Homodimer-type bispecific antibody targeting cd19 and cd3, and preparation method therefor and application thereof

Abstract

Provided is a tetravalent, homodimer-type bispecific antibody molecule that targets both immune effector cell antigen CD3 and tumor-related antigen CD19. The bispecific antibody molecule comprises first and second single-chain Fv and Fc fragments in sequence from the N-terminus to the C-terminus, wherein the first single-chain Fv can specifically bind to CD19, the second single-chain Fv can specifically bind to CD3, the first and second single-chains Fv are connected by means of a linker peptide, the second single-chain Fv and Fc fragments are directly connected to each other or connected by means of a linker peptide; and the Fc fragment does not have effector functions such as CDC, ADCC, and ADCP. The bispecific antibody can significantly inhibit or kill tumor cells, and has toxic and side effects that may be caused by excessive activation of effector cells; in addition, such bispecific antibody is of homodimer type, without the problem of heavy chain and light chain mismatch; the purification step is simple and efficient, the expression is high, and the physical and chemical properties as well as in vivo stability of the antibody are significantly improved.

1 . A bispecific antibody, which is a tetravalent homodimer formed by two identical polypeptide chains that bind to each other by a covalent bond, wherein each of the polypeptide chains comprises a first single-chain Fv that specifically binds to tumor-associated antigen CD19, a second single-chain Fv that specifically binds to effector cell antigen CD3 and an Fc fragment in sequence from N-terminus to C-terminus; wherein the first single-chain Fv is linked to the second single-chain Fv by a linker peptide, the second single-chain Fv is linked to the Fc fragment directly or by a linker peptide, and the Fc fragment has no effector functions comprising CDC, ADCC and ADCP. 
     
     
         2 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv comprises a VH domain and a VL domain that are linked by a linker peptide which has an amino acid sequence of (GGGGX) n , wherein X comprises Ser or Ala, and n is a natural number from 1 to 5, wherein the tumor-associated antigen CD19 comprises any variant, isotype, derivative and species homolog of CD19; and wherein CD19 is derived from a human, a  cynomolgus  monkey or a rhesus monkey. 
     
     
         3 . (canceled) 
     
     
         4 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv specifically binds to CD19 and contains CDR1, CDR2 and CDR3 selected from the group consisting of:
 (i) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 1, 2 and 3, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 1, 2 and 3; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 4, 5 and 6, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 4, 5 and 6;   (ii) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 9, 10 and 11, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 9, 10 and 11; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 12, 13 and 14, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 12, 13 and 14;   (iii) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 17, 18 and 19, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 17, 18 and 19; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 20, 13 and 21, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 20, 13 and 21;   (iv) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 24, 25 and 26, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 24, 25 and 26; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 27, 28 and 29, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 27, 28 and 29; and   (v) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 17, 18 and 19, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 17, 18 and 19; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 32, 33 and 21, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 32, 33 and 21.   
     
     
         5 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv specifically binds to CD19 and is selected from the group consisting of:
 (i) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 7 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 7; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 8 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 8;   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 15 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 15; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 16 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 16;   (iii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 22 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 22; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 23 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 23;   (iv) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 30 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 30; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 31 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 31;   (v) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 34 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 34; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 35 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 35;   (vi) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 36 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 36; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 37 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 37; and   (vii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 38 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 38; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 39 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 39.   
     
     
         6 . A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CD19, comprising CDR1, CDR2 and CDR3 contained in the first single-chain Fv of the bispecific antibody according to  claim 4 , which are selected from the group consisting of:
 (i) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 17, 18 and 19, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 17, 18 and 19; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 32, 33 and 21, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 32, 33 and 21; and   (ii) HCDR1, HCDR2 and HCDR3, comprised in a VH domain, that are as shown in SEQ ID NOs: 17, 18 and 19, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 17, 18 and 19; and LCDR1, LCDR2 and LCDR3, comprised in a VL domain, that are as shown in SEQ ID NOs: 20, 13 and 21, respectively or have sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 20, 13 and 21;   preferably, the monoclonal antibody is a humanized antibody;   preferably, the antigen-binding fragment is selected from scFv, Fab, Fab′, (Fab′)2, an Fv fragment or Fv linked by a disulfide bond (dsFv).   
     
