WO2023098846A1 - 抗bcma纳米抗体及其应用 - Google Patents

抗bcma纳米抗体及其应用 Download PDF

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WO2023098846A1
WO2023098846A1 PCT/CN2022/136074 CN2022136074W WO2023098846A1 WO 2023098846 A1 WO2023098846 A1 WO 2023098846A1 CN 2022136074 W CN2022136074 W CN 2022136074W WO 2023098846 A1 WO2023098846 A1 WO 2023098846A1
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seq
antibody
antigen
amino acid
bcma
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PCT/CN2022/136074
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French (fr)
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付雅媛
游术梅
殷博薇
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • This application relates to the field of biomedicine, in particular, to an anti-BCMA nanobody and its application.
  • Multiple myeloma is a malignant plasmacytoma originating in the bone marrow, a type of B-cell lymphoma, also known as plasmacytoma. It is characterized by abnormal proliferation of bone marrow plasma cells with overproduction of monoclonal immunoglobulin or light chain (M protein), and very few patients can be non-secretory MM that does not produce M protein. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and kidney damage. Due to the suppression of normal immunoglobulin production, it is prone to various bacterial infections.
  • M protein monoclonal immunoglobulin or light chain
  • multiple myeloma accounts for 1% of all tumors and 10-15% of hematologic malignancies.
  • the male to female ratio is 1.6:1, and most patients are >40 years old.
  • Treatment for multiple myeloma includes chemotherapy and hematopoietic stem cell transplantation.
  • the immunomodulators represented by lenalidomide and the protease inhibitors represented by bortezomib in the form of single drug or combination, have shown good efficacy and have become the routine treatment for patients with multiple myeloma. means.
  • multiple myeloma is still considered an incurable disease.
  • Current treatments can only alleviate the symptoms of multiple myeloma, but cannot completely remove the tumor, and almost all patients will eventually relapse. Therefore, there is an urgent need for new treatment options.
  • BCMA B-cell maturation antigen
  • CD269 also known as CD269 or TNFRSF17
  • TNFRSF17 B-cell maturation antigen
  • the receptor is mainly expressed on the surface of mature B lymphocytes and plasma cells, and is a marker protein of B lymphocyte maturation, which is hardly expressed in other tissue cells.
  • BCMA consists of three main domains: extracellular segment (aa1-54), transmembrane region (aa55-77) and intracellular segment (aa78-184).
  • BCMA B cell activating factor
  • APRIL proliferation-inducing ligand
  • BCMA such as CAR T, bispecific antibodies, and ADCs
  • the present application provides an antibody specifically binding to BCMA or an antigen-binding fragment thereof, a multispecific antigen-binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid fragment, a vector, a host cell, a pharmaceutical composition, a kit, The preparation method and its application in treating diseases and detecting BCMA.
  • the application provides an antibody or an antigen-binding fragment thereof that specifically binds BCMA, wherein the antibody or an antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, and the CDR1 comprises SEQ ID NO: 7-21
  • the CDR1 of the VHH domain shown in any one of 24 ⁇ 31 and 92 ⁇ 101 the CDR2 comprises the VHH domain shown in any one of SEQ ID NO: 7 ⁇ 21, 24 ⁇ 31 and 92 ⁇ 101 HCDR2
  • the CDR3 comprises the HCDR3 of the VHH domain shown in any one of SEQ ID NO: 7-21, 24-31 and 92-101.
  • the present application provides a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises the aforementioned antibody or an antigen-binding fragment thereof, and an antigen-binding molecule that binds to an antigen other than BCMA, or binds to Epitopes of BCMA different from the aforementioned antibodies or antigen-binding fragments thereof.
  • the present application provides an isolated nucleic acid fragment, wherein the nucleic acid fragment encodes the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multispecific antigen-binding molecule.
  • the present application provides a vector, wherein the vector comprises the aforementioned nucleic acid fragment.
  • the present application provides a host cell, wherein the host cell comprises the aforementioned nucleic acid fragment or the aforementioned vector.
  • the present application provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody or antigen-binding fragment expressed by the cell or multispecific antigen-binding molecules.
  • the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned carrier, or is prepared according to the aforementioned method obtained product.
  • the present application provides a method for treating tumor or cancer, wherein the method comprises administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid The fragment, the aforementioned carrier, the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition.
  • the present application provides the aforementioned antibodies or antigen-binding fragments thereof, the aforementioned multispecific antigen-binding molecules, the aforementioned nucleic acid fragments, the aforementioned vectors, the products prepared according to the aforementioned methods, or the aforementioned pharmaceutical compositions for the treatment of tumors or cancers. Uses in medicine.
  • the present application provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned carrier, the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition for treating tumors or Uses for cancer.
  • the present application provides a kit, wherein the kit comprises the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned carrier, the aforementioned method prepared product or the aforementioned pharmaceutical composition.
  • the present application provides a method for detecting the expression of BCMA in a biological sample, wherein the method comprises, under the condition that a complex can be formed between the aforementioned antibody or its antigen-binding fragment and BCMA, allowing the A biological sample is contacted with the antibody or antigen-binding fragment thereof.
  • the present application provides the use of the aforementioned antibodies or antigen-binding fragments thereof in the preparation of BCMA detection reagents.
  • This application provides an antibody or antigen-binding fragment thereof with higher affinity for BCMA target, which can better block the binding of BCMA and its ligand APRIL, thus providing a better choice for BCMA antibody drugs and cell therapy products. It is of great significance to fill in the lack of treatment options for multiple myeloma, especially relapsed/refractory multiple myeloma.
  • Fig. 1A is ELISA to detect the binding reaction of control antibody and human BCMA-His protein
  • Fig. 1B is the binding reaction of ELISA detection control antibody and monkey BCMA-His protein
  • Figure 2A is the FACS result of the detection of BCMA expression in H929 cells by REGN5459-hIgG1 and HPN217-hHcAb antibodies;
  • Figure 2B is the FACS result of detecting the expression of BCMA in U266 cells by REGN5459-hIgG1 and HPN217-hHcAb antibodies;
  • Figure 3 is the FACS result of detecting BCMA expression in Flp-inCHO-human BCMA cells with REGN5459-hIgG1 antibody;
  • Figure 4 is the FACS result of detecting BCMA expression in Flp-inCHO-monkey BCMA cells with REGN5459-hIgG1 antibody;
  • Fig. 5 is ELISA to detect the binding reaction of recombinant candidate antibody and human BCMA-his protein
  • Fig. 6 is ELISA to detect the binding reaction of recombinant candidate antibody and monkey BCMA-his protein
  • Figure 7 is a FACS detection of the binding reaction of recombinant candidate antibodies to H929 tumor cells
  • Figure 8 is a FACS detection of the binding reaction of recombinant candidate antibodies to U266 tumor cells
  • Figure 9 is a ligand binding competition ELISA to detect the blocking effect of recombinant candidate antibodies on the binding of ligand APRIL to human BCMA protein;
  • Figure 10 is the ELISA detection of the binding reaction of humanized candidate nanobodies to human BCMA-his protein
  • Figure 11 is the ELISA detection of the binding reaction of the humanized candidate nanobody to the monkey BCMA-his protein
  • Figure 12 is the FACS detection of the binding reaction of humanized candidate nanobodies to H929 tumor cells
  • Figure 13 is the FACS detection of the binding reaction of humanized candidate nanobodies to U266 tumor cells
  • Figure 14 is a ligand binding competition ELISA to detect the blocking effect of humanized candidate nanobodies on the binding of ligand APRIL to human BCMA protein;
  • Figure 15 is an ELISA detection of the binding reaction between the chimeric antibody and the human BCMA-his protein
  • Figure 16A is the FACS detection of the binding reaction of the chimeric antibody to H929 tumor cells
  • Figure 16B is the FACS detection of the binding reaction of the chimeric antibody to U266 tumor cells
  • Figure 17 is a ligand binding competition ELISA to detect the blocking effect of chimeric antibodies on the binding of ligand APRIL to human BCMA protein.
  • BCMA B cell maturation antigen
  • B cell maturation antigen belongs to the tumor necrosis factor receptor family member.
  • BCMA is mainly expressed on the surface of late B cells, short-lived proliferating plasmablasts and long-lived plasma cells, but not in naive B cells, CD34-positive hematopoietic stem cells and other normal tissue cells, but it is highly expressed in MM cells , plays a key role in the survival, proliferation, metastasis and drug resistance of MM cells by mediating downstream signaling pathways, so BCMA is an ideal antigen target for the treatment of MM.
  • KD equilibrium dissociation constant
  • high affinity generally means having a KD of about 10 ⁇ 6 M or lower, 10 ⁇ 7 M or lower, about 10 ⁇ 8 M or lower, about 10 ⁇ 9 M or lower.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or from the variable region of an immunoglobulin light chain to specifically bind to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • antibody includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.
  • Antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that can interact with antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgM, IgD, IgG, IgA, and IgE immunoglobulins
  • their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( Heavy-chain antibodies (HCAbs) produced by camelids such as Vicugna pacos) and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptors Ig new antigen receptors, IgNAR
  • antibody herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • heavy chain antibody herein refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • nanobody refers to a single-domain antibody composed of only the variable region of the heavy chain obtained by cloning the variable region of the heavy chain antibody (the heavy chain antibody that naturally lacks the light chain existing in camels, etc.), also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH domain and “nanobody” and “single domain antibody” (single domain antibody, sdAb) herein have the same meaning and are used interchangeably, referring to the variable region of a cloned heavy chain antibody, constructed A single domain antibody consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment.
  • the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • multispecific herein refers to the ability of an antibody or antigen-binding fragment thereof to bind, for example, different antigens or at least two different epitopes on the same antigen.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody can bind.
  • conventional monospecific IgG-type antibodies have two identical antigen-binding sites (paratopes) and thus can only bind the same epitope (rather than bind different epitopes).
  • multispecific antibodies have at least two different types of paratopes/binding sites and thus can bind at least two different epitopes.
  • complementarity determining region refers to the antigen binding site of an antibody.
  • a single “specificity” may refer to one, two, three or more than three identical CDRs in a single antibody (the actual number of CDRs/binding sites in a single antibody molecule is referred to as " price").
  • a single native IgG antibody is monospecific and bivalent because it has two identical paratopes.
  • a multispecific antibody comprises at least two (different) complementarity determining regions/binding sites.
  • the term “multispecific antibody” refers to an antibody that has more than one paratope and has the ability to bind two or more different epitopes.
  • multispecific antibody includes in particular bispecific antibodies as defined above, but generally also proteins, e.g. antibodies, scaffolds which specifically bind three or more than three different epitopes, i.e. having three or more Antibodies with more than three paratopes/binding sites.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • exemplary, "antigen-binding fragment” or “antibody fragment” herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody and single domain Antibody.
  • chimeric antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as rat, mouse, rabbit or alpaca) and derived from a different organism (such as human ) of the immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; incorporated by reference above and into this article.
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the 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 a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity or enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain variable region” can be used interchangeably with “VL” and “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • variable domains hypervariable regions
  • FR framework regions
  • amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains may be considered heterozygous hypervariable positions, as these positions may be considered within hypervariable regions under one set of criteria (such as IMGT or KABAT) but under a different set of criteria (such as KABAT or IMGT) outside the hypervariable region. One or more of these positions may also be found in extended hypervariable regions.
  • the invention includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the heavy chain variable region CDR may be abbreviated as HCDR and the light chain variable region may be abbreviated as LCDR.
  • the variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference).
  • CDR CDR
  • Kabat et al. J.Biol.Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); Chothia et al., J.Mol.Biol.196:901-917 (1987); Al-Lazikani B. et al., J.Mol.Biol., 273:927-948 (1997); MacCallum et al., J.Mol. .Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • a “heavy chain constant region” is selected from a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • typical "heavy chain constant region fragments" are selected from Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the light chain of an antibody, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region is selected from a constant kappa domain or a constant lambda domain.
  • Fc region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage whereby one or more, especially one or two amino acids are excised from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include cleavage variants of the full-length heavy chain.
  • the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index).
  • the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region includes IgG CH2 and IgG CH3 domains, optionally, on this basis, it may also include a complete or partial hinge region, but does not include a CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • a "CH3 domain” comprises the stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, Described in National Institutes of Health, Bethesda, MD, 1991.
  • Fc variant herein refers to changes in the structure or function of Fc caused by one or more amino acid substitutions, insertions or deletion mutations at appropriate positions on the Fc.
  • Interaction between Fc variants refers to the space-filling effect, electrostatic guidance, hydrogen bond interaction, hydrophobic interaction, etc. between Fc variants designed by mutation. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins.
  • a preferred mutation design is a "Knob-into-Hole” style mutation design.
  • the mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins.
  • the representative one is the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996); Heterodimer form (US 20100286374A1); Heterodimer form (SEEDbodies) formed by IgG/Ig chain exchange proposed by Jonathan H. Davis et al.
  • the Knob/Hole structure on the Fc variant fragments of the present invention means that the two Fc fragments are mutated respectively, and can be combined in a "Knob-into-Hole" form after the mutations. It is preferable to use the "knob-into-hole" model of Cater et al. to carry out site mutation transformation on the Fc region, so that the obtained first Fc variant and the second Fc variant can be in the form of "knob-into-hole” Combine together to form heterodimers.
