Double-epitope antibody for resisting cadherin-17 and antibody drug conjugate prepared from double-epitope antibody

Abstract

The invention provides an anti-CDH17 antibody or an antigen binding fragment of the anti-CDH17 antibody. The anti-CDH17 antibody can be a double-epitope antibody obtained by construction. The anti-CDH17 antibody provided by the invention can be specifically combined with CDH17 protein, has a relatively strong CDH17 expression tumor cell killing effect, can be effectively internalized on CDH17 expression tumor cells, and can be prepared into ADC (Analog to Digital Converter) which is effectively internalized on CDH17 expression tumor cells and has high killing activity; particularly, compared with an antibody combined with a CDH17 single epitope, the double-epitope antibody disclosed by the invention has more accurate and multi-dimensional antigen recognition capability and higher targeting property.

Description

Technical Field

[0001] The present invention belongs to the field of antibody drugs. Specifically, the present invention relates to an anti-cadherin-17 dual-epitope antibody, an antibody-drug conjugate targeting cadherin-17, and applications thereof. Background Art

[0002] Cadherin-17 (CDH17) is a key member of the cadherin family. It consists of seven extracellular domains (ECs), EC1 to EC7, and a very short cytoplasmic domain. CDH17 is primarily expressed in gastrointestinal epithelial cells and plays a key role in cell-cell adhesion and tissue morphology maintenance.

[0003] Studies have shown that CDH17 is abnormally overexpressed in a variety of tumors, particularly in digestive system malignancies such as gastric cancer, colorectal cancer, and pancreatic cancer. Clinical studies have found that high expression of CDH17 is often closely associated with a tumor's high degree of differentiation, invasiveness, metastasis, and poor prognosis. Its expression levels often increase significantly in the late and metastatic stages of the tumor.

[0004] Biparatopic bispecific antibodies (bpAbs) bind to two distinct, non-overlapping epitopes on an antigen. Combining the expression characteristics of CDH17 with biparatopic antibodies to develop biparatopic antibodies targeting CDH17 and further ADC drugs will provide a novel treatment 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 the present invention is to provide a new type of anti-CDH17 antibody, which should bind to CDH17 protein with high specificity and high affinity, such as CDH17 expressed by tumor cells, thereby having obvious tumor cell killing ability through efficient binding to CDH17 protein expressed by tumors. Furthermore, the present invention characterizes the binding characteristics of different anti-CDH17 antibodies to the target protein CDH17, selects antibodies that bind to different regions or epitopes on the CDH17 protein, and further constructs antibodies with dual or more CDH17 epitope binding activity. Furthermore, the antibodies with dual or more CDH17 epitope binding activity constructed by the present invention should also have a strong ability to internalize into CDH17-expressing cells, thereby being suitable for preparation into antibody-drug conjugates, and thus an antibody-drug conjugate with better therapeutic effect on CDH17-expressing tumor cells can be obtained.

[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 that has 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 solutions of the present invention are as follows.

[0008] First aspect

[0009] The present invention provides an anti-cadherin-17 (CDH17) antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof can specifically bind to CDH17, in particular human CDH17.

[0010] In the context of the present invention, unless otherwise specified, the term "CDH17" encompasses any form or structural region of CDH17.

[0011] In the context of the present 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 antigen-binding ability and corresponding biological activity of an antibody can be achieved by fragments of an 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 intact antibodies. For example, the antigen-binding fragment of the antibody can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of an intact antibody.

[0012] Specifically, the anti-CDH17 antibodies or antigen-binding fragments thereof provided herein comprise heavy chain complementarity determining regions (CDRs), i.e., heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and light chain complementarity determining regions (CDRs), i.e., light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). According to a specific embodiment of the present invention, the anti-CDH17 antibodies or antigen-binding fragments thereof comprise heavy chain CDRs derived from a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, and 15, and a light chain variable region having an amino acid sequence as shown in any one of 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 provided above are amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary anti-CDH17 antibodies provided in the "Detailed Description of the Invention" section of this application. Using any one or a combination of the definition tools for antibody heavy chain or light chain complementary determining regions 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 definition tools known in the art or conventionally used, and antibodies or fragments thereof comprising each of these heavy chain CDRs and light chain CDRs combinations are within the scope of protection of the present invention.

[0014] Preferably, the anti-CDH17 antibody or antigen-binding fragment thereof 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 pairing:

[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 divide the CDRs in the above amino acid sequence pairings, as shown in the examples of the present invention.

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

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

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

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

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

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

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

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

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

[0033] As described above, the anti-CDH17 antibodies or antigen-binding fragments thereof provided herein specifically bind to cadherin-17 (CDH17), preferably primate or rodent CDH17, such as human, monkey, or mouse CDH17. Optionally, the antibodies or antigen-binding fragments thereof provided herein may or may not have species-cross-binding activity to human, monkey (cyno or Rhesus), or mouse CDH17.

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

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

[0036] (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.2;

[0037] (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 3, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 4;

[0038] (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 6;

[0039] (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 8;

[0040] (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 10;

[0041] (6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 11, or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 12, or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 14; or

[0043] (8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.15, or comprises an amino acid sequence that is at least 75% identical to 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 comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.16.

[0044] In the context of the present invention, the term "at least 75% identity" with respect to an amino acid sequence encompasses any percentage of identity between at least 75% identity and 100% identity, for example 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. Up to 25% difference in amino acid sequence resulting from "at least 75% identity" may exist in any framework region of the heavy or light chain variable region, or in any domain or sequence other than the heavy and light chain variable regions of the antibodies or antigen-binding fragments thereof of the invention. The difference may result from deletions, additions, or substitutions of amino acids at any position, wherein the substitutions may be conservative or non-conservative.

