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HOMODIMERIC BISPECIFIC ANTIBODY, PREPARATION METHOD THEREFOR AND USE THEREOF

20210371526 · 2021-12-02

    Inventors

    Cpc classification

    International classification

    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.

    Claims

    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).sub.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).sub.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.sub.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.sub.xS.sub.y(GGGGS).sub.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).sub.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.

    Description

    BRIEF DESCRIPTION OF DRAWINGS

    [0316] FIG. 1-1 illustrates configurations of bispecific antibodies AB7K, AB7K4, AB7K5, AB7K6, AB7K7, and AB7K8 as shown in a, b, c, d, e and f, respectively.

    [0317] FIG. 1-2 illustrates an expression plasmid map of bispecific antibody AB7K7. The expression plasmid has a full length of 9293 bp and contains nine major gene fragments which are (1) an hCMV promoter, (2) target genes, (3) EMCV IRES, (4) mDHFR screening gene, (5) a Syn discontinuation sequence, (6) an SV40 promoter, (7) Kalamycin resistance gene; (8) an SV40 termination sequence, and (9) a PUC replicon.

    [0318] FIG. 1-3 illustrates SEC-HPLC detection results of a purified sample of bispecific antibody AB7K7.

    [0319] FIG. 1-4 illustrates SDS-PAGE electrophoresis results of a purified sample of bispecific antibody AB7K7.

    [0320] FIG. 1-5 illustrates SDS-PAGE results of bispecific antibody AB7K7 in an acceleration test at 25° C.

    [0321] FIG. 1-6 illustrates SDS-PAGE results of bispecific antibody AB7K7 in a freeze-thaw test.

    [0322] FIG. 2-1 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K4 to bind to tumor cells BT474.

    [0323] FIG. 2-2 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K5 to bind to tumor cells BT474.

    [0324] FIG. 2-3 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K6 to bind to tumor cells BT474.

    [0325] FIG. 2-4 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K7 to bind to tumor cells BT474.

    [0326] FIG. 2-5 illustrates an ability, detected by FACS, of bispecific antibody AB7K8 to bind to tumor cells BT474.

    [0327] FIG. 2-6 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K4 to bind to effector cells CIK.

    [0328] FIG. 2-7 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K5 to bind to effector cells CIK.

    [0329] FIG. 2-8 illustrates an ability, detected by FACS, of bispecific antibody AB7K6 to bind to effector cells CIK.

    [0330] FIG. 2-9 illustrates abilities, detected by FACS, of bispecific antibodies AB7K and AB7K7 to bind to effector cells CIK.

    [0331] FIG. 2-10 illustrates an ability, detected by FACS, of bispecific antibody AB7K8 to bind to effector cells CIK.

    [0332] FIG. 2-11 illustrates an ability, detected by FACS, of bispecific antibody AB7K to bind to cynomolgus monkey T cells.

    [0333] FIG. 2-12 illustrates abilities, detected by ELISA, of five Anti-Her2×CD3 bispecific antibodies to bind to CD3 molecules and Her2 molecules.

    [0334] FIG. 2-13 illustrates abilities, detected by a microplate reader, of five Anti-Her2×CD3 bispecific antibodies to activate Jurkat T cells of a reporter gene cell strain.

    [0335] FIG. 2-14 illustrates structural modeling of a CTP linker and anti-CD3 scFv VH.

    [0336] FIG. 2-15 illustrates structural modeling of a GS linker and anti-CD3 scFv VH.

    [0337] FIG. 2-16 illustrates a molecular docking model of anti-CD3 scFv and a CD3 epsilon chain.

    [0338] FIG. 3-1 illustrates in vivo anti-tumor effects of bispecific antibodies AB7K4 and AB7K7 in an NCG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and HCC1954 cells.

    [0339] FIG. 3-2 illustrates an in vivo anti-tumor effect of bispecific antibody AB7K7 in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human breast cancer cells HCC1954.

    [0340] FIG. 3-3 illustrates in vivo anti-tumor effects of bispecific antibodies AB7K7 and AB7K8 at different administration frequencies in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human breast cancer cells HCC1954.

    [0341] FIG. 3-4 illustrates an in vivo anti-tumor effect of bispecific antibody AB7K7 in an NPG mouse of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and SK-OV-3 cells.

    [0342] FIG. 3-5 illustrates an in vivo anti-tumor effect of bispecific antibody AB7K7 in an NPG mouse of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and HT-29 cells.

    [0343] FIG. 3-6 illustrates an in vivo anti-tumor effect of bispecific antibody AB7K7 in a CD34 immune-reconstituted NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human breast cancer cells HCC1954.

    [0344] FIG. 3-7 illustrates an in vivo anti-tumor effect of bispecific antibody AB7K7 in a PBMC immune-reconstituted NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human breast cancer cells HCC1954.

    [0345] FIG. 4-1 illustrates in vivo anti-tumor effects of bispecific antibodies AB7K4 and AB7K7 in an NCG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Raji cells.

    [0346] FIG. 4-2 illustrates an anti-tumor effect of bispecific antibody AB7K7 in an NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human breast cancer cells HCC1954.

    [0347] FIG. 4-3 illustrates changes of weights in normal cynomolgus monkeys administered with bispecific antibodies AB7K7 and AB7K8 multiple times.

    [0348] FIG. 5-1 illustrates concentration-time curves of bispecific antibody AB7K7 in SD rats by two ELISA methods.

    [0349] FIG. 5-2 illustrates concentration-time curves of bispecific antibody AB7K8 in SD rats by two ELISA methods.

    [0350] FIG. 5-3 illustrates concentration-time curves of bispecific antibodies AB7K7 and AB7K8 in cynomolgus monkeys.

    [0351] FIG. 5-4 illustrates abilities, detected at a pH of 6.0, of bispecific antibodies AB7K, AB7K5, and AB7K7 to bind to FcRn.

    [0352] FIG. 5-5 illustrates abilities, detected at a pH of 7.0, of bispecific antibodies AB7K, AB7K5, and AB7K7 to bind to FcRn.

    [0353] FIG. 6-1 illustrates an in vivo anti-tumor effect of bispecific antibody AB9K in a NOD-SCID mouse of transplanted tumor model constructed by subcutaneously co-inoculating human PBMC cells and Huh-7 cells.

    [0354] FIG. 6-2 illustrates an in vivo anti-tumor effect of bispecific antibody AB9K in a CD34 immune-reconstituted NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human liver cancer cells Huh-7.

    [0355] FIG. 6-3 illustrates an in vivo anti-tumor effect of bispecific antibody AB9K in a CD34 immune-reconstituted NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human liver cancer cells Huh-7.

    [0356] FIG. 7-1 illustrates an ability, detected by flow cytometry, of bispecific antibody AB2K to bind to CD20-positive tumor cells.

    [0357] FIG. 7-2 illustrates abilities of bispecific antibodies AB2K and AB7K7 to mediate effector cells to kill Raji-luc cells.

    [0358] FIG. 7-3 illustrates abilities, detected by reporter gene assay, of bispecific antibodies AB2K and AB7K7 to activate Jurkat NFATRE Luc cells.

    [0359] FIG. 7-4 illustrates an in vivo anti-tumor effect of bispecific antibody AB2K in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Raji cells.

    [0360] FIG. 7-5 illustrates an in vivo anti-tumor effect of bispecific antibody AB2K in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Daudi cells.

    [0361] FIG. 8 illustrates changes of leukocytes and lymphocytes in normal cynomolgus monkeys administered with bispecific antibody AB2K multiple times.

    [0362] FIG. 9-1 illustrates an ability, detected by FACS, of an Anti-CD19×CD3 bispecific antibody to bind to tumor cells Raji.

    [0363] FIG. 9-2 illustrates an ability, detected by FACS, of an Anti-CD19×CD3 bispecific antibody to bind to effector cells CIK.

    [0364] FIG. 9-3 illustrates abilities, detected by FACS, of bispecific antibodies AB1K2 and AB23P10 to bind to cynomolgus monkey T cells.

    [0365] FIG. 9-4 illustrates abilities, detected through an ELISA, of four Anti-CD19×CD3 bispecific antibodies to bind to CD3 molecules and CD19 molecules.

    [0366] FIG. 9-5 illustrates abilities, detected by a microplate reader, of bispecific antibodies AB1K2 and AB23P8 to activate Jurkat T cells of a reporter gene cell strain.

    [0367] FIG. 9-6 illustrates abilities, detected through a microplate reader, of four Anti-CD19×CD3 bispecific antibodies to activate Jurkat T cells of a reporter gene cell strain.

    [0368] FIG. 10-1 illustrates binding of AB11K to tumor cells that overexpress antigen Mucin1 and to primary T cells of human or cynomolgus monkeys.

    [0369] FIG. 10-2 illustrates an ability of AB11K to mediate expanded T cells to kill tumor cells.

    [0370] FIG. 10-3 illustrates an ability of AB11K to mediate PBMC to kill tumor cells.

    [0371] FIG. 10-4 illustrates an ability of AB11K to specifically activate T cells.

    [0372] FIG. 11 illustrates an in vivo anti-tumor effect of bispecific antibody AB8K in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human skin cancer cells A431.

    DETAILED DESCRIPTION

    [0373] The present disclosure is further described through examples that should not be construed as further limitations. All drawings, all reference documents, and the contents of patents and published patent applications cited in the entire application are expressly incorporated herein by reference.

    Example 1 Design and Preparation of Anti-Her2×CD3 Bispecific Antibodies Having Different Structures

    1.1 Design of Bispecific Antibodies Having Different Structures

    [0374] In order to screen bispecific antibodies having suitable configuration, bispecific antibodies having six different configurations were designed for Her2 and CD3, among which AB7K5, AB7K6, and AB7K8 are single-chain bivalent bispecific antibodies while AB7K, AB7K4, and AB7K7 are double-chain tetravalent bispecific antibodies (see FIG. 1-1), where only AB7K8 is free of Fc fragments. Specifically, the configuration of the bispecific antibodies with the above four configurations and their composition from the N-terminus to the C-terminus as well as their amino acid sequence numbers are shown in Table 1-1. The specific structural composition properties of the six bispecific antibodies are described below:

    [0375] Bispecific antibody AB7K consists of an anti-Her2 full-length antibody whose two heavy chains are each linked at the C-terminus to an anti-CD3 scFv domain by a linker peptide (L1). For the amino acid sequence of the intact antibody against Her2 contained in AB7K, reference is made to the sequence of monoclonal antibody Herceptin® (IMGT database INN 7637), wherein AB7K contains an Fc fragment from human IgG1 and has D356E/L358M mutations (EU numbering). The linker peptide L1 consists of a flexible peptide and a rigid peptide, wherein the composition of the flexible peptide is GS(GGGGS).sub.3 and the rigid peptide is SSSSKAPPPSLPSPSRLPGPSDTPILPQ, wherein the composition of the linker peptide L2 between VH and VL of the anti-CD3 scFv is (GGGGS).sub.3.

    [0376] Bispecific antibody AB7K4 consists of an anti-Her2 full-length antibody whose two light chains are each linked at the C-terminus to an anti-CD3 scFv domain by a linker peptide (L1). For the amino acid sequence of the heavy chain variable region of the intact antibody against Her2 contained in AB7K4, reference is made to the available region sequence of the monoclonal antibody Herceptin®, and for the light chain amino acid sequence thereof, reference is made to the light chain amino acid sequence of the monoclonal antibody Herceptin® (IMGT database INN 7637). The AB7K4 heavy chain contains an Fc fragment from human IgG1, has multiple amino acid substitutions/replacements, which are L234A, L235A, T250Q, N297A, P331S, and M428L (EU numbering), respectively, and also has a deleted/missed K447 (EU numbering) at the C-terminus of the Fc fragment. The linker peptide L1 consists of a flexible peptide and a rigid peptide, wherein the composition of the flexible peptide is G.sub.2(GGGGS).sub.3 and the rigid peptide is SSSSKAPPPS, wherein the composition of the linker peptide L2 between VH and VL of the anti-CD3 scFv is (GGGGS).sub.3.

    [0377] Bispecific antibody AB7K5 consists of an anti-Her2 scFv, an Fc fragment, a linker peptide L2 and an anti-CD3 scFv, which are sequentially connected in series, wherein VH and VL in the anti-Her2 scFv are connected by a linker peptide L1, and VH and VL in the anti-CD3 scFv are connected by a linker peptide L3. For the amino acid sequence of the scFv against Her2 contained in AB7K5, reference is made to the available region sequence of the monoclonal antibody Herceptin®. The AB7K5 contains an Fc fragment from human IgG1 and has multiple amino acid substitutions/replacements, which are C226S, C229S, L234A, L235A, T250Q, N297A, P331S, T366R, L368H, K409T, and M428L (EU numbering), respectively. Mutations at the five sites C226S, C229S, T366R, L368H, and K409T can prevent polymerization between Fc fragments, thereby promoting the formation of a single-chain bivalent bispecific antibody. ADCC and CDC activities are removed from Fc fragments carrying the mutations L234A/L235A/P331S. The mutations T250Q/M428L can enhance the binding affinity of Fc fragments for the receptor FcRn, thereby extending the half-life. The mutation N297A avoids antibody glycosylation and loses the ability to bind FcγRs. In addition, K447 (EU numbering) at the C-terminus of the Fc fragment is deleted/missed, thereby eliminating the charge heterogeneity of the antibody. The linker peptide (L2) consists of a flexible peptide and a rigid peptide, wherein the flexible peptide is G.sub.2(GGGGS).sub.3 and the rigid peptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3 inside each scFv is (GGGGS).sub.3.

    [0378] Bispecific antibody AB7K6 consists of an anti-Her2 scFv, a linker peptide L2, an anti-CD3 scFv, and an Fc fragment, which are sequentially connected in series, wherein VH and VL in the anti-Her2 scFv are connected by a linker peptide L1, and VH and VL in the anti-CD3 scFv are connected by a linker peptide L3. The AB7K6 contains an Fc fragment from human IgG1 and has multiple amino acid substitutions/replacements, which are C226S, C229S, L234A, L235A, T250Q, N297A, P331S, T366R, L368H, K409T, and M428L (EU numbering), respectively. Mutations at the five sites C226S, C229S, T366R, L368H, and K409T can prevent polymerization between Fc fragments, thereby promoting the formation of a single-chain bivalent bispecific antibody. ADCC and CDC activities are removed from Fc fragments carrying the mutations L234A/L235A/P331S. The mutations T250Q/M428L can enhance the binding affinity of Fc fragments for the receptor FcRn, thereby extending the half-life. The mutation N297A avoids antibody glycosylation and loses the ability to bind FcγRs. In addition, K447 (EU numbering) at the C-terminus of the Fc fragment is deleted/missed, thereby eliminating the charge heterogeneity of the antibody. The linker peptide (L2) consists of a flexible peptide and a rigid peptide, wherein the flexible peptide is G.sub.2(GGGGS).sub.3 and the rigid peptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3 inside each scFv is (GGGGS).sub.3.

    [0379] Bispecific antibody AB7K7 consists of an anti-Her2 scFv, a linker peptide L2, an anti-CD3 scFv, and an Fc fragment, which are sequentially connected in series, wherein VH and VL in the anti-Her2 scFv are connected by a linker peptide L1, and VH and VL in the anti-CD3 scFv are connected by a linker peptide L3. For the amino acid sequence of the scFv against Her2 contained in AB7K7, reference is made to the available region sequence of the monoclonal antibody Herceptin®. The AB7K7 contains an Fc fragment from human IgG1, and has multiple amino acid substitutions/replacements, which were L234A, L235A, T250Q, N297A, P331S, and M428L (EU numbering), respectively, and also has a deleted/missed K447 (EU numbering) at the C-terminus of the Fc fragment. The linker peptide (L2) consists of a flexible peptide and a rigid peptide, wherein the flexible peptide is G.sub.2(GGGGS).sub.3 and the rigid peptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3 inside each scFv is (GGGGS).sub.3.

    [0380] Bispecific antibody AB7K8 consists of an anti-Her2 scFv, a linker peptide L2, an anti-CD3 scFv, and a His-tag, which are sequentially connected in series, wherein VH and VL in the anti-Her2 scFv are connected by a linker peptide L1, and VH and VL in the anti-CD3 scFv are connected by a linker peptide L3. For the amino acid sequence of the scFv against Her2 contained in AB7K8, reference is made to the available region sequence of the monoclonal antibody Herceptin®. AB7K8 is added with a His-tag at the C-terminus of the anti-CD3 scFv to facilitate antibody purification, wherein the composition of the His tag is HHHHHHHH. The linker peptide (L2) consists of a flexible peptide and a rigid peptide, wherein the flexible peptide is G.sub.2(GGGGS).sub.3 and the rigid peptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3 inside each scFv is (GGGGS).sub.3.

    [0381] VH and VL amino acid sequences of the anti-CD3 scFv contained in the above six bispecific antibodies are as shown in SEQ ID NO: 247 and SEQ ID NO: 248, respectively, wherein VH and VL are connected to each other by (GGGGS).sub.3. The monoclonal antibody (designated as CD3-3) specifically binds to human and cynomolgus monkey CD3 antigens and has a weak binding affinity for CD3.

    TABLE-US-00001 TABLE 1-1 Bispecific antibodies with four different structures against Her2 and CD3 Amino acid Code Configuration Composition from N-terminus to C-terminus sequence No. Single-chain AB7K5 scFv-mFc-scFv [VH-L1-VL].sub.Her2-mFc-L2-[VH-L3-VL].sub.CD3 SEQ ID NO: 1 bivalent AB7K6 scFv-scFv-mFc [VH-L1-VL].sub.Her2 L2-[VH-L3-VL].sub.CD3-mFc SEQ ID NO: 2 bispecific AB7K8 scFv-scFv-His tag [VH-L1-VL].sub.Her2-L2-[VH-L3-VL].sub.CD3-H.sub.8 SEQ ID NO: antibody 3 Double-chain AB7K IgG(H)-scFv [VH-CH].sub.Her2-L1-[VH-L2-VL].sub.CD3 SEQ ID NO: 4 tetravalent [VL-CL].sub.Her2 SEQ ID NO: 5 bispecific AB7K4 IgG(L)-scFv [VH-CH].sub.Her2 SEQ ID NO: 6 antibody [VL-CL].sub.Her2-L1-[VH-L2-VL].sub.CD3 SEQ ID NO: 7 AB7K7 scFv-scFv-mFc [VH-L1-VL].sub.Her2-L2-[VH-L3-VL].sub.CD3-mFc SEQ ID NO: 8 Note: Ln in the table represents the linker peptides between different structural units, wherein n is numbered sequentially in the order of the linker peptides contained between different structural units from the N-terminus to the C-terminus of the bispecific antibody.