  • the selection of particular immunoglobulin Fc regions from particular immunoglobulin classes and subclasses is within the purview of those skilled in the art.
  • the Fc region of human antibody IgG1, IgG2, IgG3, IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred.
  • One of the first Fc variant or the second Fc variant is randomly selected for knob mutation and the other for hole mutation.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, gap length penalty of 12, gap penalty of 4, using the E. Meyers and W. Miller algorithm which has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) to determine the percent identity between two amino acid sequences or nucleotide sequences.
  • nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg to identify other family member sequences or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the invention in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP2101823B1).
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, Agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., and combinations thereof, which are known to those skilled in the art (see For example, Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329). Except in cases of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or lesions, such as cancers and tumors, in the subject being treated.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject herein refers to an organism receiving treatment for a particular disease or condition as described herein.
  • a “subject” includes a mammal, such as a human, a primate (eg, monkey) or a non-primate mammal, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • the present application provides an antibody specifically binding to BCMA or an antigen-binding fragment thereof, a multispecific antigen-binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid fragment, a vector, a host cell, a pharmaceutical composition, a kit, The preparation method and its application in treating diseases and detecting BCMA.
  • the application provides an antibody or an antigen-binding fragment thereof that specifically binds BCMA, the antibody or an antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises SEQ ID NO: 7-21, HCDR1 of the VHH structural domain shown in any one of the sequences 24-31 and 92-101, the CDR2 comprising the VHH structural domain shown in any one of the sequences of SEQ ID NO: 7-21, 24-31 and 92-101 HCDR2, and the CDR3 comprises HCDR3 of the VHH domain shown in any one of SEQ ID NO: 7-21, 24-31 and 92-101.
  • the HCDR1, HCDR2 and HCDR3 are identified according to the Kabat, IMGT or Chothia numbering system.
  • the HCDR1, HCDR2 and HCDR3 are selected from the amino acid sequences represented by SEQ ID NO: 34-91 and 102-157.
  • the HCDR1 comprises SEQ ID NO: 34, 37, 40, 42, 45, 48, 50, 52, 54, 56, 58, 64, 67, 71, 74, 77, 81, The amino acid sequence represented by 84, 86, 87, 104, 107, 113, 116, 119, 121, 124, 127, 131, 134, 138 or 141.
  • the HCDR2 comprises SEQ ID NO: 35, 38, 41, 43, 46, 49, 59, 61, 63, 65, 68, 70, 72, 75, 78, 79, 82, 85, 88, 89, 90, 91, 102, 105, 108, 109, 111, 114, 117, 120, 122, 125, 128, 129, 132, 135, 136, 139, 142, 143, 148, 150, The amino acid sequence represented by 152, 153, 155 or 157.
  • the HCDR3 comprises SEQ ID NO: 36, 39, 44, 47, 51, 53, 55, 57, 60, 62, 66, 69, 73, 76, 80, 83, 103, The amino acid sequence represented by 106, 110, 112, 115, 118, 123, 126, 130, 133, 137, 140, 144, 145, 146, 147, 149, 151, 154 or 156.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 35 and 36; SEQ ID NO: 37, 38 and 39; or SEQ ID NO: Amino acid sequences represented by 40, 41 and 36.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 42, 43 and 44; SEQ ID NO: 45, 46 and 47; or SEQ ID NO: Amino acid sequences represented by 48, 49 and 44.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 50, 35 and 51; SEQ ID NO: 52, 38 and 53; or SEQ ID NO: Amino acid sequences represented by 54, 41 and 51.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 35 and 55; SEQ ID NO: 56, 38 and 57; or SEQ ID NO: Amino acid sequences represented by 58, 41 and 55.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 59 and 60; SEQ ID NO: 56, 61 and 62; or SEQ ID NO: Amino acid sequences represented by 58, 63 and 60.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 64, 65 and 66; SEQ ID NO: 67, 68 and 69; or SEQ ID NO: Amino acid sequences represented by 58, 70 and 66.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 71, 72 and 73; SEQ ID NO: 74, 75 and 76; or SEQ ID NO: Amino acid sequences represented by 77, 78 and 73.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 79 and 80; SEQ ID NO: 81, 82 and 83; or SEQ ID NO: Amino acid sequences represented by 84, 85 and 80.
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences represented by SEQ ID NO: 86, 35 and 36, respectively.
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences represented by SEQ ID NO: 87, 88 and 44, respectively.
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences represented by SEQ ID NO: 42, 88 and 44, respectively.
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences represented by SEQ ID NO: 42, 89 and 44, respectively.
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences represented by SEQ ID NO: 42, 90 and 44, respectively.
  • the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences represented by SEQ ID NO: 42, 91 and 44, respectively.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 71, 102 and 103; SEQ ID NO: 104, 105 and 106; or SEQ ID NO: Amino acid sequences represented by 107, 108 and 103.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 71, 109 and 110; SEQ ID NO: 104, 111 and 112; or SEQ ID NO: Amino acid sequences represented by 107, 108 and 110.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 113, 114 and 115; SEQ ID NO: 116, 117 and 118; or SEQ ID NO: Amino acid sequences represented by 119, 120 and 115.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 121, 122 and 123; SEQ ID NO: 124, 125 and 126; or SEQ ID NO: Amino acid sequences represented by 127, 128 and 123.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 113, 129 and 130; SEQ ID NO: 131, 132 and 133; or SEQ ID NO: Amino acid sequences represented by 134, 135 and 130.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 113, 136 and 137; SEQ ID NO: 138, 139 and 140; or SEQ ID NO: Amino acid sequences represented by 141, 142 and 137.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 143 and 144; SEQ ID NO: 37, 38 and 145; or SEQ ID NO: Amino acid sequences represented by 40, 41 and 144.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 143 and 146; SEQ ID NO: 56, 38 and 147; or SEQ ID NO: Amino acid sequences represented by 58, 41 and 146.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 34, 148 and 149; SEQ ID NO: 37, 150 and 151; or SEQ ID NO: Amino acid sequences represented by 40, 152 and 149.
  • said HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 71, 153 and 154; SEQ ID NO: 104, 155 and 156; or SEQ ID NO: Amino acid sequences represented by 107, 157 and 154.
  • the CDR1, CDR2 and/or CDR3 comprise amino acid sequences in which 1, 2 or 3 mutations occur on the aforementioned HCDR1, HCDR2 and/or HCDR3; the mutations are selected from insertion, deletion And/or replacement, the replacement is preferably a conservative amino acid replacement.
  • the CDR1, CDR2 and/or CDR3 comprise at least 80, 85%, 90%, 91%, 92%, 93%, 94% of the aforementioned HCDR1, HCDR2 and/or HCDR3 , 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
  • the antibody or antigen-binding fragment thereof comprises a single domain antibody comprising the aforementioned CDR1, CDR2 and CDR3.
  • the single domain antibody comprises an amino acid sequence selected from any one of SEQ ID NO: 7-21, 24-31 and 92-101.
  • the single domain antibody comprises at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
  • a mutated amino acid sequence the mutation is selected from insertions, deletions and/or substitutions, the substitutions are preferably conservative amino acid substitutions.
  • the single domain antibody comprises at least 80%, 85%, 90%, Amino acid sequences that are 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.
  • the antibody comprises the FR region in the VHH domain shown in any one of SEQ ID NO: 7-21, 24-31 and 92-101.
  • the antibody comprises at most 15 or 14 FR regions compared with the FR regions in the VHH domains shown in any of SEQ ID NO: 7-21, 24-31 and 92-101 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid sequence of mutations selected from insertions , deletion and/or substitution, the substitution is preferably a conservative amino acid substitution;
  • the antibody comprises at least 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
  • the antibody or antigen-binding fragment thereof is selected from: (1) chimeric antibody or fragment thereof; (2) humanized antibody or fragment thereof; or (3) fully human antibody or fragment thereof.
  • the antibody or antigen-binding fragment thereof comprises or does not comprise an antibody heavy chain constant region; optionally, the antibody heavy chain constant region is selected from human, alpaca, mouse, rat, Rabbit and sheep; Optionally, the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE and IgD; Optionally, the IgG is selected from IgG1, IgG2, IgG3 and IgG4; Optionally, the The heavy chain constant region is selected from Fc region, CH3 region and complete heavy chain constant region, preferably, the heavy chain constant region is a human Fc region; preferably, the antibody or antigen-binding fragment thereof is a heavy chain antibody.
  • the antibody heavy chain constant region is selected from human, alpaca, mouse, rat, Rabbit and sheep;
  • the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE and IgD;
  • the IgG is selected from IgG1, IgG2, IgG3 and Ig
  • the antibody or antigen-binding fragment thereof is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutics, and immunomodulators, and the tracer
  • the therapeutic agent is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the antibody or its antigen-binding fragment is also linked with other functional molecules, preferably, the other functional molecules are selected from one or more of the following: signal peptides, protein tags, cell factors, angiogenesis inhibitors, and immune checkpoint inhibitors.
  • the present application also provides a multispecific antigen-binding molecule, which comprises the aforementioned antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to other antigens other than BCMA, or binds to BCMA epitopes different from the aforementioned antibodies or antigen-binding fragments thereof; optionally, other antigens other than BCMA are selected from: CD3 (preferably CD3 ⁇ ), CD16, CD137, CD258, PD-1, PD-L1, 4- 1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, Phosphatidylserine (PS), C-Met, HSA, GPRC5D, MSLN, blood-brain barrier receptor, GPC3, PSMA, CD33 , GD2, ROR1, ROR2, FR ⁇ and Gucy2C.
  • CD3 preferably CD3 ⁇
  • CD16 CD137, CD258, PD-1, PD-L1, 4- 1
  • said other antigen-binding molecule is an antibody or an antigen-binding fragment thereof.
  • the multispecific antigen binding molecule may be bispecific, trispecific or tetraspecific.
  • the multispecific antigen binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.
  • the present application also provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof or a multispecific antigen-binding molecule.
  • the present application also provides a vector, the vector comprising the aforementioned nucleic acid fragment.
  • the present application also provides a host cell, which comprises the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (such as Escherichia coli), fungi (such as yeast ), insect cells or mammalian cells (eg CHO cell line or 293T cell line).
  • a host cell which comprises the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (such as Escherichia coli), fungi (such as yeast ), insect cells or mammalian cells (eg CHO cell line or 293T cell line).
  • the present application also provides a method for preparing the aforementioned antibody or its antigen-binding fragment or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody or antigen-binding fragment expressed by the cell or multispecific antigen-binding molecules.
  • the present application also provides a pharmaceutical composition, which comprises the aforementioned antibody or its antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, carrier, or product prepared according to the aforementioned method ;
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier (carrier), diluent or adjuvant;
  • the pharmaceutical composition also includes an additional antineoplastic agent.
  • the pharmaceutically acceptable carrier is a carrier that does not weaken the viability and function of immune cells, and does not affect the specific binding of the antibody or its antigen-binding fragment to the antigen, including but not limited to cell culture medium, buffer , normal saline and balanced salt solution.
  • buffers include isotonic phosphates, acetates, citrates, borates, carbonates, and the like.
  • the pharmaceutically acceptable carrier is phosphate buffered saline containing 1% serum.
  • the anti-tumor agent is, for example, various chemotherapeutic drugs, such as alkylating agent chemotherapeutic drugs, including but not limited to cyclophosphamide, ifosfamide, melphalan, carmustine, lomustine, niger Mostine, Formostine, etc.; anti-metabolite chemotherapy drugs, including but not limited to methotrexate, fluorouracil, tegafur, carmofur, deoxyfluorouridine, capecitabine, gemcitabine, raltitrex Anticancer antibiotics, including but not limited to actinomycin D, mitomycin, bleomycin, daunorubicin, adriamycin, epirubicin, pirarubicin, etc.; plant Chemotherapy drugs, including but not limited to vincristine, vindesine, vinorelbine, irinotecan, topotecan, paclitaxel, docetaxel, etc.; miscellaneous, including but
  • the present application also provides a method for treating a tumor or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, A carrier, a product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition; optionally, the tumor or cancer is a tumor or cancer expressing BCMA, preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • BCMA preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • the present application also provides the aforementioned antibody or its antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, carrier, product or pharmaceutical composition prepared according to the aforementioned method in the preparation of the drug for treating tumors or the aforementioned cancer
  • the tumor or cancer is a tumor or cancer expressing BCMA, preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • the present application also provides the aforementioned antibodies or antigen-binding fragments thereof, multispecific antigen-binding molecules, nucleic acid fragments, vectors, products prepared according to the aforementioned methods, or the aforementioned pharmaceutical compositions for the treatment of tumors or cancers.
  • the tumor or cancer is a tumor or cancer expressing BCMA, preferably B-cell lymphoma, more preferably multiple myeloma (MM).
  • the present application also provides a kit comprising the aforementioned antibody or its antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, carrier, product prepared according to the aforementioned method, or The foregoing pharmaceutical composition.