[0045] Preferably, the anti-CDH17 antibodies provided by the present invention can be mouse antibodies, rabbit antibodies, human antibodies, or mouse antibodies, chimeric antibodies, or fully or partially humanized antibodies. The CDH17 antibodies can also be derivatized antibodies, for example, antibodies obtained by performing CDRs transplantation, affinity maturation, point mutation modification, chemical modification, etc. on the basis of the initial mouse monoclonal antibody, wherein the chemical modification includes glycosylation, acetylation, pegylation, phosphorylation, amidation, protease cleavage, connection with cellular ligands or effector molecules, protection and / or blocking of active reactive groups, etc. 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), (disulfide-stabilized variable fragment) 2 ((dsFv) 2), an antigen-binding fragment (Fab), a Fab' fragment (Fab'), (Fab' fragment) 2 (F(ab') 2) or a variable fragment (Fv), etc. Regarding the antigen-binding fragment of the antibody of the present invention, it can be any fragment of the antibody that can specifically bind to CDH17.

[0046] In addition to the heavy chain and / or light chain variable regions, the anti-CDH17 antibody or antigen-binding fragment thereof provided herein may further comprise a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or mouse heavy chain constant region and / or a light chain constant region. Preferably, the anti-CDH17 antibody or antigen-binding fragment thereof comprises an IgG, IgA, IgM, IgD, or IgE heavy chain constant region and / or a kappa or lambda 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 murine, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody comprises an IgG1 heavy chain constant region sequence, such as a human IgG1 heavy chain constant region; and / or comprises a kappa light chain constant region, such as a 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 immunoglobulin type is human IgA, IgD, IgE, IgG or IgM. More preferably, the antibody is of the human IgG1 subtype.

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

[0050] Preferably, the present invention provides an anti-cadherin-17 (CDH17) dual-epitope antibody or antigen-binding fragment thereof, the dual-epitope antibody or antigen-binding fragment thereof comprising 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 complementary determining region (CDRs) of the antibody and the light chain complementary determining region (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, the second binding domain binds to a second epitope of CDH17, and the first epitope and the second epitope may be located in the extracellular region 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, and the second binding domain binds to a second epitope located in the extracellular region EC3-4 of CDH17; vice versa.

[0051] With respect to the sequences of the heavy chain and light chain complementary determining regions contained therein, the heavy chain CDRs contained in the first binding domain and the second binding domain of the dual-epitope antibody or antigen-binding fragment provided by the present invention, respectively, can be derived from a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, and 15 and from a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, and 16.

[0052] As described above, the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.16 are amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary anti-CDH17 antibodies provided in the "Detailed Description" section of this application. Using any one or a combination of the definition tools for antibody heavy chain or light chain complementary determining regions 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 definition tools known or conventional in the art, and antibodies or fragments thereof containing each of these heavy chain CDRs and light chain CDRs combinations are within the scope of protection of the present invention.

[0053] Preferably, the anti-CDH17 bi-epitope antibody or antigen-binding fragment thereof 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 extracellular 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] Further preferably, the bi-epitope antibody or antigen-binding fragment thereof provided by the present invention comprises the first binding domain and the 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 heavy chain CDRs comprised by 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 have 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 second binding domain comprises heavy chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.11, and light chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.12; (3)

[0066] The heavy chain CDRs comprised by 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 second binding domain comprises 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; (4)

[0069] The heavy chain CDRs comprised by 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 second binding domain comprises heavy chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.11, and light chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.12; (5)

[0072] The heavy chain CDRs comprised by 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 comprised by 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 second binding domain comprises heavy chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 15, and light chain CDRs derived from the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 16; (7)

[0078] The heavy chain CDRs comprised by 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 second binding domain comprises 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; (8)

[0081] The heavy chain CDRs comprised by 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 comprised by 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 heavy chain CDRs comprised by 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; 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 heavy chain CDRs comprised by the second 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.

[0089] Preferably, the bi-epitope antibody or antigen-binding fragment thereof 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 comprising the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, in sequence;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[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 antigen-binding fragment thereof provided by the present invention may respectively comprise a heavy chain variable region (VH) and a light chain variable region (VL), and similarly, both include the above-mentioned CDRs and the framework region (FR) therebetween, and the arrangement of each region from N-terminus to 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 antigen-binding fragment thereof provided by the present invention both comprise 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 of SEQ ID NO. 11 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 of SEQ ID NO. 15 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 15; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 16 or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 dual-epitope antibody or antigen-binding fragment thereof provided by the present invention, the first binding domain and the second binding domain may comprise a heavy chain constant region (CH) and / or a light chain constant region (CL), and the constant region is as defined above.

[0153] Furthermore, in the anti-CDH17 dual-epitope antibody or antigen-binding fragment thereof provided by the present invention, the first binding domain and the second binding domain can be selected from IgG antibody and scFv fragment forms, respectively. For example, the first binding domain is an IgG antibody, the second binding domain is an scFv fragment, and the two are connected together by a linker that allows the two to bind to different epitopes of CDH17 independently of each other. The linker can be a flexible linker comprising glycine-serine, such as a (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 a scFv fragment, and the scFv fragment comprises domains arranged in the order of "VH-Linker-VL" or "VL-Linker-VH" from the amino terminus to the carboxyl terminus, wherein Linker is a flexible linker as described above. Further, the scFv fragment is connected to the carboxyl terminus of the IgG antibody at its amino terminus through a flexible linker. Exemplarily, the anti-CDH17 dual-epitope antibody may comprise two identical heavy chains and two identical light chains, wherein the heavy chain comprises domains arranged in the order of "Ab1VH-CH1-CH2-CH3-Linker-Ab2 VH-Linker-VL" or "Ab1VH-CH1-CH2-CH3-Linker-Ab2 VL-Linker-VH" from the amino terminus to the carboxyl terminus; the light chain comprises domains arranged in the order of "Ab1 VL-CL" from the amino terminus to the carboxyl terminus, thereby obtaining a tetravalent dual-epitope antibody; see Figure 9 When constructing the tetravalent bi-epitope 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 assembly of the antibody.

