WO2023088359A1 - 靶向bcma的嵌合抗原受体及其应用 - Google Patents

靶向bcma的嵌合抗原受体及其应用 Download PDF

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WO2023088359A1
WO2023088359A1 PCT/CN2022/132545 CN2022132545W WO2023088359A1 WO 2023088359 A1 WO2023088359 A1 WO 2023088359A1 CN 2022132545 W CN2022132545 W CN 2022132545W WO 2023088359 A1 WO2023088359 A1 WO 2023088359A1
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cells
seq
bcma
amino acid
optionally
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French (fr)
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白大勇
张云龙
张超
吕璐璐
周立
王永增
王瑞
丁伟
路佳兴
张其猛
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合源生物科技(天津)有限公司
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Definitions

  • the present application relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting BCMA and its application.
  • Multiple myeloma defined as the 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 has a high incidence in the elderly over 60 years old and the incidence has increased steadily in recent years. For most patients, multiple myeloma is incurable and will eventually develop into relapsed/refractory multiple myeloma. The survival period of patients with relapsed/refractory multiple myeloma who are ineffective with existing multiple myeloma treatments (such as immunomodulators, proteasome inhibitors, antibody drugs) is only about 13 months.
  • multiple myeloma treatments such as immunomodulators, proteasome inhibitors, antibody drugs
  • BCMA B Cell Maturation Antigen
  • BCMA B Cell Maturation Antigen
  • a chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is the core component of a CAR cell therapy drug, which may include a targeting moiety (for example, a part that binds a tumor-associated antigen (Tumor-Associated Antigen, TAA)), a hinge region, a spanning Membrane domains and intracellular domains.
  • CAR-T cell immunotherapy is considered to be one of the most promising means to overcome tumors.
  • CAR-T cells use genetic modification to enable T cells to express CAR proteins. This CAR protein has the ability to recognize the intact protein on the membrane surface without relying on antigen presentation, thereby causing the activation and functional effects of T cells.
  • the present application provides a chimeric antigen receptor targeting BCMA and its application.
  • the inventors constructed chimeric antigen receptor expression vectors using multiple BCMA-targeting scFvs and prepared BCMA-targeting CAR-T cells, and also verified that BCMA CAR-T cells have good tumor-inhibitory effects at the cellular and animal levels. Function and identify optimal chimeric antigen receptors targeting BCMA.
  • a chimeric antigen receptor targeting BCMA comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises an anti-BCMA scFv antibody,
  • the amino acid sequences of the VH complementarity-determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the VL complementarity-determining regions of the scFv antibody
  • the amino acid sequences of regions 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 above chimeric antigen receptor, the scFv antibody is a humanized antibody
  • the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 7
  • its VL sequence includes the following: The amino acid sequence shown in SEQ ID NO:8.
  • the above-mentioned chimeric antigen receptor, the scFv antibody is a rabbit-derived antibody
  • the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 9
  • its VL sequence includes the following: The amino acid sequence shown in SEQ ID NO:10.
  • connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 37-39.
  • the sequence of the scFv antibody is shown in SEQ ID NO: 11 or SEQ ID NO: 12.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ .
  • the above-mentioned chimeric antigen receptor, the transmembrane region is derived from one of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70 or Various.
  • the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.
  • the above-mentioned chimeric antigen receptor wherein the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, Syk one or more of.
  • the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1
  • the co-stimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1
  • ICOS LIGHT, NKG2C, NKG2D, DAP10, B7-H3, MyD88.
  • the above chimeric antigen receptor further comprises a leader peptide located at the N-terminal of the amino acid sequence of the chimeric antigen receptor; optionally, the leader peptide is derived from CD8 ⁇ .
  • the extracellular antigen recognition domain further comprises scFv antibodies against one of the following targets: CD138, NKG2D, CD38, CD19, SLAMF7, CD70, CD44v6, Lewis Y.
  • the present application also provides an isolated nucleic acid molecule comprising the nucleic acid sequence encoding the above-mentioned chimeric antigen receptor.
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the above-mentioned vector is an expression vector; in some embodiments, the vector is a viral vector; in some embodiments, it is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned carrier.
  • the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and differentiated pluripotent stem cells.
  • the above-mentioned 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 a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable auxiliary materials.
  • the pharmaceutically acceptable excipients include protective agents.
  • the pharmaceutically acceptable adjuvant includes cell cryopreservation solution.
  • the above-mentioned pharmaceutical composition is an intravenous injection.
  • the present application also provides the use of the above-mentioned chimeric antigen receptor, nucleic acid molecule, carrier or immune effector cell in the preparation of medicines for treating diseases or conditions related to the expression of BCMA.
  • the disease or disease associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or recurrent 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, myasthenia gravis or autoimmune hemolytic anemia.
  • the drug is an intravenous injection.
  • the present application also provides a method for treating a disease or a disease related to the expression of BCMA, comprising the following steps: applying an effective amount of the above-mentioned immune effector cells or a pharmaceutical composition to a disease or disease related to the expression of BCMA. subjects in need.
  • the disease or disorder associated with the expression of BCMA is cancer; optionally, the cancer is multiple myeloma; further optionally, the cancer is refractory or recurrent multiple myeloma.
  • the disease or condition associated with the 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, or autoimmune hemolytic anemia.
  • the administration is by intravenous injection.
  • the administering method is to administer an effective amount of the immune effector cells or the pharmaceutical composition to the subject in a single injection.
  • the effective amount of immune effector cells or the pharmaceutical composition is 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/kg.
  • the present application also provides the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition for treating diseases or diseases related to the expression of BCMA.
  • the above-mentioned immune effector cells or the above-mentioned pharmaceutical composition, the disease or disease related to 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, myasthenia gravis or autoimmune Immune hemolytic anemia.
  • Figure 1 shows the basic structure of CAR molecules by taking BY-02G as an example.
  • Figure 2 shows the expression of BCMA CAR molecules on the surface of CAR-T cells in Example 2, wherein: 01G-CAR ⁇ 05G-CAR represents T cells transduced with CAR, and UTD represents T cells not transduced with CAR.
  • the left peak represents UTD
  • the right peak represents 01G-CAR, 02G-CAR, 03G-CAR, 04G-CAR, and 05G-CAR, respectively.
  • Figure 3A shows the number of CAR-positive T cells in each group D0-D6 in multiple rounds of antigen stimulation experiments in Example 3;
  • Figure 3B shows the number of CAR-positive T cells in each group D8-D14 in multiple rounds of antigen stimulation experiments in Example 3 The number of T cells;
  • Figure 3C shows the CAR MFI of each group D2-D6 in multiple rounds of antigen stimulation experiments in Example 3;
  • Figure 3D shows the CAR MFI of D8-D14 in multiple rounds of antigen stimulation experiments in Example 3.
  • Figure 4A shows the proportion of CAR-positive T cells in D0-D6 in multiple rounds of antigen stimulation experiments in Example 3;
  • Figure 4B shows the proportion of CAR-positive T cells in D8-D14 in multiple rounds of antigen stimulation experiments in Example 3.
  • Figure 5 shows the expression rates of immune checkpoint proteins PD1, Lag3 and Tim3 on the cell surface after multiple rounds of antigen stimulation of BCMA CAR-T cells in Example 4. For each group of cells, the expression rates of PD1, Lag3, and Tim3 are represented from left to right.
  • Figure 6 shows the Annexin V positive ratio of CAR positive cells after multiple rounds of antigen stimulation of BCMA CAR-T cells in Example 5.
  • Figure 7 shows the release of cytokine IFN- ⁇ after multiple rounds of antigen stimulation of BCMA CAR-T cells in Example 6.
  • K562 cells and MM.1S cells from left to right, the release levels of cytokine IFN- ⁇ in T cells that were not transduced with CAR, BY-01G cells, BY-02G cells, BY-04G cells and BY-05G cells .
  • Figure 8A shows the tumor volume change curves of each group in the subcutaneous tumor-bearing animal experiment of RPMI8226 cells in Example 7;
  • Figure 8B shows the MM.1S cells in Example 7 in the tail vein tumor-bearing animal experiment of immunodeficient mice The average fluorescence imaging signal intensity change curve of each group;
  • Figure 9A shows the survival curves of the low dose BY-02G group and the bb2121 group in Example 7;
  • Figure 9B shows the survival curves of the dose BY-02G group and the bb2121 group in Example 7.
  • Figure 10 shows the average body weight change curves of each group in Example 8.
  • Figure 11 shows the specific recognition of BCMA by BCMA CAR-T cells in Example 9.
  • the first line of each target cell in the figure shows the expression of BCMA protein on the surface of the target cell, and the second line shows the expression of CD107a on the surface of CD8-positive T cells expression.
  • Figure 12 shows the specific recognition of BCMA by BCMA scFv in Example 9.
  • CAR Chimeric Antigen Receptor
  • extracellular antigen recognition domain refers to the antigen recognition domain (Antigen Recognition Domain, ARD).
  • CAR cell therapy products such as CAR-T cells
  • ARD Antigen Recognition Domain
  • antigen recognition domains have been derived from antibody single-chain variable regions (Single Chain Variable Fragment, abbreviated as scFv), or from receptor-ligand interactions, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR), the most common source is scFv antibody.
  • scFv antibody refers to the scFv antibody targeting BCMA.
  • the scFv antibody includes one, two or more than two antibody heavy chain variable regions (VH) and one, two or more than two light chain variable regions (VL), which are connected by a peptide chain, For example: the connecting sequence GSTSGSGKPGSGEGSTKG composed of 18 amino acids.