  • the present application also provides a method for detecting the expression of BCMA in a biological sample, the method comprising, under the condition that a complex can be formed between the aforementioned antibody or its antigen-binding fragment and BCMA, allowing the The biological sample is contacted with the antibody or antigen-binding fragment thereof; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of BCMA in the sample.
  • the present application also provides the use of the aforementioned antibody or antigen-binding fragment thereof in the preparation of a BCMA detection reagent.
  • Both REGN5459 and HPN217 are antibodies that recognize human BCMA protein.
  • the sequence of REGN5459 is from US Patent Publication No. US2020/0024356A1
  • the sequence of HPN217 is from US Patent Publication No. US20190112381A1.
  • the heavy chain variable region (VH) of REGN5459 was recombined into an expression vector containing the signal peptide and the human antibody IgG1 heavy chain constant region
  • the light chain variable region (VL) sequence was recombined into an expression vector containing the signal peptide and the human antibody IgG1 light chain
  • the expression vector of the constant region was obtained as a recombinant plasmid, and the synthesized antibody was named REGN5459-hIgG1.
  • the VHH sequence of HPN217 was recombined into the expression vector containing signal peptide and human antibody IgG1Fc to obtain a recombinant plasmid, and the synthesized antibody was named HPN217-hHcAb.
  • the negative control antibody hIgG1 is the antibody anti-hel-hIgG1 against Hen Egg Lysozyme chicken egg lysozyme (purchased from Baiying, item number: B117901), hereinafter referred to as hIgG1.
  • IgG1 Fc contains the C220S mutation.
  • the binding activity of the control antibody to human BCMA-His protein (purchased from Acro, product number: BCA-H522y) and monkey BCMA-His protein (purchased from Acro, product number: BCA-C52H7) was detected by ELISA.
  • the specific method is: dilute the antigenic protein with PBS to a final concentration of 1 ⁇ g/mL, and then add 50 ⁇ l per well to a 96-well ELISA plate. Seal with a plastic film and incubate at 4°C overnight, wash the plate twice with PBS the next day, add blocking solution [PBS+2% (w/v) BSA] to block at room temperature for 2 hours.
  • REGN5459-hIgG1 antibody has good binding activity to human BCMA protein and monkey BCMA protein
  • HPN217-hHcAb has good binding activity to human BCMA protein, but has good binding activity to human BCMA protein.
  • Monkey BCMA protein does not bind.
  • the H929 cells and U266 cells were expanded to the logarithmic growth phase in T-75 cell culture flasks, and the cells were pipetted to a single cell suspension. After cell counting, centrifuge, resuspend the cell pellet with FACS buffer (PBS+2% fetal calf serum) to 2 ⁇ 106 cells per milliliter, add 50 ⁇ l per well to a 96-well FACS reaction plate, use REGN5459-hIgG1 and HPN217-hHcAb antibody as primary antibody, APC-labeled goat anti-human IgG (H+L) secondary antibody (purchased from Jackson, catalog number: 109-605-088) was detected and analyzed by FACS (FACS CantoTM, purchased from BD Company) . The results are shown in Table 4 and Figures 2A and 2B, indicating that both H929 cells and U266 cells can bind to REGN5459-hIgG1 and HPN217-hHcAb antibodies.
  • FACS buffer PBS
  • the Flp-inCHO cell line (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) was transfected ( 3000 Transfection Kit (purchased from Invitrogen, catalog number: L3000-015), in 600 ⁇ g/ml hygromycin (ThermoFisher, catalog number: 10687010) containing 10% (v/v) fetal bovine serum (ExCell Bio, catalog number: FND500) Selective culture in F12K Medium (Gibco, product number: 21127030) medium for 2 weeks, using REGN5459-hIgG1 and goat anti-human IgG (H+L) antibody (Jackson, product number: 109605088) in the flow cytometer FACS CantoII (purchased from BD Biosciences) was used for detection, cells with high expression level and single peak shape were amplified, and the amplified cells were retested by flow cytometry.
  • 3000 Transfection Kit purchased from Invitrogen, catalog number: L3000-015
  • Table 5 shows that the Flp-inCHO highly expressing cell population positively expressing human BCMA and the Flp-inCHO highly expressing cell population positively expressing monkey BCMA have been prepared respectively.
  • the abscissa is the fluorescence intensity of the cells, and the ordinate is the number of cells.
  • the immunized animal was 1 alpaca (Alpaca, serial number NB254).
  • 10 mL of blood was taken as a negative control, and 0.5 mg of human BCMA (Met1-Ala54)-hFc protein (purchased from Acro, product number: BC7-H5254 ) mixed with 1 mL of complete Freund's adjuvant (CFA) and injected subcutaneously; on the 21st day for the second immunization, 0.25 mg of human BCMA-hFc protein was mixed with 1 mL of incomplete Freund's adjuvant (IFA) and injected subcutaneously; 42 days for the third immunization, mix 0.25 mg of human BCMA-hFc protein and cynomolgus monkey BCMA (Met1-Ala53)-hFc protein (purchased from Acro, product number: BCA-C5253) with 1 mL of IFA and inject subcutaneously; Three weeks in accordance with the method of three immune booster immunization once
  • the 50mL peripheral blood collected after the fourth and fifth immunizations was used to separate PBMCs according to the instructions of the lymphocyte separation medium.
  • the total RNA of NB254 four-immune and five-immune PBMCs was extracted with RNAiso Plus reagent.
  • the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, product number: 6210A) kit was used for reverse transcription, cDNA was prepared by reverse transcription according to the instructions, and a total of 5 ⁇ g RNA was transcribed.
  • the cDNA stocks of Sifang and Wufang were mixed in equal proportions, diluted 5 times for nested amplification, and VHH fragments were recovered by tapping the rubber.
  • the vector and target fragment were digested with SfiI overnight at 50°C and then recovered.
  • the vector and the target fragment are ligated at a ratio of 1:3.
  • Perform 10 times of electroshock transformation on the ligation product Immediately after the electroshock, add 1 mL of 2YT medium (preheated at 37°C) to the electroshock cup for recovery, suck out the electroshock product and wash the electroshock cup with 2YT medium, recover for 45 minutes at 37°C and 180 rpm, and take 100 ⁇ L of the gradient Dilute to 10 -3 and 10 -4 to determine the number of library transformants, spread on 90mm plates, centrifuge the rest, add 8mL 2YT medium to resuspend, spread on 8 200mm plates.
  • the calculated storage capacity was 2.14 ⁇ 10 9 . 48 clones were randomly selected for culture, and the insertion rate was 100% by PCR detection.
  • the phage library was precipitated according to the procedure on the NEB official website, and its titer was determined.
  • the microplate was coated with human BCMA-hFc protein 0.5 ⁇ g/well, a total of 8 wells; the input amount of the phage library was 2.2 ⁇ 10 11 , and the titer of the eluted product in one round was measured to be 1.85 ⁇ 10 7 .
  • the positive rate of binding to human BCMA protein by ELISA was 89.58%, the positive rate of binding to monkey BCMA protein was 4.2%, and the positive rate of cross-binding to human and monkey BCMA protein was 4.2%.
  • the eluted product of one round of panning was amplified according to the conventional procedure, and the titer of the amplified phage was determined to be 1.14 ⁇ 10 13 .
  • the microplate was coated with monkey BCMA protein 0.2 ⁇ g/well, a total of 4 wells; the amount of phage library input was 2.2 ⁇ 10 11 , and the titer of the eluted product was 1.32 ⁇ 10 7 .
  • the positive rate of binding to human BCMA protein by ELISA was 92.1%, the positive rate of binding to monkey BCMA protein was 39.3%, and the positive rate of cross-binding to human and monkey BCMA protein was 39.3%.
  • the target VHH sequence is recombined into the expression vector of human IgG1Fc to obtain a recombinant plasmid (see SEQ ID NO: 4 for the sequence of human IgG1Fc).
  • recombinant candidate antibody plasmids and transfection reagent PEI purchased from Polysciences, catalog number: 24765-1
  • OPTI-MEM OPTI-MEM
  • Expi293F cells manufactured in Polysciences, catalog number: 24765-1
  • Thermofisher catalog number: A14527
  • the SEC-HPLC Concentration determination was performed with an ultramicro spectrophotometer (Nanodrop8000, Thermo).
  • the protein samples to be tested were analyzed by SEC-HPLC to characterize the molecular size uniformity and purity of the protein.
  • the HPLC used is Agilent 1260, the chromatographic column TSKgel G3000SWXL is from Tosoh Bioscience, the mobile phase is 200mM phosphate buffer, pH 7.0/isopropanol (v/v 9:1) (batch number: 20210519001), the detection temperature is 25°C, the flow rate It is 0.5mL/min, and the detection wavelength is 280nm.
  • the SEC-HPLC data was analyzed by manual integration method, and the protein purity was calculated according to the area normalization method. The main peak was considered as a monomer, the chromatographic peak before the main peak was called an aggregate, and the chromatographic peak after the main peak was called a fragment. The results are shown in Table 7, and the final product of
  • FACS buffer PBS+2% fetal bovine serum
  • the antigenic protein was diluted with PBS to a final concentration of 1 ⁇ g/mL, and then added to a 96-well ELISA plate at 50 ⁇ l per well. Seal with a plastic film and incubate at 4°C overnight, wash the plate twice with PBS the next day, add blocking solution [PBS+2% (w/v) BSA] to block at room temperature for 2 hours. Pour off the blocking solution, wash the plate 3 times with PBS, add 200 nM serially diluted BCMA nano-recombinant chimeric antibody or negative control antibody, 50 ⁇ l per well. Dilute hAPRIL-biotin (purchased from Acro, product number: APL-H82F5) to 0.5 ⁇ g/mL, 50 ⁇ l per well.
  • Anti-BCMA recombinant candidate antibodies were captured using a Protein A chip (GE Healthcare; 29-127-558).
  • Sample and running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, then a single concentration of BCMA antigen protein was injected to record the binding and dissociation process of the antibody and antigen protein, and finally Glycine pH1.5 (GE Healthcare; BR-1003- 54) Complete chip regeneration.
  • Binding was measured by injecting different concentrations of human BCMA-His in solution for 240 seconds with a flow rate of 30 ⁇ L/min starting from 200 nM (see detailed results for actual concentrations tested), diluted 1:1 for a total of 5 concentrations.
  • the dissociation phase is monitored for up to 600 seconds and is triggered by switching from sample solution to running buffer.
  • the binding rate (K a ), dissociation rate (K dis ) and binding affinity (KD) of the recombinant candidate antibody to human BCMA protein are shown in the table, and the REGN5459-hIgG1 antibody was used as a control. As shown in Table 9, the KD values of the recombinant candidate antibody and human BCMA protein were all below 1E-7M.
  • BCMA-Lab01 and BCMA-Lab02 alpaca antibodies were selected for subsequent humanization.
  • the sequences of BCMA-Lab01-BCMA-Lab08 alpaca antibodies are shown in Table 10, and the CDR sequences analyzed by Kabat, IMGT, and Chothia methods are shown in Table 11.
  • the heavy chain variable region germline genes with high homology to VHH nanobodies were selected as templates,
  • the CDRs of the VHH Nanobodies were grafted into corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the backbone sequence were back-mutated to the corresponding amino acids of the VHH nanobody to ensure the original affinity, that is, a humanized monoclonal antibody was obtained.
  • the CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system.
  • the humanized heavy chain templates of the VHH nanobody BCMA-Lab01 are IGHV3-64*04 and IGHJ3*01, and the CDRs of the VHH nanobody BCMA-Lab01 were transplanted into their human templates to obtain the corresponding humanized versions.
  • the key amino acids in the FR region sequence of the humanized antibody of BCMA-Lab01 were back-mutated to the amino acids corresponding to the VHH nanobody to ensure the original affinity.
  • the antibody has sites that are prone to chemical modification. Point-to-point mutations have eliminated the risk of modification.
  • the specific mutation design is shown in Table 12.
  • Graft means that the VHH Nanobody CDR is implanted into the human germline template FR region sequence; H35G means that the 35th position of Graft is mutated into G, and so on.
  • the numbering of backmutated amino acids is the natural sequence numbering.
  • variable region of the BCMA-Lab01 humanized antibody is as follows:
  • amino acid sequence of the humanized heavy chain template IGHV3-64*04 is shown in SEQ ID NO: 22:
  • amino acid sequence of the humanized heavy chain template IGHJ3*01 is shown in SEQ ID NO: 23:
  • the humanized heavy chain templates of the VHH single-domain antibody BCMA-Lab02 are IGHV3-7*01 and IGHJ6*01, and the CDRs of the VHH single-domain antibody BCMA-Lab02 are transplanted into their human templates to obtain the corresponding humanization Version.
  • the key amino acids in the FR region sequence of the humanized antibody of BCMA-Lab02 were back-mutated to the amino acids corresponding to the VHH single domain antibody to ensure the original affinity.
  • the antibody has a site that is prone to chemical modification. For these Point mutations at sites have eliminated the risk of modification.
  • the specific mutation design is shown in Table 14.
  • Graft means that the VHH single domain antibody CDR is implanted into the human germline template FR region sequence; W47L means that the 47th position of Graft is mutated into L, and so on.
  • the numbering of backmutated amino acids is the natural sequence numbering.
