WO2020224606A1 - 靶向bcma的工程化免疫细胞及其用途 - Google Patents

靶向bcma的工程化免疫细胞及其用途 Download PDF

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WO2020224606A1
WO2020224606A1 PCT/CN2020/088836 CN2020088836W WO2020224606A1 WO 2020224606 A1 WO2020224606 A1 WO 2020224606A1 CN 2020088836 W CN2020088836 W CN 2020088836W WO 2020224606 A1 WO2020224606 A1 WO 2020224606A1
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car
cells
bcma
variable region
chain variable
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PCT/CN2020/088836
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English (en)
French (fr)
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张华�
石欢
沈连军
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 JP2021566227A priority Critical patent/JP2022531911A/ja
Priority to CN202080034113.7A priority patent/CN113784732B/zh
Priority to AU2020270298A priority patent/AU2020270298A1/en
Priority to CA3139346A priority patent/CA3139346A1/en
Priority to KR1020217039584A priority patent/KR20220017914A/ko
Priority to US17/041,977 priority patent/US11840575B2/en
Priority to EP20801569.3A priority patent/EP3967329A4/en
Priority to SG11202112382WA priority patent/SG11202112382WA/en
Publication of WO2020224606A1 publication Critical patent/WO2020224606A1/zh
Priority to IL287873A priority patent/IL287873A/en

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Definitions

  • the present invention relates to the field of immunotherapy, and more specifically to an engineered immune cell targeting BCMA and its use.
  • Multiple myeloma is a malignant plasma cell tumor.
  • the tumor cells originate from plasma cells in the bone marrow, and plasma cells are cells that develop to the final functional stage of B lymphocytes.
  • Multiple myeloma is basically an incurable disease with the characteristics of high morbidity and high mortality.
  • 2017 statistics there were 30,000 newly diagnosed multiple myeloma patients in the United States, and 12,000 may face death.
  • common therapies for multiple myeloma include cytotoxic drugs, protease inhibitors (bortezomib, etc.), lenalidomide, monoclonal antibodies, and corticosteroids. However, they are all partially effective and cannot last for a long time. The chance of recurrence is very high. Therefore, the improvement of multiple myeloma therapy is particularly important.
  • the purpose of the present invention is to provide an engineered immune cell targeting BCMA and its use.
  • Another object of the present invention is to provide an engineered immune cell that simultaneously targets CD19 and BCMA and its use.
  • a chimeric antigen receptor (CAR) or TCR is provided, and the antigen binding domain (scFv) of the CAR or TCR includes the variable heavy chain of the antibody shown in SEQ ID NO: 9 Region, and the variable region of the antibody light chain shown in SEQ ID NO: 10.
  • the scFv further comprises a connecting peptide located between the variable region of the heavy chain and the variable region of the light chain.
  • the scFv is represented by the following formula A or formula B:
  • V H of the antibody heavy chain variable region V L of the antibody light chain variable region; and "-" connecting peptide or a peptide bond.
  • the linker peptide between the V H and V L, 1-4 consecutive SEQ ID NO: 7 sequence (GGGGS) of preferably 1-4, more preferably Land 3-4.
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is no or signal peptide sequence
  • H is no or hinge area
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ .
  • a bispecific CAR or TCR is provided, the bispecific CAR or TCR targets BCMA and the first target,
  • the BCMA-targeting antigen binding domain (scFv) in the bispecific CAR includes the antibody heavy chain variable region shown in SEQ ID NO: 9, and the antibody light chain shown in SEQ ID NO: 10 can be Change area.
  • the first target is selected from the following group:
  • CD138 Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T
  • the first target is CD19
  • the CD19-targeting antigen binding domain (scFv) in the bispecific CAR includes the antibody heavy chain shown in SEQ ID NO: 11. Variable region, and the variable region of the antibody light chain shown in SEQ ID NO: 12.
  • the first target is CD19
  • the CD19-targeting antigen binding domain (scFv) in the bispecific CAR includes any one of SEQ ID NO: 21-30
  • the CD19 antibody heavy chain variable region (H1) shown in SEQ ID NO: 22 is shown in SEQ ID NO: 22
  • CD19 antibody heavy chain variable region (H8) shown in SEQ ID NO: 23 is CD19 antibody heavy chain variable region (H8) shown in SEQ ID NO: 23
  • CD19 antibody heavy chain variable region (H10) shown in SEQ ID NO: 24 is CD19 antibody heavy chain variable region (H10) shown in SEQ ID NO: 24
  • CD19 antibody heavy chain variable region (H2) shown in SEQ ID NO: 25 is CD19 antibody heavy chain variable region (H2) shown in SEQ ID NO: 25
  • CD19 antibody heavy chain variable region (H4) shown in SEQ ID NO: 27 is CD19 antibody heavy chain variable region (H4) shown in SEQ ID NO: 27
  • CD19 antibody heavy chain variable region (H5) shown in SEQ ID NO: 28 is CD19 antibody heavy chain variable region (H5) shown in SEQ ID NO: 28
  • the bispecific CAR includes both an antigen binding domain targeting the first target and an antigen binding domain targeting the BCMA.
  • the structure of the bispecific CAR is shown in the following formula II:
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is no or signal peptide sequence
  • I is a flexible joint
  • H is no or hinge area
  • 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, and the other is an antigen binding domain targeting BCMA.
  • the scFv1 and scFv2 can be independent of each other, can also be connected in series, or have a loop structure.
  • the scFv1 is an antigen binding domain targeting the first target
  • the scFv2 is an antigen binding domain targeting BCMA.
  • the scFv1 is an antigen binding domain targeting BCMA
  • the scFv2 is an antigen binding domain targeting the first target.
  • sequence of the flexible linker I includes 1-6, preferably 3-5 consecutive sequences shown in SEQ ID NO: 7 (GGGGS).
  • the flexible linker I has a sequence as shown in SEQ ID NO: 17, 18 or 19.
  • the structure of the antigen binding domain targeting the first target is shown in the following formula C or formula D:
  • V L1 is the variable region of the light chain of the anti-first target antibody
  • V H1 is the variable region of the heavy chain of the anti-first target antibody
  • "-" is the connecting peptide or peptide bond.
  • CD19-targeting antigen binding domain is shown in the following formula C or formula D:
  • V L1 is the variable region of the light chain of the anti-CD19 antibody
  • V H1 is the variable region of the heavy chain of the anti-CD19 antibody
  • "-" is the connecting peptide or peptide bond.
  • 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: 11.
  • the light chain variable region of the anti-CD19 antibody has an amino acid sequence as shown in SEQ ID NO: 12.
  • the structure of the antigen binding domain of the targeted BCMA is shown in the following formula A or formula B:
  • V H of the antibody heavy chain variable region V L of the antibody light chain variable region; and "-" connecting peptide or a peptide bond.
  • the scFv1 includes the antibody heavy chain variable region shown in SEQ ID NO: 11 and the antibody light chain variable region shown in SEQ ID NO: 12; and the scFv2 includes The antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10.
  • the scFv1 includes the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10; and the scFv2 includes The antibody heavy chain variable region shown in SEQ ID NO: 11 and the antibody light chain variable region shown in SEQ ID NO: 12.
  • the scFv1 and/or scFv2 are murine, human, human and murine chimeric, or fully humanized single-chain antibody variable region fragments.
  • the structure of the bispecific CAR is shown in the following formula III or III':
  • Each "-" is independently a connecting peptide or a peptide bond
  • V H3 is the first anti-target antibody heavy chain variable region, and V L3 of the first anti-target antibody light chain variable region; as a target or scFv3 binding domain to the first target antigen, V H3 is an anti-BCMA antibody heavy chain variable region, and V L3 is an anti-BCMA antibody light chain variable region.
  • the scFv3 includes the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10.
  • the V H3 has the antibody heavy chain variable region as shown in SEQ ID NO: 9 and the V L3 has the antibody light chain variable region as shown in SEQ ID NO: 10.
  • the scFv3 comprises the antibody heavy chain variable region shown in SEQ ID NO: 11 and the antibody light chain variable region shown in SEQ ID NO: 12; and the V H3 has The antibody heavy chain variable region shown in SEQ ID NO: 9, and VL3 has the antibody light chain variable region shown in SEQ ID NO: 10.
  • the scFv3 comprises the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10; and the V H3 has The antibody heavy chain variable region shown in SEQ ID NO: 11, and VL3 have the antibody light chain variable region shown in SEQ ID NO: 12.
  • the structure of the CAR is shown in FIG. 1.
  • 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: 16 or 1.
  • the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof. In another preferred embodiment, each of the H is independently a hinge region derived from CD8.
  • the H has an amino acid sequence as shown in SEQ ID NO: 8.
  • the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154, or a combination thereof.
  • the TM is each independently a transmembrane region derived from CD8 or CD28.
  • the CD8-derived transmembrane region has an amino acid sequence as shown in SEQ ID NO:7.
  • the transmembrane region derived from CD28 has an amino acid sequence as shown in SEQ ID NO:6.
  • 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.
  • the C is a costimulatory signal molecule derived from CD28 and/or 4-1BB.
  • the costimulatory signal molecule derived from 4-1BB has an amino acid sequence as shown in SEQ ID NO: 5.
  • the CD28-derived costimulatory signal molecule has an amino acid sequence as shown in SEQ ID NO: 4.
  • the CD3 ⁇ has an amino acid sequence as shown in SEQ ID NO: 3.
  • the CAR (preferably C-terminal or N-terminal) further includes a cell suicide element.
  • the cell suicide element and the L or CD3 ⁇ of the CAR or the bispecific CAR are connected via T2A.
  • a nucleic acid molecule which encodes the CAR or TCR according to the first aspect or the bispecific CAR or TCR according to the second 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 is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • the vector is a lentiviral vector.
  • a host cell which contains the vector according to the fourth aspect of the present invention, or the nucleic acid molecule according to the third aspect of the present invention integrated into the chromosome, Or express the CAR or TCR described in the first aspect of the present invention or the bispecific CAR or TCR described in the second aspect of the present invention.
  • an engineered immune cell contains the vector according to the fourth aspect of the present invention, or the chromosome integrates the foreign source according to the third aspect of the present invention Nucleic acid molecules, or express the CAR or TCR described in the first aspect of the present invention or the bispecific CAR or TCR described in the second aspect of the present invention.
  • the immune cell has one or more characteristics selected from the following group:
  • the immune cell is a T cell, and the TCR gene expression of the T cell is silenced;
  • the immune cells express exogenous cell suicide 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 signal 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 engineered immune cells are selected from the following group:
  • CAR-NK cells Chimeric antigen receptor NK cells
  • the immune cells express exogenous cellular suicide elements.
  • CAR and 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-terminus or C-terminus 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 IV:
  • 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 cell suicide 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 cell suicide element is EGFRt.
  • the engineered immune cells are used for autoimmune therapy and/or allogeneic immunotherapy.
  • the engineered immune cells can kill tumor cells with clonal proliferation ability.
  • the immune cell expressing the bispecific CAR of the second aspect has a longer survival time in vivo than the immune cell expressing the CAR of the first aspect of the present invention.
  • the body includes an autologous body or a foreign body.
  • an engineered immune cell the immune cell containing an exogenous first expression cassette and a second expression cassette, wherein the first expression cassette is used to express the first expression cassette.
  • the first CAR or the first exogenous TCR of the target, and the second expression cassette is used to express the second CAR or the second exogenous TCR targeting BCMA;
  • the immune cell expresses the first CAR or the first exogenous TCR targeting the first target and the second CAR or the second exogenous TCR targeting BCMA;
  • the antigen binding domain (scFv) targeting BCMA in the second CAR or second exogenous TCR includes the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody heavy chain variable region shown in SEQ ID NO: 10 Antibody light chain variable region;
  • the first target is selected from the following group:
  • CD138 Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T
  • the first target is CD19
  • the antigen binding domain (scFv) targeting CD19 in the first CAR includes the antibody heavy chain variable region shown in SEQ ID NO: 11, and The variable region of the antibody light chain shown in SEQ ID NO: 12.
  • the second CAR is the CAR according to the first aspect of the present invention.
  • the first CAR and the second CAR are located on the cell membrane of the immune cell.
  • a first CAR targeting CD19 and a second CAR targeting BCMA are expressed on the cell membrane of the immune cells.
  • first expression cassette and the second expression cassette are located on the same or different vectors.
  • first expression cassette and the second expression cassette are located in the same vector.
  • the structure of the first CAR is shown in the following formula V:
  • Each "-" is independently a connecting peptide or a peptide bond
  • scFv1' is an antigen binding domain that targets CD19.
  • first CAR and the second CAR are connected by a 2A peptide.
  • sequence of the 2A peptide is shown in SEQ ID NO: 2.
  • the immune cell also includes a cell suicide element.
  • the cell suicide element and the bispecific CAR are connected (or connected in series) via T2A.
  • the cell suicide element is connected to the first CAR and/or the second CAR through T2A.
  • the expression of PD1 gene of the immune cell is silenced.
  • the "PD-1 gene expression is silenced" means that the PD-1 gene is not expressed or is underexpressed.
  • the "low expression” refers to the ratio of the expression level G1 of the PD-1 gene of the immune cells to the expression level G0 of the PD-1 gene of the normal immune cells, that is, G1/G0 ⁇ 0.5, preferably Ground G1/G0 ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, most preferably 0.
  • the "low expression” refers to the ratio of the expression level G1 of the PD-1 gene of the CAR-T cell to the expression level G0 of the normal T cell PD-1 gene, that is, G1/G0 ⁇ 0.5, Preferably G1/G0 ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, most preferably 0.
  • the formulation is a liquid formulation.
  • the dosage form of the preparation is injection.
  • the concentration of the engineered immune cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml ml.
  • the CAR includes a bispecific CAR.