  • VH antibody heavy chain variable regions
  • VL light chain variable regions
  • the regions are called hypervariable regions (HVR); in the V regions of the L chain and H chain
  • HVR hypervariable regions
  • CDR division 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 input the VH and VL sequences and select the corresponding rules. CDR sequences according to different rules can be obtained. Those skilled in the art should understand that the protection scope of the present application covers the combination of CDR sequences obtained by analysis using different rules. In this application, the CDR is divided according to the IMGT rule.
  • telomere binding refers to the recognition and/or binding between CAR and a specific target, with greater affinity, avidity, easier, and/or bind the target for a greater duration.
  • humanized antibody is also referred to as a humanized engineered antibody, which is obtained by combining the complementarity-determining regions (CDRs) of non-human mammalian antibodies, such as mouse antibodies, rat antibodies, and rabbit antibodies.
  • CDRs complementarity-determining regions
  • conventional recombinant DNA techniques for preparing humanized antibodies are known (eg WO96/02576).
  • primers can be synthesized (the corresponding primers can be obtained by referring to the method described in WO98/13388) and used to link the CDRs of the rabbit antibody to the framework regions (FRs) of the human antibody.
  • FRs linked to CDRs those are selected such that the CDR regions form good antigen-binding sites.
  • hinge region refers to the link between the extracellular antigen recognition domain and the transmembrane domain. This region allows CAR to recognize antigen by giving the antigen recognition domain a certain range of motion.
  • Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ .
  • the typical hinge region also contains residues that are involved in CAR dimerization and contribute to enhanced 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, and the downstream signal transmission can be improved through interaction.
  • the transmembrane region can 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 co-stimulatory signaling regions.
  • intracellular signaling region refers to the activation of at least one normal effector function of an immune effector cell responsible for the expression of CAR.
  • 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, Syk.
  • the term "co-stimulatory signaling domain" exists because, in addition to the stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, and activation of cell activation. Effector function.
  • the CAR may also include one or more co-stimulatory signaling regions, wherein the co-stimulatory signaling regions 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, MyD88.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude artificial or synthetic substances obtained from the 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 a short peptide before the extracellular antigen recognition domain (such as scFv sequence), which functions to guide the export of recombinant protein synthesized in the cell to the outside of the cell.
  • scFv sequence extracellular antigen recognition domain
  • Commonly used guide peptides are 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 in 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 single. By selecting appropriate targets, the anti-tumor activity of CAR-T cells can be optimized.
  • the extracellular antigen recognition domain can also include an extracellular antigen recognition domain (such as: scFv antibody) targeting any of the following targets: CD138, NKG2D, CD38, CD19, SLAMF7, CD70, CD44v6, Lewis Y.
  • an extracellular antigen recognition domain such as: scFv antibody
  • the extracellular antigen recognition domain includes scFv sequences targeting two targets.
  • a scFv antibody against a single target includes an antibody heavy chain variable region (VH) and a light chain variable region (VL), which are connected by a linking sequence; scFv antibodies against two or more targets include For the VH region and VL region of different targets, different regions are also connected directly or indirectly through linking sequences, and the arrangement can be in any of the following forms: target 1VL-target 1VH-target 2VL-target Spot2VH, Target2VL-Target2VH-Target1VL-Target1VH, Target1VL-Target2VL-Target2VH-Target1VH, Target2VL-Target1VL-Target1VH-Target2VH , the above "-" represents connection through a linker sequence.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, which may be isolated from its natural environment or a synthetic analogue .
  • gene transduction/transfection methods mainly include viral and non-viral methods. Such as: through ⁇ -retroviral vectors, lentiviral vectors, adeno-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.
  • vector generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can transform, transduce or transfect the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal viruses.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses 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.
  • Vectors may also
  • transposon refers to a discontinuous DNA segment that has the ability to migrate between chromosomal sites and carry genetic information, such as: Sleeping Beauty SB system and PB system derived from Lepidoptera insects.
  • electroporation can also be used to transduce mRNA into T cells.
  • Immune effector cell generally refers to a cell that participates in an immune response, eg, promotes an immune effector response.
  • Immune effector cells may be selected from the group consisting of: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells, induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), embryonic stem cells one or more species.
  • NK cells natural killer cells
  • PBMC cells peripheral blood mononuclear cells
  • pluripotent stem cells T lymphocytes differentiated from pluripotent stem cells
  • NK cells induced pluripotent stem cells
  • iPSC induced pluripotent stem cells
  • iPSC-T T cells differentiated from induced pluripotent stem cells
  • the term "pharmaceutical composition” generally refers to a pharmaceutical composition suitable for administration to patients, 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 carrier, protective agent, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative.
  • the pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solution.
  • the pharmaceutical composition of the present application is a cell suspension or frozen cells thereof.
  • subject generally refers to human or non-human animals, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cows, sheep or monkeys.
  • the term "about” usually refers to the fluctuation range acceptable to those skilled in the art above or below the specified value, such as: within the range of ⁇ 0.5%-10%, for example, 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%, 9%, 9.5% or 10% range.
  • Chimeric antigen receptors nucleic acids, vectors, immune effector cells, pharmaceutical compositions
  • the present application provides a chimeric antigen receptor targeting BCMA, which comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain; wherein: the extracellular antigen recognition domain comprises Anti-BCMA scFv antibody, the amino acid sequences of the VH complementary determining regions CDR1, CDR2, and CDR3 of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the The amino acid sequences of CDR1, CDR2, and CDR3 of the VL complementarity determining regions of the scFv antibody include the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the scFv antibody is a humanized antibody; optionally, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 7, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: the amino acid sequence shown in 8.
  • the scFv antibody is a rabbit-derived antibody; optionally, the VH sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: 9, and the VL sequence of the scFv antibody includes the amino acid sequence shown in SEQ ID NO: NO: the amino acid sequence shown in 10.
  • connecting region between VH and VL in the scFv antibody, and the connecting region is selected from one or more of the following: SEQ ID NOs: 37-39.
  • sequence of the scFv antibody is shown in SEQ ID NO: 11 or SEQ ID NO: 12.
  • the present application also includes substitution, deletion, addition and/or insertion of one or more amino acids in the amino acid sequence of any one of the above chimeric antigen receptors, and it has the equivalent of any one of the above Chimeric antigen receptor activity; optionally, the substitutions are conservative substitutions.
  • substitutions are conservative substitutions.
  • the humanized transformation of the FR region in the scFv is different from the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 .
  • SEQ ID NO: 7 amino acid sequence shown in SEQ ID NO: 7
  • SEQ ID NO: 8 amino acid sequence shown in SEQ ID NO: 9
  • SEQ ID NO: 10 amino acid sequence shown in SEQ ID NO: 10 .
  • those skilled in the art also know that in order to make the CDR region of the modified antibody retain a suitable antigen-binding site during the process of humanization, if necessary, one, two, three or none of the CDRs More than 10% of the amino acid sequence may be substituted, deleted, added and/or inserted, and these are also included in this application.
  • 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 optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO: 13.
  • the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70; alternatively, The amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the intracellular domain comprises an intracellular signaling region; optionally, also includes a co-stimulatory signaling region; further optionally, wherein the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , one or more of CD36, CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, Syk; still further optionally, the intracellular signaling region is derived from CD3 ⁇ ,
  • the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the co-stimulatory 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 than three of NKG2D, DAP10, B7-H3, and MyD88; optionally, the costimulatory signal transduction region is derived from CD28 or 4-1BB; further optionally, the costimulatory signal transduction region
  • the amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the chimeric antigen receptor further comprises a guide peptide located at the N-terminal of the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8 ⁇ ; further optionally, The amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the chimeric antigen receptor of the present invention comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the extracellular antigen recognition domain only comprises scFv antibodies targeting a single target of BCMA.
  • the extracellular antigen recognition domain further comprises scFv antibodies against any of the following targets: CD138, NKG2D, CD38, CD19, SLAMF7, CD70, CD44v6, Lewis Y.
  • the present application also provides an isolated nucleic acid molecule comprising the nucleic acid sequence encoding the above chimeric antigen receptor.
  • the application provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, said nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 18, or the nucleotide sequence described in SEQ ID NO: 18 Nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity and encoding the same chimeric antigen receptor sequence. This sequence identity is due to the wobble (degeneracy) of the third base of the nucleic acid codon.
  • the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 33.
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • 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. .
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • the vector is an expression vector; optionally, the vector is a viral vector; further optionally, a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned carrier.
  • the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, pluripotent One or more of NK cells and embryonic stem cells differentiated from stem cells.
  • the 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 immune effector cells may express the chimeric antigen receptors described herein.
  • the present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable auxiliary materials.
  • the pharmaceutically acceptable auxiliary materials include: one or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.
  • the pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solution.
  • the pharmaceutical composition is a cell suspension or frozen cells thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the present application also provides a method for preparing immune effector cells, which includes the following steps: transducing the vector described in the present application into the immune effector cells.
  • the immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells differentiated from pluripotent stem cells, pluripotent One or more of NK cells and embryonic stem cells differentiated from stem cells.
  • the 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 use of the chimeric antigen receptor described in the present application, the nucleic acid molecule, the carrier and/or the immune effector cell for the preparation of a drug, wherein the drug For use in the treatment of diseases or conditions associated with the expression of BCMA.
