WO2024074145A1 - 一种结合baffr和cd3的双特异性抗体及其应用 - Google Patents

一种结合baffr和cd3的双特异性抗体及其应用 Download PDF

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WO2024074145A1
WO2024074145A1 PCT/CN2023/123288 CN2023123288W WO2024074145A1 WO 2024074145 A1 WO2024074145 A1 WO 2024074145A1 CN 2023123288 W CN2023123288 W CN 2023123288W WO 2024074145 A1 WO2024074145 A1 WO 2024074145A1
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seq
antigen binding
bispecific antibody
cancer
sequence
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PCT/CN2023/123288
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English (en)
French (fr)
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周冲
殷刘松
姜晓玲
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盛禾(中国)生物制药有限公司
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Publication of WO2024074145A1 publication Critical patent/WO2024074145A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody combining BAFFR and CD3 and an application thereof.
  • CD3 T cell surface glycoprotein CD3, signal transduction co-receptor of T cell receptor, which contains subunits ⁇ , ⁇ , ⁇ and ⁇
  • CD3 is a differentiation antigen expressed on the surface of all T lymphocytes, which mainly mediates the transduction of T cell activation signals. It plays an important role in the anti-infection immunity of the body's immune system.
  • the CD3 molecule forms a stable TCR-CD3 complex with the T cell antigen receptor.
  • the extracellular region recognizes and binds to the major histocompatibility complex class II molecules, enhancing the stability of the binding between the T cell antigen receptor and the MHC molecule; the intracellular region enhances the activation signal transduced by leukocyte CD3, thereby participating in and regulating the activation of the immune system.
  • the number of CD3-positive lymphocytes is an important indicator for measuring the cellular immunity of the body.
  • BAFFR The only ligand of BAFFR is BAFF.
  • BAFFR and BAFF work together to activate the NF-kB signaling pathway in B cells, promoting the proliferation and activation of B cells.
  • the BAFF-BAFFR signaling pathway and the BCR pathway are key pathways for the maturation and activation of Immature B cells. Blocking the dual pathways can completely eliminate peripheral B lymphocytes and inhibit the development and proliferation of B lymphocytes.
  • BAFFR-targeted antibody drugs C55 and C90 are as effective as Rituximab, and can completely eliminate lymphoma cells in the blood of mice and prolong the tumor-free survival of mice.
  • C90 can still completely eliminate lymphoma cells in the mouse blood and extend the tumor-free survival of mice to 100 days (rituximab survival time is within 20 days).
  • BAFFR-targeted antibodies can effectively solve the problems of ineffectiveness and recurrent drug resistance of ibrutinib.
  • the anti-tumor activity of the ADCC activity of monoclonal antibody drugs is limited.
  • T cells have been proven to be an effective strategy as effective anti-tumor cells. Therefore, the development of T cell engager therapy that can target both T cell receptors and tumor-associated antigens (TAA) is widely used.
  • TAA tumor-associated antigens
  • Anti-CD3 and anti-BAFFR bispecific antibodies can activate endogenous T cells by bispecifically binding to BAFFR on the surface of tumor cells and CD3 on the surface of T cells, leading to the directed lysis of BAFFR-positive tumor cells, thereby achieving the purpose of treating tumors.
  • the inventors have developed a bispecific antibody with good performance that can bind to BAFFR and CD3.
  • the bispecific antibody of the present invention maximizes the tumor killing effect through multivalent binding of tumor-associated antigens while controlling the toxicity of CD3: BAFFR antibody multivalently binds to BAFFR-positive tumors, and the CD3 antibody at the other end binds to T cells.
  • the bispecific antibody acts as a connector to bring T cells and tumor cells closer to form an immune synapse, allowing T cells to kill tumors; the monovalent CD3 antibody reduces toxicity and can Better form immune synapses to achieve more effective, specific and safer anti-tumor responses.
  • the present invention provides a bispecific antibody, which comprises: (a) a first antigen-binding portion that specifically binds to a first antigen, wherein the first antigen is BAFFR; (b) a second antigen-binding portion that specifically binds to a second antigen, wherein the second antigen is BAFFR; and (c) a third antigen-binding portion that specifically binds to a third antigen, wherein the third antigen is CD3.
  • the first antigen binding moiety is a full length antibody consisting of two heavy chains and two light chains.
  • the second antigen binding moiety is an antibody fragment comprising a heavy chain variable domain (VH) and/or a light chain variable domain (VL).
  • the third antigen binding moiety is an antibody fragment comprising a heavy chain variable domain (VH) and/or a light chain variable domain (VL).
  • the second antigen binding moiety is Fab, Fab', scFab, F(ab')2, Fv, dsFv or scFv.
  • the second antigen binding moiety is Fab.
  • the third antigen binding moiety is Fab, Fab', scFab, F(ab')2, Fv, dsFv or scFv.
  • the third antigen binding moiety is scFv.
  • the second antigen binding moiety is fused to the N-terminus of one heavy chain of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the C-terminus of one heavy chain of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus and C-terminus of one heavy chain of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-termini of the two heavy chains of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the C-termini of the two heavy chains of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus and C-terminus of the two heavy chains of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus of one light chain of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the C-terminus of one light chain of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus and C-terminus of one light chain of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-termini of the two light chains of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the C-termini of the two light chains of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus and C-terminus of the two light chains of the first antigen binding moiety.
  • the third antigen binding moiety replaces one or two Fab regions of the first antigen binding moiety.
  • the third antigen binding moiety replaces the Fab region of the first antigen binding moiety fused to the second antigen binding moiety.
  • the third antigen binding moiety replaces one or two Fv regions of the first antigen binding moiety.
  • the third antigen binding moiety replaces the Fv region fused to the second antigen binding moiety.
  • the Fv region of the first antigen binding moiety is not limited to one or two Fv regions of the first antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus of one heavy chain of the first antigen binding moiety and the third antigen binding moiety replaces the Fab region of the first antigen binding moiety fused to the second antigen binding moiety.
  • the second antigen binding moiety is fused to the N-terminus of one heavy chain of the first antigen binding moiety and the third antigen binding moiety replaces the Fv region of the first antigen binding moiety fused to the second antigen binding moiety.
  • the bispecific antibody comprises a first Fc region and a second Fc region.
  • the first Fc region and the second Fc region are the same or different.
  • the Fc region is selected from IgG, IgA, IgD, IgE, IgM and variants thereof.
  • the Fc region is selected from IgG1, IgG2, IgG3, IgG4 and variants thereof.
  • the Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.
  • the first Fc region is knob-Fc and the second Fc region is hole-Fc.
  • the first Fc region is hole-Fc and the second Fc region is knob-Fc.
  • the VH and VL of the third antigen binding moiety are swapped.
  • the second antigen binding moiety is fused to the N-terminus of one heavy chain of the first antigen binding moiety
  • the third antigen binding moiety replaces the Fab region of the first antigen binding moiety fused to the second antigen binding moiety
  • the VH and VL of the third antigen binding moiety are interchanged.
  • the second antigen binding moiety is fused to the N-terminus of one heavy chain of the first antigen binding moiety
  • the third antigen binding moiety replaces the Fv region of the first antigen binding moiety fused to the second antigen binding moiety
  • the VH and VL of the third antigen binding moiety are interchanged.
  • the second and third antigen binding moieties are fused to the first antigen binding moiety via a linker.
  • the linker is a peptide linker.
  • the peptide linker is a GS linker or a mutant human IgG hinge.
  • the peptide linker has an amino acid sequence as shown in (G4S) x , where x is an integer selected from 1-6; preferably, the peptide linker is (G4S) 2 , (G4S) 3 or (G4S) 4. More preferably, the peptide linker is (G4S) 2 .
  • the first antigen binding portion specifically binds to BAFFR, wherein HCDR1 of the first antigen binding portion is as shown in SEQ ID NO:3, or a sequence having at least 80% identity with SEQ ID NO:3; HCDR2 is as shown in SEQ ID NO:4, or a sequence having at least 80% identity with SEQ ID NO:4; HCDR3 is as shown in SEQ ID NO:5, or a sequence having at least 80% identity with SEQ ID NO:5; LCDR1 is as shown in SEQ ID NO:6, or a sequence having at least 80% identity with SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence having at least 80% identity with SEQ ID NO:7; LCDR3 is as shown in SEQ ID NO:8, or a sequence having at least 80% identity with SEQ ID NO:8.
  • the second antigen binding portion specifically binds BAFFR, wherein HCDR1 of the second antigen binding portion is as shown in SEQ ID NO:3, or a sequence having at least 80% identity with SEQ ID NO:3; HCDR2 is as shown in SEQ ID NO:4, or a sequence having at least 80% identity with SEQ ID NO:4; HCDR3 is as shown in SEQ ID NO:5, or a sequence having at least 80% identity with SEQ ID NO:5; LCDR1 is as shown in SEQ ID NO:6, or a sequence having at least 80% identity with SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence having at least 80% identity with SEQ ID NO:7; LCDR3 is as shown in SEQ ID NO:8, or a sequence having at least 80% identity with SEQ ID NO:8.
  • the third antigen binding portion specifically binds to CD3, wherein HCDR1 of the third antigen binding portion is as shown in SEQ ID NO:11, or a sequence having at least 80% identity with SEQ ID NO:11; HCDR2 is as shown in SEQ ID NO:12, or a sequence having at least 80% identity with SEQ ID NO:12; HCDR3 is as shown in SEQ ID NO:13, or a sequence having at least 80% identity with SEQ ID NO:13; and LCDR1 is as shown in SEQ ID NO:14, or a sequence having at least 80% identity with SEQ ID NO:14; LCDR2 is as shown in SEQ ID NO:15, or a sequence having at least 80% identity with SEQ ID NO:15; LCDR3 is as shown in SEQ ID NO:16, or a sequence having at least 80% identity with SEQ ID NO:16.
  • HCDR1 of the third antigen binding portion is as shown in SEQ ID NO:11, or a sequence having at least 80% identity with S
  • the first antigen binding portion specifically binds to BAFFR, wherein the heavy chain variable region VH of the first antigen binding portion is as shown in SEQ ID NO:1, or a sequence with at least 80% identity to SEQ ID NO:1; the light chain variable region VL is as shown in SEQ ID NO:2, or a sequence with at least 80% identity to SEQ ID NO:2.
  • the second antigen binding portion specifically binds to BAFFR, wherein the heavy chain variable region VH of the second antigen binding portion is as shown in SEQ ID NO:1, or a sequence having at least 80% identity with SEQ ID NO:1; the light chain variable region VL is as shown in SEQ ID NO:2, or a sequence having at least 80% identity with SEQ ID NO:2.
