WO2020151752A1 - Cd20组合靶向的工程化免疫细胞 - Google Patents

Cd20组合靶向的工程化免疫细胞 Download PDF

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WO2020151752A1
WO2020151752A1 PCT/CN2020/073873 CN2020073873W WO2020151752A1 WO 2020151752 A1 WO2020151752 A1 WO 2020151752A1 CN 2020073873 W CN2020073873 W CN 2020073873W WO 2020151752 A1 WO2020151752 A1 WO 2020151752A1
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car
cells
target
antibody
cell
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PCT/CN2020/073873
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English (en)
French (fr)
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张华�
石欢
沈连军
何佳平
曹卫
刘丽萍
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亘喜生物科技（上海）有限公司
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Priority to CN202080010291.6A priority Critical patent/CN113330038A/zh
Publication of WO2020151752A1 publication Critical patent/WO2020151752A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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 A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A61K2239/25—Suicide switch
    • 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 A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • 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 A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • 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 A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention relates to the field of immunotherapy, and more specifically to engineered immune cells targeted by CD20 combination.
  • Cellular immunotherapy is an emerging tumor treatment model with significant curative effect, and a new type of autoimmune anti-cancer treatment method. It is a method that uses biotechnology and biological preparations to culture and amplify the immune cells collected from the patient's body and then return it to the patient's body to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • CD19 is highly expressed on the surface of most B-cell malignant tumors.
  • the two CAR-T products approved by the FDA for the market are both for the CD19 antigen, and their indications are also expanding, such as chronic lymphocytic leukemia.
  • anti-CD19 CAR-T there are still some patients with poor therapeutic effects and prone to relapse.
  • this field still needs further research to develop a treatment method and medicine that can treat hematological tumors and solid tumors more effectively, with good specificity and less side effects, and effectively prevent recurrence.
  • the purpose of the present invention is to provide a treatment method and medicine that can treat hematological tumors and solid tumors more effectively, with good specificity and less side effects, and effectively prevent recurrence.
  • Another object of the present invention is to provide a dual-target or multi-target immunotherapy.
  • Another object of the present invention is to provide a specific CAR-T cell that specifically targets CD20 and another target.
  • Another object of the present invention is to provide an engineered immune cell with a suicide switch that simultaneously targets CD20 and another target.
  • a chimeric antigen receptor CAR targets a first target and a second target, wherein the first target is CD20, and the second target Points are selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2 , NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, folate receptor- ⁇ (FR- ⁇ ), or a combination thereof.
  • the first target is CD20
  • the second target Points are selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, Mesothe
  • the second target is CD19.
  • the CAR also targets one or more targets selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL- 1(CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, folic acid Receptor alpha, or a combination thereof.
  • targets selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL- 1(CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is an optional signal peptide sequence
  • I is a flexible joint
  • H is an optional hinge region
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ ;
  • One of scFv1 and scFv2 is an antigen binding domain targeting the first target (CD20), and the other is an antigen binding domain targeting the second target.
  • the scFv1 is an antigen binding domain targeting CD20
  • the scFv2 is an antigen binding domain targeting a second target.
  • the scFv1 and scFv2 can be independent of each other, can be connected in series, or have a loop structure.
  • the element (I-scFv0)m is m identical or different I-scFv0 in series, wherein m is a positive integer of 0-10, each I is a flexible linker, and each scFv0 is independently a target Antigen binding domains to the same or different targets.
  • the target is selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, mesothelin , EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, folate receptor alpha, or a combination thereof.
  • CD20-targeting antigen binding domain is shown in the following formula A or formula B:
  • V H1 -V L1 A
  • V L1 -V H1 B
  • V H1 is the variable region of the heavy chain of the anti-CD20 antibody
  • V L1 is the variable region of the light chain of the anti-CD20 antibody
  • "-" is the connecting peptide or peptide bond.
  • the CD20-targeting antigen binding domain includes the heavy chain variable region and the light chain variable region of monoclonal antibody Leu16.
  • the heavy chain variable region of the anti-CD20 antibody has an amino acid sequence as shown in SEQ ID NO.: 5, 9, 11, 13, or 31.
  • the light chain variable region of the anti-CD20 antibody has an amino acid sequence as shown in SEQ ID NO.: 6, 10, 12, 14, or 32.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 5, and the antibody light chain as shown in SEQ ID NO.: 6 Change area.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 9 and the antibody light chain as shown in SEQ ID NO.: 10 Change area.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 11, and the antibody light chain as shown in SEQ ID NO.: 12 Change area.
  • the CD20-targeting antigen-binding domain comprises the antibody heavy chain variable region shown in SEQ ID NO.: 13, and the antibody light chain shown in SEQ ID NO.: 14 Change area.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 31, and the antibody light chain as shown in SEQ ID NO.: 32. Change area.
  • the antibody heavy chain variable region shown in SEQ ID NO.: 31 and the antibody light chain variable region shown in SEQ ID NO.: 32 pass through SEQ ID NO.: 41 The connecting peptide shown is connected.
  • the nucleotide sequence of the antigen-binding domain targeting CD20 comprises the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 1 and the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 2 The coding sequence of the variable region of the antibody light chain is shown.
  • the structure of the antigen binding domain targeting the second target is shown in the following formula C or formula D:
  • V H2 is the variable region of the heavy chain of the antibody against the second target
  • VL2 is the variable region of the light chain of the antibody against the second target
  • "-" is the connecting peptide or peptide bond.
  • the second target is CD19.
  • the CD19-targeting antigen binding domain includes the heavy chain variable region and the light chain variable region of monoclonal antibody FMC63.
  • the heavy chain variable region of the anti-CD19 antibody has an amino acid sequence as shown in SEQ ID NO.:7.
  • the light chain variable region of the anti-CD19 antibody has an amino acid sequence as shown in SEQ ID NO.:8.
  • the CD19-targeting antigen-binding domain comprises the antibody heavy chain variable region shown in SEQ ID NO.: 7 and the antibody light chain shown in SEQ ID NO.: 8 Change area.
  • the antibody heavy chain variable region shown in SEQ ID NO.: 7 and the antibody light chain variable region shown in SEQ ID NO.: 8 pass through SEQ ID NO.: 21
  • the connecting peptide shown is connected.
  • the nucleotide sequence of the antigen-binding domain targeting CD19 comprises the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 3, and the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 4 The coding sequence of the variable region of the antibody light chain is shown.
  • the scFv1 and/or scFv2 are murine, human, human and murine chimeric, or fully humanized single-chain antibody variable region fragments.
  • the flexible linker (or connecting peptide) I may be composed of multiple sub-short peptides, and the multiple sub-short peptides may be the same or different.
  • V H1 and V L1 can be connected without or through a flexible linker (or connecting peptide) Ia.
  • V H2 and VL2 can be connected without or through a flexible linker (or connecting peptide) Ib.
  • the flexible linker (or connecting peptide) Ia may be composed of multiple sub-short peptides, and the multiple sub-short peptides may be the same or different.
  • the flexible linker (or connecting peptide) Ib may be composed of multiple sub-short peptides, and the multiple sub-short peptides may be the same or different.
  • each sub-short peptide is 0-6, preferably 0-5, more preferably 1-3.
  • the sequence of the sub-short peptide is selected from the following group: GGGGS (SEQ ID NO.: 22), EAAAK (SEQ ID NO.: 18), EXXXK (SEQ ID NO.: 39), wherein X corresponds to Xaa in the sequence table, which can be any naturally occurring amino acid), DYKDDDDK (SEQ ID NO.: 40), SSGGGGGSCPYSNPSLCSGGGGGS (SEQ ID NO.: 24), GSTSGGSGGGSGGGGSS (SEQ ID NO.: 41), GSTSGSGKPGSGEGSTKG (SEQ ID NO.: ID NO.: 42), SGGGGGS (SEQ ID NO.: 33), CPYSNPSLC (SEQ ID NO.: 34), SSGGGGGS (SEQ ID NO.: 43), or any combination thereof.
  • the flexible linker (or connecting peptide) I, Ia or Ib each independently has the structure of the following formula VII:
  • Each "-" is independently a connecting peptide or a peptide bond
  • X, Y and Z are each independently a sequence selected from the following group:
  • a, b, and c are each independently a positive integer of 0-6.
  • the X is a sequence selected from the following group: GGGGS, EAAAK, EXXXK, SSGGGGGS, GTSSGGSGGGSGGGGSS, or GTSSGSGKPGSGEGSTKG.
  • the Y is a sequence selected from the following group: GGGGS, EAAAK, EXXXK, DYKDDDDK, CPYSNPSLC, GTSSGGSGGGSGGGGSS, or GTSSGSGKPGSGEGSTKG.
  • the Z is a sequence selected from the following group: GGGGS, EAAAK, EXXXK, SGGGGGS, GTSSGGSGGGSGGGGSS, or GTSSGSGKPGSGEGSTKG.
  • the flexible linker (or connecting peptide) I, Ia or Ib each independently has a sequence selected from the following group: (GGGGS)n, (EAAAK)n, (EXXXK)n, (GGGGS )a+(EAAAK)b+(GGGGS)c, (GGGGS)a+(EXXXK)b+(GGGGS)c, (GGGGS)a+(DYKDDDDK)b+(GGGGS)c, (SSGGGGGS+CPYSNPSLC+SGGGGGS)n, GSGGGGSSYSNGGGS, GSGGGGSSGSSNGGGS, GSGGGGSSYSNGGGS, GSGGGGSSGSSNGGGS, GSGGGGSSGSSNGGGS, GSGGGGSS, GSGGGS, GGS )n, (GSTSGSGKPGSGEGSTKG)n;
  • n, a, b, and c are each independently a positive integer of 0-6.
  • the three of a, b and c are not 0 at the same time.
  • the flexible linker (or connecting peptide) I, Ia or Ib each independently has a sequence as shown in any one of SEQ ID NO.: 15-25.
  • the flexible linker disclosed in the present invention includes (specifically) a flexible linker composed of any number of any of the short peptides.
  • the structure of the CAR is shown in the following formula II or II':
  • Each "-" is independently a connecting peptide or a peptide bond
  • V H and the second is an anti-target antibody heavy chain variable region, V L and the second is an anti-target antibody light chain variable region; scFv targeting or binding to the second target antigenic domains, V H is an anti-CD20 antibody heavy chain variable region, V L and an anti-CD20 antibody light chain variable region.
  • the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • the L is a signal peptide derived from CD8.
  • the L has an amino acid sequence as shown in SEQ ID NO.:30.
  • the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.
  • the H is a hinge region derived from CD8.
  • the H has an amino acid sequence as shown in SEQ ID NO.:35.
  • the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154, or a combination thereof.
  • the TM is a transmembrane region derived from CD8.
  • the TM has an amino acid sequence as shown in SEQ ID NO.:36.
  • the C is a costimulatory signal molecule of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • the C is a costimulatory signal molecule derived from 4-1BB.
  • the C has an amino acid sequence as shown in SEQ ID NO.: 37.
  • the CD ⁇ has an amino acid sequence as shown in SEQ ID NO.:38.
  • VL and scFv can be connected without or through a flexible linker (or connecting peptide) Ic.
  • VH and scFv can be connected without or through a flexible linker (or connecting peptide) Id.
  • the flexible linker (or connecting peptide) Ic or Id can each independently be composed of multiple sub-short peptides, and the multiple sub-short peptides can be the same or different.
  • the flexible linker (or connecting peptide) Ic or Id each independently has the structure of formula VII: Xa-Yb-Zc(VII)
  • each "-" is independently a connecting peptide or a peptide bond
  • X, Y, Z, a, b, and c are as described above.
  • the flexible linker (or connecting peptide) Ic or Id each independently has a sequence selected from the following group: (GGGGS)n, (EAAAK)n, (EXXXK)n, (GGGGS)a+ (EAAAK)b+(GGGGS)c, (GGGGS)a+(EXXXK)b+(GGGGS)c, (GGGGS)a+(DYKDDDDK)b+(GGGGS)c, (SSGGGGGS+CPYSNPSLC+SGGGGGS)n, GSGGGGSCPYSNPSLCSGGGGSELPTQKPTYSGGGSGGSSGGSSGGSSNPSGGGS) , (GSTSGSGKPGSGEGSTKG)n;
  • n, a, b, and c are each independently a positive integer of 0-6.
  • the three of a, b and c are not 0 at the same time.
  • the flexible linker (or connecting peptide) Ic or Id each independently has a sequence as shown in any one of SEQ ID NO.: 15-25.
  • the structure of the CAR is LV H1 -Ia-V L1 -IV L2 -Ib-V H2 -H-TM-C-CD3 ⁇ .
  • the CAR has an amino acid sequence as shown in any one of SEQ ID NO.: 26-29.
  • amino acid sequence of the flexible linker (or connecting peptide) Ia is shown in SEQ ID NO.: 41.
  • amino acid sequence of the flexible linker (or connecting peptide) Ib is shown in SEQ ID NO.:21.
  • amino acid sequence of the flexible linker I is shown in any one of SEQ ID NO.: 15-25.
  • amino acid sequence of the flexible linker I is shown in any one of SEQ ID NO.: 15-19, 23.
  • amino acid sequence of the flexible linker I is shown in any one of SEQ ID NO.: 15, 16, 17, 23.
  • amino acid sequence of the flexible linker I is shown in SEQ ID NO.: 15.
  • amino acid sequence of the hinge region, transmembrane region and intracellular region of the CAR is shown in SEQ ID NO.:44.
  • the CAR has the CAR structure of 1#, 2#, 3#, 4#, 5#, 9# in Table 1.
  • the CAR has 1#, 2 in Table 1.
  • the CAR has the amino acid sequence shown in SEQ ID NO.:26.
  • an engineered immune cell is provided, and the immune cell has the following characteristics:
  • the immune cell expresses a chimeric antigen receptor CAR, and the CAR targets a first target and a second target, wherein the first target is CD20, and the second target is selected from Group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1 , ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, FR- ⁇ , or a combination thereof.
  • the first target is CD20
  • the second target is selected from Group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, mesothe
  • the second target is CD19.
  • the CAR also targets one or more targets selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL- 1(CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, folic acid Receptor alpha, or a combination thereof.
  • targets selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL- 1(CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA
  • an engineered immune cell having the following characteristics:
  • the immune cells express exogenous TCR, and the TCR targets a first target and a second target, wherein the first target is CD20, and the second target is selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1 , EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, FR- ⁇ , or a combination thereof.
  • the first target is CD20
  • the second target is selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, mesothelin,
  • the TCR also targets one or more targets selected from the group consisting of CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL- 1(CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, folic acid Receptor alpha, or a combination thereof.
  • targets selected from the group consisting of CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL- 1(CLEC12A), SLAMF7, mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL
  • the CAR is a single CAR, and the CAR simultaneously targets the first target and the second target.
  • the CAR includes both an antigen binding domain targeting the first target and an antigen binding domain targeting the second target.
  • the CAR further comprises an antigen binding domain of one or more targets selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1 -CAM, folate receptor alpha, or a combination thereof.
  • targets selected from the following group: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL-1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA,
  • the CAR is the CAR described in the first aspect of the present invention.
  • the CAR includes a first CAR targeting the first target and a second CAR targeting the second target.
  • the structure of the first CAR is shown in the following formula III:
  • Each "-" is independently a connecting peptide or a peptide bond
  • scFv1' is an antigen binding domain that targets CD20.
  • Each "-" is independently a connecting peptide or a peptide bond
  • scFv2' is an antigen binding domain that targets the second target.
  • the CAR also includes a CAR targeting one or more targets selected from the group consisting of CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1 , CLL-1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1- CAM, folate receptor alpha, or a combination thereof.
  • a CAR targeting one or more targets selected from the group consisting of CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1 , CLL-1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, R
  • the CAR also includes one or more CARs that are selected from the group of targets: CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL -1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP, cMET, VEGFR2, MUC16, PSMA, GD2, L1-CAM, Folate receptor alpha, or a combination thereof.
