WO2022222846A1 - 靶向cd19的嵌合抗原受体、制备方法及其应用 - Google Patents

靶向cd19的嵌合抗原受体、制备方法及其应用 Download PDF

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WO2022222846A1
WO2022222846A1 PCT/CN2022/086904 CN2022086904W WO2022222846A1 WO 2022222846 A1 WO2022222846 A1 WO 2022222846A1 CN 2022086904 W CN2022086904 W CN 2022086904W WO 2022222846 A1 WO2022222846 A1 WO 2022222846A1
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nucleic acid
cell
cells
immune cell
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French (fr)
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李建强
刘莹
王琳
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河北森朗生物科技有限公司
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • 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
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • A61K2039/80—Vaccine for a specifically defined cancer
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • 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
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    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/106—Plasmid DNA for vertebrates
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology, in particular to a chimeric antigen receptor targeting CD19, a preparation method and an application thereof.
  • Immunotherapy represented by CAR-T Chimeric Antigen Receptor T-Cell, chimeric antigen receptor-modified T cells
  • CAR-T Chimeric Antigen Receptor T-Cell, chimeric antigen receptor-modified T cells
  • its principle is mainly to modify the chimeric antigen receptor on T cells extracted from the patient's own by means of genetic engineering CAR-T cells are formed, which can specifically recognize tumor surface-associated antigens (tumor cell markers) to target and kill tumors.
  • CAR-T cells have higher targeting, killing activity and persistence, and can overcome the local immunosuppressive microenvironment of the tumor and break the host immune tolerance state.
  • the modified immune cell therapy represented by CAR-T cells has a significant effect in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatments.
  • CAR-T immunotherapy is one of the most disruptive emerging technologies at present.
  • countries in Europe, America and Asia are actively conducting clinical trials of this therapy. Among them, China has carried out the most clinical trial projects, reaching 204, accounting for the global CAR-T immunotherapy. 50% of clinical trials of therapies.
  • the University of Pennsylvania Hospital, Children's Hospital of Philadelphia, the National Cancer Institute (NCI), Fred Hutchinson Cancer Research Center, Memorial Sloan Kettering Cancer Center and Seattle Children's Hospital were among the earliest research institutions to develop CART cell therapy.
  • Emerging international biopharmaceutical companies such as Juno, Kite, Celgene, Cellectis, Bluebird, and traditional international pharmaceutical giants such as Novartis, Bristol-Myers Squibb, Pfizer, Johnson & Johnson, Gilead, etc.
  • CAR-T products targeting CD19 generally suffer from fever or neurotoxic side effects caused by cytokine release syndrome, which brings great challenges to the extensive clinical use and clinical treatment of this product.
  • the rise and application of synthetic biology technology provides more options for developing safe and efficient CAR-T for CAR-T immunotherapy of tumors, and provides inspiration for the development of new safer CAR-T therapies and drugs.
  • the present invention provides a chimeric antigen receptor targeting CD19, the chimeric antigen receptor includes the following components in sequence: extracellular antigen binding domain, spacer domain, transmembrane domain, costimulatory signal transduction domain, primary signaling domain.
  • the extracellular antigen binding domain is an scFv that specifically binds to CD19; the amino acid sequence of the scFv is shown in SEQ ID NO.1.
  • the spacer domain is an IgG4 hinge region; preferably, the amino acid sequence of the IgG4 hinge region is shown in SEQ ID NO.2.
  • the transmembrane domain is the transmembrane region of CD28; preferably, the amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO.3.
  • the costimulatory signaling domain is the intracellular signaling domain of 4-1BB; preferably, the amino acid sequence of the intracellular signaling domain of 4-1BB is shown in SEQ ID NO.4.
  • the primary signaling domain is the intracellular signaling domain of CD3 ⁇ ; preferably, the amino acid sequence of the intracellular signaling domain of CD3 ⁇ is the sequence shown in SEQ ID NO.5.
  • the chimeric antigen receptor further comprises a signal peptide at its N-terminus; preferably, the signal peptide is a granulocyte-macrophage colony-stimulating factor receptor 2 signal peptide.
  • the chimeric antigen receptor further comprises a self-cleaving peptide at its C-terminus; preferably, the self-cleaving peptide is T2A.
  • the self-cleaving peptide is attached to the C-terminus of the primary signaling domain.
  • the amino acid sequence of T2A is shown in SEQ ID NO.7.
  • the chimeric antigen receptor further comprises a cell surface marker at its C-terminus.
  • Cell surface markers are used to determine whether the chimeric antigen receptor is expressed, in particular, the cell surface markers are used to determine the transduction or engineering of the cells for expressing the chimeric antigen receptor, such as cells Truncated forms of surface receptors, such as truncated EGFR (tEGFR).
  • the marker comprises CD34, NGFR, or epidermal growth factor receptor (eg, tEGFR), or a functional variant thereof, in whole or in part (eg, a truncated form).
  • the cell surface marker is a fragment (tEGFR) containing extracellular domain III and extracellular domain IV in EGFR, preferably, the amino acid sequence of tEGFR is shown in SEQ ID NO.8.
  • amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.6.
  • Chimeric antigen receptors of embodiments of the invention may comprise synthetic amino acids in place of one or more naturally occurring amino acids.
  • synthetic amino acids include, for example, aminocyclohexanecarboxylic acid, norleucine, alpha-amino n-decanoic acid, homoserine, S-acetamidomethyl-cysteine, Trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine Amino acid, ⁇ -phenylserine, ⁇ -hydroxyphenylalanine, phenylglycine, ⁇ -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic
  • Chimeric antigen receptors of embodiments of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted to an acid addition salt and /or optionally dimerized or multimerized.
  • Chimeric antigen receptors of embodiments of the present invention can be obtained by methods known in the art.
  • Chimeric antigen receptors can be made by any suitable method for making polypeptides or proteins. Suitable methods for de novo synthesis of polypeptides and proteins are known in the art.
  • chimeric antigen receptors can be produced recombinantly using nucleic acids as described herein using standard recombinant methods as described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press (2012). .
  • the chimeric antigen receptors described herein can be synthesized commercially by companies.
  • the chimeric antigen receptors of the invention may be synthetic and/or recombinant.
  • the present invention provides a nucleic acid encoding the aforementioned chimeric antigen receptor.
  • the nucleic acid further comprises a promoter, and/or an enzyme cleavage site located behind the promoter, and/or a kozak sequence located after the enzyme cleavage site.
  • the promoter is located at the N-terminus of the nucleic acid, and the promoter is the promoter of elongation factor 1 ⁇ ; preferably, the promoter sequence is shown in SEQ ID NO.9.
  • restriction site is the recognition site of Pac I; preferably, the sequence of the restriction site is shown in positions 1-8 in SEQ ID NO.10.
  • kozak sequence is shown in positions 9-14 in SEQ ID NO.10.
  • the coding sequence of the signal peptide in the nucleic acid is shown in positions 15-78 in SEQ ID NO.10.
  • the coding sequence of scFv in the nucleic acid is shown in positions 79-822 in SEQ ID NO.10.
  • the coding sequence of the spacer domain in the nucleic acid is shown in positions 823-846 in SEQ ID NO.10.
  • the coding sequence of the transmembrane domain in the nucleic acid is shown in positions 847-939 in SEQ ID NO.10.
  • the coding sequence of the co-stimulatory signaling domain of the nucleic acid is shown in positions 940-1068 in SEQ ID NO.10.
  • the coding sequence of the primary signaling domain in the nucleic acid is shown in positions 1069-1404 in SEQ ID NO.10.
  • nucleotide sequence of the T2A is shown in positions 1405-1476 in SEQ ID NO.10.
  • nucleotide sequence of the tEGFR is shown in positions 1477-2550 in SEQ ID NO.10.
  • the coding sequence of the chimeric antigen receptor in the nucleic acid is shown in positions 15-2585 in SEQ ID NO.10.
  • the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.10.
  • the present invention provides a vector comprising the aforementioned nucleic acid.
  • the vector of the present invention can be any suitable vector and can be used to transform or transfect any suitable host cell.
  • Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
  • the vector can be selected from: pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).
  • Phage vectors such as ⁇ GT10, ⁇ GT11, ⁇ ZapII (Stratagene), ⁇ EMBL4 and ⁇ NM1149 can also be used.
  • vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • the present invention provides a recombinant microorganism comprising the aforementioned nucleic acid or the aforementioned vector; preferably, the recombinant microorganism is a recombinant virus.
  • the present invention provides an engineered immune cell that expresses the aforementioned chimeric antigen receptor; preferably, the immune cell contains the aforementioned nucleic acid or the aforementioned vector.