  • the third antigen binding portion specifically binds to CD3, wherein the heavy chain variable region VH of the third antigen binding portion is as shown in SEQ ID NO:9, or a sequence with at least 80% identity with SEQ ID NO:9; the light chain variable region VL is as shown in SEQ ID NO:10, or a sequence with at least 80% identity with SEQ ID NO:10.
  • the present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned bispecific antibodies.
  • the cancer is selected from human brain astrocytoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's
  • the present invention also provides the use of any of the above-described bispecific antibodies in the preparation of a medicament for treating an autoimmune disease.
  • the autoimmune disease is selected from graft-versus-host disease, rheumatoid arthritis, Crohn's disease, multiple sclerosis, colitis, psoriasis, autoimmune uveitis, pemphigus, epidermolysis bullosa, or type I diabetes.
  • the use is achieved by one or more of tumor immunotherapy, cell therapy, or gene therapy.
  • the present invention also provides a pharmaceutical composition, comprising any of the bispecific antibodies described above and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides an antibody-drug conjugate, comprising any of the above-mentioned bispecific antibodies.
  • the conjugated drug is selected from cytotoxins, small molecule chemical drugs or immunotoxins.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • CDR complementarity determining region in an immunoglobulin variable region
  • IgG immunoglobulin G.
  • antibody refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be reconstructed and separated from natural resources such as plasma or serum in which the antibody is naturally present, or from the culture supernatant of hybridoma cells that produce the antibody, from animal immune serum, or from phage library screening. Alternatively, it can be partially or completely synthesized using techniques such as gene recombination. Preferred antibodies include, for example, antibodies of isotypes of immunoglobulins or subclasses of these isotypes.
  • Known human immunoglobulins include 9 categories (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM.
  • antibodies of the present invention can include IgG1, IgG2, IgG3, and/or IgG4.
  • the partial antibodies used herein are immunoglobulin molecules consisting of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant domain does not directly participate in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high variability (called complementarity determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form an antigen binding site, respectively.
  • antigen binding portion or “antigen binding fragment” refers to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds.
  • antigen binding fragments include (1) Fab fragments; (2) F(ab')2 fragments; (3) Fd fragments, consisting of VH and CH1 domains; (4) Fv fragments; (5) dAb fragments, consisting of VH domains; (6) CDRs, separated complementarity determining regions.
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to connect them through synthetic linkers so that they can be produced as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions are paired to form a monovalent molecule.
  • single-chain antibodies are also intended to be included in the "antigen-binding fragment" of the term antibody.
  • Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies.
  • Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antigen-binding fragments can also be incorporated into single-chain molecules comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which form a pair of antigen-binding regions together with complementary light chain polypeptides.
  • Fab fragment consists of a complete L chain and the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain.
  • VH variable region domain
  • CH1 first constant domain
  • Fab fragments can be produced recombinantly or by papain digestion of full-length antibodies.
  • Fab' fragment differs from the Fab fragment in that a few additional residues are added at the carboxyl terminus of the CH1 domain, including
  • Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to an antigen with a reducing agent such as dithiothreitol.
  • F(ab')2 fragment was originally produced as a pair of Fab' fragments with a hinge cysteine between them.
  • F(ab')2 fragments can be produced recombinantly or by pepsin digestion of intact antibodies (which removes most of the Fc region while leaving part of the hinge region intact).
  • F(ab')2 fragments can be dissociated (into two Fab' molecules) by treatment with a reducing agent such as ⁇ -mercaptoethanol.
  • scFab refers to a single-chain Fab fragment, in which a polypeptide linker is introduced between the heavy chain variable domain (VH) and the light chain (CL) to form a single-chain Fab fragment (scFab).
  • Fv is the smallest antibody fragment containing a complete antigen recognition and binding site.
  • the fragment is composed of a dimer formed by a heavy chain variable region domain and a light chain variable region domain through tight non-covalent binding.
  • the folding of these two domains produces six hypervariable loops (3 loops from the H chain and 3 loops from the L chain), which contribute to the amino acid residues for antigen binding and give the antibody antigen binding specificity.
  • six hypervariable loops (3 loops from the H chain and 3 loops from the L chain), which contribute to the amino acid residues for antigen binding and give the antibody antigen binding specificity.
  • a single variable domain has the ability to recognize and bind antigens, its affinity is lower than that of the complete binding site.
  • the term "scFv" fragment refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form a desired structure for antigen binding.
  • the "scFv-Fc” fragment comprises an scFv connected to an Fc domain.
  • the Fc domain can be connected to the C-terminus of the scFv.
  • the Fc domain may be after VH or VL.
  • the Fc domain may be any suitable Fc domain known in the art or described herein. In some cases, the Fc domain is an IgG1 Fc domain.
  • dsFv refers to a disulfide-stabilized Fv fragment.
  • dsFv a polypeptide in which one amino acid residue in each VH and VL is replaced by a cysteine residue is connected via a disulfide bond between the cysteine residues.
  • each amino acid in the framework region of VH and VL is mutated to cysteine, which in turn forms a stable interchain disulfide bond.
  • position 44 in VH and position 100 in VL are mutated to cysteine.
  • dsFv encompasses both dsFv (molecules in which VH and VL are connected by interchain disulfide bonds rather than linker peptides) or scdsFv (molecules in which VH and VL are connected by linkers and interchain disulfide bonds) known in the art.
  • epitope refers to an antigenic determinant in an antigen, and refers to an antigenic site bound by a domain of an antigen binding molecule comprising an antibody variable region disclosed in this specification. Therefore, an epitope can be defined based on its structure. In addition, the epitope can also be defined based on the antigen binding activity in an antigen binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that form the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.
  • the term "specificity" means that one of the molecules involved in specific binding does not show any significant binding to molecules other than one or more of the binding partner molecules.
  • the term is also used when the domain containing the antibody variable region is specific for a particular epitope among multiple epitopes in an antigen.
  • the antigen-binding molecule containing the domain containing the antibody variable region can bind to various antigens having the epitope.
  • bispecific antibody refers to a protein molecule that can specifically bind to two target antigens or target antigen epitopes.
  • bispecific antigen-binding protein comprising an antibody or antigen-binding fragment (e.g., Fab, scFv, etc.) and "bispecific antibody” and “bi-antibody” can be used interchangeably.
  • knock-Fc refers to replacing an amino acid residue in the CH3 domain of the first subunit of the Fc domain with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned in the recess in the CH3 domain of the second subunit. For example, by mutating serine T at position 366 of CH3 of a heavy chain to tryptophan W, a protruding "knob"-like protrusion is formed.
  • hole-Fc refers to replacing the amino acid residues in the CH3 domain of the second subunit of the Fc domain with amino acid residues having a smaller side chain volume, thereby generating a depression in the CH3 domain of the second subunit in which the protrusion in the CH3 domain of the first subunit can be positioned.
  • hole-Fc refers to replacing the amino acid residues in the CH3 domain of the second subunit of the Fc domain with amino acid residues having a smaller side chain volume, thereby generating a depression in the CH3 domain of the second subunit in which the protrusion in the CH3 domain of the first subunit can be positioned.
  • fusion refers to the connection of two amino acid sequences into a new sequence through a linker or other technical means, thereby forming a new artificial protein or antibody.
  • linker or "L1" used to connect two protein domains refers to a connecting polypeptide sequence that is used to connect protein domains and has a certain degree of flexibility. The use of the linker will not cause the loss of the original function of the protein domain.
  • variable region or “variable domain” of an antibody refers to the variable region (VL) of an antibody light chain or the variable region (VH) of an antibody heavy chain, either alone or in combination.
  • VL variable region
  • VH variable region
  • the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions).
  • FRs framework regions
  • CDRs complementary determining regions
  • the CDRs in each chain are held together closely by the FRs and contribute to the formation of the antigen-binding site of the antibody together with the CDRs from the other chain.
  • variable refers to the fact that certain segments of the variable domain are widely different in sequence between antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen.
  • variability is not evenly distributed over the entire variable domain range. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light chain and heavy chain variable domains.
  • HVRs hypervariable regions
  • FRs framework regions
  • the variable domains of the native heavy and light chains each contain four FR regions, most of which adopt a ⁇ -folded configuration, connected by three HVRs, which form a loop connection, and in some cases form a part of the ⁇ -folded structure.
  • the HVR in each chain is closely held together by the FR region, and together with the HVRs of other chains, contribute to the formation of the antigen binding site of the antibody.
  • the constant domain is not directly involved in the combination of the antibody and the antigen, but exhibits various effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.
  • antibody drug conjugate refers to a binding protein (such as an antibody or an antigen-binding fragment thereof) connected to one or more conjugated drugs (which may optionally be a therapeutic agent or a cytotoxic agent), and its structure generally consists of three parts: an antibody or antibody-like ligand, a drug portion, and a linker that couples the antibody or antibody-like ligand and the drug.
  • ADCs generally have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs conjugated to the antibody.
  • polypeptide refers to an amino acid chain of any length, regardless of modification (e.g., phosphorylation or glycosylation).
  • the term polypeptide includes proteins and fragments thereof.
  • Polypeptides can be "exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell utilized, such as human polypeptides produced by bacterial cells.
  • Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written from left to right in the direction of the amino terminus to the carboxyl terminus. Amino acid residue sequences are named by three-letter or one-letter codes according to standard nomenclature.
  • amino acid refers to the twenty common naturally occurring amino acids.
  • amino acid also includes non-natural amino acids. Any suitable non-natural amino acid can be used.
  • the non-natural amino acid comprises a reactive portion for conjugating the agent to MIAC.
  • identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage sequence identity. Comparisons for purposes of determining percentage amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST software or FASTA packages.
  • the term "at least 80% identity” refers to the amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence.
  • the percentage of identical amino acid residues is above 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.
  • amino acid mutation refers to the presence of amino acid mutations or changes in variant proteins or polypeptides compared to the original protein or polypeptide, including the insertion, deletion or substitution of one or more amino acids in the original protein or polypeptide.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • host cell refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses a selected target gene.
  • the term includes the offspring of a parent cell, whether or not the offspring is identical to the original parent cell in morphology or genetic composition, as long as the offspring has the selected target gene.
  • Commonly used host cells include bacteria, yeast, mammalian cells, etc.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked and are referred to herein as "expression vectors.”
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers, such as phosphate-buffered saline solutions, water, and emulsions, as well as various types of wetting agents.
