Bispecific antibody combining BAFFR and CD3 and application thereof

Abstract

A bispecific antibody, the bispecific antibody comprising: (a) a first antigen binding portion specifically binding to a first antigen, the first antigen being BAFFR; (b) a second antigen binding portion specifically binding to a second antigen, the second antigen being BAFFR; and (c) a third antigen binding part specifically binding to a third antigen, wherein the third antigen is CD3. The bispecific antibody maximizes the tumor killing effect through multivalent binding of tumor-associated antigens while controlling CD3 toxicity: the BAFFR antibody is in multivalent binding with BAFFR positive tumors, the CD3 antibody at the other end is bound with T cells, the bispecific antibody serves as an adapter, the distance between the T cells and tumor cells is increased, immune synapses are formed, and the T cells kill the tumors; the monovalent CD3 antibody reduces toxicity, so that immune synapse can be better formed, and more effective, more specific and safer anti-tumor reaction is achieved.

Description

A bispecific antibody binding to BAFFR and CD3 and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody binding to BAFFR and CD3 and its application. Background Art

[0002] CD3 (T cell surface glycoprotein CD3, a signal transduction co-receptor of the T cell receptor, which contains subunits γ, δ, ε, and ζ) is a differentiation antigen expressed on the surface of all T lymphocytes, primarily mediating the transduction of T cell activation signals. It plays an important role in the body's anti-infection immunity. The CD3 molecule forms a stable TCR-CD3 complex with the T cell antigen receptor. Its extracellular domain recognizes and binds to major histocompatibility complex class II molecules, enhancing the stability of the binding between the T cell antigen receptor and the MHC molecule; the intracellular domain 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 body's cellular immunity.

[0003] BAFFR's sole ligand is BAFF. In addition to serving as a marker for specific B cell developmental stages, BAFFR and BAFF, when combined, activate the NF-κB signaling pathway in B cells, promoting B cell proliferation and activation. The BAFF-BAFFR signaling pathway and the BCR pathway are key pathways for immune B cell maturation and activation. Blocking both pathways can completely eliminate peripheral B lymphocytes and inhibit their development and proliferation.

[0004] Clinical studies have shown that approximately 30-60% of NHL patients who initially receive rituximab develop resistance to a single dose, and approximately 60% of relapsed patients develop resistance to a second dose of rituximab. The primary mechanism of resistance is insufficient CD20 expression or CD20 shedding from B lymphocytes. CD20 is not essential for B cell growth. In vitro studies have shown that B lymphocyte growth rates remain unchanged after CD20 knockout. BAFFR, which is expressed at a higher abundance than CD20, is a signaling pathway protein essential for B cell growth. Together with the BCR pathway, it is essential for B cell proliferation and activation. In vivo pharmacodynamic studies have shown that BAFFR-targeting antibodies C55 and C90 are as effective as rituximab in Z138 lymphoma cells, a cell line sensitive to rituximab, completely eliminating lymphoma cells from the blood and prolonging tumor-free survival in mice. For the rituximab-resistant cell line (CD20 gene knockout Raji), C90 can still completely eliminate lymphoma cells in the mouse blood and prolong the tumor-free survival of the mice to 100 days (rituximab survival time is within 20 days).

[0005] Therefore, as a downstream signaling pathway of BCR, BAFFR-targeted antibodies can effectively solve the problems of ineffectiveness and recurrent resistance to ibrutinib. However, the anti-tumor activity of the ADCC activity of monoclonal antibodies is limited. T cells have been proven to be an effective strategy as effective anti-tumor cells. Therefore, the development of T cell engager therapies that can target both T cell receptors and tumor-associated antigens (TAAs) has been widely used. 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 targeted lysis of BAFFR-positive tumor cells, thereby achieving the purpose of treating tumors.

[0006] Summary of the Invention

[0007] In the present application, 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 CD3 toxicity: BAFFR antibodies multivalently bind to BAFFR-positive tumors, and CD3 antibodies on the other end bind to T cells. The bispecific antibody acts as a connector to bring T cells and tumor cells closer, forming an immune synapse and enabling T cells to kill tumors; the monovalent CD3 antibody reduces toxicity and can better form an immune synapse, achieving a more effective, specific and safer anti-tumor response.

[0008] The present invention provides a bispecific antibody, comprising: (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.

[0009] In some embodiments, the first antigen binding moiety is a full length antibody consisting of two heavy chains and two light chains.

[0010] In some embodiments, the second antigen binding moiety is an antibody fragment comprising a heavy chain variable domain (VH) and / or a light chain variable domain (VL).

[0011] In some embodiments, the third antigen binding portion is an antibody fragment comprising a heavy chain variable domain (VH) and / or a light chain variable domain (VL).

[0012] In some embodiments, the second antigen binding moiety is Fab, Fab', scFab, F(ab')2, Fv, dsFv or scFv. Preferably, the second antigen binding moiety is Fab.

[0013] In some embodiments, the third antigen binding moiety is Fab, Fab', scFab, F(ab')2, Fv, dsFv or scFv. Preferably, the third antigen binding moiety is scFv.

