WO2021147929A1 - 多链嵌合抗原受体及其用途 - Google Patents

多链嵌合抗原受体及其用途 Download PDF

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WO2021147929A1
WO2021147929A1 PCT/CN2021/072980 CN2021072980W WO2021147929A1 WO 2021147929 A1 WO2021147929 A1 WO 2021147929A1 CN 2021072980 W CN2021072980 W CN 2021072980W WO 2021147929 A1 WO2021147929 A1 WO 2021147929A1
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cancer
cells
domain
cell
protein interaction
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French (fr)
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史其萍
江雯
贺小宏
任江涛
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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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of immunotherapy. Specifically, the present invention relates to a multi-chain chimeric antigen receptor (CAR) and its use, especially its use in the treatment of cancer.
  • CAR multi-chain chimeric antigen receptor
  • 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's body to kill cancer cells, thereby achieving the purpose of treating tumors.
  • T lymphocytes also known as T cells
  • B lymphocytes also known as B cells
  • natural killer cells NK cells
  • macrophages Dendritic cells, mast cells, etc.
  • T cells are the main component of lymphocytes and have a variety of biological functions, such as directly killing target cells, assisting or inhibiting the production of antibodies by B cells, responding to specific antigens, and producing cytokines.
  • the immune response generated by T cells is cellular immunity. There are two main types of cellular immunity: one is to specifically bind to target cells, destroy the target cell membrane, and directly kill the target cells; the other is to release lymphokines, which ultimately makes immunity The effect is expanded and enhanced.
  • NK cells are small, but they are essential for human innate immunity. Such immune cells do not need antibodies and Major Histocompatibility Complex (MHC) mediation for the recognition of foreign antigens, and the immune killing response of NK cells is rapid.
  • MHC Major Histocompatibility Complex
  • the broad and rapid immune killing ability of NK cells makes them an ideal immune cell in tumor immune cell therapy.
  • Macrophages have a variety of functions. They not only have phagocytosis against pathogens, but also can present antigens after ingestion. There are also a large number of tumor-associated macrophages (Tumor Associated Macrophages, TAM) in the tumor microenvironment.
  • TAM tumor-associated Macrophages
  • DC Dendritic cells
  • APC full-time antigen presenting cells
  • NK cells and macrophages have significant tumor invasion advantages, and can efficiently present antigens to T cells.
  • NK cells also have the effect of activating DC cells.
  • NK cells activating NK cells, macrophages, DC cells, etc. while carrying out CAR-T immunotherapy will help to solve CAR-T cell therapy such as immunosuppression of the tumor microenvironment, tumor heterogeneity, and difficulty in infiltration of T cells. And many other problems, and significantly improve the overall treatment effect.
  • the present invention provides a multi-chain chimeric antigen receptor comprising: (a) an Fc fusion polypeptide, the Fc fusion polypeptide comprising an antigen binding region, a first protein interaction domain and an Fc region; And (b) a chimeric receptor polypeptide, the chimeric receptor polypeptide comprising a second protein interaction domain, a transmembrane domain, and an intracellular signaling domain, wherein the first protein interaction domain is capable of interacting with The second protein interaction domain specifically binds.
  • the antigen binding region is selected from the group consisting of sdAb, Nanobody, antigen binding ligand, recombinant fibronectin domain, anticalin and DARPIN.
  • 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 contained in the chimeric receptor polypeptide 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, 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 comprised by the chimeric receptor polypeptide 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 chimeric receptor polypeptide further comprises one or more costimulatory domains.
  • the costimulatory domain is a costimulatory signal transduction domain 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 Fc region comprises a CH2 domain and a CH3 domain, preferably the CH2 and CH3 domains of IgG1.
  • the first protein interaction domain and the second protein interaction domain are a combination selected from the group consisting of Fk506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB) of mTOR, FKBP and calcineurin A (CnA), FKBP and cyclophilin (CyP), GAI and GID, Snap tag and Halo tag, glucocorticoid receptor (GR) and DHFR, PYL and ABI, cAMP-dependent protein kinase The dimerization docking domain of A (PKA) and the anchoring domain of A-kinase anchor protein (AKAP), avidin and biotin, leucine zipper domain and leucine zipper domain, and zinc finger structure Domain and nucleotide tags.
  • FKBP FKBP
  • FKBP and FKBP-rapamycin binding domain FKBP and FKBP-rapamycin binding domain (FRB) of mTOR
  • both the first protein interaction domain and the second protein interaction domain are leucine zipper domains, such as AZip and BZip, respectively.
  • the first protein interaction domain and the second protein interaction domain are respectively a zinc finger domain and a nucleotide tag.
  • the multi-chain chimeric antigen receptor of the present invention may further comprise a second Fc fusion polypeptide, the second Fc fusion polypeptide comprising a second antigen binding region, a third protein interaction domain and The second Fc region.
  • the second protein interaction domain is the first part of the nucleotide tag
  • the third protein interaction domain is the second part of the nucleotide tag, and only when the nucleoside
  • the complex can specifically bind to the zinc finger domain as the first protein interaction domain.
  • the present invention also provides a nucleic acid comprising a sequence encoding the multi-chain chimeric antigen receptor of the present invention, a vector or a vector system comprising the nucleic acid, and an immune cell comprising the nucleic acid or the vector or vector system .
  • the invention provides a nucleic acid comprising a sequence encoding the chimeric receptor polypeptide of the invention and a sequence encoding the Fc fusion polypeptide 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 a vector system comprising a first nucleic acid sequence encoding the chimeric receptor polypeptide of the present invention and a second nucleic acid sequence encoding the Fc fusion polypeptide of the present invention, the first nucleic acid sequence And the second nucleic acid sequence are located in a different vector. In another embodiment, the first nucleic acid sequence and the second nucleic acid sequence are located in the same vector.
  • the present invention provides an immune cell comprising the nucleic acid or vector or vector system of the present invention, which is capable of expressing the multi-chain 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 multi-chain chimeric antigen receptor of the present invention as defined above or its encoding nucleic acid, vector or vector system or immune cells containing them, and one or more A pharmaceutically acceptable excipient.
  • the present invention provides a method of treating a subject suffering from cancer, comprising administering to the subject an effective amount of the multi-chain chimeric antigen receptor, immune cell or Pharmaceutical composition.
  • the multiple peptide chains contained in the multi-chain chimeric antigen receptor of the present invention may be administered together or separately.
  • immune cells or pharmaceutical compositions containing the first Fc fusion polypeptide and immune cells or pharmaceutical compositions containing the chimeric receptor polypeptide can be administered to the subject, respectively.
  • the treatment may also include further administering to the subject an immune cell or pharmaceutical composition comprising a second Fc fusion polypeptide, the second Fc fusion polypeptide comprising a second antigen binding region, a third protein Interaction domain and second Fc region.
  • 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
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide whose basic structure includes an antigen binding region (for example, the antigen binding portion of an antibody), a transmembrane 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 endogenous T cell receptor
  • the extracellular binding domain of the CAR is composed of a single chain variable fragment (scFv) derived from the fusion of the variable heavy chain region and the light chain region of a murine or human or chimeric monoclonal antibody.
  • the scFv that can be used is derived from Fab (rather than from an antibody, for example, obtained from a Fab library). In various embodiments, this scFv is fused to the transmembrane domain and then to the intracellular signaling domain.
  • 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 cells self-destruct.
  • multi-chain chimeric antigen receptor or "multi-chain CAR” refers to a CAR comprising at least two peptide chains, wherein each peptide chain contains a protein interaction domain, and each peptide chain only Can perform one of the functions of target binding and signal transduction. Only when the at least two peptide chains bind to each other (for example, through the specific binding of the protein interaction domain), can signal transduction be performed while binding to the target.
  • a multi-chain CAR contains two peptide chains, one chain is the Fc fusion polypeptide responsible for target binding, and the other chain is the chimeric receptor polypeptide responsible for signal transduction. The two peptide chains pass through their respective protein interaction structures.
  • the domains are combined with each other.
  • the protein interaction domain contained in the third chain can form a complex with the protein interaction domain contained in the second chain, thereby forming a complex with the protein interaction domain contained in the first chain.
  • the protein interaction domain specifically binds and initiates the signal transduction pathway, and can also compete with the protein interaction domain contained in the second chain to bind to the protein interaction domain contained in the first chain, for example, through stronger binding activity Replace the second chain to identify new targets and conduct signal transduction.
  • the multi-chain chimeric antigen receptor of the present invention comprises: (a) a chimeric receptor comprising a first protein interaction domain, a transmembrane domain, and intracellular signaling Domain; and (b) an Fc fusion polypeptide, the Fc fusion polypeptide comprising an antigen binding region, a second protein interaction domain and an Fc region, wherein the first protein interaction domain can interact with the second protein interaction domain Specific binding.
  • protein interaction domain refers to a domain that allows two separate polypeptides to specifically bind to each other. This article provides many exemplary protein interaction domains and their combination pairings.
  • the first protein interaction domain in the multi-chain CAR may specifically bind to the second protein interaction domain.
  • specific binding occurs between two separate protein interaction domains.
  • specific binding occurs between three separate protein interaction domains. Exemplary protein interaction domains are known in the art and can be used in the embodiments described herein.
  • the first protein interaction domain and the second protein interaction domain are a combination selected from the group consisting of Fk506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB) of mTOR, FKBP and calcineurin A (CnA), FKBP and cyclophilin (CyP), GAI and GID, Snap tag and Halo tag, glucocorticoid receptor (GR) and DHFR, PYL and ABI, cAMP-dependent protein kinase The dimerization docking domain of A (PKA) and the anchoring domain of A-kinase anchor protein (AKAP), avidin and biotin, leucine zipper domain and leucine zipper domain, and zinc finger structure Domain and nucleotide tags.
  • FKBP FKBP
  • FKBP and FKBP-rapamycin binding domain FKBP and FKBP-rapamycin binding domain (FRB) of mTOR
  • both the first protein interaction domain and the second protein interaction domain are leucine zipper domains.
  • leucine zipper domain refers to a type of protein-protein interaction domain commonly found in transcription factors, which is characterized in that leucine residues are evenly spaced by an ⁇ -helix. Leucine zippers can form heterodimers or homodimers.
