Bispecific antibodies against cadherin-17 and antibody drug conjugates prepared using the same

Abstract

The present application provides an anti-CDH17 antibody or an antigen binding fragment thereof, which can be a bispecific antibody constructed. The anti-CDH17 antibody provided by the present application can specifically bind to CDH17 protein, has a relatively strong CDH17-expressing tumor cell killing effect, can be effectively internalized on CDH17-expressing tumor cells, and can be prepared into an ADC which can be effectively internalized on CDH17-expressing tumor cells and has high killing activity; in particular, compared with an antibody binding to a single epitope of CDH17, the bispecific antibody of the present application has more accurate and multi-dimensional antigen recognition ability and higher targeting.

Description

Technical Field

[0001] This invention belongs to the field of antibody drugs. Specifically, this invention relates to dual epitope antibodies against cadherin-17, antibody-drug conjugates targeting cadherin-17, and their applications. Background Technology

[0002] Cadherin-17 (CDH17) is an important member of the cadherin family, consisting of seven extracellular domains (ECs) – EC1 to EC7 – and a very short cytoplasmic domain. CDH17 is mainly expressed in gastrointestinal epithelial cells, and its main functions are to participate in cell adhesion and tissue morphology maintenance.

[0003] Studies have shown that CDH17 is abnormally highly expressed in various tumors, particularly in digestive system malignancies such as gastric cancer, colorectal cancer, and pancreatic cancer. Clinical studies have found that high CDH17 expression is often closely related to tumor differentiation, invasiveness, metastasis, and poor prognosis, especially in the late and metastatic stages of tumors, where its expression level often increases significantly.

[0004] Biparatopic antibodies (bpAbs) bind to two distinct, non-overlapping epitopes on an antigen. Combining the expression characteristics of CDH17 with biparatopic antibodies to develop CDH17-targeting biparatopic antibodies and further ADC drugs will provide a novel therapeutic strategy for tumors or cancers with high CDH17 expression (such as gastric cancer, colorectal cancer, and other digestive system tumors). Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a novel anti-CDH17 antibody that binds to the CDH17 protein, such as CDH17 expressed by tumor cells, with high specificity and high affinity, thereby exhibiting significant tumor cell killing ability through efficient binding to the tumor-expressed CDH17 protein. Furthermore, this invention characterizes the binding characteristics of different anti-CDH17 antibodies to the target protein CDH17, selecting antibodies that bind to different regions or epitopes on the CDH17 protein, and further constructing antibodies with dual or more CDH17 epitope binding activities. Furthermore, the antibodies with dual or more CDH17 epitope binding activities constructed by this invention should also have a strong ability to internalize into CDH17-expressing cells, thus being suitable for preparation into antibody-drug conjugates, thereby obtaining an antibody-drug conjugate with better therapeutic effect against CDH17-expressing tumor cells.

[0006] Therefore, one object of the present invention is to provide an antibody or fragment thereof that specifically binds to CDH17, or an antibody or fragment thereof having dual or more CDH17 epitope binding activity. Another object of the present invention is to provide an antibody-drug conjugate or a salt thereof that targets CDH17.

[0007] The technical solution of the present invention is as follows.

[0008] First aspect

[0009] The present invention provides an antibody or antigen-binding fragment thereof against cadherin-17 (CDH17), wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to CDH17, particularly human CDH17.

[0010] In the context of this invention, unless otherwise stated, the term "CDH17" covers any form or structural region of CDH17.

[0011] In the context of this invention, the term "antigen-binding fragment" encompasses various functional fragments of the antibody that specifically binds to CDH17, which retain the antibody's ability to bind to the antigen and the corresponding biological activity. It is well known in the art that the antibody's ability to bind to the antigen and the corresponding biological activity can be achieved from fragments of the intact antibody, which can be obtained using conventional techniques known to those skilled in the art and screened for functionality in the same manner as for the intact antibody. For example, antigen-binding fragments of the antibody can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody.

[0012] Specifically, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). According to a specific embodiment of the present invention, the heavy chain CDRs comprised in the anti-CDH17 antibody or its antigen-binding fragment are derived from the heavy chain variable region having any one of the amino acid sequences shown in SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, and 15, and from the light chain variable region having any one of the amino acid sequences shown in SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, and 16.

[0013] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.16 above are the amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary anti-CDH17 antibody provided in the "Detailed Description" section of this application. Using any one or a combination of antibody heavy chain or light chain complementarity-determining regions (CDRs) known in the art (e.g., Chothia, Kabat, IMGT, Contact, AbM, CCG, etc.), those skilled in the art can readily determine the heavy chain CDRs and light chain CDRs contained therein. Combinations of heavy chain CDRs and light chain CDRs can be obtained according to known or conventional definition tools in the art, and antibodies or fragments thereof containing each of these combinations of heavy chain CDRs and light chain CDRs are within the protection scope of this invention.

[0014] Preferably, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises heavy chain CDRs and light chain CDRs from the heavy chain variable region and light chain variable region shown in the following amino acid sequence pairings:

[0015] (1) SEQ ID NO.1 + SEQ ID NO.2;

[0016] (2) SEQ ID NO.3 + SEQ ID NO.4;

[0017] (3) SEQ ID NO.5 + SEQ ID NO.6;

[0018] (4) SEQ ID NO.7 + SEQ ID NO.8;

[0019] (5)SEQ ID NO.9+SEQ ID NO.10;

[0020] (6)SEQ ID NO.11+SEQ ID NO.12;

[0021] (7) SEQ ID NO.13 + SEQ ID NO.14; or

[0022] (8) SEQ ID NO.15+SEQ ID NO.16.

[0023] As described above, for example, the CCG definition can be used to classify the CDRs in the above amino acid sequence pairings, as shown in the embodiments of the present invention.

[0024] Accordingly, in the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention, the heavy chain CDRs and light chain CDRs are as follows:

[0025] (1) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48;

[0026] (2) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.52, SEQ ID NO.53, and SEQ ID NO.54;

[0027] (3) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60;

[0028] (4) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66;

[0029] (5) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72;

[0030] (6) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77;

[0031] (7) HCDR1, HCDR2, and HCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO.78, SEQ ID NO.79, and SEQ ID NO.80; and LCDR1, LCDR2, and LCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO.81, SEQ ID NO.82, and SEQ ID NO.83; or

[0032] (8) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.84, SEQ ID NO.85, and SEQ ID NO.86; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.87, SEQ ID NO.71, and SEQ ID NO.88.

[0033] As described above, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention specifically binds to cadherin-17 (CDH17), preferably primate or rodent CDH17, such as human, monkey, or mouse CDH17. Optionally, the antibody or its antigen-binding fragment provided by the present invention may or may not have species cross-binding activity with human, cyno, or mouse CDH17.

[0034] Preferably, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), both of which include the aforementioned CDRs and the framework region (FR) therebetween. The arrangement of each region from the N-terminus to the C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0035] According to specific embodiments of the present invention, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region and a light chain variable region as shown below:

[0036] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.1, or contains an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.2, or contains an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2;

[0037] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.3, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.4, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.4;

[0038] (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.5, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.6, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.6;

[0039] (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.7, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.8, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0040] (5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.9, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.9; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.10, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.10.

[0041] (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.11, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.11; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.12, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.12.

[0042] (7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 13, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 13; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 14, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 14; or

[0043] (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.15, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.15; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.16, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.16.

[0044] In the context of this invention, the term "at least 75% identity" in relation to amino acid sequences encompasses any percentage of identity between at least 75% and 100% identity, such as 75%, 80%, 85%, 90%, and even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% identity. The maximum 25% difference in amino acid sequence resulting from "at least 75% identity" can exist in any frame region within the heavy chain variable region or the light chain variable region, or in any domain or sequence outside the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment of this invention. Such differences can arise from amino acid deletions, additions, or substitutions at any position, wherein substitutions can be conservative or non-conservative.

[0045] Preferably, the anti-CDH17 antibody provided by the present invention can be a mouse antibody, rabbit antibody, or human antibody, or it can be a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody. The CDH17 antibody can also be a derivatized antibody, such as an antibody obtained by CDR transplantation, affinity maturation, point mutation modification, or chemical modification based on an initial mouse monoclonal antibody. The chemical modification includes glycosylation, acetylation, polyethylene glycol modification, phosphorylation, amidation, protease cleavage, linkage with cellular ligands or effector molecules, protection of active reactive groups, and / or blocking. Preferably, the antigen-binding fragment of the antibody can be a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized variable fragment (dsFv), (dsFv)2, an antigen-binding fragment (Fab), Fab' fragment (Fab'), (Fab' fragment)2 (F(ab')2), or a variable fragment (Fv), etc. Regarding the antigen-binding fragment of the antibody described in this invention, it can be any fragment of the antibody capable of specifically binding to CDH17.