    1.2 Construction of an Expression Vector of a Bispecific Antibody Molecule

    [0382] Genes encoding the preceding five bispecific antibodies were synthesized by conventional molecular biology method, and cDNAs encoding the obtained fusion genes were inserted into corresponding restriction endonuclease sites of eukaryotic expression plasmids pCMAB2M modified with PCDNA3.1. The heavy chains and light chains of AB7K and AB7K4 may be constructed into one vector or separately into two different vectors. For example, the expression plasmid map of AB7K7 is as shown in FIG. 1-2, wherein the plasmid contains cytomegalovirus early promoter. The promoter is an enhancer required for the high-level expression of foreign genes in mammalian cells. The plasmid pCMAB2M also contains a selective marker so that kanamycin resistance may be present in bacteria and G418 resistance may be present in mammalian cells. In addition, when host cells are deficient in the expression of DHFR genes, the pCMAB2M expression vector contains mouse dihydrofolate reductase (DHFR) genes so that target genes and the DHFR genes can be co-amplified in the presence of methotrexate (MTX) (see U.S. Pat. No. 4,399,216).

    1.3 Expression of Bispecific Antibody Molecules

    [0383] The preceding constructed expression plasmids were transfected into a mammalian host cell line to express bispecific antibodies. To maintain stable and high-level expression, the preferred host cell line is a DHFR deficient CHO-cell (see U.S. Pat. No. 4,818,679), and in this Example, the host cell was selected as the CHO-derived cell strain DXB11. A preferred transfection method is electroporation. Other methods, including calcium phosphate co-precipitation and lipofection may also be used. During electroporation, 50 μg of expression vector plasmids DNA were added to 5×10.sup.7 cells in a cuvette with a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, Calif.) with an electric field of 300 V and capacitance of 1500 μFd. After two-day transfection, the medium was changed to a growth medium containing 0.6 mg/mL G418. Transfectants were subcloned by the limiting dilution method, and the secretion rate of each cell line was determined by ELISA. Cell strains expressing bispecific antibodies at high levels were screened.

    [0384] To achieve the high-level expression of fusion proteins, DHFR genes inhibited by MTX should be used for co-amplification. The transfected fusion protein genes were co-amplified with the DHFR genes in growth media containing MTX with increasing concentrations. Subclones that were positive for DHFR expression were subjected to limiting dilution with gradually increased pressure to screen transfectants capable of growing in media with MTX of up to 6 μM. The secretion rates of the transfectants were determined and cell lines with high foreign protein expression were screened. Cell lines with a secretion rate of greater than about 5 μg/10.sup.6 (millions) cells/24 hours (preferably about 15 μg/10.sup.6 cells/24 hours) were adaptively suspended using a serum-free medium. Cell supernatants were collected and bispecific antibodies were separated and purified.

    [0385] Hereinafter, the purification process, stability, in vitro and in vivo biological functions, safety, and pharmacokinetics of the bispecific antibodies of several configurations were evaluated to screen the bispecific antibody of an appropriate configuration.

    1.4 Purification Process and Stability Detection of Bispecific Antibodies

    [0386] Antibodies are generally purified by a three-step purification strategy: crude purification (sample capture), intermediate purification, and fine purification. In the crude purification stage, the target antibodies are generally captured by affinity chromatography which can effectively remove a large number of impurities such as heterologous proteins, nucleic acids, endotoxins, and viruses from the sample. The intermediate purification is often carried out using hydrophobic chromatography or CHT hydroxyapatite chromatography to remove most of the remaining impurity proteins and polymers. Fine purification is mostly carried out using anion exchange chromatography or gel filtration chromatography (molecular sieve) to remove the small or trace amount of remaining impurity proteins whose nature is similar to the nature of the target antibodies and further to remove contaminants such as HCP and DNA.

    [0387] In the present disclosure, the culture supernatant of bispecific antibody AB7K8 fused with His-tag can be crudely purified using a metal chelation affinity chromatography column (e.g., HisTrap FF from GE). The bispecific antibodies AB7K4, AB7K5, AB7K6, AB7K, and AB7K7 containing Fc can be crudely purified using a Protein A/G affinity chromatography column (e.g., Mabselect SURE from GE). The products obtained after the above crude purification are then subjected to the intermediate purification and the fine purification to finally obtain purified target antibodies of high purity and high quality. The preservation buffers for the above bispecific antibodies are then replaced with PBS or other suitable buffers using desalination columns (e.g., HiTrap desalting from GE).

    a) Purification of Double-Chain Tetravalent Bispecific Antibody AB7K7

    [0388] Specific purification steps and solutions for such bispecific antibodies of a tetravalent homodimer configuration are illustrated below by using an example of AB7K7.

    [0389] The bispecific antibody AB7K7 was purified by three-step chromatography. The three-step chromatography included affinity chromatography, hydrophobic chromatography, and anion exchange chromatography. (The protein purifier used in this example was AKTA pure 25 M from GE, U.S. Reagents used in this example were purchased from Sinopharm Chemical Reagent Co., Ltd and had purity at an analytical grade).

    [0390] In a first step, affinity chromatography was performed. Sample capture and concentration and the removal of partial pollutants were performed using an affinity chromatography medium MabSelect Sure from GE or other commercially available affinity media (e.g., Diamond Protein A from Bestchrom). First, chromatography columns were equilibrated with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 140 mM NaCl, pH 7.4) at a linear flow rate of 100-200 cm/h. The clarified fermentation broth was loaded at a linear flow rate of 100-200 cm/h with a load not higher than 20 mg/mL. After loading, the chromatography columns were equilibrated with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 140 mM NaCl, pH 7.4) at a linear flow rate of 100-200 cm/h to remove unbound components. The chromatography columns were rinsed with 3-5 column volumes of decontamination buffer 1 (50 mM NaAc-HAc, 1 M NaCl, pH 5.0) at a linear flow rate of 100-200 cm/h to remove partial pollutants. The chromatography columns were equilibrated with 3-5 column volumes (CVs) of decontamination buffer 2 (50 mM NaAc-HAc, pH 5.0) at a linear flow rate of 100-200 cm/h. The target product was eluted using an elution buffer (40 mM NaAc-HAc, pH 3.5) at a linear flow rate not higher than 100 cm/h and target peaks were collected.

    [0391] In a second step, hydrophobic chromatography was performed. Intermediate purification was performed using Butyl HP from Bestchrom or other commercially available hydrophobic chromatography media to reduce the content of polymers. After the target proteins were polymerized, since the polymers and monomers differed in property such as charge characteristics and hydrophobicity, the polymers and the monomers could be separated on the basis of the above differences between them. First, chromatography columns were equilibrated with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 0.3 M (NH.sub.4).sub.2SO.sub.4, pH 7.0) at a linear flow rate of 100-200 cm/h. The target proteins separated through the affinity chromatography in the first step were subjected to conductivity adjustment to 40-50 ms/cm with the solution of 2 M (NH.sub.4).sub.2SO.sub.4 and then loaded with a load controlled to be less than 20 mg/mL. After loading, the chromatography columns were rinsed with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 0.3 M (NH.sub.4).sub.2SO.sub.4, pH 7.0) at a linear flow rate of 100-200 cm/h. Finally, the target proteins were eluted using 3-5 column volumes (CVs) of an elution buffer (20 mM PB, pH 7.0) with gradients of 40%, 80% and 100% at a linear flow rate not higher than 100 cm/h. Eluted fractions were collected and sent for SEC-HPLC, respectively. Target components with the percentage of monomers being greater than 90% were combined for chromatography in the next step.

    [0392] In a third step, anion exchange chromatography was performed. Fine purification was performed by using Q-HP from Bestchrom or other commercially available anion exchange chromatography media (e.g., Q HP from GE, Toyopearl GigaCap Q-650 from TOSOH, DEAE Beads 6FF from Smart-Lifesciences, Generik MC-Q from Sepax Technologies, Inc, Fractogel EMD TMAE from Merck, and Q Ceramic HyperD F from Pall) to separate structural variants and further remove pollutants such as HCP and DNA. First, chromatography columns were rinsed with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, pH 7.0) at a linear flow rate of 100-200 cm/h. The target proteins separated through the hydroxyapatite chromatography in the second step were loaded and through-flow peaks were collected. After loading, the chromatography columns were rinsed with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, pH 7.0) at a linear flow rate of 100-200 cm/h. The through-flow components were collected and sent for the detection of protein content, SEC-HPLC and electrophoresis.

    [0393] The SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in FIG. 1-3 and FIG. 1-4. The SEC-HPLC results show that the purity of the main peak of the bispecific antibody was more than 95% after three-step chromatography. The band pattern in the SDS-PAGE electrophoresis was as expected, wherein a band was shown at 180 KDa in the non-reducing electrophoresis and a clear single-chain band (90 KDa) was obtained after reduction.

    b) Purification of Single-Chain Bivalent Bispecific Antibodies AB7K5 and AB7K6

    [0394] The bispecific antibody AB7K5 was purified by Protein A affinity chromatography and hydroxyapatite (CHT) chromatography. After the SEC-HPLC test, it was found that the purity of bispecific antibody AB7K5 was low, its yield was not high, and there was a problem of extremely low expression yield.

    [0395] For another single-chain bivalent bispecific antibody AB7K6, there also was a problem of high process development difficulty. The bispecific antibody AB7K6 was subjected to two-step purification, that is, Protein A affinity chromatography and molecular sieve chromatography Superdex 200. After the SEC-HPLC test, it was found that it was difficult to quantify the purity of bispecific antibody AB7K6, and there was a significant “shoulder peak” in the main peak; in addition, the expression yield of AB7K6 was very low and very unstable. After 24 hours of standing in a refrigerator at 4° C., it was found that the peak shape in the SEC-HPLC result was changed from two peaks to one main peak, which attributed, presumably, to the conversion from the single-chain structure to the double-chain structure in AB7K6. From the above, it can be seen that the current process development difficulty of AB7K6 is too high to achieve process scale-up and industrialization.

    [0396] In summary, AB7K7 had significant advantages over AB7K5 and AB7K6 in terms of process development and had advantages such as high yield, simple and efficient purification methods, and stable downstream processes. The physicochemical stability of AB7K7 in different buffer systems and at different storage conditions was further studied.

    c) Assay on Stability of Bispecific Antibody AB7K7

    [0397] The stability of AB7K7 proteins in a citrate buffer system (20 mM citrate, pH 5.5) and a histidine buffer system (20 mM histidine, pH 5.5) was studied, respectively. AB7K7 proteins were stored for four weeks under accelerated conditions at 25° C. for the evaluation of protein stability.

    [0398] AB7K7 proteins were transferred to the preceding citrate buffer system (F2) and the histidine buffer system (F3), respectively, with the concentration adjusted to 0.5 mg/mL, wherein 8% sucrose (w/v) and 0.02% PS80 (w/v) were added to both buffer systems as excipients. The above buffer systems were filtered using a 0.22 m PES membrane needle filter, and then vialed into 2 mL penicillin bottles, respectively, 0.8 mL in each bottle. After the vialing, a stopper was immediately pressed and capped. Samples were placed in different stability chambers according to the schemes in Table 1-2. Samples were taken at each sampling point for detection and analysis, wherein the detection terms included appearance, concentration, purity (detected by SEC-HPLC), HMW %, LMW %, and turbidity (A340) of the sample.

    TABLE-US-00002 TABLE 1-2 Stability detection scheme Condition T.sub.0 Sampling point and detection term 40° C. X, Y 1 Week (W) 2 W 4 W X, Y X X, Y 25° C. 1 W 2 W 4 W X, Y X X, Y Freeze-thaw 3 cycles (−70° C./ X, Y room temperature) Note: X = appearance, concentration, SEC-HPLC, SDS-PAGE (reducing & non-reducing); Y = turbidity (A340)

    [0399] The appearance, concentration, turbidity and SEC-HPLC detection results of two preparations stored for 0-4 weeks at 25° C. are shown in Table 1-3 and Table 1-4, and SDS-PAGE (reducing/non-reducing) results thereof are shown in FIG. 1-5. There was no significant change in the appearance and concentration of the two preparations. In the SEC-HPLC results, the SEC results of the F2 and F3 preparations did not show any significant change. The purity after 4 weeks was 97.9% and 98.2%, respectively. SDS-PAGE (reducing/non-reducing) results were generally consistent with the trend of LMW % results, and F2 and F3 slightly changed.

    [0400] To know the unfolding temperature of AB7K7 proteins in the two buffer systems, the Tm (unfolding temperature) and Tmonset (the temperature at which the protein begins to unfold) in the two preparations were measured by DSF and the results are shown in Table 1-5. Both preparations had low Tmonset values, and F2 and F3 had Tmonset values less than 45° C.

    TABLE-US-00003 TABLE 1-3 Appearance, concentration, and turbidity results in the acceleration test at 25° C. Appearance Concentration Turbidity A340 T 0 1 W 2 W 4 W T 0 1 W 2 W 4 W T 0* 1 W 4 W F2 Colorless clear liquid without 0.46 0.46 0.45 0.46 0.003 0.004 0.002 F3 visible foreign matter 0.47 0.46 0.45 0.47 0.004 0.003 0.005 *T 0 turbidity: the sample to be detected was a sample subjected to 1 cycle of freeze-thaw.

    TABLE-US-00004 TABLE 1-4 SEC-HPLC results in the acceleration test at 25° C. SEC-Purity % SEC-HMW % SEC-LMW % T 0 1 W 2 W 4 W T 0 1 W 2 W 4 W T 0 1 W 2 W 4 W F2 97.5 98.3 98.5 97.9 2.5 1.5 1.5 1.5 0 0.3 0.0 0.6 F3 97.7 98.8 98.7 98.2 2.3 1.2 1.3 1.2 0 0.0 0.0 0.6

    TABLE-US-00005 TABLE 1-5 DSF results Tmonset (° C.) Tm1 (° C.) Tm2 (° C.) F2 42.0 46.0 60.5 F3 41.0 45.0 58.0

    [0401] The stability of AB7K7 proteins in the above two buffer systems during freeze−thaw (−70° C./room temperature, 3 cycles of freeze-thaw) was studied by performing 3 cycles of freeze-thaw. The preparation and detection solution of the sample were the same as those described above.

    [0402] The appearance, concentration, turbidity and SEC-HPLC detection results of samples are shown in Table 1-6, and SDS-PAGE (reducing/non-reducing) results thereof are shown in FIG. 1-6. In the SDS-PAGE (non-reducing) results, there were no significant changes in the results of each detection item of both F2 and F3 preparations subjected to three cycles of freeze-thaw.

    TABLE-US-00006 TABLE 1-6 Appearance, concentration, turbidity, and SEC-HPLC results in the freeze-thaw test Appearance Concentration Turbidity A340 SEC-Purity % SEC-HMW % SEC-LMW % T 0 FT-3 C. T 0 FT-3 C. T 0 FT-3 C. T 0 FT-3 C. T 0 FT-3 C. T 0 FT-3 C. F2 Colorless Colorless 0.46 0.46 0.003 0.005 97.5 98.4 2.5 1.6 0 0.0 F3 clear liquid clear liquid 0.47 0.48 0.004 0.005 97.7 98.5 2.3 1.4 0 0.2 without visible without visible foreign matter foreign matter *T 0 turbidity: the sample to be detected was a sample subjected to 1 cycle of freeze-thaw.

    Example 2 Evaluation of In Vitro Biological Functions of Anti-Her2×CD3 Bispecific Antibodies

    2.1 Detection of Binding Activities of Bispecific Antibodies to Effector Cells and Target Cells (FACS)

    a) Detection of Binding Activities of Bispecific Antibodies to Her2-Positive Tumor Cells BT-474 by Flow Cytometry

    [0403] Tumor cells BT-474 that were positive for Her2 expression (from the cell bank of Chinese Academy of Sciences, Shanghai) were cultured, then digested with 0.25% trypsin, and centrifuged to collect cells. The collected cells were resuspended with 1% PBSB, placed in 96-well plates after the cell density was adjusted to (2×10.sup.6) cells/ml, 100 μl (2×10.sup.5 cells) per well, and blocked for 0.5 hours at 4° C. The blocked cells were centrifuged to discard the supernatant, and a series of diluted bispecific antibodies were added to the cells. The cells were incubated for 1 hour at 4° C., then centrifuged to discard the supernatant, and washed three times using a PBS solution with 1% BSA (PBSB). Diluted AF488-labeled goat anti-human IgG antibodies or murine anti-6×His IgG antibodies were added to the cells, and the cells were incubated for 1 hour at 4° C. in the dark. The obtained cells were centrifuged to discard the supernatant, and washed twice with 1% PBSB, and cells in each well were resuspended with 100 μl of 1% paraformaldehyde (PF). The signal intensity was detected by flow cytometry. The analysis was performed with the average fluorescence intensity as the Y-axis and the antibody concentration as the X-axis through software GraphPad to calculate the EC.sub.50 value for the binding of bispecific antibodies to tumor cells BT-474.

    [0404] The results show that bispecific antibodies with different structures had good binding activity to tumor cells overexpressing Her2. FIG. 2-1 to FIG. 2-5 show binding curves of bispecific antibodies with different structures to tumor cells BT-474. As shown in Table 2-1, the EC.sub.50 for the binding of AB7K to tumor cells and the EC.sub.50 for the binding of AB7K4 to tumor cells both were around 5 nM, the EC.sub.50 for the binding of AB7K7 to tumor cells was close to 50 nM, the EC.sub.50 for the binding of AB7K5 to tumor cells and the EC.sub.50 for the binding of AB7K8 to tumor cells both were greater than 100 nM, and the EC.sub.50 for the binding of AB7K6 to tumor cells was greater than 200 nM.

    TABLE-US-00007 TABLE 2-1 Detection of abilities of Anti-Her2 × CD3 bispecific antibodies to bind to tumor cells BT474 AB7K AB7K4 AB7K5 AB7K6 AB7K7 AB7K8 EC.sub.50 (nM) 5.009 4.388 125.0 239.9 51.98 125.3

    b) Detection of Binding Activities of Bispecific Antibodies to Human T Cells by FACS

    [0405] PBMCs were prepared from fresh human blood by density gradient centrifugation. The prepared PBMCs were resuspended in a 1640 medium containing 10% heat-inactivated FBS, added with 2 μg/ml OKT3 for activation for 24 h, then added with 250 IU/ml IL-2 for amplification for 7 days, to prepare cytokine-induced killer (CIK) cells which were detected by flow cytometry to be positive for CD3 expression on the surface. The to-be-detected samples were prepared and detected in the same manner as in a) of Example 2.1. Cells resuspended with 1% PF were detected on a machine and, with the average fluorescence intensity, analyzed by software OriginPro 8 to calculate the EC.sub.50 value for the binding of each bispecific antibody to human CIK cells.

    [0406] The results show that there were great differences among the binding of each bispecific antibody to CIK cells (FIG. 2-6 to FIG. 2-10). As shown in Table 2-2, the EC.sub.50 of AB7K was about 20 nM, which was roughly equal to the EC.sub.50 of AB7K4, the EC.sub.50 of AB7K7 was more than 6 times higher than the EC.sub.50 of AB7K, and the EC.sub.50 of AB7K5, AB7K6 and AB7K8 was more than 10 times higher than the EC.sub.50 of AB7K.