[0014] In some embodiments, the second antigen binding moiety is fused to the N-terminus of one heavy chain of the first antigen binding moiety.

[0015] In some embodiments, the second antigen binding moiety is fused to the C-terminus of one heavy chain of the first antigen binding moiety.

[0016] In some embodiments, the second antigen binding moiety is fused to the N-terminus and C-terminus of one heavy chain of the first antigen binding moiety.

[0017] In some embodiments, the second antigen binding moiety is fused to the N-termini of both heavy chains of the first antigen binding moiety.

[0018] In some embodiments, the second antigen binding moiety is fused to the C-termini of both heavy chains of the first antigen binding moiety.

[0019] In some embodiments, 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.

[0020] In some embodiments, the second antigen binding moiety is fused to the N-terminus of one of the light chains of the first antigen binding moiety.

[0021] In some embodiments, the second antigen binding moiety is fused to the C-terminus of one of the light chains of the first antigen binding moiety.

[0022] In some embodiments, the second antigen binding moiety is fused to the N-terminus and C-terminus of one light chain of the first antigen binding moiety.

[0023] In some embodiments, the second antigen binding moiety is fused to the N-termini of the two light chains of the first antigen binding moiety.

[0024] In some embodiments, the second antigen binding moiety is fused to the C-termini of the two light chains of the first antigen binding moiety.

[0025] In some embodiments, 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.

[0026] In some embodiments, the third antigen binding moiety replaces one or two Fab regions of the first antigen binding moiety. Preferably, the third antigen binding moiety replaces the Fab region of the first antigen binding moiety fused to the second antigen binding moiety.

[0027] In some embodiments, the third antigen binding moiety replaces one or both Fv regions of the first antigen binding moiety. Preferably, the third antigen binding moiety replaces the Fv region of the first antigen binding moiety fused to the second antigen binding moiety.

[0028] In some embodiments, 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.

[0029] In some embodiments, 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.

[0030] In some embodiments, the bispecific antibody comprises a first Fc region and a second Fc region. Preferably, the first Fc region and the second Fc region are the same or different. Preferably, the Fc region is selected from IgG, IgA, IgD, IgE, IgM, and variants thereof. Preferably, the Fc region is selected from IgG1, IgG2, IgG3, IgG4, and variants thereof.

[0031] In some embodiments, the Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

[0032] In some embodiments, the first Fc region is knob-Fc and the second Fc region is hole-Fc.

[0033] In some embodiments, the first Fc region is hole-Fc and the second Fc region is knob-Fc.

[0034] In some embodiments, the VH and VL of the third antigen binding moiety are swapped.

[0035] In some embodiments, 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, and the VH and VL of the third antigen binding moiety are interchanged.

[0036] In some embodiments, 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, and the VH and VL of the third antigen binding moiety are interchanged.

[0037] In some embodiments, the second and third antigen binding moieties are fused to the first antigen binding moiety via a linker.

[0038] In some embodiments, the linker is a peptide linker. Preferably, the peptide linker is a GS linker or a mutant human IgG hinge. Preferably, the peptide linker has a structure such as (G4S) x The amino acid sequence shown, 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.

[0039] In some embodiments, the first antigen binding portion specifically binds BAFFR, wherein HCDR1 of the first antigen binding portion is as shown in SEQ ID NO:3, or a sequence that is at least 80% identical to SEQ ID NO:3; HCDR2 is as shown in SEQ ID NO:4, or a sequence that is at least 80% identical to SEQ ID NO:4; HCDR3 is as shown in SEQ ID NO:5, or a sequence that is at least 80% identical to SEQ ID NO:5; LCDR1 is as shown in SEQ ID NO:6, or a sequence that is at least 80% identical to SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence that is at least 80% identical to SEQ ID NO:7; and LCDR3 is as shown in SEQ ID NO:8, or a sequence that is at least 80% identical to SEQ ID NO:8.

[0040] In some embodiments, the second antigen binding portion specifically binds BAFFR, wherein HCDR1 of the second antigen binding portion is as set forth in SEQ ID NO:3, or a sequence at least 80% identical to SEQ ID NO:3; HCDR2 is as set forth in SEQ ID NO:4, or a sequence at least 80% identical to SEQ ID NO:4; HCDR3 is as set forth in SEQ ID NO:5, or a sequence at least 80% identical to SEQ ID NO:5; LCDR1 is as set forth in SEQ ID NO:6, or a sequence at least 80% identical to SEQ ID NO:6; LCDR2 is as set forth in SEQ ID NO:7, or a sequence at least 80% identical to SEQ ID NO:7; and LCDR3 is as set forth in SEQ ID NO:8, or a sequence at least 80% identical to SEQ ID NO:8.

[0041] In some embodiments, the third antigen binding portion specifically binds CD3, wherein the HCDR1 of the third antigen binding portion is as shown in SEQ ID NO:11, or a sequence at least 80% identical to SEQ ID NO:11; HCDR2 is as shown in SEQ ID NO:12, or a sequence at least 80% identical to SEQ ID NO:12; HCDR3 is as shown in SEQ ID NO:13, or a sequence at least 80% identical to SEQ ID NO:13; and LCDR1 is as shown in SEQ ID NO:14, or a sequence at least 80% identical to SEQ ID NO:14; LCDR2 is as shown in SEQ ID NO:15, or a sequence at least 80% identical to SEQ ID NO:15; and LCDR3 is as shown in SEQ ID NO:16, or a sequence at least 80% identical to SEQ ID NO:16.

