BISPECIFIC ANTIBODY AGAINST CLDN18.2 AND CD3

Abstract

The present invention relates to a bispecific antibody against CLDN18.2 and CD3. The bispecific antibody effectively binds to CLDN18.2 and CD3 antigens.

Claims

1. A bispecific antibody, comprising an antigen-binding domain specifically binding to CD3 and an antigen-binding domain specifically binding to CLDN18.2, wherein the antigen-binding domain specifically binding to CD3 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CD3 comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in any one of SEQ ID NO: 7, 10 or 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in any one of SEQ ID NO: 9, 11 or 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81 or 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 2). an antigen-binding domain specifically binding to CD3 comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; or 3). an antigen-binding domain specifically binding to CD3 comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97; and wherein the antigen-binding domain specifically binding to CLDN18.2 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CLDN18.2 comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; 2). an antigen-binding domain specifically binding to CLDN18.2 comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; or 3). an antigen-binding domain specifically binding to CLDN18.2 comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71, wherein the variants differ from the CDRs by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

2. The bispecific antibody according to claim 1, comprising an antigen-binding domain specifically binding to CD3 and an antigen-binding domain specifically binding to CLDN18.2, wherein the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, or a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, or a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; and wherein the antigen-binding domain specifically binding to CD3 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 7, or a heavy chain variable region set forth in SEQ ID NO: 7, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 9, or a light chain variable region set forth in SEQ ID NO: 9, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 2). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 10, or a heavy chain variable region set forth in SEQ ID NO: 10, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 11, or a light chain variable region set forth in SEQ ID NO: 11, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 3). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 12, or a heavy chain variable region set forth in SEQ ID NO: 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 13, or a light chain variable region set forth in SEQ ID NO: 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 4). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, or a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, or a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; or 5). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, or a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, or a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97, wherein the variants differ from the CDRs or the variable regions by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

3. The bispecific antibody according to claim 1, comprising an antigen-binding domain specifically binding to CD3 and an antigen-binding domain specifically binding to CLDN18.2, wherein the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, or a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, or a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; and wherein the antigen-binding domain specifically binding to CD3 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 7, or a heavy chain variable region set forth in SEQ ID NO: 7, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 9, or a light chain variable region set forth in SEQ ID NO: 9, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 2). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 10, or a heavy chain variable region set forth in SEQ ID NO: 10, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 11, or a light chain variable region set forth in SEQ ID NO: 11, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 3). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 12, or a heavy chain variable region set forth in SEQ ID NO: 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 13, or a light chain variable region set forth in SEQ ID NO: 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 4). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, or a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, or a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; or 5). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, or a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, or a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97, wherein the variants differ from the CDRs or the variable regions by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

4. The bispecific antibody according to claim 1, comprising an antigen-binding domain specifically binding to CD3 and an antigen-binding domain specifically binding to CLDN18.2, wherein the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, or a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, or a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; and wherein the antigen-binding domain specifically binding to CD3 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 7, or a heavy chain variable region set forth in SEQ ID NO: 7, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 9, or a light chain variable region set forth in SEQ ID NO: 9, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 2). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 10, or a heavy chain variable region set forth in SEQ ID NO: 10, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 11, or a light chain variable region set forth in SEQ ID NO: 11, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 3). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 12, or a heavy chain variable region set forth in SEQ ID NO: 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 13, or a light chain variable region set forth in SEQ ID NO: 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; 4). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, or a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, or a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; or 5). an antigen-binding domain specifically binding to CD3 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, or a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, or a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97, wherein the variants differ from the CDRs or the variable regions by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

5. The bispecific antibody according to claim 1, comprising an antigen-binding domain specifically binding to CD3 and an antigen-binding domain specifically binding to CLDN18.2, wherein (1) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 7, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 9, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; or (2) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 10, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 11, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; or (3) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; or (4) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; or (5) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 4, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 1, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 47, the CDRH2 has a sequence set forth in SEQ ID NO: 48, the CDRH3 has a sequence set forth in SEQ ID NO: 49, the CDRL1 has a sequence set forth in SEQ ID NO: 50, the CDRL2 has a sequence set forth in SEQ ID NO: 51, and the CDRL3 has a sequence set forth in SEQ ID NO: 52; or (6) wherein the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 7, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 9, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is selected from SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; or (7) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 10, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 11, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; or (8) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; or (9) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; or (10) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, preferably, the CDRH1 is selected from SEQ ID NOs: 53, 59 and 64, the CDRH2 is selected from SEQ ID NOs: 54, 60 and 65, the CDRH3 is selected from SEQ ID NOs: 55 and 61, the CDRL1 is selected from SEQ ID NOs: 56 and 62, the CDRL2 is selected from SEQ ID NOs: 57 and 63, and the CDRL3 is set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 53, the CDRH2 has a sequence set forth in SEQ ID NO: 54, the CDRH3 has a sequence set forth in SEQ ID NO: 55, the CDRL1 has a sequence set forth in SEQ ID NO: 56, the CDRL2 has a sequence set forth in SEQ ID NO: 57, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 59, the CDRH2 has a sequence set forth in SEQ ID NO: 60, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 64, the CDRH2 has a sequence set forth in SEQ ID NO: 65, the CDRH3 has a sequence set forth in SEQ ID NO: 61, the CDRL1 has a sequence set forth in SEQ ID NO: 62, the CDRL2 has a sequence set forth in SEQ ID NO: 63, and the CDRL3 has a sequence set forth in SEQ ID NO: 58; or (11) wherein the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 7, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 9, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 81, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; or (12) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 10, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 11, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; or (13) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 12, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 13, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 79, the CDRH2 has a sequence set forth in SEQ ID NO: 80, the CDRH3 has a sequence set forth in SEQ ID NO: 85, the CDRL1 has a sequence set forth in SEQ ID NO: 82, the CDRL2 has a sequence set forth in SEQ ID NO: 83, and the CDRL3 has a sequence set forth in SEQ ID NO: 84; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; or (14) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 14, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 15, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 86, the CDRH2 has a sequence set forth in SEQ ID NO: 87, the CDRH3 has a sequence set forth in SEQ ID NO: 88, the CDRL1 has a sequence set forth in SEQ ID NO: 89, the CDRL2 has a sequence set forth in SEQ ID NO: 90, and the CDRL3 has a sequence set forth in SEQ ID NO: 91; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; or (15) the antigen-binding domain specifically binding to CD3 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 16, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 17, preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 92, the CDRH2 has a sequence set forth in SEQ ID NO: 93, the CDRH3 has a sequence set forth in SEQ ID NO: 94, the CDRL1 has a sequence set forth in SEQ ID NO: 95, the CDRL2 has a sequence set forth in SEQ ID NO: 96, and the CDRL3 has a sequence set forth in SEQ ID NO: 97; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, preferably, the CDRH1 is selected from SEQ ID NOs: 66, 72 and 77, the CDRH2 is selected from SEQ ID NOs: 67, 73 and 78, the CDRH3 is selected from SEQ ID NOs: 68 and 74, the CDRL1 is selected from SEQ ID NOs: 69 and 75, the CDRL2 is selected from SEQ ID NOs: 70 and 76, and the CDRL3 is set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 66, the CDRH2 has a sequence set forth in SEQ ID NO: 67, the CDRH3 has a sequence set forth in SEQ ID NO: 68, the CDRL1 has a sequence set forth in SEQ ID NO: 69, the CDRL2 has a sequence set forth in SEQ ID NO: 70, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 72, the CDRH2 has a sequence set forth in SEQ ID NO: 73, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; more preferably, the CDRH1 has a sequence set forth in SEQ ID NO: 77, the CDRH2 has a sequence set forth in SEQ ID NO: 78, the CDRH3 has a sequence set forth in SEQ ID NO: 74, the CDRL1 has a sequence set forth in SEQ ID NO: 75, the CDRL2 has a sequence set forth in SEQ ID NO: 76, and the CDRL3 has a sequence set forth in SEQ ID NO: 71; wherein the variants differ from the CDRs by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

6. The bispecific antibody according to claim 1, wherein the antigen-binding domain specifically binding to CD3 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CD3 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 9; 2). an antigen-binding domain specifically binding to CD3 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 10, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 11; 3). an antigen-binding domain specifically binding to CD3 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 13; 4). an antigen-binding domain specifically binding to CD3 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 15; or 5). an antigen-binding domain specifically binding to CD3 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 17; wherein the antigen-binding domain specifically binding to CLDN18.2 is selected from the group consisting of: 1). an antigen-binding domain specifically binding to CLDN18.2 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 4, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 1; 2). an antigen-binding domain specifically binding to CLDN18.2 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or 3). an antigen-binding domain specifically binding to CLDN18.2 comprising the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 6, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 3, wherein the variants differ from the variable region amino acid sequences by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