  • targets CD19, CD22, BCMA, CD33, CD38, CD47, CD123, CD138, kappa light chain, CS1, CLL -1 (CLEC12A), SLAMF7, Mesothelin, EGFR, Epha2, FLT3, GPC3, Her2, NKG2D, PD1, ROR1, EGFRVIII, IL13RA, CEA, FAP,
  • the immune cell has one or more characteristics selected from the following group:
  • the immune cells express exogenous cell suicide switching elements
  • the immune cells express or secrete PD-1 antibody, PD-L1 antibody, CD47 antibody, Tim3 antibody, Lag3 antibody, Tigit antibody, OX40 antibody, ICOS antibody, IL7, CXCL19, IL21, IL15, IL2, IL18, Or a combination thereof; and
  • the cytokine-related signaling pathway of the immune cell is enhanced, wherein the cytokine is selected from the group consisting of IL7, CXCL19, IL21, IL15, IL2, IL18, or a combination thereof.
  • the immune cells are T cells and are used for allogeneic immunotherapy, the TCR gene expression of the T cells is silenced.
  • the expression of PD-1 gene of the immune cell is silenced.
  • the engineered immune cells are selected from the following group:
  • CAR-NK cells Chimeric antigen receptor NK cells
  • TCR Exogenous T cell receptor (TCR) T cells
  • the TCR gene expression of the CAR-T cell is silenced.
  • the expression of the TCR ⁇ chain gene and the expression of the TCR ⁇ chain gene of the CAR-T cell are silenced.
  • the "gene expression is silenced” means that the silenced gene is not expressed or is low expressed.
  • the "low expression” refers to the ratio of the expression level of the CAR-T cell silenced gene G1 to the corresponding gene expression level G0 of normal T cells, that is, G1/G0 ⁇ 0.5, preferably G1/ G0 ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, most preferably 0.
  • the immune cells express exogenous cell suicide elements.
  • the CAR and the cell suicide element are co-expressed in the immune cells.
  • the CAR and the cell suicide element are connected by a self-shearing element.
  • the cell suicide element is located at the N-terminal or C-terminal of the CAR.
  • the self-cleaving element includes 2A sequence or IRES sequence, preferably: P2A and T2A.
  • the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, EGFRt, or a combination thereof.
  • the structure of the cell suicide element is shown in the following formula V:
  • Each "-" is independently a connecting peptide or a peptide bond
  • L2 is an optional signal peptide sequence
  • D is the suicide switch element
  • F is the transmembrane element.
  • the signal peptide is derived from GM-CSFR.
  • the suicide switch element is selected from the group consisting of truncated epidermal growth factor receptor (EGFRt), truncated CD19 (CD19t) gene, induced caspase 9 gene (iCasp9), HSV-TK, ⁇ CD20, mTMPK, or a combination thereof.
  • EGFRt epidermal growth factor receptor
  • CD19t truncated CD19 gene
  • iCasp9 induced caspase 9 gene
  • HSV-TK induced caspase 9 gene
  • ⁇ CD20 ⁇ CD20
  • mTMPK mTMPK
  • the suicide switch element is icasp9 (FKBP12-F36V-Caspase9).
  • the endogenous HLA-I and HLA-II genes of the CAR-T cell are normally expressed.
  • the "normal gene expression” means that the HLA-I gene expression level of the CAR-T cell is the same as or substantially the same as the HLA-I gene expression level of the normal T cell.
  • the ratio (E1/E0) of the HLA-I gene expression E1 of the CAR-T cell to the HLA-I gene expression E0 of the normal T cell is 0.5-2.0, preferably 0.6-1.5, more The best place is 0.8-1.2.
  • the HLA-I includes HLA-A, HLA-B, HLA-C, HLA-E and/or HLA-G.
  • the endogenous HLA-E and/or HLA-G of the CAR-T cell is normally expressed or overexpressed.
  • the "overexpression” refers to the ratio of the expression level of the CAR-T cell gene HLA-E and/or HLA-G F1 to the corresponding gene expression level F0 of normal T cells, that is, F1/F0 ⁇ 1.5, preferably F1/F0 ⁇ 2, more preferably F1/F0 ⁇ 5.
  • the CAR-T cell expresses exogenous HLA-E and/or HLA-G.
  • the exogenous HLA-E includes HLA-E SCT (HLA-E single-chain trimer).
  • the structure of the HLA-E SCT is shown in the following formula VI:
  • Each "-" is independently a connecting peptide or a peptide bond
  • L3 is an optional signal peptide sequence
  • S is an autoantigen peptide element
  • E is the HLA-E heavy chain element.
  • the CAR-T cells can be used for the treatment of autologous and/or allogeneic tumors.
  • the CAR-T cell universal type CAR-T cell.
  • nucleic acid molecule which encodes the chimeric antigen receptor of the first aspect of the present invention.
  • a vector is provided, and the vector contains the nucleic acid molecule according to the third aspect of the present invention.
  • the vector includes DNA and RNA.
  • the vector is selected from the following group: plasmid, viral vector, transposon, or a combination thereof.
  • the vector includes DNA virus and retrovirus vector.
  • the vector is selected from the group consisting of a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is a lentivirus vector.
  • a method for preparing engineered immune cells that express the chimeric antigen receptor of the first aspect of the present invention including the following steps:
  • the nucleic acid molecule described in the aspect or the vector described in the fourth aspect of the present invention is transferred into immune cells to obtain the engineered immune cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • the immune cells are modified so that the immune cells express CARs that target the first target and the second target, thereby obtaining the immune cells of the second aspect of the present invention.
  • step (B) the endogenous PD-1 gene and/or TCR gene are knocked out using a gene editing system.
  • the gene editing system includes CRISPR-Cas9 system, zinc finger protein system, or TALEN system.
  • the CRISPR-Cas9 system includes gRNA and Cas9 protein.
  • the immune cells are T cells.
  • step (B) the T cell is modified so that the PD-1 gene expression of the CAR-T cell is silenced.
  • a gene editing system is used to knock down the PD-1 gene. Except for the PD-1 gene.
  • step (B) the T cell is modified so that the TCR gene expression of the CAR-T cell is silenced.
  • a gene editing system is used to knock out the TCR gene.
  • step (B) the T cells are modified so that the CAR-T cells express exogenous cell suicide elements.
  • a preparation which contains the engineered immune cells described in the second aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the dosage form of the preparation is injection.
  • the concentration of the genetically engineered cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml .
  • a preparation which contains a first CAR-T cell and a second CAR-T cell, and the first CAR-T cell expresses a CAR that targets CD20, so The second CAR-T cell expresses a CAR that targets the second target.
  • the first CAR-T cell expresses the aforementioned first CAR.
  • the second CAR-T cell expresses the aforementioned second CAR.
  • a CAR according to the first aspect of the present invention an engineered immune cell according to the second aspect of the present invention, a nucleic acid molecule according to the third aspect of the present invention, or the present invention
  • the use of the carrier described in the fourth aspect is to prepare a preparation for preventing and/or treating autologous tumors or allogeneic tumors.
  • the tumor is a tumor with CD20 markers on the surface of tumor cells.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B cell lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small cell lung
  • the solid tumor is selected from the group consisting of ovarian cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, endometrial cancer, or a combination thereof.
  • kits for preparing the engineered immune cells according to the second aspect of the present invention, the kit containing a container, and in the container:
  • a first nucleic acid sequence which contains a first expression cassette for expressing the CAR.
  • the kit further contains: (2) a second nucleic acid sequence, which contains a second expression cassette or a third gRNA for silencing the TCR gene.
  • the kit further contains: (3) a third nucleic acid sequence, which contains a third expression cassette or a fourth gRNA for silencing the PD-1 gene.
  • the kit further contains: (4) a fourth nucleic acid sequence, and the fourth nucleic acid sequence contains a fourth expression cassette for expressing exogenous cell suicide elements.
  • each of the nucleic acid sequences is independent or connected.
  • nucleic acid sequences are located in the same or different containers.
  • any two, three or four of the nucleic acid sequences are located in the same expression vector.
  • the kit further contains: (4) a fifth nucleic acid sequence, which contains a fifth expression cassette for expressing Cas9 protein; or Cas9 protein.
  • the eleventh aspect of the present invention there is provided a use of the engineered immune cell according to the second aspect of the present invention to prevent and/or treat autologous tumors or allogeneic tumors.
  • a method for preventing and/or treating autologous tumors or allogeneic tumors includes the steps of administering the engineered immune cell cells described in the second aspect of the present invention to a subject in need.
  • Figure 1 shows the expression of different structures of CD20-CD19-CAR on Jurkat cells (CD19 antigen test). 1#-11#CAR is expressed on Jurkat cell line and detected by CD19 antigen.
  • Figure 2 shows the expression of different structures of CD20-CD19-CAR on Jurkat cells (CD20 antigen test). 1#-11#CAR is expressed on Jurkat cell line and detected by CD20 antigen. Among them, the ability of 10# and 11#CAR to recognize CD20 antigen on Jurkat cells is weaker than other CARs.
  • FIG 3 shows the expression of CD20-CD19-CAR with different structures on primary T cells (CD20 and CD19 antigen test). The results showed that the 10# and 11# CARs have weaker ability to recognize antigens on T cells than other CARs.
  • Figure 4 shows the killing effect of different structures of CD19-CD20-CAR-T against Hela antigen overexpressing cell lines (RTCA method).
  • the results show that 1#-11#CAR-T cells have a significant ability to kill target cells, and can kill CD19 or CD20 single antigen overexpressing Hela cells (Hela-CD19, Hela-CD20), indicating that dual CAR-T Both of the above CAR molecules can function.
  • Figure 5 shows the secretion of IFN ⁇ during the killing of Hela antigen-overexpressing cells by CD19-CD20-CAR-T of different structures.
  • Figure 6 shows the killing ability of different structures of CD19-CD20-CAR-T against Raji tumor cells and knockout cell lines (Raji, Raji-KO19, Raji-KO20 and Raji-KO1920). The results showed that among the 1-9#CAR-T cells' killing ability, groups 1, 2, 3, 4, 5, and 9 showed better target cell killing ability.
  • Figure 7 shows the release of IFN ⁇ during the killing of Raji tumor cells and knockout cell lines (Raji, Raji-KO19, Raji-KO20 and Raji-KO1920) by CD19-CD20-CAR-T of different structures.
  • the results show that 1#, 2#, 3#, 9# CAR-T can release IFN ⁇ not lower than other CAR-T groups.
  • Figure 8 shows the CD4:CD8 ratio of 1#CAR-T to single CAR-T.
  • Figure 9 shows that 1#CAR-T has a higher killing efficiency on Hela overexpressing cells than single CAR-T (RTCA method).
  • the results show that 1#CAR-T has a higher killing ability against Hela-19 and Hela-20 target cells than CD19 CAR-T and CD20 CAR-T cells, and it also has a higher killing ability against Hela-CD19 CD20 target cells. Not less than the killing ability of single CAR-T cells.
  • Figure 10 shows that 1#CAR-T is more effective than single CAR-T in killing target cells.
  • the results show that 1#CAR-T is significantly better than CD19 CAR-T and CD20 CAR-T cells against CD19 and CD20 double-positive target cells Raji, and against CD19 or CD20 knockout Raji-KO19 or Raji-KO20 cells, 1#CAR-T cells kill significantly higher than the corresponding CD20 CAR-T cells or CD19 CAR-T single CAR-T cells.
  • Figure 11 shows that the amount of INFr secreted in the process of 1#CAR-T killing target cells is greater than that of single CAR-T.
  • the results show that in the process of killing Raji or Raji-KO19, Raji-KO20 target cells, the secretion of the cytokine IFN ⁇ of 1#CAR-T is also significantly higher than that of CD19 CAR-T or CD20 CAR-T single CAR-T cell.
  • Figure 12 shows that 1#CAR-T is better than single CAR-T (LDH method) in killing various target cells.
  • 1#CAR-T showed no less than CD19 CAR-T or CD20 CAR-T single CAR-T cell killing ability, especially for Pfeiffer and SU-DHL-10 The killing ability is superior to that of single CAR-T cells.
  • Figure 13 shows a schematic diagram of the structure containing the CAR of the present invention and the cell suicide element.
  • Figure 14 shows that 1#CAR-T is better than single CAR-T in up-regulating CD107a during incubation with different target cells.
  • the results showed that when 1#CAR-T cells were incubated with single-target or dual-target target cells, 1#CAR-T cells all showed higher single CAR-T than CD19 CAR-T or CD20 CAR-T.
  • the ability of T cell CD107a to up-regulate expression Among them, both the CD4 positive cell population and the CD8 positive cell population are reflected.
  • Figure 15 shows that in the subcutaneous Raji model, the cell reinfusion 1#CAR-T has better than single CAR-T's ability to eliminate tumors and inhibit expansion.
  • Experiments with subcutaneously modeled mice showed that for Raji cell modeled mice, 1#CAR-T cells showed higher tumor clearance ability than single CAR-T cells of CD19 CAR-T or CD20 CAR-T.
  • Figure 16 shows that in the subcutaneous Raji model, cell reinfusion 1#CAR-T has the ability to eliminate tumors and inhibit expansion.
  • Experiments with subcutaneously modeled mice showed that colleagues who modeled mice with Raji cells and 1#CAR-T cleared tumor cells did not show a weight loss higher than that of the NT group, indicating that it is safe and reliable.
  • a chimeric antigen receptor immune cell that simultaneously targets CD20 and CD19.
  • a cell suicide element is added to control the activity of CAR-T cells and CRS and other related toxicities, and enhance safety.
  • it has a synergistic effect, reduces the recurrence rate, and has a better curative effect.
  • the use of specific suitable flexible linkers between scFvs of the present invention can increase the elastic space of scFvs, retain the affinity of scFvs as much as possible, and reduce the mutual interference between different scFvs when targets are used in combination. On this basis, the present invention has been completed.
  • administration refers to the use of any of the various methods and delivery systems known to those skilled in the art to physically introduce the product of the present invention into a subject, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or Other parenteral routes of administration, such as by injection or infusion.
  • antibody shall include, but is not limited to, immunoglobulins, which specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigens thereof Combine parts.
  • Each H chain contains a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region contains a constant domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), which are interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • CD19 is a 95kDa glycoprotein on the surface of B cells. It is expressed from the early stage of B cell development until it differentiates into plasma cells. CD19 is a member of the immunoglobulin (Ig) superfamily. As one of the components of the B cell surface signal transduction complex, it participates in the regulation of the signal transduction process of the B cell receptor. In CD19-deficient mouse models, the number of B cells in peripheral lymphoid tissues will be significantly reduced, and the response to vaccines and mitogens will also decline, accompanied by a decrease in serum Ig levels. It is generally believed that the expression of CD19 is limited to B-cell lineage and not on the surface of pluripotent hematopoietic stem cells.
  • CD19 is also expressed on the surface of most B-cell lymphomas, mantle cell lymphomas, ALLs, CLLs, hairy cell leukemia, and some acute myeloid leukemia cells. Therefore, in the treatment of leukemia/lymphoma, CD19 is a very valuable immunotherapy target. Importantly, CD19 will not be expressed on the surface of most normal cells except B cells, including pluripotent hematopoietic stem cells. This feature makes CD19 a safe therapeutic target, which can cause patients to develop autoimmune diseases or The risk of irreversible bone marrow toxic damage is minimized.
  • anti-CD19 antibodies or scFv fragments have been developed, and their application prospects have been proven in mouse models and humans/primates.
  • CD20 antigen is a non-glycosylated cell membrane phosphoprotein with a molecular weight of 35kd. Current studies have found that CD20 antigen is a part of the signal transmission channel complex, its function is similar to calcium ion channels, and participates in regulating the growth and differentiation of B lymphocytes. The expression of CD20 antigen is strictly limited to the advanced and mature B lymphocytes of pre-B lymphocytes, and hematopoietic stem cells. There is no CD20 antigen expression in progenitor cells and other normal tissues. When B lymphocytes differentiate into plasma cells that secrete antibodies, the expression of CD20 antigen disappears. More than 95% of B cell NHLs express CD20 antigen.