  • the first protein interaction domain and the second protein interaction domain are AZip and BZip, respectively.
  • AZip has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 4
  • BZip has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6.
  • Other suitable leucine zipper domains can include SYNZIP1 to SYNZIP48, as well as BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, and HEPTAD. Many leucine zipper domains are known in the art and can be used in the present invention.
  • the first protein interaction domain and the second protein interaction domain are respectively a zinc finger domain and a nucleotide tag.
  • the zinc finger domain is composed of an ⁇ -helix and two anti-parallel ⁇ -sheets, and has the function of binding zinc ions.
  • the zinc finger domain can recognize a specific base sequence, thereby regulating the activity of a gene containing the specific base sequence.
  • the zinc finger domain binds to DNA
  • the nucleotide tag is a DNA tag, more preferably a dsDNA tag. Examples of zinc finger domains and their associated nucleotide tags are well known to those skilled in the art.
  • the nucleotide tag in the case of a zinc finger domain and a nucleotide tag, can be divided into multiple parts, such as two parts, three parts, and only when these multiple parts are combined to form a complete Only when the nucleotide tag is used can it specifically bind to the zinc finger domain.
  • the multi-chain chimeric antigen receptor of the present invention comprises: (a) a chimeric receptor polypeptide comprising a zinc finger domain, a transmembrane domain and an intracellular signaling domain; (b) a first Fc fusion polypeptide comprising a first antigen binding region, a first part of a nucleotide tag, and a first Fc region; and (c) a second Fc fusion polypeptide comprising a second antigen binding region, a nucleoside
  • the second part of the acid tag and the second Fc region only when the first part of the nucleotide tag and the second part of the nucleotide tag form a complex, the complex can specifically bind to the zinc finger domain .
  • the first part may be ssDNA and the second part may be ssDNA complementary to it, or the first part may be dsDNA with overhangs, and the second part may be dsDNA with complementary overhangs.
  • the first part and the second part can be hybridized under suitable conditions.
  • a complete dsDNA nucleotide tag required for binding to the zinc finger domain is formed.
  • the dsDNA nucleotide tag consists of three parts that are present in the three Fc fusion polypeptides, for example, the first part is ssDNA, and the second and third parts are ssDNA that are complementary to the first part. And they do not overlap with each other, and only when the nucleotide tags of these three parts form a complete nucleotide tag that can be recognized by the zinc finger domain, the complex can specifically bind to the zinc finger domain.
  • the first protein interaction domain and the second protein interaction domain are chemically induced protein interaction domains, which specifically bind to form dimers only in the presence of specific chemicals, also known as It is a chemically induced dimerization (Chmical Induced Dimerization, CID) system.
  • exemplary chemically induced protein interaction domains include, but are not limited to, the following combinations: FKBP and FRB that are bound by rapamycin and its derivatives, such as photosensitive cage rapamycin; and those that are bound by abscisic acid.
  • the first protein interaction domain and the second protein interaction domain are the dimerization docking domain (Dimerization Docking Domain, DDD) of cAMP-dependent protein kinase A (PKA) and A- Anchoring Domain (AD) of Kinase Anchored Protein (AKAP).
  • DDD dimerization Docking Domain
  • PKA cAMP-dependent protein kinase A
  • AD A- Anchoring Domain
  • AKAP Kinase Anchored Protein
  • PKA has two types of R subunits (RI and RII), and each type has ⁇ and ⁇ isotypes, so there are four types of DDD: RI ⁇ , RI ⁇ , RII ⁇ , and RII ⁇ .
  • AKAP is widely present in various species and is located in various subcellular sites, including plasma membrane, actin cytoskeleton, nucleus, mitochondria and endoplasmic reticulum.
  • the AD used to bind PKA in AKAP is an amphipathic helix with 14-18 residues.
  • the amino acid sequence of AD is quite different between AKAP.
  • the binding between AD and DDD is specific, and the affinity is very high.
  • the sequences of various AD and DDD peptides and their variants are known to those skilled in the art, such as described in Baillie et al., FEBS Letters. 2005, 579: 3264. Wong; Scott, Nat. Rev. Mol. Cell Biol. 2004 , 5:959; PCT/US03/054842, the entirety of which is incorporated herein by reference.
  • the first protein interaction domain and the second protein interaction domain are avidin and biotin, respectively.
  • Avidin is a basic glycoprotein composed of 4 identical subunits, which can resist the action of a variety of proteolytic enzymes. Commonly used ones include streptavidin, for example.
  • Biotin is widely present in various animal and plant tissues. It contains two ring structures, in which the imidazolone ring is the main part that binds to avidin. The binding interaction between avidin and biotin has good stability and strong specificity, and is not affected by reagent concentration, pH environment, or protein denaturant and other organic solvents.
  • chimeric receptor refers to a polypeptide containing a protein interaction domain located on the cell membrane. Its function is mainly to initiate a signal transduction pathway after the specific binding of the protein interaction domain, thereby activating the The activity of the immune cells of the chimeric receptor.
  • the chimeric receptor in the present invention comprises a first protein interaction domain, a transmembrane domain and an intracellular signal transduction domain, wherein the definition of the protein interaction domain is as described above.
  • transmembrane domain refers to a polypeptide that enables the chimeric receptor polypeptide to be expressed on the surface of immune cells (such as lymphocytes, NK cells, or NKT cells) and guides the 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 multi-chain chimeric antigen receptor of the present invention binds to the target antigen, the transmembrane domain can conduct 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%, more preferably at least 90%, 95%, 97% or 99% with the amino acid sequence of SEQ ID NO: 12 or 100% sequence identity.
  • the chimeric receptor polypeptide 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 can be derived from all or part of a natural molecule, such as from all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of 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 the hinge region of human CD8 ⁇ chain, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably the hinge of human CD8 ⁇ or IgG4, which has the same amino acid sequence as SEQ ID NO: 26 or 28. 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 intracellular signal transmission 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 can be cytolytic activity or accessory 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 initiate signal transduction after the antigen receptor is bound. , And any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions.
  • Intracellular signaling domains contain two different types of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals.
  • the primary cytoplasmic signal sequence 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 chimeric receptor polypeptide of the present invention may comprise a CD3 ⁇ signal domain, which has at least 70% of the amino acid sequence shown in SEQ ID NO: 16, 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 further comprises one or more costimulatory domains.
  • the costimulatory domain may be an intracellular functional signaling domain derived from a costimulatory molecule, which may include 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, more preferably at least 70%, preferably at least 80%, more preferably at least 90%, 95% with the amino acid sequence of SEQ ID NO: 14. , 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor of the present invention comprises a CD8 ⁇ transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ⁇ signaling domain. More preferably, the chimeric antigen receptor further comprises a CD28 costimulatory domain and a CD8 ⁇ hinge region or an IgG4 hinge region.
  • the term "Fc fusion polypeptide” is a recombinant polypeptide comprising a protein interaction domain, an Fc region and an antigen binding region, wherein the definition of the protein interaction domain is as described above.
  • the Fc fusion polypeptide of the present invention can bind to the Fc receptors on the surface of other immune cells such as macrophages, NK cells, dendritic cells, etc., thereby recruiting these immune cells and performing additional treatment on target cells. Kill or play the role of antigen presentation and expand the killing effect of CART cells.
  • the Fc fusion polypeptide of the present invention can also provide additional antigen binding regions, that is, provide individual target cell killing ability and diversified antigen targeting properties.
  • 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 single domain antibody (Single Domain Antibody, sdAb), Nanobody (Nb), antigen binding ligand, recombinant fibronectin domain, anticalin and DARPIN, etc., preferably selected from sdAb And Nanobodies.
  • the antigen binding region contained in the Fc fusion polypeptide of the present invention is not a single chain antibody (single chain antidoby fragment, scFv).
  • the antigen binding region can be monovalent or bivalent, and can be monospecific , Bispecific or multispecific.
  • 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.
  • 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 Fc fusion polypeptide or chimeric receptor polypeptide 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, CAIX, LMP2, gplOO , Bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS
  • 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 antigen binding region binds Claudin 18.2.
  • the antigen binding region comprises a functional variant against the above sequence, such as having the same CDR as SEQ ID NO: 8 and having at least 80%, at least 85%, or at least 90% with SEQ ID NO: 8 %, at least 95%, at least 98%, or at least 99% sequence identity.
  • the functional variant may be formed by substituting, adding or deleting one or more (for example, 1 to 10, 1 to 5, or 1 to 3) amino acid residues.
  • the functional variant has the same or similar functions and activities as SEQ ID NO: 8.
  • the multi-chain chimeric antigen receptor of the present invention may comprise two or more Fc fusion polypeptides, wherein each Fc fusion polypeptide comprises a protein interaction domain, an antigen binding region and an Fc region, wherein the antigen
  • the binding regions can bind the same or different targets, and wherein the Fc regions can be the same or different.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least part of the constant region.
  • the Fc region has no antigen binding activity and is the site where immunoglobulin interacts with effector molecules or cells.
  • the term includes native Fc regions and variant Fc regions.
  • Native Fc region refers to a molecule or sequence that contains non-antigen-binding fragments produced by digestion of intact antibodies, whether in monomeric form or in multimeric form.
  • the immunoglobulin source that produces the natural Fc region is preferably derived from humans.
  • Natural Fc fragments are composed of monomeric polypeptides that can be connected in the form of dimers or multimers through covalent linkages (such as disulfide bonds) and non-covalent linkages.
  • covalent linkages such as disulfide bonds
  • non-covalent linkages such as disulfide bonds
  • the natural Fc molecule monomer subunits have 1-4 intermolecular disulfides key.
  • An example of a natural Fc region is a dimer linked by disulfide bonds produced by digesting IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9).
  • natural Fc generally refers to monomer, dimer and multimer forms.
  • a “variant Fc region” refers to an amino acid sequence that differs from the amino acid sequence of a "natural” or “wild-type” Fc region due to at least one "amino acid modification” as defined herein, and is also referred to as a "Fc variant”. Therefore, “Fc region” also includes single-chain Fc (scFc), that is, a single-chain Fc region composed of two Fc monomers connected by a polypeptide linker, which can naturally fold into a functional dimer Fc region.
