WO2023226921A9 - 靶向bcma-cd19的双特异性嵌合抗原受体及其应用 - Google Patents

靶向bcma-cd19的双特异性嵌合抗原受体及其应用 Download PDF

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WO2023226921A9
WO2023226921A9 PCT/CN2023/095470 CN2023095470W WO2023226921A9 WO 2023226921 A9 WO2023226921 A9 WO 2023226921A9 CN 2023095470 W CN2023095470 W CN 2023095470W WO 2023226921 A9 WO2023226921 A9 WO 2023226921A9
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sequence
bcma
seq
cells
amino acid
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WO2023226921A1 (zh
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张超
张其猛
白大勇
吕璐璐
周立
王永增
张云龙
丁伟
路佳兴
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合源康华医药科技(北京)有限公司
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Publication of WO2023226921A1 publication Critical patent/WO2023226921A1/zh
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Definitions

  • This application relates to the field of biomedicine, specifically to a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application.
  • myeloma defined as a malignant proliferation of plasma cells in the bone marrow, is the second most common hematological malignancy, accounting for 1% of all cancers. Studies have shown that multiple myeloma is more common among people over 60 years old and its incidence has increased steadily in recent years. For most patients, multiple myeloma is incurable and eventually develops into relapsed/refractory multiple myeloma. The survival period of patients with relapsed/refractory multiple myeloma who are ineffective against existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, and antibody drugs) is only about 13 months.
  • BCMA B Cell Maturation Antigen
  • BCMA is a transmembrane glycoprotein that belongs to the tumor necrosis factor receptor family. BCMA is highly expressed on multiple myeloma cells and not on most other cells. Malignant tumor plasma cells usually express higher levels of BCMA than normal plasma cells. Upregulation of BCMA promotes the growth of multiple myeloma cancer cells, while downregulation of BCMA can inhibit the growth of multiple myeloma cancer cells.
  • multiple myeloma as a B-cell lineage tumor, generally does not express CD19 molecules, so CD19 is usually not used as a target for multiple myeloma treatment.
  • CD19 is usually not used as a target for multiple myeloma treatment.
  • studies in the literature suggesting that some trace amounts of drug-resistant and relapsed multiple myeloma clones also have a CD19 + phenotype.
  • Chimeric Antigen Receptor is the core component of CAR cell therapy, which can include a targeting part (for example, a part that binds to Tumor-Associated Antigen (TAA)), hinge region, trans membrane region and intracellular domain.
  • TAA Tumor-Associated Antigen
  • CAR-T cell immunotherapy is considered to be one of the most promising methods to conquer tumors.
  • CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.
  • This application provides a bispecific chimeric antigen receptor targeting BCMA-CD19 and its application.
  • the inventor constructed multiple bispecific chimeric antigen receptor expression vectors targeting BCMA-CD19 and prepared target Using BCMA-CD19 bispecific CAR-T cells, it was also verified at the cellular level that BCMA-CD19 bispecific CAR-T cells have good tumor suppressive functions.
  • a bispecific chimeric antigen receptor targeting BCMA-CD19 which includes an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain includes an anti- BCMA extracellular antigen recognition domain and anti-CD19 extracellular antigen recognition domain;
  • the anti-BCMA extracellular antigen recognition domain includes BCMA VH and BCMA VL, wherein the amino acid sequences of the BCMA VH complementary determining regions CDR1, CDR2, and CDR3 respectively include SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
  • the amino acid sequence shown in 3 the amino acid sequences of BCMA VL complementarity determining region CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.
  • the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, wherein the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2, and CDR3 respectively include as follows
  • the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and the amino acid sequences of CD19 VL complementary determining regions CDR1, CDR2, and CDR3 include SEQ ID NO:10 and SEQ ID NO:11 respectively.
  • the BCMA VH sequence includes the amino acid sequence shown in SEQ ID NO: 13
  • the BCMA VL sequence includes the amino acid sequence shown in SEQ ID NO: 14 sequence.
  • the BCMA VH sequence includes the amino acid sequence shown in SEQ ID NO: 15
  • the BCMA VL sequence includes the amino acid sequence shown in SEQ ID NO: 16 sequence.
  • the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO: 17
  • the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO: 18 sequence.
  • the extracellular antigen recognition structure of the bispecific chimeric antigen receptor includes any one selected from the following structures: CD19 VL sequence-first linkage Sequence - CD19 VH sequence - 2nd connection sequence - BCMA VL sequence - 3rd connection sequence - BCMA VH sequence, BCMA VL sequence - 4th connection sequence - BCMA VH sequence - 5th connection sequence - CD19 VL sequence - 6th connection sequence - CD19 VH sequence, BCMA VL sequence - 7th junction sequence - CD19 VL sequence - 8th junction sequence - CD19 VH sequence - 9th junction sequence - BCMA VH sequence, and CD19 VL sequence - 10th junction sequence - BCMA VL sequence - The 11th connection sequence - BCMA VH sequence - the 12th connection sequence - CD19 VH sequence.
  • the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes any one selected from the following structures: BCMA VL sequence-No. 7 Junction Sequence - CD19 VL Sequence - 8th Junction Sequence - CD19 VH Sequence - 9th Junction Sequence - BCMA VH Sequence, and CD19 VL Sequence - 10th Junction Sequence - BCMA VL Sequence - 11th Junction Sequence - BCMA VH Sequence - 12th Linker sequence - CD19 VH sequence.
  • the first linking sequence, the second linking sequence, the third linking sequence, the fourth linking sequence, the fifth linking sequence, the sixth linking sequence, and the seventh linking sequence , the 8th connection sequence, the 9th connection sequence, the 10th connection sequence, the 11th connection sequence, and the 12th connection sequence are independently selected from one or more of the following sequences: SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36.
  • the extracellular antigen recognition domain of the bispecific chimeric antigen receptor includes the amino acids shown in SEQ ID NO: 19 or SEQ ID NO: 20 sequence.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ ; optionally, the hinge region The amino acids of the region are derived from CD8 ⁇ ; further optionally, the amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO: 21.
  • the transmembrane region is derived from CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70
  • the amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 22.
  • the intracellular domain includes an intracellular signaling region; optionally, it also includes a costimulatory signaling region.
  • the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, One or more of DAP12 and Syk; optionally, the intracellular signaling region is derived from CD3 ⁇ ; further optionally, the amino acid sequence of the intracellular signaling region includes as shown in SEQ ID NO: 23 amino acid sequence.
  • the costimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, One, two or more than three of LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; Further optionally, the amino acid sequence of the costimulatory signaling region includes the amino acid sequence shown in SEQ ID NO: 24.
  • the above-mentioned dual-specific chimeric antigen receptor also includes a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8 ⁇ ; further, it can Optionally, the amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO: 25.
  • the dual-specific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned dual-specific chimeric antigen receptor.
  • the nucleotide sequence encoding the bispecific chimeric antigen receptor includes:
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the above vector is an expression vector; in some embodiments, the vector is a viral vector; in some embodiments, the vector is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which contains the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.
  • the engineered immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), multipotent Stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), Induced pluripotent stem cells differentiate into one or more of NK cells (iPSC-NK) and embryonic stem cells.
  • NK cells natural killer cells
  • PBMC cells peripheral blood mononuclear cells
  • multipotent Stem cells T cells differentiated from pluripotent stem cells
  • NK cells differentiated from pluripotent stem cells induced pluripotent stem cells (iPSC)
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • iPSC-NK Induced pluripotent stem cells differentiate into one or more of NK cells
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes. Lymphocytes.
  • This application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include protective agents.
  • pharmaceutically acceptable excipients include cell cryopreservation solution.
  • the pharmaceutical composition is an intravenous injection.
  • the present application also provides the use of the above-mentioned chimeric antigen receptor, isolated nucleic acid molecule, vector or engineered immune effector cell in the preparation of medicines for treating diseases or conditions related to the expression of BCMA.
  • the disease or condition associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed Multiple myeloma.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the present application also provides a method for treating diseases or conditions related to the expression of BCMA, comprising the following steps: applying an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical compositions to patients with a disease or disorder related to the expression of BCMA. Subjects in need of disease or condition.
  • the disease or condition associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed Multiple myeloma.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the administration is by intravenous injection.
  • the administration method is to administer an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject in a single injection.
  • the effective amount of engineered immune effector cells or pharmaceutical composition is a dose of 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/kg.
  • the present application also provides the above-mentioned engineered immune effector cells or the above-mentioned pharmaceutical composition for treating diseases or conditions related to the expression of BCMA.
  • the disease or condition associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, The cancer is refractory or relapsed multiple myeloma.
  • the disease or disorder associated with the expression of BCMA may be an autoimmune disease.
  • the autoimmune disease can be selected from the following: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, severe disease Myasthenia and autoimmune hemolytic anemia.
  • Figure 1 shows a schematic structural diagram of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CARs in Example 1 of the present application.
  • Figure 2A shows the CAR expression on the surface of CAR-T cells of two complete CARs (i.e., CD19-2A-BCMA and BCMA-2A-CD19) connected to the self-cleaving polypeptide T2A in Example 2 of the present application (6 days after infection) ;
  • the left picture in Figure 2A is the CD19-2A-BCMA group
  • the middle picture in Figure 2A is the BCMA-2A-CD19 group
  • the right picture in Figure 2A is the UTD group (T cells without CAR transduction)
  • Figure 2B to Figure 2E represent The CAR expression on the surface of the four bispecific CAR-T cells (i.e., Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, and Loop BCMA-CD19 cells) in Example 2 of the present application (after infection 6 days); among them: Figure 2B is the Tan CD19-BCMA group, Figure 2C is the Tan BCMA-CD19 group, Figure 2D is the Loop CD19-BCMA group
  • Figures 3A-3C show the cytokine release of various BCMA-CD19 bispecific CARs, CD19 CARs, and BCMA CARs after being activated by positive target cells in Example 4 of the present application; wherein: Figure 3A is the IL-2 of each group Figure 3B shows the release of IFN- ⁇ in each group, and Figure 3C shows the release of TNF- ⁇ in each group. Among them, Figure 3A, Figure 3B and Figure 3C all contain two dotted boxes.
  • the five columns from left to right represent UTD cells, TanCD19 -Cytokine release from BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells and BCMA cells after activation by K562-BCMA cells; in the second dotted box in Figure 3A, Figure 3B and Figure 3C , the five columns from left to right represent the cytokine release of UTD cells, TanCD19-BCMA cells, TanBCMA-CD19 cells, LoopCD19-BCMA cells, LoopBCMA-CD19 cells and CD19 cells after activation by K562-CD19 cells.
  • Figure 4A- Figure 4C shows the killing effect of various BCMA-CD19 bispecific CAR, CD19 CAR, BCMA CAR cells on different target cells in Example 5 of the present application; wherein: Figure 4A is the NALM6 group, and NALM6 is CD19+BCMA -Target cells; Figure 4B is the MM.1S group, MM.1S is BCMA+CD19- target cells; Figure 4C is the NALM6-KO CD19 group, NALM6-KO CD19 is a negative target cell control that knocks out CD19 and does not express BCMA. .
  • Figures 5A to 5D show the continued proliferation of CAR-T cells in each group after multiple rounds of antigen stimulation in Example 6 of the present application;
  • Figure 5A is the proliferation of CD3+ cells stimulated with MM.1S cells
  • Figure 5B is The proliferation of CD3+ cells stimulated with NALM6 cells
  • Figure 5C shows the proliferation of CAR+ cells stimulated with MM.1S cells
  • Figure 5D shows the proliferation of CAR+ cells stimulated with NALM6 cells.
  • CAR Chimeric Antigen Receptor
  • extracellular antigen recognition domains for example, binding to tumor-associated antigens (Tumor-Associated Antigen) , part of TAA), hinge region, transmembrane region and intracellular domain.
  • CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising methods to conquer tumors.
  • CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.
  • extracellular antigen recognition domain refers to the antigen recognition domain (Antigen Recognition Domain, ARD).
  • CAR cell therapy products such as CAR-T cells
  • the antigen recognition domain has been derived from the single antigen of an antibody.
  • Chain variable region Single Chain Variable Fragment, abbreviated as scFv
  • TCR single Chain Variable Fragment
  • VLR variable lymphocyte receptors
  • the scFv includes the antibody heavy chain variable region (VH region) and the light chain variable region (VL region). They are connected by a peptide chain, such as: 18
  • the connecting sequence GSTGSSGKPGSGEGSTKG consists of amino acids.
  • scFv antibodies targeting two or more targets include VH regions and VL regions targeting different targets.
