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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French (fr)
Chinese (zh)
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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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Jiangsu Simcere Pharmaceutical Co Ltd
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Jiangsu Simcere Pharmaceutical Co Ltd
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Priority to JP2024533063A priority Critical patent/JP2024541668A/ja
Priority to CN202280078809.9A priority patent/CN118696058A/zh
Priority to EP22900650.7A priority patent/EP4428156A4/en
Priority to US18/715,498 priority patent/US20250026844A1/en
Publication of WO2023098846A1 publication Critical patent/WO2023098846A1/zh
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    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117534763A (zh) * 2023-12-28 2024-02-09 康维众和(中山)生物药业有限公司 抗bcma的纳米抗体及其制备方法与应用
WO2025077315A1 (zh) * 2023-10-12 2025-04-17 浙江康佰裕生物科技有限公司 抗bcma单域抗体及其制备方法与应用
CN120329445A (zh) * 2025-06-20 2025-07-18 北京大学第一医院(北京大学第一临床医学院) 靶向bcma的单域抗体及其相关材料与应用
WO2026072709A1 (en) 2024-09-25 2026-04-02 Orna Therapeutics, Inc. Chimeric antigen receptors targeting bcma

Citations (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1994025591A1 (en) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF $i(CAMELIDAE)
WO1995004079A1 (fr) 1993-08-02 1995-02-09 Raymond Hamers Vecteur recombinant contenant une sequence d'un gene de lipoproteine pour l'expression de sequences de nucleotides
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1997049805A2 (en) 1996-06-27 1997-12-31 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recognition molecules interacting specifically with the active site or cleft of a target molecule
US5821333A (en) 1995-03-01 1998-10-13 Genetech, Inc. Method for making heteromultimeric polypeptides
WO1999037681A2 (en) 1998-01-26 1999-07-29 Unilever Plc Method for producing antibody fragments
WO2000040968A1 (en) 1999-01-05 2000-07-13 Unilever Plc Binding of antibody fragments to solid supports
WO2000043507A1 (en) 1999-01-19 2000-07-27 Unilever Plc Method for producing antibody fragments
WO2000065057A1 (en) 1999-04-22 2000-11-02 Unilever Plc Inhibition of viral infection using monovalent antigen-binding proteins
WO2001021817A1 (en) 1999-09-24 2001-03-29 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recombinant phages capable of entering host cells via specific interaction with an artificial receptor
WO2001040310A2 (en) 1999-11-29 2001-06-07 Unilever Plc Immobilisation of proteins using a polypeptide segment
WO2001044301A1 (en) 1999-11-29 2001-06-21 Unilever Plc Immobilized single domain antigen-binding molecules
EP1134231A1 (en) 2000-03-14 2001-09-19 Unilever N.V. Antibody heavy chain variable domains against human dietary enzymes, and their uses
WO2001090190A2 (en) 2000-05-26 2001-11-29 National Research Council Of Canada Single-domain antigen-binding antibody fragments derived from llama antibodies
WO2002048193A2 (en) 2000-12-13 2002-06-20 Unilever N.V. Camelidae antibody arrays
WO2003025020A1 (en) 2001-09-13 2003-03-27 Institute For Antibodies Co., Ltd. Method of constructing camel antibody library
WO2003035694A2 (en) 2001-10-24 2003-05-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
WO2003050531A2 (en) 2001-12-11 2003-06-19 Algonomics N.V. Method for displaying loops from immunoglobulin domains in different contexts
WO2003054016A2 (en) 2001-12-21 2003-07-03 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method for cloning of variable domain sequences
WO2003055527A2 (en) 2002-01-03 2003-07-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Immunoconjugates useful for treatment of tumours
WO2004041863A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Single domain antibodies directed against interferon- gamma and uses therefor
WO2004041867A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
WO2004062551A2 (en) 2003-01-10 2004-07-29 Ablynx N.V. RECOMBINANT VHH SINGLE DOMAIN ANTIBODY FROM CAMELIDAE AGAINST VON WILLEBRAND FACTOR (vWF) OR AGAINST COLLAGEN
WO2005044858A1 (en) 2003-11-07 2005-05-19 Ablynx N.V. Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses therefor
WO2006040153A2 (en) 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
WO2006079372A1 (en) 2005-01-31 2006-08-03 Ablynx N.V. Method for generating variable domain sequences of heavy chain antibodies
WO2006122825A2 (en) 2005-05-20 2006-11-23 Ablynx Nv Single domain vhh antibodies against von willebrand factor
WO2006122786A2 (en) 2005-05-18 2006-11-23 Ablynx Nv Improved nanobodies™ against tumor necrosis factor-alpha
US20100286374A1 (en) 2008-01-07 2010-11-11 Gunasekaran Kannan Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
CA2955386A1 (en) * 2014-07-21 2016-01-28 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
EP2101823B1 (en) 2007-01-09 2016-11-23 CureVac AG Rna-coded antibody
CN108350076A (zh) * 2015-08-17 2018-07-31 詹森药业有限公司 抗-bcma抗体，结合bcma和cd3的双特异性抗原结合分子及其用途
CN109134665A (zh) * 2018-08-24 2019-01-04 深圳普瑞金生物药业有限公司 一种基于单域抗体的bcma嵌合抗原受体及应用
US20190112381A1 (en) 2017-10-13 2019-04-18 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US20200024356A1 (en) 2018-07-19 2020-01-23 Regeneron Pharmaceuticals, Inc. Bispecific Anti-BCMA x Anti-CD3 Antibodies and Uses Thereof
CN112437671A (zh) * 2018-07-19 2021-03-02 瑞泽恩制药公司 具有bcma特异性的嵌合抗原受体及其用途
CN112585168A (zh) * 2019-07-30 2021-03-30 上海翰森生物医药科技有限公司 抗bcma抗体、其抗原结合片段及其医药用途
EP3819007A1 (en) * 2019-11-11 2021-05-12 Amgen Research (Munich) GmbH Dosing regimen for anti-bcma agents