[0046] In addition to the heavy chain and / or light chain variable regions, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention may also include a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably including a human or mouse heavy chain constant region and / or a light chain constant region. Preferably, the anti-CDH17 antibody or its antigen-binding fragment includes a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region.

[0047] According to a specific embodiment of the present invention, the anti-CDH17 antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain constant region sequence of IgG1, such as the human IgG1 heavy chain constant region; and / or comprises a kappa light chain constant region, such as the human kappa light chain constant region.

[0048] According to a specific embodiment of the present invention, the anti-CDH17 antibody of the present invention is a monoclonal antibody. Preferably, the anti-CDH17 antibody provided by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. More preferably, the antibody is human IgG1 subtype.

[0049] Furthermore, the present invention provides an anti-cadherin 17 (CDH17) antibody or its antigen-binding fragment thereof, which may be an anti-CDH17 antibody or its antigen-binding fragment binding to two or more epitopes of CDH17. The anti-CDH17 antibody or its antigen-binding fragment binding to two or more epitopes of CDH17 may be artificially constructed such that the antibody or its antigen-binding fragment contains at least two binding domains to bind different epitopes of CDH17, wherein the epitopes may be located in any one or more of the extracellular regions EC1 to EC7 of CDH17.

[0050] Preferably, the present invention provides a dual epitope antibody or antigen-binding fragment against cadherin-17 (CDH17), wherein the dual epitope antibody or antigen-binding fragment comprises at least a first binding domain and a second binding domain. The first binding domain and the second binding domain may respectively comprise the heavy chain complementarity-determining regions (CDRs) and the light chain complementarity-determining regions (CDRs) of the antibody as described above, and respectively bind to different epitopes located in different extracellular regions of CDH17. For example, the first binding domain binds to a first epitope of CDH17, and the second binding domain binds to a second epitope of CDH17. The first epitope and the second epitope may be located in the extracellular regions EC1-2, EC3-4, or EC5-6 of CDH17, respectively. For example, the first binding domain binds to a first epitope located in the extracellular region EC1-2 of CDH17, while the second binding domain binds to a second epitope located in the extracellular region EC3-4 of CDH17; and vice versa.

[0051] Regarding the sequences of the heavy chain and light chain complementarity-determining regions contained therein, the heavy chain CDRs contained in the first binding domain and the second binding domain of the biepisode antibody or its antigen-binding fragment provided by the present invention may be derived from the heavy chain variable region having any one of the amino acid sequences shown in SEQ ID NOs.1, 3, 5, 7, 9, 11, 13, 15 and the light chain variable region having any one of the amino acid sequences shown in SEQ ID NOs.2, 4, 6, 8, 10, 12, 14, 16.

[0052] As described above, the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.16 are the amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary anti-CDH17 antibody provided in the "Detailed Description" section of this application. Using any one or a combination of antibody heavy chain or light chain complementarity-determining regions (CDRs) known in the art (e.g., Chothia, Kabat, IMGT, Contact, AbM, CCG, etc.), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained therein. Combinations of heavy chain CDRs and light chain CDRs can be obtained according to known or conventional definition tools in the art, and antibodies or fragments thereof containing each of these combinations of heavy chain CDRs and light chain CDRs are within the protection scope of this invention.

[0053] Preferably, the anti-CDH17 dual epitope antibody or its antigen-binding fragment provided by the present invention comprises a first binding domain and a second binding domain, wherein the first binding domain and the second binding domain respectively bind to a first epitope and a second epitope located in different exosome regions of CDH17, wherein:

[0054] (1) The first epitope is located in the extracellular region EC1-2 of CDH17, and the second epitope is located in the extracellular region EC5-6 of CDH17;

[0055] (2) The first epitope is located in the extracellular region EC3-4 of CDH17, and the second epitope is located in the extracellular region EC5-6 of CDH17;

[0056] (3) The first epitope is located in the extracellular region EC5-6 of CDH17, and the second epitope is located in the extracellular region EC3-4 of CDH17; or

[0057] (4) The first epitope is located in the extracellular region EC1-2 of CDH17, and the second epitope is located in the extracellular region EC3-4 of CDH17.

[0058] More preferably, the dual epitope antibody or its antigen-binding fragment provided by the present invention comprises a first binding domain and a second binding domain as shown below: (1)

[0060] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.1, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.2.

[0061] The second binding domain contains heavy chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9, and light chain CDRs having the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 10. (2)

[0063] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.1, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.2.

[0064] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.11, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.12. (3)

[0066] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.5, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.6.

[0067] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 10. (4)

[0069] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.5, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.6.

[0070] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.11, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.12. (5)

[0072] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.11, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.12.

[0073] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.5, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.6. (6)

[0075] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.5, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.6.

[0076] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.15, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.16. (7)

[0078] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.7, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.8.

[0079] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 10. (8)

[0081] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.7, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.8.

[0082] The heavy chain CDRs contained in the second binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.11, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.12. (9)

[0084] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.3, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.4.

[0085] The second binding domain contains heavy chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9, and light chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 10; or (10)

[0087] The heavy chain CDRs contained in the first binding domain are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.3, and the light chain CDRs are derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.4.

[0088] The second binding domain contains heavy chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.7, and light chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.8.

[0089] Preferably, the dual epitope antibody or its antigen-binding fragment provided by the present invention comprises a first binding domain and a second binding domain, and: (1)

[0091] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48.

[0092] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72. (2)

[0094] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48.

[0095] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77. (3)

[0097] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60.

[0098] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72. (4)

[0100] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60.

[0101] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77. (5)

[0103] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77.

[0104] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60. (6)

[0106] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60.

[0107] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.84, SEQ ID NO.85, and SEQ ID NO.86; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.87, SEQ ID NO.71, and SEQ ID NO.88. (7)

[0109] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66.

[0110] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72. (8)

[0112] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66.

[0113] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77. (9)

[0115] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.52, SEQ ID NO.53, and SEQ ID NO.54.

[0116] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69; and LCDR1, LCDR2, and LCDR3, sequentially comprising the amino acid sequences shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72; or (10)

[0118] The first binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.52, SEQ ID NO.53, and SEQ ID NO.54.

[0119] The second binding domain comprises: HCDR1, HCDR2, and HCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63; and LCDR1, LCDR2, and LCDR3, which sequentially comprise the amino acid sequences shown in SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66.

[0120] Furthermore, the first and second binding domains of the anti-CDH17 dual epitope antibody or its antigen-binding fragment provided by the present invention may respectively include a heavy chain variable region (VH) and a light chain variable region (VL). Similarly, both include the above-mentioned CDRs and the framework region (FR) therebetween. The arrangement of each region from the N-terminus to the C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0121] According to a specific embodiment of the present invention, the first binding domain and the second binding domain of the anti-CDH17 dual epitope antibody or its antigen-binding fragment provided by the present invention both include a heavy chain variable region and a light chain variable region, and: (1)

[0123] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2;

[0124] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.10. (2)

[0126] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2;

[0127] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12. (3)

[0129] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.6.

[0130] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.10. (4)

[0132] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.6.

[0133] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12. (5)

[0135] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12.

[0136] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.6; (6)

[0138] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.6.

[0139] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 15; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 16. (7)

[0141] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0142] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.10. (8)

[0144] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0145] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12. (9)

[0147] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4;

[0148] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 10; or (10)

[0150] In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4;

[0151] In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8.

[0152] In addition to the heavy chain and / or light chain variable regions, similarly, in the anti-CDH17 biepisode antibody or its antigen-binding fragment provided by the present invention, the first binding domain and the second binding domain may include a heavy chain constant region (CH) and / or a light chain constant region (CL), as defined above.

[0153] Furthermore, in the anti-CDH17 dual epitope antibody or its antigen-binding fragment provided by the present invention, the first binding domain and the second binding domain can be selected from IgG antibodies and scFv fragments, respectively. For example, the first binding domain is an IgG antibody, and the second binding domain is an scFv fragment, which are linked together by a linker that allows them to independently bind to different epitopes of CDH17. The linker can be a flexible linker containing glycine-serine, such as the (GGGGS)n linker, where n = 2-5, particularly n = 4.