  • the different regions are connected directly or indirectly through connecting sequences, and their arrangement can be in any of the following forms: Target 1 VL - Target 1 VH - Target 2 VL - Target 2 VH, Target 2 VL - Target 2 VH - Target 1 VL - Target 1 VH, Target 1 VL - Target 2 VL - Target 2 VH-Target 1 VH, Target 2 VL-Target 1 VL-Target 1 VH-Target 2 VH, the above "-" represents connection through the connecting sequence.
  • the term "specific recognition and/or binding” refers to the recognition and/or binding between the CAR and the specific target with greater affinity, avidity, easier, and greater affinity than the CAR binding to other targets. and/or bind the target for a greater duration.
  • hinge region refers to the connecting segment between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by giving the antigen recognition domain a certain range of activity.
  • the hinge regions currently used are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ .
  • the typical hinge region also contains some residues that participate in CAR dimerization and help enhance antigen sensitivity.
  • transmembrane region refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but they are not directly involved in signal transmission. They can improve downstream signal transmission through interactions.
  • the transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.
  • intracellular domain includes intracellular signaling regions and may also include costimulatory signaling regions.
  • intracellular signaling region refers to the activation of at least one normal effector function of a CAR-expressing immune effector cell.
  • the intracellular signaling region can be derived from one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, and Syk.
  • the term "costimulatory signaling region" exists because in addition to stimulation by antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival, as well as activation of cells. Effector functions.
  • the CAR may also include one or more costimulatory signaling domains, wherein the costimulatory signaling domains may be derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4- One, two or more of 1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3 and MyD88.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolated does not exclude substances that have been artificially or synthetically obtained from their natural state by artificial means, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • guide peptide refers to the short peptide before the extracellular antigen recognition domain (such as scFv sequence), which functions to guide the recombinant protein synthesized in the cell to be exported to the outside of the cell.
  • extracellular antigen recognition domain such as scFv sequence
  • Commonly used guide peptides include human CD8 ⁇ signal peptide or human GM-CSF receptor ⁇ signal peptide.
  • BCMA B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily. Human BCMA is expressed almost exclusively on plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapeutics against multiple myeloma. However, due to the heterogeneity of specific antigens on the surface of multiple myeloma cells, the selection of its antigen target is not necessarily a single one. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized.
  • the "CD19" molecule is currently the main target for the treatment of hematological tumors derived from B lymphocytes, and is also a hot spot in CAR-T cell therapy research. Most malignant tumors derived from B cells express CD19 molecules on their cell surfaces. As a B-cell lineage tumor, multiple myeloma generally does not express CD19 molecules. Therefore, CD19 is usually not used as a target for multiple myeloma treatment. However, there are also studies in the literature suggesting that some trace amounts of drug-resistant and relapsed multiple myeloma clones also have a CD19 + phenotype.
  • dual-target CAR-T products as long as one tumor antigen target is recognized, CAR-T cells can be activated to prevent tumor antigen escape.
  • dual-target CAR immune cells have the following advantages: 1. Fewer immune cells are required, and preparation is convenient and cost-saving; 2. From the perspective of medication In terms of safety, the safety and operability of using one product are much higher than using two products.
  • linking sequence generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure/region of the chimeric antigen receptor of the invention.
  • the linking sequence can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linker sequences can be used when it is desired to ensure that two adjacent domains do not interfere with each other spatially.
  • isolated nucleic acid molecule generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, which may be isolated from its natural environment or a synthetic analog .
  • gene transduction/transfection methods mainly include viral and non-viral methods.
  • viral and non-viral methods For example: through gamma retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed.
  • the vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • Viruses (such as SV40).
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site. Vectors may also contain components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but they are not the only ones.
  • transposon refers to a discontinuous segment of DNA that has the ability to migrate and carry genetic information between chromosomal sites, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects.
  • electroporation can also be used to transduce mRNA into T cells.
  • Immune effector cells generally refers to cells that participate in immune responses, such as promoting immune effector responses.
  • Immune effector cells may be selected from the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, pluripotent stem cells differentiated into NK cells, induced pluripotent stem cells (iPSC), induced pluripotent stem cells differentiated into T cells (iPSC-T), induced pluripotent stem cells differentiated into NK cells (iPSC-NK) and One or more types of embryonic stem cells.
  • NK cells natural killer cells
  • PBMC cells peripheral blood mononuclear cells
  • pluripotent stem cells T lymphocytes differentiated from pluripotent stem cells
  • pluripotent stem cells differentiated into NK cells induced pluripotent stem cells (iPSC), induced pluripotent stem cells differentiated into T cells (iPSC-T),
  • the term "pharmaceutical composition” generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as : One or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.
  • pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solutions.
  • the pharmaceutical composition of the present application is a cell suspension or cryopreserved cells thereof.
  • subject generally refers to a human or non-human animal, including but not limited to mouse, rat, cat, dog, rabbit, horse, pig, cow, sheep, or monkey.
  • the term "about” generally refers to a range of fluctuations above or below the specified value that is acceptable to those skilled in the art, such as: within the range of ⁇ 0.5%-10%, such as 0.5 above or below the specified value. %, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, Changes within the range of 9%, 9.5% or 10%.
  • Chimeric antigen receptor nucleic acid, vector, immune effector cell, pharmaceutical composition
  • the present application provides a bispecific chimeric antigen receptor targeting BCMA-CD19, which includes an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular The antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain;
  • the anti-BCMA extracellular antigen recognition domain includes BCMA VH and BCMA VL, wherein the amino acid sequences of the BCMA VH complementary determining regions CDR1, CDR2, and CDR3 respectively include SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
  • the amino acid sequence shown in 3 the amino acid sequences of BCMA VL complementarity determining region CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.
  • CDR classification rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying the website that implements these rules, just enter the VH and VL sequences and select the corresponding rules. CDR sequences based on different rules can be obtained. In this application, IMGT rules are used to classify CDRs, but those skilled in the art should understand that the protection scope of this application covers combinations of CDR sequences obtained by analyzing using different rules.
  • the anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, wherein the amino acid sequences of the CD19 VH complementarity determining regions CDR1, CDR2, and CDR3 respectively include such as SEQ ID NO: 7, SEQ ID NO: 8.
  • the amino acid sequence shown in SEQ ID NO:9, the amino acid sequences of the CD19 VL complementary determining regions CDR1, CDR2, and CDR3 include the amino acids shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively. sequence.
  • the BCMA VH sequence includes the amino acid sequence set forth in SEQ ID NO: 13
  • the BCMA VL sequence includes the amino acid sequence set forth in SEQ ID NO: 14.
  • the BCMA VH sequence includes the amino acid sequence set forth in SEQ ID NO: 15, and the BCMA VL sequence includes the amino acid sequence set forth in SEQ ID NO: 16.
  • the CD19 VH sequence includes the amino acid sequence set forth in SEQ ID NO:17
  • the CD19 VL sequence includes the amino acid sequence set forth in SEQ ID NO:18.
  • the application also includes the substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of any of the above-mentioned dual-specific chimeric antigen receptors, and it has an effect equivalent to the above-mentioned Activity of any chimeric antigen receptor.
  • the amino acids in the FR region of the VH and VL sequences can be substituted so that the CDR region of the modified antibody can still retain a suitable antigen-binding site. Therefore, this application naturally Including different amino acid sequences obtained by humanizing the FR regions in the VH and VL sequences based on the above-mentioned CDRs of this application.
  • the extracellular antigen recognition domain of the dual-specific chimeric antigen receptor includes any one selected from the following structures: CD19 VL sequence-1st connection sequence-CD19 VH sequence-2nd connection Sequence - BCMA VL sequence - 3rd connection sequence - BCMA VH sequence, BCMA VL sequence - 4th connection sequence - BCMA VH sequence - 5th connection sequence - CD19 VL sequence - 6th connection sequence - CD19 VH sequence, BCMA VL sequence - 7th linker sequence - CD19 VL sequence - 8th linker sequence - CD19 VH sequence - 9th linker sequence - BCMA VH sequence, and CD19 VL sequence - 10th linker sequence - BCMA VL sequence - 11th linker sequence - BCMA VH sequence - No. 12 connection sequence-CD19 VH sequence.
  • the extracellular antigen recognition domain of the dual-specific chimeric antigen receptor includes any one selected from the following structures: BCMA VL sequence-7th linkage sequence-CD19 VL sequence-8th linkage Sequence - CD19 VH sequence - 9th junction sequence - BCMA VH sequence, and CD19 VL sequence - 10th junction sequence - BCMA VL sequence - 11th junction sequence - BCMA VH sequence - 12th junction sequence - CD19 VH sequence.
  • the 1st connection sequence, the 2nd connection sequence, the 3rd connection sequence, the 4th connection sequence, the 5th connection sequence, the 6th connection sequence, the 7th connection sequence, the 8th connection sequence, the 9th connection sequence , the 10th connection sequence, the 11th connection sequence, and the 12th connection sequence are independently selected from one or more of the following sequences: SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.
  • the extracellular antigen recognition domain of the dual-specific chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ ; optionally, the amino acid sequence of the hinge region is derived from CD8 ⁇ ; further, it can be Optionally, the amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO: 21.
  • the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, The amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 22.
  • the intracellular domain includes an intracellular signaling region; optionally, it also includes a costimulatory signaling region; further optionally, the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , one or more of CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signaling region is derived from CD3 ⁇ ,
  • the amino acid sequence of the intracellular signaling region includes the amino acid sequence shown in SEQ ID NO: 23.
  • the costimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, One, two or more of NKG2D, DAP10, B7-H3 and MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further optionally, the costimulatory signaling region
  • the amino acid sequence includes the amino acid sequence shown in SEQ ID NO:24.
  • a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence is also included; optionally, wherein the guide peptide is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the guide peptide Contains the amino acid sequence shown in SEQ ID NO:25.
  • the dual-specific chimeric antigen receptor includes the amino acid sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned dual-specific chimeric antigen receptor.
  • the nucleotide sequence encoding the bispecific chimeric antigen receptor comprises:
  • Vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes derived from P1 (PAC); phages such as lambda phage or M13 phage and animal viruses, etc. .
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses.
  • Viruses such as SV40
  • the vector is an expression vector; optionally, the vector is a viral vector; further optionally, the vector is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which contains the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.
  • the engineered immune effector cells are selected from the group consisting of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and T lymphocytes differentiated from pluripotent stem cells.
  • NK cells differentiated from pluripotent stem cells
  • iPSC induced pluripotent stem cells
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • iPSC-NK induced pluripotent stem cells
  • iPSC-T induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • embryonic stem cells embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.
  • the surface of the engineered immune effector cells may express or have chimeric antigen receptors described herein.
  • this application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients include: one or more of carriers, protective agents, stabilizers, and diluents.
  • pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solutions.
  • the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the present application also provides a method for preparing engineered immune effector cells, which includes the following steps: transducing the vector described in the application into the immune effector cells.
  • the engineered immune effector cells are selected from the group consisting of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and T lymphocytes differentiated from pluripotent stem cells.
  • NK cells differentiated from pluripotent stem cells
  • iPSC induced pluripotent stem cells
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • iPSC-NK induced pluripotent stem cells
  • iPSC-T induced pluripotent stem cells
  • iPSC-NK differentiated from induced pluripotent stem cells
  • embryonic stem cells embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.
  • the present application also provides the preparation of the bispecific chimeric antigen receptor, the isolated nucleic acid molecule, the vector and/or the engineered immune effector cell described in the present application.
  • the disease or disorder associated with expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma tumor.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the present application also provides a method for treating a disease or disorder associated with the expression of BCMA, the method comprising the steps of: administering an effective dose to a subject in need of treating a disease or disorder associated with the expression of BCMA.
  • the disease or disorder associated with expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or relapsed multiple myeloma tumor.
  • the disease or disorder associated with expression of BCMA may be an autoimmune disease.
  • the autoimmune disease may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis, and autoimmune hemolytic anemia.
  • the administration can be by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the method of administration may be by intravenous injection to the subject.
  • an effective dose of engineered immune effector cells or pharmaceutical compositions can be administered to the subject in a single time, or in divided doses within a certain period, such as once a week, Once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the dosage may be different for different indications; the dosage may also be different for patients with different severity of illness.
  • the administered dose may range from 1 ⁇ 10 5 CAR positive T cells/kg to 1 ⁇ 10 7 CAR positive T cells/kg, for example, 1 ⁇ 10 5 CAR positive T cells/kg to 1 ⁇ 10 6 CAR positive T cells/kg, 1 ⁇ 10 6 CAR-positive T cells/kg to 1 ⁇ 10 7 CAR-positive T cells/kg, 0.5 ⁇ 10 6 CAR-positive T cells/kg, 0.6 ⁇ 10 6 CAR-positive T cells /kg, 0.7 ⁇ 10 6 CAR-positive T cells/kg, 0.8 ⁇ 10 6 CAR-positive T cells/kg, 0.9 ⁇ 10 6 CAR-positive T cells/kg, 1.0 ⁇ 10 6 CAR-positive T cells/kg , 1.1 ⁇ 10 6 CAR-positive T cells/kg, 1.2 ⁇ 10 6 CAR-positive T cells/kg, 1.3 ⁇ 10 6 CAR-positive T cells/kg, 1.4 ⁇ 10 6 CAR-positive T cells/kg, 1.5
  • the subjects may include humans and non-human animals.
