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WO2021051390A1 - 靶向bcma的抗体及嵌合抗原受体 - Google Patents

靶向bcma的抗体及嵌合抗原受体 Download PDF

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Publication number
WO2021051390A1
WO2021051390A1 PCT/CN2019/107004 CN2019107004W WO2021051390A1 WO 2021051390 A1 WO2021051390 A1 WO 2021051390A1 CN 2019107004 W CN2019107004 W CN 2019107004W WO 2021051390 A1 WO2021051390 A1 WO 2021051390A1
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Prior art keywords
antibody
cells
antigen
seq
cell
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PCT/CN2019/107004
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English (en)
French (fr)
Inventor
杜靓
牟男
张红艳
金理娜
于跃
袁纪军
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上海吉倍生物技术有限公司
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Priority to CN201980100434.XA priority Critical patent/CN114401989B/zh
Priority to JP2022518203A priority patent/JP2023501871A/ja
Priority to EP19945687.2A priority patent/EP4032907A4/en
Priority to PCT/CN2019/107004 priority patent/WO2021051390A1/zh
Priority to US17/761,989 priority patent/US20220331363A1/en
Priority to TW109130746A priority patent/TW202115111A/zh
Publication of WO2021051390A1 publication Critical patent/WO2021051390A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A61K39/4611—
    • A61K39/4631—
    • A61K39/464417—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of disease treatment and immunology. Specifically, the present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to BCMA, and chimeric antigen receptors (CAR) containing the same.
  • the present invention also relates to nucleic acid molecules encoding such CARs, immune effector cells expressing such CARs, and methods for preparing such immune effector cells.
  • the present invention also relates to the use of these CARs and immune effector cells for the prevention and/or treatment of B-cell-related conditions (such as B-cell malignancies, autoimmune diseases) and methods for preventing and/or treating the B-cell-related conditions.
  • B-cell-related conditions such as B-cell malignancies, autoimmune diseases
  • B cells are a kind of lymphocytes, pluripotent stem cells derived from bone marrow, which play an important role in producing antibodies to mediate humoral immune response, presenting soluble antigens, and producing a large number of cytokines, participating in immune regulation, inflammation, and hematopoiesis (1).
  • Some major diseases involve B cells. Malignant transformation of B cells leads to cancer, including some lymphomas such as Hodgkin's lymphoma and multiple myeloma.
  • Abnormal B cell physiology may lead to the development of autoimmune diseases, including systemic lupus erythematosus.
  • the above two types of diseases involving B cells can be considered to be due to excessive B cell growth and/or inappropriate attack on parts of the body.
  • a possible control strategy is to use antibodies or antigen-binding parts of antibodies that target pathological B cells, or Other forms of drugs based on these antibodies or the antigen-binding portion of antibodies.
  • BCMA B-cell maturation antigen
  • TNFRSF17 or CD269 B-cell maturation antigen
  • BCMA is composed of 184 amino acids and is a type I transmembrane signal protein lacking a signal peptide.
  • BCMA is a member of the tumor necrosis factor receptor family (TNFR) and binds TNF family ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand) (2).
  • TNFR tumor necrosis factor receptor family
  • BAFF B cell activating factor
  • APRIL proliferation inducing ligand
  • BCMA knockout mice have normal lymphoid organs and immune system (5).
  • the development of B lymphocytes is normal, but the number of plasma cells is significantly reduced. This proves that BCMA plays an important role in maintaining the survival of plasma cells.
  • the main mechanism is Including the combination of BCMA and BAFF protein, conduction signals to activate the NF- ⁇ B pathway, and up-regulate the anti-apoptotic genes Bcl-2, Mcl-1 and Bclw, etc., to maintain cell growth (6).
  • BCMA is commonly expressed in B-cell malignancies such as multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL), and it plays an important role in promoting the malignant proliferation of tumor cells (7), in summary
  • B-cell malignancies such as multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL)
  • MM multiple myeloma
  • NHL non-Hodgkin’s lymphoma
  • the BCMA can be used as one of the targets of B-cell malignancies for the treatment of multiple myeloma and non-Hodgkin's lymphoma.
  • multiple myeloma is a malignant plasma cell disease, manifested by malignant clonal proliferation of bone marrow plasma cells, secreting monoclonal immunoglobulin or its fragments (M protein), causing bone, kidney and other related target organs or tissue damage, common clinical Manifested as bone pain, anemia, renal insufficiency, infection, etc. (8).
  • M protein monoclonal immunoglobulin or its fragments
  • multiple myeloma is the second largest malignant tumor of the hematological system, accounting for 13% of the hematological malignant tumors, and its incidence is increasing year by year with the increase of age. In recent years, there is a trend of younger generation (9).
  • CAR-T cells chimeric antigen receptor T cells
  • ADC antibody-drug conjugates
  • BCMA-targeted drugs the fastest progress is Blue Bird's bb2121.
  • the extracellular domain used in the drug form of bb2121 contains a mouse anti-antigen binding fragment (CN 201580073309.6) that binds to human BCMA, and its source is the anti-BCMA antibody with clone number C11D5.3 developed by Biogen Idec (CN 201510142069.2). C11D5.3 is widely used in BCMA-CAR T cell therapy.
  • bb21217 In addition to bb2121, bb21217, NCI's BCMA-CAR T, and Hengrun Dasheng's BCMA-CAR T (CN 201610932365.7) all use this cloned antigen binding fragment To achieve the binding to the BCMA target, and the mouse-derived form of this clone is used in the CAR T mentioned above.
  • Humanized antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body.
  • a classic method of mouse antibody humanization is CDR implantation.
  • the CDR of the antibody variable region is the region where the antibody recognizes and binds to the antigen, which directly determines the specificity of the antibody.
  • the CDR of the murine monoclonal antibody is transplanted to the variable region of the human antibody to replace the CDR of the human antibody, so that the human antibody obtains the antigen binding specificity of the murine monoclonal antibody while reducing its heterogeneity.
  • the antigen is mainly in contact with the CDR of the antibody, the FR region often participates in the role and affects the spatial configuration of the CDR.
  • the V region embedded between the mouse CDR and the human FR may change the CDR configuration of a single antigen, and the ability to bind to the antigen will decrease or even significantly decrease.
  • Blue Bird found in its patent application (CN 201580050638.9) that a CAR T constructed with humanized C11D5.3 as the antigen-binding part can cause significant antigen-independent release of cytokines, and the humanization was observed at the same time.
  • the introduction of CAR will induce continuous activation and exhaustion of T cells.
  • the structural modification of the non-BCMA binding part of the CAR cannot solve the above problems, making this humanized anti-C11D5.3 anti-BCMA CAR not suitable for T cell therapy.
  • the inventors conducted in-depth research and modification on the murine antibody C11D5.3 that specifically recognizes BCMA, and developed a humanized antibody of the murine antibody.
  • the humanized antibody maintained a binding equivalent to that of the murine antibody. While expressing the activity of BCMA tumor cells, the non-specific binding to normal tissue cells is significantly reduced. On this basis, the inventors have paid a lot of creative work to further construct and obtain a chimeric antigen receptor (CAR) that can specifically bind to BCMA.
  • CAR chimeric antigen receptor
  • the CAR of the present invention can direct immune effector cell specificity and reactivity to cells expressing BCMA in a non-MHC-restricted manner, so that they can be eliminated, and will not cause antigen-independent cytokine release, and will not induce T cells The continued activation and exhaustion. Therefore, the humanized antibody and CAR of the present invention have the potential to be used to prevent and/or treat B-cell-related conditions (such as B-cell malignancies, autoimmune diseases, etc.), and have great clinical value.
  • B-cell-related conditions such as B-cell malignancies, autoimmune diseases, etc.
  • the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to BCMA, the antibody or antigen-binding fragment thereof comprising:
  • VH The heavy chain variable region (VH), which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 1 or 3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • VL The light chain variable region (VL), which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 2 or 4 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH The heavy chain variable region (VH), which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO:1 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • VL The light chain variable region (VL), which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 2 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises the sequence shown in SEQ ID NO:1 or in combination therewith.
  • the VL includes the sequence shown in SEQ ID NO: 2 or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH The heavy chain variable region (VH), which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • VL The light chain variable region (VL), which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 4 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises the sequence shown in SEQ ID NO: 3 or Compared with it has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 %, or 100% sequence identity
  • the VL includes the sequence shown in SEQ ID NO: 4 or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least A sequence of 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the antibody or antigen-binding fragment thereof has one or more of the following biological functions:
  • the affinity of binding to the isolated human BCMA protein is not lower than that of the murine antibody C11D5.3; the expression "isolated” means that the protein is not contained in the cell or on the cell surface; the affinity can be determined by, for example, the term ka (from Defined by the rate constant of the binding of the antibody of the antibody/antigen complex), kD (dissociation constant) and/or KD (kD/ka);
  • the affinity for binding to cells expressing human BCMA is not lower than that of the murine antibody C11D5.3; the affinity can be determined by, for example, the terms ka (rate constant of the binding of the antibody from the antibody/antigen complex), kD (dissociation Constant) and/or KD (kD/ka) to define;
  • T cells expressing CAR containing the antibody or antigen-binding fragment thereof do not substantially cause antigen-independent cytokine release.
  • the antibody or antigen-binding fragment thereof of the present invention may further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof, and the variant is derived from it Compared with the sequence, there are one or more amino acid substitutions, deletions or additions.
  • the variant has conservative substitutions of one or more amino acids compared to the sequence from which it is derived.
  • the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has compared with the sequence from which it is derived
  • One or more amino acid substitutions, deletions, or additions for example, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; for example, 1, 2, 3, 4 Or 5 amino acid substitutions, deletions or additions; and/or,
  • the light chain of the antibody or antigen-binding fragment thereof of the present invention comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has conservative substitutions of up to 20 amino acids compared to the sequence from which it is derived (For example, conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, or 5 amino acids).
  • CL light chain constant region
  • the heavy chain constant region is selected from IgG, IgM, IgE, IgD, or IgA.
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.
  • the heavy chain constant region is a murine IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.
  • the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.
  • preferably the heavy chain constant region is a human IgG1 or IgG4 heavy chain constant region.
  • the light chain constant region is selected from kappa or lambda.
  • the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region is a murine kappa light chain constant region. In certain embodiments, the light chain constant region is a human kappa light chain constant region.
  • the antibodies of the invention are humanized antibodies.
  • the antigen-binding fragment of the present invention is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, di-scFv, (scFv) 2 .
  • the antibody or antigen-binding fragment thereof of the present invention may include variants that differ from the antibody or antigen-binding fragment from which it is derived by only one or more (for example, up to 20 Conservative substitution of 1, at most 15, at most 10, or at most 5 amino acids) conservative substitution of amino acid residues, or at least 85%, at least 90%, or at least 95% of the antibody or antigen-binding fragment from which it is derived , At least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and basically retain the above-mentioned biological functions of the antibody or antigen-binding fragment from which it is derived.
  • the antibody of the present invention can be prepared by various methods known in the art, for example, obtained by genetic engineering recombination technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into the expression vector, and then transfected into the host cell. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present invention is expressed.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium.
  • the present invention provides an isolated nucleic acid molecule comprising nucleotides encoding the antibody of the present invention or its antigen-binding fragment, or its heavy chain variable region and/or light chain variable region. sequence.
  • the isolated nucleic acid molecule encodes the antibody or antigen-binding fragment thereof of the present invention, or its heavy chain variable region and/or light chain variable region.
  • the present invention provides a vector (such as a cloning vector or expression vector), which comprises the isolated nucleic acid molecule of the present invention.
  • a vector such as a cloning vector or expression vector
  • the vectors of the present invention are, for example, plasmids, cosmids, bacteriophages and the like.
  • the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (such as a mammal, such as a human).
  • the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DG44).
  • a method for preparing the antibody or antigen-binding fragment thereof of the present invention comprises culturing the host cell of the fourth aspect under conditions that allow expression of the antibody or antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the culture of the cultured host cell.
  • the antibody or antigen-binding fragment thereof of the present invention can be derivatized, for example, linked to another molecule (for example, another polypeptide or protein).
  • another molecule for example, another polypeptide or protein.
  • the derivatization (eg, labeling) of the antibody or its antigen-binding fragment will not adversely affect its binding to BCMA (especially human BCMA). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms.
  • the antibody or antigen-binding fragment thereof of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (for example, to form Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (for example, avidin or polyhistidine tag).
  • another antibody for example, to form Bispecific antibodies
  • detection reagents for example, to form Bispecific antibodies
  • pharmaceutical reagents for example, to form Bispecific antibodies
  • proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (for example, avidin or polyhistidine tag).
  • the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a modified portion linked to the antibody or antigen-binding fragment thereof.
  • the modified moiety is a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.
  • the conjugate comprises the antibody or antigen-binding fragment thereof of the invention and a detectable label attached to the antibody or antigen-binding fragment thereof.
  • the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
  • Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radioactive nuclear (E.g., 3 H, 125 I, 35 S, 14 C, or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , Phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridine ester compounds), Magnetic beads (for example, ), calorimetric markers such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex,
  • Patents teaching the use of this marker include, but are not limited to, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference).
  • the detectable label as described above can be detected by methods known in the art. For example, a radioactive label can be detected using photographic film or a scintillation calculator, and a fluorescent label can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers.
  • such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the antibody or antigen-binding fragment thereof of the present invention can be linked to a therapeutic moiety.
  • the conjugate since the conjugate has the ability to selectively deliver one or more therapeutic agents to target tissues (for example, cells expressing BCMA), the conjugate can increase the antibody of the present invention or its The therapeutic efficacy of antigen-binding fragments in the treatment of diseases such as B cell related conditions.
  • the modified moiety is a therapeutic agent.
  • the conjugate comprises the antibody or antigen-binding fragment thereof of the present invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the conjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the therapeutic agent is a cytotoxic agent.
  • the cytotoxic agent includes any agent that is harmful to cells (e.g., kills cells).
  • the therapeutic agent is selected from alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
  • alkylating agents examples include, but are not limited to, nitrogen mustards (such as dichloroethyl methylamine, chlorambucil, melphalan, cyclophosphamide, etc.), ethylene imines (Such as cetepa, etc.), sulfates and polyols (such as busulfan, dibromomannitol), nitrosoureas (such as carmustine, lomustine, etc.), platinum anti-tumor agents (Such as cisplatin, oxaliplatin, carboplatin, etc.) and so on.
  • nitrogen mustards such as dichloroethyl methylamine, chlorambucil, melphalan, cyclophosphamide, etc.
  • ethylene imines such as cetepa, etc.
  • sulfates and polyols such as busulfan, dibromomannitol
  • nitrosoureas such as carmustine, lomustine, etc
  • mitotic inhibitors examples include, but are not limited to, maytansinoids (e.g., maytansine, maytansinol, C-3 esters of maytansinol, etc.), taxanes (e.g., poly Cetaxel, paclitaxel, or nanoparticle paclitaxel, etc.), vinca alkaloids (such as vindesine sulfate, vincristine, vinblastine or vinorelbine, etc.)
  • maytansinoids e.g., maytansine, maytansinol, C-3 esters of maytansinol, etc.
  • taxanes e.g., poly Cetaxel, paclitaxel, or nanoparticle paclitaxel, etc.
  • vinca alkaloids such as vindesine sulfate, vincristine, vinblastine or vinorelbine, etc.
  • anti-tumor antibiotics examples include, but are not limited to, actinomycin, anthracycline antibiotics (e.g. daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), Calichemycin, becinomycin, etc.
  • antimetabolites examples include, but are not limited to, folic acid antagonists (e.g., methotrexate, etc.), pyrimidine antagonists (e.g., 5-fluorouracil, fluorouridine, cytarabine, cappene Tabine, gemcitabine, etc.), purine antagonists (e.g. 6-mercaptopurine, 6-thioguanine, etc.), adenosine deaminase inhibitors (e.g., Cladribine, Fludarabine, Nelarabine, Penstat Ding et al.).
  • folic acid antagonists e.g., methotrexate, etc.
  • pyrimidine antagonists e.g., 5-fluorouracil, fluorouridine, cytarabine, cappene Tabine, gemcitabine, etc.
  • purine antagonists e.g. 6-mercaptopurine, 6-thioguanine, etc.
  • topoisomerase inhibitors examples include but are not limited to (camptothecins and their derivatives (e.g. irinotecan, topotecan, etc.), amsacrine, daunorubicin Vitamins, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, propylimine, teniposide, etc.
  • tyrosine kinase inhibitors that can be used in the conjugates of the present invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib , Imatinib, lapatinib, letutinib, nilotinib, simazanib, sunitinib, vandetanib, etc.
  • radionuclide agents examples include, but are not limited to, I 131 , In 111 , Y 90 , Lu 177 and the like.
  • the therapeutic agent is selected from platinum anti-tumor agents, anthracycline antibiotics, taxanes, nucleoside analogs, camptothecins, and analogs or homologs thereof , And any combination thereof.
  • the antibodies of the present invention or antigen-binding fragments thereof are optionally conjugated to a modified portion (e.g., a detectable label or therapeutic agent) via a linker.
  • a modified portion e.g., a detectable label or therapeutic agent
  • the cytotoxic agent can be coupled to the antibody or antigen-binding fragment thereof of the present invention by using linker technology available in the art.
  • linker technology available in the art.
  • types of linkers that have been used to couple cytotoxic agents to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers.
  • a linker can be selected that is easy to be cleaved by low pH in the lysosomal compartment or easy to be cleaved by a protease (e.g., a protease that is preferentially expressed in tumor tissue, such as cathepsin, such as cathepsin B, C, D).
  • the antibody or antigen-binding fragment thereof of the present invention can be used to construct a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the features of the CAR of the present invention include non-MHC-restricted BCMA recognition ability, which confers immune effector cells (for example, T cells, T cells, NK cells, monocytes, macrophages or dendritic cells) do not rely on antigen processing and presentation to recognize the ability of cells expressing BCMA.
  • the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, wherein the The extracellular antigen-binding domain includes the antibody of the present invention or an antigen-binding fragment thereof.
  • the CAR includes an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain from N-terminus to C-terminus.
  • the extracellular antigen binding domain contained in the CAR of the present invention confers the ability of the CAR to recognize BCMA.
  • the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises the sequence shown in SEQ ID NO:1 or a combination thereof Compared with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , Or 100% sequence identity, the VL includes the sequence shown in SEQ ID NO: 2 or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • VH heavy chain variable region
  • VL light chain variable region
  • the extracellular antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH comprises the sequence shown in SEQ ID NO: 3 or a combination thereof Compared with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , Or 100% sequence identity, the VL includes the sequence shown in SEQ ID NO: 4 or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the extracellular antigen binding domains include, but are not limited to, Fab fragments, Fab' fragments, F(ab)' 2 fragments, Fv, disulfide bond stabilized Fv protein ("dsFv"), scFv , Di-scFv, (scFv) 2 .
  • the antibody or antigen-binding fragment thereof is scFv, di-scFv or (scFv) 2 .
  • the extracellular antigen binding domain comprises a linker.
  • the VH and VL contained in the extracellular antigen binding domain are connected by a linker.
  • the linker has a sequence of SEQ ID NO: 5.
  • the transmembrane domain contained in the CAR of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in the cell membrane (especially eukaryotic cell membrane).