    TABLE-US-00008 TABLE 2-2 Detection of abilities of Anti-Her2 × CD3 bispecific antibodies to bind to effector cells CIK AB7K AB7K4 AB7K5 AB7K6 AB7K7 AB7K8 EC.sub.50 (nM) 20.51 19.44 375.2 241.7 132.3 504.1
    c) Detection of Cross-Reactivity of Bispecific Antibodies with Cynomolgus Monkey CIK Cell Membrane CD3 by FACS

    [0407] PBMCs were prepared from fresh cynomolgus monkey blood by density gradient centrifugation. The prepared PBMCs were resuspended in a 1640 medium containing 10% heat-inactivated FBS, added with 2 μg/ml OKT3 for activation for 24 h, then added with 250 IU/ml IL-2 for amplification for 7 days, to prepare cynomolgus monkey CIK cells for use. Human CIK cells and cynomolgus monkey CIK cells were collected by centrifugation, followed by the same test procedure as in the above examples. Cells resuspended with 1% paraformaldehyde solution were detected on a machine and, with the average fluorescence intensity, analyzed by software OriginPro 8 to calculate the EC.sub.50 values for the binding of bispecific antibodies to human CIK cells and the EC.sub.50 values for the binding of bispecific antibodies to cynomolgus monkey CIK cells.

    [0408] As shown in FIG. 2-11, the bispecific antibody AB7K bound well to cynomolgus monkey T cells, the ability of AB7K to bind to cynomolgus monkey T cells was roughly equal to the ability of AB7K to bind to human T cells, and the EC.sub.50 for the binding of AB7K to cynomolgus monkey T cells was approximately 26 nM as detected by flow cytometry. Bispecific antibodies AB7K4, AB7K5, AB7K6, AB7K7, and AB7K8 bound specifically to cynomolgus monkey T cells, as did AB7K.

    2.2 Detection of Abilities of Bispecific Antibodies to Bind to Antigens

    [0409] The binding of bispecific antibodies to soluble CD3 and Her2 was detected by double antigen sandwich ELISA.

    [0410] Her2 proteins (SinoBiological, Beijing, Cat. No. 10004-H08H4) were diluted with PBS to a concentration of 0.1 μg/ml and added to 96-well plates, 100 μl per well. The plates were coated at 4° C. overnight. The plates were then blocked with 1% skimmed milk powder for 1 hour at room temperature. Each bispecific antibody was diluted simultaneously with a 4-fold gradient for a total of 11 concentration gradients. The 96-well plates were then washed with PBST, and then the diluted bispecific antibodies were added. Control wells without antibodies were set. Incubated for 1 hour at room temperature. Unbound bispecific antibodies were washed away with PBST. Biotinylated CD3E and CD3D (ACRO Biosystem, Cat. No. CDD-H82W1) were mixed at 50 ng/ml with streptavdin HRP (BD, Cat. No. 554066), added in the 96-well plates, 100 μl per well, and incubated for 1 hour at room temperature. 96-well plates were washed with PBST, and TMB was added to the plates, 100 μl per wellss. Color development was performed at room temperature for 15 minutes, and then 0.2 M H.sub.2SO.sub.4 was added to stop the color development reaction. The light absorbance values at A450-620 nm were measured by a microplate reader. Analysis was performed by software OriginPro 8, and the EC.sub.50 values for the binding of bispecific antibodies to two antigens were calculated.

    [0411] The results show that each bispecific antibody bound specifically to both CD3 and Her2 molecules and exhibited good dose-dependence as the concentration of the antibodies changed (FIG. 2-12). The abilities of several bispecific antibodies to bind to soluble CD3 and Her2 are shown in Table 2-3, with EC.sub.50 values ranging from 0.03 nM to 3.8 nM which differ by two orders of magnitude. AB7K had the best binding activity, binding activities of AB7K4 and AB7K7 differed by one order of magnitude, and AB7K5 and AB7K8 had the weakest binding activity.

    TABLE-US-00009 TABLE 2-3 Detection of abilities of Anti-Her2 × CD3 bispecific antibodies to bind to CD3 and Her2 molecules AB7K AB7K4 AB7K5 AB7K7 AB7K8 EC.sub.50 (nM) 0.03128 0.1518 1.004 0.1398 3.815

    2.3 Evaluation of Abilities of Bispecific Antibodies to Activate T Cells Through Reporter Gene Cell Strains

    [0412] Jurkat T cells containing NFAT RE reporter genes (BPS Bioscience, Cat. No. 60621) can overexpress luciferase in the presence of bispecific antibodies and target cells, and the degree of activation of the Jurkat T cells can be quantified by detecting the activity of the luciferase. A four-parameter curve was fitted using the concentration of bispecific antibodies as the X-axis and the fluorescein signal as the Y-axis.

    [0413] The test results from FIG. 2-13 show that the monoclonal antibody Herceptin targeting Her2 cannot activate Jurkat T cells. T cells can be activated only in the presence of both antibodies. The ability of each antibody to activate Jurkat T cells is shown in Table 2-4. AB7K4 had the strongest ability to activate T cells, AB7K8 had the weakest ability to activate T cells, and their EC.sub.50 values differed by one order of magnitude.

    TABLE-US-00010 TABLE 2-4 Detection of abilities of Anti-Her2 × CD3 bispecific antibodies to a reporter gene cell strain that are Jurkat T cells AB7K AB7K4 AB7K5 AB7K7 AB7K8 Herceptin EC.sub.50 (nM) 0.02263 0.01338 0.05357 0.08952 0.1575 0.009907

    2.4 Abilities of Bispecific Antibodies to Mediate T Cells to Kill Tumor Cells

    [0414] Normally cultured tumor cell lines, including SK-BR-3, MCF-7, HCC1937, NCI-N87, HCC1954 cells (all purchased from the cell bank of Chinese Academy of Sciences, Shanghai), as target cells, were digested with 0.25% trypsin to prepare single-cell suspensions, added to 96-well cell culture plates after the cell density was adjusted to 2×10.sup.5 cells/ml, 100 μl per well, and cultured overnight. The antibodies were diluted according to the test design, and added to the cells, 50 μl per well, while wells without the addition of antibodies were supplemented with the same volume of the medium. Effector cells (human PBMCs or expanded CIK cells) whose number was five times larger than the number of target cells, were then added, 100 μl per well. Control wells were set, and wells without the addition of effector cells were supplemented with the same volume of the medium. After incubation for 48 hours, the supernatant was discarded from the 96-well plates. The cells were then washed three times with PBS, and a complete medium containing 10% CCK-8 was added, 100 μl per well, and the cells were incubated for 4 hours at 37° C. The light absorbance values at A450-620 nm were measured by a microplate reader. Analysis was performed by software OriginPro 8, and the ability of each bispecific antibody to mediate the killing of tumor cells and the ability of the same target monoclonal antibody Herceptin to mediate the killing of tumor cells were calculated and compared.

    [0415] The EC.sub.50 values of each bispecific antibody to mediate effector cells to kill tumor cells are shown in Table 2-5. The results show that each bispecific antibody exhibited a very significant killing effect on tumor cells (e.g., SK-BR-3, NCI-N87, and HCC1954) with high expression of Her2 in a dose-dependent manner. Each bispecific antibody, in particular AB7K7, also exhibited a good killing effect on breast cancer cells MCF-7 with low expression of Her2. Each bispecific antibody also had a good killing effect on the Herceptin-resistant cell strain HCC1954 while each bispecific antibody exhibited the killing effect on the cell strain HCC1937 that was negative for Her2 expression (with little expression) only at two highest concentrations.

    TABLE-US-00011 TABLE 2-5 EC.sub.50 values of bispecific antibodies to mediate PBMCs to kill different tumor cells EC.sub.50 (nM) AB7K7 AB7K8 AB7K5 Herceptin SK-BR-3 ~0.001 ~0.002 ~0.001  — ~0.001 0.011 — 0.067 MCF-7 ~0.005 0.079 0.055 — HCC1937 0.659 ~2.269 1.223 — 0.579 4.011 — >6.667  NCI-N87 0.015 0.034 — 0.129 HCC1954 0.002 0.018 — 0.050 Note: ~means approximately equal to, and — means that no detection is performed.
    2.5 Evaluation of the Effect of GS-CTP Linker Peptide on the Ability of Anti-CD3 scFv to Bind to CD3 Molecules by Computer Techniques

    [0416] The anti-CD3 scFv VH containing the GS-CTP linker peptide was structurally modeled using computer software and the spatial conformation of molecular docking of anti-CD3 scFv and its antigen CD3 epsilon chain was simulated and predicted.

    [0417] The sequence of the GS-CTP linker peptide between anti-Her2 scFv and anti-CD3 scFv in the bispecific antibody AB7K7 is (GGGGGGSGGGGSGGGGSSSSSKAPPPS), wherein the first half of the sequence is a GS-flexible peptide (GGGGGGSGGGGSGGGGS), and the second half is CTP-rigid peptide (SSSSKAPPPS). The rigid CTP portion (SSSSKAPPPS) is connected to the N-terminus of the anti-CD3 scFv VH. Through three-dimensional structural modeling using software phyre2, it is found that the CTP peptide fragment structurally overlays on the CDR1 region of VH of the anti-CD3 scFv (FIG. 2-14), which may hinder or disrupt the binding of the CD3 antibody to its antigen. The VH of the anti-CD3 scFv connected to the GS linker peptide (containing only the GS flexible peptide with the removal of CTP) was subjected to three-dimensional structural modeling using software phyre2, and then it is found that the GS linker peptide is far from the CDR region (FIG. 2-15) and does not affect antigen-antibody binding. Even if the GS linker peptide is close to the CDR region, the GS linker peptide can freely move away from the antigen-antibody binding region due to its own flexibility and thus does not affect antigen-antibody binding.

    [0418] Further, the molecular docking between the anti-CD3 scFv and its antigen CD3 epsilon chain was simulated by software Discovery Studio. Since the structure of the double-chain anti-CD3 FV is highly similar to the structure of the anti-CD3 scFv, the structure simulation was performed using the double-chain anti-CD3 FV instead of the anti-CD3 scFv. The simulation results show that the antigen CD3 epsilon chain binds to CDR2 and CDR3 of VH of the anti-CD3 Fv while does not bind to the CDR1 region (FIG. 2-16), which indicates that the CTP overlaying the VH CDR1 region of the anti-CD3 Fv does not interfere with the binding of the anti-CD3 scFv to the antigen. However, given that the CD3 molecule is a complex including one CD3 gamma chain, one CD3 delta chain, and two CD3 epsilon chains, the CD3 molecule, together with the TCR and Zeta chains, constitutes a T-cell receptor complex. Although the CTP peptide fragment covering the VH CDR1 of the anti-CD3 scFv does not directly interfere with the binding of the anti-CD3 scFv to its antigen CD3 epsilon chain, the CTP peptide fragment may indirectly affect the binding of the anti-CD3 scFv to its antigen CD3 epsilon chain by making spatial structural contact with a certain constituent protein of the T-cell receptor complex.

    [0419] Due to the presence of CTP covering the VH CDR1 region of the anti-CD3 scFv, the binding affinity of the anti-CD3 scFv for its antigen is greatly diminished so that there is no substantial release of cytokines caused by the overactivation of T cells, thereby avoiding some unnecessary T cell-mediated non-specific killing.

    Example 3 Pharmacodynamics Study of Anti-Her2×CD3 Bispecific Antibodies in a Mouse Transplanted Tumor Model

    3.1 NCG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Breast Cancer Cells HCC1954

    [0420] Her2-positive human breast cancer cells HCC1954 were selected to study the effect of bispecific antibodies in inhibiting tumor growth in vivo in an NCG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and HCC1954 cells.

    [0421] The peripheral blood of a normal human was subjected to density gradient centrifugation (Lymphoprep™, Lymphocytes Separation Medium, STEMCELL) to separate human PBMCs. Then the human PBMCs were resuspended in RPMI-1640 culture medium added with 10% inactivated FBS, and added with OKT3 at a final concentration of 1 μg/mL and human IL-2 at 250 IU/mL. After three days of culture, the human PBMCs were centrifuged at 300 g for 5 minutes, and the medium was changed. The cells were cultured in RPMI-1640 added with 10% inactivated FBS and added with human IL-2 at 250 IU/mL. After that, a fresh medium was then added every 2 days and CIK cells were collected on the tenth day of culture. Female NCG mice at the age of seven to eight weeks (purchased from Jiangsu GemPharmatech Co. Ltd Company) were selected and HCC1954 cells in the logarithmic growth stage were collected. 5×10.sup.6 HCC1954 cells and 5×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NCG mouse. One hour later, the mice were randomly divided into seven groups with five mice in each group according to their weights and intraperitoneally administered with corresponding drugs. All positive control groups and PBS control group were administered twice a week for a total of 3 doses, wherein the positive control groups were administered with Herceptin (from Roche) at doses of 1 mg/kg and 3 mg/kg, respectively, and the PBS control group was administered with a PBS solution of the same volume as Herceptin. The treated groups were administered with bispecific antibodies AB7K4 and AB7K7 every day at doses of 0.1 mg/kg and 1 mg/kg, respectively, for a total of 10 doses. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly with an electronic vernier caliper. The volume of the tumor was calculated using the following formula: volume (mm.sup.3)=[D×d.sup.2]/2. The tumor growth inhibition rate (TGI) was calculated for each treated group using the following formula: TGI (%)=(1−volume of the treated group/volume of the control group)×100%.

    [0422] As shown in FIG. 3-1, on Day 33 of administration, the average tumor volume of the PBS control group was 1494.61±500.28 mm.sup.3; the average tumor volume of the treated group administrated with Herceptin at a dose of 1 mg/kg was 1327.29±376.65 mm.sup.3; the average tumor volume of the treated group administrated with Herceptin at a dose of 3 mg/kg was 510.49±106.07 mm.sup.3, and the TGI was 65.84%, which was not significantly different from that of the control group.

    [0423] The average tumor volumes of treated groups administrated with AB7K4 at doses of 0.1 mg/kg and 1 mg/kg were 304.10±108.50 mm.sup.3 and 79.70±58.14 mm.sup.3, respectively, and TGIs thereof were 79.65% and 94.67%, respectively, which were significantly different from that of the PBS control group (P<0.05). The average tumor volumes of treated groups administrated with AB7K7 at doses of 0.1 mg/kg and 1 mg/kg were 385.82±95.41 mm.sup.3 and 209.98±51.74 mm.sup.3, respectively, and TGIs thereof were 74.19% and 85.95%, respectively, which were significantly different from that of the PBS control group (P<0.05). In summary, the results show that the bispecific antibodies AB7K4 and AB7K7 at different doses could inhibit the growth of tumor cells by activating human immune cells in animals and exhibited great anti-tumor effects; and at the same dose of 1 mg/kg, the anti-tumor effect of the bispecific antibody was better than that of the monoclonal antibody Herceptin.

    3.2 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Breast Cancer Cells HCC1954

    [0424] Her2-positive human breast cancer cells HCC1954 were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human breast cancer cells HCC1954.

    [0425] CIK cells were prepared in the method as described in Example 3.1. Female NPG mice at the age of seven to eight weeks (purchased from Beijing Vitalstar Biotechnology Co., Ltd.) were selected and HCC1954 cells in the logarithmic growth stage were collected. 5×10.sup.6 HCC1954 cells and 5×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. After 6 days of tumor growth, the mice were randomly divided into three groups with six mice in each group according to the tumor volumes and weights and intraperitoneally administered with corresponding drugs. Specifically, AB7K7 treated groups were administered twice a week at doses of 0.1 mg/kg and 1 mg/kg, respectively, and the control group was administered with a PBS solution of the same volume as AB7K7. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0426] As shown in FIG. 3-2, on Day 21 of administration, the average tumor volume of the PBS control group was 821.73±201.82 mm.sup.3; the average tumor volume of the treated group administrated with AB7K7 at a dose of 0.1 mg/kg was 435.60±51.04 mm.sup.3, and the TGI was 50.83%, which was not significantly different from that of the control group; the average tumor volume of the treated group administrated with AB7K7 at a dose of 1 mg/kg was 40.98±12.64 mm.sup.3, and the TGI was 95.37%, which was significantly different from that of the control group (P<0.01). The above results show that the administration of the bispecific antibody AB7K7 had a good therapeutic effect even when tumors had grown to a certain volume, wherein 50% tumor inhibition effect was achieved at the low dose of 0.1 mg/kg, and there was complete tumor regression in 4 of 6 mice in the treated group at the dose of 1 mg/kg and the tumor volumes in the other 2 mice were both less than 100 mm.sup.3, which was smaller than the tumor volume at the time of grouping (the average tumor volume of this group at the time of grouping was 161.37±18.98 mm.sup.3). Therefore, the bispecific antibody AB7K7 had a great therapeutic effect on tumors.

    [0427] In addition, the inhibiting effect of bispecific antibodies AB7K7 and AB7K8 on tumor growth in the above-described transplanted tumor model at two administration frequencies were also studied. CIK cells were prepared in the method as described above. Female NPG mice at the age of seven to eight weeks were selected, and 5×10.sup.6 HCC1954 cells and 5×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour later, the mice were randomly divided into six groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. Specifically, the control group and the Herceptin treated group were administered twice a week, wherein Herceptin was administrated at a dose of 3 mg/kg and the control group was administered with a PBS solution of the same volume as Herceptin. The bispecific antibody AB7K7 was administered at a dose of 1 mg/kg and AB7K8 was administered at a dose of 0.7 mg/kg. Two administration frequencies were set for each of the two bispecific antibodies, wherein the QD group was administered once a day for 10 consecutive days and the BIW group was administered twice a week. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The tumor volume (mm.sup.3) of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown above.

    [0428] As shown in FIG. 3-3, on Day 25 of administration, the average tumor volume of the PBS control group was 1588.12±120.46 mm.sup.3; the average tumor volume of the treated group administrated with Herceptin at a dose of 3 mg/kg was 361.72±134.70 mm.sup.3; the average tumor volumes of the QD group and the BIW group administrated with AB7K7 were 260.18±45.96 mm.sup.3 and 239.39±40.62 mm.sup.3, respectively, and TGIs were 83.62% and 84.93%, respectively, which were significantly different from that of the PBS control group (P<0.01); the average tumor volumes of the QD group and the BIW group administrated with AB7K8 were 284.98±26.62 mm.sup.3 and 647.14±118.49 mm.sup.3, respectively, and TGIs were 82.06% and 59.25%, respectively, which were significantly different from that of the PBS control group (P<0.01). As can be seen from the above results, the anti-tumor effect of the bispecific antibody AB7K7 was superior to that of the Herceptin in both the QD group and the BIW group; at equimolar doses, AB7K8 and AB7K7 in the QD group exhibited basically equal tumor inhibiting effects, while the anti-tumor effect of AB7K7 in the BIW group was significantly superior than that of AB7K8, presumably due to the fact that AB7K8 is a bispecific antibody of BiTE configuration with no Fc domain, and therefore AB7K7 has a longer half-life than AB7K8, from which it is anticipated that the clinical administration frequency of AB7K7 is reduced and AB7K7 has a better therapeutic effect.