  • the present application also provides a method for treating a disease or disorder associated with the expression of BCMA, the method comprising administering an effective dose of the present invention to a subject in need of treating a disease or disorder associated with the expression of BCMA.
  • the chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the immune effector cell are used to treat a disease or disorder associated with the expression of BCMA.
  • the administration can be performed by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the mode of administration may be administered to a subject by intravenous injection.
  • the effective dose of immune effector cells or the pharmaceutical composition can be administered to the subject once, or dividedly administered to the subject within a certain period of time, such as once a week, once every two weeks, Once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.
  • the dosage may be different; for patients with different disease severity, the dosage may also be different.
  • 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.
  • the administered dose can also be measured by the total administered dose, for example: the total administered dose does not exceed 5 ⁇ 10 8 CAR-positive T cells. In this application, the administration dose is based on the count of CAR-positive T cells, and details will not be repeated in the specific examples.
  • the subjects can include humans and non-human animals.
  • the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cows, sheep, rabbits or monkeys.
  • the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the immune effector cell, which can be used to treat diseases related to the expression of BCMA or illness.
  • the disease or condition associated with the expression of BCMA may include non-solid tumors, and optionally, the non-solid tumors are hematological tumors.
  • the disease or condition associated with the expression of BCMA can include multiple myeloma.
  • the multiple myeloma is relapsed or refractory 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, or autoimmune hemolytic anemia.
  • the following examples are only for explaining the chimeric antigen receptor, immune effector cell, preparation method and application of the present application, and are not intended to limit the scope of the present invention.
  • the Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, to insert genes encoding proteins into such vectors and plasmids, or to 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.
  • the CAR structural fragments containing BY-01G ⁇ BY-05G scFv in this application were artificially synthesized (the amino acid sequences thereof are respectively as SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: As shown in ID NO: 31, the nucleotide sequences encoding the above CAR structural fragments are respectively shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 ), and were respectively constructed into the empty lentiviral vector (manufacturer: SBI company, article number: CD500-CD800, as described in WO2021/121227 Example 1 after resistance transformation) to obtain the CAR expression vector after resistance transformation, and then The CAR expression vector and three packaging plasmids were transfected into 293T cells together, and a functional lentiviral vector was obtained after collection and purification.
  • the three packaging plasmids were PMD2.0G (purchased from Biovector, product number Biovector012259), pMDLg-pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253).
  • PBMC Peripheral blood mononuclear cells
  • the isolated T cells were treated with complete lymphocyte culture medium (X-VIVO15 medium+5%FBS+300IU/ml IL-2 or X-VIVO15 medium+5%FBS+5ng/ml IL-15+10ng/ml IL -7) Resuspend to make the final concentration (1 ⁇ 2) ⁇ 10 6 cells/ml, and add 5 ⁇ 10 ⁇ l of CD3/CD28 magnetic beads to stimulate, mix well and place in the incubator for culture, the culture condition is 37 °C + 5% CO 2 , the incubation time is at least 24 hours.
  • complete lymphocyte culture medium X-VIVO15 medium+5%FBS+300IU/ml IL-2 or X-VIVO15 medium+5%FBS+5ng/ml IL-15+10ng/ml IL -7) Resuspend to make the final concentration (1 ⁇ 2) ⁇ 10 6 cells/ml, and add 5 ⁇ 10 ⁇ l of CD3/CD28 magnetic beads to stimulate, mix well and place in the incubator for culture, the culture condition is 37 °C + 5%
  • polybrene polybrene
  • MOI lentiviral vector
  • the transduced cells were taken out, and the cell density was monitored to maintain the cells at (0.5-1) ⁇ 10 6 cells/ml for use in subsequent examples.
  • T cells obtained after transducing T cells with lentiviruses containing BY-01G ⁇ BY-05G scFv were named BCMA CAR-T cells BY-01G ⁇ BY-05G, respectively.
  • BCMA CAR-T cells BY-01G ⁇ BY-05G were named BCMA CAR-T cells BY-01G ⁇ BY-05G, respectively.
  • the CAR protein molecules expressed on the surface of the five BCMA CAR-T cells obtained in Example 1 on the 5th day after transduction were detected.
  • the results are shown in Figure 2, except that the CAR-positive ratio (10.25%) and CAR expression level of BCMA CAR-T cells BY-03G (the abscissa value of the fluorescence intensity of CAR-positive cells in Figure 2 can refer to the CAR expression level) are very high.
  • the CAR-positive ratio and CAR expression level of BCMA CAR-T cells BY-01G, BY-02G, BY-04G, BY-05G are relatively high, especially the CAR-positive ratio of BCMA CAR-T cells BY-02G reached up to 96.6%.
  • Antigen stimulation can activate CAR-T cells to proliferate CAR-T cells, and the continuous activation of T cells will lead to cell exhaustion, the proliferation ability and effector function of exhausted T cells will be reduced, and the expression levels of some immune checkpoint genes will be raised.
  • We verified the persistence of CAR-T cells by detecting the proliferation of BCMA CAR-T cells in multiple rounds of antigen stimulation experiments.
  • BCMA CAR-T cells in each group were co-cultured with BCMA-positive target cells MM.1S (human multiple myeloma cells) in a 24-well plate at an effect-target ratio of 1:1.
  • BCMA-positive target cells MM.1S human multiple myeloma cells
  • Well 2ml X-VIVO15 medium each group of cells repeated 3 wells. After 2 days, 500 ⁇ l of cells were stained with fluorescently labeled CD3 antibody (BioLegend Cat. No.
  • CD3 is a marker to distinguish whether they are T cells
  • T cells were added to MM.1S cells at an effect-to-target ratio of 1:1 for a new round of stimulation, and this was repeated until CAR-T cell proliferation stagnated, ending antigen stimulation.
  • BCMA CAR-T cells in each group proliferated significantly (as shown in Figure 3A and Figure 3B).
  • the CAR MFI value mean fluorescence intensity of CAR-positive cells in BCMA CAR-T cells after multiple rounds of antigen stimulation was detected by flow cytometry to reflect the average expression level of CAR on a single cell.
  • the results are as follows: After 7 rounds of stimulation (Day 14), the final CAR MFI value of the BCMA CAR-T cell BY-02G group was the highest, which was significantly higher than that of BY-01G, BY-04G and BY-05G (the results are shown in Figure 3C and Figure 3D), indicating that after After multiple rounds of antigen stimulation, the BCMA CAR-T cell BY-02G group had the highest average CAR expression level on a single cell.
  • the BCMA CAR-T cells in each group could continue to proliferate.
  • the results of flow cytometry showed that the CAR-positive ratio of the BCMA CAR-T cell BY-02G group was the highest, and there was little difference in the total number of CAR + cells. In the case of a large number, it indicates that its specific proliferation is the best, that is, the proliferation efficiency of CAR + T cells in the BY-02G group is better than that of non-CAR + T cells.
  • the specific results are as follows: After multiple rounds of antigen stimulation, the CAR-positive ratio of BCMA CAR-T cells in each group changed significantly.
  • Example 4 Expression rates of immune checkpoint proteins PD1, Lag3 and Tim3 on the surface of BCMA CAR-T cells after continuous proliferation
  • Example 3 After multiple rounds of antigen stimulation (day 14), we also detected the expression of immune checkpoint proteins on the surface of BCMA CAR-T cells.
  • the immune checkpoint proteins include PD1, Lag3 and Tim3, these proteins also serve as markers of cellular exhaustion, and the expression levels of these immune checkpoint genes are upregulated in exhausted T cells.
  • BCMA CAR-T cell BY-02G has the lowest PD1 ratio and lower Lag3 ratio, which may indicate better cell function.
  • Example 3 After multiple rounds of antigen stimulation (day 14), we also detected the Annexin V positive ratio of CAR-positive cells in BCMA CAR-T cells in each group. The results are shown in Figure 6.
  • the positive rate of Annexin V in BCMA CAR-T cells BY-02G was the lowest among BY-01G, BY-02G, BY-04G, and BY-05G, indicating that the level of apoptosis was lower.
  • Example 6 BCMA CAR-T cells after continuous proliferation were subjected to cytokine release experiments and cell killing experiments
  • BCMA CAR-T cells and target cells were co-cultured in X-VIVO15 medium for 24 hours according to the effect-to-target ratio of CAR-positive cells to target cells 1:1, and the target cells were MM.1S (the cells themselves are multiple Myeloma cell line expressing BCMA endogenously).
  • K562 was a negative target cell control.
  • the concentration of the cytokine IFN- ⁇ in the cell supernatant was detected by ELISA, and the results of the cytokine release experiment are shown in FIG. 7 .
  • the results showed that the CAR-T cells in each group had the ability to release IFN- ⁇ , and the BCMA CAR-T cell BY-02G released the most IFN- ⁇ after multiple rounds of antigen stimulation experiments, which was significantly higher than that of other groups.
  • the BCMA CAR-T cells developed by us can maintain the ability to proliferate and recognize and kill tumor cells under the condition of repeated antigen stimulation, and the BCMA CAR-T cells BY-02G have better performance than other candidate CAR-T Even better, it also indicates that CAR-T cells can have better persistence in the process of tumor treatment in vivo.