  • the transmembrane domain of the CAR suitable for the present invention can be derived from natural sources.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain may be a synthetic non-naturally occurring protein segment, such as a protein segment that mainly contains hydrophobic residues such as leucine and valine.
  • the transmembrane domain is a transmembrane region selected from the following proteins: alpha, beta, or zeta chain of T cell receptor, CD8 ⁇ , CD28, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10 and any combination thereof.
  • the transmembrane domain comprises the transmembrane region of CD8 ⁇ .
  • the transmembrane domain comprises a transmembrane domain of a T cell costimulatory molecule (eg, CD137 or CD28).
  • the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO:7.
  • the chimeric antigen receptor of the present invention may include a spacer domain between the extracellular antigen binding domain and the transmembrane domain.
  • the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (eg, IgG1 or IgG4).
  • an immunoglobulin eg, IgG1 or IgG4
  • CH2 and CH3 extend the antigen-binding domain of the CAR from the cell membrane of the CAR-expressing cell, and can more accurately mimic the size and domain of the natural TCR structure.
  • the spacer domain comprises a hinge domain.
  • the hinge domain may be a stretch of amino acids commonly found between two domains of a protein, which may allow the protein to have flexibility and allow one or two domains to move relative to each other. Therefore, the hinge domain may be any amino acid sequence as long as it can provide this flexibility of the extracellular antigen binding domain and this mobility relative to the transmembrane domain.
  • the hinge domain is the hinge region of a naturally occurring protein or a portion thereof.
  • the hinge domain comprises the hinge region of CD8 ⁇ or a portion thereof, for example, a fragment containing at least 15 (eg, 20, 25, 30, 35, or 40) consecutive amino acids of the hinge region of CD8 ⁇ .
  • the spacer domain comprises the amino acid sequence shown in SEQ ID NO:6.
  • the intracellular signal transduction domain contained in the CAR of the present invention participates in the signal transduction of effective antigen receptor binding (the combination of the CAR of the present invention and BCMA) into the immune effector cells, and activates at least the immune effector cells expressing the CAR.
  • effective antigen receptor binding the combination of the CAR of the present invention and BCMA
  • the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain.
  • the primary signaling domain may be any intracellular signaling domain that contains an immunoreceptor tyrosine activation motif (ITAM).
  • the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM).
  • the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, DAP10, CD79b, or CD66d.
  • the primary signaling domain comprises the intracellular signaling domain of CD3 ⁇ .
  • the costimulatory signal transduction domain may be an intracellular signal transduction domain from a costimulatory molecule.
  • the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4- 1BB), CD150 (SLAMF1), CD270 (HVEM) or DAP10.
  • the costimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137 (4-1BB), or a combination of fragments of the two.
  • the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains. In such embodiments, the two or more costimulatory signaling domains may be the same or different.
  • the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain.
  • the primary signal transduction domain and at least one costimulatory signal transduction domain may be connected in series to the carboxyl end of the transmembrane domain in any order.
  • the intracellular signaling domain may comprise the intracellular signaling domain of CD3 ⁇ and the intracellular signaling domain of CD137.
  • the intracellular signaling domain of CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO:9.
  • the intracellular signaling domain of CD137 comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the CAR of the present invention may further include a signal peptide at its N-terminus.
  • a signal peptide is a polypeptide sequence that targets the sequence linked to it to a desired site in the cell.
  • the signal peptide can target the CAR linked to it to the secretory pathway of the cell, and allow the CAR to be further integrated and anchored into the lipid bilayer.
  • Signal peptides that can be used in CAR are known to those skilled in the art.
  • the signal peptide comprises a heavy chain signal peptide (for example, the heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8 ⁇ signal peptide .
  • a heavy chain signal peptide for example, the heavy chain signal peptide of IgG1
  • GM-CSFR2 granulocyte-macrophage colony stimulating factor receptor 2
  • the present invention provides a chimeric antigen receptor capable of specifically binding to BCMA.
  • the chimeric antigen receptor comprises an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and a cellular antigen binding domain, from the N-terminus to the C-terminus. Internal signaling domain.
  • the intracellular signal transduction domain is a costimulatory signal transduction domain and a primary signal transduction domain from the N-terminus to the C-terminus.
  • the spacer domain includes the hinge region of CD8 (for example, CD8 ⁇ ), which has the sequence shown in SEQ ID NO:6.
  • the transmembrane domain comprises the transmembrane region of CD8 (for example, CD8 ⁇ ), which has the sequence shown in SEQ ID NO:7.
  • the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3 ⁇ , which has SEQ ID NO : Sequence shown in 9.
  • the costimulatory signaling domain includes the intracellular signaling domain of CD137, which has the sequence shown in SEQ ID NO: 8.
  • the CAR has an amino acid sequence selected from: (1) the amino acid sequence shown in SEQ ID NO: 10 or 12, and (2) the amino acid sequence shown in SEQ ID NO: 10 or 12
  • the amino acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (for example, it can be restricted by non-MHC
  • the method points the specificity and reactivity of immune effector cells to the ability of cells expressing BCMA).
  • nucleic acid molecule encoding CAR may include introducing at least one nucleic acid molecule encoding CAR into a cell and expressing the nucleic acid molecule in the cell.
  • the nucleic acid molecule encoding the CAR of the present invention can be included in an expression vector (e.g., a lentiviral vector), which can be expressed in a host cell such as a T cell to produce the CAR.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the present invention.
  • the isolated nucleic acid molecule encodes a chimeric antigen receptor of the invention.
  • the CAR encoded by the isolated nucleic acid molecule includes an extracellular antigen binding domain having a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: the VH consists the sequence shown in SEQ ID NO:1 or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity, the VL comprises the sequence shown in SEQ ID NO: 2 or has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • VH heavy chain variable region
  • VL light chain variable region
  • the CAR encoded by the isolated nucleic acid molecule includes an extracellular antigen binding domain having a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: the VH consists the sequence shown in SEQ ID NO: 3 or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity, the VL comprises the sequence shown in SEQ ID NO: 4 or has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • VH heavy chain variable region
  • VL light chain variable region
  • the CAR encoded by the isolated nucleic acid molecule has an amino acid sequence selected from the group consisting of: (1) the amino acid sequence shown in SEQ ID NO: 10 or 12, (2) and SEQ ID NO:
  • the amino acid sequence shown by 10 or 12 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , At least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (e.g. , Can point the specificity and reactivity of immune effector cells to the ability of cells expressing BCMA in a non-MHC-restricted manner).
  • nucleotide sequence encoding a chimeric antigen receptor of the present invention can have a variety of different sequences. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the nucleotide sequence encoding the chimeric antigen receptor of the present invention is selected from: (1) the nucleotide sequence shown in SEQ ID NO: 11 or 13; (2) and The nucleotide sequence shown in SEQ ID NO: 11 or 13 has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and The sequence basically retains at least one biological activity of the nucleotide sequence from which it is derived (for example, it can encode the ability to direct the specificity and reactivity of immune effector cells to cells expressing BCMA in a non-MHC-restricted manner CAR).
  • the present invention provides a vector (for example, a cloning vector or an expression vector), which comprises the isolated nucleic acid molecule as described above.
  • a vector for example, a cloning vector or an expression vector
  • the vector comprises a nucleotide sequence encoding the chimeric antigen receptor of the invention.
  • the CAR has an amino acid sequence selected from: (1) the amino acid sequence shown in SEQ ID NO: 10 or 12, and (2) the amino acid sequence shown in SEQ ID NO: 10 or 12
  • the amino acid sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (for example, it can be restricted by non-MHC
  • the method points the specificity and reactivity of immune effector cells to the ability of cells expressing BCMA).
  • the nucleotide sequence encoding the chimeric antigen receptor of the present invention is selected from: (1) the nucleotide sequence shown in SEQ ID NO: 11 or 13; (2) and The nucleotide sequence shown in SEQ ID NO: 11 or 13 has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and The sequence basically retains at least one biological activity of the nucleotide sequence from which it is derived (for example, it can encode the ability to direct the specificity and reactivity of immune effector cells to cells expressing BCMA in a non-MHC-restricted manner CAR).
  • the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, and a viral vector.
  • the vector is an expression vector.
  • the carrier is an episomal carrier.
  • the vector is a viral vector.
  • the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.
  • the vector is an episomal or non-integrating viral vector, such as an integration-deficient retrovirus or a lentivirus.
  • the present invention provides an immune effector cell that expresses the chimeric antigen receptor of the present invention.
  • Various methods can be used to introduce the polynucleotide encoding the CAR into the cell, and the CAR can also be synthesized in situ in the cell. Alternatively, the CAR can be produced outside the cell and then introduced into the cell.
  • the immune effector cell comprises the isolated nucleic acid molecule of the seventh aspect or the vector of the eighth aspect.
  • the isolated nucleic acid molecules or vectors as described above can be introduced into immune effector cells in various suitable ways, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposome) or viral vector-mediated transfection (such as lentivirus infection, retrovirus infection, adenovirus infection), and other methods for transferring into host cells Physical, chemical or biological means, such as transposon technology, CRISPR-Cas9 and other technologies.
  • the immune effector cells are derived from patients or healthy donors. In certain embodiments, the immune effector cells are selected from T lymphocytes, natural killer (NK) cells, monocytes, macrophages, or dendritic cells, and any combination thereof. In certain embodiments, the immune effector cells are selected from T lymphocytes and/or natural killer (NK) cells.
  • the present invention also provides a method for preparing immune effector cells expressing the chimeric antigen receptors of the present invention, which comprises: (1) providing immune effector cells; (2) separating the immune effector cells described in the sixth aspect
  • the nucleic acid molecule or the vector of the seventh aspect is introduced into the immune effector cell.
  • the isolated nucleic acid molecule or vector comprises a nucleotide sequence encoding the chimeric antigen receptor of the present invention.
  • the immune effector cells are selected from T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof.
  • the immune effector cells are pretreated; the pretreatment includes sorting, activation and/or proliferation of immune effector cells. In some embodiments, the pretreatment includes contacting immune effector cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune effector cells and inducing their proliferation, thereby generating pretreated immune effector cells.
  • nucleic acid molecules or vectors are introduced into immune effector cells through viral infection.
  • the nucleic acid molecule or vector is introduced into immune effector cells by means of non-viral vector transfection, such as a vector system via transposon, CRISPR/Cas9 vector, TALEN method, ZFN Methods, electroporation methods, calcium phosphate transfection, DEAE-dextran-mediated transfection or microinjection methods.
  • the method further includes: amplifying the immune effector cells obtained in step (2).
  • the present invention also provides a kit for preparing a chimeric antigen receptor capable of specifically binding to BCMA or expressing the chimeric antigen receptor, the kit comprising the isolation as described in the seventh aspect
  • the nucleic acid molecule, or the carrier described in the eighth aspect, and the necessary solvent for example, sterile water or physiological saline, or cell culture medium.
  • the kit optionally includes instructions for use.
  • the present invention provides the use of the above kit for preparing a chimeric antigen receptor capable of specifically binding to BCMA or a cell expressing the chimeric antigen receptor.
  • the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the conjugate according to the fifth aspect, and the chimeric composition according to the sixth aspect of the present invention.
  • the pharmaceutical composition of the invention comprises the antibody of the invention or an antigen-binding fragment thereof.
  • the pharmaceutical composition of the invention comprises the conjugate of the invention.
  • the conjugate comprises the antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the pharmaceutical composition of the present invention comprises the isolated nucleic acid molecule described in the seventh aspect, the vector described in the eighth aspect, or the immune effector cell described in the ninth aspect.
  • the nucleic acid molecule and the vector comprise a nucleotide sequence encoding the chimeric antigen receptor of the present invention, and the immune effector cell expresses the chimeric antigen receptor.
  • the pharmaceutical composition may also include additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers (e.g. gemcitabine, 5-fluorouracil, taxane, cisplatin, etc.), anti-angiogenesis agents, cytokines (e.g.
  • GM-CSF GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.
  • antibodies that specifically target tumor cells e.g., CD20 antibodies such as rituximab, Her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab , EGFR antibodies such as cetuximab, etc.
  • immune checkpoint inhibitors for example, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, etc.
  • the antibody or antigen-binding fragment, conjugate, isolated nucleic acid molecule, carrier or immune effector cell of the present invention and the other pharmaceutically active agent can be used as a separate Component or provided as a mixed component. Therefore, the antibody or antigen-binding fragment, conjugate, isolated nucleic acid molecule, carrier or immune effector cell of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
  • the antibody or antigen-binding fragment, conjugate, immune effector cell or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels , Capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition of the present invention should be sterile and stable under the conditions of production and storage.
  • a preferred dosage form is injection. Such injection may be a sterile injection solution.
  • a sterile injection solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment, conjugate, immune effector cell or pharmaceutical composition of the present invention in an appropriate solvent, and optionally , While incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization .
  • the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g. 0.9% (w/v) NaCl), Glucose solution (e.g. 5% dextrose), solution containing surfactant (e.g. 0.01% polysorbate 20), pH buffer solution (e.g. phosphate buffer solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • Glucose solution e.g. 5% dextrose
  • surfactant e.g. 0.01% polysorbate 20
  • pH buffer solution e.g. phosphate buffer solution
  • Ringer's solution e.g. phosphate buffer solution
  • the pharmaceutical composition of the present invention comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • such sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g. 0.9% (w/v) NaCl), glucose Solutions (e.g. 5% glucose), solutions containing surfactants (e.g. 0.01% polysorbate 20), pH buffered solutions (e.g. phosphate buffered solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • glucose Solutions e.g. 5% glucose
  • solutions containing surfactants e.g. 0.01% polysorbate 20
  • the antibody or antigen-binding fragment, conjugate, immune effector cell, or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, eyeball, Topical, parenteral, rectal, intralobular, intracytoplasmic reticulum, groin, intravesical, topical (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • the route and/or manner of administration will vary according to the intended purpose.
  • the antibody or antigen-binding fragment, conjugate, immune effector cell, or pharmaceutical composition of the present invention is administered by intravenous injection or bolus injection.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the conjugate according to the fifth aspect, or the eighth aspect Immune effector cells.
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease.
  • “Therapeutically effective amount” refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who have already suffered from the disease.
  • the therapeutically effective dose of the antibody or antigen-binding fragment thereof of the present invention, the conjugate of the fifth aspect, or the immune effector cell of the eighth aspect may vary according to the following factors: the severity of the disease to be treated, the patient's own The general status of the immune system, the general condition of the patient such as age, weight and gender, the way the drug is administered, and other treatments administered at the same time, etc.
  • the present invention provides a method for preventing and/or treating B cell-related conditions in a subject (such as a human), the method comprising administering to a subject in need thereof an effective amount of The antibody or antigen-binding fragment thereof according to the first aspect, the conjugate according to the fifth aspect, the immune effector cell according to the eighth aspect, or the pharmaceutical composition of the present invention.
  • the conjugate comprises the antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the method includes administering to the subject an effective amount of an antibody of the invention or an antigen-binding fragment or conjugate thereof.
  • the method includes administering to the subject an effective amount of the immune effector cell according to the eighth aspect or a pharmaceutical composition comprising the immune effector cell.
  • the method includes the following steps: (1) providing immune effector cells (such as T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells) (2) introducing a nucleic acid molecule encoding the chimeric antigen receptor of the sixth aspect of the present invention into the immune effector cell described in step (1) to obtain immune effector cells expressing the chimeric antigen receptor; (3) The immune effector cells obtained in step (2) are administered to the subject for treatment.
  • immune effector cells such as T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells
  • the step of obtaining the immune effector cells from the subject is included before step (1).
  • the immune effector cells are selected from T lymphocytes and/or NK cells.
  • peripheral blood mononuclear cells are obtained from the subject, and the PBMC are directly genetically modified to express CAR.
  • T cells are obtained from the subject.
  • T cells can be obtained from a variety of sources, including but not limited to: peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • a variety of techniques known to the skilled person such as deposition, such as FICOLLTM separation
  • FICOLLTM separation can be used to obtain T cells from blood units collected from the subject.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the product of plasmapheresis usually contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets.
  • the cells collected by the plasmapheresis method can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or medium for subsequent processing.
  • the cells can be washed with PBS or with other suitable solutions that lack calcium, magnesium and most of the divalent cations (if not all other divalent cations).
  • the washing step can be completed by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge.
  • the cells can be resuspended in a variety of biocompatible buffers or other salt solutions with or without buffers.
  • undesired components in a plasmapheresis sample can be removed in the medium in which the cells are directly resuspended.
  • T cells are separated from peripheral blood mononuclear cells (PBMC) by lysing red blood cells and depleting monocytes (eg, by centrifugation through a PERCOLLTM gradient).
  • PBMC peripheral blood mononuclear cells
  • the specific T cell subsets expressing one or more of the following markers can be further isolated by positive or negative selection techniques: CD3, CD28, CD4, CD8, CD45RA and CD45RO.
  • specific T cell subpopulations expressing CD3, CD28, CD4, CD8, CD45RA and CD45RO are further isolated by positive or negative selection techniques.
  • a combination of antibodies against specific surface markers of negatively selected cells can be used to complete the enrichment of the T cell population through negative selection.
  • An exemplary method is to perform cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry, which utilizes the presence of cell surfaces on negatively selected cells.
  • a mixture of markers of monoclonal antibodies For example, in order to enrich CD4+ cells by negative selection, the monoclonal antibody mixture usually contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.
  • Flow cytometry and cell sorting can also be used to isolate the target cell population used in the present invention.
  • immune effector cells such as T cells
  • the immune effector cells can be activated and expanded (or, in the case of progenitor cells, differentiated) in vitro before genetic modification.
  • immune effector cells such as T cells
  • are genetically modified with the chimeric antigen receptor of the present invention for example, transduced with a viral vector containing a nucleic acid encoding a CAR, and then performed in vitro Activate and expand.
  • the B-cell-related condition is a B-cell malignancy, such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • the multiple myeloma (MM) is selected from: overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic Myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.
  • the non-Hodgkin’s lymphoma is selected from: Burkitt’s lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma Tumor, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and mantle cell lymphoma.
  • the B cell-related condition is a plasma cell malignancy.
  • the B cell-related condition is an autoimmune disease.
  • the autoimmune disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura or myasthenia gravis or autoimmune hemolytic anemia.
  • the B-cell-related condition is selected from the group consisting of multiple myeloma, non-Hodgkin’s lymphoma, B-cell proliferation with uncertain malignant potential, lymphomalioid granulomatosis, post-transplant lymphoproliferative Symptoms, immunomodulatory disorders, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, nodules Arteritis, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-related small vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolysis Anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-related amyloidosis, or monoclonal gammaglobulinemia of unknown
  • the antibody or antigen-binding fragment, conjugate, immune effector cell, or pharmaceutical composition of the invention is administered in combination with additional therapies.
  • This additional therapy can be any therapy known for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.
  • This additional therapy can be administered before, at the same time or after administration of the antibody or antigen-binding fragment, conjugate, immune effector cell, or pharmaceutical composition of the present invention.
  • the subject may be a mammal, such as a human.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the conjugate according to the fifth aspect, the chimeric antigen receptor according to the sixth aspect, the second aspect or the seventh aspect are provided.
  • the isolated nucleic acid molecule described in the aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect, the immune effector cell described in the ninth aspect, or the pharmaceutical composition of the present invention are used in the preparation of a medicine.
  • the medicament is used to prevent and/or treat B cell-related conditions in a subject (e.g., a human).
