WO2021148019A1 - 病毒载体转导细胞的方法 - Google Patents

病毒载体转导细胞的方法 Download PDF

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WO2021148019A1
WO2021148019A1 PCT/CN2021/073418 CN2021073418W WO2021148019A1 WO 2021148019 A1 WO2021148019 A1 WO 2021148019A1 CN 2021073418 W CN2021073418 W CN 2021073418W WO 2021148019 A1 WO2021148019 A1 WO 2021148019A1
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cells
cell
hours
transduced
nucleic acid
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PCT/CN2021/073418
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English (en)
French (fr)
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王华茂
高慧萍
童潇
姚晔风
朱寅玉
李宗海
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科济生物医药（上海）有限公司
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Priority claimed from CN202110076311.6A external-priority patent/CN114854790A/zh
Application filed by 科济生物医药（上海）有限公司 filed Critical 科济生物医药（上海）有限公司
Priority to CN202180010727.6A priority Critical patent/CN115003818A/zh
Publication of WO2021148019A1 publication Critical patent/WO2021148019A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the invention belongs to the field of genetic engineering, and specifically relates to a method for transducing recombinant nucleic acid through virus to enter.
  • immune effector cells such as T cells, NK cells, NK T cells, etc.
  • T cells such as T cells, NK cells, NK T cells, etc.
  • NK T cells NK T cells
  • TCR T cell chimeric antigen receptor-modified CAR T cells
  • TCR T cell chimeric TCR receptor modification The TCR T cell and so on.
  • cells that recognize tumor-associated antigens are obtained by introducing a recombinant nucleic acid that can encode a foreign receptor that recognizes tumor-associated antigens into a viral vector, and then infect the transduced cells with the viral vector carrying the recombinant nucleic acid.
  • the viral vector carrying recombinant nucleic acid usually takes a long time to infect transduced cells, such as the preparation of CAR T cells.
  • Virus transduction is subsequently carried out. Virus transduction takes 1 day, and amplification is required after transduction is completed.
  • the amplification takes 1-2 weeks, which makes the preparation of CAR T cells take a long time and not only increases the preparation of cell products.
  • the time cost and reagent cost may also increase the risk of cell mutation, and due to the preparation time process, when the cell therapy product is given to the patient, some patients have already experienced tumor progression, which delays the timing of tumor treatment and affects the effect of clinical treatment.
  • the purpose of the present invention is to provide a method for transducing cells with viral vectors, which can significantly shorten the preparation time of receptor-modified cells that recognize tumor-associated antigens, without affecting or even further enhancing the efficacy of cell therapy.
  • the present invention provides a method for transducing cells with a viral vector, the method comprising:
  • Step (1) incubate the input composition containing the cells to be transduced, the cell stimulator to be transduced, and the viral vector particles carrying the recombinant nucleic acid together, and the incubation time does not exceed 72 hours,
  • Step (2) harvesting to obtain an output composition, the output composition containing cells transduced with recombinant nucleic acid;
  • the incubation time is 1 hour to 72 hours;
  • the incubation time is 2 hours to 48 hours;
  • the incubation time is 2 hours to 36 hours;
  • the incubation time is 12 hours to 36 hours;
  • the incubation time is 12 hours to 24 hours
  • the incubation time is 15 hours to 24 hours.
  • the present invention provides a method for transducing cells with a viral vector, the method comprising:
  • Step (1) incubate the input composition containing the cells to be transduced and the cell stimulator to be transduced for no more than 72 hours,
  • Step (2) then add the viral vector particles of recombinant nucleic acid to incubate, and the incubation time does not exceed 24 hours.
  • Step (3) harvesting to obtain an output composition containing cells transduced with recombinant nucleic acid
  • the total incubation time of (1) and (2) does not exceed 72 hours.
  • the total incubation time of (1) and (2) does not exceed 60h, or does not exceed 48h, or does not exceed 32h, or does not exceed 28h, or does not exceed 24h.
  • the incubation time of the step (1) is 2-72 hours
  • the incubation time of the step (1) is 2-71 hours;
  • the incubation time of the step (1) is 2-48 hours
  • the incubation time of the step (1) is 2-32 hours
  • the incubation time of the step (1) is 2-28 hours
  • the incubation time of the step (1) is 3-24 hours;
  • the incubation time of the step (1) is 5-24 hours;
  • the incubation time of the step (1) is 7-24 hours
  • the incubation time of the step (1) is 7-23 hours
  • the incubation time of the step (1) is 10-23 hours
  • the incubation time of the step (1) is 15-23 hours
  • the incubation time of the step (1) is 15-22 hours.
  • the incubation time of the step (2) is 30 minutes to 24 hours
  • the incubation time of the step (2) is 30 minutes to 21 hours;
  • the incubation time of the step (2) is 30 minutes to 17 hours;
  • the incubation time of the step (2) is 30 minutes to 12 hours;
  • the incubation time of the step (2) is 30 minutes-10 hours;
  • the incubation time of the step (2) is 30 minutes to 8 hours;
  • the incubation time of the step (2) is 1 hour to 8 hours;
  • the incubation time of the step (2) is 1 hour to 4 hours;
  • the incubation time of the step (2) is 1 hour to 3 hours.
  • the infusion composition is obtained from peripheral blood, cord blood, bone marrow and/or induced pluripotent stem cells.
  • the infusion composition is a leukocyte separation sample; preferably, the infusion The composition is enriched or isolated CD3+ T cells, enriched or isolated CD4+ T cells, or enriched or isolated CD8+ T cells, or a combination thereof.
  • the viral vector particles are derived from retroviral vectors; preferably, the viral vector particles are lentiviral vectors.
  • the multiplicity of infection of the viral vector particles is not higher than 20; preferably, the multiplicity of infection is 0.5-20; more preferably, the multiplicity of infection is 1.5-20; more preferably, the multiplicity of infection is 1.5-20; The multiplicity of infection is 3-20; more preferably, the multiplicity of infection is 3-12.
  • the number of cells to be transduced in the input composition does not exceed 1*10 10 ;
  • the number of cells to be transduced in the input composition is not less than 1*10 5 ;
  • the number of cells to be transduced in the input composition is not less than 1*10 6 .
  • the recombinant nucleic acid can encode a receptor that recognizes a specific target antigen; preferably, the receptor that recognizes a specific target antigen is a T cell receptor (TCR), a chimeric antigen receptor ( CAR), chimeric T cell receptor, or T cell antigen coupler (TAC).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric antigen receptor comprises a cell surface antigen recognition domain that specifically binds to a target antigen and an intracellular signal transduction domain comprising ITAM.
  • the intracellular signaling domain comprises the intracellular domain of the CD3-zeta (CD3 ⁇ ) chain.
  • the chimeric antigen receptor further includes a transmembrane domain connecting the extracellular domain and the intracellular signal transduction domain.
  • the transmembrane domain comprises the transmembrane portion of CD28 and/or CD8.
  • the intracellular signaling domain further comprises the intracellular signaling domain of T cell costimulatory molecules.
  • the T cell costimulatory molecule is selected from CD28 and/or 41BB.
  • the specific target antigen is a disease-related antigen or a universal label
  • the disease is cancer, autoimmune disease, or infectious disease
  • the cancer is a hematological tumor; more preferably, the hematological tumor is leukemia, myeloma, lymphoma and/or a combination thereof.
  • the specific target antigen is a tumor-associated antigen
  • the tumor-associated antigen is selected from: B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), EGFR, Her2/neu (receptor tyrosine kinase erbB2), CD19, CD20, CD22, mesothelial , CEA, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epiglin 2 (EPG-2), epiglin 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb- B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R- ⁇ , IL-13R- ⁇ 2, kinase insertion domain Receptor (kdr), L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE
  • the cell stimulator to be transduced can activate one or more intracellular signal domains of one or more components of the TCR complex and one or more cells of one or more costimulatory molecules.
  • the cell stimulator to be transduced comprises (i) a primary reagent that specifically binds to a member of the TCR complex, optionally specifically binding to CD3, and (ii) a primary reagent that specifically binds to T cell costimulatory molecules.
  • Secondary reagent optionally wherein the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40 or ICOS.
  • the cell to be transduced is an immune effector cell
  • the cells to be transduced are T cells, NK cells, NKT cells, dendritic cells, macrophages, CIK cells, and stem cell-derived immune effector cells or combinations thereof;
  • the cells to be transduced are T cells.
  • T cells are CD4+ and/or CD8+ cells.
  • the ratio of the CD4+ cells to the CD8+ cells is 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1, 1:5 , 5:1, 1:6 or 6:1.
  • a reagent 4 for the selection or enrichment is included.
  • the reagent 4 that is not bound to the T cell can be removed by centrifugation.
  • the 4 is fixed on a solid support, and preferably, the solid support is a polymer matrix.
  • the polymer matrix is a polymer nanomatrix and/or a bead reagent.
  • the bead reagent includes magnetic beads and/or microbeads.
  • the sample is a leukocyte separation technique sample.
  • the T cell is an enriched or isolated CD3+ T cell, an enriched or isolated CD4+ T cell, or an enriched or isolated CD8+ T cell.
  • the T cells have been selected or enriched from the sample from the subject.
  • the cell stimulator to be transduced comprises a CD3 binding molecule, a CD28 binding molecule, a recombinant IL-2, a recombinant IL-15, a recombinant IL-7, a recombinant IL-21, or a combination thereof;
  • the cell stimulator to be transduced comprises an anti-CD3 antibody and/or an anti-CD28 antibody.
  • the cell stimulator to be transduced before harvesting, can be removed by centrifugation.
  • the cell stimulator to be transduced is a free molecule.
  • the cell stimulator to be transduced is immobilized on a solid support
  • the solid support is a polymer matrix material
  • the polymer matrix material is a degradable polymer nanomatrix or bead reagent.
  • the bead reagent is magnetic beads or microbeads.
  • the content of cells transduced with recombinant nucleic acid in the output composition is not less than 30%, or not less than 40%, or not less than 50%, or not less than 60%, Or not less than 70%, or not less than 80%.
  • the content of cells transduced with recombinant nucleic acid in the output composition is not higher than 50%; preferably, not higher than 40%, more preferably, not higher than 38%; more preferably , Not higher than 35%; more preferably, not higher than 30%.
  • the content of immature cells in the cells transduced with the recombinant nucleic acid is lower than the content of immature cells in the cells to be transduced;
  • the content of immature cells is reduced to less than 10%;
  • the content of immature cells is reduced to less than 5%.
  • the content of memory cells in the cells transduced with the recombinant nucleic acid is higher than the content of memory cells in the cells to be transduced;
  • the memory cell is a memory stem cell
  • the memory stem cell is TSCM.
  • the content of memory stem cells in the cells transduced with the recombinant nucleic acid is about 2 times or more than the content of memory stem cells in the cells to be transduced, preferably, the memory in the cells transduced with the recombinant nucleic acid
  • the content of sex stem cells is about 3 times or more than the content of memory stem cells in the cells to be transduced.
  • the cells transduced with recombinant nucleic acid contain undifferentiated cells.
  • the input composition comprises recombinant IL-2, optionally recombinant human IL-2, the concentration of the recombinant IL-2 is 10 IU/mL to 500 IU/mL, 50 IU/mL to 250 IU/mL Or 100IU/mL to 200IU/mL; or a concentration of at least 10IU/mL, 50IU/mL, 100IU/mL, 200IU/mL, 300IU/mL, 400IU/mL or 500IU/mL; and/or
  • the input composition comprises recombinant IL-15, optionally recombinant human IL-15, the concentration of the recombinant IL-15 is 1IU/mL to 100IU/mL, 2IU/mL to 50IU/mL, or 5IU/mL to 10IU/mL mL; or the concentration is at least 1IU/mL, 2IU/mL, 5IU/mL, 10IU/mL, 25IU/mL or 50IU/mL; and/or
  • the input composition comprises recombinant IL-7, optionally recombinant human IL-7, the concentration of the recombinant IL-7 is 50IU/mL to 1500IU/mL, 100IU/mL to 1000IU/mL to 200IU/mL to 600IU/mL mL; or the concentration is at least 50IU/mL, 100IU/mL, 200IU/mL, 300IU/mL, 400IU/mL, 500IU/mL, 600IU/mL, 700IU/mL, 800IU/mL, 900IU/mL or 1000IU/mL.
  • the harvested output composition is washed to obtain cells transduced with recombinant nucleic acid.
  • the cells transduced with the recombinant nucleic acid are added to a buffer for storage; preferably, the buffer contains a cell cryopreservation agent.
  • the cells transduced with the recombinant nucleic acid do not need to be expanded in vitro after being harvested and before being administered to a subject in need.
  • the present invention provides a composition of cells transduced with recombinant nucleic acid produced by the method described in the first or second aspect.
  • the cell is an immune effector cell.
  • the cell is a T cell.
  • the ratio of TSCM in the cells transduced with the recombinant nucleic acid is higher than the ratio of TSCM in the cells to be transduced;
  • the ratio of TSCM in the cell transduced with the recombinant nucleic acid is about 2 times or more of the ratio of TSCM in the cell to be transduced;
  • the ratio of TSCM in the cell transduced with the recombinant nucleic acid is about 3 times or more of the ratio of TSCM in the cell to be transduced.
  • the proportion of TSCM in the cells transduced with recombinant nucleic acid is more than 10%, preferably more than 13%, and more preferably more than 15%.
  • the cells transduced with the recombinant nucleic acid do not need to be expanded in vitro before being administered to the subject.
  • the present invention provides a composition comprising the cell transduced with the recombinant nucleic acid described in the third aspect and a pharmaceutically acceptable carrier.
  • the present invention provides a method of adoptive cell therapy, comprising administering the composition of the fourth aspect to a subject in need thereof.
  • the present invention not only shortens the activation and/or activation steps before being exposed to retroviral vector particles, but also further shortens the incubation time after transduction, shortens the in vitro activation and transduction culture time even to 1 to 2 days, and in the activation After the transduction is completed, there is no need for expansion before the prepared cells are used for patient treatment. Under the same conditions of activation, the later the lentiviral vector is added, the transduction efficiency has a significantly higher trend, although the actual transduction duration is decreasing.
  • FIG. 1 shows the changes in transduction efficiency at different times after the CAR T cells prepared by the new process and the conventional control transduction under various activated transduction conditions.
  • FIG. 3 Shows the transduction efficiency changes at different times after the CAR T cells prepared by the new process and conventional controls are transduced under various activated transcription conditions.
  • Figure 4 Shows the anti-tumor experiment of CAR-T cells in vivo.
  • Figure 5. shows the survival of human T cells in the peripheral blood of mice.
  • Figure 6 Shows the effect of different concentrations of T cell activator on the efficiency of lentiviral transduction.
  • the inventor unexpectedly discovered that the shortening of the activation transduction time in the preparation of T cells not only does not affect the expression of recombinant nucleic acid, but instead improves the proliferation ability and survival time of T cells in vivo.
  • the present invention has been completed on this basis.
  • cell and other grammatical forms can refer to cells of human or non-human animal origin.
  • immune effector cells refers to cells that participate in immune responses and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, and macrophages , Mast cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cell may be an autologous T cell, a heterologous T cell, or an allogeneic T cell.
  • the NK cells may be allogeneic NK cells.
  • artificially modified cell with immune effector cell function refers to a cell or cell line that does not have an immune effect after being artificially modified or stimulated by a stimulus, the cell acquires the immune effector cell function.
  • 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
  • T cells may be pluripotent stem cells derived from bone marrow, which differentiate and mature into mature T cells with immunological activity in the thymus.
  • T cells can be cell populations with specific phenotypic characteristics, or mixed cell populations with different phenotypic characteristics.
  • T cells can be cells containing at least one T cell subpopulation: memory Stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells ( Tem).
  • Tscm cells memory Stem cell-like memory T cells
  • Tcm central memory T cells
  • effector T cells Tef, Tefff
  • Tregs regulatory T cells
  • Tem effector memory T cells
  • Tem effector memory T cells
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of techniques known to those skilled in the art, such as FicollTM isolation can be used to obtain T cells from blood collected from an individual.
  • cells from the circulating blood of the individual are obtained by a single blood collection.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets.
  • the cells collected by apheresis collection can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from healthy donors, from patients diagnosed with cancer.
  • chimeric antigen receptor includes extracellular antigen binding domains, transmembrane domains, and intracellular signaling domains.
  • the intracellular signaling domain includes functional signaling domains of stimulatory molecules and/or costimulatory molecules.
  • the stimulatory molecule is the zeta chain that binds to the T cell receptor complex; in one aspect, the cytoplasmic signal
  • the conduction domain further includes one or more functional signaling domains of costimulatory molecules, such as 4-1BB (ie, CD137), CD27, and/or CD28.
  • T cell receptor T cell receptor, TCR
  • MHC major histocompatibility complex
  • T cells T cell receptor, TCR
  • the classic TCR receptor is composed of two peptide chains, ⁇ and ⁇ . Each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region, etc., and its antigen
  • V region variable region
  • C region constant region
  • TCR ⁇ variable region
  • T cells expressing classic TCR can use methods such as antigen stimulation on T cells. Induces the specificity of the TCR of T cells to the target antigen.
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides constituting the TCR, which can bind to the surface antigens of target cells and interact with other polypeptides of the complete TCR complex, usually co-localized in T cell surface.
  • the chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain.
  • the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, and TCR cell
  • the stimulation domain of the intracellular signal domain of the intradomain; the TCR subunit and the antibody domain are effectively connected, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , and ,
  • the chimeric T cell receptor is integrated into the TCR expressed on the T cell.
  • T cell antigen coupler includes three functional domains: 1. Antigen binding domain, including single-chain antibodies, designed ankyrin repeat protein (DARPin) Or other targeting groups; 2. The extracellular domain, a single-chain antibody that binds to CD3, so that the TAC receptor and the TCR receptor are close to each other; 3. The transmembrane region and the intracellular region of the CD4 co-receptor, where The intracellular domain is connected to the protein kinase LCK to catalyze the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • transduction refers to the introduction of exogenous nucleic acid into eukaryotic cells.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Individuals of the present invention include, but are not limited to, humans, non-human primates (such as rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.
  • peripheral blood mononuclear cell refers to cells with mononuclear nuclei in peripheral blood, including lymphocytes and monocytes.
  • T cell activation or “T cell activation” refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • exogenous refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function of overexpression.
  • vector refers to a nucleic acid molecule that can propagate another nucleic acid molecule to which it is linked.
  • the term includes a vector that is a self-replicating nucleic acid structure as well as a vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • Vectors include viral vectors, such as retroviral vectors, such as lentivirus or gamma retroviral vectors, which have a genome that carries another nucleic acid and can be inserted into the host genome for its reproduction.
  • treatment refers to the complete or partial reduction or reduction of a disease, or related symptoms, adverse reactions or results, or phenotype.
  • the effect is therapeutic, such that it partially or completely cures the disease or the adverse symptoms attributed to it.
  • “Therapeutically effective amount” refers to an amount that is effective to achieve the desired therapeutic result (such as for the treatment of a disease or disorder) and/or the pharmacokinetic or pharmacodynamic effect of the treatment under the necessary dosage and within the necessary time period.
  • the therapeutically effective amount may vary according to factors such as the disease state, the age, sex, and weight of the subject, and the cell population administered.
  • MMI multipleplicity of infection
  • MHC major histocompatibility complex
  • the protein can interact with the peptide antigen of the polypeptide (including Peptide antigens processed by the cellular machinery) complex.
  • MHC molecules can be displayed or expressed on the cell surface, including as a complex with a peptide, that is, an MHC-peptide complex, used to present antigen receptors (such as TCR or TCR-like antibodies) on T cells. Recognizable conformation of the antigen.
  • MHC class I molecules are heterodimers that have a membrane spanning the alpha chain, in some cases three alpha domains and non-covalently associated beta 2 microglobulin.
  • MHC class II molecules are composed of two transmembrane glycoproteins ⁇ and ⁇ , both of which usually span membranes.
  • the MHC molecule may include an effective part of MHC, which contains an antigen binding site or a site for binding peptides and a sequence required for recognition by an appropriate antigen receptor.
  • MHC class I molecules deliver cytosol-derived peptides to the cell surface, where the MHC-peptide complex is composed of T cells (e.g. usually CD8+ T cells, but in some cases CD4+ T cells). ) Identification.
  • MHC class II molecules deliver peptides derived from the vesicle system to the cell surface, where the peptides are generally recognized by CD4+ T cells.
  • MHC molecules are encoded by a set of linked loci, which are collectively referred to as H-2 in mice and human leukocyte antigen (HLA) in humans. Therefore, usually human MHC can also be called human leukocyte antigen (HLA).
  • MHC-peptide complex or "peptide-MHC complex” or a variant thereof refers to a complex or association of a peptide antigen and an MHC molecule, for example, usually through the peptide in the binding groove or cleft of the MHC molecule Of non-covalent interactions.
  • the MHC-peptide complex is present or displayed on the cell surface.
  • the MHC-peptide complex can be specifically recognized by an antigen receptor (eg, TCR, TCR-like CAR, or antigen binding portion thereof).
  • peptides of the polypeptide can be associated with MHC molecules, for example for recognition by antigen receptors.
  • the peptides are derived from or based on fragments of longer biomolecules, such as polypeptides or proteins.
  • the peptide is generally about 8 to about 24 amino acids in length.
  • the peptide is 9 to 22 amino acids in length and is used for recognition in MHC class II complexes.
  • the peptide is 8 to 13 amino acids in length and is used for recognition in MHC class I complexes.
  • antigen receptors such as TCR or TCR-like CAR
  • TCR TCR or TCR-like CAR
  • the present invention provides a method for transducing a viral vector into a cell (for example, an immune effector cell).
  • the method involves the activation and transduction of the cell to be transduced.
  • the activation and transduction of the cell can be performed simultaneously, that is, the cell to be transduced is contained.
  • the input composition, the cell stimulator to be transduced, and the viral vector particles carrying the recombinant nucleic acid are incubated together, or it can be activated before transduction.
  • the input composition containing the cells to be transduced and the cell stimulator to be transduced Co-incubate for activation, then add viral vector particles carrying recombinant nucleic acid to incubate, and complete the transduction of recombinant nucleic acid and control the total time of transduction within 72 hours, preferably within 48 hours, or within 36 hours, or Completed within 24 hours.
  • the provided methods involve incubating and/or contacting retroviral vector particles (e.g., lentiviral vectors) with a population of cells (e.g., immune cells, e.g., T cells), before contacting the cells with viral particles or Before and/or at the same time and/or after the incubation, an ex vivo cell activation reagent (for example, an anti-CD3/anti-CD28 reagent) is used to activate and/or activate T cells.
  • the cells are activated first, and then viral transduction is performed.
  • the incubation time does not exceed 72 hours for harvesting to obtain the output composition
  • the output composition contains cells transduced with recombinant nucleic acid; preferably, it can be 1 hour to 72 hours; more preferably, the incubation time is 2 hours to 48 hours; more preferably, the incubation time is 2 hours to 36 hours; more preferably, the incubation time is 12 hours to 36 hours; more preferably, the incubation time is 12 hours to 24 hours; more preferably, the incubation time is 15 hours to 24 hours .
  • a pharmaceutical preparation can be prepared without further in vitro amplification and culture, that is, the drug prepared by using the output composition is used in the test The person (or patient) does not need to be expanded in vitro before.
  • the method for transducing cells with a viral vector includes the following steps: step (1), incubating the input composition containing the cells to be transduced and a stimulant for the cells to be transduced, and the incubation time does not exceed 72 hours, and step (2) ).
  • the output composition contains the cells transduced with the recombinant nucleic acid; preferably, The total incubation time of (1) and (2) does not exceed 72 hours; more preferably, the total incubation time of (1) and (2) does not exceed 60h, or does not exceed 48h, or does not exceed 32h, or It does not exceed 28h, and more preferably, the total incubation time of (1) and (2) does not exceed 24h.
  • the incubation time of step (1) is 2-72 hours; preferably, the incubation time of step (1) is 2-71 hours, and more preferably, the incubation time of step (1) More preferably, the incubation time of the step (1) is 2 to 32 hours; more preferably, the incubation time of the step (1) is 2-28 hours; more preferably, the incubation time of the step (1) is 2-28 hours; The incubation time of step (1) is 3-24 hours; more preferably, the incubation time of step (1) is 5-24 hours; more preferably, the incubation time of step (1) is 7-24 hours ; More preferably, the incubation time of the step (1) is 7-23 hours; more preferably, the incubation time of the step (1) is 10-23 hours; more preferably, the incubation time of the step (1) The incubation time is 15-23 hours; more preferably, the incubation time in the step (1) is 15-22 hours.
  • the incubation time of the step (2) is 30 minutes to 24 hours, preferably, the incubation time of the step (2) is 30 minutes to 21 hours; preferably, the step ( The incubation time of 2) is 30 minutes to 17 hours; preferably, the incubation time of step (2) is 30 minutes to 12 hours; preferably, the incubation time of step (2) is 30 minutes to 10 hours. Hours; preferably, the incubation time of step (2) is 30 minutes to 8 hours; preferably, the incubation time of step (2) is 1 hour to 8 hours; preferably, the step ( The incubation time of 2) is 1 hour to 4 hours; more preferably, the incubation time of step (2) is 1 hour to 3 hours.
  • the recombinant nucleic acid may be a receptor that encodes a specific target antigen, such as a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupling ⁇ (TAC).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupling ⁇
  • the specific target antigen is a disease-related antigen or a universal tag.
  • the disease is cancer, autoimmune disease, or infectious disease.
  • the specific target antigen is a tumor-associated antigen, such as: B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2), CD19, CD20, CD22, mesothelin, CEA, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epiglin 2 (EPG-2), epiglin 40 (EPG-40), EPHa2, erb- B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R- ⁇ , IL- 13R- ⁇ 2, kinase insertion domain receptor (kdr), L1 cell adhesion molecule (L1-CAM), melanoma
  • BCMA
  • the resulting cells transduced with recombinant nucleic acid can be used for adoptive immunotherapy.
  • the provided method can be used to prepare immune cells, such as T cells, for adoptive therapy, and the total time of activation and transduction of the method is controlled at 24 hours, or 36 hours, or 48 hours, or 72 hours. Within hours.
  • the provided methods shorten the time to engineer and/or prepare cells for adoptive cell therapy.
  • the input composition comprises a primary cell population that has been obtained from a sample of the subject and/or is enriched for a specific subset of cells (e.g., T cells).
  • the cell population e.g., input composition
  • the cell population may be a cell population that has previously been cryopreserved. In some embodiments, no more than or no more than about 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, after obtaining a sample containing primary cells (e.g., apheresis sample) from the subject Start the incubation and/or contact at 48 hours or 72 hours.
  • the method produces an output composition, wherein at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the total cells (or specific target cell types, such as T The cell) uses the viral vector to transduce and/or express the recombinant gene product encoded by it.
  • T cell activation markers Methods for evaluating the expression of T cell activation markers are known in the art. Antibodies and reagents for detecting such labels are well known in the art and are readily available. The assays and methods used to detect such markers include, but are not limited to, flow cytometry (including intracellular flow cytometry), ELISA, ELISPOT, cell counting bead arrays or other multiple methods, western blots, and other methods based on immunoaffinity . In some embodiments, the method is capable of achieving at least a specific transduction efficiency under certain conditions.
  • the provided methods may also include a cryopreservation step before or after incubating (eg, transducing) the cells with virus particles.
  • this step can provide for the preservation of cell products, such as cell preservation during transportation, or cell preservation after preparation.
  • activation or stimulation can be performed ex vivo or in vivo.
  • the cells after incubating (eg, transducing) the cells with viral particles, the cells can be infused into the patient for in vivo activation and expansion.
  • the cell activator to be transduced may be one type, two types, or a combination of multiple types.
  • T cell activators can use CD3 binding molecules (such as CD3 antibodies), CD28 binding molecules (such as CD28 antibodies), recombinant IL-2, recombinant IL-15, recombinant IL-7, recombinant IL-21, or at least two A mixture of species, such as using CD3 antibody and CD28 antibody, or using CD3 antibody, CD28 antibody or IL2.
  • the multiplicity of infection of the viral vector particles is not higher than 20; preferably, the multiplicity of infection is 0.5-20; more preferably, the multiplicity of infection is 1.5-20; more preferably, the multiplicity of infection is 1.5-20; It is 3-20; more preferably, the multiplicity of infection is 3-12.
  • the provided method may further include culturing the input composition, output composition, and/or transduced cells ex vivo, such as culturing under conditions of activated cells, to induce their proliferation And/or activate.
  • Activation is carried out in the presence of one or more activators.
  • the activator can be a CD3 binding molecule, a CD28 binding molecule, or a cytokine (such as recombinant IL-2, recombinant IL-15, recombinant IL-7, or recombinant IL-21).
  • the binding molecule is an antibody or antigen-binding fragment, such as an anti-CD3 antibody and/or an anti-CD28 antibody.
  • further culturing is performed under conditions that achieve cell expansion to produce a therapeutically effective dose of cells for administration to the subject by adoptive cell therapy.
  • the provided methods avoid the use of nucleic acid encoding recombinant receptors (such as CAR) to introduce, transfer, and/or transduce T cells to significantly change the differentiation state of T cells and/or make T cells ex vivo. Changes in T cell differentiation status are minimized.
  • memory T cells are generated according to the provided methods, including stem cell memory T cells, central memory T cells, and effector memory T cells.
  • the cell output composition of the present invention contains a ratio of cells transduced with recombinant nucleic acid (such as CAR T cells) that is lower than that of conventional processes.
  • the number of cells does not exceed 1*10 10 , 1*10 9 , 1*10 8 , 1*10 7 , 1*10 6 , 1*10 5 , or 1*10 4 .
  • the output composition of the present invention comprising cells transduced with recombinant nucleic acid has a higher content of memory cell phenotype (such as memory T cells) than conventional processes.
  • the content is at least 1.5 times, 2 times, 3 times, 4 times, or 5 times higher.
  • memory T cells are cells with a T central cell memory (TCM) phenotype, such as CD45RO+CCR7+CD62L+ T cells and/or CD45RO+CCR7+CD27+CD28+CD62L+T cells.
  • TCM T central cell memory
  • one, more, or all steps in the preparation of cells for clinical use (for example, in adoptive cell therapy) of the present invention are performed under sterile conditions.
  • one or more processes of cells being enriched, activated, transduced, or washed are performed in a closed system.
  • treating the cells ex vivo for a shorter period of time further shortens the time.
  • the recombinant nucleic acid-transduced cells (such as CAR T cells) produced by the provided methods exhibit longer persistence and/or reduced cell consumption when administered to a subject.
  • the recombinant nucleic acid-transduced cells (such as CAR T cells) produced by the provided methods exhibit better efficacy when administered to a subject.
  • the provided methods reduce cell variability during the preparation of cell therapy products.
  • eliminating the time for cell activation and transduction in vitro improves the process of preparing cells transduced with recombinant nucleic acid for adoptive immunotherapy.
  • the input composition is a composition of primary cells obtained from a subject, wherein, in some cases, a subpopulation or subset of cells has been selected and/or enriched.
  • the input composition may be a T cell population, an enriched T cell population, or PBMC.
  • the cell includes one or more nucleic acids introduced by genetic engineering according to the provided methods, thereby expressing recombinant or genetically engineered products of such nucleic acids.
  • the nucleic acid is heterologous, that is, it is not normally present in a cell or a sample obtained from a cell, such as a nucleic acid obtained from another organism or cell, for example, the nucleic acid is generally not present in the engineered cell And/or found in the organism from which this cell is derived.
  • the nucleic acid is not a naturally occurring nucleic acid, such as a nucleic acid not found in nature, and includes a nucleic acid encoding a chimeric combination of nucleic acids of various domains from multiple different cell types.
  • the processing steps of the method may include any one or more of the multiple cell processing steps alone or in combination.
  • the processing step includes transducing the cell with viral vector particles containing a retroviral vector, such as a vector encoding a recombinant product for expression in the cell.
  • the method may further and/or alternatively include other processing steps, such as isolation, separation, selection, washing, suspension, dilution, concentration, and/or formulation of cells.
  • the method may also include an ex vivo culturing step (e.g., activating the cell to, for example, induce its proliferation and/or activation).
  • the step of activating or activating cells is performed in the body after administering the cells to the subject, through antigen recognition and/or during the administration of one or more agents to strengthen or expand the cells in the subject After the amplification, activation and/or proliferation.
  • the method includes isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same subject before or after cryopreservation.
  • the method includes processing steps performed in the following order, wherein: first isolate (eg select or isolate) primary cells from a biological sample; activate the selected cells in vitro in the presence of an activating reagent , Amplify or multiply, and then add viral vector particles and incubate for transduction.
  • the total activated transduction time does not exceed or does not exceed about 24 or 36 or 48 hours, wherein the transduction time is at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours.
  • the transduced cells are activated, expanded, or proliferated ex vivo, for example, by activation in the presence of an activation reagent.
  • the method may include one or more processing steps from washing, suspending, diluting, and/or concentrating cells, which may be used in isolation (e.g., separation or selection), activation, transduction, and/or formulation Before, during, or at the same time or after the step.
  • one or more or all of the processing steps are using integrated or self-contained systems System, device or equipment in and/or in an automated or programmable manner.
  • one or more cell processing steps combined with the preparation, processing, and/or incubation of cells in conjunction with the provided transduction method can be performed in a culture bag or flask, which is compared with other available methods Can provide certain advantages.
  • the system includes a series of containers, such as bags, tubing, stopcocks, clips, connectors, and centrifuge chambers.
  • the container such as a culture bag or a culture bottle
  • the container includes one or more containers (such as a culture bag or a culture bottle), which are in the same container or a separate container (such as the same culture bag or a culture bottle; or a separate culture bag).
  • culture flask containing cells to be transduced and viral vector particles.
  • the system e.g., a closed system
  • the system is sterile.
  • the system may be disposable, such as a disposable culture bag or culture bottle.
  • the cell is usually a eukaryotic cell, such as a mammalian cell, and usually a human cell.
  • the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system, such as cells of innate or adaptive immunity, such as bone marrow or lymphocytes, including lymphocytes, usually T cells and / Or NK cells.
  • Other exemplary cells include stem cells, such as pluripotent stem cells and pluripotent stem cells, including induced pluripotent stem cells (iPSC).
  • the cells are generally primary cells such as those primary cells isolated directly from the subject and/or isolated and frozen from the subject.
  • the cells include one or more subgroups of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those subpopulations defined by: Function, activation state, maturity, possibility of differentiation, expansion, recycling, localization and/or persistence, antigen specificity, antigen receptor type, presence in specific organs or compartments, markers or cytokine secretion Characteristics and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous.
  • the methods include off-the-shelf methods.
  • the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSC).
  • the method includes isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same subject before or after cryopreservation.
  • T cells naive T cells
  • TEFF effector T cells
  • T cells memory T cells and their subtypes (such as stem cell memory T (TSCM), central memory T (TCM), effect memory T (TEM) or terminally differentiated effect memory T cells), Tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T Cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), ⁇ / ⁇ T cells and ⁇ / ⁇ T cells.
  • TIL Tumor infiltrating lymphocytes
  • immature T cells immature T cells
  • mature T cells mature T cells
  • helper T cells cytotoxic T cells
  • mucosal associated invariant T (MAIT) cells mucosal associated invariant
  • the cell is a natural killer (NK) cell.
  • the cells are monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and/or basophils .
  • the cell is derived from a cell line, for example, a T cell line.
  • the cells are obtained from a heterogeneous source, such as from mice, rats, non-human primates, and pigs.
  • the cells can be isolated from a sample, such as a biological sample, such as a sample obtained from or derived from a subject.
  • a sample such as a biological sample, such as a sample obtained from or derived from a subject.
  • the subject from which the cells are isolated is a subject suffering from a disease or in need of cell therapy or to whom cell therapy will be administered.
  • the subject is a human in need of specific therapeutic intervention (such as adoptive cell therapy, in which cells are isolated, processed, and/or engineered).
  • the cell is a primary cell, such as a primary human cell.
  • Samples include tissues, fluids and other samples taken directly from the subject, as well as samples from one or more processing steps (such as separation, centrifugation, genetic engineering (such as transduction with viral vectors), washing and/or incubation) .
  • the biological sample may be a sample obtained directly from a biological source or a processed sample.
  • Biological samples include but are not limited to body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or separated is blood or a blood-derived sample, or is or is derived from apheresis or leukocyte separation.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosal-associated lymphoid tissue, spleen, other lymphoid tissues, Liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs and/or cells derived therefrom.
  • samples include samples from autologous and allogeneic sources.
  • cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte separation.
  • the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the blood sample collected from the subject is washed, for example, to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the washing solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • the washing step is completed by an automatic or semi-automatic "flow-through" centrifuge (e.g., Cobe2991 cell processor, Baxter, MACS PLUS) according to the manufacturer's instructions.
  • the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions.
  • the cells are resuspended in a variety of biocompatible buffers (e.g., PBS without Ca++/Mg++) after washing.
  • the components of the blood cell sample are removed and the cells are directly resuspended in the culture medium.
  • the sample prior to enrichment and/or selection of cells, is contacted with serum or plasma (e.g., human serum or plasma) and/or contains the serum or plasma.
  • serum or plasma e.g., human serum or plasma
  • the serum or plasma is autologous to the subject from which the cells are obtained.
  • serum or plasma is present in the sample at the following concentrations: at least or at least about 10% (v/v), at least or at least about 15% (v/v), at least or at least about 20% (v/v) v), at least or at least about 25% (v/v), at least or at least about 30% (v/v), at least or at least about 35% (v/v), or at least or at least about 40% (v/v) .
  • a sample containing primary cells is contacted with or contains an anticoagulant.
  • the anticoagulant is or contains free citrate ions, for example, the anticoagulant citrate dextrose solution, solution A (ACD-A).
  • cells from the sample are transferred or suspended in a serum-free medium.
  • the serum-free medium is a defined and/or well-defined cell culture medium.
  • a serum-free medium is formulated to support the growth, proliferation, health, and homeostasis of cells of a certain cell type (e.g., immune cells, T cells, and/or CD4+ and CD8+ T cells).
  • the sample is maintained or kept at a temperature of 2°C to 8°C for up to 48 hours, such as up to 12 hours, 24 hours, or 36 hours.
  • the preparation method includes a step of freezing (e.g., cryopreservation) cells before or after the isolation, selection, and/or enrichment and/or incubation for transduction and engineering.
  • freezing and subsequent thawing steps remove granulocytes in the cell population, and to some extent monocytes.
  • the cells are suspended in a freezing solution to remove plasma and platelets, for example, after a washing step.
  • a variety of known freezing solutions can be used.
  • the T cells are cryopreserved in the presence of a cryoprotectant.
  • a cryoprotectant One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media.
  • HSA human serum albumin
  • the separation of cells includes one or more preparation and/or non-affinity-based cell separation steps.
  • the cells are washed, centrifuged and/or incubated in the presence of one or more reagents, for example to remove unwanted components, enrich for desired components, lyse or remove sensitive to specific reagents Cell.
  • cells are separated based on one or more characteristics (such as density, adhesion characteristics, size, sensitivity and/or resistance to specific components).
  • the separation method includes separating different cell types based on the expression or presence of one or more specific molecules (such as surface markers, such as surface proteins, intracellular markers, or nucleic acids) in the cell.
  • specific molecules such as surface markers, such as surface proteins, intracellular markers, or nucleic acids
  • the separation is based on affinity or immunoaffinity.
  • the separation includes separating cells and cell populations based on the expression or expression levels of one or more markers (usually cell surface markers) of the cells, for example by and specifically binding to such markers The antibody or binding partner is incubated with the antibody or binding partner, followed by a washing step and separation of cells that have bound the antibody or binding partner from those cells that are not bound to the antibody or binding partner.
  • Such separation steps may be based on positive selection (in which cells that have been bound to the reagent are retained for further use) and/or negative selection (in which cells that are not bound to the antibody or binding partner are retained).
  • the separation need not result in 100% enrichment or removal of specific cell populations or cells expressing specific markers.
  • positive selection or enrichment for specific types of cells refers to increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker.
  • negative selection, removal or depletion of specific types of cells refers to reducing the number or percentage of such cells, but does not need to result in the complete removal of all such cells.
  • T cells such as cells that are positive or highly expressing one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and/or CD45RO+T cells) are separated by positive or negative selection techniques.
  • Anti-CD3/anti-CD28 conjugated magnetic beads or microbeads can be used to positively select CD3+ and CD28+ T cells.
  • separation is performed by positive selection for enrichment for a specific cell population or by negative selection for depletion of a specific cell population.
  • the positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that are associated with the positive or negative selection of cells, respectively.
  • One or more surface markers specifically expressed on or at a relatively high level (marker high) (marker +) specifically binds.
  • T cells are separated from PBMC samples by negative selection of markers expressed on non-T cells (such as B cells, monocytes, or other white blood cells, such as CD14).
  • non-T cells such as B cells, monocytes, or other white blood cells, such as CD14.
  • CD4+ or CD8+ selection step is used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells.
  • Such CD4+ and CD8+ populations can be further classified into subpopulations by positive or negative selection of markers expressed on one or more naive, memory, and/or effector T cell subpopulations or at a relatively high degree.
  • CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the corresponding subpopulation.
  • central memory T (TCM) cells are enriched to increase efficacy, such as to improve long-term survival, expansion and/or transplantation after administration.
  • TCM-rich CD8+ T cells and CD4+ T cells further enhance the efficacy.
  • memory T cells exist in two subsets of CD8+ peripheral blood lymphocytes, CD62L+ and CD62L-.
  • the PBMC can be enriched or depleted against the CD62L-CD8+ and/or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.
  • the enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some aspects, it is based on positive or high expression Negative selection of cells expressing CD45RA and/or Granzyme B.
  • the isolation of a CD8+ population rich in TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD62L.