  • variable region of the BCMA-Lab02 humanized antibody is as follows:
  • BCMA-Lab02-VHH1 The amino acid sequence of BCMA-Lab02-VHH1 is shown in SEQ ID NO: 24:
  • amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown in SEQ ID NO: 32:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 33:
  • Example 3 For the specific operation method, see Example 3, The target humanized VHH sequence was recombined into the expression vector of human IgG1Fc to obtain a recombinant plasmid (see SEQ ID NO: 4 for the sequence of human IgG1Fc). The cell supernatant obtained by expressing the humanized VHH-Fc chimeric antibody was collected, and the purity of the obtained antibody was qualitatively analyzed by SEC-HPLC. The results are shown in Table 16, where "/" indicates that no detection was performed.
  • Example 4 The specific method is the same as in Example 4 (A), and the results are shown in Figure 10 and Figure 11, BCMA-Lab02-VHH1 and BCMA-Lab02-VHH2 are not combined with human BCMA and monkey BCMA proteins, and the remaining humanized candidate nanobodies are combined with Human BCMA all bind well, and have cross-binding activity with monkey BCMA protein, and the IgG subtype control is human IgG1.
  • binding rate (Ka), dissociation rate (Kdis) and binding affinity (KD) of humanized candidate nanobodies to human BCMA protein are shown in Table 18, wherein the REGN5459-hIgG1 antibody was used as a control. As shown in Table 18, the KD values of humanized candidate Nanobodies and human BCMA protein are all below 1E-8M.
  • Example 2 (C) positive clones binding to human BCMA were further panned, and a total of 10 clones from Lab09-18 were selected for construction, expression, and identification.
  • the recombinant candidate antibody of VHH-IgG1 was obtained, and the final product of the candidate antibody was identified by SEC-HPLC with high purity.
  • FIG. 15 shows that all antibodies bound to human BCMA protein comparable to the positive control antibody HPN217-hHcAb.
  • Example 4(B) FACS was used to identify the binding of recombinant candidate antibodies to endogenous cells expressing BCMA.
  • the results are shown in FIGS. 16A , 16B and Table 19.
  • the results showed that, except for BCMA-Lab09 which was weakly bound to H929 and not bound to U266, the binding of other antibodies to H929 and U266 cells was strong, which was stronger than or comparable to the positive control antibody.
  • Example 4(C) the blocking effect of the recombinant antibody on the binding of ligand APRIL to human BCMA protein was detected by ligand competition ELISA, and the results are shown in FIG. 17 .
  • the results showed that except for BCMA-Lab09 which could not block the binding of ligand APRIL and human BCMA protein, the other antibodies could well block the binding of ligand APRIL and human BCMA protein, which was equivalent to the positive control antibody.
  • Biacore was used to detect the affinity of the recombinant antibody to human BCMA protein, and the results are shown in Table 20. The results showed that all the antibodies had strong affinity with human BCMA protein.
  • the sequence of the nanobody is shown in Table 21, and its CDR sequence is shown in Table 22.
  • BCMA-Lab10 8.02E+06 5.51E-03 6.87E-10 BCMA-Lab11 5.83E+06 1.26E-03 2.17E-10 BCMA-Lab12 6.92E+05 1.03E-02 1.49E-08 BCMA-Lab13 5.78E+06 1.71E-04 2.97E-11 BCMA-Lab14 4.43E+06 2.48E-03 5.60E-10 BCMA-Lab15 2.47E+06 2.19E-03 8.87E-10 BCMA-Lab16 2.82E+06 1.57E-03 5.58E-10 BCMA-Lab17 4.15E+06 1.76E-02 4.25E-09 BCMA-Lab18 2.04E+06 2.06E-03 1.01E-09 HPN217-hHcAb 2.64E+06 3.10E-03 1.17E-09 REGN5459-hIgG1 7.39E+05 1.97E-04 2.67E-10

Abstract

一种特异性结合BCMA的抗体或其抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及用于治疗疾病的用途，例如***中的用途。

Description

抗BCMA纳米抗体及其应用
相关专利申请的交叉引用
本申请要求于2021年12月03日向中国国家知识产权局提交的，专利申请号为202111468274.X，发明名称为《BCMA纳米抗体及其应用》的中国专利申请和申请日为2022年08月19日向中国国家知识产权局提交的，专利申请号为20221100054.9，发明名称为《抗BCMA纳米抗体及其应用》的优先权。上述在先申请的全文通过引用的方式并入本申请中。
技术领域
本申请涉及生物医药领域，具体而言，涉及一种抗BCMA纳米抗体及其应用。
背景技术
多发性骨髓瘤(multiple myeloma，MM)是一种起源于骨髓中的恶性浆细胞瘤，为B细胞淋巴瘤的一种，又称浆细胞瘤。其特征为骨髓浆细胞异常增生伴有单克隆免疫球蛋白或轻链(M蛋白)过度生成，极少数患者可以是不产生M蛋白的未分泌型MM。多发性骨髓瘤常伴有多发性溶骨性损害、高钙血症、贫血、肾脏损害。由于正常免疫球蛋白的生成受抑制，因此容易出现各种细菌性感染。
多发性骨髓瘤发病率占所有肿瘤的1％，占血液恶性肿瘤的10-15％。男女比例为1.6:1，大多患者年龄>40岁。多发性骨髓瘤治疗包括化疗和造血干细胞移植。其中以来那度胺为代表的免疫调节剂和以硼替佐米为代表的蛋白酶抑制剂，以单药或联用的形式，显示出较好的药效，已成为多发性骨髓瘤病人的常规治疗手段。但多发性骨髓瘤仍被认为是无法治愈的疾病，目前治疗手段只能缓解多发性骨髓瘤的症状，均不能彻底清除肿瘤，几乎所有患者最终仍会复发。因此，对新治疗方案存在着迫切需求。
B细胞成熟抗原(BCMA)，也称CD269或TNFRSF17，是肿瘤坏死因子受体超家族的一员，于20世纪90年代初首次被发现。该受体主要表达于成熟B淋巴细胞及浆细胞表面，是一种B淋巴细胞成熟的标志蛋白，在其他组织细胞中几乎不表达。结构上BCMA由三个主要结构域组成：胞外段(aa1-54)、跨膜区(aa55-77)和胞内段(aa78-184)。B细胞活化因子(BAFF)和增殖诱导配体(APRIL)为BCMA主要配体，通过与BCMA相互作用来传导细胞刺激信号，激活TRAF依赖的NF-κB，JNK途径，增加B细胞的增殖和存活率。BCMA在MM细胞中是高度表达的，是多发性骨髓瘤理想的抗原靶点。BCMA的人猴同源性88％，筛选交叉结合抗体有一定难度。
针对BCMA的CAR T、双特异性抗体和ADC等治疗疗法取得重要进展，显示出光明前景，但开发新一代更高效的BCMA特异性抗体及基于此的生物治疗产品仍然存在迫切需求。
发明内容
本申请提供了一种特异性结合BCMA的抗体或其抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、宿主细胞、药物组合物、试剂盒、制备方法及其在治疗疾病以及检测BCMA中的应用。
在一方面，本申请提供了一种特异性结合BCMA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CDR1、CDR2和CDR3，所述CDR1包含SEQ ID NO：7～21、 24～31和92～101中任一序列所示的VHH结构域的HCDR1、所述CDR2包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR2，并且所述CDR3包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR3。
在另一方面，本申请提供了一种多特异性抗原结合分子，其中，所述多特异性抗原结合分子包含前述抗体或其抗原结合片段，以及结合BCMA以外其他抗原的抗原结合分子，或结合与前述抗体或其抗原结合片段不同的BCMA表位。
在另一方面，本申请提供了一种分离的核酸片段，其中，所述核酸片段编码前述抗体或其抗原结合片段或前述多特异性抗原结合分子。
在另一方面，本申请提供了一种载体，其中，所述载体包含前述核酸片段。
在另一方面，本申请提供了一种宿主细胞，其中，所述宿主细胞包含前述核酸片段或前述载体。
在另一方面，本申请提供了一种制备前述抗体或其抗原结合片段或前述多特异性抗原结合分子的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
在另一方面，本申请提供了一种药物组合物，其中，所述药物组合物包含前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述核酸片段、前述载体或根据前述方法制备获得的产品。
在另一方面，本申请提供了一种***或癌症的方法，其中，所述方法包括向受试者施用有效量的前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述核酸片段、前述载体、根据前述方法制备获得的产品或前述药物组合物。
在另一方面，本申请提供了前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述核酸片段、前述载体、根据前述方法制备获得的产品或前述药物组合物在制备***或癌症药物中的用途。
在另一方面，本申请提供了前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述核酸片段、前述载体、根据前述方法制备获得的产品或前述药物组合物，用于***或癌症的用途。
在另一方面，本申请提供了一种试剂盒，其中，所述试剂盒包含前述抗体或其抗原结合片段、前述多特异性抗原结合分子、前述核酸片段、前述载体、根据前述方法制备获得的产品或前述药物组合物。
在另一方面，本申请提供了一种检测生物学样品中BCMA表达的方法，其中，所述方法包括在前述抗体或其抗原结合片段与BCMA之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或其抗原结合片段接触。
在另一方面，本申请提供了前述抗体或其抗原结合片段在制备BCMA检测试剂中的用途。
本申请提供了对BCMA靶点具有较高亲和力的抗体或其抗原结合片段，能较好地阻断BCMA与其配体APRIL的结合，从而为BCMA抗体药物和细胞治疗类产品提供较好的选择， 对填补多发性骨髓瘤，特别是复发/难治型多发性骨髓瘤的治疗手段缺乏，具有重要意义。
附图说明
图1A为ELISA检测对照抗体与人BCMA-His蛋白的结合反应；
图1B为ELISA检测对照抗体与猴BCMA-His蛋白的结合反应；
图2A为REGN5459-hIgG1和HPN217-hHcAb抗体检测H929细胞BCMA表达量的FACS结果；
图2B为REGN5459-hIgG1和HPN217-hHcAb抗体检测U266细胞BCMA表达量的FACS结果；
图3为REGN5459-hIgG1抗体检测Flp-inCHO-人BCMA细胞BCMA表达量的FACS结果；
图4为REGN5459-hIgG1抗体检测Flp-inCHO-猴BCMA细胞BCMA表达量的FACS结果；
图5为ELISA检测重组候选抗体与人BCMA-his蛋白的结合反应；
图6为ELISA检测重组候选抗体与猴BCMA-his蛋白的结合反应；
图7为FACS检测重组候选抗体与H929肿瘤细胞的结合反应；
图8为FACS检测重组候选抗体与U266肿瘤细胞的结合反应；
图9为配体结合竞争ELISA检测重组候选抗体对配体APRIL与人BCMA蛋白结合的阻断作用；
图10为ELISA检测人源化候选纳米抗体与人BCMA-his蛋白的结合反应；
图11为ELISA检测人源化候选纳米抗体与猴BCMA-his蛋白的结合反应；
图12为FACS检测人源化候选纳米抗体与H929肿瘤细胞的结合反应；
图13为FACS检测人源化候选纳米抗体与U266肿瘤细胞的结合反应；
图14为配体结合竞争ELISA检测人源化候选纳米抗体对配体APRIL与人BCMA蛋白结合的阻断作用；
图15为ELISA检测嵌合抗体与人BCMA-his蛋白的结合反应；
图16A为FACS检测嵌合抗体与H929肿瘤细胞的结合反应；
图16B为FACS检测嵌合抗体与U266肿瘤细胞的结合反应；
图17为配体结合竞争ELISA检测嵌合抗体对配体APRIL与人BCMA蛋白结合的阻断作用。
发明的详细描述
术语定义和说明
除非本文另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包 括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“BCMA”全称B细胞成熟抗原(B cell maturation antigen)，属于肿瘤坏死因子受体家族成员。BCMA主要表达于晚期B细胞、短寿命增殖浆母细胞和长寿命浆细胞表面，而在初始B细胞、CD34阳性造血干细胞和其他正常组织细胞中不表达，但它在MM细胞中是高度表达的，通过介导下游信号通路，对MM细胞的存活、增殖、转移和耐药中起着关键性的作用，所以BCMA是治疗MM理想的抗原靶点。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10 -6M或更低、10 -7M或更低、约10 -8M或更低、约10 -9M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按照最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer等人，2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque等人，Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
术语“抗体”包括完整抗体及其任何抗原结合片段(即“抗原结合部分”)或单链。“抗体”是指包含通过二硫键互相连接在一起的至少两条重(H)链和两条轻(L)链的糖蛋白，或其抗原结合部分。每条重链由重链可变区(在此缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(在此缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步再分为高变区，称为互补决 定区(CDR)，CDR散布在被称为构架区(FR)的更加保守的区域中。每个VH和VL，均由三个CDR和四个FR组成，它们从氨基端向羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有可与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，该宿主组织或因子包括免疫***的各种细胞(例如效应细胞)和经典补体***的第一成分(C1q)。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文术语“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