  • positive control refers to a natural or engineered cell or antibody that can bind to or express a target protein.
  • the positive control referred to herein refers to a single-target positive control.
  • control refers to the use of the same species, subtype, dose, immunoglobulin and subtype of immunoglobulin, and the same marker as the experimental sample in the same experiment to eliminate the experimental background effect of non-specific binding samples on the experimental values, as a control to better illustrate the experimental effect.
  • affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding module of MIAC) and its binding partner (e.g., an antigen). Within each antigenic site, the variable region of the antibody “arm” interacts with the antigen at multiple amino acid sites through weak non-covalent forces; the greater the interaction, the stronger the affinity.
  • binding affinity refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as by using surface plasmon resonance (SPR) technology (e.g., an instrument) or biolayer interferometry (e.g., an instrument) to measure.
  • SPR surface plasmon resonance
  • biolayer interferometry e.g., an instrument
  • effector cell is a leukocyte that expresses one or more FcRs and performs effector functions.
  • the effector cell expresses at least Fc ⁇ RIII and performs ADCC effector functions.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.
  • PBMCs peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils effector cells can be separated from natural sources (e.g., blood). Effector cells are generally lymphocytes associated with the effector phase and are used to produce cytokines (helper T cells), cells that kill infectious pathogens (cytotoxic T cells), or secrete antibodies (differentiated B cells).
  • Figure 1 depicts an exemplary bispecific antibody, in which a full-length antibody capable of specifically recognizing a first antigen (BAFFR) is fused with a second antigen (BAFFR) binding portion and a third antigen (CD3) binding portion, wherein the second antigen binding portion is Fab, and the second antigen binding portion is fused to the N-terminus of a heavy chain of the first antigen binding portion, and the third antigen binding portion is scFv, and the third antigen binding portion replaces the Fab region of the first antigen binding portion fused with the second antigen binding portion, and the first Fc region of the bispecific antibody is knob-Fc, and the second Fc region is hole-Fc.
  • BAFFR full-length antibody capable of specifically recognizing a first antigen
  • BAFFR second antigen binding portion
  • CD3 antigen (CD3) binding portion CD3 binding portion
  • the second antigen binding portion is Fab
  • the second antigen binding portion is fused to the N-terminus of a heavy chain of
  • Figure 2 describes an exemplary bispecific antibody, in which a full-length antibody capable of specifically recognizing a first antigen (BAFFR) is fused with a second antigen (BAFFR) binding portion and a third antigen (CD3) binding portion, wherein the second antigen binding portion is Fab, and the second antigen binding portion is fused to the N-terminus of a heavy chain of the first antigen binding portion, and the third antigen binding portion is Fab, and the VH and VL of the third antigen binding portion are interchanged, and the third antigen binding portion replaces the Fab region of the first antigen binding portion fused with the second antigen binding portion, and the first Fc region of the bispecific antibody is knob-Fc, and the second Fc region is hole-Fc.
  • BAFFR full-length antibody capable of specifically recognizing a first antigen
  • BAFFR second antigen binding portion
  • CD3 antigen (CD3) binding portion CD3 binding portion
  • FIG 3 shows the structure of bispecific antibody C, in which a full-length antibody capable of specifically recognizing a first antigen (BAFFR) is fused with a second antigen (CD3) binding portion, wherein the second antigen binding portion is Fab, the VH and VL of the second antigen binding portion are interchanged, and the second antigen binding portion replaces the Fab region on one side of the first antigen binding portion, the first Fc region of the bispecific antibody is knob-Fc, and the second Fc region is hole-Fc.
  • BAFFR full-length antibody capable of specifically recognizing a first antigen
  • CD3 second antigen
  • FIG4 shows the binding activity of the bispecific antibody to BAFFR protein.
  • FIG5 shows the binding activity of bispecific antibodies to CD3 protein.
  • FIG6 shows the binding activity of bispecific antibodies to Jeko-1 cells.
  • FIG. 7 shows the binding activity of bispecific antibodies to Raji cells.
  • FIG8 shows the binding activity of bispecific antibodies to Su-DHL4 cells.
  • FIG. 9 shows the T cell-specific activation activity of the bispecific antibody against jukart-NFAT-luc reporter gene cells.
  • FIG. 10 shows the killing activity of the bispecific antibody against CHO-K1-BAFFR cells.
  • FIG. 11 shows the killing activity of the bispecific antibody against CHO-K1 cells.
  • FIG. 12 shows the killing activity of the bispecific antibody against Raji-luc cells.
  • the embodiment is to construct bispecific antibodies targeting BAFFR and CD3 targets according to Figures 1-3, and they are named Antibody A, Antibody B, and Antibody C respectively.
  • the Fc of the antibody amino acid sequence is adjusted to other IgG types, and the desired form of amino acid mutations is further designed in each heavy chain, thereby obtaining the amino acid sequence of the target antibody (see Table 1).
  • the constructed antibody amino acid sequence combination is shown in Table 2 and includes the theoretical molecular weight.
  • the above target amino acid sequences were converted into nucleotide sequences, and optimized for a series of parameters that may affect the expression of antibodies in mammalian cells: codon preference, GC content (i.e., the ratio of guanine G and cytosine C in the four bases of DNA), CpG island (i.e., the region with a higher density of CpG dinucleotides in the genome), secondary structure of mRNA, splicing site, pre-mature PolyA site, internal Chi site (a short DNA fragment in the genome, near which the probability of homologous recombination increases) or ribosome binding site, RNA unstable sequence, inverted repeat sequence and restriction enzyme sites that may interfere with cloning; at the same time, related sequences that may improve translation efficiency, such as Kozak sequence and SD sequence, were added.
  • codon preference i.e., the ratio of guanine G and cytosine C in the four bases of DNA
  • CpG island i.e., the
  • the heavy chain gene and light chain gene encoding the above antibodies were designed, and the nucleotide sequences encoding signal peptides optimized according to the amino acid sequence were designed at the 5' end of the heavy chain and light chain, respectively; in addition, stop codons were added to the 3' end of the light chain and heavy chain nucleotide sequences, respectively.
  • the pcDNA3.1-G418 vector is used as a plasmid vector for expressing the multifunctional antibody.
  • the pcDNA3.1-G418 vector contains the promoter CMVPromoter, the eukaryotic screening marker G418 tag and the prokaryotic screening marker Ampicilline.
  • the nucleotide sequence for constructing the antibody expression light chain and heavy chain is obtained by gene synthesis, and the vector and the target fragment are double-digested with HindIII and XhoI, and then enzymatically linked by DNA ligase after recovery, and transformed into Escherichia coli competent cells DH5 ⁇ , and the positive clones are selected and plasmid extraction and enzyme digestion verification are performed to obtain the antibody-containing plasmid.
  • the recombinant plasmids containing the above-mentioned target genes were transformed into Escherichia coli competent cells DH5 ⁇ , and the transformed bacteria were spread on LB plates containing 100 ⁇ g/mL ampicillin for culture.
  • the plasmid clones were selected and cultured in liquid LB medium, and the bacteria were shaken at 260 rpm for 14 hours.
  • the plasmids were extracted using an endotoxin-free plasmid extraction kit, dissolved in sterile water, and the concentration was measured using a nucleic acid protein quantifier.
  • Expi CHO was cultured at 37°C, 8% CO 2 , 100 rpm to a cell density of 6 ⁇ 10 6 cells/mL.
  • the constructed plasmids were transfected into the above cells according to the combination pairing using liposomes, the transfection plasmid concentration was 1 mg/mL, the liposome volume was determined with reference to the Expi CHO TM Expression System kit, and cultured at 32°C, 5% CO 2 , 100 rpm for 7-10 days. Feed was performed once 18-22 hours after transfection and between the 5th day.
  • the above culture product was centrifuged at 4000g, filtered through a 0.22 ⁇ m filter membrane and the culture supernatant was collected.
  • the antibody protein obtained was purified using Protein A and ion columns and the eluate was collected.
  • the specific operation steps of Protein A and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken and affinity chromatography is performed using GE's Protein A chromatography column.
  • the equilibrium buffer used for chromatography is 1 ⁇ PBS (pH7.4). After the cell supernatant is loaded and bound, it is washed with PBS until the ultraviolet returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH3.0) elution buffer, and the pH is adjusted to neutral with Tris for storage.
  • the pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units lower or higher than pI, and appropriately diluted to control the sample conductivity below 5ms/cm.
  • appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions
  • conventional ion exchange chromatography methods in the field such as anion exchange or cation exchange are used to perform NaCl gradient elution under corresponding pH conditions, and the collection tubes where the target protein is located are selected according to SDS-PAGE and combined for storage.
  • the purified eluate was ultrafiltered and exchanged into a buffer solution, and the protein was detected by SDS-polyacrylamide gel electrophoresis.
  • Human-BAFFR-His (purchased from Acro, Cat: BAR-H52H3) was diluted to 1 ⁇ g/mL using PBS buffer at pH 7.4, and 100 ⁇ L was added to each well of a 96-well ELISA plate and coated overnight at 4°C. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the constructed antibody was diluted to 100 nM with 0.5% BSA sample diluent, and this was used as the starting concentration. A 3-fold gradient dilution was performed, with a total of 11 gradients, 100 ⁇ L per well, and incubated at 37°C for 1 hour.
  • the plate was washed 3 times with PBST again, and HRP-labeled goat anti-human IgG-Fc (purchased from Jackson, Cat: 109-035-098) was diluted 1:20000 with sample diluent, 100 ⁇ L was added to each well, and incubated at room temperature for 1 hour. Negative control (irrelevant antibody) and positive control were set up.
  • the positive control was BAFFR monoclonal antibody (BAFFR monoclonal antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 2, with the constant region of human IgG1 added, see SEQ ID NO: 27 and SEQ ID NO: 28).
  • 100 ⁇ L TMB substrate was added to each well. Incubate at room temperature in the dark for 10 minutes, and 100 ⁇ L 1M HCl solution was added to each well to terminate the color reaction. Read the plate on a multifunctional microplate reader.
  • the ELISA results of the antibodies are shown in FIG4 , and the bispecific antibodies can bind to the BAFFR protein at all concentrations.
  • Human-CD3-His (purchased from Acro, Cat: CDD-H52W4) was diluted to 0.2 ⁇ g/mL using pH 7.4 PBS buffer, and 100 ⁇ L was added to each well of a 96-well ELISA plate and coated overnight at 4°C. After blocking with 1% BSA blocking solution for 1 hour. After washing the plate 3 times with PBST, the constructed expression antibody was diluted to 100 nM with 0.5% BSA sample diluent, and this was used as the starting concentration for 3-fold gradient dilution, a total of 11 gradients, and negative controls (blank wells and IgG1 isotype controls) and positive controls were set up.