  • the present invention provides an immune cell population, the immune cell population includes the aforementioned engineered immune cells; preferably, the immune cell population also includes unmodified or incompletely engineered immune cells.
  • the present invention provides a method for preparing the aforementioned engineered immune cells, which comprises introducing the aforementioned nucleic acid or the aforementioned vector into the immune cells.
  • nucleic acids and corresponding vectors into host cells.
  • Methods include, but are not limited to, calcium phosphate transfection, electroporation, lipofection, use of transfection reagents such as cationic lipids, biolistic-mediated or polymer-mediated transfer.
  • the immune cells of the present invention may be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC).
  • PBL peripheral blood lymphocytes
  • PBMC peripheral blood mononuclear cells
  • B cells B cells, natural killer (NK) cells or T cells.
  • the T cells of the present invention can be any T cells, such as cultured T cells, such as primary T cells; or T cells from cultured T cell lines, such as Jurkat, SupT1, etc.; or T cells obtained from mammals . If obtained from a mammal, T cells can be obtained from a variety of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cells can be human T cells. The T cells can be T cells isolated from humans.
  • T cells can be of any type and at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells (eg Th 1 and Th 2 cells), CD8+ T cells (eg cells Toxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc.
  • the T cells can be CD8+ T cells or CD4+ T cells.
  • the present invention provides a composition comprising the aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned vector, the aforementioned recombinant microorganism, the aforementioned engineered Immune cells, the aforementioned population of immune cells.
  • the composition comprises a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is one which is free from deleterious side effects or toxicity under the conditions of use.
  • Methods of preparing administrable medicaments are known or apparent to those skilled in the art, and are described in more detail, e.g., in Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press (2012).
  • compositions of the present invention may also comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or suitable preservatives preparation. Acceptable ingredients of the compositions are not toxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one route can be used to administer the nucleic acids, vectors, immune cells of the invention, and in some cases a particular route can provide a more direct and effective response than another route.
  • compositions of the present invention may also include other pharmaceutically active agents or drugs, such as chemotherapeutic agents, eg, asparaginase, busulfan, administered in combination with the nucleic acids, vectors, immune cells of the present invention.
  • chemotherapeutic agents eg, asparaginase, busulfan
  • Carboplatin Cisplatin, Daunomycin, Doxorubicin, Fluorouracil, Gemcitabine, Hydroxyurea, Methotrexate, Taxol, Rituximab, Vinblastine, Vincristine, etc.
  • nucleic acid, vector, immune cell of the invention When a nucleic acid, vector, immune cell of the invention is administered with one or more additional therapeutic agents, the one or more additional therapeutic agents can be co-administered to the mammal.
  • “Co-administration” means administering one or more additional therapeutic agents and a nucleic acid, vector, immune cell of the invention close enough in time so that the nucleic acid, vector, immune cell of the invention can enhance one or more effect of an additional therapeutic agent, and vice versa.
  • the nucleic acid, vector, immune cell of the invention can be administered first, followed by one or more additional therapeutic agents, or vice versa.
  • the nucleic acid, vector, immune cell and one or more additional therapeutic agents of the invention can be administered concurrently.
  • the present invention provides a kit comprising the aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned vector, the aforementioned recombinant microorganism, the aforementioned modified An immune cell, a population of immune cells as described above, a composition as described above.
  • the present invention provides an application, and the application includes the application described in any one of the following:
  • the aforementioned chimeric antigen receptor is used to prepare the aforementioned nucleic acid, the aforementioned vector, the aforementioned recombinant microorganism, the aforementioned immune cell, the aforementioned immune cell population, the aforementioned The composition, the application in the aforementioned kit;
  • the aforementioned nucleic acid is used to prepare the aforementioned vector, the aforementioned recombinant microorganism, the aforementioned immune cell, the aforementioned immune cell population, the aforementioned composition, and the aforementioned kit. application in;
  • the present invention provides a method for a disease or condition associated with CD19 expression, the method comprising administering the aforementioned nucleic acid, the aforementioned vector, the aforementioned recombinant microorganism, the aforementioned immunization to a person in need thereof A cell, the aforementioned population of immune cells, the aforementioned composition.
  • Diseases or disorders associated with CD19 expression of the present invention include tumors, autoimmune and inflammatory diseases, and infectious diseases.
  • the tumors include B cell malignancies, such as B cell leukemias and lymphomas, including B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, Common acute lymphoblastic leukemia, non-acute lymphoblastic leukemia, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, splenic and marginal zone lymphoma , mantle cell lymphoma, indolent B-cell lymphoma, Hodgkin lymphoma.
  • B cell chronic lymphocytic leukemia CLL
  • ALL acute lymphocytic leukemia
  • prolymphocytic leukemia hairy cell leukemia
  • Common acute lymphoblastic leukemia non-acute lymphoblastic leukemia
  • non-Hodgkin lymphoma non
  • the B-cell malignancy is relapsed CD19 positive leukemia.
  • Exemplary autoimmune and inflammatory diseases include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).
  • the dosing regimen may be to administer a bolus infusion
  • multiple divided doses may be administered over time, or the dose may be decreased or increased in proportion to the exigencies of the therapeutic situation.
  • the treatment regimen may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the dosing regimen includes intravenous administration, and the administered dose may range from 0.1 ⁇ 10 8 to 3 ⁇ 10 8 CAR-positive immune cells, eg, 0.15 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive immune cells, 0.5 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive immune cells, 1 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive immune cells, 0.2 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive immune cells, 0.2 ⁇ 10 8 to 1 ⁇ 10 8 CAR-positive immune cells, 0.25 ⁇ 10 8 to 1 ⁇ 10 8 CAR-positive immune cells, 0.25 ⁇ 10 8 to 0.5 ⁇ 10 8 CAR-positive immune cells, or 0.5 ⁇ 10 8 CAR-positive immune cells, or 2 ⁇ 10 8 CAR positive immune cells.
  • the dosage can be different.
  • the administered dose of the immune effector cells for relapsed and refractory adult acute lymphoblastic leukemia patients may be 0.25 ⁇ 10 8 to 0.5 ⁇ 10 8 CAR-positive immune cells, or 0.5 ⁇ 10 8 CAR-positive immune cells Immune cells, for example, may be 0.3 ⁇ 10 8 to 0.5 ⁇ 10 8 , 0.4 ⁇ 10 8 to 0.5 ⁇ 10 8 , 0.25 ⁇ 10 8 to 0.4 ⁇ 10 8 , 0.3 ⁇ 10 8 to 0.4 ⁇ 10 8 or 0.4 ⁇ 10 8 to 0.5 ⁇ 10 8 CAR-positive immune cells.
  • the administration dose of the immune effector cell therapy for relapsed and refractory adult acute lymphoblastic leukemia patients can be 0.25 ⁇ 10 8 , 0.26 ⁇ 10 8 , 0.27 ⁇ 10 8 , 0.28 ⁇ 10 8 , 0.29 ⁇ 10 8 , 0.3 ⁇ 10 8 , 0.31 ⁇ 10 8 , 0.32 ⁇ 10 8 , 0.33 ⁇ 10 8 , 0.34 ⁇ 10 8 , 0.35 ⁇ 10 8 , 0.36 ⁇ 10 8 , 0.37 ⁇ 10 8 , 0.38 ⁇ 10 8 , 0.39 ⁇ 10 8 , 0.4 ⁇ 10 8 , 0.41 ⁇ 10 8 , 0.42 ⁇ 10 8 , 0.43 ⁇ 10 8 , 0.44 ⁇ 10 8 , 0.45 ⁇ 10 8 , 0.46 ⁇ 10 8 , 0.47 ⁇ 10 8 , 0.48 ⁇ 10 8 , 0.49 ⁇ 10 8 or 0.5 ⁇ 10 8 CAR-positive immune cells.
  • the administration dose of the immune effector cells in the treatment of relapsed and refractory non-Hodgkin lymphoma patients can be 1 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive immune cells, or 2 ⁇ 10 8 CAR Positive immune cells, such as 1 ⁇ 10 8 to 1.8 ⁇ 10 8 , 1 ⁇ 10 8 to 1.5 ⁇ 10 8 , 1 ⁇ 10 8 to 1.3 ⁇ 10 8 , 1.3 ⁇ 10 8 to 2 ⁇ 10 8 , 1.3 ⁇ 10 8 to 1.5 ⁇ 10 8 , 1.5 ⁇ 10 8 to 2 ⁇ 10 8 , 1.5 ⁇ 10 8 to 1.8 ⁇ 10 8 or 1.8 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive immune cells.