[0154] According to a specific embodiment of the present invention, the first binding domain is an IgG antibody, and the second binding domain is an scFv fragment. The scFv fragment contains domains arranged in the order "VH-Linker-VL" or "VL-Linker-VH" from its amino terminus to its carboxyl terminus, wherein the Linker is a flexible linker as described above. Further, the scFv fragment is connected to the carboxyl terminus of the IgG antibody via a flexible linker at its amino terminus. Exemplarily, the anti-CDH17 biepisode antibody may contain two identical heavy chains and two identical light chains, wherein the heavy chains contain domains arranged in the order "Ab1VH-CH1-CH2-CH3-Linker-Ab2 VH-Linker-VL" or "Ab1VH-CH1-CH2-CH3-Linker-Ab2 VL-Linker-VH" from its amino terminus to its carboxyl terminus; the light chains contain domains arranged in the order "Ab1 VL-CL" from its amino terminus to its carboxyl terminus, thereby obtaining a tetravalent biepisode antibody; see also... Figure 9 In constructing this tetravalent biepisode antibody, at least one cysteine ​​residue can be deleted or mutated at a specific amino acid site in the scFv fragment, such as a G=>C mutation, to achieve efficient antibody assembly.

[0155] Alternatively, the anti-CDH17 biepisode antibody or its antigen-binding fragment provided by the present invention may be in the form of an IgG antibody, with the first binding domain and the second binding domain located in the two haptens of the IgG antibody, respectively. Exemplarily, the anti-CDH17 biepisode antibody may comprise two different heavy chains and two different light chains, with one heavy chain and one light chain respectively constituting the hapten.

[0156] According to a specific embodiment of the present invention, the anti-CDH17 biepisode antibody provided by the present invention is constructed using Crossmab. The first binding domain is located in the first half-antibody (hereinafter also referred to as antibody A portion), which comprises a heavy chain and a light chain. The heavy chain contains domains arranged in the sequence "VH-CH1-CH2-CH3" from the amino terminus to the carboxyl terminus, and the light chain contains domains arranged in the sequence "VL-CL" from the amino terminus to the carboxyl terminus. The second binding domain is located in the second half-antibody (hereinafter also referred to as antibody B portion), which comprises a heavy chain and a light chain. The heavy chain contains domains arranged in the sequence "VH-CL-CH2-CH3" from the amino terminus to the carboxyl terminus, and the light chain contains domains arranged in the sequence "VL-CH1" from the amino terminus to the carboxyl terminus, thereby obtaining a bivalent biepisode antibody. See also: Figure 10 In constructing this bivalent biepisode antibody, Knob-in-Hole modification can be further performed on both heavy chains to achieve efficient antibody assembly.

[0157] Second aspect

[0158] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody against CDH17 as described herein or an antigen-binding fragment thereof.

[0159] The phrase "encoding the antibody against CDH17 or its antigen-binding fragment according to the present invention" refers to the nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable regions, heavy chain variable regions, heavy chains, and / or light chains contained in the antibody or its antigen-binding fragment. For example, the nucleic acid molecule provided by the present invention contains nucleotide sequences encoding each of the heavy chain CDRs and light chain CDRs contained in the aforementioned antibody or its antigen-binding fragment; contains nucleotide sequences encoding the heavy chain variable regions and light chain variable regions contained in the aforementioned antibody or its antigen-binding fragment; or contains nucleotide sequences encoding the heavy chains and light chains contained in the aforementioned antibody or its antigen-binding fragment.

[0160] Third aspect

[0161] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in a third aspect, this invention also provides a vector containing the nucleic acid molecules of this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector, etc. The vectors or nucleic acid molecules of this invention can be used to transform or transfect host cells, for purposes such as preserving or expressing antibodies.

[0162] Fourth aspect

[0163] The present invention also provides a host cell comprising the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, or animal cells.

[0164] Fifth aspect

[0165] The anti-CDH17 antibody or its antigen-binding fragment provided by this invention can be obtained using any method known in the art. For example, this invention also provides a method for preparing the antibody or its antigen-binding fragment, the method comprising culturing the host cells provided by this invention while allowing the host cells to express the heavy and light chains of the antibody. Optionally, the method further includes the step of recovering the generated antibody or its antigen-binding fragment.

[0166] Sixth aspect

[0167] The anti-CDH17 antibody or its antigen-binding fragment provided by this invention can also be directly or indirectly linked to other parts, such as heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, and light chains of other antibodies. For example, the antibody or its antigen-binding fragment can be constructed together with other binding domains to form a multi-epitope antibody or a general bispecific antibody or multispecific antibody. In the context of this invention, the term "multi-epitope antibody" refers to an antibody that can bind to other epitopes of CDH17 in addition to the first epitope and second epitope described above. In the context of this invention, the terms "bispecific antibody" or "multispecific antibody" refer to antibodies that can bind to one or more other target proteins in addition to CDH17.

[0168] Alternatively, the other components may be small molecule compounds, such as cytotoxic compounds used in antibody-drug conjugates.

[0169] Accordingly, in a sixth aspect, the present invention also provides the use of the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, vector, or host cell in the preparation of antibody-drug conjugates (ADCs). For example, the antibody or its antigen-binding fragment, or an antibody or its antigen-binding fragment encoded by the nucleic acid molecule, may be directly or indirectly conjugated to a small molecule drug.

[0170] Seventh aspect

[0171] The present invention provides an antibody-drug conjugate or a salt thereof targeting CDH17, which comprises the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention.

[0172] The antibody-drug conjugate can be formed by conjugating the anti-CDH17 antibody or its antigen-binding fragment provided by this invention with a cytotoxic compound. The cytotoxic compound can be a tubulin inhibitor, a topoisomerase inhibitor, a DNA binder, etc. For example, the tubulin inhibitor can be maytansine compounds such as DM1 and DM4, sarsaparilla toxin compounds such as Monomethyl Dolastatin 10, MMAE, MMAF, tubulolysin compounds, Cryptophycin derivatives, Taltobulin, muscarine, chalcogenide, eribulin, and derivatives of the aforementioned drugs; the topoisomerase inhibitor can be camptothecin compounds such as Dxd, exatecan, and their derivatives, doxorubicin metabolite PNU-159682 derivative, and irinotecan and its metabolite SN38, etc.; the DNA binder can be PBD derivatives and Duocarmycin and its derivatives, etc.

[0173] Eighth aspect

[0174] The anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, or antibody-drug conjugate or its salt provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical formulation, for use in various purposes as needed.

[0175] Therefore, the present invention also provides a composition comprising an anti-CDH17 antibody or its antigen-binding fragment provided by the present invention, a nucleic acid molecule, a carrier, a host cell, or an antibody-drug conjugate or its salt. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients. The pharmaceutical compositions provided by the present invention can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.

[0176] Ninth aspect

[0177] This invention also provides the use of the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition in the preparation of a medicament for the prevention, treatment and / or improvement of a disease that may be associated with CDH17 expression (including overexpression), such as CDH17-positive solid tumors. The anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition may exert their effects by binding to CDH17 to exert an ADCC effect or through the cytotoxic toxicity of cytotoxic compounds in the antibody-drug conjugate, but are not limited thereto.

[0178] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0179] Tenth aspect

[0180] The present invention also provides a method for preventing, treating and / or improving a disease, the method comprising administering to a subject in need an anti-CDH17 antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or a salt thereof or a combination thereof, the disease being associated with CDH17 expression (including overexpression), such as a CDH17-positive solid tumor.

[0181] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer. The subject may be a mammal, preferably a primate or rodent, such as a human.

[0182] The methods for preventing, treating and / or improving diseases provided by the present invention depend on a variety of factors when applied, including the specific active ingredient of the pharmaceutical composition applied, the patient's age, weight, sex or physical and medical condition, the severity of the disease to be treated, the route of administration, etc.

[0183] The method provided by this invention can also be used in combination with other drugs or means. These other drugs or means refer to those that can be administered in combination with the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt, or a combination thereof, as described in this invention. Examples include small molecule drugs, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutics, and radiotherapy. The combined administration of these two methods can be carried out in any form, such as simultaneously, continuously, or at intervals.

[0184] Eleventh aspect

[0185] The present invention also provides the use of the antibody against CDH17 or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition in the preparation of reagents for diagnosing diseases that may be associated with CDH17 expression (including overexpression), such as CDH17-positive solid tumors.

[0186] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0187] Twelfth aspect

[0188] The present invention also provides a method for diagnosing a disease, the method comprising contacting an antibody against CDH17 of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a carrier, a host cell, an antibody-drug conjugate or a salt thereof or a combination thereof with a sample from a subject, wherein the disease or condition may be associated with CDH17 expression (including overexpression), such as a CDH17-positive solid tumor.

[0189] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer. The subject may be a mammal, preferably a primate or rodent, such as a human.

[0190] Thirteenth aspect

[0191] This invention provides a kit comprising an anti-CDH17 antibody of the present invention or its antigen-binding fragment, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or its salt or a combination thereof. The kit can be used for the aforementioned prevention, treatment and / or improvement, or for the aforementioned diagnosis. Depending on the intended application, the kit may also contain other reagents. For example, the kit is for detecting CDH17 expression (including overexpression) in any biological sample using an ELISA.