7. The bispecific antibody according to claim 1, comprising an antigen-binding domain specifically binding to CD3 and an antigen-binding domain specifically binding to CLDN18.2, wherein (1) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 9; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 4, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 1; or (2) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 10, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 11; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 4, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 1; or (3) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 13; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 4, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 1; or (4) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 15; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 4, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 1; or (5) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 17; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 4, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 1; or (6) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 9; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or (7) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 10, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 11; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or (8) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 13; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or (9) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 15; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or (10) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 17; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 5, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or (11) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 9; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 6, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 3; or (12) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 10, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 11; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 6, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 3; or (13) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 13; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 6, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 3; or (14) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 15; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 6, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 3; or (15) the antigen-binding domain specifically binding to CD3 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 17; and the antigen-binding domain specifically binding to CLDN18.2 comprises the following variable region amino acid sequences or variants thereof: (i) a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 6, and (ii) a light chain variable region amino acid sequence set forth in SEQ ID NO: 3; wherein the variants differ from the variable region amino acid sequences by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

8. The bispecific antibody according to claim 1, wherein the antigen-binding domain specifically binding to CLDN18.2 is in the form of an Fab fragment and the antigen-binding domain specifically binding to CD3 is in the form of an scFv, preferably, the bispecific antibody comprising: a) a first monomer, comprising a first heavy chain, wherein the first heavy chain comprises: 1) a first heavy chain variable region; 2) a first heavy chain constant region, comprising a first CH1 domain and a first Fc domain (wherein, preferably the first CH1 domain and the first Fc domain are linked by hinge region 1, and preferably the first Fc domain is selected from an IgG1 subtype, an IgG2 subtype, an IgG3 subtype and an IgG4 subtype); 3) an antigen-binding domain specifically binding to CD3 (preferably human CD3), wherein a heavy chain variable region and a light chain variable region in the antigen-binding domain are linked by a linker peptide and positions of the two are interchangeable, wherein the antigen-binding domain specifically binding to CD3 is as defined in claim 1, wherein i) the antigen-binding domain is linked to a C-terminus of the first CH1 domain and an N-terminus of the first Fc domain by a linker peptide, preferably by a linker peptide and a hinge region (e.g., hinge region 3), and preferably the antigen-binding domain is linked to the first Fc domain by a linker peptide and a hinge region (e.g., hinge region 2), or ii) the antigen-binding domain is linked to a C-terminus of the first Fc domain by a linker peptide, preferably by a linker peptide and/or a hinge region (e.g., hinge region 1, 2 or 3), or iii) the antigen-binding domain is linked to an N-terminus of the first heavy chain variable region by a linker peptide, preferably by a linker peptide and/or a hinge region (e.g., hinge region 1, 2 or 3); b) a second monomer, comprising a second heavy chain, wherein the second heavy chain comprises: a second heavy chain variable region and a second heavy chain constant region, the second heavy chain constant region comprising a second CH1 domain and a second Fc domain (preferably the second CH1 domain and the second Fc domain are linked by a hinge region (e.g., hinge region 1), and preferably the second Fc domain is selected from an IgG1 subtype, an IgG2 subtype, an IgG3 subtype and an IgG4 subtype), c) a first light chain assembled with the first heavy chain and comprising a first light chain variable region and a first light chain constant region; d) a second light chain assembled with the second heavy chain and comprising a second light chain variable region and a second light chain constant region; wherein the first light chain constant region is identical to or different from the second light chain constant regions, the first CH1 domain is identical to or different from the second CH1 domain, and the first Fc domain is identical to or different from the second Fc domain; wherein the first heavy chain variable region and the first light chain variable region form a first antigen-binding domain, the second heavy chain variable region and the second light chain variable region form a second antigen-binding domain, and a sequence of the first antigen-binding domain and the second antigen-binding domain is a sequence of the antigen-binding domain specifically binding to CLDN18.2 as defined in claim 1, and the first antigen-binding domain and the second antigen-binding domain have identical or different sequences.

9. The bispecific antibody according to claim 1, wherein the antigen-binding domain specifically binding to CLDN18.2 is in the form of an Fab fragment and the antigen-binding domain specifically binding to CD3 is in the form of an scFv, preferably, the bispecific antibody comprising: a) a first monomer, comprising a heavy chain, wherein the heavy chain comprises: 1) a first heavy chain variable region; 2) a heavy chain constant region comprising a CH1 domain and a first Fc domain (preferably the CH1 domain and the first Fc domain are linked by a hinge region (e.g., hinge region 1), and preferably the first Fc domain is selected from an IgG1 subtype, an IgG2 subtype, an IgG3 subtype and an IgG4 subtype); b) a second monomer, comprising an antigen-binding domain specifically binding to CD3 (preferably human CD3), wherein the antigen-binding domain is linked to an N-terminus of a second Fc domain (preferably the second Fc domain is selected from an IgG1 subtype, an IgG2 subtype, an IgG3 subtype and an IgG4 subtype) by a linker peptide (preferably by a linker peptide and/or a hinge region (e.g., hinge region 1, 2 or 3)), wherein a heavy chain variable region and a light chain variable region in the antigen-binding domain specifically binding to CD3 are linked by a linker peptide and positions of the two are interchangeable, wherein the antigen-binding domain specifically binding to CD3 is as defined in claim 1, c) a light chain assembled with the heavy chain and comprising a first light chain variable region and a light chain constant region; wherein the first heavy chain variable region and the first light chain variable region form the antigen-binding domain specifically binding to CLDN18.2 as defined in claim 1.

10. The bispecific antibody according to claim 9, wherein the antigen-binding domain specifically binding to CLDN18.2 is selected from the group consisting of: 1) an antigen-binding domain specifically binding to CLDN18.2 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 5, or a heavy chain variable region set forth in SEQ ID NO: 5, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 2, or a light chain variable region set forth in SEQ ID NO: 2; or 2) an antigen-binding domain specifically binding to CLDN18.2 comprising the following CDRs (or variants thereof) or variable regions (or variants thereof): (i) CDRH1, CDRH2 and CDRH3 comprised in a heavy chain variable region set forth in SEQ ID NO: 6, or a heavy chain variable region set forth in SEQ ID NO: 6, and (ii) CDRL1, CDRL2 and CDRL3 comprised in a light chain variable region set forth in SEQ ID NO: 3, or a light chain variable region set forth in SEQ ID NO: 3, wherein the variants differ from the CDRs or the variable regions by 3, 2 or 1 amino acids or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

11. The bispecific antibody according to claim 8, wherein the light chain constant region CL has a sequence set forth in SEQ ID NO: 18, 98, 99 or 100, and the CH1 domain has a sequence set forth in SEQ ID NO: 19, 101, 102 or 103, or wherein the first Fc domain or the second Fc domain has identical or different sequence, preferably selected from SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26, or wherein the hinge region 1 has a sequence set forth in SEQ ID NO: 27, the hinge region 2 has a sequence set forth in SEQ ID NO: 28, and/or the hinge region 3 has a sequence set forth in SEQ ID NO: 29, or wherein the linker peptide has a sequence selected from sequences set forth in SEQ ID NOs: 8, 30, 31 and 32.

12. The bispecific antibody according to claim 9, wherein the light chain constant region CL has a sequence set forth in SEQ ID NO: 18, 98, 99 or 100, and the CH1 domain has a sequence set forth in SEQ ID NO: 19, 101, 102 or 103, or wherein the first Fc domain or the second Fc domain has identical or different sequence, preferably selected from SEQ ID NO: 20, 21, 22, 23, 24, 25 or 26, or wherein the hinge region 1 has a sequence set forth in SEQ ID NO: 27, the hinge region 2 has a sequence set forth in SEQ ID NO: 28, and/or the hinge region 3 has a sequence set forth in SEQ ID NO: 29, or wherein the linker peptide has a sequence selected from sequences set forth in SEQ ID NOs: 8, 30, 31 and 32.

13. (canceled)

14. (canceled)

15. A nucleic acid composition, comprising a nucleic acid sequence encoding the bispecific antibody according to claim 1.