  • the conjugate comprises the antibody or antigen-binding fragment thereof of the invention and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the present invention can specifically bind to BCMA, and thus can be used to detect the presence or level of BCMA in a sample.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention.
  • the kit is used to diagnose whether a subject has a BCMA-expressing tumor.
  • the BCMA-expressing tumor is selected from B-cell malignancies, such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • the BCMA-expressing tumor is a plasma cell malignancy.
  • the antibody or antigen-binding fragment thereof of the present invention bears a detectable label.
  • the kit further includes a second antibody, which specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof.
  • the second antibody further includes a detectable label.
  • the detectable label may be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. It is particularly preferable that such a label can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).
  • immunological detection for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.
  • the present invention provides a method for detecting the presence or amount of BCMA in a sample, which includes the following steps:
  • the formation of the complex indicates the presence of BCMA or cells expressing BCMA.
  • the sample is a cell sample, that is, a sample containing cells (eg, tumor cells).
  • the complex is formed between the antibody, antigen-binding fragment or conjugate and the BCMA expressed by the cells in the sample.
  • the antibody or antigen-binding fragment thereof of the present invention also bears a detectable label.
  • a reagent with a detectable label is used to detect the antibody or antigen-binding fragment thereof of the present invention.
  • the method can be used for diagnostic purposes, or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).
  • the BCMA is human BCMA.
  • the BCMA is human BCMA.
  • the present invention provides a method for diagnosing whether a subject has a tumor expressing BCMA, which comprises using the antibody or antigen-binding fragment thereof according to the first aspect of the present invention to detect whether BCMA is derived from said The amount in the subject's sample.
  • the BCMA-expressing tumor is selected from B-cell malignancies, such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • B-cell malignancies such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • the BCMA-expressing tumor is a plasma cell malignancy.
  • the method further includes the step of comparing the amount of BCMA in the sample from the subject with a reference value.
  • the reference value refers to the level of BCMA in a sample from a subject who is known to have no BCMA-expressing tumor (also referred to as a "negative reference value”), or refers to the level of BCMA in a sample that is known to have BCMA-expressing tumors
  • the level in the subject samples sample (also referred to as the "positive reference value"). For example, if the amount of BCMA in the sample from the subject is similar to the negative reference value (or there is no significant difference), it indicates that the subject does not have a tumor expressing BCMA, and if the BCMA When the amount in the sample from the subject increases relative to the negative reference value, it indicates that the subject has a BCMA-expressing tumor. In addition, if the amount of BCMA in the sample from the subject is similar to the positive reference value (or there is no significant difference), it indicates that the subject has a tumor that expresses BCMA.
  • the amount of BCMA in a sample from the subject is detected by the following steps:
  • the antibody or antigen-binding fragment thereof of the present invention also bears a detectable label.
  • a reagent with a detectable label is used to detect the antibody or antigen-binding fragment thereof of the present invention.
  • the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically removed Tumor tissue, biopsy or fine-needle aspiration tissue), histological preparations, etc.
  • the method further comprises administering BCMA-targeted immunotherapy to a subject diagnosed with a BCMA-expressing tumor.
  • the BCMA-targeted immunotherapy includes administration of the antibody or antigen-binding fragment or conjugate thereof of the present invention.
  • the BCMA-targeted immunotherapy includes administration of immune effector cells of the present invention.
  • the BCMA-targeted immunotherapy includes administration of immune effector cells expressing the chimeric antigen receptor of the sixth aspect of the present invention.
  • the method includes the following steps: (1) providing immune effector cells; (2) introducing the nucleotide sequence encoding the chimeric antigen receptor of the present invention into the immune effector cells described in step (1) to obtain the expression of said immune effector cells.
  • the immune effector cells of the chimeric antigen receptor (3) The immune effector cells obtained in step (2) and optionally unmodified and/or unsuccessfully modified immune effector cells are administered to the subject.
  • the method before step (1), the method further includes the step of obtaining the immune effector cells from the subject.
  • BCMA B-cell maturation antigen
  • B-cell maturation antigen also known as TNFRSF17 or CD269.
  • BCMA is a member of the tumor necrosis factor receptor family (TNFR), which combines TNF family ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand), and is mainly used in terminally differentiated B cells, such as memory B cells and Expressed on plasma cells.
  • TNFR tumor necrosis factor receptor family
  • BAFF B cell activating factor
  • APRIL proliferation inducing ligand
  • the BCMA gene is encoded on chromosome 16, producing a 994-nucleotide primary mRNA transcript (NCBI accession number NM_001192.2), which encodes a protein of 184 amino acids (NP_001183.2).
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL. Constant domains are not directly involved in the binding of antibodies and antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.
  • the allocation of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 -917; Definition of Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • Each of the variable regions of the heavy chain and the light chain contains three CDRs, named CDR1, CDR2, and CDR3.
  • CDR1, CDR2, and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the Kabat, Chothia or IMGT numbering system. In certain embodiments, the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the Kabat numbering system.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody It is also called “antigen binding part” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or through the enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, di- scFv, (scFv) 2 and such polypeptides, which comprise at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody composed of two “full-length heavy chains” and two “full-length light chains.”
  • full-length heavy chain refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • HR hinge region
  • heavy chain constant region CH3 domain are composed; and, when the full-length antibody is of the IgE isotype, it optionally also includes the heavy chain constant region CH4 domain.
  • the "full-length heavy chain” is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are connected by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention includes two antigen binding sites formed by a pair of VH and VL respectively, and the two antigen binding sites specifically recognize/bind the same antigen.
  • the term “Fd” means an antibody fragment composed of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment composed of VH domains (Ward et al., Nature 341:544 546 ( 1989))
  • the term “Fab fragment” means an antibody fragment composed of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment
  • the term “Fab'fragment” means a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and heavy chain Fd Fragments (consisting of VH and CH1 domains) are composed.
  • Fv means an antibody fragment composed of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind antigen, although its affinity may be lower than the complete binding site.
  • Fc refers to a disulfide bond formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions, but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore eds, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • scFv can form di-scFv, which refers to two or more single scFv connected in series to form an antibody.
  • the scFv can form (scFv) 2 , which refers to two or more single scFvs connected in parallel to form an antibody.
  • Each of the aforementioned antibody fragments maintains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • antibody includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with the sequence of a human antibody.
  • CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from human source.
  • Humanized antibodies usually retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc.
  • the donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with desired properties (e.g., antigen specificity, affinity, reactivity, etc.).
  • Humanized antibodies can not only retain the expected properties of non-human donor antibodies (such as murine antibodies), but also can effectively reduce the immunogenicity of non-human donor antibodies (such as murine antibodies) in human subjects. Therefore, it is particularly advantageous.
  • the expected properties of the humanized antibody eg, antigen specificity, affinity, reactivity, ability to improve immune cell activity, and/or The ability to enhance immune response
  • the humanized antibody not only has the highest degree of humanization, but also retains the expected properties of the donor antibody as much as possible.
  • the prior art does not provide detailed guidance. Technicians need to explore, explore and modify specific donor antibodies, and pay a lot of creative work to obtain humanized antibodies that have a high degree of humanization and retain the expected properties of specific donor antibodies.
  • germline antibody gene or “germline antibody gene segment (germline antibody gene segment)” refers to a sequence encoding immunoglobulin that exists in the genome of an organism , It has not experienced the maturation process that can lead to genetic rearrangement and mutation of the expression of specific immunoglobulins.
  • the expression “heavy chain germline gene” refers to the germline antibody gene or gene fragment encoding the heavy chain of immunoglobulin, which includes V gene (variable), D gene (diversity), and J gene (joining) And C gene (constant); similarly, the expression “light chain germline gene” refers to germline antibody genes or gene fragments encoding immunoglobulin light chains, including V genes (variable), J genes (joining) and C gene (constant).
  • the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also referred to as "germline sequence”. Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or inquired from professional databases (for example, IMGT, UNSWIg, NCBI or VBASE2).
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both “association rate constant” (ka or kon) and “dissociation rate constant” (kdis or koff) can be calculated by the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187). The ratio of kdis/kon is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). Any effective method can be used to measure K D , kon and kdis values.
  • SPR surface plasmon resonance
  • bioluminescence interferometry or Kinexa can also be used to measure the dissociation constant.
  • chimeric antigen receptor refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain It combines the antibody-based specificity against the target antigen (for example, BCMA) with the activated intracellular domain of immune effector cells to exhibit specific immune activity against cells expressing the target antigen (for example, BCMA).
  • target antigen for example, BCMA
  • CDMA-expressing immune effector cells refers to immune effector cells that express CAR and have antigen specificity determined by the targeting domain of the CAR.
  • CARs for example, for cancer treatment
  • Methods of manufacturing CARs are known in the art, see, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., NEnglJ Med., 368:1509-1518 , 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Patent Publications WO2012/079000, WO2013/059593; and U.S. Patent Publications 2012/0213783, all of which are incorporated herein by reference in their entirety.
  • anti-BCMA CAR refers to the CAR that contains the extracellular antigen binding domain that can specifically bind to BCMA;
  • anti-BCMA CAR-T refers to the immune effector cells expressing the above CAR (such as PBMC, such as T cells).
  • humanized CAR refers to the CAR whose extracellular antigen-binding domain is derived from a humanized antibody; similarly, the expression “murine CAR” refers to the contained extracellular antigen binding domain The antigen binding domain is derived from the CAR of a murine antibody.
  • extracellular antigen binding domain refers to a polypeptide capable of specifically binding a target antigen or receptor. This domain will be able to interact with cell surface molecules. For example, an extracellular antigen binding domain can be selected to recognize an antigen as a cell surface marker of a target cell associated with a specific disease state. Typically, the extracellular antigen binding domain is an antibody-derived targeting domain.
  • intracellular signaling domain refers to the portion of a protein that conducts effect signal function signals and guides cells to perform specialized functions. Therefore, the intracellular signal transduction domain has the ability to activate at least one normal effector function of immune effector cells expressing CAR.
  • the effector function of T cells may be cytolytic activity or auxiliary activity, including the secretion of cytokines.
  • primary signaling domain refers to a protein portion capable of modulating the primary activation of the TCR complex in a stimulating manner or in an inhibitory manner.
  • the primary signaling domain that acts in a stimulating manner usually contains a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, DAP10, CD79a, CD79b, and CD66d.
  • costimulatory signaling domain refers to the intracellular signaling domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes after binding to an antigen.
  • Non-limiting examples of the costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1) CD270 ( HVEM), CD278 (ICOS), DAP10.
  • immune effector cell refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC) .
  • effector cells are cells that have hematopoietic origin and play a role in immune responses.
  • effector function refers to a specialized function of an immune effector cell, such as a function or response that enhances or promotes an immune attack on a target cell (such as killing a target cell, or inhibiting its growth or proliferation).
  • the effector function of T cells for example, may be cytolytic activity or auxiliary or activity including the secretion of cytokines.
  • immune effector cells include T cells (such as ⁇ / ⁇ T cells and ⁇ / ⁇ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.
  • the immune effector cells of the present invention can be self/autologous ("self") or non-self ("non-self", such as allogeneic, syngeneic or allogeneic).
  • self refers to cells from the same subject;
  • allogeneic refers to cells of the same species that are genetically different from the comparison cell;
  • seyngeneic refers to the genetic difference from the comparison cell The same cell from a different subject;
  • allogene refers to a cell from a different species than the compared cell.
  • the cells of the invention are allogeneic.
  • T lymphocytes Exemplary immune effector cells that can be used in the CARs described herein include T lymphocytes.
  • T cell or "T lymphocyte” is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • the T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • the T cells can be helper T cells (HTL; CD4 T cells), CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells, or any other subset of T cells.
  • T cells may include naive T cells and memory T cells.
  • immune effector cells can also be used as immune effector cells with CARs as described herein, such as NK cells.
  • immune effector cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils, and macrophages.
  • Immune effector cells also include progenitor cells of immune effector cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune effector cells.
  • immune effector cells include progenitor cells of immune effector cells, such as hematopoietic stem cells (HSC) contained in a population of CD34+ cells derived from cord blood, bone marrow, or peripheral blood, which are in the subject After mid-administration, it differentiates into mature immune effector cells, or it can be induced in vitro to differentiate into mature immune effector cells.
  • HSC hematopoietic stem cells
  • cytotoxic agent includes any agent that is harmful to cells (e.g., kills cells), such as chemotherapeutic drugs, bacterial toxins, phytotoxins, or radioisotopes.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • episomal refers to the ability of the vector to replicate without integrating into the chromosomal DNA of the host and not being gradually lost by dividing the host cell. It also means that the vector is extrachromosomal or freely copy.
  • viral vector is broadly used to refer to nucleic acid molecules (eg, transfer plasmids) that include virus-derived nucleic acid elements that typically facilitate the transfer or integration of nucleic acid molecules into the genome of a cell, or mediate nucleic acid transfer Virus particles. In addition to nucleic acids, viral particles will typically include various viral components and sometimes host cell components.
  • viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or refer to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements mainly derived from viruses.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof mainly derived from retroviruses.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof (including LTR) mainly derived from lentivirus.
  • the terms "lentiviral vector” and “lentiviral expression vector” can be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles.
  • elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.
  • integration-deficient retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that cannot integrate the viral genome into the genome of a host cell.
  • the integrase protein is mutated to specifically reduce its integrase activity.
  • the integration-deficient lentiviral vector can be obtained by modifying the pol gene encoding the integrase protein to generate a mutant pol gene encoding the integration-deficient integrase.
  • the integration-deficient viral vector has been described in the patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes) Cells, NK cells, monocytes, macrophages or dendritic cells, etc.).
  • the host cell can include a single cell or a population of cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • each molecule is the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) ; And Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancement Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugar, NaCl and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, paraben, chlorobutanol, phenol, sorbic acid and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredients in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate).
  • the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • such sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g. 0.9% (w/v) NaCl), glucose Solutions (e.g. 5% glucose), solutions containing surfactants (e.g. 0.01% polysorbate 20), pH buffered solutions (e.g. phosphate buffered solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • glucose Solutions e.g. 5% glucose
  • surfactants e.g. 0.01% polysorbate 20
  • pH buffered solutions e.g. phosphate buffered solution
  • Ringer's solution e.g. phosphate buffered solution
  • prevention refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., tumor) in a subject.
  • treatment refers to a method performed in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (ie, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the disease Status, and relief of symptoms (whether partial or full), whether detectable or undetectable.
  • treatment can also refer to prolonging survival compared to expected survival (if not receiving treatment).
  • the term “subject” refers to a mammal, such as a primate mammal, such as a human.
  • the term “subject” is meant to include living organisms in which an immune response can be elicited.
  • the subject e.g., human
  • has a B-cell-related condition e.g., a B-cell malignancy
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., a B cell related condition
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially Prevents the amount of disease and its complications in patients who have already suffered from the disease. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments that are administered at the same time and so on.
  • B-cell-related conditions refers to conditions involving improper B-cell activity and B-cell malignancies, including but not limited to B-cell malignancies or B-cell-related autoimmune diseases.
  • B-cell malignancy includes cancer types formed in B cells (immune system cell types), for example, multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL).
  • the present invention provides a humanized antibody targeting BCMA, which maintains the activity of binding and expressing BCMA tumor cells comparable to that of the murine antibody, while significantly reducing non-specific binding to normal tissue cells, which is beneficial to reduce non-specific binding. Heterosexual toxicity improves the safety of the drug.
  • the present invention further provides a chimeric antigen receptor targeting BCMA comprising the humanized antibody.
  • the immune effector cells expressing the chimeric antigen receptor of the present invention basically do not cause antigen-independent cytokine release, and do not induce the continuous activation and exhaustion of T cells, and have significantly improved safety. Therefore, the humanized antibody and CAR of the present invention have the potential to be used to prevent and/or treat B-cell-related conditions (such as B-cell malignancies, autoimmune diseases, etc.), and have great clinical value.
  • Figure 1 shows the binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and the parent murine antibody C11D5.3 to recombinant human BCMA protein.
  • Figure 2 shows the dose-dependent binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and the parent murine antibody C11D5.3 binding to human multiple myeloma cells U266.
  • Figure 3 shows the dose-dependent binding curves of the humanized antibodies C11D5.3-03, C11D5.3-04 and the parent murine antibody C11D5.3 binding to human multiple myeloma cells MM.1S.
  • Figure 4 shows the dose-dependent binding curves of the humanized antibodies C11D5.3-03, C11D5.3-04 and the parent murine antibody C11D5.3 binding to human multiple myeloma cells RPMI8226.
  • Figure 5 shows the dose-dependent binding curves of humanized antibodies C11D5.3-03, C11D5.3-04, and maternal murine antibody C11D5.3 with human Burkitt's lymphoma cells Daudi that do not express BCMA.
  • Figure 6 shows the binding curves of humanized antibodies C11D5.3-03, C11D5.3-04, and maternal murine antibody C11D5.3 with human immortalized keratinocyte Hacat.
  • Figure 7 shows the binding curves of humanized antibodies C11D5.3-03, C11D5.3-04, and maternal murine antibody C11D5.3 with human gastric mucosal epithelial cells GES.
  • Figure 8 shows the expression of BCMA-CAR on T cells transduced with C11D5.3-CAR, or humanized C11D5.3-03-CAR, C11D5.3-04-CAR.
  • Figure 9 shows the IFN ⁇ release measurement results of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T.
  • Figure 10 shows the IL2 release measurement results of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T.
  • Figure 11 shows the LDH release results of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T on U266.
  • Figure 12 shows the LDH release results of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T against Daudi.
  • Figures 13-17 show C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T, C11D5.3-04 CAR-T and untransfected PBMC cells in serum-free cells without antigen stimulation.
  • Figure 18 shows C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T, C11D5.3-04 CAR-T and untransfected PBMC cells containing 5% human serum without antigen stimulation The release levels of IL4, IL6, IL10, TNF ⁇ and IFN ⁇ in the culture system.
  • Figure 19 shows the detection results of the expression level of HLA-DR on the surface of CAR-T cells.
  • Figure 20 shows the detection results of the expression level of CD25 on the surface of CAR-T cells.
  • Figure 21 shows the detection results of the expression levels of activated Caspase-3 in C11D5.3-03 CAR-T, C11D5.3-04 CAR-T, C11D5.3 CAR-T and control PBMC.
  • SEQ ID NO description SEQ ID NO description 1 C11D5.3-03 VH 9 CD3 ⁇ ITAM 2 C11D5.3-03 VL 10 C11D5.3-03-CAR full-length amino acid sequence 3 C11D5.3-04 VH 11 C11D5.3-03-CAR encoding nucleic acid sequence 4 C11D5.3-04 VL 12 C11D5.3-04-CAR full-length amino acid sequence 5 Linker 13 C11D5.3-04-CAR encoding nucleic acid sequence 6 Hinge area 14 C11D5.3-CAR full-length amino acid sequence 7 Transmembrane zone 15 C11D5.3-CAR encoding nucleic acid sequence 8 CD137 intracellular signaling domain 16 P2A-tEGFR coding nucleic acid sequence
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer.
  • the embodiments describe the present invention by way of example, and are not intended to limit the scope of protection claimed by the present invention.