     
         7 . The monoclonal antibody or the antigen-binding fragment thereof according to  claim 6 , comprising a VH domain and a VL domain selected from the group consisting of:
 (i) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 36 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 36; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 37 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 37; and   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 38 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 38; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 39 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 39.   
     
     
         8 . The bispecific antibody according to  claim 1 , wherein the second single-chain Fv comprises a VH domain and a VL domain that are linked by a linker peptide which has an amino acid sequence of (GGGGX)n, wherein X comprises Ser or Ala, and n is a natural number from 1 to 5,
 wherein the second single-chain Fv binds to an effector cell at an EC 50  value greater than about 10 nM, or greater than 20 nM, or greater than 40 nM, or greater than about 50 nM in an in vitro binding affinity assay; and wherein, the second single-chain Fv of the bispecific antibody is capable of binding to human CD3 and specifically binding to CD3 of a  cynomolgus  monkey or a rhesus monkey.   
     
     
         9 . (canceled) 
     
     
         10 . The bispecific antibody according to  claim 1 , wherein the second single-chain Fv specifically binds to CD3; the VH domain of the second single-chain Fv contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than SEQ ID NOs: 40, 41 and 42; and the VL domain of the second single-chain Fv contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 43, 44 and 45, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than SEQ ID NOs: 43, 44 and 45. 
     
     
         11 . The bispecific antibody according to  claim 10 , wherein the second single-chain Fv specifically binds to CD3; the VH domain of the second single-chain Fv contains an amino acid sequence as shown in SEQ ID NO: 46 or has a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 46; and the VL domain of the second single-chain Fv contains an amino acid sequence as shown in SEQ ID NO: 47 or has a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 47. 
     
     
         12 . The bispecific antibody according to  claim 1 , wherein the linker peptide that links the first single-chain Fv to the second single-chain Fv consists of a flexible peptide and a rigid peptide; wherein the flexible peptide comprises two or more amino acids, and preferably selected from the following amino acids: Gly(G), Ser(S), Ala(A) and Thr(T); more preferably, the flexible peptide comprises G and S residues; most preferably, an amino acid composition structure of the flexible peptide has a general formula of GxSy(GGGGS)z, wherein x, y and z are integers greater than or equal to 0 and x+y+z≥1; the rigid peptide is derived from a full-length sequence consisting of amino acids 118 to 145 at carboxyl terminus of natural human chorionic gonadotropin β-subunit or a truncated fragment thereof; preferably, the rigid peptide comprises SSSSKAPPPS. 
     
     
         13 . The bispecific antibody according to  claim 12 , wherein the linker peptide contains an amino acid sequence as shown in SEQ ID NO: 49. 
     
     
         14 . The bispecific antibody according to  claim 1 , wherein the linker peptide that links the Fc fragment to the second single-chain Fv comprises 1-20 amino acids, and preferably selected from the following amino acids: Gly(G), Ser(S), Ala(A) and Thr(T); preferably Gly(G) and Ser(S); more preferably, the linker peptide consists of (GGGGS)n, wherein n=1, 2, 3 or 4. 
     