  • the subject may include, but is not limited to, mouse, rat, cat, dog, horse, pig, cow, sheep, rabbit, or monkey.
  • the present application also provides the chimeric antigen receptor, the isolated nucleic acid molecule, the vector and/or the engineered immune effector cell, which can be used to treat patients with Diseases or conditions associated with expression of BCMA.
  • the disease or disorder associated with expression of BCMA may include a non-solid tumor, optionally, the non-solid tumor is a hematological tumor.
  • the disease or disorder associated with expression of BCMA may include multiple myeloma.
  • the multiple myeloma is relapsed or refractory multiple myeloma.
  • the following examples are only to illustrate the chimeric antigen receptor, engineered immune effector cells, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.
  • the examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into such vectors and plasmids, or introduce plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual ,2nd edition,Cold Spring Harbor Laboratory Press.
  • BCMA-specific humanized antibody the amino acid sequence of its VH is shown in SEQ ID NO:13, the nucleotide sequence of its VH is shown in SEQ ID NO:37, and the amino acid sequence of its VL is shown in SEQ ID NO:14 is shown, the nucleotide sequence of its VL is shown as SEQ ID NO:38, and the amino acid sequences of its VH CDR1, CDR2, and CDR3 are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID respectively.
  • the amino acid sequences of its VL CDR1, CDR2, and CDR3 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively) and a CD19-specific antibody (its VH
  • the amino acid sequence is shown in SEQ ID NO:17
  • the nucleotide sequence of its VH is shown in SEQ ID NO:39
  • the amino acid sequence of its VL is shown in SEQ ID NO:18
  • the nucleotide sequence of its VL is shown in SEQ ID NO:40 is shown
  • the amino acid sequences of its VH CDR1, CDR2, and CDR3 are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively
  • the amino acid sequences of its VL CDR1, CDR2, and CDR3 are respectively As shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12), we hope to obtain a better bispecific CAR structure.
  • BCMA-CD19 bispecific CAR Taking into account changes in the order of the target BCMA and CD19, and the order of VH and VL in the extracellular antigen recognition domain of each target, there are many options for the structural design of the BCMA-CD19 bispecific CAR. We chose to screen candidate BCMA-CD19 bispecific CAR structures on the second-generation CAR structure.
  • CD8 ⁇ guide chain is used as the signal peptide (as shown in SEQ ID NO:25), the hinge region (as shown in SEQ ID NO:21) and the transmembrane region (as shown in SEQ ID NO:22).
  • FIG. 21 adopts the structure of CD8 ⁇ , uses 4-1BB as the intracellular costimulatory signal (as shown in SEQ ID NO:24), and CD3 ⁇ as the T cell activation signal (as shown in SEQ ID NO:23).
  • Loop BCMA-CD19 structure CD19 VL-BCMA VL-BCMA VH-CD19 VH;
  • CD19-2A-BCMA structure CD19 CAR-T2A-BCMA CAR (before and after the self-cleaving polypeptide T2A of this structure, there is a complete CD19 CAR structure and BCMA CAR structure respectively.
  • the complete CAR structure refers to the same structure as other groups. Signal peptide, hinge region, transmembrane region, intracellular costimulatory signal region, T cell activation signal region);
  • BCMA-2A-CD19 structure BCMA CAR-T2A-CD19 CAR (before and after the self-cleaving polypeptide T2A of this structure, there is a complete BCMA CAR structure and CD19 CAR structure respectively.
  • the complete CAR structure refers to the same structure as other groups. Signal peptide, hinge region, transmembrane region, intracellular costimulatory signal region, T cell activation signal region);
  • CD19 CAR structure CD19 VL-CD19 VH;
  • BCMA CAR structure BCMA VL-CD19 VH.
  • the above 6 BCMA-CD19 bispecific CAR structures, 1 CD19 CAR structure, and 1 BCMA CAR structure were constructed into modified empty lentiviral vectors (manufacturer: SBI Company, product number: CD500-CD800, such as WO2021/121227
  • the CAR expression vector was obtained (conventional resistance transformation as described in Example 1), and then the CAR expression vector and three packaging plasmids were transfected into 293T cells. After collection and purification, a functional lentiviral vector was obtained.
  • the three packaging plasmids are pMD2.G (purchased from Biovector Company, product number Biovector012259), pMDLg/pRRE (purchased from Biovector Company, product number Biovector012251), and pRSV-Rev (purchased from Biovector Company, product number Biovector012253).
  • the transduction experiment was performed according to conventional methods known to those skilled in the art.
  • the transduction steps are briefly described as follows:
  • PBMC Peripheral blood mononuclear cells
  • the isolated T cells were cultured with complete lymphocyte culture medium (X-VIVO15 medium + 5% FBS + 300 IU/ml IL-2 or X-VIVO15 medium + 5% FBS + 5ng/ml IL-15 + 10ng/ml IL -7) Resuspend to a final concentration of (1 ⁇ 2) ⁇ 10 6 cells/ml, add 5 ⁇ 10 ⁇ l of CD3/CD28 magnetic beads for stimulation, mix well and place in an incubator for culture.
  • the culture conditions are 37 °C + 5% CO 2 , incubation time is at least 24 hours.
  • Tan CD19-BCMA structure Tan BCMA-CD19 structure, Loop CD19-BCMA structure, Loop BCMA-CD19 structure, CD19-2A-BCMA structure, BCMA-2A-CD19 structure, CD19 CAR, and BCMA CAR respectively.
  • T cells After infecting T cells respectively, the obtained T cells were named Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, CD19-2A-BCMA cells, and BCMA-2A-CD19 cells. , CD19 cells and BCMA cells.
  • the inventors tried to individually stain the bispecific CAR-T cells obtained in Example 1 after 11 days of infection of the T cells (individual staining refers to bispecific CAR-T cells that are only labeled with FITC fluorescently labeled BCMA antigen or PE fluorescently). CD19 antigen is stained alone) and co-staining (co-staining means that bispecific CAR-T cells are stained by both FITC fluorescently labeled BCMA antigen and PE fluorescently labeled CD19 antigen) to select the staining method.
  • the above observations are not limited by the relative positions of the BCMA extracellular antigen recognition domain and the CD19 extracellular antigen recognition domain in the CAR structure.
  • the BCMA antigen is smaller and is easier to overcome the steric hindrance problem in the bispecific CAR structure and bind to the CAR protein. Therefore, in the subsequent experiments of this application, the BCMA antigen detection data is used to characterize BCMA-CD19
  • the positive proportion of bispecific CAR of course, the positive proportion of BCMA-CD19 bispecific CAR can also be characterized using the data of BCMA antigen detection in the co-staining method, but the separate staining method is simpler.
  • Cytokine release experiment 4 types of bispecific CAR-T cells obtained in Example 1 (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, CD19 CAR-T cells and UTD cells were co-cultured with target cells in X-VIVO15 medium at an effect-to-target ratio of 1:1 for 24 hours.
  • IL-2, IFN- ⁇ , and TNF- ⁇ in the cell supernatants were detected by ELISA.
  • K562 is a double-negative target cell of BCMA and CD19
  • K562-BCMA is a positive target cell that exogenously expresses BCMA but does not express CD19
  • K562-CD19 is a positive target cell that exogenously expresses CD19 but does not express BCMA. Since K562-BCMA cells do not express CD19 antigen, they were not used to detect CD19 CAR-T cells. For the same reason, K562-CD19 cells did not express BCMA antigen, so they were not used to detect BCMA CAR-T cells (in the figure In 3A, Figure 3B, and Figure 3C, the K562-BCMA group and the K562-CD19 group each lack 1 bar).
  • Example 1 -T cells and UTD cells were co-cultured with target cells in X-VIVO15 medium for 4 hours according to different effector-target ratios (0:1, 1:1, 3:1 or 9:1) of effector cells and target cells. , detect the target cell killing ratio by detecting the luciferase activity stably expressed in the target cells.
  • NALM6 human acute lymphoblastic leukemia cells
  • MM.1S human multiple myeloma cells
  • NALM6 - KO CD19 is a negative target cell control that knocks out CD19 and does not express BCMA.
  • the cell killing results are shown in Figure 4A- Figure 4C: In Figure 4A, Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and CD19 CAR-T cells all responded to endogenous NALM6, a positive target cell expressing CD19, has a good killing effect.
  • BCMA CAR-T cells and UTD cells do not have a killing effect on NALM6, a positive target cell expressing CD19 endogenously.
  • Tan CD19-BCMA cells, Tan BCMA -CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, and BCMA CAR-T cells all have good killing effects on MM.1S, a positive target cell that expresses endogenous BCMA.
  • CD19 CAR-T cells and UTD cells The positive target cell MM.1S that endogenously expresses BCMA has no killing effect; in Figure 4C, all cells have no killing effect on the negative target cell control that knocks out CD19 and does not express BCMA.
  • Antigen stimulation can activate CAR-T cells and cause CAR-T cell proliferation. However, continued activation of T cells will lead to cell exhaustion. The proliferation ability and effector function of exhausted T cells will be reduced.
  • BCMA-CD19 bispecific The proliferation of CD3+ cells (i.e., the proliferation of T cells), the proliferation of CAR+ cells, and the proportion of CAR+ cells after multiple rounds of antigen stimulation experiments of CAR-T cells verify the continued proliferation of BCMA-CD19 bispecific CAR-T cells. sex.
  • the four bispecific CAR-T cells (Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, Loop BCMA-CD19), BCMA CAR-T cells, and CD19 CAR obtained in Example 1 were -The CAR-positive proportion of T cells was adjusted by UTD to a level consistent with the group of CAR-T cells with the lowest CAR-positive proportion, including Tan CD19-BCMA cells, Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA- The CAR positive ratio of CD19 cells and BCMA CAR-T cells is based on the BCMA antigen detection data, and the CAR positive ratio of CD19 CAR-T cells is based on the CD19 antigen detection data.
  • each group of CAR-T cells were co-cultured with positive target cells in a 24-well plate at an effective-to-target ratio of 1:2, with 2 ml of X-VIVO15 culture medium per well, and each group of cells was repeated 3 times. hole.
  • the positive target cells were MM.1S and NALM6 respectively, which is equivalent to using BCMA and CD19 as antigens for multiple rounds of stimulation to test the impact of multiple rounds of stimulation with different antigens on the continued proliferation of BCMA-CD19 bispecific CAR-T cells. Since MM.1S cells do not express CD19 antigen, they were not used to detect CD19 CAR-T cells. For the same reason, NALM6 cells did not express BCMA antigen, so they were not used to detect BCMA CAR-T cells.
  • CD3 antibody Manufacturer: BioLegend, Cat. No.: 300312
  • BCMA antigen BCMA antigen
  • CD19 antigen standard for CD3 and CAR staining
  • Tan CD19 -BCMA cells Tan BCMA-CD19 cells, Loop CD19-BCMA cells, Loop BCMA-CD19 cells, BCMA CAR-T cells stained with BCMA antigen, CD19 CAR-T cells stained with CD19 antigen
  • flow cytometry Analysis showing the proportion and number of CAR-positive cells among CD3-positive cells.
  • the number of CAR-positive cells among CD3-positive cells can also be calculated based on the conversion of volume multiples (CD3 is a marker that distinguishes whether they are T cells), and then each calculation result can be used to calculate the number of CAR-positive cells among CD3-positive cells.
  • the group then took out a certain amount of CAR-T cells and added corresponding positive target cells according to the effect-to-target ratio of 1:2 for a new round of stimulation, and repeated 3-4 rounds of stimulation.
  • Tan CD19-BCMA, Tan BCMA-CD19, Loop CD19-BCMA, and Loop BCMA-CD19 structures have better expression on the surface of T cells, cytokine release, and cell killing in vitro.
  • the performance; in terms of the sustained proliferation of CAR-T, the Loop CD19-BCMA and Loop BCMA-CD19 structures are significantly better than the Tan CD19-BCMA and Tan BCMA-CD19 structures and perform better.
  • the Loop CD19-BCMA and Loop BCMA-CD19 structures perform relatively close to BCMA CAR-T and CD19 CAR-T in various experimental tests, and have complete functions for dual targets.