  • the positive control was CD3 monoclonal antibody (CD3 antibody sequence was derived from INN blinatumomab, the sequence consisted of SEQ ID NO: 9, SEQ ID NO: 10, and the constant region of human IgG1 was added.
  • the constant region of IgG1 is shown in SEQ ID NO: 27 and SEQ ID NO: 28), 100 ⁇ L per well, incubated at 37°C for 1 hour. Wash the plate three times with PBST, dilute HRP-labeled goat anti-human IgG-Fc with sample diluent at 1:20,000, add 100 ⁇ L to each well, and incubate at room temperature for 1 hour.
  • the ELISA results of the antibodies are shown in FIG5 , and the bispecific antibodies can bind to CD3 at all concentrations.
  • Example 7 Flow cytometry detection of antibody binding activity to BAFFR-positive tumor cells
  • the positive control is BAFFR monoclonal antibody (BAFFR monoclonal antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 2, and the constant region of human IgG1 is added.
  • the constant region of human IgG1 is shown in SEQ ID NO: 27 and SEQ ID NO: 28), and add 100 ⁇ L of antibody diluent.
  • the cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer.
  • the cells were resuspended in 100 ⁇ L FACS buffer, and anti-human IgG Fc fluorescent secondary antibody-FITC (Biolegend, Cat: 109306) was added to the sample, incubated for 30 minutes and washed twice with excess FACS buffer.
  • the cells were resuspended in flow cytometry buffer and then detected and analyzed by flow cytometry.
  • BAFFR-positive Jeko-1 cells (from the Chinese Academy of Sciences Cell Collection Center) were plated in 96-well plates at 2 ⁇ 10 4 /well, and then the effector cells Jurkat-NFAT-Luc were added at 5 ⁇ 10 4 /well.
  • the antibody was diluted to 30 ⁇ g/mL using PBS, diluted 5 times, and a total of 9 concentration gradients were added to the wells of the corresponding cells.
  • a negative control group was set up and cultured at 37°C for 6h. Then Bio-Lite was added to the sample wells and incubated at room temperature for 10min before reading with a multifunctional microplate reader.
  • bispecific antibodies A and B specifically activate jurkart-NFAT-Luc reporter cells expressing CD3 in the presence of BARRF-positive target cells Jeko-1. However, there is no activation activity in the absence of antibodies or target cells. This shows that after bispecific antibodies A and B recognize BAFFR-positive cells, they can specifically activate CD3-positive T cells, and then activate downstream signals that induce T cells to kill tumors.
  • Bispecific antibody C has non-specific activation and poses risks.
  • BAFFR-positive CHO-K1-BAFFR cells BAFFR overexpression engineered cell line constructed by BAFFR lentiviral transduction of CHO-K1 cells
  • BAFFR-negative CHO-K1 BAFFR-negative CHO-K1
  • bispecific antibodies and irrelevant antibodies were added, starting at 10 ⁇ g/mL and diluted 10 times, with a total of 6 concentration gradients.
  • CIK CD3+CD56+ cells
  • effector cells were added at 1 ⁇ 10 5 /well, with an effector-target ratio of 5:1. Blank control (diluent), negative control (target cells + CIK cells, no antibody), and irrelevant antibody group were set.
  • negative control 1 was BAFFR monoclonal antibody (BAFFR monoclonal antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 2, with the constant region of human IgG1 added, and the constant region of human IgG1 is shown in SEQ ID NO: 27 and SEQ ID NO: 28), and negative control 2 was anti-GPC3 and CD3 antibody (the antibody is derived from patent US11001643B2, and the amino acid sequence consists of SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31). After incubation in a cell culture incubator for 24 h, the cells were rinsed with PBS for 3 h.
  • BAFFR monoclonal antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 2, with the constant region of human IgG1 added, and the constant region of human IgG1 is shown in SEQ ID NO: 27 and SEQ ID NO: 28
  • negative control 2 was anti-GPC3 and CD3 antibody (the antibody is derived from patent US110016
  • Cell killing rate (%) (1-(OD value of sample well-OD value of blank well)/(OD value of negative well-OD value of blank well) OD value)) ⁇ 100%.
  • the bispecific antibody can kill CHO-K1-BAFFR overexpressing BAFFR, and the irrelevant antibody has no killing effect. Both the bispecific antibody and the irrelevant antibody have no killing effect on CHO-K1, indicating that the bispecific antibody can mediate the specific killing of BAFFR-positive cells by CIK cells.
  • BAFFR-positive Raji-luc (Luc lentiviral transduction of raji) was plated in 96-well plates at 1 ⁇ 10 4 /well. After culturing for 24 hours, bispecific antibodies and irrelevant antibodies were added, starting at 50 ⁇ g/mL, 10-fold dilution, a total of 6 concentration gradients, and CIK (CD3+CD56+ cells) effector cells were added at 1 ⁇ 10 5 /well, with an effector-target ratio of 10:1, and blank controls (diluent), negative controls (target cells + CIK cells, no antibodies), and irrelevant antibody groups were set.
  • negative control 1 was BAFFR monoclonal antibody (BAFFR monoclonal antibody sequence consists of SEQ ID NO:1, SEQ ID NO:2, and the constant region of human IgG1 is added, and the constant region of IgG1 is shown in SEQ ID NO:27 and SEQ ID NO:28), and negative control 2 was anti-GPC3 and CD3 bispecific antibodies (the antibody sequence is derived from patent US11001643B2, and the sequence consists of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31).
