ANTIBODY AND USE THEREOF
20230192840 · 2023-06-22
Assignee
- SICHUAN KELUN-BIOTECH BIOPHARMACEUTICAL CO., LTD. (Chengdu, Sichuan, CN)
- KLUS PHARMA INC. (Cranbury, NJ, US)
Inventors
- Haijun TIAN (Cranbury, NJ, US)
- Sujun DENG (Cranbury, NJ, US)
- Chunxia ZHAO (Cranbury, NJ, US)
- Hong Li (Cranbury, NJ)
- Dengnian LIU (Chengdu, Sichuan, CN)
- Hu LONG (Chengdu, Sichuan, CN)
- Cheng WANG (Chengdu, Sichuan, CN)
- Liang XIAO (Chengdu, Sichuan, CN)
- Tongtong XUE (Chengdu, Sichuan, CN)
- Jingyi Wang (Chengdu, Sichuan, CN)
Cpc classification
A61K31/513
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
A61K31/555
HUMAN NECESSITIES
G01N33/57492
PHYSICS
A61K2300/00
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
A61K31/704
HUMAN NECESSITIES
C07K16/464
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
A61K31/704
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
Abstract
The present application relates to the field of treatment of diseases, and in particular, to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, nucleic acid molecules for encoding said antibody and fragment, and method for preparing said antibody and fragment. The anti-CLDN18.2 antibody or the antigen-binding fragment thereof has high specificity and affinity to CLDN18.2, and can effectively bind to CLDN18.2 and mediate the killing of CLDN18.2 expressing cells. Therefore, the present application further relates to a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and use thereof in the preparation of drugs, wherein the drugs are used for the prevention and/or treatment of tumors.
Claims
1. An antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region (VH) shown in SEQ ID NO: 1; and/or CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region (VL) shown in SEQ ID NO: 2; or (b) CDR-H1, CDR-H2 and CDR-H3 of the VH shown in SEQ ID NO: 3, 39 or 40; and/or CDR-L1, CDR-L2 and CDR-L3 of the VL shown in SEQ ID NO: 4 or 41; or (c) CDR-H1, CDR-H2 and CDR-H3 of the VH shown in SEQ ID NO: 44; and/or CDR-L1, CDR-L2 and CDR-L3 of the VL shown in SEQ ID NO:45; or (d) CDR-H1, CDR-H2 and CDR-H3 of the VH shown in SEQ ID NO: 46; and/or CDR-L1, CDR-L2 and CDR-L3 of the VL shown in SEQ ID NO: 47; or (e) CDR-H1, CDR-H2 and CDR-H3 of the VH shown in SEQ ID NO: 48; and/or CDR-L1, CDR-L2 and CDR-L3 of the VL shown in SEQ ID NO: 49; or (f) CDR-H1, CDR-H2 and CDR-H3 of the VH shown in SEQ ID NO: 50; and/or CDR-L1, CDR-L2 and CDR-L3 of the VL shown in SEQ ID NO: 51; or (g) CDR-H1, CDR-H2 and CDR-H3 of the VH shown in SEQ ID NO: 52; and/or CDR-L1, CDR-L2 and CDR-L3 of the VL shown in SEQ ID NO: 53; or (h) the above-mentioned heavy chain variable region (VH) and/or a light chain variable region (VL), wherein, said heavy chain variable region (VH) and/or a light chain variable region (VL) comprises at least one CDR with a mutation compared with any of the heavy chain variable region and/or a light chain variable region in (a) to (g), said mutation is a substitution, deletion, or addition of one or several amino acids (such as a substitution, deletion, or addition of 1, 2, or 3 amino acids); preferably, the substitution is a conservative substitution; preferably, the CDR is defined according to Kabat, IMGT, Chothia or AbM numbering system; preferably, the VH and/or VL of the antibody or antigen binding fragment thereof comprises Framework Regions (FR) derived from a human or a mouse immunoglobulin; preferably, the antibody or antigen binding fragment thereof binds to human CLDN 18.2.
2. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof comprises: (1) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein CDR is defined according to the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 5, CDR-H2 with a sequence as set forth in SEQ ID NO: 6, and CDR-H3 with a sequence as set forth in SEQ ID NO: 7; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 8, CDR-L2 with a sequence as set forth in SEQ ID NO: 9, and CDR-L3 with a sequence as set forth in SEQ ID NO: 10; or (b) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 11, CDR-H2 with a sequence as set forth in SEQ ID NO: 12 or 110, and CDR-H3 with a sequence as set forth in SEQ ID NO: 13; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 14, CDR-L2 with a sequence as set forth in SEQ ID NO: 15, and CDR-L3 with a sequence as set forth in SEQ ID NO: 16; or (c) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 81, CDR-H2 with a sequence as set forth in SEQ ID NO: 82, and CDR-H3 with a sequence as set forth in SEQ ID NO: 83; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 95, CDR-L2 with a sequence as set forth in SEQ ID NO: 96, and CDR-L3 with a sequence as set forth in SEQ ID NO: 97; or (d) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 81, CDR-H2 with a sequence as set forth in SEQ ID NO: 84, and CDR-H3 with a sequence as set forth in SEQ ID NO: 85; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 95, CDR-L2 with a sequence as set forth in SEQ ID NO: 96, and CDR-L3 with a sequence as set forth in SEQ ID NO: 97; or (e) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 86, CDR-H2 with a sequence as set forth in SEQ ID NO: 87, and CDR-H3 with a sequence as set forth in SEQ ID NO: 88; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 95, CDR-L2 with a sequence as set forth in SEQ ID NO: 98, and CDR-L3 with a sequence as set forth in SEQ ID NO: 99; or (f) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 89, CDR-H2 with a sequence as set forth in SEQ ID NO: 90, and CDR-H3 with a sequence as set forth in SEQ ID NO: 91; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 95, CDR-L2 with a sequence as set forth in SEQ ID NO: 98, and CDR-L3 with a sequence as set forth in SEQ ID NO: 99; or (g) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO: 92, CDR-H2 with a sequence as set forth in SEQ ID NO: 93, and CDR-H3 with a sequence as set forth in SEQ ID NO: 94; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 95, CDR-L2 with a sequence as set forth in SEQ ID NO: 100, and CDR-L3 with a sequence as set forth in SEQ ID NO: 101; or (2) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein CDRs are defined according to the AbM numbering system: (a) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:17, CDR-H2 with a sequence as set forth in SEQ ID NO: 18, and CDR-H3 with a sequence as set forth in SEQ ID NO: 19; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 20, CDR-L2 with a sequence as set forth in SEQ ID NO: 21, and CDR-L3 with a sequence as set forth in SEQ ID NO: 22; or (b) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:23, CDR-H2 with a sequence as set forth in SEQ ID NO: 24 or 111, and CDR-H3 with a sequence as set forth in SEQ ID NO: 25; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO: 26, CDR-L2 with a sequence as set forth in SEQ ID NO: 27, and CDR-L3 with a sequence as set forth in SEQ ID NO: 28; or (c) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:58, CDR-H2 with a sequence as set forth in SEQ ID NO: 62, and CDR-H3 with a sequence as set forth in SEQ ID NO: 67; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO:72, CDR-L2 with a sequence as set forth in SEQ ID NO: 74, and CDR-L3 with a sequence as set forth in SEQ ID NO: 78; or (d) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:58, CDR-H2 with a sequence as set forth in SEQ ID NO: 63, and CDR-H3 with a sequence as set forth in SEQ ID NO: 68; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO:72, CDR-L2 with a sequence as set forth in SEQ ID NO: 74, and CDR-L3 with a sequence as set forth in SEQ ID NO: 78; or (e) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:59, CDR-H2 with a sequence as set forth in SEQ ID NO: 64, and CDR-H3 with a sequence as set forth in SEQ ID NO: 69; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO:72, CDR-L2 with a sequence as set forth in SEQ ID NO: 75, and CDR-L3 with a sequence as set forth in SEQ ID NO: 79; or (f) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:60, CDR-H2 with a sequence as set forth in SEQ ID NO: 65, and CDR-H3 with a sequence as set forth in SEQ ID NO: 70; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO:72, CDR-L2 with a sequence as set forth in SEQ ID NO: 76, and CDR-L3 with a sequence as set forth in SEQ ID NO: 79; or (g) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with a sequence as set forth in SEQ ID NO:61, CDR-H2 with a sequence as set forth in SEQ ID NO: 66, and CDR-H3 with a sequence as set forth in SEQ ID NO: 71; and/or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 with a sequence as set forth in SEQ ID NO:73, CDR-L2 with a sequence as set forth in SEQ ID NO: 77, and CDR-L3 with a sequence as set forth in SEQ ID NO: 80; or (3) the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein, said heavy chain variable region (VH) and/or light chain variable region (VL) comprises at least one CDR with a mutation compared with any of the heavy chain variable region and/or light chain variable region in (a) to (g) of (1) or (2), said mutation is a substitution, deletion, or addition of one or several amino acids (such as a substitution, deletion, or addition of 1, 2, or 3 amino acids); preferably, the substitution is a conservative substitution; preferably, the VH and/or VL of the antibody or antigen binding fragment thereof comprises Framework Regions (FRs) derived from a human or mouse immunoglobulin; preferably, the antibody or antigen binding fragment thereof binds to human CLDN 18.2.
3. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof comprises: (a) a VH sequence as shown in any one of SEQ ID NOs: 1, 29, 30, 31 and 32, and/or, a VL sequence as shown in any one of SEQ ID NOs: 2, 33, 34, 35, 36, 37 and 38; (b) a VH sequence as shown in any one of SEQ ID NOs: 3, 39, and 40, and/or, a VL sequence as shown in any one of SEQ ID NOs: 4, and 41; (c) a VH sequence as shown in SEQ ID NO: 44, and/or a VL sequence as shown in SEQ ID NO: 45; (d) a VH sequence as shown in SEQ ID NO: 46, and/or a VL sequence as shown in SEQ ID NO: 47; (e) a VH sequence as shown in SEQ ID NO: 48, and/or a VL sequence as shown in SEQ ID NO: 49; (f) a VH sequence as shown in SEQ ID NO: 50, and/or a VL sequence as shown in SEQ ID NO: 51; (g) a VH sequence as shown in SEQ ID NO: 52, and/or a VL sequence as shown in SEQ ID NO: 53; (h) a VH sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VH in any of (a) to (g); and/or a VL sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VL in any of (a) to (g); or (i) a VH sequence comprising a substitution, deletion, or addition of 1, 2, 3, 4, 5 amino acids) compared with the VH in any of (a) to (g); and/or a VL sequence comprising a substitution, deletion, or addition of 1, 2, 3, 4, 5 amino acids compared with the VL in any of (a) to (g); preferably, the substitution is a conservative substitution.
4. The antibody or antigen binding fragment thereof according to claim 1 which comprises: (a) a VH having a sequence of SEQ ID NO: 1 and a VL having a sequence of SEQ ID NO: 2; (b) a VH having a sequence of SEQ ID NO: 29 and a VL having a sequence of SEQ ID NO: 33; (c) a VH having a sequence of SEQ ID NO: 29 and a VL having a sequence of SEQ ID NO: 34; (d) a VH having a sequence of SEQ ID NO: 29 and a VL having a sequence of SEQ ID NO: 35; (e) a VH having a sequence of SEQ ID NO: 29 and a VL having a sequence of SEQ ID NO: 36; (f) a VH having a sequence of SEQ ID NO: 29 and a VL having a sequence of SEQ ID NO: 37; (g) a VH having a sequence of SEQ ID NO: 29 and a VL having a sequence of SEQ ID NO: 38; (h) a VH having a sequence of SEQ ID NO: 30 and a VL having a sequence of SEQ ID NO: 33; (i) a VH having a sequence of SEQ ID NO: 31 and a VL having a sequence of SEQ ID NO: 33; (j) a VH having a sequence of SEQ ID NO: 32 and a VL having a sequence of SEQ ID NO: 33, (k) a VH having a sequence of SEQ ID NO: 3 and a VL having a sequence of SEQ ID NO: 4; (l) a VH having a sequence of SEQ ID NO: 39 and a VL having a sequence of SEQ ID NO: 41; (m) a VH having a sequence of SEQ ID NO: 40 and a VL having a sequence of SEQ ID NO: 41; (n) a VH having a sequence of SEQ ID NO: 44 and a VL having a sequence of SEQ ID NO: 45; (o) a VH having a sequence of SEQ ID NO: 46 and a VL having a sequence of SEQ ID NO: 47; (p) a VH having a sequence of SEQ ID NO: 48 and a VL having a sequence of SEQ ID NO: 49; (q) a VH having a sequence of SEQ ID NO: 50 and a VL having a sequence of SEQ ID NO: 51; (r) a VH having a sequence of SEQ ID NO: 52 and a VL having a sequence of SEQ ID NO: 53; (s) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VH in any of (a) to (r); and/or, a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the VL in any of (a) to (r); (t) a VH comprising a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5 amino acids) compared with the VH in any of (a) to (r); and/or, a VL comprising a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5 amino acids) compared with the VL in any of (a) to (r); preferably, the substitution is a conservative substitution.
5. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof is a mouse antibody, a chimeric antibody, or a humanized antibody.
6. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, wherein said variant comprises a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared with the sequence from which it is derived; and (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein said variant comprises a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared with the sequence from which it is derived; preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of: (1) human IgG1 heavy chain constant region; (2) human IgG4 heavy chain constant region; preferably, the antibody or antigen binding fragment thereof comprising a heavy chain constant region (CH) having a sequence as set forth in SEQ ID NO: 42 or a variant thereof, the variant comprises a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, 5 amino acids); preferably, the light chain constant region is a kappa light chain constant region; preferably, the antibody or antigen binding fragment thereof comprises a light chain constant region (CL) having a sequence as set forth in SEQ ID NO: 43 or a variant thereof, the variant comprises a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, 5 amino acids); more preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) having a sequence as set forth in SEQ ID NO: 42 and a light chain constant region (CL) having a sequence as set forth in SEQ ID NO: 43.
7. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody is selected from any one of the following groups: (a) a heavy chain comprising a VH having a sequence of SEQ ID NO: 1 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 2 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (b) a heavy chain comprising a VH having a sequence of SEQ ID NO: 29 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 33 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (c) a heavy chain comprising a VH having a sequence of SEQ ID NO: 29 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 34 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (d) a heavy chain comprising a VH having a sequence of SEQ ID NO: 29 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 35 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (e) a heavy chain comprising a VH having a sequence of SEQ ID NO: 29 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 36 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (f) a heavy chain comprising a VH having a sequence of SEQ ID NO: 29 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 37 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (g) a heavy chain comprising a VH having a sequence of SEQ ID NO: 29 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 38 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (h) a heavy chain comprising a VH having a sequence of SEQ ID NO: 30 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 33 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (i) a heavy chain comprising a VH having a sequence of SEQ ID NO: 31 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 33 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (j) a heavy chain comprising a VH having a sequence of SEQ ID NO: 32 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 33 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (k) a heavy chain comprising a VH having a sequence of SEQ ID NO: 3 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 4 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (l) a heavy chain comprising a VH having a sequence of SEQ ID NO: 39 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 41 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (m) a heavy chain comprising a VH having a sequence of SEQ ID NO: 40 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 41 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (n) a heavy chain comprising a VH having a sequence of SEQ ID NO: 44 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 45 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (o) a heavy chain comprising a VH having a sequence of SEQ ID NO: 46 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 47 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (p) a heavy chain comprising a VH having a sequence of SEQ ID NO: 48 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 49 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; (q) a heavy chain comprising a VH having a sequence of SEQ ID NO: 50 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 51 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43; or (r) a heavy chain comprising a VH having a sequence of SEQ ID NO: 52 and a heavy chain constant region (CH) having a sequence of SEQ ID NO: 42, and a light chain comprising a VL having a sequence of SEQ ID NO: 53 and a light chain constant region (CL) having a sequence of SEQ ID NO: 43.
8. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment comprises: (a) a heavy chain, comprising an amino acid sequence selected from the group consisting of: (i) a sequence as set forth in SEQ ID NO: 102; (ii) a sequence having a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared with SEQ ID NO: 102; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 102; and a light chain, comprising an amino acid sequence selected from the group consisting of: (iv) a sequence as set forth in SEQ ID NO: 103; (v) a sequence having a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared with SEQ ID NO: 103; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 103; or (b) a heavy chain, comprising an amino acid sequence selected from the group consisting of: (i) a sequence as set forth in SEQ ID NO: 106; (ii) a sequence having a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared with SEQ ID NO: 106; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 106; and a light chain, comprising an amino acid sequence selected from the group consisting of: (iv) a sequence as set forth in SEQ ID NO: 107; (v) a sequence having a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, or 5 amino acids) compared with SEQ ID NO: 107; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 107; preferably, the substitution is a conservative substitution.
9. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof is selected from scFv, Fab, Fab′, (Fab′).sub.2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multi-specificity antibody.
10. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof is labeled; preferably, the antibody or antigen binding fragment thereof comprises a detectable label, for example, an enzyme (such as horseradish peroxidase), a radioactive isotope, a fluorescent substance, a luminescent substance (such as a chemiluminescent substance) or biotin.
