An antibody against cadherin-17 and antibody drug conjugate thereof

Abstract

The application provides an anti-CDH17 antibody or antigen binding fragment thereof, an antibody drug conjugate (ADC) targeting CDH17 and application thereof. The anti-CDH17 antibody provided by the application can specifically bind to CDH17 protein and has a relatively strong CDH17-expressing tumor cell killing effect, and therefore has a high specific treatment application potential for CDH17-expressing tumors. Further, the anti-CDH17 antibody provided by the application can be effectively internalized on CDH17-expressing tumor cells, and the ADC formed by coupling the antibody with a small molecule toxic compound maintains high internalization activity, strong binding capacity to the target protein CDH17, and shows high killing activity to CDH17-expressing tumor cells.

Description

Technical Field

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

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

[0003] Studies have shown that CDH17 is abnormally highly expressed in various tumors, particularly prominent in digestive system malignancies such as gastric cancer, colorectal cancer, and pancreatic cancer. Clinical studies have found that high CDH17 expression is often closely associated with tumor differentiation, invasiveness, metastasis, and poor prognosis, especially in advanced and metastatic stages of tumors, where its expression level tends to increase significantly. Compared with traditional chemotherapy drugs, CDH17-targeting adjuvants (ADCs) can reduce unnecessary side effects through their targeting mechanism, demonstrating their great potential as targeted therapies. Summary of the Invention

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

[0005] Therefore, one object of the present invention is to provide an antibody or 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 solution of the present invention is as follows.

[0007] First aspect

[0008] The present invention provides an antibody or antigen-binding fragment thereof against cadherin-17 (CDH17), wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to CDH17, particularly human CDH17.

[0009] In the context of this invention, unless otherwise stated, the term "CDH17" covers any form or structural region of CDH17.

[0010] In the context of this invention, the term "antigen-binding fragment" encompasses various functional fragments of the antibody that specifically binds to CDH17, which retain the antibody's ability to bind to the antigen and the corresponding biological activity. It is well known in the art that the antibody's ability to bind to the antigen and the corresponding biological activity can be achieved from fragments of the intact antibody, which can be obtained using conventional techniques known to those skilled in the art and screened for functionality in the same manner as for the intact antibody. For example, antigen-binding fragments of the antibody can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody.

[0011] Specifically, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3). According to a specific embodiment of the present invention, the heavy chain CDRs comprised in the anti-CDH17 antibody or its antigen-binding fragment are derived from the heavy chain variable region having any one of the amino acid sequences shown in SEQ ID NOs. 1, 3, 5, 7, 9, and 11, and from the light chain variable region having any one of the amino acid sequences shown in SEQ ID NOs. 2, 4, 6, 8, 10, 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 antibody provided in the "Detailed Description" section of this application. Using any one or a combination of antibody heavy chain or light chain complementarity-determining regions (CDRs) known in the art (e.g., Chothia, Kabat, IMGT, Contact, AbM, CCG, etc.), those skilled in the art can readily determine the heavy chain CDRs and light chain CDRs contained therein. Combinations of heavy chain CDRs and light chain CDRs can be obtained according to known or conventional definition tools in the art, and antibodies or fragments thereof containing each of these combinations of heavy chain CDRs and light chain CDRs are within the protection scope of this invention.

[0013] Preferably, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises heavy chain CDRs and light chain CDRs from the heavy chain variable region and light chain variable region shown in the following amino acid sequence pairings:

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

[0021] Accordingly, in the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention, the heavy chain CDRs and light chain CDRs are as follows:

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

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

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

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

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

[0027] (6) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48.

[0028] As described above, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention specifically binds to cadherin-17 (CDH17), preferably primate or rodent CDH17, such as human, monkey, or mouse CDH17. Optionally, the antibody or its antigen-binding fragment provided by the present invention may or may not have species cross-binding activity with human, cyno, or mouse CDH17.