  • the enrichment of central memory T (TCM) cells is performed from a negative cell fraction selected based on CD4 expression, which is negatively selected based on the expression of CD14 and CD45RA and positively selected based on CD62L.
  • a PBMC sample or other white blood cell sample is subjected to CD4+ cell selection, in which negative and positive fractions are retained. Then the negative fraction is negatively selected based on the expression of CD14 and CD45RA or CD19, and positively selected based on the marker characteristics of central memory T cells (such as CD62L or CCR7), wherein the positive and negative selections are performed in any order.
  • CD4+ cell selection in which negative and positive fractions are retained. Then the negative fraction is negatively selected based on the expression of CD14 and CD45RA or CD19, and positively selected based on the marker characteristics of central memory T cells (such as CD62L or CCR7), wherein the positive and negative selections are performed in any order.
  • CD4+T helper cells are classified into naive, central memory and effector cells.
  • CD4+ lymphocytes can be obtained by standard methods.
  • the naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells.
  • the central memory CD4+ cells are CD62L+ and CD45RO+.
  • the effector CD4+ cells are CD62L- and CD45RO-.
  • the monoclonal antibody cocktail in order to enrich CD4+ cells by negative selection, usually includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.
  • the antibody or binding partner is bound to a solid support (e.g., beads) or matrix (e.g., magnetic or paramagnetic beads or microbeads) to allow cell separation for positive and/or negative selection .
  • a solid support e.g., beads
  • matrix e.g., magnetic or paramagnetic beads or microbeads
  • immunomagnetic (or affinity magnetic) separation techniques are used to separate or separate cells and cell populations.
  • the T cell activator is a solid support (e.g., beads, including magnetic beads and/or microbeads; polymerized with anti-CD3 and/or anti-CD28 and/or anti-41-BB monoclonal antibodies).
  • Substance matrix including polymer nano matrix).
  • a sample or composition of cells to be separated is incubated with small magnetizable or magnetically responsive materials (such as magnetically responsive particles or microparticles, such as paramagnetic beads (such as Dynabeads or MACS beads, for example)).
  • the magnetic particles or beads comprise a magnetically responsive material that binds to a specific binding member (such as an antibody or other binding partner).
  • a specific binding member such as an antibody or other binding partner
  • the incubation is usually carried out under such conditions, whereby the antibody or binding partner or a molecule (such as a secondary antibody or other reagent) that specifically binds to such an antibody or binding partner attached to the magnetic particles or beads and the cell Surface molecules (if present on cells within the sample) bind specifically.
  • a molecule such as a secondary antibody or other reagent
  • the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles attached to it will be attracted to the magnet and separated from the unlabeled cells. For positive selection, keep the cells attracted by the magnet; for negative selection, keep the unattracted cells (unlabeled cells).
  • the magnetically responsive particles or beads remain attached to the cells, and the cells are subsequently incubated, cultured, and/or engineered; in some aspects, the particles or beads remain attached to the cells.
  • magnetizable or magnetically responsive particles are removed from the cell. Methods for removing magnetizable particles or beads from cells are known, and include, for example, the use of competing non-labeled antibodies and magnetizable particles or antibodies or beads conjugated to a cleavable linker.
  • the magnetizable particles are biodegradable.
  • the affinity-based selection is performed via magnetically activated cell sorting (M A C S) (Miltenyi Biotech, Auburn, CA).
  • MACS magnetically activated cell sorting
  • MACS operates in a mode in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, the cells attached to the magnetized particles remain in place, while the unattached species are eluted. Then, after completing the first elution step, the species trapped in the magnetic field and prevented from being eluted are released in some way so that they can be eluted and recovered.
  • the non-target cells are labeled and depleted from the heterogeneous cell population.
  • the method includes selecting cells, wherein all or part of the selection is performed in the lumen of the centrifuge chamber, for example, under centrifugal rotation.
  • incubating the cells with a selection reagent eg, a selection reagent based on immunoaffinity
  • selection based on immunoaffinity may depend on the favorable energy interaction between the isolated cell and a labeled molecule that specifically binds to the cell, such as an antibody on a solid (e.g., particle) or other binding partner.
  • the particles and cells are incubated in a container (e.g., tube or bag) while shaking or mixing, and the cell density is consistent with that of the particles (e.g., beads).
  • the ratio of) is constant to help promote energetically favorable interactions.
  • Such an approach may not be ideal for use in large-scale production, for example, because it may require the use of large volumes to maintain the optimal or desired cell to particle ratio while maintaining the desired number of cells. Therefore, such approaches may need to be processed in a batch mode or form, which may require increased time, number of steps, and operations, thereby increasing costs and the risk of user error.
  • the selection step is performed in a centrifuge chamber, which includes incubating the cells with selection reagents.
  • a certain volume of cells is mixed with a certain amount of the required affinity-based selection reagent, the volume and the amount are significantly less than in a tube or container according to the manufacturer’s instructions for similar selection.
  • the amount of one or more selection reagents used is the same for the same number of cells and/or the same volume of cells used to select cells in a tube or container-based incubation according to the manufacturer’s instructions.
  • the amount of one or more selection reagents is not more than 5%, not more than 10%, not more than 15%, not more than 20%, not more than 25%, not more than 50%, not more than 60%, not more than 70% Or not more than 80%.
  • incubation with one or more selection reagents as part of a selection method that can be performed in the cavity of the chamber includes the use of one or more selection reagents based on one or more specific molecules (e.g., surface markers, such as surface markers).
  • one or more selection reagents based on one or more specific molecules (e.g., surface markers, such as surface markers).
  • the expression or presence of protein, intracellular markers or nucleic acid) in or on cells, one or more different cell types are selected.
  • any known method, using one or more selection reagents can be used for separation based on such labels.
  • one or more selection reagents result in separation, which separation is based on affinity or immunoaffinity.
  • selection includes incubation with one or more reagents for separating cells and cell populations based on cell expression or expression levels of one or more markers (usually cell surface markers), such as by Antibodies or binding partners that specifically bind to such a label are incubated together, after which a washing step is usually performed and cells that have bound the antibody or binding partner are separated from those cells that are not bound to the antibody or binding partner.
  • markers usually cell surface markers
  • the cells are incubated in a chamber cavity in a composition, the composition further containing a selection buffer with a selection agent, such as Molecules, such as antibodies, that specifically bind to surface markers on cells that are desired to be enriched and/or depleted (rather than other cells in the composition), which are optionally coupled to a scaffold (e.g., a polymer or surface,
  • a selection agent such as Molecules, such as antibodies, that specifically bind to surface markers on cells that are desired to be enriched and/or depleted (rather than other cells in the composition
  • a scaffold e.g., a polymer or surface
  • beads such as magnetic beads or microbeads, such as magnetic beads or microbeads coupled with monoclonal antibodies specific for CD4 and CD8.
  • the total duration of incubation with the selection agent is 5 minutes to 6 hours, such as 30 minutes to 3 hours, such as at least 30 minutes, 60 minutes, 120 minutes, or 180 minutes.
  • the incubation is generally performed under mixing conditions, such as in the presence of rotation, usually at a relatively low force or speed, for example, the speed is lower than the speed used to precipitate the cells, for example, 600 rpm to 1700 rpm (e.g., 600 rpm to 1700 rpm).
  • the RCF is 80g to 100g (for example, at least 80g, 85g, 90g, 95g or 100g).
  • the incubated cells are forced out of the centrifugal chamber, for example, transferred from the centrifugal chamber to the system for immunoaffinity Separate the cells.
  • the system for separation based on immunoaffinity is or contains a magnetic separation column.
  • one or more other processing steps may be performed in the chamber, such as washing.
  • the CliniMACS system (Miltenyi Biotic) is used for separation and/or other steps, such as for automated separation of cells on a clinical scale in a closed and sterile system.
  • the CliniMACS Prodigy system (Miltenyi Biotec) is used for separation and/or other steps.
  • the CliniMACS Prodigy system is equipped with a cell processing complex that allows automated washing and fractionation of cells by centrifugation.
  • the cell populations described herein are collected and enriched (or depleted) by flow cytometry, where cells stained for multiple cell surface markers are carried in a fluid stream.
  • the cell populations described herein are collected and enriched (or depleted) by preparative scale (FACS) sorting.
  • FACS preparative scale
  • the antibody or binding partner is labeled with one or more detectable labels to facilitate isolation for positive and/or negative selection.
  • separation can be based on binding to fluorescently labeled antibodies.
  • the separation of cells carried in the fluid stream is based on the binding of antibodies specific to one or more cell surface markers or other binding partners, such as by fluorescence activated cell sorting (FACS), including preparation Scale (FACS) and/or microelectromechanical system (MEMS) chips, for example combined with flow cytometry systems.
  • FACS fluorescence activated cell sorting
  • FACS preparation Scale
  • MEMS microelectromechanical system
  • the screened cells are incubated and/or cultured.
  • the incubation step may include activating transduction to integrate the viral vector into the host genome of one or more cells.
  • Incubation and/or engineering can be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, petri dish, bag, or other container for culturing or cultivating cells .
  • the composition or cells are incubated in the presence of stimulating conditions or activating agents.
  • These conditions include those designed for the following conditions: conditions for inducing the proliferation, expansion, activation and/or survival of cells in the population, for simulating antigen exposure and/or for inducing cells to be genetically engineered, such as Introduction of recombinant antigen receptors.
  • the further incubation is performed under conditions for stimulation and/or activation of the cells, and the conditions may include one or more of the following: specific medium, temperature, oxygen content, carbon dioxide Content, time, medicament (e.g. nutrients), amino acids, antibiotics, ions and/or stimulating factors (e.g. cytokines, chemokines), antigens, binding partners, fusion proteins, recombinant soluble receptors and any other designed to activate cells Of medicine.
  • medicament e.g. nutrients
  • amino acids e.g. antibiotics, ions and/or stimulating factors (e.g. cytokines, chemokines), antigens, binding partners, fusion proteins, recombinant soluble receptors and any other designed to activate cells Of medicine.
  • stimulating factors e.g. cytokines, chemokines
  • the activation conditions or agents include one or more agents capable of activating the intracellular signaling domain of the TCR complex (e.g., stimulatory and/or auxiliary agents), such as ligands.
  • the agent turns on or initiates the TCR/CD3 intracellular signaling cascade in T cells, for example, is suitable for delivering primary signals such as to initiate ITAM-induced signals (such as those specific for TCR components)
  • Activating agents, and/or agents that promote costimulatory signals for example, costimulatory signals specific to T cell costimulatory receptors
  • costimulatory signals for example, costimulatory signals specific to T cell costimulatory receptors
  • anti-CD3, anti-CD28, or anti-41-BB for example, which optionally Binding to a solid support (e.g.
  • the stimulant includes anti-CD 3/anti-CD 28 beads (e.g., DYNABEADS M-4 5 0C D 3/C D 2 8T cell expansion agent and/or ExpACT Beads).
  • the activation method may further include the step of adding an anti-CD3 and/or anti-CD28 antibody, such as OKT-3, to the medium.
  • the stimulant includes IL-2 and/or IL-15 and/or IL-7, for example, the IL-2 concentration is at least about 10 units/mL.
  • the activation conditions or agents include one or more agents (eg, ligands) that are capable of activating the intracellular signaling domain of the TCR complex.
  • the agent turns on or initiates the TCR/CD3 intracellular signaling cascade in T cells.
  • agents may include, for example, antibodies bound to solid supports (such as beads, including magnetic beads or microbeads), such as antibodies specific for TCR components and/or costimulatory receptors (such as anti-CD3, anti-CD28) ; And/or one or more cytokines.
  • the amplification method may further include the step of adding anti-CD3 and/or anti-CD28 antibodies (for example, at a concentration of at least about 0.5 ng/ml) to the medium.
  • the stimulant includes IL-2 and/or IL-15 and/or IL-7, for example, the IL-2 concentration is at least about 10 units/mL, at least about 50 units/mL, at least about 100 units /mL or at least about 200 units/mL.
  • the total duration of incubation with the activator is or is between about 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, 4 hours and 36 hours. Time, between 8 hours and 30 hours, or between 12 hours and 24 hours, for example at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours.
  • the methods provided herein do not include further culturing or incubation, for example, do not include an ex vivo amplification step, or include a significantly shorter ex vivo amplification step.
  • the entire process of cell engineering is the following period of time after the sample is obtained from the subject Within: more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 1 day. It should be understood that this timing does not include any period of time during which the cells are subjected to cryopreservation.
  • the engineered cells are administered to the subject immediately or shortly after transduction without significant ex vivo expansion .
  • the engineered cell can be administered immediately after the transduction step.
  • the engineered cell may not undergo significant ex vivo expansion shortly after the activation transduction step, for example, compared to conventional methods (which may require significant in vitro activation, expansion, and/or enrichment) Or given in the case of significantly shorter ex vivo amplification.
  • the engineered cells can be administered within three days, two days, or one day of transduction.
  • the engineered cell can be within 48 hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour or less of the activation transduction step. give. In some embodiments, compared with conventional methods, the engineered cells undergo a significantly shorter in vitro expansion, such as 48 hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour or less.
  • the expansion and/or activation of the cells can be performed in vivo after exposure to the antigen, for example, the expansion of engineered cells in the subject after administration of the cells.
  • the scope, degree, or magnitude of expansion in vivo can be expanded, enhanced, or enhanced by a variety of methods, which can modulate (e.g., increase) the performance of a given cell (e.g., a cell expressing a recombinant receptor). Amplification, proliferation, survival, and/or efficacy.
  • such methods include methods involving administering to engineered cells, the cells are further modified with an agent (e.g., nucleic acid) to alter (e.g., increase or decrease) the expression or activity of the molecule, wherein such altered
  • an agent e.g., nucleic acid
  • the expression or activity expands, enhances or enhances the expansion, proliferation, survival and/or efficacy of the administered cells.
  • the expression of an agent e.g., nucleic acid
  • the expression of an agent is inducible, repressible, adjustable, and/or user-controlled, for example, by administering an inducer or other regulatory molecule.
  • such methods include methods involving combined administration (e.g., simultaneous or sequential administration) with drugs or agents capable of expanding, strengthening, or enhancing the administered cells (e.g., cells expressing recombinant receptors). ) Expansion, proliferation, survival and/or efficacy.
  • the viral vector particle is a retroviral vector particle, such as a lentiviral particle, which contains a recombinant and/or heterologous molecule (such as a recombinant or heterologous protein, such as a recombinant and/or Heterologous receptors, such as chimeric antigen receptors (CARs or other antigen receptors) nucleic acids.
  • a recombinant and/or heterologous molecule such as a recombinant or heterologous protein, such as a recombinant and/or Heterologous receptors, such as chimeric antigen receptors (CARs or other antigen receptors) nucleic acids.
  • the genome of a viral vector particle usually includes sequences other than the nucleic acid encoding the recombinant molecule. Such sequences may include sequences that allow the packaging of the genome into viral particles and/or sequences that facilitate the expression of nucleic acids encoding recombinant receptors (e
  • the viral vector particles contain a genome derived from a retroviral genome-based vector (e.g., derived from a lentiviral genome-based vector).
  • a heterologous nucleic acid encoding a recombinant receptor eg, an antigen receptor, such as a CAR is contained in and/or located between the 5'LTR and 3'LTR sequences of the vector genome.
  • the viral vector genome is a lentiviral genome, such as the HIV-1 genome or the SIV genome.
  • these viral vectors are plasmid-based or virus-based, and are configured to carry basic sequences for incorporation of foreign nucleic acid for selection and for transfer of the nucleic acid into host cells.
  • Non-limiting examples of lentiviral vectors include those derived from lentiviruses, such as human immunodeficiency virus 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human T lymphotropic virus 1 (HTLV-1), HTLV-2 or horses are infected with anemia virus (E1AV).
  • the viral genome vector may contain the sequences of the 5'and 3'LTR of a retrovirus (e.g., a lentivirus).
  • the viral genome construct can contain sequences from the 5'and 3'LTR of the lentivirus, and in particular, can contain the R and U5 sequences from the 5'LTR of the lentivirus and the inactivation or autonomy from the lentivirus. Inactivate 3'LTR.
  • the LTR sequence can be the LTR sequence of any lentivirus from any species. For example, they can be LTR sequences from HIV, SIV, FIV or BIV. Generally, the LTR sequence is the HIV LTR sequence.
  • the viral vector contains a nucleic acid encoding a heterologous recombinant protein.
  • the heterologous recombinant molecule is or includes a recombinant receptor (e.g., chimeric antigen receptor), SB transposon (e.g. for gene silencing), capsid-encapsulated transposon, homologous double Stranded nucleic acid (e.g. for genome recombination) or reporter gene (e.g. fluorescent protein, e.g. GFP) or luciferase).
  • a recombinant receptor e.g., chimeric antigen receptor
  • SB transposon e.g. for gene silencing
  • capsid-encapsulated transposon e.g. for gene silencing
  • homologous double Stranded nucleic acid e.g. for genome recombination
  • reporter gene e.g. fluorescent protein, e.g. GFP
  • the viral vector contains a nucleic acid encoding a recombinant receptor and/or a chimeric receptor (e.g., a heterologous receptor protein).
  • Recombinant receptors may include antigen receptors, such as functional non-TCR antigen receptors, including chimeric antigen receptors (CAR) and other antigen binding receptors, such as transgenic T cell receptors (TCR) .
  • Receptors may also include other receptors, such as other chimeric receptors, such as receptors that bind to specific ligands and have transmembrane and/or intracellular signaling domains similar to those present in the CAR.
  • the encoded recombinant antigen receptor is a receptor capable of specifically binding to one or more ligands on the cell or disease to be targeted, such as cancer , Infectious diseases, inflammation or autoimmune diseases or other diseases.
  • exemplary antigens are or include ⁇ v ⁇ 6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250 ), cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, CC motif chemokine ligand 1 ( CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epiglin 2 (EPG-2), epiglin 40 (EPG-40), ephrin B2, ephrin B2,
  • receptor-targeted antigens include antigens associated with B cell malignancies, such as any of many known B cell markers.
  • the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig ⁇ , Ig ⁇ , CD79a, CD79b, or CD30.
  • exemplary antigens are orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, OEPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R- ⁇ , IL-13R- ⁇ 2, kdr, ⁇ light chain, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen, ROR
  • the antigen is or includes a pathogen-specific or pathogen-expressed antigen.
  • the antigen is a viral antigen (e.g., viral antigens from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasite antigens.
  • antigen receptors including CARs and recombinant TCRs
  • their production and introduction include, for example, those described in the following documents: International Patent Application Publication Nos. WO2015172339A1, WO2016008405A1, WO2016086813A1, WO2016150400, WO2017032293A1, WO2017041749A1, WO2017080377A1 , WO2018018958A1, WO2018108106A1, WO2018045811A1, WO2018219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/149279, WO2019/170147A1, WO2019/210863, CN109385400A, CN109468279A, CN109880803A, CN110438082A, CN 110468105A, WO2019/219029, WO 200014257, WO 2013126726, WO 2012/129514,
  • the nucleic acid contained in the viral vector genome encodes a chimeric antigen receptor (CAR).
  • CAR is usually a genetically engineered receptor, which has an extracellular ligand binding domain, such as an extracellular part containing an antibody or fragment thereof, and the extracellular ligand binding domain is associated with one or more intracellular signal transduction groups. Sub-connection.
  • the chimeric antigen receptor includes a transmembrane domain and/or an intracellular domain connecting an extracellular domain and an intracellular signaling domain. Such molecules usually mimic or approximate the signals emitted by natural antigen receptors and/or the signals emitted by the combination of such receptors and co-stimulatory receptors.
  • the CAR is constructed to have specificity for a particular marker, such as a marker expressed in a particular cell type targeted by adoptive therapy, such as a cancer marker and/or any of the antigens. Therefore, a CAR usually includes one or more antigen-binding fragments, domains or portions of an antibody, or one or more antibody variable domains and/or antibody molecules. In some embodiments, the CAR includes one or more antigen binding portions of an antibody molecule, such as a variable heavy chain (VH) or antigen binding portion thereof, or a single chain antibody fragment (scFv) derived from a variable heavy chain (VH) ) And the variable light chain (VL) of the monoclonal antibody (mAb).
  • VH variable heavy chain
  • scFv single chain antibody fragment
  • engineered cells such as T cells, which express CARs specific to a specific antigen (or marker or ligand), such as an antigen expressed on the surface of a specific cell type .
  • the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule.
  • the antigen is selectively expressed or overexpressed on diseased cells, such as tumor cells or disease-causing cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or expressed on engineered cells.
  • the recombinant receptor (such as a chimeric receptor) contains intracellular signaling regions that include cytoplasmic signaling domains or regions (also interchangeably referred to as intracellular signaling Domains or regions), for example, cytoplasmic (intracellular) regions capable of inducing primary activation signals in T cells, for example, cytoplasmic signaling domains or regions of T cell receptor (TCR) components (for example, CD3-zeta( CD3 ⁇ ) chain or its functional variants or the cytoplasmic signaling domain or region of the ⁇ chain of the signaling part); and/or the intracellular signaling region contains an immunoreceptor tyrosine-based activation motif (ITAM) The cytoplasmic signaling domain or region.
  • TCR T cell receptor
  • ITAM immunoreceptor tyrosine-based activation motif
  • the chimeric receptor also contains an extracellular ligand binding domain that specifically binds to the ligand (eg, antigen) antigen.
  • the chimeric receptor is a CAR, which contains an extracellular antigen recognition domain that specifically binds to an antigen.
  • the ligand (eg, antigen) is a protein expressed on the surface of a cell.
  • the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which is similar to TCR in the context of a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • Exemplary antigen receptors include, for example, those described in the following documents: International Patent Application Publication Nos. WO2015172339A1, WO2016008405A1, WO2016086813A1, WO2016150400, WO2017032293A1, WO2017041749A1, WO2017080377A1, WO2018018958A1, WO2018108106A1, WO2018045811A1, WO2018219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/149279, WO2019/170147A1, WO2019/210863, CN109385400A, CN109468082279A, CN109880803A, CN , CN110468105A, WO2019/219029, WO 200014257, WO2013126726, WO 2012/129514, WO2014031687, WO2013/16
  • antigen receptors include CARs as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO/2014055668A1.
  • CARs include CARs as disclosed in any of the following publications such as WO2015172339A1, WO2016008405A1, WO2016086813A1, WO2016150400, WO2017032293A1, WO2017041749A1, WO2017080377A1, WO2018018958A1, WO2018108106A1, WO2018045811A1, WO2018219299, WO2018/210279, WO2019 /024933, WO2019/114751, WO2019/114762, WO2019/149279, WO2019/170147A1, WO2019/210863, CN109385400A, CN109468279A, CN109880803A, CN110438082A, CN110468105A, WO2019/219029, WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S.
  • Patent No. 7,446,190 U.S. Patent No. 8,389,282; Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10,267-276 (2013); Wang et al., (2012) J.Immunother.35(9):689-701; And Brentjens et al., Sci Transl Med. 2013 5 (177). See also WO 2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent No. 7,446,190, and US Patent No. 8,389,282.
  • the CAR is constructed to have specificity for specific antigens (or markers or ligands), such as antigens expressed in specific cell types targeted by adoptive therapy (e.g., cancer markers) and /Or antigens intended to induce an attenuated response (e.g., antigens expressed on normal or non-diseased cell types). Therefore, CAR usually includes one or more antigen-binding molecules in its extracellular part, such as one or more antigen-binding fragments, domains or parts, or one or more antibody variable domains, and/or Antibody molecule.
  • the CAR includes one or more antigen-binding portions of an antibody molecule, such as a single chain antibody derived from a variable heavy chain (VH) and a variable light chain (VL) of a monoclonal antibody (mAb) Fragment (scFv).
  • an antibody molecule such as a single chain antibody derived from a variable heavy chain (VH) and a variable light chain (VL) of a monoclonal antibody (mAb) Fragment (scFv).
  • the antibody or antigen binding portion thereof is expressed on the cell as part of a recombinant receptor (e.g., antigen receptor).
  • the antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR).
  • CARs containing antibodies or antigen-binding fragments exhibiting TCR-like specificity against peptide-MHC complexes can also be referred to as TCR-like CARs.
  • the extracellular antigen-binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular through a linker and/or one or more transmembrane domains.
  • the signal transduction component is connected.
  • such molecules can generally mimic or approach signals through natural antigen receptors (such as TCR), and optionally through such receptors in combination with co-stimulatory receptors.
  • a recombinant receptor e.g., a chimeric receptor, such as a CAR
  • a recombinant receptor includes a ligand binding domain that binds (e.g., specifically binds) to an antigen (or ligand).
  • Antigens targeted by chimeric receptors include antigens expressed in the context of diseases, disorders, or cell types targeted by adoptive cell therapy.
  • the diseases and conditions include proliferative, neoplastic and malignant diseases, including cancers and tumors, including blood cancers, cancers of the immune system, such as lymphoma, leukemia and/or myeloma, such as type B leukemia, type T leukemia and myeloid Leukemia, lymphoma and multiple myeloma.
  • the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on diseased cells (e.g., tumor or disease-causing cells) as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or expressed on engineered cells.
  • diseased cells e.g., tumor or disease-causing cells
  • the antigen is expressed on normal cells and/or expressed on engineered cells.
  • the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen expressed on the surface of a cell, such as an intact antigen.
  • the antigen or ligand
  • the antigen is a tumor antigen or cancer marker.
  • the antigen (or ligand) antigen is or includes ⁇ v ⁇ 6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NYESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif Chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), Truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epiglin 2 (EPG-2), epiglin 40 (EPG-40), eph
  • receptor-targeted antigens include antigens associated with B cell malignancies, such as any of many known B cell markers.
  • the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig ⁇ , Ig ⁇ , CD79a, CD79b, or CD30.
  • the antigen is or includes a pathogen-specific or pathogen-expressed antigen.
  • the antigen is a viral antigen (e.g., viral antigens from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasite antigens.
  • the CAR contains TCR-like antibodies, such as antibodies or antigen-binding fragments (e.g., scFv), which specifically recognize intracellular antigens (e.g., tumor-associated antigens) present on the cell surface as MHC-peptide complexes.
  • the antibody or antigen binding portion thereof that recognizes the MHC-peptide complex can be expressed on the cell as part of a recombinant receptor (eg, an antigen receptor).
  • the antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR).
  • CARs containing antibodies or antigen-binding fragments exhibiting TCR-like specificity against peptide-MHC complexes can also be referred to as TCR-like CARs.
  • an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex.
  • the peptide of the MHC-peptide complex is an epitope capable of binding to an antigen of MHC, such as a tumor antigen, such as a general tumor antigen, a myeloma antigen, or other antigens as described below.
  • an effective amount of an immunogen is then administered to the host for eliciting an immune response, wherein the immunogen maintains its three-dimensional form for a period sufficient to elicit three-dimensional targeting of the peptide in the binding groove of the MHC molecule.
  • the time of the presented immune response The serum collected from the host is then measured to determine whether the desired antibody that recognizes the three-dimensional representation of the peptide in the binding groove of the MHC molecule is produced.
  • the antibodies produced can be evaluated to confirm that the antibodies can distinguish MHC-peptide complexes from individual MHC molecules, individual target peptides, and complexes of MHC and unrelated peptides. The desired antibody can then be isolated.
  • Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.
  • the single domain antibody is a human single domain antibody.
  • the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or a cell surface antigen of a cell to be targeted or a disease (e.g., tumor cell or cancer cell), such as those described herein. Any target antigen mentioned or known.
  • Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells.
  • the antibody is a recombinantly produced fragment, such as a fragment comprising an arrangement that does not occur in nature (such as those having two or more antibody regions or chains connected by a synthetic linker (e.g., peptide linker) ), and/or may not be produced by enzymatic digestion of naturally-occurring intact antibodies.
  • the antibody fragment is a scFv.
  • chimeric antigen receptors include extracellular portions containing antibodies or antibody fragments.
  • the antibody or fragment includes scFv.
  • the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signaling region.
  • the intracellular signaling region comprises an intracellular signaling domain.
  • the intracellular signaling domain is or comprises a major signaling domain, a signaling domain capable of inducing primary activation signals in T cells, a signaling domain that is a component of T cell receptor (TCR) And/or a signal transduction domain containing an immunoreceptor tyrosine-based activation motif (ITAM).
  • TCR T cell receptor
  • ITAM immunoreceptor tyrosine-based activation motif
  • the extracellular portion of the CAR also includes a spacer, such as a spacer region between the antigen recognition component (eg, scFv) and the transmembrane domain.
  • the spacer may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region, such as an IgG4 hinge region, and/or a CH1/CL and/or Fc region.
  • the recombinant receptor further comprises a spacer and/or hinge region.
  • the constant region or portion is of human IgG such as IgG4 or IgG1.
  • a portion of the constant region serves as a spacer region between the antigen recognition component (e.g., scFv) and the transmembrane domain.
  • the spacer may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region (e.g., an IgG4 hinge region), and/or CH1/CL and/or Fc region .
  • the recombinant receptor further comprises a spacer and/or hinge region.
  • the constant region or portion is of human IgG such as IgG4 or IgG1.
  • a portion of the constant region serves as a spacer region between the antigen recognition component (e.g., scFv) and the transmembrane domain.
  • the length of the spacer can provide enhanced cell reactivity after antigen binding.
  • the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids.
  • Exemplary spacers include IgG4 hinges alone, IgG4 hinges connected to CH2 and CH3 domains, or IgG4 hinges connected to CH3 domains.
  • the extracellular ligand binding domain (for example, the antigen recognition domain) is usually connected to one or more intracellular signal transduction components, for example, the one or more intracellular signal transduction components are received by the antigen in the case of CAR.
  • Somatic complexes mimic activated signaling components and/or signals transmitted through another cell surface receptor.
  • the transmembrane domain connects the extracellular ligand binding domain with the intracellular signaling domain.
  • the antigen binding component e.g., antibody
  • the CAR includes a transmembrane domain fused to an extracellular domain.
  • a transmembrane domain that is naturally associated with one of the domains in the receptor e.g., CAR
  • the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex Interaction.
  • the transmembrane domain is derived from natural or synthetic sources.
  • the domain can be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions include those transmembrane regions derived from (ie, include at least one or more transmembrane regions of the following items): ⁇ , ⁇ , or ⁇ chains of T cell receptors, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.
  • the transmembrane domain is synthetic.
  • the synthetic transmembrane domain mainly contains hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the connection is through a linker, spacer, and/or one or more transmembrane domains.
  • a short oligopeptide or polypeptide linker exists and forms a CAR.
  • a linker of between 2 and 10 amino acids in length such as a linker containing glycine and serine, such as a glycine-serine doublet
  • Recombinant receptors generally include at least one or more intracellular signaling components.
  • the receptor includes the intracellular component of the TCR complex, such as the TCR and CD3 chain that mediates T cell activation and cytotoxicity, for example, the CD3 ⁇ chain. Therefore, in some aspects, the antigen binding portion is connected to one or more cell signaling modules.
  • the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domains.
  • the receptor e.g., CAR
  • the receptor also includes a portion of one or more additional molecules (e.g., Fc receptor gamma, CD8, CD4, CD25, or CD16).
  • additional molecules e.g., Fc receptor gamma, CD8, CD4, CD25, or CD16.
  • CAR or other chimeric receptors include chimeric molecules between CD3- ⁇ (CD3- ⁇ ) or Fc receptor ⁇ and CD8, CD4, CD25, or CD16.
  • the cytoplasmic domain and/or region or intracellular signaling domain and/or region of the receptor activate immune cells (e.g., engineered to express CAR At least one of the normal effector function or response of T cells).
  • CARs induce T cell functions, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors.
  • a truncated portion of the intracellular signaling domain of the antigen receptor component or costimulatory molecule is used instead of the complete immunostimulatory chain.
  • the intracellular signaling region (e.g., containing one or more intracellular signaling domains) includes the cytoplasmic sequence of the T cell receptor (TCR), and in some aspects also includes a co-receptor (which is in Acting in parallel with this receptor in a natural context to initiate signal transduction after antigen receptor engagement) and/or any derivative or variant of such molecules, and/or any synthetic sequence with the same functional capacity.
  • TCR T cell receptor
  • components used to generate secondary or co-stimulatory signals are also included in the CAR.
  • the CAR does not include components for generating costimulatory signals.
  • additional CARs are expressed in the same cell and provide components for generating secondary or co-stimulatory signals.
  • T cell activation has been described in some respects as being mediated by at least two types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide Those of secondary or costimulatory signals (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences those that initiate antigen-dependent primary activation via TCR
  • secondary cytoplasmic signaling sequences those that act in an antigen-independent manner to provide Those of secondary or costimulatory signals.
  • the CAR includes one or both of such signaling components.
  • the CAR includes a primary cytoplasmic signaling sequence that modulates the primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulating manner may contain signaling motifs (which are called immunoreceptor tyrosine-based activation motifs or ITAMs).
  • ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD8, CD22, CD79a, CD79b, and CD66d.
  • ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 ⁇ , FcR ⁇ , or FcR ⁇ .
  • the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain derived from CD3 ⁇ .
  • the CAR includes signaling domains and/or transmembrane portions of costimulatory receptors (eg, CD28, 4-1BB, OX40, CD27, DAP10, and ICOS). In some aspects, the same CAR includes activation or signaling regions and costimulatory components.
  • costimulatory receptors eg, CD28, 4-1BB, OX40, CD27, DAP10, and ICOS.
  • the same CAR includes activation or signaling regions and costimulatory components.
  • the activation domain is included in one CAR, and the costimulatory component is provided by another CAR that recognizes another antigen.
  • the CAR includes an activating or stimulating CAR and a co-stimulating CAR that are expressed on the same cell (see WO 2014/055668).
  • the CAR is a stimulatory or activating CAR; in other aspects, it is a costimulatory CAR.
  • the cell also includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl.
  • the intracellular signaling domain comprises a CD28 transmembrane and signaling domain connected to the CD3 intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 costimulatory domain connected to the CD3 intracellular domain.
  • the intracellular signaling domain of CD8+ cytotoxic T cells is the same as the intracellular signaling domain of CD4+ helper T cells. In some embodiments, the intracellular signaling domain of CD8+ cytotoxic T cells is different from the intracellular signaling domain of CD4+ helper T cells.
  • the CAR encompasses one or more (e.g., two or more) costimulatory domains and activation domains (e.g., primary activation domains) in the cytoplasmic portion.
  • exemplary CARs include intracellular components of CD3- ⁇ , CD28, and 4-1BB.
  • one or more recombinant receptors (such as CAR) encoded by one or more nucleic acids in the provided viral vector also include one or more markers, for example, for confirming that the receptors are to be expressed. Transduction or engineering of somatic cells and/or selection and/or targeting of cells expressing one or more molecules encoded by polynucleotides.
  • this marker can be encoded by a different nucleic acid or polynucleotide, which can also be introduced during the genetic engineering process, usually by the same method (for example, by any of the methods provided herein). Transduction, such as transduction via the same vector or the same type of vector) is introduced.
  • the marker e.g., transduction marker
  • the marker is a protein and/or a cell surface molecule.
  • Exemplary markers are truncated variants of naturally occurring (e.g., endogenous) markers (e.g., naturally occurring cell surface molecules).
  • CARs are referred to as first-generation, second-generation, and/or third-generation CARs.
  • the first-generation CAR is a CAR that only provides a signal induced by the CD3 chain after antigen binding;
  • the second-generation CAR is a CAR that provides such a signal and a costimulatory signal, for example, it includes a CAR from a costimulatory receptor (Such as CD28 or CD137) intracellular signaling domain CAR;
  • the third-generation CAR is a CAR that includes multiple costimulatory domains of different costimulatory receptors.
  • the chimeric antigen receptor includes an extracellular ligand binding portion (e.g., an antigen binding portion, such as an antibody or fragment thereof) and an intracellular domain.
  • the antibodies or fragments include scFv or single domain VH antibodies, and the intracellular domain contains ITAM.
  • the intracellular signaling domain includes the signaling domain of the zeta chain of the CD3-zeta (CD3 ⁇ ) chain.
  • the chimeric antigen receptor includes a transmembrane domain connected and/or disposed between an extracellular domain and an intracellular signaling region or domain.
  • the transmembrane domain contains the transmembrane portion of CD28.
  • the extracellular domain and the transmembrane can be directly or indirectly connected.
  • the extracellular domain and the transmembrane are connected by a spacer (such as any spacer described herein).
  • the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain.
  • the T cell costimulatory molecule is CD28 or 4-1BB.
  • the CAR contains an antibody (such as an antibody fragment), a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and a signaling portion containing CD28 or a functional variant thereof and CD3 ⁇ or its The intracellular signaling domain of the signaling part of the functional variant.
  • the CAR contains an antibody such as an antibody fragment, a transmembrane domain (which is a transmembrane portion of CD28 or a functional variant thereof or a transmembrane portion containing CD28 or a functional variant thereof) and a signal containing 4-1BB The intracellular signal transduction domain of the transduction portion or its functional variant and the CD3 ⁇ signal transduction portion or its functional variant.
  • the receptor further includes a spacer containing a portion of an Ig molecule (such as a human Ig molecule, such as an Ig hinge, such as an IgG4 hinge), such as a hinge-only spacer.
  • the transmembrane domain of the receptor is the transmembrane domain of human CD28 or a variant thereof, such as the 27 amino acid transmembrane domain of human CD28 (accession number: P10747.1) .
  • the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule.
  • the T cell costimulatory molecule is CD28 or 4-1BB.
  • the intracellular domain comprises an intracellular costimulatory signaling domain of human CD28 or a functional variant or part thereof, such as its 41 amino acid domain, and/or at position 186- of the native CD28 protein. 187 has this domain substituted from LL to GG.
  • the intracellular signaling region and/or domain comprises a human CD3 chain, optionally a CD3 ⁇ stimulatory signaling domain or a functional variant thereof, such as a homologous of human CD3 ⁇ (accession number: P20963.2)
  • a CD3 ⁇ stimulatory signaling domain or a functional variant thereof such as a homologous of human CD3 ⁇ (accession number: P20963.2)
  • the 112 AA cytoplasmic domains of type 3 or the CD3 ⁇ signaling domain as described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993.
  • the CAR includes: an extracellular ligand binding portion, such as an antigen binding portion, such as an antibody or fragment thereof, including sdAb and scFv, which specifically binds to an antigen, such as the antigen described herein; a spacer, such as any A spacer containing an Ig hinge; a transmembrane domain, which is a part of CD28 or a variant thereof; an intracellular signaling domain, which contains a signaling portion of CD28 or a functional variant thereof; and a CD3 ⁇ signaling domain or its Signaling part of functional variants.
  • an extracellular ligand binding portion such as an antigen binding portion, such as an antibody or fragment thereof, including sdAb and scFv, which specifically binds to an antigen, such as the antigen described herein
  • a spacer such as any A spacer containing an Ig hinge
  • a transmembrane domain which is a part of CD28 or a variant thereof
  • an intracellular signaling domain which contains
  • the CAR includes: an extracellular ligand binding portion, such as an antigen binding portion, such as an antibody or fragment thereof, including sdAb and scFv, which specifically binds to an antigen, such as the antigen described herein; a spacer, such as any A spacer containing an Ig hinge; a transmembrane domain, which is a part of CD28 or a variant thereof; an intracellular signaling domain, which contains a signaling portion of 4-1BB or a functional variant thereof; and a CD3 ⁇ signaling domain Or the signaling part of its functional variants.
  • an extracellular ligand binding portion such as an antigen binding portion, such as an antibody or fragment thereof, including sdAb and scFv, which specifically binds to an antigen, such as the antigen described herein
  • a spacer such as any A spacer containing an Ig hinge
  • a transmembrane domain which is a part of CD28 or a variant thereof
  • an intracellular signaling domain which contains
  • TCR T cell receptor
  • the one or more recombinant molecules encoded by the one or more nucleic acids are or include a recombinant T cell receptor (TCR).
  • TCR T cell receptor
  • the recombinant TCR is specific for an antigen, which is usually an antigen present on target cells, such as a tumor-specific target antigen, expressed on specific cell types associated with autoimmune or inflammatory diseases Antigens, or antigens derived from viral pathogens or bacterial pathogens.
  • engineered cells are provided, such as T cells, which express TCRs or antigen binding portions thereof that recognize peptide epitopes of target polypeptides (eg, antigens of tumors, viruses, or autoimmune proteins) or T cell epitopes.
  • the "T cell receptor” or “TCR” contains variable alpha and beta chains (also referred to as TCR ⁇ and TCR ⁇ , respectively) or variable gamma and delta chains (also referred to as TCR ⁇ and TCR ⁇ , respectively) A molecule or an antigen-binding portion thereof, and it can specifically bind to a peptide that binds to an MHC molecule.
  • the TCR is in the ⁇ form.
  • TCRs in the form of ⁇ and ⁇ are generally similar in structure, but the T cells expressing them can have different anatomical locations or functions.
  • the term "TCR” should be understood to encompass the complete TCR and its antigen-binding portion or antigen-binding fragment thereof.
  • the TCR is a complete or full-length TCR, including a TCR in the form of ⁇ or ⁇ .
  • the TCR is an antigen-binding portion that is less than the full-length TCR but binds to a specific peptide bound in an MHC molecule (eg, binds to an MHC-peptide complex).
  • the antigen-binding portion or fragment of the TCR may only contain a part of the structural domain of the full-length or complete TCR, but can still bind to the peptide epitope (such as MHC-peptide complex) that binds to the complete TCR.
  • the antigen binding portion contains the variable domain of the TCR (such as the variable alpha chain and the variable beta chain of the TCR), which is sufficient to form a binding site for binding to a specific MHC-peptide complex.
  • the variable chain of the TCR contains complementarity determining regions involved in the recognition of peptides, MHC and/or MHC-peptide complexes.
  • variable domain of the TCR contains a hypervariable loop or complementarity determining region (CDR), which is usually a major contributor to antigen recognition and binding capacity and specificity.