  • Bio-Lite was added, and after incubation in the incubator for 10 minutes, the OD value was detected at 450nm on a microplate reader.
  • the bispecific antibody can kill BAFFR-positive Raji, while the irrelevant antibody has no killing effect, indicating that the bispecific antibody mediates the CIK cell-specific killing of BAFFR-positive cells.

Abstract

一种双特异性抗体，所述双特异性抗体包含：(a)特异性结合第一抗原的第一抗原结合部分，所述第一抗原为BAFFR；(b)特异性结合第二抗原的第二抗原结合部分，所述第二抗原为BAFFR；(c)特异性结合第三抗原的第三抗原结合部分，所述第三抗原为CD3。双特异性抗体在控制CD3毒性的同时通过肿瘤相关抗原的多价结合最大化肿瘤杀伤效应：BAFFR抗体多价结合BAFFR阳性肿瘤，另一端CD3抗体结合T细胞，双特异性抗体作为衔接器，拉进T细胞和肿瘤细胞距离，形成免疫突触，使T细胞杀伤肿瘤；单价的CD3抗体减少了毒性，可以更好形成免疫突触，达到更有效、更特异、更安全的抗肿瘤反应。

Description

一种结合BAFFR和CD3的双特异性抗体及其应用 技术领域
本发明属于生物医药领域，具体涉及一种结合BAFFR和CD3的双特异性抗体及其应用。
背景技术
CD3(T细胞表面糖蛋白CD3，T细胞受体的信号转导共受体，其包含亚基γ、δ、ε和ζ)是表达于所有T淋巴细胞表面的一种分化抗原，主要介导T细胞活化信号的转导。在机体免疫***的抗感染免疫中起着重要作用。CD3分子与T细胞抗原受体形成稳定的TCR-CD3复合体，其胞外区识别、结合主要组织相容性复合体II类分子，增强T细胞抗原受体与MHC分子结合的稳定性；胞内区增强白细胞CD3转导的活化信号，从而参与并调节免疫***的活化。CD3阳性淋巴细胞群的数量指标是衡量机体细胞免疫情况的重要指标。
BAFFR的唯一配体为BAFF，除作为B细胞特定发育阶段的标志物，BAFFR与BAFF结合后，共同作用激活B细胞中NF-kB信号通路，促进B细胞的增殖和活化。BAFF-BAFFR信号通路和BCR通路是Immature B细胞成熟和活化的关键通路，对双通路的阻断可以完全清除外周B淋巴细胞并抑制B淋巴细胞的发育和增殖。
临床研究显示，单剂量治疗初次接受美罗华的NHL病人约有30-60％耐药，约有60％复发病人对二次美罗华单剂治疗耐药，而耐药的主要机制是CD20的表达丰度不够或者CD20从B淋巴细胞上脱落。CD20并不是B细胞生长所必要，体外研究表明B淋巴细胞在敲除CD20之后，其生长速度没有变化。BAFFR的表达丰度高于CD20，且是B细胞生长所必要的信号通路蛋白，其和BCR通路是B细胞增殖和活化的必要通路。体内药效学试验表明，对于美罗华敏感淋巴瘤细胞Z138，BAFFR靶点抗体药物C55和C90同美罗华一样有效，可以完全清除小鼠血液中的淋巴瘤细胞，延长小鼠无瘤的生存期。对于美罗华耐药细胞株(CD20基因敲除Raji)，C90仍然可以完全清除小鼠血液中的淋巴瘤细胞，延长小鼠无瘤生存期至100天(美罗华生存期20天内)。
因此，作为BCR的下游信号通路，BAFFR靶点抗体可以有效的解决依鲁替尼的无效和复发耐药问题。但是单抗药物的ADCC活性的抗肿瘤活性有限，T细胞作为有效的抗肿瘤细胞已被验证是有效的策略，因此开发既能靶向T细胞受体，又能靶向肿瘤相关抗原(TAA)的T细胞衔接器疗法被广泛应用。抗CD3和抗BAFFR双特异性抗体可以通过双特异性结合肿瘤细胞表面的BAFFR和T细胞表面的CD3，激活内源性T细胞，导致BAFFR阳性的肿瘤细胞定向裂解，从而达到***的目的。
发明内容
在本申请中，发明人开发了具有良好性能的能够与BAFFR和CD3结合的双特异性抗体，本发明的双特异性抗体在控制CD3毒性的同时通过肿瘤相关抗原的多价结合最大化肿瘤杀伤效应：BAFFR抗体多价结合BAFFR阳性肿瘤，另一端CD3抗体结合T细胞，双特异性抗体作为衔接器，拉进T细胞和肿瘤细胞距离，形成免疫突触，使T细胞杀伤肿瘤；单价的CD3抗体减少了毒性，可以 更好形成免疫突触，达到更有效、更特异、更安全的抗肿瘤反应。
本发明提供了一种双特异性抗体，所述双特异性抗体包含：(a)特异性结合第一抗原的第一抗原结合部分，所述第一抗原为BAFFR；(b)特异性结合第二抗原的第二抗原结合部分，所述第二抗原为BAFFR；(c)特异性结合第三抗原的第三抗原结合部分，所述第三抗原为CD3。
在一些实施方案中，第一抗原结合部分是由两条重链和两条轻链组成的全长抗体。
在一些实施方案中，所述第二抗原结合部分是包含重链可变域(VH)和/或轻链可变域(VL)的抗体片段。
在一些实施方案中，所述第三抗原结合部分是包含重链可变域(VH)和/或轻链可变域(VL)的抗体片段。
在一些实施方案中，所述第二抗原结合部分是Fab、Fab'、scFab、F(ab')2、Fv、dsFv或scFv。优选地，所述第二抗原结合部分是Fab。
在一些实施方案中，所述第三抗原结合部分是Fab、Fab'、scFab、F(ab')2、Fv、dsFv或scFv。优选地，所述第三抗原结合部分是scFv。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链N端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链N端和C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的两条重链的N端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的两条重链的C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的两条重链的N端和C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条轻链的N端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条轻链的C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条轻链的N端和C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的两条轻链的N端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的两条轻链的C端。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的两条轻链的N端和C端。
在一些实施方案中，所述第三抗原结合部分替换第一抗原结合部分的一个或两个Fab区。优选地，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分的Fab区。
在一些实施方案中，所述第三抗原结合部分替换第一抗原结合部分的一个或两个Fv区。优选地，所述第三抗原结合部分替换与第二抗原结合部分相融合的 第一抗原结合部分的Fv区。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链的N端，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分的Fab区。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链的N端，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分的Fv区。
在一些实施方案中，所述双特异性抗体包含第一Fc区和第二Fc区。优选地，所述第一Fc区和第二Fc区是相同或不同的。优选地，所述Fc区选自IgG、IgA、IgD、IgE、IgM及其变体。优选地，所述Fc区选自IgG1、IgG2、IgG3、IgG4及其变体。
在一些实施方案中，所述Fc区包含一个或多个氨基酸突变，优选氨基酸置换、***或缺失。
在一些实施方案中，所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc。
在一些实施方案中，所述第一Fc区为hole-Fc，所述第二Fc区为knob-Fc。
在一些实施方案中，所述第三抗原结合部分的VH和VL互换。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链的N端，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分Fab区，第三抗原结合部分的VH和VL互换。
在一些实施方案中，所述第二抗原结合部分融合到第一抗原结合部分的一条重链的N端，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分的Fv区，第三抗原结合部分的VH和VL互换。
在一些实施方案中，所述第二抗原结合部分和第三抗原结合部分通过连接子与所述第一抗原结合部分融合。
在一些实施方案中，所述连接子为肽连接子。优选地，所述肽连接子为GS连接子或突变人类IgG铰链。优选地，所述肽连接子具有如(G4S)x所示的氨基酸序列，x为选自1-6的整数；优选地，所述肽连接子为(G4S)2、(G4S)3或(G4S)4。更优选地，所述肽连接子为(G4S)2。
在一些实施方案中，所述第一抗原结合部分特异性结合BAFFR，其中，所述第一抗原结合部分的HCDR1如SEQ ID NO:3所示，或为与SEQ ID NO:3具有至少80％同一性的序列；HCDR2如SEQ ID NO:4所示，或为与SEQ ID NO:4具有至少80％同一性的序列；HCDR3如SEQ ID NO:5所示，或为与SEQ ID NO:5具有至少80％同一性的序列；LCDR1如SEQ ID NO:6所示，或为与SEQ ID NO:6具有至少80％同一性的序列；LCDR2如SEQ ID NO:7所示，或为与SEQ ID NO:7具有至少80％同一性的序列；LCDR3如SEQ ID NO:8所示，或为与SEQ ID NO:8具有至少80％同一性的序列。
在一些实施方案中，所述第二抗原结合部分特异性结合BAFFR，其中，所述第二抗原结合部分的HCDR1如SEQ ID NO:3所示，或为与SEQ ID NO:3具有至少80％同一性的序列；HCDR2如SEQ ID NO:4所示，或为与SEQ ID NO:4具有至少80％同一性的序列；HCDR3如SEQ ID NO:5所示，或为与SEQ ID NO:5具有至少80％同一性的序列；LCDR1如SEQ ID NO:6所示，或为与SEQ ID NO:6具有至少80％同一性的序列；LCDR2如SEQ ID NO:7所示，或为与SEQ ID NO:7具有至少80％同一性的序列；LCDR3如SEQ ID NO:8所示，或为与SEQ ID NO:8具有至少80％同一性的序列。
在一些实施方案中，所述第三抗原结合部分特异性结合CD3，其中，所述第三抗原结合部分的HCDR1如SEQ ID NO:11所示，或为与SEQ ID NO:11具有至少80％同一性的序列；HCDR2如SEQ ID NO:12所示，或为与SEQ ID NO:12具有至少80％同一性的序列；HCDR3如SEQ ID NO:13所示，或为与SEQ ID NO:13具有至少80％同一性的序列；以及，LCDR1如SEQ ID NO:14所示，或为与SEQ ID NO:14具有至少80％同一性的序列；LCDR2如SEQ ID NO:15所示，或为与SEQ ID NO:15具有至少80％同一性的序列；LCDR3如SEQ ID NO:16所示，或为与SEQ ID NO:16具有至少80％同一性的序列。
在一些实施方案中，第一抗原结合部分特异性结合BAFFR，其中，所述第一抗原结合部分的重链可变区VH如SEQ ID NO:1所示，或为与SEQ ID NO:1具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:2所示，或为与SEQ ID NO:2具有至少80％同一性的序列。