  • the ninth aspect of the present invention there is provided a use of the CAR or TCR according to the first or second aspect of the present invention, or the engineered immune cell according to the sixth or seventh aspect of the present invention, for preparing prevention and / Or drugs or preparations to treat cancer or tumors.
  • the tumor is a hematological tumor.
  • 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 cancer or tumor is multiple myeloma.
  • the cancer or tumor is lymphoma.
  • the lymphoma is selected from the group consisting of Hodgkin's lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukocyte (CLL) ), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and complex B-cell non-Hodgkin lymphoma.
  • HL Hodgkin's lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • CLL chronic lymphocytic leukocyte
  • SLL small lymphocytic lymphoma
  • MZL marginal zone lymphoma
  • MCL mantle cell lymphoma
  • BL Burkitt lymphoma
  • complex B-cell non-Hodgkin lymphoma complex B-cell non-Hodg
  • the cancer or tumor includes recurrent cancer or tumor.
  • the drug or preparation treats cancer or tumor by killing tumor cells with clonal proliferation ability.
  • the tumor cells with clonal proliferation ability include clone forming cells, tumor cell precursor cells, and tumor progenitor cells.
  • a method for preparing engineered immune cells is provided.
  • the engineered immune cells express the CAR or TCR described in the first or second aspect of the present invention, including the following steps:
  • the nucleic acid molecule according to the third aspect of the invention or the vector according to the fourth aspect of the invention is transferred into immune cells to obtain the engineered immune cells.
  • the immune cells are T cells or NK cells.
  • a method for preparing engineered immune cells which includes the following steps:
  • the antigen binding domain (scFv) targeting BCMA in the second CAR includes the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10 ;
  • the first target is selected from the following group:
  • CD138 Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T
  • the step (2) can be performed before, after, at the same time or alternately after the step (3).
  • step (2) or step (3) can be omitted.
  • a kit for preparing the engineered immune cells according to the sixth or seventh aspect of the present invention, and the kit contains a container and is located in The nucleic acid molecule according to the third aspect of the present invention or the vector according to the fourth aspect of the present invention in the container.
  • kits for preparing the engineered immune cells according to the sixth or seventh aspect of the present invention, and the kit contains a container and is located in Inside the container:
  • a first nucleic acid sequence, said first nucleic acid sequence containing a first expression cassette, and said first expression cassette is used to express a first CAR targeting a first target;
  • a second nucleic acid sequence which contains a second expression cassette, and the second expression cassette is used to express the second CAR that targets BCMA;
  • the antigen binding domain (scFv) targeting BCMA in the second CAR includes the antibody heavy chain variable region shown in SEQ ID NO: 9 and the antibody light chain variable region shown in SEQ ID NO: 10 ;
  • the first target is selected from the following group:
  • CD138 Kappa Light Chain, NKG2D-ligands, TACI, GPRC5D, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T
  • first and second nucleic acid sequences are located in the same or different containers.
  • first and second nucleic acid sequences are located in the same expression vector.
  • the fourteenth aspect of the present invention there is provided a use of the engineered immune cells according to the sixth or seventh aspect of the present invention to prevent and/or treat cancer or tumors.
  • the cancer or tumor is multiple myeloma.
  • a method for treating diseases comprises administering an appropriate amount of the cells according to the sixth or seventh aspect of the present invention or the preparation according to the fifth aspect of the present invention to a subject in need of treatment.
  • the disease is cancer or tumor.
  • a method for enhancing the survival ability of immune cells in vivo or enhancing the killing ability of immune cells against tumor cells with clonal proliferation ability includes (a) simultaneously expressing in said immune cells An exogenous first expression cassette and a second expression cassette, wherein the first expression cassette is used to express the first CAR targeting CD19, and the second expression cassette is used to express the second CAR targeting BCMA; or ( b) Express the bispecific CAR described in the second aspect in the immune cells.
  • the immune cells constructed by the method are as described in the sixth and seventh aspects of the present invention.
  • first expression cassette and the second expression cassette have the same meaning as the first expression cassette and the second expression cassette in the seventh aspect of the present invention.
  • the body includes an autologous body or a foreign body.
  • the seventeenth aspect of the present invention there is provided a method for enhancing the in vivo viability of engineered immune cells targeted to BCMA or killing tumor cells with clonal proliferation ability, including in the engineered
  • the first exogenous expression cassette is expressed in the immune cell, and the first expression cassette is used to express the first CAR targeting CD19.
  • the first expression cassette has the same meaning as the first expression cassette and the second expression cassette in the seventh aspect of the present invention.
  • the engineered immune cell targeting BCMA is an immune cell expressing the CAR according to the first aspect of the present invention.
  • the body includes an autologous body or a foreign body.
  • a use of a first expression cassette is provided.
  • the first expression cassette is used to express a first CAR targeting CD19 for enhancing the in vivo of engineered immune cells targeting BCMA Survivability or killing ability against tumor cells with clonal proliferation ability, or to prepare a kit for enhancing the in vivo survival ability of engineered immune cells targeting BCMA or The killing ability of tumor cells that clone the proliferation ability.
  • the body includes an autologous body or a foreign body.
  • Figure 1 shows a schematic diagram of the structure containing the CAR of the present invention and the cell suicide element. Among them, the bispecific CAR and the suicide switching element are connected through 2A.
  • Figure 2 shows the flow cytometric analysis results of CAR-BB and CAR-S1 in the present invention expressed on the surface of Jurkat cells and primary T cells, respectively.
  • FIG 3 shows the killing results (RTCA method) of CAR-BB and CAR-S1 on Hela cells and overexpressing BCMA cells (Hela-BCMA) in the present invention, and CAR-April and CAR-S1 on Hela cells and overexpressing respectively BCMA cell (Hela-BCMA) killing result (RTCA method).
  • Figure 4 shows the BCMA expression of target cells used in the present invention.
  • Figure 5 shows the in vitro killing experiment results (Luciferase method) of different batches of CAR-BB and CAR-S1 on MM.1s cells and RPMI-8226 cells, respectively.
  • Figure 6 shows the release of IFNr cytokines during the killing process of CAR-BB and CAR-S1 on Hela cells and overexpressing BCMA cells (Hela-BCMA) in the present invention.
  • Figure 7 shows the tumor elimination ability of CAR-BB and CAR-S1 of the present invention after intravenous infusion of the immunodeficiency mouse RPMI-8226 subcutaneous model model.
  • Figure 8 shows the expression of CD19-CAR and BCMA-CAR in the bispecific CAR-T cell of the present invention.
  • Figure 9 shows the expression of CD19-CAR and BCMA-CAR and the analysis of EGFRt expression in the bispecific CAR-T cell with the safety switch added in the present invention.
  • Figure 10 shows the comparison of the killing conditions of different batches of CAR-19, CAR-BCMA and bispecific CAR-T in the present invention on Hela and Hela overexpressing antigen cell lines Hela-BCMA, Hela-CD19 and Hela-BCMA-CD19
  • Figure 11 shows the in vitro killing test results (Luciferase method) of different batches of CAR-CD19, CAR-BCMA and bispecific CAR-T on MM.1s cells, RPMI-8226 cells and Nalm6 cells in the present invention.
  • Figure 12 shows the release of cytokines during the killing process of CAR-BCMA and bispecific CAR-T cells of the present invention on Hela overexpressing BCMA cells (Hela-BCMA).
  • Figure 13 Analysis of CD107a molecule expression on the surface of dual CAR-T cells co-cultured with MM.1s or Raji tumor target cells.
  • Figure 14 shows the tumor elimination ability of CAR-S1, bispecific CAR-S2 and CAR-S4 of the present invention on immunodeficiency mouse RPMI-8226 subcutaneous modeling model after intravenous infusion.
  • Figure 15 shows the tumor elimination ability of different doses of the bispecific CAR-S2 and CAR-S4 cells of the present invention after intravenous infusion of the MM.1s-luc intravenous model of immunodeficiency mice.
  • Figure 16 shows the clonogenic inhibitory ability of different CAR-T cells on CD34-negative monocytes in the bone marrow of MM patients.
  • Figure 17 shows the in vivo tumor clearance ability of different CAR-T cells in NOG mice modeled by Nalm6-Luc cells.
  • Figure 18 shows the expression of CAR and safety switches of CAR-T cells on the surface of T cells.
  • Figure 19 The killing effects of different CAR-T cells on Nalm6 or RMPI8226 cells (Luciferase method). Compared with single CAR-T cells, dual CAR pairs have stronger ability to kill target cells. Figure 20 Different CAR-T cells have no killing ability on negative target cells (K562, Raji-KO19, Nalm6-KO19, CCRF).
  • Figure 21 The tumor elimination ability of different CAR-T cells against Raji-Luc modeled NOG cells in vivo. Compared with single CAR-T cells, dual CAR pairs have stronger ability to kill target cells.
  • Figure 22 shows the results of the in vitro killing experiment of CAR-S1 on Raji lymphoma cells (Luciferase method). Among them, Figure 22A shows the expression of BCMA antigen on the surface of Raji lymphoma target cells, and Figure 22B shows the killing of CAR-S1 cells on Raji lymphoma target cells under different E:T ratios.
  • the inventors constructed a new engineered immune cell targeting BCMA for the first time, and the antigen-binding domain in the CAR contained in it was scFv derived from S.
  • the CAR-T cells constructed in the present invention have higher killing effect and tumor clearance ability.
  • the present invention also utilizes S scFv and CD19 scFv to construct dual CAR-T cells, which can simultaneously kill BCMA and CD19-positive CAR-T cells.
  • the present invention uses scFv of different BCMA antibodies to construct CAR-T cells, and compares them. It is unexpectedly found that CAR-T cells constructed from scFv from BB and BCMA binding domain from April have more High ability to kill BCMA overexpressing cells and BCMA positive tumor target cells. In vivo mouse animal models also showed higher tumor clearance ability than CAR-T derived from BB. CAR-T cells constructed with other scFv targeting BCMA that are common in the art do not show ideal in vitro and in vivo functions.
  • administration refers to the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, 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 Combined part.
  • H chain includes 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.
  • amino acids in this article are identified by internationally accepted single English letters, and the corresponding three-letter abbreviations of amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), I1e(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).
  • BCMA B cell maturation antigen
  • BCMA is a transmembrane protein expressed on the surface of mature B lymphocytes, namely plasmablasts and plasma cells. And multiple myeloma is caused by abnormal proliferation of plasma cells and invasion of bone marrow. Studies have shown that BCMA is expressed on multiple myeloma cells. Car-T cells targeting BCMA have been shown to specifically kill myeloma cells. However, some patients still have a relapse process after receiving CAR-T cell therapy targeting BCMA. For these relapsed patients, it is necessary to find a target that is different from BCMA before continuing treatment.
  • CD19 molecule is a transmembrane protein on the surface of B cells, which is closely related to B cell activation, signal transduction and growth regulation. As shown in Figure 1, CD19 is almost expressed on the surface of all B cells, and CAR-T cells targeting CD19 are currently effective in the treatment of leukemia and lymphoma. It is generally believed that 99.95% of plasma cells do not express CD19 on the surface, so the possibility of CD19 being used to treat multiple myeloma is ignored.
  • 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 needed for effective response of lymphocytes to antigens, not antigen receptors or their ligands.
  • 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 an antigen binding domain that targets BCMA (or BCMA and CD19).
  • BCMA BCMA and CD19
  • the CAR of the present invention can perform antigen recognition based on the antigen binding specificity.
  • 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.
  • 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 the antigen binding sites.
  • an Fv antibody also contains 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 antigen-binding domain includes an antibody that specifically recognizes BCMA.
  • the antigen-binding domain further includes an antibody that specifically recognizes CD19, preferably a single-chain antibody.
  • 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 interaction with the receptor complex. Interaction of other members.
  • the intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3 ⁇ .
  • the CAR of the present invention also includes a cell suicide element.
  • the scFv targeting BCMA of the present invention is S scFv, and the BB scFv and April chain in the examples are used as controls.
  • BB scFv and April chain are commonly used binding sequences targeting BCMA in this field.
  • BB scFv is described in PCT application WO 2010104949 A3, and April chain is described in CN105658671A.
  • Multiple myeloma is a malignant plasma cell tumor.
  • the tumor cells originate from plasma cells in the bone marrow, and plasma cells are cells that develop to the final functional stage of B lymphocytes.
  • Multiple myeloma is basically an incurable disease with the characteristics of high morbidity and high mortality.
  • 2017 statistics there were 30,000 newly diagnosed multiple myeloma patients in the United States, and 12,000 may face death.
  • common therapies for multiple myeloma include cytotoxic drugs, protease inhibitors (bortezomib, etc.), lenalidomide, monoclonal antibodies, and corticosteroids. However, they are all partially effective and cannot last for a long time. The chance of recurrence is very high. Therefore, the improvement of multiple myeloma therapy is particularly important.
  • CD19 is a glycoprotein with a molecular weight of 95kDa. It is expressed on the membrane surface of pre-B cells and mature B cells. It is closely related to the transmembrane conduction pathway of B cell Ca++, and has a regulatory effect on B cell proliferation and differentiation. CD19 is mainly expressed in normal B cells and cancerous B cells, with high tissue expression specificity, and is a good antibody or CAR-T immunotherapy target. However, in the course of immunotherapy, the CD19 epitope of B cells is often lost, causing patients to fail to respond to immunotherapy or relapse.
  • Bi-specificity means that the same CAR can specifically bind to and immunorecognize two different antigens, and CAR can produce an immune response when combined with any antigen.
  • the bispecific CAR targeting CD19 and BCMA is as described in the second aspect of the present invention.
  • the extracellular domain of the CAR provided by the present invention includes antigen binding domains targeting CD19 and BCMA, including anti-CD19 scFv and anti-BCMA scFv.
  • the present invention provides a bispecific chimeric antigen receptor for CD19 and BCMA antigens.