  • SEQ ID NO:1 BCMA VH CDR1;
  • SEQ ID NO:2 BCMA VH CDR2;
  • SEQ ID NO:3 BCMA VH CDR3;
  • SEQ ID NO:4 BCMA VL CDR1;
  • SEQ ID NO:5 BCMA VL CDR2 (ETS, Glu Thr Ser);
  • SEQ ID NO:6 BCMA VL CDR3;
  • SEQ ID NO:9 CD19 VH CDR3;
  • SEQ ID NO:11 CD19 VL CDR2 (SAT, Ser Ala Thr);
  • SEQ ID NO:13 BCMA VH sequence (humanized);
  • SEQ ID NO:14 BCMA VL sequence (humanized);
  • SEQ ID NO:15 BCMA VH sequence (rabbit source);
  • SEQ ID NO:16 BCMA VL sequence (rabbit source);
  • SEQ ID NO:17 CD19 VH sequence
  • SEQ ID NO:18 CD19 VL sequence
  • SEQ ID NO:19 Amino acid sequence of scFv in Loop CD19-BCMA structure
  • SEQ ID NO:20 Amino acid sequence of scFv in Loop BCMA-CD19 structure
  • SEQ ID NO:21 Amino acid sequence of hinge region
  • SEQ ID NO:22 Amino acid sequence of transmembrane region
  • SEQ ID NO:23 Amino acid sequence of intracellular signaling region
  • SEQ ID NO:24 Amino acid sequence of costimulatory signaling region
  • SEQ ID NO:25 Amino acid sequence of guide peptide
  • SEQ ID NO:26 Amino acid sequence of Tan CD19-BCMA structure
  • SEQ ID NO:27 Amino acid sequence of Tan BCMA-CD19 structure
  • SEQ ID NO:28 Amino acid sequence of Loop CD19-BCMA structure
  • SEQ ID NO:29 Amino acid sequence of Loop BCMA-CD19 structure
  • SEQ ID NO:30 Amino acid sequence of CD19-2A-BCMA structure
  • SEQ ID NO:31 Amino acid sequence of BCMA-2A-CD19 structure
  • SEQ ID NO:32 Amino acid sequence of CD19 CAR structure
  • SEQ ID NO:33 Amino acid sequence of BCMA CAR structure
  • SEQ ID NO:34 connection sequence
  • SEQ ID NO:35 connection sequence
  • SEQ ID NO:36 connection sequence
  • SEQ ID NO:37 Nucleotide sequence encoding the BCMA VH amino acid sequence shown in SEQ ID NO:13;
  • SEQ ID NO:38 Nucleotide sequence encoding the BCMA VL amino acid sequence shown in SEQ ID NO:14;
  • SEQ ID NO:39 Nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:17;
  • SEQ ID NO:40 Nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:18.

Abstract

本发明提供了靶向BCMA-CD19的双特异性嵌合抗原受体及其应用，该双特异性嵌合抗原受体包含胞外抗原识别结构域；其中：该胞外抗原识别结构域包含抗BCMA胞外抗原识别结构域和抗CD19胞外抗原识别结构域；该抗BCMA胞外抗原识别结构域包括BCMA VH和BCMA VL，其中BCMA VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列，BCMA VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示的氨基酸序列。

Description

靶向BCMA-CD19的双特异性嵌合抗原受体及其应用 技术领域
本申请涉及生物医药领域，具体涉及一种靶向BCMA-CD19的双特异性嵌合抗原受体及其应用。
 背景技术
多发性骨髓瘤被定义为骨髓中浆细胞的恶性增殖，它是第二大常见的血液恶性肿瘤，占所有癌种的1％。研究表明，多发性骨髓瘤在60岁以上老年人中高发且近年来发病率稳步上升。对于大多数患者而言，多发性骨髓瘤是不可治愈的，最终都会发展成为复发/难治性多发性骨髓瘤。现有多发性骨髓瘤治疗方法(例如免疫调节剂、蛋白酶体抑制剂、抗体类药物)均无效的复发/难治性多发性骨髓瘤患者的存活期仅约为13个月。
B细胞成熟抗原(B Cell Maturation Antigen，缩写为BCMA)是一个跨膜糖蛋白，它属于肿瘤坏死因子受体家族。BCMA在多发性骨髓瘤细胞上高度表达，在大多数其他细胞上不表达。恶性的肿瘤浆细胞通常都比正常的浆细胞BCMA表达水平要高，BCMA的上调促进了多发性骨髓瘤癌细胞的生长，而它的表达下调能够抑制多发性骨髓瘤癌细胞的生长。
另外，多发性骨髓瘤作为B细胞系肿瘤一般不表达CD19分子，因此CD19通常不作为多发性骨髓瘤治疗的靶点。但也有文献研究提示，一些微量的具有耐药性和复发性的多发性骨髓瘤克隆，也具有CD19+表型。
嵌合抗原受体(Chimeric Antigen Receptor，CAR)是CAR细胞治疗药物的核心部件，其可包括靶向部分(例如，结合肿瘤相关抗原(Tumor-Associated Antigen，TAA)的部分)、铰链区、跨膜区和细胞内结构域。CAR-T细胞免疫疗法，被认为是最有希望攻克肿瘤的手段之一。CAR-T细胞就是利用基因改造的方法使T细胞表达CAR蛋白，这种CAR蛋白有能力在不依赖于抗原提呈的情况下识别膜表面的完整蛋白，进而引起T细胞的活化和功能效应。
2021年百时美施贵宝与蓝鸟生物共同宣布美国食品药品监督管理局(FDA)已批准其靶向BCMA的CAR-T细胞疗法(bb2121)，用于4线治疗后(包括免疫调节剂、蛋白酶体抑制剂以及抗体类药物治疗)的复发或难治性多发性骨髓瘤的成年患者，这是全球首款靶向BCMA的CAR-T细胞疗法。开发更多的靶向BCMA的细胞治疗方法具有现实意义。
发明内容
本申请提供了一种靶向BCMA-CD19的双特异性嵌合抗原受体及其应用，发明人构建了多个靶向BCMA-CD19的双特异性嵌合抗原受体表达载体并制备了靶向BCMA-CD19的双特异性CAR-T细胞，还在细胞水平验证了BCMA-CD19双特异性CAR-T细胞具有良好的抑瘤功能。
一种靶向BCMA-CD19的双特异性嵌合抗原受体，其包含胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域；其中：所述胞外抗原识别结构域包含抗BCMA胞外抗原识别结构域和抗CD19胞外抗原识别结构域；
所述抗BCMA胞外抗原识别结构域包括BCMA VH和BCMA VL，其中BCMA VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列，BCMA VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述抗CD19胞外抗原识别结构域包括CD19 VH和CD19 VL，其中CD19 VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示的氨基酸序列，CD19 VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述BCMA VH序列包括如SEQ ID NO:13所示的氨基酸序列，所述BCMA VL序列包括如SEQ ID NO:14所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述BCMA VH序列包括如SEQ ID NO:15所示的氨基酸序列，所述BCMA VL序列包括如SEQ ID NO:16所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述CD19 VH序列包括如SEQ ID NO:17所示的氨基酸序列，所述CD19 VL序列包括如SEQ ID NO:18所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述双特异性嵌合抗原受体的胞外抗原识别结构包括选自以下结构中的任意一种：CD19 VL序列-第1连接序列-CD19 VH序列-第2连接序列-BCMA VL序列-第3连接序列-BCMA VH序列，BCMA VL序列-第4连接序列-BCMA VH序列-第5连接序列-CD19 VL序列-第6连接序列-CD19 VH序列，BCMA VL序列-第7连接序列-CD19 VL序列-第8连接序列-CD19 VH序列-第9连接序列-BCMA VH序列，和CD19 VL序列-第10连接序列-BCMA VL序列-第11连接序列-BCMA VH序列-第12连接序列-CD19 VH序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述双特异性嵌合抗原受体的胞外抗原识别结构域包括选自以下结构中的任意一种：BCMA VL序列-第7连接序列-CD19 VL序列-第8连接序列-CD19 VH序列-第9连接序列-BCMA VH序列，和CD19 VL序列-第10连接序列-BCMA VL序列-第11连接序列-BCMA VH序列-第12连接序列-CD19 VH序列。
上述双特异性嵌合抗原受体在某些实施方式中，第1连接序列、第2连接序列、第3连接序列、第4连接序列、第5连接序列、第6连接序列、第7连接序列、第8连接序列、第9连接序列、第10连接序列、第11连接序列、第12连接序列相互独立得选自以下序列的一种或多种：SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36。
上述双特异性嵌合抗原受体在某些实施方式中，所述双特异性嵌合抗原受体的胞外抗原识别结构域包括如SEQ ID NO:19或SEQ ID NO:20所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述铰链区来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种；可选地，所述铰链区的氨基酸来源于CD8α；进一步可选地，所述铰链区的氨基酸序列包含如SEQ ID NO:21所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述跨膜区来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种；可选地，所述跨膜区的氨基酸序列来源于CD8α；进一步可选地，所述跨膜区的氨基酸序列包含如SEQ ID NO:22所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，其中所述细胞内结构域包含胞内信号传导区；可选地，还包括共刺激信号传导区。
上述双特异性嵌合抗原受体在某些实施方式中，其中所述胞内信号传导区来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种；可选地，所述胞内信号传导区来源于CD3δ；进一步可选地，所述胞内信号传导区的氨基酸序列包含如SEQ ID NO:23所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，其中所述共刺激信号传导区来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上；可选地，所述共刺激信号传导区来源于CD28或4-1BB；进一步可选地，所述共刺激信号传导区的氨基酸序列包含如SEQ ID NO:24所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，还包含位于所述嵌合抗原受体氨基酸序列N-末端的引导肽；可选地，其中所述引导肽来源于CD8α；进一步可选地，所述引导肽的氨基酸序列包含如SEQ ID NO:25所示的氨基酸序列。
上述双特异性嵌合抗原受体在某些实施方式中，所述双特异性嵌合抗原受体包括如SEQ ID NO:28或SEQ ID NO:29所示的氨基酸序列。
本申请还提供了一种分离的核酸分子，其包含编码上述双特异性嵌合抗原受体的核苷酸序列。
上述分离的核酸分子在某些实施方式中，编码所述双特异性嵌合抗原受体的核苷酸序列包含：
1)编码如SEQ ID NO:13所示的BCMA VH氨基酸序列的核苷酸序列，其如SEQ ID NO:37所示；和编码如SEQ ID NO:14所示的BCMA VL氨基酸序列的核苷酸序列，其如SEQ ID NO:38所示；和/或
2)编码如SEQ ID NO:17所示的CD19 VH氨基酸序列的核苷酸序列，其如SEQ ID NO:39所示；和编码如SEQ ID NO:18所示的CD19 VL氨基酸序列的核苷酸序列，其如SEQ ID NO:40所示。
本申请还提供了一种载体，其包含上述分离的核酸分子。
上述载体在某些实施方式中，为表达载体；在某些实施方式中，载体为病毒载体；在某些实施方式中，载体为慢病毒载体。
本申请还提供了一种经工程化的免疫效应细胞，其包含上述嵌合抗原受体、上述分离的核酸分子，或上述载体。