  • Example 1 Humanization of anti-BCMA murine antibody and preparation of humanized antibody
  • the murine antibody C11D5.3 for its heavy chain variable region sequence and light chain variable region sequence, respectively, see Chinese patent application CN 201510142069.2 in SEQ ID NO: 3 and SEQ ID NO: 12
  • Chinese patent application CN 201510142069.2 in SEQ ID NO: 3 and SEQ ID NO: 12 for humanization design, using methods known in the art to insert mouse CDR regions into human framework sequences (see US Patent No. 5,225,539 of Winter; Queen et al. Human U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).
  • CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from human source.
  • Immunoglobulin receptor antibody
  • VH and VL of C11D5.3-03 are shown in SEQ ID NO:1 and SEQ ID NO: 2, respectively; the VH and VL of C11D5.3-04 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • a murine antibody C11D5.3 (scFv) was prepared as a reference antibody.
  • HEK293-F cells (Invitrogen, R7907) transiently transfected with the expression plasmid encoding the above antibody were cultured with Freestyle 293 expression medium (Invitrogen, 12338018), and incubated in a CO 2 incubator at 37°C for 5-7 days.
  • the cells and cell debris were removed by centrifugation, the culture supernatant was collected, filtered through a 0.22 ⁇ m membrane, and purified using a protein A column (GE Life Science, 17127901), following the manufacturer’s instructions, washing with PBS, and containing 20mM citrate, 150mM
  • the antibody was eluted in an acidic buffer of NaCl (pH 3.5), the eluate was neutralized with 1M Tris (pH 8.0), and dialyzed with PBS.
  • the antibody concentration is determined by measuring the absorbance at 280nm, and the aliquoted antibody is stored in a refrigerator at -80°C.
  • Example 2 Evaluation of the affinity of anti-BCMA humanized antibodies to recombinant human BCMA protein
  • the ELISA method was used to evaluate the antibody's binding activity to human BCMA protein, and the dose-tolerance binding curve in ELISA was used to compare the antibody efficacy.
  • the plate was washed 3 times in washing buffer, and the bound secondary antibody was detected by adding TMB (HRP substrate) and incubating the plate in the dark at room temperature for 5 to 10 minutes.
  • the enzyme reaction was stopped by adding sulfuric acid solution 1M, and the light absorption was measured at 450 nm.
  • the results are shown in Figure 1.
  • the FACS method was used to evaluate the binding activity of the humanized antibody obtained in Example 1 to cells expressing BCMA, and the dose-dependent binding curve in FACS was used to compare the antibody efficacy.
  • Digest cells human-derived myeloma cells expressing BCMA or tissue cells expressing BCMA
  • plate 500,000 cells in 50 ⁇ L PBS (FACS buffer) containing 2% FBS, using a U-shaped bottom 96-well plate.
  • a 3-fold serial dilution of the test sample was performed by diluting 1/3 volume (100 ⁇ L) in 200 ⁇ L FACS buffer, and the initial antibody concentration was 300 ⁇ g/mL (final concentration).
  • Add 50 ⁇ L of diluted antibody to each well of the cell plate and incubate at 4°C for 1 hour.
  • Figures 2, 3, 4, and 5 respectively show the dose-dependent binding curves of antibody binding to human multiple myeloma cells U266, MM.1S, RPMI8226, and human Burkitt's lymphoma cells that do not express BCMA, Daudi.
  • the specific EC50 values are as follows Table 2 shows. The results showed that for the three types of tumor cells expressing BCMA, the humanized antibodies C11D5.3-03 and C11D5.3-04 have high affinity equivalent to murine antibodies; at the same time, for tumor cells that do not express BCMA, humanized antibodies The modified antibodies C11D5.3-03 and C11D5.3-04 also exhibited low affinity comparable to murine antibodies.
  • Table 2 EC50 values of anti-BCMA antibody binding to tumor cells expressing BCMA
  • Example 4 Evaluation of non-specific binding of anti-BCMA humanized antibodies to non-tumor tissue cells
  • FIGS 6-7 respectively show the binding curves of antibodies to human immortalized keratinocytes Hacat and human gastric mucosal epithelial cells GES.
  • the murine antibody C11D5.3 binds to both Hacat and GES cells at a high concentration (100 ⁇ g/mL).
  • the two humanized antibodies C11D5.3-03 and C11D5.3-04 At the tested concentration (0.003-100 ⁇ g/mL), it did not bind to the two kinds of non-tumor tissue cells.
  • the above results indicate that the humanized antibody of the present invention has reduced non-specific binding compared to the murine antibody C11D5.3, and this technical effect is remarkable and unexpected.
  • Example 5 Construction and preparation of chimeric antigen receptor (CAR) lentiviral expression vector
  • a CAR lentiviral expression vector was further constructed.
  • the amino acid sequence of each element from the N-terminus to the C-terminus is shown in Table 3 below.
  • the CAR includes the scFv (VH+VL) of the above-mentioned humanized antibody, the CD8 ⁇ hinge region, the CD8 ⁇ transmembrane region, the intracellular domain of CD137, and the ITAM region of CD3 ⁇ .
  • the expression cassette is EF1a Promoter-BCMA CAR-P2A-tEGFR-WPRE, which can be passed Detect the expression of tEGFR to monitor the expression of BCMA CAR.
  • Table 3 The amino acid sequence of each element from the N-terminal to the C-terminal in the chimeric antigen receptor
  • PBMC cells collected from healthy people Xuanfeng Bio, SLB-HP010); 1 ⁇ g/ml CD3 (Miltenyi, 170-076-124) antibody and 1 ⁇ g/ml CD28 (Miltenyi, 170-076- 117) Antibody; add PBMC to the coated 6-well plate and culture overnight.
  • T cells transduced with mouse C11D5.3-CAR C11D5.3 CAR- T
  • T cells transduced with humanized C11D5.3-03-CAR C11D5.3-03 CAR-T
  • T cells transduced with humanized C11D5.3-04-CAR C11D5 .3-04 CAR-T
  • the expression of tEGFR was detected with Cetuximab labeled with PE fluorescence.
  • Figure 8 shows the expression of BCMA-CAR on untransfected control PBMC and the above three CAR-T cells. The results show that the above three CAR-Ts can express the corresponding anti-BCMA CAR, indicating that the anti-BCMA CAR-T was successfully constructed.
  • Cytokine release assay After collecting the anti-BCMA CAR-T cells prepared in Example 6, they were washed twice with DPBS solution and resuspended in 2% FBS RPMI1640 medium; after the tumor cells were collected, washed and reselected In the RPMI1640 medium containing 2% FBS, the tumor cells include: tumor cells that do not express BCMA (293T, K562), and tumor cells that express BCMA (M1ss, U266, RPMI8226, Daudi).
  • the supernatant was taken and the cytokine release in the supernatant was determined by BD CBA assay.
  • Control PBMC, The IFN ⁇ and IL2 release results of C11D5.3CAR-T, C11D5.3-03 CAR-T, and C11D5.3-04 CAR-T are shown in Figure 9 and Figure 10, respectively.
  • LDH CytoTox96 Non-Radioactive Cytotoxicity Assay
  • anti-BCMA CAR-T cells may release cytokines in the absence of BCMA antigen. This release varies with the structural difference of anti-BCMA CAR molecules; the toxicity caused by enhanced cytokine release is known to be anti-BCMA CAR
  • One of the toxicity problems of T cell clinical treatment It is known that CAR-T cell therapy based on the mouse antibody C11D5.3 has not found significant enhanced cytokine release in the current clinical practice.
  • CAR-T cell therapy based on the mouse antibody C11D5.3 has not found significant enhanced cytokine release in the current clinical practice.
  • certain humanized anti-BCMA CARs cause antigen-independent The release of sexual cytokines, this feature will make humanized anti-BCMA CAR not suitable for T cell therapy. For this reason, this example evaluated the antigen-independent cytokine release of humanized anti-BCMA CAR-T cells.
  • Example 6 In an AIM-V medium (Gibco, A3021002) containing 60 IU/mL IL2 (Miltenyi, 130-097-748), the CAR obtained in Example 6 was cultured statically in an incubator with 5% CO 2 at 37°C -T or PBMC control, at different culture time points to detect the release of cytokines in the absence of stimulation. Incubate the control PBMC, C11D5.3CAR-T, C11D5.3-03 CAR-T, or C11D5.3-04 CAR-T on the first day (before the first medium change), the second day, and the third day.
  • AIM-V medium Gibco, A3021002
  • human serum Corning, MT35060CI
  • BCMA antigen 1 ⁇ 10 5 control PBMC, C11D5.3 CAR-T, C11D5.3-03 CAR-T, Or C11D5.3-04 CAR-T was cultured for 48 hours, and the levels of 5 cytokines released into the supernatant, including IL4, IL6, IL10, TNF ⁇ , and IFN ⁇ , were measured using the multivariate magnetic bead method.
  • Figure 18 shows a comparison of the release levels of IL4, IL6, IL10, TNF ⁇ and IFN ⁇ in the above three CAR-T cells and untransfected PBMC cells in a culture system containing 5% human serum without antigen stimulation.
  • the CAR obtained based on the humanized antibodies C11D5.3-03 and C11D5.3-04 of the present invention can reduce the potential immunogenicity caused by the immune response of the mouse sequence, and at the same time, does not exhibit obvious antigens.
  • the independent release of cytokines and the reactivity to the new generation of proteins in human serum have significantly improved safety. Such technical effects are remarkable and unexpected.
  • Example 10 Evaluation of humanized anti-BCMA CAR T cells apoptosis
  • the failure of T cells is usually related to activation-induced cell death caused by apoptosis.
  • Measure the level of activated Caspase-3 (CaspGLOW TM Fluorescein Active Caspase-3 Staining Kit, Invitrogen) to check whether the introduction of anti-BCMA CAR can lead to higher levels of T cell apoptosis.
  • the FACS method was used to detect the expression levels of C11D5.3-03 CAR-T, C11D5.3-04 CAR-T, C11D5.3 CAR-T, and control PBMC for activated Caspase-3, and the results are shown in Figure 21, respectively.
  • the results showed that the introduction of humanized C11D5.3-03 CAR and C11D5.3-04 CAR did not affect the expression level of activated Caspase-3 in T cells, and did not lead to cell death caused by T cell apoptosis.

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Abstract

特异性结合BCMA的抗体及其抗原结合片段以及包含其的嵌合抗原受体(CAR)，以及编码这种CAR的核酸分子、表达这种CAR的免疫效应细胞以及制备这种免疫效应细胞的方法。所述CAR和免疫效应细胞用于预防和/或治疗B细胞相关病况(例如B细胞恶性肿瘤、自身免疫疾病)的用途以及预防和/或治疗所述B细胞相关病况的方法。

Description

靶向BCMA的抗体及嵌合抗原受体 技术领域
本发明涉及疾病治疗及免疫学领域，具体而言，本发明涉及特异性结合BCMA的抗体及其抗原结合片段以及包含其的嵌合抗原受体(CAR)。本发明还涉及编码这种CAR的核酸分子、表达这种CAR的免疫效应细胞以及制备这种免疫效应细胞的方法。本发明还涉及这些CAR和免疫效应细胞用于预防和/或治疗B细胞相关病况(例如B细胞恶性肿瘤、自身免疫疾病)的用途以及预防和/或治疗所述B细胞相关病况的方法。
背景技术
B细胞是一种淋巴细胞，来源于骨髓的多能干细胞，其在产生抗体介导体液免疫应答，提呈可溶性抗原，以及产生大量细胞因子、参与免疫调节、炎症反应及造血中发挥重要的作用(1)。一些重大疾病涉及B细胞。B细胞的恶性转化导致癌症，包括一些淋巴瘤如霍奇金氏淋巴瘤和多发性骨髓瘤。B细胞生理学异常可能导致自身免疫性疾病，包括系统性红斑狼疮的发展。以上这两类涉及B细胞的疾病可以被认为是因为B细胞生长过度和/或不适当的攻击身体的部分，可能的控制策略是使用靶向病理B细胞的抗体或抗体的抗原结合部分，或者是基于这些抗体或抗体的抗原结合部分的其他药物形式。