[0155] Alternatively, the anti-CDH17 bi-epitope antibody or antigen-binding fragment thereof provided herein is in the form of an IgG antibody, wherein the first binding domain and the second binding domain are respectively located in two half-antibodies of the IgG antibody. For example, the anti-CDH17 bi-epitope antibody may comprise two different heavy chains and two different light chains, wherein one heavy chain and one light chain each constitute the half-antibody.

[0156] According to a specific embodiment of the present invention, the anti-CDH17 dual-epitope antibody provided by the present invention is constructed using Crossmab, and the first binding domain is located in the first half antibody (hereinafter also referred to as the antibody A part), which comprises a heavy chain and a light chain, and the heavy chain comprises domains arranged in the order of "VH-CH1-CH2-CH3" from the amino terminus to the carboxyl terminus, and the light chain comprises domains arranged in the order of "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 the antibody B part), which comprises a heavy chain and a light chain, and the heavy chain comprises domains arranged in the order of "VH-CL-CH2-CH3" from the amino terminus to the carboxyl terminus, and the light chain comprises domains arranged in the order of "VL-CH1" from the amino terminus to the carboxyl terminus, thereby obtaining a bivalent dual-epitope antibody; see Figure 10 When constructing the bivalent bi-epitope antibody, Knob-in-Hole modification can be further performed in the two heavy chains to achieve efficient assembly of the antibody.

[0157] Second aspect

[0158] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-CDH17 antibody or antigen-binding fragment thereof of the present invention.

[0159] The term "encoding the anti-CDH17 antibody or antigen-binding fragment thereof of the present invention" refers to a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable region, heavy chain variable region, heavy chain, and / or light chain contained in the antibody or antigen-binding fragment thereof. For example, the nucleic acid molecule provided herein comprises a nucleotide sequence encoding each of the heavy chain CDRs and light chain CDRs contained in the above-mentioned antibody or antigen-binding fragment thereof; a nucleotide sequence encoding the heavy chain variable region and light chain variable region contained in the above-mentioned antibody or antigen-binding fragment thereof; or a nucleotide sequence encoding both the heavy chain and light chain contained in the above-mentioned antibody or antigen-binding fragment thereof.

[0160] The third aspect

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

[0162] The fourth aspect

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

[0164] The fifth aspect

[0165] The anti-CDH17 antibodies or antigen-binding fragments thereof provided herein can be obtained using any method known in the art. For example, the present invention also provides a method for preparing the antibody or antigen-binding fragment thereof, comprising culturing the host cell provided herein while allowing the host cell to express the heavy and light chains of the antibody. Optionally, the method further comprises the step of recovering the produced antibody or antigen-binding fragment thereof.

[0166] The sixth aspect

[0167] The anti-CDH17 antibodies or antigen-binding fragments thereof provided by the present invention may also be directly or indirectly connected to other parts, such as the 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 antigen-binding fragment thereof can be constructed together with other binding domains into a multi-epitope antibody or a general bispecific antibody or a multispecific antibody. In the context of the present 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 the second epitope described above. In the context of the present invention, the term "bispecific antibody" or "multispecific antibody" refers to an antibody that can bind to one or more other target proteins in addition to binding to CDH17.

[0168] Alternatively, the other moiety may be a small molecule compound, for example a cytotoxic compound used as an antibody drug conjugate.

[0169] Therefore, accordingly, in a sixth aspect, the present invention further provides the use of the anti-CDH17 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, or host cell in the preparation of an antibody-drug conjugate (ADC). For example, the antibody or antigen-binding fragment thereof, or the antibody or antigen-binding fragment thereof encoded by the nucleic acid molecule, is 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 an antigen-binding fragment thereof provided by the present invention.

[0172] The antibody-drug conjugate can be formed by coupling the anti-CDH17 antibody or antigen-binding fragment thereof provided by the present invention with a cytotoxic compound. The cytotoxic compound can be a tubulin inhibitor, a topoisomerase inhibitor, a DNA binder, and the like. For example, the tubulin inhibitor can be a maytansine compound such as DM1 and DM4, a dolastatin compound such as Monomethyl Dolastatin 10, MMAE, MMAF, a tubulysin compound, a cryptophycin derivative, taltobulin, amanitin, calicheamicin, eribulin, and derivatives of the above drugs; the topoisomerase inhibitor can be a camptothecin compound such as Dxd, exatecan, and derivatives thereof, a derivative of the doxorubicin metabolite PNU-159682, and an irinotecan metabolite SN38; the DNA binder can be a PBD derivative and a duocarmycin derivative, and the like.

[0173] The eighth aspect

[0174] The anti-CDH17 antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, or antibody-drug conjugates or salts thereof provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical preparation, and thus used for various purposes according to actual needs.

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

[0176] Ninth aspect

[0177] The present invention also provides the use of the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition in the preparation of a medicament for preventing, treating and / or ameliorating a disease, wherein the disease may be associated with CDH17 expression (including overexpression), such as a solid tumor that is positive for CDH17 expression. The anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition may exert its effect by binding to CDH17 to exert an ADCC effect or through the cell-killing toxicity of the cytotoxic compound in the antibody-drug conjugate, but is not limited thereto.

[0178] The disease or disorder may be a digestive system tumor or cancer, such as gastric 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 ameliorating a disease, comprising administering to a subject in need thereof an anti-CDH17 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody-drug conjugate or salt thereof or composition of the present invention, wherein the disease may be associated with CDH17 expression (including overexpression), such as a solid tumor that is positive for CDH17 expression.