  • the administered dose of the immune effector cells for relapsed and refractory Hodgkin lymphoma patients can be 1 ⁇ 10 8 , 1.1 ⁇ 10 8 , 1.2 ⁇ 10 8 , 1.3 ⁇ 10 8 , 1.4 ⁇ 10 8 , 1.5 ⁇ 10 8 , 1.6 ⁇ 10 8 , 1.7 ⁇ 10 8 , 1.8 ⁇ 10 8 , 1.9 ⁇ 10 8 or 2.0 ⁇ 10 8 CAR-positive immune cells.
  • the present invention utilizes a fully human scFv targeting CD19 to construct a CAR-T.
  • the CAR-T of the present invention has lower immunogenicity, and is especially suitable for those resistant to mouse-derived CAR-T. Patients with relapsed CD19+ leukemia.
  • Figure 1 shows a schematic diagram of the specific structure of the human H19-CAR gene
  • Figure 2 shows a schematic diagram of the specific structure of the murine M19-CAR gene
  • Figure 3 shows the CAR-T cell expansion curve
  • Figure 4 shows a statistical graph of CAR transfection rate
  • Figure 5 shows the results of the killing ability of CAR-T cells to Nalm-6 cells
  • Figure 6 shows the statistical chart of the results of cytokine secretion by CAR-T cells
  • Fig. 7 shows the image of the animal in vivo
  • Figure 8 shows a graph of fluorescence intensity statistics.
  • the scfv fragment of fully human or murine CD19 was cloned into the second-generation CAR structural backbone with 4-1BB and CD3 ⁇ , and the extracellular tEGFR domain was connected with T2A to obtain the name H19-CAR (fully human) or M19 -CAR (mouse origin).
  • the specific structure of the H19-CAR gene is antiHCD19 scFv-IgG4 hinge-CD28TM-4-1BB-CD3 ⁇ -T2A-tEGFR
  • the specific structure of the M19-CAR gene is antiMCD19 scFv-IgG4 hinge-CD28TM-4-1BB-CD3 ⁇ -T2A-tEGFR ( Figure 1 and Figure 2).
  • anti HCD19 scFv is the coding sequence of anti-fully human CD19 single-chain variable fragment (scFv)
  • antiMCD19 scFv is the coding sequence of anti-mouse CD19 single-chain variable fragment (scFv)
  • IgG4 hinge is the hinge region
  • CD28TM is The transmembrane region
  • 4-1BB is the co-stimulatory domain
  • CD3 ⁇ is the intracellular signal transduction domain
  • the extracellular region of tEGFR is expressed through T2A peptide connection, so that the expression of CAR can be detected after viral transduction.
  • the coding sequence of the H19-CAR gene coding chain is the 15-2585th position of SEQ ID NO.10, the 1-8th position in SEQ ID NO.10 is the recognition site of Pac I, and the 15th-78th position is the signal peptide.
  • Coding sequence position 79-822 is the coding sequence of fully human anti-CD19 single-chain variable fragment (scFv), position 823-846 hinge sequence, position 847-939 is the CD28TM gene sequence, position 940-1068 is 4-1BB gene sequence, the 1069-1404th position is the CD3 ⁇ gene sequence, the 1405-1476th position is the T2A gene sequence, and the 1477th-2550th position is the tEGFR gene sequence.
  • the amino acid sequence of protein H19-CAR is shown in SEQ ID NO.6 in the sequence listing.
  • the H19-CAR gene expression vector (lentiviral expression vector) is a full-sequence synthesis, named Senl-H19, and the sequence is shown in SEQ ID NO.13.
  • Senl-H19 contains the coding region of the H19-CAR gene shown in No. 79-2550 in SEQ ID NO.10, and the amino acid sequence that can express in cells is the protein H19-CAR shown in SEQ ID NO.6 in the sequence listing.
  • the successfully constructed lentiviral expression vector was identified by Pac I and SpeI digestion and sequenced correctly, and then used for lentiviral packaging.
  • Senl_H19 Replace the scfv of fully human CD19 in Senl_H19 with the scfv of mouse CD19 (the amino acid sequence is shown in SEQ ID NO.12, and the nucleotide sequence is shown in SEQ ID NO.11), and the Senl_M19 expression vector is prepared.
  • the successfully constructed lentiviral expression vector was identified by Pac I and SpeI digestion and sequenced correctly, and then used for lentiviral packaging.
  • the 293FT cells used to package the virus were taken out from the cell incubator at 37°C 5% CO 2 , the above mixture was added evenly to each plate, shaken gently, and placed in a 37° C 5% CO 2 incubator. After 4h-6h, discard the old medium, add 5mL of pre-warmed PBS to wash the cells, then add 20ml of fresh pre-warmed DMEM medium containing 10% fetal bovine serum, put it into a 37°C 5% CO 2 incubator cultivated in.
  • the culture supernatant as the virus stock solution After culturing for 48h-72h, collect the culture supernatant as the virus stock solution, collect the stock solution into a 50ml centrifuge tube, centrifuge at 2000r/min for 5min to remove the cell precipitate, and then use a 50ml syringe to filter the virus stock solution through a 0.45 ⁇ m filter to a new 50ml In a centrifuge tube, centrifuge at 18300g for 2h at 4°C. This is the virus concentrate. The virus concentrate was divided into 100 ⁇ l/tube, and another 10 ⁇ l was reserved for virus titer determination. Store the aliquoted concentrate at -80°C.
  • PBMC peripheral blood mononuclear cells
  • the peripheral blood can be either a healthy donor or the patient's own peripheral blood.
  • Disperse the cell precipitation layer add sterile saline to make the volume to 50mL, invert the centrifuge tube upside down, mix evenly, and then transfer to a centrifuge, centrifuge at 800g for 5 minutes at 4°C, and the obtained precipitate is mononuclear cells.
  • cell sorting solution DPBS+0.5%HSA
  • MSC magnetic separation beads
  • the centrifuge tube containing the sorted positive cells was mixed and centrifuged at 800 g for 5 min at 4°C. After centrifugation, the supernatant was discarded, and resuspended in TexMACS GMP serum-free medium (MACS) containing 200UI/mL IL-2, and then followed by T cells and activated magnetic beads CD3/CD28 (Thermo Fisher) 1: Add activated magnetic beads at a ratio of 1, and put them into a 37°C, 5% CO 2 cell incubator for culture.
  • cell culture mediums are TexMACS GMP serum-free medium containing 200UI/mL IL-2.
  • T cells were prepared according to the above-mentioned method for T cell isolation and activation.
  • the sorted T cells were activated by adding Dynabeads CD3/CD28 activated magnetic beads, placed in a 37°C, 5% CO 2 cell incubator for culture (recorded as Day0), and after 48 hours of culture, concentrated with the virus prepared above.
  • the solution was added to T cells, centrifuged for 2 hours (2000rpm, 35°C, speed-up 4, speed-down 4) for lentiviral transfection, after transfection was completed, put it into a 37°C, 5% CO2 cell incubator for culture, Day5 Samples were taken for CAR+ ratio detection by flow cytometry.
  • the cells were harvested from Day12 to Day14, the medium was removed by washing, and the cells were washed with 0.9% saline containing 1% HSA to obtain T cells expressing CAR targeting CD19, and CAR-T cells expressing fully human scFv were called Senl_H19 cells, CAR-T cells expressing murine scFv are called Senl_M19 cells.
  • the expansion curve of CAR-T cells is shown in Figure 3, and the CAR transfection rate is shown in Figure 4.
  • the expansion folds of Senl_H19 cells and Senl_M19 cells were not significantly different, and the expansion folds were >100 times when cultured for 14 days.
  • the CAR positive rate of Senl_H19 cells was 52% ⁇ 0.14%, which was not significantly different from the CAR positive rate of Senl_M19, 53.77% ⁇ 12.2%.
  • the results showed that, compared with murine scFv, human scFv had no significant effect on cell growth and CAR transfection.
  • an appropriate amount of effector cells were added to the target cells, mixed and incubated for 4 hours, and the cells were detected by flow cytometry kill ratio.
  • Cells were stained with 2 ⁇ L of 7AAD and Anexxin V and incubated in the dark for 15 min.
  • the killing rate (%) of CFSE-labeled target cells was the sum of the ratios of Annexin V+/7AAD-, Annexin V+/7AAD+ and Annexin V-/7AAD+.
  • the target cells take the target cells, adjust the cell density to 1 ⁇ 10 6 cells/ml, add 100 ⁇ l per well to a 96-well plate, and the well density is 1 ⁇ 10 5 cells/well.