    3.3 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Ovarian Cancer Cells SK-OV-3

    [0429] Her2-positive human ovarian cancer cells SK-OV-3 were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and SK-OV-3 cells.

    [0430] The peripheral blood of a normal human was subjected to density gradient centrifugation to separate human PBMCs. Then the human PBMCs were resuspended in McCoy's 5A culture medium added with 10% inactivated FBS, and added with OKT3 at a final concentration of 1 μg/mL and human IL-2 at 250 IU/mL. After three days of culture, the human PBMCs were centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cells were resuspended in RPMI-1640 added with 10% inactivated FBS and added with 250 IU/mL of human IL-2. After that, a fresh medium was then added every 2 days and CIK cells were collected on the tenth day of culture. Female NPG mice at the age of seven to eight weeks were selected and SK-OV-3 cells (purchased from the cell bank of Chinese Academy of Sciences, Shanghai) in the logarithmic growth stage were collected. 3×10.sup.6 SK-OV-3 cells and 3×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour after inoculation, the mice were randomly divided into seven groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. Herceptin and AB7K7 treated groups were administered twice a week at doses of 1 mg/kg, 0.2 mg/kg, and 0.04 mg/kg, respectively, and the control group was administered with a PBS solution of the same volume. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0431] As shown in FIG. 3-4, on Day 21 of administration, the average tumor volume of the PBS control group was 834.09±45.64 mm.sup.3; the average tumor volumes of the treated groups administrated with Herceptin at doses of 1 mg/kg, 0.2 mg/kg, and 0.04 mg/kg were 644.84±58.22 mm.sup.3, 884.95±38.63 mm.sup.3, and 815.79±78.39 mm.sup.3, respectively; and the tumors in all AB7K7 treated groups were completely regressed. The above results show that in the ovarian cancer SK-OV-3 model, AB7K7 enabled the tumor to completely regress even at a very low dose of 0.04 mg/kg, exhibiting an excellent anti-tumor effect.

    3.4 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Colon Cancer Cells HT-29

    [0432] Her2-positive human colon cancer cells HT-29 were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and HT-29 cells.

    [0433] CIK cells were prepared in the method as described in Example 3.1. Female NPG mice at the age of seven to eight weeks were selected and HT-29 cells (purchased from the cell bank of Chinese Academy of Sciences, Shanghai) in the logarithmic growth stage were collected. 3×10.sup.6 HT-29 cells and 3×10.sup.6 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour after inoculation, the mice were randomly divided into five groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. Specifically, Herceptin was administered at a dose of 3 mg/kg, and AB7K7 was administered at doses of 3 mg/kg, 1 mg/kg, and 0.3 mg/kg, respectively. All treated groups were administered twice a week. The control group was administered with a PBS solution of the same volume. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0434] As shown in FIG. 3-5, on Day 21 of administration, the average tumor volume of the PBS control group was 1880.52±338.26 mm.sup.3; the average tumor volume of the treated group administrated with Herceptin at a dose of 3 mg/kg was 1461.36±177.94 mm.sup.3; the average tumor volumes of the treated groups administrated with AB7K7 at doses of 3 mg/kg, 1 mg/kg, and 0.3 mg/kg were 13.94±7.06 mm.sup.3, 26.31±10.75 mm.sup.3, and 10.47±6.71 mm.sup.3, wherein tumors in four mice in the treated group at a dose of 0.3 mg/kg were completely regressed, tumors in three mice in the treated group at a dose of 1 mg/kg were completely regressed, and tumors in four mice in the treated group at a dose of 3 mg/kg were completely regressed. The above results show that in the colon cancer HT-29 model, Herceptin had few pharmacological effect on this tumor model, whereas AB7K7 exhibited complete tumor regression in mice at all three doses and exhibited excellent anti-tumor effect even at very low doses.

    3.5 CD34 Immune-Reconstituted NPG Mouse Model of Transplanted Tumor Constructed by Inoculating Human Breast Cancer Cells HCC1954

    [0435] Her2-positive human breast cancer cells HCC1954 were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in a CD34 immune-reconstituted NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human breast cancer cells HCC1954.

    [0436] CD34-positive hematopoietic stem cells were enriched from fresh umbilical cord blood using CD34-positive selective magnetic beads (purchased from Miltenyi Biotec, Germany). Female NPG mice at the age of seven to eight weeks (purchased from Beijing Vitalstar Biotechnology Co., Ltd.) were selected and injected with CD34-positive hematopoietic stem cells via the tail vein to reconstitute a human immune system in each mouse. Sixteen weeks later, blood was collected from the orbital venous plexus of mice for flow cytometry, and when the proportion of human CD45 in mice was greater than 15%, it was considered that the immune reconstitution succeeded. HCC1954 cells in the logarithmic growth stage were collected and 5×10.sup.6 HCC1954 cells were inoculated subcutaneously on the right back of the mice with successful immune reconstitution. One hour after inoculation, the mice were randomly divided into three groups with six mice in each group according to their weights. The treated groups were intraperitoneally administered with AB7K7 and Herceptin at a dose of 1 mg/kg, and the control group was administered with a PBS solution of the same volume, twice a week for a total of 6 doses. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0437] As shown in FIG. 3-6, on Day 21 of administration, the average tumor volume of the PBS control group was 475.23±58.82 mm.sup.3; the average tumor volume of the treated group administrated with Herceptin was 293.27±66.35 mm.sup.3, and the TGI was 38.29%, which was not significantly different from that of the control group; the average tumor volume of the treated group administrated with AB7K7 was 0.67±0.67 mm.sup.3, and the TGI was 99.86%, meaning that basically all tumors were regressed, which was significantly different from that of the control group (P<0.01). In summary, the above results show that the bispecific antibody AB7K7 had an excellent anti-tumor effect in the CD34 immune-reconstituted model.

    3.6 PBMC Immune-Reconstituted NPG Mouse Model of Transplanted Tumor Constructed by Inoculating Human Breast Cancer Cells HCC1954

    [0438] Her2-positive HCC1954 cells were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in a PBMC immune-reconstituted NPG mouse model of transplanted tumor constructed by inoculating human breast cancer cells HCC1954.

    [0439] The peripheral blood of a normal human was subjected to density gradient centrifugation to separate human PBMCs. Female NPG mice at the age of five to six weeks were selected and intraperitoneally injected with human PBMC cells to reconstitute a human immune system in each mouse. After seven days of PBMC injection, HCC1954 cells in the logarithmic growth stage were collected and 5×10.sup.6 HCC1954 cells were inoculated subcutaneously on the right back of each mouse. After 13 days of PBMC injection, blood was collected from the orbital venous plexus for flow cytometry, and when the proportion of human CD45 in mice was greater than 15%, it was considered that the immune reconstitution succeeded. After 14 days of PBMC injection, the mice with successful immune reconstitution were randomly divided into two groups with six mice in each group according to the tumor volumes and weights. The treated group was intraperitoneally administered with AB7K7 at a dose of 1 mg/kg, and the control group was administered with PBS, three times a week. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0440] As shown in FIG. 3-7, on Day 23 of administration, the average tumor volume of the PBS control group was 1224.05±224.39 mm.sup.3; the average tumor volume of the treated group administrated with AB7K7 was 32.00±0.00 mm.sup.3, and the TGI was 97.41%, meaning that basically all tumors were regressed, which was significantly different from that of the control group (P<0.001). In summary, the above results show that the bispecific antibody AB7K7 had an excellent anti-tumor effect in the PBMC immune-reconstituted model.

    Example 4 Evaluation of the Safety of Anti-Her2×CD3 Bispecific Antibodies

    [0441] 4.1 Bispecific Antibodies being Incapable of Mediating Non-Specific Killing on Her2-Negative Tumor Cells

    [0442] Her2-negative human Burkkit's lymphoma Raji cells were selected to study whether bispecific antibodies can inhibit tumor growth in an NCG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Raji cells.

    [0443] CIK cells were prepared in the method as described in Example 3.1. Female NCG mice at the age of seven to eight weeks were selected and Raji cells (purchased from the cell bank of Chinese Academy of Sciences, Shanghai) in the logarithmic growth stage were collected. 5×10.sup.6 Raji cells and 2×10.sup.6 CIK cells were mixed and inoculated subcutaneously on the right back of each NCG mouse. One hour after inoculation, the mice were randomly divided into three groups with five mice in each group according to their weights. The treated groups were intraperitoneally administered with AB7K4 and AB7K7 at a dose of 1 mg/kg, and the control group was administered with a PBS solution of the same volume, once a day continuously for 10 days. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0444] As shown in FIG. 4-1, on Day 25 of administration, the average tumor volume of the PBS control group was 2439.88±193.66 mm.sup.3; the average tumor volume of the treated group administrated with AB7K4 was 2408.81±212.44 mm.sup.3; the average tumor volume of the treated group administrated with AB7K7 was 2598.11±289.35 mm.sup.3; and there was no difference between the average tumor volume of each of the two treated groups and the average tumor volume of the control group. In summary, the results show that bispecific antibodies AB7K4 and AB7K7 exhibited no non-specific killing on Her2-negative cell strains, which indicates that AB7K4 and AB7K7 do not mediate T cells to kill non-target tissues in vivo (i.e., specifically dependent on binding of bispecific antibodies to corresponding target antigens), there is no off-target toxicity, and the safety is high.

    4.2 Bispecific Antibodies Killing Tumor Cells Depending on the Activation of T Cells

    [0445] Her2-positive human breast cancer cells HCC1954 were selected to study whether bispecific antibodies inhibit tumor growth in an NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human breast cancer cells HCC1954.

    [0446] Female NPG mice at the age of seven to eight weeks were selected and HCC1954 cells in the logarithmic growth stage were collected. 5×10.sup.6 HCC1954 cells and Matrigel (Corning, Cat. No. 354234) were mixed in a volume ratio of 1:1 and then inoculated subcutaneously on the right back of each NPG mouse. After 6 days of tumor growth, the mice were randomly divided into three groups with six mice in each group according to the tumor volumes and weights. The treated groups were intraperitoneally administered with Herceptin at a dose of 3 mg/kg and AB7K7 at a dose of 1 mg/kg, respectively, and the control group was administered with a PBS solution of the same volume, twice a week. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0447] As shown in FIG. 4-2, on Day 21 of administration, the average tumor volume of the PBS control group was 1311.35±215.70 mm.sup.3; the average tumor volume of the treated group administrated with Herceptin was 273.98±60.10 mm.sup.3; the average tumor volume of the treated group administrated with AB7K7 was 1243.20±340.31 mm.sup.3, which was not different from the average tumor volume of the control group. In summary, the results show that AB7K7 did not inhibit the growth of HCC1954 subcutaneous tumors in the absence of human immune cells, indicating that bispecific antibody AB7K7 needs to be mediated by immune effector cells so as to kill tumor cells, unlike Herceptin, which primarily depends on FcγR-mediated ADCC or CDC effects to kill tumor cells. Thus, it is proved that Fc variants contained in AB7K7 cannot bind to FcγR, which avoids mediating systemic activation of T cells caused by extensive expression of its receptor FcγR, resulting in higher drug safety.

    4.3 Evaluation of Toxicity of Bispecific Antibodies to Normal Cynomolgus Monkeys

    [0448] Adult cynomolgus monkeys (purchased from Guangzhou Xiangguan Biotechnology Co., Ltd.) at the age of 3-4 years and with the weight of 3-4 kg were divided into three groups with one monkey in each group, wherein the three groups were a vehicle control group, an AB7K7 treated group and an AB7K8 treated group. The groups were administrated via intravenous drip by a peristaltic pump for 1 hour on Day 0 (D0), Day 7 (D7), Day 21 (D21), and Day 28 (D28), respectively, for a total of four doses, and the drug dose was gradually increased each time. The monkeys were weighed weekly. The dose amount and volume administered are shown in Table 4-1.

    [0449] On D0, after administration, cynomolgus monkeys in the AB7K8 treated group exhibited somnolence and pupil contraction and recovered to normal the next day while there was no abnormality in the other groups. On D7, after administration, cynomolgus monkeys in the AB7K7 treated group exhibited vomiting symptoms 2-3 hours after administration and recovered to normal the next day of administration while there was no abnormality in the other groups. On D21, after administration, cynomolgus monkeys in the AB7K7 treated group exhibited symptoms of vomiting food 3 hours after administration and excreted jelly-like feces, cynomolgus monkeys in the AB7K8 treated group exhibited symptoms of vomiting food 1 hour after administration, and cynomolgus monkeys in both groups recovered to normal on the second day after administration; On D28, after administration, cynomolgus monkeys in both AB7K7 treated group and AB7K8 treated group exhibited vomiting symptoms 40 to 50 minutes later and excreted feces 3 hours later, in which jelly-like mucus was found; cynomolgus monkeys in the AB7K7 treated group excreted watery feces with fishy smelling; and 24 hours later, all the animals recovered to normal and ingested normally. The body weight change of cynomolgus monkeys is shown in FIG. 4-3, wherein the arrow represents the administration time. It can be seen that the body weight of each group does not change too much and fluctuates within the normal physiological range.

    TABLE-US-00012 TABLE 4-1 Dosing schedule for cynomolgus monkey acute toxicity evaluation To-be-tested Group drugs name Dose volume Dose amount G1 Vehicle D0: 5 mL/kg N/A control D7: 5 mL/kg group D21: 10 mL/kg D28: 10 mL/kg G2 AB7K7 D0: 5 mL/kg D0: 0.06 mg/kg D7: 5 mL/kg D7: 0.3 mg/kg D21: 10 mL/kg D21: 1.5 mg/kg D28: 10 mL/kg D28: 3 mg/kg G3 AB7K8 D0: 5 mL/kg D0: 0.04 mg/kg D7: 5 mL/kg D7: 0.2 mg/kg D21: 10 mL/kg D21: 1 mg/kg D28: 10 mL/kg D28: 2 mg/kg

    [0450] The different degree of diarrhea observed in this example may be related to the expression of related receptors in the gut, which is supposed to be caused by the imbalance of chloride ion in the gut caused by the inhibition of heterodimer of Her1/Her2 or Her2/Her3 by bispecific antibodies, which belongs to the extension of pharmacological action and can recover to normal after 24 hours of administration. Cynomolgus monkeys were still well tolerated when administrated with AB7K7 at a high dose of 3 mg/kg. The results of pharmacodynamics test in mice show that AB7K7 at a low dose shows a good anti-tumor effect, which indicates that AB7K7 has a wide treatment window and high safety.

    Example 5 Pharmacokinetics Study of Anti-Her2×CD3 Antibodies

    5.1 In Vivo Pharmacokinetics Test of Bispecific Antibody AB7K7 in SD Mice

    [0451] AB7K7 was administered to four healthy Sprague-Dawley (SD) rats (purchased from Shanghai Salccas Laboratory Animals Co., Ltd.,) via the tail vein at a dose of 1 mg/kg. The blood sampling time points were Hour 1, Hour 3, Hour 6, Hour 24, Hour 72, Hour 96, Hour 120, Hour 168, Hour 216 and Hour 264, respectively. A certain amount of whole blood was taken at each time point, the serum was separated, and then the drug concentration in the serum was detected by two ELISA methods.

    [0452] Method I. Plates were coated with the anti-AB7K7 antibody A (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 0.5 μg/mL. AB7K7 was formulated at concentrations of 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL and 1.56 ng/mL, separately. Standard curves were established. HRP-labeled anti-AB7K7 antibody B (Ampsource Biopharma Shanghai Inc., anti-herceptin-HRP) was used at a concentration of 1:5000, and developed with TMB. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 5-1.

    [0453] Method II. The drug concentration in the serum of the SD rats was detected. Plates were coated with the anti-AB7K7 antibody A (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 0.5 μg/mL. AB7K7 was formulated at concentrations of 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.156 ng/mL and 0.078 ng/mL, separately. Standard curves were established. Mouse anti-human IgG Fc-HRP (Ampsource Biopharma Shanghai Inc.) was added at a concentration of 1:5000, and developed with TMB. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 5-2.

    [0454] FIG. 5-1 shows the blood drug concentration of AB7K7 in the body of rats detected using two different detection methods. It can be seen that the blood drug concentrations obtained by detecting the concentration of AB7K7 in blood using two different detection methods were basically the same, and the calculated pharmacokinetics parameters were roughly equivalent, which indicates that AB7K7 can be metabolized in the form of intact molecules in vivo, thereby ensuring the biological function of AB7K7.

    TABLE-US-00013 TABLE 5-1 Pharmacokinetics parameters of bispecific antibody AB7K7 in SD rats (Method 1) AUC 0-inf_ob Vz_obs Cl_obs AB7K7 t.sub.1/2 (h) (ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 42.10 550236.77 0.02351 3.811E−4 parameter

    TABLE-US-00014 TABLE 5-2 Pharmacokinetics parameters of bispecific antibody AB7K7 in SD rats (Method 2) AUC 0-inf_ob Vz_obs Cl_obs AB7K7 t.sub.1/2 (h) (ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 41.02 706126.89 0.01720 2.899E−4 parameter

    5.2 In Vivo Pharmacokinetics Test on Bispecific Antibody AB7K7 in NPG Model Mice

    [0455] NPG mice (purchased from Beijing Vitalstar Biotechnology Co., Ltd.) were inoculated with HCC1954 cells (purchased from the Institute of Cells, Chinese Academy of Sciences) one week before administration, with an inoculum density of 3.5×10.sup.6/mouse. CIK cells were resuscitated two days before administration, cultured for 24 hours and then collected and injected intravenously into mice. The mice were randomly divided into three groups with four mice in each group. The three treated groups were administrated at doses of 0.3 mg/kg, 1 mg/kg and 3 mg/kg, respectively. The blood sampling time points were Hour 1, Hour 3, Hour 6, Hour 24, Hour 48, Hour 72, Hour 96, Hour 120, Hour 168, Hour 216 and Hour 264, respectively. A certain amount of whole blood was taken at each time point, the serum was separated, and then the drug concentration in the serum was detected by ELISA.

    [0456] Plates were coated with the anti-AB7K7 antibody A (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 0.5 μg/mL. AB7K7 was formulated at concentrations of 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL and 1.56 ng/mL, separately. Standard curves were established. HRP-labeled anti-AB7K7 antibody B (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) was used at a concentration of 1:5000, and developed with TMB. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 5-3. It can be seen from Table 5-3 that the pharmacokinetics parameters of AB7K7 in NPG model mice were not significantly different from those in SD rats.