  • CD20 is also one of the targets for the treatment of B-cell lymphoma and leukemia.
  • the successfully developed monoclonal antibody drug Rituximab (Rituxan, Genentech, Inc, approved by the FDA in January 2011 for NHL treatment) , Obinutuzumab (Gazyva, Genentech, Inc, FDA approved in November 2013 for CLL treatment), Ibritumomab tiuxetan (Zevalin, IDEC Pharmaceuticals Corp., FDA approved In February 2002, used for NHL treatment), Tositumomab (Bexxar, GSK, approved by the FDA in June 2003, used for NHL treatment), and Ofatumumab (Genmab, approved by the FDA) In October 2009, used for CLL treatment).
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also called antigen binding domains).
  • the intracellular domain includes the costimulatory signal transduction region and the zeta chain part.
  • the costimulatory signal transduction region refers to a part of the intracellular domain that includes costimulatory molecules.
  • Co-stimulatory molecules are cell surface molecules required for effective response of lymphocytes to antigens, not antigen receptors or their ligands.
  • the design of CARs has gone through the following process: the first generation CAR has only one intracellular signal component CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , And cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical effects.
  • the second-generation CARs introduce a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function has been greatly improved, which further strengthens the persistence of CAR-T cells and the effect on tumor cells. The killing ability. On the basis of the second-generation CARs, some new co-stimulatory molecules such as CD27 and CD134 are connected in series to develop into the third and fourth-generation CARs.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the present invention includes a binding domain that targets CD20 and a binding domain that targets a non-CD20 tumor antigen.
  • the preferred tumor antigen is CD19.
  • the CAR of the present invention can perform antigen recognition based on the antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, resulting in tumor cells not growing, being promoted to die or being affected in other ways, and causing the patient's tumor burden to shrink or eliminate.
  • the antigen binding domain is preferably fused with an intracellular domain from one or more of the costimulatory molecule and the zeta chain.
  • the antigen binding domain is fused with the intracellular domain combined with the 4-1BB signaling domain and the CD3 ⁇ signaling domain.
  • the extracellular domain of the CAR provided by the present invention also includes a signal peptide L.
  • the L is a signal peptide derived from CD8.
  • the L has an amino acid sequence as shown in SEQ ID NO.:30.
  • antigen binding domain and “single chain antibody fragment” all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments that have antigen binding activity.
  • Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • the antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1/6 that of a complete antibody.
  • the single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain.
  • the scFv includes an antibody that specifically recognizes CD19 and/or CD20, preferably a single chain antibody.
  • the CD20-targeting antigen binding domain includes the heavy chain variable region and the light chain variable region of monoclonal antibody Leu16.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 5, and the antibody light chain as shown in SEQ ID NO.: 6 Change area.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 9 and the antibody light chain as shown in SEQ ID NO.: 10 Change area.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 11, and the antibody light chain as shown in SEQ ID NO.: 12 Change area.
  • the CD20-targeting antigen-binding domain comprises the antibody heavy chain variable region shown in SEQ ID NO.: 13, and the antibody light chain shown in SEQ ID NO.: 14 Change area.
  • the CD20-targeting antigen binding domain comprises the variable region of the antibody heavy chain as shown in SEQ ID NO.: 31, and the antibody light chain as shown in SEQ ID NO.: 32. Change area.
  • the nucleotide sequence of the antigen-binding domain targeting CD20 comprises the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 1 and the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 2 The coding sequence of the variable region of the antibody light chain is shown.
  • the CD19-targeting antigen binding domain includes the heavy chain variable region and the light chain variable region of monoclonal antibody FMC63.
  • the CD19-targeting antigen-binding domain comprises the antibody heavy chain variable region shown in SEQ ID NO.: 7 and the antibody light chain shown in SEQ ID NO.: 8 Change area.
  • the nucleotide sequence of the antigen-binding domain targeting CD19 comprises the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 3, and the antibody heavy chain variable region coding sequence shown in SEQ ID NO.: 4 The coding sequence of the variable region of the antibody light chain is shown.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain that is naturally associated with one of the domains in the CAR is used.
  • transmembrane domains can be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing the interaction with the receptor complex. Interaction of other members.
  • the hinge region H is a hinge region derived from CD8.
  • the hinge region H has an amino acid sequence as shown in SEQ ID NO.:35.
  • the transmembrane region TM is a transmembrane region derived from CD8.
  • the transmembrane region TM has an amino acid sequence as shown in SEQ ID NO.: 36.
  • the C 41BB intracellular signal region
  • the CD ⁇ has an amino acid sequence as shown in SEQ ID NO.:38.
  • the CAR of the present invention also contains a flexible linker, and the flexible linker may include any connecting peptide for connecting various elements (such as antibody heavy chain variable region, light chain variable region).
  • the use of specific suitable flexible linkers between scFvs of the present invention can increase the elastic space of scFvs, retain the affinity of scFvs as much as possible, and reduce the mutual interference between different scFvs when the targets are used in combination.
  • the flexible linker (or connecting peptide) I may be composed of multiple sub-short peptides, and the multiple sub-short peptides may be the same or different.
  • the number n of each sub-short peptide is 0-6, preferably 0-5, more preferably 1-3.
  • the flexible linkers (or connecting peptides) each independently have the structure of the following formula VII:
  • each "-" is independently a connecting peptide or a peptide bond
  • X, Y and Z are each independently a sequence selected from the following group:
  • a, b, and c are each independently a positive integer of 0-6.
  • the flexible linker (or connecting peptide) I, Ia, Ib, Ic, Id each independently has a sequence as shown in any one of SEQ ID NO.: 15-25.
  • the CAR has an amino acid sequence as shown in any one of SEQ ID NO.: 26-29.
  • the CAR has an amino acid sequence as shown in any one of SEQ ID NO.:44.
  • CAR-T cell As used herein, the terms “CAR-T cell”, “CAR-T” and “CAR-T cell of the present invention” all refer to the universal CAR-T cell according to the first aspect of the present invention, which expresses A chimeric antigen receptor CAR that targets CD20 and the second target.
  • the CAR-T cell of the present invention knocks out the TCR expression in the cell through gene editing technology, so that the normal cells of the recipient will not be killed due to TCR recognition during allogeneic infusion, that is, it will not carry To GVHD response; by simultaneously targeting CD19 and CD20 CAR-T eliminates tumor cells while eliminating host T cells, avoiding host-versus graft (HVG) reactions, and increasing the presence of allogeneic CAR-T cells in the recipient In vivo survival and anti-tumor effect.
  • a suicide gene switch is added to CAR-T, and CAR-T can be cleared at any time when necessary to reduce side effects of immunotherapy.
  • the construction structure is the chimeric antigen receptor of CD20 scFv-CD19 scFv-Hinge-TM-CD28/41BB-CD3 ⁇
  • the CD20 scFv fragment is the heavy chain of the monoclonal Leu16 antibody and The light chain variable region is connected by a connecting peptide.
  • the CD19 scFV fragment is a monoclonal antibody FMC63.
  • the heavy and light chain variable regions are connected by a connecting peptide. It also includes the hinge region and transmembrane region in series, human CD28 or 41BB intracellular The costimulatory element, and the human CD3 ⁇ intracellular domain.
  • suicide gene switch As used herein, the terms “suicide gene switch”, “suicide switch”, “safety switch”, and “cellular suicide element” have the same meaning, and all refer to the ability to effectively eliminate CAR-T cells in the body under the action of exogenous drugs. element.
  • the suicide gene switch used in the present invention can be EGFRt, HSV-TK, iCasp9, CD20, mTMPK, etc.
  • HSV-TK, iCasp9 and CD20 have the same ability to clear T cells, but the clearance of iCasp9 and CD20 is faster, and HSV-TK is slower.
  • the preferred suicide gene switch is icasp9.
  • CAR-T cells are active drugs. Once imported, the physiological mechanism regulates T cell balance, memory formation and antigen-driven expansion. However, this treatment is not yet perfect. T cells will off-target and attack other tissues, or the amount of expansion will be too high for treatment. In view of the fact that CAR-T cells have been included in the scope of standard treatment, it is very useful to design a patient or drug controllable activation or shutdown mechanism to regulate the existence of CAR-T cells. Due to technical reasons, the shutdown mechanism is easier to apply to T cells.
  • the scavenging antibodies that have been used clinically can be used to make CAR-T cells express the proteins that these antibodies target at the same time, such as icasp9. After the treatment-related toxicity occurs or the treatment has been completed, the corresponding CAR-T cells can be eliminated by giving antibody drugs. T cells.
  • the CAR-T cells of the present invention all have a suicide gene switch, which can effectively eliminate CAR-T cells in the body under the action of exogenous drugs.
  • T cells block unknown or uncontrollable long-term toxicity to ensure patient safety.
  • the CAR-T cell of the present invention introduces a safety switch that is icasp9.
  • iCasp9 is a suicide gene system, which is composed of an inducible caspase9 (iCasp9) gene and a small molecule chemically inducing drug with no biological activity.
  • the small molecule chemically inducing drug is selected from AP1903 and AP20187, preferably AP1903.
  • TCR/MHC complex Tcell receptor, TCR
  • T cell receptor is a characteristic mark on the surface of all T cells, which binds to CD3 by non-covalent bonds to form a TCR-CD3 complex.
  • the role of TCR is to recognize antigens and activate T cells to play a killing effect.
  • TCR is a heterodimer composed of two different peptide chains.
  • TCR is divided into two types: TCR1 and TCR2.
  • TCR1 is composed of two chains, ⁇ and ⁇
  • TCR2 is composed of two chains, ⁇ and ⁇ .
  • TCR2 In peripheral blood, 90%-95% of T cells express TCR2.
  • Each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region; its characteristic is that the cytoplasmic region is very short.
  • TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity exists in the V region; V regions (V ⁇ , V ⁇ ) each have three hypervariable regions CDR1, CDR2, and CDR3, of which CDR3 has the largest variation, which directly determines the antigen of TCR Binding specificity.
  • V regions V ⁇ , V ⁇
  • CDR1 and CDR2 recognize and bind to the sidewall of the antigen binding groove of the MHC molecule
  • CDR3 directly binds to the antigen peptide.
  • Major histocompatibility antigen is a general term for all biocompatible complex antigens (MHC molecule), which means a molecule encoded by the MHC gene family (MHC class I, class II, class III), located in On the cell surface, the names of the encoded products of different mammalian MHC genes are different.
  • Human MHC is usually called HLA (human leucocyte antigen, HLA), that is, human leucocyte antigen.
  • HLA human leucocyte antigen
  • MHC gene located on the short arm of human chromosome 6, is highly polymorphic.
  • Immune checkpoints refer to some inhibitory signal pathways in the immune system, which avoid tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues, and participate in maintaining tolerance to self-antigens. Using the inhibitory signal pathway of immune checkpoints to inhibit T cell activity is an important mechanism for tumors to avoid immune killing. Therefore, enhancing the activation of T cells through different strategies is of great significance for tumor immunotherapy.
  • the present invention enhances T cell activation by blocking PD-1 signal.
  • PD-1 includes human PD-1 (hPD-1), variants of hPD-1 (mutated hPD-1), isotype and species homologues, and those with hPD-1 An analog with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.
  • the complete hPD-1 sequence can be found under GenBank accession number U64863.
  • CRISPR/Cas9 RNA interference technology
  • TALENs transcription activator-like (TAL) effect nucleases
  • ZFNs Zinc finger nucleases
  • the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas (CRISPR-associated) system is a natural immune system unique to prokaryotes, which is used to resist viruses or foreign plasmids.
  • Type II CRISPR/Cas system as a genome editing tool mediated by RNA has been successfully applied in many eukaryotes and prokaryotes.
  • the development of the CRISPR/Cas9 system has completely changed people's ability to edit DNA sequences and regulate the expression level of target genes, thereby providing powerful tools for precise genome editing of organisms.
  • the simplified CRISPR/Cas9 system consists of two parts: Cas9 protein and gRNA.
  • the principle of action is that gRNA forms a Cas9-gRNA complex with Cas9 protein through its Cas9 handle.
  • the base complementary pairing region sequence of gRNA in the Cas9-gRNA complex and the target sequence of the target gene are paired and combined through the principle of base complementary pairing.
  • Cas9 uses its own endonuclease activity to cut the target DNA sequence.
  • the CRISPR/Cas9 system has several obvious advantages: ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes at the same time.
  • T cell therapy by infusion of T cells is designed to rebuild immunity against pathogens and malignant tumors.
  • the amount of time required to produce T cells with desired properties and a sufficient number ex vivo is often incompatible with the patient's treatment window.
  • autologous T cells from patients with advanced disease may have impaired function and are tolerant to the desired antigen.
  • patients can be infused with allogeneic T cells to avoid immune-mediated rejection caused by host T cells that recognize different primary or secondary histocompatibility antigens on the infused cells.
  • allogeneic T cells can be infused with allogeneic T cells to avoid immune-mediated rejection caused by host T cells that recognize different primary or secondary histocompatibility antigens on the infused cells.
  • a rapid and robust method for isolating the source of TCR and HLA class I disrupted populations can be generated.
  • the nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard Technology to separate directly from cells and tissues containing the gene.
  • the gene of interest can be produced synthetically.
  • the present invention also provides a vector into which the expression cassette of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • the expression cassette or nucleic acid sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector.
  • the vector is suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector contains transcription and translation terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression construct of the present invention can also use standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, for example, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors, which include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector can be provided to the cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.
  • a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent number 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to target cells in vivo or in vitro.
  • Many retroviral systems are known in the art.
  • adenovirus vectors are used.
  • Many adenovirus vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation. Generally, these are located in the 30-110 bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible in order to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before the activity begins to decrease.
  • tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter , Myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired.
  • inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter.
  • the expression vector introduced into the cell may also contain either or both of the selectable marker gene or the reporter gene, so as to facilitate the search for the cell population to be transfected or infected by the viral vector To identify and select expressing cells.
  • the selectable marker can be carried on a single piece of DNA and used in the co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that does not exist in or is expressed by a recipient organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzyme activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct with a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be linked to a reporter gene and used to evaluate the ability of the reagent to regulate the promoter-driven transcription.
  • the vector can be easily introduced into a host cell by any method in the art, for example, mammalian, bacterial, yeast, or insect cells.
  • the expression vector can be transferred into the host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and so on. Methods of producing cells including vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, such as human cells.
  • Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, and so on. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, and beads
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids Plastid.
  • Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (for example, artificial membrane vesicles).
  • an exemplary delivery vehicle is a liposome.
  • Lipid-associated nucleic acids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, and attached via linking molecules associated with both liposomes and oligonucleotides
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in the solution.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • lipids include fat droplets, which occur naturally in the cytoplasm and in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides an engineered immune cell containing the second aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine ; Antioxidant; Chelating agent such as EDTA or glutathione; Adjuvant (for example, aluminum hydroxide); and Preservative.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • protein polypeptides or amino acids such as glycine
  • Antioxidant such as EDTA or glutathione
  • Adjuvant for example, aluminum hydroxide
  • Preservative for example, aluminum hydroxide
  • the present invention includes therapeutic applications with cells (e.g., T cells) transduced with lentiviral vectors (LV) encoding the expression cassettes of the present invention.
  • the transduced T cells can target tumor cell markers CD19 and CD20, can be used for autologous and allogeneic tumor treatment, can be prepared on a large scale, have uniform and stable quality, and can be used by any patient at any time.
  • the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, which comprises the following steps: administering the CAR-T cell of the present invention to the mammal.
  • the present invention includes a type of cell therapy in which the universal CAR-T cells modified by the present invention are directly administered to patients in need.