16. The nucleic acid composition according to claim 15, which is contained in an expression vector or a host cell.

17. (canceled)

18. A material which is selected from the group consisting of the followings: (1) a pharmaceutical composition, comprising the bispecific antibody according to claim 1 and a pharmaceutical carrier, and optionally, a drug (e.g., a small molecule drug or a macromolecular drug) for the treatment of cancer (e.g., gastric cancer, pancreatic cancer, ovarian cancer, esophageal cancer or non-small cell lung cancer); a kit, comprising the bispecific antibody according to claim 1, and optionally, a drug (e.g., a small molecule drug or a macromolecular drug) for the treatment of cancer (e.g., gastric cancer, pancreatic cancer, ovarian cancer, esophageal cancer or non-small cell lung cancer), and a bispecific antibody conjugate, comprising the bispecific antibody according to claim 1 and a further substance, wherein the further substance is selected from one or more of a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent and PEG, and preferably the therapeutic agent comprises a detectable label, e.g., a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent such as a drug or a toxin, an ultrasound enhancing agent and a nonradioactive label.

19. (canceled)

20. (canceled)

21. A method for treating cancer, comprising administering to a subject a therapeutically effective amount of the bispecific antibody according to claim 1, wherein the cancer is, for example, gastric cancer, pancreatic cancer, ovarian cancer, esophageal cancer or non-small cell lung cancer.

22. A bispecific antibody conjugate, comprising the bispecific antibody according to claim 1 and a further substance, wherein the further substance is selected from one or more of a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent and PEG, and preferably the therapeutic agent comprises a detectable label, e.g., a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent such as a drug or a toxin, an ultrasound enhancing agent and a nonradioactive label.

23. A nucleic acid composition, comprising: a) a first expression vector comprising a first nucleic acid encoding the antigen-binding domain specifically binding to CD3 as defined in claim 5; and b) a second expression vector comprising a second nucleic acid encoding the antigen-binding domain specifically binding to CLDN18.2 as defined in claim 5.

24. A nucleic acid composition, comprising: a) a first expression vector comprising a first nucleic acid encoding the antigen-binding domain specifically binding to CD3 as defined in claim 6; and b) a second expression vector comprising a second nucleic acid encoding the antigen-binding domain specifically binding to CLDN18.2 as defined in claim 6.

25. A nucleic acid composition, comprising: a) a first expression vector comprising a first nucleic acid encoding the antigen-binding domain specifically binding to CD3 as defined in claim 7; and b) a second expression vector comprising a second nucleic acid encoding the antigen-binding domain specifically binding to CLDN18.2 as defined in claim 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is an exemplary schematic diagram of the structure of a YBODY antibody (A) and the primary structure of each component protein (B).

[0040] FIG. 2 is an exemplary schematic diagram of the structure of a CSBODY antibody (A) and the primary structure of each component protein (B).

[0041] FIG. 3 is an exemplary schematic diagram of the structure of the bispecific antibody (A) and the primary structure of each component protein (B) of the present invention.

[0042] FIG. 4 is an exemplary schematic diagram of the structure of the bispecific antibody (A) and the primary structure of each component protein (B) of the present invention.

[0043] FIG. 5 shows the expression of CLDN18.2 antigen on the surface of cell strains 293 T-hCLDN18.2, NUGC4-hCLDN18.2, KATOIII-hCLDN18.2, CT26-hCLDN18.2, NUGC4, 293T-hCLDN18.1 and KATOIII.

[0044] FIG. 6 shows the binding effect of each antibody molecule to 293T-hCLDN18.1 cells.

[0045] FIG. 7 shows that the antibody-mediated hPBMC has in vitro killing effect on NUGC4 cells and NUGC4-hCLDN18.2 cells with an effector-to-target ratio of 10:1, and the hPBMC is detected after being incubated at 37° C. for 48 h; Y1, Y2, Y4, CS1 and CS2 bispecific antibodies have in vitro killing effect on NUGC4 cells (A) and NUGC4-hCLDN18.2 cells (B); Y6, Y7, Y8, CS4 and CS5 bispecific antibodies have in vitro killing effect on NUGC4 cells (C) and NUGC4-hCLDN18.2 cells (D); E+T is tumor cells mixed with effector cells without antibody added.

[0046] FIG. 8 shows that the antibody-mediated hPBMC has in vitro killing effect on 293T-hCLDN18.1 cells with an effector-to-target ratio of 10:1, and the hPBMC is detected after being incubated for 48 h; (A) Y1, Y2, Y4 and CS2 molecules have killing effect on 293T-hCLDN18.1 cells; (B) Y6, Y7, Y8, CS4 and CS5 molecules have killing effect on 293T-hCLDN18.1 cells; T+E is tumor cells mixed with effector cells without antibody added.

[0047] FIG. 9 shows that the antibody molecules induce the expression of immune cells CD69 and CD25 in the presence of NUGC4, NUGC4-hCLDN18.2 and 293T-hCLDN18.1 cells with an effector-to-target ratio of 10:1, and antibody molecules are detected after being incubated at 37° C. for 48 h; CS2, CS4, Y4 and Y6 molecules induce the expression of CD69 (A) and CD25 (D) on the surface of CD3+T cells in the presence of target cells NUGC4; CS2, CS4, Y4 and Y6 molecules induce the expression of CD69 (B) and CD25 (E) on the surface of CD3+T cells in the presence of target cells NUGC4-hCLDN18.2; CS4, Y4 and Y6 molecules induce the expression of CD69 (C) and CD25 (F) on the surface of CD3+T cells in the presence of target cells 293T-hCLDN18.1; wherein E+CS2, E+CS4, E+Y4, E+Y6 and E+isotype control groups indicate that the experimental group only has effector cells and CS2, CS4, Y4, Y6 or isotype control bispecific antibody; E+T is tumor cells mixed with effector cells without antibody added, and the effector cells are hPBMC cells.

[0048] FIG. 10 shows that the antibody molecule CS2 has proliferation promoting effect on immune cells in the presence of NUGC4, NUGC4-hCLDN18.2 and KATOIII cells; the CS2 molecule has the proliferation promoting effect on CD3+T cells in the presence of target cells NUGC4 (A), NUGC4-hCLDN182 (B) and non-target cells KATOIII (C); E is effector cell, i.e., hPBMC; Isotype is an isotype control bispecific antibody (anti-luciferase and anti-CD3); wherein E+CS2 indicates that the experimental group only has effector cells and CS2 bispecific antibody, and E+isotype control indicates that the experimental group only has effector cells and isotype control bispecific antibody; E+T is tumor cells mixed with effector cells without antibody added.

[0049] FIG. 11 shows the in vivo efficacy of the bispecific antibody CS4 in a CD3+T cell immune reconstituted B-NDG mouse subcutaneous NCI-N87-hCLDN18.2 xenograft tumor model, and the body weights (A) and tumor volumes (B) of mice in groups after the mice are randomly grouped for administration.

[0050] FIG. 12 shows the in vivo efficacy of the bispecific antibodies CS4 and Y6 in an hCD3E mouse CT26-hCLDN18.2 subcutaneous tumor model, and changes in body weights (A) and tumor volumes (B) of mice in groups after the mice are randomly grouped for administration.

[0051] FIG. 13 is an HPLC-SEC pattern of bispecific antibody CS2 molecule without acid treatment (A) and with treatment at pH 3.5 for 30 min (B), and an HPLC-SEC pattern of bispecific antibody CS4 molecule without acid treatment (C) and with treatment at pH 3.5 for 30 min (D).

DETAILED DESCRIPTION

[0052] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. The cases without the specific descriptions of techniques or conditions were carried out according to the technologies or conditions described in the literature in the art (e.g., see, Guide to Molecular Cloning Experiments, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the product manual. Reagents or instruments used are commercially available conventional products if the manufacturers thereof are not specified.

Example 1: Construction of Expression Vectors of Bispecific Antibodies

1. Construction of Expression Vector of Bispecific Antibody

[0053] The DNA sequences of coding genes of chains corresponding to the bispecific antibodies were cloned into pcDNA3.1 eukaryotic expression vectors by the molecular biological method commonly used by those skilled in the art, wherein the bispecific antibodies comprise bispecific antibody molecules Y1, Y2, Y4, Y6, Y7, Y8, Y17, Y18 and SY1 having a YBODY structure (FIG. 1), and bispecific antibody molecules CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS9, CS10, CS11 and SCS1 having a CSBODY structure (FIG. 2). The anti-CD3 scFv in bispecific antibody molecules Y1, Y2, Y4, Y6, Y7, Y8, Y17, Y18, SY1, CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS9, CS10, CS11 and SCS1 has a structure from N—C terminus of VH-linker peptide 4-VL. In the bispecific antibody molecules CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS9, CS10, CS11 and SCS1, the antigen-binding domain specifically binding to CLDN18.2 in a first heavy chain and a first light chain has VH and VL sequences identical to VH and VL sequences of the antigen-binding domain specifically binding to CLDN18.2 in a second heavy chain and a second light chain. Specific sequences of the bispecific antibodies are shown in Table 1 below.