    TABLE-US-00015 TABLE 5-3 Pharmacokinetics parameters of bispecific antibody AB7K7 in NPG model mice Parameter AUC0-inf_obs Vz_ob Cl_obs Group t.sub.1/2 (h) (ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h 0.3 mg/kg IV 39.54 79932.39 0.004872 8.968E−05 1 mg/kg IV 42.70 597036.63 0.002461 3.996E−05 3 mg/kg IV 46.03 2171649.41 0.002292 3.469E−05

    5.3 In Vivo Pharmacokinetics Test on Bispecific Antibody AB7K8 in SD Rats

    [0457] AB7K8 was administered to three healthy SD rats via the tail vein at doses of 1 mg/kg and 3 mg/kg, respectively. The blood sampling time points were Hour 0.25, Hour 0.5, Hour 1, Hour 2, Hour 3, Hour 4, Hour 5, and Hour 7, respectively. A certain amount of whole blood was taken at each time point, the serum was separated, and then the drug concentration in the serum was detected by ELISA.

    [0458] Plates were coated with the anti-AB7K8 antibody C (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 2.5 μg/mL. AB7K8 was formulated at concentrations of 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL, 1.56 ng/mL, and 0.78 ng/mL, separately. Standard curves were established. HRP-labeled anti-his antibody (Ampsource Biopharma Shanghai Inc.) was used at a concentration of 1:5000, and developed with TMB. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 5-4.

    [0459] The pharmacokinetics parameter T.sub.1/2 of AB7K8 were almost the same at two doses, which indicates that AB7K8 showed linear metabolic kinetics in SD rats. Since AB7K8 does not contain Fc, T.sub.1/2 of AB7K8 is very short, about twenty times shorter than T.sub.1/2 of AB7K7.

    TABLE-US-00016 TABLE 5-4 Pharmacokinetics parameters of bispecific antibody AB7K8 in SD rats AUC 0-inf_ob Vz_obs Cl_obs AB7K8 t.sub.1/2 (h) (ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h 1 mg/kg IV 2.27 4623.14 0.17082 0.05191 3 mg/kg IV 1.98 20608.77 0.10220 0.03579

    5.4 In Vivo Pharmacokinetics Test on Bispecific Antibody AB7K in SD Rats

    [0460] AB7K was administered to four healthy SD rats via the tail vein at a dose of 0.8 mg/kg. The blood sampling time points were Hour 2, Hour 24, Hour 48, Hour 72, Hour 96, Hour 120, Hour 144, Hour 168, Hour 216 and Hour 264, respectively. A certain amount of whole blood was taken at each time point, the serum was separated, and then the drug concentration in the serum was detected by two ELISA methods.

    [0461] Method I. Plates were coated with the anti-AB7K antibody A (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 1 μg/mL. AB7K was formulated at concentrations of 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL and 0.3125 ng/mL, separately. Standard curves were established. 25 ng/mL of biotin-labeled human CD3E&CD3D (Acro, Cat. No. CDD-H82W0) was added, incubated for 1 hour, and after that, HRP-labeled streptavidin (BD Pharmingen, Cat. No. 554066) diluted at a factor of 1:500 was added, and developed with TMB. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 5-5.

    TABLE-US-00017 TABLE 5-5 Pharmacokinetics parameters of bispecific antibody AB7K AUC 0-inf_ob Vz_obs Cl_obs AB7K t.sub.1/2 (h) (ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 60.47 1022788.69 0.01726 1.985E−4 parameter

    [0462] Method II. Plates were coated with the anti-AB7K antibody A (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 1 μg/mL. AB7K was formulated at concentrations of 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL and 0.3125 ng/mL, separately. Standard curves were established. Mouse anti-human IgG Fc-HRP (diluted at 1:10000) (Ampsource Biopharma Shanghai Inc.) was added, incubated in an incubator for 1 hour, and developed with TMB.

    [0463] FIG. 5-2 shows the blood drug concentration of AB7K in rats by using two different detection methods. From the results, it is showed that the difference between the two detection methods is large. The concentrations of the first two points (2h, 1D) on the curve were close, but after the next day, the concentrations detected by the two methods differed greatly, which is supposed to be caused by the fact that the linkage peptide between the heavy chains of anti-CD3 scFv and anti-Her2 antibodies was broken. AB7K is structurally unstable in vivo and thus can't play its biological function, while the improved AB7K7 can metabolize in complete form in vivo and thus can play its biological function normally.

    5.5 In Vivo Pharmacokinetics Test on Bispecific Antibodies AB7K7 and AB7K8 in Cynomolgus Monkeys

    [0464] Female cynomolgus monkeys (purchased from Guangzhou Xiangguan Biotechnology Co., Ltd.) with the weight of 3-4 kg were divided into three groups with one monkey in each group. The first group (G1-1) was a blank control group; the second group (G2-1) was an AB7K7 treated group administrated at a dose of 0.3 mg/kg; and the third group (G3-1) was an AB7K8 treated group administrated at a dose of 0.2 mg/kg. The blood sampling time points were Minute 15, Hour 1, Hour 3, Hour 6, Hour 24, Hour 48, Hour 72, Hour 96, Hour 144, Hour 192, Hour 240 and Hour 288, respectively, a total of 13 time points. Serum was collected from blood and frozen at −80° C. The concentration of the drug in serum was determined by ELISA.

    [0465] Plates were coated with the anti-AB7K7 antibody A (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 0.5 μg/mL. AB7K7 was formulated at concentrations of 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL and 1.56 ng/mL, separately. Standard curves were established. HRP-labeled anti-AB7K7 antibody B (Ampsource Biopharma Shanghai Inc., mouse-anti-herceptin) was used at a concentration of 1:5000, and developed with TMB. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 5-6.

    [0466] FIG. 5-3 shows the blood drug concentration of AB7K7 in rats. T.sub.1/2 of AB7K7 in the normal cynomolgus monkey was only about eight hours. Pharmacokinetics parameters of AB7K8 could not be calculated due to too few points on the concentration-time curve of AB7K8. However, it can be seen from the concentration-time curve that the half-life of AB7K7 in the normal cynomolgus monkey was much longer than the half-life of AB7K8.

    TABLE-US-00018 TABLE 5-6 Pharmacokinetics parameters of bispecific antibody AB7K7 in cynomolgus monkeys AUC 0-inf_obs Vz_obs Cl_obs AB7K7 t.sub.1/2 (h) (ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 7.95 87995.48 0.1563 0.01364 parameter

    5.6 Evaluation of Abilities of Bispecific Antibodies to Bind to FcRn by ELISA

    [0467] Each bispecific antibody was diluted with the PBS solution to a concentration of 10 μg/ml and added to 96-well plates, 100 μl per well. The plates were coated at 4° C. overnight. The plates were then blocked with 1% skimmed milk powder for 1 hour at room temperature. Biotin-labeled FcRn proteins (ACRO Biosystem, Cat. No. FCM-H8286) were diluted using diluents at pH of 6.0 and 7.0, respectively, with a 4-fold gradient for a total of 11 concentration gradients. The 96-well plates were then washed with PBST of the same pH, and then each of the bispecific antibodies diluted with the diluent at the same pH was added. Control wells without antibodies were set. Incubated for 1 hour at room temperature. Plates were washed with the PBST solution of the same pH, streptavidin-HRP (BD, Cat. No. 554066) was added to 96-well plates, 100 μl per well, and the plates were incubated for 0.5 hours at room temperature. 96-well plates were washed with PBST, and TMB was added to the plates, 100 μl per wells. Color development was performed at room temperature for 15 minutes, and then 0.2 M H.sub.2SO.sub.4 was added to stop the color development reaction. The light absorbance values at A450-620 nm were measured by a microplate reader. Analysis was performed by software OriginPro 8, and the EC.sub.50 values for the binding of bispecific antibodies to FcRn were calculated.

    [0468] The results show that the ability of each antibody to bind to FcRn was different under different pH conditions, and it is analyzed in conjunction with data of in vivo PK that the half-life of bispecific antibody AB7K7 was longer than the half-life of AB7K but shorter than the half-life of Herceptin, which may be more favorable for clinical application (FIGS. 5-4 and 5-5). Table 5-7 and Table 5-8 show the detection results of the ability of each antibody to bind to FcRn at pHs 6.0 and 7.0, respectively.

    TABLE-US-00019 TABLE 5-7 Detection of abilities of bispecific antibodies AB7K, AB7K5 and AB7K7 to bind to FcRn at pH 6.0 Herceptin AB7K AB7K5 AB7K7 EC.sub.50 (μg/ml) 2.591 0.8027 1.706 0.4630

    TABLE-US-00020 TABLE 5-8 Detection of abilities of bispecific antibodies AB7K, AB7K5 and AB7K7 to bind to FcRn at pH 7.0 Herceptin AB7K AB7K5 AB7K7 EC.sub.50 (μg/ml) −287.1 1.651 13.43 4.838

    Example 6 Preparation of Bispecific Antibody with scFv1-scFv2-Fc Configuration

    [0469] According to the above research results of six kinds of anti-Her2×CD3 bispecific antibodies, it can be determined that the bispecific antibody with scFv1-scFv2-Fc configuration such as AB7K7 is easy to be prepared, can be purified in a simple and efficient method, and has great stability in preparation and storage process. More advantageously, such a bispecific antibody has a weak non-specific killing effect on normal cells, has significant advantages of controlled toxic and side effects possibly caused by overactivation of effector cells, and has good druggability.

    [0470] With reference to the design and preparation method of the bispecific antibody AB7K7 in Example 1, a series of bispecific antibody molecules that target immune effector cell antigen CD3 and a tumor-associated antigen were constructed. Such bispecific antibody molecules are tetravalent homodimers formed by two identical polypeptide chains that bind to each other by an interchain disulfide bond in the hing region of the Fc fragment, wherein each polypeptide chain consists of, in sequence from N-terminus to C-terminus, an anti-TAA scFv, a linker peptide, an anti-CD3 scFv, and an Fc fragment. The molecular composition of each structural unit of each bispecific antibody is described below in detail.

    [0471] The tumor-associated antigen includes, but is not limited to, 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; preferably, the tumor-associated antigen is CD19, CD20, CD22, CD30, CD38, BCMA, CS1, EpCAM, CEA, Her2, EGFR, CA125, Mucin1, GPC-3, and Mesothelin.

    [0472] Some preferred amino acid sequences of the VH domain and its complementary determining regions (HCDR1, HCDR2, and HCDR3) and amino acid sequences of the VL domain and its complementary determining regions (LCDR1, LCDR2 and LCDR3) of a first single-chain Fv targeting the tumor-associated antigen are exemplified in Table 6-1, wherein the amino acid composition of the linker peptide between VH and VL of the anti-TAA scFv is (GGGGS).sub.n, wherein n=1, 2, 3, 4 or 5.