本文术语“纳米抗体”是指，克隆重链抗体(骆驼等体内存在的天然缺失轻链的重链抗体)的可变区得到的只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文术语“VHH结构域”和“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“单域抗体”、“VHH结构域”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等人，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews in Molecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO 94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文术语“多特异性”是指抗体或其抗原结合片段结合例如不同抗原或同一抗原上的至少两种不同表位的能力。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体可以结合的不同表位的数目。例如，常规的单特异性IgG型抗体具有两个相同的抗原结合位点(互补位)，因此仅可以结合相同的表位(而不是结合不同的表位)。相比之下，多特异性抗体具有至少两种不同类型的互补位/结合位点，因此可以结合至少两种不同的表位。如本文所述，“互补决定区”是指抗体的抗原结合位点。此外，单个“特异性”可以指单个抗体中的一个、两个、三个或多于三个相同的互补决定区(一个单个抗体分子中的互补决定区/结合位点的实际数量称为“价”)。例如，单个天然IgG抗体是单特异性和二价的，因为它具有两个相同的互补位。相应地，多特异性抗体包含至少两种(不同的)互补决定区/结合位点。因此，术语“多特异性抗体”是指具有多于一个互补位并具有结合两种或多于两种不同表位的能力的抗体。术语“多特异性抗体”特别地包括如上文所定义的双特异性抗体，但是通常还包括蛋白质，例如特别结合三种或多于三种不同的表位的抗体、支架，即具有三种或多于三种互补位/结合位点的抗体。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。示例性地，本文“抗原结合片段”或“抗体片段”包括但不限于Fab、F(ab’)2、Fab’、Fab’-SH、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠、小鼠、兔或羊驼)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985 J Immunol Methods 125:191-202；以上通过援引加入并入本文。
本文术语“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或部分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、提高免疫细胞活性的能力或增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。
本文术语“互补决定区”与“CDR”可互换使用，通常指在轻链和重链可变结构域中均发现的高变区(HVR)。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。重链可变区CDR可缩写为HCDR，轻链可变区可缩写为LCDR。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人，美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人，J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人，Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号***、Chothia编号***或IMGT编号***，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号***”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号***(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号***”通常是指由Chothia等人提出的免疫球蛋白编号***，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号***”通常是指基于由Lefranc等人发起的国际免疫遗传学信息***(The international ImMunoGeneTics information system(IMGT))的编号***，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”选自CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”选自Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区选自恒定κ结构域或恒定λ结构域。
本文中的术语“Fc区”用于定义抗体重链中含有恒定区的至少一部分的C端区域。该术语包括天然序列Fc区和变体Fc区。示例性地，人IgG重链Fc区可自Cys226或Pro230延伸至重链的羧基末端。然而，由宿主细胞生成的抗体可经历翻译后切割，自重链的C端切除一个或多个，特别是一个或两个氨基酸。因此，通过编码全长重链的特定核酸分子的表达由宿主细胞生成的抗体可包括全长重链，或者它可包括全长重链的切割变体。当重链的最终两个C端氨基酸是甘氨酸(G446)和赖氨酸(K447，编号方式依照Kabat EU索引)时可能就是这种情况。因此，Fc区的C端赖氨酸(Lys447)，或C端甘氨酸(Gly446)和赖氨酸(Lys447)可以存在或不存在。典型地，IgG Fc区包含IgG CH2和IgG CH3域，可选地，在此基础上还可包含完整或部分铰链区，但不包含CH1域。人IgG Fc区的“CH2域”通常自约位置231处的氨基酸残基延伸至约位置340处的氨基酸残基。在一个实施方案中，碳水化合物链附着于CH2域。本文中的CH2域可以是天然序列CH2域或变体CH2域。“CH3域”包含Fc区中在CH2域C端的那段残基(即自IgG的约位置341处的氨基酸残基至约位置447处的氨基酸残基)。本文中的CH3区可以是天然序列CH3域或变体CH3域(例如具有在其一条链中引入的“***”“节”，(knob)和在其另一条链中相应引入的“空腔”“穴”，(hole)的CH3域；参见美国专利No.5,821,333，通过援引明确收入本文)。如本文中描述的，此类变体CH3域可用于促进两条不相同抗体重链的异二聚化。
除非本文中另有规定，Fc区或恒定区中的氨基酸残基的编号依照EU编号***，也称作EU索引，如Kabat等人，Sequences of Proteins of Immunological Interest,5 th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991中描述的。
本文术语“Fc变体”是指，通过在Fc上合适的位点处存在一个或者多个氨基酸替换、***或缺失突变引起Fc结构或功能的变化。“Fc变体间作用”指的是经突变设计的Fc变体之间，可以形成空间填充效应、静电导引、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体蛋白。优选的突变设计为“Knob-into-Hole”形式的突变设计。
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(Protein Engineering vol.9 no.7 pp.617-621，1996)提出的“Knob-into-Hole”形式；Amgen公司技术人员利用静电导引(Electrostatic Steering)形成含Fc的异源二聚体形式(US 20100286374A1)；Jonathan H.Davis等人(Protein Engineering，Design&Selection pp.1-8，2010)提出的通过IgG/Ig链交换形成的异源二聚体形式(SEEDbodies)； Genmab公司DuoBody(Science，2007.317(5844))平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(mAbs 3：6，546-557；November/December 2011)；苏州康宁杰瑞公司的基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本发明所述的Fc变体片段上的Knob/Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-into-Hole”形式进行结合。优选用Cater等人的“knob-into-hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“knob-into-hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法，((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的 同一性百分数。
额外地或备选地，可以进一步使用本发明所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人，(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本发明核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本发明蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人，(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5’至3’。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本发明的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP2101823B1)。
本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色***置的染色***置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“药学上可接受的载体”包括任何和所有溶剂、分散介质、包衣材料、表面活性剂、抗氧化剂、防腐剂(例如，抗细菌剂、抗真菌剂)、等渗剂、吸收延迟剂、盐、防腐剂、药物稳定剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料等及其组合，其为本领域技术人员所知(参见例如，Remington's Pharmaceutical Sciences,第18版，MackPrinting Company，1990，第1289-1329页)。除了与活性成分不相容的情况之外，考虑任一常规载体在治疗或药物组合物中的应用。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症和肿瘤。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本文所述的特定疾病或病症的治疗的生物体。示例性地，“受试者”包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
具体实施方式
本申请提供了一种特异性结合BCMA的抗体或其抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、宿主细胞、药物组合物、试剂盒、制备方法及其在治疗疾病以及检测BCMA中的应用。
在第一方面，本申请提供了一种特异性结合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CDR1、CDR2和CDR3，所述CDR1包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR1，所述CDR2包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR2，并且所述CDR3包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR3。
在一些具体的实施方式中，所述HCDR1、HCDR2和HCDR3根据Kabat、IMGT或Chothia编号***确定。
在可选的实施方式中，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：34～91和102～157所表示的氨基酸序列。
在可选的实施方式中，所述HCDR1包含SEQ ID NO：34、37、40、42、45、48、50、52、54、56、58、64、67、71、74、77、81、84、86、87、104、107、113、116、119、121、124、127、131、134、138或141所表示的氨基酸序列。
在可选的实施方式中，所述HCDR2包含SEQ ID NO：35、38、41、43、46、49、59、61、63、65、68、70、72、75、78、79、82、85、88、89、90、91、102、105、108、109、111、114、117、120、122、125、128、129、132、135、136、139、142、143、148、150、152、153、155或157所表示的氨基酸序列。
在可选的实施方式中，所述HCDR3包含SEQ ID NO：36、39、44、47、51、53、55、57、60、62、66、69、73、76、80、83、103、106、110、112、115、118、123、126、130、133、137、140、144、145、146、147、149、151、154或156所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和36；SEQ ID NO：37、38和39；或SEQ ID NO：40、41和36所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、43和44；SEQ ID NO：45、46和47；或SEQ ID NO：48、49和44所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：50、35和51；SEQ ID NO：52、38和53；或SEQ ID NO：54、41和51所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和55；SEQ ID NO：56、38和57；或SEQ ID NO：58、41和55所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、59和60；SEQ ID NO：56、61和62；或SEQ ID NO：58、63和60所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：64、65和66；SEQ ID NO：67、68和69；或SEQ ID NO：58、70和66所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、72和73；SEQ ID NO：74、75和76；或SEQ ID NO：77、78和73所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、79和80；SEQ ID NO：81、82和83；或SEQ ID NO： 84、85和80所表示的氨基酸序列。
在优选的实施方式中，根据Kabat编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：86、35和36所表示的氨基酸序列。
在优选的实施方式中，根据Kabat编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：87、88和44所表示的氨基酸序列。
在优选的实施方式中，根据Kabat编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、88和44所表示的氨基酸序列。
在优选的实施方式中，根据Kabat编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、89和44所表示的氨基酸序列。
在优选的实施方式中，根据Kabat编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、90和44所表示的氨基酸序列。
在优选的实施方式中，根据Kabat编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、91和44所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、102和103；SEQ ID NO：104、105和106；或SEQ ID NO：107、108和103所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、109和110；SEQ ID NO：104、111和112；或SEQ ID NO：107、108和110所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：113、114和115；SEQ ID NO：116、117和118；或SEQ ID NO：119、120和115所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：121、122和123；SEQ ID NO：124、125和126；或SEQ ID NO：127、128和123所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：113、129和130；SEQ ID NO：131、132和133；或SEQ ID NO：134、135和130所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：113、136和137；SEQ ID NO：138、139和140；或SEQ ID NO：141、142和137所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、143和144；SEQ ID NO：37、38和145；或SEQ ID NO：40、41和144所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、143和146；SEQ ID NO：56、38和147；或SEQ ID  NO：58、41和146所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、148和149；SEQ ID NO：37、150和151；或SEQ ID NO：40、152和149所表示的氨基酸序列。