  • BCMA CAR-T cell BY-02G We further tested the BCMA CAR-T cell BY-02G, and used the BCMA CAR-T cell bb2121 of Bluebird Bio as a positive control (the amino acid sequence of bb2121 scFv is shown in SEQ ID NO: 40, and its nucleotide sequence is shown in SEQ ID NO: 40 As shown in ID NO: 41, except for the scFv sequence, the sequences of the other components in the structure of bb2121CAR are exactly the same as BY-02G), to verify whether the BCMA CAR-T cell BY-02G has a good anti-tumor function.
  • mice The results of tumor volume changes in mice showed that, compared with non-transduced T cells, CAR-T in each experimental group had a good tumor-inhibiting effect.
  • Subcutaneous tumors of some mice, bb2121 group and BY-02G group showed similar performance in middle dose and high dose groups.
  • MM.1S-GFP-Luc cells construct a lentiviral expression vector containing green fluorescent protein GFP and luciferase Luc coding region, then package lentivirus, transduce with lentivirus MM.1S cells, use GFP signal to sort positive monoclonal cells by flow cytometry, through culture expansion and GFP expression identification, after confirming that they are monoclonal, the cell preparation is completed) according to 5 ⁇ 10 6 cells/0.2ml/cell Tail vein inoculation in female B-NDG mice (body weight 20 ⁇ 2g).
  • mice On the 7th day after cell inoculation, the average fluorescence intensity of the group reached about 1 ⁇ 10 8 , and they were randomly divided into groups according to the fluorescence signal intensity and body weight, and CAR-T cells were reinfused. There were 6 mice in each group, a total of 6 groups, respectively: G1: bb2121 (low dose 6 ⁇ 10 5 CAR + cells/mouse), G2: bb2121 (medium dose 3 ⁇ 10 6 CAR + cells/mouse), G3: BY-02G (low dose 6 ⁇ 10 5 CAR + cells/mouse) , G4: BY-02G (medium dose 3 ⁇ 10 6 CAR + cells/mouse), G5: UTD, G6: PBS.
  • G1 bb2121 (low dose 6 ⁇ 10 5 CAR + cells/mouse)
  • G2 bb2121 (medium dose 3 ⁇ 10 6 CAR + cells/mouse)
  • G3 BY-02G (low dose 6 ⁇
  • the low-dose group BY-02G and bb2121 group performed similarly, and the medium-dose group showed the same performance as the bb2121 group.
  • the performance was slightly better, but BY-02G in the middle-dose group had better safety than the bb2121 group in terms of survival (as shown in Figure 9B).
  • the results showed that in the low dose group, the survival rate of the BY-02G group was comparable to that of the bb2121 group (as shown in Figure 9A), but in the middle dose group, all the mice in the BY-02G group remained intact until the end of the experiment. survived, while the survival rate of the bb2121 group was lower than 20% (as shown in FIG. 9B ).
  • the scFv sequence of BCMA CAR-T cells is derived from the monoclonal antibody sequence with BCMA as the antigen. We need BCMA CAR to only specifically recognize BCMA and not recognize other proteins, so as to ensure safety while ensuring effectiveness. We verified the specificity of BCMA CAR-T cells by detecting the activity of CAR-T cells after co-culture of CAR-T cells and BCMA-negative target cells.
  • different target cells including the BCMA negative cell line Huh- 7. SK-MEL-1, Ovcar3, 293T and Jurkat cells, including BCMA-positive cell line MM.1S cells
  • BCMA-negative target cells and T cells were stained, and detected and analyzed by flow cytometry to detect the expression of BCMA protein on the surface of target cells.
  • select BCMA-negative target cells and T cells to co-culture in X-VIVO15 medium according to the effect-to-target ratio of 1:2.
  • CD8-positive T cells that is, CD3 + /CD8 + T cells
  • CD107a on the cell surface
  • the expression of CD107a on the membrane is considered to be a marker of activated cytotoxic lymphocytes (CD8+T cells and NK cells), and immune activation occurs when cytotoxic lymphocytes are given specific antigen stimulation
  • CD107a therefore, can be used as a means to detect the specificity of CAR-T cells, and the detection results after co-culture of BCMA-positive target cells and T cells are used as positive controls.
  • BY-02G scFv sequence amino acid sequence shown in SEQ ID NO: 11
  • two proteins CD19 and CD319 that are B cell surface antigens like the BCMA antigen, coated the well plate, and then used the purified BY-02G scFv to detect the affinity.
  • the results are shown in Figure 12.
  • BY- The scFv of 02G can only recognize and bind BCMA antigen, showing good antibody specificity.
  • the amino acid sequences of the VH complementarity determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the VL complementarity of the scFv antibody
  • the amino acid sequences of the determining regions CDR1, CDR2, and CDR3 are the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the VH sequence is the amino acid sequence shown in SEQ ID NO: 7
  • the VL sequence of the scFv antibody is the amino acid sequence shown in SEQ ID NO: 8.
  • BCMA CAR-T cell BY-02G also has excellent performance in terms of specific recognition, tumor suppression, and safety in in vivo animal experiments.
  • Our BCMA CAR-T can effectively recognize specific antigens and target cells, express and secrete a large amount of IFN- ⁇ and CD107a, thereby mediating the corresponding immune response.
  • Our BCMA CAR-T has good safety in mice under high-dose injection. The results of in vivo anti-tumor activity studies show that our BCMA CAR-T persists in the body for a long time, and medium-dose injection can also achieve a good anti-tumor effect, suggesting that medium-dose is safe and effective.
  • Our BCMA CAR-T In the in vivo animal tumor-bearing model, it also has a good ability to recognize and kill tumor cells, and under the repeated stimulation of antigens, our BCMA CAR-T cells can still maintain a high CAR positive rate and good cell proliferation ability and lower apoptosis level, and can maintain effective killing of target cells.
  • SEQ ID NO: 1 GFSLSTYH, which is BY-02G scFv VH CDR1;
  • SEQ ID NO: 2 ISSSGST, which is BY-02G scFv VH CDR2;
  • SEQ ID NO: 3 ARDLDYVIDL, which is BY-02G scFv VH CDR3;
  • SEQ ID NO: 4 PSVYNNY, which is BY-02G scFv VL CDR1;
  • SEQ ID NO: 5 ETS, which is BY-02G scFv VL CDR2;
  • SEQ ID NO: 6 AGTYVSGDRRA, which is BY-02G scFv VL CDR3;
  • SEQ ID NO: 7 amino acid sequence of humanized BY-02G scFv VH;
  • SEQ ID NO: 8 amino acid sequence of humanized BY-02G scFv VL;
  • SEQ ID NO: 9 amino acid sequence of rabbit BY-02G scFv VH;
  • SEQ ID NO: 10 amino acid sequence of rabbit BY-02G scFv VL;
  • SEQ ID NO: 11 amino acid sequence of humanized BY-02G scFv
  • SEQ ID NO: 12 amino acid sequence of rabbit BY-02G scFv
  • SEQ ID NO: 13 the amino acid sequence of the hinge region
  • SEQ ID NO: 14 the amino acid sequence of the transmembrane region
  • SEQ ID NO: 15 the amino acid sequence of the intracellular signal transduction region
  • SEQ ID NO: 16 amino acid sequence of co-stimulatory signal transduction region
  • SEQ ID NO: 17 the amino acid sequence of the leader peptide (i.e. signal peptide);
  • SEQ ID NO: 18 nucleotide sequence encoding BY-02G scFv amino acid sequence
  • SEQ ID NO: 19 BY-01G scFv amino acid sequence
  • SEQ ID NO: 20 BY-03G scFv amino acid sequence
  • SEQ ID NO: 21 BY-04G scFv amino acid sequence
  • SEQ ID NO: 22 BY-05G scFv amino acid sequence
  • SEQ ID NO: 23 nucleotide sequence encoding BY-01G scFv amino acid sequence
  • SEQ ID NO: 24 nucleotide sequence encoding BY-03G scFv amino acid sequence
  • SEQ ID NO: 25 nucleotide sequence encoding BY-04G scFv amino acid sequence
  • SEQ ID NO: 26 nucleotide sequence encoding BY-05G scFv amino acid sequence
  • SEQ ID NO: 27 BY-01G CAR amino acid sequence
  • SEQ ID NO: 28 BY-02G CAR amino acid sequence
  • SEQ ID NO: 29 BY-03G CAR amino acid sequence
  • SEQ ID NO: 30 BY-04G CAR amino acid sequence
  • SEQ ID NO: 31 BY-05G CAR amino acid sequence
  • SEQ ID NO: 32 Nucleotide sequence encoding BY-01G CAR amino acid sequence
  • SEQ ID NO: 33 Nucleotide sequence encoding BY-02G CAR amino acid sequence
  • SEQ ID NO: 34 nucleotide sequence encoding BY-03G CAR amino acid sequence
  • SEQ ID NO: 35 nucleotide sequence encoding BY-04G CAR amino acid sequence
  • SEQ ID NO: 36 Nucleotide sequence encoding BY-05G CAR amino acid sequence
  • SEQ ID Nos: 37-39 the amino acid sequence of the linking region connecting VH and VL;
  • SEQ ID NO: 40 bb2121scFv amino acid sequence
  • SEQ ID NO: 41 Nucleotide sequence encoding the amino acid sequence of bb2121 scFv.