B细胞成熟抗原(B-cell maturation antigen，BCMA)，又称TNFRSF17或CD269，由184个氨基酸组成，属于缺少信号肽的I型跨膜信号蛋白。BCMA是肿瘤坏死因子受体家族(TNFR)的成员，并且结合TNF家族配体BAFF(B细胞激活因子)和APRIL(增殖诱导配体)(2)。多种B细胞系的分析表明BCMA表达于成熟B细胞和浆细胞表面(3),对人体正常组织的基因水平/蛋白水平研究表明BCMA不表达于除成熟B细胞和浆细胞的其他人体正常组织，且不表达于CD34+造血细胞中(4)。BCMA基因敲除小鼠具有正常的淋巴器官和免疫系统(5)，B淋巴细胞的发育正常，但浆细胞数量明显减少，证明BCMA在维持浆细胞的存活中起了重要的作用，其机制主要包括BCMA与BAFF蛋白结合，传导信号激活NF-κB通路，并上调抗凋亡基因Bcl-2，Mcl-1及Bclw等，维持细胞生长(6)。研究表明，BCMA在B细胞恶性肿瘤如多发性骨髓瘤(MM)和非霍奇金淋巴瘤(NHL)中普遍表达，并且对肿瘤细胞的恶性增生起了重要的促进作用(7)，综上所述BCMA可以作为B细胞恶性肿瘤的靶点之一用于多发性骨髓瘤和非霍奇金淋巴瘤的治疗。
BCMA靶向治疗的作用首先表现在多发性骨髓瘤的进展中。多发性骨髓瘤是一种恶性 浆细胞疾病，表现为骨髓浆细胞恶性克隆性增生，分泌单克隆免疫球蛋白或其片段(M蛋白)，导致骨骼、肾脏等相关靶器官或组织损伤，常见临床表现为骨痛、贫血、肾功能不全、感染等(8)。目前，多发性骨髓瘤为血液系统第二大恶性肿瘤，占血液系统恶性肿瘤的13％，其发病率随着年龄的增长逐年增高，近几年更是有年轻化的趋势(9)。目前，研究人员正积极参与开发针对BCMA的三种主要类型的免疫疗法，他们分别是嵌合抗原受体T细胞(CAR-T细胞)，双特异性抗体和抗体药物偶联物(ADC)，临床前和临床研究的结果证明了以BCMA为靶点的这几类疗法的安全性及有效性。
上述这些靶向BCMA的药物中进展最快的是Blue Bird的bb2121。bb2121药物形式中所用胞外结构域包含结合人BCMA的鼠抗抗原结合片段(CN 201580073309.6)，其来源为Biogen Idec公司所研发的克隆号为C11D5.3的抗BCMA抗体(CN 201510142069.2)。C11D5.3在BCMA-CAR T细胞治疗中的应用广泛，除了bb2121外，bb21217、NCI的BCMA-CAR T、以及恒润达生的BCMA-CAR T(CN 201610932365.7)都通过这个克隆的抗原结合片段来达到和BCMA靶点的结合，并且在上述提到的这些CAR T里用的都是这个克隆的鼠源形式。
人源化抗体可以大大减少异源抗体对人类机体造成的免疫副反应。鼠源抗体人源化的一个经典方法是CDR植入，抗体可变区的CDR是抗体识别和结合抗原的区域，直接决定抗体的特异性。将鼠源单抗的CDR移植至人源抗体可变区，替代人源抗体CDR，使人源抗体获得鼠源单抗的抗原结合特异性，同时减少其异源性。然而，抗原虽然主要和抗体的CDR接触，但FR区也常参与作用，影响CDR的空间构型。因此换成人源FR区后，这种鼠源CDR和人源FR相嵌的V区，可能改变了单抗原有的CDR构型，结合抗原的能力会下降甚至明显下降，此外，甚至可能带来明显的不期望的活性。例如，Blue Bird在其专利申请中(CN 201580050638.9)发现以人源化的C11D5.3为抗原结合部分构建的CAR T会引起明显的抗原非依赖性的细胞因子释放，同时观察到该人源化CAR的导入会诱导T细胞的持续激活和衰竭。对非BCMA结合部分的CAR进行结构改造并不能解决上述问题，使得此人源化的抗C11D5.3抗BCMA CAR不适用于T细胞疗法。
发明内容
本发明人对特异性识别BCMA的鼠源抗体C11D5.3进行了深入的研究和改造，开发了该鼠源抗体的人源化抗体，该人源化抗体在保持了与鼠源抗体相当的结合表达BCMA肿瘤细胞的活性的同时，对于正常组织细胞的非特异性结合明显降低。在此基础上，本发明 人又付出了大量的创造性劳动，进一步构建获得了能够特异性结合BCMA的嵌合抗原受体(CAR)。本发明的CAR能够以非MHC限制的方式将免疫效应细胞特异性和反应性指向表达BCMA的细胞从而使其被清除，并且不会引起抗原非依赖性的细胞因子释放，并且不会诱导T细胞的持续激活和衰竭。因此，本发明的人源化抗体及CAR具有用于预防和/或治疗B细胞相关病况(例如B细胞恶性肿瘤、自身免疫疾病等)的潜力，具有重大的临床价值。
本发明的抗体
因此，在第一方面，本发明提供了能够特异性结合BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:1或3所示的序列；
(ii)与SEQ ID NO:1或3所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1或3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:2或4所示的序列；
(v)与SEQ ID NO:2或4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:2或4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:1所示的序列；
(ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:2所示的序列；
(v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:1所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:2所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:3所示的序列；
(ii)与SEQ ID NO:3所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:4所示的序列；
(v)与SEQ ID NO:4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:3所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:4所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述抗体或其抗原结合片段具备以下生物学功能中的一项或多项：
(a)与分离的人BCMA蛋白结合的亲和力不低于鼠源抗体C11D5.3；表述“分离的”是指该蛋白不包含于细胞中或细胞表面；所述亲和力可以通过例如术语ka(来自抗体/抗原复合物的抗体的结合的速率常数)、kD(解离常数)和/或KD(kD/ka)来定义；
(b)以0.05μg/ml或更小(例如，0.04μg/ml，0.03μg/ml，0.02μg/ml或更小)的EC50结合分离的人BCMA蛋白；例如，所述EC50通过ELISA技术测得；
(c)与表达人BCMA的细胞结合的亲和力不低于鼠源抗体C11D5.3；所述亲和力可以通过例如术语ka(来自抗体/抗原复合物的抗体的结合的速率常数)、kD(解离常数)和/或KD(kD/ka)来定义；
(d)以8μg/ml或更小(例如，5μg/ml，4μg/ml，3μg/ml，2μg/ml，1μg/ml或更小)的EC50结合表达人BCMA的细胞；例如，所述EC50通过流式细胞技术测得；
(e)与正常组织(例如非肿瘤组织)细胞结合的亲和力低于鼠源抗体C11D5.3；
(f)表达包含所述抗体或其抗原结合片段的CAR的T细胞基本不引起抗原非依赖性的细胞因子释放。
在某些实施方案中，本发明的抗体或其抗原结合片段可以进一步包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的恒定区序列或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加。在某些实施方案中，所述变体与其所源自的序列相比具有一个或多个氨基酸的保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
本发明的抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。
在某些实施方案中，所述重链恒定区选自IgG、IgM、IgE、IgD或IgA。
在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。在某些实施方案中，所述重链恒定区是鼠IgG1、IgG2、IgG3或IgG4重链恒定区。在某些实施方案中，所述重链恒定区是人IgG1、IgG2、IgG3或IgG4重链恒定区。在某些实施方案中，优选地所述重链恒定区是人IgG1或IgG4重链恒定区。
在某些实施方案中，所述轻链恒定区选自κ或λ。
在某些实施方案中，所述轻链恒定区是κ轻链恒定区。在某些实施方案中，所述轻链恒定区是鼠κ轻链恒定区。在某些实施方案中，所述轻链恒定区是人κ轻链恒定区。
在某些实施方案中，本发明的抗体是人源化抗体。在某些实施方案中，本发明的抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、di-scFv、(scFv) 2。
在本发明中，本发明的抗体或其抗原结合片段可以包括这样的变体，所述变体与其所源自的抗体或其抗原结合片段相比差异仅在于一个或多个(例如，至多20个、至多15 个、至多10个、或至多5个氨基酸的保守置换)氨基酸残基的保守置换，或者与其所源自的抗体或其抗原结合片段具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，且基本保留了其所源自的抗体或其抗原结合片段的上述生物学功能。
抗体的制备
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’) 2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，在第二方面，本发明提供了一种分离的核酸分子，其包含编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。在某些优选的实施方案中，所述分离的核酸分子编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在第三方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些优选的实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。
在第四方面，本发明提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。在某些优选的实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO (例如CHO-K1、CHO-S、CHO DG44)。
在另一个方面，还提供了制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养第四方面所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
缀合物
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对BCMA(特别是人BCMA)的结合。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
因此，在第五方面，本发明提供了缀合物，其包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的修饰部分。
在某些实施方案中，所述修饰部分是可检测的标记，例如酶、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。在某些实施方案中，所述缀合物包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的可检测的标记。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物)、磁珠(例如，
Figure PCTCN2019107004-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检 测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
在某些实施方案中，本发明的抗体或其抗原结合片段可以与治疗部分连接。在此类实施方案中，由于该缀合物具有选择性递送一种或多种治疗剂至靶组织(例如表达BCMA的细胞)的能力，因此，该缀合物可以提高本发明的抗体或其抗原结合片段在治疗疾病(例如B细胞相关病况)中的治疗效力。
因此，在某些实施方案中，所述修饰部分是治疗剂。在某些实施方案中，所述缀合物包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
在某些实施方案中，所述缀合物是抗体-药物偶联物(ADC)。
在某些实施方案中，所述治疗剂是细胞毒剂。在本发明中，所述细胞毒剂包括对细胞有害(例如杀伤细胞)的任何试剂。
在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合。
可用于本发明的缀合物的烷化剂的实例包括但不限于氮芥类(如双氯乙基甲胺、苯丁酸氮芥、美法仑、环磷酰胺等)、乙烯亚胺类(如塞替哌等)、硫酸酯及多元醇类(如白消安、二溴甘露醇)、亚硝基脲类(如卡莫司汀、洛莫司汀等)、铂类抗肿瘤剂(如顺铂、奥沙利铂、卡铂等)等。
可用于本发明的缀合物的有丝分裂抑制剂的实例包括但不限于美登素类(例如美登素、美登醇、美登醇的C-3酯等)、紫杉烷类(例如多西他赛、紫杉醇或纳米颗粒紫杉醇等)、长春花生物碱类(例如硫酸长春地辛、长春新碱、长春花碱或长春瑞滨等)
可用于本发明的缀合物的抗肿瘤抗生素的实例包括但不限于放线菌素、蒽环类抗生素(例如柔红霉素、阿霉素、表柔比星、伊达比星等)、卡利奇霉素、倍癌霉素等。
可用于本发明的缀合物的抗代谢物的实例包括但不限于叶酸拮抗剂(例如甲氨蝶呤等)、嘧啶拮抗剂(例如5-氟尿嘧啶、氟尿苷、阿糖胞苷、卡培他滨、吉西他滨等)、嘌呤拮抗剂(例如6-巯基嘌呤、6-硫鸟嘌呤等)、腺苷脱氨酶抑制剂(例如克拉屈滨、氟达拉滨、奈拉滨、喷司他丁等)。
可用于本发明的缀合物的拓扑异构酶抑制剂的实例包括但不限于(喜树碱类及其衍生物(例如伊立替康、托泊替康等)、安吖啶、道诺霉素、阿霉素、表鬼臼毒素类、玫瑰树碱类、表柔比星、依托泊苷、丙亚胺、替尼泊苷等。
可用于本发明的缀合物的酪氨酸激酶抑制剂的实例包括但不限于阿西替尼、博舒替尼、西地尼布、达沙替尼、厄洛替尼、吉非替尼、伊马替尼、拉帕替尼、来妥替尼、尼洛替尼、司马沙尼、舒尼替尼、凡德他尼等。
可用于本发明的缀合物的放射性核素剂的实例包括但不限于于I 131、In 111、Y 90、Lu 177等。
在某些实例性实施方案中，所述治疗剂选自铂类抗肿瘤剂、蒽环类抗生素、紫杉烷类化合物、核苷类似物、喜树碱类化合物，及其类似物或同系物，及其任意组合。
在某些实施方案中，本发明的抗体或其抗原结合片段任选地通过接头与修饰部分(例如可检测的标记或治疗剂)缀合。
在本发明中，使用本领域现有的接头技术可以将细胞毒剂偶联至本发明的抗体或其抗原结合片段。已经用于将细胞毒剂偶联至抗体的接头类型的实例包括但不限于腙、硫醚、酯、二硫化物和含肽的接头。可以选择例如易于在溶酶体隔室内被低pH切割或易于被蛋白酶(例如优先在肿瘤组织中表达的蛋白酶，如组织蛋白酶，诸如组织蛋白酶B、C、D)切割的接头。
关于细胞毒剂的类型、接头及将治疗剂偶联至抗体的方法的进一步讨论还可参见Saito，G.等人(2003)Adv.Drug Deliv.Rev.55：199-215；Trail，P.A.等人(2003)Cancer Immunol.Immunother.52：328-337；Payne，G.(2003)Cancer Cell 3：207-212；Allen，T.M.(2002)Nat.Rev.Cancer 2：750-763；Pastan，I.and Kreitman，R.J.(2002)Curr.Opin.Investig.Drugs 3：1089-1091；Senter，P.D.and Springer，C.J.(2001)Adv.Drug Deliv.Rev.53：247-264。
嵌合抗原受体
本发明的抗体或其抗原结合片段可用于构建嵌合抗原受体(CAR)，本发明CAR的特征包括非MHC限制的BCMA识别能力，其赋予表达该CAR的免疫效应细胞(例如，T细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞)不依赖于抗原加工及提呈而识别表达BCMA的细胞的能力。
因此，在第六方面，本发明提供了一种嵌合抗原受体(CAR)，其包含胞外抗原结合结 构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，其中所述胞外抗原结合结构域包含本发明的抗体或其抗原结合片段。在某些实施方案中，所述CAR从N端至C端包含胞外抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域。
1、胞外抗原结合结构域
本发明的CAR中所包含的胞外抗原结合结构域赋予所述CAR识别BCMA的能力。
在某些实施方案中，所述胞外抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:1所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:2所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述胞外抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:3所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:4所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述胞外抗原结合结构域包括但不限于Fab片段、Fab'片段、F(ab)' 2片段、Fv、二硫键稳定的Fv蛋白(“dsFv”)、scFv、di-scFv、(scFv) 2。
在某些实施方案中，所述抗体或其抗原结合片段是scFv、di-scFv或(scFv) 2。
在某些实施方案中，所述胞外抗原结合结构域包含还包含连接子。在某些实施方案中，所述胞外抗原结合结构域所包含的VH和VL通过连接子连接。在某些示例性实施方案中，所述连接子具有SEQ ID NO：5的序列。
2、跨膜结构域
本发明的CAR所包含的跨膜结构域可以是本领域已知的任何蛋白结构，只要其能够在细胞膜(特别是真核细胞膜)中热力学稳定。适用于本发明的CAR的跨膜结构域可衍生自天然来源。在此类实施方案中，所述跨膜结构域可衍生自任何膜结合的或跨膜的蛋白质。或者，所述跨膜结构域可为合成的非天然存在的蛋白质区段，例如主要包含疏水残基例如亮氨酸和缬氨酸的蛋白质区段。
在某些实施方案中，所述跨膜结构域是选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD8α、CD28、CD3ε、CD3ζ、CD45、CD4、CD5、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、DAP10及其任意组合。在某些示例性实施方案中，所述跨膜结构域包含CD8α的跨膜区。在某些示例性实施方案中，所述跨膜结构域包含T细胞共刺激分子(例如CD137或CD28)的跨膜结构域。
在某些示例性实施方案中，所述跨膜结构域包含如SEQ ID NO:7所示的氨基酸序列。
3、间隔结构域
本发明的嵌合抗原受体可以在胞外抗原结合结构域与跨膜结构域之间包含间隔结构域。
在某些实施方案中，所述间隔结构域包含免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区。在此类实施方案中，不受特定理论的约束，认为CH2和CH3使所述CAR的抗原结合结构域从表达CAR的细胞的细胞膜延伸出去，并且可更精确地模拟天然TCR的大小和结构域结构。
在某些实施方案中，所述间隔结构域包含铰链结构域。铰链结构域可以是通常在蛋白质的两个结构域之间发现的氨基酸区段，其可以允许蛋白质具有柔性并且允许一个或两个结构域相对于彼此的运动。因此，所述铰链结构域可以是任何氨基酸序列，只要其能够提供胞外抗原结合结构域的这种柔性以及其相对于跨膜结构域的这种运动性。
在某些实施方案中，所述铰链结构域是天然存在的蛋白质的铰链区或其部分。在某些实施方案中，所述铰链结构域包含CD8α的铰链区或其部分，例如含有CD8α的铰链区的至少15个(例如20、25、30、35或40个)连续氨基酸的片段。在某些示例性实施方案中，所述间隔结构域包含SEQ ID NO:6所示的氨基酸序列。
4、胞内信号传导结构域
本发明的CAR中所包含的胞内信号传导结构域参与将有效的抗原受体结合(本发明的CAR与BCMA的结合)的信号传导进免疫效应细胞内部，激活表达CAR的免疫效应细胞的至少一种正常效应子功能，或增强表达CAR的免疫效应细胞的至少一种细胞因子的分泌(例如IL-2，IFN-γ)。
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和/或共刺激信号传导结构域。
在本发明中，所述初级信号传导结构域可以是包含免疫受体酪氨酸活化基序(ITAM)的任何胞内信号传导结构域。在某些实施方案中，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)。在某些实施方案中，所述初级信号传导结构域包含选自下列的蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD22、CD79a、DAP10、CD79b或CD66d。在某些实施方案中，所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域。
在本发明中，所述共刺激信号传导结构域可以是来自共刺激分子的胞内信号传导结构域。在某些实施方案中，所述共刺激信号传导结构域包含选自下列的蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)或DAP10。
在某些实施方案中，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域、或CD137(4-1BB)的胞内信号传导结构域、或二者片段的组合。
在某些实施方案中，所述胞内信号传导结构域包含一个共刺激信号传导结构域。在某些实施方案中，所述胞内信号传导结构域包含两个或更多个共刺激信号传导结构域。在此类实施方案中，所述两个或更多个共刺激信号传导结构域可以是相同的，也可以是不同的。
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域。所述初级信号传导结构域以及至少一个共刺激信号传导结构域可以以任意顺序串联至跨膜结构域的羧基端。
在某些实施方案中，所述胞内信号传导结构域可包含CD3ζ的胞内信号传导结构域和CD137的胞内信号传导结构域。在某些示例性实施方案中，所述CD3ζ的胞内信号传导结构域包含SEQ ID NO:9所示的氨基酸序列。在某些示例性实施方案中，所述CD137的胞内信号传导结构域包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方案中，本发明的CAR可进一步在其N端包含信号肽。通常，信号肽是将与其连接的序列靶向至细胞中所需位点的多肽序列。在某些实施方案中，所述信号肽可以将与其连接的CAR靶向至细胞的分泌途径，并允许该CAR进一步整合并锚定到脂质双分子层中。可用于CAR的信号肽是本领域技术人员已知的。在某些实施方案中，所述信号肽包含重链信号肽(例如IgG1的重链信号肽)、粒细胞-巨噬细胞集落刺激因子受体2(GM-CSFR2)信号肽、或CD8α信号肽。
5、全长CAR
本发明提供了能够特异性地结合BCMA的嵌合抗原受体，所述嵌合抗原受体从其N端至C端依次包含胞外抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域。在某些优选实施方案中，其中所述胞内信号传导结构域从N端到C端为共刺激信号传导结构域和初级信号传导结构域。
在某些实施方案中，所述间隔结构域包含CD8(例如CD8α)的铰链区，其具有SEQ ID NO：6所示序列。在某些实施方案中，所述跨膜结构域包含CD8(例如CD8α)的跨膜区，其具有SEQ ID NO：7所示序列。
在某些实施方案中，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中初级信号传导结构域包含CD3ζ的胞内信号传导结构域，其具有SEQ ID NO：9所示序列。共刺激信号传导结构域包含CD137的胞内信号传导结构域，其具有SEQ ID NO：8所示序列。
