Ligand-cytotoxic drug conjugates and their medical use

Abstract

This invention provides an antitumor drug with excellent therapeutic effects, exhibiting superior antitumor efficacy and safety. It also provides an anti-cadherin 17 antibody comprising a heavy chain variable region and a light chain variable region, a drug conjugate thereof, and a composition containing an anti-cadherin 17 antibody or a drug conjugate thereof, and their use as a drug.

Description

Technical Field

[0001] This invention relates to novel cadherin 17 antibodies comprising engineered heavy and light chains, or functional fragments thereof. The invention also relates to conjugates of improved cadherin 17 antibodies with small molecule drugs. Furthermore, the invention relates to the use of these antibodies and their conjugates in the preparation of medicaments for treating cancer. Background Technology

[0002] cadherin 17 (CDH17) is a cell surface marker belonging to the cadherin superfamily and possesses a unique biological structure. Compared to the classic five-repeated cadherin, it has seven extracellular cadherin repeats and a very short intracellular region of 20 amino acid residues, lacking a conserved intracellular domain (Berndorff et al., J CellBiol. 1994, 125(6):1353-1369). Although the biological function of CDH17 is not fully explored, it has been reported that CDH17 can be used to... 2+ CDH17 regulates water uptake in a dependent manner (Ahl et al., Biol. Med. Model. 2011, 8(18)), and also participates in maintaining tissue integrity by interacting with integrins within extracellular tight junctions. It is primarily expressed in the human gastrointestinal (GI) tract and pancreas.

[0003] CDH17 has been reported to be highly expressed at both the DNA and protein levels in tumors, including colorectal, gastric, and pancreatic tumors (Takamura et al., Med Mol Morphol. 2013, 46:1-7). It plays a crucial role in regulating cancer metastasis and tumor growth. The tumor activity manipulated by CDH17 involves multiple signaling pathways. One of the most important mechanisms involves CDH17-integrin interaction. It has been demonstrated that the RDG motif of CDH17 binds to α2β1 integrin, inducing β1 integrin activation, which leads to increased cancer cell proliferation and adhesion (Bartomome et al., J Biol Chem. 2014, 289(50):34801-34914). Studies have also shown that CDH17 regulates cancer invasion in GI cancers through the Wnt / β-catenin signaling pathway (Qiu et al., PloS one 2013, 8(3)) and the NFκB signaling pathway (Wang et al., Cancer biology & therapy. 2013, 14(3):262-270). The limited expression of CDH17 in normal tissues and its high expression in various cancers make CDH17 a promising cancer target.

[0004] Several CDH17-targeting antibody-based drugs have been investigated in this field. Two bispecific antibody drugs, BI905711 (Boehringer Ingelheim) and ARB202 (Arbele), are undergoing Phase I trials, while other anti-CDH17 CAR (chimeric antigen receptor) and monoclonal drugs are in preclinical trials. However, there are currently no reports of anti-CDH17 antibody-drug conjugates (ADCs). This invention presents a first-in-class anti-CDH17 ADC.

[0005] Antibody-drug conjugates (ADCs) represent a new class of therapies that involve antibodies conjugated to cytotoxic drugs via chemical linkers. The therapeutic concept of ADCs is to combine the binding ability of antibodies with that of drugs, whereby the antibody delivers the drug to tumor cells by binding to antigens on the target surface.

[0006] Therefore, there remains a need in the art for therapeutic anti-CDH17 antibodies and ADCs that can be used to treat cancers expressing CDH17. Such conjugates exhibit better biological activity, stability, homogeneity, and lower toxicity. Summary of the Invention

[0007] This invention provides anti-CDH17 antibodies and antibody-drug conjugates (ADCs), as well as methods of using them. The anticancer efficacy of antibody-drug conjugates is believed to depend on the uptake of surface antigens by cancer cells expressing them; therefore, insufficient internalization of monoclonal antibodies targeting CDH17 is a pressing issue. Generally, the development of ADCs requires consideration of all these key components, including the selection of target antigens, antibodies, toxic drugs, and adaptors.

[0008] The technical problem this invention aims to solve is to develop advanced anti-CDH17 antibody-drug conjugates that exhibit very strong anti-tumor activity in cancer. The anti-tumor activity may be due to higher DAR and robust endocytosis.

[0009] Specifically, the present invention includes the following aspects: This disclosure relates to antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof and their pharmaceutical uses, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof comprises (optionally via a linker) an anti-CDH17 antibody or an antigen-binding fragment thereof conjugated to a toxin drug.