    TABLE-US-00021 TABLE 6-1 Amino acid sequences of the anti-TAA scFv included in the bispecific antibody and amino acid sequences of its CDR regions CD19 SEQ ID NO: 9 HCDR1 SYWMN SEQ ID NO: 10 HCDR2 QIWPGDGDTNYNGKFKG SEQ ID NO: 11 HCDR3 RETTTVGRYYYAMDY SEQ ID NO: 12 LCDR1 KASQSVDYDGDSYLN SEQ ID NO: 13 LCDR2 DASNLVS SEQ ID NO: 14 LCDR3 QQSTEDPWT QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN SEQ ID NO: 15 VH WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG RYYYAMDYWGQGTTVTVSS DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDS SEQ ID NO: 16 VL YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG TKLEIK CD19 SEQ ID NO: 17 HCDR1 SNWMH SEQ ID NO: 18 HCDR2 EIDPSDSYTNYNQNFQG SEQ ID NO: 19 HCDR3 GSNPYYYAMDY SEQ ID NO: 20 LCDR1 SASSGVNYMH SEQ ID NO: 21 LCDR2 DTSKLAS SEQ ID NO: 22 LCDR3 HQRGSYT QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMH SEQ ID NO: 23 VH WVKQAPGQGLEWIGEIDPSDSYTNYNQNFQGKAKL TVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYY AMDYWGQGTSVTVSS SEQ ID NO: 24 VL EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWY QQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTDY SLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK CD19 SEQ ID NO: 25 HCDR1 TSGMGVG SEQ ID NO: 26 HCDR2 HIWWDDDKRYNPALKS SEQ ID NO: 27 HCDR3 MELWSYYFDY SEQ ID NO: 28 LCDR1 SASSSVSYMH SEQ ID NO: 29 LCDR2 DTSKLAS SEQ ID NO: 30 LCDR3 FQGSVYPFT SEQ ID NO: 31 VH QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMG VGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVT ISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSY YFDYWGQGTLVTVSS SEQ ID NO: 32 VL EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWY QQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDF TLTISSLEPEDVAVYYCFQGSVYPFTFGQGTKLEI K CD19 SEQ ID NO: 33 HCDR1 SSWMN SEQ ID NO: 34 HCDR2 RIYPGDGDTNYNVKFKG SEQ ID NO: 35 HCDR3 SGFITTVRDFDY SEQ ID NO: 36 LCDR1 RASESVDTFGISFMN SEQ ID NO: 37 LCDR2 EASNQGS SEQ ID NO: 38 LCDR3 QQSKEVPFT SEQ ID NO: 39 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSWMN WVRQAPGKGLEWVGRIYPGDGDTNYNVKFKGRFTI SRDDSKNSLYLQMNSLKTEDTAVYYCARSGFITTV RDFDYWGQGTLVTVSS SEQ ID NO: 40 VL EIVLTQSPDFQSVTPKEKVTITCRASESVDTFGIS FMNWFQQKPDQSPKLLIHEASNQGSGVPSRFSGSG SGTDFTLTINSLEAEDAATYYCQQSKEVPFTFGGG TKVEIK CD20 SEQ ID NO: 41 HCDR1 SYNMH SEQ ID NO: 42 HCDR2 AIYPGNGDTSYNQKFKG SEQ ID NO: 43 HCDR3 STYYGGDWYFNV SEQ ID NO: 44 LCDR1 RASSSVSYIH SEQ ID NO: 45 LCDR2 ATSNLAS SEQ ID NO: 46 LCDR3 QQWTSNPPT SEQ ID NO: 47 VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD WYFNVWGAGTTVTVSA SEQ ID NO: 48 VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWF QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI K CD20 SEQ ID NO: 49 HCDR1 NYYIH SEQ ID NO: 50 HCDR2 WIYPGDGNTKYNEKFKG SEQ ID NO: 51 HCDR3 DSYSNYYFDY SEQ ID NO: 52 LCDR1 RASSSVSYMH SEQ ID NO: 53 LCDR2 APSNLAS SEQ ID NO: 54 LCDR3 QQWSFNPPT SEQ ID NO: 55 VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIH WVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATL TADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYY FDYWGQGTLVTVSS SEQ ID NO: 56 VL DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWY QQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEI K CD20 SEQ ID NO: 57 HCDR1 YSWIN SEQ ID NO: 58 HCDR2 RIFPGDGDTDYNGKFKG SEQ ID NO: 59 HCDR3 NVFDGYWLVY SEQ ID NO: 60 LCDR1 RSSKSLLHSNGITYLY SEQ ID NO: 61 LCDR2 QMSNLVS SEQ ID NO: 62 LCDR3 AQNLELPYT SEQ ID NO: 63 VH QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIN WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTI TADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYW LVYWGQGTLVTVSS SEQ ID NO: 64 VL DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGI TYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGG GTKVEIK CD20 SEQ ID NO: 65 HCDR1 DYAMH SEQ ID NO: 66 HCDR2 TISWNSGSIGYADSVKG SEQ ID NO: 67 HCDR3 DIQYGNYYYGMDV SEQ ID NO: 68 LCDR1 RASQSVSSYLA SEQ ID NO: 69 LCDR2 DASNRAT SEQ ID NO: 70 LCDR3 QQRSNWPIT SEQ ID NO: 71 VH EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMH WVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTI SRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNY YYGMDVWGQGTTVTVSS SEQ ID NO: 72 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLE IK CD22 SEQ ID NO: 73 HCDR1 RSWMN SEQ ID NO: 74 HCDR2 RIYPGDGDTNYSGKFKG SEQ ID NO: 75 HCDR3 DGSSWDWYFDV SEQ ID NO: 76 LCDR1 RSSQSIVHSVGNTFLE SEQ ID NO: 77 LCDR2 KVSNRFS SEQ ID NO: 78 LCDR3 FQGSQFPYT SEQ ID NO: 79 VH EVQLVESGGGLVQPGGSLRLSCAASGYEFSRSWMN WVRQAPGKGLEWVGRIYPGDGDTNYSGKFKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARDGSSWDW YFDVWGQGTLVTVSS SEQ ID NO: 80 VL DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSVGN TFLEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCFQGSQFPYTFGQ GTKVEIK CD22 SEQ ID NO: 81 HCDR1 IYDMS SEQ ID NO: 82 HCDR2 YISSGGGTTYYPDTVKG SEQ ID NO: 83 HCDR3 HSGYGTHWGVLFAY SEQ ID NO: 84 LCDR1 RASQDISNYLN SEQ ID NO: 85 LCDR2 YTSILHS SEQ ID NO: 86 LCDR3 QQGNTLPWT SEQ ID NO: 87 VH EVQLVESASTGGGLVKPGGSLKLSCAASGFAFSIY DMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGR FTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGY GTHWGVLFAYWGQGTLVT SEQ ID NO: 88 VL DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNW YQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTD YSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLE IK CD30 SEQ ID NO: 89 HCDR1 DYYIT SEQ ID NO: 90 HCDR2 WIYPGSGNTKYNEKFKG SEQ ID NO: 91 HCDR3 YGNYWFAY SEQ ID NO: 92 LCDR1 KASQSVDFDGDSYMN SEQ ID NO: 93 LCDR2 AASNLES SEQ ID NO: 94 LCDR3 QQSNEDPWT SEQ ID NO: 95 VH QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYIT WVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATL TVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFA YWGQGTQVTVSA SEQ ID NO: 96 VL DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDS YMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSG SGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGG TKLEIK CD30 SEQ ID NO: 97 HCDR1 AYYWS SEQ ID NO: 98 HCDR2 DINHGGGTNYNPSLKS SEQ ID NO: 99 HCDR3 LTAY SEQ ID NO: 100 LCDR1 RASQGISSWLT SEQ ID NO: 101 LCDR2 AASSLQS SEQ ID NO: 102 LCDR3 QQYDSYPIT SEQ ID NO: 103 VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYYWS WIRQPPGKGLEWIGDINHGGGTNYNPSLKSRVTIS VDTSKNQFSLKLNSVTAADTAVYYCASLTAYWGQG SLVTVSS SEQ ID NO: 104 VL DIQMTQSPTSLSASVGDRVTITCRASQGISSWLTW YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYDSYPITFGQGTRLE IK EpCAM SEQ ID NO: 105 HCDR1 SYGMH SEQ ID NO: 106 HCDR2 VISYDGSNKYYADSVKG SEQ ID NO: 107 HCDR3 DMGWGSGWRPYYYYGMDV SEQ ID NO: 108 LCDR1 RTSQSISSYLN SEQ ID NO: 109 LCDR2 WASTRES SEQ ID NO: 110 LCDR3 QQSYDIPYT SEQ ID NO: 111 VH EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDMGWGSG WRPYYYYGMDVWGQGTTVTVSS SEQ ID NO: 112 VL ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNW YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTD FTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLE IK EpCAM SEQ ID NO: 113 HCDR1 NYGMN SEQ ID NO: 114 HCDR2 WINTYTGESTYADSFKG SEQ ID NO: 115 HCDR3 FAIKGDY SEQ ID NO: 116 LCDR1 RSTKSLLHSNGITYLY SEQ ID NO: 117 LCDR2 QMSNLAS SEQ ID NO: 118 LCDR3 AQNLEIPRT SEQ ID NO: 119 VH EVQLVQSGPGLVQPGGSVRISCAASGYTFTNYGMN WVKQAPGKGLEWMGWINTYTGESTYADSFKGRFTF SLDTSASAAYLQINSLRAEDTAVYYCARFAIKGDY WGQGTLLTVSS SEQ ID NO: 120 VL DIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGI TYLYWYQQKPGKAPKLLIYQMSNLASGVPSRFSSS GSGTDFTLTISSLQPEDFATYYCAQNLEIPRTFGQ GTKVELK CEA SEQ ID NO: 121 HCDR1 DTYMH SEQ ID NO: 122 HCDR2 RIDPANGNSKYADSVKG SEQ ID NO: 123 HCDR3 FGYYVSDYAMAY SEQ ID NO: 124 LCDR1 RAGESVDIFGVGFLH SEQ ID NO: 125 LCDR2 RASNLES SEQ ID NO: 126 LCDR3 QQTNEDPYT SEQ ID NO: 127 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMH WVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSD YAMAYWGQGTLVTVSS SEQ ID NO: 128 VL DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVG FLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSG SRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQG TKVEIK CEA SEQ ID NO: 129 HCDR1 TYWMS SEQ ID NO: 130 HCDR2 EIHPDSSTINYAPSLKD SEQ ID NO: 131 HCDR3 LYFGFPWFAY SEQ ID NO: 132 LCDR1 KASQDVGTSVA SEQ ID NO: 133 LCDR2 WTSTRHT SEQ ID NO: 134 LCDR3 QQYSLYRS SEQ ID NO: 135 VH EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMS WVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTI SRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPW FAYWGQGTPVTVSS SEQ ID NO: 136 VL DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAW YQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTD FTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEI K CEA SEQ ID NO: 137 HCDR1 EFGMN SEQ ID NO: 138 HCDR2 WINTKTGEATYVEEFKG SEQ ID NO: 139 HCDR3 WDFAYYVEAMDY SEQ ID NO: 140 LCDR1 KASAAVGTYVA SEQ ID NO: 141 LCDR2 SASYRKR SEQ ID NO: 142 LCDR3 HQYYTYPLFT SEQ ID NO: 143 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMN WVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTF TTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYV EAMDYWGQGTTVTVSS SEQ ID NO: 144 VL DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAW YQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKL EIK Her2 SEQ ID NO: 145 HCDR1 DTYIH SEQ ID NO: 146 HCDR2 RIYPTNGYTRYADSVKG SEQ ID NO: 147 HCDR3 WGGDGFYAMDY SEQ ID NO: 148 LCDR1 RASQDVNTAVA SEQ ID NO: 149 LCDR2 SASFLYS SEQ ID NO: 150 LCDR3 QQHYTTPPT SEQ ID NO: 151 VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSS SEQ ID NO: 152 VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE IK Her2 SEQ ID NO: 153 HCDR1 DYTMD SEQ ID NO: 154 HCDR2 DVNPNSGGSIYNQRFKG SEQ ID NO: 155 HCDR3 NLGPSFYFDY SEQ ID NO: 156 LCDR1 KASQDVSIGVA SEQ ID NO: 157 LCDR2 SASYRYT SEQ ID NO: 158 LCDR3 QQYYIYPYT SEQ ID NO: 159 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD WVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTL SVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFY FDYWGQGTLVTVSS SEQ ID NO: 160 VL DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAW YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVE IK Her2 SEQ ID NO: 161 HCDR1 DTYIH SEQ ID NO: 162 HCDR2 RIYPTNGYTRYDPKFQD SEQ ID NO: 163 HCDR3 WGGDGFYAMDY SEQ ID NO: 164 LCDR1 KASQDVNTAVA SEQ ID NO: 165 LCDR2 SASFRYT SEQ ID NO: 166 LCDR3 QQHYTTPPT SEQ ID NO: 167 VH QVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIH WVKQRPEQGLEWIGRIYPTNGYTRYDPKFQDKATI TADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFY AMDYWGQGASVTVSS SEQ ID NO: 168 VL DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAW YQQKPGHSPKLLIYSASFRYTGVPDRFTGSRSGTD FTFTISSVQAEDLAVYYCQQHYTTPPTFGGGTKVE IK EGFR SEQ ID NO: 169 HCDR1 NYGVH SEQ ID NO: 170 HCDR2 VIWSGGNTDYNTPFTS SEQ ID NO: 171 HCDR3 ALTYYDYEFAY SEQ ID NO: 172 LCDR1 RASQSIGTNIH SEQ ID NO: 173 LCDR2 YASESIS SEQ ID NO: 174 LCDR3 QQNNNWPTT SEQ ID NO: 175 VH QVQLKQSGPGLVQPSQSLSITCTVSGF SLTNYGVH WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE FAYWGQGTLVTVSA SEQ ID NO: 176 VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE LK EGFR SEQ ID NO: 177 HCDR1 SGDYYWS SEQ ID NO: 178 HCDR2 YIYYSGSTDYNPSLKS SEQ ID NO: 179 HCDR3 VSIFGVGTFDY SEQ ID NO: 180 LCDR1 RASQSVSSYLA SEQ ID NO: 181 LCDR2 DASNRAT SEQ ID NO: 182 LCDR3 HQYGSTP LT SEQ ID NO: 183 VH QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYY WSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVT MSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGV GTFDYWGQGTLVTVSS SEQ ID NO: 184 VL EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAE IK EGFR SEQ ID NO: 185 HCDR1 NYYIY SEQ ID NO: 186 HCDR2 GINPTSGGSNFNEKFKT SEQ ID NO: 187 HCDR3 QGLWFDSDGRGFDF SEQ ID NO: 188 LCDR1 RSSQNIVHSNGNTYLD SEQ ID NO: 189 LCDR2 KVSNRFS SEQ ID NO: 190 LCDR3 FQYSHVPWT SEQ ID NO: 191 VH QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIY WVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTI TVDESTNTAYMELSSLRSEDTAFYFCARQGLWFDS DGRGFDFWGQGSTVTVSS SEQ ID NO: 192 VL DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGN TYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGS GSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQ GTKLQIT GPC-3 SEQ ID NO: 193 HCDR1 DYEMH SEQ ID NO: 194 HCDR2 AIDPQTGNTAFNQKFKG SEQ ID NO: 195 HCDR3 FYSLTY SEQ ID NO: 196 LCDR1 RSSQSIVHSNGNTYLQ SEQ ID NO: 197 LCDR2 KVSNRFS SEQ ID NO: 198 LCDR3 FQGSHFPYA SEQ ID NO: 199 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMH WVKQAPGQGLEWIGAIDPQTGNTAFNQKFKGRVTL TRDKSSSTVYMELSSLRSEDTAVYYCTRFYSLTYW GQGTLVTVSS SEQ ID NO: 200 VL DVLMTQSPLSLPVTLGQPASISCRSSQSIVHSNGN TYLQWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTYFTLKISRVEAEDVGVYYCFQGSHFPYAFGG GTKVEIK Mesothelin SEQ ID NO: 201 HCDR1 IYGMH SEQ ID NO: 202 HCDR2 VIWYDGSHEYYADSVKG SEQ ID NO: 203 HCDR3 DGDYYDSGSPLDY SEQ ID NO: 204 LCDR1 RASQSVSSYLA SEQ ID NO: 205 LCDR2 DASNRAT SEQ ID NO: 206 LCDR3 QQRSNWPLT SEQ ID NO: 207 VH QVYLVESGGGVVQPGRSLRLSCAASGITFSIYGMH WVRQAPGKGLEWVAVIWYDGSHEYYADSVKGRFTI SRDNSKNTLYLLMNSLRAEDTAVYYCARDGDYYDS GSPLDYWGQGTLVTVSS SEQ ID NO: 208 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVE IK Mucin1 SEQ ID NO: 209 HCDR1 SYVLH SEQ ID NO: 210 HCDR2 YINPYNDGTQYNEKFKG SEQ ID NO: 211 HCDR3 GFGGSYGFAY SEQ ID NO: 212 LCDR1 SASSSVSSSYLY SEQ ID NO: 213 LCDR2 STSNLAS SEQ ID NO: 214 LCDR3 HQWNRYPYT SEQ ID NO: 215 VH QVQLQQSGAEVKKPGASVKVSCEASGYTFPSYVLH WVKQAPGQGLEWIGYINPYNDGTQYNEKFKGKATL TRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYG FAYWGQGTLVTVSS SEQ ID NO: 216 VL DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLY WYQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGT DFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRL EIK Mucin1 SEQ ID NO: 217 HCDR1 NYWMN SEQ ID NO: 218 HCDR2 EIRLKSNNYTTHYAESVKG SEQ ID NO: 219 HCDR3 HYYFDY SEQ ID NO: 220 LCDR1 RSSKSLLHSNGITYFF SEQ ID NO: 221 LCDR2 QMSNLAS SEQ ID NO: 222 LCDR3 AQNLELPPT SEQ ID NO: 223 VH EVQLVESGGGLVQPGGSMRLSCVASGFPFSNYWMN WVRQAPGKGLEWVGEIRLKSNNYTTHYAESVKGRF TISRDDSKNSLYLQMNSLKTEDTAVYYCTRHYYFD YWGQGTLVTVSS SEQ ID NO: 224 VL DIVMTQSPLSNPVTPGEPASISCRSSKSLLHSNGI TYFFWYLQKPGQSPQLLIYQMSNLASGVPDRFSGS GSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGQ GTKVEIK CA125 SEQ ID NO: 225 HCDR1 SYAMS SEQ ID NO: 226 HCDR2 TISSAGGYIFYSDSVQG SEQ ID NO: 227 HCDR3 QGFGNYGDYYAMDY SEQ ID NO: 228 LCDR1 KSSQSLLNSRTRKNQLA SEQ ID NO: 229 LCDR2 WASTRQS SEQ ID NO: 230 LCDR3 QQSYNLLT SEQ ID NO: 231 VH VKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSW VRLSPEMRLEWVATISSAGGYIFYSDSVQGRFTIS RDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGD YYAMDYWGQGTTVTVSS SEQ ID NO: 232 VL DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTR KNQLAWYQQKPGQSPELLIYWASTRQSGVPDRFTG SGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGP GTKLEVK BCMA SEQ ID NO: 233 HCDR1 NYWMH SEQ ID NO: 234 HCDR2 ATYRGHSDTYYNQKFKG SEQ ID NO: 235 HCDR3 GAIYDGYDVLDN SEQ ID NO: 236 LCDR1 SASQDISNYLN SEQ ID NO: 237 LCDR2 YTSNLHS SEQ ID NO: 238 LCDR3 QQYRKLPWT SEQ ID NO: 239 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMH WVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTI TADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGY DVLDNWGQGTLVTVSS SEQ ID NO: 240 VL DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW YQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLE IK

    [0473] The anti-CD3 scFv binds to an effector cell at an EC.sub.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 FACS binding affinity assay; more preferably, the second single-chain Fv of the bispecific antibody not only binds to human CD3 but also specifically binds to CD3 of cynomolgus monkeys or rhesus monkeys.

    [0474] Some preferred amino acid sequences of the VH domain and its complementary determining regions (HCDR1, HCDR2, and HCDR3) and amino acid sequences of the VL domain and its complementary determining regions (LCDR1, LCDR2 and LCDR3) of the anti-CD3 scFv are exemplified in Table 6-2, wherein the amino acid residues contained in the CDRs are defined according to the Kabat rule, wherein the amino acid composition of the linker peptide between VH and VL of the anti-CD3 scFv is (GGGGS).sub.n, wherein n=1, 2, 3, 4 or 5.

    TABLE-US-00022 TABLE 6-2 Amino acid sequences of the anti-CD3 scFv included in the bispecific antibody and amino acid sequences of its CDR regions CD3-3 SEQ ID HCDR1 TYAMN NO: 241 SEQ ID HCDR2 RIRSKYNNYATYYADSVKD NO: 242 SEQ ID HCDR3 HGNFGNSYVSWFAY NO: 243 SEQ ID LCDR1 RSSTGAVTTSNYAN NO: 244 SEQ ID LCDR2 GTNKRAP NO: 245 SEQ ID LCDR3 ALWYSNLWV NO: 246 SEQ ID VH EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMN NO: 247 WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYVSWFAYWGQGTLVTVSS SEQ ID VL ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYA NO: 248 NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLG GKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTK LTVL CD3-4 SEQ ID HCDR1 KYAMN NO: 249 SEQ ID HCDR2 RIRSKYNNYATYYADSVKD NO: 250 SEQ ID HCDR3 HGNFGNSYISYWAY NO: 251 SEQ ID LCDR1 GSSTGAVTSGYYPN NO: 252 SEQ ID LCDR2 GTKFLAP NO: 253 SEQ ID LCDR3 ALWYSNRWV NO: 254 SEQ ID VH EVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMN NO: 255 WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSS SEQ ID VL ELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYP NO: 256 NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG GKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTK LTVL

    [0475] The linker peptide that connects the anti-TAA scFv to the anti-CD3 scFv consists of a flexible peptide and a rigid peptide; preferably, the amino acid composition of the flexible peptide has a general formula G.sub.xS.sub.y(GGGGS).sub.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 (as shown in SEQ ID NO: 257) consisting of amino acids at positions 118 to 145 of the carboxy terminus of the natural human chorionic gonadotropin beta subunit, or a truncated fragment thereof, preferably, the composition of the CTP rigid peptide is SSSSKAPPPS (CTP.sup.1). Some preferred amino acid sequences of the linker peptide that connects the anti-TAA scFv and the anti-CD3 scFv are exemplified in Table 6-3.

    TABLE-US-00023 TABLE 6-3 Amino acid sequences of the linker peptide that connects the anti-TAA scFv and the anti-CD3 scFv SEQ ID NO: G.sub.2(GGGGS).sub.3CTP.sup.1 GGGGGGSGGGGSGGGGSSSSSK 258 APPPS SEQ ID NO: (GGGGS).sub.3CTP.sup.1 GGGGSGGGGSGGGGSSSSSKAP 259 PPS SEQ ID NO: GS(GGGGS).sub.2CTP.sup.1 GSGGGGSGGGGSSSSSKAPPPS 260 SEQ ID NO: (GGGGS).sub.1CTP.sup.4 GGGGSSSSSKAPPPSLPSPSRL 261 PGPSDTPILPQ

    [0476] The Fc fragment is directly connected or connected by a linker peptide to the anti-CD3 scFv, wherein the linker peptide includes 1 to 20 amino acids that are preferably selected from the following amino acids: Gly(G), Ser(S), Ala(A), and Thr(T), more preferably Gly(G) and Ser(S); and most preferably, the linker peptide consists of (GGGGS)n, wherein n=1, 2, 3 or 4.

    [0477] The Fc fragment is preferably selected from heavy chain constant regions of human IgG1, IgG2, IgG3 and IgG4 and more particularly selected from heavy chain constant regions of human IgG1 or IgG4; and Fc is mutated to modify the properties of the bispecific antibody molecule, e.g., reduced affinity to at least one of human FcγRs (FcγRJ, FcγRIIa or FcγRIIIa) and C1q, a reduced effector cell function, or a reduced complement function. In addition, the Fc fragment may also contain amino acid substitutions that change one or more other characteristics (e.g., an ability of binding to an FcRn receptor, the glycosylation of the antibody or the charge heterogeneity of the antibody).

    [0478] Some amino acid sequences of the Fc fragment with one or more amino acid mutations are exemplified in Table 6-4.

    TABLE-US-00024 TABLE 6-4 Amino acid sequences of Fc from human IgG Amino acid sequence of a constant region of an IgG1 Fc (L234A/L235A) mutant (EU numbering) SEQ ID DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT NO: 262 CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK Amino acid sequence of a constant region of an IgG1 (L234A/L235A/T250Q/N297A/ P331S/M428L/K447-)mutant (EU numbering) SEQ ID DKTHTCPPCP APEAAGGPSV FLFPPKPKDQ LMISRTPEVT NO: 263 CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYASTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA SIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVLHE ALHNHYTQKS LSLSPGK

    [0479] Amino acid sequences of some preferred bispecific antibodies and corresponding nucleotide sequences thereof are exemplified in Table 6-5.

    TABLE-US-00025 TABLE 6-5 several bispecific antibodies of scFv1-scFv2-Fc configuration Antibody Amino acid Nucleotide sequence code Target site sequence No. No. AB1K1 Anti-CD19 × CD3 SEQ ID NO: 264 SEQ ID NO: 265 AB1K2 Anti-CD19 × CD3 SEQ ID NO: 283 SEQ ID NO: 284 AB2K Anti-CD20 × CD3 SEQ ID NO: 266 SEQ ID NO: 267 AB3K Anti-CD22 × CD3 SEQ ID NO: 268 SEQ ID NO: 269 AB4K Anti-CD30 × CD3 SEQ ID NO: 270 SEQ ID NO: 271 AB5K Anti-EpCAM × CD3  SEQ ID NO: 272 SEQ ID NO: 273 AB6K .sup. Anti-CEA × CD3 SEQ ID NO: 274 SEQ ID NO: 275 AB7K7 .sup. Anti-Her2 × CD3 SEQ ID NO: 8 SEQ ID NO: 276 AB8K Anti-EGFR × CD3.sup.  SEQ ID NO: 277 SEQ ID NO: 278 AB9K Anti-GPC-3 × CD3.sup.  SEQ ID NO: 279 SEQ ID NO: 280 AB10K Anti-Mesothelin × CD3  .sup.  SEQ ID NO: 281 SEQ ID NO: 282 AB11k Anti-Mucin 1 × CD3  SEQ ID NO: 285 SEQ ID NO: 286

    Example 7 Pharmacodynamics Study of Anti-GPC-3×CD3 Bispecific Antibodies in a Mouse Transplanted Tumor Model

    7.1 NOD-SCID Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human PBMCs and Human Liver Cancer Cells Huh-7

    [0480] GPC-3-positive human liver cancer cells Huh-7 were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in a NOD-SCID mouse model of transplanted tumor constructed by subcutaneously co-inoculating human PBMC cells and Huh-7 cells.