TABLE-US-00023 TABLE 1 Sequences corresponding to bispecific antibodies Bi- specific SEQ anti- ID bodies Polypeptide Domain No. NO: Y1 Light chain VLm 175D10 1 CL CL 18 Heavy chain VHm 175D10 4 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH 2a5ga 7 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5ga 9 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y2 Light chain VLm 175D10 1 CL CL 18 Heavy chain VHm 175D10 4 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y4 Light chain VLm 1E9.2 2 CL CL 18 Heavy chain VHm 1E9.2 5 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y6 Light chain VLm 2C6.9 3 CL CL 18 Heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y7 Light chain VLm 2C6.9 3 CL CL 18 Heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH Di6-2 12 Linker peptide 4 Lin4 8 Anti-CD3 VL Di6-2 13 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y8 Light chain VLm 2C6.9 3 CL CL 18 Heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH L2K 14 Linker peptide 4 Lin4 8 Anti-CD3 VL L2K 15 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y17 Light chain VLm 1E9.2 2 CL CL 18 Heavy chain VHm 1E9.2 5 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH Di6-2 12 Linker peptide 4 Lin4 8 Anti-CD3 VL Di6-2 13 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 Y18 Light chain VLm 1E9.2 2 CL CL 18 Heavy chain VHm 1E9.2 5 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Second monomer anti-CD3 VH L2K 14 Linker peptide 4 Lin4 8 Anti-CD3 VL L2K 15 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 SY1 Light chain VLm 2C6.9 3 CL CL 18 Heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 Single chain anti-CD3 VH 2C11 16 Linker peptide 4 Lin4 8 Anti-CD3 VL 2C11 17 Linker peptide 1 Lin1 30 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS1 First light chain/ VLm 175D10 1 Second light chain CL CL 18 Second heavy VHm 175D10 4 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 175D10 4 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 First heavy chain anti-CD3 VH 2a5ga 7 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5ga 9 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS2 First light chain/ VLm 175D10 1 Second light chain CL CL 18 VHm 175D10 4 Second heavy CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 175D10 4 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 First heavy chain anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS3 First light chain/ VLm 1E9.2 2 Second light chain CL CL 18 VHm 1E9.2 5 Second heavy CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 1E9.2 5 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 First heavy chain anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS4 First light chain/ VLm 2C6.9 3 Second light chain CL CL 18 VHm 2C6.9 6 Second heavy CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 2C6.9 6 CH1 CH1 19 Hinge region 3 Hin3 29 First heavy chain Linker peptide 2 Lin2 31 anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS5 First light chain/ VLm 2C6.9 3 Second light chain CL CL 18 VHm 2C6.9 6 Second heavy CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 2C6.9 6 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 First heavy chain anti-CD3 VH 2a5ga 7 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5ga 9 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS6 First light chain/ VLm 175D10 1 Second light chain CL CL 18 VHm 175D10 4 Second heavy CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 175D10 4 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 First heavy chain anti-CD3 VH Di6-2 12 Linker peptide 4 Lin4 8 Anti-CD3 VL Di6-2 13 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS7 First light chain/ VLm 1E9.2 2 Second light chain CL CL 18 VHm 1E9.2 5 Second heavy CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 VHm 1E9.2 5 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 First heavy chain anti-CD3 VH Di6-2 12 Linker peptide 4 Lin4 8 Anti-CD3 VL Di6-2 13 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS8 First light chain/ VLm 2C6.9 3 Second light chain CL CL 18 Second heavy VHm 2C6.9 6 CH1 CH1 19 chain Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH Di6-2 12 Linker peptide 4 Lin4 8 Anti-CD3 VL Di6-2 13 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS9 First light chain/ VLm 175D10 1 Second light chain CL CL 18 Second heavy VHm 175D10 4 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 175D10 4 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH L2K 14 Linker peptide 4 Lin4 8 Anti-CD3 VL L2K 15 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS10 First light chain/ VLm 1E9.2 2 Second light chain CL CL 18 Second heavy VHm 1E9.2 5 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 1E9.2 5 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH L2K 14 Linker peptide 4 Lin4 8 Anti-CD3 VL L2K 15 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 CS11 First light chain/ VLm 2C6.9 3 Second light chain CL CL 18 Second heavy VHm 2C6.9 6 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH L2K 14 Linker peptide 4 Lin4 8 Anti-CD3 VL L2K 15 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 SCS1 First light chain/ VLm 2C6.9 3 Second light chain CL CL 18 Second heavy VHm 2C6.9 6 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 2C6.9 6 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH 2C11 16 Linker peptide 4 Lin4 8 Anti-CD3 VL 2C11 17 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 Note: the variable region sequences of Y1 and Y2 are from US8425902B2 and PCT/CN2019/075901. The antigen-binding domains specifically binding to CLDN18.2 of Y1, Y2, CS1, CS2, CS6 and CS9 are from 175D10; the antigen-binding domains specifically binding to CLDN18.2 of Y4, Y17, Y18, CS3, CS7 and CS10 are from 1E9.2; the antigen-binding domains specifically binding to CLDN18.2 of Y6, Y7, Y8, SY1, CS4, CS5, CS8, CS11 and SCS1 are from 2C6.9.

Example 2: Expression and Purification of Bispecific Antibodies

[0054] The plasmid was extracted according to a conventional plasmid extraction method and used to transfect 293 cells or CHO-S cells, and the transfection reagent may be Lipofectamine2000 (Thermo fisher, Catalog No. 11668019). The transfected cells were cultured in 293 cells or CHO cells in suspension shaking at 37° C. in a 5% CO.sub.2 shaker for 7-10 days. The supernatant was collected by centrifugation at 3000×g and filtered through a 0.22 μm filter, and then purified by protein A affinity chromatography and cation exchange chromatography to obtain the bispecific antibodies. The concentration of the purified bispecific antibody was determined by UV absorbance at 280 nm and the corresponding extinction coefficient for each protein. The purified protein was analyzed by SDS-PAGE, and the molecular weight of the bispecific antibody with a structure of YBODY was about 125 KD, and the molecular weight of the bispecific antibody with a structure of CSBODY was about 175 KD. The high-polymer content of each bispecific antibody was tested by high performance size exclusion chromatography (HPLC-SEC). The content of the bispecific antibody in the collected supernatant was 10.5-240 mg/L, and the purity of the bispecific antibody finally obtained through purification was more than 95% through HPLC-SEC detection.

Example 3: Detection of Affinity of Bispecific Antibody CLDN18.2 Terminus

[0055] The affinity of the bispecific antibody CLDN18.2 terminus included in the present invention utilized flow cytometry to detect the binding of the bispecific antibody to the target antigen CLDN18.2 (SEQ ID NO: 40) of the corresponding cell. The present invention adopted human gastric cancer cell strain NUGC4 and human CLDN18.2 over-expressed cell strains including human embryonic kidney cell strains 293T-hCLDN18.2, human gastric cancer cell strains NUGC4-hCLDN18.2, human gastric cancer cell strains KATOIII-hCLDN18.2 and mouse colon cancer cell strains CT26-hCLDN18.2 as CLDN18.2 antigen expression-positive cells, and adopted human gastric cancer cell strains KATOIII and human CLDN18.1 (SEQ ID NO: 39) over-expressed human embryonic kidney cell strains 293T-hCLDN18.1 as CLDN18.2 negative cells. The CT26-hCLDN18.2, 293T-hCLDN18.1 and 293T-hCLDN18.2 cells were purchased from KYinno Biotechnology (Beijing) Co., Ltd, the NUGC4 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd., and the KATOIII cells were purchased from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences. KATOIII-hCLDN18.2 and NUGC4-hCLDN18.2 cell strains were obtained by transfecting KATOIII and NUGC4 cells with lentivirus containing the gene encoding human CLDN18.2 and antibiotic pressure screening according to the relevant reagent instructions, for example, see the method as described in the instruction of Lenti-Pac HIV lentivirus package kit (GeneCopoeia, HPK-LvTR-20).