上述经工程化的免疫效应细胞在某些实施方式中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞、诱导性多能干细胞(Induced pluripotent stem cells,iPSC)、诱导性多能干细胞分化成的T细胞(iPSC-T)、诱导性多能干细胞分化成的NK细胞(iPSC-NK)和胚胎干细胞中的一种或多种。
上述经工程化的免疫效应细胞在某些实施方式中，所述经工程化的免疫效应细胞是T淋巴细胞；可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
本申请还提供了一种药物组合物，其包括上述经工程化的免疫效应细胞和药学上可接受的辅料。
上述药物组合物在某些实施方式中，药学上可接受的辅料包括保护剂。
上述药物组合物在某些实施方式中，药学上可接受的辅料包括细胞冻存液。
上述药物组合物在某些实施方式中，所述药物组合物为静脉注射剂。
本申请还提供上述嵌合抗原受体、分离的核酸分子、载体或经工程化的免疫效应细胞在制备药物中的应用，所述药物用于治疗与BCMA的表达相关的疾病或病症。
上述应用在某些实施方式中，与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
上述应用在某些实施方式中，所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病。
上述应用在某些实施方式中，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
本申请还提供了一种治疗与BCMA的表达相关的疾病或病症的方法，包括以下步骤：将有效量的上述经工程化的免疫效应细胞或药物组合物施用于具有治疗与BCMA的表达相关的疾病或病症的需求的受试者。
上述方法在某些实施方式中，与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
上述方法在某些实施方式中，所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病。
上述方法在某些实施方式中，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
上述方法在某些实施方式中，所述施用的方式为静脉注射。
上述方法在某些实施方式中，所述施用的方式为将有效量的经工程化的免疫效应细胞或药物组合物以单次注射的方式施用于受试者。
上述方法在某些实施方式中，有效量的经工程化的免疫效应细胞或药物组合物为1×105至1×107个细胞/kg的剂量。
本申请还提供了上述经工程化的免疫效应细胞或上述药物组合物，用于治疗与BCMA的表达相关的疾病或病症。
上述经工程化的免疫效应细胞或上述药物组合物在某些实施方式中，与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
上述经工程化的免疫效应细胞或上述药物组合物在某些实施方式中，所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病。
上述经工程化的免疫效应细胞或上述药物组合物在某些实施方式中，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
 附图说明
图1表示了本申请实施例1中各种BCMA-CD19双特异性CAR、CD19 CAR、BCMA CAR的结构示意图。
图2A表示了本申请实施例2中自裂解多肽T2A连接的两个完整CAR(即CD19-2A-BCMA和BCMA-2A-CD19)在CAR-T细胞表面的CAR表达情况(感染后6天)；其中：图2A左图是CD19-2A-BCMA组、图2A中图是BCMA-2A-CD19组、图2A右图是UTD组(未转导CAR的T细胞)；图2B-图2E表示了本申请实施例2中4种双特异性CAR-T细胞(即Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞)表面的CAR表达情况(感染后6天)；其中：图2B是Tan CD19-BCMA组、图2C是Tan BCMA-CD19组、图2D是Loop CD19-BCMA组、图2E是Loop BCMA-CD19细胞组。
图3A-图3C表示了本申请实施例4中各种BCMA-CD19双特异性CAR、CD19 CAR、BCMA CAR被阳性靶细胞激活后的细胞因子释放情况；其中：图3A是各组IL-2的释放情况、图3B是各组IFN-γ的释放情况、图3C是各组TNF-α的释放情况。其中，在图3A、图3B和图3C中均包含两个虚线框，在图3A、图3B和图3C中的第一个虚线框中，从左到右5个柱依次代表UTD细胞、TanCD19-BCMA细胞、TanBCMA-CD19细胞、LoopCD19-BCMA细胞、LoopBCMA-CD19细胞和BCMA细胞被K562-BCMA细胞激活后的细胞因子释放情况；在图3A、图3B和图3C中的第二个虚线框中，从左到右5个柱依次代表UTD细胞、TanCD19-BCMA细胞、TanBCMA-CD19细胞、LoopCD19-BCMA细胞、LoopBCMA-CD19细胞和CD19细胞被K562-CD19细胞激活后的细胞因子释放情况。
图4A-图4C表示了本申请实施例5中各种BCMA-CD19双特异性CAR、CD19 CAR、BCMA CAR细胞对不同靶细胞的杀伤效果；其中：图4A是NALM6组，NALM6是CD19+BCMA-靶细胞；图4B是MM.1S组，MM.1S是BCMA+CD19-靶细胞；图4C是NALM6-KO CD19组，NALM6-KO CD19是敲除CD19同时也不表达BCMA的阴性靶细胞对照。
图5A-图5D表示了本申请实施例6中抗原多轮刺激之后各组CAR-T细胞的持续增殖情况；其中：图5A是用MM.1S细胞刺激的CD3+细胞的增殖情况，图5B是用NALM6细胞刺激的CD3+细胞的增殖情况，图5C是用MM.1S细胞刺激的CAR+细胞的增殖情况，图5D是用NALM6细胞刺激的CAR+细胞的增殖情况。
 具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)是CAR细胞治疗药物的核心部件，其可包括胞外抗原识别结构域(例如，结合肿瘤相关抗原(Tumor-Associated Antigen，TAA)的部分)、铰链区、跨膜区和细胞内结构域。CAR-T(Chimeric Antigen Receptor T)细胞免疫疗法，被认为是最有希望攻克肿瘤的手段之一。CAR-T细胞就是利用基因改造的方法使T细胞表达CAR蛋白，这种CAR蛋白有能力在不依赖于抗原提呈的情况下识别膜表面的完整蛋白，进而引起T细胞的活化和功能效应。
在本申请中，术语“胞外抗原识别结构域”是指抗原识别结构域(Antigen Recognition Domain，ARD)。CAR细胞治疗产品(如CAR-T细胞)之所以能特异性识别和/或结合到肿瘤细胞表达的靶抗原，依赖于胞外抗原识别结构域，到目前为止，抗原识别结构域从抗体的单链可变区(Single Chain Variable Fragment，缩写为scFv)、或者从受体配体相互作用、TCR模拟物、可变的淋巴细胞受体(Variable Lymphocyte Receptors，VLR)衍生而来。到目前为止，最为常见的来源就是抗体的scFv段，scFv包括抗体重链可变区(VH区)和轻链可变区(VL区)，他们之间由一段肽链连接，如：由18个氨基酸组成的连接序列GSTSGSGKPGSGEGSTKG。针对二个或大于二个靶点的scFv抗体中包括针对不同靶点的VH区和VL区，不同区域之间通过连接序列直接连接或间接连接，其排列方式可以通过以下形式的任意一种：靶点1 VL-靶点1 VH-靶点2 VL-靶点2 VH，靶点2 VL-靶点2 VH-靶点1 VL-靶点1 VH，靶点1 VL-靶点2 VL-靶点2 VH-靶点1 VH，靶点2 VL-靶点1 VL-靶点1 VH-靶点2 VH，上述“-”代表通过连接序列连接。
本申请中，术语“特异性识别和/或结合”是指CAR和特异性靶标之间的识别和/或结合，是以比CAR结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标。
在本申请中，术语“铰链区”是指作用于胞外抗原识别结构域与跨膜结构域之间的连接段，这个区域通过给予抗原识别结构域一定的活动范围，允许CAR识别抗原。目前使用的铰链区主要来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种。此外，典型的铰链区还包含一些残基，这些残基参与CAR二聚化，有助于增强抗原的敏感性。
在本申请中，“跨膜区”是指连接着CAR结构的细胞内和细胞外成分的跨膜结构域。不同的跨膜结构域可以一定程度上影响CAR的表达和稳定性，但是并不直接参与信号传递，通过相互作用可以提高下游信号传递。所述跨膜区可以来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种。
在本申请内，术语“细胞内结构域”包括胞内信号传导区，还可以包括共刺激信号传导区。
在本申请中，术语“胞内信号传导区”是指负责表达CAR的免疫效应细胞的至少一种正常效应子功能的活化。所述胞内信号传导区可以来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种。
在本申请中，术语“共刺激信号传导区”之所以存在，是因为除了抗原特异性信号的刺激之外，很多免疫效应细胞还需要共刺激来促进细胞增殖、分化和存活，以及活化细胞的效应子功能。在一些实施例中，CAR还可以包括一个或多个共刺激信号传导区，其中，共刺激信号传导区可以来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除从天然状态下经人工手段获得后，经过人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“引导肽”，是指胞外抗原识别结构域(如scFv序列)前的短肽，其作用是引导细胞内合成的重组蛋白质输出到细胞外。常用的引导肽有人CD8α信号肽，或者人GM-CSF受体α信号肽。
在本申请中，决定CAR-免疫细胞治疗效果的关键因素之一是对肿瘤靶抗原的选择。在本申请中，术语“BCMA”是指B细胞成熟抗原，是肿瘤坏死因子受体超家族成员。人BCMA几乎排他性地在浆细胞和多发性骨髓瘤细胞中表达。BCMA可以是针对多发性骨髓瘤的免疫治疗剂的合适肿瘤抗原靶标。但由于多发性骨髓瘤细胞表面的特异性抗原具有异质性，对其抗原靶点的选择不一定是单一的。通过选择合适的靶点，能优化CAR-T细胞的抗肿瘤活性。“CD19”分子目前是治疗B淋巴细胞来源血液系统肿瘤的主要靶点，也是CAR-T细胞治疗研究的热点。大部分B细胞来源的恶性肿瘤细胞表面都表达CD19分子，多发性骨髓瘤作为B细胞系肿瘤一般不表达CD19分子，因此CD19通常不作为多发性骨髓瘤治疗的靶点。但也有文献研究提示，一些微量的具有耐药性和复发性的多发性骨髓瘤克隆，也具有CD19+表型。使用这种双靶点CAR-T产品，只要有一个肿瘤抗原靶点被识别，CAR-T细胞就可被活化，防止肿瘤抗原逃逸现象。同时，相较于分别制备针对不同靶点的CAR-免疫细胞并共同使用，双靶点的CAR免疫细胞具有如下优点：1.需要更少的免疫细胞，制备方便、节约成本；2、从用药方面，施用一种产品的安全性、可操作性要大大高于施用两种产品。
在本申请中，术语“连接序列”通常是指长度为约1至100个氨基酸的寡肽或多肽区，其将本发明的嵌合抗原受体的任何结构/区域连接在一起。连接序列可由不同的氨基酸残基(如甘氨酸和丝氨酸)组成，以便相邻的蛋白质结构域相对于彼此自由移动。当期望确保两个相邻结构域在空间上不相互干扰时，可使用较长的连接序列。
在本申请中，术语“分离的核酸分子”通常指任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，其可以是从其天然环境分离的或人工合成的类似物。
在本申请中，CAR基因转导/转染和靶基因表达时，基因转导/转染方法主要包括病毒和非病毒的方法。如：通过γ反转录病毒载体、慢病毒载体、腺病毒相关病毒载体、质粒DNA依赖的载体、转座子依赖的基因转移、mRNA介导的基因转导。
术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。术语“转座子”是指不连续的DNA片段，具有在染色体位点之间迁移和携带基因信息的能力，如：睡美人SB系统和来源于鳞翅目昆虫的PB系统。在一些实施例中，还可以使用电转的方法将mRNA转导进T细胞。
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，例如促进免疫效应应答的细胞。免疫效应细胞可以选自以下组：T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T淋巴细胞、多能干细胞分化成的NK细胞、诱导性多能干细胞(Induced pluripotent stem cells,iPSC)、诱导性多能干细胞分化成的T细胞(iPSC-T)、诱导性多能干细胞分化成的NK细胞(iPSC-NK)和胚胎干细胞中的一种或多种。
在本申请中，术语“药物组合物”通常指适合施用于患者的药物组合物，其可以包含本申请所述的免疫效应细胞，还可以包含一种或多种药学上可接受的辅料，如：载剂、保护剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂、防腐剂中的一种或多种。在一些实施例中，药学上可接受的辅料包括保护剂，如：细胞冻存液。在一些实施例中，本申请的药物组合物为细胞悬液或其冻存细胞。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于小鼠、大鼠、猫、狗、兔、马、猪、牛、羊或猴。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下本领域技术人员可接受的波动范围，如：在±0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％或10％的范围内变动。
嵌合抗原受体、核酸、载体、免疫效应细胞、药物组合物
一方面，本申请提供一种靶向BCMA-CD19的双特异性嵌合抗原受体，其包含胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域；其中：所述胞外抗原识别结构域包含抗BCMA胞外抗原识别结构域和抗CD19胞外抗原识别结构域；
所述抗BCMA胞外抗原识别结构域包括BCMA VH和BCMA VL，其中BCMA VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列，BCMA VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示的氨基酸序列。