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1994025591A1 (en) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF $i(CAMELIDAE)
WO1995004079A1 (fr) 1993-08-02 1995-02-09 Raymond Hamers Vecteur recombinant contenant une sequence d'un gene de lipoproteine pour l'expression de sequences de nucleotides
US5821333A (en) 1995-03-01 1998-10-13 Genetech, Inc. Method for making heteromultimeric polypeptides
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1997049805A2 (en) 1996-06-27 1997-12-31 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recognition molecules interacting specifically with the active site or cleft of a target molecule
WO1999037681A2 (en) 1998-01-26 1999-07-29 Unilever Plc Method for producing antibody fragments
WO2000040968A1 (en) 1999-01-05 2000-07-13 Unilever Plc Binding of antibody fragments to solid supports
WO2000043507A1 (en) 1999-01-19 2000-07-27 Unilever Plc Method for producing antibody fragments
WO2000065057A1 (en) 1999-04-22 2000-11-02 Unilever Plc Inhibition of viral infection using monovalent antigen-binding proteins
WO2001021817A1 (en) 1999-09-24 2001-03-29 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recombinant phages capable of entering host cells via specific interaction with an artificial receptor
WO2001040310A2 (en) 1999-11-29 2001-06-07 Unilever Plc Immobilisation of proteins using a polypeptide segment
WO2001044301A1 (en) 1999-11-29 2001-06-21 Unilever Plc Immobilized single domain antigen-binding molecules
EP1134231A1 (en) 2000-03-14 2001-09-19 Unilever N.V. Antibody heavy chain variable domains against human dietary enzymes, and their uses
WO2001090190A2 (en) 2000-05-26 2001-11-29 National Research Council Of Canada Single-domain antigen-binding antibody fragments derived from llama antibodies
WO2002048193A2 (en) 2000-12-13 2002-06-20 Unilever N.V. Camelidae antibody arrays
WO2003025020A1 (en) 2001-09-13 2003-03-27 Institute For Antibodies Co., Ltd. Method of constructing camel antibody library
WO2003035694A2 (en) 2001-10-24 2003-05-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
WO2003050531A2 (en) 2001-12-11 2003-06-19 Algonomics N.V. Method for displaying loops from immunoglobulin domains in different contexts
WO2003054016A2 (en) 2001-12-21 2003-07-03 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method for cloning of variable domain sequences
WO2003055527A2 (en) 2002-01-03 2003-07-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Immunoconjugates useful for treatment of tumours
WO2004041863A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Single domain antibodies directed against interferon- gamma and uses therefor
WO2004041867A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
WO2004041865A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Stabilized single domain antibodies
WO2004041862A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
WO2004062551A2 (en) 2003-01-10 2004-07-29 Ablynx N.V. RECOMBINANT VHH SINGLE DOMAIN ANTIBODY FROM CAMELIDAE AGAINST VON WILLEBRAND FACTOR (vWF) OR AGAINST COLLAGEN
WO2005044858A1 (en) 2003-11-07 2005-05-19 Ablynx N.V. Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses therefor
WO2006040153A2 (en) 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
WO2006079372A1 (en) 2005-01-31 2006-08-03 Ablynx N.V. Method for generating variable domain sequences of heavy chain antibodies
WO2006122787A1 (en) 2005-05-18 2006-11-23 Ablynx Nv Serum albumin binding proteins
WO2006122786A2 (en) 2005-05-18 2006-11-23 Ablynx Nv Improved nanobodies™ against tumor necrosis factor-alpha
WO2006122825A2 (en) 2005-05-20 2006-11-23 Ablynx Nv Single domain vhh antibodies against von willebrand factor
EP2101823B1 (en) 2007-01-09 2016-11-23 CureVac AG Rna-coded antibody
US20100286374A1 (en) 2008-01-07 2010-11-11 Gunasekaran Kannan Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
CA2955386A1 (en) * 2014-07-21 2016-01-28 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
CN108350076A (zh) * 2015-08-17 2018-07-31 詹森药业有限公司 抗-bcma抗体，结合bcma和cd3的双特异性抗原结合分子及其用途
US20190112381A1 (en) 2017-10-13 2019-04-18 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US20200024356A1 (en) 2018-07-19 2020-01-23 Regeneron Pharmaceuticals, Inc. Bispecific Anti-BCMA x Anti-CD3 Antibodies and Uses Thereof
CN112437671A (zh) * 2018-07-19 2021-03-02 瑞泽恩制药公司 具有bcma特异性的嵌合抗原受体及其用途
CN109134665A (zh) * 2018-08-24 2019-01-04 深圳普瑞金生物药业有限公司 一种基于单域抗体的bcma嵌合抗原受体及应用
CN112585168A (zh) * 2019-07-30 2021-03-30 上海翰森生物医药科技有限公司 抗bcma抗体、其抗原结合片段及其医药用途
EP3819007A1 (en) * 2019-11-11 2021-05-12 Amgen Research (Munich) GmbH Dosing regimen for anti-bcma agents