在某些示例性实施方案中，所述CAR具有选自下列的氨基酸序列：(1)SEQ ID NO:10或12所示的氨基酸序列，(2)与SEQ ID NO:10或12所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，并且所述序列基本保留了其所源自的氨基酸序列的至少一种生物学活性(例如，能够以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达BCMA的细胞的能力)。
嵌合抗原受体的制备
生成嵌合抗原受体、包含该嵌合抗原受体的免疫效应细胞(例如T细胞)的方法是本领域已知的，其详细描述可参见，例如，Brentjens等人,2010,Molecular Therapy,18:4,666-668；Morgan等人,2010,Molecular Therapy,published online February 23,2010,第1-9页；Till等人,2008,Blood,112:2261-2271；Park等人,Trends Biotechnol.,29:550-557,2011；Grupp等人,NEnglJMed.,368:1509-1518,2013；Han等人,J.Hematol Oncol.,6:47,2013；PCT专利公开文本WO2012/079000、WO2013/126726；和U.S.专利公开文本2012/0213783，其全部通过引用整体并入本文)。例如，可包括用至少一种编码CAR的核酸分子导入细胞，并在细胞中表达该核酸分子。例如，可将编码本发明的CAR的核酸分子包含于表达载体(例如，慢病毒载体)中，所述 表达载体能够在宿主细胞例如T细胞中表达，以制造所述CAR。
因此，在第七方面，本发明提供了一种分离的核酸分子，其包含编码本发明的嵌合抗原受体的核苷酸序列。在某些实施方案中，所述分离的核酸分子编码本发明的嵌合抗原受体。
在某些实施方案中，所述分离的核酸分子编码的CAR包括具有重链可变区(VH)和/或轻链可变区(VL)的胞外抗原结合结构域，其中：所述VH包含如SEQ ID NO:1所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:2所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述分离的核酸分子编码的CAR包括具有重链可变区(VH)和/或轻链可变区(VL)的胞外抗原结合结构域，其中：所述VH包含如SEQ ID NO:3所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:4所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些示例性实施方案中，所述分离的核酸分子编码的CAR具有选自下列的氨基酸序列：(1)SEQ ID NO:10或12所示的氨基酸序列，(2)与SEQ ID NO:10或12所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，并且所述序列基本保留了其所源自的氨基酸序列的至少一种生物学活性(例如，能够以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达BCMA的细胞的能力)。
本领域技术人员理解，由于遗传密码的简并性，编码一种本发明的嵌合抗原受体的核苷酸序列可以具有多种不同的序列。因此，除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括作为彼此的简并形式且编码相同氨基酸序列的所有核苷酸序列。
在某些示例性实施方案中，所述编码本发明的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:11或13所示的核苷酸序列；(2)与SEQ ID NO:11或13所示的核苷酸序列 相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达BCMA的细胞的能力的CAR)。
在第八方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。
在某些实施方案中，所述载体包含编码本发明的嵌合抗原受体的核苷酸序列。
在某些示例性实施方案中，所述CAR具有选自下列的氨基酸序列：(1)SEQ ID NO:10或12所示的氨基酸序列，(2)与SEQ ID NO:10或12所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，并且所述序列基本保留了其所源自的氨基酸序列的至少一种生物学活性(例如，能够以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达BCMA的细胞的能力)。
在某些示例性实施方案中，所述编码本发明的嵌合抗原受体的核苷酸序列选自：(1)SEQ ID NO:11或13所示的核苷酸序列；(2)与SEQ ID NO:11或13所示的核苷酸序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列，并且所述序列基本保留了其所源自的核苷酸序列的至少一种生物学活性(例如，能够编码具有以非MHC限制的方式将免疫效应细胞的特异性和反应性指向表达BCMA的细胞的能力的CAR)。
在某些实施方案中，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，病毒载体。
在某些实施方案中，所述载体是表达载体。
在某些实施方案中，所述载体是游离型载体。
在某些实施方案中，所述载体是病毒载体。
在某些示例性实施方案中，所述病毒载体是慢病毒载体、腺病毒载体或逆转录病毒载体。
在某些实施方案中，所述载体是游离型或非整合病毒载体，例如整合缺陷型逆转录病毒或慢病毒。
修饰的免疫效应细胞及制备方法
在第九方面，本发明提供了一种免疫效应细胞，其表达本发明的嵌合抗原受体。可以用多种方法将编码CAR的多核苷酸引入细胞后，也可以在细胞中原位合成CAR。或者，可以在细胞外生产CAR，然后将其引入细胞。
在某些实施方案中，所述免疫效应细胞包含第七方面所述的分离的核酸分子或第八方面所述的载体。
可以通过各种合适的方式将如上所述的分离的核酸分子或载体引入免疫效应细胞，例如磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、电穿孔、TALEN方法、ZFN方法、非病毒载体介导的转染(例如脂质体)或病毒载体介导的转染(如慢病毒感染，逆转录病毒感染，腺病毒感染)，以及其他用于转移入宿主细胞的物理、化学或生物学手段，如转座子技术，CRISPR-Cas9等技术。
在某些实施方案中，所述免疫效应细胞来源于患者或健康供体。在某些实施方案中，所述免疫效应细胞选自T淋巴细胞、自然杀伤(NK)细胞、单核细胞、巨噬细胞或树突状细胞及其任意组合。在某些实施方案中，所述免疫效应细胞选自T淋巴细胞和/或自然杀伤(NK)细胞。
在另一方面，本发明还提供了制备表达本发明的嵌合抗原受体的免疫效应细胞的方法，其包括：(1)提供免疫效应细胞；(2)将第六方面所述的分离的核酸分子或第七方面所述的载体引入所述免疫效应细胞。所述分离的核酸分子或载体包含编码本发明的嵌合抗原受体的核苷酸序列。
在某些实施方案中，所述免疫效应细胞选自T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合。
在某些实施方案中，在步骤(1)中，所述免疫效应细胞经过预处理；所述预处理包括免疫效应细胞的分选、激活和/或增殖。在某些实施方案中，所述预处理包括将免疫效应细胞与抗CD3抗体和抗CD28抗体接触，从而刺激所述免疫效应细胞并诱导其增殖，由此生成经预处理的免疫效应细胞。
在一些实施方案中，在步骤(2)中，将核酸分子或载体通过病毒感染引入免疫效应细胞。在另一些实施方案中，在步骤(2)中，将核酸分子或载体通过非病毒载体转染的方式引入免疫效应细胞，如通过转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方 法、电穿孔方法、磷酸钙转染、DEAE-葡聚糖介导的转染或显微注射等方法。
在某些实施方案中，在步骤(2)之后，所述方法还包括：扩增步骤(2)获得的免疫效应细胞。
在另一方面，本发明还提供了用于制备能够特异性结合BCMA的嵌合抗原受体或表达所述嵌合抗原受体的试剂盒，所述试剂盒包括如第七方面所述的分离的核酸分子，或第八方面所述的载体，和必要的溶剂(例如，如无菌水或生理盐水，或细胞培养液)。所述试剂盒任选地包括使用说明书。
在另一方面，本发明提供了上述试剂盒用于制备能够特异性结合BCMA的嵌合抗原受体或表达所述嵌合抗原受体的细胞的用途。
药物组合物
在第十方面，本发明提供了一种药物组合物，其含有本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的缀合物、第六方面所述的嵌合抗原受体、第二方面或第七方面所述的分离的核酸分子、第三方面或第八方面所述的载体、第四方面所述的宿主细胞或第九方面所述的免疫效应细胞，以及药学上可接受的载体和/或赋形剂。
在一些实施方案中，本发明的药物组合物包含本发明的抗体或其抗原结合片段。
在一些实施方案中，本发明的药物组合物包含本发明的缀合物。在此类实施方案中，所述缀合物包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
在一些实施方案中，本发明的药物组合物包含第七方面所述的分离的核酸分子、第八方面所述的载体、或第九方面所述的免疫效应细胞。所述核酸分子、载体包含编码本发明的嵌合抗原受体的核苷酸序列，所述免疫效应细胞表达所述嵌合抗原受体。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂(例如吉西他滨、5-氟尿嘧啶、紫杉烷、顺铂等)、抗血管生成剂、细胞因子(例如GM-CSF、IL-7、IL-12、IL-15、IL-18、IL-21等)、特异性靶向肿瘤细胞抗体(例如，CD20抗体如利妥昔单抗、Her2抗体如曲妥珠单抗、VEGF抗体如贝伐珠单抗、EGFR抗体如西妥昔单抗等)、免疫检查点抑制剂(例如，PD-1抗体、PD-L1抗 体、CTLA-4抗体、LAG-3抗体等)等。
在某些实施方案中，在所述药物组合物中，本发明的抗体或其抗原结合片段、缀合物、分离的核酸分子、载体或免疫效应细胞与所述另外的药学活性剂可以作为分离的组分或作为混合的组分提供。因此，本发明的抗体或其抗原结合片段、缀合物、分离的核酸分子、载体或免疫效应细胞与所述另外的药学活性剂可以同时、分开或相继施用。
本发明的抗体或其抗原结合片段、缀合物、免疫效应细胞或药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的抗体或其抗原结合片段、缀合物、免疫效应细胞或药物组合物，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
因此，在某些示例性实施方案中，本发明的药物组合物包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本发明的抗体或其抗原结合片段、缀合物、免疫效应细胞、或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药 (例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明的抗体或其抗原结合片段、缀合物、免疫效应细胞、或药物组合物通过静脉注射或推注给予。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的缀合物或第八方面所述的免疫效应细胞。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明的抗体或其抗原结合片段、第五方面所述的缀合物或第八方面所述的免疫效应细胞的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
治疗方法及用途
在另一方面，本发明提供了一种用于在受试者(例如人)中预防和/或治疗B细胞相关病况的方法，所述方法包括向有此需要的受试者施用有效量的第一方面所述的抗体或其抗原结合片段、第五方面所述的缀合物、第八方面所述的免疫效应细胞或本发明的药物组合物。在此类实施方案中，所述缀合物包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
在某些实施方案中，所述方法包括向所述受试者施用有效量的本发明的抗体或其抗原结合片段或缀合物。
在某些实施方案中，所述方法包括向所述受试者施用有效量的第八方面所述的免疫效应细胞或包含所述免疫效应细胞的药物组合物。
在某些实施方案中，所述方法包括以下步骤：(1)提供免疫效应细胞(例如T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突状细胞、或这些细胞的任意组合)；(2)将包含编码本发明第六方面所述的嵌合抗原受体的核酸分子导入步骤(1)所述的免疫效应细胞，以获得表达所述嵌合抗原受体的免疫效应细胞；(3)将步骤(2)中获得的免疫效应细胞施用至所述受试者以进行治疗。
在某些实施方案中，在步骤(1)之前包括从所述受试者获得所述免疫效应细胞的步骤。在某些实施方案中，所述免疫效应细胞选自T淋巴细胞和/或NK细胞。
在某些实例性实施方案中，从所述受试者获得外周血单核细胞(PBMC)，并对PBMC直接进行遗传修饰，以表达CAR。
在某些实例性实施方案中，从所述受试者获得T细胞。T细胞可获自多种来源，包括但不限于：外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织以及肿瘤。在某些实施方案中，可以利用技术人员已知的多种技术(如沉积，例如FICOLLTM分离)从收集自对象的血液单位获得T细胞。在一个实施方案中，通过血浆分离置换法(apheresis)获得来自个体循环血的细胞。血浆分离置换法的产物通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其它有核白细胞、红细胞和血小板。在一个实施方案中，可以对通过血浆分离置换法收集的细胞进行洗涤，以移除血浆级分，并将细胞置于合适的缓冲剂或介质中用于随后处理。可以用PBS或者用缺乏钙、镁以及大部分二价阳离子(如果不是所有其它二价阳离子)的其它合适的溶液洗涤细胞。如本领域普通技术人员所理解，可以通过本领域技术人员已知的方法，如通过使用半自动流通式离心机，来完成洗涤步骤。例如，Cobe 2991细胞处理器、Baxter CytoMate等。洗涤之后，可在多种生物相容的缓冲剂或者含有或不含缓冲剂的其它盐溶液中重悬细胞。在某些实施方案中，可在细胞直接重悬的培养基中移除血浆分离置换法样品中不想要的组分。
在某些实施方案中，通过裂解红细胞并耗尽单核细胞(例如，通过经PERCOLLTM梯度离心)，从外周血单核细胞(PBMC)分离T细胞。可以通过阳性或阴性选择技术进一步分离表达下述标志物中的一种或多种的特定T细胞亚群：CD3、CD28、CD4、CD8、CD45RA和CD45RO。在一个实施方案中，通过阳性或阴性选择技术进一步分离表达CD3、CD28、CD4、CD8、CD45RA和CD45RO的特定T细胞亚群。例如，可以用针对阴性选择的细胞特有的表面标志物的抗体组合来完成通过阴性选择的T细胞群的富集。一种实例性方法是经负磁性免疫粘附或流式细胞术进行细胞分选和/或选择，所述负磁性免疫粘附或流式细胞术利用针对被阴性选择的细胞上存在的细胞表面标志物的单克隆抗体的混合物。例如，为了通过阴性选择富集CD4+细胞，单克隆抗体混合物通常包含针对CD14、CD20、CD11b、CD16、HLA-DR和CD8的抗体。也可将流式细胞术和细胞分选用于分离本发明使用的目的细胞群。
在分离之后，对免疫效应细胞(如T细胞)进行遗传修饰，或者可在进行遗传修饰之前，在体外活化并扩增(或者在祖细胞的情况下，分化)免疫效应细胞。在某些实例性实施方案中，将免疫效应细胞(如T细胞)用本发明的嵌合抗原受体进行遗传修饰(例如，用包含编码CAR的核酸的病毒载体转导)，然后在体外进行活化并扩增。
在某些实施方案中，所述B细胞相关病况是B细胞恶性肿瘤，例如多发性骨髓瘤 (MM)或非霍奇金氏淋巴瘤(NHL)。在某些实施方案中，所述多发性骨髓瘤(MM)选自：明显多发性骨髓瘤、冒烟型多发性骨髓瘤、浆细胞白血病、非分泌型骨髓瘤、IgD骨髓瘤、骨硬化性骨髓瘤、骨孤立性浆细胞瘤以及髓外浆细胞瘤。在某些实施方案中，所述非霍奇金氏淋巴瘤(NHL)选自：伯基特淋巴瘤、慢性淋巴细胞白血病/小淋巴细胞淋巴瘤(CLL/SLL)、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、免疫母细胞性大细胞淋巴瘤、前体B淋巴母细胞性淋巴瘤以及套细胞淋巴瘤。
在某些实施方案中，所述B细胞相关病况是浆细胞恶性肿瘤。
在某些实施方案中，所述B细胞相关病况是自身免疫性疾病。在某些实施方案中，所述自身免疫性疾病选自：全身性红斑狼疮、风湿性关节炎、特发性血小板减少性紫癜或重症肌无力或自身免疫性溶血性贫血。
在某些实施方案中，所述B细胞相关病况选自多发性骨髓瘤、非霍奇金淋巴瘤、具有不确定的恶性潜能的B细胞增殖、淋巴瘤样肉芽肿病、移植后淋巴增生性病症、免疫调节病症、风湿性关节炎、重症肌无力、特发性血小板减少性紫癜、抗磷脂综合征、恰加斯氏病、格雷夫斯氏病、韦格纳肉芽肿、结节性多动脉炎、斯耶格伦氏综合征、寻常天疱疮、硬皮病、多发性硬化、抗磷脂综合征、ANCA相关性小血管炎、古德帕斯彻病、川崎病、自身免疫性溶血性贫血以及急进性肾小球肾炎、重链疾病、原发性或免疫细胞相关淀粉样变性或者意义未明的单克隆丙种球蛋白血症。
在某些实施方案中，将本发明的抗体或其抗原结合片段、缀合物、免疫效应细胞、或药物组合物与额外的疗法组合施用。这种额外的疗法可以是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗。这种额外的疗法可以在施用本发明的抗体或其抗原结合片段、缀合物、免疫效应细胞、或药物组合物之前、同时或之后施用。
在某些实施方案中，所述受试者可以为哺乳动物，例如人。
在另一个方面，提供了本发明第一方面所述的抗体或其抗原结合片段、第五方面所述的缀合物、第六方面所述的嵌合抗原受体、第二方面或第七方面所述的分离的核酸分子、第三方面或第八方面所述的载体、第四方面所述的宿主细胞、第九方面所述的免疫效应细胞、或本发明的药物组合物在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/或治疗B细胞相关病况。在此类实施方案中，所述缀合物包含本发明的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
检测方法和试剂盒
本发明的抗体或其抗原结合片段能够特异性结合BCMA，从而可用于检测BCMA在样品中的存在或其水平。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。
在某些实施方案中，所述试剂盒用于诊断受试者是否患有表达BCMA的肿瘤。某些实施方案中，所述表达BCMA的肿瘤选自B细胞恶性肿瘤，例如多发性骨髓瘤(MM)或非霍奇金氏淋巴瘤(NHL)。在某些实施方案中，所述表达BCMA的肿瘤是浆细胞恶性肿瘤。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记。
在某些优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。
在本发明中，所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。
在另一个方面，本发明提供了检测BCMA在样品中的存在或其量的方法，其包括以下步骤：
(1)将所述样品与本发明的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与BCMA之间复合物的形成或检测所述复合物的量。
所述复合物的形成表明存在BCMA或表达BCMA的细胞。
在某些实施方案中，所述样品是细胞样品，即包含细胞(例如肿瘤细胞)的样品。在此类实施方案中，优选地，所述复合物是由所述抗体、抗原结合片段或缀合物与所述样品中的细胞所表达的BCMA之间形成的。
在某些实施方案中，本发明的抗体或其抗原结合片段还带有可检测的标记。在某些实施方案中，在步骤(2)中，使用带有可检测的标记的试剂来检测本发明的抗体或其抗原结合片段。
所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非 来自患者的样品)。在某些实施方案中，所述BCMA是人BCMA。
在另一个方面，提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测BCMA在样品中的存在或其量。在某些实施方案中，所述BCMA是人BCMA。
在另一方面，本发明提供了一种用于诊断受试者是否患有表达BCMA的肿瘤的方法，其包括使用本发明第一方面所述的抗体或其抗原结合片段检测BCMA在来自所述受试者的样品中的量。
在某些实施方案中，所述表达BCMA的肿瘤选自B细胞恶性肿瘤，例如多发性骨髓瘤(MM)或非霍奇金氏淋巴瘤(NHL)。在某些实施方案中，所述表达BCMA的肿瘤是浆细胞恶性肿瘤。
在某些实施方案中，所述方法还包括：将所述BCMA在来自所述受试者的样品中的量与参考值进行比较的步骤。
所述参考值是指BCMA在来自已知不具有表达BCMA的肿瘤的受试者的样品中的水平(也称作“阴性参考值”)，或者是指BCMA在已知患有表达BCMA的肿瘤的受试者的样品中的水平(也称作“阳性参考值”)。例如，如果所述BCMA在来自所述受试者的样品中的量与阴性参考值相似(或者无显著差异)，则指示所述受试者未患有表达BCMA的肿瘤，以及如果所述BCMA在来自所述受试者的样品中的量相对于阴性参考值升高时，则指示所述受试者患有表达BCMA的肿瘤。此外，如果所述BCMA在来自所述受试者的样品中的量与阳性参考值相似(或者无显著差异)，则指示所述受试者患有表达BCMA的肿瘤。
在某些实施方案中，通过以下步骤检测BCMA在来自所述受试者的样品中的量：
(1)将来自所述受试者的样品与本发明的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与BCMA所形成的复合物的量。
在某些实施方案中，在步骤(1)中，本发明的抗体或其抗原结合片段还带有可检测的标记。在某些实施方案中，在步骤(2)中，使用带有可检测的标记的试剂来检测本发明的抗体或其抗原结合片段。
在某些实施方案中，所述样品可以选自尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞(即游离细胞)、组织(例如手术切除的肿瘤组织、活体组织切片或细针抽吸组织)、组织学制备物等。
在某些实施方案中，所述方法还包括给被诊断为患有表达BCMA的肿瘤的受试者施用 靶向BCMA的免疫疗法。
在某些实施方案中，所述靶向BCMA的免疫疗法包括施用本发明的抗体或其抗原结合片段、或缀合物。
在某些实施方案中，所述靶向BCMA的免疫疗法包括施用本发明的免疫效应细胞。
在某些实施方案中，所述靶向BCMA的免疫疗法包括施用表达本发明第六方面所述的嵌合抗原受体的免疫效应细胞。所述方法包括以下步骤：(1)提供免疫效应细胞；(2)将编码本发明的嵌合抗原受体的核苷酸序列导入步骤(1)所述的免疫效应细胞，以获得表达所述嵌合抗原受体的免疫效应细胞；(3)将步骤(2)中获得的免疫效应细胞以及可选的未改造和/或未成功改造的免疫效应细胞施用至所述受试者。在某些实施方案中，在步骤(1)之前，所述方法还包括从所述受试者获得所述免疫效应细胞的步骤。
在另一个方面，提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于诊断受试者是否患有表达BCMA的肿瘤或者用于检测肿瘤是否能够通过靶向BCMA的抗肿瘤疗法来治疗。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子生物学、生物化学、核酸化学、免疫学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“BCMA(B-cell maturation antigen)”是指B细胞成熟抗原,又称TNFRSF17或CD269。BCMA是肿瘤坏死因子受体家族(TNFR)的成员，结合TNF家族配体BAFF(B细胞激活因子)和APRIL(增殖诱导配体)，并且主要在终末分化的B细胞，例如记忆B细胞和浆细胞上表达。BCMA的基因在染色体16上编码，产生长度为994个核苷酸的初级mRNA转录物(NCBI登录号NM_001192.2)，其编码184个氨基酸的蛋白质(NP_001183.2)。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更 多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统确定。