[0042] In some embodiments, 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.

[0043] In some embodiments, 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 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.

[0044] In some embodiments, 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 to 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 to SEQ ID NO:10.

[0045] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-described bispecific antibodies.

[0046] The present invention also provides the use of any of the above-mentioned bispecific antibodies in the preparation of a drug for treating cancer. In some embodiments, the cancer is selected from human brain astroglioblastoma, 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, pancreatic islet cell tumor, Kaposi sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer.

[0047] The present invention also provides use of any of the above-described bispecific antibodies in the preparation of a medicament for treating an autoimmune disease. In some embodiments, 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.

[0048] In some embodiments, the use is achieved by one or more of tumor immunotherapy, cell therapy, or gene therapy.

[0049] The present invention also provides a pharmaceutical composition comprising any of the above-described bispecific antibodies and a pharmaceutically acceptable carrier, diluent or excipient.

[0050] The present invention also provides an antibody-drug conjugate comprising any of the above-described bispecific antibodies. In some embodiments, the conjugated drug is selected from a cytotoxin, a small molecule chemical drug, or an immunotoxin.

[0051] Abbreviations and Definitions

[0052] The following abbreviations are used herein: VH: antibody heavy chain variable region; VL: antibody light chain variable region; CDR: complementarity determining region in an immunoglobulin variable region; IgG: immunoglobulin G.

[0053] The term "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, in animal immune serum, or by phage library screening. Alternatively, they can be synthesized partially or completely using techniques such as genetic recombination. Preferred antibodies include, for example, antibodies of the isotype of an immunoglobulin or subclasses of these isotypes. Known human immunoglobulins include 9 categories (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the present invention can include IgG1, IgG2, IgG3, and / or IgG4.

[0054] As used herein, some antibodies 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 is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of the immunoglobulin 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 further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to 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 the antigen-binding site.

[0055] The term "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. Examples of "antigen-binding fragments" of an antibody include (1) a Fab fragment; (2) a F(ab')2 fragment; (3) an Fd fragment, consisting of the VH and CH1 domains; (4) an Fv fragment; (5) a dAb fragment, consisting of the VH domain; and (6) CDRs, which are isolated complementarity-determining regions.

[0056] In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to connect them by 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. Such 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 complete immunoglobulins. The antigen-binding fragment can also be incorporated into a single-chain molecule comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions.

[0057] The term "Fab fragment" is composed of an intact L chain, the variable region domain (VH) of the H chain, and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent for antigen binding, i.e., it has a single antigen-binding site. For example, Fab fragments can be produced recombinantly or by papain digestion of a full-length antibody.

[0058] The term "Fab' fragment" differs from the Fab fragment by the addition of a few additional residues at the carboxyl terminus of the CH1 domain, including

[0059] One or more cysteines from the hinge region of an antibody. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to an antigen with a reducing agent such as dithiothreitol.

[0060] The term "F(ab')2 fragment" originally refers to a pair of Fab' fragments having a hinge cysteine ​​residue 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 a portion of the hinge region intact. F(ab')2 fragments can be dissociated (into two Fab' molecules) by treatment with a reducing agent such as β-mercaptoethanol.

[0061] The term "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).

[0062] The term "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This 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. However, even if a single variable domain has the ability to recognize and bind antigen, its affinity is lower than that of the complete binding site.

[0063] 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 that enables the scFv to form a desired structure for antigen binding. A "scFv-Fc" fragment comprises an scFv connected to an Fc domain. For example, the Fc domain can be connected to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH), the Fc domain may be after the 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.

[0064] The term "dsFv" refers to a disulfide-stabilized Fv fragment. In dsFv, a polypeptide in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue is linked via a disulfide bond between the cysteine ​​residues. To produce such molecules, one amino acid in each framework region of VH and VL is mutated to cysteine, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine. The term dsFv encompasses both dsFv (molecules in which VH and VL are connected by an interchain disulfide bond rather than a linker peptide) or scdsFv (molecules in which VH and VL are connected by a linker and an interchain disulfide bond) known in the art.

[0065] The term "epitope" refers to an antigenic determinant in an antigen and refers to the antigenic site bound by the domains of the antigen-binding molecules comprising the antibody variable regions disclosed herein. Therefore, an epitope can be defined based on its structure. Alternatively, an epitope can be defined based on the antigen-binding activity of 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.

[0066] The term "specific" 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. In addition, this term is also used when the domain containing the antibody variable region has specificity for a particular epitope among multiple epitopes in an antigen. When the epitope bound by the domain containing the antibody variable region is contained in several different antigens, the antigen-binding molecule containing the domain containing the antibody variable region can bind to various antigens containing the epitope.