[0192] This invention provides a novel anti-CDH17 antibody. Experiments have demonstrated that the anti-CDH17 antibody provided by this invention does not specifically bind to CDH17-negative cells, but it can specifically bind to the CDH17 protein, such as recombinant cells overexpressing CDH17 protein or tumor cells endogenously expressing CDH17, thereby exhibiting highly specific tumor-targeting ability. Regarding antibody-mediated tumor-killing activity, the anti-CDH17 antibody provided by this invention has a relatively strong cell-killing effect. Furthermore, the anti-CDH17 antibody provided by this invention can be effectively internalized on CDH17-expressing tumor cells.

[0193] Furthermore, this invention provides a constructed anti-CDH17 dual-epitope antibody with CDH17 dual-epitope binding activity. Experiments have demonstrated that the anti-CDH17 dual-epitope antibody provided by this invention retains its highly efficient internalization activity on CDH17-expressing tumor cells; and, after further conjugation with a small molecule toxic compound, the resulting antibody-drug conjugate also maintains its internalization activity and strong binding ability to the target protein, and exhibits high killing activity against CDH17-expressing tumor cells. In particular, compared to antibodies binding to a single CDH17 epitope, the dual-epitope antibody provided by this invention has a higher binding ability to the target protein CDH17 and exhibits higher killing activity against CDH17-expressing tumor cells. Therefore, the dual-epitope antibody provided by this invention has more precise and multi-dimensional antigen recognition capabilities and higher targeting specificity, resulting in a stronger therapeutic effect against CDH17-related diseases.

[0194] Therefore, the anti-CDH17 antibody provided by this invention has significant application potential in tumor targeted therapy and ADC drug development. In particular, with the continuous maturation of dual epitope antibodies and ADC technology, the anti-CDH17 dual epitope ADC of this invention will become an important weapon in the field of digestive system tumor treatment, promoting the development of precision oncology. Attached Figure Description

[0195] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0196] Figure 1The results of the binding detection of the anti-CDH17 antibody provided by the present invention with recombinant human and monkey CDH17 overexpressing cells are shown; wherein, A and C: recombinant human CDH17 overexpressing cells, and B and D: recombinant monkey CDH17 overexpressing cells.

[0197] Figure 2 The results of the binding detection of the anti-CDH17 antibody provided by the present invention with CDH17-overexpressing tumor cells and CDH17-negative tumor cells are shown; wherein, A: gastric cancer cells AGS, B: pancreatic cancer cells Aspc-1, C: colorectal cancer cells SK-CO-1, and D: CDH17-negative cells.

[0198] Figure 3 The results of internalization detection of the anti-CDH17 antibody provided by the present invention on CDH17-overexpressing tumor cells are shown; wherein, A, B and D: gastric cancer cells AGS, and C: pancreatic cancer cells Aspc-1.

[0199] Figure 4 The results of the detection of the killing effect of the anti-CDH17 antibody provided by the present invention on CDH17-overexpressing tumor cells are shown.

[0200] Figure 5 The results of antibody binding detection with different domains of human CDH17 are shown.

[0201] Figure 6 A schematic diagram of the structure of the NC hIgG1-Ab2 scFv antibody is shown.

[0202] Figure 7 The binding detection results of NC hIgG1-Ab2 scFv antibodies with different construction forms to recombinant human (AD) and monkey (EH) CDH17 overexpressing cells are shown.

[0203] Figure 8 The results of the binding assays of NC hIgG1-Ab2 scFv antibodies with different configurations to SK-CO-1 in tumor-overexpressing CDH17 cells are shown.

[0204] Figure 9 A schematic diagram of the structure of the Ab1-Ab2 HLscFv antibody is shown.

[0205] Figure 10 A schematic diagram of the structure of the CrossMab antibody is shown.

[0206] Figure 11 , Figure 12 , Figure 13 The results of the binding detection of the dual epitope antibody to recombinant human and monkey CDH17-expressing cells are shown respectively.

[0207] Figure 14 , Figure 15 The results of the detection of the binding of the biepisode antibody to tumor cells are shown.

[0208] Figure 16 The results of the internalization detection of the dual epitope antibody on tumor cells are shown.

[0209] Figure 17 The results of the killing effect of the dual epitope antibody on tumor cells are shown.

[0210] Figure 18 The results of the detection of binding between the biepisode antibody-drug conjugate and tumor cells are shown.

[0211] Figure 19 The results of internalization assays of the biepisotope antibody-drug conjugate on tumor cells are shown.

[0212] Figure 20 The results of the killing effect of the biepisode antibody-drug conjugate on tumor cells are shown. Detailed Implementation

[0213] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0214] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0215] The hIgG1 or NC hIgG1 used in the examples are irrelevant antibodies that do not bind to CDH17.

[0216] Example 1 Screening of anti-CDH17 antibodies

[0217] (I) Animal Immunization and Hybridoma Screening

[0218] Female Balb / c mice aged 6-8 weeks were immunized with recombinant human CDH17 EC1-7 his protein (Acro Biosystems, CA7-H52H3) to produce specific anti-CDH17 antibodies. The immunization regimen consisted of an initial subcutaneous injection of Freund's complete adjuvant (CFA) emulsified antigen (50 μg / mouse); followed by 2-3 booster immunizations using Freund's incomplete adjuvant (IFA) emulsified antigen (25 μg / mouse). Immunizations were spaced 2-3 weeks apart, and serum antigen-binding activity was measured in mice one week after the second immunization. Mice exhibiting high antigen-binding activity were selected using ELISA or other methods.

[0219] After selecting suitable mice, they underwent further booster immunization, and then spleen cells from these mice were electrofused with mouse myeloma cells SP20. Following fusion, the cells were cultured in HAT and HT selective media for 10-14 days, and the supernatant of hybridoma cells producing specific anti-CDH17 antibodies was selected. Binding screening was performed at the protein and cellular levels using ELISA and flow cytometry (FACS), respectively.

[0220] In protein-level screening, 96-well plates (100 μl / well) were coated with 1 μg / ml of the recombinant human CDH17 EC1-7 his protein and incubated overnight at 4°C. After incubation, the plates were washed and blocked, then hybridoma supernatant (100 μl / well) was added, and incubation continued. Finally, the binding activity of the antibody to CDH17 protein in the supernatant was detected. In cell-level screening, the CHOK1 cell line overexpressing recombinant human CDH17 protein (UniProt KB accession number: Q12864) was used for initial screening. Subsequently, to further verify the binding specificity and cross-species binding ability of candidate molecules, recombinant human CDH17, monkey CDH17 (UniProt KB accession number: #A0A1D5R2B4) overexpressing cells, and tumor cells endogenously expressing CDH17 were used as screening targets to ensure the targeting, selectivity, and broad applicability of the obtained molecules.

[0221] Finally, clones that showed positive FACS results and high binding activity were selected for subclonal culture. The VH / VL sequences of these clones were extracted to further screen for high-affinity clones for scale-up culture.

[0222] (II) Extraction of candidate cloned genes, construction of chimeric expression vectors, expression and purification

[0223] Total RNA was extracted from monoclonal hybridoma cells using Trizol reagent, and then the RNA was reverse transcribed into cDNA. The variable regions (VH / VL) of the heavy and light chains were amplified by PCR, sequenced, and compared with the IMGT database to extract the sequence information of CDR1, CDR2, and CDR3, ultimately determining the effective antibody VH / VL sequences.

[0224] The final selected VH / VL sequences were cloned into the pTT5 expression vector (containing the hIgG1 heavy chain and kappa light chain constant regions) to construct heavy and light chain plasmids. The heavy and light chain plasmids were co-transfected into HEK293 cells at a 2:3 ratio using PEI transfection. After transfection, cells were cultured under suitable conditions for 5-7 days, and the culture supernatant was collected. The culture supernatant was filtered through a 0.22 μm filter, purified by affinity chromatography, and then filtered again through a 0.22 μm filter. The antibody concentration was then measured.

[0225] The heavy and light chain variable region sequences of different antibodies are shown in Table 1 (the bold and underlined parts are CDRs, which are divided according to the CCG definition).

[0226] Table 1. Anti-CDH17 antibodies

[0227]

[0228]

[0229]

[0230]

[0231] Example 2 Antibody activity identification of anti-CDH17 antibody

[0232] 1) Specific binding of anti-CDH17 antibody to CDH17 overexpressing cells

[0233] Cellular-level antibody binding was detected using CHOK1 cells (Genomeditech, GM-C25980) overexpressing human CDH17, HEK293 cells (Genomeditech, GM-C28746) overexpressing macaque CDH17, AGS gastric cancer cells, Aspc-1 pancreatic cancer cells, SK-CO-1 colorectal cancer cells, and RKO cells that were negative for CDH17.