11. The antibody or antigen binding fragment thereof according to claim 1, characterized by one or more of the following: (1) the antibody or antigen binding fragment thereof binds to CLDN18.2 (e.g., human CLDN18.2) with a KD value less than about 100 nM, for example, less than about 50 nM, 40 nM, 40 nM, 20 nM, 10 nM, 1 nM, 0.1 nM, or less; preferably, the KD value is determined by Biofilm Interference Technology (BLI); (2) the antibody or antigen binding fragment thereof binds to CLDN18.2 (e.g., human CLDN18.2) with an EC50 value less than about 500 nM, for example, less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM or less; preferably, the EC50 is determined by flow cytometry or by a competitive ELISA technique; (3) said antibody or antigen binding fragment does not bind to CLDN18.1 (e.g., human CLDN18.1); (4) said antibody or antigen binding fragment has ADCC activity and/or CDC activity; (5) said antibody or antigen binding fragment has enhanced ADCC activity and/or CDC activity.
12. An isolated nucleic acid molecule, encoding the antibody or antigen binding fragment thereof of claim 1, a heavy chain and/or light chain thereof, or a heavy chain variable region and/or light chain variable region thereof.
13. The isolated nucleic acid molecule according to claim 12, which comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein said nucleic acid molecule encoding an antibody heavy chain variable region has a sequence selected from the group consisting of: (a) a nucleotide sequence as set forth in SEQ ID NO: 54 or 56, or (b) a sequence substantially identical to the nucleotide sequence as set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or higher identity to the nucleotide sequence as set forth in (a) or having a substitution of one or several nucleotides compared with the nucleotide sequence as set forth in (a)), or (c) a sequence which differs from the nucleotide sequence as set forth in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; and/or, said nucleic acid molecule encoding an antibody light chain variable region has a sequence selected from the group consisting of: (d) a nucleotide sequence as set forth in SEQ ID NO: 55 or 57, or (e) a sequence substantially identical to the nucleotide sequence as set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or higher identity to the nucleotide sequence as set forth in (d) or having a substitution of one or several nucleotides compared with the nucleotide sequence as set forth in (d)), or (f) a sequence which differs from the nucleotide sequence as set forth in (d) by no more than 3, 6, 15, 30 or 45 nucleotides; preferably, the nucleic acid molecule encoding an antibody heavy chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 54, and/or the nucleic acid molecule encoding an antibody light chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 55; preferably, the nucleic acid molecule encoding an antibody heavy chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 56, and/or the nucleic acid molecule encoding an antibody light chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 57.
14. The isolated nucleic acid molecule according to claim 12, which comprises a nucleic acid molecule encoding an antibody heavy chain, and/or a nucleic acid molecule encoding an antibody light chain, wherein the nucleic acid molecule encoding an antibody heavy chain has a sequence selected from the group consisting of: (a) a nucleotide sequence as set forth in SEQ ID NO: 104 or 108, or (b) a sequence substantially identical to the nucleotide sequence as set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or higher identity to the nucleotide sequence as set forth in (a) or having a substitution of one or several nucleotides compared with the nucleotide sequence as set forth in (a)), or (c) a sequence which differs from the nucleotide sequence as set forth in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; and/or the nucleic acid molecule encoding an antibody light chain has a sequence selected from the group consisting of: (d) a nucleotide sequence as set forth in SEQ ID NO: 105 or 109, or (e) a sequence substantially identical to the nucleotide sequence as set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or higher identity to the nucleotide sequence as set forth in (d) or having a substitution of one or several nucleotides compared with the nucleotide sequence as set forth in (d)), or (f) a sequence which differs from the nucleotide sequence as set forth in (d) by no more than 3, 6, 15, 30 or 45 nucleotides; preferably, the nucleic acid molecule encoding an antibody heavy chain has a nucleotide sequence as set forth in SEQ ID NO: 104, and/or the nucleic acid molecule encoding an antibody light chain has a nucleotide sequence as set forth in SEQ ID NO: 105; preferably, the nucleic acid molecule encoding an antibody heavy chain has a nucleotide sequence as set forth in SEQ ID NO: 108, and/or the nucleic acid molecule encoding an antibody light chain has a nucleotide sequence as set forth in SEQ ID NO: 109.
15. A vector, which comprises an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof of claim 1, a heavy chain and/or light chain thereof, or a heavy chain variable region and/or light chain variable region thereof; preferably, the vector is a cloning vector or an expression vector.
16. A host cell, which comprises: an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof of claim 1, a heavy chain and/or light chain thereof, or a heavy chain variable region and/or light chain variable region thereof, or a vector comprising the isolated nucleic acid molecule.
17. A method for preparing the antibody or antigen binding fragment thereof of claim 1, comprising culturing a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof under conditions suitable for expression of said antibody or antigen binding fragment thereof, and recovering the antibody or antigen binding fragment thereof from host cell cultures.
18. A conjugate, which comprises an antibody or an antigen binding fragment thereof, and a conjugate moiety, wherein said antibody is the antibody or antigen binding fragment thereof of claim 1, and the conjugate moiety is selected from: a detectable label, radioisotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), nuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, proteins, receptors, ligands, and other active substance that inhibits tumor cell growth or promotes apoptosis or necrosis of tumor cells.
19. A chimeric antigen receptor, which comprises the antibody or antigen binding fragment thereof of claim 1 (e.g., scFv), a transmembrane domain, and one or multiple intracellular T cell signaling domains.
20. A multi-specific antibody, which is formed by conjugation of a first antibody or a fragment thereof with an additional antibody or a fragment thereof or with an antibody mimetic, wherein each antibody or fragment thereof or antibody mimetic retains the original binding specificity, and the first antibody or fragment thereof is the antibody or antigen binding fragment thereof of claim 1; preferably, the multi-specific antibody is a bispecific antibody or a tri-specific antibody or a tetra-specific antibody.
21. A pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and/or an excipient and comprises one of the following: the antibody or antigen binding fragment thereof of claim 1, or a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, or a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, or a conjugate comprising the antibody or antigen binding fragment thereof and a conjugate moiety, or a chimeric antigen receptor comprising the antibody or antigen binding fragment thereof, a transmembrane domain, and one or multiple intracellular T cell signaling domains, or a multi-specific antibody formed by conjugation of the antibody or antigen binding fragment thereof with an additional antibody or a fragment thereof or with an antibody mimetic; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; more preferably, the antibody or antigen binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as components of a single composition.
22. The pharmaceutical composition according to claim 21, wherein the antibody or antigen binding fragment thereof comprised therein is used in a subject to: (a) induce apoptosis in tumor cells; (b) inhibit tumor cell proliferation; (c) induce and/or increase T cell infiltration; (d) induce and/or enhance immune response; (e) induce and/or increase complement dependent cytotoxicity; (f) induce and/or increase antibody-dependent cytotoxicity; (g) increase NK cell activity; (h) inhibit the expression and activation of CLDN18.2; (i) inhibit CLDN18.2-mediated cell signaling pathway; or (j) any combination of (a) to (i).
23. The pharmaceutical composition according to claim 21, which further comprises a second antibody or a nucleic acid encoding the second antibody, the second antibody specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3 and any combination thereof.
24. A diagnostic or therapeutic kit, which comprises an instruction for use and comprises one of the following: the antibody or antigen binding fragment thereof of claim 1, or a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, or a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, or a conjugate comprising the antibody or antigen binding fragment thereof and a conjugate moiety, or a chimeric antigen receptor comprising the antibody or antigen binding fragment thereof, a transmembrane domain, and one or multiple intracellular T cell signaling domains, or a multi-specific antibody formed by conjugation of the antibody or antigen binding fragment thereof with an additional antibody or a fragment thereof or with an antibody mimetic, or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, the vector, the host cell, the conjugate, the chimeric antigen receptor, or the multi-specific antibody, and a pharmaceutically acceptable carrier and/or an excipient.
25. (canceled)
26. (canceled)
27. (canceled)
28. A method for preventing and/or treating a tumor, and/or delaying tumor progression, and/or reducing or inhibiting tumor recurrence, in a subject, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 21.
29. The method according to claim 28, which further comprises administering a second therapy to the subject, the second therapy being selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy can be administered separately, in combination, simultaneously or sequentially with the method; preferably, the chemotherapy is one or more agents selected from the group consisting of: epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel, albumin-bound paclitaxel, combination of epirubicin+oxaliplatin+5-fluorouracil, FOLFOX4, FOLFOX6, mFOLFOX6 (including oxaliplatin, leucovorin and 5-fluorouracil).