[0067] The term "bispecific antibody" refers to a protein molecule that can specifically bind to two target antigens or target antigen epitopes. In the present invention, "bispecific antigen-binding protein" comprising an antibody or antigen-binding fragment (e.g., Fab, scFv, etc.) is used interchangeably with "bispecific antibody" and "bispecific antibody".

[0068] The term "knob-Fc" refers to substitutions of amino acid residues in the CH3 domain of the first subunit of the Fc domain with amino acid residues having larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be positioned within a recess within the CH3 domain of the second subunit. For example, by mutating Serine T at position 366 of the CH3 domain of a heavy chain to Tryptophan W, a protruding "knob"-like protrusion is formed.

[0069] The term "hole-Fc" refers to substitutions of amino acid residues in the CH3 domain of the second subunit of the Fc domain with amino acid residues having smaller side chain volumes, thereby creating a recess within the CH3 domain of the second subunit into which the protrusion within the CH3 domain of the first subunit can be positioned. For example, by mutating Serine T at position 366 of the other heavy chain to Serine S, Leucine L at position 368 to Alanine A, and Tyrosine Y at position 407 to Valine V or Alanine A, a concave "hole"-like recess is formed.

[0070] The term "fusion" refers to the process of connecting two amino acid sequences to form a new sequence through technologies such as linkers, thereby forming a new artificially synthesized protein or antibody.

[0071] The term "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 does not cause the loss of the original function of the protein domain.

[0072] The term "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. As known in the art, 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). The CDRs in each chain are held together tightly by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody.

[0073] The term "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. However, variability is not evenly distributed across the entire variable domain range. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three HVRs, which form a loop connection and, in some cases, form part of the β-sheet structure. The HVRs in each chain are tightly held together by the FR region and, together with the HVRs of the other chains, contribute to the formation of the antigen binding site of the antibody. The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

[0074] The term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or antigen-binding fragment thereof) linked to one or more conjugated drugs (which may optionally be therapeutic agents or cytotoxic agents). ADCs typically consist of three components: an antibody or antibody-like ligand, a drug moiety, and a linker that couples the antibody or antibody-like ligand and drug. ADCs typically have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs conjugated to the antibody.

[0075] The term "polypeptide" refers to an amino acid chain of any length, regardless of modifications such as phosphorylation or glycosylation. The term polypeptide includes proteins and fragments thereof. Polypeptides can be "exogenous," meaning they are "heterologous," i.e., foreign to the host cell being 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 amino terminus to carboxyl terminus. Amino acid residue sequences are designated by three-letter or one-letter codes according to standard nomenclature.

[0076] The term "amino acid" refers to the twenty common naturally occurring amino acids. In some embodiments, the term "amino acid" also includes unnatural amino acids. Any suitable unnatural amino acid can be used. In some embodiments, the unnatural amino acid comprises a reactive moiety for conjugating the agent to the MIAC.

[0077] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Comparisons for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST software or the FASTA program package.

[0078] The term "at least 80% identity" means that the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence is greater than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0079] The term "amino acid mutation" refers to a mutation or change in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including insertion, deletion or substitution of one or more amino acids in the original protein or polypeptide.

[0080] The term "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.

[0081] The term "host cell" refers to a cell that has been or is capable of being transformed with a nucleic acid sequence to express a selected gene of interest. The term includes the progeny of a parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent cell, as long as the gene of interest is present in the progeny. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0082] The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors 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 operatively linked and are referred to herein as "expression vectors."

[0083] The term "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.

[0084] The term "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.

[0085] The term "negative 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, and serve as a control to better illustrate the experimental effect.

[0086] The term "affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., the antigen binding moiety of MIAC) and its binding partner (e.g., 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. Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, for example, by using surface plasmon resonance (SPR) technology (e.g., instrument) or biolayer interferometry (e.g., instrument) to measure.

[0087] The term "effector cell" refers to a leukocyte that expresses one or more FcRs and performs effector functions. In one aspect, effector cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and 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).

[0088] There are multiple methods / systems in the field for defining and describing CDRs. These systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used and defines CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of structural loop regions; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the AbM antibody modeling software from Oxford Molecular and is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in many aspects. Unless otherwise specified, this article uses the Kabat definition of CDRs. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 depicts an exemplary bispecific antibody, in which a full-length antibody capable of specifically recognizing a first antigen (BAFFR) is fused to a second antigen (BAFFR) binding portion and a third antigen (CD3) binding portion, wherein the second antigen-binding portion is a Fab, which is fused to the N-terminus of one heavy chain of the first antigen-binding portion, and the third antigen-binding portion is a scFv, which replaces the Fab region of the first antigen-binding portion fused to the second antigen-binding portion. The first Fc region of the bispecific antibody is a knob-Fc, and the second Fc region is a hole-Fc.

[0090] Figure 2 describes an exemplary bispecific antibody, in which a full-length antibody capable of specifically recognizing a first antigen (BAFFR) is fused to 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 one 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 to 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.

[0091] Figure 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 a Fab, and 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 a knob-Fc, and the second Fc region is a hole-Fc.

[0092] FIG4 shows the binding activity of bispecific antibodies to BAFFR protein.