在一些实施方案中，第二抗原结合部分特异性结合BAFFR，其中，所述第二抗原结合部分的重链可变区VH如SEQ ID NO:1所示，或为与SEQ ID NO:1具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:2所示，或为与SEQ ID NO:2具有至少80％同一性的序列。
在一些实施方案中，所述第三抗原结合部分特异性结合CD3，其中，所述第三抗原结合部分的重链可变区VH如SEQ ID NO:9所示，或为与SEQ ID NO:9具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:10所示，或为与SEQ ID NO:10具有至少80％同一性的序列。
本发明还提供一种分离的核酸分子，其包含编码上述任一项所述的双特异性抗体的核苷酸序列。
本发明还提供上述任一项所述的双特异性抗体在制备治疗癌症的药物中的用途。在一些实施方案中，所述癌选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、***、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促***增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、***状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、***癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌或子宫癌。
本发明还提供上述任一项所述的双特异性抗体在制备用于治疗自身免疫性疾病的药物中的用途。在一些实施方案中，所述自身免疫性疾病选自移植物抗宿主病、类风湿性关节炎、克罗恩病、多发性硬化症、结肠炎、牛皮癣、自身免疫性葡萄膜炎、天疱疮、大疱性表皮松解症或I型糖尿病。
在一些实施方案中，所述用途通过肿瘤免疫疗法、细胞疗法或基因疗法中的一种或多种来实现。
本发明还提供一种药物组合物，其包含上述任一项所述的双特异性抗体和药学上可接受的载体、稀释剂或赋形剂。
本发明还提供一种抗体药物偶联物，其包含上述任一项所述的双特异性抗体。在一些实施方案中，所述偶联药物选自细胞毒素、小分子化学药物或免疫毒素。
缩写及术语定义
在本文中使用以下缩写。VH：抗体重链可变区；VL：抗体轻链可变区；CDR：免疫球蛋白可变区中的互补决定区；IgG：免疫球蛋白G。
术语“抗体”是指天然的免疫球蛋白或者通过部分或完全合成而制备的免疫球蛋白。抗体可从天然存在该抗体的血浆或血清等的天然资源、或者产生抗体的杂交瘤细胞的培养上清中、动物免疫血清中、噬菌体文库筛选进行重建得到分离。备选地，可通过使用基因重组等的技术部分或完全地合成。优选的抗体包括，例如，免疫球蛋白的同种型或这些同种型的亚类的抗体。已知人免疫球蛋白包括IgGl、IgG2、IgG3、IgG4、IgAl、IgA2、IgD、IgE、IgM这9种类别(同种型)。在这些同种型中，本发明的抗体可以包括IgGl、IgG2、IgG3和/或IgG4。
本文中所使用的部分抗体由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成，或者只有轻链恒定区(CL)。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。
术语“抗原结合部分”或“抗原结合片段”是指抗体的一个或多个保留结合所述抗体结合的抗原的能力的部分。抗体的“抗原结合片段”的实例包括(1)Fab片段；(2)F(ab′)2片段；(3)Fd片段，由VH和CH1结构域组成；(4)Fv片段；(5)dAb片段，由VH结构域组成；(6)CDR，经分离互补决定区。
此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的连接子连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv))。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选的片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗原结合片段还可并入至包含一对串联Fv片段(VH-CH1-VH-CH1)的单链分子中，该对串联Fv片段连同互补轻链多肽一起形成一对抗原结合区。
术语“Fab片段”由完整的L链以及H链的可变区结构域(VH)和一条重链的第一恒定域(CH1)组成。各Fab片段对于抗原结合是单价的，即，其具有单一抗原结合位点。例如，可以重组产生或通过全长抗体的木瓜蛋白酶消化产生Fab片段。
术语“Fab'片段”不同于Fab片段之处在于在CH1域的羧基末端处添加了几个额外的残基，包括
来自抗体铰链区的一个或多个半胱氨酸。Fab'可以通过用还原剂例如二硫苏糖醇处理特异性识别并结合抗原的F(ab')2来生产。
术语“F(ab')2片段”最初作为在其间具有铰链半胱氨酸的Fab'片段对而产生。可以重组或通过胃蛋白酶消化完整的抗体(其除去大部分Fc区同时保留完整铰链区的部分)产生F(ab')2片段。通过用还原剂如β-巯基乙醇处理可以将F(ab')2片段解离(成两个Fab'分子)。
术语“scFab”是指单链Fab片段，在重链可变域(VH)和轻链(CL)之间引入多肽连接子，形成单链Fab片段(scFab)。
术语“Fv”是含有完整抗原识别和结合位点的最小抗体片段。该片段由一个重链可变区结构域与一个轻链可变区结构域通过紧密的非共价结合形成的二聚体组成。这两个结构域的折叠产生六个高变环(3个环来自H链并且3个环来自L链)，这些高变环贡献了用于抗原结合的氨基酸残基并且赋予抗体以抗原结合特异性。然而，即使单个可变域具有识别并结合抗原的能力，但与完整结合位点相比其亲和力较低。
术语“scFv”片段是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单一多肽链中。所述Fv多肽可以进一步在VH与VL结构域之间包含多肽连接子，所述多肽连接子使scFv能够形成抗原结合所希望的结构。“scFv-Fc”片段包含连接于Fc结构域的scFv。例如，Fc结构域可以连接到scFv的C末端。取决于scFv中可变结构域的取向(即VH-VL或VL-VH)，Fc结构域可以在VH或VL后。Fc结构域可以是本领域已知的或本文所述的任何合适的Fc结构域。在一些情况下，Fc结构域是IgG1Fc结构域。
术语“dsFv”是指二硫键稳定型Fv片段。在dsFv中，每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连接。为产生此类分子，将VH和VL的框架区中的各一个氨基酸突变为半胱氨酸，其反过来形成稳定的链间二硫键。典型地，VH中的位置44和VL中的位置100突变为半胱氨酸。所述术语dsFv涵盖本领域中已知的dsFv(其中VH和VL通过链间二硫键而不是连接子肽连接的分子)或scdsFv(其中VH和VL通过连接子和链间二硫键连接的分子)两者。
术语“表位”是指抗原中的抗原决定簇，并且是指在本说明书中公开的包含抗体可变区的抗原结合分子的结构域所结合的抗原位点。因此，可以根据其结构来定义表位。另外，也可以根据识别该表位的抗原结合分子中的抗原结合活性来定义该表位。当抗原是肽或多肽时，表位可以由形成表位的氨基酸残基指定；当表位是糖链时，表位可以通过其特定的糖链结构来确定。
术语“特异性”表示参与特异性结合的分子之一不显示任何与不同于结合伙伴分子中的一个或数个的分子的显著结合。此外，在含抗体可变区的结构域对抗原中的多个表位中的特定表位具有特异性时，也使用该术语。当含抗体可变区的结构域所结合的表位被包含在数个不同抗原中时，包含含抗体可变区的结构域的抗原结合分子可以结合具有所述表位的各种抗原。
术语“双特异性抗体”是指能够与两个目标抗原或目标抗原表位特异性结合的蛋白分子。在本发明中，包含抗体或抗原结合片段(例如Fab、scFv等)的“双特异性抗原结合蛋白”与“双特异性抗体”、“双抗”可以互换使用。
术语“knob-Fc”是指用具有更大侧链体积的氨基酸残基取代所述Fc结构域的第一亚基的CH3结构域中的氨基酸残基，从而在所述第一亚基的CH3结构域内产生可定位在所述第二亚基的CH3结构域内的凹陷中的凸起。例如，通过将一条重链的CH3第366位丝氨酸T突变为色氨酸W，形成一个突起的类似“杵”的凸起。
术语“hole-Fc”是指用具有更小侧链体积的氨基酸残基取代所述Fc结构域的第二亚基的CH3结构域中的氨基酸残基，从而在所述第二亚基的CH3结构域内产生所述第一亚基的CH3结构域内的凸起可定位在其中的凹陷。例如，通过将另外一条重链的第366位丝氨酸T突变为丝氨酸S，第368位亮氨酸L突变为丙氨酸A，第407位氨基酸由酪氨酸Y突变为缬氨酸V或突变为丙氨酸A，突变后形成一个凹陷的类似“臼”的凹陷。
术语“融合”是指通过连接子等技术方式将两段氨基酸序列连接组成一条新序列，从而形成新的人工合成蛋白或抗体。
术语“连接子(Linker)”或用于连接两个蛋白质结构域中间的“L1”指连接性多肽序列，用于连接蛋白质结构域，具有一定的柔性，连接子的使用不会使蛋白质结构域原有的功能丧失。
术语抗体的“可变区”或“可变域”是指单独的或组合的抗体轻链的可变区(VL)或抗体重链的可变区(VH)。如在本领域中已知的，重链和轻链的可变区各自由通过3个互补决定区(CDR)(也称为高变区)连接的4个框架区(FR)组成。每一条链中的CDR通过FR紧密地保持在一起并且与来自另一条链的CDR一起促成抗体的抗原结合部位的形成。
术语“可变”是指以下事实：可变域的某些区段在抗体之间在序列上广泛不同。V结构域介导抗原结合并限定特定抗体对于其特定抗原的特异性。然而，可变性在整个可变域范围上并非均匀分布的。相反，它集中于轻链与重链可变域内三个称为高变区(HVR)的区段中。可变域的更高度保守部分被称作框架区(FR)。天然重链与轻链的可变域各自包含四个FR区，大部分采用β-折叠构型，由三个HVR连接，其形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的HVR通过FR区紧密保持在一起，并且与其它链的HVR一起促成抗体的抗原结合位点的形成。恒定域不直接牵涉于抗体与抗原的结合中，但展现出各种效应功能，例如参与抗体的抗体依赖性细胞毒性。
术语“抗体药物偶联物”或“ADC”是指与一个或多个偶联药物(其可以任选地是治疗剂或细胞毒性剂)连接的结合蛋白(如抗体或其抗原结合片段)，其结构通常由三部分组成：抗体或抗体类配体、药物部分、以及将抗体或抗体类配体及药物偶联起来的连接子。ADC通常具有与抗体偶联的1、2、3、4、5、6、7、8、9或10个数的药物。
术语“多肽”是指任何长度的氨基酸链，而与修饰(例如磷酸化或糖基化)无关。术语多肽包括蛋白质及其片段。多肽可以是“外源的”，意指它们是“异源的”，即是所利用的宿主细胞外来的，例如由细菌细胞产生的人多肽。本文将多肽公开为氨基酸残基序列。那些序列按氨基末端到羧基末端的方向从左到右书写。根据标准命名法，氨基酸残基序列以三字母或单字母代码命名。
术语“氨基酸”是指二十种常见的天然存在的氨基酸。在一些实施方案中，术语“氨基酸”还包括非天然氨基酸。可以使用任何合适的非天然氨基酸。在一些实施方案中，非天然氨基酸包含用于将药剂与MIAC缀合的反应性部分。
术语“同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST软件或FASTA程序包。
术语“至少80％同一性”是指候选序列中与对照多肽序列中的氨基酸残基相 同的氨基酸残基的百分率为80％以上，包括80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。
术语“氨基酸突变”是指与原蛋白质或多肽相比，变体蛋白质或多肽存在氨基酸的突变或改变，包括在原蛋白质或多肽的基础上发生一个或多个氨基酸的***、缺失或替换。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达，本文称为“表达载体”。
术语“药学上可接受的载体”包括任何标准药物载体，诸如磷酸盐缓冲盐水溶液、水和乳液，以及各种类型的润湿剂。