如本文中所使用的，术语“构架区(framework region)”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、di-scFv、(scFv) 2和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为， 六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv) 2，其指的是两个或两个以上单个scFv并联而形成抗体。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。
人源化抗体既能够保留非人源供体抗体(例如鼠源抗体)的预期性质，又能够有效降低非人源供体抗体(例如鼠源抗体)在人受试者中的免疫原性，因此，是特别有利的。然而，由于供体抗体的CDR与受体抗体的FR之间的匹配问题，人源化抗体的预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)通常低于非人源供体抗体(例如鼠源抗体)。
因此，尽管本领域的研究人员已对抗体的人源化展开了深入的研究，并取得了一些进展，但是，如何对某一供体抗体进行充分的人源化，以使得所产生的人源化抗体既具有尽可能高的人源化程度，又能够尽可能地保留供体抗体的预期性质，现有技术并没有提供详尽的指导。技术人员需要针对具体供体抗体进行摸索、探究和改造，付出大量的创造性劳动才有可能获得，既具有高人源化程度、又保留具体供体抗体的预期性质的人源化抗体。
如本文中所使用的，术语“胚系抗体基因(germline antibody gene)”或“胚系抗体基因片段(germline antibody gene segment)”是指，存在于生物体的基因组中的编码免疫球蛋白的序列，其没有经历过能够导致表达特异性免疫球蛋白的遗传学重排及突变的成熟过程。在本发明中，表述“重链胚系基因”是指，编码免疫球蛋白重链的胚系抗体基因或基因片段，其包括V基因(variable)、D基因(diversity)、J基因(joining)和C基因(constant)；类似地，表述“轻链胚系基因”是指，编码免疫球蛋白轻链的胚系抗体基因或基因片段，其包括V基因(variable)、J基因(joining)和C基因(constant)。在本发明中，由所述胚系抗体基因或胚系抗体基因片段所编码的氨基酸序列也称为“胚系序列(germline sequence)”。胚系抗体基因或胚系抗体基因片段及其相应的胚系序列是本领域技术人员熟知的，并且可从专业数据库(例如，IMGT、UNSWIg、NCBI或VBASE2)获得或查询。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(K D)表示。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数K D(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量K D、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，术语“嵌合抗原受体(CAR)”是指包含至少一个细胞外抗原结合结构域、间隔结构域、跨膜结构域和胞内信号传导结构域的重组多肽构建体，其将针对目的抗原(例如BCMA)的基于抗体的特异性与免疫效应细胞活化胞内结构域组合以展现针对表达该目的抗原(例如BCMA)细胞的特异性免疫活性。在本发明中，表述“表达CAR的免疫效应细胞”是指表达CAR并且具有由该CAR的靶向结构域决定的抗原特异性的免疫效应细胞。制造CAR(例如，用于癌症治疗)的方法是本领域已知的，可参见例如，Park等人,TrendsBiotechnol.,29:550-557,2011；Grupp等人,NEnglJMed.,368:1509-1518,2013；Han等人,J.HematolOncol.,6:47,2013；PCT专利公开文本WO2012/079000、WO2013/059593；和美国专利公开文本2012/0213783，其全部通过引用整体并入本文。在本文中，表述“抗BCMA CAR”是指，所包含的细胞外抗原结合结构域能够能够特异性结合BCMA的CAR；表述“抗BCMA CAR-T”是指，表达上述CAR的免疫效应细胞(例如PBMC，例如T细胞)。在本文中，表述“人源化的CAR”是指，所包含的细胞外抗原结合结构域源自人源化抗体的CAR；类似地，表述“鼠源CAR”是指，所包含的细胞外抗原结合结构域源自鼠源抗体的CAR。
如本文中所使用的，术语“胞外抗原结合结构域”是指能够特异性结合目的抗原或受体的多肽。该结构域将能够与细胞表面分子相互作用。例如，可以选择胞外抗原 结合结构域来识别作为与特定疾病状态相关的靶细胞细胞表面标志物的抗原。典型地，所述胞外抗原结合结构域是抗体衍生的靶向结构域。
如本文中所使用的，术语“胞内信号传导结构域”是指传导效应信号功能信号并引导细胞进行专门的功能的蛋白质部分。因此，胞内信号传导结构域具有激活表达CAR的免疫效应细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
如本文中所使用的，术语“初级信号传导结构域”是指能够以刺激方式或以抑制方式调节TCR复合物的初级活化的蛋白质部分。以刺激方式作用的初级信号传导结构域通常含有已知为基于免疫受体酪氨酸的活化基序(ITAM)的信号传导基序。含有特别用于本发明中的初级信号传导结构域的ITAM的非限制性实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、DAP10、CD79a、CD79b和CD66d的那些。
如本文中所使用的，术语“共刺激信号传导结构域”是指共刺激分子的胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体以外的在结合到抗原后提供T淋巴细胞的高效活化和功能所需的第二信号的细胞表面分子。所述共刺激分子的非限制性实例包括CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)CD270(HVEM)、CD278(ICOS)、DAP10。
如本文中使用的，术语“免疫效应细胞”是指具有一种或多种效应功能(例如，细胞毒性细胞杀伤活性、细胞因子的分泌、ADCC和/或CDC的诱导)的免疫系统的任何细胞。典型地，免疫效应细胞是具有造血的起源并在免疫应答中起作用的细胞。术语“效应功能”指免疫效应细胞的特化功能，例如增强或促进对靶细胞的免疫攻击(例如对靶细胞的杀伤，或者抑制其生长或增殖)的功能或反应。T细胞的效应功能，例如，可以是细胞溶解活性或者辅助或者包括细胞因子的分泌在内的活性。免疫效应细胞的实例包括T细胞(例如α/βT细胞和γ/δT细胞)、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞和骨髓来源巨噬细胞。
本发明所述的免疫效应细胞可以是自身的/自体的(“自我”)或非自身的(“非自我”，例如同种异体的、同基因的或异基因的)。如本文中使用的，“自身的”是指来自同一受试者的细胞；“同种异体的”是指与比较细胞遗传不同的同一物种的细胞；“同基因的”是指与比较细胞遗传相同的来自不同受试者的细胞；“异基因的”是指与比较细胞来自不同物种的细胞。在优选实施例中，本发明的细胞是同种异体的。
可用于本文所述的CAR的示例性免疫效应细胞包括T淋巴细胞。术语“T细胞”或“T淋巴细胞”是本领域公知的并且意图包括胸腺细胞、未成熟的T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。T细胞可以是T辅助(Th)细胞，例如T辅助1(Th1)或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL；CD4T细胞)CD4T细胞、细胞毒性T细胞(CTL；CD8T细胞)、CD4CD8T细胞、CD4CD8T细胞或任何其它T细胞子组。在某些实施方案中，T细胞可以包括原初T细胞和记忆T细胞。
本领域技术人员将理解，其它细胞也可以用作具有如本文所述的CAR的免疫效应细胞，例如NK细胞。具体来说，免疫效应细胞还包括NK细胞、单核细胞、巨噬细胞或树突状细胞、NKT细胞、嗜中性白细胞和巨噬细胞。免疫效应细胞还包括免疫效应细胞的祖细胞，其中所述祖细胞可以在体内或体外经诱导以分化成免疫效应细胞。因此，在某些实施方案中，免疫效应细胞包括免疫效应细胞的祖细胞，例如含于衍生自脐血、骨髓或流动周边血液的CD34+细胞群体内的造血干细胞(HSC)，其在受试者中投与后分化成成熟免疫效应细胞，或其可以在体外经诱导以分化成成熟免疫效应细胞。
如本文中所使用的，术语“细胞毒剂”包括对细胞有害(例如杀死细胞)的任何试剂，例如化疗药物、细菌毒素、植物毒素或放射性同位素等。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“游离型载体”中游离型是指载体能够复制而不整合到宿主的染色体DNA中并且不由分裂宿主细胞逐渐丧失，还意指所述载体在染色体外或游离地复制。
如本文中所使用的，术语“病毒载体”广泛用以指包括典型地促进核酸分子转移或整合到细胞的基因组中的病毒衍生的核酸元件的核酸分子(例如转移质粒)，或介导 核酸转移的病毒颗粒。除了核酸之外，病毒颗粒典型地将包括各种病毒组分并且有时还包括宿主细胞组分。术语“病毒载体”可以指能够将核酸转移到细胞中的病毒或病毒颗粒，或指转移的核酸本身。病毒载体和转移质粒含有主要衍生自病毒的结构和/或功能遗传元件。
如本文中所使用的，术语“逆转录病毒载体”是指含有主要衍生自逆转录病毒的结构和功能遗传元件或其部分的病毒载体或质粒。
如本文中所使用的，术语“慢病毒载体”是指含有主要衍生自慢病毒的结构和功能遗传元件或其部分(包括LTR)的病毒载体或质粒。在某些实施方案中，术语“慢病毒载体”、“慢病毒表达载体”可以用以指慢病毒转移质粒和/或感染性慢病毒颗粒。在本文提及元件(例如克隆位点、启动子、调节元件、异源核酸等)时，应理解，这些元件的序列以RNA形式存在于本发明的慢病毒颗粒中并且以DNA形式存在于本发明的DNA质粒中。
如本文中所使用的，“整合缺陷型”逆转录病毒或慢病毒是指具有不能将病毒基因组整合到宿主细胞的基因组中的整合酶的逆转录病毒或慢病毒。在某些实施方案中，整合酶蛋白突变以特异性降低其整合酶活性。整合缺陷型慢病毒载体可以通过修饰编码整合酶蛋白的pol基因，产生编码整合缺陷型整合酶的突变pol基因而获得。所述整合缺陷型病毒载体已经描述于专利申请WO 2006/010834中，所述专利申请以全文引用的方式并入本文中。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞，免疫细胞(如T淋巴细胞、NK细胞、单核细胞、巨噬细胞或树突状细胞等)。宿主细胞可以包括单个细胞或细胞群体。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％ 的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨 基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，术语“受试者”是指包括其中可以引出免疫应答的活生物体。在某些实施方式中，所述受试者(例如人)患有B细胞相关病况(例如B细胞恶性肿瘤)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，B细胞相关病况)有效量是指，足以预防，阻止，或延迟疾病(例如，B细胞相关病况)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中使用的，术语“B细胞相关病况”是指涉及不当B细胞活性和B细胞恶性肿瘤的病况，包括但不限于B细胞恶性肿瘤或与B细胞相关的自身免疫疾病。在本文中，术语“B细胞恶性肿瘤”包括在B细胞(免疫系统细胞类型)中形成的癌症类型，例如，多发性骨髓瘤(MM)和非霍奇金氏淋巴瘤(NHL)。
发明的有益效果
本发明提供了一种靶向BCMA的人源化抗体，其在保持了与鼠源抗体相当的结合表达BCMA肿瘤细胞的活性的同时，对于正常组织细胞的非特异性结合明显降低，有利于减少非特异性毒性，提高药物的安全性。本发明进一步提供了包含所述人源化抗体的靶向BCMA的嵌合抗原受体。表达本发明的嵌合抗原受体的免疫效应细胞基本不会引起抗原非依赖性的细胞因子释放，并且不会诱导T细胞的持续激活和衰竭，具备明显提高的安全性。因此，本发明的人源化抗体及CAR具有用于预防和/或治疗B细胞相关病况(例如B细胞恶性肿瘤、自身免疫疾病等)的潜力，具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
附图说明
图1显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3对重组人源BCMA蛋白的结合曲线。
图2显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3与人多发性骨髓瘤细胞U266结合的剂量依赖性结合曲线。
图3显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3与人多发性骨髓瘤细胞MM.1S结合的剂量依赖性结合曲线。
图4显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3与人多发性骨髓瘤细胞RPMI8226结合的剂量依赖性结合曲线。
图5显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3与不表达BCMA的人Burkitt's淋巴瘤细胞Daudi结合的剂量依赖性结合曲线。
图6显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3与人永生化角质形成细胞Hacat的结合曲线。
图7显示了人源化抗体C11D5.3-03、C11D5.3-04及母本鼠源抗体C11D5.3与人胃粘膜上皮细胞GES的结合曲线。
图8显示了转导了C11D5.3-CAR、或者人源化的C11D5.3-03-CAR、C11D5.3-04-CAR的T细胞上BCMA-CAR的表达情况。
图9显示了C11D5.3 CAR-T或者人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T的IFNγ释放测定结果。
图10显示了C11D5.3 CAR-T或者人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T的IL2释放测定结果。
图11显示了C11D5.3 CAR-T或者人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T对U266杀伤的LDH释放结果。
图12显示了C11D5.3 CAR-T或者人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T对Daudi杀伤的LDH释放结果。
图13-17分别显示了C11D5.3 CAR-T或者人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T和未转染的PBMC细胞在不存在抗原刺激的无血清培养体系中的IL4(图13)、IL6(图14)、IL10(图15)、TNFα(图16)以及IFNγ(图17)释放水平检测结果。
图18显示了C11D5.3 CAR-T或者人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T和未转染的PBMC细胞在不存在抗原刺激的含有5％人血清的培养体系中的IL4、IL6、 IL10、TNFα以及IFNγ释放水平检测结果。
图19显示了CAR-T细胞表面HLA-DR的表达水平检测结果。
图20显示了CAR-T细胞表面CD25的表达水平检测结果。
图21显示了C11D5.3-03 CAR-T、C11D5.3-04 CAR-T、C11D5.3 CAR-T以及对照PBMC中的活化的Caspase-3的表达水平检测结果。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
SEQ ID NO 描述 SEQ ID NO 描述
1 C11D5.3-03 VH 9 CD3ζITAM
2 C11D5.3-03 VL 10 C11D5.3-03-CAR全长氨基酸序列
3 C11D5.3-04 VH 11 C11D5.3-03-CAR编码核酸序列
4 C11D5.3-04 VL 12 C11D5.3-04-CAR全长氨基酸序列
5 连接子 13 C11D5.3-04-CAR编码核酸序列
6 铰链区 14 C11D5.3-CAR全长氨基酸序列
7 跨膜区 15 C11D5.3-CAR编码核酸序列
8 CD137胞内信号传导结构域 16 P2A-tEGFR编码核酸序列
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：抗BCMA鼠源抗体的人源化及人源化抗体的制备
为提高抗体与人源抗体的序列的同源性，减少抗体对人的免疫原性，对鼠源抗体 C11D5.3(其重链可变区序列和轻链可变区序列分别参见中国专利申请CN 201510142069.2中的SEQ ID NO：3和SEQ ID NO：12)进行人源化设计，使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。根据以上方法，并经过大量筛选，以鼠源抗体C11D5.3的CDR为基础，共构建了2株人源化抗体(scFv)，分别命名为C11D5.3-03和C11D5.3-04。C11D5.3-03的VH和VL分别如SEQ ID NO:1和SEQ ID NO:2所示；C11D5.3-04的VH和VL分别如SEQ ID NO:3和SEQ ID NO:4所示。此外，制备鼠源抗体C11D5.3(scFv)作为参比抗体。
用Freestyle 293表达培养基(Invitrogen,12338018)培养编码上述抗体的表达质粒瞬时转染的HEK293-F细胞(Invitrogen,R7907),在37℃的CO 2培养箱中温育5-7天。离心去除细胞以及细胞碎片，收集培养上清，使用0.22μm薄膜过滤后，使用蛋白A柱(GE Life Science，17127901)进行纯化，遵循制造商指导，用PBS洗涤，在含有20mM柠檬酸盐、150mM NaCl的酸性缓冲液(pH3.5)中洗脱抗体，用1M Tris(pH8.0)中和洗脱物，使用PBS透析。通过测量280nm处吸光度来测定抗体浓度，分装好的抗体储存于-80℃冰箱。
实施例2：抗BCMA人源化抗体对重组人源BCMA蛋白的亲和力评估
使用ELISA方法评估抗体对人源BCMA蛋白的结合活性，使用ELISA中的剂量依耐性结合曲线比较抗体效能。
在PBS总稀释BCMA蛋白(Sino Biological,10620-H08H)至1μg/mL,在ELISA板上的每孔中加入100μL蛋白溶液，在4℃下过夜。将板在洗涤缓冲液(PBS，0.05％吐温20)中洗涤3次，并且通过添加200μl/w PBS+2％BSA阻断使非特异性位点饱和。加入C11D5.3-03、C11D5.3-04或鼠源抗体C11D5.3，在37℃下孵育2h。将板在洗涤缓冲液中洗涤3次，并且在室温下添加HRP偶联的山羊抗人二抗，孵育1h。将板在洗涤缓冲液中洗涤3次，通过添加TMB(HRP底物)并且在室温下在黑暗中孵育板5至10分钟来检测结合的二抗。通过添加硫酸溶液1M终止酶反应，并且在450nm处测量光吸收。
结果如图1所示，人源化抗体C11D5.3-03、C11D5.3-04分别具有0.011μg/mL和0.011μg/mL的EC50(50％活性时的有效浓度)，与鼠源抗体C11D5.3(EC50＝0.011μg/mL)具有同等的结合活性。
实施例3：抗BCMA人源化抗体对表达BCMA的肿瘤细胞的亲和力评估
使用FACS方法评估实施例1获得的人源化抗体对表达BCMA细胞的结合活性，使用FACS中的剂量依耐性结合曲线比较抗体效能。
消化细胞(BCMA表达的人源骨髓瘤细胞或者BCMA不表达的组织细胞)，将500000个细胞铺于50μL含2％FBS的PBS中(FACS buffer)，使用U形底96孔板。通过将1/3体积(100μL)稀释于200μL FACS buffer中来对测试样品实施3倍梯度稀释，起始抗体浓度为300μg/mL(终浓度)。细胞板的每一孔中加入50μL经稀释的抗体4℃孵育1小时。用FACS buffer清洗两次后，每孔加入100μL第二抗体(Abcam ab97003，5μg/mL，稀释于FACS buffer中)，4℃孵育0.5小时。染色完成后，用FACS buffer清洗两次后，每孔加入200μLFACS buffer，然后上机器(BD C6plus)进行读数。
图2、3、4、5分别显示了抗体与人多发性骨髓瘤细胞U266、MM.1S、RPMI8226，以及不表达BCMA的人Burkitt's淋巴瘤细胞Daudi结合的剂量依赖性结合曲线，具体EC50值如表2所示。结果表明，对于表达BCMA的3种肿瘤细胞，人源化抗体C11D5.3-03、C11D5.3-04均具备与鼠源抗体相当的高亲和力；同时，对于不表达BCMA的肿瘤细胞，人源化抗体C11D5.3-03、C11D5.3-04也展现了与鼠源抗体相当的低亲和力。
表2：抗BCMA抗体结合表达BCMA的肿瘤细胞的EC50值
Figure PCTCN2019107004-appb-000002
实施例4：抗BCMA人源化抗体与非肿瘤组织细胞的非特异性结合的评估
使用FACS方法评估纯化的实施例1获得的人源化抗体与非肿瘤的组织细胞之间的非特异性结合，使用BCMA不表达的组织细胞，具体方法如前文所描述。图6-7分别显示了抗体与人永生化角质形成细胞Hacat，人胃粘膜上皮细胞GES的结合曲线。如图所示，鼠源抗体C11D5.3在高浓度(100μg/mL)下与Hacat和GES细胞均存在结合，相比之下， 两个人源化抗体C11D5.3-03和C11D5.3-04在所测浓度下(0.003-100μg/mL)与两种非肿瘤组织细胞都没有结合。以上结果表明，本发明的人源化抗体相比于鼠源抗体C11D5.3具备降低的非特异性结合，这样的技术效果是显著的和出人意料的。
实施例5：嵌合抗原受体(CAR)慢病毒表达载体的构建及制备
基于上述实施例中获得的人源化抗体，进一步构建CAR慢病毒表达载体。构建的嵌合抗原受体慢病毒表达载体中,从N端到C端各元件氨基酸序列分别如下表3所示。所述CAR包含以上述人源化抗体的scFv(VH+VL)、CD8α铰链区、CD8α跨膜区、CD137的胞内结构域和CD3ζ的ITAM区。将编码上述CAR的核酸序列与编码P2A-tEGFR的序列(SEQ ID NO:16)连接并插入GV401载体(吉凯基因)，其表达框为EF1a Promoter-BCMA CAR-P2A-tEGFR-WPRE，可通过检测tEGFR表达监测BCMA CAR表达。
表3：嵌合抗原受体中从N端到C端各元件的氨基酸序列
Figure PCTCN2019107004-appb-000003
实施例6：CAR-T细胞制备
采集自健康人的PBMC细胞(轩锋生物，SLB-HP010)；在6孔板中包被1μg/ml CD3(Miltenyi，170-076-124)抗体和1μg/ml CD28(Miltenyi，170-076-117)抗体；向包被后的6孔板中加入PBMC，培养过夜。将实施例5所述的CAR慢病毒载体按照MOI＝3加至PBMC，2000rpm离心60分钟，以进行转导，分别获得转导了鼠源C11D5.3-CAR的T细胞(C11D5.3 CAR-T)、转导了人源化的C11D5.3-03-CAR的T细胞(C11D5.3-03 CAR-T)以及转导了人源化的C11D5.3-04-CAR的T细胞(C11D5.3-04 CAR-T)。培养 48小时后，用标记PE荧光的Cetuximab检测tEGFR表达。
图8显示了未经转染的对照PBMC、以及上述三种CAR-T细胞上BCMA-CAR的表达情况。结果显示，上述三种CAR-T均能够表达相应的抗BCMA CAR，表明抗BCMA CAR-T构建成功。
实施例7：抗BCMA CAR-T体外抗肿瘤能力的评估
细胞因子释放测定：将实施例6制备获得的抗BCMA CAR-T细胞收集后，用DPBS溶液清洗2次，重悬至含2％FBS RPMI1640培养基中；将肿瘤细胞收集后，清洗后重选至含2％FBS RPMI1640培养基中，所述肿瘤细胞包括：不表达BCMA的肿瘤细胞(293T，K562)、表达BCMA的肿瘤细胞(M1ss，U266，RPMI8226，Daudi)。
将抗BCMA CAR-T细胞与上述肿瘤细胞系按E:T(效应细胞/靶细胞)＝1：1混合培养16小时，取上清利用BD CBA assay测定上清中细胞因子释放，对照PBMC、C11D5.3CAR-T、C11D5.3-03 CAR-T、C11D5.3-04 CAR-T的IFNγ和IL2释放结果分别如图9和图10所示。