[0093] FIG5 shows the binding activity of bispecific antibodies to CD3 protein.

[0094] FIG6 shows the binding activity of bispecific antibodies to Jeko-1 cells.

[0095] FIG7 shows the binding activity of bispecific antibodies to Raji cells.

[0096] FIG8 shows the binding activity of bispecific antibodies to Su-DHL4 cells.

[0097] FIG9 shows the T cell-specific activation activity of the bispecific antibody against jukart-NFAT-luc reporter gene cells.

[0098] FIG10 shows the cytotoxic activity of the bispecific antibody against CHO-K1-BAFFR cells.

[0099] FIG11 shows the cytotoxicity of bispecific antibodies against CHO-K1 cells.

[0100] FIG12 shows the killing activity of the bispecific antibody against Raji-luc cells. DETAILED DESCRIPTION

[0101] The present invention will be further described below with reference to the accompanying drawings and specific examples, and the protection content of the present invention is not limited to the following examples. It should also be understood that the terms used in the examples of the present invention are for the purpose of describing specific embodiments, rather than for the purpose of limiting the scope of protection of the present invention. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be imagined by those skilled in the art are all included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0102] Example 1 Acquisition and optimization of nucleotide sequences

[0103] In the embodiment, bispecific antibodies were constructed according to Figures 1-3 targeting BAFFR and CD3, and were named Antibody A, Antibody B, and Antibody C, respectively.

[0104] As needed, the Fc of the antibody amino acid sequence is adjusted to other IgG types, and the desired amino acid mutations are further designed in each heavy chain to obtain 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.

[0105] Table 1 Antibody sequence information

[0106] Table 2 Sequence combinations of antibodies

[0107] Each of the target amino acid sequences was converted into nucleotide sequences and optimized for a range of parameters that may affect antibody expression in mammalian cells, including codon bias, GC content (i.e., the ratio of guanine G to cytosine C among the four bases in DNA), CpG islands (i.e., regions of high density of CpG dinucleotides in the genome), mRNA secondary structure, splice sites, premature PolyA sites, internal Chi sites (short DNA fragments in the genome near which homologous recombination occurs) or ribosome binding sites, RNA unstable sequences, inverted repeats, and restriction enzyme sites that may interfere with cloning. Sequences that may improve translation efficiency, such as Kozak and SD sequences, were also added. Genes encoding the heavy and light chains of the antibodies were designed, respectively. Nucleotide sequences encoding signal peptides, derived from the optimized amino acid sequences, were also designed at the 5' end of the heavy and light chains, respectively. Furthermore, stop codons were added to the 3' end of the light and heavy chain nucleotide sequences, respectively.

[0108] Example 2 Gene synthesis and construction of expression vector

[0109] The pcDNA3.1-G418 vector was used as a plasmid vector for expressing the multifunctional antibody. The pcDNA3.1-G418 vector contains the CMVPromoter promoter, the eukaryotic selection marker G418 tag, and the prokaryotic selection marker Ampicilline. Nucleotide sequences for constructing the antibody expressing light and heavy chains were obtained through gene synthesis. The vector and target fragment were double-digested with HindIII and XhoI, then recovered and ligated using DNA ligase. The fragments were then transformed into competent Escherichia coli DH5α cells. Positive clones were selected, subjected to plasmid extraction, and enzyme digestion verification to obtain a plasmid containing the antibody.

[0110] Example 3 Plasmid Extraction

[0111] The recombinant plasmids containing the above-mentioned target genes were transformed into Escherichia coli DH5α competent cells, and the transformed bacteria were spread on LB plates containing 100 μg / mL ampicillin for culture. The selected plasmid clones were cultured in liquid LB medium and 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.

[0112] Example 4 Plasmid transfection, transient expression and antibody purification

[0113] Cultivate Expi CHO at 37°C, 8% CO2, and 100 rpm to a cell density of 6 × 10 6Use liposomes to transfect the constructed plasmids into the above cells according to the combination pairing. The transfection plasmid concentration is 1 mg / mL. The liposome volume refers to Expi CHO TM Culture was performed at 32°C, 5% CO2, and 100 rpm for 7-10 days using the Expression System kit. Feeds were added 18-22 hours after transfection and again on day 5. The culture was centrifuged at 4000 g, filtered through a 0.22 μm filter, and the supernatant collected. The resulting antibody protein was purified using a Protein A column and an ion column, and the eluate was collected.

[0114] The specific operating 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 equilibration 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 light returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH3.0) as an elution buffer, and the pH is adjusted to neutral for storage using Tris. 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. Using appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions, conventional ion exchange chromatography methods in the art, such as anion exchange or cation exchange, perform NaCl gradient elution under corresponding pH conditions, and select the collection tube where the target protein is located according to SDS-PAGE and store it together.

[0115] The purified eluate was then ultrafiltered and exchanged into a buffer solution, and the protein was detected by SDS-polyacrylamide gel electrophoresis.

[0116] SDS-PAGE assay confirmed that the target bands were present in both the non-reducing gel and the reducing gel conditions, corresponding to the heavy and light chains of the desired antibody. Therefore, the plasmid transfection, transient expression, and purification confirmed that the antibody had the correct structure.