  • the concentrations of IFN- ⁇ in the supernatants of Senl_H19 cells and Senl_M19 cells were 3550.28pg/ml and 2205.11pg/ml, respectively; TNF- ⁇ was 470.86pg/ml and 505.48pg/ml; the concentrations of IL-5 were 1182.50pg/ml, respectively ml and 1483.29pg/ml; IL-13 concentrations were 3112.10pg/ml and 3253.56pg/ml; IL-2 concentrations were 1328.96pg/ml and 1286.70pg/ml; IL6 concentrations were 9.89/ml and 9.89pg/ml, respectively 13.45pg/ml; IL-10 concentrations were 31.34pg/ml and 45.04pg/ml; IL-4 concentrations were 48.73pg/ml and 30.26pg/ml, respectively.
  • a mouse tumor model was constructed with human B lymphocytic leukemia cells Raji-luc. 5 ⁇ 10 5 Raji-luc were injected into the tail vein of 6-8 week-old NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) with a body weight of 20-24 g. cell therapy.
  • mice were randomly divided into 2 groups: Senl_H19 cell group and Senl_M19 cell group, 6 mice in each group.
  • Senl_H19 cells and Senl_M19 cells were prepared by the above CAR-T cell culture method, respectively, and the cells harvested on the 14th day of culture were used as the Senl_H19 cells, Senl_M19 cells and T cell controls used in this example.
  • Each mouse was injected with 1 ⁇ 10 7 CAR+ cells/mice via tail vein. Mice survival was monitored daily and every 7 days with a small animal in vivo imager (IVIS Lumina, Series III, PE) to observe tumor growth characteristics and collect luminescent signals.
  • IVIS Lumina IVIS Lumina, Series III, PE
  • Fig. 7 is a detection picture of a small animal in vivo imager
  • Fig. 8 is a fluorescence intensity diagram. All mice survived within 28 days after dosing. The clinical symptoms of mice in the Senl_H19 cell group and Senl_M19 cell group were significantly improved, and there were no drug-related or unrelated adverse reactions during the test, indicating that Senl_H19 cells and Senl_M19 cells have anti-lymphocytic leukemia effects in mice.

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Abstract

本发明公开了靶向CD19的嵌合抗原受体、制备方法及其应用。本发明的嵌合抗原受体含有针对CD19的全人源的单链抗体。相比目前临床使用的利用针对CD19的鼠源单链抗体构建而成的CAR-T细胞，本发明的CAR-T细胞可明显降低免疫原性，可用于复发性CD19阳性白血病的治疗。

Description

靶向CD19的嵌合抗原受体、制备方法及其应用 技术领域
本发明涉及生物技术领域，尤其是涉及靶向CD19的嵌合抗原受体、制备方法及其应用。
背景技术
CAR-T(Chimeric Antigen Receptor T-Cell，嵌合抗原受体修饰的T细胞)为代表的免疫疗法，其原理主要是通过基因工程手段对病人自身提取的T细胞进行嵌合抗原受体的修饰形成CAR-T细胞，该T细胞能特异性地识别肿瘤表面相关抗原(肿瘤细胞标志物)，从而靶向杀伤肿瘤。相对于常规免疫细胞，CAR-T细胞的靶向性、杀伤活性和持久性都更高，并且可以克服肿瘤局部免疫抑制微环境并打破宿主免疫耐受状态。该以CAR-T细胞为代表的修饰免疫细胞疗法在急性白血病和非霍奇金淋巴瘤的治疗上有着显着的疗效，被认为是最有前景的肿瘤治疗方式之一。
CAR-T免疫疗法是目前最具颠覆性潜力的新兴技术之一，欧美及亚洲各国都在积极开展该疗法的临床试验，其中中国开展临床试验项目最多，达204项，占据了全球CAR-T疗法临床试验的50％。宾夕法尼亚大学医院、费城儿童医院、美国癌症研究院(NCI)、Fred Hutchinson癌症研究中心、纪念斯隆凯特琳癌症中心和西雅图儿童医院等是最早开展CART细胞疗法的研究机构。新兴国际生物制药公司如Juno、Kite、Celgene、Cellectis、Bluebird，传统国际制药巨头如诺华、百时美施贵宝、辉瑞、强生、吉利德等都投入了大量资金和人力到CAR-T疗法的研发中，其中KITE和诺华公司所研发靶向CD19CART新药产品KTE-C19和CTL-019因为突出疗效先后获得FDA批准。
然而到目前为止，靶向CD19的CAR-T产品普遍存在因细胞因子释放综合症引起的发热或神经毒性副反应，为该产品的广泛临床使用和临床治疗带来 较大的挑战。合成生物学技术的兴起和应用，为肿瘤的CAR-T免疫治疗提供更多开发安全、高效的CAR-T的选择，为开发更安全CAR-T新疗法和药物的开发提供了启示。
发明内容
本发明提供了一种靶向CD19的嵌合抗原受体，所述嵌合抗原受体顺次包括以下组件：胞外抗原结合结构域、间隔结构域、跨膜结构域、共刺激信号转导结构域、初级信号传导结构域。
优选地，胞外抗原结合结构域为特异性结合CD19的scFv；所述scFv的氨基酸序列如SEQ ID NO.1所示。
优选地，间隔结构域是IgG4铰链区；优选地，所述IgG4铰链区的氨基酸序列如SEQ ID NO.2所示。
优选地，所述跨膜结构域是CD28的跨膜区；优选地，所述CD28的跨膜区的氨基酸序列如SEQ ID NO.3所示。
优选地，所述共刺激信号传导结构域是4-1BB的胞内信号传导结构域；优选地，所述4-1BB的胞内信号传导结构域的氨基酸序列如SEQ ID NO.4所示。
优选地，所述初级信号传导结构域是CD3ζ的胞内信号传导结构域；优选地，所述CD3ζ的胞内信号传导结构域的氨基酸序列如SEQ ID NO.5所示的序列。
优选地，所述嵌合抗原受体进一步在其N端包含信号肽；优选地，所述信号肽是粒细胞-巨噬细胞集落刺激因子受体2信号肽。
优选地，所述嵌合抗原受体进一步在其C端包含自裂解肽；优选地，所述自裂解肽是T2A。自裂解肽与初级信号传导结构域的C末端连接。
T2A的氨基酸序列如SEQ ID NO.7所示。
优选地，所述嵌合抗原受体进一步在其C端包含细胞表面标志物。
细胞表面标志物用于确定嵌合抗原受体是否被表达，具体地，所述细胞表面标志物用于确定所述细胞用于表达所述嵌合抗原受体的转导或工程改造，例 如细胞表面受体的截短形式，例如截短的EGFR(tEGFR)。在一些方面中，所述标志物包括CD34、NGFR或表皮生长因子受体(例如，tEGFR)或其功能变体的全部或部分(例如，截短形式)。
优选地，所述细胞表面标志物是EGFR中的含胞外结构域III和胞外结构域IV的片段(tEGFR)，优选地，tEGFR的氨基酸序列如SEQ ID NO.8所示。
