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WO2023044633A1 - Bcma car-t在制备用于治疗自身免疫病的药物中的应用 - Google Patents

Bcma car-t在制备用于治疗自身免疫病的药物中的应用 Download PDF

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WO2023044633A1
WO2023044633A1 PCT/CN2021/119720 CN2021119720W WO2023044633A1 WO 2023044633 A1 WO2023044633 A1 WO 2023044633A1 CN 2021119720 W CN2021119720 W CN 2021119720W WO 2023044633 A1 WO2023044633 A1 WO 2023044633A1
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seq
amino acid
acid sequence
use according
car
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PCT/CN2021/119720
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English (en)
French (fr)
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汪文
胡广
蔡松柏
李大可
操昌普
谢白露
吴明
徐夏君
姚正
郭静静
庞昊
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南京驯鹿医疗技术有限公司
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Priority to PCT/CN2021/119720 priority Critical patent/WO2023044633A1/zh
Priority to PCT/CN2022/119980 priority patent/WO2023045934A1/zh
Publication of WO2023044633A1 publication Critical patent/WO2023044633A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • This application relates to the field of biomedicine, in particular to the application of CAR-T cells targeting BCMA in the preparation of drugs for the treatment of autoimmune diseases.
  • Antibody-mediated idiopathic inflammatory disease of the nervous system is an autoimmune disease in which autoimmune cells and molecules attack the nervous system as the main pathogenic mechanism.
  • pathogens that act on the nervous system autoantigens are collectively called nervous system autoantibodies, and antibody-mediated nervous system idiopathic inflammatory diseases can occur in the central nervous system, peripheral nervous system, and nerve-muscle junctions.
  • NMO Neuromyelitis optica
  • NMO sectrum disorder NMO sectrum disorder
  • AQP4-Ab is the most important pathogenic antibody of NMOSD. A large number of basic research and clinical research It has been confirmed that the antibody can cause damage to the central nervous system of animals and humans, and its diagnostic specificity can be as high as more than 90%. The positive rate of AQP4-Ab in NMOSD patients is between 40% and 90%.
  • BCMA B cell maturation antigen, B cell maturation antigen
  • B cell maturation antigen is a cell surface receptor mainly expressed in long-lived plasma cells (effector B cells), germinal center B cells and human memory B cells. BCMA detaches from the cell surface to form free BCMA (sBCMA) in peripheral blood. Studies have shown that the level of sBCMA is correlated with the level of IgG. So far, the drugs marketed in China include hormones, immunosuppressants, etc., and patients frequently relapse after using them. There is no effective therapy for the treatment of neuromyelitis optica. Drugs used for off-label diseases are not effective. Relapse and refractory treatment are still a difficult problem for the majority of medical workers. Therefore, it is necessary and urgent to find new treatments.
  • the application provides the use of immune effector cells in the preparation of drugs for the prevention and/or treatment of autoimmune diseases
  • the immune effector cells include a chimeric antigen receptor (CAR) targeting BCMA, and the immune effector cells can effectively use
  • autoimmune diseases such as neuromyelitis optica spectrum diseases.
  • the application provides the use of immune effector cells in the preparation of drugs for the prevention and/or treatment of autoimmune diseases, the immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a BCMA-targeted Antigen binding domain, transmembrane domain, co-stimulatory domain and intracellular signaling domain, wherein the antigen binding domain is a fully human antigen binding domain.
  • CAR chimeric antigen receptor
  • the autoimmune disease comprises an autoimmune inflammatory disease.
  • the inflammatory disease comprises neuritis.
  • the inflammatory disease comprises a demyelinating disease.
  • the inflammatory disease comprises an inflammatory disease of the central nervous system.
  • the inflammatory disease comprises an inflammatory demyelinating disease of the central nervous system.
  • the inflammatory disease comprises optic neuritis.
  • the inflammatory disease comprises myelitis.
  • the inflammatory disease comprises Neuromyelitis Optic Spectrum Disease (NMOSD).
  • NOSD Neuromyelitis Optic Spectrum Disease
  • the inflammatory disease comprises AQP4 (aquaporin 4)-Ab positive neuromyelitis optica spectrum disorder.
  • the autoimmune disease comprises an AQP4-Ab-mediated autoimmune disease.
  • the autoimmune disease comprises an AQP4-Ab positive disease and/or condition.
  • the AQP4-Ab positive disease and/or condition comprises central nervous system demyelinating disease.
  • the AQP4-Ab positive disease and/or condition comprises an AQP4-Ab positive neuromyelitis optica spectrum disorder.
  • the autoimmune disease comprises Neuromyelitis Optic Spectrum Disease (NMOSD).
  • NOSD Neuromyelitis Optic Spectrum Disease
  • the neuromyelitis optica spectrum disorder is relapsed refractory NMOSD.
  • the antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, Said HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the antigen binding domain comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18 , and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:7.
  • the antigen binding domain comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15.
  • the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:43.
  • the antigen binding domain comprises an antibody or fragment thereof.
  • the antigen binding domain comprises a scFv.
  • the antigen binding domain is capable of binding BCMA.
  • the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8a , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:27.
  • the co-stimulatory domain comprises a polypeptide selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS.
  • the co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:31.
  • the intracellular signaling domain comprises a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:33.
  • the CAR further comprises a hinge region linking the antigen binding domain and the transmembrane domain.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:25.
  • the CAR is also linked to a signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:3.
  • the CAR is also linked to a cleavage peptide.
  • the cleaved peptide comprises an amino acid sequence from a T2A peptide.
  • the cleaved peptide comprises the amino acid sequence shown in SEQ ID NO:35.
  • the CAR comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:51.
  • the immune effector cells comprise T cells and/or natural killer (NK) cells.
  • the medicament comprises optionally a pharmaceutically acceptable carrier.
  • the present application also provides an administration method, which comprises administering the immune effector cells described in the present application to a subject in need.
  • the administration method can be used for the prevention and/or treatment of autoimmune diseases.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25 ⁇ 10 6 to 1 ⁇ 10 6 cells/kg.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25 ⁇ 10 6 to 0.5 ⁇ 10 6 cells/kg.
  • the administration method comprises administering the immune effector cells to the subject at a dose of about 0.5 ⁇ 10 6 to 1 ⁇ 10 6 cells/kg
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25 x 106 cells/kg.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.5 x 106 cells/kg.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 1 x 106 cells/kg.
  • the method of administration comprises intravenous injection.
  • the immune effector cells are administered once.
  • the method of administration comprises treating the subject prior to infusion of the immune effector cells.
  • the treating of the subject comprises lymphoid depleting treatment.
  • said lymphoid depletion treatment is initiated about 4 days prior to infusion of said immune effector cells.
  • the period of the lymphoid depletion treatment is about 3 days.
  • the method of administration comprises a 3-day lymphodepletion treatment beginning 4 days prior to infusion of the immune effector cells.
  • the lymphoid depletion treatment comprises infusing the subject with cyclophosphamide prior to infusion of the immune effector cells.
  • the infusion dose of cyclophosphamide is about 500 mg/m 2 /day.
  • said lymphoid depletion treatment comprises infusion of said cyclophosphamide 4 days, 3 days, or 2 days prior to infusion of said immune effector cells.
  • the method of administration comprises administering fludarabine to the subject following infusion of the cyclophosphamide.
  • the fludarabine is administered at a dose of about 30 mg/m 2 .
  • the fludarabine is administered for more than about 30 minutes.
  • the lymphoid depletion treatment comprises receiving an infusion of cyclophosphamide at about 500 mg/m 2 /day 4 days, 3 days, and 2 days prior to the infusion of the immune effector cells, and infusion of cyclophosphamide Administration of about 30 mg/ m2 of fludarabine over about 30 minutes immediately following the amide.
  • the present application provides a pharmaceutical kit, which includes: 1) immune effector cells, the immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain targeting BCMA, transmembrane domain, co-stimulatory domain and intracellular signal transduction domain, wherein the antigen binding domain is a fully human antigen binding domain; and 2) a depleting reagent.
  • CAR chimeric antigen receptor
  • the depleting agent includes cyclophosphamide, and/or fludarabine.
  • the immune effector cells in the pharmaceutical kit comprise CAR
  • the antigen binding domain of the CAR comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises amino acids shown in SEQ ID NO:9
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the immune effector cells in the pharmaceutical kit comprise a CAR
  • the antigen binding domain of the CAR comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises SEQ ID NO: 17 Amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the immune effector cells in the pharmaceutical kit comprise a CAR
  • the antigen binding domain of the CAR comprises a heavy chain variable region
  • the heavy chain variable region comprises SEQ ID NO: 7 Amino acid sequence shown.
  • the immune effector cells in the pharmaceutical kit comprise a CAR
  • the antigen binding domain of the CAR comprises a light chain variable region comprising SEQ ID NO: 15 Amino acid sequence shown.
  • the immune effector cells in the pharmaceutical kit comprise CAR, and the antigen binding domain of the CAR comprises the amino acid sequence shown in SEQ ID NO:43.
  • Figures 1A-1C show the results of serum AQP4-IgG concentration changes in NMOSD patients after reinfusion of BCMA CAR-T cells described in this application.
  • Figure 2 shows the results of serum sBCMA detection of NMOSD patients after reinfusion of BCMA CAR-T cells described in this application.
  • Figures 3A-3B show the changes in the BCMA CAR-T cell concentration-time curve and VCN-time curve described in this application.
  • chimeric antigen receptor Chimeric Antigen Receptor, CAR
  • CAR Chimeric Antigen Receptor
  • TAA chimeric Antigen receptor T cells
  • the CAR can be a genetically engineered chimeric protein capable of redirecting the cytotoxicity of immune effector cells to B cells, which combines antibody-based antigen (such as BCMA) specificity with T cell receptor activation
  • B cells which combines antibody-based antigen (such as BCMA) specificity with T cell receptor activation
  • the intracellular domains are grouped together.
  • T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • BCMA and “B-cell maturation antigen” are used interchangeably, a member of the tumor necrosis factor receptor family.
  • the BCMA may be human BCMA, whose GenBank accession number is BAB60895.1.
  • BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) characteristic of TNFR family members in the extracellular domain (ECD), which forms a ligand-binding motif.
  • CCD cysteine-rich domain
  • ECD extracellular domain
  • BCMA proteins may also include fragments of BCMA, such as the extracellular domain and fragments thereof.
  • BCMA binding domain generally refers to a domain that can specifically bind to a BCMA protein or a fragment thereof.
  • the BCMA extracellular binding domain may comprise a chimeric antigen receptor capable of specifically binding to a human BCMA polypeptide expressed on a B cell, an anti-BCMA antibody or an antigen-binding fragment thereof.
  • binding domain "binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “extracellular antigen-specific binding domain” are used interchangeably in this application used, and a CAR having the ability to specifically bind a target antigen of interest (eg, BCMA) is provided.
  • BCMA binding domains may be of natural, synthetic, semi-synthetic or recombinant origin.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), As well as antibody fragments (eg, Fab, Fab' and (Fab)2 fragments) that bind to the antigen.
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • single-chain antibody may be an antibody formed by linking the heavy chain variable region and the light chain variable region through a linker peptide.
  • transmembrane domain (Transmembrane Domain) generally refers to the domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals.
  • co-stimulatory domain generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective response of lymphocytes to antigens.
  • the costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.
  • hinge region generally refers to the structure used to connect the antigen recognition region and the transmembrane region.
  • HA-tag generally refers to a protein tag based on human influenza virus hemagglutinin (Human influenza hemagglutinin) antigen. short amino acid sequence. After the HA-tag sequence is joined to one end of the target protein by means of molecular biology, the specific antibody against the HA-tag can be used to bind the recombinant protein, which is beneficial to the carrying out of experiments such as immunohistochemistry (IHC) and Western Blotting (see Schembri, Laura et al. The HA tag is cleaved and loses immunoreactivity during apoptosis. Nature Methods. February 2007, 4(2):107-108).
  • IHC immunohistochemistry
  • Western Blotting see Schembri, Laura et al. The HA tag is cleaved and loses immunoreactivity during apoptosis. Nature Methods. February 2007, 4(2):107-108).
  • intracellular signaling domain generally refers to the component of CAR located in intracellular signal transduction, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: its It may be selected from CD3 ⁇ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.
  • signal peptide generally refers to a short (5-30 amino acids in length) peptide chain that directs the transfer of newly synthesized proteins to the secretory pathway.
  • cleaved peptide refers to self-cleaved 2A peptide, which can realize the function of cleaved protein through ribosome jumping instead of proteolytic hydrolysis, which may include T2A, F2A and P2A, etc.
  • the term "marker detection signal” generally refers to a gene, protein or other molecule with known function or sequence that can function as a specific marker and emit a detectable signal.
  • the label detection signal can be a fluorescent protein, such as: GFP, RFP, YFP and the like.
  • the marker detection signal may be EGFRt.
  • the term "EGFRt” refers to the gene encoding a truncated human epidermal growth factor receptor polypeptide that lacks the distal membrane EGF-binding domain and cytoplasmic signaling tail, but retains extracellular epitopes recognized by anti-EGFR antibodies.
  • EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker for the function of genetically modified cells. In this application, it can be used as a marker molecule for CAR-T cells, and it can be used to clear CAR-T cells in vivo if necessary through the cetuximab mediated ADCC pathway (see US8802374B2).