30. The method according to claim 28, wherein the tumor is a solid tumor, a hematological tumor, or a metastatic, refractory or recurrent lesion of cancer; preferably, the tumor or cancer is selected from the group consisting of esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, or leukemia; preferably, the tumor is selected from the group consisting of gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, or lung cancer (e.g., non-small cell lung cancer); preferably, the tumor is gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, such as a locally advanced unresectable gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, or a metastatic gastric cancer or GEJ adenocarcinoma; preferably, the tumor is CLDN 18.2 positive, more preferably the tumor is HER2 negative.
31. A method of detecting the presence or level of CLDN18.2 in a sample, comprising contacting the sample with the antibody or antigen binding fragment thereof of claim 1 under conditions which permit formation of a complex between the antibody or antigen binding fragment thereof and CLDN 18.2, and detecting the formation of a complex between the antibody or antigen binding fragment thereof and CLDN 18.2.
32. A method for diagnosing or differentially diagnosing a tumors or tumor metastasis, comprising using the antibody or antigen binding fragment thereof of claim 1 or a conjugate or multispecific antibody comprising the antibody or antigen binding fragment thereof, wherein the tumor is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (for example, non-small cell lung cancer).
33. The antibody or antigen-binding fragment thereof of claim 6, comprising a heavy chain constant region (CH) having a sequence as set forth in SEQ ID NO: 42 and a light chain constant region (CL) having a sequence as set forth in SEQ ID NO: 43.
34. The pharmaceutical composition of claim 21, which further comprises an additional pharmaceutically active agent having antitumor activity.
35. The pharmaceutical composition of claim 34, wherein the additional pharmaceutically active agent is interferon, interleukin-2 or a chemotherapy drug.
36. The pharmaceutical composition of claim 35, wherein the additional pharmaceutically active agent is one or more agents selected from the group consisting of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel, albumin-bound paclitaxel, combination of epirubicin+oxaliplatin+5-fluorouracil, FOLFOX4, FOLFOX6, mFOLFOX6 (including oxaliplatin, leucovorin and 5-fluorouracil).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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SEQUENCE INFORMATION
[0427] The sequence information involved in the invention is provided in the table below.
TABLE-US-00001 SEQ ID NO Description 1 heavy chain variable region of mouse antibody 1E9.2 2 light chain variable region of mouse antibody 1E9.2 3 heavy chain variable region of mouse antibody 2C6.9 4 light chain variable region of mouse antibody 2C6.9 5 IMGT 1E9.2 CDR-H1 6 IMGT 1E9.2 CDR-H2 7 IMGT 1E9.2 CDR-H3 8 IMGT 1E9.2 CDR-L1 9 IMGT 1E9.2 CDR-L2 10 IMGT 1E9.2 CDR-L3 11 IMGT 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-H1 12 IMGT 2C6.9/2C6.9-hz11 CDR-H2 13 IMGT 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-H3 14 IMGT 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-L1 15 IMGT 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-L2 16 IMGT 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-L3 17 AbM 1E9.2 CDR-H1 18 AbM 1E9.2 CDR-H2 19 AbM 1E9.2 CDR-H3 20 AbM 1E9.2 CDR-L1 21 AbM 1E9.2 CDR-L2 22 AbM 1E9.2 CDR-L3 23 AbM 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-H1 24 AbM 2C6.9/2C6.9-hz11 CDR-H2 25 AbM 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-H3 26 AbM 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-L1 27 AbM 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-L2 28 AbM 2C6.9/2C6.9-hz11/2C6.9-hz21 CDR-L3 29 heavy chain variable region of humanized antibody 1E9.2hz11/1E9.2hz12/1E9.2hz13/1E9.2hz14/1E9.2hz15/1E9.2hz17 30 heavy chain variable region of humanized antibody 1E9.2hz21 31 heavy chain variable region of humanized antibody 1E9.2hz31 32 heavy chain variable region of humanized antibody 1E9.2hz41 33 light chain variable region of humanized antibody 1E9.2hz11/1E9.2hz21/1E9.2hz31/1E9.2hz41 34 light chain variable region of humanized antibody 1E9.2hz12 35 light chain variable region of humanized antibody 1E9.2hz13 36 light chain variable region of humanized antibody 1E9.2hz14 37 light chain variable region of humanized antibody 1E9.2hz15 38 light chain variable region of humanized antibody 1E9.2hz17 39 Heavy chain variable region of humanized antibody 2C6.9hz11 40 heavy chain variable region of humanized antibody 2C6.9hz21 41 light chain variable region of humanized antibody 2C6.9hz11/2C6.9hz21 42 human IgG1 heavy chain constant region 43 human k light chain constant region 44 heavy chain variable region sequence of mouse antibody 19H11.6 45 light chain variable region sequence of mouse antibody 19H11.6 46 heavy chain variable region sequence of mouse antibody 16A9.11 47 light chain variable region sequence of mouse antibody 16A9.11 48 heavy chain variable region sequence of mouse antibody 9C8.1 49 light chain variable region sequence of mouse antibody 9C8.1 50 heavy chain variable region sequence of mouse antibody 6B9.22 51 light chain variable region sequence of mouse antibody 6B9.22 52 heavy chain variable region sequence of mouse antibody 19G10.14 53 light chain variable region sequence of mouse antibody 19G10.14 54 heavy chain variable region sequence of mouse antibody 1E9.2 55 light chain variable region sequence of mouse antibody 1E9.2 56 heavy chain variable region sequence of mouse antibody 2C6.9 57 light chain variable region sequence of mouse antibody 2C6.9 58 AbM 19H11.6/16A9.11 CDR-H1 59 AbM 9C8.1 CDR-H1 60 AbM 6B9.22 CDR-H1 61 AbM 19G10.14 CDR-H1 62 AbM 19H11.6CDR-H2 63 AbM 16A9.11 CDR-H2 64 AbM 9C8.1 CDR-H2 65 AbM 6B9.22 CDR-H2 66 AbM 19G10.14 CDR-H2 67 AbM 19H11.6 CDR-H3 68 AbM 16A9.11 CDR-H3 69 AbM 9C8.1 CDR-H3 70 AbM 6B9.22 CDR-H3 71 AbM 19G10.14 CDR-H3 72 AbM 19H11.6/16A9.11/9C8.1/6B9.22 CDR-L1 73 AbM 19G10.14 CDR-L1 74 AbM 19H11.6/16A9.11 CDR-L2 75 AbM 9C8.1 CDR-L2 76 AbM 6B9.22 CDR-L2 77 AbM 19G10.14 CDR-L2 78 AbM 19H11.6/16A9.11 CDR-L3 79 AbM 9C8.1/6B9.22 CDR-L3 80 AbM 19G10.14 CDR-L3 81 IMGT 19H11.6/16A9.11 CDR-H1 82 IMGT 19H11.6CDR-H2 83 IMGT 19H11.6 CDR-H3 84 IMGT 16A9.11 CDR-H2 85 IMGT 16A9.11 CDR-H3 86 IMGT 9C8.1 CDR-H1 87 IMGT 9C8.1 CDR-H2 88 IMGT 9C8.1 CDR-H3 89 IMGT 6B9.22 CDR-H1 90 IMGT 6B9.22 CDR-H2 91 IMGT 6B9.22 CDR-H3 92 IMGT 19G10.14 CDR-H1 93 IMGT 19G10.14 CDR-H2 94 IMGT 19G10.14 CDR-H3 95 IMGT 19H11.6/16A9.11/9C8.1/6B9.22/19G10.14 CDR-L1 96 IMGT 19H11.6/16A9.11 CDR-L2 97 IMGT 19H11.6/16A9.11 CDR-L3 98 IMGT 9C8.1/6B9.22 CDR-L2 99 IMGT 9C8.1/6B9.22 CDR-L3 100 IMGT 19G10.14 CDR-L2 101 IMGT 19G10.14 CDR-L3 102 heavy chain amino acid sequence of humanized antibody 1E9.2 hz11 103 light chain amino acid sequence of humanized antibody 1E9.2 hz11 104 heavy chain nucleotide sequence of humanized antibody 1E9.2 hz11 105 light chain nucleotide sequence of humanized antibody 1E9.2 hz11 106 heavy chain amino acid sequence of humanized antibody 2C6.9 hz21 107 light chain amino acid sequence of humanized antibody 2C6.9 hz21 108 heavy chain nucleotide sequence of humanized antibody 2C6.9 hz21 109 light chain nucleotide sequence of humanized antibody 2C6.9 hz21 110 IMGT 2C6.9-hz21 CDR-H2 111 AbM 2C6.9-hz21 CDR-H2
EXAMPLES
[0428] The invention will now be described with reference to the following examples, which are intended to illustrate, but not to limit the invention.