     
         15 . The bispecific antibody according to  claim 1 , wherein the Fc fragment comprises a hinge region, a CH2 domain and a CH3 domain from a human immunoglobulin heavy chain constant region; preferably, the Fc fragment is selected from heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; more preferably, the Fc fragment is selected from heavy chain constant regions of human IgG1, IgG2, IgG3 and IgG4; further preferably, the Fc fragment is selected from a heavy chain constant region of human IgG1 or IgG4; and compared to a natural sequence from which the Fc fragment is derived, the Fc fragment has one or more amino acid substitutions, deletions or additions selected from the group consisting of:
 (i) amino acid substitutions L234A/L235A/P331S that are determined according to an EU numbering system   (ii) amino acid substitutions M428L, T250Q/M428L, M428L/N434S or M252Y/S254T/T256E determined according to the EU numbering system;   (iii) an amino acid substitution N297A determined according to the EU numbering system; and   (iv) an amino acid deletion K447 determined according to the EU numbering system.   
     
     
         16 - 19 . (canceled) 
     
     
         20 . The bispecific antibody according to  claim 15 , wherein the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 54 that has six amino acid substitutions or replacements L234A/L235A/N297A/P331S/T250Q/M428L determined according to the EU numbering system and a deleted or removed K447 determined according to the EU numbering system compared to the natural sequence from which the Fc fragment is derived. 
     
     
         21 . The bispecific antibody according to  claim 1 , wherein the bispecific antibody binds to human CD19 and CD3 and has an amino acid sequence comprising:
 group (a):
 (i) a sequence as shown in SEQ ID NO: 55; 
 (ii) a sequence having one or more substitutions, deletions or additions relative to the sequence as shown in SEQ ID NO: 55; or 
 (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity relative to the sequence as shown in SEQ ID NO: 55; 
   group (b):
 (i) a sequence as shown in SEQ ID NO: 57; 
 (ii) a sequence having one or more substitutions, deletions or additions relative to the sequence as shown in SEQ ID NO: 57; or 
 (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity relative to the sequence as shown in SEQ ID NO: 57; 
   group (c):
 (i) a sequence as shown in SEQ ID NO: 59; 
 (ii) a sequence having one or more substitutions, deletions or additions relative to the sequence as shown in SEQ ID NO: 59; or 
 (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity relative to the sequence as shown in SEQ ID NO: 59; 
   group (d):
 (i) a sequence as shown in SEQ ID NO: 61; 
 (ii) a sequence having one or more substitutions, deletions or additions relative to the sequence as shown in SEQ ID NO: 61; or 
 (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity relative to the sequence as shown in SEQ ID NO: 61. 
   
     
     
         22 . A DNA molecule encoding the bispecific antibody according to  claim 1 , which has a nucleotide sequence as shown in SEQ ID NO: 56, 58, 60 or 62. 
     
     
         23 - 25 . (canceled) 
     
     
         26 . A pharmaceutical composition, comprising the bispecific antibody according to  claim 1  and a pharmaceutically acceptable excipient, carrier or diluent;
 preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; 
 preferably, the additional pharmaceutically active agent is a drug for treating an immune-related disease; 
 preferably, the additional pharmaceutically active agent is a drug having antitumor activity; 
 preferably, the additional pharmaceutically active agent is a drug for treating an autoimmune disease or an inflammatory disease; 
 preferably, the additional pharmaceutically active agent is a drug for treating a disease or disorder related to transplant rejection; 
 preferably, the antibody and the additional pharmaceutically active agent are provided as separate components or as components of a same composition. 
 
     
     
         27 . The pharmaceutical composition according to  claim 26 , further comprising a pharmaceutically active agent administered before, after or while the pharmaceutical composition is administered to a subject, wherein the pharmaceutically active agent is selected from an antibody, an antibody fragment, a drug, an enzyme, a cytotoxic agent, a toxin, an antibiotic, a hormone, an immunomodulator, a cytokine, a chemokine or a radioisotope. 
     
     
         28 - 31 . (canceled) 
     
     
         32 . A method for enhancing or stimulating an immune response or function, comprising administering a therapeutically effective amount of the bispecific antibody according to  claim 1  to an individual. 
     