[0010] This disclosure relates to antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof and their pharmaceutical uses, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof comprises (optionally via a linker) an anti-CDH17 antibody or antigen-binding fragment thereof conjugated to a toxin drug, wherein the anti-CDH17 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region of the antibody, wherein: a) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 01, SEQ ID NO: 11, and SEQ ID NO: 22, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 32, SEQ ID NO: 43, and SEQ ID NO: 48, respectively; or b) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 02, SEQ ID NO: 12, and SEQ ID NO: 23, respectively; and the light chain variable region comprises amino acid sequences as shown in SEQ ID NO: 01, SEQ ID NO: 11, and SEQ ID NO: 22, respectively; and the light chain variable region comprises amino acid sequences as shown in SEQ ID NO: 02, SEQ ID NO: 12, and SEQ ID NO: 23, respectively; and the light chain variable region comprises amino acid sequences as shown in SEQ ID NO: 02, SEQ ID NO: 11, and SEQ ID NO: 2 ... LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 49; or c) the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 03, SEQ ID NO: 13, and SEQ ID NO: 24, respectively; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 43, and SEQ ID NO: 50, respectively; or d) the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 04, SEQ ID NO: 14, and SEQ ID NO: 25, respectively; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 35, SEQ ID NO: 43, and SEQ ID NO: 49, respectively; or e) the heavy chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 05, SEQ ID NO: 15, and SEQ ID NO: 49, respectively. HCDR1, HCDR2, and HCDR3 with the amino acid sequences shown in SEQ ID NO: 26; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively; or f) the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 06, SEQ ID NO: 16, and SEQ ID NO: 27, respectively;and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 43, and SEQ ID NO: 52, respectively; or g) the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; or h) the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 01, SEQ ID NO: 18, and SEQ ID NO: 22, respectively; and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 43, and SEQ ID NO: 48, respectively; or i) the heavy chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 43, and SEQ ID NO: 52, respectively; and i) the heavy chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 43, and SEQ ID NO: 48, respectively; and i) the heavy chain variable region includes LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 08, SEQ ID NO: 19, and SEQ ID NO: 29; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 40, SEQ ID NO: 46, and SEQ ID NO: 51, respectively; or j) the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 09, SEQ ID NO: 20, and SEQ ID NO: 30, respectively; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 52, respectively; or k) the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 21, and SEQ ID NO: 31, respectively; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 47, and SEQ ID NO: 51, respectively. The amino acid sequences shown in NO:54 are LCDR1, LCDR2, and LCDR3.

[0011] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, is a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a multispecific antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a recombinant antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0012] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, comprises a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 55-65 or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 66-76 or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith.

[0013] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, comprises a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 65, 61, 59 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; and / or a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 76, 72, 70 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith.

[0014] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, comprises: a) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 55 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 66 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or b) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 56 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 67 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or c) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 5 ... The amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or d) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or e) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 69 ... NO: 70, having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence thereon; or f) a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence thereon; and / or a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence thereon; or g) a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence thereon;And / or light chain variable region having an amino acid sequence as shown in SEQ ID NO: 72 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or h) heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 62 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or light chain variable region having an amino acid sequence as shown in SEQ ID NO: 73 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or i) heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 63 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or light chain variable region having an amino acid sequence as shown in SEQ ID NO: 74 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or j) heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 72 ... The amino acid sequence shown in SEQ ID NO: 64 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 75 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 76 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it.

[0015] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprises: a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 55; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 66; or b) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 56; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 67; or c) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 57; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 68; or d) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 69; or e) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 70; or f) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 69; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 70; or f) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 69; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 69; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 70; and ... The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 60; and / or the light chain variable region of the amino acid sequence shown in SEQ ID NO: 71; or g) the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 61; and / or the light chain variable region of the amino acid sequence shown in SEQ ID NO: 72; or h) the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 62; and / or the light chain variable region of the amino acid sequence shown in SEQ ID NO: 73; or i) the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 63; and / or the light chain variable region of the amino acid sequence shown in SEQ ID NO: 74; or j) the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 64; and / or the light chain variable region of the amino acid sequence shown in SEQ ID NO: 75; or k) the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 65; and / or the light chain variable region of the amino acid sequence shown in SEQ ID NO: 76.