  • the variant Fc region and the natural Fc region have at least about 80%, at least about 85%, at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or at least about 99% The sequence identity.
  • the Fc region contained in the Fc fusion polypeptide of the present invention is preferably derived from IgG.
  • human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4. Among them, IgG1 has the highest abundance in serum.
  • the constant region sequences of these four subtypes are highly homologous, but each subtype is specific for antigen binding, immune complex formation, complement activation, triggering effector cells, half-life, and placental transport characteristics.
  • the affinity of IgG1 and IgG3 to Fc receptors is higher than that of IgG2 and IgG4, and has a stronger ability to activate antibody-dependent cytotoxicity and complement-dependent cytotoxicity; IgG2 and IgG4 subtypes are It has the function of hindering or inhibiting the effect. Therefore, in a preferred embodiment, the Fc region contained in the Fc fusion polypeptide of the present invention is preferably derived from IgG1 to enhance the affinity between the Fc region and the receptor, thereby improving the recruitment efficiency of other immune cells.
  • the Fc region of the present invention refers to a constant region that does not include CH1.
  • the Fc region in the case of IgA, IgD, and IgG, the Fc region contains constant domains CH2 and CH3; in the case of IgE and IgM, the Fc region contains constant domains CH2, CH3, and CH4.
  • the Fc region may also include a lower hinge region between CH1 and CH2. Therefore, preferably, the Fc region of the present invention includes CH2 and CH3 of IgG1, and more preferably also includes the lower hinge region between CH1 and CH2.
  • the Fc region has the same or similar receptor binding activity as the amino acid sequence shown in SEQ ID NO: 10, and has at least 70% of the amino acid sequence shown in SEQ ID NO: 10, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the present invention also provides a nucleic acid comprising a sequence encoding the chimeric receptor polypeptide of the present invention and a sequence encoding the Fc fusion polypeptide 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.
  • the present invention also provides a vector system comprising a first nucleic acid sequence encoding a chimeric receptor polypeptide and a second nucleic acid sequence encoding an Fc fusion polypeptide; the first nucleic acid sequence and the second nucleic acid sequence are located in the same vector or different vectors .
  • 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 a hybrid recombinase using a specific targeting site sequence.
  • An “expression vector” is a vector used for the transcription of heterologous nucleic acid sequences (such as those encoding the Fc fusion polypeptide or chimeric 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 first nucleic acid sequence encoding the chimeric receptor polypeptide and the second nucleic acid sequence encoding the Fc fusion polypeptide are located in the same vector.
  • the chimeric receptor polypeptide and the Fc fusion polypeptide of the present invention can be expressed independently without affecting each other.
  • the term "2A peptide” is a cis-hydrolase action element (CHYSEls), originally found in foot-and-mouth disease virus (FMDV).
  • the average length of the 2A peptide is 18-22 amino acids.
  • the 2A peptide can be broken from the C-terminus of the last two amino acids of itself through ribosome jumping. Specifically, the peptide chain binding group between glycine and proline is damaged at position 2A, which can trigger ribosome jumping and start translation from the second codon, thereby making two proteins in one transcription unit Independent expression.
  • This 2A peptide-mediated cleavage is widespread in eukaryotic animal cells. Using the higher shearing efficiency of 2A peptide and the ability to promote balanced expression of upstream and downstream genes can improve the expression efficiency of heterologous polyproteins (such as cell surface receptors, cytokines, immunoglobulins, etc.).
  • Conventional 2A peptides include: P2A, T2A, E2A, F2A, etc.
  • the first nucleic acid sequence encoding the chimeric receptor polypeptide and the second nucleic acid sequence encoding the Fc fusion polypeptide are located in different vectors.
  • the present invention provides engineered immune cells, which comprise a chimeric receptor polypeptide or its encoding nucleic acid, and an Fc fusion polypeptide or its encoding nucleic acid, and are also referred to herein as Fite CAR (Fc induced target cell engaging Chimeric Antigen Receptor) cells. Therefore, in one embodiment, the engineered immune cell of the invention comprises a first nucleic acid sequence encoding the chimeric receptor polypeptide of the invention and a second nucleic acid sequence encoding the Fc fusion polypeptide of the invention.
  • 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 receptor polypeptides and Fc fusion polypeptides.
  • 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.
  • transfection 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. Techniques for plant transformation include Agrobacterium-mediated transfer (such as by A. tumefaciens), rapidly advanced tungsten or gold microprojectiles, electroporation, microinjection, and polyethylene glycol mediation. Guided intake.
  • Agrobacterium-mediated transfer such as by A. tumefaciens
  • the immune cell of the present invention further comprises at least one inactivating gene selected from the following: CD52, GR, TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247 ⁇ , HLA-I, HLA-II genes , Immune checkpoint genes such as PD1 and CTLA-4. More specifically, the immune cell may contain at least one selected TCR ⁇ or TCR ⁇ gene inactivating gene. 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 multi-chain chimeric antigen receptor, nucleic acid, carrier, system 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 multi-chain chimeric antigen receptor, nucleic acid, vector, system 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.
  • a pharmaceutical composition comprising, for example, the immune cells described herein, a multi-chain chimeric antigen receptor, or a nucleic acid or vector encoding the same is usually provided in the form of a solution, and preferably contains 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 receptor polypeptide and Fc fusion polypeptide of the present invention or the nucleic acid sequences encoding both of the two into immune cells, so that the immune cells express the Chimeric receptor polypeptide and Fc fusion polypeptide.
  • 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, comprising administering to the subject an effective amount of the multi-chain chimeric antigen receptor, immune cell or 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) in vitro combining the multi-stranded chimeric receptor of the present invention or its encoding nucleic acid or vector or vector system
  • the immune cells are introduced to obtain modified immune cells, and (c) the modified immune cells are administered 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. ).
  • immune cells capable of expressing the chimeric receptor polypeptide and Fc fusion polypeptide of the present invention and exerting the desired biological effect function as described herein can also be used.
  • selected immune cells are compatible with the immune system of the subject, that is, it is preferred that the immune cells do not elicit an immunogenic response.
  • "universal acceptor cells” can be used, that is, universally compatible lymphocytes that can grow and expand in vitro that perform the desired biological effect function. The use of such cells will not require obtaining and/or providing the subject's own lymphocytes.
  • 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.
  • a viral vector being the aforementioned lentiviral vector, adenoma Viral vector, adeno-associated virus vector or retroviral vector.
  • Other conceivable methods include the use of transfection reagents (such as liposomes) or transient RNA transfection.
  • the two or more peptide chains contained in the multi-chain chimeric antigen receptor of the present invention may be administered together or separately.
  • an immune cell or pharmaceutical composition containing the first Fc fusion polypeptide and an immune cell or pharmaceutical composition containing the chimeric receptor polypeptide can be administered to the subject separately to activate the signal transduction pathway at an appropriate time as needed. Activate the killing function of immune cells.
  • the treatment may also include further administering to the subject an immune cell or pharmaceutical composition comprising a second Fc fusion polypeptide, the second Fc fusion polypeptide comprising a second antigen binding region, a third protein Interaction domain and second Fc region.
  • the treatment method further includes administering to the subject a chemical that can induce the protein interaction domain to bind to each other.
  • the treatment method also includes the administration of gibberellin; when the protein interaction domains are the Snap tag and the Halo tag, the treatment method also includes the administration of HaXS;
  • the treatment method also includes administration of rapamycin and its derivatives such as photosensitive cage rapamycin;
  • the treatment method also includes administration of shedding Acid;
  • the protein interaction domains are FKBP and CyP
  • the treatment method also includes administration of FKCsA; when the protein interaction domains are FKBP and CnA, the treatment method also includes administration of FK506; when the protein interaction domains are respectively For GR and DHFR, the treatment method also includes the administration of Dex-Mtx.
  • 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: 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, digestive system cancer, 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, leukemia, 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), melanoma, my
  • the disease that can be treated with the multi-chain 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 , Stomach cancer, etc.
  • 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 Design schematic diagram of a preferred embodiment of the present invention.
  • Figure 2 shows the scFv expression levels of Fite-CAR-1s and Fite-CAR-2s T cells.
  • Figure 3 Shows the killing effect of Fite-CAR-1s and Fite-CAR-2s T cells on target cells.
  • Figure 4 shows the secretion level of scFv-Fc fusion polypeptide in Fite-CAR-1s and Fite-CAR-2s T 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, and ** indicates that the P value is less than 0.01, both reaching a significant level.
  • Figure 5 shows the expression of AZip and Fc fusion polypeptides in Fite-CAR-2s T cells.
  • Figure 6 Shows the secretion level of sdAb-Fc in Fite-CARX-1s and Fite-CARX-2s T cells (A) and the killing effect on target cells (B). 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, and ** indicates that the P value is less than 0.01, both reaching a significant level.
  • Figure 7 Shows the IFN- ⁇ release levels of Fite-CARX-1s and Fite-CARX-2s T cells.
  • Figure 8 shows the NK cell killing effect of Fite-CARX-1s and Fite-CARX-2s T 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, and ** indicates that the P value is less than 0.01, both reaching a significant level.
  • SEQ ID NO describe SEQ ID NO: 1 Nucleotide sequence of Claudin18.2-scFv SEQ ID NO: 2 Amino acid sequence of Claudin18.2-scFv SEQ ID NO: 3 Nucleotide sequence of AZip SEQ ID NO: 4 Nucleotide sequence of AZip SEQ ID NO: 5 Nucleotide sequence of BZip SEQ ID NO: 6 Nucleotide sequence of BZip SEQ ID NO: 7 The nucleotide sequence of Claudin 18.2 sdAb SEQ ID NO: 8 Amino acid sequence of Claudin 18.2 sdAb SEQ ID NO: 9 Nucleotide sequence of Fc region SEQ ID NO: 10 Amino acid sequence of Fc region SEQ ID NO: 11 The nucleotide sequence of transmembrane domain CD8 ⁇ SEQ ID NO: 12 Amino acid sequence of transmembrane domain CD8 ⁇ SEQ ID NO: 13 Nucleotide sequence of co-activation
  • 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.