[0429] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are generally referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. It will be understood by a person skilled in the art that the examples are given for the purpose of illustration and are not to be construed to limit the scope of the present invention.
Example 1. Construction and Identification of Human Claudin18.2 and Human Claudin 18.1 Overexpression Cell Lines
1.1 Construction of Human Claudin18.2 and Human Claudin18.1 Overexpression Cell Lines
[0430] To determine the specificity and function of anti-human Claudin18.2 antibody, the complete coding sequences of human Claudin18.2 (Gene accession number: NM_001002026.2, synthesized by Nanjing Genscript Biotech Corporation) and human Claudin18.1 (Gene accession number: NM_016369.3, synthesized by Nanjing Genscript Biotech Corporation) were cloned into lentivirus vector pLVX-IRES-puro and the viruses were prepared by lentivirus packaging system according to the published method (Mohammadi Z etl., Mol Biotechnol. 2015 September; 57(9):793-800.). The viruses obtained were used to infect HEK293T, L929, KATOIII and NCI-N87 cells. Monoclonal stable cell lines of HEK293T-Claudin 18.1, HEK293T-Claudin 18.2, L929-Claudin 18.2, KATOIII-Claudin 18.2, and NCI-N87-Claudin 18.2 were obtained by puromycin screening and single clone selection. BaF/3 cells (DSMZ, Cat #ACC300) were transfected with plasmids coding human Claudin18.2 or human Claudin18.1 using 4D-Nucleofector X transfection kit (Lonza, Cat #V4XC-3012). 48h after transfection, cells were screened by addition of 1.25 mg/mL hygromycin (Thermo Fisher Sci. Cat #10687010). Single clones were selected 12 days later, thereby obtaining monoclonal cell lines BaF/3-Claud18.1 and BaF/3-Claud18.2.
1.2 Detection of Human Claudin18.2 and Human Claudin18.1 Overexpression Cell Lines
[0431] Western Blot was used to detect HEK293T-Claudin 18.1 (Detection Antibody: Proteintech, 66167-1-Ig) and FACS was used to detect other cell lines (Flow cytometer: Beckman, CytoFlex; Detection Antibody: IMAB362, of which the sequences are from patent: CN 101312989 B). As shown in
Example 2. Preparation of Mouse Anti-Human Claudin18.2 Monoclonal Antibodies
[0432] DNA/cell immunization was performed in wild type mice to generate mouse anti-human Claudin 18.2 monoclonal antibodies. Each Balb/c mouse was injected with 100 μg plasmid containing the complete coding sequence of human Claudin 18.2 through tail vein. After the fourth and sixth immunizations, serum titers were determined by FACS. Mice with high serum titers were boosted with BaF/3-Claudin18.2 overexpression cell lines 3-5 days prior to fusion. PEG mediated fusion of mouse splenocytes and mouse myeloma cell line Sp2/0 (ATCC, Cat #CRL-1581) was performed with standard fusion protocol, followed by HAT selection. FACS screening was carried out 10-14 days after fusion.
[0433] Supernatants of about 6000 hybridomas were screened using flow cytometry (available from Sartorius, as Model iQue Screener Plus) and 43 positive hybridomas binding HEK293T-Claudin18.2 cell line were obtained and sub-cloned. 14 hybridomas which specifically bound to human Claudin18.2 but not to human Claudin18.1 were selected by FACS using HEK293T-Claudin18.2 and HEK293T-Claudin18.1 cell lines. Single clones were obtained by limited dilution and subclone selection.
[0434] Human gastric cancer cell line NUGC4 (purchased from Japan JCRB cell bank, catalog number: JCRB0834) expresses Claudin18.2 endogenously and is widely used to evaluate the binding between antibodies and endogenous Claudin18.2 and develop functional assays. NUGC4 cells were used to evaluate the candidate clones and 7 sub-clones were finally selected, which are named 1E9.2, 2C6.9, 6B9.22, 9C8.1, 16A9.11, 19G10.14 and 19H11.6 respectively.
Example 3. Determination of the Affinity of Mouse Anti-Human Claudin18.2 Antibodies
[0435] The affinity of the candidates to Claudin18.2 was measured using FACS. Single hybridoma clone 1E9.2, 2C6.9, 6B9.22, 9C8.1, 16A9.11, 19G10.14 and 19H11.6 were cultured in serum-free medium and mouse antibodies were purified from 100 mL supernatant by protein A chromatography (MabSelect SuRe, GE). Ba/F3-Claudin18.1 and Ba/F3-Claudin18.2 cells were incubated with the purified mouse antibodies at various concentrations on ice for 30 min and mouse IgG was used as negative control (Thermo Fisher, Cat: 31903), followed by washing with FACS buffer (PBS+2% FBS) twice. The cells were then incubated with a fluorescent goat-anti-mouse secondary antibody (Jackson ImmunoResearch, Cat: 115-605-071) on ice for 30 min before FACS analysis.
[0436] As shown in
TABLE-US-00002 TABLE 1 Determination of the affinity of mouse anti-human Claudin 18.2 antibodies to Ba/F3-Claudin 18.2 cells Clone Number EC50 (nM) Max MFI* (×10.sup.6) 1E9.2 8.506 5.085 19H11.6 10.94 6.008 16A9.11 10.2 5.815 9C8.1 9.399 4.969 6B9.22 3.64 3.624 19G10.14 16.27 3.501 2C6.9 10.57 6.111 Control IgG No binding No binding *Max MFI is Max Mean Fluorescence Intensity
Example 4. Identification of the Subtype of Mouse Anti-Human Claudin18.2 Monoclonal Antibody and Amplification of Variable Regions
[0437] In order to identify the antibody subtype of the candidate hybridoma clones, Pierce Rapid Isotyping kit (Thermo Fisher Sci. Cat #26179) was used to identify the antibody subtype of the seven candidate clones. The results showed that all candidates have IgG1 heavy chain and Kappa light chain.
[0438] Hybridoma cells were expanded and around 8000 cells were harvested and lysed. The first cDNA chain was synthesized by cDNA reverse transcription kit (Thermo Fisher Sci. Cat #18080-200). VH and VK (VL Kappa) genes were amplified by PCR from the cDNA using primers. The PCR products were purified by DNA purification kit (Qiagen, Cat #28104) and ligated to TOPO vector (Thermo Fisher Sci. Cat #K457540). About twelve clones were picked from each ligation reaction and sequenced. The sequences were then analyzed by Vector NTI 11.5 (Thermo Fisher Sci.) and Sequencer 5.4.6 (Genecodes). The variable region and CDR sequences of mouse anti-human Claudin18.2 antibody were shown in Table 2.
TABLE-US-00003 TABLE 2 The variable region and CDR amino acid sequences of mouse anti-human Claudin18.2 antibody s Heavy chain Light chain variable variable Heavy chain Heavy chain Heavy chain Light chain Light chain Light chain Clone region (SEQ region (SEQ Definition CDR1 (SEQ CDR2 (SEQ CDR3 (SEQ CDR1 (SEQ CDR2 (SEQ CDR3 (SEQ Number ID NO:) ID NO:) mode ID NO:) ID NO:) ID NO:) ID NO:) ID NO:) ID NO:) 1E9.2 1 2 IMGT 5 6 7 8 9 10 AbM 17 18 19 20 21 22 2C6.9 3 4 IMGT 11 12 13 14 15 16 AbM 23 24 25 26 27 28 19H11.6 44 45 IMGT 81 82 83 95 96 97 AbM 58 62 67 72 74 78 16A9.11 46 47 IMGT 81 84 85 95 96 97 AbM 58 63 68 72 74 78 9C8.1 48 49 IMGT 86 87 88 95 98 99 AbM 59 64 69 72 75 79 6B9.22 50 51 IMGT 89 90 91 95 98 99 AbM 60 65 70 72 76 79 19G10.14 52 53 IMGT 92 93 94 95 100 101 AbM 61 66 71 73 77 80
Example 5. Determination of the Affinity of Anti-Human Claudin18.2 Chimeric Antibodies
[0439] The amino acid sequence of the light chain variable region of 1E9.2, 2C6.9, 6B9.22, 9C8.1, 16A9.11, 19G10.14 or 19H11.6 was linked to the amino acid sequence of the light chain constant region κ (SEQ ID NO: 43) respectively; the amino acid sequence of the heavy chain variable region was linked to the amino acid sequence of IgG1 heavy chain constant region (SEQ ID NO: 42) respectively, to construct the chimeric antibody. Codon optimized cDNA was synthesized and cloned to pcDNA3.4 (synthesized by Nanjing Genscript Biotech Corporation). The pcDNA3.4 plasmids coding the light chain and heavy chain of each antibody were co-transfected to Expi293F cells (purchased from Thermo Fisher). The antibodies were purified from culture supernatant by protein A (MabSelect SuRe, GE). And the purified chimeric antibodies were named 1E9.2-hz00, 2C6.9-hz00, 6B9.22-hz00, 9C8.1-hz00, 16A9.11-hz00, 19G10.14-hz00 and 19H11.6-hz00.