  • the CAR structural components that target both CD19 and BCMA can include signal peptides, anti-CD19 scFv, anti-BCMA scFv, hinge region, transmembrane region, and intracellular T cell signaling region, where CD19scFv and BCMAscFv pass through a short peptide Segments (G4S)xN are connected.
  • G4S short peptide Segments
  • the CD19 and BCMA bispecific CAR of the present invention has a single structure and contains scFv against CD19 and BCMA.
  • the CAR includes CD19 scFv and BCMA scFv, and the order and hinge of CD19 scFv and BCMA scFv are the main influencing factors of its function.
  • the present invention optimizes the sequence of the BCMA scFv, the BCMA scFv (S scFv) has high affinity with BCMA, good specificity, and can specifically target the full-length BCMA antigen and extracellular area.
  • (G4S)x3 is used to connect CD19scFv and BCMAscFv, when CAR has the best activity and lethality.
  • the present invention uses CARs that target CD19 and BCMA bispecifically. Compared with CARs that target a single antigen, the affinity is significantly enhanced, the activity of immune cells is significantly increased, and it has a synergistic effect. In addition, due to the uneven expression levels of CD19 and BCMA in tumor cells, dual-targeting CAR-T treatment has a wider range. CAR-immune cells targeting CD19 and BCMA at the same time can reduce the possibility of antigen escape caused by the down-regulation or deletion of a single surface antigen.
  • the bispecific CAR-T of CD19 and BCMA has significantly better than single CAR-T's ability to inhibit the in vitro clone formation of CD34-negative monocytes in the bone marrow of myeloma patients, indicating that it is significantly better than single CAR-T The ability of cells to inhibit tumor progenitor cells.
  • the addition of CD19 antigen can increase the survival ability of the bispecific CAR-T of CD19 and BCMA.
  • CAR-T cell As used herein, the terms “CAR-T cell”, “CAR-T”, and “CAR-T cell of the present invention” include the CAR-T cell included in the third aspect of the present invention.
  • CAR-T cells have the following advantages over other T cell-based therapies: (1) The action process of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, they target a certain tumor Once the CAR gene construction of the antigen is completed, it can be widely used; (3) CAR can use both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) using the patient's own body Cells reduce the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • CAR-NK cells Chimeric antigen receptor NK cells
  • CAR-NK cell As used herein, the terms “CAR-NK cell”, “CAR-NK”, and “CAR-NK cell of the present invention” all refer to the CAR-NK cell included in the third aspect of the present invention.
  • the CAR-NK cells of the present invention can be used to treat tumors with high BCMA expression, such as multiple myeloma.
  • Natural killer (NK) cells are a major type of immune effector cells that protect the body from virus infection and tumor cell invasion through non-antigen-specific ways.
  • the engineered (gene modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, for example: (1) They directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) They release A small amount of cytokines reduces the risk of cytokine storm; (3) It is easy to expand and develop into "off-the-shelf" products in vitro. Otherwise, it is similar to CAR-T cell therapy.
  • 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. , To block unknown or uncontrollable long-term toxicity to ensure patient safety.
  • the suicide switch used in the present invention can be the herpes simplex virus thymidine kinase (HSV-TK), inducible caspase9 (iCasp9), CD20, mutation Type human thymidylate kinase (mutated human thymidylate kinase, mTMPK) and so on.
  • HSV-TK, iCasp9 and CD20 have the same clearance ability on CAR-cells, but the clearance of iCasp9 and CD20 is faster, and HSV-TK is slower.
  • the iCasp9 suicide switch contains the FKBP12-F36V domain, which can be connected to Caspase-9 via a flexible linker, which does not contain a recruitment domain.
  • FKBP12-F36V contains a FKBP domain with phenylalanine substituted for valine at the 36th amino acid residue position. It has high selectivity and sub-nanomolar affinity, and can bind to dimerization to form a ligand, such as other inert small molecules AP1903. When a small molecule is added, it can promote its dimerization, thereby inducing cell apoptosis, but it is not effective for normal cells that do not carry a suicide switch.
  • Induction of safety switch caspase9 uses human caspase9 fusion FK506 binding protein (FKBP), so that it can be induced to form dimers with chemical inducers (AP1903/Rimiducid, Bellicum Pharmaceutical), leading to apoptosis of cells expressing the fusion protein.
  • FKBP human caspase9 fusion FK506 binding protein
  • CD19 and BCMA are highly expressed in tumor cells, they are also expressed in normal B cells.
  • the engineered immune cells of the present invention can attack normal B cells in vivo.
  • 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, directly isolated from the 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 transgenes and their 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 constructs 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, U.S. 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 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 No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into the 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 30-110bp 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
  • individual elements can act cooperatively or independently to initiate transcription.
  • 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, Ruth 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 Identification and selection of 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).
  • the vector can be easily introduced into a host cell by any method in the art, for example, a mammalian, bacterial, yeast, or insect cell.
  • 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.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids Plastid.
  • 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 (e.g., artificial membrane vesicles).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with lipids.
  • 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 present invention provides a CAR-T cell containing the first 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 (eg, T cells) transduced with lentiviral vectors (LV) encoding the expression cassettes of the present invention.
  • the transduced T cells can target tumor cell markers BCMA and/or CD19, and coordinately activate T cells to cause T cell immune response, thereby significantly improving its killing efficiency on tumor cells.
  • 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 patient's autologous T cells (or heterologous donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • the probability of suffering from graft-versus-host disease is extremely low, and the antigen is recognized by T cells in a non-MHC-restricted manner.
  • 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.
  • CAR-T cells against BCMA and/or CD19 elicit a specific immune response against cells expressing BCMA and/or CD19.
  • the present invention should be construed as including the construction of Any number of changes in each of the body components.
  • 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 CAR-modified T cells of the present invention.
  • 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 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 (e.g., before, simultaneously, or after) patient.
  • chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (e.g., before, simultaneously, or after) patient.
  • the subject can 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 by, for example, intravenous reinfusion for each treatment or course of treatment. .
  • CAR-T cells containing S scFv constructed in the present invention have higher tumor killing and functional activities in vivo and in vitro than BB and April CAR-T.
  • the bispecific CAR-T constructed in the present invention can simultaneously recognize two or more targets including BCMA.
  • Example 1 Isolation of PBMC and expansion of T cells from donor blood
  • Hela cells expressing BCMA, CD19 and BCMA/CD19 at the same time are stable transfected cell lines obtained by transferring BCMA and CD19 antigens through lentiviral vectors, which can specifically express BCMA or/and CD19 protein molecules, MM .1s-ffluc cells and RPMI8226-ffluc cells are stable cell lines screened after infection with firefly luciferase lentivirus.
  • a single CAR targeting BCMA and a dual CAR targeting BCMA and CD19 were designed and constructed.
  • the schematic structure is shown in Figure 1.
  • CAR, CD19CAR and suicide switch-EGFRt element are connected by 2A peptide.
  • the CAR structure involved in the present invention is shown in Figure 1, and the naming and composition are shown in Table 1.
  • the CAR genes in Table 1 were cloned into the FUW lentiviral vector backbone to construct a complete lentiviral expression vector that can be used to infect T cells.
  • the BCMA CAR gene is placed under the EF1 ⁇ (EF-1 ⁇ ) promoter to form Fuw-EF1 ⁇ -BCMA CAR, and Fuw-EF1 ⁇ -BCMA CAR, lentivirus package
  • the membrane plasmid pMD2.G Additional membrane plasmid pMD2.G (Addgene, Plasmid #12259) and the lentiviral packaging plasmid psPAX2 (Addgene, Plasmid #12260) were transferred into 293T using Lipofectamine3000 to prepare the complete lentiviral expression vector; the viral supernatant was collected at 48h and 72h, Concentration by ultracentrifugation; the concentrated virus can be used to infect T cells.
  • the experimental method is as follows:
  • RTCA method test The results are shown in Figure 3 (RTCA method test).
  • Figure 4 shows the antigen expression on the surface of target cells.
  • Figure 5 shows that under the same E:T ratio, NT cells have no killing function.
  • CAR-S1 cells have no effect on MM.1S-Luc cells (MM.1S cells transferred with luciferase gene) and RPMI8226-Luc cells (transferred with fluorescence).
  • the RPMI8226 cells with the sulfidase gene have a dose-dependent killing effect, and CAR-S1 cells show better killing ability than CAR-BB and CAR-April.
  • CAR-T cells by using a variety of targeted BCMA scFv commonly used in the art. After testing, none of these CAR-T cells showed ideal killing function.
  • CAR-T cells are co-cultured with target cells (BCMA overexpressing cells, BCMA-positive tumor cells, MM.1s-Luc and RPMI8226 cells)
  • target cells BCMA overexpressing cells, BCMA-positive tumor cells, MM.1s-Luc and RPMI8226 cells
  • the target cells can be lysed by CAR-T cells targeting BCMA
  • CAR-S1 shows higher lethality than CAR-BB.
  • some CAR-T cells constructed by scFv targeting BCMA that are common in the art did not show ideal killing function.
  • Example 4 CAR T cells (CAR-S1 CAR-T cells and CAR-BB CAR-T cells) targeting BCMA were mixed with tumor cells (Hela, Hela-BCMA, Hela-CD19, Hela-BCMA-CD19) , RPMI medium was placed in each cell density of 1X10 4 th formulated / ml, CAR-T cells and tumor cells in each lOOul placed in 96-well plates were cultured overnight, the supernatant was collected, the supernatant after centrifugation detection The release level of cytokines IFN- ⁇ , etc. Elisa kit was used for detection.
  • CAR-T cells that simultaneously target BCMA and CD19 are involved.
  • the CAR structure is shown in Figure 1 (CAR S2, CAR S3, CAR S4, CAR S5, CAR S6, and CAR S7).
  • BCMA CAR, CD19CAR and suicide switch-EGFRt element are connected by 2A peptide.
  • the scFv in the BCMA CAR structure is composed of the heavy chain and the light chain of the scFv of S and BB, where S scFv is composed of SEQ ID NO: 9 and SEQ ID NO: 10; BB scFv is composed of SEQ ID NO: 13 and SEQ ID NO: 14 Composition; CD19 scFv consists of SEQ ID NO: 11 and SEQ ID NO: 12; In addition, scFv can be replaced by the BCMA binding region composed of the April partial sequence (SEQ ID NO: 15) to form a new CAR structure.
  • Lentiviral infection Two days after the isolated and purified primary T cells are activated, use the lentivirus constructed as above to infect the lentiviral vector at MOI (1-10), transfer to a cell culture flask, and place it at 37°C, 5% CO 2 a thermostat incubator.
  • Cell proliferation and CAR positive rate detection On the 3rd day after infection and before cryopreservation, the number of cells and the proportion of BCMA/CD19 double positive cells are sampled, that is, the CAR positive rate of T cells is detected. Replace half of the culture every 2-3 days base.
  • Figure 9 shows that the expression of BCMA CAR, CD19 CAR and EGFRt can be simultaneously detected on the surface of CAR-S6 and CAR-S7 cells.
  • the CAR-T cells obtained in Example 8 were subjected to in vitro killing experiments.
  • By transferring the luciferase gene into target cells stable transfected cell lines (RPMI8226, MM.1s and Nalm6) were obtained after clonal screening.
  • luciferase reacts with luciferin to produce fluorescence.
  • the intensity of fluorescence the activity of luciferase can be measured, and the survival ratio of cells can be detected to obtain the killing effect of CART cells.
  • Figure 11 shows that dual CAR-T can significantly kill BCMA single-positive tumor target cells MM.1s and RPMI8226. And it can kill CD19 positive tumor target cells Raji and Nalm6 significantly. It shows that the dual CAR combined with BCMA and CD19 has a killing effect on BCMA and CD19-positive tumor target cells.
  • BCMA-CD19 CAR-T cells obtained in Example 8
  • tumor cells Hela-BCMA
  • the cell density was prepared as 1 ⁇ 10 4 cells/ml
  • CAR-T cells and tumor cells were each 100ul, placed in a 96-well plate, co-cultured overnight, collected the supernatant, centrifuged to take the supernatant to detect the cytokine release level, using the CBA method for detection.
  • BCMA-CD19 CAR-T can secrete a large amount of cytokines after being stimulated by BCM-positive target cells, and NT only secretes a small amount of cytokines. It shows that BCMA-CD19 CAR-T can be activated by BCMA.
  • the CAR-T cells obtained in Example 8 were subjected to flow cytometry analysis of CD107a expression changes after activation, and a co-incubation activation experiment was performed using tumor cell lines expressing CD19 or BCMA.
  • the cells after co-incubation were labeled with antibodies for CD3, CD8 and CD107a, and then subjected to flow cytometry analysis.
  • mice selected 6-12 weeks and inject 1 ⁇ 10 7 RPMI8226 cells subcutaneously. Two days later, the tumor graft load was measured. After 10 days, they were divided into groups with equivalent tumor load. CAR-T cells were injected one day after grouping. After CAR-T treatment, the tumor volume load of mice was evaluated twice a week.
  • Fig. 15 show that compared with the control group, the tumor burden of the mice injected with dual CAR-T cells was significantly reduced until disappeared, indicating that BCMA-CD19 CAR-T cells have significant anti-tumor effects.
  • MM myeloma
  • the clinical manifestations are generally that most of the tumor cells can be eliminated, and the tumor cells with clonal proliferation that lead to tumor recurrence tend to have relatively higher drug resistance.
  • this study established the method of myeloma clonal formation experiment and investigated CAR -T's ability to inhibit clone formation.
  • MM tumor cells with proliferation ability will grow in the clonal proliferation medium, but the interference of CD34+ hematopoietic stem cells with proliferation ability on the experiment needs to be removed during the experiment, and the cells dominated by tumor proliferation cells are harvested. This experiment requires An important issue of control.