  • Kozak sequence generally refers to the (gcc)gccRccAUGG sequence shared in eukaryotic mRNA, which plays an important role in the initiation of the translation process and is recognized by the ribosome as a translation initiation site (see De Angioletti M et al. A novel silent beta-thalassaemia mutation, the first in the Kozak sequence. Br J Haematol. 2004, 124(2):224–31.).
  • sequence identity generally refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-by-amino acid basis over a comparison window.
  • Percent sequence identity can be calculated by comparing two optimally aligned sequences over a comparison window and determining the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size) and the result multiplied by 100 to yield the percent sequence identity.
  • Optimal alignment for purposes of determining percent sequence identity can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.
  • variant in relation to an antibody is used in this application to refer to an antibody that has been substituted by at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids, Antibodies with deletions and/or insertions of amino acid alterations to regions of the antibody of interest (e.g., heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region), wherein the variant substantially retains the antibody prior to the alteration Biological properties of molecules.
  • this application encompasses variants of any of the antibodies described in this application.
  • the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the pre-altered antibody.
  • the alterations do not result in the antibody variant losing binding to the antigen, but optionally may confer properties such as increased affinity for the antigen and different effector functions.
  • the antibody heavy chain variable region or light chain variable region, or each CDR region can be altered individually or in combination.
  • the amino acid changes may be amino acid substitutions, such as conservative substitutions.
  • the antibody variant has at least 80%, 85%, 90% or 95% or 99% or more amino acid identity to the parent antibody over the antibody sequence region of interest.
  • immune effector cells generally refers to immune cells involved in clearing foreign antigens and performing effector functions in an immune response.
  • immune effector cells For example, plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.
  • pharmaceutically acceptable carrier generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation.
  • additives are well known to those skilled in the art.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, the situation consisting of the mentioned elements, integers or steps is also covered.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • autoimmune disease is used interchangeably with “autoimmune disease” and generally refers to any disease and/or condition in which the body mounts an immune system response to some component of its own tissues.
  • the immune system loses its ability to recognize a tissue or system in the body as itself, targeting and attacking it as if it were foreign.
  • the autoimmune disease may include autoimmune inflammatory disease.
  • inflammatory disease generally includes immune-mediated inflammatory processes, eg, components of the immune system in the body that cause, mediate or otherwise contribute to an inflammatory response, contributing to a disease or disorder.
  • the term "AQP4-Ab positive” can be used interchangeably with “AQP4 antibody positive” and "AQP4-IgG positive”, and generally refers to the detection of AQP4-Ab by one or more detection means.
  • an in vitro diagnostic kit enzyme-linked immunoassay
  • the positive judgment value can be determined independently through experiments according to each kit.
  • the AQP4-Ab positive may include normal or pathologically significant references established in the clinic. For example, it can be determined whether it is AQP4-Ab positive by testing according to the positive determination part in the instruction manual of the ElisaRSRTM AQP4 Ab Version kit.
  • AQP4-Ab-mediated diseases and/or disorders generally refers to diseases and/or disorders associated with AQP4-Ab expression.
  • the disease and/or condition can be caused by AQP4-Ab binding to AQP4, which binding can induce complement-dependent cytotoxicity and/or antibody-dependent cytotoxicity.
  • the binding can result in inflammation, oligodendrocyte damage demyelination, and/or neuronal loss.
  • the term "neuromyelitis optica spectrum disorder” can be used interchangeably with “NMOSD”, and generally refers to an autoimmune disease of the central nervous system, which can include two groups of symptoms of the optic nerve and spinal cord.
  • NMOSD a autoimmune disease of the central nervous system
  • the term can cover AQP4-Ab positive NMOSD as well as AQP4-Ab negative NMOSD.
  • AQP4-Ab and “AQP4-IgG” generally refer to anti-AQP4 antibodies.
  • the present application provides the use of immune effector cells comprising chimeric antigen receptors in the preparation of medicaments for preventing and/or treating autoimmune diseases.
  • the immune effector cells comprise an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.
  • the present application provides immune effector cells for preventing and/or treating autoimmune diseases.
  • the present application provides a method for preventing and/or treating an autoimmune disease, which comprises administering the immune effector cells described in the present application to a subject in need.
  • the autoimmune disease may include autoimmune inflammatory disease.
  • the inflammatory disease may include neuritis.
  • the inflammatory diseases may include demyelinating diseases.
  • the inflammatory diseases may include central nervous system inflammatory diseases.
  • the inflammatory disease may include central nervous system inflammatory demyelinating disease.
  • the inflammatory disease may include optic neuritis.
  • the inflammatory disease may include myelitis.
  • the inflammatory disease may comprise neuromyelitis optica (NMO).
  • the inflammatory disease may comprise Neuromyelitis Optic Spectrum Disease (NMOSD).
  • the inflammatory disease may comprise AQP4 (aquaporin 4)-Ab positive neuromyelitis optica spectrum disorder.
  • the autoimmune disease may include AQP4-Ab-mediated autoimmune disease.
  • the autoimmune disease may include AQP4-Ab positive diseases and/or disorders.
  • the AQP4-Ab-positive diseases and/or conditions may include central nervous system demyelinating diseases.
  • the AQP4-Ab-positive diseases and/or disorders may comprise AQP4-Ab-positive neuromyelitis optica spectrum disorders.
  • the autoimmune disease may comprise neuromyelitis optica spectrum disease (NMOSD).
  • NOSD neuromyelitis optica spectrum disease
  • a method of reducing the titer of AQP4-Ab in AQP4-Ab positive subjects in need of treatment for NMOSD is provided.
  • a method of reducing the occurrence and/or progression of NMOSD-related diseases and/or disorders in a subject in need of treatment for NMOSD is provided.
  • Neuromyelitis optica also known as Devic's disease or Devic's syndrome, is an autoimmune inflammatory disease in which a person's own immune system attacks the optic nerve and spinal cord. The disease causes inflammation of the optic nerve (optic neuritis) and spinal cord (myelitis).
  • Neuromyelitis optica is an acute or subacute inflammatory demyelinating disease of the central nervous system, usually an antibody-mediated idiopathic inflammatory disease of the nervous system.
  • NMO sectrum disorder is marked by the presence of NMO-IgG antibodies in serum, covering NMO and NMO-related diseases.
  • AQP4-Ab is the most important pathogenic antibody of NMOSD. A large number of basic and clinical studies have confirmed that this antibody can cause damage to the central nervous system of animals and humans, and its diagnostic specificity can be as high as 90%. NMOSD patients The positive rate of AQP4-Ab was between 40% and 90%.
  • AQP4-Ab is the pathogenic antibody of NMOSD.
  • AQP4-IgG in serum and plasma cells producing AQP4-IgG infiltrate into the central nervous system, causing AQP4-IgG to bind to aquaporin 4 (AQP4) channels on astrocytes.
  • AQP4 aquaporin 4
  • the immune effector cells may comprise a chimeric antigen receptor, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
  • the antigen-binding domain of the CAR may include a fully human antigen-binding domain.
  • the extracellular domain of the CAR may comprise the single-chain scFv antibody of the present invention.
  • the single chain antibody may be linked to the transmembrane domain via a hinge region, such as the CD8 hinge.
  • the CAR can be used to transduce immune effector cells (such as T cells) and expressed on the cell surface.
  • the chimeric antigen receptor may comprise a BCMA binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
  • the BCMA-binding domain may comprise an antibody or fragment thereof that specifically binds BCMA.
  • the antigen binding domain may comprise heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), the amino acids of the HCDR1-3
  • the antibody may comprise light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), the LCDR1
  • the amino acid sequence of -3 is shown in SEQ ID No:17-19.
  • the antigen-binding domain may comprise a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7.
  • the antibody may comprise a light chain variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15.
  • the antigen-binding domain may comprise a single-chain antibody.
  • the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 43 or a functional variant thereof.
  • the single chain antibody may comprise the amino acid sequence shown in SEQ ID NO:43.
  • the CAR described in the present application may include a transmembrane domain, which may comprise a polypeptide from a protein selected from the group consisting of ⁇ , ⁇ or ⁇ chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5 , CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain may comprise the amino acid sequence shown by xx or a functional variant thereof.
  • the transmembrane domain of the present application may include CD8a, the sequence of which is shown in SEQ ID NO:27.
  • the co-stimulatory domain may comprise a polypeptide from a protein selected from CD28, 4-1BB, OX40 and ICOS.
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31 or a functional variant thereof.
  • the CAR described herein can include an intracellular signaling domain, which can comprise a signaling domain from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 33 or a functional variant thereof.
  • the CAR described in the present application may include a hinge region, and the hinge region may connect the antibody and the transmembrane domain.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 25 or a functional variant thereof.
  • the CAR described in the present application may also include an HA-tag, and the HA-tag may be located at the N-terminal of the CAR.
  • the HA-tag may comprise the amino acid sequence shown in SEQ ID NO: 5 or a functional variant thereof.
  • the expression of the CAR described in this application can be detected by using an anti-HA antibody to specifically bind it and used to enrich CAR-T cells for functional research.
  • the CAR described in the present application may include a signal peptide, and the signal peptide may include the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof.
  • the signal peptide can be a CD8a signal peptide, the sequence of which is shown in SEQ ID NO:3.
  • the CAR may include CAR0037, CAR0085, CAR0087.
  • the CAR can also be linked to a cleavage peptide.
  • the cleaved peptide may comprise an amino acid sequence derived from a T2A peptide.
  • the cleaved peptide may comprise the amino acid sequence shown in SEQ ID NO: 35 or a functional variant thereof.
  • the cleaved peptide can be T2A, the sequence of which is shown in SEQ ID NO:35.
  • the CAR may include CAR0037, CAR0087.
  • the CAR can also be connected with a marker detection signal, and the marker detection signal can be located at the C-terminal of the CAR.
  • the label detection signal can be a fluorescent protein, which can be selected from the following group: GFP, RFP and YFP.
  • the expression of CAR molecules can be evaluated indirectly by detecting the signal of GFP.
  • the CAR may include CAR0037, the marker detection signal sequence of which is shown in SEQ ID NO:37.
  • the marker detection signal may be EGFRt.
  • the CAR may include CAR0087, the marker detection signal sequence of which is shown in SEQ ID NO:39.
  • the vector expressing the CAR may include the coding sequence of Kozak, the nucleotide sequence of which is shown in SEQ ID NO:2.
  • the vector expressing the CAR may include the coding sequence of Kozak.
  • the CAR may include CAR0037, CAR0085, CAR0087.
  • the CAR may comprise the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 51 or a functional variant thereof.
  • the CAR can be selected from CAR0037, the sequence of which is shown in SEQ ID NO:49.
  • the CAR can be selected from CAR0085, whose sequence is shown in SEQ ID NO:51; the CAR can be selected from CAR0087, whose sequence is shown in SEQ ID NO:51.
  • the CAR described in the present application may sequentially include a BCMA-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus.
  • the CAR may include a BCMA binding domain, and the sequence of the BCMA binding domain is shown in SEQ ID NO:43.
  • the BCMA binding domain may include HCDR1-3, its sequence is shown in sequence as SEQ ID NO:9-11; and, the BCMA binding domain may include LCDR1-3, its sequence is as shown in SEQ ID NO:17- 19.
  • the CAR may include CAR0037 or the CAR described in this application having the same LCDR1-3 and HCDR1-3.
  • the BCMA-binding domain can include a heavy chain variable region, whose sequence is shown in SEQ ID NO:7; and, the BCMA-binding domain can also include a light chain variable region, whose sequence is as shown in SEQ ID NO:15 shown.
  • the CAR may include CAR0037 or the CAR described in the present application having the same light chain variable region and heavy chain variable region as CAR0037.
  • a connecting peptide may also be included between the light chain variable region and the heavy chain variable region, and its sequence is as shown in SEQ ID NO:23.
  • the CAR may include CAR0037 or the CAR described in the present application having the same linking peptide as CAR0037.
  • the transmembrane domain may comprise a transmembrane domain from CD8a, and its sequence may be as shown in SEQ ID NO:27.
  • the CAR may include CAR0037 or the CAR described in the present application having the same transmembrane domain as CAR0037.
  • the co-stimulatory domain may comprise a co-stimulatory structure from CD28, and its sequence may be as shown in SEQ ID NO:29.
  • the CAR may include CAR0037 or the CAR described in the present application having the same costimulatory domain as CAR0037.
  • the intracellular signaling domain may comprise a signaling domain from CD3 ⁇ , the sequence of which is shown in SEQ ID NO:33.
  • the CAR may include CAR0037 or the CAR described in the present application having the same intracellular signal transduction domain as CAR0037.
  • the CAR may also include a hinge region, which may be located at the C-terminus of the BCMA-binding domain and at the N-terminus of the transmembrane domain, the sequence of which is shown in SEQ ID NO: 25.
  • the CAR may include CAR0037 or the CAR described in this application having the same hinge region as CAR0037.
  • the CAR can also include an HA-tag, and the HA-tag can be located at the N-terminus of the BCMA-binding domain, and its sequence is shown in SEQ ID NO:5.
  • the CAR may include CAR0037 or the CAR described in this application having the same HA-tag as it.
  • the CAR can also be connected with a signal peptide, which can be located at the N-terminus of the CAR, and its sequence can be as shown in SEQ ID NO:3.
  • the CAR can also be linked to a cleavage peptide, such as T2A.