[0440] The affinity of the anti-Claudin18.2 chimeric antibodies to Ba/F3-Claudin18.2 cells were determined. Specifically, Ba/F3-Claudin18.2 cells (60000 in 10 ul buffer) were incubated with serially diluted anti-human Claudin18.2 chimeric antibodies or the control antibody IMAB362 on ice for 30 min, followed by washing with FACS buffer (PBS+2% FBS) twice and incubating with fluorescent goat-anti-human secondary antibody (Thermo Fisher Sci., Cat: A-21445) on ice for another 30 min. The cells were then analyzed by FACS.
[0441] As shown in Table 3, the values of EC50 of 1E9.2-hz00, 16A9.11-hz00 or 19H11.6-hz00 was lower than that of the positive control IMAB362, indicating that the above anti-human Claudin 18.2 chimeric antibodies had stronger affinity than IMAB362. All 1E9.2-hz00, 6B9.22-hz00, 9C8.1-hz00, 16A9.11-hz00, or 19H11.6-hz00 showed stronger maximal mean fluorescence intensity than IMAB362, indicating that the amount of the antibodies of present invention bound to antigen in a saturated state was higher than that of IMAB362, and the antibody of the invention had stronger binding activity than IMAB362.
TABLE-US-00004 TABLE 3 Measurement of affinity of anti-human Claudin 18.2 chimeric antibodies to cells Antibody EC50 (nM) Max MFI (×10.sup.6) 1E9.2-hz00 11.03 11.8 6B9.22-hz00 18.24 16.70 9C8.1-hz00 23.95 14.41 16A9.11-hz00 10.48 12.40 19H11.6-hz00 11.64 12.97 IMAB362 18.03 8.97
Example 6. Humanization of Anti-Human Claudin18.2 Mouse Antibodies
[0442] CDR-grafting method was used to humanize the two mouse antibodies 1E9.2 and 2C6.9. Briefly, the humanization process was involved in the following steps: the amino acid sequences of mouse monoclonal antibodies were aligned with the amino acid sequences of human germline antibody to identify human germline frameworks with high homology and good physical-chemical properties; the affinity to HLA-DR was determined and the human germline frameworks with low affinity to HLA-DR were then selected; the six CDR regions of mouse antibodies were then grafted to the selected heavy chain and light chain frameworks.
[0443] Specifically, the heavy chain and light chain CDRs of mouse antibodies 1E9.2 and 2C6.9 were grafted to the frameworks (FR) of the corresponding humanization templates. The humanization template for the heavy chain of 1E9.2 is human germline sequence IGHV3-21*04 (IMGT reference number HM855688). The humanization templates for light chain are human germline sequences IGKV4-1*01 (IMGT reference number Z00023) and IGKV6-21*02 (IMGT reference number KM455568). The humanization templates for heavy chain and light chain of 2C6.9 are human germline sequence IGHV4-59*01 (IMGT reference number AB019438) and IGKV4-1*01 (IMGT reference number Z00023), respectively.
[0444] Moreover, with computer simulation, molecular docking was performed to analyze the variable region and its surrounding framework amino acid sequences, in order to determine the spatial stereoscopic binding mode of the antibodies. By calculating electrostatic forces, Van der Waals forces, hydrophobic interactions and entropy, critical amino acid residues which may interact with Claudin18.2 or maintain spatial structure in the amino acid sequence of the mouse antibody were identified, and these mouse amino acids were maintained after grafting. In other words, a series of back mutations were taken in the FR region residues of humanization template so that the affinities of the mouse antibodies could be kept in humanized antibodies to the maximal extent.
[0445] Using the previously described methods, 9 humanized antibodies were constructed based on the CDRs of mouse antibody 1E9.2, which were named 1E9.2hz11, 1E9.2hz12, 1E9.2hz13, 1E9.2hz14, 1E9.2hz15, 1E9.2hz17, 1E9.2hz21, 1E9.2hz31 and 1E9.2hz41, respectively. 2 humanized antibodies were constructed based on the CDRs of mouse antibody 2C6.9, which were named 2C6.9hz11 and 2C6.9hz21, respectively. The heavy chain constant regions of the constructed antibodies are the same as wild-type human IgG1 heavy chain constant region (SEQ ID NO: 42). The light chain constant regions are all the same as wild-type human IgG1 light chain constant region (SEQ ID NO: 43).
[0446] The amino acid sequences of variable region and constant region of the above humanized antibodies are shown in Table 4.
TABLE-US-00005 TABLE 4 The amino acid sequences of variable region and constant region of the humanized antibodies Variable Variable Constant Constant Region of Region of Region of Region of Heavy Light Heavy Light Chain Chain Chain Chain Antibody (SEQ ID (SEQ ID (SEQ ID (SEQ ID Name NO:) NO:) NO:) NO:) 1E9.2hz11 29 33 42 43 1E9.2hz12 29 34 1E9.2hz13 29 35 1E9.2hz14 29 36 1E9.2hz15 29 37 1E9.2hz17 29 38 1E9.2hz21 30 33 1E9.2hz31 31 33 1E9.2hz41 32 33 2C6.9hz11 39 41 2C6.9hz21 40 41
Example 7. Evaluation of the Affinity and ADCC Activity of the Anti-Human Claudin18.2 Humanized Antibodies
[0447] In order to evaluate the anti-Claudin18.2 humanized antibodies, codon optimized cDNAs of the heavy chains and the light chains of the candidate humanized antibodies from Example 6 were synthesized and ligated into pcDNA3.4 (by Nanjing Genscript Biotech Corporation). Expi293F cells (purchased from Thermo Incorporation) were co-transfected with pcDNA3.4 coding the heavy chain and light chain of each antibody. The antibodies were purified from the supernatant using protein A (MabSelect SuRe, GE). The affinity of the humanized antibodies to Claudin18.2 was determined using Ba/F3-Claudin18.2 cells which over-expressed human Claudin18.2. Ba/F3-Claudin18.2 cells (60000 in 10 ul buffer) were incubated with purified humanized antibodies on ice for 30 min, followed by washing with buffer (PBS+2% FBS) twice and addition of Alexa647-labeled goat-anti-human IgG Fc antibody (1:500 dilution). The cells were incubated on ice for another 30 min and analyzed by IntelliCyt.
[0448] The results of affinity detected with Ba/F3-Claudin 18.2 cells over-expressing human Claudin 18.2 are shown in Table 5. The Max MFI showed that all the chimeric antibodies and humanized antibodies of 1E9.2 and control antibody IMAB362 could bind to Claudin18.2. The Max MFI of each of the chimeric antibody and humanized antibodies was stronger than that of control antibody IMAB362, indicating that the amount of the candidate antibody bound to antigen in a saturated state was higher than that of IMAB362; in another word, the antibody of present invention has stronger binding activity to the antigen.
TABLE-US-00006 TABLE 5 Measurement of affinity for anti-human Claudin 18.2 chimeric and humanized candidate antibodies to Ba/F3-Claudin 18.2 cells Candidate Antibody Max MFI (×10.sup.6) 1E9.2-hz00 11.17 1E9.2-hz11 11.34 1E9.2-hz21 14.56 1E9.2-hz31 10.64 1E9.2-hz41 11.28 1E9.2-hz12 6.71 1E9.2-hz13 6.02 1E9.2-hz14 5.36 1E9.2-hz15 8.99 1E9.2-hz17 5.90 IMAB362 4.99* *represents that the value is the average of two determinations.
[0449] The affinity of 2C6.9-hz21 to human claudin 18.2 on cell membrane was detected using HEK293T-Claudin 18.2 cells, as follows. HEK293T-Claudin 18.2 cells were detached and centrifuged, followed by washing for twice with PBS. And then the cells were resuspended in PBS containing 1% BSA and were plated into 96-well tip bottom plate at 300000 cells per well in a volume of 50 μl for 20 wells in total. 2C6.9-hz21 or IMAB362 antibody was then added with 11 concentrations starting from 1000 nM with three-fold dilution. Human IgG was served as negative control. The reactions were mixed well and incubated for 1 h at 4° C. in the dark. Following by three times of washing with PBS, the FITC-labelled anti-human Fc secondary antibody (Biolegend, 409322) was added and incubated for 0.5h in the dark. Detection was performed by flow cytometry (Beckman, Cytoflex) after washing for three times with PBS.