[0029] Preferably, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), both of which include the aforementioned CDRs and the framework region (FR) therebetween. The arrangement of each region from the N-terminus to the C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0030] According to a specific embodiment of the present invention, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region and a light chain variable region as shown below:

[0031] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.1, or contains an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.2, or contains an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2;

[0032] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.3, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.4, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.4;

[0033] (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.5, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.5; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.6, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.6;

[0034] (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.7, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.8, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0035] (5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 10, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 10; or

[0036] (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.11, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.11; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.12, or contains an amino acid sequence that has at least 75% identity with the amino acid sequence shown in SEQ ID NO.12.

[0037] In the context of this invention, the term "at least 75% identity" in relation to amino acid sequences encompasses any percentage of identity between at least 75% and 100% identity, such as 75%, 80%, 85%, 90%, and even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% identity. The maximum 25% difference in amino acid sequence resulting from "at least 75% identity" can exist in any frame region within the heavy chain variable region or the light chain variable region, or in any domain or sequence outside the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment of this invention. Such differences can arise from amino acid deletions, additions, or substitutions at any position, wherein substitutions can be conservative or non-conservative.

[0038] Preferably, the anti-CDH17 antibody provided by the present invention can be a mouse antibody, rabbit antibody, or human antibody, or it can be a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody. The CDH17 antibody can also be a derivatized antibody, such as an antibody obtained by CDR transplantation, affinity maturation, point mutation modification, or chemical modification based on an initial mouse monoclonal antibody. The chemical modification includes glycosylation, acetylation, polyethylene glycol modification, phosphorylation, amidation, protease cleavage, linkage with cellular ligands or effector molecules, protection of active reactive groups, and / or blocking. Preferably, the antigen-binding fragment of the antibody can be a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized variable fragment (dsFv), (dsFv)2, an antigen-binding fragment (Fab), Fab' fragment (Fab'), (Fab' fragment)2 (F(ab')2), or a variable fragment (Fv), etc. Regarding the antigen-binding fragment of the antibody described in this invention, it can be any fragment of the antibody capable of specifically binding to CDH17.

[0039] In addition to the heavy chain and / or light chain variable regions, the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention may also include a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably including a human or mouse heavy chain constant region and / or a light chain constant region. Preferably, the anti-CDH17 antibody or its antigen-binding fragment includes a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region.

[0040] According to a specific embodiment of the present invention, the anti-CDH17 antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain constant region sequence of IgG1, such as the human IgG1 heavy chain constant region; and / or comprises a kappa light chain constant region, such as the human kappa light chain constant region.

[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 type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. More preferably, the antibody is 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 its antigen-binding fragment described herein.

[0044] The term "nucleotide sequence encoding the anti-CDH17 antibody or its antigen-binding fragment according to the present invention" refers to a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable regions, heavy chain variable regions, heavy chains, and / or light chains contained in the antibody or its antigen-binding fragment. For example, the nucleic acid molecule provided by the present invention contains nucleotide sequences encoding each of the heavy chain CDRs and light chain CDRs contained in the aforementioned antibody or its antigen-binding fragment; contains nucleotide sequences encoding the heavy chain variable regions and light chain variable regions contained in the aforementioned antibody or its antigen-binding fragment; or contains nucleotide sequences encoding the heavy chains and light chains contained in the aforementioned antibody or its antigen-binding fragment.

[0045] Third aspect

[0046] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in a third aspect, this invention also provides a vector containing the nucleic acid molecules of this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector, etc. The vectors or nucleic acid molecules of this invention can be used to transform or transfect host cells, for purposes such as preserving or expressing antibodies.

[0047] Fourth aspect

[0048] The present invention also provides a host cell comprising the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, or animal cells.

[0049] Fifth aspect

[0050] The anti-CDH17 antibody or its antigen-binding fragment provided by this invention can be obtained using any method known in the art. For example, this invention also provides a method for preparing the anti-CDH17 antibody or its antigen-binding fragment, the method comprising culturing the host cells provided by this invention while allowing the host cells to express the heavy and light chains of the antibody. Optionally, the method further includes the step of recovering the generated anti-CDH17 antibody.