1. Detection of Expression of CLDN18.2 on the Surface of Cells

[0056] Cells were collected and resuspended in buffer (PBS+1% FBS) at 1×10.sup.4 cells/well in a 96-well plate at 100 μL per well. Then the mixture was centrifuged at 350×g for 5 min, and the supernatant was removed. Anti-CLDN18.2 monoclonal antibody 2C6.9 (with a light chain sequence set forth in SEQ ID NO: 33 and a heavy chain sequence set forth in SEQ ID NO: 34, prepared by Wuhan YZY Biopharma Co. Ltd.) and anti-CLDN18.2 monoclonal antibody 1E9.2 (with a light chain sequence set forth in SEQ ID NO: 104 and a heavy chain sequence set forth in SEQ ID NO: 105, prepared by Wuhan YZY Biopharma Co. Ltd.) were diluted to 1000 nM with buffer, added to a 96-well plate, resuspended, incubated at 4° C. in the dark for 1 h and centrifuged to remove supernatant; the plate was washed twice with buffer, resuspended in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304), incubated at 4° C. in the dark for 30 min, then washed twice with buffer, resuspended in 100 μL buffer, and detected on a flow cytometer (BD Accuri™ C6). As shown in FIG. 5, both anti-CLDN18.2 monoclonal antibody 2C6.9 and monoclonal antibody 1E9.2 could effectively bind to 293T-hCLDN18.2 but not to 293T-hCLDN18.1 cells, which indicated specificity of binding of anti-CLDN18.2 monoclonal antibodies 2C6.9 and 1E9.2 to the CLDN18.2 antigen. Meanwhile, NUGC4-hCLDN18.2, KATOIII-hCLDN18.2 and CT26-hCLDN18.2 cells after detection had strong binding effect to anti-CLDN18.2 monoclonal antibody 2C6.9, which indicated that the cell strains highly expressed human CLDN18.2 antigen; the human gastric cancer cell strain NUGC4 had certain binding effect to the CLDN18.2 monoclonal antibody 2C6.9, which indicated that moderately expressed human CLDN18.2 antigen; however, the human gastric cancer cell strains KATOIII had no binding effect to the CLDN18.2 monoclonal antibody, which indicated that the human gastric cancer cell strains KATOIII did not express the human CLDN18.2 antigen.

2. Detection of Binding Activity of Bispecific Antibodies to CLDN18.2 Positive Cells Using Flow Cytometry Analysis

[0057] NUGC4 and NUGC4-hCLDN18.2 cells were cultured, digested with pancreatin and centrifuged to collect cells. The cells were collected and resuspended in buffer (PBS+1% FBS) at 1×10.sup.4 cells/well in a 96-well plate at 100 μL per well. Then the mixture was centrifuged at 350×g for 5 min, and the supernatant was removed. The bispecific antibody was diluted to 3000 nM with buffer and serially 3- or 4-fold diluted to the 11.sup.th concentration, and then the bispecific antibody was added to a 96-well plate at 100 μL/well, resuspended, incubated at 4° C. in the dark for 1 h and centrifuged to remove supernatant; the plate was washed twice with buffer, resuspended in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304), incubated at 4° C. in the dark for 30 min, then washed twice with buffer, resuspended in 100 μL buffer, and detected on a flow cytometer (BD Accuri™ C6). Y4, Y6, Y7, Y8, CS3, CS4, CS5, SY1 and SCS1 all had significant binding effects to NUGC4 and NUGC4-hCLDN18.2 cells, and specific binding EC.sub.50 values are shown in Table 2. In the same experimental system, other bispecific antibodies such as Y17, Y18, CS6, CS7, CS8, CS9, CS10 and CS11 of the present invention all had binding EC.sub.50 values of less than 50 nM to NUGC4-hCLDN18.2 cells; Y17, Y18, CS7, CS8, CS10 and CS11 all had binding EC.sub.50 values of less than 100 nM to NUGC4 cells; IMAB362 monoclonal antibody (specifically binding to CLDN18.2, with a light chain sequence set forth in SEQ ID NO: 35 and a heavy chain sequence set forth in SEQ ID NO: 36, with reference to U.S. Pat. No. 8,425,902B2, prepared by Wuhan YZY Biopharma Co. Ltd.) did not significantly bind to NUGC4 cells, and had a binding EC.sub.50 value of 10.79 nM to NUGC4-hCLDN18.2 cells. Y4, Y6, Y7, Y8, Y17, Y18, CS3, CS4, CS5, CS7, CS8, CS10, CS11, SY1 and SCS1 had superior binding effect to NUGC4 cells than IMAB362.

TABLE-US-00024 TABLE 2 Binding ability of bispecific antibodies to NUGC4 and NUGC4-hCLDN18.2 cells Bispecific EC.sub.50 (nM) antibodies NUGC4 NUGC4~hCLDN18.2 Y1 No significant binding effect Not reach plateau Y2 No significant binding effect Not reach plateau Y4 58.94 24.36 Y6 183.5 45.91 Y7 127.4 25.42 Y8 75.11 46.93 CS1 Not reach plateau 14.63 CS2 Not reach plateau 35.97 CS3 63.95 31.48 CS4 23.42 18.87 CS5 25.72 18.18 SY1 303.8 125.1 SCSI 29.12 25.37

3. Detection of Binding Activity of Bispecific Antibodies to CLDN18.2 Negative Cells Using Flow Cytometry Analysis

[0058] The step 3.2 was repeated for detection by using 293T-hCLDN18.1 cells. As shown in FIG. 6, bispecific antibodies had no significant binding effect to 293T-hCLDN18.1 cells, and had weak binding effect under the condition of high dose, and possibly non-specific binding effect. Under the same detection conditions, other bispecific antibodies such as Y17, Y18, CS6, CS7, CS8, CS9, CS10 and CS11 of the present invention had no significant binding effect with 293T-hCLDN18.1 cells.

Example 4: Detection of Affinity of Bispecific Antibody CD3 Terminus by BIACORE

[0059] A human CD3 antigen (SEQ ID NO: 45) was fixed on a CM5 chip by adopting an amino coupling method, wherein the antigen coupling amount was 1500 RU, when the binding activity of the CD3 antigen terminus was detected, a sample was diluted to an initial concentration by adopting 1×HBS-EP+buffer, and then serially 2-fold diluted to the 4.sup.th concentration, and then the samples were added to Biacore T200 from a low concentration to a high concentration, wherein the binding flow rate was 30 μL/min, the binding time was 120 s, and the dissociation time was 300 s; the chip was regenerated by adopting a pH1.5 Glycine solution, the regeneration flow rate was 10 μL/min, and the regeneration time was 30 s. After the detection was completed, data fitting was carried out on the result pattern by adopting Biacore T200 Evaluation Software in a 1:1 binding fitting mode to obtain an equilibrium dissociation constant (KD). As shown in Table 3, Y2, Y4, Y6, Y7, Y8, CS2, CS3 and CS4 all had strong binding effect to human CD3 antigen. In the same experiment system, other bispecific antibodies such as Y17, Y18, CS6, CS7, CS8, CS9, CS10 and CS11 of the present invention had stronger binding effect to human CD3 antigen with KD value less than 15 nM.

TABLE-US-00025 TABLE 3 Detection of binding ability of bispecific antibodies to human CD3 antigen by BIACORE Bispecific antibodies Y1 Y2 Y4 Y6 Y7 Y8 CS2 CS3 CS4 KD 1975 20.32 5.742 5.773 4.856 22.22 3.100 10.00 12.03 (nM)

Example 5: Detection of Affinity of Bispecific Antibody CD3 Terminus by BIACORE

[0060] The experiment in Example 4 was repeated using the murine CD3 antigen (SEQ ID NO: 46). The results are shown in Table 4, wherein SY1 and SCS1 had binding KD values of 79.30 nM and 94.10 nM to murine CD3 antigen, respectively.

TABLE-US-00026 TABLE 4 Detection of binding ability of bispecific antibodies to murine CD3 antigen by BIACORE Bispecific antibodies KD (nM) SY1 79.30 SCS1 94.10

Example 6: Detection of In Vitro Killing Effect Mediated by Bispecific Antibodies

1. Isolation of Human Peripheral Blood Mononuclear Cells

[0061] The collected peripheral blood was transferred to a 50 mL centrifuge tube, and added with PBS in a volume ratio 1:1 for dilution, and then the mixture was gently mixed. A 50 mL centrifuge tube was added with 10-15 mL of Ficoll solution, then the diluted blood was gently added dropwise on the Ficoll upper layer of the centrifuge tube, and then centrifuged at 400×g for 30 min (the rising speed was set as 1, and the falling speed was set as 0). After the centrifugation was completed, the middle white peripheral blood mononuclear cell (PBMC) layer was sucked into another clean 50 mL centrifuge tube, the tube was added with 2-fold volume of PBS and centrifuged at 400×g for 10 min to remove the supernatant; the above operation was repeated, and the tube was added with red blood cell lysis buffer, gently pipetted and mixed well, and then lysed for 4-5 min at room temperature. The tube was added with PBS and washed 1-2 times. The cells were resuspended in culture medium according to the needs of the experiment and counted for subsequent experiments.