[0450] As shown in
TABLE-US-00007 TABLE 6 Measurement of affinity for humanized antibody 2C6.9-hz21 to HEK293T-Claudin 18.2 by flow cytometry Antibody Name EC50 (nM) Max MFI 2C6.9-hz21 5.767 165866.3 IMAB362 8.217 156715.7
[0451] In order to evaluate the ADCC activity, ten thousand Ba/F3-Claudin18.2 cells were cultured in 40 μl NK-92 media. Antibodies were serially diluted in 10 μl PBS. Fifty thousand NK-92MI_mCD16 cells in 50 μl NK-92 media were added and the reactions were incubated at 37° C. for 6h. 33.3 μl CytoTox-Glo (Promega, G9290) was added to each reaction and absorbance was read on a plate reader. As shown in Table 7, the EC50 of ADCC killing activity and max killing rate of 1E9.2 chimeric antibody and all 1E9.2 humanized antibodies against Ba/F3-Claudin 18.2 cells were both stronger than that of IMAB362. The results showed that the chimeric antibody and humanized antibody of 1E9.2 had stronger ADCC activity.
TABLE-US-00008 TABLE 7 Evaluation of ADCC activity of the chimeric and humanized antibodies against human Claudin 18.2 ADCC activity on Ba/F3-Claudin 18.2 cells Antibody Name EC50 (μg/mL) Max killing rate % 1E9.2-hz00 0.049* 59.83* 1E9.2-hz11 0.041* 60.17* 1E9.2-hz41 0.048 55.43 1E9.2-hz12 0.091 64.36 1E9.2-hz13 0.068 63.78 1E9.2-hz14 0.097 60.45 1E9.2-hz15 0.080 66.22 1E9.2-hz17 0.113 59.64 IMAB362 0.351* 54.17* *represents that the value is the average of two determinations.
Example 8 Determination of the Affinity and Specificity of Anti-Human Claudin18.2 Humanized Antibody
[0452] Human Claudin18.2 is a four transmembrane protein and has a complex structure. Cellular ELISA was thus carried out to maintain the structure of Claudin18.2. The stable cell line L929-Claudin 18.2 constructed in Example 1 were used for detection. Specifically, L929-Claudin 18.2 adherent cells were detached by 2 mM EDTA treatment. The cells were resuspended and adjusted to 2×10.sup.5/mL and 100 μL of the resuspension was plated into 96 well plate and incubated overnight at 37° C. The next day, the media was removed, and the plate was washed with PBS once. 100 μL/well 4% formaldehyde was added to the plate. After 30 min of incubation at room temperature, formaldehyde was removed, followed by washing twice with PBS. Blocking buffer (PBS containing 2% BSA) was then added to the plate at 100 μL/well, and incubated at 37° C. for 2 h. After removal of blocking buffer, 100 μL/well of serially diluted antibodies (starting from 1 μM, with 4-fold dilution, for a total of 11 concentrations) were added to the corresponding wells, which were incubated at 37° C. for 2h afterwards. The plate was washed with 250 μL PBST for 5 times, and was allowed to stand for 2 min each time. 100 μL/well of horseradish peroxidase labeled anti-Human IgG secondary antibody (HRP-anti-Human IgG, Jackson ImmunoResearch, 109-035-003) diluted 1:10000 in PBS (containing 2% BSA) was added to the plate. The plate was incubated at 37° C. for 1 h and washed with 250 μL PB ST 6 times, wherein the plate was allowed to stand for 2 min each time. 100 μL/well of TMB solution (Thermo, 34029) was added. The reactions were incubated at 37° C. for 20 min and stopped with 50 μL 2 mol/L H2504. The absorbance at 450 nm was read on a plate reader (MD, SpectraMax M2) and the results were subjected to curve fitting by Graphpad Prism.
[0453] The results of multiple experiments are shown in
TABLE-US-00009 TABLE 8 The affinity of the humanized antibody 1E9.2-hz1 1 binding to L929-Claudin 18.2 cells using Cellular ELISA Antibody Name EC50 (nM) Max Signal Value (OD450) 1E9.2-hz11 0.1 1 IMAB362 0.18 0.82
TABLE-US-00010 TABLE 9 The affinity of the humanized antibody 2C6.9-hz21 binding to L929-Claudin 18.2 cells using Cellular ELISA Max Signal Value Antibody Name EC50 (nM) (OD450) 2C6.9-hz21 0.12 0.92 IMAB362 0.15 0.53
TABLE-US-00011 TABLE 10 The affinity of the humanized antibody binding to L929-Claudin 18.2 cells using Cellular ELISA Antibody Name EC50 (nM) Max Signal Value (OD450) 1E9.2-hz11 0.09 0.8 2C6.9-hz11 0.16 0.8 2C6.9-hz21 0.18 0.8
[0454] To determine binding affinity of the candidate antibodies to cell lines endogenously expressing human Claudin18.2, FACS analysis was performed to detect human Claudin18.2 expression level on NUGC-4 cells (purchased from Japan JCRB cell bank, catalog number: JCRB0834) which endogenously expresses human Claudin18.2. As shown in
TABLE-US-00012 TABLE 11 The affinity of the humanized antibody binding to NUGC-4 cells Antibody Name EC50 (nM) OD450 1E9.2-hz11 0.4129 0.5 IMAB362 ≈663.7 Not detected
[0455] The specificity of candidate antibodies was detected by FACS. The steps are as follows, HEK293T, HEK293T-human Claudin 18.1 and HEK293T-human Claudin18.2 cells were detached and then centrifuged. After washing twice with PBS, the cells were resuspended with PBS containing 1% BSA. 300,000 cells for each cell line were added with the candidate antibodies at a final concentration of 1000 nM, mixed well, and incubated for 1 h at 4° C., protected from light. Then the cells were washed for three times with PBS and FITC-labeled anti-human Fc secondary antibody (BioLegend, 409322) was added, followed by 0.5h of incubation at 4° C. in the dark. Detection was performed by flow cytometry (Beckman, Cytoflex) after washing with PBS for three times.
[0456] As shown in
Example 9. Determination of Complement Dependent Cytotoxicity (CDC) of Anti-Claudin18.2 Humanized Antibody
[0457] 1E9.2-hz11 belongs to IgG1 subtype, which can activate classical complement pathway effectively, and induce complement dependent cytotoxicity (CDC). Guinea pig serum (purchased from Zhengzhou Baiji, catalog number S0001) which is rich in complements was used in our research in order to determine the CDC activity of 1E9.2-hz11. The procedure is described as follows: HEK293T-Claudin 18.2 cells were harvested and centrifuged followed by adjustment of cell density. 5×10.sup.4/well of cells were plated on a plate and incubated overnight. DMEM containing 20% guinea pig serum was prepared the next day and was used to dilute 1E9.2-hz11 and IMAB362. Starting from 20 μg/mL, 10 concentrations were prepared with two-fold dilution. The original cell culture media for HEK293T-Claudin 18.2 cells was removed, and the antibody dilutions were added to the corresponding wells, 100 μL/well. 10 μl/well lysis buffer was provided as the positive control. The reactions were left to stand in incubator at 37° C., 5% CO.sub.2 and incubated for 3 h. Then, CCK8 (Rhinogen, QDY-003-D) was added at 20 μl/well and allowed to react for 2h. Absorbance at 450 nm were taken on a plate reader (MD, SpectraMax M2). The results were subjected to curve fitting by Graphpad Prism.
[0458] As shown in
[0459] The CDC detection method of antibody 2C6.9-hz21 is similar to that of 1E9.2-hz11. The cells were left to stand in incubator at 37° C., 5% CO.sub.2 and incubated for 3h, with the medium containing guinea pig serum. Subsequently, CellTiter-Glo Luminescent (CTG, Purchased from Promega, Item No.: G7573) was added at 50 μl/well for staining followed by a 30-second mixing and left to stand for 1 min at room temperature. The fluorescence signal value was then determined by a microplate reader (MD, SpectraMax M2), the results of which were imported into Graphpad Prism for curve fitting.