在优选的实施方式中，根据Kabat、IMGT或Chothia编号***，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、153和154；SEQ ID NO：104、155和156；或SEQ ID NO：107、157和154所表示的氨基酸序列。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含在前述HCDR1、HCDR2和/或HCDR3上发生1个、2个或3个突变的氨基酸序列；所述突变选自***、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含与前述HCDR1、HCDR2和/或HCDR3相比具有至少80、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些具体的实施方式中，所述抗体或其抗原结合片段包含单域抗体，所述单域抗体包含前述CDR1、CDR2和CDR3。
在一些具体的实施方式中，所述单域抗体包含选自SEQ ID NO：7～21、24～31和92～101任一序列所示的氨基酸序列。
在可选的实施方式中，所述单域抗体包含与SEQ ID NO：7～21、24～31和92～101任一序列所示的序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列，所述突变选自***、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在可选的实施方式中，所述单域抗体包含与SEQ ID NO：7～21、24～31和92～101任一序列所示的序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些具体的实施方式中，所述抗体包含SEQ ID NO：7～21、24～31和92～101任一序列所示VHH结构域中的FR区。
在可选的实施方式中，所述抗体包含与SEQ ID NO：7～21、24～31和92～101任一序列所示的VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列，所述突变选自***、缺失和/或替换，所述替换优选为保守氨基酸的替换；
在可选的实施方式中，所述抗体包含与SEQ ID NO：7～21、24～31和92～101任一序列所示的VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些具体的实施方式中所述抗体或其抗原结合片段选自：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些具体的实施方式中，所述抗体或其抗原结合片段包含或不包含抗体重链恒定区； 可选地，所述抗体重链恒定区选自人、羊驼、小鼠、大鼠、兔和羊；可选地，所述抗体重链恒定区选自IgG、IgM、IgA、IgE和IgD；可选地，所述IgG选自IgG1，IgG2，IgG3和IgG4；可选地，所述重链恒定区选自Fc区、CH3区和完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述抗体或其抗原结合片段为重链抗体。
在一些具体的实施方式中，所述抗体或其抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药和免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在一些具体的实施方式中，所述抗体或其抗原结合片段还连接有其他功能性分子，优选地，所述其他功能性分子选自以下的一种或多种：信号肽、蛋白标签、细胞因子、血管生成抑制剂和免疫检查点抑制剂。
在第二方面，本申请还提供了一种多特异性抗原结合分子，所述多特异性抗原结合分子包含前述的抗体或其抗原结合片段，以及结合BCMA以外其他抗原的抗原结合分子，或结合与前述抗体或其抗原结合片段不同的BCMA表位；可选地，所述BCMA以外的其他抗原选自：CD3(优选CD3ε)、CD16、CD137、CD258、PD-1、PD-L1、4-1BB、CD40、CD64、EGFR、VEGF、HER2、HER1、HER3、IGF-1R、磷脂酰丝氨酸(Phosphatidylserine，PS)、C-Met、HSA、GPRC5D、MSLN、血脑屏障受体、GPC3、PSMA、CD33、GD2、ROR1、ROR2、FRα和Gucy2C。
在优选的实施方式中，所述其他抗原结合分子为抗体或其抗原结合片段。
在优选的实施方式中，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性。
在优选的实施方式中，所述多特异性抗原结合分子可为二价、三价、四价、五价或六价。
在第三方面，本申请还提供了一种分离的核酸片段，所述核酸片段编码前述抗体或其抗原结合片段或多特异性抗原结合分子。
在第四方面，本申请还提供了一种载体(vector)，所述载体包含前述核酸片段。
在第五方面，本申请还提供了一种宿主细胞，所述宿主细胞包含前述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(例如大肠杆菌)、真菌(例如酵母)、昆虫细胞或哺乳动物细胞(例如CHO细胞系或293T细胞系)。
在第六方面，本申请还提供了一种制备前述抗体或其抗原结合片段或多特异性抗原结合分子的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
在第七方面，本申请还提供了一种药物组合物，所述药物组合物包含前述的抗体或其抗原结合片段、多特异性抗原结合分子、核酸片段、载体或根据前述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
在一些实施方式中，所述药学上可接受的运载体为不减弱免疫细胞活力以及功能、不影响抗体或其抗原结合片段与抗原特异性结合的载体，包括但不限于细胞培养基、缓冲液、生理盐水和平衡盐溶液等。缓冲液的实例包括等渗磷酸盐、醋酸盐、柠檬酸盐、硼酸盐以及碳酸盐等。在具体的实施方式中，所述药学上可接受的运载体为含1％血清的磷酸盐缓冲液。
所述抗肿瘤剂例如为各种化疗药物，例如烷化剂化疗药物，包括但不限于环磷酰胺、异环磷酰胺、苯丙氨酸氮芥、卡莫斯汀、洛莫斯汀、尼莫斯汀、福莫斯汀等；抗代谢类化疗药物，包括但不限于甲氨蝶呤、氟尿嘧啶、替加氟、卡莫氟、脱氧氟尿苷、卡培他滨、吉西他滨、雷替曲塞等；抗癌抗生素，包括但不限于放线菌素D、丝裂霉素、博来霉素、柔红霉素、阿霉素、表阿霉素、吡喃阿霉素等；植物类化疗药物，包括但不限于长春新碱、长春地辛、长春瑞滨、伊立替康、拓扑替康、紫杉醇、多烯紫杉醇等；杂类，包括但不限于达卡巴嗪、顺铂、卡铂、奥沙利铂、奈达铂等。
在第八方面，本申请还提供了一种***或癌症的方法，所述方法包括向受试者施用有效量的前述的抗体或其抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或前述药物组合物；任选地，所述肿瘤或癌症为表达BCMA的肿瘤或癌症，优选为B细胞淋巴瘤，更优选为多发性骨髓瘤(MM)。
在第九方面，本申请还提供了前述抗体或其抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或药物组合物在制备***或前述癌症药物中的用途；任选地，所述肿瘤或癌症为表达BCMA的肿瘤或癌症，优选为B细胞淋巴瘤，更优选为多发性骨髓瘤(MM)。
在第十方面，本申请还提供了前述抗体或其抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或前述药物组合物，用于***或癌症的用途；任选地，所述肿瘤或癌症为表达BCMA的肿瘤或癌症，优选为B细胞淋巴瘤，更优选为多发性骨髓瘤(MM)。
在第十一方面，本申请还提供了一种试剂盒，所述试剂盒包含前述的抗体或其抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或前述药物组合物。
在第十二方面，本申请还提供了一种检测生物学样品中BCMA表达的方法，所述方法包括在前述的抗体或其抗原结合片段与BCMA之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或其抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中BCMA的存在或表达水平。
在第十三方面，本申请还提供了前述抗体或其抗原结合片段在制备BCMA检测试剂中的用途。
实施例
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本文的实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请的技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
实施例1对照抗体制备、内源细胞鉴定和过表达细胞株的制备
(A)对照抗体的制备
REGN5459和HPN217均是识别人BCMA蛋白的抗体，其中REGN5459序列来自美国专利公开号US2020/0024356A1，HPN217序列来自美国专利公开号US20190112381A1。将REGN5459的重链可变区(VH)重组到包含信号肽和人源抗体IgG1重链恒定区的表达载体，轻链可变区(VL)序列重组到包含信号肽和人源抗体IgG1轻链恒定区的表达载体，得到重组质粒，合成的抗体命名为REGN5459-hIgG1。将HPN217的VHH序列重组到包含信号肽和人源抗体IgG1Fc的表达载体中，得重组质粒，合成的抗体命名为HPN217-hHcAb。阴性对照抗体hIgG1为针对Hen Egg Lysozyme鸡卵溶菌酶的抗体anti-hel-hIgG1(购自百英，货号：B117901)，以下简称hIgG1。
表1对照抗体序列表
Figure PCTCN2022136074-appb-000001
注：IgG1 Fc包含C220S突变。
对照抗体与人BCMA-His蛋白(购自Acro，货号：BCA-H522y)和猴BCMA-His蛋白(购自Acro，货号：BCA-C52H7)的结合活性用ELISA进行检测。具体方法为：将抗原蛋白 用PBS稀释到终浓度1μg/mL，然后以50μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS洗板2次，加入封闭液[PBS+2％(w/v)BSA]室温封闭2小时。倒掉封闭液，加入100nM梯度稀释的对照抗体或阴性对照抗体50μl每孔。37℃孵育2小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Merck，货号：AP113P)，37℃孵育1小时后，用PBS洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值。结果如表2、3和图1A、1B所示，REGN5459-hIgG1抗体与人BCMA蛋白和猴BCMA蛋白均有很好的结合活性，HPN217-hHcAb与人BCMA蛋白有很好的结合活性，但与猴BCMA蛋白不结合。
表2 ELISA检测对照抗体与人BCMA-his蛋白的结合反应
Figure PCTCN2022136074-appb-000002
表3 ELISA检测对照抗体与猴BCMA-his蛋白的结合反应
Figure PCTCN2022136074-appb-000003
(B)内源性表达BCMA蛋白的细胞株鉴定
将H929细胞和U266细胞在T-75细胞培养瓶中扩大培养至对数生长期，吹打细胞至单细胞悬液。细胞计数后，离心，将细胞沉淀用FACS缓冲液(PBS+2％胎牛血清)重悬至2×10 6细胞每毫升，按每孔50μl加入到96孔FACS反应板中，用REGN5459-hIgG1和HPN217-hHcAb抗体作为一抗，APC标记的山羊抗人IgG(H+L)二抗(购自Jackson，货号：109-605-088)经FACS(FACS CantoTM，购自BD公司)检测和分析。结果如表4以及图2A和2B所示，说明H929细胞和U266细胞与REGN5459-hIgG1和HPN217-hHcAb抗体均可结合。
表4 内源细胞系H929和U266的FACS检测结果
Figure PCTCN2022136074-appb-000004
(C)人BCMA蛋白和猴BCMA蛋白的Flp-inCHO重组细胞株的制备
将编码人BCMA和猴BCMA氨基酸序列的核苷酸序列分别克隆到pcDNA5/FRT载体(购自Clontech)。人BCMA NCBI Gene ID：608，猴BCMA NCBI Gene ID：712212。分别对Flp-inCHO细胞系(购自中国科学院典型培养物保藏委员会细胞库)进行转染(
Figure PCTCN2022136074-appb-000005
3000 Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含600μg/ml  hygromycin(ThermoFisher,货号：10687010)的含10％(v/v)胎牛血清(ExCell Bio，货号：FND500)的F12K Medium(Gibco,货号：21127030)培养基中选择性培养2周，用REGN5459-hIgG1和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)在流式细胞仪FACS CantoII(购自BD Biosciences)上进行检测，对表达量高且峰形单一的细胞进行扩增，对扩增后的细胞经流式细胞分析法进行复测。选择长势较好、荧光强度较高、均一性较好的阳性细胞群继续扩大培养并液氮冻存。表5说明，已经分别制得人BCMA阳性表达的Flp-inCHO高表达细胞群和猴BCMA阳性表达的Flp-inCHO高表达细胞群。图3和图4中，横坐标为细胞荧光强度，纵坐标为细胞数。
表5表达人BCMA蛋白的Flp-inCHO重组细胞系FACS检测结果
Figure PCTCN2022136074-appb-000006
实施例2针对BCMA的羊驼纳米抗体的制备
(A)动物免疫和血清效价测定
免疫动物为1只羊驼(Alpaca，编号为NB254)，免疫前预先取血10mL为阴性对照，初免采用0.5mg人BCMA(Met1-Ala54)-hFc蛋白(购自Acro，货号：BC7-H5254)与弗氏完全佐剂(CFA)1mL混匀后皮下注射；第21天进行二免，将0.25mg人BCMA-hFc蛋白与弗氏不完全佐剂(IFA)1mL混匀后皮下注射；第42天进行三免，将人BCMA-hFc蛋白和食蟹猴BCMA(Met1-Ala53)-hFc蛋白(购自Acro，货号：BCA-C5253)各0.25mg与IFA 1mL混匀后皮下注射；此后每隔三周按照三免时的方法加强免疫一次。
每次加强免疫1周后采血，用ELISA检测血清抗体效价和特异性，结果如表6所示。说明经人BCMA-hFc和食蟹猴BCMA-hFc蛋白免疫后的羊驼的血清对免疫原均有不同程度的结合，呈现抗原抗体反应。其中空白对照为1％(w/v)BSA，其中四免和五免分别指第四次和第五次免疫后第七天的羊驼血清，表中的数据为OD450nm值，k表示1000。
表6 ELISA检测蛋白免疫后的羊驼血清抗体效价
Figure PCTCN2022136074-appb-000007
(B)噬菌体文库构建
将采集的四免和五免之后的50mL外周血，按照淋巴细胞分离液使用说明分离PBMC。用RNAiso Plus试剂提取NB254四免和五免PBMC总RNA。采用PrimeScript TMII 1st Strand  cDNA Synthesis Kit(Takara，货号：6210A)试剂盒逆转录，按照说明逆转录制备cDNA，共转录5μg RNA。将四免和五免cDNA原液等比例混合后稀释5倍进行巢式扩增，割胶回收VHH片段。载体与目的片段分别用SfiI进行50℃过夜酶切后回收。载体与目的片段以1:3比例连接。对连接产物进行10次电击转化，电击之后立即向电击杯中加入1mL 2YT培养基(37℃预热)复苏，吸出电击产物并用2YT培养基洗净电击杯，37℃180rpm复苏45min，取100μL梯度稀释至10 -3和10 -4测定库转化子数目，涂布于90mm的平板上，其余离心，加入8mL 2YT培养基重悬，涂布于8块200mm的平板上。第二天计数计算库容为2.14×10 9。随机挑取48个克隆培养，PCR检测其***率为100％。
将大肠杆菌文库接入2×300mL 2YT(Amp:100μg/ml、Glu:1％)培养基中至其初始OD600＝0.1-0.2,37℃、230rpm培养至OD600＝0.8以上。依据OD600计算添加的辅助噬菌体M13K07的量(M13K07:大肠杆菌＝20:1)，37℃静置侵染30min后37℃180rpm培养30min。5000rpm离心去上清，更换2YT(100μg/mL Amp+50μg/mL Kan)培养基于30℃，220rpm过夜培养。依据NEB官网流程沉淀噬菌体文库，并测定其滴度。
(C)针对BCMA纳米抗体淘选
微孔板包被人BCMA-hFc蛋白0.5μg/孔，共8个孔；噬菌体文库的投入量为2.2×10 11，测得一轮洗脱产物滴度为1.85×10 7。经ELISA鉴定一轮淘选产物与人BCMA蛋白结合的阳性率为89.58％，与猴BCMA蛋白结合的阳性率为4.2％，与人、猴BCMA蛋白交叉结合的阳性率为4.2％。按照常规流程扩增一轮淘选洗脱产物，测定其扩增噬菌体的滴度为1.14×10 13。二轮淘选微孔板包被猴BCMA蛋白0.2μg/孔，共4个孔；噬菌体文库投入量为2.2×10 11，测得洗脱产物滴度为1.32×10 7。经ELISA鉴定二轮淘选产物与人BCMA蛋白结合的阳性率为92.1％，与猴BCMA蛋白结合的阳性率为39.3％，与人、猴BCMA蛋白交叉结合的阳性率为39.3％。
(D)测序
经淘选共得到人猴交叉阳性克隆70个，将阳性克隆送测序分析，根据VHH编码蛋白序列构建进化树序列，经聚类分析发现70条序列所属3个种系，分别挑取Lab01、02、03、04、05、06、07和08号共8个克隆构建、表达、鉴定。