[0051] Sixth aspect

[0052] The anti-CDH17 antibody or its antigen-binding fragment provided by the present invention can also be directly or indirectly linked to other parts, such as heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, and light chains of other antibodies; or, such as small molecule compounds, for example, cytotoxic compounds used in antibody-drug conjugates; or, such as cell surface receptors, sugars, polymers, etc., that modify the antibody or its antigen-binding fragment.

[0053] Accordingly, in a sixth aspect, the present invention also provides the use of the said anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, vector, or host cell in the preparation of antibody-drug conjugates (ADCs).

[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 its antigen-binding fragment provided by the present invention.

[0056] The antibody-drug conjugate can be formed by conjugating the anti-CDH17 antibody or its antigen-binding fragment provided by this invention with a cytotoxic compound. The cytotoxic compound can be a tubulin inhibitor, a topoisomerase inhibitor, a DNA binder, etc. For example, the tubulin inhibitor can be maytansine compounds such as DM1 and DM4, sea hare toxin compounds such as Monomethyl Dolastatin 10, MMAE, and MMAF, tubulolysin compounds, Cryptophycin derivatives, Taltobulin, muscarine, chalcogenide, eribulin, and derivatives of the aforementioned drugs; the topoisomerase inhibitor can be camptothecin compounds such as Dxd, exatecan, and their derivatives, doxorubicin metabolite PNU-159682 derivative, and irinotecan and its metabolite SN38, etc.; the DNA binder can be PBD derivatives and Duocarmycin and its derivatives, etc.

[0057] Eighth aspect

[0058] The anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell or antibody-drug conjugate or its salt provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical formulation, for use in various purposes as needed.

[0059] Therefore, the present invention also provides a composition comprising the anti-CDH17 antibody or its antigen-binding fragment provided by the present invention, a nucleic acid molecule, a carrier, a host cell, or an antibody-drug conjugate or its salt. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients. The pharmaceutical compositions provided by the present invention can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.

[0060] Ninth aspect

[0061] This invention also provides the use of the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition in the preparation of a medicament for the prevention, treatment and / or improvement of a disease that may be associated with CDH17 expression (including overexpression), such as CDH17-positive solid tumors. The anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition may exert their effects by binding to CDH17 to exert an ADCC effect or by the cytotoxic toxicity of cytotoxic compounds in the antibody-drug conjugate, but are not limited thereto.

[0062] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0063] Tenth aspect

[0064] The present invention also provides a method for preventing, treating and / or improving a disease, the method comprising administering to a subject in need an anti-CDH17 antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or a salt thereof or a combination thereof, the disease being associated with CDH17 expression (including overexpression), such as a CDH17-positive solid tumor.

[0065] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer. The subject may be a mammal, preferably a primate or rodent, such as a human.

[0066] The methods for preventing, treating and / or improving diseases provided by the present invention depend on a variety of factors when applied, including the specific active ingredient of the pharmaceutical composition applied, the patient's age, weight, sex or physical and medical condition, the severity of the disease to be treated, the route of administration, etc.

[0067] The method provided by this invention can also be used in combination with other drugs or methods. These other drugs or methods refer to those that can be administered in combination with the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition described in this invention, such as small molecule chemical drugs, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiotherapy. The combined administration of these two methods can be carried out in any form, such as simultaneously, continuously, or at intervals.

[0068] Eleventh aspect

[0069] The present invention also provides the use of the anti-CDH17 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition in the preparation of reagents for diagnosing diseases that may be associated with CDH17 expression (including overexpression), such as CDH17-positive solid tumors.

[0070] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0071] Twelfth aspect

[0072] The present invention also provides a method for diagnosing a disease, the method comprising contacting an anti-CDH17 antibody of the present invention or its antigen-binding fragment, a nucleic acid molecule, a carrier, a host cell, an antibody-drug conjugate or its salt or a combination thereof with a sample from a subject, wherein the disease or condition may be associated with CDH17 expression (including overexpression), such as a CDH17-positive solid tumor.

[0073] The disease or condition may be a tumor or cancer of the digestive system, such as stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer. The subject may be a mammal, preferably a primate or rodent, such as a human.

