Anti-cadhein-17 antibody and antibody drug conjugate thereof

Abstract

The invention provides an anti-CDH17 antibody or an antigen binding fragment thereof, a CDH17-targeted antibody drug conjugate (ADC) and application thereof. The anti-CDH17 antibody provided by the invention can be specifically combined with CDH17 protein, and has a relatively strong CDH17 expression tumor cell killing effect, so that the anti-CDH17 antibody has high-specificity treatment application potential of CDH17 expression tumor. Furthermore, the anti-CDH17 antibody provided by the invention can be effectively internalized on CDH17 expression tumor cells, ADC formed by coupling the anti-CDH17 antibody with a small-molecule toxic compound keeps high internalization activity and strong binding capacity to target protein CDH17, and shows high killing activity to the CDH17 expression tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of antibody drugs, and in particular, relates to anti-cadherin-17 antibodies and antibody-drug conjugates thereof, as well as applications thereof. Background Art

[0002] Cadherin-17 (CDH17) is a key member of the cadherin family, consisting 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 various tumors, particularly in digestive system malignancies such as gastric cancer, colorectal cancer, and pancreatic cancer. Clinical studies have found that elevated CDH17 expression is often closely associated with a tumor's high degree of differentiation, invasiveness, metastasis, and poor prognosis. Its expression levels are particularly elevated in the late and metastatic stages of the tumor. Compared to traditional chemotherapy drugs, ADCs targeting CDH17 can reduce unwanted side effects through their targeted mechanism, demonstrating their significant potential as targeted therapeutics. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a novel anti-CDH17 antibody that should bind to CDH17 proteins, such as CDH17 expressed by tumor cells, with high specificity and high affinity, thereby having significant tumor cell killing ability through efficient binding to CDH17 proteins expressed by tumors; in addition, the antibody should also have a strong ability to internalize into CDH17-expressing cells, thereby being suitable for preparation into antibody-drug conjugates.

[0005] Therefore, one object of the present invention is to provide an antibody or a fragment thereof that specifically binds to CDH17. Another object of the present invention is to provide an antibody-drug conjugate or a salt thereof that targets CDH17.

[0006] The technical solutions of the present invention are as follows.

[0007] First aspect

[0008] 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.

[0009] In the context of the present invention, unless otherwise specified, the term "CDH17" encompasses any form or structural region of CDH17.

[0010] 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.

[0011] 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 set forth in any one of SEQ ID NOs. 1, 3, 5, 7, 9, and 11, and a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs. 2, 4, 6, 8, 10, and 12.

[0012] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.12 provided above are the 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.

[0013] 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:

[0014] (1) SEQ ID NO. 1 + SEQ ID NO. 2;

[0015] (2) SEQ ID NO. 3 + SEQ ID NO. 4;

[0016] (3) SEQ ID NO. 5 + SEQ ID NO. 6;

[0017] (4) SEQ ID NO. 7 + SEQ ID NO. 8;

[0018] (5) SEQ ID NO. 9 + SEQ ID NO. 10; or

[0019] (6) SEQ ID NO.11+SEQ ID NO.12.

[0020] 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.

[0021] 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:

[0022] (1) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, in sequence;

[0023] (2) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, in sequence;

[0024] (3) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO. 28, SEQ ID NO. 29, and SEQ ID NO. 30, in sequence;

[0025] (4) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, in sequence;

[0026] (5) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.39, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42, in sequence; or

[0027] (6) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, respectively; 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, respectively.

[0028] 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.

[0029] 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.

[0030] 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:

[0031] (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;

[0032] (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;

[0033] (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;

[0034] (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;

[0035] (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; or

[0036] (6) 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Second aspect

[0043] 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.

[0044] The "nucleotide sequence 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.

[0045] The third aspect

[0046] 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.

[0047] The fourth aspect

[0048] 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.

[0049] The fifth aspect

[0050] The anti-CDH17 antibodies or antigen-binding fragments thereof provided herein can be obtained by any method known in the art. For example, the present invention also provides a method for preparing the anti-CDH17 antibodies or antigen-binding fragments thereof, the method comprising culturing the host cells provided herein in a manner that allows the host cells to express the heavy and light chains of the antibodies. Optionally, the method further comprises the step of recovering the produced anti-CDH17 antibodies.

[0051] The sixth aspect

[0052] The anti-CDH17 antibodies or antigen-binding fragments thereof provided by the present invention may also be directly or indirectly linked 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; or, such as small molecule compounds, such as cytotoxic compounds used as antibody-drug conjugates; or, such as cell surface receptors, carbohydrates, polymers, etc., that modify the antibody or antigen-binding fragment thereof.