优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.6所示。
本发明的实施方案的嵌合抗原受体可以包含合成的氨基酸代替一个或多个天然存在的氨基酸。此类合成的氨基酸为本领域已知的，并且包括例如，氨基环己羧酸、正亮氨酸、α-氨基n-癸酸、高丝氨酸、S-乙酰氨甲基-半胱氨酸、反式-3-羟脯氨酸和反式-4-羟脯氨酸、4-氨基苯丙氨酸、4-硝基苯丙氨酸、4-氯苯丙氨酸、4-羧基苯丙氨酸、β-苯基丝氨酸、β-羟基苯丙氨酸、苯基甘氨酸、α-萘基丙氨酸、环己基丙氨酸、环己基甘氨酸、吲哚啉-2-羧酸、1,2,3,4-四氢异喹啉-3-羧酸、氨基丙二酸、氨基丙二酸单酰胺、N’-苯甲基-N’-甲基-赖氨酸、N’,N’-二苄基-赖氨酸、6-羟赖氨酸、鸟氨酸、α-氨基环戊烷羧酸、α-氨基环己羧酸、α-氨基环庚烷羧酸、α-(2-氨基-2-降莰烷)-羧酸、α,γ-二氨基丁酸、α,β-二氨基丙酸、高苯丙氨酸以及α-叔-丁基甘氨酸。
本发明的实施方案的嵌合抗原受体可以被糖基化、酰胺化、羧酸化、磷酸化、酯化、N-酰化、经由例如二硫桥环化、或者转变为酸加成盐和/或任选地二聚化或多聚化。
本发明的实施方案的嵌合抗原受体可以通过本领域已知的方法获得。可以通过制备多肽或蛋白质的任何合适的方法制得嵌合抗原受体。从头合成多肽和蛋白质的合适方法为本领域已知的。另外，可以使用例如Green and Sambrook,Molecular Cloning:A Laboratory Manual(第4版),Cold Spring Harbor Laboratory Press(2012)中所述的标准重组方法，使用本文所述的核酸重组产生嵌合抗原受体。可选地，本文所述的嵌合抗原受体可以通过公司商业合成。在这方面，本发明的嵌合抗原受体可以是合成的和/或重组的。
本发明提供了一种核酸，所述核酸编码前面所述的嵌合抗原受体。
进一步，所述核酸还包含启动子、和/或位于启动子之后的酶切位点、和/或位于酶切位点之后的kozak序列。
更进一步，启动子位于所述核酸的N端，所述启动子是延长因子1α的启动子；优选地，所述启动子序列如SEQ ID NO.9所示。
更进一步，所述酶切位点是Pac I的识别位点；优选地，所述酶切位点的序列如SEQ ID NO.10中第1-8位所示。
更进一步，所述kozak序列如SEQ ID NO.10中第9-14位所示。
优选地，所述核酸中信号肽的编码序列如SEQ ID NO.10中第15-78位所示。
优选地，所述核酸中scFv的编码序列如SEQ ID NO.10中第79-822位所示。
优选地，所述核酸中间隔结构域的编码序列如SEQ ID NO.10中第823-846位所示。
优选地，所述核酸中跨膜结构域的编码序列如SEQ ID NO.10中第847-939位所示。
优选地，所述核酸中共刺激信号传导结构域的编码序列如SEQ ID NO.10中第940-1068位所示。
优选地，所述核酸中初级信号传导结构域的编码序列如SEQ ID NO.10中第1069-1404位所示。
优选地，所述T2A的核苷酸序列如SEQ ID NO.10中第1405-1476位所示。
优选地，所述tEGFR的核苷酸序列如SEQ ID NO.10中第1477-2550位所示。
优选地，所述核酸中嵌合抗原受体的编码序列如SEQ ID NO.10中第15-2585位所示。
优选地，所述核酸包括SEQ ID NO.10所示的核苷酸序列。
本发明提供了一种载体，所述载体包含前面所述的核酸。
在实施方案中，本发明的载体可以是任何合适的载体，并且可以被用于转化或转染任何合适的宿主细胞。合适的载体包括设计以用于增殖和扩增或者用 于表达或者用于这两种的那些载体，如质粒和病毒。载体可以选自：pUC系列(Fermentas Life Sciences,Glen Burnie,MD)、pBluescript系列(Stratagene,LaJolla,CA)、pET系列(Novagen,Madison,WI)、pGEX系列(Pharmacia Biotech,Uppsala,Sweden)以及pEX系列(Clontech,Palo Alto,CA)。也可使用诸如λGT10、λGT11、λZapII(Stratagene)、λEMBL4和λNM1149的噬菌体载体。载体的实例包括pEUK-Cl、pMAM和pMAMneo(Clontech)。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
本发明提供了一种重组微生物，所述重组微生物包含前面所述的核酸或前面所述的载体；优选地，所述重组微生物是重组病毒。
本发明提供了一种经改造的免疫细胞，所述经改造的免疫细胞表达前面所述的嵌合抗原受体；优选地，所述免疫细胞含有前面所述的核酸或前面所述的载体。
本发明提供了一种免疫细胞群体，所述免疫细胞群体包括前面所述的经改造的免疫细胞；优选地，所述免疫细胞群体还包括未经改造的或改造不完全的免疫细胞。
本发明提供了一种前面所述的经改造的免疫细胞的制备方法，所述制备方法包括将前面所述的核酸或前面所述的载体导入免疫细胞中。
现有技术已知有数种合适的方法用于将核酸和相应的载体导入宿主细胞。方法包括但不限于磷酸钙转染、电穿孔、脂质体转染、利用转染试剂，例如阳离子脂质，生物射弹介导或多聚体介导的转移。
本发明的免疫细胞可以是外周血淋巴细胞(PBL)或外周血单核细胞(PBMC)。本发明的免疫细胞可以是B细胞、自然杀伤(NK)细胞或T细胞。
本发明的T细胞可以是任何T细胞，如培养的T细胞，例如原代T细胞；或者是来自培养的T细胞系的T细胞，例如Jurkat、SupT1等；或者是获自哺乳动物的T细胞。如果获自哺乳动物，T细胞可以获自多种来源，包括但不限 于血液、骨髓、***、胸腺或者其它组织或液体。T细胞也可以是富集的或纯化的。T细胞可以是人T细胞。T细胞可以是分离自人的T细胞。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Th 1和Th 2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、初始T细胞等。T细胞可以是CD8+T细胞或CD4+T细胞。
本发明提供了一种组合物，所述组合物包括前面所述的嵌合抗原受体、前面所述的核酸、前面所述的载体、前面所述的重组微生物、前面所述的经改造的免疫细胞、前面所述的免疫细胞群体。
优选地，所述组合物包括药物组合物，所述药物组合物还包括药剂学上可接受的载体。
药学上可接受的载体是在使用条件下，无有害副作用或毒性的载体。制备可施用药物的方法为本领域技术人员已知的或显而易见的，并且更详细地描述于，例如，Remington:The Science and Practice of Pharmacy，第22版，Pharmaceutical Press(2012)。
本发明的药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。
载体的选择将部分由具体的本发明的核酸、载体、免疫细胞以及由用于施用本发明的核酸、载体、免疫细胞的具体方法决定。因此，存在多种合适的本发明的药物组合物的制剂。合适的制剂可以包括用于肠胃外、皮下、静脉内、肌肉内、动脉内、鞘内、瘤内或腹膜内施用的那些制剂中的任何制剂。可以使用多于一种途径来施用本发明的核酸、载体、免疫细胞，并且在某些情况下，特定途径可以比另一途径提供更直接以及更有效的应答。
本发明的药物组合物还可包括与本发明的核酸、载体、免疫细胞联合施用的其它药学活性剂或药物，所述其它药学活性剂或药物如化疗剂，例如天冬酰胺酶、白消安、卡铂、顺铂、道诺霉素、多柔比星、氟尿嘧啶、吉西他滨、羟 基脲、甲氨蝶呤、紫杉酚、利妥昔单抗、长春花碱、长春新碱等。
当将本发明的核酸、载体、免疫细胞与一种或多种另外的治疗剂一起施用时，一种或多种另外的治疗剂可以共施用至哺乳动物。“共施用”意为在时间上足够接近地施用一种或多种另外的治疗剂和本发明的核酸、载体、免疫细胞，以使本发明的核酸、载体、免疫细胞可以增强一种或多种另外的治疗剂的作用，反之亦然。就这点而言，可以首先施用本发明的核酸、载体、免疫细胞，之后施用一种或多种另外的治疗剂，反之亦然。可选地，可以同时施用本发明的核酸、载体、免疫细胞和一种或多种另外的治疗剂。
本发明提供了一种试剂盒，所述试剂盒包括前面所述的嵌合抗原受体、前面所述的核酸、前面所述的载体、前面所述的重组微生物、前面所述的经改造的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物。
本发明提供了一种应用，所述应用包括以下任一项所述的应用：
1)前面所述的嵌合抗原受体、前面所述的核酸、前面所述的载体、前面所述的重组微生物、前面所述的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物在制备治疗与CD19表达相关的疾病或病症的药物中的应用；