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Abstract

本发明提供了一种靶向BCMA的嵌合抗原受体，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的scFv抗体，所述scFv抗体的VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的氨基酸序列，所述scFv抗体的VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示的氨基酸序列。

Description

靶向BCMA的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的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。
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、效果更好的靶向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的嵌合抗原受体及其应用。发明人利用多条靶向BCMA的scFv分别构建了嵌合抗原受体表达载体并制备了靶向BCMA的CAR-T细胞，还在细胞水平和动物水平验证BCMA CAR-T细胞具有良好的抑瘤功能并确定最优的靶向BCMA的嵌合抗原受体。
一种靶向BCMA的嵌合抗原受体，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的scFv抗体，所述scFv抗体的VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的氨基酸序列，所述scFv抗体的VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，所述scFv抗体为人源化抗体，可选地所述scFv抗体的VH序列包括如SEQ ID NO：7所示的氨基酸序列，其VL序列包括如SEQ ID NO：8所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，所述scFv抗体为兔源抗体，可选地所述scFv抗体的VH序列包括如SEQ ID NO：9所示的氨基酸序列，其VL序列包括如SEQ ID NO：10所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，所述scFv抗体中VH和VL之间具有连接区，所述连接区选自以下的一种或多种：SEQ ID NOs：37-39。
上述嵌合抗原受体在某些实施方式中，所述scFv抗体的序列如SEQ ID NO：11或SEQ ID NO：12所示。
上述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。
上述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。
上述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CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种。
上述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。
上述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。
上述嵌合抗原受体在某些实施方式中，所述胞外抗原识别结构域还包含抗以下一种靶点的scFv抗体：CD138、NKG2D、CD38、CD19、SLAMF7、CD70、CD44v6、Lewis Y。
本申请还提供了一种分离的核酸分子，其包含编码上述嵌合抗原受体的核酸序列。
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，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、上述经分离的核酸分子，或上述载体。
上述免疫效应细胞在某些实施方式中，所述免疫效应细胞选自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(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)、胚胎干细胞中的一种或多种。
上述免疫效应细胞在某些实施方式中，所述免疫效应细胞是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，为静脉注射剂。
本申请还提供上述嵌合抗原受体、核酸分子、载体或免疫效应细胞在制备药物中的用途，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、重症肌无力或自身免疫性溶血性贫血。
上述应用在某些实施方式中，所述药物为静脉注射剂。
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、重症肌无力或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，有效量的免疫效应细胞或药物组合物为1×10 5至1×10 7个细胞/kg的计量。
本申请还提供了上述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、重症肌无力或自身免疫性溶血性贫血。
附图说明
图1以BY-02G为例展示了CAR分子的基本结构。
图2表示了实施例2中BCMA CAR分子在CAR-T细胞表面的表达，其中：01G-CAR～05G-CAR代表转导CAR的T细胞，UTD代表未转导CAR的T细胞。在各图中，左侧峰代表UTD，右侧峰分别代表01G-CAR、02G-CAR、03G-CAR、04G-CAR、05G-CAR。
图3A表示了实施例3中在多轮抗原刺激实验中各组D0-D6的CAR阳性T细胞数量；图3B表示了实施例3中在多轮抗原刺激实验中各组D8-D14的CAR阳性T细胞数量；图3C表示了实施例3中在多轮抗原刺激实验中各组D2-D6的CAR MFI图3D表示了实施例3中在多轮抗原刺激实验中D8-D14的CAR MFI。
图4A表示了实施例3中在多轮抗原刺激实验中D0-D6的CAR阳性T细胞比例；图4B表示了实施例3中在多轮抗原刺激实验中D8-D14的CAR阳性T细胞比例。
图5表示了实施例4中BCMA CAR-T细胞多轮抗原刺激之后细胞表面免疫检查点蛋白PD1、Lag3、Tim3的表达率。对于各组细胞，从左至右依次代表PD1、Lag3、Tim3的表达率。
图6表示了实施例5中BCMA CAR-T细胞多轮抗原刺激之后CAR阳性细胞的Annexin V阳性比例。
图7表示了实施例6中BCMA CAR-T细胞多轮抗原刺激之后的细胞因子IFN-γ的释放情况。针对K562细胞和MM.1S细胞，从左至右依次为未转导CAR的T细胞、BY-01G细胞、BY-02G细胞、BY-04G细胞和BY-05G细胞的细胞因子IFN-γ释放水平。
图8A表示了实施例7中RPMI8226细胞的皮下荷瘤动物实验中各组的肿瘤体积变化曲线；图8B表示了实施例7中MM.1S细胞在免疫缺陷小鼠进行尾静脉荷瘤动物实验中各组的平均荧光成像信号强度变化曲线；
图9A表示了实施例7低剂量BY-02G组与bb2121组的生存曲线；图9B表示了 实施例7中剂量BY-02G组与bb2121组的生存曲线。
图10表示了实施例8中各组平均体重变化曲线。
图11表示了实施例9中BCMA CAR-T细胞对BCMA的特异性识别，图中每种靶细胞的第一行显示靶细胞表面BCMA蛋白的表达情况，第二行显示CD8阳性T细胞表面CD107a的表达情况。
图12表示了实施例9中BCMA scFv对BCMA的特异性识别。
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，最为常见的来源就是scFv抗体。本申请中如无特别指明，scFv抗体是指靶向BCMA的scFv抗体。scFv抗体包括一个、两个或大于两个的抗体重链可变区(VH)和一个、两个或大于两个的轻链可变区(VL)，二者之间由一段肽链连接，如：由18个氨基酸组成的连接序列GSTSGSGKPGSGEGSTKG。在抗体可变区内有一小部分氨基酸残基变化特别强烈，这些氨基酸的残基组成和排列顺序更易发生变异区域称高变区 (hypervariable regions，HVR)；在L链、H链的V区中各有三个高变区，该部位因在空间结构上可与抗原决定簇形成精密的互补，故高变区又称互补性决定区(complementarity determining region，CDR)。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。本申请中，通过IMGT规则对CDR进行划分。
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。
本申请中，术语“人源化抗体”也称为经过人源化改造的抗体，其通过将非人哺乳动物的抗体，例如小鼠抗体、大鼠抗体、兔抗体的互补决定区(CDR)移植到人抗体的CDR中进行制备，制备人源化抗体的常规重组DNA技术是已知的(如：WO96/02576)。例如，在CDR获白兔抗体的情况下，可合成引物(参考WO98/13388中描述的方法可以获得相应引物)，将其用于将兔抗体的CDR与人抗体的框架区(FR)连接。对于与CDR相连接的人抗体FR而言，要选择能使得CDR区形成良好的抗原结合位点的那些。
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CD3ζ、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。因此胞外抗原识别结构域还可以包含靶向以下任一种靶点的胞外抗原识别结构域(如：scFv抗体)：CD138、NKG2D、CD38、CD19、SLAMF7、CD70、CD44v6、Lewis Y。如：在双靶点CAR-T产品中胞外抗原识别结构域就包括针对二个靶点的scFv序列。针对单一靶点的scFv抗体包括抗体重链可变区(VH)和轻链可变区(VL)，二者之间通过连接序列连接；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，不同区域之间也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1VL-靶点1VH-靶点2VL-靶点2VH，靶点2VL-靶点2VH-靶点1VL-靶点1VH，靶点1VL-靶点2VL-靶点2VH-靶点1VH，靶点2VL-靶点1VL-靶点1VH-靶点2VH，上述“-”代表通过连接序列连接。
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(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)、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的嵌合抗原受体，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的scFv抗体，所述scFv抗体的VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的氨基酸序列，所述scFv抗体的VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示的氨基酸序列。
在一些实施例中，所述scFv抗体为人源化抗体；可选地，所述scFv抗体的VH序列包括如SEQ ID NO：7所示的氨基酸序列，所述scFv抗体的VL序列包括如SEQ ID NO：8所示的氨基酸序列。