  • the CAR-T cells of the present invention can eliminate tumor cells by simultaneously targeting CD19 and CD20 CAR-T.
  • a suicide gene switch is added to CAR-T, and CAR-T can be cleared at any time when necessary to reduce side effects of immunotherapy.
  • one CAR-T can treat all cancers that express the antigen.
  • CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the present invention can undergo stable T cell expansion in vivo and last for an extended amount of time.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • anti-CD19 CAR-T cells elicit a specific immune response against CD19-expressing cells.
  • Cancers that can be treated include tumors that have not been vascularized or have not been substantially vascularized, as well as vascularized tumors.
  • the cancer may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or may include solid tumors.
  • the types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcoma, carcinoma, and melanoma. Also includes adult tumor/cancer and childhood tumor/cancer.
  • Hematological cancer is cancer of the blood or bone marrow.
  • leukemias include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic type , Monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non- Hodgkin's lymphoma (painless and high-grade form), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such as acute lymphoblastic leukemia, acute myeloid leuk
  • a solid tumor is an abnormal mass of tissue that does not usually contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and cancer include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.
  • the CAR-modified T cell of the present invention can also be used as a type of vaccine for ex vivo immunity and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells are isolated from mammals (preferably humans) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing the CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits.
  • the mammalian recipient can be a human, and the CAR-modified cell can be autologous relative to the recipient.
  • the cell may be allogeneic, syngeneic, or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides a method of treating tumors, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.
  • the CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical composition of the present invention may include the target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelate Mixtures such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelate Mixtures such as EDTA or glutathione
  • adjuvants for example, aluminum hydroxide
  • preservatives for example, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the number and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.
  • the precise amount of the composition of the present invention to be administered can be determined by the physician, who considers the patient (subject ) Individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. May generally indicated: including those described herein, the pharmaceutical compositions of T cells may be 104 to 109 doses cells / kg body weight, preferably 105 to 106 cells / kg body weight doses (including all integers within that range Value) application. The T cell composition can also be administered multiple times at these doses.
  • the cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation.
  • the compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previously , At the same time or after) administration to the patient, the treatment form includes but not limited to treatment with the following agents: the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients.
  • the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies Or other immunotherapeutics.
  • the cell composition of the present invention is administered to bone marrow transplantation using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (for example, before, simultaneously, or after) patient.
  • chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (for example, before, simultaneously, or after) patient.
  • chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide
  • the subject may undergo the standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of the expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratio for human administration can be implemented according to the practice accepted in the art.
  • 1 ⁇ 10 6 to 1 ⁇ 10 10 modified T cells (for example, CAR-T20 cells) of the present invention can be administered to the patient per treatment or course of treatment, for example, by intravenous infusion .
  • Combining targets can expand the range of CAR-T cells to recognize target cells, reduce the chance of tumor cell escape, and reduce the chance of recurrence.
  • a suitable linker can retain the affinity of each scFv in the dual target to the greatest extent, and reduce the influence of steric hindrance on the structure and function of CAR.
  • Adding a safety switch can increase the safety of CAR-T cells.
  • CAR-T cells were co-cultured with tumor cells in vitro to detect the release of cytokines IFN ⁇ and IL-2. At the same time, in vivo pharmacodynamic experiments were performed to detect the regression of the tumor after the tumor-bearing mice were injected with CAR-T cells.
  • Example 1 Isolation of PBMC and expansion of T cells from donor blood
  • the Hela cells expressing CD20, CD19 and CD20/CD19 simultaneously express CD20 and CD19 antigens through lentiviral vector, and then obtain stable transfection cell line after monoclonal screening, which can express CD20 and CD19 specifically
  • the protein molecules were obtained after screening of the ffluc cells of Raji, Daudi and Nalm6 using fireflyluciferase lentivirus infection.
  • CD20-CD19Bi-CAR The structure of the CAR that simultaneously targets CD20 and CD19 in the present invention is shown in Figure 13 (hereinafter referred to as CD20-CD19Bi-CAR).
  • CD20 CAR, CD19 CAR and suicide switch icasp9 (FKBP12-F36V-Caspase9) components are connected through T2A.
  • the present invention designs CARs targeting CD20 and CD19 with various structures.
  • the elements and sequences contained in each CAR are shown in Table 1 below.
  • the number in the table is the sequence number in the sequence table, that is, 1 means SEQ ID NO.:1.
  • the linker design of 2# and 3#CAR introduces proline and glycine, which can theoretically change the spatial conformation of CD19 and CD20 CAR to recognize antigens, and increase the ability of dual CARs to better recognize target antigens.
  • the 9#CAR linker has added the DYKDDDDK (SEQ ID NO.: 40) sequence, which can be recognized by specific antibodies, which can increase the convenience of CAR-T cell detection.
  • the designed CD20-CD19 CAR gene was cloned into the FUW lentiviral vector backbone and placed under the EF1 ⁇ (EF-1 ⁇ ) promoter to form Fuw-EF1 ⁇ -CD20-CD19-icasp9 CAR, and Fuw-EF1 ⁇ -CD20- CD19-icasp9CAR and lentivirus packaging system helper plasmids were transferred into 293T using Lipofectamine3000 to prepare a complete lentiviral expression vector; the virus supernatant was collected at 48h and 72h, and the supernatant was concentrated (Merck) Millipore; the concentrated virus can be used to infect T cell.
  • EF-1 ⁇ EF-1 ⁇
  • the constructed virus was used to infect Jurkat cells.
  • the flow cytometry results are shown in Figures 1 and 2.
  • the expression of CD20 CAR and CD19 CAR can be detected on the surface of Jurkat cells at the same time, indicating that the designed CAR gene can produce CD20 CAR and CD19 at the same time.
  • Lentiviral vector for CAR In the figure, the definitions of 1#-11# and CD19 CAR-T, CD20 CAR-T and CD20-2 CAR-T are as described in Table 1.
  • the expression of 1#-11#CAR on Jurkat can be detected by CD19 antigen, while the ability of 10# and 11#CAR to recognize CD20 antigen on Jurkat cells is weaker than that of other CARs.
  • the experimental method is as follows:
  • Example 3 Two days after the isolated and purified primary T cells were activated, the lentivirus constructed in Example 3 was used to carry out lentiviral vector infection at MOI (1-10), transferred to a cell culture flask, and placed at 37°C, 5% CO 2 constant temperature Cultivate in an incubator.
  • CD20-CD19 CAR-T cells were successfully constructed.
  • Example 4 An in vitro killing experiment was performed on each CAR-T cell obtained in Example 4.
  • luciferase gene By transferring the luciferase gene into target cells, stable transfected cell lines are obtained after clonal screening.
  • luciferase and luciferin can react to produce fluorescence.
  • the intensity of fluorescence By detecting the intensity of fluorescence, the activity of luciferase can be measured, and the survival rate of cells can be detected to obtain the killing effect of CART cells.
  • the target cells for the experiment include: Hela, Hela-CD19, Hela-CD20, Hela-CD19-CD20.
  • Figure 9 separately shows the results of 1#CAR-T cell and single CAR-T cell killing Hela target cells.
  • 1#CAR-T has higher killing target cell efficiency than single CAR-T on Hela overexpressing cells, indicating 1 #CAR-T kills the target cells of Hela-19 and Hela-20. It can be seen that it has a higher killing ability than CD19 CAR-T and CD20 CAR-T cells, and the killing of Hela-CD19 CD20 target cells is not low. The killing ability of single CAR-T cells.
  • the Raji target cells were transfected with Luciferase, and CD19 or CD20 was knocked out on this basis to construct Raji-KO19, Raji-KO20, and the Raji cell line Raji-KO1920 double knocked by CD19 and CD20.
  • the Luciferase-labeled cells that survived the killing, the Luciferin substrate was catalyzed, and the bioluminescence of the 1-9#CAR-T cells was analyzed using a microplate reader to analyze the killing of Raji target cells.
  • 1-9#CAR-T cells all have significant ability to kill target cells. Among them, for the killing ability of 1-9#CAR-T cells, 1#, 2#, 3#, 4#, 5#, 9# showed better target cell killing ability.
  • Figure 10 shows the killing effect of 1#CAR-T on Raji target cells.
  • 1#CAR-T is significantly better than CD19 CAR-T and CD20 CAR-T cells against CD19 and CD20 double-positive target cells, while it is targeted at CD19 or CD20.
  • 1#CAR-T cells kill significantly higher than the corresponding CD20 CAR-T cells or CD19 CAR-T single CAR-T cells.
  • each CAR-T cell obtained in Example 4 and Hela target cells (Hela, Hela-CD19, Hela-CD20 and Hela-CD19-CD20) or Raji target cells (Raji, Raji-KO19, Raji-KO20 and Raji-KO1920) were co-cultured at a certain ratio, and the supernatant was collected overnight. After centrifugation, the supernatant was taken to detect the release level of cytokine IFN- ⁇ , and the Elisa kit (Biolegend) was used for detection.
  • 1#CAR-T cells showed higher IFN ⁇ release, which should be further analyzed.
  • Figure 11 shows the amount of INFr secreted in the process of 1#CAR-T killing Raji target cells, and the secretion of the cytokine IFN ⁇ of 1#CAR-T during the killing process of Raji or Raji-KO19, Raji-KO20 target cells It is significantly higher than the single CAR-T cell of CD19 CAR-T or CD20 CAR-T.
  • Example 8 Using LDH enzyme to detect cell killing
  • the LDH enzyme released during cell lysis was used to detect the killing of different target cells by 1#CAR-T cells and CD19 and CD20 single CAR-T cells.
  • the target cells include Pfeiffer cells, Farage cells, SU-DHL-4 cells, SU-DHL-6 cells, and SU-DHL-10 cells.
  • the cell mixture is transferred to a 96-well culture plate according to 100 ⁇ l per well, and each group is set to re-holes.
  • the target cell natural release hole (negative control) does not add effector cells and only adds 100 ⁇ l culture medium;
  • Killing activity (%) [(experimental group A value-total natural release A value) / (maximum release group A value-total natural release A value)] ⁇ 100%.
  • CD19 CAR-T and CD20 CAR-T cells were subjected to flow cytometry analysis of CD107a expression changes after activation, and the tumor cell lines expressing CD19 or/and CD20 were used for co-incubation and activation experiments.
  • the cells after co-incubation were labeled with antibodies for CD3, CD4, and CD107a, and then flow cytometric analysis was performed.
  • Example 4 In order to further verify the elimination of CART cells mediated by iCasp9 gene, the similar methods in Example 3, Example 4 and Example 7 were used to construct CD20-CD19 CAR-T/T cells that did not contain icasp9 gene as a control. experiment.
  • the drug-induced in vitro proliferation experiment of CAR-T cells in vitro detection of the proliferation activity of CD20-CD19-icasp9-CAR-T cells after the inducing drug is added. Add 10 nM AP1903 to the CFSE-labeled CD20-CD19-icasp9-CAR-T/CD20-CD19 CAR-T/T cell culture medium, and measure the number of dead and live cells at different times, and calculate the proportion of drug-induced cell death.
  • In vivo function experiment to verify the in vivo proliferation activity of CAR-T cells after drug induction.
  • Mice were injected with 5*10 6 T cells, FFLuc-CD20-CD19-icasp9-CAR-T and FFLuc-CD20-CD19 CAR-T cells, and AP1903 (50mg/mouse) was injected intraperitoneally on the 7th day, respectively on Day0/ Inject 3mg d-luciferin intraperitoneally on 2 days, take pictures with Xenogen IVIS Imaging System, and calculate the amount of photons/s/cm 2 /spherical angle (p/s/cm 2 /sr).
  • the RNP complex composed of gRNA and cas9 protein was transferred to CART by CRISPR technology. After knocking out TRAC, B2M, and PD1 genes, the universal CD20-CD19-CAR-T in vitro (CD20-CD19-UCAR-T) was tested. ) The ability to kill target cells. The results show that CD20-CD19-UCAR-T has higher or similar activity compared with CD20-CD19-CAR-T.
  • the expression of scFv that secretes PD-1 through T2A linkage allows CD20-CD19-CAR-T to express CAR and secrete scFv against PD-1.
  • the test can improve the killing ability of CD20-CD19-CAR-T in vitro.