[0074] Thirteenth aspect

[0075] This invention provides a kit comprising the anti-CDH17 antibody of the present invention or its antigen-binding fragment, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or its salt or a combination thereof. The kit can be used for the above-mentioned prevention, treatment and / or improvement, or for the above-mentioned diagnosis. Depending on the intended application, the kit may also contain other reagents. For example, the kit is for detecting CDH17 expression (including overexpression) in any biological sample using ELISA.

[0076] This invention provides an anti-CDH17 antibody. The antibody provided by this invention does not specifically bind to CDH17-negative cells, but it can specifically bind to the CDH17 protein, such as recombinant cells overexpressing CDH17 protein or tumor cells endogenously expressing CDH17, thereby exhibiting highly specific tumor-targeting ability. Regarding the antibody's own mediated tumor-killing activity, the anti-CDH17 antibody provided by this invention has a relatively strong cell-killing effect.

[0077] Furthermore, experiments have demonstrated that the anti-CDH17 antibody provided by this invention can be effectively internalized on CDH17-expressing tumor cells. After conjugation with a small molecule toxic compound, the resulting antibody-drug conjugate maintains this highly efficient internalization activity, strong binding ability to the target protein, and exhibits high killing activity against CDH17-expressing tumor cells. Therefore, the anti-CDH17 antibody provided by this invention has significant application potential in tumor targeted therapy and ADC drug development. Attached Figure Description

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

[0079] Figure 1The results of the detection of the binding of the anti-CDH17 antibody provided by the present invention to recombinant human and monkey CDH17 overexpressing cells are shown; wherein, A and C: recombinant human CDH17 overexpressing cells, and B and D: recombinant monkey CDH17 overexpressing cells.

[0080] Figure 2 The results of the binding detection of the anti-CDH17 antibody provided by the present invention with CDH17-overexpressing tumor cells and CDH17-negative tumor cells are shown; wherein, A: gastric cancer cells AGS, B: pancreatic cancer cells Aspc-1, C: colorectal cancer cells SK-CO-1, and D: CDH17-negative cells.

[0081] Figure 3 The results of internalization detection of the anti-CDH17 antibody provided by the present invention on CDH17-overexpressing tumor cells are shown; wherein, A, B and D: gastric cancer cells AGS, and C: pancreatic cancer cells Aspc-1.

[0082] Figure 4 The results of the detection of the killing effect of the anti-CDH17 antibody provided by the present invention on CDH17-overexpressing tumor cells are shown.

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

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

[0085] Figure 7 The results of the binding detection of the antibody-drug conjugate targeting CDH17 provided by the present invention with recombinant CDH17-expressing cells and tumor cells are shown; wherein, A and 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: binding of CHOK1 empty cells.

[0086] Figure 8 The results of the detection of the internalization of the antibody-drug conjugate targeting CDH17 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 detection of the killing effect of the antibody-drug conjugate targeting CDH17 provided by the present invention on tumor cells SK-CO-1 are shown. Detailed Implementation

[0088] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0089] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0090] The hIgG1 used in the examples is an irrelevant 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 immunizations: the first was a subcutaneous injection of Freund's complete adjuvant (CFA) emulsified antigen (50 μg / mouse), followed by two injections of Freund's incomplete adjuvant (IFA) emulsified antigen (25 μg / mouse). The immunization intervals were 2-3 weeks, and blood samples were collected one week after immunization to detect antigen binding activity. Mice showing high binding activity in their serum were selected for hybridoma fusion.

[0093] After booster immunization of the selected mice, spleen cells were electrofused with mouse myeloma cells SP20. The cells were then cultured in HAT and HT media for 10-14 days, and hybridoma supernatant was obtained. Protein or cellular binding in the hybridoma supernatant was screened using ELISA or FACS methods, 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. After incubation, the plates were washed and blocked, then hybridoma supernatant (100 μL / well) was added and incubated before detection. For cell-level screening, CHOK1 cells overexpressing human CDH17 (UniProtKB accession number: Q12864) were used. After positive clones were selected, further screening was performed using recombinant human CDH17, monkey CDH17 (UniProtKB accession number: A0A1D5R2B4) expressing cells, and tumor cells endogenously expressing CDH17.