[0016] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprises: a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 55; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 66; or b) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 56; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 67; or c) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 57; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 68; or d) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 69; or e) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 70; or f) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 6 ... The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 60; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 71; or g) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 72; or h) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 62; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 73; or i) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 63; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 74; or j) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 64; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 75; or k) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 76.

[0017] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, comprises a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region of the human antibody constant region is selected from the κ and λ chain constant regions of human antibodies and their conventional variants; more preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 99 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith, and the human light chain constant region of SEQ ID NO: 100 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; further preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 99 and the human light chain constant region of SEQ ID NO: 100.

[0018] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 97 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and a light chain having an amino acid sequence as shown in SEQ ID NO: 98 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or Heavy chains having the amino acid sequence shown in SEQ ID NO: 89 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it, and light chains having the amino acid sequence shown in SEQ ID NO: 90 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or Heavy chains having an amino acid sequence as shown in SEQ ID NO: 85 or having an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the sequence therein, and light chains having an amino acid sequence as shown in SEQ ID NO: 86 or having an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the sequence therein.

[0019] In some embodiments of this disclosure, the anti-CDH17 antibody or antigen-binding fragment according to any of the foregoing embodiments, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, comprises: a) a heavy chain having the amino acid sequence shown in SEQ ID NO: 77 and a light chain having the amino acid sequence shown in SEQ ID NO: 78; or b) a heavy chain having the amino acid sequence shown in SEQ ID NO: 79 and a light chain having the amino acid sequence shown in SEQ ID NO: 80; or c) a heavy chain having the amino acid sequence shown in SEQ ID NO: 81 and a light chain having the amino acid sequence shown in SEQ ID NO: 82; or d) a heavy chain having the amino acid sequence shown in SEQ ID NO: 83 and a light chain having the amino acid sequence shown in SEQ ID NO: 84; or e) a heavy chain having the amino acid sequence shown in SEQ ID NO: 85 and a light chain having the amino acid sequence shown in SEQ ID NO: 86; or f) a heavy chain having the amino acid sequence shown in SEQ ID NO: 87 and a light chain having the amino acid sequence shown in SEQ ID NO: 88; or g ...9 and a light chain having the amino acid sequence shown in SEQ ID NO: 80; or c) a heavy chain having the amino acid sequence shown in SEQ ID NO: 81 and a light chain having the amino acid sequence shown in SEQ ID NO: 82; or d) a heavy chain having the amino acid sequence shown in SEQ ID NO: 83 and a light chain having the amino acid sequence shown in SEQ ID NO: 84; or e) a heavy chain having the amino acid sequence shown in SEQ ID NO: 85 and a light chain having the amino acid sequence shown in SEQ ID NO: 86; or f) a heavy The heavy chain having the amino acid sequence shown in SEQ ID NO: 89 and the light chain having the amino acid sequence shown in SEQ ID NO: 90; or h) the heavy chain having the amino acid sequence shown in SEQ ID NO: 91 and the light chain having the amino acid sequence shown in SEQ ID NO: 92; or i) the heavy chain having the amino acid sequence shown in SEQ ID NO: 93 and the light chain having the amino acid sequence shown in SEQ ID NO: 94; or j) the heavy chain having the amino acid sequence shown in SEQ ID NO: 95 and the light chain having the amino acid sequence shown in SEQ ID NO: 96; or k) the heavy chain having the amino acid sequence shown in SEQ ID NO: 97 and the light chain having the amino acid sequence shown in SEQ ID NO: 98.

[0020] In some embodiments of this disclosure, an anti-CDH17 antibody or antigen-binding fragment is contained in an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CDH17 antigen-binding fragment is selected from: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (dimeric antibody), disulfide-stabilized Fv (dsFv), and peptides containing CDR.

[0021] In some embodiments of this disclosure, the toxic agent in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments is selected from microtubule inhibitors, topoisomerase inhibitors, DNA intercalators, and RNA polymerase inhibitors or their pharmaceutically acceptable salts, esters, or analogs; preferably, the toxic agent is selected from olipattin analogs, camptothecin derivatives, and maytansine analogs; more preferably, the toxic agent is selected from MMAE, MMAF, exatecan, MMAD, DM1, DM4, eribulin, pyrrolobenzodiazepines (PBD), DGN-549-C, S N-38, irinotecan, topotecan, belotecan, rubitecan, doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, α-amanitin, or pharmaceutically acceptable salts, esters, or analogs thereof.