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Abstract

本发明提供了CD20组合靶向，特别是同时靶向CD20和CD19的嵌合抗原受体免疫细胞，还添加了细胞自杀元件，以控制CAR-T细胞的活性和CRS等相关毒性，增强安全性；在scFv间使用了特定的接头增加弹性空间，最大可能保留scFv的亲和力，并减少不同scFv之间的相互干扰。

Description

CD20组合靶向的工程化免疫细胞 技术领域
本发明涉及免疫治疗领域，更具体地涉及CD20组合靶向的工程化免疫细胞。
背景技术
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法，来激发、增强机体自身免疫功能，从而达到治疗肿瘤的目的。
大部分的B细胞性恶性肿瘤表面都高表达CD19。目前，FDA批准上市的两款CAR-T产品,均针对CD19抗原,其适应证也在扩大,如慢性淋巴细胞白血病。尽管抗CD19 CAR-T的疗效突出,但仍有部分患者的治疗效果不佳,容易复发。
因此，本领域仍然需要进一步的研究，开发一种能更有效、特异性好、副作用小地治疗血液肿瘤和实体瘤，并且有效防止复发的治疗方法和药物。
发明内容
本发明的目的在于提供能更有效、特异性好、副作用小地治疗血液肿瘤和实体瘤，并且有效防止复发的治疗方法和药物。
本发明的另一目的是提供一种靶向双靶点或多靶点免疫疗法。
本发明的又一目的是提供一种特异性靶向CD20及另一靶点的特异性CAR-T细胞。
本发明的又一目的是提供一种具有自杀开关的同时靶向CD20和另一种靶点的工程化免疫细胞。
在本发明的第一方面，提供了一种嵌合抗原受体CAR，所述的CAR靶向第一靶点和第二靶点，其中所述第一靶点为CD20，所述第二靶点选自下组：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α(folate receptor-α)(FR-α)、或其组合。
在另一优选例中，所述第二靶点为CD19。
在另一优选例中，所述CAR还靶向选自下组中的一个或多个靶点：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在另一优选例中，所述CAR的结构如下式I所示：
L-scFv1-I-scFv2-H-TM-C-CD3ζ    (I)
式中，
各“-”独立地为连接肽或肽键；
L为任选的信号肽序列；
I为柔性接头；
H为任选的铰链区；
TM为跨膜结构域；
C为共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
scFv1和scFv2两者中一个为靶向所述第一靶点(CD20)的抗原结合结构域，另一个为靶向所述第二靶点的抗原结合结构域。
在另一优选例中，所述的scFv1为靶向CD20的抗原结合结构域，所述scFv2为靶向第二靶点的抗原结合结构域。
在另一优选例中，所述的scFv1和scFv2可以是各自独立的，也可以是串联的，或者是loop的结构。
在另一优选例中，所述scFv2和H之间为无或连接有元件(I-scFv0)m。
在另一优选例中，所述元件(I-scFv0)m为串联的m个相同或不同的I-scFv0，其中m为0-10的正整数,各I为柔性接头，各scFv0独立为靶向相同或不同靶点的抗原结合结构域。
在另一优选例中，所述靶点选自下组：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在另一优选例中，所述靶向CD20的抗原结合结构域的结构如下式A或式B所示：
V H1-V L1  (A)；V L1-V H1  (B)；
式中，V H1为抗CD20抗体重链可变区；V L1为抗CD20抗体轻链可变区；“-”为连接肽或肽键。
在另一优选例中，所述靶向CD20的抗原结合结构域包括单克隆号Leu16抗体的重链可变区和轻链可变区。
在另一优选例中，所述抗CD20抗体重链可变区具有如SEQ ID NO.:5、9、11、13、或31所示的氨基酸序列。
在另一优选例中，所述抗CD20抗体轻链可变区具有如SEQ ID NO.:6、10、12、14、或32所示的氨基酸序列。
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:5所示的抗 体重链可变区，和如SEQ ID NO.:6所示的抗体轻链可变区。
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:9所示的抗体重链可变区，和如SEQ ID NO.:10所示的抗体轻链可变区。
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:11所示的抗体重链可变区，和如SEQ ID NO.:12所示的抗体轻链可变区。
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:13所示的抗体重链可变区，和如SEQ ID NO.:14所示的抗体轻链可变区。
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:31所示的抗体重链可变区，和如SEQ ID NO.:32所示的抗体轻链可变区。
在另一优选例中，所述的如SEQ ID NO.:31所示的抗体重链可变区和如SEQ ID NO.:32所示的抗体轻链可变区通过SEQ ID NO.:41所示的连接肽相连。
在另一优选例中，所述靶向CD20的抗原结合结构域的核苷酸序列包含如SEQ ID NO.:1所示的抗体重链可变区编码序列，和如SEQ ID NO.:2所示的抗体轻链可变区编码序列。
在另一优选例中，所述靶向第二靶点的抗原结合结构域的结构如下式C或式D所示：
V H2-V L2  (C)；V L2-V H2  (D)；
式中，V H2为抗所述第二靶点抗体重链可变区；V L2为抗所述第二靶点抗体轻链可变区；“-”为连接肽或肽键。
在另一优选例中，所述的第二靶点为CD19。
在另一优选例中，所述靶向CD19的抗原结合结构域包括单克隆号FMC63抗体的重链可变区和轻链可变区。
在另一优选例中，所述抗CD19抗体重链可变区具有如SEQ ID NO.:7所示的氨基酸序列。
在另一优选例中，所述抗CD19抗体轻链可变区具有如SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，所述靶向CD19的抗原结合结构域包含如SEQ ID NO.:7所示的抗体重链可变区，和如SEQ ID NO.:8所示的抗体轻链可变区。
在另一优选例中，所述的如SEQ ID NO.:7所示的抗体重链可变区和如SEQ ID NO.:8所示的抗体轻链可变区通过SEQ ID NO.:21所示的连接肽相连。
在另一优选例中，所述靶向CD19的抗原结合结构域的核苷酸序列包含如SEQ ID NO.:3所示的抗体重链可变区编码序列，和如SEQ ID NO.:4所示的抗体轻链可变区编码序列。
在另一优选例中，所述的scFv1和/或scFv2为鼠源、人源、人源和鼠源嵌合、或者全人源化的单链抗体可变区片段。
在另一优选例中，所述的柔性接头(或连接肽)I可以由多个子短肽构成，所述多个子短肽可以相同或不同。
在另一优选例中，所述的V H1和V L1之间可以为无或通过柔性接头(或连接肽)Ia相连。
在另一优选例中，所述的V H2和V L2之间可以为无或通过柔性接头(或连接肽)Ib相连。
在另一优选例中，所述的柔性接头(或连接肽)Ia可以由多个子短肽构成，所述多个子短肽可以相同或不同。
在另一优选例中，所述的柔性接头(或连接肽)Ib可以由多个子短肽构成，所述多个子短肽可以相同或不同。
在另一优选例中，当多个子短肽不相同时，各子短肽的数量n为0-6，较佳地0-5，更较佳地1-3。
在另一优选例中，所述子短肽的序列选自下组：GGGGS(SEQ ID NO.:22)、EAAAK(SEQ ID NO.:18)、EXXXK(SEQ ID NO.:39)，其中X对应于序列表中Xaa，可以是任何天然存在的氨基酸)、DYKDDDDK(SEQ ID NO.:40)、SSGGGGGSCPYSNPSLCSGGGGGS(SEQ ID NO.:24)、GSTSGGGSGGGSGGGGSS(SEQ ID NO.:41)、GSTSGSGKPGSGEGSTKG(SEQ ID NO.:42)、SGGGGGS(SEQ ID NO.:33)、CPYSNPSLC(SEQ ID NO.:34)、SSGGGGGS(SEQ ID NO.:43)、或其任意组合。
在另一优选例中，所述的柔性接头(或连接肽)I、Ia或Ib各自独立地具有下式VII的结构：
Xa-Yb-Zc    (VII)
式中，
各“-”独立地为连接肽或肽键；
X、Y和Z各自独立为选自下组的序列：
Figure PCTCN2020073873-appb-000001
a、b和c各自独立地为0-6的正整数。
在另一优选例中，所述X为选自下组的序列：GGGGS、EAAAK、EXXXK、SSGGGGGS、GSTSGGGSGGGSGGGGSS、或GSTSGSGKPGSGEGSTKG。
在另一优选例中，所述Y为选自下组的序列：GGGGS、EAAAK、EXXXK、DYKDDDDK、CPYSNPSLC、GSTSGGGSGGGSGGGGSS、或GSTSGSGKPGSGEGSTKG。
在另一优选例中，所述Z为选自下组的序列：GGGGS、EAAAK、EXXXK、SGGGGGS、GSTSGGGSGGGSGGGGSS、或GSTSGSGKPGSGEGSTKG。
在另一优选例中，所述的柔性接头(或连接肽)I、Ia或Ib各自独立地具有选自下组的序列：(GGGGS)n、(EAAAK)n、(EXXXK)n、(GGGGS)a+(EAAAK)b+(GGGGS)c、(GGGGS)a+(EXXXK)b+(GGGGS)c、(GGGGS)a+(DYKDDDDK)b+(GGGGS)c、(SSGGGGGS+CPYSNPSLC+SGGGGGS)n、GSGGGGSCPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLC、(GSTSGGGSGGGSGGGGSS)n、(GSTSGSGKPGSGEGSTKG)n；
其中，n、a、b和c各自独立地为0-6的正整数。
在另一优选例中，所述a、b和c三者不同时为0。
在另一优选例中，所述的柔性接头(或连接肽)I、Ia或Ib各自独立地具有如SEQ ID NO.:15-25中任一所示的序列。
在另一优选例中，本发明公开的柔性接头包括(具体的)由任意数量的任意所述子短肽构成的柔性接头。
在另一优选例中，所述CAR的结构如下式II或II’所示：
L-V L-scFv-V H-H-TM-C-CD3ζ    (II)
L-V H-scFv-V L-H-TM-C-CD3ζ    (II’)
式中，
各“-”独立地为连接肽或肽键；
元件L、H、TM、C和CD3ζ如上所述；
scFv为靶向CD20的抗原结合结构域，V H为抗所述第二靶点抗体重链可变区，且V L为抗所述第二靶点抗体轻链可变区；或者scFv为靶向所述第二靶点的抗原结合结构域，V H为抗CD20抗体重链可变区，且V L为抗CD20抗体轻链可变区。
在另一优选例中，所述L为选自下组的蛋白的信号肽：CD8、CD28、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述L为CD8来源的信号肽。
在另一优选例中，所述L具有如SEQ ID NO.:30所示的氨基酸序列。
在另一优选例中，所述H为选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述H为CD8来源的铰链区。
在另一优选例中，所述H具有如SEQ ID NO.:35所示的氨基酸序列。
在另一优选例中，所述TM为选自下组的蛋白的跨膜区：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述TM为CD8来源的跨膜区。
在另一优选例中，所述TM具有如SEQ ID NO.:36所示的氨基酸序列。
在另一优选例中，所述C为选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
在另一优选例中，所述C为4-1BB来源的共刺激信号分子。
在另一优选例中，所述C具有如SEQ ID NO.:37所示的氨基酸序列。
在另一优选例中，所述CDζ具有如SEQ ID NO.:38所示的氨基酸序列。
在另一优选例中，所述的V L和scFv之间可以为无或通过柔性接头(或连接肽)Ic相 连。
在另一优选例中，所述的V H和scFv之间可以为无或通过柔性接头(或连接肽)Id相连。
在另一优选例中，所述的柔性接头(或连接肽)Ic或Id各自独立地可以由多个子短肽构成，所述多个子短肽可以相同或不同。
在另一优选例中，所述的柔性接头(或连接肽)Ic或Id各自独立地具有式VII的结构：Xa-Yb-Zc(VII)
式中，各“-”独立地为连接肽或肽键；
X、Y、Z、a、b和c如上所述。
在另一优选例中，所述的柔性接头(或连接肽)Ic或Id各自独立地具有选自下组的序列：(GGGGS)n、(EAAAK)n、(EXXXK)n、(GGGGS)a+(EAAAK)b+(GGGGS)c、(GGGGS)a+(EXXXK)b+(GGGGS)c、(GGGGS)a+(DYKDDDDK)b+(GGGGS)c、(SSGGGGGS+CPYSNPSLC+SGGGGGS)n、GSGGGGSCPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLC、(GSTSGGGSGGGSGGGGSS)n、(GSTSGSGKPGSGEGSTKG)n；
其中，n、a、b和c各自独立地为0-6的正整数。
在另一优选例中，所述a、b和c三者不同时为0。
在另一优选例中，所述的柔性接头(或连接肽)Ic或Id各自独立地具有如SEQ ID NO.:15-25中任一所示的序列。
在另一优选例中，所述CAR的结构为L-V H1-Ia-V L1-I-V L2-Ib-V H2-H-TM-C-CD3ζ。
在另一优选例中，所述CAR具有如SEQ ID NO.:26-29中任一所示的氨基酸序列。
在另一优选例中，所述的柔性接头(或连接肽)Ia的氨基酸序列如SEQ ID NO.:41所示。
在另一优选例中，所述的柔性接头(或连接肽)Ib的氨基酸序列如SEQ ID NO.:21所示。
在另一优选例中，所述的柔性接头I的氨基酸序列如SEQ ID NO.:15-25中任一所示。
在另一优选例中，所述的柔性接头I的氨基酸序列如SEQ ID NO.:15-19、23中任一所示。
在另一优选例中，所述的柔性接头I的氨基酸序列如SEQ ID NO.:15、16、17、23中任一所示。
在另一优选例中，所述的柔性接头I的氨基酸序列如SEQ ID NO.:15所示。
在另一优选例中，所述CAR的铰链区、跨膜区和胞内区的氨基酸序列如SEQ ID NO.:44所示。
在另一优选例中，所述CAR具有表1中1#、2#、3#、4#、5#、9#的CAR结构，较佳地，所述CAR具有表1中1#、2#、3#、9#的CAR结构，更佳地，所述CAR具有表1中1# 所示的CAR结构。
在另一优选例中，所述CAR具有SEQ ID NO.:26所示的氨基酸序列。
在本发明的第二方面，提供了一种工程化的免疫细胞，所述免疫细胞具有以下特征：
(a)所述免疫细胞表达嵌合抗原受体CAR，且所述CAR靶向第一靶点和第二靶点，其中所述第一靶点为CD20，所述第二靶点选自下组：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、FR-α、或其组合。
在另一优选例中，所述第二靶点为CD19。
在另一优选例中，所述CAR还靶向选自下组中的一个或多个靶点：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在本发明的另一方面，提供了一种工程化的免疫细胞，所述免疫细胞具有以下特征：
(a)所述免疫细胞表达外源性TCR，且所述TCR靶向第一靶点和第二靶点，其中所述第一靶点为CD20，所述第二靶点选自下组：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、FR-α、或其组合。
在另一优选例中，所述TCR还靶向选自下组中的一个或多个靶点：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在另一优选例中，所述CAR为单一CAR，并且所述的CAR同时靶向所述第一靶点和所述第二靶点。
在另一优选例中，所述CAR同时包含靶向所述第一靶点的抗原结合结构域和靶向所述第二靶点的抗原结合结构域。
在另一优选例中，所述CAR还包含选自下组中的一个或多个靶点的抗原结合结构域：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在另一优选例中，所述CAR为本发明第一方面所述的CAR。
在另一优选例中，所述CAR包括靶向所述第一靶点的第一CAR和靶向所述第二靶点 的第二CAR。
在另一优选例中，所述第一CAR的结构如下式III所示：
L-scFv1’-H-TM-C-CD3ζ    (III)
式中，
各“-”独立地为连接肽或肽键；
元件L、H、TM、C和CD3ζ如上所述；
scFv1’为靶向CD20的抗原结合结构域。
在另一优选例中，所述第二CAR的结构如下式IV所示：
L-scFv2’-H-TM-C-CD3ζ    (IV)
式中，
各“-”独立地为连接肽或肽键；
元件L、H、TM、C和CD3ζ如上所述；
scFv2’为靶向所述第二靶点的抗原结合结构域。
在另一优选例中，所述CAR还包括靶向选自下组中的一个或多个靶点的CAR：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在另一优选例中，所述CAR还包括靶向选自下组靶点的一个或多个CAR：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
在另一优选例中，所述免疫细胞具有选自下组的一种或多种特征：
(a)所述免疫细胞的PD-1基因表达是被沉默的；