实施例3重组候选抗体构建、表达
将目标的VHH序列重组到人IgG1Fc的表达载体中，得到重组质粒(人IgG1Fc的序列参见SEQ ID NO:4)。将重组候选抗体质粒和转染试剂PEI(购自Polysciences，货号：24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293F细胞(厂家：Thermofisher，货号：A14527)中，放入5％CO 2，120rpm，37℃摇床培养。转染第二天，加入OPM-293 ProFeed(上海奥浦迈，货号：F081918-001)和6g/L葡萄糖(厂家：Sigma，货号:G7528)。转染第六天，收集细胞上清。收集培养上清，通过Protein A亲和进行纯化，所用填料为Mabselect SuRe(编号10054120，购自GE Healthcare)，缓冲液为Mab亲和平衡液(PBS Buffer)、Mab亲和淋洗液(含1M NaCl的PBS缓冲液)以及Mab亲和洗脱液(柠檬酸盐缓冲液)。用超微量分光光度计(Nanodrop8000，Thermo)进行浓度测定。应用SEC-HPLC法分析待测蛋白样品以表征蛋白的分子大小均一性和纯度。所用的HPLC为Agilent  1260，色谱柱TSKgel G3000SWXL来自Tosoh Bioscience，流动相为200mM磷酸缓冲液，pH 7.0/异丙醇(v/v 9:1)(批号：20210519001)，检测温度为25℃，流速为0.5mL/min，检测波长为280nm。SEC-HPLC数据采用手动积分法分析色谱图，按照面积归一法计算蛋白纯度，主峰认为是单体，主峰前的色谱峰称为聚集体，主峰后的色谱峰称为碎裂体。结果如表7所示，候选抗体最终产品纯度较高。
表7 BCMA VHH-hFc候选抗体表达量和SEC纯度结果
Figure PCTCN2022136074-appb-000008
实施例4重组候选抗体的鉴定
(A)酶联免疫吸附实验(ELISA)检测BCMA重组候选抗体与人BCMA蛋白的结合以及与猴BCMA蛋白的交叉结合
具体方法为：将人BCMA蛋白(购自Acro，货号：BCA-H522y)和猴BCMA蛋白(购自Acro，货号：BCA-C52H7)分别用PBS稀释到终浓度1μg/mL，然后以50μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS洗板2次，加入封闭液[PBS+2％(w/v)BSA]室温封闭2小时。倒掉封闭液，PBS洗板3次，加入100nM梯度稀释的BCMA纳米重组嵌合抗体或阴性对照抗体50μl每孔。37℃孵育2小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Merck，货号：AP113P)，37℃孵育1小时后，用PBS洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值。结果如图5、图6所示，所有抗体与均人BCMA蛋白结合，BCMA-Lab06和BCMA-Lab07与猴BCMA蛋白没有交叉结合活性，其他重组候选抗体均与猴BCMA蛋白具有交叉结合活性，IgG亚型对照为人IgG1。
(B)流式细胞实验(FACS)检测BCMA重组候选抗体与表达BCMA的内源性细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，吹打细胞至单细胞悬液。细胞计数后，离心，将细胞沉淀用FACS缓冲液(PBS+2％胎牛血清)重悬至2×10 6细胞每毫升，按每孔50μl加入到96孔FACS反应板中，加入候选抗体待测样品每孔50μl，4℃孵育1小时。用PBS缓冲液离心洗涤3次，加入每孔50μl二抗anti-hIgG(Fc)Alexa 647(购自Jackson，货号：109-605-098)，冰上孵育1小时。用PBS缓冲液离心洗涤3次，100μl用FACS(FACS CantoTM，购自BD公司)检测和分析结果。通过软件(FlowJo)进行数据分析，得到细胞的平均荧光强度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50。结果如表8和图7、图8所示，BCMA-Lab05、BCMA-Lab06、BCMA-Lab08与H929和U266细胞结合较差，其余抗体表现较好。
表8 FACS检测重组候选抗体与H929和U266细胞结合反应
Figure PCTCN2022136074-appb-000009
(C)配体结合竞争ELISA检测重组候选抗体对配体APRIL与人BCMA蛋白结合的阻断作用
将抗原蛋白用PBS稀释到终浓度1μg/mL，然后以50μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBS洗板2次，加入封闭液[PBS+2％(w/v)BSA]室温封闭2小时。倒掉封闭液，PBS洗板3次，加入200nM梯度稀释的BCMA纳米重组嵌合抗体或阴性对照抗体，50μl每孔。将hAPRIL-biotin(购自Acro，货号：APL-H82F5)稀释至0.5μg/mL，50μl每孔。37℃孵育1.5小时后，用PBS洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma，货号：S2438-250μg)，37℃孵育1小时后，用PBS洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值。结果如图9所示，BCMA-Lab05、BCMA-Lab06和BCMA-Lab08阻断效果一般，其余抗体阻断效果良好。
实施例5 BCMA重组候选抗体的亲和力检测
使用Protein A芯片(GE Healthcare；29-127-558)捕获抗BCMA重组候选抗体。样品和运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃。样品块设置为16℃。两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的BCMA抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Healthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的人BCMA-His持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1:1稀释，总共5 个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5)以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异。也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1:1模型。重组候选抗体与人BCMA蛋白的结合速率(K a)、解离速率(K dis)及结合亲和力(KD)如表所示，其中REGN5459-hIgG1抗体作为对照。如表9所示，重组候选抗体与人BCMA蛋白的KD值均在1E-7M以下。
表9重组候选抗体与人BCMA蛋白的结合亲和力
Figure PCTCN2022136074-appb-000010
经一系列实验筛选后，选取BCMA-Lab01和BCMA-Lab02羊驼抗体进行后续人源化改造。BCMA-Lab01～BCMA-Lab08羊驼抗体序列如表10所示，通过Kabat、IMGT、Chothia方式分析的CDR序列如表11所示。
表10 BCMA羊驼抗体氨基酸序列
Figure PCTCN2022136074-appb-000011
表11 Kabat、IMGT、Chothia编号分析CDR序列
Figure PCTCN2022136074-appb-000012
Figure PCTCN2022136074-appb-000013
实施例6纳米抗体的人源化
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选与VHH纳米抗体同源性高的重链可变区种系基因作为模板，将VHH纳米抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中关键氨基酸回复突变为VHH纳米抗体对应的氨基酸，以保证原有的亲和力，即得到人源化单克隆抗体。其中抗体的CDR氨基酸残基由Kabat编号***确定并注释。
(A)BCMA-Lab01抗体的人源化
VHH纳米抗体BCMA-Lab01人源化重链模板为IGHV3-64*04和IGHJ3*01，将VHH纳米抗体BCMA-Lab01的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将BCMA-Lab01的人源化抗体的FR区序列中关键氨基酸进行回复突变为VHH纳米抗体对应的氨基酸，以保证原有的亲和力，抗体存在易发生化学修饰的位点，对这些位点进行点突变已消除修饰风险。具体突变设计见表12。
表12 BCMA-Lab01的人源化抗体突变设计
Figure PCTCN2022136074-appb-000014
注：Graft代表将VHH纳米抗体CDR植入人种系模板FR区序列；H35G表示将Graft第35位H突变成G，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
BCMA-Lab01人源化抗体可变区具体序列如下：
BCMA-Lab01-VHH1氨基酸序列如SEQ ID NO：15所示：
Figure PCTCN2022136074-appb-000015
BCMA-Lab01-VHH2氨基酸序列如SEQ ID NO：16所示：
Figure PCTCN2022136074-appb-000016
BCMA-Lab01-VHH3氨基酸序列如SEQ ID NO：17所示：
Figure PCTCN2022136074-appb-000017
BCMA-Lab01-VHH4氨基酸序列如SEQ ID NO：18所示：
Figure PCTCN2022136074-appb-000018
BCMA-Lab01-VHH5氨基酸序列如SEQ ID NO：19所示：
Figure PCTCN2022136074-appb-000019
BCMA-Lab01-VHH6氨基酸序列如SEQ ID NO：20所示：
Figure PCTCN2022136074-appb-000020
BCMA-Lab01-VHH7氨基酸序列如SEQ ID NO：21所示：
Figure PCTCN2022136074-appb-000021
人源化重链模板IGHV3-64*04氨基酸序列如SEQ ID NO：22所示：
Figure PCTCN2022136074-appb-000022
人源化重链模板IGHJ3*01氨基酸序列如SEQ ID NO：23所示：
WGQGTMVTVSS
根据Kabat编号***，上述人源化纳米抗体VHH序列分析结果如表13所示。
表13 BCMA-Lab01人源化纳米抗体VHH序列的Kabat分析结果
Figure PCTCN2022136074-appb-000023
(B)BCMA-Lab02抗体的人源化
VHH单域抗体BCMA-Lab02人源化重链模板为IGHV3-7*01和IGHJ6*01，将VHH单域抗体BCMA-Lab02的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将BCMA-Lab02的人源化抗体的FR区序列中关键氨基酸进行回复突变为VHH单域抗 体对应的氨基酸，以保证原有的亲和力，抗体存在易发生化学修饰的位点，对这些位点进行点突变已消除修饰风险。具体突变设计见表14。
表14 BCMA-Lab02的人源化抗体突变设计
Figure PCTCN2022136074-appb-000024
注：Graft代表将VHH单域抗体CDR植入人种系模板FR区序列；W47L表示将Graft第47位W突变成L，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
BCMA-Lab02人源化抗体可变区具体序列如下：
BCMA-Lab02-VHH1氨基酸序列如SEQ ID NO：24所示：
Figure PCTCN2022136074-appb-000025
BCMA-Lab02-VHH2氨基酸序列如SEQ ID NO：25所示：
Figure PCTCN2022136074-appb-000026
BCMA-Lab02-VHH3氨基酸序列如SEQ ID NO：26所示：
Figure PCTCN2022136074-appb-000027
BCMA-Lab02-VHH4氨基酸序列如SEQ ID NO：27所示：
Figure PCTCN2022136074-appb-000028
BCMA-Lab02-VHH5氨基酸序列如SEQ ID NO：28所示：
Figure PCTCN2022136074-appb-000029
BCMA-Lab02-VHH6氨基酸序列如SEQ ID NO：29所示：
Figure PCTCN2022136074-appb-000030
Figure PCTCN2022136074-appb-000031
BCMA-Lab02-VHH7氨基酸序列如SEQ ID NO：30所示：
Figure PCTCN2022136074-appb-000032
BCMA-Lab02-VHH8氨基酸序列如SEQ ID NO：31所示：
Figure PCTCN2022136074-appb-000033
人源化重链模板IGHV3-7*01氨基酸序列如SEQ ID NO：32所示：
Figure PCTCN2022136074-appb-000034
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：33所示：
WGQGTTVTVSS
根据Kabat编号***，上述人源化单域抗体VHH序列分析结果如表15所示。
表15 BCMA-Lab02人源化单域抗体VHH序列的Kabat分析结果
Figure PCTCN2022136074-appb-000035
实施例7人源化候选纳米抗体的构建、表达和纯度测定
具体操作方法参见实施例3，将目标的人源化VHH序列重组到人IgG1Fc的表达载体中，得到重组质粒(人IgG1Fc的序列参见SEQ ID NO:4)。将人源化VHH-Fc嵌合抗体表达收集得到的细胞上清，通过SEC-HPLC对得到的抗体进行纯度定性分析，结果如表16所示，其中“/”表示未进行检测。
表16 BCMA人源化候选纳米抗体表达量和SEC纯度结果
Figure PCTCN2022136074-appb-000036
Figure PCTCN2022136074-appb-000037
实施例8 BCMA人源化候选纳米抗体的鉴定
(A)酶联免疫吸附实验(ELISA)检测BCMA人源化候选纳米抗体与人BCMA蛋白的结合以及与猴BCMA蛋白的交叉结合
具体方法同实施例4(A)，结果如图10、图11所示，BCMA-Lab02-VHH1和BCMA-Lab02-VHH2与人BCMA和猴BCMA蛋白均不结合，其余人源化候选纳米抗体与人BCMA均结合良好，与猴BCMA蛋白均有交叉结合活性，IgG亚型对照为人IgG1。
(B)流式细胞实验(FACS)检测人源化候选纳米抗体与表达BCMA的内源性细胞的结合
具体方法同实施例4(B)，结果如表17和图12、图13所示，BCMA-Lab02-VHH1和BCMA-Lab02-VHH2与H929和U266细胞不结合，其余人源化候选纳米抗体与H929和U266细胞结合良好。
表17 FACS检测人源化候选纳米抗体H929与U266细胞结合反应
Figure PCTCN2022136074-appb-000038
Figure PCTCN2022136074-appb-000039
(C)配体结合竞争ELISA检测人源化候选纳米抗体对配体APRIL与人BCMA蛋白结合的阻断作用
具体方法同实施例4(C)，结果如图14所示，BCMA-Lab02-VHH1和BCMA-Lab02-VHH2不能阻断配体APRIL与人BCMA蛋白的结合，BCMA-Lab01VHH1和BCMA-Lab01VHH2对配体APRIL与人BCMA蛋白结合的阻断作用较差，其余人源化候选纳米抗体的阻断作用良好。
实施例9 BCMA人源化候选纳米抗体的亲和力检测
具体操作方法参见实施例5。人源化候选纳米抗体与人BCMA蛋白的结合速率(Ka)、解离速率(Kdis)及结合亲和力(KD)如表18所示，其中REGN5459-hIgG1抗体作为对照。如表18所示，人源化候选纳米抗体与人BCMA蛋白的KD值均在1E-8M以下。
表18人源化候选纳米抗体与人BCMA蛋白的结合亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
BCMA-Lab01-VHH1 1.19E+06 8.41E-03 7.07E-09
BCMA-Lab01-VHH2 1.04E+06 3.61E-03 3.48E-09
BCMA-Lab01-VHH3 1.15E+06 1.38E-03 1.20E-09
BCMA-Lab01-VHH4 1.14E+06 1.37E-03 1.20E-09
BCMA-Lab01-VHH5 1.20E+06 1.41E-03 1.17E-09
BCMA-Lab01-VHH6 1.31E+06 8.02E-04 6.13E-10
BCMA-Lab01-VHH7 1.11E+06 1.47E-03 1.32E-09
BCMA-Lab01 1.28E+06 6.81E-04 5.33E-10
BCMA-Lab02-VHH4 9.37E+05 1.37E-04 1.46E-10
BCMA-Lab02-VHH5 7.84E+05 1.03E-04 1.32E-10
BCMA-Lab02-VHH7 8.05E+05 7.10E-05 8.82E-11
BCMA-Lab02 7.38E+05 4.47E-05 6.06E-11
REGN5459-hIgG1 7.81E+05 1.62E-04 2.08E-10
实施例10针对BCMA的纳米抗体淘选与重组候选抗体鉴定
(A)针对BCMA的纳米抗体淘选
根据实施例2(C)的方法，进一步淘选结合人BCMA的阳性克隆，挑取Lab09～18共10个克隆构建、表达、鉴定。
(B)重组候选抗体构建、表达
根据实施例3中的方法，得到VHH-IgG1的重组候选抗体，同时经SEC-HPLC鉴定，候选抗体最终产品纯度均较高。
(C)ELISA检测BCMA重组候选抗体与人BCMA蛋白的结合
参考实施例4(A)中的方法，使用ELISA检测重组候选抗体和人BCMA的结合，结果如图15所示。图15显示，所有抗体与均人BCMA蛋白结合，且与阳性对照抗体HPN217-hHcAb相当。
(D)FACS检测BCMA重组候选抗体与表达BCMA的内源性细胞的结合
