞(193F)分别铺于96孔板中，然后以1:1的比例将bbzg-CAR T、con-CAR T(阳性对照)和NT细胞(阴性对照)分别与靶细胞或非靶细胞共培养，18-24小时后收集细胞共培养上清液。
(2)ELISA检测上清中IFNγ分泌量
使用捕获抗体Purified anti-human IFN-γ Antibody(Biolegend，货号506502)包被96孔板4℃孵育过夜，然后移除抗体溶液，加入250μL含有2％BSA(sigma，货号V900933-1kg)的PBST(含0.1％吐温的1XPBS)溶液，37℃孵育2小时。然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。每孔加入50μL细胞共培养上清液或标准品，并在37℃孵育1小时，然后用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。然后向各孔分别加入50μL检测抗体Anti-Interferon gamma抗体[MD-1](Biotin)(abcam，货号ab25017)，在37℃孵育1小时后，用250μL PBST(含0.1％吐温的1XPBS)清洗板3次。再加入HRP Streptavidin(Biolegend，货号405210)，在37℃孵育30分钟后，弃上清液，加入250μL PBST(含0.1％吐温的1XPBS)，清洗5次。向各孔加入50μL TMB底物溶液。使反应在室温下于暗处发生30分钟，之后向各孔中加入50μL1mol/L H 2SO 4以停止反应。在停止反应的30分钟内，使用酶标仪检测450nm处吸光度，并根据标准曲线(根据标准品的读值和浓度绘制)计算细胞因子的含量，结果如图3所示。
可以看出，在非靶细胞293F中均没有检测到IFNγ的释放，表明bbz-CAR T细胞和bbzg-CAR T细胞的杀伤都是特异性的。并且，在杀伤靶细胞时，bbzg-CAR T细胞的IL2释放水平显著低于传统CAR T细胞，但其IFN-γ的释放水平却显著高于传统CAR T细胞。总体上，本发明的bbzg-CAR T细胞的细胞因子释放与传统CAR-T细胞相当。
实施例3 CAR-T细胞的肿瘤抑制效果验证
在小鼠模型体内验证CAR-T细胞对肿瘤的抑制效果。
将20只8周龄的健康雌性NCG小鼠分成四组：PBS组、NT组(阴性对照)、bbz-CART 组(阳性对照)和bbzg-CAR T组。在第0天(D0)，向每只小鼠尾静脉注射1x10 6个Nalm6细胞。7天后(D7)，根据分组情况向每只小鼠尾静脉注射PBS溶液或者2x10 6个NT细胞、con-CAR T细胞或bbzg-CAR T细胞。每周评估小鼠的存活率和肿瘤负荷的变化。
采用活体动物体内光学成像技术评估每组小鼠的肿瘤负荷变化。于D7、D14、D21、D28、D35、D42、D49检测小鼠肿瘤负荷并以Photons/s表示，结果如图4所示。
可以看出，在PBS和NT组中，小鼠体内的肿瘤负荷进展迅速，在D21即达到最高值(随即死亡)。bbz-CAR组的小鼠在接受CAR-T细胞处理后，肿瘤负荷迅速下降，但在D28或D35又逐渐反弹。与此相反，bbzg-CAR T组的小鼠不仅在接受处理后肿瘤负荷迅速下降，而且将低水平维持至D49没有复发。这表明，本发明的bbzg-CAR T细胞能够有效抑制肿瘤生长，并且效果显著优于传统的bbz-CAR T细胞。
发明人还监测了接受bbz-CART细胞和bbzg-CAR T细胞处理的两组小鼠在第21天体内的T细胞扩增情况。具体地，在D21对小鼠颌下静脉取血，进行Trucount FACS分析(hCD3、hCD8、hCD4的表达水平)，结果如图5所示。
可以看出，在bbz-CAR T组和bbzg-CAR T组的小鼠体内均检测到T细胞扩增。虽然两者CD4+T细胞的扩增程度相当，但bbzg-CAR组T的CD3+和CD8+T细胞的扩增则明显高于bbz-CAR T组。因此，虽然两组在第21天的肿瘤负荷情况差不多(图4)，但由于bbzg-CAR T组小鼠体内的T细胞扩增显著更多，使其可以将肿瘤负荷一直抑制在较低水平；相反，由于T细胞的扩增较少并且持续耗竭，bbz-CAR T组小鼠的肿瘤负荷在之后出现反弹。
此外，发明人还统计了截至本实验结束(即，接种肿瘤细胞Nalm6后第105天)，各组小鼠的存活百分比(图6)。其中，PBS和NT组小鼠全部死亡，用bbz-CAR T细胞处理的小鼠仅存活一只(20％)，而用bbzg-CAR T细胞处理的小鼠仍有60％存活。这再次表明，本发明的bbzg-CAR T细胞能够有效抑制肿瘤，提高存活率。
综上，与传统的CAR T细胞相比，本发明的bbzg-CAR T细胞由于引入了细胞因子受体通用γ链，使得可以极大促进T细胞的扩增，从而提高对肿瘤细胞持续的杀伤效果，改善体内的肿瘤抑制效果，增加小鼠的存活。
实施例4.制备不同结构CAR-T细胞并验证其功能
将γc链胞内区(SEQ ID NO：65)插入到bbz-CAR质粒的4-1BB共刺激结构域和CD3ζ初级信号传导结构域之间，获得bbgz-CAR质粒，其与bbzg-CAR质粒的区别仅在 于γc链胞内区的位置不同。根据实施例1的方法制备bbgz-CAR T细胞。
根据实施例2所述的方法检测CAR-T细胞对靶细胞的杀伤效果，结果如图7所示。可以看出，bbgz-CAR T细胞对靶细胞的杀伤效果与传统的bbz-CAR T细胞相当，但都显著低于bbzg-CAR T细胞的杀伤效果。这表明，额外信号传导区(即，γc链或其胞内区)在CAR结构中的位置对CAR T细胞的杀伤活性有重要影响。