  • the cleavage peptide may be located at the C-terminus of the intracellular signal transduction domain, and its sequence may be as shown in SEQ ID NO:35.
  • the CAR can also be linked with a label detection signal, which can be located at the C-terminus of the CAR (or, the cleaved peptide).
  • the marker detection signal can be selected from the following groups: GFP, RFP and YFP, the sequence of which is shown in SEQ ID NO:37.
  • the CAR described in this application can be CAR0037, the amino acid sequences of its LCDR1-3 are respectively shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; the amino acid sequence of VL is shown in SEQ ID NO: 15; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 7; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID NO: 23; its hinge region is shown in SEQ ID NO: 25; its transmembrane domain is shown in SEQ ID NO: 27; its costimulatory domain is a CD28 costimulatory structure domain, as shown in SEQ ID NO:29; its CD3 ⁇ intracellular signaling domain as shown in SEQ ID NO:33; the CAR0037 can also comprise a cleavage peptide as shown in SEQ ID NO:35, and as shown in SEQ ID NO
  • the CAR described in this application can be CAR0085, the amino acid sequences of its LCDR1-3 are respectively shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; the amino acid sequence of VL is shown in SEQ ID NO: 15; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 7; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID NO: 23; its hinge region is shown in SEQ ID NO: 25; its transmembrane domain is shown in SEQ ID NO: 27; its co-stimulatory domain is 4-1BB co- Stimulatory domain, as shown in SEQ ID NO: 31; Its CD3 ⁇ intracellular signaling domain is shown in SEQ ID NO: 33;
  • the vector expressing CAR0085 can also include the KOZAK sequence shown in SEQ ID NO: 1 ;
  • the CAR described in this application can be CAR0087, the amino acid sequences of its LCDR1-3 are respectively shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; the amino acid sequence of VL is shown in SEQ ID NO: 15; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 7; between VH and VL
  • the sequence of the connecting peptide is shown in SEQ ID NO: 23; its hinge region is shown in SEQ ID NO: 25; its transmembrane domain is shown in SEQ ID NO: 27; its co-stimulatory domain is 4-1BB co-
  • the stimulating domain is shown in SEQ ID NO:31; its CD3 ⁇ intracellular signaling domain is shown in SEQ ID NO:33; the vector expressing CAR0087 can also include the KOZAK sequence shown in SEQ ID NO:2 ;
  • the CAR0087 can
  • NMOSD can be evaluated with reference to the EDSS neurological scale score, the number of active lesions on MRI, the annual recurrence rate and the number of inactive lesions, the annual recurrence rate, and the patient-free ratio.
  • the measured values of serum AQP4-Ab titers of NMOSD patients before and after treatment can be analyzed by descriptive statistics method, and the changes from the baseline can be used to evaluate the treatment effect.
  • the AQP4-Ab titer in the subject's serum can be detected by ELISA method to measure the change of AQP4-Ab in the subject's serum.
  • the medicament for preventing and/or treating autoimmune diseases may comprise the immune effector cells described in the present application and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionic surface Active agents, etc.
  • the drug can be formulated for intravenous injection.
  • the present application provides an administration method, the method comprising administering the immune effector cells described in the present application to a subject in need.
  • the subject in need includes a subject suffering from an autoimmune disease.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25 ⁇ 10 6 to 1 ⁇ 10 6 cells/kg.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25 x 106 cells/kg.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.5 x 106 cells/kg. In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.395 x 106 cells/kg. In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.425 x 106 cells/kg. In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.495 x 106 cells/kg.
  • the method of administering comprises administering the immune effector cells to the subject at a dose of about 1 x 106 cells/kg.
  • the administration method may comprise intravenous injection.
  • the number of administration of the immune effector cells may be one time.
  • the method of administration comprises treating the subject prior to infusion of the immune effector cells.
  • the treating of the subject comprises lymphoid depleting treatment.
  • said lymphoid depletion treatment is initiated about 4 days prior to infusion of said immune effector cells.
  • the period of the lymphoid depletion treatment is about 3 days.
  • the method of administration comprises a 3-day lymphodepletion treatment beginning 4 days prior to infusion of the immune effector cells.
  • the lymphoid depletion treatment comprises infusing the subject with cyclophosphamide prior to infusion of the immune effector cells.
  • the infusion dose of cyclophosphamide is about 500 mg/m 2 /day.
  • said lymphoid depletion treatment comprises infusion of said cyclophosphamide 4 days, 3 days, or 2 days prior to infusion of said immune effector cells.
  • the method of administration comprises administering fludarabine to the subject following infusion of the cyclophosphamide.
  • the fludarabine is administered at a dose of about 30 mg/m 2 .
  • the fludarabine is administered for more than about 30 minutes.
  • the lymphoid depletion treatment comprises receiving an infusion of cyclophosphamide at about 500 mg/m 2 /day 4 days, 3 days, or 2 days prior to infusion of the immune effector cells, and Immediately thereafter, administer about 30 mg/ m2 of fludarabine over about 30 minutes.
  • the present application also provides a pharmaceutical kit, which includes: 1) immune effector cells, the immune effector cells comprise a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding antigen targeting BCMA domain, transmembrane domain, co-stimulatory domain and intracellular signaling domain, wherein the antigen-binding domain is a fully human antigen-binding domain; and a depleting reagent.
  • CAR chimeric antigen receptor
  • the rinsing agent may include cyclophosphamide and/or fludarabine.
  • the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen binding domain of the CAR may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO:9 Amino acid sequence, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen binding domain of the CAR may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise SEQ ID NO: 17
  • the amino acid sequence of the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 19.
  • the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen binding domain of the CAR may comprise a heavy chain variable region, and the heavy chain variable region may comprise SEQ ID NO: The amino acid sequence shown in 7.
  • the immune effector cells in the pharmaceutical kit may comprise a CAR, and the antigen binding domain of the CAR may comprise a light chain variable region, and the light chain variable region may comprise SEQ ID NO: The amino acid sequence shown in 15.
  • the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen-binding domain of the CAR may comprise the amino acid sequence shown in SEQ ID NO:43.
  • the lentiviral vector PLVX-EF1alpha-IRES-Puro was double digested with NotI and MluI, and the vector fragment was recovered.
  • the candidate scFv plasmid PXL0026 (its nucleotide sequence is SEQ ID NO: 44) was amplified by PCR, and the NotI restriction site (including protective bases), CD8a signal peptide, HA -label; hinge region, transmembrane region, CD28 co-stimulatory factor, CD3 ⁇ intracellular signaling domain gene synthesis, PCR amplification; T2A cleavage peptide and eGFP PCR amplified from plasmid pMy-BirA-T2A-eGFP, 3 Carry MluI restriction site and protective base at the ' end; then use overlap PCR to obtain a PCR fragment with a NotI restriction site at the 5' end and a MluI site at the 3' end, and perform NotI and MluI
  • the CAR plasmid numbered PXL0085 was obtained in a similar way, the lentiviral vector PLVX-EF1alpha-IRES-Puro was double-digested with NotI and MluI, and the vector fragment was recovered.
  • the candidate scFv plasmid PXL0026 was amplified by PCR, and the NotI restriction site (including protective bases), CD8a signal peptide, hinge region, transmembrane region, and 4-1BB co-stimulatory factor ( Its nucleotide sequence is SEQ ID NO: 32), CD3 ⁇ intracellular signaling domain gene synthesis, PCR amplification; then use overlap PCR to obtain the 5' end band NotI restriction site 3' end band MluI site For the PCR fragment, NotI and MluI double enzyme digestion was performed on the fragment, and the fragment was recovered. T4 connection was constructed to obtain the CAR plasmid numbered PXL0085 (the nucleotide sequence of the CAR molecular part of CAR0085 is shown in SEQ ID NO: 52).
  • the CAR plasmid numbered PXL0087 was obtained in a similar way, the lentiviral vector PLVX-EF1alpha-IRES-Puro was double digested with NotI and MluI, and the vector fragment was recovered.
  • the candidate scFv plasmid PXL0026 was amplified by PCR, and the NotI restriction site (including protective bases), CD8a signal peptide, hinge region, transmembrane region, and 4-1BB co-stimulatory factor ( Its nucleotide sequence is SEQ ID NO:32), CD3 ⁇ intracellular signaling domain, T2A cleavage peptide, EGFRt (its nucleotide sequence is SEQ ID NO:40) gene synthesis, PCR amplification; Then use overlap A PCR fragment with a NotI restriction site at the 5' end and a MluI site at the 3' end was obtained by PCR, and the fragment was double digested with NotI and MluI and recovered. T4 connection was constructed to obtain the CAR plasmid numbered PXL0087 (the nucleotide sequence of the CAR molecular part of CAR0087 is shown in SEQ ID NO: 52).
  • PBMCs were isolated from peripheral blood collected from healthy donors, and T cells were further sorted using CD3 MicroBeads.
  • the sorted T cells were activated using CD3/CD28 Dynabeads, and then transduced with LV0007, LV0020, and LV0021 lentiviruses.
  • the density of T cells during transduction was about 1.5 ⁇ 106 cells/ml.
  • a medium change was performed on the transduced T cells.
  • LV0007-CAR-T, LV0020-CAR-T and LV0021-CAR-T cells were obtained.
  • the preparation process of CAR-T cells is shown in the table below. The product must pass the quality inspection before it can be released. Before the release of each batch of finished products, the quality control department must collect and evaluate all the test records related to the batch of finished products. In the form of frozen suspension, filled in specific infusion bags for storage and transportation.
  • the preparation process of CAR-T cells is shown in Table 1.
  • Embodiment 2 Clearing lymph (lymphatic depletion) pretreatment
  • the pretreatment process for clearing stranguria is as follows: intravenous infusion of 500 mg/m 2 cyclophosphamide, once a day for 3 consecutive days, each time over 30 minutes, and immediately after the completion of cyclophosphamide intravenous infusion, intravenous infusion of 30 mg/m 2 Fludarabine, once a day for 3 consecutive days, each time more than 30 minutes.
  • intravenous infusion of 500 mg/m 2 cyclophosphamide once a day for 3 consecutive days, each time over 30 minutes, and immediately after the completion of cyclophosphamide intravenous infusion, intravenous infusion of 30 mg/m 2 Fludarabine, once a day for 3 consecutive days, each time more than 30 minutes.
  • subject Neu-003 as an example, the process of clearing stranguria is described in detail, and the pretreatment process of clearing stranguria is shown in Table 2. The degreasing treatment of other subjects was carried out in a similar manner.
  • the subjects were AQP4-IgG positive relapsed refractory NMOSD patients.
  • Three subjects were given BCMA CAR-T cells at a dose of 0.5 ⁇ 10 6 CAR+ cells/kg, and the actual infusion dose was 0.395 ⁇ 10 6 CAR+ cells /kg, 0.425 ⁇ 10 6 CAR+ cells/kg and 0.495 ⁇ 10 6 CAR+ cells/kg of BCMA CAR-T cells (containing the sequence of CAR0087).
  • the BCMA CAR-T cells were infused intravenously two days after the end of the depletion preconditioning, and the number of infusions was once.
  • the timing of venous blood collection for changes in serum AQP4-IgG titer before and after treatment is as follows: screening period (D42-D20), twice a week from the screening period to before clearing the leprosy, 12 to 5 days before cell reinfusion (time window: no later than 1 minute before delineation), 1 day before cell reinfusion (time window: before reinfusion), 3 days (time window 8 hours), 7 days (time window 12 hours), 10 days (time window ⁇ 12 hours ), 14 days (time window ⁇ 1 day), 21 days (time window ⁇ 1 day), 28 days (time window ⁇ 3 days), 56 days (time window ⁇ 3 days), 84 days (time window ⁇ 7 days ), every 3 months (time window ⁇ 14 days) within 2 years after 84 days.
  • CSF AQP4-IgG collection time D12 to D5, 14 days (time window ⁇ 1 day), 28 days (time window ⁇ 3 days), 84 days (time window ⁇ 7 days), 84 days later Every 3 months (time window ⁇ 14 days) within 2 years and at the time of group visit.
  • the second-generation aquaporin 4 (AQP4) autoantibody ELISA detection kit (source: RSR company, batch number: 2KAQE66) was used for detection, and the absorbance value (OD value) was read at 450nm and 405nm on a microplate reader, and the OD value was obtained by fitting Values were compared with a standard curve to calculate the concentration levels of AQP4-IgG.
  • Descriptive statistics were used to analyze the measured values of AQP4-IgG concentrations in serum and cerebrospinal fluid of NMOSD patients before and after treatment.
  • the serum AQP4-IgG concentration-time curves of NMOSD patients were drawn at each time point.
  • the detection results of AQP4-IgG in cerebrospinal fluid are shown in Table 3.
  • the AQP4-IgG in Neu-003 patients was 1.279u/mL before reinfusion, and it could be reduced to 0.9628u/mL after BCMA CAR-T cell reinfusion.
  • the concentration of AQP4-IgG in the cerebrospinal fluid reached 26.59u/mL 9 days before the cell reinfusion, and decreased significantly to 3.14u/mL on the 14th day after the cell reinfusion. down to 0.65u/mL.
  • the AQP4-IgG in Neu-008 patient was 5.17u/mL before reinfusion, and it could be reduced to 3.88u/mL after cell reinfusion.
  • the decrease of AQP4-IgG level after cell reinfusion indicates that CAR-T cell therapy is effective.