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

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

[0183] The above method provided by the present invention can also be used in combination with other drugs or means. The other drugs or means refer to other drugs or means 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 composition of the present invention, such as small molecule drugs, targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADC, oncolytic viruses, gene and nucleic acid therapeutic drugs and radiotherapy. The combined administration of the two can be carried out in any form, such as simultaneously, continuously or at intervals of a certain time.

[0184] Aspect 11

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

[0186] The disease or disorder may be a digestive system tumor or cancer, such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0187] Aspect 12

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

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

[0190] Thirteenth Aspect

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

[0192] The present invention provides a novel anti-CDH17 antibody. Experiments have demonstrated that the anti-CDH17 antibody provided by the present invention does not nonspecifically bind to CDH17-negative cells, but can specifically bind to CDH17 protein, such as cells recombinantly overexpressing CDH17 protein or tumor cells endogenously expressing CDH17, thereby exhibiting highly specific tumor targeting capabilities. Furthermore, the anti-CDH17 antibody provided by the present invention exhibits a relatively strong cell-killing effect mediated by the antibody itself. Furthermore, the anti-CDH17 antibody provided by the present invention can be effectively internalized on CDH17-expressing tumor cells.

[0193] Furthermore, the present invention provides a constructed anti-CDH17 dual-epitope antibody with CDH17 dual-epitope binding activity. Experiments have shown that the anti-CDH17 dual-epitope antibody provided by the present invention retains efficient internalization activity on CDH17-expressing tumor cells; and, after further coupling with a small molecule toxic compound, the resulting antibody-drug conjugate also maintains internalization activity and strong binding ability to the target protein, and shows high killing activity against CDH17-expressing tumor cells. In particular, compared to antibodies that bind to a single epitope of CDH17, the dual-epitope antibody provided by the present invention has a higher binding ability to the target protein CDH17 and shows a higher killing activity against CDH17-expressing tumor cells. It can be seen from this that the dual-epitope antibody provided by the present invention has more accurate and multi-dimensional antigen recognition ability and higher targeting, and has a stronger therapeutic effect on CDH17-related diseases.

[0194] Therefore, the anti-CDH17 antibodies provided by the present invention have significant application potential in tumor targeted therapy, ADC drug development, and other areas. In particular, as bi-epitope antibody and ADC technologies continue to mature, the anti-CDH17 bi-epitope ADCs of the present invention will become an important weapon in the treatment of digestive system tumors, promoting the development of precision cancer medicine. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0199] Figure 4 The figures show the killing test results of the anti-CDH17 antibodies provided by the present invention on CDH17-overexpressing tumor cells.

[0200] Figure 5 The results of the antibody binding assay to different domain proteins 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 figures show the binding test results of NC hIgG1-Ab2 scFv antibodies with different constructs to recombinant human (AD) and monkey (EH) CDH17 overexpressing cells.

[0203] Figure 8 The results of the binding assay of NC hIgG1-Ab2 scFv antibodies of different constructs to the tumor-overexpressing CDH17 cell line SK-CO-1 are shown.

[0204] Figure 9 A schematic diagram of the structure of Ab1-Ab2 HLscFv antibodies 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 binding test results 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 binding detection of the dual-epitope antibody to tumor cells are shown respectively.

[0208] Figure 16 Shown are the results of internalization assays of the bi-epitope antibody on tumor cells.

[0209] Figure 17 Shown are the results of killing assays of the bi-epitope antibody on tumor cells.

[0210] Figure 18 The results of the binding assay of the bi-epitope antibody drug conjugate to tumor cells are shown.

[0211] Figure 19 Shown are the results of internalization assays of the bi-epitope antibody drug conjugate on tumor cells.

[0212] Figure 20 Shown are the results of a killing assay of a bi-epitope antibody drug conjugate on tumor cells. DETAILED DESCRIPTION

[0213] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0214] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

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

[0216] Example 1 Screening of anti-CDH17 antibodies

[0217] (1) Animal immunization and hybridoma screening

[0218] Six- to eight-week-old female Balb / c mice were immunized with recombinant human CDH17 EC1-7 his protein (Acro Biosystems, CA7-H52H3) to produce specific anti-CDH17 antibodies. The immunization protocol consisted of a first subcutaneous immunization with 50 μg of antigen emulsified in complete Freund's adjuvant (CFA) followed by two to three booster immunizations with 25 μg of antigen emulsified in incomplete Freund's adjuvant (IFA). Immunizations were separated by 2-3 weeks, and serum samples were collected one week after the second immunization to assess antigen-binding activity. Mice with sera exhibiting high binding activity were screened by ELISA or other methods.

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

[0220] For protein-level screening, 96-well plates (100 μl / well) were coated with 1 μg / ml of recombinant human CDH17 EC1-7 his protein and incubated overnight at 4°C. Following incubation, the plates were washed and blocked, and hybridoma supernatant (100 μl / well) was added for further incubation. The supernatant was then assayed for binding activity to the CDH17 protein. For cell-based screening, a CHOK1 cell line overexpressing recombinant human CDH17 (UniProt B accession number: Q12864) was used for preliminary screening. Subsequently, to further validate the binding specificity and cross-species binding ability of the candidate molecules, cells overexpressing recombinant human CDH17, monkey CDH17 (UniProtKB accession number: #A0A1D5R2B4), and tumor cells endogenously expressing CDH17 were used as screening targets to ensure the targeting, selectivity, and broad applicability of the resulting molecules.

[0221] Finally, clones that tested positive for FACS and had high binding activity were selected for subcloning and culture. The VH / VL sequences of these clones were extracted and further selected for high-affinity clones for expansion culture.