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

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

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

[0098] Total RNA was extracted from monoclonal hybridoma cells using Trizol reagent, and then the RNA was reverse transcribed into cDNA. The variable regions (VH / VL) of the heavy and light chains were amplified by PCR, sequenced, and compared with the IMGT database to extract the sequence information of CDR1, CDR2, and CDR3, ultimately determining the effective antibody VH / VL sequences.

[0099] The final selected VH / VL sequences were cloned into the pTT5 expression vector (containing the hIgG1 heavy chain and kappa light chain constant regions) to construct heavy and light chain plasmids. The heavy and light chain plasmids were co-transfected into HEK293 cells at a 2:3 ratio using PEI transfection. After 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 then filtered again through a 0.22 μm filter. The antibody concentration was then determined.

[0100] The heavy and light chain variable region sequences of different antibodies are shown in Table 1 (the bold and underlined parts are CDRs, which are divided according to the CCG definition).

[0101] Table 1. Anti-CDH17 antibodies

[0102]

[0103]

[0104]

[0105] (II) Identification of antibody activity of anti-CDH17 antibody

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

[0107] Cellular-level antibody binding was detected using CHOK1 cells (Genomeditech, GM-C25980) overexpressing human CDH17, HEK293 cells (Genomeditech, GM-C28746) overexpressing macaque CDH17, AGS gastric cancer cells, Aspc-1 pancreatic cancer cells, SK-CO-1 colorectal cancer cells, and RKO cells that were negative for CDH17.

[0108] CDH17 expression cells in good growth condition were collected, seeded into 96-well plates, and incubated with serially diluted antibodies at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with PBS, then a 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed three more times and resuspended in PBS. Finally, flow cytometry readings were performed using iQue Screener PLUS, and the data were analyzed using GraphPad Prism. The control antibody was PTA001-A4 (patent US20160039933A1), which was constructed and expressed according to the corresponding VH / VL sequence in the patent.

[0109] like Figure 1 and Figure 2 As shown, the results indicate that the antibodies provided by this invention can specifically bind to CDH17 overexpressing cells, but not to CDH17 negative expression cells (RKO).

[0110] 2) Internalization of anti-CDH17 antibody on CDH17-overexpressing tumor cells

[0111] The internalization effect of antibodies was assessed using a commercially available antibody internalization kit (Sartorius, 90565).

[0112] First, the concentrations of both the test antibody and the internalization reagent were adjusted to 100 μg / ml. Then, a mixture of 1 μl of test antibody, 1 μl of internalization reagent, and 48 μl of diluent was prepared and incubated at 37°C for 15 minutes. After incubation, the mixture was serially diluted twofold. Separately, the cell density of CDH17-expressing tumor cells was adjusted to 2E6 / ml, and 20 μl / well was seeded into 96-well plates, along with the diluted antibody-internalization reagent mixture (20 μl / well). The 96-well plates were then incubated at 37°C with 5% CO2 for 24 hours. Cells were collected the following day, and flow cytometry analysis was performed using iQue Screener PLUS, with data analysis conducted using GraphPadPrism.

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

[0114] 3) Killing of CDH17-overexpressing tumor cells by anti-CDH17 antibodies

[0115] The cytotoxic activity of the antibody was assessed using the commercially available reagent αHFc-CL-MMAE (Moradec, AH-102AE-50).

[0116] First, adjust the concentration of CDH17-expressing cells (e.g., SK-CO-1) to 1E5 cells / ml, then seed 50 μl / well into 96-well blank plates and incubate for 4 hours. Next, add serially diluted antibody, 10 μl per well; simultaneously add 40 μl of diluted αHFc-CL-MMAE (2.5 μg / ml). Then, incubate the 96-well blank plates at 37°C and 5% CO2 for 4 days. After incubation, add 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) to each well, and then shake the plate on a shaker for 5 minutes. Detect relative fluorescence units (RLU) using a Spectra M5e instrument. Kill percentage % = (RLU) 未处理细胞 -RLU Sample ) / RLU 未处理细胞 ×100%.