    [0481] The peripheral blood of a normal human was subjected to density gradient centrifugation to separate human PBMCs. Female NOD-SCID mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) at the age of seven to eight weeks were selected and Huh-7 cells in the logarithmic growth stage were collected. 3×10.sup.6 Huh-7 cells and 3×10.sup.6 PBMCs were mixed and inoculated subcutaneously on the right back of each NOD-SCID mouse. One hour after inoculation, the mice were randomly divided into two groups with six mice in each group according to their weights. The treated group was intraperitoneally administered with AB9K at a dose of 1 mg/kg, and the control group was administered with a PBS solution of the same volume, once a day continuously for 6 days. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0482] As shown in FIG. 6-1, on Day 21 of administration, the average tumor volume of the PBS control group was 1311.03±144.89 mm.sup.3; the average tumor volume of the treated group administrated with AB9K was 60.83±12.63 mm.sup.3, and the TGI was 95.36%, wherein the tumor in one mouse was completely regressed, which was significantly different from that of the control group (P<0.001). The above results show that most of PBMCs are inactivated primary T cells, the bispecific antibody AB9K can activate the primary T cells in the animals and draw the distance between the T cells and the target cell Huh-7 so that the T cells can directly kill the tumor cells and inhibit the growth of the tumor, and thus AB9K has a very good anti-tumor effect at a dose of 1 mg/kg.

    7.2 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Raji Cells

    [0483] GPC-3-negative human Burkkit's lymphoma Raji cells were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Raji cells.

    [0484] CIK cells were prepared in the method as described in Example 3.1. Female NPG mice at the age of seven to eight weeks were selected, and Raji cells in the logarithmic growth stage were collected. 5×10.sup.6 Raji cells and 2×10.sup.6 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour after inoculation, the mice were randomly divided into three groups with five mice in each group according to their weights. The treated group was intraperitoneally administered with AB9K at a dose of 1 mg/kg, and the control group was administered with a PBS solution of the same volume, once a day continuously for 10 days. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0485] As shown in FIG. 6-2, on Day 26 of administration, the average tumor volume of the PBS control group was 2636.66±196.62 mm.sup.3; the average tumor volume of the treated group administrated with AB9K was 2739.57±220.13 mm.sup.3, which was not significantly different from that of the control group. In summary, the results show that bispecific antibody AB9K exhibited no non-specific killing on GPC-3-negative cell strains, which indicates that the bispecific antibody does not mediate T cells to kill non-target tissues in vivo, there is no drug toxicity, and the safety is high.

    7.3 CD34 Immune-Reconstituted NPG Mouse Model of Transplanted Tumor Constructed by Inoculating Human Liver Cancer Huh-7 Cells

    [0486] GPC-3-positive human liver cancer Huh-7 cells were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in a CD34 immune-reconstituted NPG mouse model of transplanted tumor constructed by subcutaneously inoculating human liver cancer Huh-7 cells.

    [0487] CD34 immune-reconstituted NPG mice were prepared in the method as described in Example 3.5. Huh-7 cells in the logarithmic growth stage were collected and 2.5×10.sup.6 Huh-7 cells were inoculated subcutaneously on the right back of the immune-reconstituted mice. Four days after inoculation, the mice were randomly divided into two groups with seven mice in each group according to the tumor volumes and weights. The treated group was intraperitoneally administered with AB9K at a dose of 1 mg/kg, and the control group was administered with a PBS solution of the same volume, once a day until the test was completed. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0488] As shown in FIG. 6-3, on Day 21 of administration, the average tumor volume of the PBS control group was 2102.84±275.71 mm.sup.3; the average tumor volume of the treated group administrated with AB9K at the dose of 1 mg/kg was 325.01±282.21 mm.sup.3, and the TGI was 86.53%, wherein the tumor in four mice was completely regressed, which was significantly different from that of the control group (P<0.001). The above results show that the bispecific antibody AB9K had an excellent anti-tumor effect in the CD34 immune-reconstituted model.

    Example 8 In Vitro Biological Function Evaluation of Anti-CD20×CD3 Bispecific Antibodies and Pharmacodynamics Study of Anti-CD20×CD3 Bispecific Antibodies in a Mouse Transplanted Tumor Model

    8.1 Detection of the Binding Activity of AB2K to CD20-Positive Tumor Cells by Flow Cytometry

    [0489] Raji cells (purchased from the cell bank of Chinese Academy of Sciences) were cultured and collected by centrifugation. The collected cells were resuspended with 1% PBSB and placed in 96-well plates, 100 μl (i.e., 2×10.sup.5 cells) per well, after the cell density was adjusted to (2×10.sup.6) cells/ml. Diluted bispecific antibodies with a series of concentrations were added and incubated for 1 hour at 4° C. The cells were centrifuged to discard the supernatant and then washed three times using a PBS solution with 1% BSA (PBSB). Diluted AF488-labeled goat anti-human IgG antibodies (Jackson Immuno Research Inc., Cat. No. 109-545-088) or mouse anti-6×His IgG antibodies (R&D Systems, Cat. No. IC050P) were added to the cells, and the cells were incubated for 1 hour at 4° C. in the dark. The obtained cells were centrifuged to discard the supernatant and then washed twice with 1% PBSB, and cells in each well were resuspended with 100 μl of 1% paraformaldehyde. The signal intensity was detected by flow cytometry. The analysis was performed with the average fluorescence intensity as the Y-axis and the antibody concentration as the X-axis through software GraphPad to calculate the EC.sub.50 value for the binding of AB2K to Raji cells.

    [0490] As shown in FIG. 7-1, AB2K bound well to CD20-positive cells, the signal intensity was proportional to the antibody concentration, and the EC.sub.50 value for AB2K binding to Raji cells was calculated, which was about 69.97 nM.

    8.2 AB2K Mediating Effector Cells to Target and Kill CD20-Positive Tumor Cells

    [0491] Normally cultured Raji-luc cells (purchased from Beijing Biocytogen Biotechnology Co., Ltd.) were added to 96-well white plates after the cell density was adjusted to 1×10.sup.5 cells/ml, 40 μl per well. AB2K antibodies were diluted into a series of gradients and added to the 96-well white plates. After the CIK cell density was adjusted to 5×10.sup.5 cells/ml, the CIK cells were added to the 96-well white plates, 40 μl per well, to make the effector:target ratio (E:T) equal to 5:1, and cultured for 24 hours at 37° C. After 24 hours, the white plates were taken out, 100 μl of One-Glo (Promega, Cat. No. E6120) solution was added to each well, and then the white plates were placed for at least three minutes at room temperature. The luminescence value was measured by a microplate reader. The analysis was performed with the fluorescence intensity as the Y-axis and the antibody concentration as the X-axis through software GraphPad to calculate the EC.sub.50 value of AB2K killing Raji-luc cells.

    [0492] As shown in FIG. 7-2, the EC.sub.50 for AB2K mediating effector cells to kill Raji-luc cells was only 42.8 ng/ml and AB2K had target specificity, while the EC.sub.50 of AB7K7 as a negative control was 229.5 ng/ml and AB7K7 had little killing effect on Raji-luc cells.

    8.3 Evaluation of Abilities of Bispecific Antibodies to Activate T Cells Through Reporter Gene Cell Strains

    [0493] Jurkat T cells containing NFAT RE reporter genes (BPS Bioscience, Cat. No. 60621) can overexpress luciferase in the presence of bispecific antibodies and CD20-positive Raji cells, and the degree of activation of the Jurkat T cells can be quantified by detecting the activity of the luciferase. A four-parameter curve was fitted using the concentration of bispecific antibody as the X-axis and the fluorescein signal as the Y-axis.

    [0494] As shown in FIG. 7-3, AB2K can specifically activate Jurkat NFATRE Luc cells, wherein the EC.sub.50 value was 0.2006 μg/ml and its concentration was proportional to signal intensity, while AB7K7 as a negative control had little ability to activate T cells.

    8.4 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Raji Cells

    [0495] CD20-positive human Burkkit's lymphoma Raji cells were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma cells Raji.

    [0496] CIK cells were prepared in the method as described in Example 3.1. Female NPG mice (purchased from Beijing Vitalstar Biotechnology Co., Ltd.) at the age of seven to eight weeks were selected, and Raji cells in the logarithmic growth stage were collected. 4×10.sup.6 Raji cells and 8×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour after inoculation, the mice were randomly divided into five groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. Specifically, all treated groups were administered with Rituxan (from Roche) and bispecific antibody AB2K, respectively, at doses of 1 mg/kg and 0.1 mg/kg, twice a week. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0497] As shown in FIG. 7-4, on Day 24 of administration, the average tumor volume of the PBS control group was 1766.84±155.62 mm.sup.3; the average tumor volume of the treated group administrated with Rituxan at a dose of 1 mg/kg was 647.92±277.11 mm.sup.3, and TGI was 63.33%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with Rituxan at a dose of 0.1 mg/kg was 1893.81±186.99 mm.sup.3, and Rituxan herein exhibited no efficacy; the average tumor volume of the treated group administrated with AB2K at a dose of 1 mg/kg was 116.18±39.50 mm.sup.3, and TGI was 93.42%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with AB2K at a dose of 0.1 mg/kg was 1226.03±340.05 mm.sup.3, and TGI was 30.61%, which was not significantly different from that of the control group. The results show that the bispecific antibody AB2K could inhibit the growth of tumor cells by activating human immune cells in animals; and at the same dose, the efficacy of the bispecific antibody was better than the efficacy of the monoclonal antibody Rituxan, and the bispecific antibody exhibited great anti-tumor effects.

    8.5 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Daudi Cells

    [0498] CD20-positive human Burkkit's lymphoma Daudi cells were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Daudi cells.

    [0499] CIK cells were prepared in the method as described in Example 3.1. Female NPG mice at the age of seven to eight weeks were selected, and Daudi cells in the logarithmic growth stage were collected. 4×10.sup.6 Daudi cells and 8×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour later, the mice were randomly divided into five groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. All treated groups were administrated twice a week. Rituxan and bispecific antibody AB2K were both administered at doses of 1 mg/kg and 0.1 mg/kg, respectively. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0500] As shown in FIG. 7-5, on Day 30 of administration, the average tumor volume of the PBS control group was 889.68±192.13 mm.sup.3; the average tumor volume of the treated group administrated with Rituxan at a dose of 1 mg/kg was 241.51±44.91 mm.sup.3, and TGI was 72.85%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with Rituxan at a dose of 0.1 mg/kg was 746.11±299.71 mm.sup.3, which was not significantly different from that of the control group; the average tumor volume of the treated group administrated with AB2K at a dose of 1 mg/kg was 72.05±11.89 mm.sup.3, and TGI was 91.9%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with AB2K at a dose of 0.1 mg/kg was 75.36±11.81 mm.sup.3, and TGI was 91.53%, which was significantly different from that of the control group (P<0.01). The results show that the bispecific antibody AB2K could inhibit the growth of tumor cells by activating human immune cells in animals; and at the same dose, the efficacy of the bispecific antibody was better than the efficacy of the monoclonal antibody Rituxan, and AB2K exhibited good anti-tumor effects even at a low dose.

    Example 9 Evaluation of the Safety of Anti-CD20×CD3 Bispecific Antibodies

    [0501] The toxicity of AB2K was evaluated to determine appropriate dose ranges and observation indicators for subsequent toxicity tests. Adult Female cynomolgus monkeys (purchased from Guangzhou Xiangguan Biotechnology Co., Ltd.) at the age of 3-4 years and with the weight of 3-4 kg were divided into two groups with one mouse in each group, wherein the two groups were a vehicle control group and an AB2K treated group. The groups were administrated via intravenous drip by a peristaltic pump for 1 hour. The dose amount and volume administered are shown in Table 7. The groups were administrated on Day 0 (D0), Day 7 (D7), Day 21 (D21), and Day 28 (D28), respectively, for a total of four doses, and the drug dose was gradually escalated each time. The monkeys were weighed weekly.

    TABLE-US-00026 TABLE 7 Dosing schedule for cynomolgus monkey acute toxicity evaluation To-be-tested Group drugs name Dose volume Dose amount G1 Vehicle D0: 5 mL/kg N/A control D7: 5 mL/kg group D21: 10 mL/kg D28: 10 mL/kg G2 AB2K D0: 5 mL/kg D0: 0.06 mg/kg D7: 5 mL/kg D7: 0.3 mg/kg D21: 10 mL/kg D21: 1.5 mg/kg D28: 10 mL/kg D28: 3 mg/kg

    [0502] During the test, animals were periodically monitored for clinical symptoms, body weight, food consumption, body temperature, electrocardiogram, blood pressure, clinicopathological indexes (blood cell count, coagulation function measure, and blood biochemistry), lymphocyte subsets, cytokines, drug plasma concentration measure, and toxicokinetics analyses. After administration of AB2K, the physical signs of cynomolgus monkeys exhibited no abnormal reaction, the body weight was relatively stable, the body temperature fluctuation was similar to the body temperature fluctuation of the vehicle control group, and no death or impending death was observed among animals during the administration period. As shown in FIG. 8, after administration, the white blood cell changes of cynomolgus monkeys in the AB2K group were similar to the white blood cell changes in the control group; the first administration of AB2K at a dose of 0.06 mg/kg had little effect on lymphocytes; 1 hour to 6 hours after the second administration, the number of lymphocytes in the animals of the treated group decreased sharply and recovered to normal after 24 hours; as the number of administrations increased, the effect of AB2K on the decrease in the number of lymphocytes was weaker and weaker despite increasing doses. In addition, after the first administration of AB2K, the release of IL-2, IL-6 and TNF-α factors was promoted and the release of IL-5 was slightly stimulated, but the release of IFN-γ was not stimulated; as the number of administrations increased, the release-promoting effect of AB2K on cytokines became less and less significant, indicating that the body had already been adapted to the stimulation by bispecific antibodies.

    Example 10 Pharmacokinetics Evaluation of Anti-CD20×CD3 Bispecific Antibodies

    [0503] Female cynomolgus monkeys with the weight of 3-4 kg were divided into two groups with one in one monkey in each group. The first group was a blank control group, and the second group was an AB2K treated group administrated at a dose of 0.3 mg/kg. The blood sampling time points were Minute 15, Hour 1, Hour 3, Hour 6, Hour 10, Hour 24, Hour 30, Hour 48, Hour 54, Hour 72, Hour 96, and Hour 144, respectively, a total of 13 time points. Serum was collected from blood and frozen at −80° C.

    [0504] The drug concentration of AB2K in serum was determined by ELISA. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 8. The results show that T.sub.1/2 of AB2K in normal cynomolgus monkeys was about 8.5 hours.

    TABLE-US-00027 TABLE 8 Pharmacokinetics parameters of bispecific antibody AB2K in cynomolgus monkeys AUC 0-inf_obs Vz_obs Cl_obs AB2K t.sub.1/2 (h) (μg/mL*h) (μg/kg)/(μg/mL) (μg/kg)/(μg/mL)/h Pharmacokinetics 8.45 168.63 21.68 1.78 parameter

    Example 11 Evaluation of In Vitro Biological Functions of Anti-CD19×CD3 Bispecific Antibodies

    11.1 Detection of Binding Activities of Bispecific Antibodies to Effector Cells and Target Cells (FACS)

    a) Detection of Binding Activities of Bispecific Antibodies to CD19-Positive Tumor Raji Cells by Flow Cytometry

    [0505] CD19-positive tumor cells Raji cells were cultured and collected by centrifugation. The collected cells were resuspended with 1% PBSB, placed in 96-well plates after the cell density was adjusted to (2×10.sup.6) cells/ml, 100 μl (2×10.sup.5 cells) per well, and blocked for 0.5 hours at 4° C. The blocked cells were centrifuged to discard the supernatant, and then diluted bispecific antibodies AB1K2 with a series of concentrations and isotype CD19 bispecific antibodies AB23P8, AB23P9 and AB23P10 were added and incubated for 1 hour at 4° C. The cells were centrifuged to discard the supernatant and then washed three times using PBSB with 1% BSA. Diluted AF647-labeled goat anti-human IgG antibodies were added to the cells, and the cells were incubated for 1 hour at 4° C. in the dark. The obtained cells were centrifuged to discard the supernatant and washed twice with 1% PBSB, and cells in each well were resuspended with 100 μl of 1% PF. The signal intensity was detected by flow cytometry. The analysis was performed with the average fluorescence intensity as the Y-axis and the antibody concentration as the X-axis through software GraphPad to calculate the EC.sub.50 value for the binding of bispecific antibodies to tumor cells Raji.

    [0506] The results show that bispecific antibodies with different structures had a good binding activity to tumor cells over-expressing CD19. FIG. 9-1 shows binding curves of bispecific antibodies with different structures to tumor cells Raji. As shown in Table 9-1, EC.sub.50 for the binding of each of four bispecific antibodies to tumor cells Raji was at the nM level.

    TABLE-US-00028 TABLE 9-1 Detection of abilities of Anti-CD19 × CD3 bispecific antibodies to bind to tumor cells Raji AB1K2 AB23P8 AB23P9 AB23P10 EC.sub.50 (nM) 1.393 1.924 2.600 2.678

    b) Detection of Binding Activities of Bispecific Antibodies to Human T Cells by FACS

    [0507] PBMCs were prepared from fresh human blood by density gradient centrifugation. The prepared PBMCs were resuspended in a 1640 medium containing 10% heat-inactivated FBS, added with 2 μg/ml of CD3 antibody for activation for 24 h, then added with 250 IU/ml of IL-2 for amplification for 7 days, to prepare expanded T cells which were detected by flow cytometry to be positive for CD3 expression on the surface. The to-be-detected sample was prepared and detected in the same manner as in a) of Example 11.1. Cells resuspended with 1% PF were detected on a machine and, with the average fluorescence intensity, analyzed by software GraphPad to calculate EC.sub.50 value for the binding of each bispecific antibody to human T cells.

    [0508] The results in FIG. 9-2 show that each bispecific antibody had a good binding activity to CIK. As shown in Table 9-2, the EC.sub.50 of AB1K2 was about 16 nM, which was roughly equal to the EC.sub.50 of AB23P8, and the EC.sub.50 of AB23P9 and AB23P10 were about 50 nM and 30 nM, respectively.