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

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

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

[0225] The heavy chain 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) as follows.

[0226] Table 1. Anti-CDH17 Antibodies

[0227]

[0228]

[0229]

[0230]

[0231] Example 2 Antibody activity characterization of anti-CDH17 antibodies

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

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

[0234] CDH17-expressing cells with good growth status were collected, plated into 96-well plates and incubated with serially diluted antibodies at 4°C for 1 hour. After incubation, the cell plates 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 incubated at 4°C in the dark for 30 minutes. The cells were washed again three times and resuspended in PBS. Finally, cell 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 the present invention can specifically bind to CDH17 overexpressing cells, but not to CDH17 negative expression cells RKO.

[0236] 2) Internalization of anti-CDH17 antibodies in CDH17-overexpressing tumor cells

[0237] The internalization effect of the antibody was assessed using a commercial antibody internalization kit (Sartorius, 90565).

[0238] First, the concentrations of the test antibody and the internalization reagent were adjusted to 100 μg / ml. Then, 1 μl of the test antibody, 1 μl of the internalization reagent, and 48 μl of the diluent were mixed and incubated at 37°C for 15 minutes. After incubation, the mixture was diluted 2 times. In addition, the cell density of CDH17-expressing tumor cells was adjusted to 2E6 / ml, and then plated into a 96-well plate at 20 μl / well, and the above-mentioned diluted antibody-internalization reagent mixture (20 μl / well) was added. The 96-well plate was then incubated at 37°C, 5% CO2 for 24 hours. The cells were collected the next day, and cell flow cytometry analysis was performed using iQue Screener PLUS, and data analysis was performed by GraphPadPrism.

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

[0240] 3) Anti-CDH17 antibodies kill CDH17-overexpressing tumor cells

[0241] The killing activity of the antibody was evaluated using the commercial reagent αHFc-CL-MMAE (Moradec, AH-102AE-50).

[0242] First, the cell concentration of CDH17-expressing cells (such as SK-CO-1) was adjusted to 1E5 cells / ml, and then plated into a 96-well all-white plate at a volume of 50 μl / well and incubated for 4 hours. Next, add gradiently diluted antibodies, 10 μl per well; at the same time, add 40 μl of diluted αHFc-CL-MMAE (2.5 μg / ml). The 96-well all-white plate was then cultured at 37°C and 5% CO2 for 4 days. After the incubation, 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) was added to each well, and the culture plate was shaken on a shaker for 5 minutes. The relative fluorescence value (RLU, Relative Luminescence Unit) was detected using a Spectra M5e instrument. Killing 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 the present invention exhibit good killing activity on tumor cells and are superior to the control antibodies.

[0244] Table 2. Antibody killing activity test results

[0245]

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

[0247] The binding properties of the antibodies to different domains of human CDH17 protein were evaluated by ELISA binding assay.

[0248] First, the different domain recombinant proteins of human CDH17, CDH17 EC1-2 his (Kactusbio, CDH-HM1D5), CDH17 EC3-4 mFc (Kactusbio, CDH-HM3D3), CDH17 EC5-7 his (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 with 1× PBST containing 1% BSA and incubated at 37°C for 1 hour, and then washed three times with 1× PBST. Next, a serial dilution of the test antibody was added and incubated at 37°C for 1 hour. A 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, 109-035-098) was then added and incubated at 37°C for 1 hour. The 96-well plate was then washed with 1× PBST and color was developed with TMB substrate. The reaction was terminated with 1N HCl. OD values ​​were measured at 450 nm to quantitatively assess antibody binding to different domains of the human CDH17 protein.

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

[0250] Example 4 Construction and purification of a tetravalent bi-epitope antibody

[0251] A symmetrical Full IgG1 Ab1-Ab2 scFv tetravalent bi-epitope antibody was constructed using anti-CDH17 antibodies that bind to different epitopes on CDH17, particularly antibodies that bind to EC1-2 and EC5-6, and EC3-4 and EC5-6, respectively.

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

[0253] The schematic diagram of the structure of NC hIgG1-Ab2 scFv antibody is shown in 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 Figures A and B show the two constructs of "VH-VL" (HL scFv) and "VL-VH" (LH scFv) arranged from N-terminus to C-terminus, respectively.

[0254] The heavy and light chain variable region scFvs at the C-terminus (Ab2) included VH-VL (HL scFv) and VL-VH (LH scFv) combinations from the 8E8D4, 37G1F1, 66G9D2, and 82G3C12 antibody molecules, respectively. For each combination, amino acid position 44 of the VH of antibody Ab2 was mutated from G, R, or other mutations to C, and amino acid position 100 of the VL was mutated from G, A, or other mutations to C, according to the Kabat numbering. This was done to form a disulfide bond between the scFv attached to the C-terminus of the NC hIgG1 heavy chain and the scFv attached to the C-terminus of the NC hIgG1 light chain, thereby stabilizing the molecular structure. Using AscI seamless cloning technology, 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] The 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. Seven days after transfection, the cell supernatant was collected by centrifugation and filtered through a 0.22 μm filter to remove cell debris. TM MabSelect TM The cell supernatant was purified using a SuRe column (GE, Cat#11003493), filtered, and quantified before use.

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

[0257] Table 3. scFv sequences of two constructs used to assemble the heavy chain of NC hIgG1-Ab2 scFv antibody (the bold part is the linker sequence)

[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 antibodies

[0262]

[0263] (II) Specific binding of NC hIgG1-Ab2 scFv antibodies with different constructs

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

[0265] The CDH17-expressing cells were plated into 96-well plates and then incubated with gradient dilutions of the test antibodies at different concentrations at 4°C for 1 hour. The 96-well plates were centrifuged and washed three times with PBS, followed by the addition of a 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) and 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 then performed using iQue Screener PLUS, and data analysis was performed using GraphPad Prism.