根据实施例4(B)中的方法，使用FACS鉴定重组候选抗体与表达BCMA的内源细胞的结合，结果如图16A、16B和表19所示。结果显示，除BCMA-Lab09与H929结合较弱、与U266不结合外，其余抗体与H929和U266细胞的结合均较强，强于阳性对照抗体或与阳性对照抗体相当。
表19 FACS检测重组候选抗体与H929和U266细胞结合反应
Figure PCTCN2022136074-appb-000040
(E)配体结合竞争ELISA检测重组候选抗体对配体APRIL与人BCMA蛋白结合的阻断作用
根据实施例4(C)中的方法，使用配体竞争ELISA检测重组抗体对配体APRIL与人BCMA蛋白结合的阻断作用，结果如图17所示。结果显示，除BCMA-Lab09不能阻断配体APRIL与人BCMA蛋白结合外，其余抗体均能较好的阻断配体APRIL与人BCMA蛋白结合，且与阳性对照抗体相当。
实施例11 BCMA重组候选抗体的亲和力检测
根据实施例5中的方法，使用Biacore检测重组抗体与人BCMA蛋白的亲和力，结果如表20所示。结果显示，所有抗体与人BCMA蛋白亲和力均较强。纳米抗体的序列如表21所示，其CDR序列如表22所示。
表20嵌合抗体与人BCMA蛋白的结合亲和力
抗体名称 ka(1/Ms) kd(1/s) KD(M)
BCMA-Lab09 5.00E+04 3.99E-03 7.97E-08
BCMA-Lab10 8.02E+06 5.51E-03 6.87E-10
BCMA-Lab11 5.83E+06 1.26E-03 2.17E-10
BCMA-Lab12 6.92E+05 1.03E-02 1.49E-08
BCMA-Lab13 5.78E+06 1.71E-04 2.97E-11
BCMA-Lab14 4.43E+06 2.48E-03 5.60E-10
BCMA-Lab15 2.47E+06 2.19E-03 8.87E-10
BCMA-Lab16 2.82E+06 1.57E-03 5.58E-10
BCMA-Lab17 4.15E+06 1.76E-02 4.25E-09
BCMA-Lab18 2.04E+06 2.06E-03 1.01E-09
HPN217-hHcAb 2.64E+06 3.10E-03 1.17E-09
REGN5459-hIgG1 7.39E+05 1.97E-04 2.67E-10
表21 BCMA重组纳米抗体序列表
Figure PCTCN2022136074-appb-000041
表22 Kabat、IMGT、Chothia编号分析CDR序列
Figure PCTCN2022136074-appb-000042
Figure PCTCN2022136074-appb-000043

Claims (22)

  1. 一种特异性结合BCMA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CDR1、CDR2和CDR3，所述CDR1包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR1、所述CDR2包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR2，并且所述CDR3包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域的HCDR3。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中所述HCDR1、HCDR2和HCDR3根据Kabat、IMGT或Chothia编号***确定；
    可选地，所述HCDR1包含SEQ ID NO：34、37、40、42、45、48、50、52、54、56、58、64、67、71、74、77、81、84、86、87、104、107、113、116、119、121、124、127、131、134、138或141所表示的氨基酸序列；
    可选地，所述HCDR2包含SEQ ID NO：35、38、41、43、46、49、59、61、63、65、68、70、72、75、78、79、82、85、88、89、90、91、102、105、108、109、111、114、117、120、122、125、128、129、132、135、136、139、142、143、148、150、152、153、155或157所表示的氨基酸序列；
    可选地，所述HCDR3包含SEQ ID NO：36、39、44、47、51、53、55、57、60、62、66、69、73、76、80、83、103、106、110、112、115、118、123、126、130、133、137、140、144、145、146、147、149、151、154或156所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和36；SEQ ID NO：37、38和39；或SEQ ID NO：40、41和36所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、43和44；SEQ ID NO：45、46和47；或SEQ ID NO：48、49和44所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别选自SEQ ID NO：50、35和51；SEQ ID NO：52、38和53；或SEQ ID NO：54、41和51所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、35和55；SEQ ID NO：56、38和57；或SEQ ID NO：58、41和55所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、59和60；SEQ ID NO：56、61和62；或SEQ ID NO：58、63和60所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：64、65和66；SEQ ID NO：67、68和69；或SEQ ID NO：58、70和66所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、72和73；SEQ ID NO：74、75和76；或SEQ ID NO：77、78和73所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、79和80；SEQ ID NO：81、82和83；或SEQ ID NO：84、85和80所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：86、35和36所表示 的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：87、88和44所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、88和44所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、89和44所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、90和44所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：42、91和44所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、102和103；SEQ ID NO：104、105和106；或SEQ ID NO：107、108和103所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、109和110；SEQ ID NO：104、111和112；或SEQ ID NO：107、108和110所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：113、114和115；SEQ ID NO：116、117和118；或SEQ ID NO：119、120和115所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：121、122和123；SEQ ID NO：124、125和126；或SEQ ID NO：127、128和123所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：113、129和130；SEQ ID NO：131、132和133；或SEQ ID NO：134、135和130所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：113、136和137；SEQ ID NO：138、139和140；或SEQ ID NO：141、142和137所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、143和144；SEQ ID NO：37、38和145；或SEQ ID NO：40、41和144所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、143和146；SEQ ID NO：56、38和147或SEQ ID NO：58、41和146所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：34、148和149；SEQ ID NO：37、150和151；或SEQ ID NO：40、152和149所表示的氨基酸序列；
    优选地，所述HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：71、153和154；SEQ ID NO：104、155和156；或SEQ ID NO：107、157和154所表示的氨基酸序列。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其中，所述CDR1、CDR2和/或CDR3包含在权利要求1或2所述HCDR1、HCDR2和/或HCDR3上发生1个、2个或3个突变的氨基酸序列；所述突变选自***、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  4. 根据权利要求1或2所述的抗体或其抗原结合片段，其中，所述CDR1、CDR2和/或CDR3包含与前述HCDR1、HCDR2和/或HCDR3相比具有至少80、85％、90％、91％、92％、 93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
  5. 根据权利要求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包含单域抗体，所述单域抗体包含权利要求1～4任一项所述CDR1、CDR2和CDR3；
    优选地，所述单域抗体包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的氨基酸序列；
    可选地，所述单域抗体包含与SEQ ID NO：7～21、24～31和92～101中任一序列所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列，所述突变选自***、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    可选地，所述单域抗体包含与SEQ ID NO：7～21、24～31和92～101中任一序列所示的氨基酸序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
  6. 根据权利要求1～5任一项所述的抗体或其抗原结合片段，其中，所述抗体包含SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域中的FR区；
    可选地，所述抗体包含与SEQ ID NO：7～21、24～31和92～101中任一序列所示VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的氨基酸序列，所述突变选自***、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    可选地，所述抗体包含与SEQ ID NO：7～21、24～31和92～101中任一序列所示的VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
  7. 根据权利要求1～6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：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
  8. 根据权利要求1～7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包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区选自人、羊驼、小鼠、大鼠、兔和羊；可选地，所述抗体重链恒定区选自IgG、IgM、IgA、IgE和IgD；可选地，所述IgG选自IgG1，IgG2，IgG3和IgG4；可选地，所述重链恒定区选自Fc区、CH3区和完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述抗体或其抗原结合片段为重链抗体。
  9. 根据权利要求1～8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还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药和免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  10. 根据权利要求1～9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还连接有其他功能性分子，优选地，所述其他功能性分子选自以下的一种或多种：信号肽、蛋白标签、细胞因子、血管生成抑制剂和免疫检查点抑制剂。
  11. 一种多特异性抗原结合分子，其中，所述多特异性抗原结合分子包含权利要求1～10任一项所述的抗体或其抗原结合片段，以及结合BCMA以外其他抗原的抗原结合分子，或结合与权利要求1～10任一项所述的抗体或其抗原结合片段不同的BCMA表位；可选地，所述BCMA以外的其他抗原选自：CD3(优选CD3ε)、CD16、CD137、CD258、PD-1、PD-L1、4-1BB、CD40、CD64、EGFR、VEGF、HER2、HER1、HER3、IGF-1R、磷脂酰丝氨酸(Phosphatidylserine，PS)、C-Met、HSA、GPRC5D、MSLN、血脑屏障受体、GPC3，PSMA，CD33，GD2，ROR1，ROR2，FRα和Gucy2C；
    优选地，所述其他抗原结合分子为抗体或其抗原结合片段；
    优选地，所述多特异性抗原结合分子为双特异性、三特异性或四特异性；
    优选地，所述多特异性抗原结合分子为二价、三价、四价、五价或六价。
  12. 一种分离的核酸片段，其中，所述核酸片段编码权利要求1～10任一项所述的抗体或其抗原结合片段或权利要求11所述的多特异性抗原结合分子。
  13. 一种载体，其中，所述载体包含权利要求12所述的核酸片段。
  14. 一种宿主细胞，其中，所述宿主细胞包含权利要求12所述的核酸片段或权利要求13所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(例如大肠杆菌)、真菌(例如酵母)、昆虫细胞或哺乳动物细胞(例如CHO细胞系或293T细胞系)。
  15. 一种制备权利要求1～10所述的抗体或其抗原结合片段或权利要求11所述的多特异性抗原结合分子的方法，所述方法包括培养权利要求14所述的细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
  16. 一种药物组合物，其中，所述药物组合物包含权利要求1～10所述的抗体或其抗原结合片段、权利要求11所述的多特异性抗原结合分子、权利要求12所述的核酸片段、权利要求13所述的载体或根据权利要求15所述的方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
  17. 一种***或癌症的方法，其中，所述方法包括向受试者施用有效量的权利要求1～10所述的抗体或其抗原结合片段、权利要求11所述的多特异性抗原结合分子、权利要求12所述的核酸片段、权利要求13所述的载体、根据权利要求15所述的方法制备获得的产品或权利要求16所述的药物组合物；任选地，所述肿瘤或癌症为表达BCMA的肿瘤或癌症，优选地，所述肿瘤或癌症为B细胞淋巴瘤；更优选为多发性骨髓瘤(MM)。
  18. 权利要求1～10所述的抗体或其抗原结合片段、权利要求11所述的多特异性抗原结合分子、权利要求12所述的核酸片段、权利要求13所述的载体、根据权利要求15所述的方法制备获得的产品或权利要求16所述的药物组合物在制备***或癌症药物中的用途；任选地，所述肿瘤或癌症为表达BCMA的肿瘤或癌症，优选地，所述肿瘤或癌症为B细胞淋巴瘤；更优选为多发性骨髓瘤(MM)。
  19. 权利要求1～10所述的抗体或其抗原结合片段、权利要求11所述的多特异性抗原结合分子、权利要求12所述的核酸片段、权利要求13所述的载体、根据权利要求15所述的方法制备获得的产品或权利要求16所述的药物组合物，用于***或癌症的用途；任选地， 所述肿瘤或癌症为表达BCMA的肿瘤或癌症，优选地，所述肿瘤或癌症为B细胞淋巴瘤；更优选为多发性骨髓瘤(MM)。
  20. 一种试剂盒，其中，所述试剂盒包含权利要求1～10所述的抗体或其抗原结合片段、权利要求11所述的多特异性抗原结合分子、权利要求12所述的核酸片段、权利要求13所述的载体、根据权利要求15所述的方法制备获得的产品或权利要求16所述的药物组合物。
  21. 一种检测生物学样品中BCMA表达的方法，其中，所述方法包括在权利要求1～10任一项所述的抗体或其抗原结合片段与BCMA之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或其抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中BCMA的存在或表达水平。
  22. 权利要求1～10任一项所述的抗体或其抗原结合片段在制备BCMA检测试剂中的用途。
PCT/CN2022/136074 2021-12-03 2022-12-02 抗bcma纳米抗体及其应用 WO2023098846A1 (zh)

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