[0117] Example 5 ELISA to detect the affinity of antibodies to BAFFR protein

[0118] Human-BAFFR-His (purchased from Acro, Cat: BAR-H52H3) was diluted to 1 μg / mL in PBS buffer (pH 7.4). 100 μL was added to each well of a 96-well ELISA plate and coated overnight at 4°C. Blocking was performed with 1% BSA blocking buffer for 1 hour. After washing the plate three times with PBST, the constructed antibody was diluted to 100 nM in 0.5% BSA sample diluent. This concentration was used as the starting concentration for a series of 11 3-fold dilutions, with 100 μL added to each well. The plate was incubated at 37°C for 1 hour. The plate was then washed three times with PBST. HRP-conjugated goat anti-human IgG-Fc (purchased from Jackson, Cat: 109-035-098) was diluted 1:20,000 in sample diluent and 100 μL added to each well. The plate was incubated at room temperature for 1 hour. A negative control (irrelevant antibody) and a 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). After washing the plate four times with PBST, 100 μL of TMB substrate was added to each well and incubated in the dark at room temperature for 10 minutes. The color development reaction was terminated by adding 100 μL of 1 M HCl solution to each well. The plate was read on a multi-function microplate reader.

[0119] The ELISA results of the antibodies are shown in FIG4 , and the bispecific antibody can bind to the BAFFR protein at all concentrations.

[0120] Example 6 ELISA to detect the affinity of antibodies to CD3

[0121] Human-CD3-His (purchased from Acro, Cat: CDD-H52W4) was diluted to 0.2 μg / mL in PBS buffer (pH 7.4). 100 μL was added to each well of a 96-well ELISA plate and coated overnight at 4°C. The plate was blocked with 1% BSA blocking buffer for 1 hour. After washing the plate three times with PBST, the constructed expressed antibody was diluted to 100 nM in 0.5% BSA sample diluent. This concentration was used as the starting concentration for a three-fold serial dilution, resulting in 11 steps. A negative control (blank well and IgG1 isotype control) and a positive control (CD3 monoclonal antibody derived from INN blinatumomab, consisting of SEQ ID NO:9 and SEQ ID NO:10, with the human IgG1 constant region added (see SEQ ID NO:27 and SEQ ID NO:28)) were also included. 100 μL was added to each well and incubated at 37°C for 1 hour. Wash the plate three times with PBST and add 100 μL of HRP-conjugated goat anti-human IgG-Fc diluted 1:20,000 in sample diluent to each well. Incubate at room temperature for 1 hour. Wash the plate four times with PBST and add 100 μL of TMB substrate to each well. Incubate at room temperature in the dark for 10 minutes. Stop the color development reaction by adding 100 μL of 1 M HCl to each well. Measure the absorbance of each well in the 96-well plate on a multi-function microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm.

[0122] The ELISA results of the antibodies are shown in FIG5 , and the bispecific antibody can bind to CD3 at all concentrations.

[0123] Example 7 Flow cytometry detection of antibody binding activity to BAFFR-positive tumor cells

[0124] Jeko-1, Raji, and Su-DHL4 BAFFR-positive tumor cells (from the Chinese Academy of Sciences Cell Collection Center) with normal morphology and growth in logarithmic phase were transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. After the cells were resuspended in diluent, 3 × 10 5 / well was added to a 96-well cell culture plate. The purified antibody was diluted to 20 μg / mL with FACS buffer. This was used as the starting concentration and a 3-fold serial dilution was performed for a total of 6 gradients. An irrelevant antibody negative control and a 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. The constant region of human IgG1 is shown in SEQ ID NO: 27 and SEQ ID NO: 28). 100 μL of antibody dilution was added. The cells were incubated at 4°C for 60 minutes and then washed twice with excess FACS buffer. The cells were resuspended in 100 μL FACS buffer and a fluorescent secondary antibody against human IgG Fc - 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.

[0125] The FACs test results of the antibody binding activity to BAFFR-positive tumor cells Jeko-1, Raji, and SuDHL4 are shown in Figures 6-8. Both bispecific antibody A and antibody B can specifically bind to BAFFR-positive tumor cells over multiple concentration ranges.

[0126] Example 8 Construction of antibodies for specific activation of T cells

[0127] BAFFR-positive Jeko-1 cells (from the Chinese Academy of Sciences Cell Collection Center) were used at a concentration of 2 × 10 4 / well were plated in 96-well plates, and then 5×10 4 Add effector cells, Jurkat-NFAT-Luc, to each well. Dilute the antibody in PBS to a starting concentration of 30 μg / mL and then, using a 5-fold dilution series (9 concentrations total), add the antibody to the corresponding wells. Set up a negative control group and incubate at 37°C for 6 hours. Then, add Bio-Lite to the sample wells and incubate at room temperature for 10 minutes before reading on a multi-function microplate reader.