[0460] As shown in
TABLE-US-00013 TABLE 12 The CDC activity determination of anti-Claudin 18.2 humanizec antibody Antibody Name EC50 value (ng/mL) 2C6.9-hz21 1363 IMAB362 3317
Example 10. Determination of the Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of Anti-Human Claudin18.2 Humanized Antibody
[0461] 1E9.2-hz11 belongs to IgG1 subtype and has relative strong antibody-dependent cell-mediated cytotoxicity (ADCC). NK cell mediated killing assay was used to determine the ADCC activity of 1E9.2-hz11. The steps were as follows: HEK293T-Claudin 18.2, L929-Claudin 18.2, KATOIII-Claudin 18.2 and NUGC-4 cells were harvested and centrifuged followed by adjustment of cell density. 1×10.sup.4/well of cells were plated on a plate and incubated overnight. The medium was removed on the next day. NK92MI-CD16a cells (Huabo Biopharm) were centrifuged, resuspended in MEMA medium and adjusted to 1×10.sup.6/mL, then 50 μL/well cells were added to the corresponding wells. 1E9.2-hz11 and IMAB362 antibodies were diluted with MEMA medium. For HEK293T-Claudin18.2, L929-Claudin18.2 and KATOIII-Claudin18.2 cells, ten antibody concentrations were tested, starting from 40 μg/mL with 5-fold dilution. For NUGC-4 cells, eleven antibody concentrations were tested, starting from 2 mg/mL with 5-fold dilution. 50 μL/well of the diluted antibodies were added to the corresponding wells and the reactions were left to stand in incubator at 37° C., 5% CO.sub.2 and incubated for 5.5h. Lysis buffer was then added to the positive control well and incubated for another 0.5h. 50 μL/well of Lactate Dehydrogenase (LDH) detection agent (DOJINDO LABORATORISE, CK12) were added to the wells and the absorbance at 490 nm were taken every 10 min on a plate reader (MD, SpectraMax M2). The results were imported into Graphpad Prism for curve fitting.
[0462] As shown in
TABLE-US-00014 TABLE 13 The ADCC activity of the anti-human Claudin 18.2 humanized antibody antibodies 1E9.2-hz11 IMAB362 Max. Max. EC50 Cytotoxicity EC50 Cytotoxicity Cell lines (ng/mL) (%) (ng/mL) (%) HEK293T-Claudin 18.2 52.83 83 33.58 55 L929-Claudin 18.2 2.388 46 78.07 52 KATOIII-Claudin 18.2 3.586 64 10.10 49 NUGC-4 133.6 43 1219 35
[0463] Antibody 2C6.9-hz21 is also of IgG1 subtype, so the method for detecting its ADCC activity is the same as that of antibody 1E9.2-hz11. The cell used was HEK293T-Claudin 18.2. Results are shown in
TABLE-US-00015 TABLE 14 The ADCC activity of the anti-Claudin 18.2 humanized antibody 2C6.9-hz21 Antibody Name EC50 value (ng/mL) Max Cytotoxicity (%) 2C6.9-hz21 67.67 30 IMAB362 57.66 20
Example 11. Determination of the Pharmacokinetics (PK) Properties of Anti-Human Claudin18.2 Humanized Antibody in Mouse Model
[0464] To determine the PK properties of 1E9.2-hz11 and IMAB362 in mouse model, 10 mg/kg of 1E9.2-hz11 or IMAB362 were intravenously injected into SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), 2 mice per group. Blood samples were taken 8 h, 1 d, 3 d, 8 d and 15 d after injection and serum antibody concentrations were determined by Cellular ELISA as described in Example 8. Standard curves were prepared using 1E9.2-hz11 and IMAB362 standards by Graphpad Prism. The data of mouse serum was subjected to curve fitting. The PK properties of 1E9.2-hz11 and IMAB362 were determined.
[0465] As shown in Table 15, the half-life of 1E9.2-hz11 and IMAB362 in mouse are 42.5h and 35.77h, respectively. 1E9.2-hz11 is better than IMAB362.
TABLE-US-00016 TABLE l5 Determination of the PK properties of anti-human Claudin 18.2 humanized antibody Half-life (hours) AUC (h*μg/mL) 1E9.2-hz11 42.50 9088.87 IMAB362 35.77 7391.22
Example 12. In Vivo Efficacy of Anti-Claudin18.2 Humanized Antibody
[0466] In vivo efficacy of anti-Claudin18.2 humanized antibody was demonstrated through the anti-tumor effect thereof on subcutaneous transplantation tumor model of human gastric cancer cell line NCI-N87-Claudin 18.2 (an engineered NCI-N87 cell line overexpressing human Claudin 18.2) in Balb/c nude mice. Specifically, the anti-Claudin18.2 humanized antibody was injected into a tumor-bearing mouse model subcutaneously implanted with NCI-N87-Claudin 18.2 through tail vein injection, and the tumor volume and the change in animal weight were measured regularly to evaluate the in vivo efficacy of anti-human Claudin18.2 humanized antibody (tumor inhibition efficacy).
[0467] Specific steps were as follows: NCI-N87-Claudin18.2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37° C. and 5% CO.sub.2. Cells in exponential growth phase were collected and resuspended in PBS to a suitable concentration, and then inoculated subcutaneously into female Balb/c nude mice (Biocytogen Jiangsu Co., Ltd, permit no. SOCK (Su) 2016-0004) at an amount of 5×10.sup.6 cells per mouse in 0.1 mL PBS, to establish the subcutaneous transplantation tumor model. The day of inoculation was record as P0. The experiments were performed with three groups: intravenous immunoglobulin (Negative Control, Chengdu Rongsheng Pharmaceuticals Co. Ltd., abbreviated as Human IgG), combination of Epirubicin+Oxaliplatin+5-fluorouracil (abbreviated as EOF chemicals), and combination group of 1E9.2-hz11+EOF chemicals. On day 4, 11, 18 and 25, EOF chemicals (Epirubicin 1.25 mg/kg, Oxaliplatin 3.25 mg/kg, 5-fluorouracil 56.25 mg/kg) were administered intraperitoneally (once a week for 4 weeks); for the combination group of 1E9.2-hz11+EOF chemicals, on the basis of EOF administration, 1E9.2-hz11 antibody (10 mg/kg, 2 times a week for 4 weeks) was further administered via the tail vein on Day 5, 8, 12, 15, 19, 22, 26, and 29. The tumor volume and body weight of mice after administration was observed and measured regularly.
[0468] The tumor diameters were measured by vernier caliper. The tumor volume was calculated according to the following formula: V=0.5 a×b.sup.2, in which a and b represent the long diameter and short diameter of the tumor respectively. The death of animals was observed and record every day.
[0469] The anti-tumor efficacy of antibody expressed as the tumor growth inhibition rate (TGI) was calculated as following formula:
TGI(%)=[1−(V.sub.TEnd−V.sub.TInt)/(V.sub.CEnd−V.sub.CInt)]×100%
In which V.sub.TEnd means the mean tumor volume of the treatment group at the end of study;
[0470] V.sub.TInt means the mean tumor volume of the treatment group at the beginning of study; [0471] V.sub.CEnd means the mean tumor volume of the negative control group at the end of study;
[0472] V.sub.CInt means the mean tumor volume of the negative control group at the beginning of study.
[0473] The anti-tumor efficacy of antibody expressed as the relative tumor growth rate (T/C) was calculated as following formula:
T/C(%)=(T.sub.t/T.sub.0)/(C.sub.t/C.sub.0)×100%
In which T0 means the average tumor volume of the treatment group at the time of initiation (i.e., P0);
[0474] T.sub.t means the average tumor volume of treatment group at the time of each measurement;
[0475] C.sub.0 means the average tumor volume of the negative control group at the time of initiation (i.e., P0);
[0476] C.sub.t means the average tumor volume of the negative control group at the time of each measurement.
[0477] The results are shown in Table 16 and
TABLE-US-00017 TABLE 16 NCI-N87-Claudin18.2 + Balb/c nude mouse model P29 Tumor Dose Volume (mm.sup.3) TGI T/C P Value No Group (mg/kg) (
[0478] The mouse subcutaneous xenograft tumor model of NCI-N87-Claudin 18.2 was established by the method mentioned above. When the average volume of the tumors was about 110 mm.sup.3, the tumor-bearing mice were grouped randomly according to tumor size. There were 3 groups: the group of anti-chicken lysozyme human IgG1 of isotype control (Negative Control, Chengdu Kelun-Biotech Co. Ltd., abbreviated as IgG1 WT), the group of paclitaxel for injection (Albumin Bound, Hunan Kelun), and the combination group of 2C6.9-hz21+Paclitaxel for injection. All samples were injected via tail vein twice a week for 3 weeks. After administration, the tumor volume and body weight of mice was observed and measured regularly, and the data analysis method is as described above.
[0479] The results are shown in Table 17 and
TABLE-US-00018 TABLE 17 NCI-N87-Claudin 18.2 + Balb/c nude mouse model P21 Tumor Volume DOSE (mm.sup.3) TGI T/C P Value No Group (mg/kg)
[0480] In summary, the combination of humanized antibodies such as 1E9.2-hz11 and 2C6.9-hz21 with chemotherapy drugs (such as EOF or paclitaxel) can effectively inhibit tumor growth.
[0481] Although the examples of this invention have been described in detail, the researchers in this area should understand: following the guidance of the published method, modifications and variations of the example details can be made and all these modifications are protected within the scope of this patent.