  • CDRs of the TCR or a combination thereof form all or substantially all of the antigen binding site of a given TCR molecule.
  • the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged.
  • the TCR can be a heterodimer of two chains alpha and beta (or optionally gamma and delta), or it can be a single chain TCR construct.
  • the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR does contain a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR is capable of forming a TCR complex with CD3. In some embodiments, any TCR (including dTCR or scTCR) can be linked to a signaling domain that produces an active TCR on the surface of T cells. In some embodiments, the TCR is expressed on the surface of the cell.
  • one or more nucleic acids encoding TCR can be amplified by PCR, cloning or other suitable methods, and cloned into a suitable expression vector.
  • the expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.
  • the vector may be the following series of vectors: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, California), pET series (Novagen, Madison, Wisconsin), pGEX series (Pharmacia Biotech) , Uppsala, Sweden) or pEX series (Clontech, Palo Alto, California).
  • phage vectors such as ⁇ G10, ⁇ GT11, ⁇ Za pII (Stratagene), ⁇ EMBL4 and ⁇ NM1149 can also be used.
  • plant expression vectors can be used and include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • viral vectors are used, such as retroviral vectors.
  • the vector may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host (eg, bacteria, fungi, plants, or animals) into which the vector is introduced.
  • the vector may contain a non-natural promoter that is operably linked to a nucleotide sequence encoding a TCR or antigen binding portion (or other MHC-peptide binding molecule).
  • the promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter and those found in the long terminal repeats of murine stem cell viruses Promoter.
  • CMV cytomegalovirus
  • the cells and methods include multi-targeting strategies, such as the expression of two or more genetically engineered receptors on the cell, each receptor recognizing the same or different antigens, and typically each includes a different cell Internal signaling components.
  • the nucleic acid sequence encoding a recombinant receptor such as an antigen receptor, such as CAR
  • a recombinant receptor such as an antigen receptor, such as CAR
  • other genetic elements such as transcription regulatory sequences, including promoters) Or enhancer
  • transcriptional regulatory sequences are those that are temporally and/or spatially regulated in terms of activity.
  • Expression control elements that can be used to regulate the expression of components are known and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, and other regulatory elements.
  • the nucleic acid sequence contained in the viral vector genome contains multiple expression control elements, which control the encoded different components, such as different receptor components and/or signal transduction components, so that the receptor is recombined. And/or the expression, function, and/or activity of engineered cells (e.g., cells expressing engineered receptors) can be adjusted, for example, inducible, repressible, adjustable, and/or user-controlled.
  • one or more vectors may contain one or more nucleic acid sequences that contain one or more expression control elements and/or one or more encoded components such that the nucleic acid Together, the sequences can modulate the expression, activity, and/or function of the encoded component (e.g., recombinant receptor) or engineered cell.
  • the encoded component e.g., recombinant receptor
  • a nucleic acid sequence encoding a recombinant receptor is operably linked to an internal promoter/enhancer regulatory sequence.
  • the promoter used can be constitutive, tissue-specific, inducible and/or can be used to direct high-level expression of the introduced DNA segment under appropriate conditions.
  • the promoter can be heterologous or endogenous.
  • promoters and/or enhancers are produced synthetically.
  • promoters and/or enhancers are produced using recombinant cloning and/or nucleic acid amplification techniques.
  • the nucleic acid sequence encoding the recombinant receptor contains a signal sequence encoding a signal peptide.
  • the signal sequence can encode a signal peptide derived from a natural polypeptide.
  • the signal sequence can encode a heterologous or non-natural signal peptide.
  • the nucleic acid sequence encoding a recombinant receptor such as a chimeric antigen receptor (CAR) contains a signal sequence encoding a signal peptide.
  • the polynucleotide encoding the recombinant receptor contains at least one promoter that is operably linked to control the expression of the recombinant receptor. In some instances, the polynucleotide contains two, three or more promoters that are operably linked to control the expression of the recombinant receptor.
  • each polypeptide chain may be encoded by a separate nucleic acid molecule.
  • two separate nucleic acids are provided, and each can be separately transferred to or introduced into a cell for expression in the cell.
  • the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are operably linked to the same promoter, and optionally through an internal ribosome entry site (IRES) or encoding a self-cleaving peptide or causing a ribosome
  • the jumping peptide (which is optionally T2A, P2A, E2A, or F2A) separates the nucleic acid.
  • the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters.
  • the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the cell genome.
  • the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, for example, by retroviral transduction, transfection, or transformation.
  • the oligonucleotide primer contains a tag, where the tag is not specific to the target sequence.
  • tags may be called universal tags or universal tags.
  • the method includes labeling the target sequence or fragments thereof in the sample with a label that is non-specific to the target sequence. In some cases, tags are not specific to sequences on human chromosomes. Alternatively or additionally, the method includes contacting the sample with a tag and at least one oligonucleotide primer comprising a sequence corresponding to the target sequence, wherein the tag is separated from the oligonucleotide primer.
  • the tag is incorporated into the amplification product by extending the oligonucleotide primer.
  • the tags can be oligonucleotides, small molecules or peptides.
  • the marker is a transduction marker or a surrogate marker. Transduction markers or surrogate markers can be used to detect cells into which polynucleotides (e.g., polynucleotides encoding recombinant receptors) have been introduced. In some embodiments, the transduction marker can indicate or confirm the modification of the cell.
  • the surrogate marker is a protein prepared on the cell surface to be co-expressed with a recombinant receptor (eg, CAR). In certain embodiments, this surrogate marker is a surface protein that has been modified to have little or no activity. In some embodiments, the surrogate marker is encoded by the same polynucleotide encoding the recombinant receptor.
  • the nucleic acid sequence encoding the recombinant receptor is operably linked to the nucleic acid sequence encoding the marker, optionally via an internal ribosome entry site (IRES) or a peptide encoding a self-cleaving peptide or ribosome jumping (for example, 2A sequences, such as T2A, P2A, E2A, or F2A) separate nucleic acids.
  • IRS internal ribosome entry site
  • a peptide encoding a self-cleaving peptide or ribosome jumping For example, 2A sequences, such as T2A, P2A, E2A, or F2A
  • extrinsic marker genes can be used in conjunction with engineered cells to allow detection or selection of cells, and in some cases can also be used to promote cell suicide.
  • the promoter and/or enhancer may be a promoter and/or enhancer naturally associated with the nucleic acid sequence, for example, the 5'non-coding sequence located upstream of the coding segment and/or exon may be separated And get.
  • the promoter may be a tissue-specific promoter and/or a target cell-specific promoter.
  • the regulatory elements may include regulatory elements and/or systems that allow for adjustable expression and/or activity of recombinant receptors (e.g., CARs).
  • the adjustable expression and/or activity is achieved by configuring the recombinant receptor to contain or be controlled by specific regulatory elements and/or systems.
  • Viral vector genomes are usually constructed in the form of plasmids, which can be transfected into packaging cell lines or production cell lines. Any of a variety of known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome.
  • at least two components are involved in the preparation of a virus-based gene delivery system: first, the packaging plasmid, including structural proteins and enzymes necessary for the production of viral vector particles, and second, the viral vector itself, that is, to transfer Genetic material. Biosafety protection measures can be introduced when designing one or two of these components.
  • the packaging plasmid may contain all retroviral (such as HIV-1) proteins except for envelope proteins (Naldini et al., 1998).
  • a lentiviral vector (such as an HIV-based lentiviral vector) contains only the genes of the three parent viruses: gag, pol, and rev, which reduces or eliminates the possibility of reconstitution of the wild-type virus through recombination.
  • the viral vector genome is introduced into a packaging cell line that contains all the components required to package the viral genomic RNA transcribed from the viral vector genome into viral particles.
  • the packaging cell line is transfected with one or more plasmid vectors containing the components required to produce the particles.
  • a plasmid containing the viral vector genome including LTR, cis-acting packaging sequence, and target sequence, that is, nucleic acid encoding an antigen receptor (such as CAR)); and encoding viral enzymes and/or structural components (
  • helper plasmids such as Gag, pol and/or rev
  • the packaging cell line provides the components required to package viral genomic RNA into lentiviral vector particles in trans, including viral regulatory proteins and structural proteins.
  • the packaging cell line can be any cell line capable of expressing lentiviral proteins and producing functional lentiviral vector particles.
  • suitable packaging cell lines include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL34), BHK (ATCC CCL-10), and Cf2Th (ATCC CRL 1430) Cells.
  • the viral vector and packaging plasmid and/or helper plasmid are introduced into the packaging cell line by transfection or infection.
  • the packaging cell line produces viral vector particles containing the viral vector genome.
  • the methods used for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection.
  • a retroviral vector such as a lentiviral vector
  • a packaging cell line such as the exemplary HEK 293T cell line
  • packaging cells are transfected and/or contain polynucleotides encoding gag and pol, as well as polynucleotides encoding recombinant receptors (eg, antigen receptors, such as CAR).
  • the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding the rev protein.
  • the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a non-natural envelope glycoprotein (eg, VSV-G).
  • a non-natural envelope glycoprotein eg, VSV-G
  • the cell supernatant contains a recombinant lentiviral vector that can be recovered and titrated.
  • the recovered and/or produced retroviral vector particles can be used to transduce target cells using the method as described.
  • the viral RNA Once in the target cell, the viral RNA is reverse transcribed, enters the nucleus and stably integrates into the host genome.
  • the expression of recombinant proteins eg, antigen receptors, such as CAR
  • CAR antigen receptors
  • the provided methods involve methods of transducing cells by contacting (1) viral particles with an input composition comprising a plurality of cells (eg, incubating).
  • the input composition comprises primary cells obtained from the subject, such as cells enriched and/or selected from the subject.
  • the input composition comprises primary cells obtained from the subject.
  • the sample is a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, apheresis product, or a leukocyte separation product.
  • PBMC peripheral blood mononuclear cell
  • a sample containing primary cells is contacted with or contains serum or plasma at the following concentration in vitro: at least or at least about 10% (v /v), at least or at least about 15% (v/v), at least or at least about 20% (v/v), at least or at least about 25% (v/v), at least or at least about 30% (v/v ), at least or at least about 35% (v/v), at least or at least about 40% (v/v), or at least or at least about 50%.
  • the sample contains serum or plasma, and the concentration of said serum or plasma is or is approximately about or at least about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32% , 33%, 34% or 35% (v/v).
  • the serum or plasma is human.
  • the serum or plasma is autologous to the subject.
  • a sample containing primary cells is contacted with or contains an anticoagulant prior to selecting and/or transducing cells.
  • a sample containing primary cells is contacted with or contains an anticoagulant prior to selecting and/or transducing cells, a sample containing primary cells is contacted with or contains an anticoagulant.
  • the anticoagulant is or contains free citrate ions, for example, the anticoagulant citrate dextrose solution, solution A (ACD-A).
  • the sample prior to cell selection and/or transduction, is maintained at a temperature of 2°C to 8°C for up to 48 hours, for example up to 12 hours, 24 hours, or 36 hours.
  • the input composition comprises and/or is enriched in T cells, which include CD4+ and/or CD8+ T cells.
  • enrichment can be performed by affinity-based selection by incubating primary cells with one or more selection or affinity reagents that specifically bind to primary cell subpopulations. Cell surface molecules expressed on the population, thereby enriching primary cells based on binding to selection reagents.
  • enrichment can be performed by incubating cells with antibody-coated particles (e.g., microbeads, polymeric nanomatrix).
  • the input composition comprises greater than or greater than about 75%, 80%, 85%, 90%, 95%, or more T cells obtained from a sample of the subject.
  • T cells obtained from a sample of the subject.
  • no more than 5%, 10%, 20%, 30%, or 40% of the T cells in the input composition are activated cells, expressing selected from HLA-DR, CD25, CD69, CD71, CD40L and The surface marker of 4-1BB; contains intracellular expression of cytokines selected from IL-2, IFN- ⁇ , and TNF- ⁇ , in the G1 phase or later stage of the cell cycle, and/or capable of proliferation.
  • the input composition may include one or more cytokines during the incubation and/or contact or during at least a portion of the incubation and/or contact.
  • the cytokine is selected from IL-2, IL-7, or IL-15.
  • the cytokine is a recombinant cytokine.
  • the concentration of the cytokine in the input composition is independently 1IU/mL to 1500IU/mL, for example, 1IU/mL to 100IU/mL, 2IU/mL to 50IU/mL, 5IU/mL to 10IU/mL , 10IU/mL to 500IU/mL, 50IU/mL to 250IU/mL or 100IU/mL to 200IU/mL, 50IU/mL to 1500IU/mL, 100IU/mL to 1000IU/mL or 200IU/mL to 600IU/mL.
  • the concentration of the cytokine in the input composition is independently at least or at least about 1IU/mL, 5IU/mL, 10IU/mL, 50IU/mL, 100IU/mL, 200IU/mL, 500IU/mL, 1000IU /mL or 1500IU/mL.
  • an agent capable of activating the intracellular signaling domain of the TCR complex e.g., anti-CD3 and/or anti-CD28 antibody
  • the input composition may include serum during the incubation and/or contact or during at least a portion of the incubation and/or contact.
  • the serum is human serum.
  • the serum is present in the input composition at a concentration of 0.5% to 25% (v/v), 1.0% to 10% (v/v), or 2.5% to 5.0% (v/v) , Each contains end values.
  • the input composition is free and/or substantially free of serum during the incubation and/or contact or during at least a portion of the incubation and/or contact. In some embodiments, the input composition is incubated and/or contacted in the absence of serum during the incubation and/or contact or during at least a portion of the incubation and/or contact. In certain embodiments, the input composition is incubated and/or contacted in a serum-free medium during the incubation and/or contacting or during at least a portion of the incubation and/or contacting. In some embodiments, the serum-free medium is a defined and/or well-defined cell culture medium. In some embodiments, a serum-free medium is formulated to support the growth, proliferation, health, and homeostasis of cells of a certain cell type (e.g., immune cells, T cells, and/or CD4+ and CD8+ T cells).
  • a certain cell type e.g., immune cells, T cells, and/or CD4+ and CD8+ T
  • the cell concentration of the input composition is 1.0 ⁇ 10 5 cells/mL to 1.0 ⁇ 10 10 titer.
  • transduction may be less than 100, such as generally less than 60, 50, 40, 30, The multiplicity of infection (MOI) of 20, 10, 5, 4, 3, 2, 1 or less is achieved.
  • MOI multiplicity of infection
  • the method involves contacting or incubating cells with viral particles, such as mixing.
  • the contacting is carried out for 30 minutes to 72 hours, such as 30 minutes to 48 hours, 30 minutes to 24 hours, or 1 hour to 24 hours, such as at least 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours or more.
  • the contacting is in solution.
  • the cells and virus particles are contacted in a volume of 0.5 mL to 500 mL, for example, 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5mL to 500mL, 5mL to 200mL, 5mL to 100mL, 5mL to 50mL, 5mL to 10mL, 10mL to 500mL, 10mL to 200mL, 10mL to 100mL, 10mL to 50mL, 50mL to 500mL, 50mL to 200mL, 50mL to 100mL, 100mL to 500mL, 100mL to 200mL, or 200mL to 500mL.
  • contacting can be achieved by centrifugation, such as spin inoculation (e.g., centrifugal inoculation).
  • the composition containing cells, virus particles, and reagents can be rotated, usually at a relatively low force or speed, for example, at a speed lower than that used to pellet the cells, for example, 600 rpm to 1700 rpm (e.g., It is at least 600rpm, 1000rpm or 1500rpm or 1700rpm).
  • the rotation is performed with a force (e.g., relative centrifugal force) of 100g to 3200g (e.g., at least 100g, 200g, 300g, 400g, 500g, 1000g, 1500g, 2000g, 2500g, 3000g, or 3200g) , As measured, for example, at the inner or outer wall of a chamber or cavity.
  • a force e.g., relative centrifugal force
  • RCF relative centrifugal force
  • RCF is generally understood as being applied to an object or substance (such as a cell, sample or pellet and/or a rotating chamber at a specific point in space as compared to the axis of rotation, relative to the gravity of the earth). Or other points in the container).
  • the value can be determined using a well-known formula that takes into account gravity, rotation speed, and radius of rotation (distance from the axis of rotation and the object, substance, or particle for which the RCF is measured).
  • the incubation of cells with viral vector particles results in or produces an export composition comprising cells transduced with viral vector particles.
  • the process of preparing the cells may further include washing or preparing the cells. Therefore, the processing steps may include formulating such compositions.
  • the cells and composition are administered to the subject in the form of a pharmaceutical composition or formulation (e.g., a composition comprising a cell or cell population and a pharmaceutically acceptable carrier or excipient).
  • a pharmaceutical composition or formulation e.g., a composition comprising a cell or cell population and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutical formulation refers to a formulation that is in a form that makes the biological activity of the active ingredient contained therein effective and does not contain additional components that have unacceptable toxicity to the subject to which the formulation is administered.
  • the pharmaceutical composition additionally contains other pharmaceutically active agents or drugs, such as chemotherapeutics, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil , Gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutics such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil , Gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the agent is administered in the form of a salt, such as a pharmaceutically acceptable salt.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and those derived from organic acids.
  • Organic acids are, for example, tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acid (for example, p-toluenesulfonic acid).
  • “Pharmaceutically acceptable carrier” refers to ingredients in a pharmaceutical formulation that are not toxic to the subject except for the active ingredients.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the choice of vector depends in part on the particular cell and/or method of administration. Therefore, there are many suitable formulations.
  • the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is generally present in an amount of about 0.0001% to about 2% by weight of the total composition.
  • the pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dose and concentration used, and includes, but is not limited to: buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine Acid; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, Such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glut
  • buffering agents are included in the composition.
  • Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.
  • a mixture of two or more buffers is used.
  • the buffering agent or mixture thereof is generally present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known.
  • the formulation may include an aqueous solution.
  • the formulation or composition may also contain more than one active ingredient that can be used for specific indications and diseases for the treatment of cells, preferably those ingredients that have complementary activities to the cells, wherein the respective activities do not produce each other. Negative Effects.
  • active ingredients are present in a suitable combination in an amount effective for the intended purpose.
  • the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as chemotherapeutics, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin , Fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine and/or vincristine.
  • chemotherapeutics such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin , Fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine and/or vincristine.
  • the pharmaceutical composition contains cells in an amount effective to treat or prevent a disease (e.g., a therapeutically effective amount or a prophylactically effective amount).
  • a disease e.g., a therapeutically effective amount or a prophylactically effective amount.
  • the efficacy of treatment or prevention is monitored by periodically evaluating the subject being treated.
  • the required dose can be delivered by a single bolus injection of cells, by multiple bolus injections of cells, or by continuous infusion of cells.
  • the composition is provided as a sterile liquid formulation (e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects can be buffered to a selected pH).
  • a sterile liquid formulation e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects can be buffered to a selected pH.
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions.
  • the liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium, which contains, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the cells into a solvent.
  • additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers.
  • the product of the method is used in a method of treatment, for example, a therapeutic method, for example, for administering cells and compositions to a subject in adoptive cell therapy.
  • a therapeutic method for example, for administering cells and compositions to a subject in adoptive cell therapy.
  • the provided methods generally involve administering cells or compositions (e.g., export compositions and/or formulated compositions) to a subject.
  • the cell expresses a recombinant receptor (e.g., CAR) or other antigen receptor (e.g., a transgenic TCR).
  • a recombinant receptor e.g., CAR
  • other antigen receptor e.g., a transgenic TCR
  • Such cells are usually administered to a subject suffering from a disease associated with a ligand specifically recognized by the receptor.
  • the cell expresses a recombinant receptor or a chimeric receptor (e.g., an antigen receptor, such as CAR or TCR), which specifically binds to disease-related or expressed by its cells or tissues Ligand.
  • the receptor is an antigen receptor
  • the ligand is a disease-specific and/or disease-related antigen.
  • Administration usually achieves improvement in one or more symptoms of the disease and/or treatment or prevention of the disease or its symptoms.
  • Diseases and conditions include tumors, including solid tumors, hematological malignancies and melanomas, and include local and metastatic tumors; infectious diseases, such as infections by viruses or other pathogens, such as HIV, HCV, HBV, CMV, and parasitic diseases ; And autoimmune and inflammatory diseases.
  • the disease is a tumor, cancer, malignant tumor, tumor, or other proliferative disease.
  • diseases include but are not limited to leukemia, lymphoma (e.g. chronic lymphocytic leukemia)
  • CLL non-Hodgkin's lymphoma
  • acute myeloid leukemia multiple myeloma
  • refractory follicular lymphoma mantle cell lymphoma
  • indolent B-cell lymphoma B-cell malignancies
  • colon Cancer lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic cancer, Hodgkin’s lymphoma, cervical cancer, nodule Rectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma.
  • such diseases include, but are not limited to, leukemia, lymphoma, such as acute myeloid (or myeloid) leukemia (AML), chronic myeloid (or myeloid) leukemia (CML), acute lymphocytic (or Lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma Tumor, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma (NHL)), anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM).
  • AML acute myeloid (or myeloid) leukemia
  • CML chronic my
  • the disease is a B-cell malignancy selected from the group consisting of acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin’s lymphoma (NHL ) And diffuse large B-cell lymphoma (DLBCL).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • NHL non-Hodgkin’s lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • the disease is NHL
  • the NHL is selected from aggressive NHL, diffuse large B-cell lymphoma (DLBCL), NOS (de novo and transformed from indolence), primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histiocytic-rich large B-cell lymphoma (TCHRBCL), Burkitt’s lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally, 3B Grade follicular lymphoma (FL3B).
  • DLBCL diffuse large B-cell lymphoma
  • NOS de novo and transformed from indolence
  • PMBCL primary mediastinal large B-cell lymphoma
  • TCHRBCL T-cell/histiocytic-rich large B-cell lymphoma
  • Burkitt’s lymphoma mantle cell lymphoma (MCL) and/or follicular lymphoma (FL
  • the disease is an infectious disease, such as but not limited to viruses, retroviruses, bacterial and protozoan infections, immunodeficiency, cytomegalovirus (CMV), Epstein-Barrvirus (EBV) ), adenovirus, BK polyoma virus.
  • the disease is an autoimmune or inflammatory disease, such as arthritis (e.g., rheumatoid arthritis (RA)), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, Psoriasis, scleroderma, autoimmune thyroid disease, Graves’ disease, Crohn’s disease, multiple sclerosis, asthma, and/or transplant-related diseases.
  • arthritis e.g., rheumatoid arthritis (RA)
  • type I diabetes e.g., systemic lupus erythematosus (SLE), inflammatory bowel disease, Psoriasis, scleroderma, autoimmune thyroid disease, Grav
  • treatment refers to the complete or partial improvement or reduction of a disease, or symptoms, adverse effects or results or phenotype associated therewith.
  • the ideal therapeutic effect includes but is not limited to preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the speed of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.
  • the term does not imply complete cure of the disease or complete elimination of any symptoms or influence on all symptoms or results.
  • delaying the development of a disease means delaying, hindering, slowing, delaying, stabilizing, inhibiting, and/or delaying the development of a disease (e.g., cancer). This delay can have different lengths of time, depending on the medical history and/or the individual being treated. It is obvious to those skilled in the art that a sufficient or significant delay can actually cover prevention because the individual will not develop the disease. For example, it may delay the development of advanced cancers, such as metastases.
  • prevention includes providing prevention of the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease.
  • the provided cells and compositions are used to delay or delay the progression of a disease.
  • inhibiting a function or activity is to reduce the function or activity when compared to the originally identical condition except for the condition or parameter of interest, or when compared to another condition.
  • cells that inhibit tumor growth reduce the growth rate of the tumor compared to the tumor growth rate in the absence of the cells.
  • Methods of administration of cells for adoptive cell therapy are known and can be used with the provided methods and compositions.
  • the disease to be treated can be any disease in which the expression of the antigen is related to and/or involved in the etiology of the disease condition, such as causing, exacerbating the disease or condition or participating in it in other ways.
  • diseases and disorders may include diseases associated with malignant tumors or cell transformation (e.g. cancer), autoimmune diseases or inflammatory diseases, or infectious diseases such as caused by bacteria, viruses, or other pathogens.
  • exemplary antigens are described above, which include antigens associated with various diseases and disorders that can be treated.
  • the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease.
  • the cells and compositions can be administered using standard administration techniques, formulations and/or devices.
  • Cellular administration can be autologous or heterologous, such as allogeneic.
  • immune response cells or progenitor cells can be obtained from a subject and administered to the same subject or different compatible subjects.
  • Peripheral blood-derived immune response cells or their progeny e.g., derived in vivo, ex vivo, or in vitro
  • can be administered by local injection including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration.
  • a therapeutic composition for example, a pharmaceutical composition containing genetically modified immune response cells
  • it is usually formulated into a unit-dose injectable form (solution, suspension, emulsion).
  • cell therapy e.g., adoptive cell therapy, for example, adoptive T cell therapy
  • adoptive cell therapy is performed by autologous transfer, separated and/or from a subject to receive cell therapy or from a sample derived from this subject
  • the cells are derived from the subject (eg, patient) and the cells in need of treatment, and after isolation and processing, the cells are administered to the same subject.
  • the cells can be administered by any suitable method, such as by bolus infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection Injection, intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjuntival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral deliver.
  • they are administered parenterally, intrapulmonarily, and intranasally, and (if needed for local treatment) intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • a given dose is administered by a single bolus injection of cells, administered by multiple bolus injections of cells, or administered by continuous infusion of cells.
  • the administration of a given "dose" of cells includes the administration of a given amount or quantity in a single composition and/or a single uninterrupted administration (for example, in a single injection or continuous infusion) It also includes the administration of a given amount or number of cells in divided doses or multiple compositions provided in multiple separate compositions or infusions within a specified time period of, for example, no more than 3 days. Therefore, in some cases, the dose is a single or continuous administration of a specified number of cells, given or started at a single point in time. However, in some cases, the dose is given by multiple injections or infusions over a period of no more than three days, such as once a day for three or two days or by multiple infusions over a period of one day.
  • the dose of cells is administered in a single pharmaceutical composition.
  • the dose of cells is administered in multiple compositions that collectively contain the dose of cells.
  • divided dose refers to a dose divided so that it is administered over a period of more than one day. This type of administration is included in the method and is considered a single dose. In some embodiments, divided doses of cells are administered in multiple compositions that collectively contain doses of cells over a period of no more than three days.
  • the dose of cells may be administered by administering multiple compositions or solutions (e.g., first and second, optionally more), each containing some cells in the dose.
  • multiple compositions are administered separately or independently, optionally within a certain period of time, each composition containing a different cell population and/or cell subtype.
  • cell populations or cell subtypes may include CD8 + and CD4 + T cells, and/or CD8 + and CD4 + -enriched populations, respectively, such as CD4 + and/or CD8 + T cells, each of which separately includes Cells genetically engineered to express recombinant receptors.
  • the administration of the dose includes administering a first composition comprising a certain dose of CD8 + T cells or a certain dose of CD4 + T cells, and administering a second composition comprising another dose of CD4 + T cells and CD8 + T cells.
  • the dose or composition of the cells includes a defined or target ratio of CD4+ cells expressing the recombinant receptor to CD8+ cells expressing the recombinant receptor and/or CD4 + cells to CD8+ cells, the ratio optionally being The ground is about 1:1, or between about 1:3 and about 3:1, such as about 1:1.
  • the administration of a composition or dose of a different cell population with a target or desired ratio e.g., CD4+:CD8+ ratio or CAR+CD4+:CAR+CD8+ ratio, e.g.
  • 1:1) involves administering a composition or dose containing one of the populations The cell composition, and subsequent administration of a separate cell composition comprising another said population, wherein said administration is performed at or approximately at a target or desired ratio.
  • the administration of a dose or composition of cells at a defined ratio results in improved expansion, durability, and/or anti-tumor activity of T cell therapy.
  • the cells are administered in a desired dose, which in some aspects includes a desired dose or number of cells or one or more cell types and/or a desired ratio of cell types. Therefore, in some embodiments, the cell dose is based on the total number of cells (or the number of cells per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the cell dose is based on the desired total number of cells or individual cell types in a single population (or number of cells per kg body weight). In some embodiments, the dosage is based on a combination of such characteristics, such as the total number of cells required, the required ratio, and the total number of cells in a single population required.
  • the cells are administered under or within the tolerance range of the desired output ratio of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes.
  • the desired ratio can be a specific ratio or can be a series of ratios.
  • the ratio of CD4+ to CD8+ cells is between 1:5 and 5:1, or between 1:3 and 3:1, such as between 2:1 and 1:5.
  • the tolerance difference is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, About 35%, about 40%, about 45%, about 50%, including any value between these ranges.
  • the number and/or concentration of cells refers to the number of cells expressing a recombinant receptor (e.g., CAR). In other embodiments, the number and/or concentration of cells refers to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMC) administered.
  • a recombinant receptor e.g., CAR
  • the number and/or concentration of cells refers to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMC) administered.
  • PBMC peripheral blood mononuclear cells
  • the size of the dose is determined based on one or more criteria, such as the subject’s response to existing treatments such as chemotherapy, the subject’s disease burden such as tumor burden, volume, size or degree, degree of metastasis, or Type, stage, and/or likelihood or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or against the cells and/or recombinant receptors administered The host immune response.
  • the subject s response to existing treatments such as chemotherapy
  • the subject’s disease burden such as tumor burden, volume, size or degree, degree of metastasis, or Type, stage, and/or likelihood or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or against the cells and/or recombinant receptors administered The host immune response.
  • relatively low doses of cells may be administered, such as suboptimal doses of cells or less than a therapeutically effective dose of cells, which are stimulated in vivo (for example, by endogenous antigens or exogenous agents) Time can result in an enhancement (e.g., increase or expansion) of the number of engineered cells present in the subject.
  • expansion and/or activation of the cells can occur with exposure to the antigen in vivo, for example, expansion of engineered cells in the subject after administration of the cells.
  • the scope, degree, or magnitude of expansion in vivo can be expanded, enhanced, or enhanced by a variety of methods, which can modulate (e.g., increase) the performance of a given cell (e.g., a cell expressing a recombinant receptor). Amplification, proliferation, survival, and/or efficacy.
  • the biological activity of the cell population is measured by any of a number of known methods.
  • the parameters to be evaluated include the specific binding of the cell to the antigen, which is evaluated in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry.
  • the ability of a cell to destroy a target cell can be measured using any suitable method known in the art.
  • the biological activity of cells can also be measured by measuring the expression and/or secretion of certain cytokines such as CD107a, IFN ⁇ , IL-2 and TNF.
  • biological activity is measured by evaluating clinical outcomes, such as reduction in tumor burden or burden.
  • the toxicity results, the persistence and/or expansion of the cells, and/or the presence or absence of the host immune response are assessed.
  • compositions and formulations are provided.
  • the cells comprising recombinant antigen receptors such as CAR or TCR are provided in the form of a composition or formulation, such as a pharmaceutical composition or formulation.
  • a composition or formulation such as a pharmaceutical composition or formulation.
  • Such compositions can be used according to the provided methods and/or together with the provided articles or compositions, for example for the prevention or treatment of diseases and disorders, or for detection, diagnosis and prognostic methods.
  • pharmaceutical formulation refers to a formulation that is in a form that makes the biological activity of the active ingredient contained therein effective and does not contain additional components that have unacceptable toxicity to the subject to which the formulation is administered.
  • “Pharmaceutically acceptable carrier” refers to ingredients in a pharmaceutical formulation that are not toxic to the subject except for the active ingredients.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Therefore, there are many suitable formulations.
  • the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used.
  • the pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dose and concentration used, and includes, but is not limited to: buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine Acid; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, Such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glut
  • buffering agents are included in the composition.
  • Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.
  • a mixture of two or more buffers is used.
  • the buffering agent or mixture thereof is generally present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known.
  • the formulation or composition may also contain more than one active ingredient, which can be used for specific indications and diseases to be prevented or treated with cells or agents, wherein the respective activities do not adversely affect each other.
  • active ingredients are present in a suitable combination in an amount effective for the intended purpose.
  • the pharmaceutical composition further comprises other pharmaceutically active agents or drugs such as chemotherapeutics, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, Fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the agent or cell is administered in the form of a salt (e.g., a pharmaceutically acceptable salt).
  • Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid) and organic acids (such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, Those salts of fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, such as p-toluenesulfonic acid).
  • the pharmaceutical composition contains an agent or cell in an amount effective to treat or prevent a disease (eg, a therapeutically effective amount or a prophylactically effective amount).
  • a disease eg, a therapeutically effective amount or a prophylactically effective amount.
  • the efficacy of treatment or prevention is monitored by periodically evaluating the subject being treated. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.
  • the required dose can be delivered by administering the composition by a single bolus injection, by administering the composition by multiple bolus injections, or by administering the composition by continuous infusion.
  • the agent or cell can be administered by any suitable method, such as by bolus infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, Subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival injection, suboculofascial injection, retrobulbar injection, peribulbar injection, or posterior proximal scleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal administration, and intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • a given dose is administered by a single bolus administration of cells or agents. In some embodiments, a given dose is administered by, for example, multiple bolus injections of cells or agents over a period of no more than 3 days or by continuous infusion of cells or agents.
  • the appropriate dosage may depend on the type of disease to be treated, the type of one or more agents, the type of cells or recombinant receptors, the severity and course of the disease, and the administration of agents or cells for prevention sexual goals are also therapeutic goals, previous therapies, the subject’s clinical history and response to drugs or cells, and the decision of the attending physician.
  • the composition is suitable for administration to a subject at one time or over a series of treatments.
  • Standard administration techniques, formulations and/or equipment can be used to administer the cells or agents.
  • Formulations and devices (such as syringes and vials) for storing and administering the composition are provided.
  • the administration can be autologous or heterologous.
  • immune response cells are obtained from a subject and administered to the same subject or different compatible subjects.
  • the peripheral blood-derived immune response cells or their progeny can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration.
  • a therapeutic composition for example, a pharmaceutical composition containing genetically modified immune response cells or an agent for treating or ameliorating neurotoxic symptoms
  • it is usually formulated in a unit-dose injectable form.
  • the administered cells are modified to expand, enhance, or enhance the expansion, proliferation, survival, and/or efficacy of the administered cells.
  • the administered cell e.g., a cell engineered to express a recombinant receptor
  • the administered cell is modified so that the expansion, proliferation, survival, and/or control of the engineered cell can be modulated and/or controlled, for example, by administering an agent. Or efficacy.
  • the method includes an in vivo step of reducing, inhibiting, and/or minimizing the effect of the inhibitory factor in inhibiting the proliferation, expansion, and/or survival of the engineered cell in vivo. In some embodiments, the method includes in vivo steps that promote, support, and/or enhance the proliferation, expansion, and/or survival of engineered cells in vivo.
  • the additional agent is a small molecule, peptide, polypeptide, antibody or antigen-binding fragment thereof, antibody mimetics, aptamer or nucleic acid molecule (e.g., siRNA), lipid, polysaccharide, or any combination thereof.
  • the additional agent is an inhibitor or activator of a specific factor, molecule, receptor, function, and/or enzyme.
  • the additional agent is an agonist or antagonist of a specific factor, molecule, receptor, function, and/or enzyme.
  • the additional agent is an analog or derivative of one or more factors and/or metabolites.
  • the additional agent is a protein or polypeptide.
  • the additional agent is a cell, such as an engineered cell.
  • the method includes, for example, administering an agent other than the administered cell (e.g., a cell engineered to express a recombinant receptor) in a combination therapy.
  • the agent specifically expands, enhances, or enhances the expansion, proliferation, survival, and/or efficacy of engineered cells due to specific modulation of a transgene (eg, a transgene encoding a recombinant receptor).
  • the agent specifically targets a transgene, such as a recombinant receptor.
  • the agent specifically binds, activates, and/or enhances the activity of the recombinant receptor and/or other functions of all or part of the recombinant molecule encoded by the transgene.
  • administration of an agent in combination with recombinant cells can enhance, enhance, or expand the proliferation, expansion, and/or survival of the administered cells, for example, enhance cell expansion in vivo.
  • exemplary methods or agents for specific amplification of transgenes include endogenous antigen exposure, vaccination, anti-idiotypic antibodies or antigen-binding fragments thereof, and/or adjustable recombinant receptors.
  • methods for specific amplification of transgenes include vaccination methods.
  • the method for specific amplification of the transgene includes the administration of anti-idiotypic antibodies.
  • Anti-idiotypic antibodies include their antigen-binding fragments
  • specifically recognize, specifically target and/or specifically bind to antibodies or their antigen-binding fragments e.g., recombinant receptors (such as chimeric antigen receptors (CAR)) Is unique to the antigen binding domain).
  • a unique site is any single antigenic determinant or epitope within the variable portion of an antibody.
  • the anti-idiotypic antibody or antigen-binding fragment thereof is an agonist and/or exhibits specific activity for stimulating cell expression of a specific antibody, and the specific antibody includes a conjugate containing the antibody or antigen-binding fragment thereof Or recombinant receptor.
  • the method includes modulation of the expansion, proliferation, survival, and/or activity of immune cells or immune functions (typically including the administered engineered cells).
  • the method includes generally immunostimulatory or generally promotes, enhances, expands, and/or strengthens the expansion and proliferation of immune cells (including administered cells) in vivo (e.g., in a subject) , Survival and/or activity steps.
  • the agent can reduce, inhibit, and/or minimize the effect of the inhibitory factor in inhibiting the proliferation, expansion, and/or survival of immune cells (eg, administered cells) in vivo.
  • the method includes modulating the expansion of engineered cells, for example, by inhibiting the proliferation, expansion, and/or activation of a negative regulator of the administered cells (eg, engineered immune cells).
  • a negative regulator of the administered cells eg, engineered immune cells.
  • the administered cells expressing the recombinant receptor may encounter an environment that suppresses or inhibits cell growth, proliferation, expansion, and/or survival, such as an immunosuppressive environment.
  • the immunosuppressive environment can contain immunosuppressive cytokines, regulatory modulators, and co-suppressive receptors.
  • additional agents include immunomodulators, immune checkpoint inhibitors, metabolic pathway modulators, adenosine pathway or adenosine receptor antagonists or agonists, and signal transduction pathway modulators (e.g., kinase inhibitors) ).
  • the additional agent is an immunomodulator, such as an immune checkpoint inhibitor.
  • the additional agent increases, enhances, or amplifies the expansion and/or proliferation of the administered cells, thereby increasing, enhancing, or amplifying the immune response by blocking immune checkpoint proteins (ie, immune checkpoint inhibitors).
  • the additional agent is an agent that enhances the activity of engineered cells (eg, recombinant receptor expressing cells), and is a molecule that inhibits immunosuppressive molecules or immune checkpoint molecules.
  • immunosuppressive molecules include PD-1, PD-L1, CTLA4, TEVI3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 And TGFR ⁇ .
  • CEACAM e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5
  • the immune checkpoint inhibitor may be an antibody against immune checkpoint protein, such as cytotoxic T lymphocyte antigen 4 (CTLA4 or CD152), programmed cell death protein 1 (PD-1), or programmed cell death protein 1 (PD-1). Antibody against cell death protein 1 ligand 1 (PD-L1).
  • CTL4 or CD152 cytotoxic T lymphocyte antigen 4
  • PD-1 programmed cell death protein 1
  • PD-L1 Antibody against cell death protein 1 ligand 1
  • the method includes contacting a cell expressing a recombinant receptor with an agent that inhibits an inhibitory cell surface receptor, such as transforming growth factor beta receptor (TGF beta R).
  • TGF beta R transforming growth factor beta receptor
  • the administered cells e.g., recombinant receptor expressing cells
  • the additional agent is an anti-TGF ⁇ antibody or an anti-TGF ⁇ R antibody.
  • the additional agent modulates the metabolism, signaling, and/or transport of immunosuppressive factors (e.g., adenosine).
  • the additional agent is an inhibitor of extracellular adenosine or adenosine receptors, or an agent that causes a decrease or decrease in the level of extracellular adenosine, such as preventing the formation of extracellular adenosine, degrading extracellular adenosine , An agent that inactivates and/or reduces extracellular adenosine.
  • the additional agent is an adenosine receptor antagonist, such as A2a, A2b, and/or A3 receptors.
  • the additional agent is an adenosine receptor antagonist or agonist, such as an antagonist or agonist of one or more of adenosine receptors A2a, A2b, A1, and A3.
  • the method includes administering an additional agent that is immunostimulatory.
  • the additional agent can generally promote the proliferation, expansion, survival, and/or efficacy of immune cells.
  • the additional agent may specifically promote the administered cells, such as recombinant receptor expressing cells.
  • the additional agent is a cytokine.
  • the additional agent is a ligand.
  • the additional agent is an immunostimulatory ligand, such as CD40L.
  • the additional agent is a cytokine, such as IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, granulocyte macrophage Colony stimulating factor (GM-CSF), alpha, beta or gamma interferon (IFN) and erythropoietin (EPO).
  • GM-CSF granulocyte macrophage Colony stimulating factor
  • IFN alpha
  • EPO erythropoietin
  • the provided methods may further include, for example, administering one or more lymphocytes to eliminate before or at the same time as the administration of the initiating cell (e.g., recombinant receptor expressing cell).
  • lymphocyte clearance therapy includes the administration of cyclophosphamide.
  • lymphocyte depletion therapy includes administration of fludarabine. In some embodiments, lymphocyte depletion therapy is not administered.
  • Pretreatment of the subject with immune clearance (eg, lymphocyte clearance) therapy can improve the effect of adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • lymphocyte scavengers including a combination of cyclosporine and fludarabine
  • TIL metastatic tumor infiltrating lymphocytes
  • the provided methods further involve administering lymphocyte depletion therapy to the subject.
  • the method involves administering lymphocyte depletion therapy to the subject before administering the cell dose.