[0128] As shown in Figure 9, the experimental results show that bispecific antibodies A and B specifically activated CD3-expressing Jurkart-NFAT-Luc reporter cells in the presence of BAFFR-positive target cells, Jeko-1. However, in the absence of antibodies or target cells, no activation activity was observed. This suggests that bispecific antibodies A and B, after recognizing BAFFR-positive cells, can specifically activate CD3-positive T cells, subsequently activating downstream signaling that induces T cells to kill tumors. Bispecific antibody C exhibits nonspecific activation, posing a risk.

[0129] Example 9 Construction of Antibodies to Specific Killing of CHO-K1-BAFFR Cells

[0130] BAFFR-positive CHO-K1-BAFFR cells (BAFFR-overexpressing engineered cell line constructed by transducing CHO-K1 cells with BAFFR lentivirus) and BAFFR-negative CHO-K1 were used at a rate of 2×10 4 / well were plated in a 96-well plate. After culturing for 24 h, bispecific antibodies and irrelevant antibodies were added, starting at 10 μg / mL and diluted 10-fold, with a total of 6 concentration gradients. At the same time, 1×10 CIK (CD3+CD56+ cells) effector cells were added. 5 The cells were plated at 4% PBS / well with an effector-target ratio of 5:1. A blank control (diluent), a negative control (target cells + CIK cells, no antibody), and an unrelated antibody group were also set up. Negative control 1 consisted of a BAFFR monoclonal antibody (BAFFR monoclonal antibody sequence consisting of SEQ ID NO:1 and SEQ ID NO:2, with the addition of the human IgG1 constant region (see SEQ ID NO:27 and SEQ ID NO:28), and negative control 2 consisted of an anti-GPC3 and anti-CD3 antibody (derived from patent US11001643B2, with the amino acid sequence consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31). After incubation for 24 hours in a cell culture incubator, the cells were rinsed three times with PBS and 100 μL of culture medium containing 10% CCK-8 (Cat: CK04) was added. After incubation for 3 hours in a culture incubator, the cells were measured at 450 nm using a microplate reader. The cell killing rate was calculated using the following formula: Cell killing rate (%) = (1-(OD value of sample well-OD value of blank well) / (OD value of negative well-OD value of blank well)) x 100%.

[0131] As shown in Figures 10-11, the bispecific antibody can kill BAFFR-overexpressing CHO-K1-BAFFR, while 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.

[0132] Example 10 Construction of antibodies to specifically kill Raji-luc cells

[0133] BAFFR-positive Raji-luc (Luc lentivirus transduced Raji) was used at 1×10 4 / well were plated in a 96-well plate. After culturing for 24 h, bispecific antibodies and irrelevant antibodies were added, starting at 50 μg / mL and diluted 10-fold, with a total of 6 concentration gradients. At the same time, 1×10 CIK (CD3+CD56+ cells) effector cells were added 5 / well, with an effector-target ratio of 10:1, and set up a blank control (diluent), a negative control (target cells + CIK cells, no antibody), and an unrelated antibody group. In the negative control group, negative control 1 was a BAFFR monoclonal antibody (BAFFR monoclonal antibody sequence consists of SEQ ID NO: 1 and SEQ ID NO: 2, with the addition of the constant region of human IgG1, the constant region of IgG1 is shown in SEQ ID NO: 27 and SEQ ID NO: 28), and negative control 2 was an anti-GPC3 and CD3 bispecific antibody (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). After incubation in a cell culture incubator for 24 hours, Bio-Lite was added, and after incubation in an incubator for 10 minutes, the OD value was detected on a microplate reader at 450 nm. The cell killing rate was calculated using the following formula: Cell killing rate (%) = (1-(OD value of sample well-OD value of blank well) / (OD value of negative well-OD value of blank well)) x 100%.

[0134] As shown in FIG12 , 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.

[0135] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody 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 moiety that specifically binds a second antigen, wherein the second antigen is BAFFR; (c) a third antigen-binding portion that specifically binds a third antigen, wherein the third antigen is CD3.

2. The bispecific antibody according to claim 1, characterized in that The first antigen-binding portion is a full-length antibody consisting of two heavy chains and two light chains.

3. The bispecific antibody according to claim 1 or 2, characterized in that The second antigen binding moiety and / or the third antigen binding moiety is an antibody fragment comprising a heavy chain variable domain (VH) and / or a light chain variable domain (VL).

4. The bispecific antibody according to claim 3, characterized in that The second antigen binding moiety and / or the third antigen binding moiety is Fab, Fab', scFab, F(ab')2, Fv, dsFv or scFv.

5. The bispecific antibody according to any one of claims 1 to 4, characterized in that The second antigen binding moiety is fused to the N-terminus and / or C-terminus of one or both heavy chains of the first antigen binding moiety.

6. The bispecific antibody according to any one of claims 1 to 4, characterized in that The second antigen binding moiety is fused to the N-terminus and / or C-terminus of one or both light chains of the first antigen binding moiety.

7. The bispecific antibody according to claim 5 or 6, characterized in that The third antigen binding moiety replaces one or both Fab regions or Fv regions of the first antigen binding moiety.

8. The bispecific antibody according to any one of claims 1 to 7, characterized in that The bispecific antibody comprises a first Fc region and a second Fc region.