  • Ficoll was used to isolate and extract bone marrow mononuclear cells and perform flow phenotyping analysis.
  • the second step is to use CD34+ cell sorting kit to remove CD34+ cells.
  • the obtained cells use different groups of CAR-T cells (dual CAR-T, single CAR-T) for killing experiments. After killing, use a T cell removal and sorting kit to remove CAR-T cells .
  • the fourth step is to use semi-solid clonal enhancement medium for clonal growth, and perform statistics, counting, and summary results after 1 week to 2 weeks.
  • CAR-S2 and CAR-S4 have more significant advantages in killing clone-forming cells or tumor cell precursors than CAR-19 and CAR-S1, indicating that they have a higher inhibition of bone marrow compared with single CAR.
  • the role of tumor cell clone formation ability is shown in Figure 16.
  • CAR-T containing EGFRt elements was stained with EGFR antibody and analyzed by flow cytometry, and CAR expression was analyzed at the same time.
  • Examples 3 and 4 were used to construct humanized CAR-T cells (CAR-h19) and humanized dual CAR-T cells (CAR-hS2, CAR-hS4).
  • the structure of humanized CAR-T cells is similar to that of CAR-19 is similar.
  • the structure of humanized dual CAR-T cell CAR-hS2 is similar to CAR-S2, and the structure of CAR-hS4 is similar to CAR-S4.
  • humanized CD19 scFv is used to replace the original structure.
  • Mouse source scFv The humanized CD19 scFv includes the antibody heavy chain variable region shown in any one of SEQ ID NO: 21-30, and the antibody light chain variable region shown in any one of SEQ ID NO: 31-36.
  • Example 9 The method of Example 9 was used to detect the in vitro killing effect of humanized dual CAR-T cells.
  • mice selected NOG mice aged 6-12 weeks and inject 3 ⁇ 10 5 Raji cells subcutaneously. Six days later, the tumor graft load was measured and divided into groups with equivalent tumor load. The double CAR-T cells prepared above were injected one day after grouping. After CAR-T treatment, the tumor volume load of mice was evaluated. Each mouse was intraperitoneally injected with 3 mg of d-luciferin (Perkin Elmer Life Sciences), four minutes later, it was photographed using Xenogen IVIS Imaging System (Perkin Elmer Life Sciences) and exposed for 30 seconds. The bioluminescence signal is calculated according to the amount of photons emitted. The amount of photons is normalized by exposure time and surface area, and finally the amount of photons/s/cm 2 /spherical angle (p/s/cm 2 /sr) is obtained.
  • d-luciferin Perkin Elmer Life Sciences
  • Example 18 The killing of Raji lymphoma cells by CAR-T cells
  • luciferase-labeled Raji lymphoma target cells to detect the killing ability.
  • the stable transfected cell line Raji-Luc was obtained after clonal screening.
  • luciferase reacts with luciferin to produce fluorescence.
  • the intensity of fluorescence the activity of luciferase can be measured, and the survival ratio of cells can be measured to obtain the CAR-T cell Killing effect.

Abstract

本发明提供了靶向BCMA的工程化免疫细胞及其用途。具体地，本发明提供了一种特异性靶向BCMA的CAR，其包含的抗原结合结构域为S来源的scFv，具有SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区。本发明还提供了包含该CAR的CAR-T细胞，以及包含S来源的scFv的双CAR和CAR T细胞及其相关应用。与利用其他scFv所构建的CAR-T细胞相比，本发明构建的CAR-T细胞具有更高的杀伤效果和肿瘤清除能力。

Description

靶向BCMA的工程化免疫细胞及其用途 技术领域
本发明涉及免疫治疗领域，更具体地涉及一种靶向BCMA的工程化免疫细胞及其用途。
背景技术
多发性骨髓瘤(MM)是一种恶性浆细胞肿瘤，其肿瘤细胞起源于骨髓中的浆细胞，而浆细胞是B淋巴细胞发育到最终功能阶段的细胞。多发性骨髓瘤基本是一个无法治愈的疾病，具有高发病率及高致死率的特征。在2017年的统计数据中，美国有3万例新诊断出来的多发性骨髓瘤患者，而可能12000例将面临死亡。目前多发性骨髓瘤常见疗法有细胞毒药物治疗，蛋白酶抑制剂(硼替佐米等)、来那度胺、单克隆抗体及皮质类固醇等。但是都是部分有效，不能持久缓解，复发的几率很大。因此，多发性骨髓瘤疗法的改进就显得尤为重要。
因此，本领域急切地需要一种有效、复发机率小且安全的多发性骨髓瘤疗法。
发明内容
本发明的目的在于提供一种靶向BCMA的工程化免疫细胞及其用途。
本发明的另一目的是提供一种同时靶向CD19和BCMA的工程化免疫细胞及其用途。
在本发明的第一方面，提供了一种嵌合抗原受体(CAR)或TCR，所述CAR或TCR的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区。
在另一优选例中，所述scFv还包含位于重链可变区和轻链可变区之间的连接肽。
在另一优选例中，所述scFv如下式A或式B所示：
V H-V L,   (A)；V L-V H,   (B)
式中，V H为所述抗体重链可变区；V L为所述抗体轻链可变区；“-”为连接肽或肽键。
在另一优选例中，所述的V H和V L之间的连接肽为1-4个连续的SEQ ID NO:7(GGGGS)所示的序列，较佳地1-4个，更佳地3-4个。
在另一优选例中，所述CAR的结构如下式I所示：
L-scFv-H-TM-C-CD3ζ   (I)
式中，
各“-”独立地为连接肽或肽键；
L为无或信号肽序列；
H为无或铰链区；
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。
在本发明的第二方面，提供了一种双特异性CAR或TCR，所述双特异性CAR或TCR靶向BCMA和第一靶点，
其中，所述双特异性CAR中的靶向BCMA的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区。
并且，所述的第一靶点选自下组：
CD138、Kappa Light Chain、NKG2D-ligands、TACI、GPRC5D、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、Frizzled、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、Folate receptorα、Folate receptorβ、GPC2、CD70、BAFF-R、TROP-2、或其组合。在另一优选例中，所述双特异性CAR或TCR中包含靶向CD19的抗原结合结构域。
在另一优选例中，所述的第一靶点为CD19，并且所述双特异性CAR中的靶向CD19的抗原结合结构域(scFv)包括SEQ ID NO:11所示的抗体重链可变区，和SEQ ID NO:12所示的抗体轻链可变区。
在另一优选例中，所述的第一靶点为CD19，并且所述双特异性CAR中的靶向CD19的抗原结合结构域(scFv)包括SEQ ID NO:21-30中任一所示的抗体重链可变区，和SEQ ID NO:31-36中任一所示的抗体轻链可变区。
具体序列如下所示
SEQ ID NO:21所示的CD19抗体重链可变区(H9)
Figure PCTCN2020088836-appb-000001
SEQ ID NO:22所示的CD19抗体重链可变区(H1)
Figure PCTCN2020088836-appb-000002
SEQ ID NO:23所示的CD19抗体重链可变区(H8)
Figure PCTCN2020088836-appb-000003
SEQ ID NO:24所示的CD19抗体重链可变区(H10)
Figure PCTCN2020088836-appb-000004
SEQ ID NO:25所示的CD19抗体重链可变区(H2)
Figure PCTCN2020088836-appb-000005
SEQ ID NO:26所示的CD19抗体重链可变区(H3)
Figure PCTCN2020088836-appb-000006
SEQ ID NO:27所示的CD19抗体重链可变区(H4)
Figure PCTCN2020088836-appb-000007
SEQ ID NO:28所示的CD19抗体重链可变区(H5)
Figure PCTCN2020088836-appb-000008
SEQ ID NO:29所示的CD19抗体重链可变区(H6)
Figure PCTCN2020088836-appb-000009
SEQ ID NO:30所示的CD19抗体重链可变区(H7)
Figure PCTCN2020088836-appb-000010
SEQ ID NO:31所示的CD19抗体轻链可变区(L5)
Figure PCTCN2020088836-appb-000011
SEQ ID NO:32所示的CD19抗体轻链可变区(L1)
Figure PCTCN2020088836-appb-000012
SEQ ID NO:33所示的CD19抗体轻链可变区(L6)
Figure PCTCN2020088836-appb-000013
SEQ ID NO:34所示的CD19抗体轻链可变区(L2)
Figure PCTCN2020088836-appb-000014
SEQ ID NO:35所示的CD19抗体轻链可变区(L3)
Figure PCTCN2020088836-appb-000015
SEQ ID NO:36所示的CD19抗体轻链可变区(L4)
Figure PCTCN2020088836-appb-000016
在另一优选例中，所述双特异性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BCMA的抗原结合结构域。
在另一优选例中，所述双特异性CAR的结构如下式II所示：
L-scFv1-I-scFv2-H-TM-C-CD3ζ     (II)
式中，
各“-”独立地为连接肽或肽键；
L为无或信号肽序列；
I为柔性接头；
H为无或铰链区；
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第一靶点的抗原结合结构域，另一个为靶向BCMA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，所述的scFv1为靶向第一靶点的抗原结合结构域，所述scFv2为靶向BCMA的抗原结合结构域。
在另一优选例中，所述的scFv1为靶向BCMA的抗原结合结构域，所述scFv2为靶向第一靶点的抗原结合结构域。
在另一优选例中，所述柔性接头I的序列包含1-6个，较佳地为3-5个连续的SEQ ID NO:7(GGGGS)所示的序列。
在另一优选例中，所述柔性接头I具有如SEQ ID NO:17、18或19所示的序列。
在另一优选例中，所述靶向第一靶点的抗原结合结构域的结构如下式C或式D所示：
V L1-V H1   (C)；V H1-V L1    (D)
其中，V L1为抗第一靶点抗体轻链可变区；V H1为抗第一靶点抗体重链可变区；“-”为连接肽或肽键。
在另一优选例中，所述靶向CD19的抗原结合结构域的结构如下式C或式D所示：
V L1-V H1  (C)；V H1-V L1   (D)
其中，V L1为抗CD19抗体轻链可变区；V H1为抗CD19抗体重链可变区；“-”为连接肽或肽键。
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:11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:12所示的氨基酸序列。
在另一优选例中，所述靶向BCMA的抗原结合结构域的结构如下式A或式B所示：
V H-V L,   (A)；V L-V H,  (B)
式中，V H为所述抗体重链可变区；V L为所述抗体轻链可变区；“-”为连接肽或肽键。
在另一优选例中，所述scFv1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；且所述scFv2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scFv1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；且所述scFv2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，所述双特异性CAR的结构如下式III或III’所示：
L-V L3-scFv3-V H3-H-TM-C-CD3ζ   (III)
L-V H3-scFv3-V L3-H1-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；
scFv3为靶向BCMA的抗原结合结构域，V H3为抗所述第一靶点抗体重链可变区，且V L3为抗所述第一靶点抗体轻链可变区；或者scFv3为靶向所述第一靶点的抗原结合结构域，V H3为抗BCMA抗体重链可变区，且V L3为抗BCMA抗体轻链可变区。
在另一优选例中，所述scFv3包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区。
在另一优选例中，所述V H3具有如SEQ ID NO:9所示的抗体重链可变区，和V L3具有如SEQ ID NO:10所示的抗体轻链可变区。
在另一优选例中，所述scFv3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；且所述V H3具有如SEQ ID NO:9所示的抗体重链可变区，和V L3具有如SEQ ID NO:10所示的抗体轻链可变区。
在另一优选例中，所述scFv3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；且所述V H3具有如SEQ ID NO:11所示的抗体重链可变区，和V L3具有如SEQ ID NO:12所示的抗体轻链可变区。
在另一优选例中，所述CAR的结构如图1所示。