[0022] In some embodiments of this disclosure, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments is a drug conjugate of general formula (A): Wherein: L1 and L2 are connecting units; y is a number selected from 1 to 10, preferably from 2 to 8, and most preferably 2, 4, 5, 6, 7, 8; Ab is the above-mentioned anti-CDH17 antibody or its antigen-binding fragment.

[0023] In some embodiments of this disclosure, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments is an antibody-drug conjugate of general formula (I): , Wherein: L1 and L2 are connecting units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, and most preferably 2, 4, 6, 8; Ab is the above-mentioned anti-CDH17 antibody or its antigen-binding fragment.

[0024] In some embodiments of this disclosure, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments, wherein L1 is selected from: and .

[0025] In some embodiments of this disclosure, in the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments, wherein L2 is -L a -L b -L c -L d -,in The L a As shown in general formula (II-a): , Where: s 1 and s 2 Each is an integer selected independently from 0-8, preferably, s 1 and s 2 Independently selected from 1, 2, 3, 4, 5, or 6; or, s 1 s is an integer from 1 to 8. 2 The value is 0, preferably s 1 Selected from 4, 5, 6, 7 or 8, s 2 It is 0; or, s 2 Integers selected from 2 to 8, s 1 The value is 2, preferably s 2 Selected from 2, 3, 4, 5, or 6, s 1 2; L b For chemical bonds; L c It is a tetrapeptide residue; preferably, L c It is a tetrapeptide residue of glycine-glycine-phenylalanine-glycine (GGFG); L d For –NR1(CR2R3)s 3 - where R1, R2, and R3 may be the same or different, and each is independently hydrogen or alkyl, s 3 It is 1 or 2; where L a The end connects to Ab, L d The end is connected to L1.

[0026] In some embodiments of this disclosure, antibody-drug conjugates of general formula (I), or pharmaceutically acceptable salts or solvates thereof, ; in: L1 and L2 are connection units; where L2 is -La-Lb-Lc-Ld-, La is shown in general formula (II-b): s1 is selected from 4, 5, 6, 7 or 8; Lb is a chemical bond; Lc is a tetrapeptide residue; preferably, Lc is a tetrapeptide residue of glycine-glycine-phenylalanine-glycine (GGFG). Ld is –NR1(CR2R3)s3-, where R1, R2, and R3 are the same or different and are each independently hydrogen or alkyl, and s3 is 1 or 2; wherein the La end is connected to Ab, and the Ld end is connected to L1. y is a number selected from 1 to 10, preferably from 2 to 8, and most preferably 2, 4, 6, or 8; Ab is an anti-CDH17 antibody or its antigen-binding fragment, wherein the anti-CDH17 antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region of the antibody, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 21, and SEQ ID NO: 31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 47, and SEQ ID NO: 54, respectively; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 05, SEQ ID NO: 15, and SEQ ID NO: 26, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively.

[0027] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a multispecific antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a recombinant antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0028] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 65, 61, 59 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; and / or a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 76, 72, 70 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith.

[0029] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 76 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 61 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 72 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 59 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 65 ... NO:70, the light chain variable region of the amino acid sequence shown or the amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it.

[0030] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 76; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 72; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59; and / or a light chain variable region having the amino acid sequence shown in SEQ ID NO: 70.

[0031] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 65 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 76; or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 61 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 72; or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 59 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 70.

[0032] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment further comprises a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region of the human antibody constant region is selected from the κ chain and λ chain constant regions of human antibodies and their conventional variants; more preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 99 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith, and the human light chain constant region of SEQ ID NO: 100 or having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; more preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 99 and the human light chain constant region of SEQ ID NO: 100.

[0033] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 97 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and a light chain having an amino acid sequence as shown in SEQ ID NO: 98 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or a heavy chain having an amino acid sequence as shown in SEQ ID NO: 89 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and a light chain having an amino acid sequence as shown in SEQ ID NO: 90 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith; or a heavy chain having an amino acid sequence as shown in SEQ ID NO: 85 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and a light chain having an amino acid sequence as shown in SEQ ID NO: 98. NO: 86, or a light chain of an amino acid sequence that has at least 80%, 85%, 90%, 95%, or 99% sequence identity with it.

[0034] In some embodiments of this disclosure, the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain having the amino acid sequence shown in SEQ ID NO: 97 and a light chain having the amino acid sequence shown in SEQ ID NO: 98; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 89 and a light chain having the amino acid sequence shown in SEQ ID NO: 90; or a heavy chain having the amino acid sequence shown in SEQ ID NO: 85 and a light chain having the amino acid sequence shown in SEQ ID NO: 86.