术语“阳性对照”是指能够结合靶点蛋白或是表达靶点蛋白的天然或工程化细胞或抗体，本文所指的阳性对照是指单靶点阳性对照。
术语“阴性对照”是指在同一实验中，使用与实验样品相同种属来源、相同亚型、相同剂量、相同的免疫球蛋白及亚型的免疫球蛋白、相同标记等，用于消除实验中非特异结合样品对实验数值产生的实验背景影响，作为一种更加说明实验效果的对照。
术语“亲和力”是指分子的单个结合位点(例如，MIAC的抗原结合模块)与其结合配偶体(例如，抗原)之间的非共价相互作用的总和的强度。在各抗原位点内，抗体“臂”的可变区通过弱非共价力与抗原在多个氨基酸位点处相互作用；相互作用愈大，亲和力愈强。除非另外指示，否则如本文所用的“结合亲和力”是指反映结合对的成员(例如，抗体与抗原)之间的1:1相互作用的固有结合亲和力。分子X对其搭配物Y的亲和力一般可由解离常数(Kd)表示。亲和力可通过本领域中已知的常用方法测量，例如通过使用表面等离子体共振(SPR)技术(例如仪器)或生物层干涉测量法(例如，仪器)来测量。
术语“效应细胞”是表达一个或多个FcR并执行效应功能的白细胞。在一个方面，效应细胞至少表达FcγRIII并执行ADCC效应功能。介导ADCC的人类白细胞的实例包括外周血单核细胞(PBMC)、自然杀手(NK)细胞、单核细胞、细胞毒性T细胞和嗜中性白细胞。效应细胞可从天然来源(例如血液)中分离。效应细胞一般是与效应期相关的淋巴细胞，并且用于产生细胞因子(辅助T细胞)、杀灭感染病原体的细胞(细胞毒性T细胞)或分泌抗体(分化的B细胞)。
本领域中有多种方法/***来定义和描述CDR，这些***和/或定义已经开发和精制多年，包括Kabat、Chothia、IMGT、AbM和Contact。Kabat是最常用的，基于序列变异性定义CDR；Chothia基于结构循环区域的位置基于序列变异性定义CDR；IMGT***基于可变域结构内的序列变异性和位置定义CDR；AbM是基于牛津分子公司的AbM抗体建模软件进行定义，是Kabat和Chothia之间的折衷；Contact基于对复杂晶体结构的分析定义CDR，在多个方面与Chothia类 似。除非特殊说明，否则本文使用Kabat定义CDR。
附图说明
图1为描述示例性双特异性抗体，能够特异性识别第一抗原(BAFFR)的全长抗体与第二抗原(BAFFR)结合部分、第三抗原(CD3)结合部分融合，所述第二抗原结合部分是Fab，第二抗原结合部分融合到第一抗原结合部分的一条重链的N端，所述第三抗原结合部分是scFv，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分Fab区，双特异性抗体的第一Fc区为knob-Fc，第二Fc区为hole-Fc。
图2为描述示例性双特异性抗体，能够特异性识别第一抗原(BAFFR)的全长抗体与第二抗原(BAFFR)结合部分、第三抗原(CD3)结合部分融合，所述第二抗原结合部分是Fab，第二抗原结合部分融合到第一抗原结合部分的一条重链的N端，所述第三抗原结合部分是Fab，第三抗原结合部分的VH和VL互换，所述第三抗原结合部分替换与第二抗原结合部分相融合的第一抗原结合部分Fab区，双特异性抗体的第一Fc区为knob-Fc，第二Fc区为hole-Fc。
图3为双特异性抗体C的结构，能够特异性识别第一抗原(BAFFR)的全长抗体与第二抗原(CD3)结合部分融合，所述第二抗原结合部分是Fab，第二抗原结合部分的VH和VL互换，所述第二抗原结合部分替换第一抗原结合部分的一侧Fab区，双特异性抗体的第一Fc区为knob-Fc，第二Fc区为hole-Fc。
图4为双特异性抗体对BAFFR蛋白结合活性。
图5为双特异性抗体对CD3蛋白结合活性。
图6为双特异性抗体对Jeko-1细胞结合活性。
图7为双特异性抗体对Raji细胞结合活性。
图8为双特异性抗体对Su-DHL4细胞结合活性。
图9为双特异性抗体对jukart-NFAT-luc报告基因细胞的T细胞特异激活活性。
图10为双特异性抗体对CHO-K1-BAFFR细胞的杀伤活性。
图11为双特异性抗体对CHO-K1细胞的杀伤活性。
图12为双特异性抗体对Raji-luc细胞的杀伤活性。
具体实施方式
以下结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。还应该理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求及其任何等同物为本发明的保护范围。
实施例1核苷酸序列的获得与优化
实施例为针对BAFFR、CD3靶点，根据图1-3构建双特异性抗体，依次命名为抗体A、抗体B、抗体C。
根据需要，调整所述抗体氨基酸序列的Fc为其他IgG类型，并进一步在各重链中设计所需形式的氨基酸突变，由此得到目标抗体的氨基酸序列(见表1)，构建的抗体氨基酸序列组合见表2并包含理论分子量。
表1抗体序列信息
表2抗体的序列组合
将上述各目标氨基酸序列转化为核苷酸序列，并针对可能影响抗体在哺乳动物细胞中表达的一系列参数：密码子偏好性、GC含量(即DNA的4种碱基中鸟嘌呤G和胞嘧啶C所占的比率)、CpG岛(即CpG双核苷酸在基因组中密度较高的区域)、mRNA的二级结构、拼接位点、前成熟PolyA位点、内部Chi位点(基因组中一段短的DNA片段，在该位点附近发生同源重组的几率增加)或者核糖体结合位点、RNA不稳定序列、反向重复序列及可能干扰克隆的限制性酶切位点等进行优化；同时增加了可能会提高翻译效率的相关序列，例如Kozak序列、SD序列。设计得到分别编码上述抗体的重链基因和轻链基因，另外在重链和轻链的5’端分别设计根据氨基酸序列优化而得的编码信号肽的核苷酸序列；此外，还对轻链和重链核苷酸序列的3’端分别加上终止密码子。
实施例2基因合成与表达载体的构建
采用pcDNA3.1-G418载体作为表达所述多功能抗体的质粒载体。pcDNA3.1-G418载体含有启动子CMVPromoter、真核筛选标记G418标签和原核筛选标签Ampicilline。基因合成得到构建抗体表达轻链和重链的核苷酸序列，用HindIII和XhoI对载体和目的片段进行双酶切，回收后通过DNA连接酶进行酶连，并转化大肠杆菌感受态细胞DH5α，挑选出阳性克隆并进行质粒提取和酶切验证，获得含所述抗体质粒。
实施例3质粒抽提
将含有上述各目的基因的重组质粒转化至大肠杆菌感受态细胞DH5α中，将转化细菌涂布在含100μg/mL氨苄青霉素的LB平板上培养，挑选质粒克隆至液体LB培养基中培养，260rpm摇菌14小时，由无内毒素质粒大抽试剂盒抽提质粒，用无菌水溶解并用核酸蛋白定量仪进行浓度测定。
实施例4质粒转染、瞬转表达与抗体纯化
在37℃、8％CO2、100rpm下培养Expi CHO至细胞密度6×106个/mL。使用脂质体将构建的质粒按照组合配对转染到上述细胞中，转染质粒浓度为1mg/mL，脂质体体积参照Expi CHOTM Expression System试剂盒确定，在32℃、5％CO2，100rpm下培养7-10天。转染18-22h之后和第5天之间分别补料一次。4000g离心上述培养产物，0.22μm滤膜过滤并收集培养基上清液，采用Protein A、离子柱纯化所得的抗体蛋白并收集洗脱液。
Protein A、离子柱纯化的具体操作步骤为：细胞培养液经过高速离心后取上清，利用GE的Protein A层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后利用洗脱缓冲液0.1M甘氨酸(pH3.0)洗脱目的蛋白，利用Tris调节pH至中性保存。将亲和层析所得产物调节pH至低于或者高于pI1-2个pH单位，适当稀释以控制样本电导在5ms/cm以下。利用合适的对应pH缓冲液如磷酸缓冲液、醋酸缓冲液等条件，利用本领域内常规的离子交换层析方法如阴离子交换或者阳离子交换进行对应pH条件下NaCl梯度洗脱，根据SDS-PAGE选择目的蛋白所在的收集管合并保存。
然后，将纯化后所得的洗脱液超滤换液至缓冲液中。通过SDS-聚丙烯酰胺凝胶电泳测定检测蛋白质。
经SDS-PAGE测定证明，非还原胶条件含有目的条带，还原胶下目标抗体均含有目的条带，对应于所需抗体的重链以及轻链。因此，经所述质粒转染、瞬转表达和纯化，证明得到结构正确抗体。
实施例5 ELISA检测抗体对BAFFR蛋白的亲和力
采用pH7.4的PBS缓冲液将Human-BAFFR-His(购于Acro，Cat：BAR-H52H3)稀释至1μg/mL，每孔100μL加入到96孔ELISA板中，4℃包被过夜。用1％BSA封闭液封闭1小时后。PBST洗板3次后，将构建抗体用0.5％BSA样品稀释液稀释至100nM，以此为起始浓度，进行3倍梯度稀释，共11个梯度，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的山羊抗人IgG-Fc(购于Jackson，Cat：109-035-098)用样品稀释液按1:20000稀释，每孔加入100μL，室温孵育1小时。设置阴性对照(无关抗体)与阳性对照，阳性对照为BAFFR单抗(BAFFR单抗序列由SEQ ID NO:1、SEQ ID NO:2组成，加入了人IgG1的恒定区，见SEQ ID NO:27和SEQ ID NO:28)，PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL 1M HCl液终止显色反应。在多功能酶标仪上读数。
抗体的ELISA结果如图4所示，双特异性抗体在各浓度下均可与BAFFR蛋白结合。
实施例6 ELISA检测抗体对CD3的亲和力
采用pH7.4的PBS缓冲液将Human-CD3-His(购于Acro，Cat：CDD-H52W4)稀释至0.2μg/mL，每孔100μL加入到96孔ELISA板中，4℃包被过夜。用1％的BSA封闭液封闭1小时后。PBST洗板3次后，将构建的表达抗体用0.5％的BSA样品稀释液稀释至100nM，以此为起始浓度，进行3倍梯度稀释，共11个梯度，并设阴性对照(空白孔与IgG1同型对照)与阳性对照，阳性对照为CD3单抗(CD3抗体序列来源于INN blinatumomab，序列由SEQ ID NO:9、SEQ ID NO:10组成，加入了人IgG1的恒定区，IgG1的恒定区见SEQ ID NO:27和SEQ ID NO:28)，每孔100μL，37℃孵育1h。再用PBST洗板3次，将HRP标记的山羊抗人IgG-Fc用样品稀释液按1:20000稀释，每孔加入100μL，室温孵育1小 时。PBST洗板4次后，每孔加入100μL TMB底物，室温避光孵育10分钟，每孔加入100μL 1M HCl液终止显色反应。在多功能酶标仪上选择波长450nm，参比波长570nm测定96孔板中各孔的吸光值。
抗体的ELISA结果如图5所示，双特异性抗体在各浓度下均可与CD3结合。
实施例7流式细胞术检测抗体对BAFFR阳性肿瘤细胞的结合活性
取对数期生长、形态正常的BAFFR阳性肿瘤细胞Jeko-1、Raji、Su-DHL4(来源于中科院细胞保藏中心)，转至离心管1000rpm离心5min，稀释液重悬细胞后，取3×105/孔加入96孔细胞培养板中。将纯化得到的抗体用FACS缓冲液稀释至20μg/mL，以此为起始浓度，进行3倍梯度稀释，共6个梯度，并设无关抗体阴性对照与阳性对照，阳性对照为BAFFR单抗(BAFFR单抗序列由SEQ ID NO:1、SEQ ID NO:2组成，加入了人IgG1的恒定区，人IgG1的恒定区见SEQ ID NO:27和SEQ ID NO:28)，并添加100μL抗体稀释液。将细胞在4℃下孵育60分钟，然后用过量FACS缓冲液洗涤两次。将细胞重新悬浮于100μL FACS缓冲液中，并将抗人IgG Fc的荧光二抗-FITC(Biolegend，Cat：109306)添加到样品中，孵育30分钟并用过量FACS缓冲液洗涤两次。将细胞在流式缓冲液中重悬，随后通过流式细胞仪检测并进行分析。
抗体与BAFFR阳性肿瘤细胞Jeko-1、Raji、SuDHL4的细胞结合活性的FACs检测结果如图6-8所示，双特异性抗体A和抗体B在多浓度范围下均可与BAFFR阳性肿瘤细胞特异结合。
实施例8构建抗体对T细胞的特异激活
使用BAFFR阳性的Jeko-1细胞(来源于中科院细胞保藏中心)以2×104/孔铺于96孔板，而后按5×104/孔加入效应细胞Jurkat-NFAT-Luc。抗体使用PBS稀释至30μg/mL起始，5倍稀释，共9个浓度梯度，加入相应细胞的孔板里。设置阴性无关对照组，37℃培养6h。而后加入Bio-Lite至样品孔中，室温孵育10min后多功能酶标仪读数。