[0234] CDH17 expression cells in good growth condition were collected, seeded into 96-well plates, and incubated with serially diluted antibodies at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with PBS, then a 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed three more times and resuspended in PBS. Finally, flow cytometry readings were performed using iQue Screener PLUS, and the data were analyzed using GraphPad Prism. The control antibody was PTA001-A4 (patent US20160039933A1), which was constructed and expressed according to the corresponding VH / VL sequence in the patent.

[0235] like Figure 1 and Figure 2 As shown, the results indicate that the antibodies provided by this invention can specifically bind to CDH17 overexpressing cells, but not to CDH17 negative expression cells (RKO).

[0236] 2) Internalization of anti-CDH17 antibody on CDH17-overexpressing tumor cells

[0237] The internalization effect of antibodies was assessed using a commercially available antibody internalization kit (Sartorius, 90565).

[0238] First, the concentrations of both the test antibody and the internalization reagent were adjusted to 100 μg / ml. Then, a mixture of 1 μl of test antibody, 1 μl of internalization reagent, and 48 μl of diluent was prepared and incubated at 37°C for 15 minutes. After incubation, the mixture was serially diluted twofold. Separately, the cell density of CDH17-expressing tumor cells was adjusted to 2E6 / ml, and 20 μl / well was seeded into 96-well plates, along with the diluted antibody-internalization reagent mixture (20 μl / well). The 96-well plates were then incubated at 37°C with 5% CO2 for 24 hours. Cells were collected the following day, and flow cytometry analysis was performed using iQue Screener PLUS, with data analysis conducted using GraphPadPrism.

[0239] like Figure 3 As shown, the results indicate that the antibodies provided by this invention can all be effectively internalized.

[0240] 3) Killing of CDH17-overexpressing tumor cells by anti-CDH17 antibodies

[0241] The cytotoxic activity of the antibody was assessed using the commercially available reagent αHFc-CL-MMAE (Moradec, AH-102AE-50).

[0242] First, adjust the concentration of CDH17-expressing cells (e.g., SK-CO-1) to 1E5 cells / ml, then seed 50 μl / well into 96-well blank plates and incubate for 4 hours. Next, add serially diluted antibody, 10 μl per well; simultaneously add 40 μl of diluted αHFc-CL-MMAE (2.5 μg / ml). Then, incubate the 96-well blank plates at 37°C and 5% CO2 for 4 days. After incubation, add 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) to each well, and then shake the plate on a shaker for 5 minutes. Detect relative fluorescence units (RLU) using a Spectra M5e instrument. Kill percentage % = (RLU) 未处理细胞 -RLU Sample ) / RLU 未处理细胞 ×100%.

[0243] like Figure 4As shown in Table 2, the results indicate that the antibodies provided by this invention exhibit good killing activity on tumor cells, and are superior to the control antibodies.

[0244] Table 2. Results of antibody cytotoxic activity assay

[0245]

[0246] Example 3 Binding of anti-CDH17 antibodies to different domains of CDH17

[0247] The binding characteristics of the antibody to different domains of the human CDH17 protein were evaluated using an ELISA binding assay.

[0248] First, recombinant proteins of different domains of human CDH17, namely CDH17 EC1-2 his (Kactusbio, CDH-HM1D5), CDH17 EC3-4 mFc (Kactusbio, CDH-HM3D3), CDH17 EC5-7his (Kactusbio, CDH-HM1D4), CDH17 EC1-6 his (Kactusbio, CDH-HM1D1), and CDH17 EC1-7 his (Acro Biosystems, CA7-H52H3), were coated into 96-well plates at a concentration of 1 μg / ml, 100 μL per well, and incubated overnight at 4°C. The next day, the 96-well plates were washed three times with 1×PBST, then blocked by adding 1×PBST containing 1% BSA and incubating at 37°C for 1 hour, followed by washing three times with 1×PBST. Next, serially diluted test antibodies of different concentrations were added and incubated at 37°C for 1 hour. Then, a 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson Immuno Research, 109-035-098) was added and incubated at 37°C for 1 hour. The 96-well plate was then washed with 1×PBST, followed by the addition of TMB substrate for color development, and finally the reaction was terminated with 1N HCl. OD values ​​were measured at 450 nm to quantitatively assess the binding of the antibody to different domains of the human CDH17 protein.

[0249] like Figure 5 As shown, the detection results indicate that the antibodies provided by this invention specifically bind to human CDH17EC1-7 his protein; furthermore, antibodies 1D12H10, 1F3C8, and 5B3A6 bind to the CDH17 EC1-2 domain protein, antibody 47E1G1 binds to the CDH17 EC3-4 domain protein, while antibodies 8E8D4, 37G1F1, 66G9D2, and 82G3C12 can bind to the CDH17EC5-6 domain protein.

[0250] Example 4 Construction and purification of tetravalent biepisotope antibodies

[0251] Using anti-CDH17 antibodies that bind to different epitopes on CDH17, especially antibodies that bind to EC1-2 and EC5-6, and EC3-4 and EC5-6 respectively, a symmetrical Full IgG1 Ab1-Ab2 scFv tetravalent biepisode antibody was constructed.

[0252] (I) Construction and purification of NC hIgG1-Ab2 scFv antibody

[0253] A schematic diagram of the structure of the NC hIgG1-Ab2 scFv antibody is shown below. Figure 6 .exist Figure 6 In the two structures shown, the N-terminus of the antibody is NC hIgG1, and the C-terminus (Ab2) is a scFv composed of the variable region of the anti-CDH17 antibody. Figure 6 A and B in the figure show two construction forms with the N-terminal to C-terminal domains arranged as “VH-VL” (HL scFv) and “VL-VH” (LH scFv), respectively.

[0254] The C-terminal (Ab2) heavy and light chain variable region scFv includes VH-VL (HL scFv) and VL-VH (LH scFv) combinations from antibody molecules 8E8D4, 37G1F1, 66G9D2, and 82G3C12. For each combination, the 44th amino acid of VH in antibody Ab2 was mutated from G, R, or other to C, and the 100th amino acid of VL was mutated from G, A, or other to C. This amino acid position is encoded according to Kabat. The purpose is to form a disulfide bond between the scFv linked to the C-terminus of the NC hIgG1 heavy chain and the scFv linked to the C-terminus of the NC hIgG1 light chain, thereby stabilizing the molecular structure. Using the AscI seamless cloning technique, different HL scFv or LH scFv sequences were inserted into the C-terminus of the PTT5-NC hIgG1 HC sequence to construct heavy chain expression plasmids containing the corresponding scFv sequences.

[0255] Heavy chain plasmid and light chain plasmid (containing the PTT5-NC hIgG1 LC sequence) were co-transfected into HEK293 cells using the PEI transfection method. On day 7 post-transfection, cell supernatant was collected by centrifugation and filtered through a 0.22 μm filter to remove cell debris. The AKTAxpress system and HiTrap were used for cell supernatant transfection. TM MabSelect TM The cell supernatant was purified using a SuRe column (GE, Cat#11003493), filtered, quantified, and then used for later use.

[0256] The scFv sequences of two construction forms used to assemble the NC hIgG1-Ab2 scFv antibody heavy chain are shown in Table 3; the N-terminus of the scFv sequence is linked to the C-terminus of the NC hIgG1 HC sequence via a linker sequence (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO.89) to form the heavy chain of the NC hIgG1-Ab2 scFv antibody.

[0257] Table 3. scFv sequences for two construction methods used to assemble the NC hIgG1-Ab2 scFv antibody heavy chain (bold parts are linker sequences).

[0258]

[0259]

[0260] The heavy and light chains of different NC hIgG1-Ab2 scFv antibodies are shown in Table 4.

[0261] Table 4. NC hIgG1-Ab2 scFv Antibody

[0262]

[0263] (II) Specific binding of NC hIgG1-Ab2 scFv antibodies in different construction forms

[0264] Cellular-level binding of antibodies was detected using CHOK1 cells (Genomeditech, GM-C25980) overexpressing CDH17, HEK293 cells (Genomeditech, GM-C28746) overexpressing CDH17 from rhesus monkeys, and SK-CO-1 colorectal cancer cells.

[0265] The CDH17-expressing cells were seeded into 96-well plates and incubated with serially diluted test antibodies at 4°C for 1 hour. The 96-well plates were then centrifuged and washed three times with PBS. A 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added, and the plates were incubated at 4°C in the dark for 30 minutes. The cells were then washed three times with PBS and resuspended. Flow cytometry analysis was performed using iQue Screener PLUS, and data analysis was performed using GraphPad Prism.