2. Detection of In Vitro Killing Effect of Bispecific Antibody on Target Cells by PI Single-Staining Method

[0062] Bispecific antibody mediated in vitro killing effect was detected by using the human PBMC (hPBMC) obtained by isolation as effector cells and CLDN18.2 expressing cells as target cells. The target cells were digested into single-cell suspensions with pancreatin, and centrifuged at 300×g for 5 min to collect cells; the cells were stained with 5 μM of 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (37° C., 15 min), washed twice with complete medium and counted on a Vi-Cell cell counter (Beckman); then the cells were added to a 96-well plate at 2×10.sup.4 cells/100 μL/well according to the experimental design. The prepared 4×antibody molecule was added at 50 μL/well and the hPBMC was counted on a Cellometer cell counter and then placed in a 96-well plate (2×10.sup.5 cells/50 μL/well at an effector-to-target ratio of 10:1). The cell culture plate was placed in a cell incubator for culturing for 48 h, after the cells were digested into single cell suspension, propidium iodide (PI) solution with a final concentration of 1 μg/mL was added, after the cells were incubated for 10 min, the cells were detected on a flow cytometer (BD Accuri™ C6), and the percentage of CFSE+PI+double positive cells in CFSE+positive cells was analyzed. Anti-CD3 monoclonal antibody (with a light chain sequence set forth in SEQ ID NO: 37 and a heavy chain sequence set forth in SEQ ID NO: 38, prepared by Wuhan YZY Biopharma Co. Ltd.) was used as the CD3 monoclonal antibody control.

[0063] As shown in FIG. 7 and Table 5, bispecific antibodies Y4, Y6, Y7, Y8, CS3, CS4 and CS5 all had strong killing effects on NUGC4-hCLDN18.2 cells and NUGC4 cells, which were significantly stronger than IMAB362. CS1 and CS2 also had strong killing effect on NUGC4-hCLDN18.2 cells, which are significantly stronger than IMAB362. However, the anti-CD3 monoclonal antibody did not show killing effect in the same experiment system. According to the same experimental procedures, other bispecific antibodies such as Y17, Y18, CS6, CS7, CS8, CS9, CS10 and CS11 of the present invention had an EC.sub.50 value of less than 10 pM for killing effect on NUGC4-hCLDN18.2 cells; Y17, Y18, CS7, CS8, CS10 and CS11 had an EC.sub.50 value of less than 10 pM for killing effect on NUGC4 cells.

TABLE-US-00027 TABLE 5 In vitro killing effect of antibody-mediated hPBMC on NUGC4 cells and NUGC4-hCLDN18.2 cells Bispecific EC.sub.50 (pM) antibodies NUGC4 NUGC4~hCLDN18.2 Y1 No significant killing effect No significant killing effect Y2 No significant killing effect Not reach plateau Y4 136.6 0.3781 Y6 7.993 1.070 Y7 5.833 0.6550 Y8 57.83 4.088 CS1 Not reach plateau 428.4 CS2 Not reach plateau 2.170 CS3 0.3326 0.6984 CS4 0.9397 0.3064 CS5 433.2 132.5

3. In Vitro Killing Effect of Bispecific Antibody on Non-Target Cells

[0064] The procedures in 6.2 were repeated using hPBMC obtained by isolation as effector cells and CLDN18.1 expressing cells as non-target cells to detect bispecific antibody mediated in vitro killing effect. As shown in FIG. 8, Y1, Y2, Y4, Y6, Y7, Y8, CS2, CS4 and CS5 all did not have significant killing effect on non-target cells 293T-hCLDN18.1. Under the same experimental conditions, other bispecific antibodies such as Y17, Y18, CS1, CS3, CS6, CS7, CS8, CS9, CS10 and CS11 of the present invention did not show significant killing effect on non-target cells 293T-hCLDN18.1.

Example 7: Bispecific Antibody-Mediated Activation on Immune Cells

1. Activation on Immune Cells

[0065] The isolated human peripheral blood mononuclear cells were taken out of the incubator, and digested into a single cell suspension by using pancreatin; 1 mL of the single cell suspension was taken and counted on a VI-Cell counter, and added into a 96-well plate at 2×10.sup.4 cells/100 μL/well according to the experimental design. The prepared 4×antibody molecules were added at 50 μL/well. The hPBMC was counted on a Cellometer cell counter and then placed in a 96-well plate at 2×10.sup.5 cells/50 μL/well with an effector-to-target ratio of 10:1; the cell culture plate was placed in a cell incubator for culturing for 48 h. The cell suspension in the cell culture plate was mixed and transferred to another 96-well plate, and after centrifugation at 400×g for 5 min, the hPBMC was resuspended in 100 μL buffer (containing 1.5 μL FITC-labeled anti-CD3 antibody (Biolegend, 300306); 1.5 μL APC-labeled anti-CD25 antibody (Biolegend, 302610) and 1.5 μL PE-labeled anti-CD69 antibody (Biolegend, 310906)). After incubation at 4° C. for 30 min, the cells were washed once with buffer and resuspended in 100 μL buffer, and detected by flow cytometry (BD Accuri™ C6). As shown in FIG. 9 and Table 6, in the presence of the target cells NUGC4, Y4, Y6, Y7, Y8, CS2, CS3, CS4 and CS5 could activate the expression of CD69 molecules (with an EC.sub.50 value in a range of 0.4460-140.6 pM) and CD25 molecules (with an EC.sub.50 value in a range of 2.377-480.8 pM) on the surface of CD3+T cells; in the presence of target cells NUGC4-hCLDN18.2, Y4, Y6, Y7, Y8, CS2, CS3, CS4 and CS5 could activate the expression of CD69 molecules (with an EC.sub.50 value in a range of 0.1956-70.47 pM) and CD25 molecules (with an EC.sub.50 value in a range of 0.9623-142.3 pM) on the surface of CD3+T cells; bispecific antibodies had no activation effect on T cells in the presence of the non-target cells 293T-hCLDN18.1. The above results indicated the targeting activation of the bispecific antibodies. According to the same experimental procedures, other bispecific antibodies such as Y17, Y18, CS1, CS6, CS7, CS8, CS9, CS10 and CS11 of the present invention all had good activation effect on T cells in the presence of target cells, and no activation effect on T cells in the absence of target cells.

TABLE-US-00028 TABLE 6 Activation of bispecific antibodies on immune cells in the presence of gastric cancer cells NUGC4, NUGC4-hCLDN18.2 and non-target cells 293T-hCLDN18.1 Induction of expression of CD69 Induction of expression of CD25 EC.sub.50 (pM) EC.sub.50 (pM) Bispecific NUGC4~ 293T~ NUGC4~ 293T~ antibodies NUGC4 hCLDN18.2 hCLDN18.1 NUGC4 hCLDN18.2 hCLDN18.1 Y4 0.6097 0.6698 n/a 12.23 0.9623 n/a Y6 4.865 0.5916 n/a 17.22 2.318 n/a Y7 4.133 0.4647 n/a 15.71 1.782 n/a Y8 33.27 2.981 n/a 181.9 16.52 n/a CS2 29.52 0.3892 n/a 215.5 1.210 n/a CS3 0.4614 1.448 n/a 5.384 5.447 n/a CS4 0.4460 0.1956 n/a 2.377 1.189 n/a CS5 140.6 70.47 n/a 480.8 142.3 n/a Note: n/a represents no significant killing effect.