在一些实施例中，所述scFv抗体为兔源抗体；可选地，所述scFv抗体的VH序列包括如SEQ ID NO：9所示的氨基酸序列，所述scFv抗体的VL序列包括如SEQ ID NO：10所示的氨基酸序列。
在一些实施例中，所述scFv抗体中VH和VL之间具有连接区，所述连接区选自以下的一种或多种：SEQ ID NOs：37-39。
在一些实施例中，所述scFv抗体的序列如SEQ ID NO：11或SEQ ID NO：12所示。
在一些实施例中，本申请还包含了上述任一项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；可选地，所属取代是保守取代。本领域技术人员知晓，在人源化改造的过程中，scFv FR区中的氨基酸可被取代使得经过改造的抗体的CDR区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scFv中的FR区进行人源化改造所获得的不同于SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示的氨基酸序列。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：13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：14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CD3ζ、CD3γ、CD36、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CD3ζ，如：所述胞内信号传导区的氨基酸序列包含如SEQ ID NO：15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：16所示的氨基酸序列。
在一些实施例中，所述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，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：17所示的氨基酸序列。
在一些实施例中，本发明的嵌合抗原受体包含SEQ ID NO：28所示的氨基酸序列。
在一些实施例中，所述胞外抗原识别结构域只包含靶向BCMA单靶点的scFv抗体。
在一些实施例中，所述胞外抗原识别结构域还包含抗以下任一种靶点的scFv抗体：CD138、NKG2D、CD38、CD19、SLAMF7、CD70、CD44v6、Lewis Y。
另一方面，本申请还提供了一种分离的核酸分子，其包含编码上述嵌合抗原受体的核酸序列。
另一方面，本申请提供一种编码嵌合抗原受体的分离的核酸分子，所述核酸分子包含如SEQ ID NO：18所示的核苷酸序列、或与SEQ ID NO：18所述的核苷酸序列具有至少 70％、75％、80％、85％、90％、95％、96％、97％、98％或99％序列同一性并且编码相同嵌合抗原受体的核苷酸序列。这种序列同一性是由于核酸密码子第三位碱基的摆动性(简并性)而产生的。可选地，所述核酸分子包含SEQ ID NO：33所示的核苷酸序列。
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，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、上述经分离的核酸分子，或上述载体。
在一些实施例中，所述免疫效应细胞选自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、胚胎干细胞中的一种或多种。
在一些实施例中，所述免疫效应细胞是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，所述免疫效应细胞的表面可以表达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、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，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。
制备方法和用途
另一方面，本申请还提供了制备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、胚胎干细胞中的一种或多种。
在一些实施例中，所述免疫效应细胞是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。
另一方面，本申请还提供了本申请所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与BCMA的表达相关的疾病或病症。
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、所述的核酸分子、所述的载体和/或所述的免疫效应细胞。
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1×10 5个CAR阳性T细胞/kg至1×10 7个CAR阳性T细胞/kg，例如，1×10 5个CAR阳性T细胞/kg至1×10 6个CAR阳性T细胞/kg、1×10 6个CAR阳性T细胞/kg至1×10 7个CAR阳性T细胞/kg。施用剂量还可以通过施用总剂量进行衡量，如：施用总剂量不超过5×10 8个CAR阳性T细胞。在本申请中，施用剂量均是以CAR阳性T细胞计数，具体实施例中不再赘述。
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、所述的核酸分子、所述的载体和/或所述的免疫效应细胞，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。
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、重症肌无力或自身免疫性溶血性贫血。
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、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
实施例1、BCMA CAR-T细胞的获得
我们已有5条BCMA特异性的scFv序列(其scFv的氨基酸序列分别如SEQ ID NO：19、SEQ ID NO：11、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22所示，这5条scFv的氨基酸序列在本申请中也分别用编号BY-01G、BY-02G、BY-03G、BY-04G、BY-05G作为简称。编码上述BY-01G至BY-05G scFv的核苷酸序列分别如SEQ ID NO：23、SEQ ID NO：18、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26所示)。由于蛋白水平scFv的功能验证，不能反映其在细胞水平的功能，我们选择在二代CAR结构上对候选BCMA scFv序列进行筛选。CAR结构示意图如图1所示，我们采用CD8α引导链为信号肽，以BCMA scFv为胞外肿瘤抗原识别区域，铰链区和跨膜区采用CD8α的结构，以4-1BB为胞内共刺激信号，CD3ζ为T细胞激活信号。
1、慢病毒载体的构建
分别人工合成包含本申请中BY-01G～BY-05G scFv的CAR结构片段(其氨基酸序列分别如SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31所示，编码上述CAR结构片段的核苷酸序列分别如SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36所示)，并分别构建到经过抗性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中记载的经过抗性改造)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PMD2.0G(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5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)×10 6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％CO 2，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％CO 2，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)×10 6个细胞/ml，以备后续实施例使用。
将使用包含有BY-01G～BY-05G scFv的慢病毒转导T细胞后获得的T细胞分别命名为BCMA CAR-T细胞BY-01G～BY-05G。接下来，我们在细胞水平和动物水平对5条CAR结构进行筛选，以最终确定一个最优的候选scFv序列。
实施例2、BCMA CAR-T细胞BY-01G～BY-05G表面表达的CAR分子的检测
对实施例1中获得的转导后第5天的5种BCMA CAR-T细胞表面表达的CAR蛋白分子进行检测。我们使用FITC标记的BCMA抗原(ACRO Biosystems Cat.No.BCA-HF254)对细胞进行染色，通过流式细胞术进行检测分析。结果如图2所示，除了BCMA CAR-T细胞BY-03G的CAR阳性比例(10.25％)和CAR表达水平(图2中CAR阳性细胞的荧光强度横坐标值可以指代CAR表达水平)都非常低以外，BCMA CAR-T细胞BY-01G、BY-02G、BY-04G、BY-05G的CAR阳性比例和CAR表达水平都比较高，尤其是BCMA CAR-T细胞BY-02G的CAR阳性比例达到了96.6％。
另外，由于实验中使用了BCMA抗原进行染色，这一结果也提示了这些CAR蛋白 特异识别BCMA抗原的能力有所区别。在后续实验中，我们会对表达较好的BCMA CAR-T细胞BY-01G、BY-02G、BY-04G、BY-05G进行各方面的功能检测，而不对表达最差的BY-03G进行更多的研究。
实施例3、BCMA 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，同时一些免疫检查点基因的表达水平会上调。我们通过检测BCMA CAR-T细胞在多轮抗原刺激实验中的增殖情况，验证CAR-T细胞的持续性。
抗原刺激之前，将各组BCMA CAR-T细胞的CAR阳性比例都利用未转导T细胞调整到一致。在多轮抗原刺激实验中，将各组BCMA CAR-T细胞分别与BCMA阳性靶细胞MM.1S(人多发性骨髓瘤细胞)按照效靶比1∶1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。2天后取500μl细胞用荧光标记的CD3抗体(BioLegend Cat.No.317318)和BCMA抗原(同实施例2)进行CD3和CAR的染色，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、数量和荧光强度，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CAR-T细胞按照效靶比1∶1加入MM.1S细胞进行新一轮刺激，如此重复直到CAR-T细胞增殖出现停滞，结束抗原刺激。
经过多轮抗原刺激之后，各组BCMA CAR-T细胞都有明显、大量的增殖(如图3A和图3B所示)。用流式细胞仪检测多轮抗原刺激后BCMA CAR-T细胞中CAR阳性细胞的CAR MFI值(平均荧光强度)以反映CAR在单个细胞上的平均表达水平，结果如下：在经过7轮刺激后(第14天)，最终BCMA CAR-T细胞BY-02G组的CAR MFI值最高，显著高于BY-01G、BY-04G和BY-05G(结果如图3C和图3D所示)，说明经过多轮抗原刺激之后，BCMA CAR-T细胞BY-02G组在单个细胞上的平均CAR表达水平最高。