  • lymphocyte clearance therapy contains chemotherapeutic agents.
  • the method includes administering to the subject a pretreatment agent, such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, before administering the cell dose.
  • a pretreatment agent such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof
  • the pretreatment agent may be administered to the subject at least 2 days before the first or subsequent dose, such as at least 3, 4, 5, 6, or 7 days.
  • the pretreatment agent is administered to the subject no more than 7 days, such as no more than 6, 5, 4, 3, or 2 days before administering the cell dose.
  • the subject is pretreated with cyclophosphamide at a dose of or between about 20 mg/kg and 100 mg/kg, such as at or between about 40 mg/kg and 80 mg/kg .
  • fludarabine can be given in a single dose or can be given in multiple doses, such as daily, every other day, or every three days.
  • fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days.
  • the subject is given about 30 mg/m2 of fludarabine every day for 3 days before starting cell therapy.
  • cyclophosphamide is administered once a day for one or two days.
  • the subject before receiving the first dose, the subject receives lymphocytic depletion pretreatment chemotherapy of cyclophosphamide and fludarabine (cy/flu), which is the first dose of CAR expressing
  • the cells are administered at least two days before and usually no more than 7 days before the cells are administered.
  • the subject is given the dose of CAR-expressing T cells as described above.
  • administration of the pretreatment agent prior to the infusion of the cell dose improves the outcome of the treatment.
  • pretreatment improves the efficacy of treatment with a dose or increases the persistence of cells expressing recombinant receptors (e.g., CAR-expressing cells, such as CAR-expressing T cells) in the subject.
  • the pretreatment treatment increases disease-free survival, such as the percentage of subjects who survive, and do not exhibit minimal residual or molecularly detectable disease after a given period of time after the cell dose. In some embodiments, the time to median disease-free survival is increased.
  • the biological activity of the engineered cell population is measured by any of a number of known methods.
  • the parameters to be evaluated include the specific binding of engineered or natural T cells or other immune cells to the antigen, which is evaluated in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry.
  • the biological activity of cells can also be measured by measuring the expression and/or secretion of certain cytokines such as CD107a, IFN ⁇ , IL-2 and TNF.
  • biological activity is measured by evaluating clinical outcomes, such as reduction in tumor burden or burden.
  • the toxicity results, the persistence and/or expansion of the cells, and/or the presence or absence of the host immune response are assessed.
  • administration of the pretreatment agent prior to the infusion of the cell dose improves the outcome of the treatment (e.g., by improving the efficacy of the dose treatment), or increases the cells expressing the recombinant receptor (e.g., CAR-expressing cells, such as CAR T cells) persistence in subjects.
  • the recombinant receptor e.g., CAR-expressing cells, such as CAR T cells
  • the cells are modified in any number of ways so as to increase their therapeutic or preventive efficacy and/or can modulate expansion, proliferation, survival, and/or efficacy.
  • the cells are modified so that expansion, proliferation, survival, and/or efficacy can be modulated (e.g., enhanced, strengthened, and/or expanded) after administration to a subject.
  • the cells are modified so that the expression of transgenes and/or immunoregulatory factors can be regulated and/or controlled.
  • the cell is modified to modulate the expression and/or activity of specific components of the recombinant receptor.
  • the cell is modified to increase or decrease the expression of an agent (e.g., a nucleic acid, such as an inhibitory nucleic acid).
  • the cell is modified to express and/or secrete an agent.
  • the engineered recombinant receptor (e.g., CAR) expressed by the engineered cell can be conjugated to the targeting moiety either directly or indirectly via a linker.
  • the method includes modulating the administered cell by contacting the cell with an agent that reduces the expression of a negative regulator of the administered cell (e.g., an engineered T cell expressing a recombinant receptor) or is capable of The reduction in expression is achieved.
  • a negative regulator of the administered cell e.g., an engineered T cell expressing a recombinant receptor
  • Negative regulators of cells include any of those described herein, such as immune checkpoint inhibitors, inhibitory receptors, and/or adenosine modulators.
  • an agent that reduces the expression of a negative regulator or is capable of achieving the reduction in expression includes being or comprising an inhibitory nucleic acid molecule (e.g., complementary to, targeting, inhibiting, and/or binding to a gene or nucleic acid encoding a negative regulator) Said gene or nucleic acid molecule).
  • the agent is or comprises a complex comprising a ribonucleoprotein (RNP) complex, the ribonucleoprotein (RNP) complex including Cas9 (e.g., in some cases enzymatically lost Live Cas9) and gRNAs targeting genes encoding negative regulators.
  • the inhibitory nucleic acid molecule includes an RNA interfering agent.
  • the inhibitory nucleic acid is or contains or encodes small interfering RNA (siRNA), microRNA adapted shRNA, short hairpin RNA (shRNA), hairpin siRNA, precursor microRNA (pre-miRNA) Or microRNA (miRNA).
  • the engineered cell is subjected to genetic modification or gene editing, which targets a locus encoding a gene involved in immune regulation, negative regulation of immune cells, and/or immunosuppression.
  • gene editing results in insertions or deletions at the targeted locus, or "knockout" of the targeted locus and elimination of the expression of the encoded protein.
  • gene editing is achieved by using the CRISPR/Cas9 system for non-homologous end joining (NHEJ).
  • one or more guide RNA (gRNA) molecules can interact with one or more Cas9 nucleases, Cas9 nickases, enzymatically inactivated Cas9 or variants thereof, or engineered zinc fingers or TALE. The system is used together.
  • the cell e.g., recombinant receptor expressing cell
  • the cell is further modified to express and/or secrete an additional agent that promotes, enhances, strengthens, and/or expands the administration of the cell Proliferation, expansion, survival and/or efficacy.
  • recombinant receptor expressing cells e.g., CAR-expressing cells
  • additional agents that overcome immunosuppressive effects and/or enhance the expansion of T cells and recombinant receptors And/or function.
  • the cells can be engineered to express cytokines that promote expansion of the administered cells.
  • such additional agents may be operably linked to an inducible expression system (e.g., an inducible promoter).
  • the cells administered may be modified to express and/or secrete agents that inhibit immunosuppressive factors (such as any of the ones described herein) and/or stimulate immunostimulatory factors.
  • the additional agent expressed by the administered cells reduces or prevents immunosuppression of the cells in the tumor microenvironment.
  • the additional agent encoded and/or secreted by the administered cell may include any additional agent described herein.
  • the additional agent encoded by the administered cell is soluble and secreted.
  • the additional agent is a soluble scFv.
  • the additional agent is a cytokine.
  • the method includes modifying the cell to allow regulatable expression and/or activity of a recombinant receptor (e.g., CAR), thereby modulating the signal through the recombinant receptor.
  • regulatable expression and/or activity is achieved by configuring the recombinant receptor to contain or be controlled by specific regulatory elements and/or systems (such as any of those described herein).
  • administration of engineered cells into a subject and/or exposure to specific ligands can modulate the expression and/or activity of recombinant receptors (e.g., CARs).
  • the regulation of the expression and/or activity of the recombinant receptor is achieved by administering an additional agent that can modulate the expression of the recombinant receptor (eg, CAR).
  • an additional agent that can modulate the expression of the recombinant receptor eg, CAR
  • the regulated expression of a recombinant receptor is achieved through an adjustable transcription factor release system, or by administering additional agents that can induce polypeptides (e.g., recombinant receptors).
  • Conformational changes and/or multimerization is a chemical inducer.
  • subject includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans and non-human animals, including farm animals, sports animals, rodents, and pets.
  • enrichment refers to, for example, compared to the total number of cells in the composition or the volume of the composition or relative to other cell types, such as by Positive selection of markers expressed by the population or cells, or by negative selection based on markers that are not present on the cell population or cells to be depleted, to increase the number or percentage of the cell types or population.
  • the term does not require complete removal of other cells, cell types, or populations from the composition, and does not require the cells so enriched to be present at equal to or even close to 100% in the enriched composition.
  • a cell or cell population is "positive” or "+” for a particular marker refers to the detectable presence of a particular marker (usually a surface marker) on or in the cell.
  • a surface marker it refers to the presence of surface expression as detected by flow cytometry in some embodiments, for example by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein The staining is detectable by flow cytometry at a level that is substantially higher than the staining detected by the same procedure with an isotype matched control under otherwise identical conditions, and/or the level It is substantially similar to the level of cells known to be positive for the marker, and/or the level is substantially higher than the level of cells known to be negative for the marker.
  • a cell or cell population is "negative" for a particular marker means that the particular marker (usually a surface marker) is not substantially detectable on or in the cell.
  • a surface label refers to the absence of surface expression as detected by flow cytometry (for example, by staining with an antibody that specifically binds to the label and detecting the antibody) in some embodiments , wherein the staining is not detected by flow cytometry at the following level: significantly higher than the level of staining detected by the same procedure with an isotype-matched control under otherwise identical conditions, and/or significantly lower At the level of cells known to be positive for the marker, and/or at a substantially similar level as compared to cells known to be negative for the marker.
  • percent amino acid sequence identity %
  • percent identity is defined as when sequences are aligned and gaps are introduced when necessary to achieve maximum sequence identity
  • the percentage of amino acid residues in the candidate sequence eg, Vpx or Vpr protein
  • the alignment used to determine the percentage of amino acid sequence identity can be achieved in a variety of ways well known in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve maximum alignment over the full length of the compared sequences.
  • composition refers to any mixture of two or more products, substances, or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.
  • the purpose of the present invention is to provide a new method for preparing CAR-T cells. This method only takes about 1 day for the preparation of CAR-T cells, which greatly shortens the in vitro culture compared with the conventional preparation method of CAR T cells (about 2 weeks). At the same time, it will be able to better maintain the memory phenotype of CAR-T cells, and enhance the tumor-killing function of CAR T cells and their survival time in the body.
  • Leukocyte separation is used to collect leukocyte-rich samples from subjects, and Ficoll density gradient centrifugation is used to collect the albuginea layer to obtain high-purity peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • PBMC phosphate buffered saline
  • PBS phosphate buffered saline
  • EDTA EDTA
  • human serum albumin The PBMC are sorted to obtain a CD4+CD8+ enriched T cell population.
  • T cell activator anti-CD3 and/or anti-CD28 bead reagents and lentiviral vector particles.
  • the viral vector particles contain coding chimera Antigen receptor (CAR-BCMA, see SEQ ID NO: 2 for amino acid sequence, and SEQ ID NO: 1 for nucleic acid sequence).
  • CAR-BCMA coding chimera Antigen receptor
  • the lentiviral vector particles are added according to the multiplicity of infection (MOI) of the virus at 3. After 24 hours of culture, The culture broth is centrifuged to change the solution, washed with physiological saline, and then added to the cryoprotective solution for cryopreservation. This preparation process is called a new process.
  • mononuclear cells obtained by leukocyte separation or T cell populations obtained after enrichment are activated by adding anti-CD3 and/or anti-CD28-conjugated bead reagents for 24 hours or 48 hours, and then according to the multiplicity of viral infection ( MOI) Add lentiviral vector for 1-3 and continue to culture for 7 days or more.
  • MOI multiplicity of viral infection
  • CAR T cells targeting GPC3 were selected for cell transduction research, referring to the aforementioned method, PBMC or enriched T cell population, T cell activator and lentiviral vector particles were incubated for 24 hours for harvesting.
  • Transduction was carried out with different multiplicity of infection (MOI) of the virus, and the transduction efficiency of CAR was tested 96 hours after transduction (see Table 2 for the results).
  • MOI multiplicity of infection
  • the new process was studied with MOI of 3, and the cells harvested for 24 hours were transduced. The detection showed that the transduction efficiency of CAR was almost undetectable, only 0.2%.
  • the cells were continued to be supplemented with 2% AB serum, 300IU/mL IL- 2 was cultured in AIM-V medium, and it was found that with the extension of in vitro culture time, the detected transduction efficiency continued to increase (as shown in Table 3).
  • the amino acid sequence of the CAR of the CAR T cell targeting GPC3 is shown in SEQ ID NO: 3.
  • Example 2 Evaluation of the transduction efficiency of primary T cell activation and transduction culture for 24-48 hours
  • Leukocyte separation is used to collect leukocyte-rich samples from subjects, and the albuginea layer is collected by Ficoll density gradient centrifugation to obtain high-purity peripheral blood mononuclear cells (PBMC), which are added to the washed leukocytes in the centrifuge chamber Separation technique sample.
  • PBMC peripheral blood mononuclear cells
  • the cells are then passed from the transfer bag through a sterile system of closed tubing and separation column in the presence of a magnetic field using standard methods to separate the cells bound to the CD4-specific reagent and/or CD8-specific reagent.
  • the enriched T cells were resuspended in the culture medium, and the bead reagents of CD3 and CD28 were added for incubation first, and after incubation for different times, the lentiviral vector containing the recombinant nucleic acid encoding GPC3CAR was added for transduction.
  • the amino acid sequence of GPC3CAR is shown in SEQ ID NO: 3.
  • the process of activation transduction is: resuspend about 2x10 8 T cells in X-VIVO 15 medium with a total volume of 140 mL, inoculate them into culture flasks/bags, add anti-CD3 and CD28 bead reagents for activation, and activate them at different times.
  • the multiplicity of infection (MOI) of the virus is 3 and the lentiviral vector is added for transduction.
  • the total culture time for activation and transduction is 24h. After the end, the harvest is carried out. The culture medium is centrifuged and replaced with saline and washed with saline and resuspended in the freezing solution. Store frozen.
  • the cells are cultured at 37°C and 5% CO 2 and the incubation times for activation and transduction in different protocols are as follows:
  • Control The control cells are the conventional process, activated for 48 hours, add lentiviral vector at MOI of 1.5 for transduction for 24 hours, centrifuge and change the medium to remove the episomal vector, continue to expand the cell culture and harvest on the 8th day .
  • the longer the activation time the easier the cells are to transfect.
  • the lentivirus can enter quickly.
  • the process may take less than 2 hours, and the transduction efficiency level is basically the same as the conventional control level.
  • CD19CAR The preparation process of CD19CAR is activated and transduced according to the following scheme.
  • Cells are cultured at 37°C and 5% CO 2 and the MOI of scheme one to scheme five is 3:
  • control cells are the conventional process, first activated for 48 hours, add lentiviral vector for 24 hours at MOI of 2 for transduction, centrifuge to change the medium to remove the free vector, continue to expand the cell culture and harvest on the 8th day .
  • the cells harvested from Scheme 1 to Scheme 5 were cryopreserved and resuscitated, and then inoculated into the culture medium and continued to be cultured for 144 hours before transduction efficiency was tested.
  • the results are shown in Figure 3.
  • lentiviral vectors were added for transduction within the range of 16 hours to 23 hours of activation, and the total culture time was 24 hours.
  • the transduction efficiency of T cells was not much different, which was slightly higher than that of conventional controls, indicating that after T cells were activated to a certain state , The shortest time for the lentivirus to enter the cell is only 1 hour.
  • CAR T cells prepared by the new process In order to evaluate the anti-tumor activity of CAR T cells prepared by the new process, we compared CAR T cells prepared by the conventional process (48h after activation and then cultured in vitro for more than 7 days after transduction) as a control, and compared the CAR T cells prepared by the new process with different doses.
  • CAR-BCMA T cells prepared by conventional processes were collected on D7 (cultured for 7 days after transduction, defined as conventional process 1) and D11 (transduced for 11 days, defined as conventional process 2).
  • the CAR-BCMA prepared by the new process T cell activation and transduction of BCMA is carried out at the same time, transduction is carried out at MOI of 3, and samples are collected and stored frozen after 24 hours of culture.
  • the relevant phenotypes are shown in Table 4 and the positive rate (the new process is the detection of cryopreservation and resuscitation for 168 hours), which is shown in Table 5. .
  • TN CD95 - CD62L + CD45RA + CCR7 + CD45RO -
  • TSCM CD3 + CD95 + CD62L + CD45RA + CCR7 + CD45RO -
  • the CART cells prepared by the above-mentioned new process were used to evaluate their anti-tumor efficacy in NPG mice bearing human multiple myeloma cells RPMI-8226 subcutaneously transplanted tumors, as well as their survival in the peripheral blood of the mice, and tumor cells
  • the day of inoculation is recorded as D0, and the specific dosage and experimental design are shown in Table 6.
  • CAR T cell infusion was performed on D12 after tumor inoculation.
  • the tumor volume in the vehicle control group exceeded 2000 mm 3 .