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 (18)

  1. 一种嵌合抗原受体，其包含抗原结合区、跨膜结构域、共刺激结构域、胞内信号传导结构域和额外信号传导区，其中所述额外信号传导区由γc链或其胞内区组成。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述共刺激结构域、胞内信号传导结构域和额外信号传导区按照与细胞膜的距离从近到远依次排列。
  3. 根据权利要求1或2所述的嵌合抗原受体，其中所述γc链的氨基酸序列如SEQ ID NO：14所示；其胞内区的氨基酸序列如SEQ ID NO：16所示。
  4. 根据权利要求1所述的嵌合抗原受体，其中所述抗原结合区选自sdAb、纳米抗体、抗原结合配体、重组纤连蛋白结构域、anticalin和DARPIN。
  5. 根据权利要求1所述的嵌合抗原受体，其中所述抗原结合区选自单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠源抗体和嵌合抗体。
  6. 根据权利要求1-3任一项所述的嵌合抗原受体，其中所述抗原结合区结合的靶标选自：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、Folate受体α、ERBB2(Her2/neu)、MUC1、EGFR、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。
  7. 根据权利要求1-6任一项所述的嵌合抗原受体，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、CD8、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  8. 根据权利要求1-7任一项所述的嵌合抗原受体，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。
  9. 根据权利要求1-8任一项所述的嵌合抗原受体，其中所述嵌合受体多肽包含一个或多个共刺激结构域，其中所述共刺激结构域是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)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD270(HVEM)、CD272(BTLA)、CD273(PD-L2)、CD274(PD-L1)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、LAT、NKG2C、SLP76、LIGHT、TRIM以及ZAP70。
  10. 一种核酸，其包含编码根据权利要求1-9任一项所述的嵌合抗原受体。
  11. 一种载体，包含根据权利要求10所述的核酸。
  12. 一种免疫细胞，其包含根据权利要求1-9任一项所述的嵌合抗原受体、根据权利要求10所述的核酸，或根据权利要求11所述的载体。
  13. 根据权利要求12所述的免疫细胞，其中所述载体是线性核酸分子、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)、噬菌体、粘粒或人工染色体。
  14. 根据权利要求12-13任一项所述的免疫细胞，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。
  15. 根据权利要求14所述的免疫细胞，其中所述免疫细胞是选自以下的T细胞：CD4+/CD8+双阳性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细胞和αβ-T细胞。
  16. 根据权利要求12所述的免疫细胞，所述免疫细胞还包含至少一种选自以下的失活基因：CD52、GR、TCRα、TCRβ、CD3γ、CD3δ、CD3ε、CD247ζ、HLA-I、HLA-II基因、免疫检查点基因如PD1和CTLA-4。
  17. 一种药物组合物，包含根据权利要求1-9任一项所述的嵌合抗原受体、根据权利要求10所述的核酸、根据权利要求11所述的载体或根据权利要求12-16任一项 所述的免疫细胞，和一种或多种药学上可接受的赋型剂。
  18. 根据权利要求17所述的药物组合物，所述药物组合物用于治疗选自以下的癌症：胚细胞瘤、肉瘤、白血病、基底细胞癌、胆道癌、膀胱癌、骨癌、脑和CNS癌症、乳腺癌、腹膜癌、宫颈癌、绒毛膜癌、结肠和直肠癌、结缔组织癌症、消化系统的癌症、子宫内膜癌、食管癌、眼癌、头颈癌、胃癌、胶质母细胞瘤(GBM)、肝癌、肝细胞瘤、上皮内肿瘤、肾癌、喉癌、白血病、肝肿瘤、肺癌、淋巴瘤、黑色素瘤、骨髓瘤、神经母细胞瘤、口腔癌、卵巢癌、胰腺癌、前列腺癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸系统的癌症、唾液腺癌、皮肤癌、鳞状细胞癌、胃癌、睾丸癌、甲状腺癌、子宫或子宫内膜癌、泌尿系统的恶性肿瘤、外阴癌以及其它癌和肉瘤、以及B细胞淋巴瘤、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)。
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