[0266] like Figure 7 and Figure 8As shown, the results indicate that both NC hIgG1-Ab2 HLscFv and NC hIgG1-Ab2 LHscFv antibodies can specifically bind to human CDH17-overexpressing cells, rhesus monkey CDH17-overexpressing cells, and SK-CO-1 tumor cells. However, the binding strength of NC hIgG1-Ab2 HLscFv antibody is comparable to or similar to that of its corresponding monoclonal antibody, while the binding strength of NC hIgG1-Ab2 LHscFv antibody is relatively weak. Therefore, the NC hIgG1-Ab2 HLscFv form was chosen for further evaluation and construction.

[0267] (III) Construction of the Ab1-Ab2 HLscFv tetravalent biepisode antibody

[0268] The VH and VL values ​​of the N-terminal NC hIgG1 portion of the NC hIgG1-Ab2 HLscFv antibody were replaced with the VH and VL values ​​of the anti-CDH17 antibody molecule to construct the Ab1-Ab2 HLscFv tetravalent biepisode antibody. A schematic diagram of the antibody structure is shown below. Figure 9 .

[0269] Using the same construction method as described above, the HL scFv coding sequence of the selected Ab2 antibody molecule was inserted into the C-terminus of the HC sequence in the heavy chain expression vector (PTT5-Ab1, see the antibody heavy chain expression vector in Example 1); wherein, the Ab1 molecule is EC1-2 binding antibody 1D12H10 or 5B3A6, EC3-4 binding antibody 47E1G1, or EC5-6 binding antibody 37G1F1. The Ab2 antibody molecule is EC3-4 binding antibody 47E1G1 or EC5-6 binding antibody 8E8D4, 37G1F1. A heavy chain expression plasmid containing the corresponding HL scFv sequence, namely Ab1-Ab2 HL scFv plasmid, was constructed. In this plasmid, following the construction method of NChIgG1-Ab2 HLscFv antibody mentioned above, the amino acids at position 44 of VH and position 100 of VL of Ab2 (amino acid positions according to Kabat encoding) were mutated to cysteine, thereby forming a disulfide bond between the scFv of the heavy chain and the light chain to stabilize the structure of the antibody molecule.

[0270] Heavy chain plasmid and light chain plasmid (see antibody light chain expression vector in Example 1) were co-transfected into HEK293 cells using the PEI transfection method. Following the above description, multiple Ab1-Ab2 HLscFv antibodies were finally obtained after expression and purification.

[0271] The heavy chain and light chain sequences used to assemble the Ab1-Ab2 HLscFv tetravalent biepitaxy antibody are shown in Table 5.

[0272] Table 5. Sequences used for assembling the Ab1-Ab2 HLscFv tetravalent biepisode antibody (bold parts are linker sequences)

[0273]

[0274]

[0275]

[0276]

[0277]

[0278] The heavy and light chains of different Ab1-Ab2 HLscFv tetravalent biepisode antibodies are shown in Table 6.

[0279] Table 6. Ab1-Ab2 HLscFv tetravalent biepisode antibodies

[0280]

[0281]

[0282] Example 5 Construction and purification of CrossMab bivalent biepitaxy antibody

[0283] Using the CrossMab method, antibody A was kept unchanged while the CH1 and CL domains of antibody B were interchanged to construct CrossMab antibodies binding to different epitopes on CDH17. Antibody molecules binding to the EC1-2 domain of CDH17 were selected as antibody A, and antibody molecules binding to the EC3-4 or EC5-6 domain of CDH17 were selected as antibody B. The structural diagrams are shown below. Figure 10 .

[0284] As described above, through expression and purification, multiple CrossMab bivalent biepithelial antibodies capable of binding to two epitopes simultaneously were finally obtained.

[0285] The heavy chain and light chain sequences used to assemble the CrossMab bivalent biepitaxy antibody are shown in Table 7.

[0286] Table 7. Sequences used for assembling CrossMab bivalent biepitope antibodies

[0287]

[0288]

[0289] The heavy and light chains of different CrossMab bivalent biepithelial antibodies are shown in Table 8.

[0290] Table 8. CrossMab bivalent biepisotope antibodies

[0291]

[0292] Example 6 Identification of biepisode antibodies

[0293] The activity of the Ab1-Ab2 HLscFv tetravalent biepisode antibody obtained in Example 4 and the Crossmab bivalent biepisode antibody obtained in Example 5 was evaluated.

[0294] (I) Binding of biepisotope antibodies to recombinant human and monkey CDH17-expressing cells

[0295] Referring to the method described in Example 4, “(II) Specific binding of NC hIgG1-Ab2 scFv antibodies with different construction forms”, different cells were incubated with serially diluted antibodies. After incubation with secondary antibodies and washing, cell flow cytometry analysis was performed using iQueScreener PLUS, and data analysis was performed using GraphPad Prism.

[0296] like Figure 11 , Figure 12 and Figure 13 As shown, the results indicate that the dual epitope antibodies provided by this invention can specifically bind to recombinant human and monkey CDH17-expressing cells.

[0297] (II) Binding of biepisotope antibodies to CDH17-overexpressing tumor cells

[0298] Referring to the method described in Example 2, "(II) Specific binding of NC hIgG1-Ab2 scFv antibodies with different construction forms", SK-CO-1 and NCI-H508 cells were incubated with serially diluted antibodies. After incubation with secondary antibodies and washing, cell flow cytometry analysis was performed using iQue Screener PLUS, and the data were analyzed using GraphPad Prism.

[0299] like Figure 14 and Figure 15 As shown, the results indicate that the dual epitope antibody of the present invention specifically binds to tumor cells overexpressing CDH17, and is superior to monoclonal antibodies.

[0300] (III) Internalization of biepisotope antibodies on CDH17-overexpressing tumor cells

[0301] The internalization effect of antibodies was assessed using a commercially available antibody internalization kit (Sartorius, 90565).

[0302] First, the concentrations of both the antibody and the internalization reagent were adjusted to 100 μg / mL. Then, a mixture of 1 μl of antibody, 1 μl of internalization reagent, and 48 μl of diluent was prepared and incubated at 37°C for 15 minutes. After incubation, the mixture was serially diluted twofold. Next, the density of CDH17-expressing tumor cells was adjusted to 2E6 / ml, and 20 μl of the diluted antibody-internalization reagent mixture was seeded into 96-well plates. The diluted antibody-internalization reagent mixture (20 μl / well) was then added. The 96-well plates were incubated at 37°C and 5% CO2 for 24 hours. Cells were collected the following day, and flow cytometry analysis was performed using iQue Screener PLUS. Data analysis was then performed using GraphPadPrism.

[0303] like Figure 16 As shown, the results indicate that all detected biepisode antibodies can be effectively internalized, and are superior to monoclonal antibodies.

[0304] (iv) Killing of CDH17-overexpressing tumor cells by biepisode antibodies

[0305] The cytotoxic activity of the antibody was assessed using the commercially available reagent αHFc-CL-MMAE (Moradec, AH-102AE-50).

[0306] First, the concentration of CDH17-expressing cells (such as NCI-H508 or SK-CO-1) was adjusted to 1E5 cells / ml, and then seeded into 96-well blank plates at a rate of 50 μl / well, and incubated for 4 hours. Next, serially diluted antibody was added at 10 μl per well; simultaneously, 40 μl of diluted αHFc-CL-MMAE (2.5 μg / ml) was added per well. The 96-well blank plates were then incubated at 37°C and 5% CO2 for 4 days. After incubation, 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) was added to each well, and the plate was shaken on a shaker for 5 minutes. The relative fluorescence unit (RLU) was detected using a Spectra M5e instrument.

[0307] Damage percentage % = (RLU) 未处理细胞 -RLU Sample ) / RLU 未处理细胞 *100%.

[0308] like Figure 17 As shown in Tables 9-1, 9-2, and 9-3, the results indicate that the tested biepisode antibodies all exhibited good killing activity on tumor cells, and were superior to monoclonal antibodies 8E8D4 and 37G1F1, as well as the corresponding control antibodies of the same conformation.

[0309] Table 9-1 Detection of the killing effect of biepisode antibodies on NCI-H508 tumor cells

[0310]

[0311] Table 9-2 Detection of the killing effect of biepisode antibodies on SK-CO-1 tumor cells

[0312]

[0313] Table 9-3 Detection of the killing effect of biepisode antibodies on SK-CO-1 tumor cells

[0314]

[0315] Example 7 Preparation of biepisode antibody-drug conjugates

[0316] (I) Preparation of ADC

[0317] The BL20E (Linker+MMAE payload) was synthesized according to the method of patent CN110088086B.