  • the tumor volume and tumor regression of each group are as follows:
  • the tumor volume inhibition rate was 56.05%, and there was no tumor regression in mice.
  • the tumor volume of the mice in the above groups is shown in Figure 4 and the mouse body weight changes over time.
  • the new process 1 group is slightly later than the conventional process 1 and conventional process 2
  • the new process 2 group and the new process 3 group have a later onset time, and the different doses of CAR T and the onset time in the new process group There is a dose correlation.
  • the new technology 1 group and the new technology 2 group all achieved complete tumor clearance during the observation period (D39 days after tumor inoculation).
  • the new technology group 3 we also observed that the tumor volume from D32 to D35 was nearly stable, and began to decrease at D39.
  • the new technology group 3 group is retained Animals to continue to observe the sustained efficacy. Consistent with expectations, the tumor volume of mice in the new process group 3 continued to decrease during the subsequent observation period. On D68 days after tumor inoculation, the tumor volume inhibition rate of this dose group was 100%, and the tumors of 5 mice were almost all Fading, showing an encouraging anti-tumor effect. In terms of toxicity, during the experiment, except for tumor-influencing factors, the weight of the mice did not change much, suggesting that CART has no obvious side effects on mice.
  • Leukocyte separation is used to collect leukocyte-rich samples from subjects, and Ficoll density gradient centrifugation is used to collect the albuginea layer to obtain high-purity peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the PBMC were washed and resuspended in a buffer, and sorted based on immunoaffinity, the buffer containing phosphate buffered saline (PBS), EDTA and human serum albumin.
  • PBS phosphate buffered saline
  • EDTA EDTA
  • human serum albumin phosphate buffered saline
  • T cell sorting based on immunoaffinity the washed cells in the sorting buffer are incubated with a bead reagent coupled to a monoclonal antibody at room temperature for 30 minutes, and a magnetic separation column is used for sorting.
  • the enriched T cells were resuspended in X-VIVO15 medium, and different T cell activators were added to activate them.
  • the lentiviral vector containing the nucleic acid encoding the chimeric antigen receptor (CAR-CD19) was added according to the multiplicity of viral infection (MOI) is 3 for addition. After culturing for 24 hours, it was replaced with AIM-V medium supplemented with 2% AB serum and 300 IU/mL IL-2, and the culture time was extended to 144 hours. The transduction efficiency at this time was used as the basis for evaluating activation conditions.
  • CD19-CAR T cells Activate according to the following reagent conditions and concentrations for 22 hours and then transduce for 2 hours to prepare CD19-CAR T cells.
  • the MOI of transduction schemes one to four is 3, and the culture conditions are 37°C and 5% CO 2 culture.

Abstract

提供了病毒载体转导细胞的方法，还提供了用重组或异源基因转导的所获得细胞及其组合物，以及将其用于过继免疫治疗的方法。在不影响重组核酸的表达的前提下，该方法缩短了基因工程化细胞的制备过程中活化、转导的时间。

Description

病毒载体转导细胞的方法 技术领域
本发明属于基因工程领域，具体涉及通过病毒转导重组核酸进入的方法。
背景技术
免疫效应细胞(如T细胞、NK细胞、NK T细胞等)在肿瘤免疫治疗中的作用日益受到重视。近年来，人们对免疫效应细胞进行外源受体的修饰，获得特异性识别肿瘤相关抗原的T细胞，进而进行肿瘤治疗，如嵌合抗原受体修饰的CAR T细胞、嵌合TCR受体修饰的TCR T细胞等。
通常，识别肿瘤相关抗原的细胞的获得是将可以编码识别肿瘤相关抗原的外源受体的重组核酸导入病毒载体，再用携带重组核酸的病毒载体感染转导细胞。然而由于携带重组核酸的病毒载体感染转导细胞通常需要的时间较长，如CAR T细胞的制备，在病毒载体转导外源核酸进入T细胞常规的工艺中，通常，T细胞活化需要一天以上，随后进行病毒转导，病毒转导需要1天，在转导完成后还需要进行扩增，扩增需要1-2周，使得CAR T细胞的制备耗时很长，不仅增加了细胞产品制备的时间成本和试剂成本，还可能增加细胞变异的风险，并且由于制备时间过程，再给予患者细胞治疗产品时，有些患者已经发生了肿瘤进展，从而延误肿瘤治疗的时机，影响临床治疗的效果。
发明内容
本发明的目的在于提供了一种病毒载体转导细胞的方法，可以显著缩短识别肿瘤相关抗原的受体修饰细胞的制备时间，且不影响甚至还进一步增强细胞治疗的疗效。
在第一方面，本发明提供一种病毒载体转导细胞的方法，所述方法包括：
步骤(1)、将包含待转导细胞的输入组合物、待转导细胞刺激剂、和携带重组核酸的病毒载体颗粒共同孵育，孵育时间不超过72小时，
步骤(2)、进行收获，获得输出组合物，所述输出组合物含有转导有重组核酸的细胞；
优选的，所述孵育时间为1小时-72小时；
更优选的，所述孵育时间为2小时-48小时；
更优选的，所述孵育时间为2小时-36小时；
更优选的，所述孵育时间为12小时-36小时；
更优选的，所述孵育时间为12小时-24小时；
更优选的，所述孵育时间为15小时-24小时。
在第二方面，本发明提供一种病毒载体转导细胞的方法，所述方法包括：
步骤(1)、将包含待转导细胞的输入组合物和待转导细胞刺激剂，孵育时间不超过72h，
步骤(2)、再加入重组核酸的病毒载体颗粒进行孵育，孵育时间不超过24小时，
步骤(3)、进行收获，获得输出组合物，所述输出组合物含有转导有重组核酸的细胞；
优选的，所述(1)和(2)的总孵育时间不超过72小时。
在具体的实施方式中，所述(1)和(2)的总孵育时间不超过60h，或不超过48h，或不超过32h，或不超过28h，或不超过24h。
在具体的实施方式中，所述步骤(1)的孵育时间为2-72小时；
优选的，所述步骤(1)的孵育时间为2-71小时；
更优选的，所述步骤(1)的孵育时间为2-48小时；
更优选的，所述步骤(1)的孵育时间为2-32小时；
更优选的，所述步骤(1)的孵育时间为2-28小时；
更优选的，所述步骤(1)的孵育时间为3-24小时；
更优选的，所述步骤(1)的孵育时间为5-24小时；
更优选的，所述步骤(1)的孵育时间为7-24小时；
更优选的，所述步骤(1)的孵育时间为7-23小时；
更优选的，所述步骤(1)的孵育时间为10-23小时；
更优选的，所述步骤(1)的孵育时间为15-23小时；
更优选的，所述步骤(1)的孵育时间为15-22小时。
在具体的实施方式中，所述步骤(2)的孵育时间为30分钟-24小时；
优选的，所述步骤(2)的孵育时间为30分钟-21个小时；
优选的，所述步骤(2)的孵育时间为30分钟-17个小时；
优选的，所述步骤(2)的孵育时间为30分钟-12个小时；
优选的，所述步骤(2)的孵育时间为30分钟-10个小时；
优选的，所述步骤(2)的孵育时间为30分钟-8个小时；
优选的，所述步骤(2)的孵育时间为1小时-8个小时；
优选的，所述步骤(2)的孵育时间为1小时-4个小时；
更优选的，所述步骤(2)的孵育时间为1个小时-3个小时。
在具体的实施方式中，所述输入组合物从外周血、脐血、骨髓和/或诱导多能干细胞中获得，优选地，所述输入组合物是白细胞分离术样品；优选地，所述输入组合物是富集或分离的CD3+T细胞，是富集或分离的CD4+T细胞或者是富集或分离的CD8+T细胞或其组合。
在具体的实施方式中，所述病毒载体颗粒源自逆转录病毒载体；优选的，所述病毒载体颗粒是慢病毒载体。
在具体的实施方式中，所述病毒载体颗粒的感染复数不高于20；优选的，所述感染复数为0.5-20；更优选的，所述感染复数为1.5-20；更优选的，所述感染复数为3-20；更优选的，所述感染复数为3-12。
在具体的实施方式中，所述输入组合物中待转导细胞的数量不超过1*10 10；
优选的，所述输入组合物中待转导细胞的数量不低于1*10 5；
更优选的，所述输入组合物中待转导细胞的数量不低于1*10 6。
在具体的实施方式中，所述重组核酸能够编码识别特异性靶抗原的受体；优选的，所述识别特异性靶抗原的受体是T细胞受体(TCR)、嵌合抗原受体(CAR)、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
在具体的实施方式中，所述嵌合抗原受体(CAR)包含特异性地结合至靶抗原的细胞表面抗原识别结构域和包含ITAM的细胞内信号传导结构域。
在优选的实施方式中，所述细胞内信号传导结构域包含CD3-zeta(CD3ζ)链的细胞内结构域。
在优选的实施方式中，所述嵌合抗原受体(CAR)还包括连接所述细胞外结构域与所述细胞内信号传导结构域的跨膜结构域。
在优选的实施方式中，所述跨膜结构域包含CD28和/或CD8的跨膜部分。
在优选的实施方式中，所述细胞内信号传导结构域还包含T细胞共刺激分子的细胞内信号传导结构域。
在优选的实施方式中，所述T细胞共刺激分子选自CD28和/或41BB。
在优选的实施方式中，所述特异性靶抗原是与疾病相关的抗原或通用标签；
优选的，所述疾病是癌症、自身免疫性疾病、或感染性疾病；
优选地，所述癌症是血液肿瘤；更优选地，所述血液肿瘤是白血病、骨髓瘤、淋巴瘤及/或其组合。
在具体的实施方式中，所述特异性靶抗原是肿瘤相关抗原；
优选的，所述肿瘤相关抗原选自：B细胞成熟抗原(BCMA)、碳酸酐酶9(CAIX)、EGFR、Her2/neu(受体酪氨酸激酶erbB2)、CD19、CD20、CD22、间皮素、CEA、CD23、CD24、CD30、CD33、CD38、CD44、EGFR、上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、EPHa2、erb-B2、erb-B3、erb-B4、erbB二聚体、EGFR vIII、叶酸结合蛋白(FBP)、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、HMW-MAA、IL-22R-α、IL-13R-α2、激酶插入结构域受体(kdr)、L1细胞粘附分子(L1-CAM)、黑色素瘤相关抗原(MAGE)、TAG72、B7-H6、IL-13受体α2(IL-13Ra2)、CA9、GD3、HMW-MAA、CD171、G250/CAIX、HLA-AI MAGEA1、HLA-A2、PSCA、叶酸受体、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、CD44v6、间皮素、粘蛋白1(MUC1)、MUC16、PSCA、NKG2D、NY-ESO-1、MART-1、gp100、癌胚胎抗原、G蛋白偶联受体5D(GPCR5D)、ROR1、TAG72、VEGF-R2、癌胚抗原(CEA)、前列腺特异性抗原、PSMA、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、Wilms肿瘤1(WT-1)、细胞周期蛋白、CCL-1、CD138，Claudin18.2、GPC3。
在具体的实施方式中，所述待转导细胞刺激剂能够激活TCR复合物的一个或多个组分的一个或多个细胞内信号域和一个或多个共刺激分子的一个或多个细胞内信号域；
优选地，所述待转导细胞刺激剂包含(i)特异性结合到TCR复合物成员的一级试剂，任选地特异性结合到CD3和(ii)特异性结合到T细胞共刺激分子的二级试剂，任选地其中所述共刺激分子选自CD28，CD137(4-1-BB)、0X40或ICOS。
在具体的实施方式中，所述待转导细胞是免疫效应细胞；
优选的，所述待转导细胞是T细胞，NK细胞，NKT细胞，树突细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞或其组合；
更优选的，所述待转导细胞是T细胞。
在优选的实施方式中，其中所述T细胞是CD4+和/或CD8+细胞。
在优选的实施方式中，所述CD4+细胞与所述CD8+细胞的比率为1:1、1:2、2:1、1:3、3:1、1:4、4:1、1:5、5:1、1:6或6:1。
在优选的实施方式中，包括用于所述选择或富集的试剂4。
在优选的实施方式中，未与所述T细胞结合的所述试剂4可以离心去除。
在优选的实施方式中，所述4固定在固体支持物，优选地，所述固体支持物是聚合物基质。
在优选的实施方式中，所述聚合物基质为聚合物纳米基质和/或珠试剂。
在优选的实施方式中，所述珠试剂包括磁珠和/或微珠。
在优选的实施方式中，其中激活和/或扩增是在体内进行的。
在优选的实施方式中，所述样品是白细胞分离术样品。
在优选的实施方式中，所述T细胞是，是富集或分离的CD3+T细胞，是富集或分离的CD4+T细胞，或者是富集或分离的CD8+T细胞。
在优选的实施方式中，已经从来自所述受试者的所述样品选择或富集所述T细胞。
在具体的实施方式中，所述待转导细胞刺激剂包含CD3结合分子、CD28结合分子、重组IL-2、重组IL-15、重组IL-7、重组IL-21或其组合物；
优选的，所述待转导细胞刺激剂包含抗CD3抗体和/或抗CD28抗体。
在具体的实施方式中，在收获之前，所述待转导细胞刺激剂可离心去除。
在具体的实施方式中，所述待转导细胞刺激剂为游离分子。
在具体的实施方式中，所述待转导细胞刺激剂固定在固体支持物上；
优选的，所述固体支持物是聚合物基质材料；
更优选的，所述聚合物基质材料为可降解的聚合物纳米基质或珠试剂。
在具体的实施方式中，所述珠试剂为磁珠或微珠。
在具体的实施方式中，所述输出组合物中，转导有重组核酸的细胞的含量不低于30％、或不低于40％、或不低于50％、或不低于60％、或不低于70％、或不低于80％。
在具体的实施方式中，所述输出组合物中，转导有重组核酸的细胞的含量不高于50％；优选的，不高于40％，更优选的，不高于38％；更优选的，不高于35％；更优选的，不高于30％。
在具体的实施方式中，所述转导有重组核酸的细胞中幼稚细胞的含量相比待转导细胞中幼稚细胞的含量降低；
优选的，所述幼稚细胞含量降低至10％以下；
更优选的，所述幼稚细胞含量降低至5％以下。
在具体的实施方式中，所述转导有重组核酸的细胞中记忆性细胞的含量相比待转导细胞中记忆性细胞的含量提高；
优选的，所述记忆性细胞是记忆性干细胞；
更优选的，所述记忆性干细胞是TSCM。
在具体的实施方式中，转导有重组核酸的细胞中记忆性干细胞的含量是待转导细胞中记忆性干细胞的含量的约2倍或以上，优选的，转导有重组核酸的细胞中记忆性干细胞的含量是待转导细胞中记忆性干细胞的含量的约3倍或以上。
在具体的实施方式中，所述转导有重组核酸的细胞含有未分化的细胞。
在具体的实施方式中，所述输入组合物包含重组IL-2，任选重组人IL-2，所述重组IL-2的浓度为10IU/mL至500IU/mL、50IU/mL至250IU/mL或100IU/mL至200IU/mL；或浓度为至少10IU/mL、50IU/mL、100IU/mL、200IU/mL、300IU/mL、400IU/mL或500IU/mL；和/或
所述输入组合物包含重组IL-15，任选重组人IL-15，所述重组IL-15的浓度为1IU/mL至100IU/mL、2IU/mL至50IU/mL或5IU/mL至10IU/mL；或浓度为至少1IU/mL、2IU/mL、5IU/mL、10IU/mL、25IU/mL或50IU/mL；和/或
所述输入组合物包含重组IL-7，任选重组人IL-7，所述重组IL-7的浓度为50IU/mL至1500IU/mL、100IU/mL至1000IU/mL至200IU/mL至600IU/mL；或浓度为至少50IU/mL、100IU/mL、200IU/mL、300IU/mL、400IU/mL、500IU/mL、600IU/mL、700IU/mL、800IU/mL、900IU/mL或1000IU/mL。
在具体的实施方式中，收获的所述输出组合物经洗涤获得转导有重组核酸的细胞。
在具体的实施方式中，将转导有重组核酸的细胞加入缓冲液中进行保存；优选的，所述缓冲液中含有细胞冻存剂。
在具体的实施方式中，所述转导有重组核酸的细胞在收获之后，给予有需要的受试者之前，不需要体外扩增。
在第三方面，本发明提供通过第一或第二方面所述的方法产生的转导有重组核酸的细胞的组合物。
在具体的实施方式中，所述细胞是免疫效应细胞。
在具体的实施方式中，所述细胞是T细胞。
在具体的实施方式中，所述转导有重组核酸的细胞中TSCM的比例高于待转导细胞中的TSCM的比例；
优选的，所述转导有重组核酸的细胞中TSCM的比例是待转导细胞中的TSCM的比例的约2倍或以上；
更优选的，所述转导有重组核酸的细胞中TSCM的比例是待转导细胞中的TSCM的比例的约3倍或以上。
在具体的实施方式中，所述转导有重组核酸的细胞中TSCM的比例在10％以上，优选的，在13％以上，更优选的，在15％以上。
在具体的实施方式中，所述转导有重组核酸的细胞在给予受试者之前不需要进行体外扩增。
在第四方面，本发明提供一种组合物，包含第三方面所述的转导有重组核酸的细胞和药学上可接受的载体。
在第五方面，本发明提供一种过继细胞治疗的方法，包括给与有此需要的受试者第四方面所述的组合物。
本发明的技术效果：
本发明不仅缩短在暴露于逆转录病毒载体颗粒之前的激活和/或活化步骤，还进一步缩短转导后的孵育时间，将体外活化和转导培养时间甚至缩短至1～2天，并且在活化和转导完成之后，在将做制备的细胞用于患者治疗之前，不需要进行的扩增。同样都进行活化的条件下，加入慢病毒载体的时间越晚，转导效率有明显增高趋势，尽管实际转导持续时间是递减的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1.新工艺制备CAR T细胞与常规对照转导后不同时间转导效率变化。
图2.显示了各种活化转导条件下的，新工艺制备CAR T细胞与常规对照转导后不同时间转导效率变化。
图3.显示了各种活化转录条件下的，新工艺制备CAR T细胞与常规对照转导后不同时间转导效率变化。
图4.显示了CAR-T细胞体内抗肿瘤实验。
图5.显示了人T细胞在小鼠外周血的存活情况。
图6.显示了不同浓度T细胞激活剂对慢病毒转导效率的影响。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现T细胞制备中活化转导时间缩短不但不影响重组核酸的表达，反而提高T细胞在体内的增殖能力及存活时间。在此基础上完成了本发明。
术语
术语“细胞”及其语法上的其他形式可以指人或非人动物来源的细胞。
术语“免疫效应细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。 在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中央记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。或者，可以从健康供体，来自诊断患有癌症的患者衍生细胞。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域，在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链；在一个方面，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。
术语“T细胞受体(T cell receptor，TCR)”介导T细胞对特异性主要组织相容性复合物(MHC)-限制性肽抗原进行识别，包括经典的TCR受体和优化的TCR受体。经典的TCR受体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等，其抗原特异性存在于V区，V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，在一个方面，表达经典的TCR的T细胞可以通过对T细胞采用如抗原刺激等方式，诱导T细胞的TCR对靶抗原的特异性。
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞 外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该嵌合T细胞受体整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“转导”是指将外源核酸引入真核细胞。
术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
术语“T细胞活化”或“T细胞激活”指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。
术语“载体”是指能传播其所连接的另一核酸分子的核酸分子。所述术语包括作为自我复制核酸结构的载体以及掺入已引入其的宿主细胞基因组中的载体。某些载体能够指导它们可操作地连接的核酸的表达。此类载体在本文中称为“表达载体”。载体包括病毒载体，例如逆转录病毒载体，例如慢病毒或γ逆转录病毒载体，其具有携带另一种核酸并且能够插入宿主基因组中以供其繁殖的基因组。
术语“治疗”是指疾病，或与其相关的症状、不良反应或结果、或表型的完全或部分减轻或减少。在某些实施方案中，效果是治疗性的，使得其部分或完全治愈疾病或归因于其的不良症状。
“治疗有效量”是指在必要的剂量下和必要的时间段内有效实现所需治疗结果(如针对治疗疾病、病症)和/或治疗的药代动力学或药效动力学作用的量。所述治疗有效量可以根据诸如疾病状态、受试者的年龄、性别和体重以及给予的细胞群等因素而变化。
术语“感染复数(MOI)”指在一个系统中感染病毒的细胞数和总细胞数之比。
术语“主要组织相容性复合物”(MHC)是指含有多态性肽结合位点或结合沟的蛋白质，通常是糖蛋白，在一些情况下，所述蛋白质可以与多肽的肽抗原(包括由细胞机构处理的肽抗原)复合。在一些情况下，MHC分子可以在细胞表面上展示或表达，包括作为与肽的复合物，即MHC-肽复合物，用于呈现具有T细胞上的抗原受体(例如TCR或TCR样抗体)可识别的构象的抗原。通常，MHC I类分子是异二聚体，其具有跨越α链的膜，在一些情况下具有三个α结构域和非共价缔合的β2微球蛋白。通常，MHC II类分子由两种跨膜糖蛋白α和β组成，两者通常都跨越膜。MHC分子可以包括MHC的有效部分，其含有抗原结合位点或 用于结合肽的位点以及由适当抗原受体识别所需的序列。在一些实施方案中，MHC I类分子将源自胞质溶胶的肽递送至细胞表面，其中MHC-肽复合物由T细胞(例如通常CD8+T细胞，但在一些情况下是CD4+T细胞)识别。在一些实施方案中，MHC II类分子将源自囊泡系统的肽递送至细胞表面，其中所述肽通常由CD4+T细胞识别。通常，MHC分子由一组连锁基因座编码，其在小鼠中统称为H-2并且在人中统称为人白细胞抗原(HLA)。因此，通常人MHC也以可称为人白细胞抗原(HLA)。
术语“MHC-肽复合物”或“肽-MHC复合物”或其变体是指肽抗原与MHC分子的复合物或缔合物，例如通常通过所述肽在MHC分子的结合沟或裂缝中的非共价相互作用来形成。在一些实施方案中，MHC-肽复合物存在或展示于细胞表面上。在一些实施方案中，MHC-肽复合物可由抗原受体(例如TCR、TCR样CAR或其抗原结合部分)特异性地识别。
在一些实施方案中，多肽的肽(例如肽抗原或表位)可以与MHC分子缔合，例如用于由抗原受体识别。通常，所述肽源自或基于较长生物分子(例如多肽或蛋白质)的片段。在一些实施方案中，所述肽的长度通常为约8至约24个氨基酸。在一些实施方案中，肽的长度为9至22个氨基酸，用于在MHC II类复合物中识别。在一些实施方案中，肽的长度为8至13个氨基酸，用于在MHC I类复合物中识别。在一些实施方案中，在识别MHC分子(例如MHC-肽复合物)背景中的肽后，抗原受体(例如TCR或TCR样CAR)产生或触发激活信号至T细胞，诱导T细胞应答，如T细胞增殖、细胞因子产生、细胞毒性T细胞应答或其他应答。
本发明提供了病毒载体转导到细胞(例如免疫效应细胞)中的方法，所述方法涉及待转导细胞的活化和转导，细胞的活化和转导可以同时进行，即将包含待转导细胞的输入组合物、待转导细胞刺激剂、和携带重组核酸的病毒载体颗粒共同孵育，也可以先活化再转导，如将包含待转导细胞的输入组合物，和待转导细胞刺激剂共同孵育进行活化，再加入携带重组核酸的病毒载体颗粒孵育，进行重组核酸的转导活化和转导总时间控制72小时之内完成，优选的，在48小时内，或在36小时内、或在24小时内完成。
在一些实施方案中，所提供的方法涉及将逆转录病毒载体颗粒(例如慢病毒载体)与细胞(例如免疫细胞，例如T细胞)群一起孵育和/或接触，在将细胞与病毒颗粒接触或孵育之前和/或同时和/或之后，使用离体细胞激活试剂(例如抗CD3/抗CD28试剂)激活和/或活化T细胞。优选先活化细胞，再进行病毒转导。
在一具体实施方式中，将包含待转导细胞的输入组合物、待转导细胞刺激剂、和携带重组核酸的病毒载体颗粒共同孵育时，孵育时间不超过72小时进行收获，获得输出组合物，所述输出组合物含有转导有重组核酸的细胞；优选的，可以为1小时-72小时；更优选的，所述孵育时间为2小时-48小时；更优选的，所述孵育时间为2小时-36小时；更优选的，所述孵育时间为12小时-36小时；更优选的，所述孵育时间为12小时-24小时；更优选的，所述孵育时间为15小时-24小时。在一具体实施方式中，所述输出组合物经洗涤、离心等纯化处理后，不经进一步体外扩增培养即可制备药用制剂，即采用所述输出组合物制备的药品在用于受试者(或患者)之前不需要体外扩增。
在一具体实施方式中，病毒载体转导细胞的方法包含以下步骤：步骤(1)、将包含待转 导细胞的输入组合物和待转导细胞刺激剂，孵育时间不超过72h，步骤(2)、再加入重组核酸的病毒载体颗粒进行孵育，孵育时间不超过24小时，步骤(3)、进行收获，获得输出组合物，所述输出组合物含有转导有重组核酸的细胞；优选的，所述(1)和(2)的总孵育时间不超过72小时；更优选的，所述(1)和(2)的总孵育时间不超过60h，或不超过48h，或不超过32h，或不超过28h，更优选的，所述(1)和(2)的总孵育时间不超过24h。在一具体实施方案中，步骤(1)的孵育时间为2-72小时；优选的，所述步骤(1)的孵育时间为2-71小时更优选的，所述步骤(1)的孵育时间为2-48小时；更优选的，所述步骤(1)的孵育时间为2-32小时；更优选的，所述步骤(1)的孵育时间为2-28小时；更优选的，所述步骤(1)的孵育时间为3-24小时；更优选的，所述步骤(1)的孵育时间为5-24小时；更优选的，所述步骤(1)的孵育时间为7-24小时；更优选的，所述步骤(1)的孵育时间为7-23小时；更优选的，所述步骤(1)的孵育时间为10-23小时；更优选的，所述步骤(1)的孵育时间为15-23小时；更优选的，所述步骤(1)的孵育时间为15-22小时。在一具体实施方案中，所述步骤(2)的孵育时间为30分钟-24小时，优选的，所述步骤(2)的孵育时间为30分钟-21个小时；优选的，所述步骤(2)的孵育时间为30分钟-17个小时；优选的，所述步骤(2)的孵育时间为30分钟-12个小时；优选的，所述步骤(2)的孵育时间为30分钟-10个小时；优选的，所述步骤(2)的孵育时间为30分钟-8个小时；优选的，所述步骤(2)的孵育时间为1小时-8个小时；优选的，所述步骤(2)的孵育时间为1小时-4个小时；更优选的，所述步骤(2)的孵育时间为1个小时-3个小时。
在一些实施方案中，重组核酸可以是编码识别特异性靶抗原的受体，如T细胞受体(TCR)、嵌合抗原受体(CAR)、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
在一些实施方案中，所述特异性靶抗原是与疾病相关的抗原或通用标签。
在一些实施方案中，所述疾病是癌症、自身免疫性疾病、或感染性疾病。
在一些实施方案中，所述特异性靶抗原是肿瘤相关抗原，如：B细胞成熟抗原(BCMA)、碳酸酐酶9(CAIX)、tEGFR、Her2/neu(受体酪氨酸激酶erbB2)、CD19、CD20、CD22、间皮素、CEA、CD23、CD24、CD30、CD33、CD38、CD44、EGFR、上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、EPHa2、erb-B2、erb-B3、erb-B4、erbB二聚体、EGFR vIII、叶酸结合蛋白(FBP)、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、HMW-MAA、IL-22R-α、IL-13R-α2、激酶插入结构域受体(kdr)、L1细胞粘附分子(L1-CAM)、黑色素瘤相关抗原(MAGE)、TAG72、B7-H6、IL-13受体α2(IL-13Ra2)、CA9、GD3、HMW-MAA、CD171、G250/CAIX、HLA-AI MAGEA1、HLA-A2、PSCA、叶酸受体、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、CD44v6、间皮素、粘蛋白1(MUC1)、MUC16、PSCA、NKG2D、NY-ESO-1、MART-1、gp100、癌胚胎抗原、G蛋白偶联受体5D(GPCR5D)、ROR1、TAG72、VEGF-R2、癌胚抗原(CEA)、前列腺特异性抗原、PSMA、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、Wilms肿瘤1(WT-1)、细胞周期蛋白、CCL-1、CD138、Claudin18.2、GPC3。
所得的转导有重组核酸的细胞可用于过继免疫疗法。在一些此类实施方案中，所提供的方法可用于制备用于过继疗法的免疫细胞，例如T细胞，所述方法活化转导总时间控制在 24小时、或36小时、或48小时、或72小时内。在一些方面，所提供的方法缩短了工程化和/或制备用于过继细胞疗法的细胞时间。
在一些实施方案中，输入组合物包含原代细胞群，所述原代细胞群已从受试者的样品获得和/或针对特定的细胞子集(例如T细胞)富集。在一些实施方案中，细胞群(例如输入组合物)可以是先前已经进行冷冻保存的细胞群。在一些实施方案中，在从受试者获得含有原代细胞的样品(例如单采术样品)后不超过或不超过约1小时、3小时、6小时、12小时、18小时、24小时、48小时或72小时开始孵育和/或接触。在一些实施方案中，所述方法产生输出组合物，其中输出组合物中至少25％、至少30％、至少40％、至少50％或至少75％的总细胞(或特定靶细胞类型，例如T细胞)用所述病毒载体转导和/或表达由其编码的重组基因产物。在一些实施方案中，细胞群(例如输出组合物)中至少5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或95％的细胞(例如T细胞)是根据所提供的方法用逆转录病毒载体颗粒转导。
用于评价T细胞激活标记的表达的方法是本领域已知的。用于检测此类标记的抗体和试剂是本领域熟知的，并且容易获得。用于检测此类标记的测定和方法包括但不限于流式细胞术(包括细胞内流式细胞术)、ELISA、ELISPOT、细胞计数珠阵列或其他多重方法、蛋白质印迹和其他基于免疫亲和力的方法。在一些实施方案中，所述方法能够在某些条件下实现至少特定的转导效率。
在一些实施方案中，所提供的方法还可以包括在用病毒颗粒孵育(例如转导)细胞之前或之后的冷冻保存步骤。在一些实施方案中，这个步骤可以提供细胞产品的保存，如运输中的细胞保存，或制备完成后的细胞保存。
在一些实施方案中，激活或刺激可以离体进行或在体内进行。在一些实施方案中，在用病毒颗粒孵育(例如转导)细胞后，可以将细胞输注到患者体内以进行体内激活和扩增。
在一些实施方案中，使用的待转导细胞激活剂可以是一种，也可以是两种，或者多种的组合。如T细胞激活剂可以采用CD3结合分子(如CD3的抗体)、CD28结合分子(如CD28的抗体)、重组IL-2、重组IL-15、重组IL-7、重组IL-21、或至少两种的混合物，如采用CD3的抗体和CD28的抗体，或采用CD3的抗体、CD28的抗体或IL2。
在一些实施方案中，病毒载体颗粒的感染复数不高于20；优选的，所述感染复数为0.5-20；更优选的，所述感染复数为1.5-20；更优选的，所述感染复数为3-20；更优选的，所述感染复数为3-12。
在一些实施方案中，在孵育期间或之后，所提供的方法可以还包括离体培养输入组合物、输出组合物和/或转导的细胞，例如在活化细胞的条件下培养，以诱导其增殖和/或激活。活化是在一种或多种激活剂的存在下进行。在一些实施方案中，激活剂可以是CD3结合分子、CD28结合分子，或是细胞因子(如重组IL-2、重组IL-15、重组IL-7、或重组IL-21)。在一些实施方案中，结合分子是抗体或抗原结合片段，例如抗CD3抗体和/或抗CD28抗体。在一些实施方案中，进一步培养是在实现细胞扩增的条件下进行，以产生治疗有效剂量的细 胞用于通过过继细胞疗法给予至受试者。
在一些实施方案中，所提供的方法避免在用编码重组受体(例如CAR)的核酸引入、转移和/或转导T细胞的过程中离体地显著改变T细胞的分化状态和/或使T细胞分化状态的变化最小化。在一些实施方案中，根据所提供的方法产生记忆型T细胞，包括干细胞记忆T细胞、中央记忆T细胞、效应记忆T细胞。
在一些实施方案中，本发明的细胞的输出组合物中，含有的转导有重组核酸的细胞(例如CAR T细胞)的比例低于常规工艺的比例，在具体实施方式中，细胞数不超过1*10 10，1*10 9，1*10 8，1*10 7，1*10 6，1*10 5，或1*10 4。
在一些实施方案中，本发明包含转导有重组核酸的细胞的输出组合物中的具有记忆细胞表型(如记忆T细胞)的含量高于常规工艺。在一些实施方案中，所述含量高至少1.5倍、2倍、3倍、4倍或5倍。
在一些实施方案中，记忆T细胞是具有T中枢细胞记忆(TCM)表型的细胞，例如CD45RO+CCR7+CD62L+T细胞和/或CD45RO+CCR7+CD27+CD28+CD62L+T细胞。
在一些实施方案中，本发明用于临床用途(例如过继细胞疗法中)的细胞的制备中的一个、多个或所有步骤是在无菌条件下进行。在一些实施方案中，细胞被富集、活化、转导、或洗涤、的一个或多个过程在封闭系统内进行。
在一些实施方案中，将细胞离体处理较短的时间，进一步缩短时间。
在一些实施方案中，所提供的方法产生的转导有重组核酸的细胞(如CAR T细胞)，在给予受试者时，展现出更久的持久性和/或降低的细胞消耗。
在一些实施方案中，所提供的方法产生的转导有重组核酸的细胞(如CAR T细胞)在给予受试者时，展现出更好的效力。
在一些实施方案中，所提供的方法降低了细胞治疗产品制备过程中细胞的变异性。
在一些实施方案中，消除细胞的离体激活和转导的时间，改善了制备用于过继免疫疗法的转导有重组核酸的细胞的过程。
活化转导方法
本文提供了一种将输入组合物(包括待转导细胞)与逆转录病毒载体颗粒(例如，慢病毒载体颗粒)一起孵育或接触的方法。在一些方面，输入组合物是从受试者获得的原代细胞的组合物，其中，在一些情况下，细胞的亚群或子集已经被选择和/或富集。例如，当待转导细胞是T细胞时，输入组合物可以是T细胞群、也可以是富集后的T细胞群，也可以是PBMC。
在一些实施方案中，细胞包括根据所提供的方法通过基因工程化引入的一种或多种核酸，从而表达此类核酸的重组或基因工程化产物。在一些实施方案中，核酸是异源的，即通常不存在于细胞或从细胞获得的样品中，如从另一种生物或细胞获得的核酸，例如，所述核酸通常不在被工程化的细胞和/或这种细胞所来源的生物中发现。在一些实施方案中，核酸不是天然存在的如自然界中未发现的核酸，包括编码来自多种不同细胞类型的各种结构域的核酸的嵌合组合的核酸。
所述方法的处理步骤可包括多个细胞处理步骤中单独的或组合的任何一个或多个步骤。在特定实施方案中，处理步骤包括用含有逆转录病毒载体的病毒载体颗粒转导细胞，例如编码用于在细胞中表达的重组产物的载体。所述方法可以进一步和/或可替代地包括其他处理步骤，例如细胞的隔离、分离、选择、洗涤、悬浮、稀释、浓缩和/或配制的步骤。在一些情况下，所述方法还可以包括离体培养步骤(例如，活化细胞以例如诱导其增殖和/或激活)。在其他情况下，活化或激活细胞的步骤是在将细胞给予至受试者后在体内进行，通过抗原识别进行和/或在给予一种或多种药剂以加强或扩大细胞在受试者体内的扩增、激活和/或增殖后进行。在一些实施方案中，所述方法包括从受试者分离细胞、制备、加工、培养和/或将它们工程化，并在冷冻保存之前或之后将它们重新引入同一受试者中。
在一些实施方案中，所述方法包括按以下顺序进行的处理步骤，其中：首先从生物样品中隔离(例如选择或分离)原代细胞；将所选择细胞在活化试剂的存在下进行离体激活、扩增或增殖，再加入病毒载体颗粒一起孵育进行转导，活化转导时间总共不超过或不超过约24或36或48小时内，其中转导时间至少或至少约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24小时。
在一些情况下，例如通过在活化试剂的存在下的活化对转导的细胞进行离体激活、扩增或增殖。在一些实施方案中，所述方法可以包括来自洗涤、悬浮、稀释和/或浓缩细胞中的一个或多个处理步骤，其可以在隔离(例如分离或选择)、活化、转导和/或配制步骤之前、期间或同时或之后进行。
在一些实施方案中，一个或多个或所有处理步骤(例如隔离、选择和/或富集、处理、活化、与转导和工程化结合的孵育)和配制步骤是使用集成或自含式系统中的系统、装置或设备进行和/或以自动化或可编程方式进行。
在一些实施方案中，与结合所提供的转导方法的制备、处理和/或孵育细胞结合的一个或多个细胞处理步骤可以在培养袋或培养瓶中进行，这与其他可用的方法相比可以提供某些优点。
在一些实施方案中，所述系统包含一系列容器，例如袋子、管路、旋塞、夹子、连接器和离心室。在一些实施方案中，容器(例如培养袋或培养瓶)包括一个或多个容器(例如培养袋或培养瓶)，其在同一容器或单独容器(例如同一培养袋或培养瓶；或单独培养袋或培养瓶)中含有待转导的细胞和病毒载体颗粒。
在一些实施方案中，所述系统(例如封闭的系统)是无菌的。
在一些实施方案中，所述系统可以是可弃式的，例如一次性培养袋或培养瓶。
A.样品和细胞制备
细胞通常是真核细胞，例如哺乳动物细胞，并且通常是人细胞。在一些实施方案中，所述细胞源自血液、骨髓、淋巴或淋巴器官，是免疫系统的细胞，如先天或适应性免疫的细胞，例如骨髓或淋巴细胞，包括淋巴细胞，通常为T细胞和/或NK细胞。其他示例性细胞包括干细胞，如多潜能干细胞和多能干细胞，包括诱导多能干细胞(iPSC)。
细胞通常是原代细胞如直接从受试者分离和/或从受试者分离并冷冻的那些原代细胞。 在一些实施方案中，细胞包括T细胞或其他细胞类型的一个或多个亚组，如整个T细胞群、CD4+细胞、CD8+细胞及其亚群，如由以下各项所定义的那些亚群：功能、激活状态、成熟度、分化的可能性、扩增、再循环、定位和/或持久能力、抗原特异性、抗原受体类型、在特定器官或区室中的存在、标记或细胞因子分泌特征和/或分化程度。关于待治疗的受试者，细胞可以是同种异体的和/或自体的。所述方法包括现成的方法。在一些方面，如对于现有技术，所述细胞是多能和/或多潜能的，如干细胞，如诱导多能干细胞(iPSC)。在一些实施方案中，所述方法包括从受试者分离细胞、制备、加工、培养和/或将它们工程化，并在冷冻保存之前或之后将它们重新引入同一受试者中。
在T细胞和/或CD4+和/或CD8+T细胞的亚型和亚群中，有幼稚T(TN)细胞(或称为
Figure PCTCN2021073418-appb-000001
T细胞)、效应T细胞(TEFF)、记忆T细胞及其亚型(如干细胞记忆T(TSCM)、中枢记忆T(TCM)，效应记忆T(TEM)或终末分化效应记忆T细胞)、肿瘤浸润淋巴细胞(TIL)、未成熟T细胞、成熟T细胞、辅助T细胞、细胞毒性T细胞、粘膜相关不变T(MAIT)细胞、天然存在和适应性调节T(Treg)细胞、辅助T细胞(如TH1细胞、TH2细胞、TH3细胞、TH17细胞、TH9细胞、TH22细胞、滤泡辅助细胞T细胞)、α/βT细胞和δ/γT细胞。
在一些实施方案中，所述细胞是天然杀伤(NK)细胞。在一些实施方案中，所述细胞是单核细胞或粒细胞，例如骨髓细胞、巨噬细胞、嗜中性粒细胞、树突细胞、肥大细胞、嗜酸性粒细胞和/或嗜碱性粒细胞。
在一些实施方案中，细胞源自于细胞系，例如，T细胞系。在一些实施方案中，细胞获得自异种来源，例如获得自小鼠、大鼠、非人灵长类动物和猪。
在一些实施方案中，细胞可以从样品中分离，例如生物样品，例如从受试者获得或源自受试者的样品。在一些实施方案中，分离出细胞的受试者是患有疾病或需要细胞疗法或将对其给予细胞疗法的受试者。在一些实施方案中，所述受试者是需要特定治疗性干预(如过继细胞疗法，其中细胞被分离、加工和/或工程化)的人。
在一些实施方案中，细胞是原代细胞，例如原代人细胞。样品包括直接取自受试者的组织、流体和其他样品，以及来自一个或多个加工步骤(如分离、离心、基因工程化(例如用病毒载体转导)、洗涤和/或孵育)的样品。所述生物样品可以是直接从生物来源获得的样品或经过加工的样品。生物样品包括但不限于体液(如血液、血浆、血清、脑脊液、滑液、尿液和汗液)、组织和器官样品，包括由其衍生的加工样品。
在一些方面，细胞从其中衍生或分离的样品是血液或血液衍生的样品，或者是或源自单采术或白细胞分离术产物。示例性样品包括全血、外周血单核细胞(PBMC)、白细胞、骨髓、胸腺、组织活检、肿瘤、白血病、淋巴瘤、淋巴结、肠相关淋巴组织、粘膜相关淋巴组织、脾、其他淋巴组织、肝、肺、胃、肠、结肠、肾、胰腺、乳房、骨、前列腺、子宫颈、睾丸、卵巢、扁桃体或其他器官和/或由其衍生的细胞。在细胞疗法(例如过继细胞疗法)的背景下，样品包括来自自体和同种异体来源的样品。
在一些例子中，来自受试者的循环血液的细胞例如通过单采术或白细胞分离术获得。在一些方面，所述样品含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核 白细胞、红细胞和/或血小板，并且在一些方面含有除红细胞和血小板之外的细胞。
在一些实施方案中，洗涤从受试者收集的血液样品，例如以去除血浆级分并将细胞置于适当的缓冲液或介质中以用于随后的加工步骤。在一些实施方案中，用磷酸盐缓冲盐水(PBS)洗涤所述细胞。在一些实施方案中，所述洗涤溶液缺乏钙和/或镁和/或许多或所有二价阳离子。在一些方面，根据制造商的说明书，通过自动或半自动“流通”离心机(例如，Cobe2991细胞加工器、Baxter、MACS PLUS)完成洗涤步骤。在一些方面，根据制造商的说明书，通过切向流过滤(TFF)完成洗涤步骤。在一些实施方案中，洗涤后将所述细胞重悬于多种生物相容性缓冲液(例如像不含Ca++/Mg++的PBS)中。在某些实施方案中，去除血细胞样品的组分并将所述细胞直接重悬于培养基中。
