Homodimeric bispecific antibody, preparation method therefor and use thereof

Abstract

Provided is a tetravalent homodimeric bispecific antibody molecule simultaneously targeting an immune effector cell antigen CD3 and a tumor-associated antigen, wherein the bispecific antibody molecule contains, in order from N-terminus to C-terminus, a first single chain Fv, a second single chain Fv and a Fc fragment; wherein the first single chain Fv can specifically bind to the tumor-associated antigen, the second single chain Fv can specifically bind to CD3, and the first and the second single chain Fvs are connected by a linker peptide, while the second single chain Fv and the Fc fragment are directly connected or connected by a linker peptide; and the Fc fragment does not have effector functions such as CDC, ADCC and ADCP.

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 an tumor-associated antigen, a second single-chain Fv that specifically bind 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 of 1 to 5. 
     
     
         3 . The bispecific antibody according to  claim 1 , wherein the tumor-associated antigen comprises CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD39, CD40, CD47, CD52, CD73, CD74, CD123, CD133, CD138, BCMA, CA125, CEA, CS1, DLL3, DLL4, EGFR, EpCAM, FLT3, gpA33, GPC-3, Her2, MEGE-A3, NYESO1, PSMA, TAG-72, CIX, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR2, VEGFR3, Cadherin, Integrin, Mesothelin, Claudin18, αVβ3, α5β1, ERBB3, c-MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, B7 protein family, Mucin family, FAP, and Tenascin. 
     
     
         4 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv specifically binds to CD19 and comprises a VH domain and a VL domain selected from the group consisting of:
 (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 9, 10, and 11, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 9, 10, and 11; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 12, 13, and 14, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 12, 13, and 14;   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 17, 18, and 19, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 17, 18, and 19; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 20, 21, and 22, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 20, 21, and 22;   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 25, 26, and 27, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 25, 26, and 27; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 28, 29, and 30, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 28, 29, and 30; and   (iv) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 33, 34, and 35, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 33, 34, and 35; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 36, 37, and 38, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 36, 37, and 38;   or the first single-train Fv specifically binds to CD20 and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 41, 42, and 43, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 41, 42, and 43; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 44, 45, and 46, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 44, 45, and 46;   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 49, 50, and 51, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 49, 50, and 51; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 52, 53, and 54, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 52, 53, and 54;   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 57, 58, and 59, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 57, 58, and 59; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 60, 61, and 62, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 60, 61, and 62; and   (iv) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 65, 66, and 67, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 33, 34, and 35; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 68, 69, and 70, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 68, 69, and 70;   or the first single-train Fv specifically binds to CD22 and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 73, 74, and 75, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 73, 74, and 75; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 76, 77, and 78, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 76, 77, and 78;   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 81, 82, and 83, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 81, 82, and 83; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 84, 85, and 86, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 84, 85, and 86; or the first single-train Fv specifically binds to CD30 and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 89, 90, and 91, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 89, 90, and 91; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 92, 93, and 94, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 92, 93, and 94; and   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 97, 98, and 99, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 97, 98, and 99; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 100, 101, and 102, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 100, 101, and 102;   or the first single-train Fv specifically binds to ECAM and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 105, 106, and 107, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 105, 106, and 107; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 108, 109, and 110, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 108, 109, and 110; and   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 113, 114, and 115, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 113, 114, and 115; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 116, 117, and 118, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 116, 117, and 118; or   the first single-train Fv specifically binds to CEA and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 121, 122, and 123, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 121, 122, and 123; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 124, 125, and 126, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 124, 125, and 126;   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 129, 130, and 131, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 129, 130, and 131; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 132, 133, and 134, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 132, 133, and 134;   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 137, 138, and 139, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 137, 138, and 139; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 140, 141, and 142, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 140, 141, and 142; or the first single-train Fv specifically binds to Her2 and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 145, 146, and 147, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 145, 146, and 147; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 148, 149, and 150, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 148, 149, and 150;   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 153, 154, and 155, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 153, 154, and 155; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 156, 157, and 158, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 156, 157, and 158; and   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 161, 162, and 163, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 16, 162, and 163; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 164, 165, and 166, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 164, 165, and 166; or   the first single-train Fv specifically binds to EGFR and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 169, 170, and 171, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 169, 170, and 171; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 172, 173, and 174, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 172, 173, and 174;   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 177, 178, and 179, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 177, 178, and 179; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 180, 181, and 182, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 180, 181, and 182; and   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 185, 186, and 187, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 185, 186, and 187; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 188, 189, and 190, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 188, 189, and 190; or   the first single-train Fv specifically binds to GPC-3 and comprises a VH domain and a VL domain selected from:   a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 193, 194, and 195, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 193, 194, and 195; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 196, 197, and 198, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 196, 197, and 198; or   the first single-train Fv specifically binds to Mesothelin and comprises a VH domain and a VL domain selected from:   a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 201, 202, and 203, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 201, 202, and 203; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 204, 205, and 206, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 204, 205, and 206; or the first single-train Fv specifically binds to Mucin1 and comprises a VH domain and a VL domain selected from the group consisting of:   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 209, 210, and 211, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 209, 210, and 211; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 212, 213, and 214, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 212, 213, and 214; and   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 217, 218, and 219, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 217, 218, and 219; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 220, 221, and 222, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 220, 221, and 2222; or the first single-train Fv specifically binds to CA125 and comprises a VH domain and a VL domain selected from:   a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 225, 226, and 227, respectively or having sequences that are substantially identical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 225, 226, and 227; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 228, 229, and 230, respectively or having sequences that are substantially identical the (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions (for example, conservative substitutions) than) any of SEQ ID NOs: 228, 229, and 230.   
     