[0035] In some embodiments of this disclosure, the anti-CDH17 antigen-binding fragment is selected from: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerized domain V (biantibody), disulfide bond-stabilized Fv (dsFv), and peptides containing CDR.

[0036] In some embodiments of this disclosure, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments has the following structure: , y is a number selected from 1 to 10, preferably from 2 to 8, and most preferably 2, 4, 5, 6, 7, or 8; Ab is the above-mentioned anti-CDH17 antibody or its antigen-binding fragment.

[0037] In some embodiments of this disclosure, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any of the foregoing embodiments is selected from the following compounds: Wherein: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8, further preferably a number selected from 4 to 6 or 6 to 8, and most preferably a number selected from 4, 5, 6, 7 or 8.

[0038] In another aspect, this disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate according to any of the foregoing embodiments and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0039] In another aspect, this disclosure provides the use of an antibody-drug conjugate or a pharmaceutical composition comprising the conjugate according to any of the foregoing embodiments as a medicament.

[0040] In another aspect, this disclosure provides the use of antibody-drug conjugates or pharmaceutical compositions comprising them according to any of the foregoing embodiments in the preparation of medicaments for treating and / or preventing CDH17-mediated diseases or conditions.

[0041] In some embodiments of this disclosure, the CDH17-mediated disease or condition is a tumor or cancer. The tumor or cancer is associated with high CDH17 expression. Alternatively, the tumor or cancer is associated with moderate CDH17 expression.

[0042] In another aspect, this disclosure provides the use of an antibody-drug conjugate or a pharmaceutical composition comprising the conjugate according to any of the foregoing embodiments in the preparation of a medicament for treating or preventing tumors or cancer; preferably, the tumor or cancer is gastrointestinal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, gastric cancer, intestinal cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, or glioblastoma.

[0043] On the other hand, this disclosure also relates to methods for treating and / or preventing tumors or cancer, wherein the method includes administering a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutical composition comprising the conjugate to a patient in need of the conjugate; preferably, the tumor or cancer is associated with high CDH17 expression, or the tumor or cancer is associated with moderate CDH17 expression.

[0044] On the other hand, this disclosure also relates to methods for treating or preventing tumors or cancer, wherein the method comprises administering to a patient in need a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutical composition comprising the conjugate according to any of the foregoing embodiments; preferably, the tumor or cancer is gastrointestinal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, gastric cancer, intestinal cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, or glioblastoma.

[0045] The active compound (e.g., a ligand-drug conjugate according to this disclosure, or a pharmaceutically acceptable salt or solvate thereof) can be formulated in a form suitable for administration via any suitable route, preferably in a unit dose or in a single dose form that can be administered by the subject. The unit dose of this disclosure can be a tablet, capsule, sac, vial, powder, granules, lozenge, suppository, regenerated powder, or liquid formulation.

[0046] The dosage of the active compound or composition used in the treatment methods disclosed herein will generally vary depending on the severity of the disease, the subject's weight, and the efficacy of the active compound. However, as a general guideline, a suitable unit dose may be from 0.01 to 1000 mg.

[0047] In addition to the active compound, the pharmaceutical compositions disclosed herein may contain one or more excipients selected from the group consisting of fillers, diluents, binders, wetting agents, disintegrants, and other excipients. Depending on the method of administration, the composition may contain 0.01 to 99.9% by weight of the active compound.

[0048] Beneficial effects of the invention: The CDH17 antibody and antibody-drug conjugate disclosed herein exhibit good affinity for cell surface antigens, good endocytosis efficiency, high tumor inhibition efficiency, and a wider drug application window, making them suitable for clinical drug applications. Attached Figure Description

[0049] Figure 1 In vitro binding characterization of hybridoma clones with AsPC1 (A), GP2d (B), and SW480 (C) cells by flow cytometry analysis.

[0050] Figure 2 Indirect killing assays were used to characterize the cellular internalization activity of selected hybridoma clones.

[0051] Figure 3 The in vitro binding characteristics of the anti-CDH17 recombinant antibody to AsPC1 cells (A) and SW480 cells (B) were determined by flow cytometry, with HBMAB81 as a positive control and B12 as an isotype control antibody (C).

[0052] Figure 4 The dose-response curve of internalization of the anti-CDH17 recombinant antibody was determined by indirect killing assay.