在多轮抗原刺激之后，各组BCMA CAR-T细胞都能持续增殖，流式细胞术结果显示BCMA CAR-T细胞BY-02G组的CAR阳性比例最高，在总的CAR +细胞数量相差不太大的情况下，说明其特异性增殖最好，即BY-02G组CAR +T细胞的增殖效率要优于非CAR +T细胞的增殖效率。具体结果如下：在多轮抗原刺激之后，各组BCMA CAR-T细胞的CAR阳性比例发生了明显变化。从第3轮刺激(第6天)至第7轮刺激(第14天)，BCMA CAR-T细胞BY-02G的CAR阳性比例显著高于BY-01G、BY-04G和BY-05G，在经过7轮刺激 后(第14天)，最终BCMA CAR-T细胞BY-02G的CAR阳性比例最高，显著高于BY-01G、BY-04G和BY-05G(结果如图4A和图4B所示)。
实施例4、持续增殖后的BCMA CAR-T细胞表面免疫检查点蛋白PD1、Lag3和Tim3的表达率
按照实施例3中的实验方法，在结束多轮抗原刺激之后(第14天)，我们还检测了免疫检查点蛋白在BCMA CAR-T细胞表面的表达情况，所述免疫检查点蛋白包括PD1、Lag3和Tim3，这些蛋白也可以作为细胞耗竭的标记，耗竭的T细胞中这些免疫检查点基因的表达水平会上调。我们用荧光标记的PD1抗体(BioLegend Cat.No.329914)、Lag3抗体(BioLegend Cat.No.369306)、Tim3抗体(BioLegend Cat.No.345012)和BCMA抗原(同实施例2)对细胞进行染色，通过流式细胞术进行检测分析。
结果如图5所示，结果显示了各组CAR阳性细胞中PD1、Lag3和Tim3的阳性比例。BCMA CAR-T细胞BY-02G有着最低的PD1比例以及较低的Lag3比例，可能预示着其具有更好的细胞功能。
实施例5、持续增殖后的BCMA CAR-T细胞的凋亡水平
有研究表明，抗原刺激之后的CAR-T细胞增殖会受到细胞凋亡的影响。在结束抗原刺激之后，我们也检测了各组BCMA CAR-T细胞的凋亡水平。在正常细胞中，磷脂酰丝氨酸只分布在细胞膜磷脂双分子层的内侧，细胞发生凋亡早期，磷脂酰丝氨酸会由细胞膜内侧翻向外侧。Annexin V作为一种磷脂结合蛋白，与磷脂酰丝氨酸有高度亲合力，被用于检测细胞凋亡。我们用荧光标记的AnnexinV(BioLegend Cat.No.640920)和BCMA抗原(同上)对细胞进行染色，通过流式细胞术进行检测分析。
按照实施例3中的实验方法，在结束多轮抗原刺激之后(第14天)，我们还检测了各组BCMA CAR-T细胞中CAR阳性细胞的Annexin V阳性比例。结果如图6所示，BCMA CAR-T细胞BY-02G中的Annexin V阳性比例在BY-01G、BY-02G、BY-04G、BY-05G中最低，预示着其细胞凋亡水平更低。
实施例6、持续增殖后的BCMA CAR-T细胞进行细胞因子释放实验和细胞杀伤实验
为了进一步验证BCMA CAR-T细胞功能的持续性，我们将按照实施例3中实验方法结束多轮抗原刺激的CAR-T细胞进行细胞因子释放实验和细胞杀伤实验。
1)将BCMA CAR-T细胞与靶细胞按照CAR阳性细胞比靶细胞的效靶比1∶1在X-VIVO15培养基中共培养24小时，靶细胞为MM.1S(这种细胞本身就是多发性骨 髓瘤的细胞系，内源表达BCMA)。K562为阴性靶细胞对照。通过ELISA的方法检测细胞上清中细胞因子IFN-γ的浓度，细胞因子释放实验结果如图7所示。结果显示各组CAR-T细胞都有释放IFN-γ的能力，其中BCMA CAR-T细胞BY-02G在多轮抗原刺激实验之后释放IFN-γ的量最多，显著高于其他各组。
2)将各组BCMA CAR-T细胞与上述靶细胞分别按照效靶比1∶4在X-VIVO15培养基中共培养48小时，通过检测靶细胞中稳定表达的荧光素酶活性检测靶细胞存活比例，细胞杀伤实验结果显示在经过抗原反复刺激后，各组BCMA CAR-T细胞均可将BCMA阳性靶细胞MM.1S进行100％杀伤。CAR-T细胞对阴性靶细胞K562没有明显的杀伤效果。
综合以上的实验结果可以说明，我们研发的BCMA CAR-T细胞可以在抗原反复刺激的条件下维持增殖和识别杀伤肿瘤细胞的能力，其中BCMA CAR-T细胞BY-02G比其他候选CAR-T表现更优，也预示着CAR-T细胞可以在体内肿瘤治疗的过程中具有较好的持续性。
实施例7、BCMA CAR-T细胞在小鼠体内的抗肿瘤活性
基于体外药效学检测结果，我们还进行了初步的体内药效学研究。在小鼠体内进行药效学检测，体内实验中我们选择了两种动物模型(人多发性骨髓瘤RPMI8226细胞在免疫缺陷小鼠进行皮下荷瘤的动物模型及使用人多发性骨髓瘤MM.1S细胞在免疫缺陷小鼠进行尾静脉荷瘤的动物模型)。我们对BCMA CAR-T细胞BY-02G进行了进一步检测，并以蓝鸟生物的BCMA CAR-T细胞bb2121作为阳性对照(bb2121scFv氨基酸序列如SEQ ID NO：40所示，其核苷酸序列如SEQ ID NO：41所示，除scFv序列以外，bb2121CAR的构造中其余各组件的序列与BY-02G完全相同)，验证BCMA CAR-T细胞BY-02G是否具有良好的抑瘤功能。
使用RPMI8226细胞的皮下荷瘤动物实验中，我们设置了bb2121组和BY-02G组的中剂量(4×10 6CAR +细胞/鼠)和高剂量(8×10 6CAR +细胞/鼠)两个CAR-T的剂量梯度。在每只雄性NPI小鼠皮下接种混合了等体积基质胶的5×10 6RPMI8226细胞进行皮下荷瘤，当平均皮下肿瘤体积接近或达到110mm 3时，进行CAR-T细胞的单次尾静脉注射，此后，每周两次测量肿瘤体积的变化，肿瘤体积变化曲线如图8A所示。小鼠肿瘤体积变化结果显示，与未转导的T细胞相比，各实验组CAR-T均有很好的抑瘤作用，中剂量组在CAR-T注射27天左右，已经完全消除了大部分小鼠的皮下肿瘤，bb2121组和BY-02G组的中剂量和高剂量组均表现相当。
将MM.1S-GFP-Luc细胞(MM.1S-GFP-Luc细胞制备方法：构建包含绿色荧光蛋白GFP和荧光素酶Luc编码区的慢病毒表达载体，然后包装慢病毒，用慢病毒转导MM.1S细胞，利用GFP信号通过流式细胞仪分选阳性单克隆细胞，通过培养扩增、GFP表达鉴定，确定为单克隆之后，细胞制备完成)按照5×10 6个/0.2ml/只尾静脉接种于雌性B-NDG小鼠(体重为20±2g)。细胞接种后第7天，组平均荧光强度达到1×10 8左右，按照荧光信号强度和体重随机分组并回输CAR-T细胞，每组6只小鼠，共6组，分别为：G1：bb2121(低剂量6×10 5CAR +细胞/鼠)、G2：bb2121(中剂量3×10 6CAR +细胞/鼠)、G3：BY-02G(低剂量6×10 5CAR +细胞/鼠)、G4：BY-02G(中剂量3×10 6CAR +细胞/鼠)、G5：UTD、G6：PBS。分组后对肿瘤生长情况和小鼠体重分别观察，每周2次测量并记录测量值。在接种7天后，组平均荧光强度达到1×10 8左右时，进行CAR-T的单次尾静脉注射的方式回输CAR-T细胞，此后，每周两次测量荧光信号强度的变化，至第28天荧光信号强度变化曲线如图8B所示(图8B中的D0是按照进行CAR-T单次注射的时间开始计算，即接种后7天)。结果显示，与未转导的T细胞相比，各实验组CAR-T均有很好的抑瘤作用，在D28时，低剂量组BY-02G和bb2121组表现相当，中剂量组中bb2121组表现略好，但中剂量组的BY-02G从生存期考虑相对于bb2121组具有更优秀的安全性(如图9B所示)。在生存期观察中，结果显示，在低剂量组，BY-02G组与bb2121组生存率相当(如图9A所示)，但在中剂量组，BY-02G组至实验终止时全部小鼠依然存活，而bb2121组的生存率已低于20％(如图9B所示)。
实施例8、BCMA CAR-T细胞的安全性
为进一步确定我们研发的BCMA CAR-T细胞的安全性，我们在CAR-T细胞注射后，对小鼠的体重进行监测。以CAR-T注射剂量更高的RPMI8226皮下荷瘤动物模型为例，结果显示(图10)，同未转导T细胞或PBS组相比，BCMA CAR-T细胞注射的小鼠体重没有明显差别。动物可耐受剂量大于4×10 8CAR +细胞/kg。
BCMA CAR-T细胞的安全性研究结果表明，在较高剂量处理中，与对照组未转导T细胞或PBS组相比，各实验CAR-T组的小鼠没有观察到异常体重下降，提示该CAR-T细胞在较高剂量注射下的安全性。而在上述抑制肿瘤生长有效性的研究中，较低剂量下，我们的BCMA CAR-T已经具有显著抑制肿瘤的效果，同时CAR-T细胞存续时间较长，说明我们研发的BCMA CAR-T在体内良好的持续性。
实施例9、BCMA CAR-T细胞的特异性
BCMA CAR-T细胞的scFv序列来源于以BCMA为抗原的单克隆抗体序列，我 们需要BCMA CAR只能特异识别BCMA而不能识别其他蛋白，这样才能在保证有效性的同时确保安全性。我们通过CAR-T细胞与BCMA阴性靶细胞共培养之后检测CAR-T细胞的活性，验证BCMA CAR-T细胞的特异性。
首先，使用荧光标记的BCMA特异性抗体(BioLegend Cat.No.357506)与相同荧光标记的同源非特异性IgG(BioLegend Cat.No.400322)对不同靶细胞(包括BCMA表达阴性的细胞系Huh-7、SK-MEL-1、Ovcar3、293T和Jurkat细胞，也包括BCMA表达阳性的细胞系MM.1S细胞)进行染色，通过流式细胞术进行检测分析，检测靶细胞表面BCMA蛋白的表达情况。然后，选取BCMA表达阴性的靶细胞与T细胞按照效靶比1∶2在X-VIVO15培养基中共培养，共培养4h后通过流式细胞术检测CD8阳性T细胞(即CD3 +/CD8 +T细胞)表面CD107a的表达情况，CD107a在膜上的表达被认为是活化细胞毒性淋巴细胞(CD8+T细胞和NK细胞)的标志物，当细胞毒性淋巴细胞给予特定抗原刺激即发生免疫激活，表达CD107a，因此可作为检测CAR-T细胞特异性的手段，，BCMA阳性靶细胞与T细胞共培养之后的检测结果作为阳性对照。
如图11所示，在BCMA蛋白表达情况的检测结果中，各种靶细胞用BCMA特异性抗体染色结果与相同荧光标记的同源非特异性IgG染色结果相比(如图11中的第1行和第3行所示)，只有MM.1S细胞作为多发性骨髓瘤细胞系，有明显的BCMA蛋白表达(在MM.1S多发性骨髓瘤的图中出现BCMA特异性抗体染色和相同荧光标记的同源非特异性IgG染色的双峰)。在CD107a的检测结果中(如图11中的第2行和第4行所示)，BCMA阴性靶细胞与未转导的UTD组T细胞共培养之后，CD8阳性T细胞CD107a表达水平几乎完全一致，而MM.1S细胞与BY-02G CAR-T细胞共培养之后有很大比例的CD107a阳性T细胞，说明BY-02G CAR-T细胞不能识别BCMA阴性靶细胞，具有很好的靶点特异性。
进一步地，我们又用ELISA的方法，对BY-02G的scFv序列(SEQ ID NO：11所示的氨基酸序列)的特异性做进一步的确认。我们选取两个与BCMA抗原一样同为B细胞表面抗原的蛋白(CD19和CD319)，进行孔板包被，然后用纯化的BY-02G scFv进行亲合力检测，结果如图12所示，BY-02G的scFv只能识别和结合BCMA抗原，显示出很好的抗体特异性。
综上，在体外药效学检测表明，BY-02G CAR在T细胞表面的表达优于其他候选；BCMA CAR-T细胞BY-02G在CAR-T持续增值性、细胞耗竭水平、细胞凋亡水平、细胞因子释放、体外细胞杀伤等方面，皆优于其他候选CAR-T。BY-02G scFv中，VH互补决 定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的氨基酸序列，所述scFv抗体的VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别为SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示的氨基酸序列。BY-02G scFv中，VH序列为SEQ ID NO：7所示的氨基酸序列，所述scFv抗体的VL序列为SEQ ID NO：8所示的氨基酸序列。