  • Neu-003 and Neu-004 patient data cut off within 90 days after cell reinfusion.
  • the data of Neu-008 patients were cut off within 28 days after cell reinfusion.
  • the subjects were AQP4-IgG positive relapsed refractory NMOSD patients.
  • Three subjects were given BCMA CAR-T cells at a dose of 0.5 ⁇ 10 6 CAR+ cells/kg, and the actual infusion dose was 0.395 ⁇ 10 6 CAR+ cells /kg, 0.425 ⁇ 10 6 CAR+ cells/kg and 0.495 ⁇ 10 6 CAR+ cells/kg of BCMA CAR-T cells (containing the sequence of CAR0087).
  • the BCMA CAR-T cells were infused intravenously two days after the end of the depletion preconditioning, and the number of infusions was once.
  • the collection time of peripheral blood free BCMA (sBCMA) before and after treatment is as follows: D12 to D5, 7 days (time window 12 hours), 14 days (time window 1 day), 21 days (time window 1 day) day), 28 days (time window 3 days), 56 days (time window ⁇ 7 days), 84 days (time window 7 days), 84 days to 2 years every 3 months (time window 14 days) once and out of the group When visiting.
  • the Human BCMA/TNFRSF17 DuoSet ELISA detection kit (source: R&R company, article number: CAT#DY193) was used for detection, and the absorbance value (OD value) was read at 450nm and 570nm on the SpectraMax i3 microplate reader, and 570nm was used as the corrected wavelength. Fit the OD value to the standard curve to calculate the concentration level of sBCMA.
  • the sBCMA results and changing trends of the three patients are shown in Figure 2.
  • the sBCMA concentration level was high before cell reinfusion, and the sBCMA level decreased significantly after reinfusion.
  • Neu-004 and Neu-008 had sBCMA levels within 28 days after reinfusion.
  • the concentration level dropped below the detection limit, suggesting that BCMA CAR-T cells can significantly reduce sBCMA in patients and indirectly reflect the reduction of plasma cells.
  • the data of Neu-008 patients were cut off within 14 days after cell reinfusion.
  • Embodiment 5 Pharmacokinetic (PK) experiment and result
  • the concentration of BCMA CAR-T cells was detected by flow cytometry, and the main reagents and instruments involved were Anti-CD45 (PercP-labeled) and Anti-CD3, FITC-labeled BCMA protein from Acro BIOSYSTEMS, and Agilent’s Novocyte flow cytometer.
  • the concentration of BCMA CAR-T cells is presented as the percentage (%) of CAR-T in lymphocytes.
  • the vector copy number (Vector copy number, VCN) of BCMA CAR-T cells was detected by droplet digital PCR (ddPCR).
  • the main reagents and instruments involved are: Genomic DNA extraction kit from Qiagen, Thermo Fisher's NanoDrop Nucleic Acid Quantitative Instrument, BIO-RAD's droplet generator oil, droplet generator card and pad, ddPCR Supermix for Probes (no dUTP), droplet read oil and QX200 droplet digital PCR system.
  • each BCMA CAR-T cell CD3 + CAR + cell
  • Sampling time 12 to 5 days before the reinfusion of BCMA CAR-T cells (time window: before clearing lymph pretreatment), 1 day after reinfusion (time window ⁇ 8 hours), 3 days (time window ⁇ 8 hours), 5 days Days (time window ⁇ 12 hours), 7 days (time window ⁇ 12 hours), 10 days (time window ⁇ 12 hours), 14 days (time window ⁇ 1 day), 21 days (time window ⁇ 1 day), 28 days
  • Blood samples were collected every 3 months (time window ⁇ 14 days) after 84 days (time window ⁇ 14 days) until CAR -2 years after the peak value was detected, no VCN was detected by droplet digital PCR (ddPCR), the disease worsened, and the group was discharged or reinfused.
  • ddPCR droplet digital PCR
  • 1 day after reinfusion means 24 hours after reinfusion time
  • 3 days after reinfusion means 72 hours after reinfusion time, and so on for subsequent visits. Try to conduct blood routine sampling at the same time as PK sampling, and the time between blood routine sampling and PK blood collection should not exceed 24 hours at most.
  • Plasma drug concentration-time data analysis draw the concentration-time curves of BCMA CAR-T cells in peripheral blood and VCN-time curves at each time point; T concentrations and VCN values.
  • PK parameter analysis Calculate the arithmetic mean, standard deviation coefficient of variation, median, and maximum value of PK parameters (C max , T max , AUC 0 ⁇ 28d , and AUC 0 ⁇ last , etc.) through VCN and BCMA CAR-T cell concentrations , Minimum, Geometric Mean, and Geometric Mean Coefficient of Variation.
  • Figure 3A-3B shows the results of changes in BCMA CAR-T cell concentration and VCN.
  • concentration of CAR-T cells and VCN levels rise rapidly at first, and then decline slowly after reaching the peak.
  • CAR-T cells expand in the patient's body. The increase was good, and VCN could still be detected 52 days after reinfusion.
  • Neu-003 and Neu-004 patient data cut off within 90 days after cell reinfusion.
  • the data of Neu-008 patients were cut off within 28 days after cell reinfusion.
  • the PK parameters are shown in Table 4.
  • the median T max of BCMA CAR-T cell concentration and VCN was 10 days, and the median C max of BCMA CAR-T cell concentration and VCN were 77.78% and 55300 Copies/ ⁇ g DNA, respectively.
  • the median AUC 0-28 of VCN is 329184.4 Days ⁇ Copies/ ⁇ g DNA (range 275819.600 ⁇ 1500890.100 Days ⁇ Copies/ ⁇ g DNA), the median AUC 0-last of VCN is 329184.4 Days ⁇ Copies/ ⁇ g DNA (range 275819.600 ⁇ 17060986.7 Days ⁇ Copies/ ⁇ g DNA), it can be seen that the peak time of BCMA CAR-T cells in NMOSD patients is fast, and the cell expansion is good.
  • the concentration of ferritin was detected by particle-enhanced immunoturbidimetry, the concentration of IL-6 was detected by electrochemiluminescence three-antigen sandwich turbidimetry, the concentration of C-reactive protein was detected by immune projection turbidimetry, and the concentration of C-reactive protein was detected by electrochemiluminescence three-antigen sandwich turbidimetry. Measure procalcitonin concentration.
  • the results of inflammatory factors in patients with Neu-003 are shown in Table 5.
  • the highest concentration of ferritin after cell reinfusion was 654.6ug/mL
  • the highest concentration of IL-6 after cell reinfusion was 31.11pg/mL
  • the concentration of C-reactive protein in cells The highest concentration after reinfusion was 16.7mg/L, and the concentration of procalcitonin after cell reinfusion was lower than that before reinfusion, and the concentration of various inflammatory factors did not increase after administration, which usually caused side effects.
  • the results showed that BCMA CAR - Less risk of cytokine release syndrome from T cell therapy.

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Abstract

提供了一种免疫效应细胞在制备预防和/或治疗自身免疫病的药物中的用途，该免疫效应细胞包含嵌合抗原受体(CAR)，其中该CAR包含靶向BCMA的抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中该抗原结合结构域为全人源抗原结合结构域。

Description

BCMA CAR-T在制备用于治疗自身免疫病的药物中的应用 技术领域
本申请涉及生物医药领域，具体的涉及靶向BCMA的CAR-T细胞在制备用于治疗自身免疫病的药物中的应用。
背景技术
抗体介导的神经系统特发性炎症疾病(又名自身免疫)是以自身免疫细胞、分子等攻击神经系统为主要致病机制的自身免疫性疾病。在免疫反应中，作用于神经系统自身抗原的致病体统称为神经系统自身抗体，抗体介导的神经系统特发性炎症疾病可生在中枢、周围神经系统及神经-肌肉接头处。