  • CD8 ⁇ signal peptide SEQ ID NO: 19
  • anti-Claudin 18.2 scFv SEQ ID NO: 1
  • CD8 ⁇ hinge region SEQ ID NO: 25
  • CD8 ⁇ transmembrane region SEQ ID NO: 11
  • 4-1BB costimulatory domain SEQ ID NO: 13
  • CD3 ⁇ intracellular signaling domain SEQ ID NO: 15
  • 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/CD28 CTSTM (Gibco, catalog number 40203D), and cultured at 37°C and 5% CO2 for 1 day. Then, the concentrated lentivirus was added, and after continuous culture for 3 days, CAR T (ie con-CAR T) cells targeting Claudin 18.2 were obtained.
  • CD3/CD28 CTSTM Gabco, catalog number 40203D
  • 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, combined, and ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated Fite-CAR lentivirus.
  • T cells were activated with DynaBeads CD3/CD28CTSTM (Gibco, catalog number 40203D), and cultured at 37°C and 5% CO2 for 1 day. Then, the concentrated Fite-CAR lentivirus was added, and after continuous culture for 3 days, Fite-CAR T cells were obtained.
  • DynaBeads CD3/CD28CTSTM Gabco, catalog number 40203D
  • the two Fite-CAR T cells of the present invention can effectively express scFv, indicating that the chimeric receptor polypeptide can specifically bind to the Fc fusion polypeptide, and the expression level is equivalent to con-CAR.
  • 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.
  • the 293T-Claudin 18.2 target cells administered in this example are Claudin 18.2 positive monoclonal cells selected by flow cytometry after infecting 293T cells with a lentivirus expressing Claudin 18.2.
  • the two Fite-CAR T cells of the present invention can effectively kill target cells, and their killing effect is equivalent to that of Con-CAR T cells.
  • Fite-CAR T cells can effectively secrete the scFv-Fc region, they can be recognized by immune effector cells expressing Fc receptors (FcR) including NK cells, macrophages, dendritic cells, etc., so as to recruit these immune effector cells , Further enhance the killing effect on target cells. Therefore, the inventors used enzyme-linked immunosorbent assay (ELISA) to detect the scFv-Fc secretion level of Fite-CAR T cells.
  • FcR Fc receptors
  • Fite-CAR-1s T cells, Fite-CAR-2s T cells, Con-CAR T and NT cells in x-vivo 15 medium (Lonza, catalog number 04-418Q) that does not contain IL-2, respectively, in 37 Cultivation at °C and 5% CO2. After 24 hours, the culture was collected and centrifuged at 4°C and 1600 rpm for 5 minutes to obtain the cell culture supernatant.
  • Fite was detected by a combination of antibodies anti-c-Fos (BOSTER, article number PA1318), Biotin-goat anti-rabbit IgG (BOSTER, article number BA1003), and APC Streptavidin (BD Pharmingen, article number 554067) by flow cytometry -The chimeric receptor expression on CAR T cells (ie, the detection of AZip expression), the use of the antibody PE anti-Human IgG Fc (Biolegend, catalog number 409304) to detect the expression of Fc fusion polypeptide on Fite-CAR-2s T cells, the result As shown in Figure 5.
  • the Fc-positive cells in the Fite-CAR-2s T cell population are basically AZip-negative at the same time (35.1%), indicating that the detected Fc fusion polypeptides have passed
  • the interaction between BZip and AZip binds to the chimeric receptor polypeptide, causing the binding site of AZip to be occupied and unable to bind the detection antibody.
  • there are very few AZip-positive and Fc-negative (0.31%) cells in the Fite-CAR-2s T cell population which also indicates that there is basically no single unbound chimeric receptor polypeptide in the Fite-CAR-2s T cell population.
  • Fite-CARX-1s T cells and Fite-CARX-2s T cells and their connection sequence used in this example are the same as those of Fite-CAR-1s T cells and Fite-CAR-2s T cells.
  • the only difference is Replace Claudin18.2scFv (SEQ ID NO:1) with Claudin18.2sdAb (SEQ ID NO: 7).
  • both Fite-CARX T supernatants can detect significantly secreted sdAb-Fc fusion polypeptides, indicating that the single-domain antibody structure can effectively avoid scFv interactions. Adhesion, thereby promoting the secretion of Fc fusion polypeptide.
  • the two Fite-CARX T cells can effectively kill target cells, and the killing effect is equivalent to that of Con-CAR T cells.
  • ELISA enzyme-linked immunosorbent assay
  • Example 7 Fite-CARX T cells mediate the killing effect of NK cells on target cells
  • Fite-CARX T cells can efficiently kill target cells and significantly secrete sdAb-Fc fusion polypeptides, the inventors further tested whether they can mediate NK cells to kill tumors.
  • the NK cells used in this example were obtained by the following method: After grinding the mouse spleen, adding mouse spleen lymphocyte separation solution (TBD, article number LTS1092PK-200), and centrifuging to obtain buffy coat cells. Then, PE anti-mouse NK1.1 (Biolegend, catalog number 108701) and Anti-PE Microbeads (Miltenyi Biotec, catalog number 130-048-801) were added, and positive screening was performed on the magnetic stand to obtain NK1.1 positive cells.
  • TBD mouse spleen lymphocyte separation solution
  • PE anti-mouse NK1.1 Biolegend, catalog number 108701
  • Anti-PE Microbeads (Miltenyi Biotec, catalog number 130-048-801) were added, and positive screening was performed on the magnetic stand to obtain NK1.1 positive cells.
  • the NUGC4-Claudin 18.2 target cells administered in this example are Claudin 18.2 positive monoclonal cells selected by flow cytometry after infecting NUGC4 cells with a lentivirus expressing Claudin 18.2.
  • the NUGC4-Claudin18.2 target cells carrying the fluorescein gene were plated into a 96-well plate at 1 ⁇ 10 4 /well. Then, Fite-CARX T cell supernatant and fresh medium (media) were used to resuspend the NK cells, and the resuspended NK cells were resuspended with an effect-to-target ratio of 4:1 (that is, the ratio of effector NK cells to target cells) Add a 96-well plate for co-cultivation, and measure the fluorescence value with a microplate reader after 16-18 hours. 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. 8.