如图9所示，实验结果表明双特异性抗体A和抗体B在BARRF阳性的靶细胞Jeko-1存在下，特异激活表达CD3的jurkart-NFAT-Luc报告基因细胞。而无抗体或无靶标细胞存在情况下，无激活活性。表明双特异性抗体A和抗体B识别BAFFR阳性细胞后，可特异性激活CD3阳性的T细胞，继而激活诱导T细胞杀伤肿瘤的下游信号。双特异性抗体C存在非特异性激活，存在风险。
实施例9构建抗体对CHO-K1-BAFFR细胞的特异性杀伤
使用BAFFR阳性的CHO-K1-BAFFR细胞(BAFFR慢病毒转导CHO-K1细胞构建而成的BAFFR过表达工程细胞株)以及BAFFR表达阴性的CHO-K1以2×104/孔铺于96孔板，培养24h后，加入双特异性抗体和无关抗体，10μg/mL起始，10倍稀释，共6个浓度梯度，同时加入CIK(CD3+CD56+细胞)效应细胞1×105/孔，效靶比为5:1，并设置空白对照(稀释液)、阴性对照(靶标细胞+CIK细胞，无抗体)、无关抗体组。阴性对照组中阴性对照1为BAFFR单抗(BAFFR单抗序列由SEQ ID NO:1、SEQ ID NO:2组成，加入了人IgG1的恒定区，人IgG1的恒定区见SEQ ID NO:27和SEQ ID NO:28)，阴性对照2为抗GPC3和CD3抗体(抗体来源于专利US11001643B2，氨基酸序列由SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:31组成)。于细胞培养箱孵育24h后，PBS润洗3 次，加入含10％CCK-8(Cat：CK04)培养基100μL，于培养箱孵育3h后，于酶标仪OD值450nm进行检测。计算细胞杀伤率，公式如下：
细胞杀伤率(％)＝(1-(样品孔OD值-空白OD值)/(阴性孔OD值-空白
OD值))×100％。
由图10-11可知，双特异性抗体可杀伤BAFFR过表达的CHO-K1-BAFFR，无关抗体不具有杀伤作用，而双特异性抗体与无关抗体均对CHO-K1无杀伤作用，表明双特异性抗体可介导CIK细胞特异性杀伤BAFFR阳性的细胞。
实施例10构建抗体对Raji-luc细胞的特异性杀伤
使用BAFFR阳性的Raji-luc(Luc慢病毒转导raji而成)以1×104/孔铺于96孔板，培养24h后，加入双特异性抗体和无关抗体，50μg/mL起始，10倍稀释，共6个浓度梯度，同时加入CIK(CD3+CD56+细胞)效应细胞1×105/孔，效靶比为10:1，并设置空白对照(稀释液)、阴性对照(靶标细胞+CIK细胞，无抗体)、无关抗体组。阴性对照组中阴性对照1为BAFFR单抗(BAFFR单抗序列由SEQ ID NO:1、SEQ ID NO:2组成，加入了人IgG1的恒定区，IgG1的恒定区见SEQ ID NO:27和SEQ ID NO:28)，阴性对照2为抗GPC3和CD3双抗(抗体序列来源于专利US11001643B2，序列由SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:31组成)。于细胞培养箱孵育24h后，加入Bio-Lite，于培养箱孵育10min后，于酶标仪OD值450nm进行检测。计算细胞杀伤率，公式如下：
细胞杀伤率(％)＝(1-(样品孔OD值-空白OD值)/(阴性孔OD值-空白
OD值))×100％。
由图12可知，双特异性抗体可杀伤BAFFR阳性的Raji，无关抗体不具有杀伤作用，表明双特异性抗体介导CIK细胞特异性杀伤BAFFR阳性的细胞。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (33)

  1. 一种双特异性抗体，其特征在于，所述双特异性抗体包含：
    (a)特异性结合第一抗原的第一抗原结合部分，所述第一抗原为BAFFR；
    (b)特异性结合第二抗原的第二抗原结合部分，所述第二抗原为BAFFR；
    (c)特异性结合第三抗原的第三抗原结合部分，所述第三抗原为CD3。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，第一抗原结合部分是由两条重链和两条轻链组成的全长抗体。
  3. 根据权利要求1或2所述的双特异性抗体，其特征在于，所述第二抗原结合部分和/或第三抗原结合部分是包含重链可变域(VH)和/或轻链可变域(VL)的抗体片段。
  4. 根据权利要求3所述的双特异性抗体，其特征在于，所述第二抗原结合部分和/或第三抗原结合部分是Fab、Fab'、scFab、F(ab')2、Fv、dsFv或scFv。
  5. 根据权利要求1-4任一项所述的双特异性抗体，其特征在于，所述第二抗原结合部分融合到第一抗原结合部分的一条重链或两条重链的N端和/或C端。
  6. 根据权利要求1-4任一项所述的双特异性抗体，其特征在于，所述第二抗原结合部分融合到第一抗原结合部分的一条轻链或两条轻链的N端和/或C端。
  7. 根据权利要求5或6所述的双特异性抗体，其特征在于，所述第三抗原结合部分替换第一抗原结合部分的一个或两个Fab区或Fv区。
  8. 根据权利要求1-7任一项所述的双特异性抗体，其特征在于，所述双特异性抗体包含第一Fc区和第二Fc区。
  9. 根据权利要求8所述的双特异性抗体，其特征在于，所述第一Fc区和第二Fc区是相同或不同的。
  10. 根据权利要求8或9所述的双特异性抗体，其特征在于，所述Fc区选自IgG、IgA、IgD、IgE、IgM及其变体。
  11. 根据权利要求10所述的双特异性抗体，其特征在于，所述Fc区选自IgG1、IgG2、IgG3、IgG4及其变体。
  12. 根据权利要求10或11所述的双特异性抗体，其特征在于，所述Fc区包含一个或多个氨基酸突变，优选氨基酸置换、***或缺失。
  13. 根据权利要求8-12任一项所述的双特异性抗体，其特征在于，所述第一Fc区为knob-Fc，所述第二Fc区为hole-Fc。
  14. 根据权利要求8-12任一项所述的双特异性抗体，其特征在于，所述第一Fc区为hole-Fc，所述第二Fc区为knob-Fc。
  15. 根据权利要求1-14任一项所述的双特异性抗体，其特征在于，所述第三抗原结合部分的VH和VL互换。
  16. 根据权利要求1-15任一项所述的双特异性抗体，其特征在于，所述第二抗原结合部分和第三抗原结合部分通过连接子与所述第一抗原结合部分融合。
  17. 根据权利要求16所述的双特异性抗体，其特征在于，所述连接子为肽连接子。
  18. 根据权利要求17所述的双特异性抗体，其特征在于，所述肽连接子为GS连接子或突变人类IgG铰链。
  19. 根据权利要求1-18任一项所述的双特异性抗体，其特征在于，所述第一抗原结合部分特异性结合BAFFR，其中，所述第一抗原结合部分的HCDR1如SEQ ID NO:3所示，或为与SEQ ID NO:3具有至少80％同一性的序列；HCDR2如SEQ ID NO:4所示，或为与SEQ ID NO:4具有至少80％同一性的序列；HCDR3如SEQ  ID NO:5所示，或为与SEQ ID NO:5具有至少80％同一性的序列；LCDR1如SEQ ID NO:6所示，或为与SEQ ID NO:6具有至少80％同一性的序列；LCDR2如SEQ ID NO:7所示，或为与SEQ ID NO:7具有至少80％同一性的序列；LCDR3如SEQ ID NO:8所示，或为与SEQ ID NO:8具有至少80％同一性的序列。
  20. 根据权利要求1-19任一项所述的双特异性抗体，其特征在于，所述第二抗原结合部分特异性结合BAFFR，其中，所述第二抗原结合部分的HCDR1如SEQ ID NO:3所示，或为与SEQ ID NO:3具有至少80％同一性的序列；HCDR2如SEQ ID NO:4所示，或为与SEQ ID NO:4具有至少80％同一性的序列；HCDR3如SEQ ID NO:5所示，或为与SEQ ID NO:5具有至少80％同一性的序列；LCDR1如SEQ ID NO:6所示，或为与SEQ ID NO:6具有至少80％同一性的序列；LCDR2如SEQ ID NO:7所示，或为与SEQ ID NO:7具有至少80％同一性的序列；LCDR3如SEQ ID NO:8所示，或为与SEQ ID NO:8具有至少80％同一性的序列。
  21. 根据权利要求1-20任一项所述的双特异性抗体，其特征在于，所述第三抗原结合部分特异性结合CD3，其中，所述第三抗原结合部分的HCDR1如SEQ ID NO:11所示，或为与SEQ ID NO:11具有至少80％同一性的序列；HCDR2如SEQ ID NO:12所示，或为与SEQ ID NO:12具有至少80％同一性的序列；HCDR3如SEQ ID NO:13所示，或为与SEQ ID NO:13具有至少80％同一性的序列；以及，LCDR1如SEQ ID NO:14所示，或为与SEQ ID NO:14具有至少80％同一性的序列；LCDR2如SEQ ID NO:15所示，或为与SEQ ID NO:15具有至少80％同一性的序列；LCDR3如SEQ ID NO:16所示，或为与SEQ ID NO:16具有至少80％同一性的序列。
  22. 根据权利要求19-21任一项所述的双特异性抗体，其特征在于，第一抗原结合部分特异性结合BAFFR，其中，所述第一抗原结合部分的重链可变区VH如SEQ ID NO:1所示，或为与SEQ ID NO:1具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:2所示，或为与SEQ ID NO:2具有至少80％同一性的序列。
  23. 根据权利要求19-22任一项所述的双特异性抗体，其特征在于，第二抗原结合部分特异性结合BAFFR，其中，所述第二抗原结合部分的重链可变区VH如SEQ ID NO:1所示，或为与SEQ ID NO:1具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:2所示，或为与SEQ ID NO:2具有至少80％同一性的序列。
  24. 根据权利要求19-23任一项所述的双特异性抗体，其特征在于，所述第三抗原结合部分特异性结合CD3，其中，所述第三抗原结合部分的重链可变区VH如SEQ ID NO:9所示，或为与SEQ ID NO:9具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:10所示，或为与SEQ ID NO:10具有至少80％同一性的序列。
  25. 一种分离的核酸分子，其包含编码权利要求1-24任一项所述的双特异性抗体的核苷酸序列。
  26. 权利要求1-24任一项所述的双特异性抗体在制备治疗癌症的药物中的用途。
  27. 根据权利要求26所述的用途，其特征在于，所述癌选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、***、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促***增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤 /恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、***状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、***癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌或子宫癌。
  28. 权利要求1-24任一项所述的双特异性抗体在制备用于治疗自身免疫性疾病的药物中的用途。
  29. 根据权利要求28所述的用途，其特征在于，所述自身免疫性疾病选自移植物抗宿主病、类风湿性关节炎、克罗恩病、多发性硬化症、结肠炎、牛皮癣、自身免疫性葡萄膜炎、天疱疮、大疱性表皮松解症或I型糖尿病。
  30. 根据权利要求26-29任一项所述的用途，其特征在于，所述用途通过肿瘤免疫疗法、细胞疗法或基因疗法中的一种或多种来实现。
  31. 一种药物组合物，其包含权利要求1-24任一项所述的双特异性抗体和药学上可接受的载体、稀释剂或赋形剂。
  32. 一种抗体药物偶联物，其包含权利要求1-24任一项所述的双特异性抗体。
  33. 根据权利要求32所述的抗体药物偶联物，其特征在于，所述偶联药物选自细胞毒素、小分子化学药物或免疫毒素。
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