BCMA CAR-T细胞BY-02G在体内动物实验中，特异性识别、肿瘤抑制、安全性的方面也有非常优异的表现。我们的BCMA CAR-T能有效识别特定抗原及靶细胞，表达并分泌大量IFN-γ和CD107a，从而介导相应的免疫反应。我们的BCMA CAR-T在高剂量注射下对小鼠具备较好的安全性。体内抗肿瘤活性研究结果显示，我们的BCMA CAR-T在体内有较长时间的存续，且中剂量注射也能达到很好的抑瘤效果，提示中剂量即安全有效，我们的BCMA CAR-T在体内动物荷瘤模型中，也有很好的识别和杀伤肿瘤细胞的能力，且在抗原的反复刺激下，我们的BCMA CAR-T细胞仍然可以保持较高的CAR阳性率、较好的细胞增殖能力和较低的凋亡水平，并且能保持对靶细胞的有效杀伤。
序列描述
SEQ ID NO：1：GFSLSTYH，其为BY-02G scFv VH CDR1；
SEQ ID NO：2：ISSSGST，其为BY-02G scFv VH CDR2；
SEQ ID NO：3：ARDLDYVIDL，其为BY-02G scFv VH CDR3；
SEQ ID NO：4：PSVYNNY，其为BY-02G scFv VL CDR1；
SEQ ID NO：5：ETS，其为BY-02G scFv VL CDR2；
SEQ ID NO：6：AGTYVSGDRRA，其为BY-02G scFv VL CDR3；
SEQ ID NO：7：人源化BY-02G scFv VH氨基酸序列；
SEQ ID NO：8：人源化BY-02G scFv VL氨基酸序列；
SEQ ID NO：9：兔源BY-02G scFv VH氨基酸序列；
SEQ ID NO：10：兔源BY-02G scFv VL氨基酸序列；
SEQ ID NO：11：人源化BY-02G scFv氨基酸序列；
SEQ ID NO：12：兔源BY-02G scFv氨基酸序列；
SEQ ID NO：13：铰链区的氨基酸序列；
SEQ ID NO：14：跨膜区的氨基酸序列；
SEQ ID NO：15：胞内信号传导区的氨基酸序列；
SEQ ID NO：16：共刺激信号传导区的氨基酸序列；
SEQ ID NO：17：引导肽(即信号肽)的氨基酸序列；
SEQ ID NO：18：编码BY-02G scFv氨基酸序列的核苷酸序列；
SEQ ID NO：19：BY-01G scFv氨基酸序列；
SEQ ID NO：20：BY-03G scFv氨基酸序列；
SEQ ID NO：21：BY-04G scFv氨基酸序列；
SEQ ID NO：22：BY-05G scFv氨基酸序列；
SEQ ID NO：23：编码BY-01G scFv氨基酸序列的核苷酸序列；
SEQ ID NO：24：编码BY-03G scFv氨基酸序列的核苷酸序列；
SEQ ID NO：25：编码BY-04G scFv氨基酸序列的核苷酸序列；
SEQ ID NO：26：编码BY-05G scFv氨基酸序列的核苷酸序列；
SEQ ID NO：27：BY-01G CAR氨基酸序列；
SEQ ID NO：28：BY-02G CAR氨基酸序列；
SEQ ID NO：29：BY-03G CAR氨基酸序列；
SEQ ID NO：30：BY-04G CAR氨基酸序列；
SEQ ID NO：31：BY-05G CAR氨基酸序列；
SEQ ID NO：32：编码BY-01G CAR氨基酸序列的核苷酸序列；
SEQ ID NO：33：编码BY-02G CAR氨基酸序列的核苷酸序列；
SEQ ID NO：34：编码BY-03G CAR氨基酸序列的核苷酸序列；
SEQ ID NO：35：编码BY-04G CAR氨基酸序列的核苷酸序列；
SEQ ID NO：36：编码BY-05G CAR氨基酸序列的核苷酸序列；
SEQ ID NOs：37-39：连接VH和VL的连接区的氨基酸序列；
SEQ ID NO：40：bb2121scFv氨基酸序列；
SEQ ID NO：41：编码bb2121scFv氨基酸序列的核苷酸序列。

Claims (25)

  1. 一种靶向BCMA的嵌合抗原受体，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的scFv抗体，所述scFv抗体的VH互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示的氨基酸序列，所述scFv抗体的VL互补决定区CDR1、CDR2、CDR3的氨基酸序列分别包括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所述的嵌合抗原受体，其中所述scFv抗体为人源化抗体，可选地，所述scFv抗体的VH序列包括如SEQ ID NO：7所示的氨基酸序列，其VL序列包括如SEQ ID NO：8所示的氨基酸序列；
    或，所述scFv抗体为兔源抗体，可选地，所述scFv抗体的VH序列包括如SEQ ID NO：9所示的氨基酸序列，其VL序列包括如SEQ ID NO：10所示的氨基酸序列。
  3. 根据权利要求2所述的嵌合抗原受体，其中所述scFv抗体中VH和VL之间具有连接区，所述连接区选自以下的一种或多种：SEQ ID NOs：37-39。
  4. 根据权利要求1所述的嵌合抗原受体，其中所述scFv抗体的序列如SEQ ID NO：11或SEQ ID NO：12所示。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，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包含如SEQ ID NO：13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包含如SEQ ID NO：14所示的氨基酸序列；
    和/或，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。
  6. 根据权利要求5中所述的嵌合抗原受体，其中所述胞内信号传导区来源于CD3ζ、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CD3ζ；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：15所示的氨基酸序列；
    和/或，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：16所示的氨基酸序列。
  7. 根据权利要求1-4中任一项所述的嵌合抗原受体，所述嵌合抗原受体还包含位于其氨基酸序列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：17所示的氨基酸序列。
  8. 根据权利要求1或4所述的嵌合抗原受体，其包含SEQ ID NO：28所示的氨基酸序列。
  9. 根据权利要求1-4中任一项所述的嵌合抗原受体，其中所述胞外抗原识别结构域还包含抗以下靶点中一种的scFv抗体：CD138、NKG2D、CD38、CS1、CD19、SLAMF7、CD70、CD44v6、Lewis Y。
  10. 一种分离的核酸分子，其包含编码权利要求1-9中任一项所述的嵌合抗原受体的核苷酸序列；可选地，所述核酸分子包含SEQ ID NO：18、所示的核苷酸序列、或与SEQ ID NO：18所述的核苷酸序列具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％序列同一性并且编码相同嵌合抗原受体的核苷酸序列；进一步可选地，所述核酸分子包含SEQ ID NO：33所示的核苷酸序列。
  11. 一种载体，其包含权利要求10所述的核酸分子；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；更进一步可选地，所述载体为慢病毒载体。
  12. 一种经工程化的免疫效应细胞，其包含权利要求1-9中任一项所述的嵌合抗原受体、权利要求10所述的经分离的核酸分子，或权利要求11所述的载体。
  13. 根据权利要求12所述的免疫效应细胞，其选自T淋巴细胞、自然杀伤细胞、外周血单核细胞、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、胚胎干细胞中的一种或多种；可选地，所述免疫效应细胞是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。
  14. 一种药物组合物，其包含权利要求12或13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，所述药学上可接受的辅料包括保护剂；进一步可选地，所述保护剂包括细胞冻存液。
  15. 根据权利要求14所述的药物组合物，所述药物组合物为静脉注射剂。
  16. 权利要求1-9中任一项所述的嵌合抗原受体、权利要求10所述的经分离的核酸分子、权利要求11所述的载体、或权利要求12或13所述的经工程化的免疫效应细胞用于制备治疗与BCMA的表达相关的疾病或病症的药物的用途。
  17. 根据权利要求16所述的用途，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，所述多发性骨髓瘤是难治性或复发性的多发性骨髓瘤。
  18. 根据权利要求16所述的用途，所述与BCMA的表达相关的疾病或病症是自身免疫疾病；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、重症肌无力或自身免疫性溶血性贫血。
  19. 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：将有效量的权利要求12或13所述的经工程化的免疫效应细胞或权利要求14或15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。
  20. 根据权利19所述的方法，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。
  21. 根据权利19所述的方法，其中所述与BCMA的表达相关的疾病或病症是自身免疫疾病；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、重症肌无力或自身免疫性溶血性贫血。
  22. 根据权利19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12或13所述的经工程化的免疫效应细胞或权利要求14或15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，所述有效量的免疫效应细胞或药物组合物为1×10 5至1×10 7个细胞/kg的计量。
  23. 权利要求12或13所述的经工程化的免疫效应细胞或权利要求14或15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。
  24. 根据权利要求23所述的经工程化的免疫效应细胞或药物组合物，用于治疗与BCMA的表达相关的疾病或病症，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。
  25. 根据权利要求23所述的经工程化的免疫效应细胞或药物组合物，用于治疗与BCMA的表达相关的疾病或病症，其中所述与BCMA的表达相关的疾病或病症是 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、重症肌无力或自身免疫性溶血性贫血。
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