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Bispecific molecules formed by Genmab's DuoBody", SCIENCE, vol. 317, no. 5844, 2007, pages 91
"Remington's Pharmaceutical Sciences", 1990, MACK PRINTING COMPANY, pages: 1289 - 1329
ABHINANDANMARTIN, MOL. IMMUNOL., vol. 45, 2008, pages 3832 - 3839
AL-LAZIKANI B. ET AL., J.MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
CATER, PROTEIN ENGINEERING, vol. 9, no. 7, 1996, pages 617 - 621
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL.BIOL., vol. 196, 1987, pages 901 - 917
E. MEYERSW. MILLER, CABIOS, vol. 4, 1989, pages 11 - 17
GILLIES ET AL., J IMMUNOL METHODS, vol. 125, 1985, pages 191 - 202
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446 - 8
HONEGGERPLUCKTHUN, J. MOL. BIOL., vol. 309, 2001, pages 657 - 670
JONATHAN H. DAVIS ET AL., PROTEIN ENGINEERING, DESIGN & SELECTION, 2010, pages 1 - 8
KABAT ET AL., J. BIOL. CHEM., vol. 252, 1977, pages 6609 - 6616
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES
KORNDORFER ET AL., PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS, vol. 53, no. 1, 2003, pages 121 - 129
LEFRANC ET AL., DEV. COMPARAT. IMMUNOL., vol. 27, 2003, pages 55 - 77
LEFRANC M.P. ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
MABS, vol. 3, no. 6, 2011, pages 546 - 557
MACCALLUM ET AL., J. MOL.BIOL., vol. 262, 1996, pages 732 - 745
MORRISON, SCIENCE, vol. 229, no. 4719, 1985, pages 1202 - 7
MUYLDERMANS, REVIEWS IN MOLECULAR BIOTECHNOLOGY, vol. 74, 2001, pages 277 - 302
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
OI ET AL., BIO TECHNIQUES, vol. 4, 1986, pages 214 - 221
ROQUE ET AL., BIOTECHNOL. PROG., vol. 20, 2004, pages 639 - 654
STADLER ET AL., NATURE MEDICINE, 2017

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025077315A1 (zh) * 2023-10-12 2025-04-17 浙江康佰裕生物科技有限公司 抗bcma单域抗体及其制备方法与应用
CN117534763A (zh) * 2023-12-28 2024-02-09 康维众和(中山)生物药业有限公司 抗bcma的纳米抗体及其制备方法与应用
CN117534763B (zh) * 2023-12-28 2024-03-26 康维众和(中山)生物药业有限公司 抗bcma的纳米抗体及其制备方法与应用
WO2026072709A1 (en) 2024-09-25 2026-04-02 Orna Therapeutics, Inc. Chimeric antigen receptors targeting bcma
CN120329445A (zh) * 2025-06-20 2025-07-18 北京大学第一医院(北京大学第一临床医学院) 靶向bcma的单域抗体及其相关材料与应用

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