  • the two Fite-CARX T cell supernatants can effectively mediate the killing of NK cells on the NUGC4-Claudin 18.2 target cells, and its effect is significantly higher than that of the fresh medium control group.

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Abstract

本发明提供了一种多链嵌合抗原受体，其包含：(a)嵌合受体，该嵌合受体包含第一蛋白质相互作用结构域、跨膜结构域和胞内信号传导结构域；和(b)Fc融合多肽，该Fc融合多肽包含抗原结合区、第二蛋白质相互结构域和Fc区，其中第一蛋白质相互作用结构域能够和第二蛋白质相互作用结构域特异性结合。还提供了包含本发明的多链嵌合抗原受体的工程化免疫细胞及其药物组合物，以及该工程化免疫细胞/药物组合物在治疗癌症中的用途。

Description

多链嵌合抗原受体及其用途 技术领域
本发明涉及免疫治疗领域，具体地，本发明涉及多链嵌合抗原受体(Chimeric Antigen Receptor,CAR)及其用途，尤其是在治疗癌症方面的用途。
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***的目的。
人体内参与免疫应答或与免疫应答相关的细胞有很多种，包括T淋巴细胞(也称为T细胞)、B淋巴细胞(也称为B细胞)、自然杀伤细胞(NK细胞)、巨噬细胞、树突状细胞、肥大细胞等。其中T细胞是淋巴细胞的主要组分，具有多种生物学功能，如直接杀伤靶细胞、辅助或抑制B细胞产生抗体、对特异性抗原的应答反应以及产生细胞因子等。T细胞产生的免疫应答是细胞免疫，细胞免疫的效应形式主要有两种：一种是与靶细胞特异性结合，破坏靶细胞膜，直接杀伤靶细胞；另一种是释放淋巴因子，最终使免疫效应扩大和增强。NK细胞数量较少，但是对人体先天免疫而言必不可少。这类免疫细胞对异体抗原的识别不需要抗体和主要组织相容性复合体(Major Histocompatibility Complex，MHC)的介导，并且NK细胞的免疫杀伤反应迅速。NK细胞所具有的这种广泛而快速的免疫杀伤能力，让它们成为肿瘤免疫细胞疗法中的一种理想免疫细胞。巨噬细胞具有多种功能，既对病原体等具有噬菌作用，亦能对抗原进行摄取后起到呈递作用。肿瘤微环境中还遍布着大量的肿瘤相关巨噬细胞(Tumor Associated Macrophages，TAM)。它们与肿瘤细胞、肿瘤干细胞、表皮细胞、成纤维细胞以及T细胞、B细胞、NK细胞等都存在高度的相互作用。树突状细胞(Dendritic cells,DC)则是机体功能最强的专职抗原呈递细胞(Antigen Presenting Cells,APC)，能够高效地摄取、加工处理和呈递抗原。NK细胞与巨噬细胞具有显著的肿瘤浸润优越性，同时可高效呈递抗原至T细胞。并且，NK细胞同时还有激活DC细胞的作用。
因此，在进行CAR-T免疫治疗的同时激活NK细胞、巨噬细胞、DC细胞等将有助于解决CAR-T细胞治疗存在的例如肿瘤微环境的免疫抑制、肿瘤异质、T细胞难以浸润等诸多问题，并显著提高整体治疗效果。
发明概述
在第一个方面，本发明提供一种多链嵌合抗原受体，其包含：(a)Fc融合多肽，所述Fc融合多肽包含抗原结合区、第一蛋白质相互作用结构域和Fc区；和 (b)嵌合受体多肽，所述嵌合受体多肽包含第二蛋白质相互作用结构域、跨膜结构域和胞内信号传导结构域，其中所述第一蛋白质相互作用结构域能够和第二蛋白质相互作用结构域特异性结合。
在一个实施方案中，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。
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、***蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、***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，嵌合受体多肽包含的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，嵌合受体多肽包含的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，所述嵌合受体多肽还包含一个或多个共刺激结构域。优选地，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)、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。
在一个实施方案中，Fc区包含CH2结构域和CH3结构域，优选IgG1的CH2和CH3结构域。
在一个实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域是选自以下的组合：Fk506结合蛋白(FKBP)和mTOR的FKBP-雷帕霉素结合结构域(FRB)、FKBP和钙调磷酸酶A(CnA)、FKBP和亲环素(CyP)、GAI和GID、Snap标签和Halo标签、糖皮质激素受体(GR)和DHFR、PYL和ABI、cAMP依赖性的蛋白激酶A(PKA)的二聚化对接结构域和A-激酶锚定蛋白(AKAP)的锚定域、亲和素和生物素、亮氨酸拉链结构域和亮氨酸拉链结构域以及锌指结构域和核苷酸标签。优选地，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域均是亮氨酸拉链结构域，例如分别为AZip和BZip。或者，优选地，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是锌指结构域和核苷酸标签。在一个具体的实施方案中，本发明的多链嵌合抗原受体还可以进一步包含第二Fc融合多肽，所述第二Fc融合多肽包含第二抗原结合区、第三蛋白质相互作用结构域和第二Fc区。例如，在一个具体的实施方案中，第二蛋白质相互作用结构域是核苷酸标签的第一部分，第三蛋白质相互作用结构域是核苷酸标签的第二部分，并且仅当所述核苷酸标签的第一部分和核苷酸标签的第二部分形成复合物时，所述复合物才能与作为第一蛋白质相互作用结构域的锌指结构域特异性结合。
在第二个方面，本发明还提供包含编码本发明的多链嵌合抗原受体的序列的核酸、包含所述核酸的载体或载体***、以及包含所述核酸或载体或载体***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受体多肽的序列和编码本发明的Fc融合多肽的序列。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，本发明提供一种载体***，其包含编码本发明的嵌合受 体多肽的第一核酸序列和编码本发明的Fc融合多肽的第二核酸序列，所述第一核酸序列和第二核酸序列位于不同载体。在另一个实施方案中，所述第一核酸序列和第二核酸序列位于同一载体。
在一个实施方案中，本发明提供包含本发明的核酸或载体或载体***的免疫细胞，其能够表达本发明的多链嵌合抗原受体。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多链嵌合抗原受体或其编码核酸、载体或载体***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多链嵌合抗原受体、免疫细胞或药物组合物。
在一个实施方案中，本发明的多链嵌合抗原受体包含的多个肽链可以一起或分别施用。例如，可以向受试者分别施用包含第一Fc融合多肽的免疫细胞或药物组合物，和包含嵌合受体多肽的免疫细胞或药物组合物。在另一个实施方案中，所述治疗还可以包括向受试者进一步施用包含第二Fc融合多肽的免疫细胞或药物组合物，所述第二Fc融合多肽包含第二抗原结合区、第三蛋白质相互作用结构域和第二Fc区。
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、***、绒毛膜癌、结肠和直肠癌、***癌症、消化***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、***癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸***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***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)。
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。
多链嵌合抗原受体
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。通常地，CAR的细胞外结合结构域由源自将鼠源或人源或嵌合的单克隆抗体的可变重链区和轻链区进行融合的单链可变片段(scFv)组成。或者，可使用的scFv源自Fab(而不是来自抗体，例如，从Fab文库获得)。在各种实施方式中，将这种scFv融合至跨膜结构域，并随后融合至细胞内信号传导结构域。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)，***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***基因以在需要时使CAR细胞自我毁灭。
如本文所用，术语“多链嵌合抗原受体”或“多链CAR”是指包含至少两条肽链的CAR，其中每条肽链均包含蛋白质相互作用结构域，并且每条肽链仅能进行靶标结合和信号传导中的其中一个功能。只有当这至少两条肽链相互结合(例如，通过蛋白质相互作用结构域的特异性结合)时，才能在结合靶标的同时进行信号传导。例如，当多链CAR包含两条肽链时，一条链是负责靶标结合的Fc融合多肽，另一条链是负责信号传导的嵌合受体多肽，两条肽链通过各自包含的蛋白质相互作用结构域彼此结合。当多链CAR包含三条或更多条肽链时，第三条链包含的蛋白质相互作用结构域可以与第二条链包含的蛋白质相互作用结构域形成复合物，从而与第一条链包含的蛋白质相互作用结构域进行特异性结合并启动信号传导通路，也可以与第二条链包含的蛋白质相互作用结构域竞争结合第一条链包含的蛋白质相互作用结构域，例如通过更强的结合活性取代第二条链，从而识别新的靶标并进行信号传导。
在一个实施方案中，本发明的多链嵌合抗原受体包含：(a)嵌合受体，所述嵌合受体包含第一蛋白质相互作用结构域、跨膜结构域和胞内信号传导结构域；和(b)Fc融合多肽，所述Fc融合多肽包含抗原结合区、第二蛋白质相互结构域和Fc区，其中所述第一蛋白质相互作用结构域能够和第二蛋白质相互作用结构域特异性结合。
如本文所用，术语“蛋白质相互作用结构域”是指允许两个分开的多肽彼此特异性结合的结构域。本文提供了许多示例性的蛋白质相互作用结构域及其组合配对。在一些实施方式中，多链CAR中的第一蛋白质相互作用结构域可以特异性地结合至第二蛋白质相互作用结构域。在一些实施方式中，特异性结合发生在两 个分开的蛋白质相互作用结构域之间。在一些实施方式中，特异性结合发生在三个分开的蛋白质相互作用结构域之间。示例性的蛋白质相互作用结构域在本领域中是已知的并且可以用于本文所述的实施方式中。
在一个实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域是选自以下的组合：Fk506结合蛋白(FKBP)和mTOR的FKBP-雷帕霉素结合结构域(FRB)、FKBP和钙调磷酸酶A(CnA)、FKBP和亲环素(CyP)、GAI和GID、Snap标签和Halo标签、糖皮质激素受体(GR)和DHFR、PYL和ABI、cAMP依赖性的蛋白激酶A(PKA)的二聚化对接结构域和A-激酶锚定蛋白(AKAP)的锚定域、亲和素和生物素、亮氨酸拉链结构域和亮氨酸拉链结构域以及锌指结构域和核苷酸标签。
在一个优选的实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域均是亮氨酸拉链结构域。如本文所用，术语“亮氨酸拉链结构域”是指一类通常见于转录因子中的蛋白质-蛋白质相互作用结构域，其特征在于通过α-螺旋将亮氨酸残基均匀地间隔开。亮氨酸拉链可以形成异源二聚体或同源二聚体。在一个具体的实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是AZip和BZip。在一个实施方案中，AZip与SEQ ID NO:4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，BZip与SEQ ID NO:6所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。其他合适的亮氨酸拉链结构域可以包括SYNZIP1至SYNZIP48，以及BATF、FOS、ATF4、ATF3、BACH1、JUND、NFE2L3和HEPTAD。很多亮氨酸拉链结构域是本领域已知的，并且可以用于本发明。
在另一个优选的实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是锌指结构域和核苷酸标签。锌指结构域由一个α-螺旋和两个反平行的β-折叠组成，具有结合锌离子的功能。锌指结构域能够识别特定碱基序列，从而调控包含该特定碱基序列的基因的活性。优选地，锌指结构域结合DNA，并且核苷酸标签是DNA标签，更优选dsDNA标签。锌指结构域及其结合的核苷酸标签的实例是本领域技术人员熟知的。