[0117] like Figure 4 As shown in Table 2, the results indicate that the antibodies provided by this invention exhibit good killing activity on tumor cells, and are superior to the control antibodies.

[0118] Table 2. Results of antibody cytotoxic activity assay

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

[0124] First, recombinant proteins of different domains of human CDH17, namely CDH17 EC1-2 his (Kactusbio, CDH-HM1D5), CDH17 EC3-4 mFc (Kactusbio, CDH-HM3D3), CDH17 EC5-7his (Kactusbio, CDH-HM1D4), CDH17 EC1-6 his (Kactusbio, CDH-HM1D1), and CDH17 EC1-7 his (Acro Biosystems, CA7-H52H3), were coated into 96-well plates at a concentration of 1 μg / ml, 100 μL per well, and incubated overnight. The next day, the 96-well plates were washed three times with 1×PBST, then blocked by adding 1×PBST containing 1% BSA and incubating at 37°C for 1 hour, followed by washing three times with 1×PBST. Next, serially diluted test antibodies of different concentrations were added and incubated at 37°C for 1 hour. Then, a 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, 109-035-098) was added and incubated at 37°C for 1 hour. The 96-well plate was then washed with 1×PBST, followed by the addition of TMB substrate for color development, and finally the reaction was terminated with 1N HCl. OD values ​​were measured at 450 nm.

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

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

[0127] The binding properties of antibodies to different monkey CDH17 proteins were evaluated using ELISA binding assays.

[0128] First, different monkey CDH17 proteins (Rhesus CDH17 [Kactusbio, CDH-RM117], Cyno CDH17 [Kactusbio, CDH-CM127]) were coated into 96-well plates at a concentration of 1 μg / ml, 100 μL per well, and incubated overnight. The next day, the 96-well plates were washed three times with 1×PBST, then blocked by adding 1×PBST containing 1% BSA and incubating at 37°C for 1 hour, followed by three more washes with 1×PBST. Then, serially diluted test antibodies were added and incubated at 37°C for 1 hour; finally, a 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson Immuno Research, 109-035-098) was added and incubated at 37°C for 1 hour. The 96-well plate was then washed with 1×PBST, followed by the addition of TMB substrate for color development, and finally the reaction was terminated with 1N HCl. The OD value was measured at 450 nm.

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

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

[0131] The hydrophobicity of the anti-CDH17 antibody was evaluated using the HIC-HPLC method.

[0132] A Waters HPLC system equipped with a TSK gel Butyl-NPR column (2.5 μm, 4.6 mm × 35 mm, TOSOH) was used. The antibody to be tested was diluted to 1.0 mg / mL in PBS, and 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 point of absorption peak appearance. Hydrophobicity was calculated using the following formula: (NH4)2SO4 concentration (M) = (23 - time + 1.25) × 0.101, where "time" is the time in minutes for the peak to appear (retention time). According to this formula, when the calculated concentration of (NH4)2SO4 exceeds 0.8M, the tested antibody is considered to be hydrophilic.

[0133] As shown in Table 3, the results indicate that the antibody provided by this invention has good hydrophilicity.

[0134] Table 3. Results of antibody hydrophobicity assessment

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

[0139]

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

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

[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℃;

[0146] Sample loading volume: 20 μL;

[0147] Washing 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 assessment of antibody-drug conjugates

[0154] Using cells with different CDH17 expression levels, the binding ability, internalization properties, and cytotoxic activity of the prepared antibody-drug conjugates were evaluated.

[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 antibody to CDH17 overexpressing cells", different cells were incubated with serially diluted ADCs. After incubation with secondary antibody and washing, cell flow cytometry analysis was performed using iQue ScreenerPLUS, and the data were analyzed using GraphPad Prism.

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

[0158] (II) Internalization of ADCs on tumor cells

[0159] Referring to the method described in Example 2 (II) "2) Internalization of anti-CDH17 antibody on CDH17 overexpressing tumor cells", the internalization of ADC on CDH17 overexpressing tumor cells was evaluated, wherein the mixture of antibody with 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 molecule provided by this invention exhibits good internalization activity at both 2 hours and 24 hours.