在另一优选例中，所述的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:16或1所示的氨基酸序列。
在另一优选例中，所述的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，所述的H各自独立地为CD8来源的铰链区。
在另一优选例中，所述H具有如SEQ ID NO:8所示的氨基酸序列。
在另一优选例中，所述的TM为选自下组的蛋白的跨膜区：CD28、CD3epsilon、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，所述的TM各自独立地为CD8或CD28来源的跨膜区。在另一优选例中，所述CD8来源的跨膜区具有如SEQ ID NO:7所示的氨基酸序列。
在另一优选例中，所述CD28来源的跨膜区具有如SEQ ID NO:6所示的氨基酸序列。
在另一优选例中，所述的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，所述的C为CD28和/或4-1BB来源的共刺激信号分子。
在另一优选例中，所述4-1BB来源的共刺激信号分子具有如SEQ ID NO:5所示的氨基酸序列。
在另一优选例中，所述CD28来源的共刺激信号分子具有如SEQ ID NO:4所示的氨基酸序列。
在另一优选例中，所述CD3ζ具有如SEQ ID NO:3所示的氨基酸序列。
在另一优选例中，所述CAR(优选地C端或N端)还包括细胞自杀元件。
在另一优选例中，所述细胞自杀元件与所述CAR或所述双特异性CAR的L或CD3ζ通过T2A连接。
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所述的CAR或TCR或第二方面所述的双特异性CAR或TCR。
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，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、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，提供了一种宿主细胞，所述的宿主细胞含有本发明第四方面所述的载体、或染色体中整合有外源的本发明第三方面所述的核酸分子、或表达本发明第一所述的CAR或TCR或第二方面所述的双特异性CAR或TCR。
在本发明的第六方面，提供了一种工程化的免疫细胞，所述的免疫细胞含有本发明第四方面所述的载体、或染色体中整合有外源的本发明第三方面所述的核酸分子、或表达本发明第一方面所述的CAR或TCR或第二方面所述的双特异性CAR或TCR。
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；
(b)所述免疫细胞为T细胞，且所述T细胞的TCR基因表达是被沉默的；和
(c)所述免疫细胞表达外源性细胞自杀元件；
(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)；或
(ii)嵌合抗原受体NK细胞(CAR-NK细胞)。
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IV所示：
L2-D-F(IV)
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，所述的细胞自杀元件选自下组：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，所述的细胞自杀元件为EGFRt。
在另一优选例中，所述的工程化的免疫细胞用于自体免疫治疗和/或异体免疫治疗。
在另一优选例中，所述的工程化的免疫细胞可以杀伤具有克隆增值能力的肿瘤细胞。
在另一优选例中，与表达本发明第一方面所述CAR的免疫细胞相比，表达第二方面所述的双特异性CAR的免疫细胞具有更长的体内存活时间。
在另一优选例中，所述的体内包括自体体内或异体体内。
在本发明的第七方面，提供了一种工程化的免疫细胞，所述免疫细胞含有外源的第一表达盒和第二表达盒，其中所述第一表达盒用于表达靶向第一靶点的第一CAR或第一外源TCR，所述第二表达盒用于表达靶向BCMA的第二CAR或第二外源TCR；
或所述免疫细胞表达所述靶向第一靶点的第一CAR或第一外源TCR和所述靶向BCMA的第二CAR或第二外源TCR；
其中，所述第二CAR或第二外源TCR中靶向BCMA的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区；
并且，所述的第一靶点选自下组：
CD138、Kappa Light Chain、NKG2D-ligands、TACI、GPRC5D、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、 ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、Frizzled、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、Folate receptorα、Folate receptorβ、GPC2、CD70、BAFF-R、TROP-2、或其组合。
在另一优选例中，所述的第一靶点为CD19，并且第一CAR中靶向CD19的抗原结合结构域(scFv)包括SEQ ID NO:11所示的抗体重链可变区，和SEQ ID NO:12所示的抗体轻链可变区。
在另一优选例中，所述第二CAR为本发明第一方面所述的CAR。
在另一优选例中，所述第一CAR和第二CAR定位于所述免疫细胞的细胞膜。
在另一优选例中，所述免疫细胞的细胞膜上表达有靶向CD19的第一CAR和靶向BCMA的第二CAR。
在另一优选例中，所述的第一表达盒和第二表达盒位于相同或不同的载体上。
在另一优选例中，所述的第一表达盒和第二表达盒位于同一载体。
在另一优选例中，所述第一CAR的结构如下式V所示：
L-scFv1’-H-TM-C-CD3ζ   (V)
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CD19的抗原结合结构域。
在另一优选例中，所述第一CAR和第二CAR通过2A肽连接。
在另一优选例中，所述2A肽的序列如SEQ ID NO:2所示。
在另一优选例中，所述免疫细胞内还包括细胞自杀元件。
在另一优选例中，所述的细胞自杀元件与所述双特异性CAR通过T2A连接(或串联)。
在另一优选例中，所述的细胞自杀元件与所述第一CAR和/或所述第二CAR通过T2A连接。
在另一优选例中，所述免疫细胞的PD1基因表达是被沉默的。
在另一优选例中，所述“PD-1基因表达是被沉默的”指PD-1基因不表达或低表达。
在另一优选例中，所述“低表达”指所述免疫细胞PD-1基因的表达量G1与正常免疫细胞PD-1基因的表达量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“低表达”指所述CAR-T细胞PD-1基因的表达量G1与正常T细胞PD-1基因的表达量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。
在本发明的第八方面，提供了一种制剂，所述制剂含有本发明第一或二方面所述的CAR或TCR、或本发明第六或七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，所述制剂中所述工程化的免疫细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
在另一优选例中，所述CAR包括双特异性CAR。
在本发明的第九方面，提供了一种本发明第一或二方面所述的CAR或TCR、或本发明第六或七方面所述的工程化的免疫细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤为血液肿瘤。
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，所述癌症或肿瘤为多发性骨髓瘤。
在另一优选例中，所述癌症或肿瘤为淋巴瘤。
在另一优选例中，所述的淋巴瘤选自下组：霍奇金淋巴瘤(HL)、弥漫大B细胞淋巴瘤(DLBCL)、滤泡淋巴瘤(FL)、慢性淋巴细胞白细胞(CLL)、小淋巴细胞淋巴瘤(SLL)、边缘区淋巴瘤(MZL)、套细胞淋巴瘤(MCL)、伯基特淋巴瘤(BL)和复杂B细胞非霍奇金淋巴瘤。
在另一优选例中，所述癌症或肿瘤包括复发性癌症或肿瘤。
在另一优选例中，所述药物或制剂通过杀伤具有克隆增值能力的肿瘤细胞治疗癌症或肿瘤。
在另一优选例中，所述的具有克隆增值能力的肿瘤细胞包括克隆形成细胞、肿瘤细胞前体细胞、肿瘤祖细胞。
在本发明的第十方面，提供了一种制备工程化免疫细胞的方法，所述的工程化免疫细胞表达本发明第一方面或第二方面所述的CAR或TCR，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免疫细胞。
在另一优选例中，所述免疫细胞为T细胞或NK细胞。
在本发明的第十一方面，提供了一种制备工程化免疫细胞的方法，包括以下步骤：
(1)提供一待改造的免疫细胞；和
(2)将用于表达靶向第一靶点的第一CAR的第一表达盒导入到所述免疫细胞；和
(3)将用于表达靶向BCMA的第二CAR的第二表达盒导入到所述免疫细胞，从而获得所述的工程化免疫细胞，
其中，所述第二CAR中靶向BCMA的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区；
并且，所述的第一靶点选自下组：
CD138、Kappa Light Chain、NKG2D-ligands、TACI、GPRC5D、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、Frizzled、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、Folate receptorα、Folate receptor β、GPC2、CD70、BAFF-R、TROP-2、或其组合。
在另一优选例中，所述步骤(2)可在步骤(3)之前、之后、同时或交替进行。
在另一优选例中，当步骤(1)中的待改造的免疫细胞已经表达第一CAR或第二CAR时，则步骤(2)或步骤(3)可以省略。
在本发明的第十二方面，提供了一种试剂盒，所述的试剂盒用于制备本发明第六或七方面所述的工程化的免疫细胞，且所述试剂盒含有容器，以及位于容器内的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。
在本发明的第十三方面，提供了一种试剂盒，所述的试剂盒用于制备本发明第六或七方面所述的工程化的免疫细胞，且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第一表达盒，所述的第一表达盒用于表达靶向第一靶点的第一CAR；和
(2)第二核酸序列，所述第二核酸序列含有第二表达盒，所述的第二表达盒用于表达所述靶向BCMA的的第二CAR；
其中，所述第二CAR中靶向BCMA的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区；
并且，所述的第一靶点选自下组：
CD138、Kappa Light Chain、NKG2D-ligands、TACI、GPRC5D、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、Frizzled、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、Folate receptorα、Folate receptor  β、GPC2、CD70、BAFF-R、TROP-2、或其组合。
在另一优选例中，所述的第一和第二核酸序列位于相同或不同的容器内。
在另一优选例中，所述的第一和第二核酸序列位于同一表达载体中。
在本发明的第十四方面，提供了一种本发明第六或七方面所述的工程化的免疫细胞的用途，用于预防和/或治疗癌症或肿瘤。
在另一优选例中，所述癌症或肿瘤为多发性骨髓瘤。
在本发明的第十五方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第六或七方面所述的细胞、或本发明第五方面所述的制剂。
在另一优选例中，所述疾病为癌症或肿瘤。
在本发明的第十六方面，提供了一种增强免疫细胞体内存活能力或增强免疫细胞对具有克隆增值能力的肿瘤细胞的杀伤能力的方法，包括(a)同时在所述的免疫细胞中表达外源的第一表达盒和第二表达盒，其中所述第一表达盒用于表达靶向CD19的第一CAR，所述第二表达盒用于表达靶向BCMA的第二CAR；或(b)在所述的免疫细胞中表达第二方面所述的双特异性CAR。
在另一优选例中，所述方法构建的免疫细胞如本发明第六和第七方面所述。
在另一优选例中，所述的第一表达盒和第二表达盒与本发明第七方面中的第一表达盒和第二表达盒具有相同含义。
在另一优选例中，所述的体内包括自体体内或异体体内。
在本发明的第十七方面，提供了一种增强靶向BCMA的工程化的免疫细胞的体内存活能力或对具有克隆增值能力的肿瘤细胞的杀伤能力的方法，包括在所述的工程化的免疫细胞中表达外源的第一表达盒，所述第一表达盒用于表达靶向CD19的第一CAR。
在另一优选例中，所述的第一表达盒与本发明第七方面中的第一表达盒和第二表达盒具有相同含义。
在另一优选例中，所述靶向BCMA的工程化的免疫细胞为表达本发明第一方面所述的CAR的免疫细胞。
在另一优选例中，所述的体内包括自体体内或异体体内。
在本发明的第十八方面，提供了一种第一表达盒用途，所述第一表达盒用于表达靶向CD19的第一CAR，用于增强靶向BCMA的工程化的免疫细胞的体内存活能力或对具有克隆增值能力的肿瘤细胞的杀伤能力，或者，用于制备一试剂盒，所述的试剂盒用于用于增强靶向BCMA的工程化的免疫细胞的体内存活能力或对具有克隆增值能力的肿瘤细胞的杀伤能力。
在另一优选例中，所述的体内包括自体体内或异体体内。
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显示了含有本发明CAR和细胞自杀元件的结构示意图。其中，双特异性CAR及自杀开关元件通过2A连接。
图2显示了本发明中CAR-BB和CAR-S1分别在Jurkat细胞和原代T细胞表面表达的流式细胞仪分析结果。
图3显示了本发明中CAR-BB和CAR-S1分别对Hela细胞和过表达BCMA细胞(Hela-BCMA)的杀伤结果(RTCA法)以及CAR-April和CAR-S1分别对Hela细胞和过表达BCMA细胞(Hela-BCMA)的杀伤结果(RTCA法)。
图4显示了本发明中所使用靶细胞的BCMA表达情况。
图5显示了本发明中不同批次CAR-BB和CAR-S1分别对MM.1s细胞和RPMI-8226细胞的体外杀伤实验结果(Luciferase法)。
图6显示了本发明中CAR-BB和CAR-S1分别对Hela细胞和过表达BCMA细胞(Hela-BCMA)的杀伤过程中IFNr细胞因子的释放。
图7显示了本发明中CAR-BB和CAR-S1对免疫缺陷小鼠RPMI-8226皮下造模模型静脉回输后的肿瘤消除能力。
图8显示了本发明中双特异CAR-T细胞中CD19-CAR及BCMA-CAR的表达情况。
图9显示了本发明中增加了安全开关的双特异CAR-T细胞中CD19-CAR及BCMA-CAR的表达情况及EGFRt表达情况分析。
图10显示了本发明中不同批次CAR-19、CAR-BCMA及双特异CAR-T对Hela及Hela过表达抗原细胞系Hela-BCMA、Hela-CD19和Hela-BCMA-CD19的杀伤情况比较
图11显示了本发明中不同批次CAR-CD19、CAR-BCMA及双特异CAR-T对MM.1s细胞、RPMI-8226细胞以及Nalm6细胞的体外杀伤实验结果(Luciferase法)。
图12显示了本发明中CAR-BCMA和双特异CAR-T细胞对Hela过表达BCMA细胞(Hela-BCMA)的杀伤过程中细胞因子的释放。
图13双CAR-T细胞与MM.1s或Raji肿瘤靶细胞共培养后表面的CD107a分子表达情况分析。
图14显示了本发明中CAR-S1和双特异性CAR-S2、CAR-S4对免疫缺陷小鼠RPMI-8226皮下造模模型静脉回输后的肿瘤消除能力。
图15显示了不同剂量的本发明的双特异性CAR-S2、CAR-S4细胞对免疫缺陷小鼠MM.1s-luc静脉造模模型进行静脉回输后的肿瘤消除能力。
图16显示了不同的CAR-T细胞对MM患者骨髓中的CD34阴性单核细胞的克隆形成抑制能力。
图17显示了不同的CAR-T细胞体内对Nalm6-Luc细胞造模NOG小鼠的体内肿瘤的清除能力。
图18显示了CAR-T细胞的CAR及安全开关在T细胞表面的表达情况。
图19不同CAR-T细胞对Nalm6或RMPI8226细胞的杀伤作用(Luciferase法)，双CAR 对相对于单CAR-T细胞具有更强的杀伤靶细胞能力。图20不同CAR-T细胞对阴性靶细胞(K562、Raji-KO19、Nalm6-KO19、CCRF)不具有杀伤能力。
图21不同CAR-T细胞对Raji-Luc造模的NOG细胞体内肿瘤消除能力。双CAR对相对于单CAR-T细胞具有更强的杀伤靶细胞能力。
图22显示了CAR-S1对Raji淋巴瘤细胞的体外杀伤实验结果(Luciferase法)。其中，图22A显示了Raji淋巴瘤靶细胞表面的BCMA抗原表达情况，图22B显示了CAR-S1细胞对Raji淋巴瘤靶细胞在不同E:T比例下的杀伤情况。
具体实施方式
本发明人经过广泛而深入地研究，首次构建了一种新的靶向BCMA的工程化免疫细胞，其包含的CAR中的抗原结合结构域为S来源的scFv。实验表明，与利用BB scFv及April来源的BCMA结合域所构建的CAR-T细胞相比，本发明构建的CAR-T细胞具有更高的杀伤效果和肿瘤清除能力。本发明还利用S scFv和CD19scFv构建的双CAR-T细胞，可以同时杀伤BCMA和CD19阳性的CAR-T细胞。