2)前面所述的嵌合抗原受体在制备前面所述的核酸、前面所述的载体、前面所述的重组微生物、前面所述的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物、前面所述的试剂盒中的应用；
3)前面所述的核酸在制备前面所述的载体、前面所述的重组微生物、前面所述的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物、前面所述的试剂盒中的应用；
4)前面所述的载体在制备前面所述的重组微生物、前面所述的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物、前面所述的试剂盒中的应用；
5)前面所述的重组微生物在制备前面所述的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物、前面所述的试剂盒中的应用；
6)前面所述的免疫细胞在制备前面所述的免疫细胞群体、前面所述的组合物、前面所述的试剂盒中的应用；
7)前面所述的免疫细胞群体在制备前面所述的组合物、前面所述的试剂盒中的应用；
8)前面所述的组合物在制备前面所述的试剂盒中的应用。
本发明提供了一种与CD19表达相关的疾病或病症的方法，所述方法包括给有需要者施用前面所述的核酸、前面所述的载体、前面所述的重组微生物、前面所述的免疫细胞、前面所述的免疫细胞群体、前面所述的组合物。
本发明的与CD19表达相关的疾病或病症包括肿瘤、自身免疫和炎性疾病、感染性疾病。
进一步，所述肿瘤包括B细胞恶性肿瘤，例如B细胞白血病和淋巴瘤，包括B细胞慢性淋巴细胞性白血病(CLL)、急性淋巴细胞性白血病(ALL)、幼淋巴细胞性白血病、毛细胞白血病、常见急性淋巴细胞性白血病、非急性淋巴母细胞性白血病、非霍奇金淋巴瘤、弥散性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤、滤泡性淋巴瘤、脾和边缘带淋巴瘤、套细胞淋巴瘤、惰性B细胞淋巴瘤、霍奇金淋巴瘤。
优选地，所述B胞恶性肿瘤是复发性CD19阳性白血病。
示例性的自身免疫和炎性疾病包括多发性硬化、类风湿性关节炎，和全身性红斑狼疮(SLE)。
在本申请中，如果给需要者输注前面所述的免疫细胞或免疫细胞群体，或者输注包含前面所述的免疫细胞或免疫细胞群体的药物组合物，则给药方案可以是施用快速灌注剂，可以随时间推移施用多个分剂量，或者剂量可以随治疗情况的危急程度成比例降低或提高。
在某些实施方式中，治疗方案可以是每周施用一次、两周一次、三周一次、四周一次、一个月一次、3个月一次、或3-6个月一次。在某些实施方式中，给药方案包括静脉内施用，施用剂量范围可以是0.1×10 8至3×10 8CAR阳性免疫细胞，例如，0.15×10 8至2×10 8CAR阳性免疫细胞、0.5×10 8至2×10 8CAR阳性免疫细胞、1×10 8至2×10 8CAR阳性免疫细胞、0.2×10 8至2×10 8CAR阳性免疫细胞、0.2×10 8至1×10 8CAR阳性免疫细胞、0.25×10 8至1×10 8CAR 阳性免疫细胞、0.25×10 8至0.5×10 8CAR阳性免疫细胞，或0.5×10 8CAR阳性免疫细胞，或2×10 8CAR阳性免疫细胞。
针对不同的适应症，给药剂量可以不同。
在某些实施方式中，所述免疫效应细胞治疗复发难治的成人急性淋巴细胞白血病患者的给药剂量可以为0.25×10 8至0.5×10 8CAR阳性免疫细胞，或0.5×10 8CAR阳性免疫细胞，例如可以是0.3×10 8至0.5×10 8、0.4×10 8至0.5×10 8、0.25×10 8至0.4×10 8、0.3×10 8至0.4×10 8或0.4×10 8至0.5×10 8CAR阳性免疫细胞。在某些实施方式中，所述免疫效应细胞治疗复发难治的成人急性淋巴细胞白血病患者的给药剂量可以为0.25×10 8、0.26×10 8、0.27×10 8、0.28×10 8、0.29×10 8、0.3×10 8、0.31×10 8、0.32×10 8、0.33×10 8、0.34×10 8、0.35×10 8、0.36×10 8、0.37×10 8、0.38×10 8、0.39×10 8、0.4×10 8、0.41×10 8、0.42×10 8、0.43×10 8、0.44×10 8、0.45×10 8、0.46×10 8、0.47×10 8、0.48×10 8、0.49×10 8或0.5×10 8CAR阳性免疫细胞。
在另一些实施方式中，所述免疫效应细胞治疗复发难治的非霍奇金淋巴瘤患者的给药剂量可以为1×10 8至2×10 8CAR阳性免疫细胞，或2×10 8CAR阳性免疫细胞，例如1×10 8至1.8×10 8、1×10 8至1.5×10 8、1×10 8至1.3×10 8、1.3×10 8至2×10 8、1.3×10 8至1.5×10 8、1.5×10 8至2×10 8、1.5×10 8至1.8×10 8或1.8×10 8至2×10 8CAR阳性免疫细胞。在另一些实施方式中，所述免疫效应细胞治疗复发难治的霍奇金淋巴瘤患者的给药剂量可以为1×10 8、1.1×10 8、1.2×10 8、1.3×10 8、1.4×10 8、1.5×10 8、1.6×10 8、1.7×10 8、1.8×10 8、1.9×10 8或2.0×10 8CAR阳性免疫细胞。
本发明的优点和有益效果：
本发明利用全人源的靶向CD19的scFv构建CAR-T，相比鼠源的scFv，本发明的CAR-T具有更低的免疫原性，尤其适用于对鼠源CAR-T耐受的CD19+白血病的复发患者。
附图说明
图1显示人源H19-CAR基因具体结构示意图；
图2显示鼠源M19-CAR基因具体结构示意图；
图3显示CAR-T细胞扩增曲线图；
图4显示CAR转染率的统计图；
图5显示CAR-T细胞对Nalm-6细胞杀伤能力的结果图；
图6显示CAR-T细胞分泌细胞因子的结果统计图；
图7显示动物活体成像图；
图8显示荧光强度统计图。
具体实施方式
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列举的目的，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。
实施例1 CAR-T构建
1、构建慢病毒载体
将全人源或者鼠源CD19的scfv片段克隆至具有4-1BB和CD3ζ的第二代CAR结构骨架中，以T2A连接胞外tEGFR结构域，得到名称为H19-CAR(全人源)或者M19-CAR(鼠源)。H19-CAR基因具体结构为antiHCD19 scFv-IgG4 hinge-CD28TM-4-1BB-CD3ζ-T2A-tEGFR，M19-CAR基因具体结构为antiMCD19 scFv-IgG4 hinge-CD28TM-4-1BB-CD3ζ-T2A-tEGFR(如图1和图2)。其中，anti HCD19 scFv是抗全人源CD19单链可变片段(scFv)的编码序列，antiMCD19 scFv是抗鼠源CD19单链可变片段(scFv)的编码序列，IgG4 hinge为铰链区，CD28TM是跨膜区，4-1BB是共刺激结构域，CD3ζ为胞内信号转导结构域，通过T2A肽连接表达tEGFR胞外区域，以便病毒转导后检测CAR的表达。
H19-CAR基因编码链的编码序列是SEQ ID NO.10的第15-2585位，SEQ ID NO.10中第1-8位为Pac I的识别位点，第15-78位为信号肽的编码序列，第79-822位为全人源抗CD19单链可变片段(scFv)的编码序列，第823-846位hinge序列，第847-939位为CD28TM基因序列，第940-1068位为4-1BB基因序列，第 1069-1404位为CD3ζ基因序列，第1405-1476位为T2A基因序列，第1477-2550位tEGFR基因序列。蛋白质H19-CAR氨基酸序列是序列表中SEQ ID NO.6所示。
H19-CAR基因表达载体(慢病毒表达载体)，为全序列合成，将其命名为Senl-H19，序列是SEQ ID NO.13所示。Senl-H19含有SEQ ID NO.10中第79-2550所示的H19-CAR基因的编码区，能在细胞中表达氨基酸序列是序列表中SEQ ID NO.6所示的蛋白质H19-CAR。
构建成功的慢病毒表达载体经Pac I和SpeI酶切鉴定及序列测定正确后，用于慢病毒包装。
将Senl_H19中全人源CD19的scfv替换成鼠源CD19的scfv(氨基酸序列如SEQ ID NO.12所示，核苷酸序列如SEQ ID NO.11所示)，即制备而成Senl_M19表达载体。
构建成功的慢病毒表达载体经Pac I和SpeI酶切鉴定及序列测定正确后，用于慢病毒包装。
2、慢病毒的制备