在一个实施方案中，在锌指结构域和核苷酸标签的情况下，所述核苷酸标签可以分成多个部分，例如两个部分、三个部分，仅当这多个部分结合形成完整的核苷酸标签时，才能与锌指结构域特异性结合。因此，在一个具体的实施方案中，本发明的多链嵌合抗原受体包含：(a)嵌合受体多肽，其包含锌指结构域、跨膜结构域和胞内信号传导结构域；(b)第一Fc融合多肽，其包含第一抗原结合区、核苷酸标签的第一部分和第一Fc区；和(c)第二Fc融合多肽，其包含第二抗原结合区、核苷酸标签的第二部分和第二Fc区，仅当所述核苷酸标签的第一部分和核苷酸标签的第二部分形成复合物时，所述复合物才能与锌指结构域特异性结合。所述第一部分可以是ssDNA，第二部分是与其互补的ssDNA，或者第一部分是具有悬端的dsDNA，第二部分是具有互补悬端的dsDNA，第一部分和第二部分可以在合适的条件下通过杂交形成与锌指结构域结合所需的完整的dsDNA核苷酸标 签。在另一个具体的实施方案中，dsDNA核苷酸标签由分别存在于三个Fc融合多肽的三个部分组成，例如第一部分是ssDNA，第二部分和第三部分是分别与第一部分互补的ssDNA并且彼此之间互相不重叠，并且仅当这三个部分的核苷酸标形成能够被锌指结构域识别的完整核苷酸标签时，所述复合物才能与锌指结构域特异性结合。
在一个实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域是化学诱导的蛋白质相互作用结构域，其仅在特定化学物的存在下特异性结合形成二聚体，也称为化学诱导二聚化(Chmical Induced Dimerization，CID)***。例如，示例性的化学诱导的蛋白质相互作用结构域包括但不限于以下组合：由雷帕霉素及其衍生物例如光敏笼状雷帕霉素诱导结合的FKBP和FRB；由脱落酸诱导结合的PYL和ABI；由赤霉素诱导的GID和GAI；由FK506诱导结合的FKBP和钙调磷酸酶A(CnA)；由FKCsA诱导结合的FKBP和亲环素(CyP)；由HaXS诱导结合的Snap标签和Halo标签；由***-甲氨蝶呤(Dex-Mtx)诱导结合的糖皮质激素受体(GR)和二氢叶酸还原酶(DHFR)。本领域技术人员已知很多CID***，例如参见Voβ S等，Current Opinion in Chemical Biology,2015,28:194-201。
在一个实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是cAMP依赖性的蛋白激酶A(PKA)的二聚化对接结构域(Dimerization Docking Domain,DDD)和A-激酶锚定蛋白(AKAP)的锚定域(Anchoring Domain,AD)。PKA具有两种类型的R亚基(RI和RII)，并且各类型具有α和β同种型，因此有四种类型的DDD：RIα、RIβ、RIIα和RIIβ。AKAP广泛存在于各种物种中，并且定位于各种亚细胞位点，包括质膜、肌动蛋白细胞骨架、细胞核、线粒体和内质网。AKAP中用于结合PKA的AD是具有14-18个残基的两亲性螺旋。AD的氨基酸序列在AKAP之间十分不同。AD与DDD之间的结合是特异性的，且亲和力非常高。各种AD和DDD肽及其变体的序列是本领域技术人员已知的，例如描述于Baillie等，FEBS Letters.2005,579:3264.Wong；Scott，Nat.Rev.Mol.Cell Biol.2004,5:959；PCT/US03/054842，其整体通过引用并入本文。
在一个实施方案中，第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是亲和素和生物素。亲和素是由4个相同亚基组成的碱性糖蛋白，能够抵抗多种蛋白水解酶的作用，常用的包括例如链霉亲和素。生物素广泛存在于各种动物和植物组织中，其包含两个环状结构，其中的咪唑酮环是与亲和素结合的主要部分。亲和素和生物素之间的结合相互作用稳定性好，特异性强，不受试剂浓度、PH环境、抑或蛋白变性剂等有机溶剂影响。
嵌合受体多肽
如本文所用，术语“嵌合受体”是指位于细胞膜上的包含蛋白质相互作用结构域的多肽，其功能主要是在蛋白质相互作用结构域的特异性结合之后启动信号传导通路，从而激活包含该嵌合受体的免疫细胞的活性。
本发明中的嵌合受体包含第一蛋白质相互作用结构域、跨膜结构域和胞内信号传导结构域，其中蛋白质相互作用结构域的定义如上所述。
如本文所用，术语“跨膜结构域”是指能够使嵌合受体多肽在免疫细胞(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。当本发明的多链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人CD8α链，其与SEQ ID NO:12的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合受体多肽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。铰链区可以源自全部或部分的天然分子，如源自全部或部分的CD8、CD4或CD28的胞外区，或源自全部或部分的抗体恒定区。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，所述铰链区包含人CD8α链、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选人CD8α或IgG4的铰链，其与SEQ ID NO:26或28的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。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物活性，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包含两种不同类型的细胞质信号序列：引发抗原依赖性初级活化的那些，以及以不依赖抗原的方式起作用以提供次级或共刺激信号的那些。初级细胞质信号序列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单不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在优选的实施方式中，本发明嵌合受体多肽的信号转导结构域可以包含CD3ζ信号结构域，该信号结构域与SEQ ID NO:16所示的氨基酸序列具有至少70％，优选至少80％，更优选至少 90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体还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，其可以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:14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。
在一个优选的实施方案中，本发明的嵌合抗原受体包含CD8α跨膜结构域、4-1BB共刺激结构域和CD3ζ信号传导结构域。更优选地，所述嵌合抗原受体进一步包含CD28共刺激结构域和CD8α铰链区或IgG4铰链区。
Fc融合多肽
如本文所用，术语“Fc融合多肽”是包含蛋白质相互作用结构域、Fc区和抗原结合区的重组多肽，其中蛋白质相互作用结构域的定义如上所述。当正常表达并分泌时，本发明的Fc融合多肽能够与其他免疫细胞例如巨噬细胞、NK细胞、树突状细胞等表面的Fc受体结合，从而招募这些免疫细胞，对靶细胞进行额外的杀伤或者发挥抗原呈递作用，扩大CART细胞的杀伤效果。此外，本发明的Fc融合多肽还可以提供额外的抗原结合区，即，提供单独的靶细胞杀伤能力，以及多样化的抗原靶向性。
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sdAb和纳米抗体。在一个实施方案中，本发明的Fc融合多肽包含的抗原结合区不是单链抗体(Single chain antidoby fragment,scFv)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。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。
“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***)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Fc融合多肽或嵌合受体多肽中。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。
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、***蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、***特异性蛋白、存活蛋白和端粒酶、PCTA-l/Galectin8、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。
根据一个具体的实施方式，抗原结合区结合Claudin18.2。根据另一个具体的实施方式，抗原结合区包含抗上述序列的功能性变体，例如与SEQ ID NO：8具有相同的CDR且与SEQ ID NO：8具有至少80％、至少85％、至少90％、至少95％、至少98％或至少99％的序列同一性。所述功能性变体可以通过取代、添加或缺失一个或多个(例如1至10、1至5或1至3个)氨基酸残基而形成。特别地，所述功能性变体与SEQ ID NO：8具有相同或相似的功能和活性。
在一个实施方案中，本发明的多链嵌合抗原受体可以包含两个或多个Fc融合多肽，其中每个Fc融合多肽均包含蛋白质相互作用结构域、抗原结合区和Fc区，其中抗原结合区可以结合相同或不同的靶标，并且其中Fc区可以相同或不同。
如本文所用，术语“Fc区”是指免疫球蛋白重链的C端区域，其含有至少部分恒定区。Fc区没有抗原结合活性，是免疫球蛋白与效应分子或细胞相互作用的部位。该术语包括天然Fc区和变体Fc区。“天然Fc区”是指包含通过消化完整抗体产生的、无论是单体形式或是多聚体形式的非抗原结合片段的分子或序列。产生天然Fc区的免疫球蛋白源优选来源于人类。天然Fc片段由可以通过共价连接(例如二硫键)和非共价连接而连接为二聚体或多聚体形式的单体多肽构成。根据类别(例如IgG、IgA、IgE、IgD、IgM)或亚型(例如IgG1、IgG2、IgG3、IgA1、IgGA2)的不同，天然Fc分子单体亚基之间具有1-4个分子间二硫键。天然Fc区的一个实例是通过用木瓜蛋白酶消化IgG产生的二硫键连接的二聚体(参见Ellison等(1982)，Nucleic Acids Res.10：4071-9)。本文所用的术语“天 然Fc”一般是指单体、二聚体和多聚体形式。“变体Fc区”是指由于至少一个本文定义的“氨基酸修饰”而与“天然”或“野生型”Fc区的氨基酸序列不同的氨基酸序列，也称为“Fc变体”。因此，“Fc区”也包括单链Fc(scFc)，即，由多肽接头连接的两个Fc单体组成的单链Fc区，其能够自然折叠成功能性二聚体Fc区域。在一个实施方案中，变体Fc区与天然Fc区具有至少约80％、至少约85％、至少约90％，更优选至少约95％、96％、97％、98％或至少约99％的序列同一性。
在一个具体的实施方案中，本发明的Fc融合多肽包含的Fc区优选来源于IgG。根据IgG分子中的r链抗原性差异，人IgG有四个亚型：IgG1、IgG2、IgG3、IgG4，其中IgG1在血清中的分布丰度最高。这四种亚型的恒定区序列高度同源，但是各亚型与抗原结合、免疫复合物的形成、补体激活、触发效应细胞、半衰期和胎盘转运特性均具有特异性。一般而言，在结构与功能效应上IgG1和IgG3与Fc受体的亲和力高于IgG2和IgG4，具有更强的激活抗体依赖的细胞毒性及补体依赖的细胞毒性的能力；IgG2和IgG4亚型则具有阻碍或抑制的效应功能。因此，在一个优选的实施方案中，本发明的Fc融合多肽包含的Fc区优选来源于IgG1，以增强Fc区与受体的亲和力，从而提高对其他免疫细胞的招募效率。
在一个实施方案中，本发明的Fc区是指不包含CH1的恒定区。例如，在IgA、IgD和IgG的情况下，Fc区包含恒定结构域CH2和CH3；在IgE和IgM的情况下，Fc区包含恒定结构域CH2、CH3和CH4。此外，对于IgG，Fc区还可以包含CH1和CH2之间的下铰链区。因此，优选地，本发明的Fc区包含IgG1的CH2和CH3，更优选还包含CH1和CH2之间的下铰链区。在一个具体的实施方案中，Fc区与SEQ ID NO:10所示的氨基酸序列具有相同或相似的受体结合活性，并且与SEQ ID NO:10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。
核酸
本发明还提供一种核酸，其包含编码本发明的嵌合受体多肽的序列和编码本发明的Fc融合多肽的序列。
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。
本发明还提供一种载体***，其包含编码嵌合受体多肽的第一核酸序列和编码Fc融合多肽的第二核酸序列；所述第一核酸序列和第二核酸序列位于同一载体或不同载体。
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Fc融合多肽或嵌合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***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。
一个实施方案中，编码嵌合受体多肽的第一核酸序列和编码Fc融合多肽的第二核酸序列位于同一载体。例如，可以通过在两个核酸序列之间***编码2A肽的核酸，使得本发明的嵌合受体多肽和Fc融合多肽可以独立表达而互不影响。如本文所用，术语“2A肽”是一种cis-水解酶作用元件(CHYSEls)，最初在***病毒(FMDV)中发现。2A肽的平均长度为18～22氨基酸。在蛋白翻译时，2A肽可以通过核糖体跳跃从自身最后2个氨基酸C末端断裂。具体地，甘氨酸和脯氨酸之间的肽链结合群在2A位点是受损的，能引发核糖体跳跃而从第2个密码子开始翻译，从而使1个转录单元里的2个蛋白独立表达。这种2A肽介导的剪切广泛存在于真核动物细胞中。利用2A肽较高的剪切效率及促使上下游基因平衡表达的能力，可以改进异源多聚蛋白(如细胞表面受体、细胞因子、免疫球蛋白等)的表达效率。常规2A肽包含：P2A、T2A、E2A、F2A等。在另一个实施方案中，编码嵌合受体多肽的第一核酸序列和编码Fc融合多肽的第二核酸序列位于不同载体。