抗体中，常见的划分CDR规则有Kabat、AbM、Chothia、Contact、IMGT，这些规则为本领域技术人员熟知的，当应用执行这些规则的网站时，只要将VH和VL序列输入并选择相应规则，即可得到依据不同规则的CDR序列。本申请中，采用IMGT规则划分CDR，但本领域技术人员应当理解，本申请的保护范围涵盖了通过采用不同规则分析获得的CDR序列的组合。
在一些实施例中，所述抗CD19胞外抗原识别结构域包括CD19 VH和CD19 VL，其中CD19 VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示的氨基酸序列，CD19 VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12所示的氨基酸序列。
在一些实施例中，所述BCMA VH序列包括如SEQ ID NO:13所示的氨基酸序列，所述BCMA VL序列包括如SEQ ID NO:14所示的氨基酸序列。
在一些实施例中，所述BCMA VH序列包括如SEQ ID NO:15所示的氨基酸序列，所述BCMA VL序列包括如SEQ ID NO:16所示的氨基酸序列。
在一些实施例中，所述CD19 VH序列包括如SEQ ID NO:17所示的氨基酸序列，所述CD19 VL序列包括如SEQ ID NO:18所示的氨基酸序列。
在一些实施例中，本申请还包含了上述任一项双特异性嵌合抗原受体的氨基酸序列中的一个或多个氨基酸被取代、缺失、添加和/或插入，且其具有相当于上述任一项嵌合抗原受体的活性。本领域技术人员知晓，在人源化改造的过程中，VH和VL序列中FR区中的氨基酸可被取代使得经过改造的抗体的CDR区仍能保有合适的抗原结合位点，因此本申请当然包括在基于本申请上述CDR的情况下，对VH和VL序列中的FR区进行人源化改造所获得的不同的氨基酸序列。并且本领域技术人员还知晓，在人源化改造的过程中为了使得经过改造的抗体的CDR区能保有合适的抗原结合位点，如果必要，CDR中的1个、2个、3个或不超过10％的氨基酸序列可能被取代、缺失、添加和/或插入，这些内容也被包含在本申请中。
在一些实施例中，所述双特异性嵌合抗原受体的胞外抗原识别结构域包括选自以下结构中的任意一种：CD19 VL序列-第1连接序列-CD19 VH序列-第2连接序列-BCMA VL序列-第3连接序列-BCMA VH序列，BCMA VL序列-第4连接序列-BCMA VH序列-第5连接序列-CD19 VL序列-第6连接序列-CD19 VH序列，BCMA VL序列-第7连接序列-CD19 VL序列-第8连接序列-CD19 VH序列-第9连接序列-BCMA VH序列，和CD19 VL序列-第10连接序列-BCMA VL序列-第11连接序列-BCMA VH序列-第12连接序列-CD19 VH序列。
在一些实施例中，所述双特异性嵌合抗原受体的胞外抗原识别结构域包括选自以下结构中的任意一种：BCMA VL序列-第7连接序列-CD19 VL序列-第8连接序列-CD19 VH序列-第9连接序列-BCMA VH序列，和CD19 VL序列-第10连接序列-BCMA VL序列-第11连接序列-BCMA VH序列-第12连接序列-CD19 VH序列。
在一些实施例中，第1连接序列、第2连接序列、第3连接序列、第4连接序列、第5连接序列、第6连接序列、第7连接序列、第8连接序列、第9连接序列、第10连接序列、第11连接序列、第12连接序列相互独立得选自以下序列的一种或多种：SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36。
在一些实施例中，所述双特异性嵌合抗原受体的胞外抗原识别结构域包括如SEQ ID NO:19或SEQ ID NO:20所示的氨基酸序列。
在一些实施例中，所述铰链区来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种；可选地，所述铰链区的氨基酸序列来源于CD8α；进一步可选地，所述铰链区的氨基酸序列包含如SEQ ID NO:21所示的氨基酸序列。
在一些实施例中，所述跨膜区来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种；可选地，所述跨膜区的氨基酸序列来源于CD8α；进一步可选地，所述跨膜区的氨基酸序列包含如SEQ ID NO:22所示的氨基酸序列。
在一些实施例中，其中所述细胞内结构域包含胞内信号传导区；可选地，还包括共刺激信号传导区；进一步可选地，其中所述胞内信号传导区来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种；再更进一步可选地，所述胞内信号传导区来源于CD3δ，如：所述胞内信号传导区的氨基酸序列包含如SEQ ID NO:23所示的氨基酸序列。
在一些实施例中，其中所述共刺激信号传导区来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上；可选地，共刺激信号传导区来源于CD28或4-1BB；进一步可选地，所述共刺激信号传导区的氨基酸序列包含如SEQ ID NO:24所示的氨基酸序列。
在一些实施例中，还包含位于所述嵌合抗原受体氨基酸序列N-末端的引导肽；可选地，其中所述引导肽来源于CD8α；进一步可选地，所述引导肽的氨基酸序列包含如SEQ ID NO:25所示的氨基酸序列。
在一些实施例中，所述双特异性嵌合抗原受体包括如SEQ ID NO:28或SEQ ID NO:29所示的氨基酸序列。
另一方面，本申请还提供了一种分离的核酸分子，其包含编码上述双特异性嵌合抗原受体的核苷酸序列。
在一些实施例中，编码所述双特异性嵌合抗原受体的核苷酸序列包含：
1)编码如SEQ ID NO:13所示的BCMA VH氨基酸序列的核苷酸序列，其如SEQ ID NO:37所示；和编码如SEQ ID NO:14所示的BCMA VL氨基酸序列的核苷酸序列，其如SEQ ID NO:38所示；和/或
2)编码如SEQ ID NO:17所示的CD19 VH氨基酸序列的核苷酸序列，其如SEQ ID NO:39所示；和编码如SEQ ID NO:18所示的CD19 VL氨基酸序列的核苷酸序列，其如SEQ ID NO:40所示。
另一方面，本申请还提供了一种载体，其包含上述分离的核酸分子。载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。
在一些实施例中，载体为表达载体；可选地，载体为病毒载体；进一步可选地，载体为慢病毒载体。
另一方面，本申请还提供了一种经工程化的免疫效应细胞，其包含上述嵌合抗原受体、上述分离的核酸分子，或上述载体。
在一些实施例中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞、诱导性多能干细胞(Induced pluripotent stem cells,iPSC)、诱导性多能干细胞分化成的T细胞(iPSC-T)、诱导性多能干细胞分化成的NK细胞(iPSC-NK)和胚胎干细胞中的一种或多种。
在一些实施例中，所述经工程化的免疫效应细胞是T淋巴细胞；可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
在一些实施例中，所述经工程化的免疫效应细胞的表面可以表达或表达有本申请所述的嵌合抗原受体。
另一方面，本申请还提供了一种药物组合物，其包括上述经工程化的免疫效应细胞和药学上可接受的辅料。药学上可接受的辅料包括：载剂、保护剂、稳定剂、稀释剂、中的一种或多种。
在一些实施例中，药学上可接受的辅料包括保护剂，如：细胞冻存液。
在一些实施例中，药物组合物为细胞悬液或其冻存细胞。
在一些实施例中，药物组合物为静脉注射剂。
制备方法
另一方面，本申请还提供了制备经工程化的免疫效应细胞的方法，其包括以下的步骤：向免疫效应细胞中转导本申请所述的载体。
在一些实施例中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞、诱导性多能干细胞(Induced pluripotent stem cells,iPSC)、诱导性多能干细胞分化成的T细胞(iPSC-T)、诱导性多能干细胞分化成的NK细胞(iPSC-NK)和胚胎干细胞中的一种或多种。
在一些实施例中，所述经工程化的免疫效应细胞是T淋巴细胞；可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
用途
另一方面，本申请还提供了本申请所述的双特异性嵌合抗原受体、所述的分离的核酸分子、所述的载体和/或所述的经工程化的免疫效应细胞在制备药物中的应用，其中所述药物用于治疗与BCMA的表达相关的疾病或病症。
在一些实施例中，与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
在一些实施例中，所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病。
在一些实施例中，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
另一方面，本申请还提供了治疗与BCMA的表达相关的疾病或病症的方法，所述方法包括以下步骤：向有治疗与BCMA的表达相关的疾病或病症需要的受试者施用有效剂量的本申请所述的嵌合抗原受体、所述的分离的核酸分子、所述的载体和/或所述的经工程化的免疫效应细胞。
在一些实施例中，与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
在一些实施例中，所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病。
在一些实施例中，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
在一些实施例中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。例如，施用的方式可以通过静脉注射的方式施用于受试者。在一些实施例中，有效剂量的经工程化的免疫效应细胞或药物组合物可以单次施用于受试者，也可以在一定期间内分次施用于受试者，如：每周施用一次、两周一次、三周一次、四周一次、一个月一次、3个月一次、或3-6个月一次。
在一些实施例中，针对不同的适应症，给药剂量可以不同；针对病情严重程度不同的患者，给药剂量也可以不同。施用剂量范围可以是1×105个CAR阳性T细胞/kg至1×107个CAR阳性T细胞/kg，例如，1×105个CAR阳性T细胞/kg至1×106个CAR阳性T细胞/kg、1×106个CAR阳性T细胞/kg至1×107个CAR阳性T细胞/kg、0.5×106个CAR阳性T细胞/kg、0.6×106个CAR阳性T细胞/kg、0.7×106个CAR阳性T细胞/kg、0.8×106个CAR阳性T细胞/kg、0.9×106个CAR阳性T细胞/kg、1.0×106个CAR阳性T细胞/kg、1.1×106个CAR阳性T细胞/kg、1.2×106个CAR阳性T细胞/kg、1.3×106个CAR阳性T细胞/kg、1.4×106个CAR阳性T细胞/kg、1.5×106个CAR阳性T细胞/kg、1.6×106个CAR阳性T细胞/kg、1.7×106个CAR阳性T细胞/kg、1.8×106个CAR阳性T细胞/kg、1.9×106个CAR阳性T细胞/kg、2.0×106个CAR阳性T细胞/kg。
在一些实施例中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于小鼠、大鼠、猫、狗、马、猪、牛、羊、兔或猴。
另一方面，本申请还提供了所述的嵌合抗原受体、所述的分离的核酸分子、所述的载体和/或所述的经工程化的免疫效应细胞，其可以用于治疗与BCMA的表达相关的疾病或病症。
在一些实施例中，所述与BCMA的表达相关的疾病或病症可以包括非实体瘤，可选地，所述非实体瘤为血液瘤。
在一些实施例中，所述与BCMA的表达相关的疾病或病症可以包括多发性骨髓瘤。
在一些实施例中，所述多发性骨髓瘤为复发性或难治性多发性骨髓瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的嵌合抗原受体、经工程化的免疫效应细胞、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press。
实施例1、BCMA-CD19双特异性CAR-T细胞的获得
我们已有1条BCMA特异性的人源化抗体(其VH的氨基酸序列如SEQ ID NO:13所示，其VH的核苷酸序列如SEQ ID NO:37所示，其VL的氨基酸序列如SEQ ID NO:14所示，其VL的核苷酸序列如SEQ ID NO:38所示，其VH CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示，其VL CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示)和1条CD19特异性的抗体(其VH的氨基酸序列如SEQ ID NO:17所示，其VH的核苷酸序列如SEQ ID NO:39所示，其VL的氨基酸序列如SEQ ID NO:18所示，其VL的核苷酸序列如SEQ ID NO:40所示，其VH CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示，其VL CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12所示)，希望获得较优的双特异性CAR结构。考虑到靶点BCMA和CD19的前后顺序、每个靶点胞外抗原识别结构域中VH和VL的前后顺序等变化，BCMA-CD19双特异性CAR的结构设计有多种选择。我们选择在二代CAR结构上对候选BCMA-CD19双特异性CAR结构进行筛选。
我们尝试了如图1所示的6种BCMA-CD19双特异性CAR结构，并以BCMA CAR结构和CD19 CAR结构作为对照。在各个CAR结构中，均采用了CD8α引导链为信号肽(如SEQ ID NO:25所示)，铰链区(如SEQ ID NO:21所示)和跨膜区(如SEQ ID NO:22所示)采用CD8α的结构，以4-1BB为胞内共刺激信号(如SEQ ID NO:24所示)，CD3δ为T细胞激活信号(如SEQ ID NO:23所示)。
1、慢病毒载体的构建
