WO2021147929A1 - Récepteur antigénique chimérique à chaînes multiples et son utilisation - Google Patents

Récepteur antigénique chimérique à chaînes multiples et son utilisation 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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Chinese (zh)
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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
    • C07—ORGANIC CHEMISTRY
    • 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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    • A61K35/14—Blood; Artificial blood
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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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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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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

La présente invention concerne un récepteur antigénique chimérique à chaînes multiples, qui comprend : (a) un récepteur chimérique, comprenant un premier domaine d'interaction avec des protéines, un domaine transmembranaire et un domaine de signalisation intracellulaire ; et (b) un polypeptide de fusion Fc, comprenant une région de liaison à un antigène, un second domaine d'interaction avec des protéines et une région Fc, le premier domaine d'interaction avec des protéines pouvant se lier spécifiquement au second domaine d'interaction avec des protéines. L'invention concerne également une cellule immunitaire modifiée comprenant le récepteur antigénique chimérique à chaînes multiples selon la présente invention et une composition pharmaceutique associée, ainsi que l'utilisation de la cellule immunitaire modifiée/composition pharmaceutique dans le traitement d'un cancer.
PCT/CN2021/072980 2020-01-21 2021-01-21 Récepteur antigénique chimérique à chaînes multiples et son utilisation WO2021147929A1 (fr)

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CN111234033B (zh) * 2020-01-21 2021-05-11 南京北恒生物科技有限公司 多链嵌合抗原受体及其用途
CN111826400A (zh) * 2020-07-21 2020-10-27 中科宝承生物医学科技有限公司 一种双特异性抗体nk细胞制备方法及其细胞和应用
WO2022104267A1 (fr) * 2020-11-16 2022-05-19 Ab Therapeutics, Inc. Anticorps multispécifiques et leurs utilisations
WO2022179562A1 (fr) * 2021-02-24 2022-09-01 Hangzhou Qihan Biotechnology Co., Ltd. Récepteurs chimériques à l'antigène dans des cellules immunitaires
CN115216449A (zh) * 2021-04-16 2022-10-21 南京北恒生物科技有限公司 工程化免疫细胞及其用途
CN113337514B (zh) * 2021-08-05 2021-10-29 卡瑞济(北京)生命科技有限公司 Tcr表达构建体以及其制备方法和用途
CN113980138B (zh) * 2021-08-11 2023-08-11 卡瑞济(北京)生命科技有限公司 EphA2嵌合抗原受体以及其用途
CN113896801B (zh) * 2021-10-08 2022-05-17 南京凯地医疗技术有限公司 靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用
CN114225008B (zh) * 2021-12-29 2024-03-08 中国医学科学院医学生物学研究所 转录因子btb-cnc同源体1在非霍奇金淋巴瘤治疗中的应用
CN114621355A (zh) * 2022-02-13 2022-06-14 黄凯旋 一种靶向特异性b淋巴细胞的嵌合抗原受体及其应用
WO2024114767A1 (fr) * 2022-12-01 2024-06-06 南京北恒生物科技有限公司 Cellule modifiée résistante au rejet immunitaire

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015090229A1 (fr) * 2013-12-20 2015-06-25 Novartis Ag Récepteur d'antigène chimérique régulable
WO2015166056A1 (fr) * 2014-05-02 2015-11-05 Cellectis Récepteur antigénique chimérique à chaînes multiples spécifique de cs1
CN107406518A (zh) * 2015-03-23 2017-11-28 Ucl商务股份有限公司 嵌合抗原受体
CN108495927A (zh) * 2015-11-23 2018-09-04 波士顿大学董事会 嵌合抗原受体相关的方法和组合物
CN111234033A (zh) * 2020-01-21 2020-06-05 南京北恒生物科技有限公司 多链嵌合抗原受体及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013167153A1 (fr) * 2012-05-09 2013-11-14 Ganymed Pharmaceuticals Ag Anticorps utiles dans le diagnostic du cancer
US10093736B2 (en) * 2012-11-13 2018-10-09 Biontech Ag Agents for treatment of claudin expressing cancer diseases
WO2014145252A2 (fr) * 2013-03-15 2014-09-18 Milone Michael C Ciblage de cellules cytotoxiques par des récepteurs chimériques pour une immunothérapie adoptive
CN109206524B (zh) * 2018-09-25 2022-04-05 山东兴瑞生物科技有限公司 抗Claudin18A2嵌合抗原受体、其修饰的T细胞及T细胞制备方法和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015090229A1 (fr) * 2013-12-20 2015-06-25 Novartis Ag Récepteur d'antigène chimérique régulable
WO2015166056A1 (fr) * 2014-05-02 2015-11-05 Cellectis Récepteur antigénique chimérique à chaînes multiples spécifique de cs1
CN107406518A (zh) * 2015-03-23 2017-11-28 Ucl商务股份有限公司 嵌合抗原受体
CN108495927A (zh) * 2015-11-23 2018-09-04 波士顿大学董事会 嵌合抗原受体相关的方法和组合物
CN111234033A (zh) * 2020-01-21 2020-06-05 南京北恒生物科技有限公司 多链嵌合抗原受体及其用途

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