2. Proliferation Effect on Immune Cells

[0066] The isolated human peripheral blood mononuclear cells were taken out of the incubator, and digested into a single cell suspension by using pancreatin; 1 mL of the single cell suspension was taken and counted on a VI-Cell counter, and added into a 96-well plate at 2×10.sup.4 cells/100 μL/well according to the experimental design. The prepared 4×antibody molecules were added at 50 μL/well. The hPBMC cells were stained with 5 μM of CFSE (37° C., 15 min), washed twice with complete medium and counted on a Cellometer cell counter; then the cells were added to a 96-well plate at 2×10.sup.5 cells/50 μL/well with an effector-to-target ratio of 10:1 according to the experimental design. The cell culture plate was placed in a cell incubator for culturing for 5 days. The cell suspension in the cell culture plate was mixed well, transferred to another 96-well plate, and centrifuged at 400×g for 5 min. The hPBMC was resuspended in 100 μL buffer (containing 1.5 μL of the APC-labeled anti-CD3 antibody) and incubated at 4° C. for 30 min, washed once with buffer, resuspended in 100 μL buffer, and detected by flow cytometry (BD Accuri™ C6). The percentage of the cell population with reduced fluorescence intensity after CFSE staining in CD3+T cell population was analyzed. As shown in FIG. 10, in the presence of target cells NUGC4 and NUGC4-hCLDN18.2, the CS2 bispecific antibody could effectively promote the proliferation effect on CD3+T cells; in the presence of non-target cells KATOIII, only weak proliferation-promoting effect was observed at high dose, which was probably related to weak non-specific adsorption of CS2 molecules to T cells at high dose. Under the same experimental conditions, Y4, Y6, Y7, Y8, CS3 and CS4 could effectively promote the proliferation effect on CD3+T cells in the presence of target cells NUGC4 and NUGC4-hCLDN18.2, while had no T cell proliferation promotion effect in the presence of non-target cells KATOIII. According to the same experimental procedures, other bispecific antibodies such as Y17, Y18, CS1, CS6, CS7, CS8, CS9, CS10 and CS11 all had good proliferation effect on T cells in the presence of target cells, and no proliferation effect on T cells in the absence of target cells.

Example 8: In Vivo Efficacy of Bispecific Antibodies in Subcutaneous Xenograft Tumors

1. In Vivo Efficacy of Bispecific Antibodies in CD3+T Cell Reconstituted Subcutaneous Tumor Model

[0067] The in vivo efficacy of bispecific antibodies was evaluated using an immune reconstituted B-NDG mouse NCI-N87-hCLDN18.2 human gastric cancer cell subcutaneous xenograft tumor model (for example, using CS4 bispecific antibody as an example, other antibodies could achieve similar effects according to the experimental procedures). NCI-N87-hCLDN18.2 cell strains are human gastric cancer cell NCI-N87 over-expressing human CLDN18.2 (NCI-N87 purchased from China Center for Type Culture Collection). A sufficient amount of NCI-N87-hCLDN18.2 cells were cultured under culture conditions, and when the cells were in exponential phase, the cells were collected and counted. Each mouse was inoculated subcutaneously at the right anterior scapula with 5×10.sup.6 human CLDN18.2 overexpression human gastric cancer cells NCI-N87-hCLDN18.2 (suspended in 0.1 mL of PBS). When the tumors grew to 100-200 mm.sup.3 (about 10 days after tumor bearing) after inoculation, the tumor-bearing mice were randomly grouped according to the body weight and the tumor volume of the mice, and the experiment was divided into a normal saline group, a CD3.sup.+ T cell group (5×10.sup.6 cells/mouse), a CD3.sup.+ T cell+CS4 (0.2 mg/kg) group (5×10.sup.6 cells/mouse). Cyclophosphamide was administered for lymphodepletion treatment on the grouping day, and the mice in the test groups were subjected to tail vein injection with CD3+T cells on the next day to reconstruct the immune system of B-NDG. CS4 bispecific antibody intervention was initiated on day 11 after CD3+T cell injection, injected with CS4 twice a week for 6 times. Mice were weighed 2 times weekly, and tumor volumes were measured, and calculated as tumor volume=½×major axis×minor axis×minor axis (mm.sup.3). Tumor growth inhibition (TGI) (%)=100%−(Tt−T0)/(Ct−C0)×100%; tumor growth inhibition T/C (%)=(Tt/T0)/(Ct/C0)×100%, wherein TO is average tumor volume of the test antibody group when administered in cages; Tt is average tumor volume of the test antibody group at each measurement; C0 is average tumor volume of the saline group when administered in cages; Ct is average tumor volume of the saline group at each measurement. As shown in FIG. 11 and Table 7, the bispecific antibody CS4 had better tumor inhibition effect in a CD3.sup.+ T cell immune reconstituted B-NDG mouse subcutaneous NCI-N87-hCLDN18.2 xenograft tumor model, and the tumor growth inhibition (TGI) was 80.11% and the T/C was 24.04% at day 49 after treatment. No significant change of the body weight of the mouse was seen in the treatment process. According to the same experimental procedures, other bispecific antibodies such as Y17, Y18, SY1, CS1, CS6, CS7, CS8, CS9, CS10, CS11 and SCS1 could effectively inhibit tumor growth and were tolerated by the animals.

TABLE-US-00029 TABLE 7 In vivo efficacy statistics of the bispecific antibody CS4 in a CD3.sup.+T cell immune reconstructed B-NDG mouse subcutaneous NCI-N87-hCLDN18.2 xenograft tumor model, and statistical analysis performed according to the tumor volume at day 49 Day 49 Tumor Group- volume (mm.sup.3) TGI T/C.sup.a P ing Groups (mean ± SD) (%) (%) value.sup.b Group 1 Normal saline 1851.67 ± 583.11 / / / group Group 2 CD3.sup.+T (5 × 10.sup.6) 1822.97 ± 412.26  1.67% 97.82% 0.9306 Group 3 CD3.sup.+T(5 × 10.sup.6) +  444.80 ± 108.29 80.11% 24.04% 0.0007 CS4 Note: .sup.aT/C is less than or equal to 40%, and p <0.05 is effective after statistical treatment; .sup.bP value is calculated by comparing the data of the group with the saline group.
2. In Vivo Efficacy of Bispecific Antibodies in hCD3E Mouse Subcutaneous Tumor Model

[0068] The in vivo efficacy of the bispecific antibodies was evaluated by using a mouse CT26-hCLDN18.2 cell subcutaneous tumor model constructed from BALB/cJGpt-Tg(CD3E BAC)/Gpt mice. BALB/cJGpt-Tg(CD3E BAC)/Gpt mice were purchased from Jiangsu GemPharmatech Co., Ltd., and T lymphocytes thereof expressed human CD3c. A sufficient amount of CT26-hCLDN18.2 cells were cultured, and when the cells were in exponential phase, the cells were collected and counted. After 3-7 days of adaptation period, 2×10.sup.5 CT26-hCLDN18.2 cells were inoculated subcutaneously to each mouse, and the cells were mixed with Matrigel (volume ratio 1:1) before inoculation, and the total volume of inoculation was 100 μL. The day of inoculation was defined SD0. When the average tumor volume was grown to about 100 mm.sup.3, the mice were grouped (6 mice/group) and administered intraperitoneally. The specific groups were as follows: a vehicle group: each mouse was injected with 0.005% (v/v) TW80-saline; a CS4 high dose group (11.7 mg/kg), a CS4 low dose group (2.3 mg/kg), a Y6 high dose group (8.3 mg/kg), a Y6 low dose group (1.7 mg/kg) and an IMAB362 group (10 mg/kg). All tested drugs were formulated with 0.005% TW80-saline. The administration was performed at SD3, SD5, SD7, SD10, SD12 and SD14, and the dose was unchanged. The body weight and tumor volume of the mice were measured before and 2-3 times a week after the administration, and the volume was calculated as tumor volume=½×major axis×minor axis×minor axis (mm.sup.3). Tumor growth inhibition (TGI) (%)=100%−(Tt−T0)/(Ct−C0)×100%, and tumor growth inhibition T/C (%)=(Tt/T0)/(Ct/C0)×100%, wherein TO is average tumor volume of the test antibody group when administered in cages; Tt is average tumor volume of the test antibody group at each measurement; C0 is average tumor volume of the vehicle group when administered in cages; Ct is average tumor volume of the vehicle group at each measurement. As shown in FIG. 12 and Table 8, although different levels of body weight reduction occurred after both the high and low doses of the test drugs were administered, the body weights of most mice recovered after the fifth administration on SD12, which exhibited a certain toxicity tolerance. The CS4 (11.7 mg/kg and 2.3 mg/kg) group and Y6 (8.3 mg/kg and 1.7 mg/kg) group all showed good tumor inhibition effects, and showed significant difference with the vehicle group. The IMAB362 (10 mg/kg) group did not show significant tumor inhibition in this experimental system. Other bispecific antibodies such as Y17, Y18, SY1, CS1, CS6, CS7, CS8, CS9, CS10, CS11 and SCS1 were also effective in inhibiting tumor growth according to the same experimental procedures, and were significantly superior to IMAB362, while exhibited good safety.