(c)所述免疫细胞表达外源性细胞自杀开关元件；
(d)所述免疫细胞表达或分泌PD-1抗体、PD-L1抗体、CD47抗体、Tim3抗体、Lag3抗体、Tigit抗体、OX40抗体、ICOS抗体、IL7、CXCL19、IL21、IL15、IL2、IL18、或其组合；和
(e)所述免疫细胞的细胞因子相关信号通路被增强，其中所述细胞因子选自下组：IL7、CXCL19、IL21、IL15、IL2、IL18、或其组合。
在另一优选例中，所述的免疫细胞为T细胞并且用于异体免疫治疗，则所述T细胞的TCR基因表达是被沉默的。
在另一优选例中，所述免疫细胞的PD-1基因表达是被沉默的。
在另一优选例中，所述的工程化的免疫细胞选自下组：
(i)嵌合抗原受体T细胞(CAR-T细胞)；
(ii)嵌合抗原受体NK细胞(CAR-NK细胞)；或
(iii)外源T细胞受体(TCR)T细胞(TCR-T细胞)。
在另一优选例中，所述CAR-T细胞的TCR基因表达是被沉默的。
在另一优选例中，所述CAR-T细胞的TCRα链基因表达、TCRβ链基因表达是被沉默的。
在另一优选例中，所述“基因表达是被沉默的”指被沉默基因不表达或低表达。
在另一优选例中，所述“低表达”指所述CAR-T细胞被沉默基因表达量G1与正常T细胞相应基因表达量G0的比值，即G1/G0≤0.5，较佳地G1/G0≤0.3，更佳地≤0.2，更佳地≤0.1，最佳地为0。
在另一优选例中，所述免疫细胞表达外源性细胞自杀元件。
在另一优选例中，所述的免疫细胞中CAR与细胞自杀元件共表达。
在另一优选例中，所述的CAR与细胞自杀元件通过自剪切元件相连接。
在另一优选例中，所述的细胞自杀元件位于CAR的N端或C端。
在另一优选例中，所述的自剪切元件包括2A序列或IRES序列，优选为：P2A和T2A。
在另一优选例中，所述的细胞自杀元件选自下组：HSV-TK、iCasp9、ΔCD20、mTMPK、ΔCD19、RQR8、EGFRt、或其组合。
在另一优选例中，所述的细胞自杀元件的结构如下式V所示：
L2-D-F  (V)
式中，
各“-”独立地为连接肽或肽键；
L2为任选的信号肽序列；
D为自杀开关元件；
F为跨膜元件。
在另一优选例中，所述的信号肽为来源GM-CSFR的信号肽。
在另一优选例中，所述的自杀开关元件选自下组：截短的表皮生长因子受体(EGFRt)、截短的CD19(CD19t)基因、诱导的胱天蛋白酶9基因(iCasp9)、HSV-TK、ΔCD20、mTMPK、或其组合。
在另一优选例中，所述的自杀开关元件为icasp9(FKBP12-F36V-Caspase9)。
在另一优选例中，所述CAR-T细胞的内源性HLA-I和HLA-II基因正常表达。
在另一优选例中，所述的“基因正常表达”指所述CAR-T细胞的HLA-I基因表达量与正常T细胞的HLA-I基因表达量相同或基本相同。典型地，所述CAR-T细胞的HLA-I基因的表达量E1与正常T细胞的HLA-I基因表达量E0的比值(E1/E0)为0.5-2.0，较佳地0.6-1.5，更佳地0.8-1.2。
在另一优选例中，所述的HLA-I包括HLA-A、HLA-B、HLA-C、HLA-E和/或HLA-G。
在另一优选例中，所述CAR-T细胞的内源性HLA-E和/或HLA-G正常表达或过表达。
在另一优选例中，所述“过表达”指所述CAR-T细胞基因HLA-E和/或HLA-G表达量 F1与正常T细胞相应基因表达量F0的比值，即F1/F0≥1.5，较佳地F1/F0≥2，更佳地F1/F0≥5。
在另一优选例中，所述CAR-T细胞表达外源性HLA-E和/或HLA-G。
在另一优选例中，所述外源性HLA-E包括HLA-E SCT(HLA-E单链三聚体)。
在另一优选例中，所述的HLA-E SCT的结构如下式VI所示：
L3-S-B-E(VI)
式中，
各“-”独立地为连接肽或肽键；
L3为任选的信号肽序列；
S为自身抗原肽元件；
B为突变的β2M元件；
E为HLA-E重链元件。
在另一优选例中，所述CAR-T细胞可以用于自体和/或异体肿瘤的治疗。
在另一优选例中，所述CAR-T细胞通用型CAR-T细胞。
在本发明的第三方面，提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的嵌合抗原受体。
在本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸分子。
在另一优选例中，所述的载体包括DNA、RNA。
在另一优选例中，所述的载体选自下组：质粒、病毒载体、转座子、或其组合。
在另一优选例中，所述的载体包括DNA病毒、逆转录病毒载体。
在另一优选例中，所述的载体选自下组：慢病毒载体、腺病毒载体、腺相关病毒载体、或其组合。
在另一优选例中，所述载体为慢病毒载体。
在本发明的第五方面，提供了一种制备工程化的免疫细胞的方法，所述的免疫细胞表达本发明第一方面所述的嵌合抗原受体，包括以下步骤：将本发明第三方面所述的核酸分子或本发明第四方面所述的载体转导入免疫细胞内，从而获得所述工程化的免疫细胞。
在另一优选例中，所述的方法还包括对获得的工程化的免疫细胞进行功能和有效性检测的步骤。
在本发明的第六方面，提供了一种制备本发明第二方面所述的免疫细胞的方法，包括以下步骤：
(A)提供一待改造的免疫细胞；和
(B)对所述的免疫细胞进行改造，使得所述的免疫细胞表达靶向所述第一靶点和所述第二靶点的CAR，从而获得本发明第二方面所述的免疫细胞。
在另一优选例中，在另一优选例中，在步骤(B)中，利用基因编辑系统敲除内源性的PD-1基因和/或TCR基因。
在另一优选例中，所述的基因编辑系统包括CRISPR-Cas9系统、锌指蛋白系统、或TALEN系统。
在另一优选例中，所述的CRISPR-Cas9系统包括gRNA和Cas9蛋白。
在另一优选例中，所述免疫细胞为T细胞。
在另一优选例中，在步骤(B)中，对所述的T细胞进行改造，使得所述CAR-T细胞的PD-1基因表达是被沉默的，较佳地，利用基因编辑系统敲除PD-1基因。
在另一优选例中，在步骤(B)中，对所述的T细胞进行改造，使得所述CAR-T细胞的TCR基因表达是被沉默的，较佳地，利用基因编辑系统敲除TCR基因。
在另一优选例中，在步骤(B)中，对所述的T细胞进行改造，使得所述CAR-T细胞表达外源性细胞自杀元件。
在本发明的第七方面，提供了一种制剂，所述制剂含有本发明第二方面所述的工程化的免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型为注射剂。
在另一优选例中，所述制剂中所述基因工程化细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
在本发明的第八方面，提供了一种制剂，所述制剂含有第一CAR-T细胞和第二CAR-T细胞，且所述的第一CAR-T细胞表达靶向CD20的CAR，所述的第二CAR-T细胞表达靶向所述第二靶点的CAR。
在另一优选例中，所述的第一CAR-T细胞表达前述的第一CAR。
在另一优选例中，所述的第二CAR-T细胞表达前述的第二CAR。
在本发明的第九方面，提供了一种本发明第一方面所述的CAR、本发明第二方面所述的工程化的免疫细胞、本发明第三方面所述的核酸分子、或本发明第四方面所述的载体的用途，用于制备预防和/或治疗自体肿瘤或异体肿瘤的制剂。
在另一优选例中，所述肿瘤为肿瘤细胞表面具有CD20标记的肿瘤。
在另一优选例中，所述的肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤 (MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在另一优选例中，所述实体瘤选自下组：卵巢癌、间皮瘤、肺癌、胰腺癌、乳腺癌、子宫内膜癌、或其组合。
在本发明的第十方面，提供了一种用于制备本发明第二方面所述的工程化的免疫细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的：
(1)第一核酸序列，所述第一核酸序列含有用于表达所述CAR的第一表达盒。
在另一优选例中，所述的试剂盒还含有：(2)第二核酸序列，所述第二核酸序列含有用于沉默TCR基因的第二表达盒或第三gRNA。
在另一优选例中，所述的试剂盒还含有：(3)第三核酸序列，所述第三核酸序列含有用于沉默PD-1基因的第三表达盒或第四gRNA。
在另一优选例中，所述的试剂盒还含有：(4)第四核酸序列，所述第四核酸序列含有用于表达外源性细胞自杀元件的第四表达盒。
在另一优选例中，所述的各核酸序列为独立的或相连的。
在另一优选例中，所述的各核酸序列位于相同或不同的容器内。
在另一优选例中，所述的各核酸序列中的任何二个、三个或四个位于同一表达载体。
在另一优选例中，所述的试剂盒还含有：(4)第五核酸序列，所述第五核酸序列含有用于表达Cas9蛋白的第五表达盒；或Cas9蛋白。
在本发明的第十一方面，提供了一种本发明第二方面所述的工程化的免疫细胞的用途，用于预防和/或治疗自体肿瘤或异体肿瘤。
在本发明的第十二方面，提供了一种预防和/或治疗自体肿瘤或异体肿瘤的方法，包括步骤给需要的对象施用本发明第二方面所述的工程化的免疫细胞细胞。
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显示了不同结构的CD20-CD19-CAR在Jurkat细胞上的表达(CD19抗原测试)。1#-11#CAR在Jurkat细胞系上表达并被CD19抗原所检测。
图2显示了不同结构的CD20-CD19-CAR在Jurkat细胞上的表达(CD20抗原测试)。1#-11#CAR在Jurkat细胞系上表达并被CD20抗原所检测，其中，10#和11#CAR在Jurkat细胞上识别CD20抗原的能力弱于其他结构的CAR。
图3显示了不同结构的CD20-CD19-CAR在原代T细胞上的表达情况(CD20及CD19抗原测试)。结果显示，10#和11#CAR在T细胞上识别抗原的能力弱于其他结构的CAR。
图4显示了不同结构的CD19-CD20-CAR-T针对Hela抗原过表达细胞系的杀伤作用(RTCA法)。结果显示，1#-11#CAR-T细胞均具有显著的杀伤靶细胞的能力，并且能杀伤CD19或CD20单抗原的过表达Hela细胞(Hela-CD19，Hela-CD20)，说明双CAR-T上面的两个CAR分子均能发挥功能。
图5显示了不同结构的CD19-CD20-CAR-T杀伤Hela抗原过表达细胞过程中的IFNγ分泌。
图6显示了不同结构的CD19-CD20-CAR-T针对Raji肿瘤细胞及敲除细胞系(Raji、Raji-KO19、Raji-KO20和Raji-KO1920)的杀伤能力。结果显示，1-9#CAR-T细胞的杀伤能力中，1、2、3、4、5、9组表现出更好的靶细胞杀伤能力。
图7显示了不同结构的CD19-CD20-CAR-T杀伤Raji肿瘤细胞及敲除细胞系(Raji、Raji-KO19、Raji-KO20和Raji-KO1920)过程中的IFNγ释放。结果显示，1#、2#、3#、9#CAR-T能释放不低于其他组CAR-T的IFNγ。
图8显示了1#CAR-T与单CAR-T的CD4：CD8的比例。
图9显示1#CAR-T比单CAR-T对Hela过表达细胞具有更高的杀伤靶细胞效率(RTCA法)。结果显示，1#CAR-T针对Hela-19和Hela-20靶细胞的杀伤具有相对CD19 CAR-T及CD20 CAR-T细胞更高的杀伤能力，针对Hela-CD19 CD20的靶细胞的杀伤也具有不低于单CAR-T细胞的杀伤能力。
图10显示1#CAR-T杀伤靶细胞的效果优于单CAR-T。结果表明，1#CAR-T针对CD19和CD20双阳靶细胞Raji的杀伤显著优于CD19 CAR-T及CD20 CAR-T细胞，而针对CD19或CD20敲除后的Raji-KO19或Raji-KO20细胞，1#CAR-T细胞的杀伤显著高于对应的CD20CAR-T细胞或者CD19 CAR-T的单CAR-T细胞。
图11显示1#CAR-T杀伤靶细胞过程中分泌的INFr量大于单CAR-T。结果表明，在对Raji或者Raji-KO19、Raji-KO20的靶细胞杀伤过程中，1#CAR-T的细胞因子IFNγ的分泌也显著高于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞。
图12显示1#CAR-T杀伤各种不同靶细胞的能力优于单CAR-T(LDH法)。结果表明，针对不同淋巴瘤细胞系，1#CAR-T表现出不低于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞杀伤能力，特别是在对Pfeiffer和SU-DHL-10的杀伤上表现出优于单CAR-T细胞的杀伤能力。
图13显示了含有本发明CAR和细胞自杀元件的结构示意图。
图14显示1#CAR-T与不同靶细胞孵育的过程中上调CD107a的能力优于单CAR-T。结果表明，1#CAR-T细胞在与单靶点或者双靶点的靶细胞共孵育的时候，1#CAR-T细胞均表现出高于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞CD107a上调表达的能力。其中，CD4阳性细胞群及CD8阳性细胞群均有体现。
图15显示皮下Raji造模中，细胞回输1#CAR-T具有优于单CAR-T的清除肿瘤及抑制扩增的能力。通过皮下造模小鼠实验表明，针对Raji细胞造模小鼠，1#CAR-T细胞表现出高于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞的肿瘤清除能力。
图16显示皮下Raji造模中，细胞回输1#CAR-T具有清除肿瘤及抑制扩增的能力。通过皮下造模小鼠实验表明，针对Raji细胞造模小鼠，1#CAR-T清除肿瘤细胞的同事，没有表现出高于NT组的体重下降，说明其安全可靠。
具体实施方式
本发明人经过广泛而深入地研究，发现一种同时靶向CD20和CD19的嵌合抗原受体免疫细胞。同时在确保靶向CD20和CD19的CAR-T细胞的活性的基础上，添加细胞自杀元件，用于控制CAR-T细胞的活性和CRS等相关毒性，增强安全性。相对于单靶向的嵌合抗原受体免疫细胞疗法，具有协同效应，复发率降低，而疗效更佳。此外，本发明在scFv间使用特定的合适的柔性接头具有增加scFv弹性空间，最大可能保留scFv亲和力的作用，并且减少靶点组合使用时不同scFv之间的相互干扰。在此基础上，完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
术语“抗体”(Ab)应包括但不限于免疫球蛋白，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以 下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
CD19
CD19是一种B细胞表面的95kDa的糖蛋白，从B细胞发育的早期即开始表达，直至其分化为浆细胞。CD19是免疫球蛋白(Ig)超家族的成员之一，作为B细胞表面信号转导复合物的组成元素之一，参与调控了B细胞受体的信号转导过程。在CD19缺陷的小鼠模型中，外周淋巴组织中B细胞的数量会出现明显的减少，对疫苗和丝裂原的应答也会下降，同时伴有血清Ig水平的减低。通常认为，CD19的表达只限于B细胞系(B-cell lineage)，而不表达于多能造血干细胞表面。CD19还表达于大多数B细胞淋巴瘤、套细胞淋巴瘤、ALLs、CLLs、多毛细胞白血病，和一部分急性髓性白血病细胞的表面。因此，在对白血病/淋巴瘤的治疗中，CD19是一种非常有价值的免疫治疗靶点。重要的是，CD19不会表达于除B细胞外的大多数正常细胞表面，包括多能造血干细胞，这一特征使CD19可以作为一种安全的治疗靶点，可将患者发生自身免疫性疾病或不可逆性骨髓毒性损伤的风险降至最低。当前，已经研制出了抗CD19的抗体或scFv片段，并且在小鼠模型和人类/灵长类动物中证明了其应用的前景。
CD20
CD20抗原是一种非糖基化的细胞膜磷蛋白，其分子量为35kd。目前研究发现，CD20抗原是信号传递通道复合物的一部分，其功能类似于钙离子通道，参与调节B淋巴细胞的生长和分化。CD20抗原的表达严格限制在前B淋巴细胞的晚期和成熟B淋巴细胞，在造血干细胞。祖细胞和其他正常组织中没有CD20抗原的表达。当B淋巴细胞分化为分泌抗体的浆细胞时，CD20抗原的表达消失。95％以上的B细胞NHL有CD20抗原的表达。
CD20也是治疗B细胞淋巴瘤和白血病的靶点之一，所开发成功的单抗药物利妥昔单抗(Rituximab)(Rituxan,Genentech,Inc，FDA批准于2011年1月，用于NHL治疗)、奥匹妥珠单抗(Obinutuzumab)(Gazyva，Genentech,Inc，FDA批准于2013年11月，用于CLL治疗)、替伊莫单抗(Ibritumomab tiuxetan)(Zevalin,IDEC Pharmaceuticals Corp.,FDA批准于2002年2月，用于NHL治疗)、托西单抗(Tositumomab)(Bexxar，GSK，FDA批准于2003年6月，用于NHL治疗)和阿法单抗(Ofatumumab)(Genmab，FDA批准于2009年10月，用于CLL治疗)。
嵌合抗原受体(CAR)
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
CARs的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI 分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CARs相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CARs。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向CD20的结合结构域，同时还包括靶向非CD20肿瘤抗原的结合结构域，优选的肿瘤抗原为CD19。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域还包括信号肽L。在另一优选例中，所述L为CD8来源的信号肽。