在一些实施方案中，在富集和/或选择细胞之前，使样品与血清或血浆(例如人血清或血浆)接触和/或含有所述血清或血浆。在一些实施方案中，血清或血浆对于从其获得细胞的受试者是自体的。在一些实施方案中，血清或血浆在样品中按以下浓度存在：至少或至少约10％(v/v)、至少或至少约15％(v/v)、至少或至少约20％(v/v)、至少或至少约25％(v/v)、至少或至少约30％(v/v)、至少或至少约35％(v/v)或至少或至少约40％(v/v)。在一些实施方案中，在选择和/或转导细胞之前，使含有原代细胞的样品与抗凝血剂接触或含有抗凝血剂。在一些实施方案中，抗凝血剂是或含有游离柠檬酸根离子，例如，抗凝血剂柠檬酸盐右旋糖溶液，溶液A(ACD-A)。
在一些实施方案中，在富集和/或选择细胞之前，将来自样品的细胞转移或悬浮于无血清培养基中。在一些实施方案中，无血清培养基是定义的和/或明确定义的细胞培养基。在一些实施方案中，配制无血清培养基以支持某种细胞类型(例如免疫细胞、T细胞和/或CD4+和CD8+T细胞)的细胞的生长、增殖、健康、稳态。
在一些实施方案中，将样品维持在或保持在为2℃至8℃的温度下长达48小时，例如长达12小时、24小时或36小时。
在一些实施方案中，制备方法在分离、选择和/或富集和/或用于转导和工程化的孵育之前或之后包括冷冻(例如冷冻保存)细胞的步骤。在一些实施方案中，所述冷冻和后续解冻步骤去除所述细胞群中的粒细胞，并且在一定程度上去除单核细胞。在一些实施方案中，例如在洗涤步骤之后将所述细胞悬浮在冷冻溶液中以去除血浆和血小板。在一些方面，可以使用多种已知的冷冻溶液。在一些实施例中，在有冷冻保护剂的存在下冷冻保存所述T细胞。一个例子涉及使用含有20％DMSO和8％人血清白蛋白(HSA)的PBS，或其他合适的细胞冷冻培养基。然后通常将细胞冷冻至-80℃并储存在液氮储罐的气相中。
在一些实施方案中，细胞的分离包括一个或多个制备和/或基于非亲和力的细胞分离步骤。在一些例子中，将细胞在一种或多种试剂的存在下洗涤、离心和/或孵育，例如以去除不需要的组分、针对所需组分进行富集、裂解或去除对特定试剂敏感的细胞。在一些例子中，基于一种或多种特性(如密度、粘附特性、尺寸、对特定组分的敏感性和/或抗性)分离细胞。
在一些实施方案中，分离方法包括基于细胞中一种或多种特定分子(如表面标记，例如表面蛋白、细胞内标记或核酸)的表达或存在来分离不同细胞类型。在一些实施方案中，可 以使用任何已知的基于此类标记的用于分离的方法。在一些实施方案中，所述分离是基于亲和力或免疫亲和力的分离。例如，在一些方面，所述分离包括基于所述细胞的一种或多种标记(通常为细胞表面标记)的表达或表达水平来分离细胞和细胞群，例如通过和与此类标记特异性结合的抗体或结合配偶体一起孵育，然后通常是洗涤步骤和从那些未与所述抗体或结合配偶体结合的细胞中分离已结合所述抗体或结合配偶体的细胞。
此类分离步骤可以基于阳性选择(其中保留已经结合所述试剂的细胞以供进一步使用)和/或阴性选择(其中保留未与所述抗体或结合配偶体结合的细胞)。所述分离不需要导致100％富集或去除特定细胞群或表达特定标记的细胞。例如，针对特定类型的细胞(如表达标记的那些)的阳性选择或富集是指增加此类细胞的数量或百分比，但不需要导致不表达所述标记的细胞的完全不存在。同样地，特定类型的细胞(如表达标记的那些)的阴性选择、去除或耗尽是指减少此类细胞的数量或百分比，但不需要导致所有此类细胞的完全去除。
例如，在一些方面，T细胞的特定亚群，如对一种或多种表面标记呈阳性或高水平表达的细胞(例如CD28+、CD62L+、CCR7+、CD27+、CD127+、CD4+、CD8+、CD45RA+和/或CD45RO+T细胞)通过阳性或阴性选择技术来分离。可以使用抗CD3/抗CD28缀合的磁珠或微珠(例如M-450CD3/CD28T Cell Expander)阳性选择CD3+,CD28+T细胞。
在一些实施方案中，通过阳性选择针对特定细胞群富集或者通过阴性选择针对特定细胞群耗尽来进行分离。在一些实施方案中，通过将细胞与一种或多种抗体或其他结合剂一起孵育来完成阳性或阴性选择，所述一种或多种抗体或其他结合剂与分别在阳性或阴性选择的细胞上表达或以相对较高水平(标记高)(标记+)的一种或多种表面标记特异性结合。
在一些实施方案中，通过阴性选择在非T细胞(如B细胞、单核细胞或其他白细胞，如CD14)上表达的标记，将T细胞与PBMC样品分离。在一些方面，CD4+或CD8+选择步骤用于分离CD4+辅助T细胞和CD8+细胞毒性T细胞。通过对在一种或多种幼稚、记忆和/或效应T细胞亚群上表达或以相对较高程度表达的标记的阳性或阴性选择，可以将此类CD4+和CD8+群体进一步分类成亚群。
在一些实施方案中，如通过基于与相应子群体相关的表面抗原进行阳性或阴性选择，将CD8+细胞针对幼稚、中枢记忆、效应子记忆和/或中枢记忆干细胞进一步富集或耗尽。在一些实施方案中，针对中枢记忆T(TCM)细胞进行富集以增加功效，如以改善给予后的长期存活、扩增和/或移植，在一些实施方案中，组合富含TCM的CD8+T细胞与CD4+T细胞进一步增强功效。
在实施方案中，记忆T细胞存在于CD8+外周血淋巴细胞的CD62L+和CD62L-两个子集中。可以例如使用抗CD8和抗CD62L抗体将PBMC针对CD62L-CD8+和/或CD62L+CD8+级分进行富集或耗尽。
在一些实施方案中，中枢记忆T(TCM)细胞的富集是基于CD45RO、CD62L、CCR7、CD28、CD3和/或CD 127的阳性或高表面表达；在一些方面，它是基于对表达或高度表达CD45RA和/或颗粒酶B的细胞的阴性选择。在一些方面，通过表达CD4、CD14、CD45RA的细胞的耗尽和表达CD62L的细胞的阳性选择或富集来进行富含TCM细胞的CD8+群体的 分离。在一个方面，中枢记忆T(TCM)细胞的富集从基于CD4表达所选择的阴性细胞级分开始进行，所述阴性细胞级分基于CD14和CD45RA的表达进行阴性选择且基于CD62L进行阳性选择。
在特定例子中，PBMC样品或其他白细胞样品进行CD4+细胞的选择，其中保留了阴性和阳性级分。然后所述阴性级分基于CD14和CD45RA或CD19的表达进行阴性选择，并基于中枢记忆T细胞(如CD62L或CCR7)的标记特征进行阳性选择，其中以任何顺序进行所述阳性和阴性选择。
通过鉴定具有细胞表面抗原的细胞群，将CD4+T辅助细胞分类为幼稚、中枢记忆和效应细胞。CD4+淋巴细胞可通过标准方法获得。在一些实施方案中，幼稚CD4+T淋巴细胞是CD45RO-、CD45RA+、CD62L+、CD4+T细胞。在一些实施方案中，中枢记忆CD4+细胞是CD62L+和CD45RO+。在一些实施方案中，效应CD4+细胞是CD62L-和CD45RO-。
在一个例子中，为了通过阴性选择富集CD4+细胞，单克隆抗体混合剂通常包括针对CD14、CD20、CD11b、CD16、HLA-DR和CD8的抗体。在一些实施方案中，所述抗体或结合配偶体与固体支持物(例如珠)或基质(如磁珠或顺磁珠或微珠)结合，以允许细胞分离以用于阳性和/或阴性选择。例如，在一些实施方案中，使用免疫磁性(或亲和磁性)分开技术来分开或分离细胞和细胞群。
在一些实施例中，T细胞激活剂是偶联有抗CD3和/或抗CD28和/或抗41-BB单克隆抗体的固体支持物(例如，珠，包括磁珠和/或微珠；聚合物基质，包括聚合物纳米基质)。
在一些方面，将待分离的细胞的样品或组合物与小的可磁化或磁响应材料(如磁响应颗粒或微粒，如顺磁珠(例如像Dynabeads或MACS珠))一起孵育。在一些实施方案中，所述磁性颗粒或珠包含与特异性结合成员(如抗体或其他结合配偶体)结合的磁响应材料。有许多在磁分离方法中使用的熟知的磁响应材料。
所述孵育通常在这样的条件下进行，由此抗体或结合配偶体或者与附着于磁性颗粒或微珠的此类抗体或结合配偶体特异性结合的分子(如二抗或其他试剂)与细胞表面分子(如果存在于所述样品内的细胞上的话)特异性结合。
在一些方面，将所述样品置于磁场中，并且具有附着于其上的磁响应或可磁化颗粒的那些细胞将被吸引到磁体并与未标记的细胞分离。对于阳性选择，保留被磁铁吸引的细胞；对于阴性选择，保留未被吸引的细胞(未标记的细胞)。
在一些实施方案中，所述磁响应颗粒或微珠保持附着于所述细胞，所述细胞随后被孵育，培养和/或工程化；在一些方面，所述颗粒或微珠保持附着于所述细胞以用于给予患者。在一些实施方案中，从所述细胞中去除可磁化或磁响应颗粒。从细胞中去除可磁化颗粒或微珠的方法是已知的，并且包括例如使用竞争的非标记抗体和与可切割接头缀合的可磁化颗粒或抗体或微珠。在一些实施方案中，可磁化颗粒是可生物降解的。
在一些实施方案中，基于亲和力的选择是经由磁激活的细胞分选(M A C S)(Miltenyi Biotech，Auburn，CA)进行的。磁激活细胞分选(MACS)系统能够高纯度选择附着有磁化颗粒的细胞。在某些实施方案中，MACS以这样的模式操作，其中在施加外部磁场之后依次洗 脱非靶标和靶标种类。也就是说，附着于磁化颗粒的细胞保持在适当的位置，而未附着的种类被洗脱。然后，在完成第一次洗脱步骤之后，以某种方式释放被捕获在磁场中并被阻止洗脱的种类，使得它们可以被洗脱和回收。在某些实施方案中，所述非靶细胞被标记并从异质细胞群中耗尽。
在一些实施方案中，所述方法包括选择细胞，其中全部或部分选择是在离心室的内腔中进行，例如在离心旋转下进行。在一些实施方案中，将细胞与选择试剂(例如基于免疫亲和力的选择试剂)一起孵育是在离心室中进行。例如，基于免疫亲和力的选择可取决于所分离细胞与特异性地结合至细胞上的标记的分子(例如固体(例如颗粒)上的抗体或其他结合配偶体)之间的有利的能量相互作用。在某些用于使用颗粒(例如珠)的基于亲和力的分离的可用方法中，将颗粒和细胞在容器(例如管或袋)中孵育，同时振荡或混合，且细胞密度与颗粒(例如，珠)的比率是恒定的，以帮助促进能量上有利的相互作用。此类途径对于用于大规模生产可能不是理想的，例如，因为其可能需要使用大体积以维持最佳或所需的细胞与颗粒的比率，同时维持所需的细胞数量。因此，此类途径可能需要以分批模式或形式进行处理，这可能需要增加时间、步骤数和操作，从而增加成本和用户错误的风险。
在一些实施方案中，至少一部分选择步骤是在离心室中进行，其包括将细胞与选择试剂一起孵育。在此类工艺的一些方面，将一定体积的细胞与一定量所需的基于亲和力的选择试剂混合，所述体积和所述量显著少于在管或容器中根据制造商的说明书进行类似选择以选择相同数量的细胞和/或相同体积的细胞时通常所用的体积和量。在一些实施方案中，所采用的一种或多种选择试剂的量为根据制造商的说明针对相同数量的细胞和/或相同体积的细胞在基于管或容器的孵育中用于选择细胞的相同的一种或多种选择试剂的量的不超过5％、不超过10％、不超过15％、不超过20％、不超过25％、不超过50％、不超过60％、不超过70％或不超过80％。
例如作为可以在室空腔中进行的选择方法的部分的与一种或多种选择试剂一起孵育包括使用一种或多种选择试剂，基于一种或多种特定分子(例如表面标记，例如表面蛋白、细胞内标记或核酸)在细胞中或细胞上的表达或存在，选择一种或多种不同的细胞类型。在一些实施方案中，可以使用任何已知的方法，使用一种或多种选择试剂，基于此类标记进行分离。在一些实施方案中，一种或多种选择试剂导致分离，所述分离是基于亲和力或免疫亲和力的分离。例如，在一些方面，选择包括与一种或多种试剂一起孵育，用于基于一种或多种标记(通常是细胞表面标记)的细胞表达或表达水平来分离细胞和细胞群，例如通过与特异性地结合至此类标记的抗体或结合配偶体一起孵育，之后通常进行洗涤步骤并分离已结合抗体或结合配偶体的细胞与未结合至抗体或结合配偶体的那些细胞。
在一些实施方案中，对于细胞的选择，例如基于免疫亲和力的选择，将细胞在室空腔中在组合物中孵育，所述组合物还含有具有选择试剂的选择缓冲液，所述选择试剂例如特异性地结合至希望富集和/或耗尽的细胞上(而不是组合物中其他细胞上)的表面标记的分子，例如抗体，其任选地偶联到支架(例如聚合物或表面，例如珠，例如磁珠或微珠，例如与对CD4和CD8具有特异性的单克隆抗体偶联的磁珠或微珠)。在一些实施方案中，与选择试剂一起 孵育的总持续时间为5分钟至6小时，例如30分钟至3小时，例如至少30分钟、60分钟、120分钟或180分钟。在一些实施方案中，孵育通常在混合条件下进行，例如在旋转的存在下，通常以相对低的力或速度旋转，例如速度低于用于使细胞沉淀的速度，例如为600rpm至1700rpm(例如，为至少600rpm、1000rpm或1500rpm或1700rpm)，例如在样品或室壁或其他容器壁处的一定RCF下，所述RCF为80g至100g(例如，至少80g、85g、90g、95g或100g)。
在一些实施方案中，在与选择试剂一起孵育后，将所孵育的细胞(包括其中已经结合选择试剂的细胞)从离心室压出，例如从离心室转移到系统中，用于基于免疫亲和力来分离细胞。在一些实施方案中，用于基于免疫亲和力的分离的系统是或含有磁分离柱。在一些实施方案中，在分离之前，可以在室中进行一个或多个其他处理步骤，例如洗涤。
在一些方面，使用CliniMACS系统(Miltenyi Biotic)进行分离和/或其他步骤，例如用于在封闭和无菌系统中在临床规模水平上的细胞的自动化分离。
在某些实施方案中，使用CliniMACS Prodigy系统(Miltenyi Biotec)进行分离和/或其他步骤。在一些方面，CliniMACS Prodigy系统配备有细胞加工联合体，其允许通过离心自动化洗涤和分级分离细胞。在一些实施方案中，通过流式细胞术收集和富集(或耗尽)本文描述的细胞群，其中针对多种细胞表面标记染色的细胞在流体流中载携。在一些实施方案中，通过制备规模(FACS)分类收集和富集(或耗尽)本文描述的细胞群。
在一些实施方案中，用一种或多种可检测标记来标记抗体或结合配偶体，以促进分离供阳性和/或阴性选择。例如，分离可以基于与荧光标记的抗体的结合。在一些例子中，基于对一种或多种细胞表面标记具特异性的抗体或其他结合配偶体的结合来分离细胞在流体流中载携，如通过荧光激活细胞分选(FACS)，包括制备规模(FACS)和/或微机电系统(MEMS)芯片，例如与流式细胞检测系统组合。此类方法允许基于多种标记同时进行阳性和阴性选择。
细胞的转导前或转导同时的激活和/或扩增
在一些实施方案中，结合基因工程化，将经筛选后的细胞(例如输入组合物)进行孵育和/或培养。所述孵育步骤可以包括活化转导，以使病毒载体整合到一个或多个细胞的宿主基因组中。孵育和/或工程化可以在培养容器中进行，所述培养容器是如单元、室、孔、柱、管、管组、阀、小瓶、培养皿、袋或其他用于培养或培育细胞的容器。在一些实施方案中，在刺激条件或活化剂的存在下孵育所述组合物或细胞。这些条件包括针对以下而设计的那些条件：用于诱导群体中细胞的增殖、扩增、激活和/或存活的条件，用于模拟抗原暴露和/或用于引发细胞进行基因工程化，如以引入重组抗原受体。
在一些实施方案中，进一步孵育是在用于细胞的刺激和/或活化的条件下进行，所述条件可以包括以下各项中的一种或多种：特定培养基、温度、氧含量、二氧化碳含量、时间、药剂(例如营养素)、氨基酸、抗生素、离子和/或刺激因子(例如细胞因子、趋化因子)、抗原、结合配偶体、融合蛋白、重组可溶性受体和任何其他设计为激活细胞的药剂。
在一些实施方案中，活化条件或药剂包括一种或多种能够激活TCR复合物的细胞内信号传导结构域的药剂(例如刺激性和/或辅助性药剂)，例如配体。在一些方面，所述药剂开启 或启动T细胞中的TCR/CD3细胞内信号传导级联，例如是适于递送初级信号以例如启动ITAM诱导的信号(例如对TCR组分具有特异性的那些)的激活的药剂，和/或促进共刺激信号(例如对T细胞共刺激受体具有特异性的共刺激信号)的药剂，例如抗CD3、抗CD28或抗41-BB(例如，其任选地结合至固体支持物(例如珠))和/或一种或多种细胞因子。所述刺激剂包括抗C D 3/抗C D 2 8珠(例如，DYNABEADS
Figure PCTCN2021073418-appb-000002
M-4 5 0C D 3/C D 2 8T细胞扩增剂和/或ExpACT
Figure PCTCN2021073418-appb-000003
珠)。任选地，活化方法可以还包括向培养基中添加抗CD3和/或抗CD28抗体的步骤，例如OKT-3。在一些实施方案中，刺激剂包括IL-2和/或IL-15和/或IL-7，例如，IL-2浓度为至少约10单位/mL。
在一些实施方案中，活化条件或药剂包括一种或多种药剂(例如配体)，其能够激活TCR复合物的细胞内信号传导结构域。在一些方面，所述药剂在T细胞中开启或启动TCR/CD3细胞内信号传导级联。此类药剂可以包括例如结合至固体支持物(如珠，包括磁珠或微珠)的抗体，如对TCR组分和/或共刺激受体具有特异性的抗体(例如抗CD3、抗CD28)；和/或一种或多种细胞因子。任选地，扩增方法还可以包括向培养基中添加抗CD3和/或抗CD28抗体(例如，以至少约0.5ng/ml的浓度)的步骤。在一些实施方案中，刺激剂包括IL-2和/或IL-15和/或IL-7，例如，IL-2浓度为至少约10单位/mL、至少约50单位/mL、至少约100单位/mL或至少约200单位/mL。
在一些实施方案中，例如与活化剂一起孵育的总持续时间在或在约1小时与96小时之间、1小时与72小时之间、1小时与48小时之间、4小时与36小时之间、8小时与30小时之间或12小时与24小时之间，例如至少6小时、12小时、18小时、24小时、36小时或72小时。
在一些实施方案中，本文所提供的方法不包括进一步培养或孵育，例如，不包括离体扩增步骤，或者包括显著更短的离体扩增步骤。
在一些实施方案中，使细胞工程化的整个过程(例如选择和/或富集、孵育与活化转导相结合和/或进一步培养或培育)是在从受试者获得样品后的以下时间段内进行：超过9天、不超过8天、不超过7天、不超过6天、不超过5天、不超过4天、不超过3天、不超过2天或不超过1天。应理解，这种定时不包括使细胞经历低温保存的任何时间段。
在本文所提供的方法的一些实施方案中，将工程化细胞(例如输出组合物或配制组合物)在转导后在不进行显著的离体扩增的情况下立即或不久给予至受试者。在一些实施方案中，工程化细胞可以在转导步骤后立即给予。在一些实施方案中，工程化细胞可以在活化转导步骤后不久，在例如与常规方法(这可能需要显著的体外激活、扩增和/或富集)相比不进行显著的离体扩增或进行显著更短的离体扩增的情况下给予。例如，在本文所提供的方法的一些实施方案中，工程化细胞可以在转导的三天、两天或一天内给予。在一些实施方案中，工程化细胞可以在活化转导步骤的48小时、36小时、24小时、20小时、16小时、12小时、8小时、4小时、2小时、1小时或更短时间内给予。在一些实施方案中，与常规方法相比，使工程化细胞经历显著更短的体外扩增，例如48小时、36小时、24小时、20小时、16小时、12小时、8小时、4小时、2小时、1小时或更短时间。
在任何此类实施方案中，细胞的扩增和/或激活可以在暴露于抗原后在体内进行，例如， 在给予细胞后在受试者体内进行工程化细胞的扩增。在一些实施方案中，体内扩增的范围、程度或量值可以通过多种方法来扩大、加强或增强，所述方法能够调节(例如增加)所给予细胞(例如表达重组受体的细胞)的扩增、增殖、存活和/或功效。
在一些实施方案中，此类方法包括涉及给予工程化细胞的方法，将所述细胞用药剂(例如核酸)进一步修饰，以改变(例如增加或减少)分子的表达或活性，其中这种改变的表达或活性扩大、加强或增强所给予细胞的扩增、增殖、存活和/或功效。在一些实施方案中，药剂(例如核酸)的表达例如通过给予诱导物或其他调节分子是可诱导的、可抑制的、可调节的和/或用户控制的。
在一些实施方案中，此类方法包括涉及与药物或药剂的组合给予(例如同时或依次给予)的方法，所述药物或药剂能够扩大、加强或增强所给予细胞(例如表达重组受体的细胞)的扩增、增殖、存活和/或功效。
在一些实施方案中，病毒载体颗粒是逆转录病毒载体颗粒，例如慢病毒颗粒，其在病毒载体的基因组中含有编码重组和/或异源分子(例如重组或异源蛋白，例如重组和/或异源受体，例如嵌合抗原受体(CAR)或其他抗原受体)的核酸。病毒载体颗粒的基因组通常包括除了编码重组分子的核酸以外的序列。此类序列可包括允许将基因组包装到病毒颗粒中的序列和/或促进编码重组受体(例如CAR)的核酸表达的序列。
在一些实施方案中，病毒载体颗粒含有源自基于逆转录病毒基因组的载体(例如源自基于慢病毒基因组的载体)的基因组。在所提供的病毒载体的一些方面，编码重组受体(例如抗原受体，例如CAR)的异源核酸含于和/或位于载体基因组的5'LTR与3'LTR序列之间。
在一些实施方案中，病毒载体基因组是慢病毒基因组，例如HIV-1基因组或SIV基因组。在一些实施方案中，这些病毒载体是基于质粒的或基于病毒的，并且被配置为携带用于掺入外来核酸的基本序列，用于选择和用于将所述核酸转移到宿主细胞中。
慢病毒载体的非限制性例子包括源自慢病毒，例如人免疫缺陷病毒1(HIV-1)、HIV-2、猿猴免疫缺陷病毒(SIV)、人嗜T淋巴细胞病毒1(HTLV-1)、HTLV-2或马感染贫血病毒(E1AV)。在一些实施方案中，病毒基因组载体可以含有逆转录病毒(例如慢病毒)的5'和3'LTR的序列。在一些方面，病毒基因组构建体可含有来自慢病毒的5'和3'LTR的序列，并且具体而言可以含有来自慢病毒的5'LTR的R和U5序列以及来自慢病毒的失活或自失活3'LTR。LTR序列可以是来自任何物种的任何慢病毒的LTR序列。例如，它们可以是来自HIV、SIV、FIV或BIV的LTR序列。通常，LTR序列是HIV LTR序列。
在一些实施方案中，病毒载体含有编码异源重组蛋白的核酸。在一些实施方案中，异源重组分子是或包括重组受体(例如，嵌合抗原受体)、SB转座子(例如用于基因沉默)、衣壳包封的转座子、同源双链核酸(例如，用于基因组重组)或报告基因(例如荧光蛋白，例如GFP)或萤光素酶)。
在一些实施方案中，病毒载体含有编码重组受体和/或嵌合受体(例如异源受体蛋白)的核酸。重组受体(例如异源受体)可以包括抗原受体，例如功能性非TCR抗原受体，包括嵌合抗原受体(CAR)和其他抗原结合受体，例如转基因T细胞受体(TCR)。受体还可以包括其他 受体，例如其他嵌合受体，例如与特定配体结合并具有与CAR中存在的那些类似的跨膜和/或细胞内信号传导结构域的受体。
在一些实施方案中，所编码的重组抗原受体(例如CAR)是能够特异性地结合至要靶向的细胞或疾病上的一种或多种配体的受体，所述疾病例如为癌症、感染性疾病、炎症或自身免疫性疾病或其他疾病。
在一些实施方案中，示例性抗原是或包括αvβ6整合素(avb6整合素)、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、碳酸酐酶9(CA9，也称为CAIX或G250)、癌症-睾丸抗原、癌症/睾丸抗原1B(CTAG，也称为NY-ESO-1和LAGE-2)、癌胚抗原(CEA)、细胞周期蛋白、C-C基序趋化因子配体1(CCL-1)、CD19、CD20、CD22、CD23、CD24、CD30、CD33、CD38、CD44、CD44v6、CD44v7/8、CD123、CD138、CD171、表皮生长因子蛋白(EGFR)、截短的表皮生长因子蛋白(tEGFR)、III型表皮生长因子受体突变(EGFR vIII)、上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、肝配蛋白B2、肝配蛋白受体A2(EPHa2)、雌激素受体、Fc受体样5(FCRL5；也称为Fc受体同源物5或FCRH5)、胎儿乙酰胆碱受体(胎儿AchR)、叶酸结合蛋白(FBP)、叶酸受体α、神经节苷脂GD2、O-乙酰化GD2(OGD2)、神经节苷脂GD3、糖蛋白100(gp100)、G蛋白偶联受体5D(GPCR5D)、Her2/neu(受体酪氨酸激酶erb-B2)、Her3(erb-B3)、Her4(erb-B4)、erbB二聚体、人高分子量黑色素瘤相关抗原(HMW-MAA)、乙型肝炎表面抗原、人白细胞抗原A1(HLAA1)、人白细胞抗原A2(HLA-A2)、IL-22受体α(IL-22Ra)、IL-13受体α2(IL-13Ra2)、激酶插入结构域受体(kdr)、κ轻链、L1细胞粘附分子(L1-CAM)、L1-CAM的CE7表位、含有富含亮氨酸的重复序列的8家族成员A(LRRC8A)、路易斯Y、黑色素瘤相关抗原(MAGE)-A1、MAGE-A3、MAGEA6、间皮素、c-Met、鼠类巨细胞病毒(CMV)、粘蛋白1(MUC1)、MUC16、自然杀伤2族成员D(NKG2D)配体、黑色素A(MART-1)、神经细胞粘附分子(NCAM)、癌胚胎抗原、优先表达的黑色素瘤抗原(PRAME)、孕酮受体、前列腺特异性靶抗原、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、存活蛋白、滋养层糖蛋白(TPBG，也称作5T4)、肿瘤相关糖蛋白72(TAG72)、血管内皮生长因子受体(VEGFR)、血管内皮生长因子受体2(VEGFR2)、Wilms肿瘤1(WT-1)、病原体特异性靶抗原或与通用标签相关的抗原、和/或生物素化的分子、和/或由HIV、HCV、HBV或其他病原体表达的分子。在一些实施方案中，受体靶向的抗原包括与B细胞恶性肿瘤相关的抗原，如许多已知B细胞标记中的任何一种。在一些实施方案中，抗原是或包括CD20、CD19、CD22、ROR1、CD45、CD21、CD5、CD33、Igκ、Igλ、CD79a、CD79b或CD30。
在一些实施方案中，示例性抗原是孤儿酪氨酸激酶受体ROR1、tEGFR、Her2、L1-CAM、CD19、CD20、CD22、间皮素、CEA和乙型肝炎表面抗原、抗叶酸受体、CD23、CD24、CD30、CD33、CD38、CD44、EGFR、EGP-2、EGP-4、0EPHa2、ErbB2、3或4、FBP、胎儿乙酰胆碱受体、GD2、GD3、HMW-MAA、IL-22R-α、IL-13R-α2、kdr、κ轻链、路易斯Y、L1细胞粘附分子、MAGE-A1、间皮素、MUC1、MUC16、PSCA、NKG2D配体、NY-ESO-1、MART-1、gp100、癌胚胎抗原、ROR1、TAG72、VEGF-R2、癌胚抗原(CEA)、前列腺特异 性靶抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、CS-1、c-Met、GD-2和MAGE A3、CE7、Wilms肿瘤1(WT-1)、细胞周期蛋白(例如细胞周期蛋白A1(CCNA1))，和/或生物素化的分子，和/或由HIV、HCV、HBV、HPV和/或其他病原体表达的分子和/或具有HIV、HCV、HBV、HPV和/或其他病原体的特征的分子或对HIV、HCV、HBV、HPV和/或其他病原体具有特异性的分子，和/或其致癌形式。
在一些实施方案中，抗原是或包括病原体特异性或病原体表达的抗原。在一些实施方案中，抗原是病毒抗原(例如来自HIV、HCV、HBV等的病毒抗原)、细菌抗原和/或寄生虫抗原。
在一些实施方案中，抗原受体(包括CAR和重组TCR)及其产生和引入包括例如以下文献中所述的那些：国际专利申请公开号WO2015172339A1、WO2016008405A1、WO 2016086813A1、WO2016150400、WO2017032293A1、WO2017041749A1、WO2017080377A1、WO2018018958A1、WO2018108106A1、WO2018045811A1、WO 2018219299、WO 2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/149279、WO 2019/170147A1、WO 2019/210863、CN109385400A、CN109468279A、CN109880803A、CN 110438082A、CN 110468105A、WO2019/219029、WO 200014257、WO 2013126726、WO 2012/129514、WO 2014031687、WO 2013/166321、WO 2013/071154、WO 2013/123061、美国专利申请公开号US 2002131960、US 2013287748、US 20130149337、美国专利号6,451,995、7,446,190、8,252,592、8,339,645、8,398,282、7,446,179、6,410,319、7,070,995、7,265,209、7,354,762、7,446,191、8,324,353和8,479,118，以及欧洲专利申请号EP 2537416和/或以下文献中所述的那些：Sadelain等,Cancer Discov.2013年4月,3(4):388-398；Davila等,(2013)PLoS ONE 8(4):e61338；Turtle等,Curr.Opin.Immunol.,2012年10月；24(5):633-39；Wu等,Cancer,2012年3月18日(2):160-75。
a.嵌合抗原受体
在一些实施方案中，病毒载体基因组中所含的核酸编码嵌合抗原受体(CAR)。CAR通常是基因工程化受体，其具有细胞外配体结合结构域，例如含有抗体或其片段的细胞外部分，所述细胞外配体结合结构域与一种或多种细胞内信号传导组分连接。在一些实施方案中，嵌合抗原受体包括连接细胞外结构域和细胞内信号传导结构域的跨膜结构域和/或细胞内结构域。此类分子通常模拟或接近通过天然抗原受体发出的信号和/或通过这种受体与共刺激受体的组合发出的信号。
在一些实施方案中，CAR被构建具有对特定标记的特异性，例如在过继疗法所靶向的特定细胞类型中表达的标记，例如癌症标记和/或任何所述抗原。因此，CAR通常包括抗体的一个或多个抗原结合片段、结构域或部分，或一个或多个抗体可变结构域和/或抗体分子。在一些实施方案中，CAR包括抗体分子的一个或多个抗原结合部分，例如可变重链(VH)或其抗原结合部分，或源自可变重链(VH)的单链抗体片段(scFv)和单克隆抗体(mAb)的可变轻链(VL)。
在一些实施方案中，提供了工程化细胞，例如T细胞，其表达对特定抗原(或标记或配 体)具有特异性的CAR，所述特定抗原例如为在特定细胞类型的表面上表达的抗原。在一些实施方案中，抗原是多肽。在一些实施方案中，它是碳水化合物或其他分子。在一些实施方案中，与正常或非靶向细胞或组织相比，抗原在疾病的细胞例如肿瘤细胞或致病细胞上选择性表达或过表达。在其他实施方案中，抗原在正常细胞上表达和/或在工程化细胞上表达。
在特定实施方案中，重组受体(如嵌合受体)含有细胞内信号传导区域，所述细胞内信号传导区域包括细胞质信号传导结构域或区域(也可互换地称为细胞内信号传导结构域或区域)，例如能够在T细胞中诱导初级激活信号的细胞质(细胞内)区域，例如，T细胞受体(TCR)组分的细胞质信号传导结构域或区域(例如，CD3-zeta(CD3ζ)链或其功能变体或信号传导部分的ζ链的细胞质信号传导结构域或区域)；和/或所述细胞内信号传导区域包含基于免疫受体酪氨酸的激活基序(ITAM)的细胞质信号传导结构域或区域。
在一些实施方案中，嵌合受体还含有特异性地结合至配体(例如抗原)抗原的细胞外配体结合结构域。在一些实施方案中，嵌合受体是CAR，其含有特异性地结合至抗原的细胞外抗原识别结构域。在一些实施方案中，配体(如抗原)是在细胞表面上表达的蛋白质。在一些实施方案中，CAR是TCR样CAR，并且抗原是加工过的肽抗原，如细胞内蛋白的肽抗原，其与TCR一样在主要组织相容性复合物(MHC)分子的背景下在细胞表面上被识别。
示例性抗原受体(包括CAR)以及将此类受体工程化并引入细胞的方法包括例如以下文献中所述的那些：国际专利申请公开号WO2015172339A1、WO2016008405A1、WO 2016086813A1、WO2016150400、WO2017032293A1、WO2017041749A1、WO2017080377A1、WO2018018958A1、WO2018108106A1、WO2018045811A1、WO 2018219299、WO 2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/149279、WO 2019/170147A1、WO 2019/210863、CN109385400A、CN109468279A、CN109880803A、CN 110438082A、CN110468105A、WO2019/219029、WO 200014257、WO2013126726、WO 2012/129514、WO2014031687、WO2013/166321、WO2013/071154、WO2013/123061、美国专利申请公开号US 2002131960、US 2013287748、US 20130149337、美国专利号6,451,995、7,446,190、8,252,592、8,339,645、8,398,282、7,446,179、6,410,319、7,070,995、7,265,209、7,354,762、7,446,191、8,324,353和8,479,118以及欧洲专利申请号EP 2537416和/或以下文献中所述的那些：Sadelain等，Cancer Discov.2013年4月,3(4):388-398；Davila等,(2013)PLoS ONE 8(4):e61338；Turtle等,Curr.Opin.Immunol.,2012年10月；24(5):633-39；Wu等,Cancer,2012年3月18日(2):160-75。在一些方面，抗原受体包括如美国专利号7,446,190中所述的CAR，以及国际专利申请公开号WO/2014055668A1中所述的那些。
CAR的实例包括如在任何下述出版物中披露的CAR，所述出版物例如WO2015172339A1、WO2016008405A1、WO2016086813A1、WO2016150400、WO2017032293A1、WO 2017041749A1、WO2017080377A1、WO2018018958A1、WO2018108106A1、WO2018045811A1、WO 2018219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/149279、WO2019/170147A1、WO 2019/210863、CN109385400A、CN109468279A、CN109880803A、CN 110438082A、CN 110468105A、WO2019/219029、WO 2014031687、 US 8,339,645、US 7,446,179、US 2013/0149337、美国专利号7,446,190、美国专利号8,389,282；Kochenderfer等,2013,Nature Reviews Clinical Oncology,10,267-276(2013)；Wang等,(2012)J.Immunother.35(9):689-701；和Brentjens等,Sci Transl Med.2013 5(177)。还参见WO 2014031687、US 8,339,645、US 7,446,179、US 2013/0149337、美国专利号7,446,190和美国专利号8,389,282。
在一些实施方案中，CAR被构建为具有对特定抗原(或标记或配体)的特异性，所述特定抗原例如为在过继疗法靶向的特定细胞类型中表达的抗原(例如癌症标记)和/或旨在诱导衰减应答的抗原(例如在正常或未患病细胞类型上表达的抗原)。因此，CAR通常在其细胞外部分中包括一种或多种抗原结合分子，例如一种或多种抗原结合片段、结构域或部分，或一种或多种抗体可变结构域，和/或抗体分子。在一些实施方案中，所述CAR包括抗体分子的一个或多个抗原结合部分，如源自单克隆抗体(mAb)的可变重链(VH)和可变轻链(VL)的单链抗体片段(scFv)。
在一些实施方案中，抗体或其抗原结合部分作为重组受体(例如抗原受体)的部分在细胞上表达。所述抗原受体包括功能性非TCR抗原受体，如嵌合抗原受体(CAR)。通常，含有针对肽-MHC复合物表现出TCR样特异性的抗体或抗原结合片段的CAR也可以称为TCR样CAR。在一些实施方案中，在一些方面，对TCR样CAR的MHC-肽复合物具有特异性的细胞外抗原结合结构域通过接头和/或一个或多个跨膜结构域与一个或多个细胞内信号传导组分连接。在一些实施方案中，此类分子通常可以通过天然抗原受体(如TCR)模拟或接近信号，并且任选地通过这种受体与共刺激受体组合模拟或接近信号。
在一些实施方案中，重组受体(例如嵌合受体，例如CAR)包括与抗原(或配体)结合(例如特异性结合)的配体结合结构域。嵌合受体靶向的抗原包括在通过过继细胞疗法靶向的疾病、病症或细胞类型的情况下表达的抗原。所述疾病和病症包括增殖性、肿瘤性和恶性疾病，包括癌症和肿瘤，包括血液癌、免疫系统癌症，如淋巴瘤、白血病和/或骨髓瘤，如B型白血病、T型白血病和骨髓性白血病、淋巴瘤和多发性骨髓瘤。
在一些实施方案中，抗原(或配体)是多肽。在一些实施方案中，它是碳水化合物或其他分子。在一些实施方案中，如与正常或非靶向细胞或组织相比，抗原(或配体)在疾病的细胞(例如肿瘤或致病细胞)上选择性表达或过表达。在其他实施方案中，抗原在正常细胞上表达和/或在工程化细胞上表达。
在一些实施方案中，CAR含有抗体或抗原结合片段(例如scFv)，其特异性识别在细胞表面上表达的抗原，例如完整抗原。在一些实施方案中，抗原(或配体)是肿瘤抗原或癌症标记。在一些实施方案中，抗原(或配体)抗原是或包括αvβ6整合素(avb6整合素)、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、碳酸酐酶9(CA9，也称为CAIX或G250)、癌症-睾丸抗原、癌症/睾丸抗原1B(CTAG，也称为NYESO-1和LAGE-2)、癌胚抗原(CEA)、细胞周期蛋白、细胞周期蛋白A2、CC基序趋化因子配体1(CCL-1)、CD19、CD20、CD22、CD23、CD24、CD30、CD33、CD38、CD44、CD44v6、CD44v7/8、CD123、CD138、CD171、表皮生长因子蛋白(EGFR)、截短的表皮生长因子蛋白(tEGFR)、III型表皮生长因子受体突变(EGFR vIII)、 上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、肝配蛋白B2、肝配蛋白受体A2(EPHa2)、雌激素受体、Fc受体样5(FCRL5；也称为Fc受体同源物5或FCRH5)、胎儿乙酰胆碱受体(胎儿AchR)、叶酸结合蛋白(FBP)、叶酸受体α、神经节苷脂GD2、O-乙酰化GD2(OGD2)、神经节苷脂GD3、糖蛋白100(gp100)、G蛋白偶联受体5D(GPCR5D)、Her2/neu(受体酪氨酸激酶erb-B2)、Her3(erb-B3)、Her4(erb-B4)、erbB二聚体、人高分子量黑色素瘤相关抗原(HMW-MAA)、乙型肝炎表面抗原、人白细胞抗原A1(HLA-A1)、人白细胞抗原A2(HLAA2)、IL-22受体α(IL-22Ra)、IL-13受体α2(IL-13Ra2)、激酶插入结构域受体(kdr)、κ轻链、L1细胞粘附分子(L1-CAM)、L1-CAM的CE7表位、含有富含亮氨酸的重复序列的8家族成员A(LRRC8A)、路易斯Y、黑色素瘤相关抗原(MAGE)-A1、MAGE-A3、MAGE-A6、间皮素、c-Met、鼠类巨细胞病毒(CMV)、粘蛋白1(MUC1)、MUC16、自然杀伤2族成员D(NKG2D)配体、黑色素A(MART-1)、神经细胞粘附分子(NCAM)、癌胚胎抗原、优先表达的黑色素瘤抗原(PRAME)、孕酮受体、前列腺特异性靶抗原、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、存活蛋白、滋养层糖蛋白(TPBG，也称为5T4)、肿瘤相关糖蛋白72(TAG72)、血管内皮生长因子受体(VEGFR)、血管内皮生长因子受体2(VEGFR2)、Wilms肿瘤1(WT-1)、病原体特异性靶抗原或与通用标签相关的抗原，和/或生物素化分子，和/或由HIV、HCV、HBV或其他病原体表达的分子。在一些实施方案中，受体靶向的抗原包括与B细胞恶性肿瘤相关的抗原，如许多已知B细胞标记中的任何一种。在一些实施方案中，抗原是或包括CD20、CD19、CD22、ROR1、CD45、CD21、CD5、CD33、Igκ、Igλ、CD79a、CD79b或CD30。
在一些实施方案中，抗原是或包括病原体特异性或病原体表达的抗原。在一些实施方案中，抗原是病毒抗原(例如来自HIV、HCV、HBV等的病毒抗原)、细菌抗原和/或寄生虫抗原。在一些实施方案中，CAR含有TCR样抗体，例如抗体或抗原结合片段(例如scFv)，其特异性识别作为MHC-肽复合物存在于细胞表面上的细胞内抗原(例如肿瘤相关抗原)。在一些实施方案中，识别MHC-肽复合物的抗体或其抗原结合部分可以作为重组受体的部分(例如抗原受体)在细胞上表达。所述抗原受体包括功能性非TCR抗原受体，如嵌合抗原受体(CAR)。通常，含有针对肽-MHC复合物表现出TCR样特异性的抗体或抗原结合片段的CAR也可以称为TCR样CAR。
在一些实施方案中，特异性地结合至MHC-肽复合物的抗体或其抗原结合部分可以通过用有效量的含有特定MHC-肽复合物的免疫原对宿主进行免疫来产生。在一些情况下，MHC-肽复合物的肽是能够结合至MHC的抗原的表位，例如肿瘤抗原，例如通用肿瘤抗原、骨髓瘤抗原或如下文所述的其他抗原。在一些实施方案中，然后向宿主给予有效量的免疫原以用于引发免疫应答，其中所述免疫原保持其三维形式持续一段足以引发针对所述肽在所述MHC分子的结合沟中的三维呈递的免疫应答的时间。然后测定从宿主收集的血清以确定是否产生了识别MHC分子结合沟中的肽的三维呈现的所需抗体。在一些实施方案中，可以评估所产生的抗体以确认所述抗体可以区分MHC-肽复合物与单独的MHC分子、单独的目标肽以及MHC与无关肽的复合物。然后可以分离所需的抗体。
单结构域抗体是包含抗体的全部或部分重链可变结构域或者全部或部分轻链可变结构域的抗体片段。在某些实施方案中，单结构域抗体是人单结构域抗体。在一些实施方案中，CAR包含特异性地结合抗原的抗体重链结构域，所述抗原例如癌症标记或待靶向的细胞或疾病(例如肿瘤细胞或癌细胞)的细胞表面抗原，例如本文所述或已知的任何靶抗原。
抗体片段可以通过各种技术制备，包括但不限于完整抗体的蛋白水解消化以及通过重组宿主细胞产生。在一些实施方案中，所述抗体是重组产生的片段，如包含天然不存在的排列的片段(如具有通过合成接头(例如，肽接头)连接的两个或更多个抗体区或链的那些)，和/或可不通过酶消化天然存在的完整抗体产生的片段。在一些实施方案中，抗体片段是scFv。
在一些实施方案中，嵌合抗原受体(包括TCR样CAR)包括含有抗体或抗体片段的细胞外部分。在一些实施方案中，抗体或片段包括scFv。在一些方面，嵌合抗原受体包括含有抗体或片段的细胞外部分和细胞内信号传导区域。在一些实施方案中，细胞内信号传导区域包含细胞内信号传导结构域。在一些实施方案中，细胞内信号传导结构域是或包含主要信号传导结构域、能够在T细胞中诱导初级激活信号的信号传导结构域、T细胞受体(TCR)组分的信号传导结构域和/或包含基于免疫受体酪氨酸的激活基序(ITAM)的信号传导结构域。
在一些实施方案中，CAR的细胞外部分(例如其抗体部分)还包括间隔子，例如抗原识别组分(例如scFv)与跨膜结构域之间的间隔子区域。间隔子可以是或包括免疫球蛋白恒定区或其变体或经修饰形式的至少一部分，例如铰链区，例如IgG4铰链区，和/或CH1/CL和/或Fc区。在一些实施方案中，重组受体还包含间隔子和/或铰链区。在一些实施方案中，恒定区或部分是人IgG如IgG4或IgG1的。在一些方面，所述恒定区的部分用作抗原识别组分(例如，scFv)与跨膜结构域之间的间隔子区。
在一些实施方案中，间隔子可以是或包括免疫球蛋白恒定区或其变体或经修饰形式的至少一部分，例如铰链区(例如IgG4铰链区)、和/或CH1/CL和/或Fc区。在一些实施方案中，重组受体还包含间隔子和/或铰链区。在一些实施方案中，恒定区或部分是人IgG如IgG4或IgG1的。在一些方面，所述恒定区的部分用作抗原识别组分(例如，scFv)与跨膜结构域之间的间隔子区。与不存在间隔子的情况下相比，间隔子的长度可以提供抗原结合后增强的细胞反应性。在一些实施方案中，间隔子区具有约12个或更少的氨基酸、约119个或更少的氨基酸或约229个或更少的氨基酸。示例性间隔子包括单独的IgG4铰链、与CH2和CH3结构域连接的IgG4铰链或与CH3结构域连接的IgG4铰链。细胞外配体结合结构域(例如抗原识别结构域)通常与一个或多个细胞内信号传导组分连接，所述一个或多个细胞内信号传导组分例如为在CAR的情况下通过抗原受体复合物(例如TCR复合物)模拟激活的信号传导组分和/或通过另一细胞表面受体传导的信号。在一些实施方案中，跨膜结构域连接细胞外配体结合结构域与细胞内信号传导结构域。在一些实施方案中，抗原结合组分(例如，抗体)与一个或多个跨膜和细胞内信号传导区域连接。在一些实施方案中，CAR包括与细胞外结构域融合的跨膜结构域。在一个实施方案中，使用天然地与受体(例如CAR)中的结构域之一相关的跨膜结构域。在一些情形中，通过氨基酸取代选择或修饰所述跨膜结构域以避免此类结构域与相同或不同表面膜蛋白的跨膜结构域结合，以最小化与所述受体复合物的其他成员的相 互作用。
在一些实施方案中，所述跨膜结构域源自天然或合成来源。当来源是天然的时，在一些方面，所述结构域可以源自任何膜结合蛋白或跨膜蛋白。跨膜区包括源自以下项的那些跨膜区(即，包括以下项的至少一个或多个跨膜区)：T细胞受体的α、β或ζ链，CD28，CD3ε，CD45，CD4，CD5，CD8，CD9，CD16，CD22，CD33，CD37，CD64，CD80，CD86，CD134，CD137或CD154。在一些实施方案中，跨膜结构域是合成的。在一些方面，合成跨膜结构域主要包含疏水性残基，例如亮氨酸和缬氨酸。在一些方面，将在合成跨膜结构域的每个末端发现苯丙氨酸、色氨酸和缬氨酸的三联体。在一些实施方案中，连接是通过接头、间隔子和/或一个或多个跨膜结构域。
在一些实施方案中，短的寡肽或多肽接头(例如，长度在2与10个之间的氨基酸的接头，如含有甘氨酸和丝氨酸的接头，例如甘氨酸-丝氨酸双联体)存在并形成CAR的跨膜结构域与细胞质信号传导结构域之间的连接。
重组受体(例如CAR)通常包括至少一种或多种细胞内信号传导组分。在一些实施方案中，受体包括TCR复合物的细胞内组分，如介导T细胞激活和细胞毒性的TCR CD3链，例如，CD3ζ链。因此，在一些方面，所述抗原结合部分与一个或多个细胞信号传导模块连接。在一些实施方案中，细胞信号传导模块包括CD3跨膜结构域、CD3细胞内信号传导结构域和/或其他CD跨膜结构域。在一些实施方案中，受体(例如CAR)还包括一种或多种另外的分子(例如Fc受体γ、CD8、CD4、CD25或CD16)的一部分。例如，在一些方面，CAR或其他嵌合受体包括CD3-ζ(CD3-ζ)或Fc受体γ与CD8、CD4、CD25或CD16之间的嵌合分子。