分别人工合成如图1所示的6种BCMA-CD19双特异性CAR结构以及BCMA CAR结构和CD19 CAR结构作为对照，Tan CD19-BCMA结构、Tan BCMA-CD19结构、Loop CD19-BCMA结构、Loop BCMA-CD19结构、CD19-2A-BCMA结构、BCMA-2A-CD19结构、CD19 CAR结构、BCMA CAR结构的氨基酸序列分别如SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33所示。如下文字表述仅为简单概要的展示CAR结构主要区别部分，未全面展示信号肽、连接序列、铰链区、跨膜区、胞内共刺激信号区、T细胞激活信号区，各结构的顺序从图1中可以看出：SP表示信号肽区，Hinge代表铰链区，TM代表跨膜区，VL、VH之间的是连接序列。
1)Tan CD19-BCMA结构：CD19 VL-CD19 VH-BCMA VL-BCMA VH；
2)Tan BCMA-CD19结构：BCMA VL-BCMA VH-CD19 VL-CD19 VH；
3)Loop CD19-BCMA结构：BCMA VL-CD19 VL-CD19 VH-BCMA VH；
4)Loop BCMA-CD19结构：CD19 VL-BCMA VL-BCMA VH-CD19 VH；
5)CD19-2A-BCMA结构：CD19 CAR-T2A-BCMA CAR(该结构的自裂解多肽T2A前后，分别有一个完整的CD19 CAR结构和BCMA CAR结构，完整的CAR结构指包括与其他组相同的信号肽、铰链区、跨膜区、胞内共刺激信号区、T细胞激活信号区)；
6)BCMA-2A-CD19结构：BCMA CAR-T2A-CD19 CAR(该结构的自裂解多肽T2A前后，分别有一个完整的BCMA CAR结构和CD19 CAR结构，完整的CAR结构指包括与其他组相同的信号肽、铰链区、跨膜区、胞内共刺激信号区、T细胞激活信号区)；
7)CD19 CAR结构：CD19 VL-CD19 VH；
8)BCMA CAR结构：BCMA VL-CD19 VH。
将上述6种BCMA-CD19双特异性CAR结构、1种CD19 CAR结构、1种BCMA CAR结构分别构建到经过改造的空慢病毒载体(厂家：SBI公司，货号：CD500-CD800，如WO2021/121227实施例1中记载的进行常规抗性改造)中获得CAR表达载体，随后将CAR表达载体和三种包装质粒一起转染293T细胞，经过收集纯化之后得到有功能性的慢病毒载体。三种包装质粒分别是pMD2.G(购自Biovector公司，产品号Biovector012259)，pMDLg/pRRE(购自Biovector公司，产品号Biovector012251)，pRSV-Rev(购自Biovector公司，产品号Biovector012253)。
2、通过慢病毒转导的方式制备相应6种BCMA-CD19双特异性CAR-T细胞、1种CD19 CAR-T细胞、1种BCMA CAR-T细胞。
转导实验按照本领域技术人员已知的常规方法进行，简述转导步骤如下：
1)分选T细胞
从人单采血细胞中分离获得外周血单个核细胞(PBMC)，然后从PBMC细胞中分选获得T细胞。
2)对T细胞进行激活处理
将分离的T细胞用完全淋巴细胞培养液(X-VIVO15培养基+5％FBS+300IU/ml IL-2或X-VIVO15培养基+5％FBS+5ng/ml IL-15+10ng/ml IL-7)进行重悬，使终浓度为(1～2)×106个细胞/ml，并加入5～10μl的CD3/CD28磁珠刺激，混匀后置于培养箱培养，培养条件为37℃+5％CO2，培养时间至少24小时。
3)慢病毒转导T细胞
取出激活培养的T细胞，加入终浓度为8μg/ml的聚凝胺(polybrene)，混匀，并按MOI＝2缓慢加入慢病毒载体，混匀后将其置于离心机中，1500rpm，离心1.5小时。然后将其置于培养箱培养，培养条件为37℃+5％CO2，培养时间至少24小时。
4)转导后T细胞的扩增培养
取出转导后的细胞，监测细胞密度，使细胞维持在(0.5～1)×106个细胞/ml，以备后续实施例使用。
使用分别包含有Tan CD19-BCMA结构、Tan BCMA-CD19结构、Loop CD19-BCMA结构、Loop BCMA-CD19结构、CD19-2A-BCMA结构、BCMA-2A-CD19结构、CD19 CAR、BCMA CAR的慢病毒分别感染T细胞后，获得的T细胞分别命名为Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞、CD19-2A-BCMA细胞、BCMA-2A-CD19细胞、CD19细胞和BCMA细胞。接下来，我们在细胞水平对6条双特异性CAR结构进行筛选，以确定各个CAR结构的特点并选出较优的双特异性CAR结构。
实施例2、BCMA-CD19双特异性CAR-T细胞表面表达的CAR分子的检测
对实施例1中获得的6种双特异性CAR-T细胞表面表达的CAR蛋白分子进行检测，我们用PE荧光标记的CD19抗原(厂家：ACRO Biosystems，货号：CD9-HP2H3)和FITC荧光标记的BCMA抗原(厂家：ACRO Biosystems，货号：BCA-HF254)对实施例1中获得的6种BCMA-CD19双特异性CAR-T细胞和UTD细胞(未转导CAR的T细胞)进行染色，并通过流式细胞术进行CAR分子阳性比例检测分析。发明人首先发现：如图2A所示，对于CD19-2A-BCMA细胞和BCMA-2A-CD19细胞，在感染T细胞6天后，T2A后面所连接的CAR分子表达很差，所以CD19-2A-BCMA细胞和BCMA-2A-CD19细胞中的这两种CAR结构首先被淘汰。如图2B、图2C、图2D、图2E所示：在感染T细胞6天后，其他4种双特异性CAR-T细胞Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞的CAR表达率分别为42.98％、66.71％、53.46％、61.63％(以BCMA抗原染色后的结果计，原因详见实施例3)。
实施例3、BCMA-CD19双特异性CAR-T细胞表面CAR分子的检测
发明人尝试了对实施例1所得感染T细胞11天后的双特异性CAR-T细胞进行单独染色(单独染色是指双特异性CAR-T细胞只采用FITC荧光标记的BCMA抗原或PE荧光标记的CD19抗原单独进行染色)和共同染色(共同染色是指双特异性CAR-T细胞既被FITC荧光标记的BCMA抗原染色，也被PE荧光标记的CD19抗原染色)，以选择染色方式。因为BCMA胞外部分和CD19胞外部分构建在一条scFv上，总是共同表达，所以理论上双特异性CAR-T细胞无论采用单独染色还是共同染色时，结果差异应该控制在误差范围内。但我们发现选用不同抗原进行染色对双特异性CAR-T细胞的CAR+细胞计算时有较大影响，结果如表1(尤其是阴影部分)所示：
[根据细则26改正 25.01.2024]
表1、单独染色方式和共同染色方式的考察
以对Tan BCMA-CD19细胞进行CAR+细胞比例表征为例，采用BCMA抗原单独染色时和采用BCMA抗原+CD19抗原共同染色时，通过BCMA抗原检测到的CAR+比例都在误差范围内。但采用CD19抗原单独染色时和采用BCMA抗原+CD19抗原共同染色时，通过CD19抗原检测到数值波动较大，已远远超出误差范围；并且通过CD19抗原检测到的CAR+细胞比例既显著低于通过BCMA抗原检测到的CAR+细胞比例，同时以CD19抗原检测时，单独染色和共同染色时检测到的CAR+细胞比例之间也具有显著差异。同时上述观察也不受CAR结构中BCMA胞外抗原识别结构域和CD19胞外抗原识别结构域相对位置的限制。我们推测：与CD19抗原相比，BCMA抗原更小一些，更容易克服双特异性CAR结构中的空间位阻问题与CAR蛋白结合，所以本申请后续试验中用BCMA抗原检测的数据表征BCMA-CD19双特异性CAR的阳性比例；当然，用共同染色法中BCMA抗原检测的数据同样可以表征BCMA-CD19双特异性CAR的阳性比例，但单独染色法更为简单。
实施例4、BCMA-CD19双特异性CAR-T细胞进行细胞因子释放实验
细胞因子释放实验：将实施例1中获得的4种双特异性CAR-T细胞(Tan CD19-BCMA、Tan BCMA-CD19、Loop CD19-BCMA、Loop BCMA-CD19)、BCMA CAR-T细胞、CD19 CAR-T细胞以及UTD细胞分别与靶细胞按照效靶比1:1的条件在X-VIVO15培养基中共培养24h后，通过ELISA方法检测细胞上清中IL-2、IFN-γ、TNF-α的浓度；其中：K562是BCMA和CD19的双阴性靶细胞、K562-BCMA是外源表达BCMA但不表达CD19的阳性靶细胞，K562-CD19是外源表达CD19但不表达BCMA的阳性靶细胞。由于K562-BCMA细胞不表达CD19抗原，因此未使用其对CD19 CAR-T细胞进行检测，同理由于K562-CD19细胞不表达BCMA抗原，因此未使用其对BCMA CAR-T细胞进行检测(在图3A、图3B、图3C中K562-BCMA组和K562-CD19组各缺少1个bar)。
细胞因子IL-2、IFN-γ、TNF-α的释放实验结果分别如图3A、图3B、图3C所示，图3A、图3B、图3C中箭头指向细胞因子释放水平较低的CAR-T细胞。从图中可以看出：相对于Tan CD19-BCMA结构和Tan BCMA-CD19结构，Loop CD19-BCMA结构和Loop BCMA-CD19结构的双特异性CAR释放各种细胞因子的水平整体较高。尤其是，从Tan CD19-BCMA结构和Tan BCMA-CD19结构的细胞释放因子水平也能看出，Tan CD19-BCMA结构和Tan BCMA-CD19结构中远离细胞膜的scFv受到抗原刺激时，CAR-T细胞释放细胞因子的水平相对较低。
实施例5、BCMA-CD19双特异性CAR-T细胞进行细胞杀伤实验
细胞杀伤实验：将实施例1中获得的4种双特异性CAR-T细胞(Tan CD19-BCMA、Tan BCMA-CD19、Loop CD19-BCMA、Loop BCMA-CD19)、BCMA CAR-T细胞、CD19 CAR-T细胞以及UTD细胞分别与靶细胞按照效应细胞与靶细胞不同效靶比(0:1、1:1、3:1或9:1)的条件在X-VIVO15培养基中共培养4小时后，通过检测靶细胞中稳定表达的荧光素酶活性检测靶细胞杀伤比例，NALM6(人急性淋巴细胞白血病细胞)是CD19+BCMA-靶细胞、MM.1S(人多发性骨髓瘤细胞)是BCMA+CD19-靶细胞，NALM6-KO CD19是敲除CD19同时也不表达BCMA的阴性靶细胞对照。细胞杀伤结果如图4A-图4C所示：在图4A中，Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞、CD19 CAR-T细胞都对内源表达CD19的阳性靶细胞NALM6具有较好的杀伤效果，BCMA CAR-T细胞和UTD细胞对内源表达CD19的阳性靶细胞NALM6不具有杀伤效果；在图4B中，Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞、BCMA CAR-T细胞都对内源表达BCMA的阳性靶细胞MM.1S具有较好的杀伤效果，CD19 CAR-T细胞和UTD细胞对内源表达BCMA的阳性靶细胞MM.1S不具有杀伤效果；在图4C中，所有细胞均对敲除CD19同时也不表达BCMA的阴性靶细胞对照不具有杀伤效果。
实施例6、BCMA-CD19双特异性CAR-T细胞的持续增殖性
抗原刺激可以激活CAR-T细胞使CAR-T细胞增殖，而T细胞的持续激活会导致细胞耗竭，耗竭的T细胞的增殖能力和效应功能都会有所下降，我们通过检测BCMA-CD19双特异性CAR-T细胞在多轮抗原刺激实验后CD3+细胞的增殖情况(即T细胞的增殖情况)、CAR+细胞的增殖情况以及CAR+细胞的比例，验证BCMA-CD19双特异性CAR-T细胞的持续增殖性。
抗原刺激之前，将实施例1中获得的4种双特异性CAR-T细胞(Tan CD19-BCMA、Tan BCMA-CD19、Loop CD19-BCMA、Loop BCMA-CD19)、BCMA CAR-T细胞、CD19 CAR-T细胞的CAR阳性比例都利用UTD调整到了与CAR阳性比例最低的一组CAR-T细胞一致的水平，其中Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞、BCMA CAR-T细胞的CAR阳性比例以BCMA抗原检测的数据为准，CD19 CAR-T细胞的CAR阳性比例以CD19抗原检测的数据为准。在多轮抗原刺激实验中，将各组CAR-T细胞分别与阳性靶细胞按照效靶比1:2在24孔板中进行共培养，每孔2ml X-VIVO15培养基，每组细胞重复3孔。阳性靶细胞分别采用MM.1S和NALM6，相当于分别用BCMA和CD19作为抗原进行多轮刺激以检验不同抗原多轮刺激对BCMA-CD19双特异性CAR-T细胞持续增殖性的影响。由于MM.1S细胞不表达CD19抗原，因此未使用其对CD19 CAR-T细胞进行检测，同理由于NALM6细胞不表达BCMA抗原，因此未使用其对BCMA CAR-T细胞进行检测。
CAR-T细胞与阳性靶细胞共培养3天后取500μl细胞用荧光标记的CD3抗体(厂家：BioLegend，货号：300312)和BCMA抗原、CD19抗原(同实施例2)进行CD3和CAR染色(Tan CD19-BCMA细胞、Tan BCMA-CD19细胞、Loop CD19-BCMA细胞、Loop BCMA-CD19细胞、BCMA CAR-T细胞用BCMA抗原染色，CD19 CAR-T细胞用CD19抗原染色)，通过流式细胞术进行检测分析，显示CD3阳性细胞中CAR阳性细胞的比例和数量，还可以根据体积倍数的换算计算CD3阳性细胞中CAR阳性的细胞数(CD3是区分是否为T细胞的标记物)，然后根据计算结果各组再取出一定量CAR-T细胞按照效靶比1:2加入对应的阳性靶细胞进行新一轮刺激，如此重复进行3-4轮刺激。
多轮抗原刺激实验后，分别用MM.1S和NALM6细胞刺激的CD3+细胞的增殖情况(即T细胞的增殖情况)的结果如图5A、图5B所示；分别用MM.1S和NALM6细胞刺激的CAR+细胞的增殖情况的结果如图5C、图5D所示。如图5A和图5C所示，多轮抗原刺激实验后，用MM.1S细胞刺激的Tan BCMA-CD19细胞中CD3+细胞数(即T细胞的增殖情况)和CAR+细胞数显著低于其他各组CAR-T细胞；如图5B和图5D所示，多轮抗原刺激实验后，用NALM6细胞刺激的Tan CD19-BCMA细胞中CD3+细胞数和CAR+细胞数显著低于其他各组CAR-T细胞；图5A和图5B相比、图5C和图5D相比可以发现，CAR-T细胞受到MM.1S细胞刺激的增殖能力强于受到NALM6细胞刺激的情况。以上说明，Tan结构的BCMA-CD19双特异性CAR-T细胞中远离细胞膜的scFv受到抗原刺激时，CAR-T细胞的持续增殖能力相对较差；而Loop结构CAR-T细胞的持续增殖能力整体强于Tan结构，受到BCMA和CD19两种抗原刺激时都具有持续增殖的能力，其中受到BCMA抗原刺激的增殖能力较强。