[0053] Therefore, accordingly, in a sixth aspect, the present invention further provides 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).

[0054] Seventh aspect

[0055] 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.

[0056] 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, and the cytotoxic compound can be a tubulin inhibitor, a topoisomerase inhibitor, a DNA binder, etc. 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, etc.

[0057] The eighth aspect

[0058] The anti-CDH17 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell or antibody-drug conjugate or salt 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.

[0059] Therefore, the present invention also provides a composition comprising the anti-CDH17 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, or antibody-drug conjugate or 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.

[0060] Ninth aspect

[0061] 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.

[0062] 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.

[0063] Tenth Aspect

[0064] The present invention also provides a method for preventing, treating and / or ameliorating a disease, comprising administering 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 to a subject in need thereof, wherein the disease may be associated with CDH17 expression (including overexpression), such as a solid tumor that is positive for CDH17 expression.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Aspect 11

[0069] 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.

[0070] 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.

[0071] Aspect 12

[0072] 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.

[0073] 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.

[0074] Thirteenth Aspect

[0075] 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.

[0076] The present invention provides anti-CDH17 antibodies. These antibodies do 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 antibodies provided by the present invention exhibit relatively strong cell-killing effects mediated by the antibodies themselves.

[0077] Furthermore, experiments have demonstrated that the anti-CDH17 antibodies provided herein are effectively internalized by CDH17-expressing tumor cells. After conjugation with a small molecule toxic compound, the resulting antibody-drug conjugate maintains this efficient internalization activity and strong binding to the target protein, while also exhibiting high cytotoxic activity against CDH17-expressing tumor cells. Therefore, the anti-CDH17 antibodies provided herein have significant potential for application in tumor targeted therapy and ADC drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Figure 4 The figures show the killing test results of the anti-CDH17 antibodies provided by the present invention on CDH17-overexpressing tumor cells.

[0083] Figure 5 The results of binding detection between the anti-CDH17 antibodies provided by the present invention and different domain proteins of human CDH17 are shown.

[0084] Figure 6 The results of the binding test between the anti-CDH17 antibody provided by the present invention and monkey CDH17 are shown.

[0085] Figure 7 The results of binding detection of the CDH17-targeting antibody-drug conjugate provided by the present invention with recombinant CDH17-expressing cells and tumor cells are shown; among them, A, B, and C: monkey CDH17-overexpressing cells, D and E: gastric cancer cells AGS, F: colorectal cancer cells NCI-H508, G: colorectal cancer cells SK-CO-1, and H: CHOK1 null cells.

[0086] Figure 8 The internalization detection results of the CDH17-targeting antibody drug conjugate provided by the present invention on tumor cells are shown; wherein, A and C: 2 hours, B and D: 24 hours.

[0087] Figure 9 The results of the killing test of the CDH17-targeting antibody-drug conjugate provided by the present invention on the tumor cells SK-CO-1 are shown. DETAILED DESCRIPTION

[0088] 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.

[0089] 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.

[0090] The hIgG1 used in the examples is an unrelated antibody that does not bind to CDH17.

[0091] Example 1 Animal immunization and hybridoma screening

[0092] Female Balb / c mice aged 6-8 weeks were immunized with recombinant human CDH17 EC1-7 his protein (Acro Biosystems, CA7-H52H3). The immunization process consisted of three subcutaneous immunizations: the first immunization was administered subcutaneously with 50 μg of antigen emulsified in complete Freund's adjuvant (CFA) and the second two immunizations were administered subcutaneously with 25 μg of antigen emulsified in incomplete Freund's adjuvant (IFA). Immunizations were separated by 2-3 weeks, and blood was collected one week after immunization for antigen binding activity. Mice with sera demonstrating high binding activity were selected for hybridoma fusion.

[0093] After boosting immunization of selected mice, spleen cells were harvested and electrofused with murine myeloma SP20 cells. After culturing in HAT and HT media for 10-14 days, hybridoma supernatants were obtained. Hybridoma supernatants were screened for protein or cellular binding using ELISA or FACS, respectively.

[0094] For protein-level screening, 96-well plates (100 μl / well) were coated with 1 μg / mL of human CDH17 EC1-7 his protein and incubated overnight. Following incubation, the plates were washed and blocked, then hybridoma supernatant (100 μl / well) was added for incubation and detection. For cell-level screening, CHOK1 cells overexpressing human CDH17 (UniProtKB Accession No.: Q12864) were used for screening. After positive clones were identified, further screening was performed using cells expressing recombinant human CDH17, monkey CDH17 (UniProtKB Accession No.: A0A1D5R2B4), and tumor cells endogenously expressing CDH17.