[0318]

[0319] To further evaluate the activity of the anti-CDH17 biepisode antibody, the biepisode antibody was conjugated with compound BL20E to prepare an ADC targeting CDH17, following the method described in patent application publication WO2022228563A1. A specific exemplary method is as follows:

[0320] Sample Reduction and Coupling: The antibody sample was replaced with a Sephadex G-25 carrier-based NAP-25 desalting column to a pH 7.4 buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, and the antibody concentration was diluted to 10 mg / ml. 10 ml of a total of 100 mg of antibody sample was taken, and 10 mg / ml of TCEP (Sigma-Aldrich) aqueous solution was added at an antibody-reducing agent molar ratio of 1:10. After incubation for 2 hours, the reaction solution was replaced with a Sephadex G25 desalting column to a pH 7.0 buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate.

[0321] The reduced antibody was diluted to 5 mg / mL, and 1.33 mL of N,N-dimethylacetamide (DMA) (7.4% of the total reaction volume) was added as a presolvent. A DMA-drug linker mixture containing 10 mg / mL of drug linker was added at an antibody-small molecule drug molar ratio of 1:5.5. The mixture was stirred at room temperature for 60 minutes. The reaction solution was then replaced with a pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer using a Sephadex G-25 carrier NAP-25 desalting column to remove excess drug linker. The mixture was then heated in a 37°C water bath for 3 hours. Mass spectrometry analysis of the conjugate structures was performed before and after water bath heating. The analysis results of different conjugates all showed that the conjugate before water bath heating was the structure of formula Ia, and after water bath heating, the completely ring-opening structure of formula Ib was obtained, i.e., 100% hydrolysis.

[0322] Sample purification: The above sample was concentrated using AMICOM ultrafiltration centrifuge tubes to approximately 15 mg / mL. A 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution was added until the conductivity reached 74 mS / cm. The sample was loaded onto a Butyl-Sepharose 4FF hydrophobic column (purchased from GE Healthcare), using phase A (50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.45 M ammonium sulfate) and phase B (50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution). Elution was performed using a 0-80% linear gradient of 12 column volumes followed by a 100% isocratic gradient in phase B, and the main peak was collected.

[0323] The final sample was transferred to 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using AMICOM ultrafiltration centrifuge tubes and filtered through a 0.22 μm filter membrane (Sartorius stedim Ministart). The antibody-drug conjugate was then purified.

[0324] (II) Characterization of the physicochemical properties of ADC drugs

[0325] a. Determination of drug-antibody conjugation ratio by ultraviolet spectrophotometry (UV-DAR method)

[0326] According to the literature [Clin Cancer Res. 2004 Oct 15; 10(20):7063-70], DAR is calculated according to the formula:

[0327] DAR=(εAb 280-A280 / Az×εAb Z) / (A280 / Az×εD Z-εD 280),

[0328] Wherein, εAb 280 is the molar absorption coefficient of the antibody at 280 nm, A280 is the ultraviolet absorbance of the antibody-drug conjugate at 280 nm, Az is the ultraviolet absorbance of the antibody-drug conjugate at the characteristic absorption wavelength Z nm of the drug-containing linker, εAb Z is the molar absorption coefficient of the antibody at the characteristic absorption wavelength Z nm of the drug-containing linker, εD Z is the molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm, and εD280 is the molar absorption coefficient of the drug-containing linker at 280 nm.

[0329] b. Size exclusion high-performance liquid chromatography (SE-HPLC) for the analysis of molecular size heterogeneity

[0330] Perform SE-HPLC with the following parameters:

[0331] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5μm, 7.8mm*300mm;

[0332] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0333] Flow rate: 0.6 mL / min;

[0334] Detection wavelength: 280nm;

[0335] Column temperature: 30℃;

[0336] Sample loading volume: 20 μL;

[0337] Washing time: 20 min;

[0338] Elution gradient: isocratic elution.

[0339] The drug-antibody conjugate ratio (DAR) was determined by ultraviolet spectrophotometry in the characterization section of the physicochemical properties of ADC drugs in this application, and molecular size heterogeneity was analyzed by size exclusion high performance liquid chromatography (SEC-HPLC).

[0340] The results are shown in Table 10.

[0341] Table 10. Antibody-Drug Conjugates

[0342]

[0343]

[0344] Example 6 Activity assessment of biepisode antibody-drug conjugates

[0345] (I) Evaluation of the binding activity of the biepisode antibody BL20E ADC

[0346] Referring to the method described in Example 2 (II) "1. Specific binding of NC hIgG1-Ab2 scFv antibodies with different construction forms", colorectal cancer cells SK-CO-1, NCI-H508 and CL40 were incubated with serially diluted antibodies. After incubation with secondary antibodies and washing, cell flow cytometry analysis was performed using iQue Screener PLUS, and the data were analyzed using GraphPad Prism.

[0347] like Figure 18 As shown in Tables 11-1 and 11-2, the results indicate that the dual epitope antibody-drug conjugates can specifically bind to tumor cells with different CDH17 expression levels.

[0348] Table 11-1 Results of binding detection of Ab1-Ab2 HLscFv biepisotope antibody-drug conjugates to tumor cells

[0349]

[0350] Table 11-2 Results of binding detection of CrossMab biepitope antibody-drug conjugate to tumor cells

[0351]

[0352] (II) Internalization of biepisotope antibody-drug conjugates

[0353] The internalization of the biepisode antibody-drug conjugate was evaluated using the method described in Example 4 (III).

[0354] like Figure 19 As shown, the results indicate that both biepisode antibody-drug conjugates can be effectively internalized, and their internalization activity is superior to that of monoclonal antibody-drug conjugates.

[0355] (III) Killing of CDH17-overexpressing tumor cells by biepisotope antibody-drug conjugates

[0356] CDH17-overexpressing colorectal cancer cells SK-CO-1, HT55, CL40, and NCI-H508 were seeded into 96-well white plates. Serially diluted ADC was added, and the plates were incubated at 37°C in CO2 for 7 days. After incubation, 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) was added to each well, and the plates were shaken for 5 minutes. Relative fluorescence units (RLUs) were measured using a Spectra M5e instrument, and the data were analyzed using a GraphPad Prism.

[0357] like Figure 20As shown in Tables 12-1 and 12-2, the results indicate that the dual epitope antibody-drug conjugates exhibited effective killing activity on different CDH17-overexpressing tumor cells, and 5B3A6-8E8D4 HLscFv BL20E showed superior activity to 5B3A6 BL20E on all three tumor cell types; 1F3C8(Knob)-8E8D4(CrHole)BL20E showed superior activity to monoclonal antibody 8E8D4 ADC on SK-CO-1 cells.

[0358] Table 12-1 Results of tumor cell killing assay using HLscFv biepisode antibody-drug conjugates Ab1-Ab2

[0359]

[0360] Table 12-2 Results of the killing effect of CrossMab biepitope antibody-drug conjugate on tumor cells

[0361]

[0362] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An antibody against cadherin-17 (CDH17) or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising heavy chain complementarity-determining regions (CDRs), namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and light chain complementarity-determining regions (CDRs), namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), wherein the heavy chain CDRs and light chain CDRs are as follows: (1) Containing, in sequence, the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69; and, in sequence, the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72; or (2) Contains, in sequence, amino acid sequences such as SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74, namely HCDR1, HCDR2, and HCDR3; and contains, in sequence, amino acid sequences such as SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, namely LCDR1, LCDR2, and LCDR3.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-CDH17 antibody or its antigen-binding fragment specifically binds to human or monkey CDH17.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-CDH17 antibody or its antigen-binding fragment has species-cross-binding activity against human or monkey CDH17.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-CDH17 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, as shown below: (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 10; or (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO. 11, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 12, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.

12.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The anti-CDH17 antibody is a mouse anti-antibody, a chimeric antibody, or a partially humanized antibody. Alternatively, the antigen-binding fragment of the anti-CDH17 antibody may be a fragment of the antibody's scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region (CH) and / or a light chain constant region (CL).

7. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further comprises a human or mouse heavy chain constant region and / or a light chain constant region.

8. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The anti-CDH17 antibody is a monoclonal antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The anti-CDH17 antibody is an immunoglobulin.

11. The antibody or its antigen-binding fragment according to claim 10, characterized in that, The immunoglobulin type is human IgA, IgD, IgE, IgG, or IgM.

12. The antibody or its antigen-binding fragment according to claim 10, characterized in that, The antibody is a human IgG1 or IgG4 subtype.