在一些实施方案中，在连接CAR或其他嵌合受体后，受体的细胞质结构域和/或区域或细胞内信号传导结构域和/或区域激活免疫细胞(例如，工程化以表达CAR的T细胞)的正常效应子功能或应答中的至少一种。例如，在一些情况下，CAR诱导T细胞的功能，例如细胞溶解活性或T辅助活性，例如细胞因子或其他因子的分泌。在一些实施方案中，使用抗原受体组分或共刺激分子的细胞内信号传导结构域的截短部分(例如如果其转导效应子功能信号的话)代替完整的免疫刺激链。在一些实施方案中，细胞内信号传导区域(例如包含一个或多个细胞内信号传导结构域)包括T细胞受体(TCR)的胞质序列，并且在一些方面还包括共受体(其在天然背景下与这种受体并行起作用以在抗原受体接合后启动信号转导)和/或此类分子的任何衍生物或变体，和/或具有相同功能能力的任何合成序列。
在天然TCR的情况下，完全激活通常不仅需要通过TCR进行信号传导，还需要共刺激信号。因此，在一些实施方案中，为了促进完全激活，用于生成次级或共刺激信号的组分也被包括在所述CAR中。在其他实施方案中，所述CAR不包括用于生成共刺激信号的组分。在一些方面，另外的CAR在同一细胞中表达，并且提供用于生成次级或共刺激信号的组分。
T细胞激活在一些方面被描述为由至少如下两类细胞质信号传导序列介导：通过TCR启动抗原依赖性初级激活的那些(初级细胞质信号传导序列)，以及以非抗原依赖性方式起作用以提供次级或共刺激信号的那些(次级细胞质信号传导序列)。在一些方面，CAR包括此类信号传导组分中的一种或两种。
在一些方面，所述CAR包括调节所述TCR复合物的初级激活的初级胞质信号传导序列。以刺激方式起作用的初级胞质信号传导序列可以含有信号传导基序(其被称为基于免疫受体酪氨酸的激活基序或ITAM)。含有初级细胞质信号传导序列的ITAM的例子包括源自以下项的那些：TCR或CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD8、CD22、CD79a、CD79b和CD66d。在某些实施方案中，含有初级细胞质信号传导序列的ITAM包括源自TCR或CD3ζ、FcRγ或FcRβ的那些。在一些实施方案中，所述CAR中的胞质信号传导分子含有源自CD3ζ的胞质信号传导结构域。
在一些实施方案中，CAR包括共刺激受体(例如CD28、4-1BB、OX40、CD27、DAP10和ICOS)的信号传导结构域和/或跨膜部分。在一些方面，相同的CAR包括激活或信号传导区域和共刺激组分。
在一些实施方案中，激活结构域包括于一个CAR内，而共刺激组分是由识别另一种抗原的另一种CAR提供。在一些实施方案中，所述CAR包括在同一细胞上表达的激活或刺激CAR和共刺激CAR(参见WO 2014/055668)。在一些方面，CAR是刺激性或激活性CAR；在其他方面，其是共刺激性CAR。在一些实施方案中，细胞还包括抑制性CAR(iCAR，参见Fedorov等,Sci.Transl.Medicine,5(215)(2013年12月)，例如识别不同抗原的CAR，其中通过识别第一抗原的CAR递送的激活信号通过抑制性CAR与其配体的结合而被减少或抑制，例如，以减少脱靶效应。
在一些实施方案中，细胞内信号传导结构域包含与CD3细胞内结构域连接的CD28跨膜和信号传导结构域。在一些实施方案中，细胞内信号传导结构域包含与CD3细胞内结构域连接的嵌合CD28和CD137共刺激结构域。
在一些实施方案中，CD8+细胞毒性T细胞的细胞内信号传导结构域与CD4+辅助性T细胞的细胞内信号传导结构域相同。在一些实施方案中，CD8+细胞毒性T细胞的细胞内信号传导结构域不同于CD4+辅助性T细胞的细胞内信号传导结构域。
在一些实施方案中，CAR在细胞质部分中涵盖一个或多个(例如，两个或更多个)共刺激结构域以及激活结构域(例如，初级激活结构域)。示例性CAR包含CD3-ζ、CD28和4-1BB的细胞内组分。
在一些实施方案中，由所提供的病毒载体内的一个或多个核酸编码的一种或多种重组受体(例如CAR)还包括一种或多种标记，例如，用于确认要表达受体的细胞的转导或工程化和/或对表达由多核苷酸编码的一种或多种分子的细胞的选择和/或靶向的目的。在一些方面，这种标记可以由不同的核酸或多核苷酸编码，所述核酸或多核苷酸也可以在基因工程化过程期间被引入，通常通过相同的方法(例如通过本文所提供的任何方法转导，例如通过相同的载体或相同类型的载体转导)被引入。
在一些方面，所述标记(例如转导标记)是蛋白质和/或是细胞表面分子。示例性标记是天然存在的(例如内源性)标记(例如天然存在的细胞表面分子)的截短的变体。
在一些情况下，CAR被称为第一代、第二代和/或第三代CAR。在一些方面，第一代CAR是在抗原结合后仅提供CD3链诱导的信号的CAR；在一些方面，第二代CAR是提供 这种信号和共刺激信号的CAR，例如包括来自共刺激受体(如CD28或CD137)的细胞内信号传导结构域的CAR；在一些方面，第三代CAR是包括不同共刺激受体的多个共刺激结构域的CAR。
在一些实施方案中，嵌合抗原受体包括细胞外配体结合部分(例如抗原结合部分，例如抗体或其片段)和细胞内结构域。在一些实施方案中，抗体或片段包括scFv或单一结构域VH抗体，并且细胞内结构域含有ITAM。在一些方面，所述细胞内信号传导结构域包括CD3-zeta(CD3ζ)链的ζ链的信号传导结构域。在一些实施方案中，嵌合抗原受体包括连接和/或设置在细胞外结构域与细胞内信号传导区域或结构域之间的跨膜结构域。
在一些方面，跨膜结构域含有CD28的跨膜部分。所述细胞外结构域和跨膜可以直接或间接连接。在一些实施方案中，所述细胞外结构域和跨膜通过间隔子(如本文描述的任何间隔子)连接。在一些实施方案中，所述嵌合抗原受体含有T细胞共刺激分子的细胞内结构域，如在所述跨膜结构域和细胞内信号传导结构域之间。在一些方面，所述T细胞共刺激分子是CD28或4-1BB。
在一些实施方案中，CAR含有抗体(例如抗体片段)、是或含有CD28或其功能变体的跨膜部分的跨膜结构域，以及含有CD28或其功能变体的信号传导部分和CD3ζ或其功能变体的信号传导部分的细胞内信号传导结构域。在一些实施方案中，CAR含有抗体例如抗体片段，跨膜结构域(其是CD28的跨膜部分或其功能变体或含有CD28的跨膜部分或其功能变体)以及含有4-1BB的信号传导部分或其功能变体和CD3ζ的信号传导部分或其功能变体的细胞内信号传导结构域。在一些实施方案中，受体进一步包括含有Ig分子(如人Ig分子，如Ig铰链，例如IgG4铰链)的一部分的间隔子如仅含铰链的间隔子。
在一些实施方案中，受体(例如CAR)的跨膜结构域是人CD28或其变体的跨膜结构域，例如人CD28(登录号：P10747.1)的27个氨基酸的跨膜结构域。
在一些实施方案中，嵌合抗原受体含有T细胞共刺激分子的细胞内结构域。在一些方面，所述T细胞共刺激分子是CD28或4-1BB。
在一些实施方案中，细胞内结构域包含人CD28或其功能变体或部分的细胞内共刺激信号传导结构域，例如其41个氨基酸的结构域，和/或在天然CD28蛋白的位置186-187处具有LL至GG取代的这种结构域。
在一些实施方案中，细胞内信号传导区域和/或结构域包含人CD3链、任选地CD3ζ刺激性信号传导结构域或其功能变体，例如人CD3ζ(登录号：P20963.2)的同种型3的112个AA的细胞质结构域或如美国专利号7,446,190或美国专利号8,911,993中所述的CD3ζ信号传导结构域。
在一些实施方案中，CAR包括：细胞外配体结合部分，例如抗原结合部分，例如抗体或其片段，包括sdAb和scFv，其特异性结合抗原，例如本文所述的抗原；间隔子，例如任何含有Ig铰链的间隔子；跨膜结构域，其是CD28或其变体的一部分；细胞内信号传导结构域，其含有CD28或其功能变体的信号传导部分；以及CD3ζ信号传导结构域或其功能变体的信号传导部分。在一些实施方案中，CAR包括：细胞外配体结合部分，例如抗原结合部 分，例如抗体或其片段，包括sdAb和scFv，其特异性结合抗原，例如本文所述的抗原；间隔子，例如任何含有Ig铰链的间隔子；跨膜结构域，其是CD28或其变体的一部分；细胞内信号传导结构域，其含有4-1BB或其功能变体的信号传导部分；以及CD3ζ信号传导结构域或其功能变体的信号传导部分。
b.T细胞受体(TCR)
在一些实施方案中，由一种或多种核酸编码的一种或多种重组分子是或包括重组T细胞受体(TCR)。在一些实施方案中，重组TCR对抗原具有特异性，所述抗原通常是存在于靶细胞上的抗原，例如肿瘤特异性靶抗原，在与自身免疫或炎性疾病相关的特定细胞类型上表达的抗原，或源自病毒病原体或细菌病原体的抗原。在一些实施方案中，提供了工程化细胞，例如T细胞，其表达识别靶多肽(例如肿瘤、病毒或自身免疫蛋白的抗原)的肽表位或T细胞表位的TCR或其抗原结合部分。
在一些实施方案中，“T细胞受体”或“TCR”是含有可变α和β链(也分别称为TCRα和TCRβ)或可变γ和δ链(也分别称为TCRα和TCRβ)的分子或其抗原结合部分，并且其能够特异性结合至与MHC分子结合的肽。在一些实施方案中，所述TCR呈αβ形式。通常，以αβ和γδ形式存在的TCR一般在结构上相似，但是表达它们的T细胞可以具有不同的解剖位置或功能。
除非另有说明，否则术语“TCR”应理解为涵盖完整的TCR以及其抗原结合部分或其抗原结合片段。在一些实施方案中，所述TCR是完整或全长TCR，包括呈αβ形式或γδ形式的TCR。在一些实施方案中，所述TCR是这样的抗原结合部分，其少于全长TCR但与在MHC分子中结合的特定肽结合(如与MHC-肽复合物结合)。在一些情况下，TCR的抗原结合部分或片段可以仅含有全长或完整TCR的结构性结构域的一部分，但是仍能够结合与完整TCR结合的肽表位(如MHC-肽复合物)。在一些情况下，抗原结合部分含有TCR的可变结构域(如TCR的可变α链和可变β链)，足以形成用于与特定MHC-肽复合物结合的结合位点。通常，TCR的可变链含有参与肽、MHC和/或MHC-肽复合物的识别的互补决定区。
在一些实施方案中，TCR的可变结构域含有超变环或互补决定区(CDR)，其通常是抗原识别和结合能力和特异性的主要贡献者。在一些实施方案中，TCR的CDR或其组合形成给定TCR分子的全部或基本上全部的抗原结合位点。
在一些实施方案中，所述TCR链含有跨膜结构域。在一些实施方案中，所述跨膜结构域带正电荷。
在一些实施方案中，TCR可以是两条链α和β(或者任选地γ和δ)的异二聚体，或者其可以是单链TCR构建体。
在一些实施方案中，TCR含有对应于跨膜序列的序列。在一些实施方案中，所述TCR确实含有对应于胞质序列的序列。在一些实施方案中，所述TCR能够与CD3形成TCR复合物。在一些实施方案中，任何TCR(包括dTCR或scTCR)可以与在T细胞的表面上产生活性TCR的信号传导结构域连接。在一些实施方案中，所述TCR在细胞的表面上表达。
在一些实施方案中，编码TCR(如α和β链)的一种或多种核酸可以通过PCR、克隆或 其他合适的方法扩增，并且克隆到合适的表达载体中。所述表达载体可以是任何合适的重组表达载体，并且可以用于转化或转染任何合适的宿主。合适的载体包括设计用于繁殖和扩增或用于表达或用于两者的那些，如质粒和病毒。
在一些实施方案中，载体可以是以下系列的载体：pUC系列(Fermentas Life Sciences)、pBluescript系列(Stratagene，加利福尼亚州拉霍亚)、pET系列(Novagen，威斯康星州麦迪逊)、pGEX系列(Pharmacia Biotech，瑞典乌普萨拉)或pEX系列(Clontech，加利福尼亚州帕罗奥图)。在一些情况下，也可以使用噬菌体载体，如λG10、λGT11、λZa pII(Stratagene)、λEMBL4和λNM1149。在一些实施方案中，可以使用植物表达载体，并且包括pBI01、pBI101.2、pBI101.3、pBI121和pBIN19(Clontech)。在一些实施方案中，动物表达载体包括pEUK-Cl、pMAM和pMAMneo(Clontech)。在一些实施方案中，使用病毒载体，如逆转录病毒载体。
在一些实施方案中，可以使用标准重组DNA技术来制备所述重组表达载体。在一些实施方案中，载体可以含有调节序列，如转录和翻译起始和终止密码子，其对引入载体的宿主(例如，细菌、真菌，植物或动物)的类型具特异性。在一些实施方案中，载体可以含有非天然启动子，其可操作地连接至编码TCR或抗原结合部分(或其他MHC-肽结合分子)的核苷酸序列。在一些实施方案中，所述启动子可以是非病毒启动子或病毒启动子，如巨细胞病毒(CMV)启动子、SV40启动子、RSV启动子和在鼠干细胞病毒的长末端重复序列中发现的启动子。
在一些实施方案中，细胞和方法包括多靶向策略，例如在细胞上表达两种或更多种基因工程化受体，每种受体识别相同或不同的抗原，并且通常各自包括不同的细胞内信号传导组分。
在本文提供的方法和组合物的一些实施方案中，病毒载体基因组中所含的编码重组受体(例如抗原受体，例如CAR)的核酸序列与其他遗传元件(例如转录调节序列，包括启动子或增强子)以功能关系可操作地连接，以特定方式调节目标序列的表达。在某些情况下，此类转录调节序列是在活性方面在时间上和/或空间上受调节的那些。可用于调节组分表达的表达控制元件是已知的，并且包括但不限于诱导型启动子、组成型启动子、分泌信号、增强子和其他调节元件。在一些实施方案中，病毒载体基因组中所含的核酸序列含有多个表达控制元件，其控制所编码的不同组分，例如不同的受体组分和/或信号传导组分，使得重组受体和/或工程化细胞(例如表达工程化受体的细胞)的表达、功能和/或活性可以被调节，例如是可诱导的、可抑制的、可调节的和/或用户控制的。在一些实施方案中，一种或多种载体可含有一种或多种核酸序列，所述核酸序列含有一种或多种表达控制元件和/或一种或多种所编码组分，使得核酸序列一起可调节所编码组分(例如重组受体)或工程化细胞的表达、活性和/或功能。
在一些实施方案中，编码重组受体(例如抗原受体，例如CAR)的核酸序列与内部启动子/增强子调节序列可操作地连接。所用的启动子可以是组成型的、组织特异性的、诱导型的 和/或在适当条件下可用于引导所引入DNA区段的高水平表达。启动子可以是异源的或内源的。在一些实施方案中，启动子和/或增强子是以合成方式产生。在一些实施方案中，启动子和/或增强子是使用重组克隆和/或核酸扩增技术产生。
在一些情况下，编码重组受体的核酸序列含有编码信号肽的信号序列。在一些方面，信号序列可以编码源自天然多肽的信号肽。在其他方面，信号序列可以编码异源或非天然信号肽。在一些情况下，编码重组受体(例如嵌合抗原受体(CAR))的核酸序列含有编码信号肽的信号序列。
在一些实施方案中，编码重组受体的多核苷酸含有至少一个启动子，所述启动子可操作连接以控制重组受体的表达。在一些例子中，多核苷酸含有两个、三个或更多个启动子，所述启动子可操作地连接以控制重组受体的表达。
在核酸分子编码两种或更多种不同多肽链(例如重组受体和标记)的某些情况下，每条多肽链可以由单独的核酸分子编码。例如，提供了两个单独的核酸，并且每一个可以单独转移到或引入细胞中以在细胞中表达。在一些实施方案中，编码重组受体的核酸和编码标记的核酸与相同的启动子可操作地连接，并且任选地通过内部核糖体进入位点(IRES)或编码自切割肽或导致核糖体跳跃的肽(其任选地是T2A、P2A、E2A或F2A)的核酸分开。在一些实施方案中，编码标记的核酸和编码重组受体的核酸可操作地连接至两个不同的启动子。在一些实施方案中，编码标记的核酸和编码重组受体的核酸存在或插入于细胞基因组内的不同位置。在一些实施方案中，例如通过逆转录病毒转导、转染或转化将编码重组受体的多核苷酸引入含有培养细胞的组合物中。
在一些实施例中，寡核苷酸引物包含标签，其中标签对靶序列不是特异性的。这样的标签可以被称为通用标签或通用标记。在一些情况下，方法包括用对靶序列非特异性的标签标记样品中的靶序列或其片段。在一些情况下，标签对人类染色体上的序列不具有特异性。备选地或附加地，方法包括使样品与标签和包含与靶序列相对应的序列的至少一种寡核苷酸引物接触，其中标签与寡核苷酸引物分离。在一些情况下，在寡核苷酸引物与靶序列杂交后，通过延伸寡核苷酸引物将标签掺入扩增产物中。标签可以是寡核苷酸、小分子或肽。在一些实施方案中，标记是转导标记或替代标记。转导标记或替代标记可用于检测已经引入多核苷酸(例如编码重组受体的多核苷酸)的细胞。在一些实施方案中，转导标记可以指示或确认对细胞的修饰。在一些实施方案中，替代标记是经制备在细胞表面上与重组受体(例如，CAR)共表达的蛋白质。在特定实施方案中，这种替代标记是已经修饰以具有极少或无活性的表面蛋白。在一些实施方案中，替代标记是由编码重组受体的相同多核苷酸编码。在一些实施方案中，编码重组受体的核酸序列与编码标记的核酸序列可操作地连接，任选地通过内部核糖体进入位点(IRES)或编码自切割肽或引起核糖体跳跃的肽(例如2A序列，例如T2A、P2A、E2A或F2A)的核酸分开。在一些情况下，外在标记基因可以结合工程化细胞用于允许检测或选择细胞，并且在一些情况下还可以用于促进细胞自杀。
在一些实施方案中，启动子和/或增强子可以是与核酸序列天然相关的启动子和/或增强子，如可以通过分离位于编码区段和/或外显子上游的5'非编码序列而获得。
在一些实施方案中，启动子可以是组织特异性启动子和/或靶细胞特异性启动子。
在一些实施方案中，调节元件可以包括允许重组受体(例如CAR)的可调节表达和/或活性的调节元件和/或系统。在一些实施方案中，可调节的表达和/或活性是通过将重组受体配置成含有特定调节元件和/或系统或受所述调节元件和/或系统控制来实现。
病毒载体颗粒的制备
病毒载体基因组通常以质粒形式构建，其可以转染到包装细胞系或生产细胞系中。可以使用多种已知方法中的任何一种来产生逆转录病毒颗粒，其基因组含有病毒载体基因组的RNA拷贝。在一些实施方案中，至少两种组分参与制备基于病毒的基因传递系统：第一，包装质粒，包括结构蛋白以及产生病毒载体颗粒所必需的酶，第二，病毒载体本身，即，要转移的遗传物质。可以在设计这些组分中的一个或两个时引入生物安全保护措施。
在一些实施方案中，包装质粒可以含有除了包膜蛋白以外的所有逆转录病毒(如HIV-1)蛋白(Naldini等人，1998)。在一些实施方案中，慢病毒载体(例如基于HIV的慢病毒载体)仅包含三种亲本病毒的基因：gag、pol和rev，这减少或消除了野生型病毒通过重组而重构的可能性。
在一些实施方案中，将病毒载体基因组引入包装细胞系中，所述细胞系含有将从病毒载体基因组转录的病毒基因组RNA包装至病毒颗粒中所需的所有组分。
在一些实施方案中，用一种或多种含有产生所述颗粒所需组分的质粒载体转染包装细胞系。在一些实施方案中，用含有病毒载体基因组(包括LTR、顺式作用包装序列和目标序列，即编码抗原受体(例如CAR)的核酸)的质粒；以及编码病毒酶和/或结构组分(例如Gag、pol和/或rev)的一种或多种辅助质粒转染包装细胞系。
在一些实施方案中，包装细胞系提供将病毒基因组RNA包装至慢病毒载体颗粒中反式作用所需的组分，包括病毒调节蛋白和结构蛋白。在一些实施方案中，包装细胞系可以是能够表达慢病毒蛋白并产生功能性慢病毒载体颗粒的任何细胞系。在一些方面，合适的包装细胞系包括293(ATCC CCL X)、293T、HeLA(ATCC CCL 2)、D17(ATCC CCL 183)、MDCK(ATCC CCL34)、BHK(ATCC CCL-10)和Cf2Th(ATCC CRL 1430)细胞。
在一些实施方案中，将病毒载体和包装质粒和/或辅助质粒通过转染或感染引入包装细胞系中。包装细胞系产生含有病毒载体基因组的病毒载体颗粒。用于转染或感染的方法是熟知的。非限制性例子包括磷酸钙、DEAE-葡聚糖和脂质转染方法、电穿孔和显微注射。
在一些实施方案中，可以通过引入质粒以允许产生慢病毒颗粒，而在包装细胞系(例如示例性HEK 293T细胞系)中产生逆转录病毒载体，例如慢病毒载体。在一些实施方案中，包装细胞被转染和/或含有编码gag和pol的多核苷酸，以及编码重组受体(例如抗原受体，例如CAR)的多核苷酸。在一些实施方案中，包装细胞系被任选地和/或另外用编码rev蛋白的多核苷酸转染和/或含有所述多核苷酸。在一些实施方案中，包装细胞系被任选地和/或另外用编码非天然包膜糖蛋白(例如VSV-G)的多核苷酸转染和/或含有所述多核苷酸。在一些此类实施方案中，在转染细胞(例如，HEK 293T细胞)后大约两天，细胞上清液含有可以回收并滴定的重组慢病毒载体。
所回收和/或产生的逆转录病毒载体颗粒可以用于使用如所述的方法转导靶细胞。一旦进入靶细胞中，病毒RNA就被逆转录，进入细胞核中并稳定整合到宿主基因组中。在病毒RNA整合后一天或两天，可以检测到重组蛋白(例如抗原受体，例如CAR)的表达。
孵育
在一些实施方案中，所提供的方法涉及通过使包含多个细胞的输入组合物与(1)病毒颗粒接触(例如孵育)来转导细胞的方法。在一些实施方案中，输入组合物包含从受试者获得的原代细胞，例如从受试者富集和/或选择的细胞。
在一些实施方案中，输入组合物包含从受试者获得的原代细胞。在一些方面，样品是全血样品、血沉棕黄层样品、外周血单核细胞(PBMC)样品、未分级T细胞样品、淋巴细胞样品、白细胞样品、单采术产物或白细胞分离术产物。
在一些实施方案中，在细胞的选择和/或转导之前，使含有原代细胞的样品与以下浓度的血清或血浆离体接触或含有所述血清或血浆：至少或至少约10％(v/v)、至少或至少约15％(v/v)、至少或至少约20％(v/v)、至少或至少约25％(v/v)、至少或至少约30％(v/v)、至少或至少约35％(v/v)、至少或至少约40％(v/v)、或者至少或至少约50％。在一些实施方案中，样品含有血清或血浆，所述血清或血浆的浓度为或大致为约或至少约25％、26％、27％、28％、29％、30％、31％、32％、33％、34％或35％(v/v)。在一些实施方案中，血清或血浆是人的。在一些实施方案中，血清或血浆对于受试者是自体的。在一些实施方案中，在选择和/或转导细胞之前，使含有原代细胞的样品与抗凝血剂接触或含有抗凝血剂。在一些实施方案中，抗凝血剂是或含有游离柠檬酸根离子，例如，抗凝血剂柠檬酸盐右旋糖溶液，溶液A(ACD-A)。
在一些实施方案中，在细胞的选择和/或转导之前，将样品维持在为2℃至8℃的温度下长达48小时，例如长达12小时、24小时或36小时。
在一些实施方案中，输入组合物包含和/或富含T细胞，所述T细胞包括CD4+和/或CD8+T细胞。在一些方面，富集可以通过基于亲和力的选择，通过将原代细胞与一种或多种选择或亲和试剂一起孵育来进行，所述选择或亲和试剂特异性地结合至原代细胞亚群上表达的细胞表面分子，从而基于与选择试剂的结合来富集原代细胞。在一些实施方案中，富集可以通过将细胞与抗体包被的颗粒(例如微珠、聚合纳米基质)一起孵育来进行。
在一些实施方案中，输入组合物包含大于或大于约75％、80％、85％、90％、95％或更多的从受试者的样品获得的T细胞。在一些方面，在孵育之前，输入组合物中不超过5％、10％、20％、30％或40％的T细胞是激活细胞，表达选自HLA-DR、CD25、CD69、CD71、CD40L和4-1BB的表面标记；包含选自IL-2、IFN-γ、TNF-α的细胞因子的细胞内表达，处于细胞周期的G1期或较后期，和/或能够增殖。
在一些实施方案中，在孵育和/或接触期间或在孵育和/或接触的至少一部分期间，输入组合物可以包含一种或多种细胞因子。在一些实施方案中，所述细胞因子选自IL-2、IL-7或IL-15。在一些实施方案中，所述细胞因子是重组细胞因子。在一些实施方案中，输入组合物中细胞因子的浓度独立地为1IU/mL至1500IU/mL，例如为1IU/mL至100IU/mL、2IU/mL至50IU/mL、5IU/mL至10IU/mL、10IU/mL至500IU/mL、50IU/mL至250IU/mL或100IU/mL 至200IU/mL、50IU/mL至1500IU/mL、100IU/mL至1000IU/mL或200IU/mL至600IU/mL。在一些实施方案中，输入组合物中细胞因子的浓度独立地为至少或至少约1IU/mL、5IU/mL、10IU/mL、50IU/mL、100IU/mL、200IU/mL、500IU/mL、1000IU/mL或1500IU/mL。在一些方面，也可以在孵育期间或在孵育的至少一部分期间或在孵育之后，包括能够激活TCR复合物的细胞内信号传导结构域的药剂(例如抗CD3和/或抗CD28抗体)。
在一些实施方案中，在孵育和/或接触期间或在孵育和/或接触的至少一部分期间，输入组合物可以包含血清。在一些实施方案中，所述血清是人血清。在一些实施方案中，所述血清是以为0.5％至25％(v/v)、1.0％至10％(v/v)或2.5％至5.0％(v/v)的浓度存于输入组合物中，每个都包含端值。
在一些实施方案中，在孵育和/或接触期间或在孵育和/或接触的至少一部分期间，输入组合物不含和/或基本上不含血清。在一些实施方案中，在孵育和/或接触期间或在孵育和/或接触的至少一部分期间，输入组合物是在血清不存在下进行孵育和/或接触。在特定实施方案中，在孵育和/或接触期间或在孵育和/或接触的至少一部分期间，输入组合物是在无血清培养基中进行孵育和/或接触。在一些实施方案中，无血清培养基是定义的和/或明确定义的细胞培养基。在一些实施方案中，配制无血清培养基以支持某种细胞类型(例如免疫细胞、T细胞和/或CD4+和CD8+T细胞)的细胞的生长、增殖、健康、稳态。
在一些实施方案中，输入组合物的细胞浓度为1.0x10 5个细胞/mL至1.0x10 10滴度在一些实施方案中，转导可以按小于100、例如通常小于60、50、40、30、20、10、5、4、3、2、1或更小的感染复数(MOI)实现。
在一些实施方案中，所述方法涉及使细胞与病毒颗粒接触或孵育，例如混合。在一些实施方案中，接触进行30分钟至72小时，例如30分钟至48小时、30分钟至24小时或1小时至24小时，例如至少30分钟、1小时、2小时、6小时、12小时、24小时、36小时或更长时间。
在一些实施方案中，接触是在溶液中进行。在一些实施方案中，使细胞和病毒颗粒以为0.5mL至500mL的体积接触，所述体积例如为0.5mL至200mL、0.5mL至100mL、0.5mL至50mL、0.5mL至10mL、0.5mL至5mL、5mL至500mL、5mL至200mL、5mL至100mL、5mL至50mL、5mL至10mL、10mL至500mL、10mL至200mL、10mL至100mL、10mL至50mL、50mL至500mL、50mL至200mL、50mL至100mL、100mL至500mL、100mL至200mL或200mL至500mL。
在一些实施方案中，接触可以通过离心、例如旋转接种(例如离心接种)来实现。在一些实施方案中，可以使含有细胞、病毒颗粒和试剂的组合物旋转，通常以相对较低的力或速度旋转，例如速度低于用于沉淀细胞的速度，例如为600rpm至1700rpm(例如，为至少600rpm、1000rpm或1500rpm或1700rpm)。在一些实施方案中，旋转是以为100g至3200g(例如，为至少为100g、200g、300g、400g、500g、1000g、1500g、2000g、2500g、3000g或3200g)的力(例如，相对离心力)来进行，如例如在室或空腔的内壁或外壁处所测量。术语“相对离心力”或RCF通常被理解为在如与旋转轴相比在空间的特定点处，相对于地球的重力，施 加在物体或物质(例如细胞、样品或团粒和/或所旋转的室或其他容器中的点)上的有效力。所述值可以使用熟知的公式来确定，所述公式考虑到重力、旋转速度和旋转半径(与旋转轴的距离以及测量RCF的物体、物质或颗粒)。
在一些实施方案中，细胞与病毒载体颗粒的孵育导致或产生包含用病毒载体颗粒转导的细胞的输出组合物。
在一些实施方案中，在进一步孵育之后，制备细胞的过程可以还包括洗涤或配制细胞。因此，处理步骤中可包括配制此类组合物。
在一些实施方案中，将细胞和组合物以药物组合物或配制品的形式(例如包含细胞或细胞群和药学上可接受的载体或赋形剂的组合物)给予至受试者。
术语“药物配制品”是指这样的制剂，其处于使得其中所含活性成分的生物活性有效的形式，并且不含对给予配制品的受试者具有不可接受的毒性的另外的组分。
在一些实施方案中，药物组合物另外包含其他药学活性药剂或药物，例如化学治疗剂，例如天冬酰胺酶、白消安、卡铂、顺铂、柔红霉素、多柔比星、氟尿嘧啶、吉西他滨、羟基脲、甲氨蝶呤、紫杉醇、利妥昔单抗、长春碱、长春新碱等。在一些实施方案中，所述药剂是以盐形式例如药学上可接受的盐给予。合适的药学上可接受的酸加成盐包括源自无机酸的那些，所述无机酸是如盐酸、氢溴酸、磷酸、偏磷酸、硝酸和硫酸，以及源自有机酸的那些，所述有机酸是如酒石酸、乙酸、柠檬酸、苹果酸、乳酸、富马酸、苯甲酸、乙醇酸、葡萄糖酸、琥珀酸和芳基磺酸(例如，对甲苯磺酸)。
“药学上可接受的载体”是指药物配制品中除了活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲液、赋形剂、稳定剂或防腐剂。
在一些方面，载体的选择部分取决于特定细胞和/或给予方法。因此，存在多种合适的配制品。例如，所述药物组合物可以含有防腐剂。合适的防腐剂可以包括例如对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸钠和苯扎氯铵。在一些方面，使用两种或更多种防腐__剂的混合物。所述防腐剂或其混合物通常以按总组合物的重量计约0.0001％至约2％的量存在。药学上可接受的载体在所用的剂量和浓度下通常对接受者无毒，并且包括但不限于：缓冲剂，如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如十八烷基二甲基苄基氯化铵；六甲氯铵；苯扎氯铵；苄索氯铵；苯酚、丁醇或苄醇；烷基对羟基苯甲酸酯，如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖类，如蔗糖、甘露醇、海藻糖或山梨糖醇；成盐抗衡离子，如钠；金属络合物(例如锌-蛋白质络合物)；和/或非离子表面活性剂，如聚乙二醇(PEG)。
在一些方面，缓冲剂被包括在所述组合物中。合适的缓冲剂包括例如柠檬酸、柠檬酸钠、磷酸、磷酸钾和各种其他酸和盐。在一些方面，使用两种或更多种缓冲剂的混合物。所述缓冲剂或其混合物通常以按总组合物的重量计约0.001％至约4％的量存在。用于制备可给 予的药物组合物的方法是已知的。
配制品可以包括水溶液。所述配制品或组合物还可含有可用于用细胞治疗的特定适应症、疾病的多于一种活性成分，优选具有与所述细胞互补的活性的那些成分，其中各自的活性不会相互产生不利影响。此类活性成分以有效用于既定目的的量以合适的方式组合存在。因此，在一些实施方案中，药物组合物还包括其他药物活性药剂或药物，例如化学治疗剂，例如天冬酰胺酶、白消安、卡铂、顺铂、柔红霉素、多柔比星、氟尿嘧啶、吉西他滨、羟基脲、甲氨蝶呤、紫杉醇、利妥昔单抗、长春碱和/或长春新碱。
在一些实施方案中，药物组合物包含有效治疗或预防疾病的量(例如治疗有效量或预防有效量)的细胞。在一些实施方案中，通过定期评估所治疗的受试者来监测治疗或预防功效。所需剂量可以通过细胞的单次推注给予、通过细胞的多次推注给予或通过细胞的连续输注给予来递送。
在一些实施方案中，组合物作为无菌液体制剂(例如，等渗水溶液、悬浮液、乳液、分散体或粘性组合物，其在一些方面可以缓冲至所选pH)提供。液体制剂一般比凝胶、其他粘性组合物和固体组合物制备起来更容易。液体或粘性组合物可以包含载体，其可以是溶剂或分散介质，其含有例如水、盐水、磷酸盐缓冲盐水、多元醇(例如，甘油、丙二醇、液体聚乙二醇)及其合适的混合物。
无菌可注射溶液可以通过将细胞掺入溶剂中来制备。
可以添加各种增强所述组合物的稳定性和无菌性的添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。
用于给予的治疗方法和组合物
在一些方面，所述方法的产物用于治疗方法，例如，治疗性方法，例如用于在过继细胞疗法中将细胞和组合物给予至受试者。还提供了此类方法和通过所述方法处理和产生的细胞的用途，以及用于其中的药物组合物和配制品。所提供的方法通常涉及将细胞或组合物(例如输出组合物和/或配制的组合物)给予至受试者。
在一些实施方案中，所述细胞表达重组受体(例如CAR)或其他抗原受体(例如转基因TCR)。通常将此类细胞给予至患有与由所述受体特异性识别的配体相关的疾病的受试者。在一个实施方案中，所述细胞表达重组受体或嵌合受体(例如抗原受体，例如CAR或TCR)，所述受体特异性地结合至与疾病相关或由其细胞或组织表达的配体。例如，在一些实施方案中，所述受体是抗原受体，并且所述配体是疾病特有的和/或与所述疾病相关的抗原。给予通常实现疾病的一种或多种症状的改善和/或治疗或预防疾病或其症状。
疾病、病症包括肿瘤，包括实体瘤、血液学恶性肿瘤和黑色素瘤，并且包括局部和转移性肿瘤；感染性疾病，如病毒或其他病原体的感染，例如HIV、HCV、HBV、CMV和寄生虫病；以及自身免疫和炎性疾病。在一些实施方案中，疾病是肿瘤、癌症、恶性肿瘤、肿疡或其他增殖性疾病。此类疾病包括但不限于白血病、淋巴瘤(例如慢性淋巴细胞白血病
(CLL)、ALL、非霍奇金淋巴瘤、急性髓样白血病、多发性骨髓瘤、难治性滤泡性淋巴瘤、 套细胞淋巴瘤、惰性B细胞淋巴瘤、B细胞恶性肿瘤)、结肠癌、肺癌、肝癌、乳腺癌、前列腺癌、卵巢癌、皮肤癌、黑色素瘤、骨癌和脑癌、卵巢癌、上皮癌、肾细胞癌、胰腺癌、霍奇金淋巴瘤、宫颈癌、结直肠癌、胶质母细胞瘤、神经母细胞瘤、尤因肉瘤、髓母细胞瘤、骨肉瘤、滑膜肉瘤和/或间皮瘤。
在一些实施方案中，此类疾病包括但不限于白血病、淋巴瘤，例如急性髓样(或髓性)白血病(AML)、慢性髓样(或髓性)白血病(CML)、急性淋巴细胞(或成淋巴细胞)白血病(ALL)、慢性淋巴细胞白血病(CLL)、毛细胞白血病(HCL)、小淋巴细胞淋巴瘤(SLL)、套细胞淋巴瘤(MCL)、边缘区淋巴瘤、伯基特淋巴瘤、霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL))、间变性大细胞淋巴瘤(ALCL)、滤泡性淋巴瘤、难治性滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤(DLBCL)和多发性骨髓瘤(MM)。在一些实施方案中，疾病是选自以下各项的B细胞恶性肿瘤：急性成淋巴细胞性白血病(ALL)、成人ALL、慢性成淋巴细胞性白血病(CLL)、非霍奇金淋巴瘤(NHL)和弥漫性大B细胞淋巴瘤(DLBCL)。在一些实施方案中，疾病是NHL，并且NHL选自侵袭性NHL、弥漫性大B细胞淋巴瘤(DLBCL)、NOS(从头的和从惰性转化的)、原发性纵隔大B细胞淋巴瘤(PMBCL)、富含T细胞/组织细胞的大B细胞淋巴瘤(TCHRBCL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)和/或滤泡性淋巴瘤(FL)，任选地，3B级滤泡性淋巴瘤(FL3B)。
在一些实施方案中，疾病是感染性疾病，例如但不限于病毒、逆转录病毒、细菌和原生动物感染、免疫缺陷、巨细胞病毒(CMV)、埃-巴二氏病毒(Epstein-Barrvirus，EBV)、腺病毒、BK多瘤病毒。在一些实施方案中，所述疾病是自身免疫性或炎性疾病，如关节炎(例如类风湿性关节炎(RA))、I型糖尿病、系统性红斑狼疮(SLE)、炎性肠病、银屑病、硬皮病、自身免疫性甲状腺疾病、格雷夫斯病、克罗恩病、多发性硬化症、哮喘和/或与移植相关的疾病。
如本文所用，“治疗”是指疾病、或者与之相关的症状、不良效果或结果或表型的完全或部分改善或减轻。理想的治疗效果包括但不限于预防疾病的发生或复发、症状的缓解、疾病的任何直接或间接病理后果的减少、预防转移、降低疾病进展的速度、改善或缓解疾病状态以及缓解或改善预后。所述术语并不暗示完全治愈疾病或完全消除任何症状或对所有症状或结果的影响。
如本文所用，“延迟疾病的发展”意指推迟、阻碍、减缓、延缓、稳定、抑制和/或延期疾病(例如癌症)的发展。此延迟可以具有不同的时间长度，这取决于病史和/或所治疗的个体。对于本领域技术人员显而易见的是，足够或显著的延迟实际上可以涵盖预防，因为个体不会患上疾病。例如，可能延迟晚期癌症，如转移的发展。
如本文所用，“预防”包括提供关于受试者的疾病的发生或复发的预防，所述受试者可能易患所述疾病但尚未被诊断患有所述疾病。在一些实施方案中，所提供的细胞和组合物用于延迟疾病的发展或延缓疾病的进展。
如本文所用，“抑制”功能或活性是当与除了感兴趣的条件或参数以外的原本相同的条件相比时，或者与另一种条件相比时，减少功能或活性。例如，与不存在所述细胞的情况 下的肿瘤生长速率相比，抑制肿瘤生长的细胞降低了肿瘤的生长速率。
用于过继细胞疗法的细胞的给予方法是已知的，并且可以与所提供的方法和组合物一起使用。
所治疗的疾病可以是任何疾病，其中抗原的表达与疾病状况的病因学相关和/或参与其中，例如导致、加剧这种疾病、病症或以其他方式参与其中。示例性疾病和病症可以包括与恶性肿瘤或细胞转化(例如癌症)、自身免疫性疾病或炎性疾病或例如由细菌、病毒或其他病原体引起的感染性疾病相关的疾病。上文描述了示例性抗原，其包括与可以治疗的各种疾病和病症相关的抗原。在具体实施方案中，所述嵌合抗原受体或转基因TCR与和所述疾病相关的抗原特异性结合。
细胞和组合物可以使用标准给予技术、配制品和/或装置来给予。细胞给予可以是自体的或异源的，例如同种异体的。例如，免疫应答细胞或祖细胞可以获得自一名受试者，并且给予至同一受试者或不同的相容受试者。外周血源免疫应答细胞或其后代(例如，体内、离体或体外衍生的)可以通过局部注射给予，包括导管给予、全身注射、局部注射、静脉内注射或肠胃外给予。在给予治疗性组合物(例如，含有遗传修饰的免疫应答细胞的药物组合物)时，通常将其配制成单位剂量可注射形式(溶液、悬浮液、乳液)。
在一些实施方案中，细胞疗法(例如过继细胞疗法，例如过继T细胞疗法)是通过自体转移来进行，从要接受细胞疗法的受试者或从源自这个受试者的样品分离和/或以其他方式制备细胞。因此，在一些方面，细胞源自需要治疗的受试者(例如，患者)和细胞，并且在分离和处理后，将细胞给予至同一受试者。
所述细胞可以通过任何合适的方式给予，例如通过推注输注，通过注射例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房注射、结膜下(subconjectval)注射、结膜下(subconjuntival)注射、眼球筋膜囊下(sub-Tenon)注射、球后注射、球周注射或后近巩膜(posteriorjuxtascleral)递送。在一些实施方案中，它们通过肠胃外、肺内和鼻内给予以及(如果需要用于局部治疗的话)病灶内给予。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给予。在一些实施方案中，给定剂量是通过细胞的单次推注给予、通过细胞的多次推注给予、或通过细胞的连续输注给予。
在过继细胞疗法的中，给定“剂量”的细胞的给予包括以单一组合物和/或单次不间断给药的方式(例如以单次注射或连续输注的方式)给予给定量或数量的细胞，并且还包括在例如不超过3天的指定时间段内以在多个单独组合物或输注中提供的分割剂量或多个组合物的方式给予给定量或数量的细胞。因此，在一些情况下，剂量是指定数量的细胞的单次或连续给药，在单个时间点给予或开始。然而，在一些情况下，剂量在不超过三天的时间段内以多次注射或输注的方式给予，如每天一次持续三天或两天或者通过在一天的时间内多次输注。
因此，在一些方面，所述剂量的细胞以单一药物组合物给予。在一些实施方案中，所述剂量的细胞以共同含有所述剂量的细胞的多种组合物给予。
术语“分割剂量”是指分割的剂量，使其在超过一天的时间内给予。这种类型的给药包括在本方法中并且被认为是单一剂量。在一些实施方案中，分割剂量的细胞在不超过三天 的时间段内以共同包含剂量的细胞的多种组合物来给予。
在一些实施方案中，所述剂量的细胞可以通过给予多个组合物或溶液(例如第一和第二，任选地更多)来给予，各自含有所述剂量的一些细胞。在一些方面，任选地在一定时间段内，分开地或独立地给予多个组合物，每个组合物含有不同细胞群和/或细胞亚型。例如，细胞群或细胞亚型可以分别包括CD8 +和CD4 +T细胞，和/或分别包含富集CD8 +和CD4 +的群体，例如CD4 +和/或CD8 +T细胞，其各自单独地包括基因工程化以表达重组受体的细胞。在一些实施方案中，所述剂量的给予包括给予第一组合物，其包含一定剂量的CD8 +T细胞或一定剂量的CD4 +T细胞，以及给予第二组合物，其包含另一剂量的CD4 +T细胞和CD8 +T细胞。
在一些实施方案中，细胞的剂量或组合物包括表达重组受体的CD4+细胞与表达重组受体的CD8 +细胞和/或CD4 +细胞与CD8+细胞的定义的或目标比率，所述比率任选地为约1:1，或者在大约1:3与大约3:1之间，例如大约1:1。在一些方面，具有目标或所需比率的不同细胞群(例如CD4+:CD8+比率或CAR+CD4+:CAR+CD8+比率，例如1:1)的组合物或剂量的给予涉及给予含有一个所述群体的细胞组合物，和随后给予包含另一所述群体的单独细胞组合物，其中所述给予是以或大约以目标或所需比率来进行。在一些方面，定义比率的细胞的剂量或组合物的给予导致改善T细胞疗法的扩增、持久性和/或抗肿瘤活性。
在一些实施方案中，细胞以所需剂量给予，所述所需剂量在一些方面包括所需剂量或数量的细胞或一种或多种细胞类型和/或所需比率的细胞类型。因此，在一些实施方案中，细胞剂量基于细胞总数(或每kg体重的细胞数量)和所需的单独群体或亚型的比率，如CD4+与CD8+的比率。在一些实施方案中，细胞剂量基于所需的单独群体中的细胞或单独细胞类型的总数(或每kg体重的细胞数量)。在一些实施方案中，剂量基于这种特征的组合，如所需的总细胞数量、所需比率和所需的单独群体中的细胞总数。
在一些实施方案中，细胞在多种细胞群或亚型(如CD4+和CD8+细胞或亚型)的所需输出比率的耐受范围下或耐受范围内给予。在一些方面，所需比率可以是特定比率或可以是一系列比率。例如，在一些实施方案中，CD4+与CD8+细胞的比率在1:5和5:1之间，或在1:3和3:1之间，如在2:1和1:5之间在一些方面，耐受差异在所需比率的约1％、约2％、约3％、约4％、约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％，包括这些范围之间的任何值。
在具体实施方案中，细胞的数量和/或浓度是指表达重组受体(例如，CAR)的细胞的数量。在其他实施方案中，细胞的数量和/或浓度是指给予的所有细胞、T细胞或外周血单核细胞(PBMC)的数量或浓度。
在一些方面，剂量的大小基于一个或多个标准来确定，如受试者对现有治疗例如化学疗法的反应、受试者的疾病负荷如肿瘤负担、体积、尺寸或程度、转移的程度或类型、分期和/或受试者发生毒性结果的可能性或发生率，例如CRS、巨噬细胞激活综合征、肿瘤溶解综合征、神经毒性和/或针对所给予的细胞和/或重组受体的宿主免疫应答。
在一些实施方案中，可以给予相对较低剂量的细胞，例如次最佳剂量的细胞或低于治疗有效量的剂量的细胞，其在体内刺激(例如通过内源性抗原或外源性药剂)时可以导致受试 者中存在的工程化细胞的数量的加强(例如增加或扩增)。在任何此类实施方案中，细胞的扩增和/或激活可以与在体内暴露于抗原一起发生，例如，在给予细胞后，受试者体内工程化细胞的扩增。在一些实施方案中，体内扩增的范围、程度或量值可以通过多种方法来扩大、加强或增强，所述方法能够调节(例如增加)所给予细胞(例如表达重组受体的细胞)的扩增、增殖、存活和/或功效。
一旦将细胞给予至受试者(例如人)，在一些方面通过许多已知方法中的任何一种来测量细胞群的生物活性。要评价的参数包括细胞与抗原的特异性结合，在体内例如通过成像来评价，或离体例如通过ELISA或流式细胞术来评价。在某些实施方案中，细胞破坏靶细胞的能力可以使用本领域已知的任何合适的方法来测量。在某些实施方案中，还可以通过测定某些细胞因子如CD107a、IFNγ、IL-2和TNF的表达和/或分泌来测量细胞的生物活性。在一些方面，通过评估临床结果(如肿瘤负荷或负担的减少)来测量生物活性。在一些方面，评估毒性结果、细胞的持久性和/或扩增和/或宿主免疫应答的存在或不存在。
组合物和配制品
在一些实施方案中，以组合物或配制品例如药物组合物或配制品的方式提供包含用重组抗原受体例如CAR或TCR工程化的细胞。此类组合物可以根据所提供的方法和/或与所提供的制品或组合物一起使用，例如用于预防或治疗疾病、病症，或用于检测、诊断和预后方法。
术语“药物配制品”是指这样的制剂，其处于使得其中所含活性成分的生物活性有效的形式，并且不含对给予配制品的受试者具有不可接受的毒性的另外的组分。
“药学上可接受的载体”是指药物配制品中除了活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲液、赋形剂、稳定剂或防腐剂。
在一些方面，载体的选择部分地由特定细胞或药剂和/或通过给药方法确定。因此，存在多种合适的配制品。例如，所述药物组合物可以含有防腐剂。合适的防腐剂可以包括例如对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸钠和苯扎氯铵。在一些方面，使用两种或更多种防腐剂的混合物。药学上可接受的载体在所用的剂量和浓度下通常对接受者无毒，并且包括但不限于：缓冲剂，如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如十八烷基二甲基苄基氯化铵；六甲氯铵；苯扎氯铵；苄索氯铵；苯酚、丁醇或苄醇；烷基对羟基苯甲酸酯，如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖类，如蔗糖、甘露醇、海藻糖或山梨糖醇；成盐抗衡离子，如钠；金属络合物(例如锌-蛋白质络合物)；和/或非离子表面活性剂，如聚乙二醇(PEG)。
在一些方面，缓冲剂被包括在所述组合物中。合适的缓冲剂包括例如柠檬酸、柠檬酸钠、磷酸、磷酸钾和各种其他酸和盐。在一些方面，使用两种或更多种缓冲剂的混合物。所述缓冲剂或其混合物通常以按总组合物的重量计约0.001％至约4％的量存在。用于制备可给予的药物组合物的方法是已知的。
配制品或组合物还可含有多于一种活性成分，其可用于用细胞或药剂预防或治疗的特定适应症、疾病，其中各自的活性不会相互产生不利影响。此类活性成分以有效用于既定目的的量以合适的方式组合存在。因此，在一些实施方案中，药物组合物进一步包含其他药物活性剂或药物如化学治疗剂，例如天冬酰胺酶、白消安、卡铂、顺铂、柔红霉素、多柔比星、氟尿嘧啶、吉西他滨、羟基脲、甲氨蝶呤、紫杉醇、利妥昔单抗、长春碱、长春新碱等。在一些实施方案中，所述药剂或细胞以盐(例如药学上可接受的盐)的形式给予。合适的药学上可接受的酸加成盐包括源自无机酸(如盐酸、氢溴酸、磷酸、偏磷酸，硝酸和硫酸)和有机酸(如酒石酸、乙酸、柠檬酸、苹果酸、乳酸、富马酸、苯甲酸、乙醇酸、葡萄糖酸、琥珀酸和芳基磺酸，例如对甲苯磺酸)的那些盐。