     
         33 . (canceled) 
     
     
         34 . A method for preventing/treating, delaying development of, or reducing/inhibiting recurrence of a disease including an immune-related disease, a tumor, an autoimmune disease, an inflammatory disease or a transplant rejection-related disease or disorder, comprising administering an effective amount of the bispecific antibody according to  claim 1  to an individual suffering from the disease or disorder,
 wherein the tumor comprises acute myeloid leukemia (AML), chronic myeloid leukemia (CML), B acute lymphocytic leukemia (B-ALL), B chronic lymphocytic leukemia (B-CLL), B-cell lymphoma (BCL), T-cell lymphoma (TCL) (such as skin), myelodysplastic syndrome (MDS), small lymphocytic lymphoma (SLL), hairy cell leukemia (HCL), marginal zone lymphoma (MZL) (such as extranodal or splenic), follicular lymphoma (FL) (such as pediatric or gastrointestinal), B-cell prolymphocytic leukemia (B-PLL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL)/Waldenstrom's macroglobulinemia (WM), lymphoblastic leukemia (ALL) (such as B cell), lymphoblastic lymphoma (LBL) (such as B cell), plasmablastic lymphoma (PBL) (such as B cell), Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL) (for example, primary or inflammation-related), Burkitt's lymphoma (BL), multiple myeloma, anaplastic large-cell lymphoma and HIV-related lymphoma, 
 the autoimmune or inflammatory disease is selected from rheumatoid arthritis (RA), osteoarthritis, reactive arthritis, systemic lupus erythematosus (SLE), Crohn's disease, multiple sclerosis, scleroderma, psoriasis, psoriatic arthritis, ulcerative colitis (such as chronic), insulin-dependent diabetes (such as juvenile), thyroiditis (such as chronic), hyperthyroidism, asthma, allergic diseases, sarcoidosis, autoimmune hemolytic anemia, pernicious anemia, graft-versus-host disease, dermatomyositis, chronic hepatitis, microscopic renal vasculitis, chronic active hepatitis, uveitis, intestinal synovitis, autoimmune intestinal disease, idiopathic leukopenia, autoimmune glomerulonephritis, autoimmune hemolytic anemia, autoimmune hepatitis, interstitial pneumonia, chronic pemphigus, pemphigus vulgaris, arteritis, polyarteritis  nodosa  and ankylosing spondylitis, 
 the transplant rejection comprises acute, hyperacute or chronic transplant rejection and the transplant rejection comprises rejection to an organ, tissue or cell transplant which comprises blood transfusion, a vascular transplant, an epithelial transplant, an endothelial transplant, a muscle transplant, a connective tissue transplant, a joint transplant, a heart transplant, a lung transplant, a liver transplant, a kidney transplant, a pancreas transplant, a skin transplant, an intestinal transplant, a corneal transplant, a bone transplant, a bone marrow transplant, a graft-versus-host disease or a host-versus-graft disease, 
 optionally, one or more additional therapies are applied, wherein the additional therapies are selected from the group consisting of a surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and a combination thereof. 
 
     
     
         35 . (canceled) 
     
     
         36 . A method for preparing the bispecific antibody according to  claim 1 , comprising: (a) obtaining a fusion gene of the bispecific antibody, and constructing an expression vector of the bispecific antibody; (b) transfecting the expression vector into a host cell by a genetic engineering method; (c) culturing the host cell under conditions that allow the bispecific antibody to be produced; (d) separating and purifying the bispecific antibody;
 wherein the expression vector in step (a) is one or more selected from a plasmid, a bacterium and a virus; preferably, the expression vector is a pCDNA3.4 vector;   wherein the host cell into which the constructed vector is transfected by the genetic engineering method in step (b) comprises a prokaryotic cell, a yeast cell or a mammalian cell, such as a CHO cell, an NS0 cell or another mammalian cell, preferably a CHO cell; and   wherein the bispecific antibody is separated and purified in step (d) by a conventional immunoglobulin purification method comprising protein A affinity chromatography and ion exchange, hydrophobic chromatography or molecular sieve.   
     
     
         37 - 42 . (canceled)