[0095] Finally, FACS-positive clones were selected for subcloning culture and VH / VL sequences were extracted, and clones with high binding activity were screened for expansion culture and subsequent analysis.

[0096] Example 2 Production and characterization of anti-CDH17 antibodies

[0097] (I) Extraction of candidate cloned genes, construction of chimeric expression vectors, expression, and purification

[0098] 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.

[0099] 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 heavy and light chain plasmids. The heavy and light chain plasmids were co-transfected into HEK293 cells at a 2:3 ratio using PEI transfection. After 5-7 days of culture, the culture supernatant was collected. The 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.

[0100] 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).

[0101] Table 1. Anti-CDH17 Antibodies

[0102]

[0103]

[0104]

[0105] (II) Antibody activity identification of anti-CDH17 antibodies

[0106] 1) Specific binding of anti-CDH17 antibodies to CDH17-overexpressing cells

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 2) Internalization of anti-CDH17 antibodies in CDH17-overexpressing tumor cells

[0111] The internalization effect of the antibody was assessed using a commercial antibody internalization kit (Sartorius, 90565).

[0112] 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.

[0113] like Figure 3 As shown, the results indicate that the antibodies provided by the present invention can be effectively internalized.

[0114] 3) Anti-CDH17 antibodies kill CDH17-overexpressing tumor cells

[0115] The killing activity of the antibody was evaluated using the commercial reagent αHFc-CL-MMAE (Moradec, AH-102AE-50).

[0116] 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%.

[0117] like Figure 4 As 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.

[0118] Table 2. Antibody killing activity test results

[0119]

[0120]

[0121] Example 3 Epitope analysis and species cross-binding identification of anti-CDH17 antibodies

[0122] (I) Binding of anti-CDH17 antibodies to different domains of human CDH17

[0123] The binding properties of the antibodies to different domains of human CDH17 protein were evaluated by ELISA binding assay.

[0124] 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, with 100 μL per well, and incubated overnight. 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 the cells were 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 the cells were incubated at 37°C for 1 hour. The 96-well plate was then washed with 1× PBST, and TMB substrate was added for color development. The reaction was terminated with 1N HCl. OD values ​​were measured at 450 nm.

[0125] like Figure 5 As shown, the results indicate that the antibodies provided by the present invention all specifically bind to human CDH17EC1-7 his protein; further, antibodies 1D12H10, 1F3C8, 5B3A6, 7G3G6 and 89B1H10 all bind to CDH17 EC1-2 domain protein, and antibody 47E1G1 binds to CDH17 EC3-4 domain protein.

[0126] (II) Binding of anti-CDH17 antibodies to monkey CDH17

[0127] The binding properties of the antibodies to different monkey CDH17 proteins were evaluated by ELISA binding assay.

[0128] First, different monkey CDH17 proteins (Rhesus CDH17 [Kactusbio, CDH-RM117], Cyno CDH17 [Kactusbio, CDH-CM127]) were coated onto 96-well plates at a concentration of 1 μg / ml, with 100 μL per well incubated overnight. The next day, the 96-well plates were washed three times with 1× PBST and blocked with 1% BSA in 1× PBST for 1 hour at 37°C. The plates were then washed three times with 1× PBST. A serial dilution of the test antibody was then added, and the plates were 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. Afterwards, the 96-well plate was washed with 1×PBST, TMB substrate was added for color development, and the reaction was terminated with 1N HCl. OD values ​​were measured at a wavelength of 450 nm.

[0129] like Figure 6 As shown, the results indicate that the antibodies provided by the present invention specifically bind to monkey CDH17 protein.

[0130] Example 4 Hydrophobicity assessment of anti-CDH17 antibodies

[0131] The hydrophobicity of anti-CDH17 antibodies was evaluated using HIC-HPLC.

[0132] A Waters HPLC system was used, equipped with a TSK gel Butyl-NPR column (2.5 μm, 4.6 mm × 35 mm, TOSOH). The test antibody was diluted to 1.0 mg / mL in PBS, and a sample load of 70 μg was loaded onto the column. A gradient elution mode was used, with the mobile phase transitioning from phase B (20 mM histidine solution, pH 6.0) to phase A (20 mM histidine solution containing 1.6 M ammonium sulfate ((NH₄)₂SO₄), pH 6.0) at a flow rate of 0.7 mL / min. UV absorbance was monitored at 280 nm. The hydrophobicity of the sample was assessed by measuring the (NH₄)₂SO₄ concentration corresponding to the time of the absorption peak. Hydrophobicity was calculated using the following formula: (NH₄)₂SO₄ concentration (M) = (23 - time + 1.25) × 0.101, where "time" is the time in minutes at which the peak (retention time) of the absorption peak appears. According to this formula, when the calculated (NH4)2SO4 concentration exceeds 0.8M, the tested antibody is considered hydrophilic.