具体地，本发明利用不同BCMA抗体的scFv构建CAR-T细胞，并对其进行对比，意外发现S来源的scFv较BB的scFv及April来源的BCMA结合域所构建的CAR-T细胞，具有更高的杀伤BCMA过表达细胞及BCMA阳性的肿瘤靶细胞的能力。在体内小鼠动物模型中也表现出较BB来源的CAR-T更高的清除肿瘤能力。利用另外一些本领域常见的靶向BCMA的scFv构建的CAR-T细胞没有表现出理想的体外和体内功能。
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。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、 Gly(G)、His(H)、I1e(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
B细胞成熟抗原(B cell maturation antigen,BCMA)
BCMA是一种跨膜蛋白，表达于成熟的B淋巴细胞表面，即浆母细胞及浆细胞表面。而多发性骨髓瘤正是由于浆细胞不正常的增生并侵犯骨髓导致。研究表明，BCMA表达于多发性骨髓瘤细胞上。靶向BCMA的Car-T细胞经证实能够特异性杀死骨髓瘤细胞。但是一些患者接受靶向BCMA的CAR-T细胞治疗后，依然会有复发的过程。针对这些再复发的病人，就需要再找到一个有别于BCMA的靶点，才能继续治疗。
CD19
CD19分子是B细胞表面的跨膜蛋白，它与B细胞活化、信号传导及生长调节密切相关。如图1所示，CD19几乎表达于所有B细胞的表面，靶向CD19的CAR-T细胞目前在白血病及淋巴瘤的治疗中效果显著。一般认为99.95％的浆细胞表面是不表达CD19的，因此都忽略了CD19用于治疗多发性骨髓瘤的可能性。
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。
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BCMA(或BCMA和CD19)的抗原结合结构域。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。
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，所述抗原结合结构域包含特异性识别BCMA的抗体，任选地，所述抗原结合结构域还包含特异性识别 CD19的抗体，较佳地为单链抗体。
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。
本发明的CAR中的胞内结构域包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
优选地，本发明的CAR中还包括有细胞自杀元件。
优选地，本发明的靶向BCMA的scFv为S scFv，实施例中的BB scFv以及April链用作对照。BB scFv以及April链均为本领域较为常用的靶向BCMA的结合序列，BB scFv在PCT申请WO 2010104949 A3中有记载，April链在CN105658671A中有记载。
靶向CD19和BCMA的双特异性CAR
多发性骨髓瘤(MM)是一种恶性浆细胞肿瘤，其肿瘤细胞起源于骨髓中的浆细胞，而浆细胞是B淋巴细胞发育到最终功能阶段的细胞。多发性骨髓瘤基本是一个无法治愈的疾病，具有高发病率及高致死率的特征。在2017年的统计数据中，美国有3万例新诊断出来的多发性骨髓瘤患者，而可能12000例将面临死亡。目前多发性骨髓瘤常见疗法有细胞毒药物治疗，蛋白酶抑制剂(硼替佐米等)、来那度胺、单克隆抗体及皮质类固醇等。但是都是部分有效，不能持久缓解，复发的几率很大。因此，多发性骨髓瘤疗法的改进就显得尤为重要。
CD19是分子量95kDa的糖蛋白,表达于前B细胞和成熟B细胞膜表面,与B细胞Ca++的跨膜传导通路密切相关,对B细胞的增殖和分化具有调节作用。CD19主要表达在正常B细胞和癌变B细胞中，组织表达特异性较高，是一个很好的抗体或CAR-T免疫治疗靶点。但在免疫治疗过程中，经常会出现B细胞的CD19表位丢失情况，造成病人对免疫治疗无反应或者复发。
双特异性是指同一个CAR可以特异结合、免疫识别两个不同的抗原，CAR结合任意一个抗原都能产生免疫反应。
在另一优选例中，所述靶向CD19和BCMA的双特异性CAR如本发明第二方面所述。
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CD19和BCMA的抗原结合结构域，包括抗CD19的scFv和抗BCMA的scFv。
在另一优选例中，本发明提供一个针对CD19和BCMA抗原的双特异性嵌合抗原受体。同时靶向CD19和BCMA的CAR结构组分可以包括信号肽，抗CD19的scFv，抗BCMA的scFv，铰链区，跨膜区，和胞内T细胞信号区，其中CD19scFv和BCMAscFv通过一个短的肽段(G4S)xN相连。同时靶向CD19和BCMA的CAR结构如本发明第二方面所述。
在另一优选例中，本发明的CD19和BCMA双特异性的CAR为单一结构，包含抗CD19和BCMA的scFv。其中CAR包含CD19 scFv和BCMA scFv，CD19 scFv和BCMA scFv的排序和铰链是其功能的主要影响因素。
在另一优选例中，本发明对所述BCMA scFv的序列进行了优化，所述BCMA scFv(S scFv) 与BCMA的亲和力高，特异性好，能特异性靶向BCMA全长抗原及胞外区域。
在本发明的一个优选实施方式中，使用(G4S)x3连接CD19scFv和BCMAscFv，此时CAR的活性和杀伤力最佳。
本发明使用双特异靶向CD19和BCMA的CAR，与靶向单抗原的CAR相比，亲和力显著增强，免疫细胞的活性显著增加，具有协同效应。此外，由于CD19和BCMA在肿瘤细胞中的表达水平不均一，双靶向CAR-T治疗范围更广泛。同时靶向CD19和BCMA的CAR-免疫细胞可以减少因单一表面抗原下调或者缺失造成的抗原逃逸的可能性。另外，CD19和BCMA的双特异性CAR-T具有显著优于单CAR-T的抑制骨髓瘤患者骨髓中的CD34阴性单核细胞的体外克隆形成的能力，说明其具有显著优于单CAR-T细胞的抑制肿瘤祖细胞的能力。最后，CD19抗原的加入可以增加CD19和BCMA的双特异性CAR-T的持续存活能力。
嵌合抗原受体T细胞(CAR-T细胞)
如本文所用，术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”包括本发明第三方面中包含的CAR-T细胞。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤抗原，针对某一种肿瘤抗原的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质抗原，又可利用糖脂类非蛋白质抗原，扩大了肿瘤抗原的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
嵌合抗原受体NK细胞(CAR-NK细胞)
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明第三方面中包含的CAR-NK细胞。本发明CAR-NK细胞可用于治疗BCMA高表达的肿瘤，如多发性骨髓瘤等。
自然杀伤(NK)细胞是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与自体CAR-T细胞相比，CAR-NK细胞还具有一下优点，例如：(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞，而对机体正常的细胞没有杀伤作用；(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险；(3)体外极易扩增及发展为“现成的”产品。除此之外，与CAR-T细胞治疗类似。
自杀基因开关
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CART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。
本发明中所用自杀开关可以为单纯疱疹病毒胸苷激酶(the herpes symplex virus thymidine kinase，HSV-TK)、可诱导的半胱氨酸天冬氨酸蛋白酶9(inducible caspase9,iCasp9)、CD20、突变型人胸苷酸激酶(mutated human thymidylate kinase，mTMPK) 等。比较而言，HSV-TK、iCasp9和CD20对CAR-细胞的清除能力等同，但是iCasp9和CD20的清除较迅速，HSV-TK清除速度较慢。
iCasp9自杀开关包含FKBP12-F36V结构域，可通过柔性接头连接半胱氨酸天冬氨酸蛋白酶9，后者不含募集结构域。FKBP12-F36V包含一个FKBP结构域，在第36个氨基酸残基位点上苯丙氨酸替代了缬氨酸。它具有高选择性和亚纳摩尔亲和力，能够结合二聚合成配基，如其他惰性小分子AP1903。当加入小分子后，能够促使其二聚话，从而诱导细胞的凋亡，而对未携带自杀开关的正常细胞无效用。
诱导安全开关caspase9(iCasp9)使用人的caspase9融合FK506结合蛋白(FKBP)，使其可以用化学诱导剂(AP1903/Rimiducid，Bellicum Pharmaceutical)诱导形成二聚体，导致表达融合蛋白的细胞凋亡。
CD19和BCMA虽然在肿瘤细胞中高表达，在正常B细胞也有表达，本发明工程化的免疫细胞在体内会攻击正常B细胞。
如何控制CAR-细胞的安全性一直都是急需解决的问题。在CAR-细胞上加入安全开关，是用于终止CAR-细胞活性最安全的方式。在CAR-细胞产生严重毒性(CRS/神经毒性)或者在病人达到长期持续缓解后，可诱导的iCasp9安全开关控制CAR-细胞清除。
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一方面所述的CAR-T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BCMA和/或CD19，协同激活T细胞，引起T细胞免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
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，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。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BCMA和/或CD19的CAR-T细胞引起抗表达BCMA和/或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抗-BCMA和/或CD19scFv、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：
(a)本发明构建的包含S scFv的CAR-T细胞具有比BB和April CAR-T更高的体内、体外肿瘤杀伤及功能活性。
(b)本发明构建的双特异性的CAR-T可以同时识别包括BCMA在内的两个或以上靶点。
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，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。
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(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BCMA的细胞系MM.1s和RPMI8226,MM.1s-ffluc细胞、RPMI8226-ffluc细胞，单表达BCMA、CD19和同时表达BCMA/CD19的Hela细胞，以上细胞均使用RPMI 1640培养基培养；293T(人肾上皮细胞系细胞，
Figure PCTCN2020088836-appb-000017
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BCMA、CD19和同时表达BCMA/CD19的Hela细胞是通过慢病毒载体将BCMA和CD19抗原转入后获得的稳转细胞系，能够特异性的表达BCMA或/和CD19蛋白分子，MM.1s-ffluc细胞和RPMI8226-ffluc细胞是使用firefly luciferase的慢病毒感染后筛选得到的稳转细胞系。
实施例3 CAR结构设计与转导
设计构建靶向BCMA的单CAR以及同时靶向BCMA及CD19的双CAR，结构示意如图1所示。其中，CAR、CD19CAR及自杀开关–EGFRt元件通过2A肽连接。具体地，本发明中涉及的CAR结构如图1所示，命名及组成如表1所示。
表1 CAR的结构
结构命名 结构组成 CAR-T名称
S1 单个S scFv CAR-S1
S2 平行的CD19 CAR+S CAR(双CAR) CAR-S2
S3 平行的S CAR+CD19 CAR(双CAR) CAR-S3
S4 Loop结构的CD19 scFv+S scFv CAR-S4
S5 串联的CD19 scFv+S scFv CAR-S5
BB 单个BB scFv CAR-BB
April 单个April链 CAR-April
19 单个CD19 scFv CAR-19
S6 平行的CD19 CAR+S CAR+EGFRt CAR-S6
S7 平行的S CAR+CD19 CAR+EGFRt CAR-S7
图1及表1所述的CAR中涉及的各元件的具体序列如下所示：
S scFv(S scFv)重链
Figure PCTCN2020088836-appb-000018
S scFv(S scFv)轻链
Figure PCTCN2020088836-appb-000019
BB scFv重链
Figure PCTCN2020088836-appb-000020
BB scFv轻链
Figure PCTCN2020088836-appb-000021
April链
Figure PCTCN2020088836-appb-000022
CD8信号肽
Figure PCTCN2020088836-appb-000023
(G4S)3链接肽
Figure PCTCN2020088836-appb-000024
(G4S)5链接肽
Figure PCTCN2020088836-appb-000025
218链接肽
Figure PCTCN2020088836-appb-000026
CD8铰链区
Figure PCTCN2020088836-appb-000027
或KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKP(SEQ ID NO:37)
或SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:38)
CD8跨膜区
Figure PCTCN2020088836-appb-000028
CD28跨膜区
Figure PCTCN2020088836-appb-000029
41BB信号区
Figure PCTCN2020088836-appb-000030
CD28信号区
Figure PCTCN2020088836-appb-000031
CD3z信号区
Figure PCTCN2020088836-appb-000032
或
Figure PCTCN2020088836-appb-000033
2A肽
Figure PCTCN2020088836-appb-000034
FMC63 scFv(CD19 scFv)重链
Figure PCTCN2020088836-appb-000035
FMC63 scFv(CD19 scFv)轻链
Figure PCTCN2020088836-appb-000036
GM-CSF信号肽
Figure PCTCN2020088836-appb-000037
EGFRt序列
Figure PCTCN2020088836-appb-000038
将表1中的各CAR基因克隆至FUW慢病毒载体骨架中，构建可用于感染T细胞的完整慢病毒表达载体。具体地，以BCMA CAR基因为例，进行具体说明，将BCMA CAR基因置于EF1α(EF-1α)的启动子下，形成Fuw-EF1α-BCMA CAR，将Fuw-EF1α-BCMA CAR、慢病毒包膜质粒pMD2.G(Addgene，Plasmid #12259)和慢病毒包装质粒psPAX2(Addgene，Plasmid #12260)三个质粒使用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，超速离心进行浓缩；浓缩后的病毒即可用于感染T细胞。
流式细胞分析的结果显示，构建的CAR基因能够制备出表达BCMA CAR的慢病毒载体。
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，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
感染第3天后及冻存前取样使用BCMA原检测细胞数量及BCMA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。
结果显示，利用实施例3中构建的慢病毒载体，成功构建了各CAR-T细胞，命名参见表1。
具体地，BCMA CAR-T细胞的构建结果如图2所示，病毒转染后的CAR-BB和CAR-S1 CAR-T细胞均能检测到BCMA CAR的表达，CAR表达可达50％以上。
实施例5 细胞体外杀伤