TABLE-US-00030 TABLE 8 In vivo efficacy statistics of bispecific antibodies CS4 and Y6 in an hCD3E mouse CT26-hCLDN18.2 subcutaneous tumor model, and statistical analysis performed according to the tumor volume at day 21 Day 21 Tumor Group- volume (mm.sup.3) TGI T/C.sup.a P ing Groups (mean ± SD) (%) (%) value.sup.b Group 1 Vehicle group 2371.72 ± 1235.68 / / / Group 2 CS4 11.7 201.85 ± 101.37 96.05 8.91 0.0107 mg/kg Group 3 CS4 2.3 301.01 ± 112.50 91.80 12.91 0.0136 mg/kg Group 4 Y6 8.3 mg/kg 142.75 ± 18.95  98.85 6.10 0.0089 Group 5 Y6 1.7 mg/kg 178.32 ± 32.56  97.25 7.65 0.0106 Group 6 IMAB362 1880.12 ± 1830.86 21.67 81.56 0.7418 10 mg/kg Note: .sup.aT/C is less than or equal to 40%, and p <0.05 is effective after statistical treatment; .sup.bP value is calculated by comparing the data of the group with the vehicle group.

Example 9: Evaluation of Acid Stability of Bispecific Antibodies

[0069] Bispecific antibodies were evaluated according to a conventional acid stability evaluation method: when the antibody molecules were subjected to protein A affinity chromatography, the eluted antibody solution was not neutralized in the acid elution step (using a citric acid buffer at pH 3.5), and after a certain period of time in this buffer, 1/10 volume of 1 M Tris-HCl (pH 8.0) was added at the 30.sup.th min for neutralization and HPLC-SEC detection was performed on this sample. As shown in FIG. 13, the bispecific antibody molecules CS2 and CS4 showed no aggregation or degradation after 30 min of treatment at pH 3.5, and they had a purity of >95%, indicating that the antibody molecules can maintain stability in acidic environment. Other bispecific antibodies Y4, Y6, Y7, Y8, Y17, Y18, CS1, CS3, CS5, CS6, CS7, CS8, CS9, CS10, CS11, SY1 and SCS1 followed the same experimental procedures, showing no aggregation or degradation after 30 min of treatment at pH 3.5, and they had a purity of >95%.

Example 10: Bispecific Antibodies with Altered Fc Fragments

[0070] Fc1 (SEQ ID NO: 20) and Fc2 (SEQ ID NO: 21) in the bispecific antibody in Example 1 were substituted with SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and the sequences of the other portions were unaltered. The expression and purification method and detection method are the same as in Examples 2-8. The results showed that the affinity of the bispecific antibody obtained after Fc substitution for CLDN18.2 and CD3, the in vitro killing effect thereof on CLDN18.2 positive target cells, and the activation effect thereof on immune cells were comparable to those of the bispecific antibody in Example 1. The in vivo efficacy results were slightly stronger than those of the bispecific antibody in Example 1 and had no significant difference.

[0071] Both Fc1 (SEQ ID NO: 20) and Fc2 (SEQ ID NO: 21) in the bispecific antibody in Example 1 were substituted with SEQ ID NO: 24, and the sequences of the other portions were unaltered. The expression and purification method and detection method are the same as in Examples 2-8. The results showed that the affinity of the bispecific antibody obtained after Fc substitution for CLDN18.2 and CD3, the in vitro killing effect thereof on CLDN18.2 positive target cells, and the activation effect thereof on immune cells were comparable to those of the bispecific antibody in Example 1. The in vivo efficacy results were slightly stronger than those of the bispecific antibody in Example 1 and had no significant difference.

[0072] Both Fc1 (SEQ ID NO: 20) and Fc2 (SEQ ID NO: 21) in the bispecific antibody in Example 1 were substituted with SEQ ID NO: 25, and the sequences of the other portions were unaltered. The expression and purification method and detection method are the same as in Examples 2-8. The results showed that the affinity of the bispecific antibody obtained after Fc substitution for CLDN18.2 and CD3, the in vitro killing effect thereof on CLDN18.2 positive target cells, the activation effect thereof on immune cells and in vivo efficacy thereof were all comparable to those of the bispecific antibody in Example 1, and had no significant difference.

[0073] Both Fc1 (SEQ ID NO: 20) and Fc2 (SEQ ID NO: 21) in the bispecific antibody in Example 1 were substituted with SEQ ID NO: 26, and the sequences of the other portions were unaltered. The expression and purification method and detection method are the same as in Examples 2-8. The results showed that the affinity of the bispecific antibody obtained after Fc substitution for CLDN18.2 and CD3, the in vitro killing effect thereof on CLDN18.2 positive target cells, the activation effect thereof on immune cells and in vivo efficacy thereof were all comparable to those of the bispecific antibody in Example 1, and had no significant difference.

Example 11: Other Bispecific Antibodies

[0074] Bispecific antibody molecules YCS2 and YCS4 were constructed according to the method in Example 1. Specifically, the YCS2 was obtained by substituting VHm (SEQ ID NO: 4) of a second heavy chain and VLm (SEQ ID NO: 1) of a second light chain in the bispecific antibody molecule CS2 with the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 3, respectively, with the sequences of the other portions unaltered. Similarly, the YCS4 was obtained by substituting VHm (SEQ ID NO: 6) of a first heavy chain and VLm (SEQ ID NO: 3) of a first light chain in the bispecific antibody molecule CS4 with the sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 2, respectively, with the sequences of the other portions unaltered. Specific sequences of YCS2 and YCS4 are shown in Table 9 below.

TABLE-US-00031 TABLE 9 Sequences of YCS2 and YCS4 SEQ Bispecific ID antibodies Polypeptide Domain No. NO: YCS2 First light chain VLm 175D10 1 CL CL 18 Second light chain VLm 2C6.9 3 CL CL 18 Second heavy VHm 2C6.9 6 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 175D10 4 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21 YCS4 First light chain VLm 1E9.2 2 CL CL 18 Second light chain VLm 2C6.9 3 CL CL 18 Second heavy VHm 2C6.9 6 chain CH1 CH1 19 Hinge region 1 Hin1 27 Fc1 G2D1 20 First heavy chain VHm 1E9.2 5 CH1 CH1 19 Hinge region 3 Hin3 29 Linker peptide 2 Lin2 31 anti-CD3 VH 2a5 10 Linker peptide 4 Lin4 8 Anti-CD3 VL 2a5 11 Linker peptide 3 Lin3 32 Hinge region 2 Hin2 28 Fc2 G2D2 21

[0075] The expression and purification method and detection method are the same as in Examples 2-8. The results showed that the in vitro killing effect of YCS2 on the tumor cells with low expression of CLDN18.2, the activation effect thereof on immune cells and the in vivo efficacy thereof were superior to that of the bispecific antibody CS2. The binding activity of YCS4 to CLDN18.2 and CD3, the in vitro killing effect thereof on CLDN18.2 positive target cells, the activation effect thereof on immune cells and in vivo efficacy thereof were all comparable to those of bispecific antibody CS4.

[0076] Bispecific antibody NCS3 having a structure shown in FIG. 3 and bispecific antibody CCS3 having a structure shown in FIG. 4 were constructed. The sequences of each portion in NCS3 and CCS3 were identical to those of bispecific antibody CS3, except that the anti-CD3 ScFv was different from linker peptides of VHm or Fc2. In bispecific antibody NCS3, the linker peptide of anti-CD3 ScFv and VHm were linker peptide 2 (SEQ ID NO: 31), and the hinge region between CH1 and FC2 was hinge region 1 (SEQ ID NO: 27). In bispecific antibody CCS3, the linker peptide of anti-CD3 ScFv and VHm was linker peptide 3 (SEQ ID NO: 32), and the hinge region between CH1 and FC2 was hinge region 1 (SEQ ID NO: 27). The expression and purification method and detection method are the same as in Examples 2-8.

[0077] The results showed that the affinity of the bispecific antibodies NCS3 and CCS3 for CLDN18.2 and CD3, the in vitro killing effect thereof on CLDN18.2 positive target cells, the activation effect thereof on immune cells and in vivo efficacy thereof were comparable to those of bispecific antibody CS3 and even superior to those of bispecific antibody CS3.

[0078] All documents mentioned in the present invention are incorporated by reference, just as each document is cited separately as a reference. In addition, it should be understood that various modifications or changes may be made by those skilled in the art after reading the above teachings of the present invention, and these equivalent forms also fall within the scope defined by the claims appended hereto.