9. The bispecific antibody according to claim 8, characterized in that The first and second Fc regions are the same or different.

10. The bispecific antibody according to claim 8 or 9, characterized in that The Fc region is selected from the group consisting of IgG, IgA, IgD, IgE, IgM and variants thereof.

11. The bispecific antibody according to claim 10, characterized in that The Fc region is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and variants thereof.

12. The bispecific antibody according to claim 10 or 11, characterized in that The Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

13. The bispecific antibody according to any one of claims 8 to 12, characterized in that The first Fc region is knob-Fc, and the second Fc region is hole-Fc.

14. The bispecific antibody according to any one of claims 8 to 12, characterized in that The first Fc region is hole-Fc, and the second Fc region is knob-Fc.

15. The bispecific antibody according to any one of claims 1 to 14, characterized in that The VH and VL of the third antigen binding portion are exchanged.

16. The bispecific antibody according to any one of claims 1 to 15, characterized in that The second antigen binding moiety and the third antigen binding moiety are fused to the first antigen binding moiety via a linker.

17. The bispecific antibody according to claim 16, characterized in that The linker is a peptide linker.

18. The bispecific antibody according to claim 17, characterized in that The peptide linker is a GS linker or a mutant human IgG hinge.

19. The bispecific antibody according to any one of claims 1 to 18, characterized in that The first antigen binding portion specifically binds BAFFR, wherein HCDR1 of the first antigen binding portion is as shown in SEQ ID NO:3, or a sequence that is at least 80% identical to SEQ ID NO:3; HCDR2 is as shown in SEQ ID NO:4, or a sequence that is at least 80% identical to SEQ ID NO:4; HCDR3 is as shown in SEQ ID NO:5, or a sequence that is at least 80% identical to SEQ ID NO:5; LCDR1 is as shown in SEQ ID NO:6, or a sequence that is at least 80% identical to SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence that is at least 80% identical to SEQ ID NO:7; and LCDR3 is as shown in SEQ ID NO:8, or a sequence that is at least 80% identical to SEQ ID NO:

8.

20. The bispecific antibody according to any one of claims 1 to 19, characterized in that The second antigen binding portion specifically binds to BAFFR, wherein HCDR1 of the second antigen binding portion is as shown in SEQ ID NO:3, or a sequence that is at least 80% identical to SEQ ID NO:3; HCDR2 is as shown in SEQ ID NO:4, or a sequence that is at least 80% identical to SEQ ID NO:4; HCDR3 is as shown in SEQ ID NO:5, or a sequence that is at least 80% identical to SEQ ID NO:5; LCDR1 is as shown in SEQ ID NO:6, or a sequence that is at least 80% identical to SEQ ID NO:6; LCDR2 is as shown in SEQ ID NO:7, or a sequence that is at least 80% identical to SEQ ID NO:7; and LCDR3 is as shown in SEQ ID NO:8, or a sequence that is at least 80% identical to SEQ ID NO:

8.

21. The bispecific antibody according to any one of claims 1 to 20, characterized in that The third antigen binding portion specifically binds CD3, wherein HCDR1 of the third antigen binding portion is as shown in SEQ ID NO:11, or a sequence at least 80% identical to SEQ ID NO:11; HCDR2 is as shown in SEQ ID NO:12, or a sequence at least 80% identical to SEQ ID NO:12; HCDR3 is as shown in SEQ ID NO:13, or a sequence at least 80% identical to SEQ ID NO:13; and LCDR1 is as shown in SEQ ID NO:14, or a sequence at least 80% identical to SEQ ID NO:14; LCDR2 is as shown in SEQ ID NO:15, or a sequence at least 80% identical to SEQ ID NO:15; and LCDR3 is as shown in SEQ ID NO:16, or a sequence at least 80% identical to SEQ ID NO:

16.

22. The bispecific antibody according to any one of claims 19 to 21, characterized in that 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.

23. The bispecific antibody according to any one of claims 19 to 22, characterized in that 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 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.

24. The bispecific antibody according to any one of claims 19 to 23, characterized in that 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 having 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 having at least 80% identity with SEQ ID NO:

10.

25. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1-24.

26. Use of the bispecific antibody according to any one of claims 1 to 24 in the preparation of a medicament for treating cancer.

27. The use according to claim 26, characterized in that The cancer is selected from the group consisting of glioblastoma, astrocytoma, cystic fibrosis, fibromyalgia, fibrosarcoma, fibromyalgia ... , kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid cancer, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, or uterine cancer.

28. Use of the bispecific antibody according to any one of claims 1 to 24 in the preparation of a medicament for treating an autoimmune disease.

29. The use according to claim 28, characterized in that 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.

30. The use according to any one of claims 26 to 29, characterized in that The use is achieved through one or more of tumor immunotherapy, cell therapy or gene therapy.

31. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 24 and a pharmaceutically acceptable carrier, diluent or excipient.

32. An antibody-drug conjugate comprising the bispecific antibody according to any one of claims 1 to 24.

33. The antibody drug conjugate according to claim 32, characterized in that The conjugated drug is selected from cytotoxins, small molecule chemical drugs or immunotoxins.

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