    TABLE-US-00029 TABLE 9-2 Detection of abilities of Anti-CD19 × CD3 bispecific antibodies to bind to effector cells CIK AB1K2 AB23P8 AB23P9 AB23P10 EC.sub.50 (nM) 15.69 16.69 49.52 32.41
    c) Detection of Cross-Reactivity of Bispecific Antibodies with CD3 on the Surface of Cynomolgus Monkey CIK Cell Membrane by FACS

    [0509] PBMCs were prepared from fresh cynomolgus monkey blood by density gradient centrifugation. The prepared PBMCs were resuspended in a 1640 medium containing 10% heat-inactivated FBS, added with 2 μg/ml of OKT3 for activation for 24 h, then added with 250 IU/ml of IL-2 for amplification for 7 days to prepare cynomolgus monkey CIK cells for use. Human CIK cells and cynomolgus monkey CIK cells were collected by centrifugation. The to-be-detected sample was prepared and detected in the same manner as in a) of Example 11.1. Cells resuspended with 1% paraformaldehyde solution were detected on a machine and, with the average fluorescence intensity, analyzed by software GraphPad to calculate the EC.sub.50 values for the binding of bispecific antibodies to human CIK cells and the EC.sub.50 values for the binding of bispecific antibodies to cynomolgus monkey CIK cells.

    [0510] As shown in FIG. 9-3, there was no difference between the binding ability of the bispecific antibody AB1K2 to cynomolgus monkey T cells and the ability of the bispecific antibody AB23P10 to cynomolgus monkey T cells, the EC.sub.50 for the binding of each of both bispecific antibodies to cynomolgus monkey T cells was approximately 5.5 nM as detected by flow cytometry, and the ability of the both bispecific antibodies to cynomolgus monkey T cells was stronger than the ability of the both bispecific antibodies to human T cells.

    11.2 Detection of Abilities of Bispecific Antibodies to Bind to Antigens

    [0511] The binding of bispecific antibodies to soluble CD3 and CD19 was detected by double antigen sandwich ELISA.

    [0512] CD19 proteins (ACRO Biosystems, Cat. No. CD9-H5251) were diluted with PBS to a concentration of 1 μg/ml and added to 96-well plates, 100 μl per well. The plates were coated at 4° C. overnight. The plates were then blocked with 1% skimmed milk powder for 1 hour at room temperature. Each bispecific antibody was diluted with a 5-fold gradient for a total of 10 concentration gradients. The 96-well plates were then washed with PBST, and then the diluted bispecific antibodies were added. Control wells without antibodies were set. Incubated for 2 hours at room temperature. Unbound bispecific antibodies were washed away with PBST. Biotinylated CD3E&CD3D (ACRO Biosystem, Cat. No. CDD-H82W1) were mixed at 50 ng/ml with streptavdin HRP (BD, Cat. No. 554066), added in 96-well plates, 100 μl per well, and incubated for 1 hour at room temperature. 96-well plates were washed with PBST, and TMB was added to the plates, 100 μl per wells. Color development was performed at room temperature for 15 minutes, and then 0.2 M H.sub.2SO.sub.4 was added to stop the color development reaction. The light absorbance values at A450-620 nm were measured by a microplate reader. Analysis was performed by software GraphPa, and the EC.sub.50 values for the binding of bispecific antibodies to two antigens were calculated.

    [0513] The results show that each bispecific antibody bound specifically to both CD3 and CD19 molecules and exhibited good dose-dependence as the concentration of the antibodies changed (FIG. 9-4). The abilities of several bispecific antibodies to bind to soluble CD3 and CD19 are shown in Table 9-3, with EC.sub.50 values ranging from 0.19 nM to 0.47 nM, and there was little difference between binding activities at both ends.

    TABLE-US-00030 TABLE 9-3 Detection of abilities of Anti-CD19 × CD3 bispecific antibodies to bind to CD3 and CD19 molecules AB1K2 AB23P8 AB23P9 AB23P10 EC.sub.50 (nM) 0.2185 0.1925 0.2211 0.4704

    11.3 Evaluation of Abilities of Bispecific Antibodies to Activate T Cells Through Reporter Gene Cell Strains

    [0514] Jurkat T cells containing NFAT RE reporter genes can overexpress luciferase in the presence of bispecific antibodies and target cells Raji, and the degree of activation of the Jurkat T cells can be quantified by detecting the activity of the luciferase. A four-parameter curve was fitted using the concentration of bispecific antibody as the X-axis and the fluorescein signal as the Y-axis.

    [0515] The test results in FIGS. 9-5 and 9-6 show that Jurkat T cells can hardly be activated in the absence of target cells overexpressing CD19, and T cells can be activated only in the presence of both the bispecific antibody and the target cells at both ends. The ability of each bispecific antibody to activate Jurkat T cells is shown in Table 9-4, and the ability of each bispecific antibody to activate Jurkat T cells was almost equivalent to each other.

    TABLE-US-00031 TABLE 9-4 Detection of abilities of Anti-CD19 × CD3 bispecific antibodies to activate a reporter gene cell strain that are Jurkat T cells AB1K2 AB23P8 AB23P9 AB23P10 Blincyto EC.sub.50 (nM) 1.080 1.123 0.8527 0.7093 2.714

    11.4 Abilities of Bispecific Antibodies to Mediate T Cells to Kill Tumor Cells

    [0516] Normally cultured tumor cell lines, including Raji-Luc, NALM6 and Reh cells (all purchased from the cell bank of Chinese Academy of Sciences, Shanghai) were used as target cells, and cell suspensions were collected and centrifuged, added to 96-well cell culture plates after the cell density was adjusted to 2×10.sup.5 cells/ml, 100 μl per well, and cultured overnight. The antibodies were diluted according to the test design, and added to the cells, 50 μl per well, while wells without the addition of antibodies were supplemented with the same volume of the medium. Effector cells (human PBMCs or expanded CIK cells) whose number was five times larger than the number of target cells, were then added, 100 μl per well. Control wells were set, and wells without the addition of effector cells were supplemented with the same volume of the medium. After 48 hours of culture, Raji-Luc cells were detected by Steady-Glo Luciferase Assay System (Promega) and other cells were detected by CytoTox96 Non-Radio Cytotoxicity Assay (Promega). The analysis was performed with the detection results as the Y-axis and the bispecific antibody concentration as the X-axis through software GraphPad to calculate and compare the ability of each bispecific antibody to mediate the killing on Raji-luc cells.

    [0517] The EC.sub.50 values of each bispecific antibody to mediate effector cells to kill tumor cells are shown in Tables 9-5 to 9-7. The results show that each bispecific antibody exhibited a very significant killing effect on tumor cells with high expression of CD19 in a dose-dependent manner, wherein EC.sub.50 of each bispecific antibody reached the pM level.

    TABLE-US-00032 TABLE 9-5 EC.sub.50 values of bispecific antibodies to mediate CIK to kill tumor cells EC.sub.50 (pM) AB1K2 AB23P8 AB23P9 AB23P10 Blincyto Raji-LUC 0.6988 0.5861 0.1480 0.1280 0.5952 0.2024 — — 0.4834 5.654 Note: —means that no detection is performed.

    TABLE-US-00033 TABLE 9-6 EC.sub.50 values of bispecific antibodies to mediate PBMCs to kill tumor cells EC.sub.50 (pM) AB1K2 AB23P8 AB23P9 AB23P10 Blincyto Raji-LUC 1.225 1.025 1.014 0.9462 5.452 1.254 — — 1.254 21.22 — — — 4.176 22.58 Note: —means that no detection is performed.

    TABLE-US-00034 TABLE 9-7 EC.sub.50 values of bispecific antibodies to mediate CIK to kill different tumor cells EC.sub.50 (pM) AB1K2 AB23P10 Blincyto NALM6 — 4.402 77.29 Reh 1.709 1.640 11.87 Note: —means that no detection is performed.

    Example 12 Evaluation of In Vitro Biological Functions of Anti-Mucin1×CD3 Bispecific Antibodies

    12.1 Binding Activities of AB11K to Tumor Cells Over-Expressing Mucin1 and to Human or Cynomolgus Monkey Primary T Cells

    [0518] Human breast cancer cells MCF-7, BT-549, HCC70, T-47D and HCC1954, human ovarian cancer cell SK-OV-3, human cervical cancer cell Hela and human colon cancer cell HT-29 were cultured, wherein MCF-7, BT-549, T-47D, HCC1954, SK-OV-3, Hela and HT-29 cells were purchase from the cell bank of Chinese Academy of Sciences, and HCC70 cells were purchase from Nanjing Cobioer Biotechnology Co., Ltd. Each kind of the above cells was digested with trypsin, collected by centrifugation, resuspended with 1% PBSB, placed in 96-well plates after the cell density of each kind of cells was adjusted to 5×10.sup.5 cells/ml, 100 μl per well, and blocked for 30 minutes at 4° C. Human or cynomolgus monkey primary T cells were collected by centrifugation, resuspended with 1% PBSB, placed in 96-well plates after the cell density of each kind of cells was adjusted to 5×10.sup.5 cells/ml, 100 μl per well, and blocked for 30 minutes at 4° C. The cells were washed once with 1% PBSB. Diluted AB11K with a series of concentrations was added at 100 μl per well and incubated for 1 hour at 4° C. The cells were centrifuged to discard the supernatant and then washed twice with 1% PBSB. Diluted AF647 goat anti human IgG (H+L) antibodies (Jackson Immuno Research Inc., diluted at 1:250) were added, 100 μl per well, and then the cells were incubated for 1 hour at 4° C. in the dark. The cells were centrifuged to discard the supernatant. The plates were washed, and after that, 4% PFA was added, 150 μl per well, to resuspend the cells. The signal intensity was detected by flow cytometry. The analysis was performed with the average fluorescence intensity as the Y-axis and the antibody molar concentration as the X-axis through software GraphPad Prism 6 to calculate the EC.sub.50 values for the binding of AB11K to the above tumor cells and human or cynomolgus monkey primary T cells.

    [0519] As shown in FIG. 10-1 and Table 10-1, at the cellular level, AB11K bound to the above tumor cells and human or cynomolgus monkey primary T cells, and the signal intensity was proportional to the antibody concentration, and EC.sub.50 calculated for the binding of AB11K to the above tumor cells ranged from 5 nM to 300 nM, wherein the binding to T-47D and Hela was strongest, followed by the binding to HCC70, HCC1954, SKOV-3 and BT-549, while the binding to MCF-7 and HT-29 was the weakest, which did not reach the upper platform. The EC.sub.50 values for the binding of AB7K to human or cynomolgus monkey T cells were 13.43 nM and 9.996 nM, respectively, and the ability of AB11K to bind to cynomolgus monkey T cells was roughly equivalent to the ability of AB11K to bind to human T cells.

    TABLE-US-00035 TABLE 10-1 EC.sub.50 results for the binding of AB11K to tumor cells over-expressing Mucin 1 and to human or cynomolgus monkey primary T cells Cell name EC.sub.50 (nM) MCF-7 / BT-549 287.2 HCC70 58.98 T-47D 5.053 HCC1954 81.24 Hela 5.515 SK-OV-3 93.72 HT-29 / Human T cells 13.43 cynomolgus monkey 9.996 T cells

    12.2 Ability of AB11K to Mediate T Cells to Kill Tumor Cells

    [0520] Normally cultured cells MCF-7, BT-549, HCC70, T-47D, HCC1954, SK-OV-3, Hela and HT-29 were used as target cells, respectively. Each kind of cells was digested with trypsin, placed in 96-well cell culture plates after the cell density of each kind of cells was adjusted to 2×10.sup.5 cells/ml, 100 μl per well, and cultured overnight at 37° C. with 5% CO.sub.2. Effector cells (expanded T cells) whose number was five times larger than the number of corresponding target cells were added as T cell group, and effector cells (PBMCs from healthy volunteers) whose number was ten times larger than the number of corresponding target cells were added as PBMC groups, 100 μl per well. Blank wells and wells without the addition of effector cells were set. AB11K was diluted to 50 μg/mL with a medium, after the 4-fold dilution, added to 96-well plates, 50 μl per well, and incubated for 48 hours at 37° C. with 5% CO.sub.2. The cell culture plates were washed three times with PBS and the suspended cells were removed. A medium containing 10% CCK-8 was added, 100 μl per well, and incubated for 4 hours at 37° C. with 5% CO.sub.2. Readings at 450 nm and 620 nm were obtained. The specific killing rates of the antibodies were calculated according to values at [OD450-OD620] using the formula as follows:

    [00001] Antibody ⁢ ⁢ specific ⁢ ⁢ killing ⁢ ⁢ rate ⁢ ⁢ ( % ) = [ OD ⁢ ⁢ 450 - OD ⁢ ⁢ 620 ] ( effector ⁢ ⁢ cells + target ⁢ ⁢ cells ) - [ OD ⁢ ⁢ 450 - OD ⁢ ⁢ 620 ] ( antibody ⁢ ⁢ treated ⁢ ⁢ group ) [ OD ⁢ ⁢ 450 - OD ⁢ ⁢ 620 ] ( effector ⁢ ⁢ cells + target ⁢ ⁢ cells ) - [ OD ⁢ ⁢ 450 - OD ⁢ ⁢ 620 ] ( blank ⁢ ⁢ group ) × 100

    [0521] The analysis was performed with the specific killing rate (%) as the Y-axis and the antibody molar concentration as the X-axis through software GraphPad Prism 6 to calculate the EC.sub.50 value for AB11K to mediate the killing on tumor target cells.

    [0522] As shown in FIGS. 10-2 and 10-3 and Table 10-2, the bispecific antibody AB11K exhibited very significant killing effects on tumor cells highly expressing Mucin1 through its mediation on effector cells. When expanded T cells were used as effector cells, the maximum specific killing of AB11K reached 99% or more, wherein the specific killing effects on MCF-7, BT-549, HCC70 and T-47D were the best with EC.sub.50 ranging from 100 pM to 200 pM, followed by the specific killing effects on Hela, HCC1954 and SK-OV-3, while the specific killing effect on HT-29 was the weakest with a relatively large EC.sub.50, about 1577 pM. When PBMCs were used as effector cells, the specific killing effects of AB11K on MCF-7 and BT-549 were the best with the maximum specific killing of 95% or more and EC.sub.50 of 131.2 pM and 955.9 pM, respectively, followed by the specific killing effects on HCC1954 and HCC70, and EC.sub.50 for specifically killing Hela and HT-29 were relatively large, which were 4810 pM and 9550 pM, respectively.

    TABLE-US-00036 TABLE 10-2 EC.sub.50 results of AB11K to mediate effector cells to kill tumor cells T cell killing EC.sub.50 PBMC killing EC.sub.50 Cell name (pM) (pM) MCF-7 152.7 131.2 BT-549 140.9 955.9 HCC70 185.4 595.2 T-47D 84.53 / HCC1954 280.9 1893 Hela 278.2 4810 SK-OV-3 689.4 / HT-29 1577 9550

    12.3 Evaluation of Abilities of Bispecific Antibodies to Activate T Cells

    [0523] Jurkat T cells containing NFAT RE reporter genes (purchased from BPS Bioscience) can overexpress luciferase in the presence of bispecific antibodies and Mucin1-positive cells, and the degree of activation of the Jurkat T cells can be quantified by detecting the activity of the luciferase.

    [0524] Specifically, cells MCF-7, BT-549, HCC70, T-47D, HCC1954, SK-OV-3, Hela and HT-29 were digested with trypsin, placed in 96-well cell culture plates after the cell density of each kind of cells was adjusted to 2×10.sup.5 cells/ml, 50 μl per well, and cultured overnight at 37° C. with 5% CO.sub.2. The cell density of Jurkat-NFAT cells was adjusted to 2.5×10.sup.6 cells/ml, 40 μl per well. AB11K was diluted to 400 μg/mL with a medium, after the 4-fold dilution, added to 96-well plates, 10 μl per well, and incubated for 48 hours at 37° C. with 5% CO.sub.2 in an incubator. Steady-Glo® Luciferase was added, 100 μl per well and reacted for 5 minutes. After that, the luminescence value was measured by a microplate reader. The analysis was performed with the fluorescein intensity as the Y-axis and the antibody molar concentration as the X-axis through software GraphPad Prism 6 to calculate the EC.sub.50 for bispecific antibodies to activate T cells.

    [0525] As shown in FIG. 10-4 and Table 10-3, AB11K specifically activated Jurkat-NFAT cells, wherein the EC.sub.50 value was at the nM level and its concentration was proportional to signal intensity.

    TABLE-US-00037 TABLE 10-3 EC.sub.50 results of the ability of AB11K to activate T cells T cell activation EC.sub.50 Cell name (nM) MCF-7 14.22 BT-549 10.49 HCC70 3.016 T-47D 0.6294 HCC1954 5.599 Hela 7.241 SK-OV-3 10.37 HT-29 6.711

    Example 13 Pharmacodynamics Study of Anti-EGFR×CD3 Bispecific Antibodies in a Mouse Transplanted Tumor Model

    [0526] A mouse transplanted tumor model of human skin cancer A431 cells that highly expressed EGFR was selected to perform the pharmacodynamics study of Anti-EGFR×CD3 bispecific antibodies AB8K, AB2K and Erbitux (from Merck KGaA) on the in vivo inhibition of tumor growth.

    [0527] CIK cells were prepared in the method as described in Example 3.1. A431 cells in the logarithmic growth stage were collected. Female NPG mice at the age of seven to eight weeks were selected, and 3×10.sup.6 A431 cells and 1×10.sup.6 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour later, the mice were randomly divided into five groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. All treated groups and the PBS control group were administrated twice a week, wherein AB2K and Erbitux were administrated at a dose of 1 mg/kg. AB8K was administrated at doses of 1 mg/kg and 0.1 mg/kg. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 3.1.

    [0528] As shown in FIG. 11, on Day 17 of administration, the average tumor volume of the PBS control group was 1370.76±216.35 mm.sup.3; the average tumor volume of the treated group administrated with Erbitux at a dose of 1 mg/kg was 1060.35±115.86 mm.sup.3, which was not significantly different from that of the control group; the average tumor volume of the treated group administrated with AB2K at a dose of 1 mg/kg was 877.76±120.38 mm.sup.3, which was not significantly different from that of the control group; the average tumor volumes of the treated groups administrated with AB8K at doses of 0.1 mg/kg and 1 mg/kg were 233.30±135.51 mm.sup.3 and 8.14±8.14 mm.sup.3, respectively, and TGIs were 82.98% and 98.36%, respectively, which were significantly different from that of the control group (P<0.01), wherein the tumors in five of six mice of the treated group administrated with AB8K at a dose of 1 mg/kg exhibited complete regression. AB2K was an isotype control of AB8K. A431 cells did not express CD20. AB2K exhibited no pharmacological effect in this model, indicating that the structure of the bispecific antibody is relatively safe and does not cause non-specific killing. More than 90% of CIK cells were activated T cells. AB8K inhibited and killed tumor cells by activating human immune cells in animals, and completely inhibited tumor growth at a dose of 1 mg/kg and exhibited a good anti-tumor effect even at a dose of 0.1 mg/kg.

    [0529] All the publications mentioned in the present invention are incorporated herein by reference as if each publication is separately incorporated herein by reference. In addition, it should be understood that those skilled in the art, who have read the disclosure, can make various changes or modifications on the present disclosure, and these equivalent forms fall within the scope of the appended claims.