[0266] like Figure 7 and Figure 8As shown in the figure, the results showed that both NC hIgG1-Ab2 HLscFv and NC hIgG1-Ab2 LHscFv antibodies could specifically bind to human CDH17-overexpressing cells, rhesus macaque CDH17-overexpressing cells, and SK-CO-1 tumor cells; however, the binding strength of NC hIgG1-Ab2 HLscFv antibody was comparable or similar to that of the corresponding monoclonal antibody, while the binding of NC hIgG1-Ab2 LHscFv antibody was relatively weak. Therefore, the NC hIgG1-Ab2 HLscFv format was selected for subsequent evaluation and construction.

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

[0268] The VH and VL of the N-terminal NC hIgG1 part of the NC hIgG1-Ab2 HLscFv antibody were replaced with the VH and VL of the anti-CDH17 antibody molecule to construct the Ab1-Ab2 HLscFv tetravalent bi-epitope antibody. The schematic diagram of the antibody structure is shown in 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 of the heavy chain expression vector of the Ab1 antibody molecule (PTT5-Ab1, see the antibody heavy chain expression vector in Example 1); wherein the Ab1 molecule is the EC1-2 binding antibody 1D12H10 or 5B3A6, the EC3-4 binding antibody 47E1G1, or the EC5-6 binding antibody 37G1F1. The Ab2 antibody molecule is the EC3-4 binding antibody 47E1G1 or the EC5-6 binding antibody 8E8D4 or 37G1F1. A heavy chain expression plasmid containing the corresponding HL scFv sequence, namely the Ab1-Ab2 HL scFv plasmid, was constructed; referring to the construction method of the NChIgG1-Ab2 HL scFv antibody above, the amino acid at position 44 of VH and position 100 of VL of Ab2 (amino acid position according to Kabat numbering) were similarly mutated to cysteine, thereby forming a disulfide bond between the heavy chain and light chain scFv to stabilize the structure of the antibody molecule.

[0270] The heavy chain plasmid and light chain plasmid (see the antibody light chain expression vector in Example 1) were co-transfected into HEK293 cells by PEI transfection. As described above, multiple Ab1-Ab2 HLscFv antibodies were finally obtained through expression and purification.

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

[0272] Table 5. Sequences used to assemble the Ab1-Ab2 HLscFv tetravalent bi-epitope antibody (the bold part is the linker sequence)

[0273]

[0274]

[0275]

[0276]

[0277]

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

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

[0280]

[0281]

[0282] Example 5 Construction and purification of CrossMab bivalent bi-epitope antibody

[0283] The CrossMab method was used to keep antibody A unchanged while swapping the CH1 and CL domains of antibody B to construct CrossMab antibodies that bind to different epitopes on CDH17. Among them, the antibody molecule that binds to the EC1-2 domain of CDH17 was selected as the antibody A part, and the antibody molecule that binds to the EC3-4 or EC5-6 domain of CDH17 was selected as the antibody B part. The structural diagram is shown in Figure 10 .

[0284] Referring to the above description, after expression and purification, multiple CrossMab bivalent bi-epitope antibodies capable of simultaneously binding to two epitopes were finally obtained.

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

[0286] Table 7. Sequences used to assemble CrossMab bivalent bi-epitope antibodies

[0287]

[0288]

[0289] The heavy and light chains of different CrossMab bivalent, bi-epitope antibodies are shown in Table 8.

[0290] Table 8. CrossMab bivalent bi-epitope antibodies

[0291]

[0292] Example 6 Identification of bi-epitope antibodies

[0293] The activities of the Ab1-Ab2 HLscFv tetravalent bi-epitope antibody obtained in Example 4 and the Crossmab bivalent bi-epitope antibody obtained in Example 5 were evaluated.

[0294] (I) Binding of bi-epitope 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 of different construction forms", different cells were incubated with gradient dilutions of antibodies. After secondary antibody incubation 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 the present invention can specifically bind to recombinant human and monkey CDH17 expressing cells.

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

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

[0300] (III) Internalization of the bi-epitope antibody in CDH17-overexpressing tumor cells

[0301] The internalization effect of the antibody was assessed using a commercial antibody internalization kit (Sartorius, 90565).

[0302] First, the concentrations of the test antibody and the internalization reagent were adjusted to 100 μg / mL, and then 1 μl of the test antibody, 1 μl of the internalization reagent and 48 μl of the diluent were mixed and incubated at 37°C for 15 minutes. After incubation, the mixture was diluted 2 times. In addition, the cell density of CDH17-expressing tumor cells was adjusted to 2E6 / ml, and then plated into a 96-well plate at 20 μl / well, and the above-mentioned diluted antibody-internalization reagent mixture (20 μl / well) was added. The 96-well plate was then incubated at 37°C, 5% CO2 for 24 hours. The cells were collected the next day, and cell flow cytometry analysis was performed using iQue Screener PLUS, and data analysis was performed by GraphPadPrism.

[0303] like Figure 16 As shown, the results showed that all the tested dual-epitope antibodies could be internalized efficiently and were better than monoclonal antibodies.

[0304] (IV) Bi-epitope Antibodies Kill CDH17-Overexpressing Tumor Cells

[0305] The killing activity of the antibody was evaluated using the commercial reagent αHFc-CL-MMAE (Moradec, AH-102AE-50).