以上结果表明，人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T与鼠源C11D5.3 CAR-T类似，均能够特异性识别BCMA+细胞系，并释放IFNγ和IL2。
肿瘤细胞裂解实验：将表达BCMA细胞系(U266、Daudi)与抗BCMA CAR-T细胞分别按照E:T＝30:1、10:1、3:1混合培养4小时，取上清检测LDH释放(CytoTox96 Non Radioactive Cytotoxicity Assay)，对照PBMC、C11D5.3 CAR-T、C11D5.3-03 CAR-T、C11D5.3-04 CAR-T对U266和Daudi杀伤的LDH释放结果分别如图11和图12所示。
以上结果表明，人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T与鼠源C11D5.3 CAR-T类似，均可识别并杀伤BCMA+肿瘤细胞。
实施例8：人源化的抗BCMA CAR-T细胞的细胞因子释放
研究发现，抗BCMA CAR-T细胞可能在不存BCMA抗原的情况下释放细胞因子，这一释放随抗BCMA CAR分子的结构差异水平不同；强化的细胞因子释放引起的毒性是已知抗BCMA CAR T细胞临床治疗的毒性问题之一。已知基于鼠源抗体C11D5.3的CAR-T细胞治疗在目前进行的临床中未发现显著的强化的细胞因子释放问题，然而研究进一步发现，某些人源化的抗BCMA CAR引起抗原非依赖性的细胞因子释放，该特征将使得人源化的抗BCMA CAR不适用于T细胞疗法。为此，本实施例评价了人源化的抗BCMA CAR-T细胞的抗原非依赖性的细胞因子释放情况。
在含有60 IU/mL的IL2(Miltenyi，130-097-748)的AIM-V培养基(Gibco， A3021002)中，于5％CO 2，37℃培养箱中静置培养实施例6获得的CAR-T或PBMC对照，在不同的培养时间点检测不存在刺激情况下的细胞因子释放。将对照PBMC、C11D5.3CAR-T、C11D5.3-03 CAR-T、或C11D5.3-04 CAR-T进行培养，在第一天(第一次换液前)、第二天、第三天、第五天(第二次换液前)、第七天、以及第九天取上清，使用多元磁珠方法(TH1/TH2 cytokine CBA kit，BD，#550749)测定释放至上清液中的5种细胞因子，包括IL4、IL6、IL10、TNFα以及IFNγ的水平。图13-17分别显示了上述三种CAR-T细胞和未转染的PBMC细胞在不存在抗原刺激的无血清培养体系中在不同培养时间点的IL4、IL6、IL10、TNFα以及IFNγ释放水平的一个比较。可以看到，不存在刺激情况下，人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T的一型炎症因子的释放相比较未转染的PBMC细胞并没有明显变化。
以上结果表明，本发明的人源化的抗BCMA CAR-T细胞没有引发明显的抗原非依赖性的细胞因子释放。
在含有人血清(Corning，MT35060CI)但没有BCMA抗原的AIM-V培养基(Gibco，A3021002)中将1×10 5的对照PBMC、C11D5.3 CAR-T、C11D5.3-03 CAR-T、或C11D5.3-04 CAR-T培养48小时，使用多元磁珠方法测定释放至上清液中的5种细胞因子，包括IL4、IL6、IL10、TNFα以及IFNγ的水平，对比上述三种CAR-T细胞的释放的细胞因子区别，评估为人源化对C11D5.3进行的序列改变是否会引入在小鼠抗BCMA CAR-T细胞中未见的对人血清中蛋白的新生的反应性。图18显示了上述三种CAR-T细胞和未转染的PBMC细胞在不存在抗原刺激的含有5％人血清的培养体系中的IL4、IL6、IL10、TNFα以及IFNγ释放水平的一个比较。可以看到，两种人源化抗BCMA CAR-T细胞的一型炎症因子的释放相比较鼠源CAR-T细胞并没有显著变化，并且与未转染的PBMC细胞在一型炎症因子释放方面也没有显著变化。
上述结果表明，C11D5.3-03和C11D5.3-04的人源化对C11D5.3进行的序列改变不会引入在小鼠抗BCMA CAR-T细胞中未见的对人血清中蛋白的新生的反应性。
综上，基于本发明的人源化抗体C11D5.3-03和C11D5.3-04获得的CAR在能够降低由小鼠序列的免疫反应引起的潜在的免疫原性的同时，未展现明显的抗原非依赖性的细胞因子释放以及对人血清中蛋白的新生的反应性，具备显著提高的安全性。这样的技术效果是显著的和出人意料的。
实施例9：人源化的抗BCMA CAR T细胞的激活状态评价
在实施例8所述的培养体系中培养实施例6制备获得的CAR-T或PBMC对照，在培养 第五天和第九天(终点)检测三种CAR-T细胞表面的T细胞活化表型标志物的表达区别，以评估人源化对C11D5.3进行的序列改变是否会引入在小鼠抗BCMA CAR-T细胞中未见的CAR-T细胞的过度活化。使用FACS方法检测CAR T细胞表面HLA-DR(APC anti-human HLA-DR Antibody，Biolegend)和CD25(FITC anti-human CD25 Antibody，Biolegend)的表达水平。
结果分别如图19和图20所示。结果显示，人源化的C11D5.3-03 CAR-T、C11D5.3-04 CAR-T相比于鼠源C11D5.3 CAR-T未出现CAR-T细胞的过度活化。
实施例10：人源化的抗BCMA CAR T细胞的凋亡评价
T细胞的衰竭通常与细胞凋亡引起的活化诱导的细胞死亡有关。测量活化的Caspase-3(CaspGLOW TM Fluorescein Active Caspase-3 Staining Kit，Invitrogen)的水平，检查抗BCMA CAR的导入是否可导致更高的T细胞凋亡水平。使用FACS方法检测C11D5.3-03 CAR-T、C11D5.3-04 CAR-T、C11D5.3 CAR-T以及对照PBMC的活化的Caspase-3的表达水平，结果分别如图21所示。结果显示，人源化的C11D5.3-03 CAR、C11D5.3-04 CAR的导入都不影响T细胞中的活化Caspase-3的表达水平，不导致T细胞凋亡引起的细胞死亡。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
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Claims (26)

  1. 能够特异性结合BCMA的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:1或3所示的序列；
    (ii)与SEQ ID NO:1或3所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1或3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:2或4所示的序列；
    (v)与SEQ ID NO:2或4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:2或4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换。
  2. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:1所示的序列；
    (ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:2所示的序列；
    (v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:1所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:2所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
  3. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:3所示的序列；
    (ii)与SEQ ID NO:3所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:4所示的序列；
    (v)与SEQ ID NO:4所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:3所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:4所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含重链恒定区(CH)和轻链恒定区(CL)；
    优选地，所述重链恒定区选自IgG、IgM、IgE、IgD或IgA；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
    优选地，所述轻链恒定区选自κ或λ；
    优选地，所述轻链恒定区是κ轻链恒定区。
  5. 权利要求1-4任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自scFv、di-scFv、(scFv) 2、Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv。
  6. 嵌合抗原受体(CAR)，其包含胞外抗原结合结构域、间隔结构域、跨膜结构域以及胞内信号传导结构域，其中所述胞外抗原结合结构域包含权利要求1-5任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段选自Fab片段、Fab'片段、F(ab)' 2片段、Fv、二硫键稳定的Fv蛋白(“dsFv”)、scFv、di-scFv、(scFv) 2；
    优选地，所述抗体或其抗原结合片段选自scFv、di-scFv、(scFv) 2。
  7. 权利要求6所述的嵌合抗原受体，其中，所述胞外抗原结合结构域包含重链可变 区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:1所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:2所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述VH和VL通过连接子连接；优选地，所述连接子具有SEQ ID NO：5所示的序列。
  8. 权利要求6所述的嵌合抗原受体，其中，所述胞外抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述VH包含如SEQ ID NO:3所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，所述VL包含如SEQ ID NO:4所示的序列或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述VH和VL通过连接子连接；优选地，所述连接子具有SEQ ID NO：5所示的序列。
  9. 权利要求6-8任一项所述的嵌合抗原受体，其中，所述间隔结构域选自铰链结构域和/或免疫球蛋白(例如IgG1或IgG4)的CH2和CH3区；
    优选地，所述间隔结构域包含CD8α的铰链区；
    优选地，所述间隔结构域包含SEQ ID NO:6所示的序列。
  10. 权利要求6-9任一项所述的嵌合抗原受体，其中，所述跨膜结构域是选自下列蛋白的跨膜区：T细胞受体的α、β或ζ链、CD8α、CD28、CD3ε、CD3ζ、CD45、CD4、CD5、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、DAP10；
    优选地，所述跨膜结构域包含CD8α的跨膜区；
    优选地，所述跨膜结构域包含SEQ ID NO:7所示的序列。
  11. 权利要求6-10任一项所述的嵌合抗原受体，其中，所述胞内信号传导结构域包含初级信号传导结构域以及任选的共刺激信号传导结构域；
    优选地，所述胞内信号传导结构域从N端到C端依次包含共刺激信号传导结构域和初级信号传导结构域；
    优选地，所述胞内信号传导结构域包含初级信号传导结构域以及至少一个共刺激信号传导结构域；
    优选地，所述初级信号传导结构域包含免疫受体酪氨酸活化基序(ITAM)；
    优选地，所述初级信号传导结构域包含选自以下蛋白的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD22、CD79a、DAP10、CD79b或CD66d；优选地，所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域；优选地，所述初级信号传导结构域包含SEQ ID NO:9所示的序列；
    优选地，所述共刺激信号传导结构域包含选自下列蛋白的胞内信号传导结构域：CARD11、CD2、CD7、CD27、CD28、CD30、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD270(HVEM)、CD278(ICOS)或DAP10；优选地，所述共刺激信号传导结构域选自CD28的胞内信号传导结构域或CD137(4-1BB)的胞内信号传导结构域或其组合；优选地，所述共刺激信号传导结构域包含SEQ ID NO:8所示的序列。
  12. 权利要求6-11任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体从其N端至C端依次包含所述胞外抗原结合结构域、间隔结构域、跨膜结构域、胞内信号传导结构域；
    优选地，所述间隔结构域包含CD8α的铰链区(例如，如SEQ ID NO：6所示序列)；
    优选地，所述跨膜结构域包含CD8α的跨膜区(例如，如SEQ ID NO：7所示序列)；
    优选地，所述胞内信号传导结构域包含初级信号传导结构域和共刺激信号传导结构域，其中所述初级信号传导结构域包含CD3ζ的胞内信号传导结构域(例如，如SEQ ID NO：9所示序列)，所述共刺激信号传导结构域包含CD137的胞内信号传导结构域(例如，如SEQ ID NO：8所示序列)；
    优选地，所述嵌合抗原受体具有选自下列的氨基酸序列：(1)SEQ ID NO:10或12所示的氨基酸序列；(2)与SEQ ID NO:10或12所示的氨基酸序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
  13. 分离的核酸分子，其包含编码权利要求1-5任一项所述的抗体或其抗原结合片段的核苷酸序列，或者编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列；
    优选地，所述分离的核酸分子包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列，所述分离的核酸分子包含选自下列的核苷酸序列：(1)SEQ ID NO:11或13所示的核苷酸序列；(2)与SEQ ID NO:11或13所示的核苷酸序列相比具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％序列同一性的序列。
  14. 载体，其包含权利要求13所述的分离的核酸分子；
    优选地，所述载体包含编码权利要求1-5任一项所述的抗体或其抗原结合片段的核苷酸序列；
    优选地，所述载体包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列；优选地，所述载体选自DNA载体，RNA载体，质粒，转座子载体，CRISPR/Cas9载体，或病毒载体；优选的，所述载体是表达载体；优选地，所述载体是游离型载体；优选地，所述载体是病毒载体，例如慢病毒载体，腺病毒载体或逆转录病毒载体。
  15. 宿主细胞，其包含权利要求13所述的分离的核酸分子，或权利要求14所述的载体；
    优选地，所述宿主细胞包含编码权利要求1-5任一项所述的抗体或其抗原结合片段的核苷酸序列或包含所述核苷酸序列的载体；优选地，所述宿主细胞包含大肠杆菌，酵母，昆虫细胞，或哺乳动物细胞；
    优选地，所述宿主细胞包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列或包含所述核苷酸序列的载体；优选地，所述宿主细胞包含免疫细胞(例如人免疫细胞)；更优选地，所述免疫细胞选自T淋巴细胞、NK细胞，单核细胞、巨噬细胞或树突状细胞及其任意组合。
  16. 制备权利要求1-5任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求15所述的宿主细胞，和从培 养的宿主细胞培养物中回收所述抗体或其抗原结合片段；其中所述宿主细胞包含编码权利要求1-5任一项所述的抗体或其抗原结合片段的核苷酸序列或包含所述核苷酸序列的载体。
  17. 免疫效应细胞，其表达权利要求6-12任一项所述的嵌合抗原受体；
    优选地，所述免疫效应细胞包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列或包含所述核苷酸序列的载体；
    优选地，所述免疫效应细胞选自T淋巴细胞、NK细胞，单核细胞、巨噬细胞或树突状细胞及其任意组合；
    优选地，所述免疫效应细胞选自T淋巴细胞和/或NK细胞。
  18. 制备权利要求17所述的免疫效应细胞的方法，其包括：(1)提供免疫效应细胞；(2)将权利要求13所述的分离的核酸分子或权利要求14所述的载体引入所述免疫效应细胞；其中，所述分离的核酸分子或载体包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列；
    优选地，所述免疫效应细胞选自T淋巴细胞、NK细胞、单核细胞、树突状细胞、巨噬细胞及其任意组合；优选地，所述免疫效应细胞选自T淋巴细胞和/或NK细胞；
    优选地，在步骤(1)中，所述免疫效应细胞经预处理，所述预处理包括免疫效应细胞的分选、激活和/或增殖；优选地，所述预处理包括将免疫效应细胞与抗CD3抗体和抗CD28抗体接触；
    优选地，在步骤(2)中将所述核酸分子或载体通过病毒感染引入免疫效应细胞；
    优选地，在步骤(2)中将所述核酸分子或载体通过非病毒载体转染的方式引入免疫效应细胞，如通过磷酸钙转染、DEAE-葡聚糖介导的转染、显微注射、转座子的载体系统、CRISPR/Cas9载体、TALEN方法、ZFN方法或电穿孔方法；
    优选地，在步骤(2)之后还包括扩增步骤(2)获得的免疫效应细胞的步骤。
  19. 试剂盒，其包括权利要求13所述的分离的核酸分子或权利要求14所述的载体；
    优选地，所述分离的核酸分子或载体包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列。
  20. 权利要求19所述的试剂盒用于制备特异性结合BCMA的嵌合抗原受体或表达所述嵌合抗原受体的细胞的用途；
    优选地，所述细胞是免疫效应细胞，例如T淋巴细胞和/或NK细胞。
  21. 缀合物，其包含权利要求1-5任一项所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的修饰部分；
    优选地，所述修饰部分选自可检测的标记，例如酶、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述修饰部分选自治疗剂，例如具有抗肿瘤活性的药物或细胞毒剂。
  22. 药物组合物，其含有权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求6-12任一项所述的嵌合抗原受体，或权利要求13所述的分离的核酸分子，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求17所述的免疫效应细胞，或权利要求21所述的缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物还包含另外的药学活性剂；优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、特异性靶向肿瘤细胞抗体或免疫检查点抑制剂。
  23. 权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求6-12任一项所述的嵌合抗原受体，或权利要求13所述的分离的核酸分子，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求17所述的免疫效应细胞，或权利要求21所述的缀合物，或权利要求22所述的药物组合物，在制备用于在受试者(例如人)中预防和/或治疗B细胞相关病况的药物中的用途；
    优选地，所述B相关病况是B细胞恶性肿瘤，例如，多发性骨髓瘤(MM)或非霍奇金淋巴瘤(NHL)；
    优选地，所述B细胞相关病况是自身免疫性疾病，例如全身性红斑狼疮、风湿性关节炎、特发性血小板减少性紫癜或重症肌无力或自身免疫性溶血性贫血；
    优选地，所述B细胞相关病况选自多发性骨髓瘤、非霍奇金淋巴瘤、具有不确定的恶性潜能的B细胞增殖、淋巴瘤样肉芽肿病、移植后淋巴增生性病症、免疫调节病症、 风湿性关节炎、重症肌无力、特发性血小板减少性紫癜、抗磷脂综合征、恰加斯氏病、格雷夫斯氏病、韦格纳肉芽肿、结节性多动脉炎、斯耶格伦氏综合征、寻常天疱疮、硬皮病、多发性硬化、抗磷脂综合征、ANCA相关性小血管炎、古德帕斯彻病、川崎病、自身免疫性溶血性贫血以及急进性肾小球肾炎、重链疾病、原发性或免疫细胞相关淀粉样变性或者意义未明的单克隆丙种球蛋白血症。
  24. 用于在受试者(例如人)中预防和/或治疗B细胞相关病况的方法，所述方法包括向有此需要的受试者施用有效量的权利要求1-5任一项所述的抗体或其抗原结合片段、权利要求17所述的免疫效应细胞、或权利要求21所述的缀合物、或权利要求22所述的药物组合物；
    优选地，所述B相关病况是B细胞恶性肿瘤，例如，多发性骨髓瘤(MM)或非霍奇金淋巴瘤(NHL)；
    优选地，所述B细胞相关病况是自身免疫性疾病，例如全身性红斑狼疮、风湿性关节炎、特发性血小板减少性紫癜或重症肌无力或自身免疫性溶血性贫血；
    优选地，所述B细胞相关病况选自多发性骨髓瘤、非霍奇金淋巴瘤、具有不确定的恶性潜能的B细胞增殖、淋巴瘤样肉芽肿病、移植后淋巴增生性病症、免疫调节病症、风湿性关节炎、重症肌无力、特发性血小板减少性紫癜、抗磷脂综合征、恰加斯氏病、格雷夫斯氏病、韦格纳肉芽肿、结节性多动脉炎、斯耶格伦氏综合征、寻常天疱疮、硬皮病、多发性硬化、抗磷脂综合征、ANCA相关性小血管炎、古德帕斯彻病、川崎病、自身免疫性溶血性贫血以及急进性肾小球肾炎、重链疾病、原发性或免疫细胞相关淀粉样变性或者意义未明的单克隆丙种球蛋白血症。
  25. 用于在受试者(例如人)中预防和/或治疗B细胞相关病况的方法，所述方法包括以下步骤：
    (1)提供免疫效应细胞；
    (2)将包含编码权利要求6-12任一项所述的嵌合抗原受体的核苷酸序列的分离的核酸分子或载体导入步骤(1)所述的免疫效应细胞，以获得表达所述嵌合抗原受体的免疫效应细胞；
    (3)将步骤(2)中获得的免疫效应细胞施用至所述受试者以进行治疗；
    优选地，所述免疫效应细胞选自T淋巴细胞、NK细胞、单核细胞、巨噬细胞、树突 状细胞、或这些细胞的任意组合；
    优选地，在步骤(1)之前，所述方法还包括从所述受试者获得所述免疫效应细胞的步骤。
  26. 用于诊断受试者(例如人)是否患有表达BCMA的肿瘤的方法，其包括使用权利要求1-5任一项所述的抗体或其抗原结合片段检测BCMA在来自所述受试者的样品中的量；
    优选地，所述方法还包括：将所述BCMA在来自所述受试者的样品中的量与参考值进行比较的步骤；
    优选地，通过以下步骤检测BCMA在来自所述受试者的样品中的量：
    (1)将来自所述受试者的样品与权利要求1-5任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与BCMA所形成的复合物的量；
    优选地，在步骤(1)中，所述抗体或其抗原结合片段还带有可检测的标记；
    优选地，所述样品选自尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞、组织(例如手术切除的肿瘤组织、活体组织切片或细针抽吸组织)、组织学制备物；
    优选地，所述表达BCMA的肿瘤选自B细胞恶性肿瘤，例如多发性骨髓瘤(MM)或非霍奇金氏淋巴瘤(NHL)；
    优选地，所述方法还包括给被诊断为患有表达BCMA的肿瘤的受试者施用靶向BCMA的免疫疗法；优选地，所述靶向BCMA的免疫疗法为权利要求24或25所述的方法。
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