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，其包含嵌合受体多肽或其编码核酸，和Fc融合 多肽或其编码核酸，在本文中也称为Fite CAR(Fc induced target cell engaging Chimeric Antigen Receptor)细胞。因此，在一个实施方案中，本发明的工程化免疫细胞包含编码本发明的嵌合受体多肽的第一核酸序列和编码本发明的Fc融合多肽的第二核酸序列。
如本文所用，术语“免疫细胞”是指免疫***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、***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受体多肽和Fc融合多肽。
采用本领域已知的常规方法(如通过转导、转染、转化等)可以将编码嵌合受体多肽的第一核酸序列和编码Fc融合多肽的第二核酸序列引入免疫细胞，使其同时表达本发明的嵌合受体多肽和Fc融合多肽。“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。对于植物转化的技术包括土壤杆菌(Agrobacterium)介导的转移(诸如通过根瘤土壤杆菌(A.tumefaciens))、被快速推进的钨或金微弹、电穿孔、显微注射和聚乙二醇介导的摄取。
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，免疫细胞可以包含至少一种选择的TCRα或TCRβ基因的失活基因。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***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多链嵌合抗原受体、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，本发明还涵盖所述多链嵌合抗原受体、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、***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、多链嵌合抗原受体或其编码核酸或载体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。
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受体多肽和Fc融合多肽或这两者的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受体多肽和Fc融合多肽。
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***，如大分子复合物、纳米胶囊、微球、珠粒以及基于脂质的***，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，已经成为将基因***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多链嵌合抗原受体、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)在体外将本发明的多链嵌合受体或其编码核酸或载体或载体***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受体多肽和Fc融合多肽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***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，本发明的多链嵌合抗原受体所包含的两个或更多个肽链可以一起或分开施用。例如，可以向受试者分别施用包含第一Fc融合多肽的免疫细胞或药物组合物，和包含嵌合受体多肽的免疫细胞或药物组合物，以根据需要在合适的时候激活信号传导通路，活化免疫细胞的杀伤功能。在另一个实施方案中，所述治疗还可以包括向受试者进一步施用包含第二Fc融合多肽的免疫细胞或药物组合物，所述第二Fc融合多肽包含第二抗原结合区、第三蛋白质相互作用结构域和第二Fc区。
在一个实施方案中，当蛋白质相互作用结构域是化学诱导二聚化结构域时，治疗方法还包括向受试者施用可以诱导蛋白质相互作用结构域互相结合的化学物。例如，当蛋白质相互作用结构域分别是GIA和GID时，治疗方法还包括施用赤霉素；当蛋白质相互作用结构域分别是Snap标签和Halo标签时，治疗方法还包括施用HaXS；当蛋白质相互作用结构域分别是FRB和FKBP时，治疗方法还包括施用雷帕霉素及其衍生物例如光敏笼状雷帕霉素；当蛋白质相互作用结构域分别是PYL和ABI时，治疗方法还包括施用脱落酸；当蛋白质相互作用结构域分别是FKBP和CyP时，治疗方法还包括施用FKCsA；当蛋白质相互作用结构域分别是FKBP和CnA时，治疗方法还包括施用FK506；当蛋白质相互作用结构域分别是GR和DHFR时，治疗方法还包括施用Dex-Mtx。
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：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、***、绒毛膜癌、结肠和直肠癌、***癌症、消化***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、白血病、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、***癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸***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***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，可以用本发明的多链嵌合抗原受体、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。
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：本发明的一个优选实施方案的设计示意图。
图2：示出了Fite-CAR-1s和Fite-CAR-2s T细胞的scFv表达水平。
图3：示出了Fite-CAR-1s和Fite-CAR-2s T细胞对靶细胞的杀伤效果。
图4：示出了Fite-CAR-1s和Fite-CAR-2s T细胞中scFv-Fc融合多肽的分泌水平。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，**表示P值小于0.01，均达到显著水平。
图5：示出了Fite-CAR-2s T细胞中AZip和Fc融合多肽的表达。
图6：示出了Fite-CARX-1s和Fite-CARX-2s T细胞中sdAb-Fc的分泌水平(A)和对靶细胞的杀伤效果(B)。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，**表示P值小于0.01，均达到显著水平。
图7：示出了Fite-CARX-1s和Fite-CARX-2s T细胞的IFN-γ释放水平。
图8：示出了Fite-CARX-1s和Fite-CARX-2s T细胞的NK细胞杀伤效果。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，**表 示P值小于0.01，均达到显著水平。
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 Claudin18.2-scFv的核苷酸序列
SEQ ID NO：2 Claudin18.2-scFv的氨基酸序列
SEQ ID NO：3 AZip的核苷酸序列
SEQ ID NO：4 AZip的核苷酸序列
SEQ ID NO：5 BZip的核苷酸序列
SEQ ID NO：6 BZip的核苷酸序列
SEQ ID NO：7 Claudin18.2 sdAb的核苷酸序列
SEQ ID NO：8 Claudin18.2 sdAb的氨基酸序列
SEQ ID NO：9 Fc区的核苷酸序列
SEQ ID NO：10 Fc区的氨基酸序列
SEQ ID NO：11 跨膜结构域CD8α的核苷酸序列
SEQ ID NO：12 跨膜结构域CD8α的氨基酸序列
SEQ ID NO：13 共激活结构域4-1BB的核苷酸序列
SEQ ID NO：14 共激活结构域4-1BB的氨基酸序列
SEQ ID NO：15 信号传导结构域CD3ζ的核苷酸序列
SEQ ID NO：16 信号传导结构域CD3ζ的氨基酸序列
SEQ ID NO：17 连接肽IgG的核苷酸序列
SEQ ID NO：18 连接肽IgG的氨基酸序列
SEQ ID NO：19 信号肽CD8α的核苷酸序列
SEQ ID NO：20 信号肽CD8α的氨基酸序列
SEQ ID NO：21 信号肽GM-CSFRα的核苷酸序列
SEQ ID NO：22 信号肽GM-CSFRα的氨基酸序列
SEQ ID NO：23 F2A肽的核苷酸序列
SEQ ID NO：24 F2A肽的氨基酸序列
SEQ ID NO：25 CD8α铰链区的核苷酸序列
SEQ ID NO：26 CD8α铰链区的氨基酸序列
SEQ ID NO：27 IgG4铰链区的核苷酸序列
SEQ ID NO：28 IgG4铰链区的氨基酸序列
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。
实施例1：构建传统型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：19)、抗Claudin18.2scFv(SEQ ID NO：1)、CD8α铰链区(SEQ ID NO：25)、CD8α跨膜区(SEQ ID NO：11)、4-1BB共刺激结构域(SEQ ID NO：13)、CD3ζ胞内信号传导结构域(SEQ ID NO：15)，获得CAR质粒，并通过测序确认目标序列的正确***。
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/CD28 CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2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Claudin18.2的CAR T(即con-CAR T)细胞。
实施例2：构建Fite-CAR T细胞
构建Fite-CAR质粒：将CD8α信号肽(SEQ ID NO：19)、Claudin18.2-scFv(SEQ ID NO：1)、BZip(SEQ ID NO：5)、IgG连接肽(SEQ ID NO：17)、Fc区(SEQ ID NO：9)、F2A肽(SEQ ID NO：23)、GM-CSFRα信号肽(SEQ ID NO：21)、AZip(SEQ ID NO：3)、CD8α铰链区(SEQ ID NO：25)、CD8α跨膜区(SEQ ID NO：11)、4-1BB共刺激结构域(SEQ ID NO：13)、CD3ζ胞内信号传导结构域(SEQ ID NO：15)的编码序列克隆至pGEM-T Easy载体(Promega，货号A1360)，获得Fite-CAR-1s质粒，并通过测序确认目标序列的正确***。用同样的方法获得Fite-CAR-2s质粒，其包含的元件与Fite-CAR-1s相同，唯一的区别在于铰链区来自IgG4(SEQ ID NO：27)。
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Fite-CAR慢病毒。
用DynaBeads CD3/CD28CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2下培养1天。然后，加入浓缩的Fite-CAR慢病毒，持续培养3天后，获得Fite-CAR T细胞。
在37℃和5％CO 2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2所示(NT是未经修饰的野生型T细胞)。
可以看出，本发明的两种Fite-CAR T细胞均可有效表达scFv，表明嵌合受体多肽能够和Fc融合多肽特异性结合，并且表达水平与con-CAR相当。
实施例3：Fite-CAR T细胞的功能验证和优化
3.1检测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。
本实施例施用的293T-Claudin18.2靶细胞是用表达Claudin18.2的慢病毒感染293T细胞后，用流式细胞术筛选出的Claudin18.2阳性单克隆细胞。
为了检测Fite-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293T-Claudin18.2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Fite-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3所示。
可以看出，与NT相比，本发明的两种Fite-CAR T细胞均能够有效杀伤靶细胞，并且其杀伤效果与Con-CAR T细胞相当。
3.2检测scFv-Fc的分泌水平
如果Fite-CAR T细胞能够有效分泌scFv-Fc区，其就能被表达Fc受体(FcR)的免疫效应细胞包括NK细胞、巨噬细胞、树突状细胞等识别，从而招募这些免疫效应细胞，进一步增强对靶细胞的杀伤效果。因此，发明人使用酶联免疫吸附(ELISA)来检测Fite-CAR T细胞的scFv-Fc分泌水平。