综上，在体外药效学检测表明，Tan CD19-BCMA、Tan BCMA-CD19、Loop CD19-BCMA、Loop BCMA-CD19结构在T细胞表面的表达、在细胞因子释放、体外细胞杀伤都有较好的表现；在CAR-T持续增殖性方面，Loop CD19-BCMA、Loop BCMA-CD19结构显著优于Tan CD19-BCMA、Tan BCMA-CD19结构，表现更佳。并且Loop CD19-BCMA、Loop BCMA-CD19结构在各种实验检测中与BCMA CAR-T、CD19 CAR-T的表现比较接近，具有针对双靶点的完整功能。
序列描述
SEQ ID NO:1：BCMA VH CDR1；
SEQ ID NO:2：BCMA VH CDR2；
SEQ ID NO:3：BCMA VH CDR3；
SEQ ID NO:4：BCMA VL CDR1；
SEQ ID NO:5：BCMA VL CDR2(ETS，Glu Thr Ser)；
SEQ ID NO:6：BCMA VL CDR3；
SEQ ID NO:7：CD19 VH CDR1；
SEQ ID NO:8：CD19 VH CDR2；
SEQ ID NO:9：CD19 VH CDR3；
SEQ ID NO:10：CD19 VL CDR1；
SEQ ID NO:11：CD19 VL CDR2(SAT，Ser Ala Thr)；
SEQ ID NO:12：CD19 VL CDR3；
SEQ ID NO:13：BCMA VH序列(人源化)；
SEQ ID NO:14：BCMA VL序列(人源化)；
SEQ ID NO:15：BCMA VH序列(兔源)；
SEQ ID NO:16：BCMA VL序列(兔源)；
SEQ ID NO:17：CD19 VH序列；
SEQ ID NO:18：CD19 VL序列；
SEQ ID NO:19：Loop CD19-BCMA结构中scFv的氨基酸序列；
SEQ ID NO:20：Loop BCMA-CD19结构中scFv的氨基酸序列；
SEQ ID NO:21：铰链区的氨基酸序列；
SEQ ID NO:22：跨膜区的氨基酸序列；
SEQ ID NO:23：胞内信号传导区的氨基酸序列；
SEQ ID NO:24：共刺激信号传导区的氨基酸序列；
SEQ ID NO:25：引导肽的氨基酸序列；
SEQ ID NO:26：Tan CD19-BCMA结构的氨基酸序列；
SEQ ID NO:27：Tan BCMA-CD19结构的氨基酸序列；
SEQ ID NO:28：Loop CD19-BCMA结构的氨基酸序列；
SEQ ID NO:29：Loop BCMA-CD19结构的氨基酸序列；
SEQ ID NO:30：CD19-2A-BCMA结构的氨基酸序列；
SEQ ID NO:31：BCMA-2A-CD19结构的氨基酸序列；
SEQ ID NO:32：CD19 CAR结构的氨基酸序列；
SEQ ID NO:33：BCMA CAR结构的氨基酸序列；
SEQ ID NO:34：连接序列；
SEQ ID NO:35：连接序列；
SEQ ID NO:36：连接序列；
SEQ ID NO:37：编码如SEQ ID NO:13所示的BCMA VH氨基酸序列的核苷酸序列；
SEQ ID NO:38：编码如SEQ ID NO:14所示的BCMA VL氨基酸序列的核苷酸序列；
SEQ ID NO:39：编码如SEQ ID NO:17所示的CD19 VH氨基酸序列的核苷酸序列；
SEQ ID NO:40：编码如SEQ ID NO:18所示的CD19 VL氨基酸序列的核苷酸序列。

Claims (27)

  1. 一种靶向BCMA-CD19的双特异性嵌合抗原受体，其包含胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域；其中：所述胞外抗原识别结构域包含抗BCMA胞外抗原识别结构域和抗CD19胞外抗原识别结构域；
    所述抗BCMA胞外抗原识别结构域包括BCMA VH和BCMA VL，其中BCMA VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列，BCMA VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示的氨基酸序列。
  2. 根据权利要求1所述的双特异性嵌合抗原受体，其中所述抗CD19胞外抗原识别结构域包括CD19 VH和CD19 VL，其中CD19 VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9所示的氨基酸序列，CD19 VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括如SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12所示的氨基酸序列。
  3. 根据权利要求1或2所述的双特异性嵌合抗原受体，其中所述BCMA VH序列包括如SEQ ID NO:13所示的氨基酸序列，所述BCMA VL序列包括如SEQ ID NO:14所示的氨基酸序列；或者
    所述BCMA VH序列包括如SEQ ID NO:15所示的氨基酸序列，所述BCMA VL序列包括如SEQ ID NO:16所示的氨基酸序列。
  4. 根据权利要求2所述的双特异性嵌合抗原受体，其中所述CD19 VH序列包括如SEQ ID NO:17所示的氨基酸序列，所述CD19 VL序列包括如SEQ ID NO:18所示的氨基酸序列。
  5. 根据权利要求1所述的双特异性嵌合抗原受体，其中所述双特异性嵌合抗原受体的胞外抗原识别结构域包括选自以下结构中的任意一种：CD19 VL序列-第1连接序列-CD19 VH序列-第2连接序列-BCMA VL序列-第3连接序列-BCMA VH序列，BCMA VL序列-第4连接序列-BCMA VH序列-第5连接序列-CD19 VL序列-第6连接序列-CD19 VH序列，BCMA VL序列-第7连接序列-CD19 VL序列-第8连接序列-CD19 VH序列-第9连接序列-BCMA VH序列，和CD19 VL序列-第10连接序列-BCMA VL序列-第11连接序列-BCMA VH序列-第12连接序列-CD19 VH序列；
    可选地，所述双特异性嵌合抗原受体的胞外抗原识别结构域包括选自以下结构中的任意一种：BCMA VL序列-第7连接序列-CD19 VL序列-第8连接序列-CD19 VH序列-第9连接序列-BCMA VH序列，和CD19 VL序列-第10连接序列-BCMA VL序列-第11连接序列-BCMA VH序列-第12连接序列-CD19 VH序列；
    进一步可选地，第1连接序列、第2连接序列、第3连接序列、第4连接序列、第5连接序列、第6连接序列、第7连接序列、第8连接序列、第9连接序列、第10连接序列、第11连接序列、第12连接序列相互独立地选自以下序列的一种或多种：SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36。
  6. 根据权利要求5所述的双特异性嵌合抗原受体，其中所述双特异性嵌合抗原受体的胞外抗原识别结构域包括如SEQ ID NO:19或SEQ ID NO:20所示的氨基酸序列。
  7. 根据权利要求1所述的双特异性嵌合抗原受体，其中所述铰链区来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种；可选地，所述铰链区的氨基酸序列来源于CD8α；进一步可选地，所述铰链区的氨基酸序列包含如SEQ ID NO:21所示的氨基酸序列；和/或
    所述跨膜区来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种；可选地，所述跨膜区的氨基酸序列来源于CD8α；进一步可选地，所述跨膜区的氨基酸序列包含如SEQ ID NO:22所示的氨基酸序列。
  8. 根据权利要求1所述的双特异性嵌合抗原受体，其中所述细胞内结构域包含胞内信号传导区；可选地，所述胞内信号传导区来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种；进一步可选地，所述胞内信号传导区来源于CD3δ；再更进一步可选地，所述胞内信号传导区的氨基酸序列包含如SEQ ID NO:23所示的氨基酸序列。
  9. 根据权利要求1所述的双特异性嵌合抗原受体，其中所述细胞内结构域还包括共刺激信号传导区；可选地，所述共刺激信号传导区来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上；进一步可选地，所述共刺激信号传导区来源于CD28或4-1BB；再更进一步可选地，所述共刺激信号传导区的氨基酸序列包含如SEQ ID NO:24所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的双特异性嵌合抗原受体，还包含位于所述嵌合抗原受体氨基酸序列N-末端的引导肽；可选地，所述引导肽来源于CD8α；进一步可选地，所述引导肽的氨基酸序列包含如SEQ ID NO:25所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的双特异性嵌合抗原受体，其中所述双特异性嵌合抗原受体包括如SEQ ID NO:28或SEQ ID NO:29所示的氨基酸序列。
  12. 一种分离的核酸分子，其包含编码权利要求1-11中任一项所述的双特异性嵌合抗原受体的核苷酸序列；
    可选地，编码所述双特异性嵌合抗原受体的核苷酸序列包含：
    1)编码如SEQ ID NO:13所示的BCMA VH氨基酸序列的核苷酸序列，其如SEQ ID NO:37所示；和编码如SEQ ID NO:14所示的BCMA VL氨基酸序列的核苷酸序列，其如SEQ ID NO:38所示；和/或
    2)编码如SEQ ID NO:17所示的CD19 VH氨基酸序列的核苷酸序列，其如SEQ ID NO:39所示；和编码如SEQ ID NO:18所示的CD19 VL氨基酸序列的核苷酸序列，其如SEQ ID NO:40所示。
  13. 一种载体，其包含权利要求12所述的分离的核酸分子；
    可选地，所述载体为表达载体；
    进一步可选地，所述载体为病毒载体；
    再更进一步可选地，所述载体为慢病毒载体。
  14. 一种经工程化的免疫效应细胞，其包含权利要求1-11中任一项所述的嵌合抗原受体、权利要求12所述的分离的核酸，或权利要求13所述的载体。
  15. 根据权利要求14所述的经工程化的免疫效应细胞，其中所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞、诱导性多能干细胞(Induced pluripotent stem cells,iPSC)、诱导性多能干细胞分化成的T细胞(iPSC-T)、诱导性多能干细胞分化成的NK细胞(iPSC-NK)和胚胎干细胞中的一种或多种；
    可选地，所述经工程化的免疫效应细胞是T淋巴细胞；
    进一步可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
  16. 一种药物组合物，其包括权利要求14或15所述的经工程化的免疫效应细胞 和药学上可接受的辅料；可选地，药学上可接受的辅料包括保护剂；可选地，药学上可接受的辅料包括细胞冻存液。
  17. 根据权利要求16所述的药物组合物，其中所述药物组合物为静脉注射剂。
  18. 权利要求1-11中任一项所述的嵌合抗原受体、权利要求12所述的分离的核酸、权利要求13所述的载体或权利要求14或15所述的经工程化的免疫效应细胞在制备药物中的应用，所述药物用于治疗与BCMA的表达相关的疾病或病症。
  19. 根据权利要求18所述的用途，其中所述与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
  20. 根据权利要求18所述的用途，其中所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病；可选地，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
  21. 一种治疗与BCMA的表达相关的疾病或病症的方法，其包括以下步骤：将有效量的权利要求14或15所述的经工程化的免疫效应细胞或权利要求16或17所述的药物组合物施用于具有治疗与BCMA的表达相关的疾病或病症的需求的受试者。
  22. 根据权利要求21所述的方法，其中所述与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
  23. 根据权利要求21所述的方法，其中所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病；可选地，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
  24. 根据权利要求21-23中任一项所述的方法，其中所述施用的方式为静脉注射；
    可选地，所述施用的方式为将有效量的权利要求14或15所述的经工程化的免疫效应细胞或权利要求16或17所述的药物组合物以单次注射的方式施用于受试者；
    进一步可选地，有效量的权利要求14或15所述的经工程化的免疫效应细胞或权利要求16或17所述的药物组合物为1×105至1×107个细胞/kg的剂量。
  25. 权利要求14或15所述的经工程化的免疫效应细胞或权利要求16或17所述的药物组合物，用于治疗与BCMA的表达相关的疾病或病症。
  26. 根据权利要求25所述的经工程化的免疫效应细胞或药物组合物，其中与BCMA的表达相关的疾病或病症为癌症；可选地，所述癌症是多发性骨髓瘤；进一 步可选地，所述癌症是难治性或复发性的多发性骨髓瘤。
  27. 根据权利要求25所述的经工程化的免疫效应细胞或药物组合物，其中所述与BCMA的表达相关的疾病或病症可以是自身免疫疾病；可选地，所述自身免疫疾病可以选自以下：全身性红斑狼疮、类风湿性关节炎、特发性血小板减少性紫癜、重症肌无力和自身免疫性溶血性贫血。
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