[0133] As shown in Table 3, the results indicate that the antibodies provided by the present invention have good hydrophilicity.

[0134] Table 3. Antibody hydrophobicity evaluation results

[0135] Antibody Retention time (min) <![CDATA[(NH4)2SO4 concentration (M)]]> 1D12H10 18.04 0.63 1F3C8 15.64 0.87 5B3A6 14.48 0.99 50A12C8 14.64 0.97 58G2E8 18.87 0.54 69B2A2 13.33 1.10 89B1H10 16.92 0.74 112F7A3 14.92 0.94 47E1G1 13.17 1.12

[0136] Example 5 Preparation of Antibody Drug Conjugates

[0137] (I) Preparation of ADC

[0138] BL20E (Linker + MMAE payload) was synthesized according to the method of patent CN110088086B.

[0139]

[0140] To further evaluate the activity of anti-CDH17 antibodies, the antibody was conjugated to compound BL20E to prepare an ADC targeting CDH17, according to the method described in patent application publication WO2022228563A1. The drug-antibody conjugation ratio (UV-DAR method) was determined by ultraviolet spectrophotometry according to the literature [Clin Cancer Res. 2004 Oct 15; 10(20): 7063-70]. The average DAR value of the ADC was detected, and molecular size heterogeneity was analyzed by size exclusion high performance liquid chromatography (SEC-HPLC). SEC-HPLC was performed according to the following parameters:

[0141] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5 μm, 7.8 mm*300 mm;

[0142] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0143] Flow rate: 0.6 mL / min;

[0144] Detection wavelength: 280nm;

[0145] Column temperature: 30°C;

[0146] Sample volume: 20uL;

[0147] Elution time: 20 min;

[0148] Elution gradient: isocratic elution.

[0149] The results are shown in Table 4.

[0150] Table 4. Antibody-drug conjugates targeting CDH17 and their characterization results

[0151]

[0152]

[0153] Example 7 Activity evaluation of antibody drug conjugates

[0154] The binding capacity, internalization properties, and cytotoxicity of the prepared antibody-drug conjugates were evaluated using cells with different CDH17 expression levels.

[0155] (I) Binding of ADC to CDH17-expressing cells

[0156] Referring to the method described in Example 2 (ii) "1) Specific binding of anti-CDH17 antibodies to CDH17-overexpressing cells", different cells were incubated with gradient dilutions of ADC, incubated with secondary antibodies and washed, and then flow cytometry analysis was performed using iQue ScreenerPLUS, and the data were analyzed by GraphPad Prism.

[0157] like Figure 7 As shown, the results indicate that the ADC molecules provided by the present invention specifically bind to recombinant monkey CDH17 expressing cells, and simultaneously bind to gastric cancer cells AGS and colorectal cancer cells NCI-H508.

[0158] (II) Internalization of ADC in tumor cells

[0159] Referring to the method described in Example 2 (ii) "2) Internalization of anti-CDH17 antibodies in CDH17-overexpressing tumor cells", the internalization of ADC in CDH17-overexpressing tumor cells was evaluated, wherein a mixture of the antibody, internalization reagent, and diluent was incubated with tumor cells SK-CO-1 for different times (2 hours and 24 hours).

[0160] like Figure 8 As shown, the results indicate that the ADC molecules provided by the present invention exhibit good internalization activity at both 2 hours and 24 hours.

[0161] (III) ADC killing of tumor cells

[0162] SK-CO-1 colorectal cancer cells overexpressing CDH17 were plated in 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.

[0163] like Figure 9 As shown, the results indicate that the ADC molecules provided by the present invention exhibit significant killing activity on SK-CO-1 cells.

[0164] 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.13, SEQ ID NO.14, and SEQ ID NO.15, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, in sequence; (2) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, in sequence; (3) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO. 28, SEQ ID NO. 29, and SEQ ID NO. 30, in sequence; (4) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, in sequence; (5) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.39, in sequence; and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42, in sequence; or (6) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, respectively; 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, 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; or (6) 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. 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 . A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to claim 1 . A vector comprising the nucleic acid molecule according to claim 5 .

7. A host cell comprising the nucleic acid molecule according to claim 5 or the vector according to claim 6, or transformed or transfected with the nucleic acid molecule according to claim 5 or the vector according to claim 6.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the host cell according to claim 7 in the preparation of an antibody-drug conjugate (ADC).

9. 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 4; 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.

10. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7, or the antibody-drug conjugate or a salt thereof according to claim 9, and optionally a pharmaceutically acceptable excipient.

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