13. A dual epitope antibody against cadherin-17 (CDH17) or an antigen-binding fragment thereof, said dual epitope antibody or antigen-binding fragment thereof comprising at least a first binding domain and a second binding domain, wherein either the first binding domain or the second binding domain comprises: (1) Containing, in sequence, the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69; and, in sequence, the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72; or (2) Containing, in sequence, amino acid sequences as shown in SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74, namely HCDR1, HCDR2, and HCDR3; and containing, in sequence, amino acid sequences as shown in SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, namely LCDR1, LCDR2, and LCDR3; Furthermore, the first binding domain and the second binding domain bind to different epitopes located in different exoregions of CDH17.

14. The dual epitope antibody or its antigen-binding fragment according to claim 13, characterized in that: (1) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 43, SEQ ID NO. 44, and SEQ ID NO. 45 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 46, SEQ ID NO. 47, and SEQ ID NO. 48 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69, respectively; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72, respectively. (2) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 43, SEQ ID NO. 44, and SEQ ID NO. 45 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 46, SEQ ID NO. 47, and SEQ ID NO. 48 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74, respectively; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, respectively. (3) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 55, SEQ ID NO. 56, and SEQ ID NO. 57 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 58, SEQ ID NO. 59, and SEQ ID NO. 60 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69, respectively; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72, respectively. (4) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 55, SEQ ID NO. 56, and SEQ ID NO. 57 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 58, SEQ ID NO. 59, and SEQ ID NO. 60 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74, respectively; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, respectively. (5) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 sequentially comprising amino acid sequences as shown in SEQ ID NO. 55, SEQ ID NO. 56, and SEQ ID NO. 57; and LCDR1, LCDR2, and LCDR3 sequentially comprising amino acid sequences as shown in SEQ ID NO. 58, SEQ ID NO. 59, and SEQ ID NO. 60; (6) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 61, SEQ ID NO. 62, and SEQ ID NO. 63 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 64, SEQ ID NO. 65, and SEQ ID NO. 66 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69, respectively; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72, respectively. (7) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 61, SEQ ID NO. 62, and SEQ ID NO. 63 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 64, SEQ ID NO. 65, and SEQ ID NO. 66 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 sequentially comprising amino acid sequences as shown in SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74; and LCDR1, LCDR2, and LCDR3 sequentially comprising amino acid sequences as shown in SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77; or (8) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 49, SEQ ID NO. 50, and SEQ ID NO. 51 in sequence; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 52, SEQ ID NO. 53, and SEQ ID NO. 54 in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69, respectively; and LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72, respectively.

15. The dual epitope antibody or its antigen-binding fragment according to claim 13, characterized in that, The dual epitope antibody or its antigen-binding fragment specifically binds to human or monkey CDH17.

16. The dual epitope antibody or its antigen-binding fragment according to claim 13, characterized in that, The dual epitope antibody or its antigen-binding fragment has species cross-binding activity against human or monkey CDH17.

17. The dual epitope antibody or its antigen-binding fragment according to claim 14, characterized in that, Both the first and second binding structural domains contain heavy chain variable regions and light chain variable regions, and: (1) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 2; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

10. (2) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 2; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12; (3) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 6; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

10. (4) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 6; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12; (5) In the first binding domain, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 6; (6) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 8; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

10. (7) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 8; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 12; or (8) In the first binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 3; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 4; In the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

10.

18. The dual epitope antibody or its antigen-binding fragment according to claim 17, characterized in that, The first or second binding domain further includes a heavy chain constant region (CH) and / or a light chain constant region (CL).

19. The antibody or antigen-binding fragment thereof according to claim 17, characterized in that, The first or second binding domain further includes a heavy chain constant region and / or a light chain constant region of human or mouse.

20. The antibody or antigen-binding fragment thereof according to claim 17, characterized in that, The first or second binding domain further includes a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

21. The biepisode antibody or its antigen-binding fragment according to any one of claims 13 to 20, characterized in that, The first binding domain and the second binding domain are selected from IgG antibody and scFv fragment, respectively.

22. The dual epitope antibody or its antigen-binding fragment according to claim 21, characterized in that, The first binding domain is an IgG antibody, and the second binding domain is an scFv fragment. The scFv fragment contains domains arranged in the order of "VH-Linker-VL" or "VL-Linker-VH" from the amino terminus to the carboxyl terminus, wherein the Linker is a flexible linker containing glycine-serine.

23. The dual epitope antibody or its antigen-binding fragment according to claim 22, characterized in that, The scFv fragment is linked to the carboxyl terminus of the IgG antibody via a flexible linker at its amino terminus.

24. The dual epitope antibody or its antigen-binding fragment according to claim 22, characterized in that, The dual epitope antibody comprises two identical heavy chains and two identical light chains, wherein the heavy chain contains domains arranged in the order "Ab1 VH-CH1-CH2-CH3-Linker-Ab2 VH-Linker-VL" or "Ab1 VH-CH1-CH2-CH3-Linker-Ab2 VL-Linker-VH" from the amino terminus to the carboxyl terminus; and the light chain contains domains arranged in the order "Ab1 VL-CL" from the amino terminus to the carboxyl terminus.

25. The biepisode antibody or its antigen-binding fragment according to any one of claims 22 to 24, characterized in that, The flexible joint is a (GGGGS)n joint, where n is an integer from 2 to 5.

26. The biepisode antibody or its antigen-binding fragment according to any one of claims 13 to 20, characterized in that, The dual epitope antibody or its antigen-binding fragment is itself in the form of an IgG antibody, and the first binding domain and the second binding domain are respectively located in the two halves of the IgG antibody.

27. The dual epitope antibody or its antigen-binding fragment according to claim 26, characterized in that, The first binding domain is located in the first half-antibody and comprises a heavy chain and a light chain. The heavy chain contains domains arranged in the sequence "VH-CH1-CH2-CH3" from the amino terminus to the carboxyl terminus, and the light chain contains domains arranged in the sequence "VL-CL" from the amino terminus to the carboxyl terminus. The second binding domain is located in the second half-antibody and comprises a heavy chain and a light chain. The heavy chain contains domains arranged in the sequence "VH-CL-CH2-CH3" from the amino terminus to the carboxyl terminus, and the light chain contains domains arranged in the sequence "VL-CH1" from the amino terminus to the carboxyl terminus.

28. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 12, or a biepisode antibody or antigen-binding fragment thereof as claimed in any one of claims 13 to 27.

29. A carrier comprising the nucleic acid molecule of claim 28.

30. A host cell comprising the nucleic acid molecule of claim 28 or the vector of claim 29, or transformed or transfected by the nucleic acid molecule of claim 28 or the vector of claim 29.

31. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the biepisode antibody or antigen-binding fragment thereof of any one of claims 13 to 27, the nucleic acid molecule of claim 28, the vector of claim 29, or the host cell of claim 30 in the preparation of antibody-drug conjugates (ADCs).

32. An antibody-drug conjugate or a salt thereof targeting CDH17, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the biepisode antibody or antigen-binding fragment thereof of any one of claims 13 to 27.

33. The antibody-drug conjugate or its salt according to claim 32, characterized in that, The antibody-drug conjugate is formed by conjugating the antibody or its antigen-binding fragment, or the biepisode antibody or its antigen-binding fragment, with a cytotoxic compound.

34. The antibody-drug conjugate or its salt according to claim 33, characterized in that, The cytotoxic compound is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder.

35. A composition comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 12, a biepisode antibody or antigen-binding fragment thereof as claimed in any one of claims 13 to 27, a nucleic acid molecule as claimed in claim 28, a vector as claimed in claim 29, a host cell as claimed in claim 30, or an antibody-drug conjugate or a salt thereof as claimed in any one of claims 32 to 34.

36. The composition according to claim 35, characterized in that, The composition is a pharmaceutical composition and further comprises pharmaceutically acceptable excipients.

37. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the biepisode antibody or antigen-binding fragment thereof of any one of claims 13 to 27, the nucleic acid molecule of claim 28, the vector of claim 29, the host cell of claim 30, the antibody-drug conjugate or salt thereof of any one of claims 32 to 34, or the composition of claim 35 or 36 in the preparation of a medicament for treating a disease, said disease being gastric cancer or colorectal cancer.

38. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, or the biepisode antibody or antigen-binding fragment thereof of any one of claims 13 to 27, in the preparation of a reagent for diagnosing a disease, said disease being gastric cancer or colorectal cancer.

39. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 12, a biepisode antibody or antigen-binding fragment thereof as claimed in any one of claims 13 to 27, a nucleic acid molecule as claimed in claim 28, a vector as claimed in claim 29, a host cell as claimed in claim 30, an antibody-drug conjugate or a salt thereof as claimed in any one of claims 32 to 34, or a composition as claimed in claim 35 or 36.

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