     
         5 - 15 . (canceled) 
     
     
         16 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv specifically binds to CD19 and comprises 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: 15 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative 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 substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 16;   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 23 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 23; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 24 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 24;   (iii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 31 or having a sequence that is substantially identical (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) to SEQ ID NO: 31; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 32 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 32; and   (iv) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 39 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 39; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 40 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 40;   or   the first single-chain Fv specifically binds to CD20 and comprises 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: 47 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 15; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 48 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 48;   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 55 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 55; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 24 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 56;   (iii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 63 or having a sequence that is substantially identical (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) to SEQ ID NO: 63; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 64 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 64; and   (iv) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 71 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 71; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 72 or having a sequence substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 72;   or   the first single-chain Fv specifically binds to CD22 and comprises 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: 79 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 79; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 80 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 80;   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 87 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 87; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 88 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 88;   or   the first single-chain Fv specifically binds to CD30 and comprises 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: 95 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 95; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 96 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 96; and   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 103 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 103; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 104 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 104;   or   the first single-chain Fv specifically binds to EpCAM and comprises 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: 111 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 111; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 112 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 112; and   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 119 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 119; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 120 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 120;   
       or
 the first single-chain Fv specifically binds to CEA and comprises 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: 127 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 127; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 128 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 128; 
 (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 135 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 135; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 136 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 136; and 
 (iii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 143 or having a sequence that is substantially identical (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 143; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 144 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 144; 
 or 
 the first single-chain Fv specifically binds to Her2 and comprises 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: 151 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 151; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 152 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 152; 
 (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 159 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 159; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 160 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 160; and 
 (iii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 167 or having a sequence that is substantially identical (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 167; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 168 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 168; 
 or 
 the first single-chain Fv specifically binds to EGFR and comprises 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: 175 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 175; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 152 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 176; 
 (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 183 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 183; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 184 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 184; and 
 (iii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 191 or having a sequence that is substantially identical (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 191; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 168 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 192; 
 or 
 the first single-chain Fv specifically binds to GPC-3 and comprises a VH domain and a VL domain selected from: 
 a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 199 or having a sequence substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 199; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 200 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 200; 
 or 
 the first single-chain Fv specifically binds to Mesothelin and comprises a VH domain and a VL domain selected from: 
 a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 207 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 207; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 208 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 208; 
 or 
 the first single-chain Fv specifically binds to Mucin1 and comprises 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: 215 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 215; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 216 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 216; and 
 (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 223 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 159; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 160 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 224; and 
 or 
 the first single-chain Fv specifically binds to CA125 and comprises a VH domain and a VL domain selected from: 
 a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 231 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 231; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 232 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 232. 
 
     
     
         17 - 27 . (canceled) 
     
     
         28 . 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, preferably Ser, and n is a natural number of 1 to 5, preferably 3,
 wherein the single chain Fv binds to an effector cell at an EC 50  value greater than about 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 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.   
     
     
         29 . (canceled) 
     
     
         30 . The bispecific antibody according to  claim 28 , wherein the second single-chain Fv comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 241, 242, and 243, 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: 241, 242, and 243; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 244, 245, and 246, 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: 244, 245, and 246, or
 the second single-chain Fv comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 249, 250, and 251, 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: 249, 250, and 251; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 252, 253, and 254, 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 any of SEQ ID NOs: 252, 253, and 254.   
     
     
         31 . (canceled) 
     
     
         32 . The bispecific antibody according to  claim 30 , wherein the second single-chain Fv specifically binds to CD3; the VH domain of the second single-chain Fv comprises an amino acid sequence as shown in SEQ ID NO: 247 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 247; and the VL domain of the second single-chain Fv comprises an amino acid sequence as shown in SEQ ID NO: 248 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 248; or the second single-chain Fv specifically binds to CD3; the VH domain of the second single-chain Fv comprises an amino acid sequence as show in SEQ ID NO: 255 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than) SEQ ID NO: 255; and the VL domain of the second single-chain Fv comprises an amino acid sequence as shown in SEQ ID NO: 256 or having a sequence that is substantially identical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions (for example, conservative substitutions) than SEQ ID NO: 256. 
     
     
         33 . (canceled) 
     
     
         34 . The bispecific antibody according to  claim 31 , 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 G x S y (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 at positions 118 to 145 at carboxyl terminus of the natural human chorionic gonadotropin beta-subunit, or a truncated fragment thereof; preferably, the rigid peptide comprises SSSSKAPPPS. 
     