将已长到80％-90％的293FT细胞培养瓶(T175)从37℃、5％CO 2的细胞培养箱中取出，加入2mL TrypLE TM EXPRESS消化后收集洗涤细胞，每145mm细胞培养皿中加入9×10 6个细胞，加20mL DMEM培养基(Thermo Fisher)，轻轻摇匀，放入37℃，5％CO 2培养箱中培养。
24h小时后，按照每个145mm平皿，375μl无血清DMEM培养基、15μg慢病毒表达载体Senl_H19或Senl_M19、7.5μg编码Rev蛋白的包装质粒psPAX2、3.75μg编码VSV-G蛋白的包膜质粒pMD2.G的量将以上溶液混合均匀，组成溶液A，315μl无血清DMEM培养基中加入60μl PEI转染试剂，混合均匀，组成溶液B，随后将溶液B加入到溶液A中混合均匀，室温静置20min。将用于包装病毒的293FT细胞从37℃5％CO 2的细胞培养箱中取出，将上述混合液平均加到每平皿中，轻轻摇匀，放入37℃5％CO 2培养箱中。4h-6h后，弃旧培养基，加入5mL已预热的PBS清洗细胞，再加入20ml新鲜的已预热的含10％胎牛血清的DMEM培养基，放入37℃5％CO 2培养箱中培养。继续培养48h-72h后收取培养上清作为病毒原液，将原液收集到50ml离心管中，2000r/min，离心5min 去除细胞沉淀，随后用50ml注射器将病毒原液通过0.45μm滤膜过滤到新的50ml离心管中，4℃，18300g高速离心2h。此即为病毒浓缩液。将病毒浓缩液按100μl/管分装，另外留取10μl进行病毒滴度测定。将分装好的浓缩液置于-80℃保存。
3、外周血单个核细胞(PBMC)的制备
本外周血可以是健康供者也可以是患者本身的外周血。
将外周血与无菌生理盐水1：1混匀稀释后，缓慢加入到单个核细胞分离液(东方华辉)中(稀释血：分离液＝2:1)，800g，离心20min，升7降4，进行密度梯度离心。离心结束后将白膜层轻轻吸出转移至新的50mL离心管中，加入无菌生理盐水重悬后800g，4℃离心5min，离心结束后将上清液弃掉，用涡旋振荡器震散细胞沉淀层，加入无菌生理盐水定容至50mL，上下颠倒离心管、混匀后移入离心机，800g，4℃离心5min，得到的沉淀即为单个核细胞。
4、T细胞的分离
离心结束后向单个核细胞沉淀中加入细胞分选液(DPBS+0.5％HSA)(1×10 7个细胞加80μl细胞分选液)和分选磁珠(MASC)(1×10 7个细胞加20μl分选磁珠)，加入分选磁珠(健康供者外周血分选磁珠为CD3分选磁珠，患者自体外周血根据流式检测结果而定，可以是CD3分选磁珠、CD4和CD8分选磁珠等)，吹吸混匀后放入4℃冰箱避光孵育15分钟，孵育完成后按照每1×10 7PBMC加入1～2mL细胞分选液，混匀后800g，4℃离心5min。按照1×10 8PBMC加入500μL缓冲液，吹吸混匀细胞沉淀层。然后将细胞悬液加入已经准备好的磁力柱中，让其自由滴下，等到液体滴完后再加入3mL细胞分选液清洗磁力柱，重复2遍。留在磁力柱中的细胞即为阳性细胞。将磁力柱从磁力架中移出，加入5mL细胞分选液用磁力柱中的活塞用力挤压磁力柱中缓冲液，将磁力柱中的阳性细胞冲洗至离心管中，即为分选阳性细胞。
5、T细胞的活化培养
将装有分选阳性细胞的离心管混匀后800g，4℃离心5min。离心结束后将上清液弃掉，用含有200UI/mL IL-2的TexMACS GMP无血清培养基(MACS)重悬混匀，然后按照T细胞与活化磁珠CD3/CD28(Thermo Fisher)1:1的比例加入活化磁珠，放入37℃、5％CO 2细胞培养箱进行培养。
如无特殊说明，下述细胞培养的培养基均为含有200UI/mL IL-2的TexMACS GMP无血清培养基。
5、表达靶向CD19的CAR的T细胞制备
按照上述T细胞分离活化的方法制备T细胞。将分选出来的T细胞，加入Dynabeads CD3/CD28活化磁珠进行激活，放入37℃、5％CO 2细胞培养箱进行培养(记为Day0)，培养48小时后，用上述制备的病毒浓缩液加入到T细胞中，离心2小时(2000rpm，35℃，升速4，降速4)进行慢病毒转染，转染完成后放入37℃，5％CO 2细胞培养箱进行培养，Day5取样，通过流式细胞术进行CAR+比例检测。Day12到Day14收获细胞，通过洗涤去除培养基，并使用含1％HSA的0.9％生理盐水进行冲悬细胞获得表达靶向CD19的CAR的T细胞，表达全人源scFv的CAR-T细胞称为Senl_H19细胞，表达鼠源scFv的CAR-T细胞称Senl_M19细胞。
6、结果
CAR-T细胞的扩增曲线见图3、CAR转染率见图4。和对照Senl_M19细胞相比，Senl_H19细胞和Senl_M19细胞两者的扩增倍数没有明显差异，培养14天时扩增倍数均＞100倍。培养到14天时，Senl_H19细胞的CAR阳性率为52％±0.14％，与Senl_M19的CAR阳性率53.77％±12.2％无显著差异。结果表明，与鼠源的scFv相比，人源的scFv对细胞的生长和CAR转染没有显著影响。
实施例2 CAR-T细胞的体外功能研究
一、步骤
用培养第14天收获细胞作为效应细胞，用Nalm-6细胞(购买于通派生物细胞库)作为靶细胞。
首先收集靶细胞，2000rpm离心5min，DPBS重悬计数，按照2×10 6个细胞添加100μl的2μM CFSE工作液的比例加入适量的2μM CFSE工作液，轻轻混匀。在37℃下避光孵育20min。孵育结束后，加入5倍体积的完全培养基(含10％FBS的1640)终止染色，2000rpm离心5min，用完全培养基重悬，37℃下避光孵育10min，孵育结束后2000rpm离心5min，完全培养基重悬细胞，调整靶细 胞密度为1×10 6个/ml，加入96孔板中，每孔100μl，孔密度为1×10 5个/孔。然后根据不同效靶比下(E：T＝0.3:1、1:1、3:1、10:1)向靶细胞中添加适量的效应细胞，混合后孵育4小时，流式细胞术检测细胞杀伤比例。用2μL 7AAD和Anexxin V对细胞进行染色，避光孵育15min。CFSE标记的靶细胞的杀伤率(％)为Annexin V+/7AAD–、Annexin V+/7AAD+和Annexin V–/7AAD+三者比例之和。
另外取靶细胞，调整细胞密度为1×10 6个/ml，加入96孔板中，每孔100μl，孔密度为1×10 5个/孔，按照效应细胞E:T＝3:1，向每孔靶细胞中添加适量的效应细胞，混匀共同孵育18小时，取上清检测细胞因子的分泌(LEGENDplex HU Th1/Th2 Panel(8-plex)(Biolegend)。
二、结果
结果显示如图5和图6，Senl_H19细胞和Senl_M19细胞对Nalm-6细胞均具有强的杀伤力，在效靶比为0.3：1、1：1、3：1、10：1时，Senl_H19细胞对Nalm-6细胞的杀伤均值分别为17.8％、43.6％、76.5％和91.1％；Senl_M19细胞对Nalm-6细胞的杀伤均值分别为16.9％、39.8％、60.1％和89.0％。Senl_H19细胞和Senl_M19细胞上清中IFN-γ的浓度分别为3550.28pg/ml和2205.11pg/ml；TNF-α分别为470.86pg/ml和505.48pg/ml；IL-5的浓度分别为1182.50pg/ml和1483.29pg/ml；IL-13的浓度分别为3112.10pg/ml和3253.56pg/ml；IL-2的浓度分别为1328.96pg/ml和1286.70pg/ml；IL6的浓度分别为9.89/ml和13.45pg/ml；IL-10的浓度分别为31.34pg/ml和45.04pg/ml；IL-4的浓度分别为48.73pg/ml和30.26pg/ml。
实施例3 CAR-T细胞的体内功能研究
一、步骤
用人B淋巴细胞白血病细胞Raji-luc构建小鼠肿瘤模型。将5×10 5个Raji-luc注射到6-8周龄、体重为20-24g的NCG小鼠(江苏集萃药康生物科技有限公司)的尾静脉中，活体成像检测成瘤后，回输细胞治疗。
将小鼠随机分为2组：Senl_H19细胞组和Senl_M19细胞组，每组6只。用上述CAR-T细胞培养方法分别制备Senl_H19细胞、Senl_M19细胞，将培养第14天收获的细胞做为本实施例使用的Senl_H19细胞、Senl_M19细胞和T细胞对照。每只小鼠通过尾静脉注射1×10 7个CAR+细胞/只。每天监测小鼠存活情况和每7天用小动物活体成像仪(IVIS Lumina,Series III，PE)观察肿瘤的生长特性并采集发光信号。
二、结果
图7为小动物活体成像仪检测图片，图8为荧光强度图。小鼠在给药后28天内全部存活。Senl_H19细胞组和Senl_M19细胞组小鼠临床症状明显改善，试验期间未出现与药物相关或无关的不良反应，说明Senl_H19细胞和Senl_M19细胞在小鼠体内具有抗淋巴细胞白血病的作用。
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 (15)