[0306] First, the cell concentration of CDH17-expressing cells (such as NCI-H508 or SK-CO-1) was adjusted to 1E5 cells / ml, and then plated into a 96-well all-white plate at a volume of 50 μl / well and incubated for 4 hours. Next, a gradient dilution of the antibody was added, 10 μl per well; at the same time, 40 μl / well of the diluted αHFc-CL-MMAE (2.5 μg / ml) was added. The 96-well all-white plate was then placed in a 37°C, 5% CO2 and cultured for 4 days. After the incubation, 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) was added to each well, and the culture plate was shaken on a shaker for 5 minutes. The relative fluorescence value (RLU, Relative Luminescence Unit) was detected using a Spectra M5e instrument.

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

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

[0309] Table 9-1 Killing test of bi-epitope antibody against tumor cells NCI-H508

[0310]

[0311] Table 9-2 Killing test of dual-epitope antibody against tumor cells SK-CO-1

[0312]

[0313] Table 9-3 Killing test of dual-epitope antibody against tumor cells SK-CO-1

[0314]

[0315] Example 7 Preparation of bi-epitope antibody-drug conjugates

[0316] (I) Preparation of ADC

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

[0318]

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

[0320] Sample reduction and coupling: Antibody samples were desalted using a Sephadex G-25-supported NAP-25 desalting column in 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. To a 100 mg antibody sample (10 ml), a 10 mg / ml aqueous solution of TCEP (Sigma-Aldrich) was added at an antibody-reducing agent molar ratio of 1:10. After incubation for 2 hours, the reaction solution was desalted using a Sephadex G25 desalting column in 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. 1.33 mL of N,N-dimethylacetamide (DMA) was added as a presolvent (7.4% of the total reaction volume). A DMA-drug linker mixture containing 10 mg / mL of the drug linker was then added at a molar ratio of 1:5.5 of antibody to small molecule drug. The mixture was stirred at room temperature for 60 minutes. The reaction solution was then desalted using a Sephadex G-25 NAP-25 desalting column into a pH 8.0 sodium dihydrogen phosphate / sodium dihydrogen phosphate buffer solution to remove excess drug linker. The reaction solution was then heated in a 37°C water bath for 3 hours. Mass spectrometry was performed before and after heating in the water bath to analyze the structure of the conjugated product. The results for each conjugated product showed that the product before heating in the water bath exhibited the structure of Formula Ia described above, while after heating in the water bath, the fully ring-opened structure of Formula Ib was obtained, indicating 100% hydrolysis.

[0322] Sample purification: The above sample was concentrated using an AMICOM ultrafiltration centrifuge tube to about 15 mg / mL. Add 50 mM disodium hydrogen phosphate - sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution until the conductivity is 74 ms / cm. Load the sample onto a Butyl-Sepharose 4FF filler (purchased from GE Healthcare) hydrophobic column, using phase A of 50 mM disodium hydrogen phosphate - sodium dihydrogen phosphate + 0.45 M ammonium sulfate; phase B is a buffer solution of 50 mM disodium hydrogen phosphate - sodium dihydrogen phosphate. Phase B was 0-80% linear gradient 12 times the column volume, phase B was 100% isocratic gradient elution, and the main peak was collected.

[0323] The final sample was replaced with 50 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using an AMICOM ultrafiltration centrifuge tube and filtered using a 0.22 μm filter membrane (Sartorius stedim Ministart). Finally, the antibody-drug conjugate was purified.

[0324] (II) Characterization of ADC drug physicochemical properties

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

[0326] According to the literature [Clin Cancer Res. 2004 Oct 15; 10(20): 7063-70], DAR was 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 absorption value of the antibody-drug conjugate at 280 nm, Az is the ultraviolet absorption value 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. Analysis of molecular size heterogeneity by size exclusion high performance liquid chromatography (SE-HPLC)

[0330] SE-HPLC was performed according to 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°C;

[0336] Sample volume: 20uL;

[0337] Elution time: 20 min;

[0338] Elution gradient: isocratic elution.

[0339] The drug-antibody coupling ratio (DAR) was determined by UV spectrophotometry in the characterization of the physicochemical properties of the ADC drug in this application, and the 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 evaluation of bi-epitope antibody drug conjugates

[0345] (I) Binding activity evaluation of the bi-epitope antibody BL20E ADC

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

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

[0348] Table 11-1 Binding test results of Ab1-Ab2 HLscFv bi-epitope antibody drug conjugate to tumor cells

[0349]

[0350] Table 11-2 Results of binding assays of CrossMab bi-epitope antibody-drug conjugates to tumor cells

[0351]

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

[0353] The internalization of the bi-epitope antibody drug conjugate was evaluated by referring to the method described in Example 4 (iii).

[0354] like Figure 19 As shown, the results showed that the dual-epitope antibody drug conjugates could be effectively internalized, and the internalization activity was better than that of the monoclonal antibody drug conjugates.

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

[0356] Colorectal cancer cells (SK-CO-1, HT55, CL40, and NCI-H508) overexpressing CDH17 were plated into 96-well white plates. Serial dilutions of ADC were added and the cells were cultured at 37°C in a CO2 atmosphere 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 on a shaker for 5 minutes. Relative fluorescence units (RLU) were measured using a Spectra M5e instrument, and data were analyzed using GraphPad Prism.