对实施例4获得CAR-S1 CAR-T细胞、CAR-BB CAR-T细胞和CAR-April CAR-T细胞进行体外杀伤实验。使用RTCA法测试CAR-T细胞对靶细胞过表达BCMA的Hela细胞系的杀伤。
结果如图3所示(RTCA法测试)，NT对照组(未转染T细胞对照组)和培养基对照组(空白对照组)对Hela-BCMA细胞没有杀伤，而CAR-S1细胞可以发挥BCMA特异性的杀伤功能，且CAR-S1细胞在杀伤BCMA阳性的Hela-BCMA细胞方面表现出优于CAR-BB细胞的结果。
使用荧光素酶标记的肿瘤靶细胞进行杀伤能力的检测。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MM.1s-Luc及RPMI8226-Luc。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，即可得到各CAR-T细胞的杀伤效应。
图4显示了靶细胞表面的抗原表达情况。图5显示相同E:T比例下，NT细胞未见杀伤功能，CAR-S1细胞对MM.1S-Luc细胞(转入荧光素酶基因的MM.1S细胞)和RPMI8226-Luc细胞(转入荧光素酶基因的RPMI8226细胞)具有剂量依赖的杀伤作用，而且CAR-S1细胞表现出优于CAR-BB和CAR-April的杀伤能力。
此外，申请人还利用本领域常见的多种靶向BCMA scFv构建了CAR-T细胞，经测试，这些CAR-T细胞均没有表现出理想的杀伤功能。
综上，CAR-T细胞与靶细胞(BCMA过表达的细胞，以及BCMA阳性的肿瘤细胞，MM.1s-Luc和RPMI8226细胞)共培养后，靶细胞可被靶向BCMA的CAR-T细胞裂解，而且CAR-S1表现出较CAR-BB更高的杀伤能力。另外一些本领域常见的靶向BCMA的scFv构建的CAR-T细胞没有表现出理想的杀伤功能。
实施例6 细胞因子释放检测
对实施例4获得靶向BCMA的CAR T细胞(CAR-S1 CAR-T细胞和CAR-BB CAR-T细胞)与肿瘤细胞(Hela、Hela-BCMA、Hela-CD19、Hela-BCMA-CD19)混合，置于RPMI培养基中，各细胞密度配制为1X10 4个/ml，CAR-T细胞与肿瘤细胞各100ul，置于96孔板中，共培养过夜，收集上清，离心后取上清检测细胞因子IFN-γ等的释放水平。采用Elisa试剂盒进行检测。
结果如图6所示，CAR-S1被Hela-BCMA靶细胞共刺激后，细胞因子INF-γ的分泌显著高于CAR-BB，而NT和Medium组未见明显分泌。
实施例7 体内药效研究
选取6-12周大的NOG小鼠，皮下注射1×10 7RPMI8226细胞。两天后检测肿瘤移植物的负荷，10天后分组，分组后一天分别注射CAR-S1 CAR-T细胞和CAR-BB CAR-T细胞，CAR-T处理后第每周两次评估小鼠肿瘤体积负荷。
结果如图7所示，相比对照组，注射CAR-S1细胞的小鼠肿瘤负荷显著受到抑制，并具有略高于CAR-BB的抗肿瘤效果。
实施例8 双CAR-T细胞的制备
实验方法如下：
本实施例中涉及同时靶向BCMA和CD19的CAR-T细胞，CAR结构示意如图1所示(CAR S2、CAR S3、CAR S4、CAR S5、CAR S6和CAR S7)。其中，BCMA CAR、CD19CAR及自杀开关–EGFRt元件通过2A肽连接。其中BCMA CAR结构中scFv为S、BB的scFv重链及轻链组成，其中S scFv由SEQ ID NO:9和SEQ ID NO:10组成；BB scFv由SEQ ID NO:13和SEQ ID NO:14组成；CD19scFv由SEQ ID NO:11和SEQ ID NO:12组成；另外，scFv可以由April部分序列(SEQ ID NO:15)组成的BCMA结合区替换组成新的CAR结构。
将BCMA-CD19CAR基因克隆至载体骨架中，置于EF1α(EF-1α)的启动子下，形成EF1α-BCMA-CD19-EGFRt CAR，将EF1α-BCMA-CD19-EGFRt CAR、慢病毒包膜质粒使用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；浓缩后的病毒即可用于感染T细胞。
慢病毒感染：分离纯化的原代T细胞在激活2天后，利用如上构建的慢病毒，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。
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BCMA/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。
结果显示，利用BCMA-CD19 CAR慢病毒载体，成功构建了BCMA-CD19 CAR-T细胞，具体如图1和表1中所示。
结果如图8显示，病毒转染后的T细胞表面能够同时使用BCMA抗原和CD19抗原检测到BCMA CAR及CD19 CAR的表达。
图9显示CAR-S6及CAR-S7细胞表面可以同时检测到BCMA CAR、CD19 CAR及EGFRt的表达。
实施例9 细胞体外杀伤
对实施例8获得的CAR-T细胞进行体外杀伤实验。使用过表达BCMA和CD19的Hela过表达细胞系进行RTCA或者使用荧光素酶标记的肿瘤靶细胞进行检测。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(RPMI8226、MM.1s和Nalm6)。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。
结果显示，将CAR-T细胞与各靶细胞(CD19/BCMA双阳、CD19单阳、BCMA单阳)共培养 后，靶细胞都会裂解，表明BCMA-CD19 CAR-T对CD19/BCMA双阳、CD19单阳、BCMA单阳的细胞都有杀伤作用。
具体结果如图10显示，双特异CAR-T对单阳CD19阳性靶细胞(Hela-CD19)或单阳的BCMA阳性靶细胞(Hela-BCMA)均有显著杀伤，并对CD19和BCMA双阳的靶细胞Hela-BCMA-CD19也有显著杀伤。说明BCMA与CD19组合的双特异性CAR-T细胞对单靶和双靶细胞都有杀伤作用。而单CAR-T(CAR-19或CAR-S1)只能对一种靶抗原发挥杀伤作用。
图11显示，双CAR-T可以显著对BCMA单阳肿瘤靶细胞MM.1s和RPMI8226产生显著杀伤。并对CD19阳性肿瘤靶细胞Raji和Nalm6有显著杀伤。说明BCMA与CD19组合的双CAR对BCMA和CD19阳性的肿瘤靶细胞都有杀伤作用。
实施例10 细胞因子释放检测
将BCMA-CD19 CAR-T细胞(实施例8获得)与肿瘤细胞(Hela-BCMA)混合，置于RPMI培养基中，各细胞密度配制为1X10 4个/ml，CAR-T细胞与肿瘤细胞各100ul，置于96孔板中，共培养过夜，收集上清，离心后取上清检测细胞因子释放水平，采用CBA方法进行检测。
结果如图12所示，BCMA-CD19 CAR-T被BCM阳性的靶细胞刺激后能大量分泌细胞因子，NT只少量分泌细胞因子分泌。表明BCMA-CD19 CAR-T能被BCMA激活。
实施例11 CD107接受刺激后的上调
对实施例8获得的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BCMA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、CD8及CD107a后，进行流式分析。
结果如图13所示，双CAR-T细胞和BCMA阳性肿瘤靶细胞MM.1s和、CD19阳性Raji共培养后，CAR-T细胞表面的CD107a分子被显著上调。
实施例12 体内药效研究
选取6-12周大的NOG小鼠，皮下注射1×10 7RPMI8226细胞。两天后检测肿瘤移植物的负荷，10天后分成肿瘤负荷相当的组，分组后一天分别注射CAR-T细胞，CAR-T处理后每周两次评估小鼠肿瘤体积负荷。
图14结果表明，CAR-S2和CAR-S4可以消除RPMI8226细胞皮下造模小鼠的肿瘤，表明其显著的抗肿瘤功效。
同时，选取6-12周大的NOG小鼠，静脉注射1×10 7MM.1s细胞。检测肿瘤移植物的负荷，并按照肿瘤负荷平均分组，分组后一天分别注射CAR-T细胞，CAR-T处理后评估小鼠肿瘤负荷，每只小鼠腹腔注射3mg d-luciferin(Perkin Elmer Life Sciences)，四分钟后使用Xenogen IVIS Imaging System(Perkin Elmer Life Sciences)拍照，曝光30s。生物发光的信号按照发出的光子量计算，光子量使用曝光时间、表面积归一化，最后得出光子量/s/cm 3/球面角度(p/s/cm 2/sr)。
图15结果表明，相比对照组，注射双CAR-T细胞的小鼠肿瘤负荷显著减小直到消失，表明BCMA-CD19 CAR-T细胞具有显著的抗肿瘤效果。
实施例13 肿瘤形成细胞的杀伤研究
骨髓瘤(MM)患者复发是临床常见现象，临床表现一般为大多数的肿瘤细胞可以被清除，而导致肿瘤复发的具有克隆增殖能力的肿瘤细胞往往具有相对更高的抗药性。为了考察CAR-T细胞对具有克隆增值能力的肿瘤细胞的杀伤能力，并比较双特异性CAR-T与单CAR-T细胞的优势，本研究建立了骨髓瘤克隆形成实验的方法，并考察CAR-T对克隆形成的抑制能力。
具有增值能力的MM肿瘤细胞会在克隆增值培养基中生长，但实验过程中需要去除具有增值能力的CD34+的造血干细胞对实验的干扰，收获到以肿瘤增值细胞为主的细胞，为本实验需要控制的重要问题。
具体实验方法如下：
第一步，使用Ficoll分离提取骨髓单个核细胞，并进行流式表型分析。第二步，使用CD34+细胞分选试剂盒，去除CD34+细胞。第三步，获得的细胞使用不同组别的CAR-T细胞(双CAR-T、单CAR-T)进行杀伤实验，杀伤结束后，使用T细胞去除分选试剂盒，去除掉CAR-T细胞。第。第四步，使用半固体克隆增值培养基进行克隆生长，1周-2周后进行统计、计数、结果汇总。
结果如图16所示，CAR-S2、CAR-S4具有相对于CAR-19和CAR-S1更显著的杀伤克隆形成细胞或肿瘤细胞前体细胞优势，说明其具有相对单CAR更高的抑制骨髓瘤细胞克隆形成能力的作用。
实施例14 Nalm6体内静脉造模实验
选取6-12周大的NOG小鼠，静脉注射1×10 7Nalm6细胞。6天后检测肿瘤移植物的负荷，并按照肿瘤负荷平均分组，分组后一天分别注射CAR-T细胞，CAR-T处理后评估小鼠肿瘤负荷，每只小鼠腹腔注射3mg d-luciferin(Perkin Elmer Life Sciences)，四分钟后使用Xenogen IVIS Imaging System(Perkin Elmer Life Sciences)拍照，曝光30s。生物发光的信号按照发出的光子量计算，光子量使用曝光时间、表面积归一化，最后得出光子量/s/cm 2/球面角度(p/s/cm 2/sr)。
结果如图17所示，注射CAR-S2、CAR-S4的小鼠肿瘤负荷显著减小直到消失，BCMA-CD19CAR-T细胞相对于CAR-19具有更显著的抗CD19阳性肿瘤的效果。
实施例15 CAR-T细胞的安全开关实验
含有EGFRt元件的CAR-T使用EGFR抗体染色后流式细胞术分析，并同时对CAR表达进行分析。
结果如图18所示，CAR-T细胞中检测到安全开关的表达。
实施例16 人源化CAR-T细胞的制备和杀伤作用检测
采用实施例3和4的方法构建人源化CAR-T细胞(CAR-h19)和人源化双CAR-T细胞(CAR-hS2、CAR-hS4)，人源化CAR-T细胞的结构与CAR-19类似，人源化双CAR-T细胞CAR-hS2的结构与CAR-S2类似，CAR-hS4的结构与CAR-S4类似，其区别仅在于使用人源化CD19 scFv替换原结构中的鼠源scFv。人源化CD19 scFv包括SEQ ID NO:21-30中任 一所示的抗体重链可变区，和SEQ ID NO:31-36中任一所示的抗体轻链可变区。
采用实施例9的方法检测人源化双CAR-T细胞的体外杀伤作用。
体外杀伤结果如图19和图20所示，人源化CAR-T细胞和人源化双CAR-T细胞对靶细胞表现出显著的杀伤效果，而不会杀伤非靶细胞。
实施例17 人源化CAR-T细胞的体内药效研究
选取6-12周大的NOG小鼠，皮下注射3×10 5Raji细胞。6天后检测肿瘤移植物的负荷，分成肿瘤负荷相当的组别，分组后一天分别注射上述制备的双CAR-T细胞，CAR-T处理后评估小鼠肿瘤体积负荷。每只小鼠腹腔注射3mg d-luciferin(Perkin Elmer Life Sciences)，四分钟后使用Xenogen IVIS Imaging System(Perkin Elmer Life Sciences)拍照，曝光30s。生物发光的信号按照发出的光子量计算，光子量使用曝光时间、表面积归一化，最后得出光子量/s/cm 2/球面角度(p/s/cm 2/sr)。
结果如图21所示，人源化CAR-hS2细胞比CAR-S2具有更强的消除Raji细胞造模小鼠的肿瘤能力，表明其显著的抗肿瘤功效。
实施例18 CAR-T细胞对Raji淋巴瘤细胞的杀伤情况
使用荧光素酶标记的Raji淋巴瘤靶细胞进行杀伤能力的检测。通过将荧光素酶基因转入Raji靶细胞，克隆筛选后获得稳转细胞株Raji-Luc。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，即可得到各CAR-T细胞的杀伤效应。
结果如图22所示，NT细胞未见杀伤功能，CAR-S1细胞对Raji-Luc细胞(转入荧光素酶基因的Raji细胞)具有剂量依赖的杀伤作用，说明其对淋巴瘤适应症的潜在应用价值。
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 (13)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述CAR的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区。
  2. 如权利要求1所述的CAR，其特征在于，所述scFv如下式A或式B所示：
    V H-V L,(A)；V L-V H,(B)
    式中，V H为所述抗体重链可变区；V L为所述抗体轻链可变区；“-”为连接肽或肽键。
  3. 一种双特异性CAR，其特征在于，所述双特异性CAR靶向BCMA和第一靶点，
    其中，所述双特异性CAR中的靶向BCMA的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区。
    并且，所述的第一靶点选自下组：
    CD138、Kappa Light Chain、NKG2D-ligands、TACI、GPRC5D、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、Frizzled、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、Folate receptorα、Folate receptorβ、GPC2、CD70、BAFF-R、TROP-2、或其组合。
  4. 如权利要求3所述的双特异性CAR，其特征在于，所述的第一靶点为CD19，并且所述双特异性CAR中的靶向CD19的抗原结合结构域(scFv)包括SEQ ID NO:11、21-30中任一所示的抗体重链可变区，和SEQ ID NO:12、31-36中任一所示的抗体轻链可变区。
  5. 如权利要求3所述的双特异性CAR，其特征在于，所述双特异性CAR的结构如下式II所示：
    L-scFv1-I-scFv2-H-TM-C-CD3ζ  (II)
    式中，
    各“-”独立地为连接肽或肽键；
    L为无或信号肽序列；
    I为柔性接头；
    H为无或铰链区；
    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第一靶点的抗原结合结构域，另一个为靶向BCMA的抗原结合结构域。
  6. 如权利要求3所述的双特异性CAR，其特征在于，所述双特异性CAR的结构如下式III或III’所示：
    L-V L3-scFv3-V H3-H-TM-C-CD3ζ  (III)
    L-V H3-scFv3-V L3-H1-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；
    scFv3为靶向BCMA的抗原结合结构域，V H3为抗所述第一靶点抗体重链可变区，且V L3为抗所述第一靶点抗体轻链可变区；
    或者scFv3为靶向所述第一靶点的抗原结合结构域，V H3为抗BCMA抗体重链可变区，且V L3为抗BCMA抗体轻链可变区。
  7. 一种核酸分子，所述核酸分子编码权利要求1所述的CAR或权利要求3所述的双特异性CAR。
  8. 一种载体，所述的载体含有权利要求7所述的核酸分子。
  9. 一种工程化的免疫细胞，所述的免疫细胞含有权利要求8所述的载体、或染色体中整合有外源的权利要求7所述的核酸分子、或表达权利要求1所述的CAR或权利要求3所述的双特异性CAR。
  10. 一种工程化的免疫细胞，所述免疫细胞含有外源的第一表达盒和第二表达盒，其中所述第一表达盒用于表达靶向第一靶点的第一CAR，所述第二表达盒用于表达靶向BCMA的第二CAR；
    或所述免疫细胞表达所述靶向第一靶点的第一CAR和所述靶向BCMA的第二CAR；
    其中，所述第二CAR中靶向BCMA的抗原结合结构域(scFv)包括SEQ ID NO:9所示的抗体重链可变区，和SEQ ID NO:10所示的抗体轻链可变区；
    并且，所述的第一靶点选自下组：