将Fite-CAR-1s T细胞、Fite-CAR-2s T细胞、Con-CAR T和NT细胞分别在不包含IL-2的x-vivo 15培养基(Lonza，货号04-418Q)中，于37℃和5％CO2条件下培养。24小时后，收集培养物，并于4℃、1600rpm离心5分钟，获得细胞培养上清液。
使用捕获抗体Recombinant Human Claudin-18.2(N-8His)(近岸生物Novoprotein，货号CR53)包被96孔板4℃孵育过夜，然后移除上清液，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。移除上清液后，加入250μL PBST(含0.1％吐温的1XPBS)，清洗3次。然后每孔加入50μL细胞培养上清液，并在37℃孵育1小时。移除上清液，然后加入250μL PBST(含0.1％吐温的1x PBS)，清洗3次。然后向各孔分别加入50μL检测抗体HRP Goat anti-mouse IgG(Biolegend,货号405306)，在37℃孵育30分钟。弃上清液，加入250μL PBST(含0.1％吐温的1x PBS)，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50μL 1mol/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，并通过与NT细胞培养上清液的读值的比值来计算上清液中scFv-Fc融合多肽的相对表达水平，结果如图4所示。
出乎意料地，与Con-CAR T和NT细胞相比，Fite-CAR-1s T细胞和Fite-CAR-2s T细胞上清液中均没有检测到显著的scFv-Fc的表达。这可能是由于Fc融合多肽包含的scFv结构互相之间有黏连作用，从而通过蛋白质相互作用结构域之间的特异性结合被固定到了细胞膜上，最终影响了scFv-Fc的正常分泌。
为了验证上述假设，通过流式细胞仪，使用抗体anti-c-Fos(BOSTER，货号PA1318)、Biotin-羊抗兔IgG(BOSTER，货号BA1003)、APC Streptavidin(BD Pharmingen，货号554067)组合检测Fite-CAR T细胞上的嵌合受体表达(即，检测AZip的表达)，使用抗体PE anti-Human IgG Fc(Biolegend，货号409304)检测Fite-CAR-2s T细胞上Fc融合多肽的表达，结果如图5所示。
可以看出，与NT和con-CAR的结果相比，Fite-CAR-2s T细胞群中Fc阳性的细胞基本上也同时是AZip阴性(35.1％)，表明所检测到的Fc融合多肽均通过BZip和AZip之间的相互作用与嵌合受体多肽结合，导致AZip的结合位点被占据从而无法结合检测抗体。此外，Fite-CAR-2s T细胞群中AZip阳性且Fc阴性(0.31％)的细胞极少，也表明Fite-CAR-2s T细胞群中基本上不存在单独的未结合嵌合受体多肽。
以上结果证实，scFv-Fc融合多肽无法分泌是由蛋白质相互作用结构域之间的特异性结合以及scFv结构之间的黏连作用造成的。由于Fite-CAR-1s T细胞和Fite-CAR-2s T细胞无法有效分泌scFv-Fc，因此不能招募其他免疫细胞来增强CAR T细胞对靶细胞的杀伤效果。
实施例4：构建Fite-CARX T细胞
由于单域抗体(sdAb)独特的VHH结构，只含有重链区而不含轻链区，使 其有望解决实施例3中发现的由于潜在的VL与VH结构域的相互黏连而导致的scFv-Fc无法分泌的问题。因此，发明人用单域抗体(sdAb)替换scFv，获得Fite-CARX T细胞。
本实施例中使用的Fite-CARX-1s T细胞和Fite-CARX-2s T细胞包含的元件及其连接顺序分别与Fite-CAR-1s T细胞和Fite-CAR-2s T细胞相同，唯一区别在于用Claudin18.2sdAb(SEQ ID NO：7)替换Claudin18.2scFv(SEQ ID NO：1)。
实施例5：Fite-CARX T细胞的功能验证
根据实施例3.2所述的方法，检测Fite-CARX T细胞中的sdAb-Fc融合多肽的分泌水平，结果如图6A所示。
可以看出，与Con-CAR T细胞和NT细胞相比，两种Fite-CARX T上清液中均可以检测到显著分泌的sdAb-Fc融合多肽，表明单域抗体结构可有效避免scFv的相互黏连，从而促进Fc融合多肽的分泌。
另外，根据实施例3.1所述的方法，检测Fite-CARX T细胞对293T-Claudin18.2靶细胞的杀伤效果，结果如图6B所示。
可以看出，与NT相比，两种Fite-CARX T细胞能够有效杀伤靶细胞，并且杀伤效果与Con-CAR T细胞相当。
实施例6：Fite-CARX 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293T-Claudin18.2和非靶细胞293T分别铺于96孔板中，然后以1:1的比例将Fite-CARX T、Con-CAR T(阳性对照)和NT细胞(阴性对照)分别与靶细胞和非靶细胞共培养，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-γAntibody(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50μL 1mol/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7所示。
可以看出，在非靶细胞293T中均没有检测到IFNγ的释放，表明con-CAR T细胞和Fite-CARX T细胞的杀伤都是特异性的。并且，在杀伤靶细胞时，两种Fite-CARX T细胞的细胞因子IFN-γ的释放水平与Con-CAR T细胞相当。
实施例7：Fite-CARX T细胞介导NK细胞对靶细胞的杀伤效果
由于Fite-CARX T细胞能够高效杀伤靶细胞，并且显著分泌sdAb-Fc融合多肽，因此发明人进一步检测其是否能够介导NK细胞进行肿瘤杀伤。
通过以下方法获得本实施例使用的NK细胞：将小鼠脾脏研磨后，加入小鼠脾脏淋巴细胞分离液(TBD，货号LTS1092PK-200)，离心获得白膜层细胞。然后，加入PE anti-mouse NK1.1(Biolegend，货号108701)和Anti-PE Microbeads(美天旎，货号130-048-801)，在磁力架上进行阳性筛选，获得NK1.1阳性细胞。
本实施例施用的NUGC4-Claudin18.2靶细胞是用表达Claudin18.2的慢病毒感染NUGC4细胞后，用流式细胞术筛选出的Claudin18.2阳性单克隆细胞。
以1x10 4/孔将携带荧光素基因的NUGC4-Claudin18.2靶细胞铺入96孔板中。然后，分别使用Fite-CARX T细胞上清液和新鲜培养基(media)重悬NK细胞，并以4:1的效靶比(即效应NK细胞与靶细胞之比)将重悬的NK细胞加入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8所示。
可以看出，与新鲜培养基相比，两种Fite-CARX T细胞上清液均可以有效介导NK细胞对NUGC4-Claudin18.2靶细胞的杀伤，其效果显著高于新鲜培养基对照组。
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. 一种多链嵌合抗原受体，其包含：
    (a)Fc融合多肽，所述Fc融合多肽包含抗原结合区、第一蛋白质相互作用结构域和Fc区；和
    (b)嵌合受体多肽，所述嵌合受体多肽包含第二蛋白质相互作用结构域、跨膜结构域和胞内信号传导结构域，
    其中所述第一蛋白质相互作用结构域能够和第二蛋白质相互作用结构域特异性结合。
  2. 根据权利要求1所述的多链嵌合抗原受体，其中所述第一抗原结合区选自sdAb、纳米抗体、抗原结合配体、重组纤连蛋白结构域、anticalin和DARPIN。
  3. 根据权利要求1所述的多链嵌合抗原受体，其中所述第一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。
  4. 根据权利要求1-3任一项所述的多链嵌合抗原受体，其中所述第一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、***蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、***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。
  5. 根据权利要求4所述的多链嵌合抗原受体，其中所述靶标选自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。
  6. 根据权利要求1-5任一项所述的多链嵌合抗原受体，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。
  7. 根据权利要求1-6任一项所述的多链嵌合抗原受体，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。
  8. 根据权利要求1-7任一项所述的多链嵌合抗原受体，其中所述嵌合受体多肽还包含一个或多个共刺激结构域。
  9. 根据权利要求8所述的多链嵌合抗原受体，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. 根据权利要求1-9任一项所述的多链嵌合抗原受体，其中所述第一Fc区包含CH2结构域和CH3结构域。
  11. 根据权利要求1-10任一项所述的多链嵌合抗原受体，其中所述第一蛋白质相互作用结构域和第二蛋白质相互作用结构域是选自以下的组合：Fk506结合蛋白(FKBP)和mTOR的FKBP-雷帕霉素结合结构域(FRB)、FKBP和钙调磷酸酶A(CnA)、FKBP和亲环素(CyP)、GAI和GID、Snap标签和Halo标签、糖皮质激素受体(GR)和DHFR、PYL和ABI、cAMP依赖性的蛋白激酶A(PKA)的二聚化对接结构域和A-激酶锚定蛋白(AKAP)的锚定域、亲和素和生物素、亮氨酸拉链结构域和亮氨酸拉链结构域以及锌指结构域和核苷酸标签。
  12. 根据权利要求11所述的多链嵌合抗原受体，其中所述第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是AZip和BZip。
  13. 根据权利要求11所述的多链嵌合抗原受体，其中所述第一蛋白质相互作用结构域和第二蛋白质相互作用结构域分别是锌指结构域和核苷酸标签。
  14. 根据权利要求1所述的多链嵌合抗原受体，进一步包含第二Fc融合多肽，所述第二Fc融合多肽包含第二抗原结合区、第三蛋白质相互作用结构域和第二Fc区。
  15. 根据权利要求14所述的多链嵌合抗原受体，其中所述第一蛋白质相互作用结构域是锌指结构域，所述第二蛋白质相互作用结构域是核苷酸标签的第一部分，所述第三蛋白质相互作用结构域是核苷酸标签的第二部分，并且仅当所述核苷酸标签的第一部分和核苷酸标签的第二部分形成复合物时，所述复合物才能与所述锌指结构域特异性结合。
  16. 一种核酸，其包含编码嵌合受体多肽的第一核酸序列和编码Fc融合多肽的第二核酸序列，所述嵌合受体多肽和所述Fc融合多肽构成根据权利要求1-15任一项所述的多链嵌合抗原受体。
  17. 一种载体，包含根据权利要求16所述的核酸。
  18. 一种载体***，其包含编码嵌合受体多肽的第一核酸序列和编码Fc融合多肽的第二核酸序列，所述嵌合受体多肽和所述Fc融合多肽构成根据权利要求1-15任一项所述的多链嵌合抗原受体，所述第一核酸序列和第二核酸序列位于不同载体。
  19. 一种免疫细胞，其包含根据权利要求1-15任一项所述的多链嵌合抗原受体、根据权利要求16所述的核酸、根据权利要求17所述的载体，或根据权利要求18所述的载体***。
  20. 根据权利要求19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。
  21. 根据权利要求19-20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。
  22. 根据权利要求21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。
  23. 一种药物组合物，包含根据权利要求1-15任一项所述的多链嵌合抗原受体、根据权利要求16所述的核酸、根据权利要求17所述的载体、根据权利要求18所述的载体***或根据权利要求19-22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。
  24. 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权利要求1-15任一项所述的多链嵌合抗原受体、根据权利要求16所述的核酸、根据权利要求17所述的载体、根据权利要求18所述的载体***或根据权利要求 19-22任一项所述的免疫细胞或者根据权利要求23所述的药物组合物。
  25. 根据权利要求24所述的方法，其中所述癌症选自：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、***、绒毛膜癌、结肠和直肠癌、***癌症、消化***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、***癌、视网膜母细胞瘤、横纹肌肉瘤、直肠癌、呼吸***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***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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