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

[0358] Table 12-1 Results of the anti-tumor cell killing test of Ab1-Ab2 HLscFv bi-epitope antibody drug conjugate

[0359]

[0360] Table 12-2 Results of CrossMab dual-epitope antibody-drug conjugates' killing of tumor cells

[0361]

[0362] The above description of the 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. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

Claims

1. An anti-cadherin-17 (CDH17) antibody or an antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (CDRs), i.e., heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and light chain complementarity determining regions (CDRs), i.e., 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) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, in sequence; (2) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.52, SEQ ID NO.53, and SEQ ID NO.54, in sequence; (3) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, in sequence; (4) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66, in sequence; (5) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 69, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO. 70, SEQ ID NO. 71, and SEQ ID NO. 72, in sequence; (6) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO. 67, SEQ ID NO. 73, and SEQ ID NO. 74, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, in sequence; (7) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.78, SEQ ID NO.79, and SEQ ID NO.80, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.81, SEQ ID NO.82, and SEQ ID NO.83, in sequence; or (8) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.84, SEQ ID NO.85, and SEQ ID NO.86, respectively; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.87, SEQ ID NO.71, and SEQ ID NO.88, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-CDH17 antibody or antigen-binding fragment thereof specifically binds to cadherin-17 (CDH17), preferably primate or rodent CDH17, such as human, monkey or mouse CDH17; Optionally, the anti-CDH17 antibody or antigen-binding fragment thereof has or does not have species cross-binding activity to human (human), monkey (cyno or Rhesus), or mouse (mouse) CDH17; Preferably, the anti-CDH17 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are as follows: (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.2; (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 3, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 4; (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 5, or comprises an amino acid sequence that is at least 75% identical to 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 comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 6; (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 8; (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9, or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 10; (6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 11, or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 12, or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 12; (7) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 13, or comprises an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO. 14; or (8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.15, or comprises an amino acid sequence that is at least 75% identical to 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 comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.

16.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The anti-CDH17 antibody is a mouse antibody, a rabbit antibody, a human antibody, or a mouse antibody, a chimeric antibody, a fully or partially humanized antibody, or a derivatized antibody; Alternatively, the antigen-binding fragment of the anti-CDH17 antibody is a fragment of any form of the antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; Optionally, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or mouse heavy chain constant region and / or a light chain constant region; preferably, the antibody or its fragment comprises an IgG, IgA, IgM, IgD or IgE heavy chain constant region and / or a κ or λ type light chain constant region.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The anti-CDH17 antibody is a monoclonal antibody; Preferably, the anti-CDH17 antibody is an immunoglobulin, for example, the immunoglobulin type is human IgA, IgD, IgE, IgG or IgM; preferably, the antibody is of human IgG1 or IgG4 subtype.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that The antibody or antigen-binding fragment thereof is an anti-CDH17 antibody or antigen-binding fragment thereof that binds to two or more epitopes of CDH17; Optionally, the anti-CDH17 antibody or its antigen-binding fragment that binds to two or more epitopes of CDH17 is artificially constructed, so that the antibody or its antigen-binding fragment contains at least two binding domains to respectively bind to different epitopes of CDH17, and the epitopes can be located in any one or more of the extracellular regions EC1 to EC7 of CDH17.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that The antibody or antigen-binding fragment thereof is an anti-cadherin-17 (CDH17) bi-epitope antibody or antigen-binding fragment thereof, the bi-epitope antibody or antigen-binding fragment thereof comprises at least a first binding domain and a second binding domain, the first binding domain and the second binding domain respectively comprising one of the heavy chain CDRs and light chain CDRs as defined in claim 1, and respectively binding to different epitopes located in different extracellular regions of CDH17; Preferably, the bi-epitope antibody or antigen-binding fragment thereof 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 extracellular regions of CDH17, wherein: (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; (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; (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 (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.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that The antibody or antigen-binding fragment thereof is a bi-epitopic antibody or antigen-binding fragment thereof and comprises a first binding domain and a second binding domain, and: (1) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72, in sequence; (2) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77, in sequence; (3) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72, in sequence; (4) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77, in sequence; (5) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, in sequence; (6) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.55, SEQ ID NO.56, and SEQ ID NO.57, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.84, SEQ ID NO.85, and SEQ ID NO.86, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.87, SEQ ID NO.71, and SEQ ID NO.88, in sequence; (7) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences 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 comprising the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72, in sequence; (8) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.61, SEQ ID NO.62, and SEQ ID NO.63, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.67, SEQ ID NO.73, and SEQ ID NO.74, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.75, SEQ ID NO.76, and SEQ ID NO.77, in sequence; (9) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.52, SEQ ID NO.53, and SEQ ID NO.54, in sequence; The second binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.67, SEQ ID NO.68, and SEQ ID NO.69 in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences shown in SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72 in sequence; or (10) The first binding domain comprises: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences 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, 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.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that The antibody or antigen-binding fragment thereof is a bi-epitopic antibody or antigen-binding fragment thereof and comprises a first binding domain and a second binding domain, each of which comprises a heavy chain variable region and a light chain variable region, 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 of SEQ ID NO. 5 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 5; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 6 or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to 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 of SEQ ID NO. 5 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 5; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 6 or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.12; (5) In the first binding domain, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 11; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to 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 of SEQ ID NO. 5 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 5; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 6 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 6; In the second binding domain, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 15 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 15; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 16 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 16; (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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.10; (8) In the first binding domain, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 7 or an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO. 7; and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 8 or an amino acid sequence that is at least 75% identical to the amino acid sequence of 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 that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.12; (9) 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.10; or (10) 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 that is at least 75% identical to 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 that is at least 75% identical to 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.7 or an amino acid sequence that is at least 75% identical to 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 that is at least 75% identical to the amino acid sequence shown in SEQ ID NO.

8.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that The antibody or antigen-binding fragment thereof is a dual-epitope antibody or antigen-binding fragment thereof and comprises a first binding domain and a second binding domain, wherein the first binding domain and the second binding domain are respectively selected from the form of an IgG antibody and a scFv fragment; or the antibody or antigen-binding fragment thereof 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 half antibodies of the IgG antibody.

10. An antibody-drug conjugate targeting CDH17 or a salt thereof, comprising the anti-CDH17 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the antibody-drug conjugate is formed by coupling the anti-CDH17 antibody or its antigen-binding fragment with a cytotoxic compound; preferably, the cytotoxic compound is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder.

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