视神经脊髓炎(neuromyelitis optica,NMO)是中枢神经系统急性或亚急性炎性脱髓鞘疾病，是一种抗体介导的神经系统特发性炎性疾病。NMO谱系疾病(NMO sectrum disorder,NMOSD)是以血清中存在NMO-IgG抗体为标志，涵盖了NMO及NMO相关疾病，AQP4-Ab是NMOSD最重要的致病性抗体，大量的基础研究和临床研究均证实该抗体可以导致动物和人类中枢神经系统出现病例损伤，其诊断特异性可高达90％以上，NMOSD患病人群AQP4-Ab阳性率在40％～90％之间。
BCMA(B cell maturation antigen，B细胞成熟抗原)是一种细胞表面受体，主要表达于长期生存的浆细胞(效应B细胞)，生发中心B细胞及人类记忆B细胞。BCMA会脱离细胞表面，在外周血中形成游离BCMA(sBCMA)。有研究表明，sBCMA水平与IgG的水平具有相关性。到目前为止，国内上市的药物包括激素、免疫抑制剂等，患者使用后频繁复发。尚无针对治疗视神经脊髓炎系疾病的有效疗法，超适应症使用的药物疗效不佳，复发难治仍然是困扰广大医务工作者的难题，因此寻找新型疗法是必要且迫切的。
发明内容
本申请提供了免疫效应细胞在制备预防和/或治疗自身免疫病的药物中的用途，所述免疫效应细胞包含靶向BCMA的嵌合抗原受体(CAR)，所述免疫效应细胞能够有效用于预防和/或治疗例如视神经脊髓炎谱系疾病等的自身免疫病。
一方面，本申请提供了免疫效应细胞在制备预防和/或治疗自身免疫病的药物中的用途，所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述CAR包含靶向BCMA的抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述抗原结合结构域为全人 源抗原结合结构域。
在某些实施方式中，所述自身免疫病包含自身免疫性炎性疾病。
在某些实施方式中，所述炎性疾病包含神经炎。
在某些实施方式中，所述炎性疾病包含脱髓鞘疾病。
在某些实施方式中，所述炎性疾病包含中枢神经系统炎性疾病。
在某些实施方式中，所述炎性疾病包含中枢神经系统炎性脱髓鞘疾病。
在某些实施方式中，所述炎性疾病包含视神经炎。
在某些实施方式中，所述炎性疾病包含脊髓炎。
在某些实施方式中，所述炎性疾病包含视神经脊髓炎谱系病(NMOSD)。
在某些实施方式中，所述炎性疾病包含AQP4(水通道蛋白4)-Ab阳性视神经脊髓炎谱系病。
在某些实施方式中，所述自身免疫病包含AQP4-Ab介导的自身免疫病。
在某些实施方式中，所述自身免疫病包含AQP4-Ab阳性的疾病和/或病症。
在某些实施方式中，所述AQP4-Ab阳性的疾病和/或病症包含中枢神经系统脱髓鞘疾病。
在某些实施方式中，所述AQP4-Ab阳性的疾病和/或病症包含AQP4-Ab阳性视神经脊髓炎谱系疾病。
在某些实施方式中，所述自身免疫病包含视神经脊髓炎谱系疾病(NMOSD)。
在某些实施方式中，所述视神经脊髓炎谱系疾病(NMOSD)为复发性难治性NMOSD。
在某些实施方式中，所述抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:10所示的氨基酸序列，切所述HCDR3包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述抗原结合结构域包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:17所示的氨基酸序列，所述LCDR2包含SEQ ID NO:18所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述抗原结合结构域包含重链可变区，所述重链可变区包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述抗原结合结构域包含轻链可变区，所述轻链可变区包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述抗原结合结构域包含SEQ ID NO:43所示的氨基酸序列。
在某些实施方式中，所述抗原结合结构域包含抗体或其片段。
在某些实施方式中，所述抗原结合结构域包含scFv。
在某些实施方式中，所述抗原结合结构域能够结合BCMA。
在某些实施方式中，所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
在某些实施方式中，所述跨膜结构域包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述共刺激结构域包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。
在某些实施方式中，所述共刺激结构域包含SEQ ID NO:29或SEQ ID NO:31所述的氨基酸序列。
在某些实施方式中，所述胞内信号传导结构域包含来自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导结构域包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述CAR还包含铰链区，所述铰链区链接所述抗原结合结构域和所述跨膜结构域。
在某些实施方式中，所述铰链区包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述CAR还连接信号肽。
在某些实施方式中，所述信号肽包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述CAR还连接剪切肽。
在某些实施方式中，所述剪切肽包含来自T2A肽的氨基酸序列。
在某些实施方式中，所述剪切肽包含SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述CAR包含SEQ ID NO:49或SEQ ID NO:51所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞包含T细胞和/或自然杀伤(NK)细胞。
在某些实施方式中，所述药物包含任选地药学上可接受的载剂。
另一方面，本申请还提供了一种施用方法，其包含向有需要的受试者施用本申请所述的免疫效应细胞。在本申请中，所述施用方法可以用于预防和/或治疗自身免疫病。
在某些实施方式中，所述施用方法包含向所述受试者以约0.25×10 6～1×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约0.25×10 6～0.5×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约0.5×10 6～1×10 6个细胞/kg的剂量施用所述免疫效应细胞
在某些实施方式中，所述施用方法包含向所述受试者以约0.25×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约0.5×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约1×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含静脉注射。
在某些实施方式中，施用所述免疫效应细胞的次数为一次。
在某些实施方式中，所述施用方法包含在输注所述免疫效应细胞前对受试者进行处理。
在某些实施方式中，所述对受试者进行的处理包括淋巴耗竭处理。
在某些实施方式中，所述淋巴耗竭处理在输注所述免疫效应细胞前约4天开始进行。
在某些实施方式中，所述淋巴耗竭处理周期为约3天。
在某些实施方式中，所述施用方法包含在输注所述免疫效应细胞前4天开始接受为期3天的淋巴耗竭处理。
在某些实施方式中，所述淋巴耗竭处理包含在输注所述免疫效应细胞前向受试者输注环磷酰胺。
在某些实施方式中，所述环磷酰胺的输注剂量为约500mg/m 2/天。
在某些实施方式中，所述淋巴耗竭处理包含在输注所述免疫效应细胞前4天、3天、2天进行所述环磷酰胺输注。
在某些实施方式中，所述施用方法包含在输注所述环磷酰胺后向受试者给与氟达拉滨。
在某些实施方式中，所述氟达拉滨的给药剂量为约30mg/m 2。
在某些实施方式中，所述氟达拉滨的给药时间超过约30分钟。
在某些实施方式中，所述淋巴耗竭处理包含在输注所述免疫效应细胞前4天、3天、和2天接受环磷酰胺约500mg/m 2/天的输注，并在环磷酰胺后立即给予超过约30分钟约30mg/m 2的氟达拉滨。
另一方面，本申请提供了药物试剂盒，其包括：1)免疫效应细胞，所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述CAR包含靶向BCMA的抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述抗原结合结构域为全人源抗原结合结构域； 以及2)清淋试剂。
在某些实施方式中，所述清淋试剂包括环磷酰胺、和/或氟达拉滨。
在某些实施方式中，所述药物试剂盒中的所述免疫效应细胞包含CAR，所述CAR的抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO：10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO：11所示的氨基酸序列。
在某些实施方式中，所述药物试剂盒中的所述免疫效应细胞包含CAR，所述CAR的抗原结合结构域包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO：17所示的氨基酸序列，所述LCDR2包含SEQ ID NO：18所示的氨基酸序列，且所述LCDR3包含SEQ ID NO：19所示的氨基酸序列。
在某些实施方式中，所述药物试剂盒中的所述免疫效应细胞包含CAR，所述CAR的抗原结合结构域包含重链可变区，所述重链可变区包含SEQ ID NO：7所示的氨基酸序列。
在某些实施方式中，所述药物试剂盒中的所述免疫效应细胞包含CAR，所述CAR的抗原结合结构域包含轻链可变区，所述轻链可变区包含SEQ ID NO：15所示的氨基酸序列。
在某些实施方式中，所述药物试剂盒中的所述免疫效应细胞包含CAR，所述CAR的抗原结合结构域包含SEQ ID NO:43所示的氨基酸序列。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A-1C显示的是本申请所述BCMA CAR-T细胞回输后的NMOSD患者血清AQP4-IgG浓度变化结果。
图2显示的是本申请所述BCMA CAR-T细胞回输后的NMOSD患者血清sBCMA检测结果。
图3A-3B显示的是本申请所述BCMA CAR-T细胞浓度-时间曲线和VCN-时间曲线变化结果。
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，本领域技术人员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。
术语定义
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)是单链抗体的可变区和T细胞信号分子的融合蛋白。它使T细胞可以通过非MHC限制性的方式识别特异性抗原，发挥杀伤作用。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其包括抗原(如肿瘤相关抗原，tumor-associated antigen，TAA)结合区、跨膜结构域、共刺激结构域和胞内信号结构域。在本申请中，所述CAR可以是一种能够将免疫效应细胞的细胞毒性重定向至B细胞的基因工程嵌合蛋白，其将基于抗体的抗原(例如BCMA)特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。
在本申请中，术语“BCMA”和“B-细胞成熟抗原”可互换地使用，是肿瘤坏死因子受体家族成员。在本申请中，所述BCMA可以为人BCMA，其GenBank登录号为BAB60895.1。BCMA是III型跨膜蛋白，在胞外结构域(ECD)中具有TNFR家族成员所特征性的富半胱氨酸结构域(CRD)，该结构域形成配体结合基序。BCMA作为一种B细胞生物标志物，在肿瘤细胞(例如多发性骨髓瘤细胞)中表达，或位于肿瘤细胞(例如存在于多发性骨髓瘤恶性浆细胞)的表面。BCMA蛋白也可包括BCMA的片段，诸如胞外结构域及其片段。
在本申请中，术语“BCMA结合结构域”通常是指可以与BCMA蛋白或其片段特异性结合的结构域。例如，所述BCMA胞外结合结构域可包含能特异性结合B细胞上表达的人BCMA多肽的嵌合抗原受体，抗BCMA抗体或其抗原结合片段。在本申请中使用的术语“结合结构域”、“胞外结构域”、“胞外结合结构域”、“抗原特异性结合结构域”和“胞外抗原特异性结合结构域”可互换使用，并且提供了具有特异性结合目标靶抗原(例如BCMA)的能力的CAR。BCMA结合结构域可以为天然来源、合成来源、半合成来源或重组来源。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球 蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单域抗体(例如，dAb)，单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。
在本申请中，术语“单链抗体”可以是由所述重链可变区和所述轻链可变区通过连接肽连接而成的抗体。
在本申请中，术语“跨膜结构域”(Transmembrane Domain)通常是指CAR中穿过细胞膜的结构域，其与细胞内信号转导结构域相连接，起着传递信号的作用。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。
在本申请中，术语“铰链区”通常是指用于连接抗原识别区和跨膜区的结构。
在本申请中，术语“HA-标签”通常是指一种基于人流感病毒血凝素(Human influenza hemagglutinin)抗原的蛋白标签，其化学本质为一段来自人流感病毒血凝素98-106号氨基酸的短氨基酸序列。使用分子生物学手段将HA-标签序列接合到目的蛋白的一端后，就可以使用抗HA-标签的特异性抗体结合该重组蛋白，利于免疫组织化学(IHC)、Western Blotting等实验的进行(参见Schembri,Laura等人The HA tag is cleaved and loses immunoreactivity during apoptosis.Nature Methods.February 2007，4(2):107–108)。
在本申请中，术语“胞内信号传导结构域”通常是指CAR位于细胞内信号传导的组分，其包含信号传导结构域和特异性结合所述受体组分的结构域，例如：其可选自CD3ζ胞内域，CD28胞内域，CD28胞内域，4-1BB胞内域和OX40胞内域。
在本申请中，术语“信号肽”(Signal peptide)通常是指引导新合成的蛋白质向分泌通路 转移的短(长度5-30个氨基酸)肽链。
在本申请中，术语“剪切肽”是指自剪切2A肽，其可经核糖体跳跃而非蛋白酶水解来实现剪切蛋白的功能，其可包括T2A，F2A和P2A等。
在本申请中，术语“标记检测信号”通常是指已知功能或序列的能够起到特异性标记作用，发出可以被检测到的信号的基因、蛋白质或其他分子。所述标记检测信号可以为荧光蛋白，如：GFP、RFP和YFP等。所述标记检测信号可以为EGFRt。术语“EGFRt”是指编码截短的人表皮生长因子受体多肽的基因，其缺乏远端膜EGF结合域和细胞质信号传导尾，但保留了由抗EGFR抗体识别的细胞外表位。EGFRt可用作具有遗传修饰细胞功能的非免疫原性选择工具以及追踪标记。在本申请中，其可作为CAR-T细胞的标记分子，用于必要时清除体内的CAR-T细胞西妥昔单抗介导的ADCC途径(cetuximab mediated ADCC pathway)(参见US8802374B2)。
术语“Kozak序列”通常是指在真核生物的mRNA中共有的(gcc)gccRccAUGG序列，其在翻译过程的启动中有重要作用，被核糖体识别为翻译起始位点(参见De Angioletti M等人a novel silent beta-thalassaemia mutation,the first in the Kozak sequence.Br J Haematol.2004,124(2):224–31.)。