  1. 一种靶向CD19的嵌合抗原受体，其特征在于，所述嵌合抗原受体顺次包括以下组件：胞外抗原结合结构域、间隔结构域、跨膜结构域、共刺激信号转导结构域、初级信号传导结构域；
    优选地，所述嵌合抗原受体顺次包括以下组件：胞外抗原结合结构域、间隔结构域、跨膜结构域、共刺激信号转导结构域、初级信号传导结构域、自裂解肽、细胞表面标志物；
    优选地，胞外抗原结合结构域为特异性结合CD19的scFv；所述scFv的氨基酸序列如SEQ ID NO.1所示；
    优选地，间隔结构域是IgG4铰链区；优选地，所述IgG4铰链区的氨基酸序列如SEQ ID NO.2所示；
    优选地，所述跨膜结构域是CD28的跨膜区；优选地，所述CD28的跨膜区的氨基酸序列如SEQ ID NO.3所示；
    优选地，所述共刺激信号传导结构域是4-1BB的胞内信号传导结构域；优选地，所述4-1BB的胞内信号传导结构域的氨基酸序列如SEQ ID NO.4所示；
    优选地，所述初级信号传导结构域是CD3ζ的胞内信号传导结构域；优选地，所述CD3ζ的胞内信号传导结构域的氨基酸序列如SEQ ID NO.5所示的序列；
    优选地，所述自裂解肽是T2A；优选地，T2A的氨基酸序列如SEQ ID NO.7所示；
    优选地，细胞表面标志物是tEGFR；优选地，tEGFR的氨基酸序列如SEQ ID NO.8所示；
    优选地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.6所示。
  2. 一种核酸，所述核酸编码权利要求1的嵌合抗原受体；
    优选地，所述核酸还包含启动子、和/或位于启动子之后的酶切位点、和/或位于酶切位点之后的kozak序列；
    优选地，启动子位于所述核酸的N端，所述启动子是延长因子1α的启动子；优选地，所述启动子序列如SEQ ID NO.9所示；
    优选地，所述酶切位点是Pac I的识别位点；优选地，所述酶切位点的序列如SEQ ID NO.10中第1-8位所示；
    优选地，所述kozak序列如SEQ ID NO.10中第9-14位所示；
    优选地，所述核酸中信号肽的编码序列如SEQ ID NO.10中第15-78位所示；
    优选地，所述核酸中scFv的编码序列如SEQ ID NO.10中第79-822位所示；
    优选地，所述核酸中间隔结构域的编码序列如SEQ ID NO.10中第823-846位所示；
    优选地，所述核酸中跨膜结构域的编码序列如SEQ ID NO.10中第847-939位所示；
    优选地，所述核酸中共刺激信号传导结构域的编码序列如SEQ ID NO.10中第940-1068位所示；
    优选地，所述核酸中初级信号传导结构域的编码序列如SEQ ID NO.10中第1069-1404位所示；
    优选地，所述核酸中嵌合抗原受体的编码序列如SEQ ID NO.10中第15-2585位所示；
    优选地，所述核酸中T2A的编码序列如SEQ ID NO.10中第1405-1476位所示；
    优选地，所述核酸中tEGFR的编码序列如SEQ ID NO.10中第1477-2550位所示；
    优选地，所述核酸包括SEQ ID NO.10所示的核苷酸序列。
  3. 一种载体，其特征在于，所述载体包含权利要求2所述的核酸；优选地，所述载体序列如SEQ ID NO.13所示。
  4. 一种重组微生物，其特征在于，所述重组微生物包含权利要求2所述的核酸或权利要求3所述的载体；优选地，所述重组微生物是重组病毒。
  5. 一种经改造的免疫细胞，其特征在于，所述经改造的免疫细胞表达权利要求1所述的嵌合抗原受体；优选地，所述免疫细胞含有权利要求2所述的核酸或权利要求3所述的载体。
  6. 一种免疫细胞群体，所述免疫细胞群体包括权利要求5所述的经改造的免疫细胞；优选地，所述免疫细胞群体还包括未经改造的或改造不完全的免疫细胞。
  7. 一种权利要求5所述的经改造的免疫细胞的制备方法，其特征在于，所述制备方法包括将权利要求2所述的核酸或权利要求3所述的载体导入免疫细胞中；
  8. 一种组合物，所述组合物包括权利要求1所述的嵌合抗原受体、权利要求2所述的核酸、权利要求3所述的载体、权利要求4所述的重组微生物、权利要求5所述的经改造的免疫细胞、权利要求6所述的免疫细胞群体；优选地，所述组合物还包括药剂学上可接受的载体。
  9. 一种试剂盒，所述试剂盒包括权利要求1所述的嵌合抗原受体、权利要求2所述的核酸、权利要求3所述的载体、权利要求4所述的重组微生物、权利要求5所述的经改造的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物。
  10. 一种与CD19表达相关的疾病或病症的方法，其特征在于，所述方法包括给有需要者施用权利要求2所述的核酸、权利要求3所述的载体、权利要求4所述的重组微生物、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物。
  11. 根据权利要求10所述的方法，其特征在于，所述与CD19表达相关的疾病或病症包括肿瘤、自身免疫和炎性疾病、感染性疾病。
  12. 根据权利要求11所述的方法，其特征在于，所述肿瘤包括B细胞恶性肿瘤，例如B细胞白血病和淋巴瘤，包括B细胞慢性淋巴细胞性白血病(CLL)、急性淋巴细胞性白血病(ALL)、幼淋巴细胞性白血病、毛细胞白血病、 常见急性淋巴细胞性白血病、非急性淋巴母细胞性白血病、非霍奇金淋巴瘤、弥散性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤、滤泡性淋巴瘤、脾和边缘带淋巴瘤、套细胞淋巴瘤、惰性B细胞淋巴瘤、霍奇金淋巴瘤。
  13. [根据细则91更正 09.06.2022]　
    根据权利要求12所述的方法，其特征在于，所述B胞恶性肿瘤是复发性CD19阳性白血病。
  14. 根据权利要求11所述的方法，其特征在于，所述自身免疫和炎性疾病包括多发性硬化、类风湿性关节炎，和全身性红斑狼疮。
  15. 一种应用，所述应用包括以下任一项所述的应用：
    1)权利要求1所述的嵌合抗原受体、权利要求2所述的核酸、权利要求3所述的载体、权利要求4所述的重组微生物、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物在制备治疗与CD19表达相关的疾病或病症的药物中的应用；
    2)权利要求1所述的嵌合抗原受体在制备权利要求2所述的核酸、权利要求3所述的载体、权利要求4所述的重组微生物、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物、权利要求9所述的试剂盒中的应用；
    3)权利要求2所述的核酸在制备权利要求3所述的载体、权利要求4所述的重组微生物、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物、权利要求9所述的试剂盒中的应用；
    4)权利要求3所述的载体在制备权利要求4所述的重组微生物、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物、权利要求9所述的试剂盒中的应用；
    5)权利要求4所述的重组微生物在制备权利要求5所述的免疫细胞、权利要求6所述的免疫细胞群体、权利要求8所述的组合物、权利要求9所述的试剂盒中的应用；
    6)权利要求5所述的免疫细胞在制备权利要求6所述的免疫细胞群体、权利要求8所述的组合物、权利要求9所述的试剂盒中的应用；
    7)权利要求6所述的免疫细胞群体在制备权利要求8所述的组合物、权利要求9所述的试剂盒中的应用；
    8)权利要求8所述的组合物在制备权利要求9所述的试剂盒中的应用；
    优选地，与CD19表达相关的疾病或病症包括肿瘤、自身免疫和炎性疾病、感染性疾病；
    优选地，所述肿瘤包括B细胞恶性肿瘤；
    优选地，所述B胞恶性肿瘤包括B细胞白血病和淋巴瘤；
    优选地，所述B胞恶性肿瘤包括B细胞慢性淋巴细胞性白血病(CLL)、急性淋巴细胞性白血病(ALL)、幼淋巴细胞性白血病、毛细胞白血病、常见急性淋巴细胞性白血病、非急性淋巴母细胞性白血病、非霍奇金淋巴瘤、弥散性大B细胞淋巴瘤(DLBCL)、多发性骨髓瘤、滤泡性淋巴瘤、脾和边缘带淋巴瘤、套细胞淋巴瘤、惰性B细胞淋巴瘤、霍奇金淋巴瘤；
    优选地，所述B胞恶性肿瘤是复发性CD19阳性白血病。
PCT/CN2022/086904 2021-04-22 2022-04-14 靶向cd19的嵌合抗原受体、制备方法及其应用 WO2022222846A1 (zh)

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