    CD138、Kappa Light Chain、NKG2D-ligands、TACI、GPRC5D、CD2、CD3、CD4、CD5、CD7、CD8、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD38、CD40、CD44V6、CD47、CD52、CD56、CD57、CD58、CD79b、CD80、CD86、CD81、CD123、CD133、CD137、CD151、CD171、CD276、CLL1、B7H4、BCMA、VEGFR-2、EGFR、GPC3、PMSA、CEACAM6、c-Met、EGFRvIII、ErbB2/HER2、ErbB3、HER-2、HER3、ErbB4/HER-4、EphA2、IGF1R、GD2、O-acetyl GD2、O-acetyl GD3、GHRHR、GHR、Flt1、KDR、Flt4、Flt3、CEA、CA125、CTLA-4、GITR、BTLA、TGFBR1、TGFBR2、TGFBR1、IL6R、gp130、Lewis、TNFR1、TNFR2、PD1、PD-L1、PD-L2、PSCA、HVEM、MAGE-A、MSLN、NY-ESO-1、PSMA、RANK、RORl、TNFRSF4、TWEAK-R、LTPR、 LIFRP、LRP5、MUC1、MUC16、TCRα、TCRβ、TLR7、TLR9、PTCH1、WT-1、Robol、Frizzled、OX40、Notch-1-4、APRIL、CS1、MAGE3、Claudin 18.2、Folate receptorα、Folate receptorβ、GPC2、CD70、BAFF-R、TROP-2、或其组合。11.一种制剂，所述制剂含有权利要求1所述的CAR、权利要求3所述的双特异性CAR、或权利要求9或10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。
  11. 一种权利要求1所述的CAR、权利要求3所述的双特异性CAR、或权利要求9或10所述的工程化的免疫细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
  12. 如权利要求12所述的用途，其特征在于，所述的药物或制剂通过杀伤具有克隆增值能力的肿瘤细胞治疗癌症或肿瘤。
  13. 如权利要求13所述的用途，其特征在于，所述的具有克隆增值能力的肿瘤细胞包括克隆形成细胞、肿瘤细胞前体细胞、肿瘤祖细胞。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021223720A1 (zh) * 2020-05-06 2021-11-11 亘喜生物科技（上海）有限公司 人源化cd19抗体及其应用
CN114317607A (zh) * 2021-12-31 2022-04-12 西安桑尼赛尔生物医药有限公司 融合一代靶向cd7 car和二代靶向bcma的双靶点通用car-t细胞及制备方法
CN116444669A (zh) * 2023-04-04 2023-07-18 上海科棋药业科技有限公司 靶向bcma car-t细胞人源化抗体

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10086055B2 (en) * 2016-06-01 2018-10-02 Promab Biotechnologies, Inc. Adherent cancer cell line expressing a hematological tumor antigen
WO2023200796A1 (en) * 2022-04-13 2023-10-19 The Regents Of The University Of California Immune cell inhibition by immune checkpoint engagers
WO2023230581A1 (en) * 2022-05-25 2023-11-30 Celgene Corporation Methods of manufacturing t cell therapies
CN116063556B (zh) * 2022-08-18 2023-06-16 再少年(北京)生物科技有限公司 Ips来源的car-nk细胞及其治疗癌症的用途
CN116814553A (zh) * 2023-03-30 2023-09-29 广州百吉生物制药有限公司 杀伤和存续能力增强型car-t细胞及其制备和应用

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
WO2001096584A2 (en) 2000-06-12 2001-12-20 Akkadix Corporation Materials and methods for the control of nematodes
WO2010104949A2 (en) 2009-03-10 2010-09-16 Biogen Idec Ma Inc. Anti-bcma antibodies
CN105142677A (zh) * 2013-02-15 2015-12-09 加利福尼亚大学董事会 嵌合抗原受体及其使用方法
CN105658671A (zh) 2013-10-10 2016-06-08 Ucl商务股份有限公司 嵌合抗原受体
WO2018151817A2 (en) * 2017-02-17 2018-08-23 Unum Therapeutics Inc. Co-use of anti-bcma antibody and antibody-coupled t celll receptor (actr) in cancer therapy and b cell disorders
CN108778329A (zh) * 2016-02-17 2018-11-09 西雅图基因公司 Bcma抗体和其用以治疗癌症和免疫病症的用途

Family Cites Families (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL104570A0 (en) 1992-03-18 1993-05-13 Yeda Res & Dev Chimeric genes and cells transformed therewith
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
CZ121599A3 (cs) 1998-04-09 1999-10-13 Aventis Pharma Deutschland Gmbh Jednořetězcová molekula vázající několik antigenů, způsob její přípravy a léčivo obsahující tuto molekulu
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
US20130266551A1 (en) 2003-11-05 2013-10-10 St. Jude Children's Research Hospital, Inc. Chimeric receptors with 4-1bb stimulatory signaling domain
US20090155275A1 (en) 2007-07-31 2009-06-18 Medimmune, Llc Multispecific epitope binding proteins and uses thereof
US20100260668A1 (en) 2008-04-29 2010-10-14 Abbott Laboratories Dual Variable Domain Immunoglobulins and Uses Thereof
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
CN106220739A (zh) * 2010-12-09 2016-12-14 宾夕法尼亚大学董事会 嵌合抗原受体‑修饰的t细胞治疗癌症的用途
AU2013221672B2 (en) 2012-02-13 2017-11-09 Seattle Children's Hospital D/B/A Seattle Children's Research Institute Bispecific chimeric antigen receptors and therapeutic uses thereof
US9765342B2 (en) 2012-04-11 2017-09-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting B-cell maturation antigen
US20130280220A1 (en) 2012-04-20 2013-10-24 Nabil Ahmed Chimeric antigen receptor for bispecific activation and targeting of t lymphocytes
AU2014236769B2 (en) 2013-03-15 2018-09-27 Amgen Inc. Heterodimeric bispecific antibodies
US10519251B2 (en) 2013-12-30 2019-12-31 Epimab Biotherapeutics, Inc. Fabs-in-tandem immunoglobulin and uses thereof
ES2767423T3 (es) * 2014-01-13 2020-06-17 Hope City Receptores de antígenos quiméricos (CARS) con mutaciones en la región del espaciador Fc y métodos para su uso
WO2015187528A1 (en) 2014-06-02 2015-12-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting cd-19
US10174095B2 (en) * 2014-07-21 2019-01-08 Novartis Ag Nucleic acid encoding a humanized anti-BCMA chimeric antigen receptor
RU2018119706A (ru) 2015-10-30 2019-12-02 Глэксосмитклайн Интеллекчуал Проперти Дивелопмент Лимитед Способ прогноза
MA43955B1 (fr) 2016-02-03 2022-02-28 Amgen Inc Anticorps anti-bcma et anti-cd3 bispécifiques de format bite
BR112018067679A2 (pt) 2016-03-04 2019-01-15 Novartis Ag células que expressam múltiplas moléculas do receptor de antígeno quimérico (car) e seu uso
US20190119636A1 (en) 2017-10-23 2019-04-25 Poseida Therapeutics, Inc. Modified stem cell memory t cells, methods of making and methods of using same
US10550183B2 (en) 2016-11-22 2020-02-04 National University Of Singapore Blockade of CD7 expression and chimeric antigen receptors for immunotherapy of T-cell malignancies
BR112019011207A2 (pt) 2016-12-05 2019-10-08 Juno Therapeutics Inc produção de células modificadas para terapia celular adotiva
CN108395478A (zh) * 2017-02-04 2018-08-14 上海恒润达生生物科技有限公司 靶向bcma的嵌合抗原受体并对其双重修饰的方法及其用途
EP3641768A4 (en) 2017-06-21 2021-07-07 iCell Gene Therapeutics LLC CHIMERIC ANTIGEN RECEPTORS (CARS); COMPOSITIONS AND METHOD OF USE THEREOF
CN109593721B (zh) * 2017-09-30 2022-11-01 亘喜生物科技（上海）有限公司 具有自杀基因开关的靶向人间皮素的工程化免疫细胞
JP2021525524A (ja) 2018-05-31 2021-09-27 ワシントン・ユニバーシティＷａｓｈｉｎｇｔｏｎ Ｕｎｉｖｅｒｓｉｔｙ がんを治療するためのキメラ抗原受容体ｔ細胞（ｃａｒ−ｔ）
JP2021533746A (ja) 2018-08-09 2021-12-09 ジュノー セラピューティクス インコーポレイテッド 操作された細胞およびその組成物を生成するための方法
CA3109959A1 (en) 2018-08-31 2020-03-05 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
SG11202104524YA (en) 2018-11-01 2021-05-28 Gracell Biotechnologies Shanghai Co Ltd Compositions and methods for t cell engineering
CN109485734B (zh) * 2018-12-30 2020-05-12 广州百暨基因科技有限公司 一种靶向bcma和cd19的双特异性嵌合抗原受体及其应用
US20220168389A1 (en) 2019-04-12 2022-06-02 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
CN113784733A (zh) * 2019-05-07 2021-12-10 亘喜生物科技（上海）有限公司 靶向bcma的工程化免疫细胞及其用途
CN114761037A (zh) 2019-11-26 2022-07-15 诺华股份有限公司 结合bcma和cd19的嵌合抗原受体及其用途

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
WO2001096584A2 (en) 2000-06-12 2001-12-20 Akkadix Corporation Materials and methods for the control of nematodes
WO2010104949A2 (en) 2009-03-10 2010-09-16 Biogen Idec Ma Inc. Anti-bcma antibodies
CN105142677A (zh) * 2013-02-15 2015-12-09 加利福尼亚大学董事会 嵌合抗原受体及其使用方法
CN105658671A (zh) 2013-10-10 2016-06-08 Ucl商务股份有限公司 嵌合抗原受体
CN108778329A (zh) * 2016-02-17 2018-11-09 西雅图基因公司 Bcma抗体和其用以治疗癌症和免疫病症的用途
WO2018151817A2 (en) * 2017-02-17 2018-08-23 Unum Therapeutics Inc. Co-use of anti-bcma antibody and antibody-coupled t celll receptor (actr) in cancer therapy and b cell disorders

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ROSENBERG ET AL., NEW ENG. J. OF MED., vol. 319, 1988, pages 1676
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
See also references of EP3967329A4
UI-TEI ET AL., FEBS LETTERS, vol. 479, 2000, pages 79 - 82
ZHANG, FENG ET AL.: "Development in Bispecific Antibody", CHINESE JOURNAL OF PHARMACEUTICAL ANALYSIS, vol. 39, no. 1, 31 January 2019 (2019-01-31), XP009522265, DOI: 10.16155/j.0254-1793.2019.01.10 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021223720A1 (zh) * 2020-05-06 2021-11-11 亘喜生物科技（上海）有限公司 人源化cd19抗体及其应用
CN114317607A (zh) * 2021-12-31 2022-04-12 西安桑尼赛尔生物医药有限公司 融合一代靶向cd7 car和二代靶向bcma的双靶点通用car-t细胞及制备方法
CN116444669A (zh) * 2023-04-04 2023-07-18 上海科棋药业科技有限公司 靶向bcma car-t细胞人源化抗体
CN116444669B (zh) * 2023-04-04 2024-02-13 科弈(浙江)药业科技有限公司 靶向bcma car-t细胞人源化抗体

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