在本申请中，“序列同一性”通常是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
与抗体相关的术语“变体”在本申请中指，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域(例如重链可变区或轻链可变区或重链CDR区或轻链CDR区)的抗体，其中变体基本上保持改变之前的抗体分子的生物学特性。在一方面，本申请涵盖在本申请中所述的任何抗体的变体。在某些实施方式中，抗体变体保持改变前抗体的至少60％，70％，80％， 90％，或100％的生物学活性(例如抗原结合能力)。在某些实施方式中，所述改变不会导致抗体变体丧失对抗原的结合，但任选地可以赋予诸如提高的抗原亲和力和不同的效应子功能等性质。可以理解的，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在某些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。在某些实施方式中，所述氨基酸改变可以为氨基酸取代，例如可以为保守取代。在某些实施方式中，抗体变体与亲本抗体在目的抗体序列区域上具有至少80％、85％、90％或95％或99％或更高的氨基酸同一性。
在本申请中，术语“免疫效应细胞”通常是指在免疫应答中参与清除异物抗原和行使效应功能的免疫细胞。例如浆细胞、细胞毒性T细胞、NK细胞、APSC多能细胞、肥大细胞等。
在本申请中，术语“药学上可接受的载剂”通常是指药学可接受的制剂载体、溶液或加强制剂特性的添加剂。此类添加剂是所属领域技术人员熟知的。
术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
如本申请中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本申请中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本申请中，术语“约”通常是指在指定数值以上或以下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。
在本申请中，术语“自身免疫病”可以与“自体免疫性疾病”互换使用，通常指身体对其自身组织的某些成分产生免疫系统应答的任何疾病和/或病症。例如，免疫系统失去了其将身体内的某组织或系统识别为自身的能力，将其如同外来的那样对其靶向和攻击。在本申请中，所述自身免疫病可以包含自身免疫性炎性疾病。
在本申请中，术语“炎性疾病”通常包括免疫介导的炎性过程，例如，体内免疫系统的成分引起、介导或以其他方式促成炎性应答、促成疾病或紊乱。
在本申请中，术语“AQP4-Ab阳性”可以与“AQP4抗体阳性”、“AQP4-IgG阳性”互换使用，通常指可以通过一种或多种检测手段检测到AQP4-Ab。例如，在本申请中，可以采用体外诊断试剂盒(酶联免疫法)对AQP4-Ab进行定性检测，阳性判断值可以根据每种试剂盒独立通过试验确定。在本申请中，所述AQP4-Ab阳性可以包括在临床中建立的正常或具有病理意义的参考。例如，可以根据ElisaRSRTM AQP4 Ab Version试剂盒说明书中阳性判定部分 通过试验判断是否为AQP4-Ab阳性。
在本申请中，术语“AQP4-Ab介导的疾病和/或病症”通常指与AQP4-Ab表达相关的疾病和/或病症。例如，所述疾病和/或病症可以由AQP4-Ab与AQP4结合引起，所述结合可以诱发补体依赖性细胞毒性和/或抗体依赖的细胞毒性。例如，所述结合可以导致炎症、少突胶质细胞损伤脱髓鞘和/或神经元丢失。
在本申请中，术语“视神经脊髓炎谱系疾病”可以与“NMOSD”互换使用，通常指一种中枢神经系统自身免疫性疾病，其可以包括视视神经和脊髓两大组症侯。在本申请中，该术语可以涵盖AQP4-Ab阳性的NMOSD，也可以涵盖AQP4-Ab阴性的NMOSD。在本申请中，术语“AQP4-Ab”和“AQP4-IgG”通常指抗AQP4抗体。
发明详述
一方面，本申请提供包含免疫效应细胞在制备预防和/或治疗自身免疫病的药物中的应用，所述免疫效应细胞包含嵌合抗原受体。在某些实施方式中，所述免疫效应细胞包含抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导域。
另一方面，本申请提供了免疫效应细胞，其用于预防和/或治疗自身免疫病。
另一方面，本申请提供了预防和/或治疗自身免疫病的方法，其包括向有需要的受试者施用本申请所述的免疫效应细胞。
在本申请中，所述自身免疫病可以包括自身免疫性炎性疾病。在本申请中，所述炎性疾病可以包含神经炎。
在本申请中，所述炎性疾病可以包含脱髓鞘疾病。
在本申请中，所述炎性疾病可以包含中枢神经系统炎性疾病。在本申请中，所述炎性疾病可以包含中枢神经系统炎性脱髓鞘疾病。
在本申请中，所述炎性疾病可以包含视神经炎。
在本申请中，所述炎性疾病可以包含脊髓炎。
在本申请中，所述炎性疾病可以包含视神经脊髓炎(NMO)。在本申请中，所述炎性疾病可以包含视神经脊髓炎谱系病(NMOSD)。
在本申请中，所述炎性疾病可以包含AQP4(水通道蛋白4)-Ab阳性视神经脊髓炎谱系病。
在本申请中，所述自身免疫病可以包含AQP4-Ab介导的自身免疫病。在本申请中，所述自身免疫病可以包含AQP4-Ab阳性的疾病和/或病症。在本申请中，所述AQP4-Ab阳性的疾病和/或病症可以包含中枢神经系统脱髓鞘疾病。在本申请中，所述AQP4-Ab阳性的疾病和/ 或病症可以包含AQP4-Ab阳性视神经脊髓炎谱系疾病。
在本申请中，所述自身免疫病可以包含视神经脊髓炎谱系疾病(NMOSD)。
在本申请中，提供了一种在需要治疗NMOSD的AQP4-Ab阳性的受试者中降低AQP4-Ab滴度的方法。在本申请中，提供了一种在需要治疗NMOSD的受试者中降低NMOSD相关疾病和/或病症产生和/或发展的方法。
视神经脊髓炎(NMO)，也称为Devic氏病或Devic氏综合征，是一种自身免疫性炎性疾病，其中，人的自身免疫系统攻击视神经和脊髓。该病导致视神经(视神经炎)和脊髓(脊髓炎)的炎症。视神经脊髓炎是一种中枢神经系统急性或亚急性炎性脱髓鞘疾病，通常是由抗体介导的神经系统特发性炎性疾病。
NMO谱系疾病(NMO sectrum disorder,NMOSD)是以血清中存在NMO-IgG抗体为标志，涵盖了NMO及NMO相关疾病。AQP4-Ab是NMOSD最重要的致病性抗体，大量的基础研究和临床研究均正式该抗体可以导致动物和人类中枢神经系统出现病例损伤，其诊断特异性可高达90％以上，NMOSD患病人群AQP4-Ab阳性率在40％～90％之间。
AQP4-Ab为NMOSD致病抗体，血清中AQP4-IgG以及产生AQP4-IgG的浆细胞渗入中枢神经系统，导致AQP4-IgG与星型胶质细胞上的水通道蛋白4(AQP4)通道结合。诱发补体依赖性细胞毒性和抗体依赖的细胞毒性导致炎症、少突胶质细胞损伤脱髓鞘和神经元丢失。
在本申请中，所述免疫效应细胞可以包含嵌合抗原受体，所述嵌合抗原受体可以包含抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导域。
在本申请中，所述CAR的抗原结合结构域可以包含全人源抗原结合结构域。
在本申请中，所述CAR的胞外结构域可以包含本发明的单链scFv抗体。例如，所述单链抗体可以通过铰链区，例如CD8铰链，与跨膜结构域连接。在本申请中，所述CAR可以用于转导免疫效应细胞(例如T细胞)并在细胞表面表达。
在本申请中，所述嵌合抗原受体(CAR)可包含BCMA结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。在本申请中，所述BCMA结合结构域可包含特异性结合BCMA的抗体或其片段。在本申请中，所述抗原结合结构域可包含重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，所述HCDR1-3的氨基酸序列如SEQ ID NO：9-11所示，所述抗体可包含轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，所述LCDR1-3的氨基酸序列如SEQ ID No:17-19所示。在本申请中，所述抗原结合结构域可包含重链可变区，所述重链可变区的氨基酸序列如SEQ ID NO:7所示。在本申请中，所述抗体可包含轻链可变区，所述轻链可变区的氨基 酸序列如SEQ ID NO:15所示。
在本申请中，所述抗原结合结构域可包含单链抗体。在某些实施方案中，所述抗原结合结构域可包含SEQ ID NO:43所示的氨基酸序列或其功能性变体。例如，所述单链抗体可以包含SEQ ID NO:43所示的氨基酸序列。
本申请所述CAR可包括跨膜结构域，所述跨膜结构域可包含来自选自下述蛋白的多肽：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。在本申请中，所述跨膜结构域可包含xx所示的氨基酸序列或其功能性变体。例如，本申请的跨膜结构域可包括CD8a，其序列如SEQ ID NO:27所示。
在本申请中，所述共刺激结构域可包含来自选自下述蛋白的多肽：CD28、4-1BB、OX40和ICOS。在本申请中，所述共刺激结构域可包含SEQ ID NO:29或SEQ ID NO:31所示的氨基酸序列或其功能性变体。
本申请所述CAR可包括胞内信号传导结构域，所述胞内信号传导结构域可包含来自CD3ζ的信号传导结构域。在本申请中，所述胞内信号传导结构域可包含SEQ ID NO:33所示的氨基酸序列或其功能性变体。
本申请所述CAR可包括铰链区，所述铰链区可连接所述抗体和所述跨膜结构域。在本申请中，所述铰链区可包含SEQ ID NO:25所示的氨基酸序列或其功能性变体。
本申请所述CAR还可包括HA-标签，所述HA-标签可位于所述CAR的N端。在本申请中，所述HA-标签可包含SEQ ID NO:5所示的氨基酸序列或其功能性变体。在本申请中，可以通过运用抗HA抗体与其特异性结合来检测本申请所述CAR的表达并用于富集CAR-T细胞以进行功能性研究。
本申请所述CAR可包括信号肽，所述信号肽可包含SEQ ID NO:3所示的氨基酸序列或其功能性变体。例如，所述信号肽可为CD8a信号肽，其序列如SEQ ID NO:3所示。例如，所述CAR可包括CAR0037、CAR0085、CAR0087。
在本申请中，所述CAR还可连接剪切肽。在本申请中，所述剪切肽可包含来自T2A肽的氨基酸序列。在本申请中，所述剪切肽可包含SEQ ID NO:35所示的氨基酸序列或其功能性变体。例如，所述剪切肽可为T2A，其序列如SEQ ID NO:35所示。例如，所述CAR可包括CAR0037、CAR0087。
在本申请中，所述CAR还可连接标记检测信号，所述标记检测信号可位于所述CAR的C端。在本申请中，所述标记检测信号可为荧光蛋白，其可选自以下组：GFP、RFP和YFP。 在本申请中，可以通过检测GFP的信号来间接评估CAR分子的表达情况。例如，所述CAR可包括CAR0037，其标记检测信号序列如SEQ ID NO:37所示。在本申请中，所述标记检测信号可为EGFRt。例如，所述CAR可包括CAR0087，其标记检测信号序列如SEQ ID NO:39所示。
在本申请中，表达所述CAR的载体可包括Kozak的编码序列，其核苷酸序列如SEQ ID NO:2所示。在本申请中，表达所述CAR的载体可包括Kozak的编码序列。例如，所述CAR可包括CAR0037、CAR0085、CAR0087。
在本申请中，所述CAR可包含SEQ ID NO:49或SEQ ID NO:51所示的氨基酸序列或其功能性变体。例如，所述CAR可选自CAR0037，其序列如SEQ ID NO:49所示。又例如，所述CAR可选自CAR0085，其序列如SEQ ID NO:51所示；所述CAR可选自CAR0087，其序列如SEQ ID NO:51所示。
在本申请中，所述CAR0037、CAR0085和CAR0087的结构如下表所示：
Figure PCTCN2021119720-appb-000001
在某些实施方式中，本申请所述CAR可自N端依次包括BCMA结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域。其中，所述CAR可包括BCMA结合结构域，所述BCMA结合结构域序列如SEQ ID NO:43所示。其中，所述BCMA结合结构域可包括HCDR1-3，其序列依次如SEQ ID NO:9-11所示；并且，BCMA结合结构域可包括LCDR1-3，其序列依次如SEQ ID NO:17-19所示。例如，所述CAR可包括CAR0037或与其具有相同的LCDR1-3及HCDR1-3的本申请所述的CAR。所述BCMA结合结构域可包括重链可变区，其序列如SEQ ID NO:7所示；并且，所述BCMA结合结构域还可包括轻链可变区，其序列如SEQ ID NO:15所示。例如，所述CAR可包括CAR0037或与其具有相同的轻链可变区及重链可变区的本申请所述的CAR。所述轻链可变区和所述重链可变区之间还可包括连接 肽，其序列如SEQ ID NO:23所示。例如，所述CAR可包括CAR0037或与其具有相同的连接肽的本申请所述的CAR。所述跨膜结构域的可包含来自CD8a的跨膜结构域，其序列可以如SEQ ID NO:27所示。例如，所述CAR可包括CAR0037或与其具有相同的跨膜结构域的本申请所述的CAR。所述共刺激结构域可包含来自CD28的共刺激结构，其序列可以如SEQ ID NO:29所示。例如，所述CAR可包括CAR0037或与其具有相同的共刺激结构域的本申请所述的CAR。所述胞内信号传导结构域可包含来自CD3ζ的信号传导结构域，其序列如SEQ ID NO:33所示。例如，所述CAR可包括CAR0037或与其具有相同的胞内信号转导结构域的本申请所述CAR。
所述CAR还可包含铰链区，所述铰链区可位于所述BCMA结合结构域的C端且位于所述跨膜结构域的N端，其序列如SEQ ID NO:25所示。例如，所述CAR可包括CAR0037或与其具有相同的铰链区的本申请所述CAR。
所述CAR还可包含HA-标签，所述HA-标签可位于所述BCMA结合结构域的N端，其序列如SEQ ID NO:5所示。例如，所述CAR可包括CAR0037或与其具有相同的HA-标签的本申请所述CAR。
所述CAR还可连接信号肽，其可位于所述CAR的N端，其序列可以如SEQ ID NO:3所示。
所述CAR还可连接剪切肽，例如：T2A。所述剪切肽可以位于所述胞内信号转导域的C端，其序列可以如SEQ ID NO:35所示。所述CAR还可连接标记检测信号，其可位于所述CAR(或者，所述剪切肽)的C端。所述标记检测信号可选自以下组：GFP、RFP和YFP，其序列如SEQ ID NO:37所示。
例如，本申请所述的CAR可以为CAR0037，其LCDR1-3的氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示；VL的氨基酸序列如SEQ ID NO:15所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示；VH的氨基酸序列如SEQ ID NO:7所示；VH与VL之间的连接肽的序列如SEQ ID NO:23所示；其铰链区如SEQ ID NO:25所示；其跨膜结构域如SEQ ID NO:27所示；其共刺激结构域为CD28共刺激结构域，如SEQ ID NO:29所示；其CD3ζ胞内信号传导结构域如SEQ ID NO:33所示；所述CAR0037还可包含如SEQ ID NO:35所示的剪切肽，以及如SEQ ID NO:37所示的GFP标记检测信号；所述CAR0037还可包含如SEQ ID NO:1所示的KOZAK序列，如SEQ ID NO:3所示的CD8a信号肽，如SEQ ID NO:5所示的HA-标签。
例如，本申请所述的CAR可以为CAR0085，其LCDR1-3的氨基酸序列分别如SEQ ID  NO:17、SEQ ID NO:18和SEQ ID NO:19所示；VL的氨基酸序列如SEQ ID NO:15所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示；VH的氨基酸序列如SEQ ID NO:7所示；VH与VL之间的连接肽的序列如SEQ ID NO:23所示；其铰链区如SEQ ID NO:25所示；其跨膜结构域如SEQ ID NO:27所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID NO:31所示；其CD3ζ胞内信号传导结构域如SEQ ID NO:33所示；所述表达CAR0085的载体还可包含如SEQ ID NO:1所示的KOZAK序列；所述CAR0085如SEQ ID NO:3所示的CD8a信号肽；所述CAR0085还可包含如SEQ ID NO:35所示的剪切肽，以及如SEQ ID NO:39所示的EGFRt标记检测信号。