[0161] (III) Killing of tumor cells by ADCs

[0162] CDH17-overexpressing colorectal cancer cells SK-CO-1 were seeded into 96-well blank plates. Serially diluted ADC was added, and the plates were incubated at 37°C in CO2 for 7 days. After incubation, 100 μl of Bio-Lite Luciferase Assay System reagent (Vazyme, DD1201-03) was added to each well, and the plates were shaken for 5 minutes. Relative fluorescence units (RLUs) were measured using a Spectra M5e instrument, and the data were analyzed using a GraphPad Prism.

[0163] like Figure 9 As shown, the results indicate that the ADC molecule provided by this invention exhibits significant killing activity in SK-CO-1 cells.

[0164] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof against cadherin-17 (CDH17), comprising heavy chain complementarity determining regions (CDRs), i.e. heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), and light chain complementarity determining regions (CDRs), i.e. light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), light chain CDR3 (LCDR3), which are as follows: (1) HCDR1, HCDR2, HCDR3 comprising the amino acid sequences of SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 in sequence; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 in sequence; (2) HCDR1, HCDR2, HCDR3 comprising the amino acid sequences of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 in sequence; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24 in sequence; (3) HCDR1, HCDR2, HCDR3 comprising the amino acid sequences of SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 in sequence; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30 in sequence; (4) HCDR1, HCDR2, HCDR3 comprising the amino acid sequences of SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 in sequence; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 in sequence; (5) HCDR1, HCDR2, HCDR3 comprising the amino acid sequences of SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 in sequence; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences of SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42 in sequence; or ​ ​ ​ ​ ​ (6) HCDR1, HCDR2, HCDR3 comprising the amino acid sequences set forth in SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, respectively; and, LCDR1, LCDR2, LCDR3 comprising the amino acid sequences set forth in SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-CDH17 antibody or antigen-binding fragment thereof specifically binds to cadherin-17 (CDH17).

3. The antibody or antigen-binding fragment thereof of claim 2, wherein, The CDH17 is primate or rodent CDH17.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein, The CDH17 is human, monkey, or murine CDH17.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-CDH17 antibody or antigen-binding fragment thereof has or does not have species cross-binding activity to human, monkey, or murine CDH17.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-CDH17 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, which are as follows: (1) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 1; and, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 2; (2) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 3; and, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 4; (3) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 5; and, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 6; (4) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 7; and, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 8; (5) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 9; and, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 10; or (6) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 11; and, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO.

12.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein, The anti-CDH17 antibody is a murine 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 scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv fragment of the antibody.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein, The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL).

9. The antibody or antigen-binding fragment thereof of claim 7, wherein, The antibody or antigen-binding fragment thereof further comprises a human or murine heavy chain constant region and / or a light chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 7, wherein, The antibody or fragment thereof further comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a kappa or lambda type light chain constant region.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein, The anti-CDH17 antibody is a monoclonal antibody.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein, The anti-CDH17 antibody is an immunoglobulin.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein, The immunoglobulin is of the type human IgA, IgD, IgE, IgG or IgM.

14. The antibody or antigen-binding fragment thereof of claim 12, wherein, The antibody is of the subtype human IgG1 or IgG4.

15. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising or being transformed or transfected with the nucleic acid molecule of claim 15 or the vector of claim 16.

18. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the nucleic acid molecule of claim 15, the vector of claim 16 or the host cell of claim 17 for the manufacture of an antibody drug conjugate (ADC).

19. An antibody drug conjugate targeting CDH17 or a salt thereof, comprising the anti- CDH17 antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

20. The antibody drug conjugate or salt thereof according to claim 19, characterized by The antibody drug conjugate further comprises a tubulin inhibitor.

21. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17 or the antibody drug conjugate or salt thereof of claim 19 or 20, and optionally a pharmaceutically acceptable excipient.

22. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17, the antibody drug conjugate or salt thereof of claim 19 or 20 or the composition of claim 21 for the manufacture of a medicament for the treatment of a disease, which disease is gastric cancer, colorectal cancer or pancreatic cancer.

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