在一些实施方案中，药物组合物含有有效治疗或预防疾病的量(如治疗有效量或预防有效量)的药剂或细胞。在一些实施方案中，通过定期评估所治疗的受试者来监测治疗或预防功效。对于数天或更长时间的重复给予，取决于病症，重复所述治疗直至出现所需疾病症状的抑制。然而，其他剂量方案可能是有用的并且可以被确定。所需剂量可以通过单次推注给予所述组合物、通过多次推注给予所述组合物或通过连续输注给予所述组合物来递送。
所述药剂或细胞可以通过任何合适的方式给予，例如通过推注输注，通过注射例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房注射、结膜下注射、眼球筋膜囊下注射、球后注射、球周注射或后近巩膜递送。在一些实施方案中，它们通过肠胃外、肺内和鼻内给予以及病灶内给予。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给予。在一些实施方案中，给定剂量通过细胞或药剂的单次推注给药来给予。在一些实施方案中，给定剂量是通过例如在不超过3天的时间段内对细胞或药剂的多次推注给予或通过细胞或药剂的连续输注给予。
对于疾病的预防或治疗，适当的剂量可取决于待治疗的疾病类型、一种或多种药剂的类型、细胞或重组受体的类型、疾病的严重程度和病程、给予药剂或细胞用于预防性目的还是治疗性目的、先前疗法、受试者的临床病史和对药剂或细胞的反应、以及主治医师的决断。在一些实施方案中，所述组合物适合一次或在一系列治疗中给予受试者。
可以使用标准给药技术、配制品和/或设备给予细胞或药剂。提供了用于储存和给予所述组合物的配制品和装置(如注射器和小瓶)。关于细胞，给药可以是自体的或异源的。例如，免疫应答细胞从受试者获得，并且给予至同一受试者或不同的相容受试者。外周血衍生的免疫应答细胞或其后代可以经由局部注射给予，包括导管给药、全身注射、局部注射、静脉内注射或肠胃外给药。当给予治疗性组合物(例如，含有基因修饰的免疫应答细胞或治疗或改善神经毒性症状的药剂的药物组合物)时，通常将其配制成单位剂量可注射形式。
在一些实施方案中，所给予的细胞(例如工程化以表达重组受体的细胞)被修饰以扩大、加强或增强所给予细胞的扩增、增殖、存活和/或功效。在一些实施方案中，所给予的细胞(例如，工程化以表达重组受体的细胞)被修饰，使得可以例如通过给予药剂来调节和/或控制工程化细胞的扩增、增殖、存活和/或功效。
在一些实施方案中，所述方法包括减少、抑制和/或最小化抑制因子在体内抑制工程化 细胞的增殖、扩增和/或存活的效应的体内步骤。在一些实施方案中，所述方法包括促进、支持和/或增强工程化细胞在体内的增殖、扩增和/或存活的体内步骤。
在一些实施方案中，另外的药剂是小分子、肽、多肽、抗体或其抗原结合片段、抗体模拟物、适体或核酸分子(例如siRNA)、脂质、多糖或其任一组合。在一些实施方案中，另外的药剂是特定因子、分子、受体、功能和/或酶的抑制剂或激活剂。在一些实施方案中，另外的药剂是特定因子、分子、受体、功能和/或酶的激动剂或拮抗剂。在一些实施方案中，另外的药剂是一种或多种因子和/或代谢物的类似物或衍生物。在一些实施方案中，另外的药剂是蛋白质或多肽。在一些实施方案中，另外的药剂是细胞，例如工程化细胞。
用于转基因特异性扩增的药剂
在一些实施方案中，所述方法包括例如在组合疗法中给予除了所给予细胞(例如工程化以表达重组受体的细胞)以外的药剂。在一些实施方案中，所述药剂由于特异性调节转基因(例如编码重组受体的转基因)而特异性扩大、加强或增强工程化细胞的扩增、增殖、存活和/或功效。在一些实施方案中，所述药剂特异性靶向转基因，例如重组受体。在一些实施方案中，所述药剂特异性结合、激活和/或增强重组受体的活性和/或由转基因编码的全部或部分重组分子的其他功能。在一些实施方案中，将药剂与重组细胞组合给予可以增强、加强或扩大所给予细胞的增殖、扩增和/或存活，例如，增强细胞的体内扩增。
在一些实施方案中，用于转基因特异性扩增的示例性方法或药剂包括内源性抗原暴露、疫苗接种、抗独特型抗体或其抗原结合片段和/或可调节的重组受体。例如，在一些实施方案中，用于转基因特异性扩增的方法包括疫苗接种方法。在一些实施方案中，用于转基因特异性扩增的方法包括给予抗独特型抗体。抗独特型抗体(包括其抗原结合片段)特异性地识别、特异性地靶向和/或特异性地结合至抗体或其抗原结合片段(例如重组受体(如嵌合抗原受体(CAR))的抗原结合结构域)的独特位。独特位是抗体可变部分内的任何单一抗原决定簇或表位。在一些实施方案中，抗独特型抗体或其抗原结合片段是激动剂和/或展现刺激细胞表达特定抗体的特异性活性，所述特定抗体包括含有所述抗体或其抗原结合片段的缀合物或重组受体。
在一些实施方案中，所述方法包括对免疫细胞或免疫功能(通常包括所给予的工程化细胞)的扩增、增殖、存活和/或活性的调节。在一些实施方案中，所述方法包括通常是免疫刺激性或通常促进、增强、扩大和/或加强免疫细胞(包括所给予的细胞)在体内(例如在受试者体内)的扩增、增殖、存活和/或活性的步骤。在一些实施方案中，所述药剂可以减少、抑制和/或最小化抑制因子在体内抑制免疫细胞(例如所给予细胞)的增殖、扩增和/或存活的效应。
在一些实施方案中，所述方法包括调节工程化细胞的扩增，例如，通过抑制所给予细胞(例如工程化免疫细胞)的增殖、扩增和/或激活的负调节物。在受试者体内的特定环境中，所给予的表达重组受体的细胞可能遇到压抑或抑制细胞生长、增殖、扩增和/或存活的环境，例如免疫抑制环境。例如，免疫抑制环境可以含有免疫抑制性细胞因子、调节性调节剂和共抑制受体。
在一些实施方案中，另外的药剂包括免疫调节剂、免疫检查点抑制剂、代谢途径调节剂、腺苷途径或腺苷受体拮抗剂或激动剂以及信号传导途径的调节剂(例如激酶抑制剂)。
在一些实施方案中，另外的药剂是免疫调节剂，例如免疫检查点抑制剂。在一些实例中，另外的药剂增加、增强或扩大所给予细胞的扩增和/或增殖，从而通过阻断免疫检查点蛋白(即免疫检查点抑制剂)来增加、增强或扩大免疫应答。在一些实施方案中，另外的药剂是增强工程化细胞(例如重组受体表达细胞)的活性的药剂，是抑制免疫抑制分子或免疫检查点分子的分子。免疫抑制分子的例子包括PD-1、PD-L1、CTLA4、TEVI3、CEACAM(例如、CEACAM-1、CEACAM-3和/或CEACAM-5)、LAG3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4和TGFRβ。
在一些实施方案中，免疫检查点抑制剂可以是针对免疫检查点蛋白的抗体，例如针对细胞毒性T淋巴细胞抗原4(CTLA4或CD152)、程序性细胞死亡蛋白1(PD-1)或程序性细胞死亡蛋白1配体1(PD-L1)的抗体。
在一些实施方案中，所述方法包括使表达重组受体的细胞与抑制抑制性细胞表面受体(例如转化生长因子β受体(TGFβR))的药剂接触。在一些实施方案中，所给予的细胞(例如重组受体表达细胞)可以被工程化以抵抗可以抑制其效应子功能的免疫抑制性细胞因子的效应。在一些实施方案中，另外的药剂是抗TGFβ抗体或抗TGFβR抗体。
在一些实施方案中，另外的药剂调节免疫抑制因子(例如腺苷)的代谢、信号传导和/或转运。在一些实施方案中，另外的药剂是细胞外腺苷或腺苷受体的抑制剂，或引起细胞外腺苷水平降低或减小的药剂，例如防止细胞外腺苷形成、降解细胞外腺苷，使细胞外腺苷失活和/或减少细胞外腺苷的药剂。在一些实施方案中，另外的药剂是腺苷受体拮抗剂，例如A2a、A2b和/或A3受体。
在一些实施方案中，另外的药剂是腺苷受体拮抗剂或激动剂，例如腺苷受体A2a、A2b、A1和A3中的一种或多种的拮抗剂或激动剂。
在一些实施方案中，所述方法包括给予具有免疫刺激性的另外的药剂。在一些实施方案中，另外的药剂通常可以促进免疫细胞的增殖、扩增、存活和/或功效。在一些实施方案中，另外的药剂可以特异性地促进所给予细胞，例如重组受体表达细胞。在一些实施方案中，另外的药剂是细胞因子。在一些实施方案中，另外的药剂是配体。
在一些实施方案中，另外的药剂是免疫刺激性配体，例如CD40L。在一些实施方案中，另外的药剂是细胞因子，例如IL-2、IL-3、IL-6、IL-11、IL-7、IL-12、IL-15、IL-21、粒细胞巨噬细胞集落刺激因子(GM-CSF)、α、β或γ干扰素(IFN)和促红细胞生成素(EPO)。
在一些方面，所提供的方法可以还包括，例如在启动细胞(例如重组受体表达细胞)的给予之前或同时，给予一种或多种淋巴细胞清除。在一些实施方案中，淋巴细胞清除疗法包括给予环磷酰胺。在一些实施方案中，淋巴细胞清除疗法包括给予氟达拉滨。在一些实施方案中，不给予淋巴细胞清除疗法。
用免疫清除(例如，淋巴细胞清除)疗法预处理受试者可以改善过继细胞疗法(ACT)的效果。用淋巴细胞清除剂(包括环孢霉素和氟达拉滨的组合)预处理已经有效地改善转移的肿瘤浸润淋巴细胞(TIL)在细胞疗法中的功效，包括改善转移细胞的应答和/或持久性。
在一些实施方案中，提供的方法还涉及向受试者给予淋巴细胞清除疗法。在一些实施 方案中，所述方法涉及在给予细胞剂量之前，向受试者给予淋巴细胞清除疗法。在一些实施方案中，淋巴细胞清除疗法含有化学治疗剂。
在一些实施方案中，所述方法包括在给予细胞剂量之前向受试者给予预处理剂如淋巴细胞清除剂或化学治疗剂，如环磷酰胺、氟达拉滨或其组合。例如，可以在第一或后续剂量之前至少2天如至少3、4、5、6或7天向所述受试者给予预处理剂。在一些实施方案中，在给予细胞剂量之前不超过7天如不超过6天、5天、4天、3天或2天向受试者给予预处理剂。
在一些实施方案中，将所述受试者用在或在约20mg/kg与100mg/kg之间、如在或在约40mg/kg与80mg/kg之间的剂量的环磷酰胺进行预处理。
在一些实施方案中，氟达拉滨可以按单一剂量给予或者可以按多个剂量给予，如每天给药、每隔一天给药或每三天给药。在一些实施方案中，每天给予氟达拉滨，如持续1-5天，例如持续3至5天。在一些情况下，在开始细胞疗法之前每天向所述受试者给予约30mg/m2的氟达拉滨，持续3天。在一些实施方案中，环磷酰胺每天给予一次，持续一天或两天。
在一个示例性剂量方案中，在接受第一剂量之前，受试者接受环磷酰胺和氟达拉滨(cy/flu)的淋巴细胞清除预处理化疗，其是在第一剂量的表达CAR的细胞之前至少两天且通常在给予细胞之前不超过7天给予。在预处理治疗后，对受试者给予如上所述的表达CAR的T细胞的剂量。
在一些实施方案中，在输注细胞剂量之前给予预处理剂改进了治疗的结果。例如，在一些方面，预处理改进了用剂量治疗的功效或增加了表达重组受体的细胞(例如表达CAR的细胞，如表达CAR的T细胞)在受试者中的持久性。在一些实施方案中，预处理治疗增加了无病生存率，例如存活的受试者的百分比，并在细胞剂量之后的给定时间段后未展现出最小残留的或分子可检测的疾病。在一些实施方案中，增加了到中值无病生存率的时间。
在将所述细胞给予受试者(例如人)后，在一些方面工程化细胞群的生物活性通过许多已知方法中的任何一种来测量。待评估的参数包括工程化或天然T细胞或其他免疫细胞与抗原的特异性结合，其在体内例如通过成像进行评估，或离体例如通过ELISA或流式细胞术进行评估。在某些实施方案中，还可以通过测定某些细胞因子如CD107a、IFNγ、IL-2和TNF的表达和/或分泌来测量细胞的生物活性。在一些方面，通过评估临床结果(如肿瘤负荷或负担的减少)来测量生物活性。在一些方面，评估毒性结果、细胞的持久性和/或扩增和/或宿主免疫应答的存在或不存在。
在一些实施方案中，在输注细胞剂量之前给予预处理剂改进了治疗的结果(例如通过改进用剂量治疗的功效)，或增加了表达重组受体的细胞(例如表达CAR的细胞，如表达CAR的T细胞)在受试者中的持久性。
在某些实施方案中，细胞以任何数量的方式被修饰，使得增加其治疗或预防功效和/或可以调节扩增、增殖、存活和/或功效。在一些实施方案中，细胞被修饰使得在给予至受试者后可以在调节(例如增强、加强和/或扩大)扩增、增殖、存活和/或功效。在一些实施方案中，细胞被修饰使得可以调节和/或控制转基因和/或免疫调节因子的表达。在一些实施方案中，细胞被修饰以调节重组受体的特定组分的表达和/或活性。在一些实施方案中，细胞被修饰以 增加或减少药剂(例如，核酸，例如抑制性核酸)的表达。在一些实施方案中，细胞被修饰以表达和/或分泌药剂。
在一些实施方案中，工程化细胞表达的工程化重组受体(例如CAR)可以直接或者通过接头间接缀合至靶向部分。
在一些实施方案中，所述方法包括通过使细胞与药剂接触来调节所给予的细胞，所述药剂减少所给予细胞(例如表达重组受体的工程化T细胞)的负调节物的表达或能够实现所述表达减少。细胞的负调节物包括本文所述的任一种，例如免疫检查点抑制剂、抑制性受体和/或腺苷调节剂。在一些实施方案中，减少负调节物的表达或能够实现所述表达减少的药剂包括是或包含抑制性核酸分子(例如与编码负调节物的基因或核酸互补、靶向、抑制和/或结合所述基因或核酸的核酸分子)的药剂。在一些实施方案中，所述药剂是或包含复合物，所述复合物包含核糖核蛋白(RNP)复合物，所述核糖核蛋白(RNP)复合物包括Cas9(例如在一些情况下酶促失活的Cas9)和靶向编码负调节物的基因的gRNA。
在一些任何此类实施方案中，抑制性核酸分子包括RNA干扰剂。在一些任何此类实施方案中，抑制性核酸是或含有或编码小干扰RNA(siRNA)、微小RNA适应的shRNA、短发夹RNA(shRNA)、发夹siRNA、前体微小RNA(前miRNA)或微小RNA(miRNA)。
在一些实施方案中，使工程化细胞经历基因改变或基因编辑，其靶向编码参与免疫调节、免疫细胞的负调节和/或免疫抑制的基因的基因座。在一些实施方案中，基因编辑导致所靶向基因座处的插入或缺失，或所靶向基因座的“敲除”以及所编码蛋白质的表达的消除。在一些实施方案中，基因编辑是通过使用CRISPR/Cas9系统进行非同源末端接合(NHEJ)来实现的。在一些实施方案中，一种或多种引导RNA(gRNA)分子可以与一种或多种Cas9核酸酶、Cas9切口酶、酶促失活的Cas9或其变体或工程化的锌指或TALE系统一起使用。
在一些实施方案中，所述细胞(例如，重组受体表达细胞)被进一步修饰以表达和/或分泌另外的药剂，所述另外的药剂促进、增强、加强和/或扩大所给予的细胞的增殖、扩增、存活和/或功效。例如，重组受体表达细胞(例如表达CAR的细胞)可以进一步工程化以表达和/或分泌另外的药剂，所述另外的药剂克服免疫抑制效应和/或增强T细胞和重组受体的扩增和/或功能。在一些实施方案中，所述细胞可以工程化以表达促进所给予细胞的扩增的细胞因子。在一些实施方案中，此类另外的药剂可以与诱导型表达系统(例如诱导型启动子)可操作地连接。
在一些实施方案中，所给予的细胞可以被修饰以表达和/或分泌药剂，所述药剂抑制免疫抑制因子(例如本文所述的任一种)和/或刺激免疫刺激因子。在一些实施方案中，由给予的细胞表达的另外的药剂减少或防止所述细胞在肿瘤微环境中的免疫抑制。在一些实施方案中，由所给予的细胞编码和/或分泌的另外的药剂可以包括本文所述的任何另外的药剂。
在一些实施方案中，由所给予的细胞编码的另外的药剂是可溶的并且被分泌。在一些实施方案中，另外的药剂是可溶性scFv。在一些实施方案中，另外的药剂是细胞因子。
在一些实施方案中，所述方法包括修饰细胞以允许重组受体(例如CAR)的可调节的表达和/或活性，从而通过重组受体调节信号。在一些实施方案中，可调节的表达和/或活性是 通过将重组受体配置成含有特定调节元件和/或系统(例如本文所述的任一种)或受其控制来实现。在一些实施方案中，将工程化细胞给予至受试者体内和/或暴露于特定配体可以调节重组受体(例如CAR)的表达和/或活性。在一些实施方案中，重组受体的表达和/或活性的调节是通过给予可以调节重组受体(例如CAR)的表达的另外的药剂来实现。在一些实施方案中，重组受体(例如CAR)的受调节的表达是通过可调节的转录因子释放系统，或通过给予另外的药剂来实现，所述另外的药剂可以诱导多肽(例如重组受体)的构象变化和/或多聚化。在一些实施方案中，另外的药剂是化学诱导剂。
除非另外定义，否则本文使用的所有领域术语、符号和其他技术和科学术语或命名旨在具有与所要求保护的主题所属领域的普通技术人员通常理解的含义相同的含义。在一些情况下，为了清楚和/或为了便于参考而在本文中定义具有通常理解的含义的术语，并且本文中包含的此类定义不应被解释为表示与本领域通常理解的实质性差异。
术语“约”或“大约”是指本技术领域的技术人员容易知道的相应值的通常误差范围。本文对“约”某一值或参数的提及包括(并描述)针对所述值或参数本身的实施方案。例如，关于“约X”的描述包括“X”的描述。在一些实施例中，“约X”或“大约X”包括50％X-150％X范围、或60％X-140％X范围、或70％X-130％X范围、或80％X-120％X范围、或90％X-110％X范围、或95％-105％X、或97％X-103％X范围。例如，“约4％”或“大约4％”包括4％、或2％-6％、或2.4％-5.6％、或2.8％-5.2％、或3.2％-4.8％、或3.6％-4.4％、或3.88％-4.12％。
术语“受试者”包括任何活的生物体，如人和其他哺乳动物。哺乳动物包括但不限于人和非人动物，包括农场动物、运动动物、啮齿动物和宠物。
如本文所用，当提及一种或多种特定细胞类型或细胞群时，“富集”是指例如与组合物中的细胞总数或组合物体积相比或者相对于其他细胞类型，如通过基于所述群体或细胞表达的标记的阳性选择，或通过基于不存在于待耗尽的所述细胞群或细胞上的标记的阴性选择来增加所述细胞类型或群体的数量或百分比。所述术语不需要从组合物中完全去除其他细胞、细胞类型或群体，并且不需要如此富集的细胞在富集的组合物中以等于或甚至接近100％存在。
如本文所用，细胞或细胞群针对特定标记是“阳性”或“+”的陈述是指，特定标记(通常是表面标记)在细胞上或细胞中的可检测存在。当提及表面标记时，是指如在一些实施方案中通过流式细胞术检测到的，表面表达的存在，例如通过用与所述标记特异性结合的抗体进行染色并检测所述抗体，其中所述染色通过流式细胞术以如下水平是可检测的，所述水平基本上高于在其他方面相同的条件下用同种型匹配对照进行相同程序检测到的染色，和/或所述水平基本上与已知对所述标记呈阳性的细胞的水平相似，和/或所述水平基本上高于已知对所述标记呈阴性的细胞的水平。
如本文所用，细胞或细胞群对特定标记呈“阴性”的陈述是指特定标记(通常为表面标记)在细胞上或细胞中不存在实质上可检测的存在。当提及表面标记时，是指在一些实施方案中，如通过流式细胞术(例如通过用特异性地结合至所述标记的抗体染色并检测所述抗体)检测到的表面表达的不存在，其中所述染色未通过流式细胞术按以下水平检测到：显著高于在其他方面都相同的条件下用同种型匹配的对照进行相同程序检测到的染色的水平，和/或显 著低于已知对所述标记呈阳性的细胞的水平，和/或如与已知对所述标记呈阴性的细胞相比基本上类似的水平。
如本文所用，在关于氨基酸序列(参考多肽序列)使用时，“氨基酸序列同一性百分比(％)”和“同一性百分比”定义为，在比对序列并在必要时引入空位以实现最大序列同一性百分比并且不将任何保守取代视作序列同一性的一部分之后，候选序列(例如，Vpx或Vpr蛋白)中与参考多肽序列中的氨基酸残基相同的氨基酸残基的百分比。用于确定氨基酸序列同一性百分比的比对可以以本领域熟知的多种方式来实现，例如，使用公众可获得的计算机软件，如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适当参数，包括在所比较序列的全长中实现最大比对所需的任何算法。
如本文所用的，组合物是指两种或更多种产物、物质或化合物(包括细胞)的任何混合物。其可以是溶液、悬浮液、液体、粉末、糊剂、水性、非水性或其任何组合。
本申请中提及的所有出版物(包括专利文献、科学文章和数据库)出于所有目的通过引用以其整体并入，在程度上如同每个单独的出版物通过引用单独并入。如果本文所述的定义与通过引用并入本文的专利、申请、公开的申请和其他出版物中所述的定义相反或在其他方面不一致，则本文所述的定义优先于通过引用并入本文的定义。
本发明的优点：
本发明的目的在于提供一种制备CAR-T细胞的新方法，该方法CAR-T制备的时间仅需1天左右，较CAR T细胞常规制备方法(约2周)，极大地缩短了体外培养时间的同时，将能够更好地维持CAR-T细胞的memory表型，增强了CAR T细胞对肿瘤的杀伤功能及其在体内的存活时间。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.慢病毒转导效率的评价
使用白细胞分离术从受试者采集富含白细胞的样品，使用Ficoll密度梯度离心法收集白膜层，获得纯度较高的外周血单个核细胞(PBMC)。
洗涤PBMC并重悬于缓冲液中，缓冲液含有磷酸盐缓冲盐水(PBS)、EDTA和人血清白蛋白，将PBMC经分选得到CD4+CD8+富集的T细胞群。
将PBMC或富集的T细胞群于X-VIVO 15培养基(购自Lonza)中，加入T细胞激活剂抗CD3和/或抗CD28珠试剂和慢病毒载体颗粒，病毒载体颗粒含有编码嵌合抗原受体(CAR-BCMA，氨基酸序列见SEQ ID NO：2，核酸序列见SEQ ID NO：1)的核酸，慢病毒载体颗粒按病毒感染复数(MOI)为3进行添加，培养24小时后，将培养液离心换液，以生理盐 水洗涤后加入冷冻保护液中冷冻保存，该制备工艺称为新工艺。
SEQ ID NO：1
Figure PCTCN2021073418-appb-000004
SEQ ID NO：2
Figure PCTCN2021073418-appb-000005
作为常规对照，经白细胞分离术获得的单个核细胞或富集后得到的T细胞群，加入偶联有抗CD3和/或抗CD28的珠试剂活化24小时或48小时，然后按病毒感染复数(MOI)为1-3加入慢病毒载体，持续培养7天或以上。
使用流式细胞术测定CAR的转导的效率(或称CAR T细胞阳性率)。本方法以及常规对 照转导后不同时间点CAR T细胞阳性率结果见下表1及图1。如表1及图1所示，新工艺培养24小时收获的CAR T的转导效率较低，仅为16.0％，将其冷冻保存后解冻，用添加了2％AB血清、300IU/mL IL-2的AIM-V培养基培养发现，前期检测的转导效率显著低于常规对照，但随着培养时间的延长，检测的转导效率不断增高，培养至144小时后趋于平稳，且与常规对照基本一致。该结果提示新工艺在收获时由于CAR尚未完全表达至细胞表面，检测的转染效率很低，但当回输至人体内或体外持续培养一定时间后，CAR能完全表达至细胞表面，阳性率水平达到常规对照水平。
表1.新工艺与常规对照转导后不同时间转导效率变化(单位：％)
Figure PCTCN2021073418-appb-000006
为了进一步验证上述结果，选择靶向GPC3的CAR T细胞进行细胞转导研究，参照前述方法，将PBMC或富集的T细胞群、T细胞激活剂和慢病毒载体颗粒共同孵育24h进行收获，首先以不同病毒感染复数(MOI)进行转导，在转导后96小时检测CAR的转导效率(结果见表2)。
以MOI为3进行新工艺研究，转导24h收获的细胞进行，检测，CAR的转导效率几乎检测不到，仅为0.2％，将细胞继续用添加了2％AB血清、300IU/mL IL-2的AIM-V培养基培养，发现随着体外培养时间的延长，检测到的转导效率不断增高(如表3所示)。
表2.不同MOI的转导效率(单位：％)
MOI值 96h的转导效率
3 21.7
6 31.6
9 37.9
12 39.8
表3.新工艺转导后不同时间转导效率变化(单位：％)
Figure PCTCN2021073418-appb-000007
靶向GPC3的CAR T细胞的CAR的氨基酸序列如SEQ ID NO：3所示。
Figure PCTCN2021073418-appb-000008
实施例2：原代T细胞活化转导培养24-48小时的转导效率评价
使用白细胞分离术从受试者采集富含白细胞的样品，使用Ficoll密度梯度离心法收集白膜层，获得纯度较高的外周血单个核细胞(PBMC)，将其添加至离心室内的经洗涤白细胞分离术样品中。然后使用标准方法在磁场的存在下使细胞从转移袋通过封闭的管路管线和分离柱的无菌系统，以分离结合至CD4特异性试剂和/或CD8特异性试剂的细胞。
将富集的T细胞重悬于培养基中，加入CD3和CD28的珠试剂先进行孵育，孵育不同时间后加入包含编码GPC3CAR的重组核酸的慢病毒载体进行转导。GPC3CAR的氨基酸序列如SEQ ID NO：3所示。
活化转导的流程为：将约2x10 8个T细胞重悬至X-VIVO 15培养基，总体积为140mL,接种至培养瓶/袋，加入抗CD3和CD28珠试剂活化，在活化不同时间以病毒感染复数(MOI)为3加入慢病毒载体进行转导,活化和转导的总培养时间为24h，结束后进行收获，将培养液离心换液以生理盐水洗涤后重悬至冻存液进行冷冻保存。
在37℃，5％CO 2下培养细胞，不同方案活化的孵育时间和转导的孵育时间如下：
方案一(Tx_No_Act)：不加入激活剂进行活化，直接转导，培养24小时后收获。
方案二(Act_Tx_0)：细胞活化与转导同时进行，培养24小时后收获。
方案三(Act 0_Tx 3)：在细胞活化3小时后转导21小时，于总培养时间为24小时后收获。
方案四(Act 0_Tx 7)：在细胞活化7小时后转导17小时，于总培养时间为24小时后收获。
方案五(Act 0_Tx 16)：在细胞活化16小时后转导8小时，于总培养时间为24小时后收获。
方案六(Act 0_Tx 20)：在细胞活化20小时后转导4小时，于总培养时间为24小时后收获。
方案七(Act 0_Tx 22)：在细胞活化22小时后转导2小时，于总培养时间为24小时后收获。
方案八(Act 0_Tx 48；对照)：对照细胞为常规工艺，先活化48h，按MOI为1.5加入慢病毒载体转导24h，离心换液去除游离载体，继续将细胞扩大培养并在第8天收获。
我们将方案一～方案七收获的细胞冻存复苏后接种至培养基中继续培养至144h后进行转导效率的检测，相关结果见图2。如图2所示，与活化转导同时进行的T细胞相比，未进行活化直接进行转导的T细胞几乎未检出CAR的表面表达，表明未进行活化的T细胞很难直接被慢病毒载体转导。另外，同样都进行活化的条件下，加入慢病毒载体的时间越晚，转导效率有明显增高趋势，尽管实际转导持续时间是递减的。该结果表明T细胞的活化状态决定细胞被慢病毒转染的难易程度，在一定范围内，活化时间越长，细胞越易转染，当细胞达到易转染状态后，慢病毒可迅速进入细胞，该过程可能少于2小时，而转导效率水平与常规对照水平基本一致。
制备靶向CD19的CAR T细胞(CD19CAR)，CAR的氨基酸序列如SEQ ID NO：4所示。
SEQ ID NO：4：
Figure PCTCN2021073418-appb-000009
CD19CAR的制备过程按照以下方案进行活化和转导，在37℃，5％CO 2下培养细胞， 方案一-方案五使用MOI均为3：
方案一(Act_Tx_0)：细胞活化与转导同时进行，培养24小时后收获。
方案二(Act 0_Tx 16)：在细胞活化16小时后转导8小时，于总培养时间为24小时后收获。
方案三(Act 0_Tx 20)：在细胞活化20小时后转导4小时，于总培养时间为24小时后收获。
方案四(Act 0_Tx 22)：在细胞活化22小时后转导2小时，于总培养时间为24小时后收获。
方案五(Act 0_Tx 23)：在细胞活化23小时后转导1小时，于总培养时间为24小时后收获。
方案六(Act 0_Tx 48；对照)：对照细胞为常规工艺，先活化48h，按MOI为2加入慢病毒载体转导24h,离心换液去除游离载体，继续将细胞扩大培养并在第8天收获。
方案一～方案五收获的细胞冻存复苏后接种至培养基中继续培养至144h后进行转导效率的检测，结果如图3所示。另外，活化16小时至23小时范围内加入慢病毒载体进行转导，总培养时间在24小时，T细胞的转导效率差异不大，略高于常规对照，表明在T细胞活化至一定状态后，慢病毒进入细胞的时间最短仅需要1小时。
实施例3：体内抗肿瘤疗效评估
为评估新工艺制备的CAR T细胞的抗肿瘤活性，我们以常规工艺(活化48h后再转导，转导后体外培养7天以上)的CAR T细胞作为对照，对比考察不同剂量新工艺制备的CAR-BCMA T细胞在荷瘤小鼠体内的抗肿瘤疗效。常规工艺制备的CAR-BCMA T细胞分别于D7(转导后培养7天，定义为常规工艺1)和D11(转导培养11天，定义为常规工艺2)收样，新工艺制备的CAR-BCMA的T细胞活化转导同时进行，按MOI为3进行转导，于培养24h收样冻存，相关表型见表4以及阳性率(新工艺为冻存复苏后培养168h检测)见表5。
表4.各类型T细胞比例
Figure PCTCN2021073418-appb-000010
表5.动物实验用CAR T细胞阳性率
Figure PCTCN2021073418-appb-000011
表型结果显示，以新工艺制备获得的BCMA-CART细胞表型与制备前的PBMC相比，TN(CD3 +CD95 -CD62L +CD45RA +CCR7 +CD45RO -)的比例由25.4％降低至检测不到，TSCM(CD3 +CD95 +CD62L +CD45RA +CCR7 +CD45RO -)的比例由5.3％升高至17.7％，提示
Figure PCTCN2021073418-appb-000012
T细胞(TN)在活化24h后向TSCM转化。常规工艺培养至D7和D11的TSCM的比例随培养时间明显下降，分别为12.3％和3.1％，提示TSCM随着培养时间的延长会向Tcm转化。因此，为了尽可能减少CAR T细胞的终末分化，维持效应功能，我们应该尽可能缩短CAR T 细胞的培养时间，而新工艺制备正是将培养时间缩短至1～2天。另外，阳性率结果显示，新工艺制备的CAR T细胞阳性率可达到常规CAR T水平。
将上述新工艺制备的CART细胞用于评估其在荷人多发性骨髓瘤细胞RPMI-8226皮下移植瘤的NPG小鼠中的抗肿瘤疗效，以及其在小鼠外周血中的存活情况，肿瘤细胞接种当天记为D0，具体剂量及实验设计见表6。
表6.动物实验剂量设计表
Figure PCTCN2021073418-appb-000013
肿瘤接种后D12进行CAR T细胞输注，至肿瘤接种后D39，溶媒对照组肿瘤体积超过2000mm 3。与溶媒对照组相比，各组肿瘤体积移植瘤及肿瘤消退情况如下：
a)常规工艺1组，肿瘤体积抑制率100％，5只小鼠肿瘤全部消退。
b)常规工艺2组，肿瘤体积抑制率为100％，5只小鼠肿瘤全部消退。
c)新工艺1组，肿瘤体积抑制率为100％，5只小鼠肿瘤全部消退。
d)新工艺2组，肿瘤体积抑制率为100％，5只小鼠肿瘤全部消退。
e)新工艺3组，肿瘤体积抑制率为56.05％，无小鼠肿瘤消退。
上述各组小鼠肿瘤体积见图4及小鼠体重随时间变化趋势。从起效时间看，新工艺1组略晚于常规工艺1和常规工艺2，新工艺2组和新工艺3组的起效时间更晚，新工艺组中不同剂量的CAR T与起效时间存在剂量相关性。从最终疗效看，新工艺1组和新工艺2组在观察期内(肿瘤接种后D39天)全部达到肿瘤完全清除。同时，新工艺3组，我们也观察到从D32至D35肿瘤体积接近平稳，至D39开始出现减小，考虑溶媒对照组肿瘤体积按照动物福利要求已达人道主义终点，因此保留新工艺3组组动物以继续观察持续疗效。与预期一致，新工艺3组组的小鼠肿瘤体积在随后的观察时间里持续减小，在肿瘤接种后D68天，该剂量组肿瘤体积抑制率接100％，5只小鼠的肿瘤几乎全部消退，显示了让人备受鼓舞的抗肿瘤疗效。毒性方面，实验期间，排除肿瘤的影响因素外，小鼠体重变化不大，提示CART对小鼠没有明显的毒副作用。
基于上述动物实验中新工艺制备的CAR-T巨大的抗肿瘤潜力，我们推测，细胞输注剂量减少至常规工艺细胞用量的1/25，在肿瘤体积增至大于1000mm3条件下仍能将肿瘤细胞快速清除，提示新工艺制备较常规工艺具有明显的优越性。
另外，我们分别在CAR T细胞输注后的D14和D21检测了各组小鼠外周血中人T细胞的存活情况，相关结果见图5。结果显示，在D21，新工艺不同剂量组外周血中CD3+T细胞的数目均值显著高于常规工艺组，且与CAR T注射D14天相比，CD3+T细胞数目均有 所增加，提示新工艺制备的CART细胞在体内的增殖能力显著优于常规工艺组。此外，新工艺3组从肿瘤接种后D12给药，至肿瘤接种后D68几乎清除肿瘤，该过程持续时间长达56天，也提示新工艺制备的CAR-T细胞在体内的存活持久性更强。
实施例4.不同T细胞激活剂及浓度对慢病毒转导的评价
使用白细胞分离术从受试者采集富含白细胞的样品，使用Ficoll密度梯度离心法收集白膜层，获得纯度较高的外周血单个核细胞(PBMC)。
洗涤PBMC并重悬于缓冲液中，基于免疫亲和力进行分选，所述缓冲液含有磷酸盐缓冲盐水(PBS)、EDTA和人血清白蛋白。对于基于免疫亲和力进行的T细胞分选，将分选缓冲液中的经洗涤的细胞与偶联至单克隆抗体的珠试剂一起在室温下孵育30分钟，并使用磁分离柱进行分选。
将富集的T细胞重悬于X-VIVO15培养基中，加入不同的T细胞激活剂进行活化，同时将含有编码嵌合抗原受体(CAR-CD19)核酸的慢病毒载体按病毒感染复数(MOI)为3进行添加。培养24h后替换为添加了2％AB血清、300IU/mL IL-2的AIM-V培养基，延长培养时间至144h，以此时转导效率作为评价活化条件的度量依据。
按以下试剂条件及浓度活化22小时后转导2小时，制备CD19-CAR T细胞，转导方案一～四的MOI均为3，培养条件是37℃，5％CO 2下培养。
方案一：细胞活化采用偶联有抗CD3和抗CD28的珠试剂活化。
方案二：细胞活化采用抗CD3抗体，使用终浓度为500ng/mL。
方案三：细胞活化采用抗CD3抗体，使用终浓度为1000ng/mL。
方案四：细胞活化采用抗CD3抗体，使用终浓度为2000ng/mL。
将方案一～方案四的细胞在转导2小时后离心换液，继续培养至144h，检测转导效率，结果如表7及图6所示，采用上述两种T细胞激活剂以及三种不同浓度条件下均能有效制备CAR T细胞。
表7.转导效率(单位：％)
不同条件 转导效率
方案一 58.1
方案二 41.1
方案三 38.7
方案四 36.8
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (37)

  1. 病毒载体转导细胞的方法，其特征在于，所述方法包括：
    步骤(1)、将包含待转导细胞的输入组合物、待转导细胞刺激剂、和携带重组核酸的病毒载体颗粒共同孵育，孵育时间不超过72小时，
    步骤(2)、进行收获，获得输出组合物，所述输出组合物含有转导有重组核酸的细胞；
    优选的，所述孵育时间为1小时-72小时；
    更优选的，所述孵育时间为2小时-48小时；
    更优选的，所述孵育时间为2小时-36小时；
    更优选的，所述孵育时间为12小时-36小时；
    更优选的，所述孵育时间为12小时-24小时；
    更优选的，所述孵育时间为15小时-24小时。
  2. 病毒载体转导细胞的方法，其特征在于，所述方法包括：
    步骤(1)、将包含待转导细胞的输入组合物和待转导细胞刺激剂，孵育时间不超过72h，
    步骤(2)、再加入重组核酸的病毒载体颗粒进行孵育，孵育时间不超过24小时，
    步骤(3)、进行收获，获得输出组合物，所述输出组合物含有转导有重组核酸的细胞；
    优选的，所述(1)和(2)的总孵育时间不超过72小时。
  3. 如权利要求2所述的方法，其特征在于，所述(1)和(2)的总孵育时间不超过60h，或不超过48h，或不超过32h，或不超过28h，或不超过24h。
  4. 如权利要求2所述的方法，其特征在于，所述步骤(1)的孵育时间为2-72小时；
    优选的，所述步骤(1)的孵育时间为2-71小时；
    更优选的，所述步骤(1)的孵育时间为2-48小时；
    更优选的，所述步骤(1)的孵育时间为2-32小时；
    更优选的，所述步骤(1)的孵育时间为2-28小时；
    更优选的，所述步骤(1)的孵育时间为3-24小时；
    更优选的，所述步骤(1)的孵育时间为5-24小时；
    更优选的，所述步骤(1)的孵育时间为7-24小时；
    更优选的，所述步骤(1)的孵育时间为7-23小时；
    更优选的，所述步骤(1)的孵育时间为10-23小时；
    更优选的，所述步骤(1)的孵育时间为15-23小时；
    更优选的，所述步骤(1)的孵育时间为15-22小时。
  5. 如权利要求2所述的方法，其特征在于，所述步骤(2)的孵育时间为30分钟-24小时；
    优选的，所述步骤(2)的孵育时间为30分钟-21个小时；
    优选的，所述步骤(2)的孵育时间为30分钟-17个小时；
    优选的，所述步骤(2)的孵育时间为30分钟-12个小时；
    优选的，所述步骤(2)的孵育时间为30分钟-10个小时；
    优选的，所述步骤(2)的孵育时间为30分钟-8个小时；
    优选的，所述步骤(2)的孵育时间为1小时-8个小时；
    优选的，所述步骤(2)的孵育时间为1小时-4个小时；
    更优选的，所述步骤(2)的孵育时间为1个小时-3个小时。
  6. 如权利要求1或2所述的方法，其特征在于，所述输入组合物从外周血、脐血、骨髓和/或诱导多能干细胞中获得，优选地，所述输入组合物是白细胞分离术样品；优选地，所述输入组合物是富集或分离的CD3+T细胞，是富集或分离的CD4+T细胞或者是富集或分离的CD8+T细胞或其组合。
  7. 如权利要求1或2所述的方法，其特征在于，所述病毒载体颗粒源自逆转录病毒载体；优选的，所述病毒载体颗粒是慢病毒载体。
  8. 如权利要求1-7中任一所述方法，其特征在于，所述病毒载体颗粒的感染复数不高于20；优选的，所述感染复数为0.5-20；更优选的，所述感染复数为1.5-20；更优选的，所述感染复数为3-20；更优选的，所述感染复数为3-12。
  9. 如权利要求1-8中任一所述方法，其特征在于，所述输入组合物中待转导细胞的数量不超过1*10 10；
    优选的，所述输入组合物中待转导细胞的数量不低于1*10 5；
    更优选的，所述输入组合物中待转导细胞的数量不低于1*10 6。
  10. 如权利要求1-9中任一所述方法，其特征在于，所述重组核酸能够编码识别特异性靶抗原的受体；优选的，所述识别特异性靶抗原的受体是T细胞受体(TCR)、嵌合抗原受体(CAR)、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
  11. 如权利要求10所述的方法，其特征在于，所述特异性靶抗原是与疾病相关的抗原或通用标签；
    优选的，所述疾病是癌症、自身免疫性疾病、或感染性疾病；
    优选地，所述癌症是血液肿瘤；更优选地，所述血液肿瘤是白血病、骨髓瘤、淋巴瘤及/或其组合。
  12. 如权利要求10所述的方法，其特征在于，所述特异性靶抗原是肿瘤相关抗原；
    优选的，所述肿瘤相关抗原选自：B细胞成熟抗原(BCMA)、碳酸酐酶9(CAIX)、EGFR、Her2/neu(受体酪氨酸激酶erbB2)、CD19、CD20、CD22、间皮素、CEA、CD23、CD24、CD30、CD33、CD38、CD44、EGFR、上皮糖蛋白2(EPG-2)、上皮糖蛋白40(EPG-40)、EPHa2、erb-B2、erb-B3、erb-B4、erbB二聚体、EGFR vIII、叶酸结合蛋白(FBP)、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、HMW-MAA、IL-22R-α、IL-13R-α2、激酶插入结构域受体(kdr)、L1细胞粘附分子(L1-CAM)、黑色素瘤相关抗原(MAGE)、TAG72、B7-H6、IL-13受体α2(IL-13Ra2)、CA9、GD3、HMW-MAA、CD171、G250/CAIX、HLA-AI MAGEA1、HLA-A2、PSCA、叶酸受体、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、CD44v6、间皮素、粘蛋白1(MUC1)、MUC16、PSCA、NKG2D、NY-ESO-1、MART-1、gp100、癌胚胎抗原、G蛋白偶联受体5D(GPCR5D)、ROR1、TAG72、 VEGF-R2、癌胚抗原(CEA)、前列腺特异性抗原、PSMA、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、Wilms肿瘤1(WT-1)、细胞周期蛋白、CCL-1、CD138，Claudin18.2、GPC3。
  13. 如权利要求1-12任一所述的方法，其特征在于，所述待转导细胞刺激剂能够激活TCR复合物的一个或多个组分的一个或多个细胞内信号域和一个或多个共刺激分子的一个或多个细胞内信号域；
    优选地，所述待转导细胞刺激剂包含(i)特异性结合到TCR复合物成员的一级试剂，任选地特异性结合到CD3和(ii)特异性结合到T细胞共刺激分子的二级试剂，任选地其中所述共刺激分子选自CD28，CD137(4-1-BB)、0X40或ICOS。
  14. 如权利要求1-13任一所述的方法，其特征在于，所述待转导细胞是免疫效应细胞；
    优选的，所述待转导细胞是T细胞，NK细胞，NKT细胞，树突细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞或其组合；
    更优选的，所述待转导细胞是T细胞。
  15. 如权利要求1-14任一所述的方法，其特征在于，所述待转导细胞刺激剂包含CD3结合分子、CD28结合分子、重组IL-2、重组IL-15、重组IL-7、重组IL-21或其组合物；
    优选的，所述待转导细胞刺激剂包含抗CD3抗体和/或抗CD28抗体。
  16. 如权利要求1-15任一所述的方法，其特征在于，在收获之前，所述待转导细胞刺激剂可离心去除。
  17. 如权利要求1-16任一所述的方法，其特征在于，所述待转导细胞刺激剂为游离分子。
  18. 如权利要求1-16任一所述的方法，其特征在于，所述待转导细胞刺激剂固定在固体支持物上；
    优选的，所述固体支持物是聚合物基质材料；
    更优选的，所述聚合物基质材料为可降解的聚合物纳米基质或珠试剂。
  19. 如权利要求18所述的方法，其特征在于，所述珠试剂为磁珠或微珠。
  20. 如权利要求1或2所述方法，其特征在于，所述输出组合物中，转导有重组核酸的细胞的含量不低于30％、或不低于40％、或不低于50％、或不低于60％、或不低于70％、或不低于80％。
  21. 如权利要求1或2所述的方法，其特征在于，所述输出组合物中，转导有重组核酸的细胞的含量不高于50％；优选的，不高于40％，更优选的，不高于38％；更优选的，不高于35％；更优选的，不高于30％。
  22. 如权利要求20或21所述的方法，其特征在于，所述转导有重组核酸的细胞中幼稚细胞的含量相比待转导细胞中幼稚细胞的含量降低；
    优选的，所述幼稚细胞含量降低至10％以下；
    更优选的，所述幼稚细胞含量降低至5％以下。
  23. 如权利要求20或21所述的方法，其特征在于，所述转导有重组核酸的细胞中记忆 性细胞的含量相比待转导细胞中记忆性细胞的含量提高；
    优选的，所述记忆性细胞是记忆性干细胞；
    更优选的，所述记忆性干细胞是TSCM。
  24. 如权利要求23所述的方法，其特征在于，转导有重组核酸的细胞中记忆性干细胞的含量是待转导细胞中记忆性干细胞的含量的约2倍或以上，优选的，转导有重组核酸的细胞中记忆性干细胞的含量是待转导细胞中记忆性干细胞的含量的约3倍或以上。
  25. 如权利要求20-24任一所述的方法，其特征在于，所述转导有重组核酸的细胞含有未分化的细胞。
  26. 如权利要求1-25中任一所述的方法，其特征在于，
    所述输入组合物包含重组IL-2，任选重组人IL-2，所述重组IL-2的浓度为10IU/mL至500IU/mL、50IU/mL至250IU/mL或100IU/mL至200IU/mL；或浓度为至少10IU/mL、50IU/mL、100IU/mL、200IU/mL、300IU/mL、400IU/mL或500IU/mL；和/或
    所述输入组合物包含重组IL-15，任选重组人IL-15，所述重组IL-15的浓度为1IU/mL至100IU/mL、2IU/mL至50IU/mL或5IU/mL至10IU/mL；或浓度为至少1IU/mL、2IU/mL、5IU/mL、10IU/mL、25IU/mL或50IU/mL；和/或
    所述输入组合物包含重组IL-7，任选重组人IL-7，所述重组IL-7的浓度为50IU/mL至1500IU/mL、100IU/mL至1000IU/mL至200IU/mL至600IU/mL；或浓度为至少50IU/mL、100IU/mL、200IU/mL、300IU/mL、400IU/mL、500IU/mL、600IU/mL、700IU/mL、800IU/mL、900IU/mL或1000IU/mL。
  27. 如权利要求1-26任一所述的方法，其特征在于，收获的所述输出组合物经洗涤获得转导有重组核酸的细胞。
  28. 如权利要求27所述的方法，其特征在于，将转导有重组核酸的细胞加入缓冲液中进行保存；优选的，所述缓冲液中含有细胞冻存剂。
  29. 如权利要求1-28任一所述的方法，其特征在于，所述转导有重组核酸的细胞在收获之后，给予有需要的受试者之前，不需要体外扩增。
  30. 通过权利要求1-29中任一所述的方法产生的转导有重组核酸的细胞的组合物。
  31. 如权利要求30所述的转导有重组核酸的细胞的组合物，其特征在于，所述细胞是免疫效应细胞。
  32. 如权利要求31所述的转导有重组核酸的细胞的组合物，其特征在于，所述细胞是T细胞。
  33. 如权利要求32所述的转导有重组核酸的细胞的组合物，其特征在于，所述转导有重组核酸的细胞中TSCM的比例高于待转导细胞中的TSCM的比例；
    优选的，所述转导有重组核酸的细胞中TSCM的比例是待转导细胞中的TSCM的比例的约2倍或以上；
    更优选的，所述转导有重组核酸的细胞中TSCM的比例是待转导细胞中的TSCM的比例的约3倍或以上。
  34. 如权利要求32所述的转导有重组核酸的细胞的组合物，其特征在于，所述转导有重组核酸的细胞中TSCM的比例在10％以上，优选的，在13％以上，更优选的，在15％以上。
  35. 如权利要求30-34任一所述的转导有重组核酸的细胞的组合物，其特征在于，所述转导有重组核酸的细胞在给予受试者之前不需要进行体外扩增。
  36. 一种组合物，包含权利要求30-34任一所述的转导有重组核酸的细胞和药学上可接受的载体。
  37. 一种过继细胞治疗的方法，包括给与有此需要的受试者权利要求36所述的组合物。
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