例如，本申请所述的CAR可以为CAR0087，其LCDR1-3的氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示；VL的氨基酸序列如SEQ ID NO:15所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示；VH的氨基酸序列如SEQ ID NO:7所示；VH与VL之间的连接肽的序列如SEQ ID NO:23所示；其铰链区如SEQ ID NO:25所示；其跨膜结构域如SEQ ID NO:27所示；其共刺激结构域为4-1BB共刺激结构域，如SEQ ID NO:31所示；其CD3ζ胞内信号传导结构域如SEQ ID NO:33所示；所述表达CAR0087的载体还可包含如SEQ ID NO:2所示的KOZAK序列；所述CAR0087还可包含如SEQ ID NO:3所示的CD8a信号肽。
在本申请中，NMOSD可以参考EDSS神经量表评分、MRI活动病灶数、年复发率和无活动病灶数、年复发率和无患者比进行评估。
在本申请中，可以采用描述统计方法分析治疗前后NMOSD患者血清AQP4-Ab滴度的测量值，及其较基线的变化对治疗效果进行评估。
在本申请中，可以采用ELISA方法检测受试者血清AQP4-Ab滴度来测量受试者血清中AQP4-Ab的变化。
在本申请中，所述用于制备预防和/或治疗自身免疫病的药物可以包含本申请所述的免疫效应细胞和任选地药学上可接受地载剂。所述药学上可接受的载剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。在本申请中，所述药物可被配制静脉注射。
另一方面，本申请提供了一种施用方法，所述方法包含向有需要的受试者施用本申请所述的免疫效应细胞。在本申请中，所述有需要的受试者包含患有自身免疫疾病的受试者。
在某些实施方式中，所述施用方法包含向所述受试者以约0.25×10 6～1×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约0.25×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约0.5×10 6个细胞/kg的剂量施用所述免疫效应细胞。在某些实施方式中，所述施用方法包含向所述受试者以约0.395×10 6个细胞/kg的剂量施用所述免疫效应细胞。在某些实施方式中，所述施用方法包含向所述受试者以约0.425×10 6个细胞/kg的剂量施用所述免疫效应细胞。在某些实施方式中，所述施用方法包含向所述受试者以约0.495×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在某些实施方式中，所述施用方法包含向所述受试者以约1×10 6个细胞/kg的剂量施用所述免疫效应细胞。
在本申请中，所述施用方法可以包含静脉注射。在本申请中，施用所述免疫效应细胞的次数可以为一次。
在某些实施方式中，所述施用方法包含在输注所述免疫效应细胞前对受试者进行处理。
在某些实施方式中，所述对受试者进行的处理包括淋巴耗竭处理。
在某些实施方式中，所述淋巴耗竭处理在输注所述免疫效应细胞前约4天开始进行。
在某些实施方式中，所述淋巴耗竭处理周期为约3天。
在某些实施方式中，所述施用方法包含在输注所述免疫效应细胞前4天开始接受为期3天的淋巴耗竭处理。
在某些实施方式中，所述淋巴耗竭处理包含在输注所述免疫效应细胞前向受试者输注环磷酰胺。
在某些实施方式中，所述环磷酰胺的输注剂量为约500mg/m 2/天。
在某些实施方式中，所述淋巴耗竭处理包含在输注所述免疫效应细胞前4天、3天、2天进行所述环磷酰胺输注。
在某些实施方式中，所述施用方法包含在输注所述环磷酰胺后向受试者给与氟达拉滨。
在某些实施方式中，所述氟达拉滨的给药剂量为约30mg/m 2。
在某些实施方式中，所述氟达拉滨的给药时间超过约30分钟。
在某些实施方式中，所述淋巴耗竭处理包含在输注所述免疫效应细胞前4天、3天、2天接受环磷酰胺约500mg/m 2/天的输注，并在环磷酰胺后立即给予超过约30分钟约30mg/m 2的氟达拉滨。
另一方面，本申请还提供了一种药物试剂盒，其包括：1)免疫效应细胞，所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述CAR包含靶向BCMA的抗原结合结构域、跨膜结 构域、共刺激结构域和胞内信号传导结构域，其中所述抗原结合结构域为全人源抗原结合结构域；以及清淋试剂。
在本申请中，所述清淋试剂可以包括环磷酰胺、和/或氟达拉滨。
在本申请中，所述药物试剂盒中的所述免疫效应细胞可以包含CAR，所述CAR的抗原结合结构域可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO：10所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO：11所示的氨基酸序列。
在本申请中，所述药物试剂盒中的所述免疫效应细胞可以包含CAR，所述CAR的抗原结合结构域可以包含LCDR1，LCDR2和LCDR3，其中所述LCDR1可以包含SEQ ID NO：17所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO：18所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO：19所示的氨基酸序列。
在本申请中，所述药物试剂盒中的所述免疫效应细胞可以包含CAR，所述CAR的抗原结合结构域可以包含重链可变区，所述重链可变区可以包含SEQ ID NO：7所示的氨基酸序列。
在本申请中，所述药物试剂盒中的所述免疫效应细胞可以包含CAR，所述CAR的抗原结合结构域可以包含轻链可变区，所述轻链可变区可以包含SEQ ID NO：15所示的氨基酸序列。
在本申请中，所述药物试剂盒中的所述免疫效应细胞可以包含CAR，所述CAR的抗原结合结构域可以包含SEQ ID NO:43所示的氨基酸序列。
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发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1 制备BCMA CAR-T细胞
将慢病毒载体PLVX-EF1alpha-IRES-Puro用NotI和MluI进行双酶切，回收载体片段。对候选scFv质粒PXL0026(其核苷酸序列为SEQ ID NO:44)进行PCR扩增，并用延伸PCR在5’端依次带上NotI酶切位点(含保护碱基)、CD8a信号肽、HA-标签；铰链区、跨膜区、CD28共刺激因子、CD3ζ胞内信号传导结构域基因合成，PCR扩增；T2A剪切肽和eGFP从质粒pMy-BirA-T2A-eGFP PCR扩增出，3’端带上MluI酶切位点及保护碱基；然后用overlap PCR得到5’端带NotI酶切位点3’端带有MluI位点的PCR片段，对该片段进行NotI及 MluI双酶切，并回收。T4连接构建得到编号为PXL0037的CAR质粒(CAR0037的核苷酸序列如SEQ ID NO:50所示)。
用类似的方法获得编号为PXL0085的CAR质粒，将慢病毒载体PLVX-EF1alpha-IRES-Puro用NotI和MluI进行双酶切，回收载体片段。对候选scFv质粒PXL0026进行PCR扩增，并用延伸PCR在5’端依次带上NotI酶切位点(含保护碱基)、CD8a信号肽、铰链区、跨膜区、4-1BB共刺激因子(其核苷酸序列为SEQ ID NO:32)、CD3ζ胞内信号传导结构域基因合成，PCR扩增；然后用overlap PCR得到5’端带NotI酶切位点3’端带有MluI位点的PCR片段，对该片段进行NotI及MluI双酶切，并回收。T4连接构建得到编号为PXL0085的CAR质粒(CAR0085的CAR分子部分的核苷酸序列如SEQ ID NO:52所示)。
用类似的方法获得编号为PXL0087的CAR质粒，将慢病毒载体PLVX-EF1alpha-IRES-Puro用NotI和MluI进行双酶切，回收载体片段。对候选scFv质粒PXL0026进行PCR扩增，并用延伸PCR在5’端依次带上NotI酶切位点(含保护碱基)、CD8a信号肽、铰链区、跨膜区、4-1BB共刺激因子(其核苷酸序列为SEQ ID NO:32)、CD3ζ胞内信号传导结构域、T2A剪切肽、EGFRt(其核苷酸序列为SEQ ID NO:40)基因合成，PCR扩增；然后用overlap PCR得到5’端带NotI酶切位点3’端带有MluI位点的PCR片段，对该片段进行NotI及MluI双酶切，并回收。T4连接构建得到编号为PXL0087的CAR质粒(CAR0087的CAR分子部分的核苷酸序列如SEQ ID NO:52所示)。
通过慢病毒载体PLVX-EF1alpha-IRES-Puro构建得到编号为PXL0037、PXL0085、PXL0087的CAR质粒后(其中CAR0037的氨基酸序列为：SEQ ID NO:49，其中CAR0085的氨基酸序列为：SEQ ID NO:51，其中CAR0087的氨基酸序列为：SEQ ID NO:51)，将CAR质粒转染细胞进行慢病毒包装得到LV0007、LV0020、LV0021(对应PXL0037、PXL0085、PXL0087的CAR质粒)慢病毒。采集健康供者的外周血分离得到PBMC，进一步使用CD3 MicroBeads分选获得T细胞。分选后的T细胞使用CD3/CD28 Dynabeads进行活化，加入LV0007、LV0020、LV0021慢病毒转导，转导时T cells密度约为1.5×106个细胞/ml。在第3天时，对转导后的T细胞进行一次换液。根据上述过程，得到了LV0007-CAR-T、LV0020-CAR-T和LV0021-CAR-T细胞。CAR-T细胞的制备过程如下表所示，产品必须通过质量检测才能放行，每批成品放行前，质控部门要收集并评价所有与该批成品相关的检测记录。以冷冻悬浮液的形式，灌装在特定的输液袋中储存和运输。CAR-T细胞的制备过程见表1。
表1.CAR-T细胞的制备过程
Figure PCTCN2021119720-appb-000002
Figure PCTCN2021119720-appb-000003
实施例2 清淋(淋巴耗竭)预处理
清淋预处理开始前1周内完成各项检查作为基线，在治疗开始前4天～前2天进行清淋预处理治疗。清淋预处理过程如下：静脉输注500mg/m 2环磷酰胺，每天1次连续3天，每次时间超过30分钟，在环磷酰胺静脉输注完成后，马上静脉输注30mg/m 2氟达拉滨，每天1次连续3天，每次时间超过30分钟。以受试者Neu-003为例，对清淋过程进行具体说明，其清淋预处理过程见表2。其他受试者的清淋处理以类似方式进行。
表2清淋预处理情况
药物名称 给药日期 开始时间 结束时间 剂量 单位
环磷酰胺 2020.12.31 16:05 16:38 1020 mg
氟达拉滨 2020.12.31 16:42 17:20 61 mg
环磷酰胺 2021.01.01 11:40 12:26 1020 mg
氟达拉滨 2021.01.01 12:30 13:18 61 mg
环磷酰胺 2021.01.02 10:16 10:50 1020 mg
氟达拉滨 2021.01.02 10:55 11:32 61 mg
实施例3 AQP4-IgG的ELISA检测方法及结果
(1)给药过程
受试者为AQP4-IgG阳性的复发性难治性NMOSD患者。向3名受试者(Neu-003，Neu-004，Neu-008)给予0.5×10 6个CAR+细胞/kg剂量的BCMA CAR-T细胞，实际输注剂量分别为0.395×10 6个CAR+细胞/kg，0.425×10 6个CAR+细胞/kg和0.495×10 6个CAR+细胞/kg的BCMA CAR-T细胞(包含CAR0087的序列)。在清淋预处理结束后两天静脉输注BCMA CAR-T细胞，输注次数为一次。
治疗前后的血清AQP4-IgG滴度变化的静脉血采集时间如下：筛选期(D42～D20)、筛选期至清淋前每周2次、细胞回输前12至5天(时间窗：不晚于清淋前1分钟)、细胞回输前1天(时间窗：至回输前)、3天(时间窗8小时)、7天(时间窗12小时)、10天(时间窗±12小时)、14天(时间窗±1天)、21天(时间窗±1天)、28天(时间窗±3天)、56天(时间窗±3天)、84天(时间窗±7天)、84天后2年内每3个月(时间窗±14天)一次。
脑脊液AQP4-IgG采集时间：清淋预处理前基线期D12至D5、14天(时间窗±1天)、28天(时间窗±3天)、84天(时间窗±7天)、84天后2年内每3个月(时间窗±14天)一次和出组访视时。
(2)检测方法
采用第2代水通道蛋白4(AQP4)自身抗体ELISA检测试剂盒(来源：RSR公司，批号：2KAQE66)进行检测，酶标仪450nm和405nm处读取吸光度值(OD值)，通过拟合OD值与标准曲线来计算AQP4-IgG的浓度水平。
采用描述统计方法分析治疗前后NMOSD患者血清和脑脊液的AQP4-IgG浓度的测量值。绘制各时间点NMOSD患者血清AQP4-IgG浓度-时间曲线。
(3)结果
对三名患者血清中AQP4-IgG浓度水平进行检测，图1A-1C依次为患者Neu-003，Neu-004，Neu-008血清中AQP4-IgG变化趋势情况。在细胞回输前AQP4-IgG浓度水平较高，回输后从第14天起AQP4-IgG浓度水平有下降趋势，提示BCMA CAR-T细胞可降低患者体内AQP4-IgG，有明显的治疗效果。Neu-003和Neu-004患者数据截止到细胞回输后90天内。Neu-008患者数据截止到细胞回输后14天内。
脑脊液中AQP4-IgG的检测结果见表3所示，Neu-003患者的AQP4-IgG回输前为1.279u/mL，BCMA CAR-T细胞回输后可降到0.9628u/mL。Neu-004患者在细胞回输9天前的AQP4-IgG浓度水平在脑脊液可达26.59u/mL，细胞回输后第14天显著下降到3.14u/mL，细胞回输后第38天浓度达下降到0.65u/mL。Neu-008患者的AQP4-IgG回输前为5.17u/mL，细胞回输后可降到3.88u/mL。细胞回输后AQP4-IgG水平下降提示CAR-T细胞治疗有效。Neu-003和Neu-004患者数据截止到细胞回输后90天内。Neu-008患者数据截止到细胞回输后28天内。
表3.BCMA CAR-T细胞回输后NMOSD患者脑脊液的AQP4-IgG浓度水平检测结果
Figure PCTCN2021119720-appb-000004
Figure PCTCN2021119720-appb-000005
实施例4 sBCMA的ELISA检测方法及结果
(1)给药过程
受试者为AQP4-IgG阳性的复发性难治性NMOSD患者。向3名受试者(Neu-003，Neu-004，Neu-008)给予0.5×10 6个CAR+细胞/kg剂量的BCMA CAR-T细胞，实际输注剂量分别为0.395×10 6个CAR+细胞/kg，0.425×10 6个CAR+细胞/kg和0.495×10 6个CAR+细胞/kg的BCMA CAR-T细胞(包含CAR0087的序列)。在清淋预处理结束后两天静脉输注BCMA CAR-T细胞，输注次数为一次。
治疗前后的外周血游离BCMA(sBCMA)检测采集时间如下：清淋预处理前基线期D12至D5、7天(时间窗12小时)、14天(时间窗1天)、21天(时间窗1天)、28天(时间窗3天)、56天(时间窗±7天)、84天(时间窗7天)、84天至2年内每3个月(时间窗14天)一次和出组访视时。
(2)检测方法
采用Human BCMA/TNFRSF17 DuoSet ELISA检测试剂盒(来源：R&R公司，货号：CAT#DY193)进行检测，SpectraMax i3酶标仪450nm和570nm处读取吸光度值(OD值)，以570nm为修正波长，通过拟合OD值与标准曲线来计算sBCMA的浓度水平。