     
         35 . The bispecific antibody according to  claim 34 , wherein the linker peptide comprises an amino acid sequence as shown in SEQ ID NO: 258. 
     
     
         36 . The bispecific antibody according to  claim 1 , wherein the linker peptide that links the Fc fragment to the second single-chain Fv comprises 1 to 20 amino acids, and preferably selected from the following amino acids: Gly(G), Ser(S), Ala(A), and Thr(T); more preferably Gly(G) and Ser(S); further preferably, the linker peptide consists of (GGGGS) n , wherein n=1, 2, 3 or 4. 
     
     
         37 . 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 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, M248L/N434S or M252Y/S254T/T25E determined according to the EU numbering system;   (iii) 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.   
     
     
         38 - 41 . (canceled) 
     
     
         42 . The bispecific antibody according to  claim 37 , wherein the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 263 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. 
     
     
         43 . The bispecific antibody according to  claim 1 , wherein
 the bispecific antibody binds to human CD19 and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 264; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 264; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 264; 
   or
 the bispecific antibody binds to human CD19 and CD3 and has an amino acid sequence as follows: 
 (i) a sequence as shown in SEQ ID NO: 283; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 283; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 283; 
   or
 the bispecific antibody binds to human CD20 and CD3 and has an amino acid sequence as follows: 
 (i) a sequence as shown in SEQ ID NO: 266; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 266; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 266; 
   or
 the bispecific antibody binds to human CD22 and CD3 and has an amino acid sequence as follows: 
 (i) a sequence as shown in SEQ ID NO: 268; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 268; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 268; 
   or   the bispecific antibody binds to human CD30 and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 270; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 270; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 270; or 
   the bispecific antibody binds to human EpCAM and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 272; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 272; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 272; or 
   the bispecific antibody binds to human CEA and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 274; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 274; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 274; 
   or   the bispecific antibody binds to human Her2 and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 8; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 8; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 8; 
   or   the bispecific antibody binds to human EGFR and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 277; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 277; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 277; 
   or   the bispecific antibody binds to human GPC-3 and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 279; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 279; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 279; 
   or   the bispecific antibody binds to human Mesothelin and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 281; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 281; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 281; 
   or   the bispecific antibody binds to human Mucin1 and CD3 and has an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 285; 
 (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence as shown in SEQ ID NO: 285; or 
 (iii) a sequence with 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 to the sequence as shown in SEQ ID NO: 285. 
   
     
     
         44 - 54 . (canceled) 
     
     
         55 . A DNA molecule encoding the bispecific antibody according to  claim 1 , which has a nucleotide sequence as shown in SEQ ID NO: 265, 267, 269, 271, 273, 275, 276, 278, 280, 282, 284 or 286. 
     
     
         56 - 58 . (canceled) 
     
     
         59 . A pharmaceutical composition, comprising the bispecific antibody according to  claim 1  and a pharmaceutically acceptable excipient, carrier or diluent. 
     
     
         60 . A method for preparing the bispecific antibody according to m  claim 1 , comprising: (a) obtaining a fusion gene of the bispecific antibody to construct 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 generated; and (d) separating and purifying the generated bispecific antibody;
 wherein the expression vector in step (a) is one or more selected from plasmids, bacteria, and viruses, and 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 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.   
     
     
         61 . (canceled) 
     
     
         62 . A method for enhancing or stimulating an immune response or function, comprising administering to a patient, subject or individual a therapeutically effective amount of the bispecific antibody of  claim 1 . 
     
     
         63 . A method for treating, delaying development, or reducing/or inhibiting recurrence of a tumor, comprising: giving or administering an effective amount of the bispecific antibody of  claim 1  to an individual suffering from cancer, wherein the cancer comprises mesothelioma, squamous cell carcinoma, myeloma, osteosarcoma, glioblastoma, neuroglioma, malignant epithelial tumours, adenocarcinoma, melanoma, sarcoma, acute and chronic leukemia, lymphoma and meningioma, Hodgkin's lymphoma, Sezary syndrome, multiple myeloma, lung cancer, non-small cell lung cancer, small cell lung cancer, laryngeal cancer, breast cancer, head and neck cancer, bladder cancer, uterine cancer, skin cancer, prostate cancer, cervical cancer, vaginal cancer, gastric cancer, renal cell carcinoma, renal carcinoma, pancreatic cancer, colorectal cancer, endometrial carcinoma, esophageal carcinoma, hepatobiliary cancer, bone cancer, skin cancer and blood cancer, and carcinoma of nasal cavity and sinus, nasopharyngeal carcinoma, oral cancer, oropharyngeal cancer, laryngeal cancer, sublaryngeal cancer, salivary cancer, mediastinal cancer, cervical cancer, small intestine cancer, colon cancer, cancer of rectum and anal regions, ureter cancer, urethral cancer, penile cancer, testicular cancer, vulva cancer, cancer of endocrine system, cancer of central nervous system, and plasmocytoma.