在另一优选例中，所述L具有如SEQ ID NO.:30所示的氨基酸序列。
GSATMALPVTALLLPLALLLHAARP(SEQ ID NO.:30)
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述scFv包含特异性识别CD19和/或CD20的抗体，较佳地为单链抗体。
在另一优选例中，所述靶向CD20的抗原结合结构域包括单克隆号Leu16抗体的重链可变区和轻链可变区。
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:5所示的抗体重链可变区，和如SEQ ID NO.:6所示的抗体轻链可变区。
Figure PCTCN2020073873-appb-000002
Figure PCTCN2020073873-appb-000003
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:9所示的抗体重链可变区，和如SEQ ID NO.:10所示的抗体轻链可变区。
Figure PCTCN2020073873-appb-000004
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:11所示的抗体重链可变区，和如SEQ ID NO.:12所示的抗体轻链可变区。
Figure PCTCN2020073873-appb-000005
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:13所示的抗体重链可变区，和如SEQ ID NO.:14所示的抗体轻链可变区。
Figure PCTCN2020073873-appb-000006
在另一优选例中，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:31所示的抗体重链可变区，和如SEQ ID NO.:32所示的抗体轻链可变区。
Figure PCTCN2020073873-appb-000007
在另一优选例中，所述靶向CD20的抗原结合结构域的核苷酸序列包含如SEQ ID NO.:1所示的抗体重链可变区编码序列，和如SEQ ID NO.:2所示的抗体轻链可变区编码序列。
Figure PCTCN2020073873-appb-000008
Figure PCTCN2020073873-appb-000009
在另一优选例中，所述靶向CD19的抗原结合结构域包括单克隆号FMC63抗体的重链可变区和轻链可变区。
在另一优选例中，所述靶向CD19的抗原结合结构域包含如SEQ ID NO.:7所示的抗体重链可变区，和如SEQ ID NO.:8所示的抗体轻链可变区。
Figure PCTCN2020073873-appb-000010
在另一优选例中，所述靶向CD19的抗原结合结构域的核苷酸序列包含如SEQ ID NO.:3所示的抗体重链可变区编码序列，和如SEQ ID NO.:4所示的抗体轻链可变区编码序列。
Figure PCTCN2020073873-appb-000011
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
在另一优选例中，所述铰链区H为CD8来源的铰链区。
在另一优选例中，所述铰链区H具有如SEQ ID NO.:35所示的氨基酸序列。
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.:35)
在另一优选例中，所述跨膜区TM为CD8来源的跨膜区。
在另一优选例中，所述跨膜区TM具有如SEQ ID NO.:36所示的氨基酸序列。
IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO.:36)
在另一优选例中，所述C(41BB胞内信号区)具有如SEQ ID NO.:37所示的氨基酸序列。
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.:37)
在另一优选例中，所述CDζ具有如SEQ ID NO.:38所示的氨基酸序列。
Figure PCTCN2020073873-appb-000012
本发明CAR中还含有柔性接头，所述柔性接头可以包括任何用于连接各元件(如抗体重链可变区、轻链可变区)的连接肽。
在另一优选例中，本发明在scFv间使用特定的合适的柔性接头具有增加scFv弹性空间，最大可能保留scFv亲和力的作用，并且减少靶点组合使用时不同scFv之间的相互干扰。
在另一优选例中，所述的柔性接头(或连接肽)I可以由多个子短肽构成，所述多个子短肽可以相同或不同。当多个子短肽不相同时，各子短肽的数量n为0-6，较佳地0-5，更较佳地1-3。
在另一优选例中，所述的柔性接头(或连接肽)各自独立地具有下式VII的结构：
Xa-Yb-Zc    (VII)
式中，各“-”独立地为连接肽或肽键；
X、Y和Z各自独立为选自下组的序列：
Figure PCTCN2020073873-appb-000013
a、b和c各自独立地为0-6的正整数。
在另一优选例中，所述的柔性接头(或连接肽)I、Ia、Ib、Ic、Id各自独立地具有如SEQ ID NO.:15-25中任一所示的序列。
Figure PCTCN2020073873-appb-000014
在另一优选例中，所述CAR具有如SEQ ID NO.:26-29中任一所示的氨基酸序列。
Figure PCTCN2020073873-appb-000015
Figure PCTCN2020073873-appb-000016
在另一优选例中，所述CAR具有如SEQ ID NO.:44中任一所示的氨基酸序列。
Figure PCTCN2020073873-appb-000017
CAR-T细胞
如本文所用，术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”均指本发明第一方面所述的通用型CAR-T细胞，所述CAR-T细胞表达嵌合抗原受体CAR，所述CAR靶向CD20和所述第二靶点。
在另一优选例中，本发明的CAR-T细胞是通过基因编辑技术敲除细胞中的TCR表达，使得异体输注时不会因为TCR的识别而杀伤受体的正常细胞，即不会带来GVHD反应；通过同时靶向CD19和CD20 CAR-T在清除肿瘤细胞的同时清除了宿主T细胞，避免宿主抗移植物的反应(host versus graft，HVG)，提高异体CAR-T细胞在受体体内的存活及抗肿瘤效果。CAR-T上增加自杀基因开关，必要时可随时清除CAR-T，减少免疫治疗副作用。
具体地，在本发明的一个技术方案中，构建结构为CD20 scFv-CD19 scFv-Hinge-TM-CD28/41BB-CD3ζ的嵌合抗原受体，CD20 scFv片段为单克隆号Leu16抗体的重链和轻链可变区由连接肽连接，CD19 scFV片段为单克隆号FMC63抗体的重链和轻链可变区由连接肽连接，还包括串联的铰链区和跨膜区、人CD28或41BB胞内的共刺激元件，以及人的CD3ζ胞内结构域。
自杀基因开关
如本文所用，术语“自杀基因开关”、“自杀开关”、“安全开关”、“细胞自杀元件”具有相同含义，均指在外源性药物的作用下，可以有效清除体内的CAR-T细胞的元件。本发明中所用自杀基因开关可以为EGFRt、HSV-TK、iCasp9、CD20、mTMPK等。比较而言，HSV-TK、iCasp9和CD20对T细胞的清除能力等同，但是iCasp9和CD20的清除较迅速，HSV-TK清楚速度较慢。在本发明中，优选的自杀基因开关为icasp9。
CAR-T细胞的一大优点是它们是活性药物，一旦输入，生理机制会调控T细胞的平 衡、记忆形成和抗原驱动的扩增。然而，这种治疗尚未完善，T细胞会脱靶而攻击其他的组织，或扩增量过高，超出治疗所需。鉴于CAR-T细胞已被纳入标准治疗范围，设计病人或药物可控的启动或关闭机制来调控CAR-T细胞的存在是非常有用的。由于技术原因，关闭机制更易应用于T细胞。可利用临床上已经使用的清除性抗体，使CAR-T细胞同时表达这些抗体针对的蛋白，如icasp9，在治疗相关的毒性反应产生或是治疗已经完成后，通过给予抗体药物清除相应的CAR-T细胞。
为进一步控制CAR-T细胞非肿瘤靶向和细胞因子释放综合征等不良，本发明中的CAR-T细胞皆带有自杀基因开关，在外源性药物的作用下，可以有效清除体内的CAR-T细胞，阻断未知的或不可控的远期毒性，以保证患者的安全。
具体地，本发明的CAR-T细胞引入了安全开关即icasp9。iCasp9是一种自杀基因系统，所述自杀基因系统由inducible caspase9(iCasp9)基因和无生物活性的小分子化学诱导药物组成。其中，小分子化学诱导药物选自AP1903、AP20187，优选AP1903。
TCR/MHC复合物(Tcell receptor,TCR)
T细胞表面受体(T cell receptor,TCR)为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR—CD3复合物。TCR的作用是识别抗原，并激活T细胞，发挥杀伤作用。TCR是由两条不同肽链构成的异二聚体，TCR分为两类：TCR1和TCR2。TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。外周血中，90％-95％的T细胞表达TCR2。每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等几部分；其特点是胞质区很短。
TCR分子属于免疫球蛋白超家族，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，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时，CDR1、CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。
主要组织相容性抗原(major histocompatibility complex，MHC)是所有生物相容复合体抗原的一种统称(MHC molecule)，表示由MHC基因家族(MHC classⅠ,classⅡ,classⅢ)编码而成的分子，位于细胞表面，不同种类哺乳动物MHC基因的编码产物的名称各异。人类的MHC通常被称为HLA(human leucocyte antigen，HLA)，即人类白细胞抗原。MHC基因(MHC gene)，定位于人类第六号染色体短臂，呈高度多态性。
免疫检查点
免疫检查点是指免疫系统中存在的一些抑制性信号通路，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受。利用免疫检查点的抑制性信号通路抑制T细胞活性是肿瘤逃避免疫杀伤的重要机制。因此，通过不同的策略增强T细胞的激活对肿瘤免疫治疗具有重要意义。本发明通过阻断PD-1信号来增强T细胞激活。如本文使用，术语“PD-1”包括人PD-1(hPD-1)，hPD-1的变体(突变的hPD-1)、同种型和物种同源物，以及与hPD-1具有至少80％、85％、90％、95％、96％、97％、98％、99％ 相同性的类似物。完整的hPD-1序列可以在GenBank登录号U64863下找到。
基因沉默方法
目前常用的基因沉默方法有CRISPR/Cas9、RNA干扰技术、TALENs(transcription activator-like(TAL)effector nucleases)和Zinc finger nucleases(ZFNs)，其中CRISPR/Cas9目前应用前景和效果最好。
CRISPR(clustered regularly interspersed short palindromic repeats)/Cas(CRISPR-associated)系统是原核生物特有的一种天然免疫系统，用于抵抗病毒或外源性质粒的侵害。Ⅱ型CRISPR/Cas系统作为RNA直接介导的基因组编辑工具已经在许多真核生物和原核生物体内成功应用。CRISPR/Cas9系统的发展彻底改变了人们编辑DNA序列和调控目标基因表达水平的能力，从而为生物体的精确基因组编辑提供了有力的工具。简化后的CRISPR/Cas9系统由两部分组成：Cas9蛋白和gRNA。其作用原理为gRNA通过自身的Cas9把手与Cas9蛋白形成Cas9-gRNA复合体，Cas9-gRNA复合体中gRNA的碱基互补配对区序列与目标基因的靶序列通过碱基互补配对原则进行配对结合，Cas9利用自身的核酸内切酶活性对目标DNA序列进行切割。与传统的基因组编辑技术相比，CRISPR/Cas9系统具有几大明显的优势：易用性、简便性、低成本、可编程性以及可同时编辑多个基因。
防止GVHD和HVG
为了致力于用于恶性肿瘤和感染性疾病的“现成的(off-the-shelf)”同种异体t细胞疗法，通过输注T细胞的细胞疗法被设计以针对病原体和恶性肿瘤重建免疫力。离体制造具有期望性质和足够数目的T细胞需要的时间量通常与患者的治疗窗口不相容。此外，来自患有晚期疾病的患者的自体T细胞可能具有受损的功能并且对期望的抗原是耐受的。
为了处理此问题，患者可以输注同种异体T细胞以避免通过识别输注的细胞上不同的主要或次要组织相容性抗原的宿主T细胞引起的免疫-介导的排斥。为了扩大T细胞疗法的应用，并且为了进一步的同种异体移植，可以生成用于分离TCR和HLA-I类破坏的群的来源的快速和稳健的方法。
当使用HLA不匹配的肿瘤细胞系攻击细胞时，T细胞上TCRα与β链和HLA-A分子的完全消融完全地废除非特异性杀伤。在长时期的共培养(5天)后，消除HLA-A分子活化的NK细胞。当这些细胞在IFNγElispot测定中被同种异体全血PBMC攻击24小时时没有观察到脱靶活性。缺少脱靶活性表明T细胞可以在遭遇同种异体细胞后在急性免疫应答中发挥主要作用。所有结果表明CRISPR/CAS9编辑的TCRα链和HLA-A分子T细胞可以充当通用效应供体细胞的来源。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯 斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第二方面所述的工程化的免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物CD19和CD20，可以用于自体和异体肿瘤治疗，可以大规模制备，质量均一稳定，随时可调用给任何患者使用。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，给需要的患者直接施用本发明改造的通用型CAR-T细胞。本发明的CAR-T细胞通过同时靶向CD19和CD20 CAR-T在清除肿瘤细胞。CAR-T上增加自杀基因开关，必要时可随时清除CAR-T，减少免疫治疗副作用。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗CD19 CAR-T细胞引 起抗表达CD19的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括icasp9，CD19-scFv、CD20-scFv、铰链和跨膜区、和4-1BB/CD28和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌、。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋 形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明经修饰的T细胞(如，CAR-T20细胞)，通过例如静脉回输的方式， 施用于患者。
本发明的主要优点包括：
1)组合靶点可以扩大CAR-T细胞的识别靶细胞范围，减少肿瘤细胞逃逸的机会，减小复发几率。
2)合适的linker可以最大限度保留识别双靶点中每个scFv的亲和力，减少空间位阻对CAR结构功能的影响。
3)增加安全开关可以增加CAR-T细胞的安全性。
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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明技术方案
根据scFv序列设计同时靶向CD20和CD19的CAR结构，构建慢病毒包装质粒，并制备CAR慢病毒表达载体，然后从供体血液中分离和激活T细胞，通过慢病毒表达载体感染激活后的T细胞，感染后检测T细胞CAR阳性率。对制备好的CAR-T细胞进行检测与功能验证。CAR-T细胞在体外与肿瘤细胞共培养，检测细胞因子IFNγ和IL-2释放。同时也进行体内药效学实验，检测肿瘤负荷小鼠输入CAR-T细胞后，肿瘤消褪情况。
实施例1从供体血液中分离PBMC和扩增T细胞
从供体血液中分离单个核细胞，使用Histopaque-1077(Sigma-Aldrich)进行密度梯度离心，并富集T细胞(EasySep人T细胞富集试剂盒，Stemcell Technologies)，使用偶联anti-CD3/anti-CD28的磁珠激活培养和扩增T细胞；培养基使用X-vivo15(含5％FBS，2mM L-glutamine，1mM丙酮酸钠，300IU/ml rhIL2)；所有细胞均置于37℃，5％CO 2恒温培养箱中培养。
实施例2细胞培养及构建
表达CD19和CD20阳性的细胞系Raji、Daudi和Nalm6细胞、单表达CD20、CD19和同时表达CD20/CD19的Hela细胞，以上细胞均使用ATCC来源，且使用ATCC推荐培养条件；293T(人肾上皮细胞系细胞，
Figure PCTCN2020073873-appb-000018
CRL-3216)使用DMEM培养基培养。所有培养基均添加10％(v/v)胎牛血清和100U/ml的青霉素和链霉素，2mML-谷氨酰胺，1mM丙酮酸钠。
其中，单表达CD20、CD19和同时表达CD20/CD19的Hela细胞是通过慢病毒载体将CD20和CD19抗原转入，再经过单克隆筛选后获得的稳转细胞系，能够特异性的表达CD20 和CD19蛋白分子，Raji、Daudi和Nalm6的ffluc细胞时使用fireflyluciferase的慢病毒感染细胞筛选后得到。