采用描述统计方法分析治疗前后NMOSD患者sBCMA浓度的测量值。绘制各时间点NMOSD患者sBCMA浓度-时间曲线。
(3)结果
该3例患者的sBCMA结果及变化趋势如图2所示，在细胞回输前sBCMA浓度水平较高，回输后sBCMA水平显著下降，其中Neu-004和Neu-008在回输后28天内sBCMA浓度水平下降至检测限以下，提示BCMA CAR-T细胞可显著降低患者体内sBCMA，并间接反映浆细胞的减少。Neu-003和Neu-004患者数据截止到细胞回输后90天内。Neu-008患者数据截止到细胞回输后14天内。
实施例5 药代动力学(PK)实验及结果
(1)实验方法
采用流式细胞术对BCMA CAR-T细胞浓度进行检测，所涉及的主要试剂和主要仪器有Anti-CD45(PercP标记)和Anti-CD3，Acro BIOSYSTEMS公司的FITC标记的BCMA蛋白，和安捷伦公司的Novocyte流式细胞仪。BCMA CAR-T细胞浓度以CAR-T占淋巴细胞百分比(％)呈现。
采用微滴式数字PCR(ddPCR)对BCMA CAR-T细胞的载体拷贝数(Vector copy number,VCN)进行检测，所涉及的主要试剂和主要仪器有：Qiagen公司的基因组DNA抽提取试剂盒，Thermo Fisher公司的NanoDrop核酸定量仪，BIO-RAD公司的微滴发生油，微滴发生卡和垫，ddPCR Supermix for Probes(no dUTP)，微滴读取油和QX200型微滴式数字PCR系统。理论上，每个BCMA CAR-T细胞(CD3 +CAR +细胞)含有2个CAR VCN。VCN的数量增加可以体现CAR-T细胞的扩增。
采样时间：BCMA CAR-T细胞回输前12至5天(时间窗：清淋预处理前)、回输后1天(时间窗±8小时)、3天(时间窗±8小时)、5天(时间窗±12小时)、7天(时间窗±12小时)、10天(时间窗±12小时)、14天(时间窗±1天)、21天(时间窗±1天)、28天(时间窗±3天)、56天(时间窗±7天)、84天(时间窗±7天)、84天以后每3个月(时间窗±14天)进行一次血样采集，直至CAR-T监测到峰值后连续2次经微滴式数字PCR(ddPCR)检测不到VCN、疾病恶化、出组或者回输后2年。其中：回输后1天代表从回输时间开始计算后24小时，回输后3天代表从回输时间开始计算后72小时，之后访视点以此类推。尽量在进行PK采样同时进行血常规采样，血常规采样与PK采血的时间最长不能超过24小时。
计算已回输细胞患者的PK参数。
·血药浓度–时间数据分析：分别绘制各时间点外周血中BCMA CAR-T细胞浓度-时间曲线和VCN-时间曲线；并采用清单列出各受试者各时间点外周血中BCMA CAR-T浓度和VCN值。
·PK参数分析：通过VCN和BCMA CAR-T细胞浓度计算PK参数(C max、T max、AUC 0～ 28d和AUC 0～last等)的算术均值、标准差变异系数、中位数、最大值、最小值、几何均数和几何均值变异系数。
(2)结果
图3A-3B显示了BCMA CAR-T细胞浓度和VCN变化结果，在给药后CAR-T细胞浓度以及VCN水平首先快速上升，达到峰值后缓慢下降，给药后CAR-T细胞在患者体内扩增良 好，VCN在回输后52天依然可被测出。Neu-003和Neu-004患者数据截止到细胞回输后90天内。Neu-008患者数据截止到细胞回输后28天内。
PK参数如表4所示，BCMA CAR-T细胞浓度和VCN的中位T max均为10天，BCMA CAR-T细胞浓度和VCN的中位C max分别为77.78％和55300 Copies/μg DNA，VCN的中位AUC 0-28为329184.4 Days×Copies/μg DNA(范围275819.600～1500890.100 Days×Copies/μg DNA)，VCN的中位AUC 0-last为329184.4 Days×Copies/μg DNA(范围275819.600～1706986.700 Days×Copies/μg DNA)，可见BCMA CAR-T细胞在NMOSD患者体内达峰时间快，细胞扩增良好。
表4.BCMA CAR-T细胞回输后的PK参数
Figure PCTCN2021119720-appb-000006
Figure PCTCN2021119720-appb-000007
实施例6 炎症因子检测方法及结果
(1)检测方法
采用颗粒增强免疫比浊方法检测铁蛋白浓度，采用电化学发光三抗原夹心比浊方法检测IL-6浓度，采用免疫投射比浊方法检测C反应蛋白浓度，采用电化学发光三抗原夹心比浊方法检测降钙素原浓度。
(2)炎症因子检测结果
Neu-003患者的炎症因子结果如表5所示，铁蛋白在细胞回输后浓度最高达654.6ug/mL，IL-6在细胞回输后浓度最高达31.11pg/mL，C反应蛋白在细胞回输后浓度最高达16.7mg/L，降钙素原在细胞回输后浓度均低于回输前浓度，各炎症因子在给药后未出现常见导致副作用的浓度升高，结果表明BCMA CAR-T细胞治疗引起细胞因子释放综合征的风险较小。
表5.炎症因子检测结果
Figure PCTCN2021119720-appb-000008
Figure PCTCN2021119720-appb-000009
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (65)

  1. 免疫效应细胞在制备预防和/或治疗自身免疫病的药物中的用途，所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述CAR包含靶向BCMA的抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述抗原结合结构域为全人源抗原结合结构域。
  2. 根据权利要求1所述的用途，其中所述自身免疫病包含自身免疫性炎性疾病。
  3. 根据权利要求2所述的用途，其中所述炎性疾病包含神经炎。
  4. 根据权利要求2-3中任一项所述的用途，其中所述炎性疾病包含脱髓鞘疾病。
  5. 根据权利要求2-4中任一项所述的用途，其中所述炎性疾病包含中枢神经系统炎性疾病。
  6. 根据权利要求2-5中任一项所述的用途，其中所述炎性疾病包含中枢神经系统炎性脱髓鞘疾病。
  7. 根据权利要求2-6中任一项所述的用途，其中所述炎性疾病包含视神经炎。
  8. 根据权利要求2-7中任一项所述的用途，其中所述炎性疾病包含脊髓炎。
  9. 权利要求2-8中任一项所述的用途，其中所述炎性疾病包含视神经脊髓炎谱系病(NMOSD)。
  10. 根据权利要求2-9中任一项所述的用途，其中所述炎性疾病包含AQP4(水通道蛋白4)-Ab阳性视神经脊髓炎谱系病。
  11. 根据权利要求1所述的用途，其中所述自身免疫病包含AQP4-Ab介导的自身免疫病。
  12. 根据权利要求1-11中任一项所述的用途，其中所述自身免疫病包含AQP4-Ab阳性的疾病和/或病症。
  13. 根据权利要求12所述的用途，其中所述AQP4-Ab阳性的疾病和/或病症包含中枢神经系统脱髓鞘疾病。
  14. 根据权利要求12-13中任一项所述的用途，其中所述AQP4-Ab阳性的疾病和/或病症包含AQP4-Ab阳性视神经脊髓炎谱系疾病。
  15. 根据权利要求1-14中任一项所述的用途，其中所述自身免疫病包含视神经脊髓炎谱系疾病(NMOSD)。
  16. 根据权利要求1-15中任一项所述的用途，其中所述抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO：10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO：11所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的用途，其中所述抗原结合结构域包含LCDR1， LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO：17所示的氨基酸序列，所述LCDR2包含SEQ ID NO：18所示的氨基酸序列，且所述LCDR3包含SEQ ID NO：19所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的用途，其中所述抗原结合结构域包含重链可变区，所述重链可变区包含SEQ ID NO：7所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的用途，其中所述抗原结合结构域包含轻链可变区，所述轻链可变区包含SEQ ID NO：15所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的用途，其中所述抗原结合结构域包含SEQ ID NO:43所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的用途，其中所述抗原结合结构域包含抗体或其片段。
  22. 根据权利要求1-21中任一项所述的用途，其中所述抗原结合结构域包含scFv。
  23. 根据权利要求1-22中任一项所述的用途，其中所述抗原结合域能够结合BCMA。
  24. 根据权利要求1-23中任一项所述的用途，其中所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8a、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  25. 根据权利要求1-24中任一项所述的用途，其中所述跨膜结构域包含SEQ ID NO:27所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的用途，其中所述共刺激结构域包含选自下述蛋白的多肽：CD28、4-1BB、OX-40和ICOS。
  27. 根据权利要求1-26中任一项所述的用途，其中所述共刺激结构域包含SEQ ID NO:29或SEQ ID NO:31所述的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的用途，其中所述胞内信号传导结构域包含来自CD3ζ的信号传导结构域。
  29. 根据权利要求1-28中任一项所述的用途，其中所述胞内信号传导结构域包含SEQ ID NO:33所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的用途，其中所述CAR还包含铰链区，所述铰链区链接所述抗原结合结构域和所述跨膜结构域。
  31. 根据权利要求30中任一项所述的用途，其中所述铰链区包含SEQ ID NO:25所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的用途，其中所述CAR还连接信号肽。
  33. 根据权利要求32所述的用途，其中所述信号肽包含SEQ ID NO:3所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的用途，其中所述CAR还连接剪切肽。
  35. 根据权利要求34所述的用途，其中所述剪切肽包含来自T2A肽的氨基酸序列。
  36. 根据权利要求34-35中任一项所述的用途，其中所述剪切肽包含SEQ ID NO:35所示的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的用途，其中所述CAR包含SEQ ID NO:49或SEQ ID NO:51所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的用途，其中所述免疫效应细胞包含T细胞和/或自然杀伤(NK)细胞。
  39. 根据权利要求1-38中任一项所述的用途，其中所述药物包含任选地药学上可接受的载剂。
  40. 施用方法，其包含向有需要的受试者施用权利要求1-39中任一项所述的免疫效应细胞。
  41. 根据权利要求40所述的施用方法，其包含向所述受试者以约0.25×10 6～1×10 6个细胞/kg的剂量施用所述免疫效应细胞。
  42. 根据权利要求40-41中任一项所述的施用方法，其包含向所述受试者以约0.25×10 6个细胞/kg的剂量施用所述免疫效应细胞。
  43. 根据权利要求40-41中任一项所述的施用方法，其包含向所述受试者以约0.5×10 6个细胞/kg的剂量施用所述免疫效应细胞。
  44. 根据权利要求40-41中任一项所述的施用方法，其包含向所述受试者以约1×10 6个细胞/kg的剂量施用所述免疫效应细胞。
  45. 根据权利要求40-44中任一项所述的施用方法，其包含静脉注射。
  46. 根据权利要求40-45中任一项所述的施用方法，其施用所述免疫效应细胞的次数为一次。
  47. 根据权利要求40-46中任一项所述的施用方法，其包含在输注所述免疫效应细胞前对受试者进行处理。
  48. 根据权利要求47所述的施用方法，其中所述处理包括淋巴耗竭处理。
  49. 根据权利要求48所述的施用方法，其中所述淋巴耗竭处理在输注所述免疫效应细胞前约4天开始进行。
  50. 根据权利要求48-49中任一项所述的施用方法，其中所述淋巴耗竭处理周期为约3天。
  51. 根据权利要求48-50中任一项所述的施用方法，其包含在输注所述免疫效应细胞前4天开始接受为期3天的淋巴耗竭处理。
  52. 根据权利要求48-51中任一项所述的施用方法，其中所述淋巴耗竭处理包含在输注所述免疫效应细胞前向受试者输注环磷酰胺。
  53. 根据权利要求52所述的施用方法，其中所述环磷酰胺的输注剂量为约500mg/m 2/天。
  54. 根据权利要求52-53中任一项所述的施用方法，其中所述淋巴耗竭处理包含在输注所述免疫效应细胞前4天、3天、和2天进行所述环磷酰胺输注。
  55. 根据权利要求52-54中任一项所述的施用方法，其在输注所述环磷酰胺后向受试者给与氟达拉滨。
  56. 根据权利要求55所述的施用方法，其中所述氟达拉滨的给药剂量为约30mg/m 2。
  57. 根据权利要求55-56中任一项所述的施用方法，其中所述氟达拉滨的给药时间超过约30分钟。
  58. 根据权利要求55-57中任一项所述的施用方法，其中所述淋巴耗竭处理包含在输注所述免疫效应细胞前4天、3天、2天接受环磷酰胺约500mg/m 2/天的输注，并在环磷酰胺后立即给予超过约30分钟约30mg/m 2的氟达拉滨。
  59. 药物试剂盒，包括：
    1)免疫效应细胞，所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述CAR包含靶向BCMA的抗原结合结构域、跨膜结构域、共刺激结构域和胞内信号传导结构域，其中所述抗原结合结构域为全人源抗原结合结构域；以及
    2)清淋试剂。
  60. 根据权利要求59所述的药物试剂盒，所述的清淋试剂包括环磷酰胺、和/或氟达拉滨。
  61. 根据权利要求59-60中任一项所述的药物试剂盒，其中所述抗原结合结构域包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO：10所示的氨基酸序列，且所述HCDR3包含SEQ ID NO：11所示的氨基酸序列。
  62. 根据权利要求59-61中任一项所述的药物试剂盒，其中所述抗原结合结构域包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO：17所示的氨基酸序列，所述LCDR2包含SEQ ID NO：18所示的氨基酸序列，且所述LCDR3包含SEQ ID NO：19所示的氨基酸序列。
  63. 根据权利要求59-62中任一项所述的药物试剂盒，其中所述抗原结合结构域包含重链可变区，所述重链可变区包含SEQ ID NO：7所示的氨基酸序列。
  64. 根据权利要求59-63中任一项所述的药物试剂盒，其中所述抗原结合结构域包含轻链可变区，所述轻链可变区包含SEQ ID NO：15所示的氨基酸序列。
  65. 根据权利要求59-64中任一项所述的药物试剂盒，其中所述抗原结合结构域包含SEQ ID NO:43所示的氨基酸序列。
PCT/CN2021/119720 2021-09-22 2021-09-22 Bcma car-t在制备用于治疗自身免疫病的药物中的应用 WO2023044633A1 (zh)

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