实施例3 CAR结构设计与转导
本发明中同时靶向CD20及CD19的CAR结构示意如图13所示(后续简称CD20-CD19Bi-CAR)。其中，CD20 CAR、CD19 CAR及自杀开关icasp9(FKBP12-F36V-Caspase9)元件通过T2A连接。
本发明设计了多种结构的靶向CD20及CD19的CAR，各个CAR中包含的各元件及其序列如下表1所示。
表1各个CAR的各元件及其序列编号
Figure PCTCN2020073873-appb-000019
注,表格中的编号为序列表中的序列编号，即1表示SEQ ID NO.:1。
上述构建的CAR中，2#和3#CAR的linker设计引入了脯氨酸和甘氨酸，理论上可以改变CD19和CD20 CAR识别抗原的空间构象，有增加双CAR更好识别靶抗原的能力。9#CAR的linker中加入了DYKDDDDK(SEQ ID NO.:40)序列，该序列可以被特异性的抗体识别，可以增加CAR-T细胞检测的便利性。
将设计好的CD20-CD19 CAR基因克隆至FUW慢病毒载体骨架中，置于EF1α(EF-1α)的启动子下，形成Fuw-EF1α-CD20-CD19-icasp9 CAR，将Fuw-EF1α-CD20-CD19-icasp9CAR、慢病毒包装系统辅助质粒使用Lipofectamine3000转入293T中制备慢病毒完整表达载体；在48h和72h收集病毒上清，超离进行浓缩(Merck Millipore)；浓缩后的病毒即可用于感染T细胞。
利用构建好的病毒感染Jurkat细胞，流式结果如图1和2所示，Jurkat细胞表面能够同时检测到CD20 CAR及CD19 CAR的表达，表明上述设计的CAR基因能够制备出同时表达CD20 CAR和CD19 CAR的慢病毒载体。图中，1#-11#以及CD19 CAR-T、CD20 CAR-T和CD20-2 CAR-T的定义如表1所述。1#-11#CAR在Jurkat上的表达均能被CD19抗原所检测，而10#和11#CAR在Jurkat细胞上识别CD20抗原的能力弱于其他结构的CAR。
实施例4 CAR-T细胞制备
实验方法如下：
4.1慢病毒感染
分离纯化的原代T细胞在激活2天后，利用实施例3构建的慢病毒，按MOI(1-10)进行慢病毒载体感染，转移至细胞培养瓶，置于37℃，5％CO 2恒温培养箱中培养。
4.2细胞增殖及CAR阳性率检测
感染后第3天及冻存前取样检测细胞数量及CD20/CD19双阳性细胞得占比，即检测T细胞的CAR阳性率，每隔2-3天更换一半培养基。
利用实施例3中构建的各个CD20-CD19 CAR慢病毒载体，成功构建了CD20-CD19 CAR-T细胞。
流式结果显示如图3所示，病毒转染后的T细胞表面能够同时检测到CD20 CAR及CD19CAR的表达，其中，10#和11#CAR在T细胞上识别抗原的能力弱于其他结构的CAR。图中，1#-11#以及CD19 CAR-T、CD20 CAR-T和CD20-2 CAR-T的定义如表1所述。
实施例5 Hela细胞体外杀伤实验
对实施例4获得的各个CAR-T细胞进行体外杀伤实验。通过将荧光素酶基因转入靶细胞，克隆筛选后获得稳转细胞株。进行实验时，加入荧光素底物，荧光素酶与荧光素反应即可产生荧光，通过检测荧光的强度可以测定荧光素酶的活性，检测细胞的存活比率，即可得到CART细胞的杀伤效应。其中，进行实验的靶细胞包括：Hela、Hela-CD19、Hela-CD20、Hela-CD19-CD20。
结果如图4所示，将各个CAR-T细胞与靶细胞(CD19/CD20双阳、CD19单阳、CD20单阳)按照一定比例共培养后，1#-11#CAR-T细胞均具有显著的杀伤靶细胞的能力，并且能杀伤CD19或CD20单抗原的过表达Hela细胞(Hela-CD19，Hela-CD20)，说明双CAR-T细胞上面的两个CAR分子均能发挥功能。
1#CAR-T细胞表现出较佳的杀伤能力，对其进行进一步的分析。
图9单独显示了1#CAR-T细胞与单CAR-T细胞杀伤Hela靶细胞的结果，1#CAR-T 比单CAR-T对Hela过表达细胞具有更高的杀伤靶细胞效率，说明1#CAR-T对Hela-19和Hela-20的靶细胞杀伤可见其具有相对CD19 CAR-T及CD20 CAR-T细胞更高的杀伤能力，针对Hela-CD19 CD20的靶细胞的杀伤也具有不低于单CAR-T细胞的杀伤能力。
实施例6 Raji细胞体外杀伤实验
利用Luciferase转染Raji靶细胞，并在此基础上敲除CD19或CD20构建Raji-KO19、Raji-KO20，以及CD19和CD20双敲的Raji细胞系Raji-KO1920。根据杀伤后存活下来的Luciferase标记细胞催化Luciferin底物，利用酶标仪测定其生物发光的方法分析1-9#CAR-T细胞对Raji靶细胞的杀伤。
结果如图6所示，1-9#CAR-T细胞均具有显著的杀伤靶细胞的能力。其中，对于1-9#CAR-T细胞的杀伤能力，1#、2#、3#、4#、5#、9#表现出更好的靶细胞杀伤能力。
1#CAR-T细胞表现出更好的杀伤能力，对其进行进一步的分析。
图10显示了1#CAR-T杀伤Raji靶细胞效果，1#CAR-T针对CD19和CD20双阳靶细胞Raji的杀伤显著优于CD19 CAR-T及CD20 CAR-T细胞，而针对CD19或CD20敲除后的Raji-KO19或Raji-KO20细胞，1#CAR-T细胞的杀伤显著高于对应的CD20 CAR-T细胞或者CD19 CAR-T的单CAR-T细胞。
实施例7细胞因子释放检测
利用RTCA技术或Luciferase法，将实施例4获得的各个CAR-T细胞与Hela靶细胞(Hela、Hela-CD19、Hela-CD20和Hela-CD19-CD20)或Raji靶细胞(Raji、Raji-KO19、Raji-KO20和Raji-KO1920)按照一定比例共培养后，过夜，收集上清，离心后取上清检测细胞因子IFN-γ的释放水平，采用Elisa试剂盒(Biolegend)进行检测。
与Hela靶细胞共培养后的IFN-γ释放结果如图5所示，1-9#CAR-T细胞在杀伤不同Hela靶细胞的过程中均有IFNγ释放。其中，10#、11#的IFNγ释放较弱，不再进行后续实验。
与Raji靶细胞共培养后的IFN-γ释放结果如图7所示，1-9#CAR-T细胞杀伤肿瘤细胞Raji、Raji-KO19、Raji-KO20和Raji-KO1920过程中均有IFNγ释放。其中，对靶细胞表现出更好杀伤能力的1#、2#、3#、9#CAR-T的IFNγ释放与其他CAR-T的IFNγ释放相当。
1#CAR-T细胞表现出更高的IFNγ释放，对其进行进一步的分析。
图11显示了1#CAR-T杀伤Raji靶细胞过程中分泌的INFr的量，在对Raji或者Raji-KO19、Raji-KO20的靶细胞杀伤过程中，1#CAR-T的细胞因子IFNγ的分泌显著高于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞。
实施例8利用LDH酶检测细胞杀伤情况
利用细胞裂解时释放的LDH酶，检测1#CAR-T细胞和CD19、CD20单CAR-T细胞杀伤不同靶细胞的情况。其中，靶细胞包括Pfeiffer细胞、Farage细胞、SU-DHL-4细胞、SU-DHL-6细胞、SU-DHL-10细胞。
使用promega公司CytoTox
Figure PCTCN2020073873-appb-000020
Non-Radioactive Cytotoxicity Assay试剂盒(货号：G1780)，按照说明书测定CAR-T细胞杀伤靶细胞的能力。具体操作如下：
1.将培养液调整靶细胞浓度至1×10 5/ml；
2.浓度调整完成后的细胞混合液按照每孔100μl转移至96孔培养板中，每组设定复孔,靶细胞自然释放孔(阴性对照)不加效应细胞只加100μl培养液；
3.将试剂盒中裂解液加入到最大释放孔(阳性对照)中代替培养液；
4.在每个实验孔中加入效应细胞100μl，设置效靶比；
5.效应细胞自然释放对照孔用100μl培养液代替靶细胞；
6.完成后把96孔培养板放置于培养箱中37℃、二氧化碳浓度5ml/dl培养4-6小时；
7.培养结束后用自动生化分析仪检测LDH数值计算活性:杀伤活性(％)＝[(实验组A值-总自然释放A值)/(最大释放组A值-总自然释放A值)]×100％。
结果如图12所示,针对不同淋巴瘤细胞系，1#CAR-T表现出不低于CD19 CAR-T或CD20CAR-T的单CAR-T细胞杀伤能力，特别是在对Pfeiffer和SU-DHL-10的杀伤上表现出优于单CAR-T细胞的杀伤能力。
实施例9 CD107a的测定
对1#CAR-T、CD19 CAR-T和CD20 CAR-T细胞进行激活后CD107a表达变化的流式分析，使用表达CD19或/和CD20的肿瘤细胞系进行共孵育激活实验。共孵育后的细胞用抗体标记CD3、CD4及CD107a后，进行流式分析。
结果如图14所示，1#CAR-T细胞在与单靶点或者双靶点的靶细胞共孵育的时候，1#CAR-T细胞均表现出高于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞CD107a上调表达的能力。其中，CD4阳性细胞群及CD8阳性细胞群均有体现。
实施例10 CD4与CD8比例的测定
利用流式细胞术，对1#CAR-T、CD19 CAR-T和CD20 CAR-T细胞表面的CD4和CD8表达进行测定，并计算其比例。
结果如图8所示，1#CAR-T与单CAR-T的CD4：CD8的比例不同，说明1#CAR-T的比例接近与NT的比例，CD20单CAR影响CD4：CD8比例较大。
实施例11体内药效研究
选取6-12周大的NOD-Prkdcscid IL2rgnull(NOG)小鼠，尾静脉注射3×10 5 Raji细胞。实验期间使用游标卡尺测量肿瘤长宽径，计算肿瘤体积。20天后将小鼠分成肿瘤负荷相当的组别，分组后分别注射不同的CAR-T细胞。CAR-T处理后每周测量肿瘤体积评估小鼠肿瘤负荷，并称重。利用流式细胞术，取外周血测量其中人CD45及CD2和CD8阳性的细胞。
结果如图15，图16所示，1#CAR-T具有优于单CAR-T的抑制肿瘤的能力。通过皮下造模小鼠实验表明针对Raji细胞造模小鼠，1#CAR-T细胞表现出高于CD19 CAR-T或CD20 CAR-T的单CAR-T细胞的肿瘤清除能力；并且，1#CAR-T清除肿瘤细胞的同时，体 重下降情况与NT组相当，说明其安全可靠。
实施例12 iCasp9基因介导的CART细胞的清除
为进一步验证iCasp9基因介导的CART细胞的清除，利用实施例3、实施例4和实施例7中的类似方法，构建不包含icasp9基因的CD20-CD19 CAR-T/T细胞，作为对照，进行实验。
具体地，药物诱导的CAR-T细胞体外增殖实验：体外检测加入诱导药物后CD20-CD19-icasp9-CAR-T细胞的增殖活性。分别向CFSE标记的CD20-CD19-icasp9-CAR-T/CD20-CD19 CAR-T/T细胞培养基中加入10nM AP1903，测定不同时间死细胞和活细胞的数目，计算药物诱导细胞死亡比例。
体内功能实验：验证药物诱导后CAR-T细胞的体内增殖活性。使用FFLuc标记CAR-T细胞，用于标记体内T细胞的活性。向小鼠分别注射5*10 6 T细胞、FFLuc-CD20-CD19-icasp9-CAR-T和FFLuc-CD20-CD19 CAR-T细胞，第7天腹腔注射AP1903(50mg/鼠)，分别在Day0/2天腹腔注射3mg d-luciferin，使用Xenogen IVIS Imaging System拍照，计算得出光子量/s/cm 2/球面角度值(p/s/cm 2/sr)。
结果显示，在注射AP1903之后，FFLuc-CD20-CD19 CAR-T细胞及T细胞组在加药后保持高强度荧光，而FFLuc-CD20-CD19-icasp9-CAR-T组，Day2小鼠体内基本检测不到荧光，FFLuc-CD20-CD19-icasp9-CAR-T细胞基本被清除，表明AP1903可快速诱导带自杀开关的移植CAR-T细胞。
实施例13敲除TCR基因的CD20-CD19 UCAR-T细胞的体外活性研究
通过CRISPR技术将设计的gRNA与cas9蛋白组成的RNP复合物电转到CART，敲除TRAC、B2M、PD1基因后，测试其体外的通用型CD20-CD19-CAR-T(CD20-CD19-UCAR-T)对靶细胞的杀伤能力，结果显示，具有与CD20-CD19-CAR-T相比，CD20-CD19-UCAR-T具有更高或类似活性。
实施例14分泌型CAR-T细胞
通过T2A链接表达分泌PD-1的scFv，使CD20-CD19-CAR-T可以表达CAR的同时分泌抗PD-1的scFv，通过测试可以提高体外CD20-CD19-CAR-T的杀伤能力。
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 (15)

  1. 一种嵌合抗原受体CAR，其特征在于，所述的CAR靶向第一靶点和第二靶点，其中所述第一靶点为CD20，所述第二靶点选自下组：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、叶酸受体α、或其组合。
  2. 如权利要求1所述的CAR，其特征在于，所述CAR的结构如下式I所示：
    L-scFv1-I-scFv2-H-TM-C-CD3ζ(I)
    式中，
    各“-”独立地为连接肽或肽键；
    L为任选的信号肽序列；
    I为柔性接头；
    H为任选的铰链区；
    TM为跨膜结构域；
    C为共刺激信号分子；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    scFv1和scFv2两者中一个为靶向所述第一靶点(CD20)的抗原结合结构域，另一个为靶向所述第二靶点的抗原结合结构域。
  3. 如权利要求2所述的CAR，其特征在于，scFv1为靶向CD20的抗原结合结构域，scFv2为靶向CD19的抗原结合结构域。
  4. 如权利要求3所述的CAR，其特征在于，所述靶向CD20的抗原结合结构域包含如SEQ ID NO.:31所示的抗体重链可变区，和如SEQ ID NO.:32所示的抗体轻链可变区。
  5. 如权利要求3所述的CAR，其特征在于，所述靶向CD19的抗原结合结构域包含如SEQ ID NO.:7所示的抗体重链可变区，和如SEQ ID NO.:8所示的抗体轻链可变区。
  6. 如权利要求2所述的CAR，其特征在于，所述的柔性接头I的氨基酸序列如SEQ ID NO.:15-25中任一所示。
  7. 如权利要求2所述的CAR，其特征在于，所述的柔性接头I的氨基酸序列如SEQ ID NO.:15、16、17、23中任一所示。
  8. 如权利要求1所述的CAR，其特征在于，所述CAR的结构如下式II或II’所示：
    L-V L-scFv-V H-H-TM-C-CD3ζ (II)
    L-V H-scFv-V L-H-TM-C-CD3ζ (II’)
    式中，
    各“-”独立地为连接肽或肽键；
    元件L、H、TM、C和CD3ζ如权利要求2所述；
    scFv为靶向CD20的抗原结合结构域，V H为抗所述第二靶点抗体重链可变区，且V L为抗所述第二靶点抗体轻链可变区；或者scFv为靶向所述第二靶点的抗原结合结构域，V H为抗CD20抗体重链可变区，且V L为抗CD20抗体轻链可变区。
  9. 一种工程化的免疫细胞，其特征在于，所述免疫细胞具有以下特征：
    (a)所述免疫细胞表达嵌合抗原受体CAR，且所述CAR靶向第一靶点和第二靶点，其中所述第一靶点为CD20，所述第二靶点选自下组：CD19、CD22、BCMA、CD33、CD38、CD47、CD123、CD138、kappa轻链、CS1、CLL-1(CLEC12A)、SLAMF7、间皮素、EGFR、Epha2、FLT3、GPC3、Her2、NKG2D、PD1、ROR1、EGFRVIII、IL13RA、CEA、FAP、cMET、VEGFR2、MUC16、PSMA、GD2、L1-CAM、FR-α、或其组合。
  10. 如权利要求9所述的免疫细胞，其特征在于，所述CAR为单一CAR，并且所述的CAR同时靶向所述第一靶点和所述第二靶点。
  11. 如权利要求9所述的免疫细胞，其特征在于，所述CAR包括靶向所述第一靶点的第一CAR和靶向所述第二靶点的第二CAR。
  12. 如权利要求9所述的免疫细胞，其特征在于，所述免疫细胞具有选自下组的一种或多种特征：
    (a)所述免疫细胞的PD-1基因表达是被沉默的；
    (b)所述免疫细胞表达外源性细胞自杀开关元件；
    (c)所述免疫细胞表达或分泌PD-1抗体或抗体片段、PD-L1抗体或抗体片段、CD47抗体或抗体片段、Tim3抗体或抗体片段、Lag3抗体或抗体片段、Tigit抗体或抗体片段、OX40抗体或抗体片段、ICOS抗体或抗体片段、IL7、CXCL19、IL21、IL15、IL2、IL18、或其组合；和
    (d)所述免疫细胞的细胞因子相关信号通路被增强，其中所述细胞因子选自下组：IL7、CXCL19、IL21、IL15、IL2、IL18、或其组合。
  13. 一种制备权利要求9所述的免疫细胞的方法，包括以下步骤：
    (A)提供一待改造的免疫细胞；和
    (B)对所述的免疫细胞进行改造，使得所述的免疫细胞表达靶向所述第一靶点和所述第二靶点的CAR，从而获得权利要求1所述的免疫细胞。
  14. 一种制剂，其特征在于，所述制剂含有权利要求9所述的免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  15. 一种权利要求1所述的CAR、权利要求9所述的免疫细胞的用途，用于制备预防和/或治疗自体肿瘤或异体肿瘤的制剂。
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