[0053] Figure 5 Competitive combinations: (A) 20B4; (B) HBMAB81.

[0054] Figure 6 Dose-response curves of the in vitro cytotoxic activity of ADCs against tumor cells.

[0055] Figure 7 Comparison of in vitro cytotoxicity of anti-CDH17 ADCs with different DAR values ​​in GP2d cell lines.

[0056] Figure 8 Comparison of in vitro cytotoxicity of anti-CDH17 ADCs with different DAR values ​​in the AsPC1 cell line.

[0057] Figure 9 Comparison of in vitro cytotoxicity of anti-CDH17 ADCs with different DAR values ​​in the SK-CO-1 cell line.

[0058] Figure 10 Comparison of in vitro cytotoxicity of anti-CDH17 ADCs with different DAR values ​​in the AsPC1 cell line.

[0059] Figure 11 Anti-CDH17 ADC inhibits tumor growth in AsPC1-bearing mice.

[0060] Figure 12 Anti-CDH17 ADC inhibits tumor growth in GP2d tumor-bearing mice.

[0061] Figure 13 Anti-CDH17 ADC inhibited tumor growth in SNU16 tumor-bearing mice.

[0062] Figure 14 Pharmacokinetic assays for anti-CDH17 ADC. Serum concentration levels of total antibody (A) and ADC (B).

[0063] Figure 15 Pharmacokinetic assays of the anti-CDH17 ADC. Serum concentration levels of total antibody and ADC. Detailed Implementation

[0064] Definition of terminology This invention is based on the development of an antibody that can specifically bind to CDH17. The antibody of this invention can optionally be conjugated to a growth inhibitor or a cytotoxic agent, such as a toxin, including, for example, a topoisomerase inhibitor or olistatin.

[0065] The headings used in this section are for illustrative purposes only and do not limit the invention. Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Furthermore, unless the context specifically requires it, singular forms include plural forms, and plural forms include singular forms. Abbreviations for amino acid residues are standard three-letter and / or single-letter codes used in the art, representing one of the 20 common L-amino acids.

[0066] The terms “CDH17” and “CDH17 antigen” are used interchangeably herein and include any variant, isotype, and species homolog of human CDH17 expressed naturally in cells or on cells transfected with the CDH17 gene.

[0067] The term “CDH17” as a target should be interpreted broadly in this article, aiming to encompass various forms of CDH17 molecules at different stages in mammals (such as humans), including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, translation, and modification of the CDH17 gene (e.g., precursor CDH17, mature CDH17, membrane-expressed CDH17, CDH17 splice variants, modified CDH17, or fragments thereof). The term also covers artificially prepared or in vitro expressed CDH17.

[0068] The term "antibody" or "antibodies" has a broad meaning and includes immunoglobulin molecules, including monoclonal antibodies (including mouse, human, humanized, and chimeric monoclonal antibodies), full-length antibodies, antigen-binding fragments, multispecific antibodies (such as bispecific, trispecific, tetraspecific, etc.), dimer, tetramer, or multimer antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of immunoglobulin molecules containing an antigen-binding site with desired specificity.

[0069] "Bispecificity" refers to the specific binding of an antibody to two different antigens or two different epitopes within the same antigen. Bispecific antibodies can exhibit cross-reactivity with other related antigens, such as the same antigen from other species (homologs), like humans or monkeys (e.g., cynomolgus macaques). cynomolgus cyno) or common chimpanzee ( Pan troglodytes (), or can combine epitopes shared by two or more different antigens.

[0070] The term "antibody" refers to a protein, or its antigen-binding portion, comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0071] As used herein, the term “antigen-binding fragment” for antibodies refers to one or more fragments of an antibody that retain the ability to bind specifically to an antigen (e.g., CDH17). It has been shown that antigen-binding functionality of antibodies can be achieved through fragments of full-length antibodies. Examples of binding fragments covered within the term “antigen-binding fragment” for antibodies include: (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments comprising two Fab fragments linked by disulfide bonds in hinge regions; (iii) Fd fragments consisting of VH and CHI domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341: 544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via synthetic linkers using recombination methods, enabling the fabrication of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USÅ 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding moiety" of antibodies.

[0072] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0073] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) or hybridomas prepared therefrom (further described in Section 1 below), (b) antibodies isolated from host cells transformed to express antibodies, such as from transfected tumors, (c) antibodies isolated from recombinant combined human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be mutagenized in vitro (or, when using animals transgenic with human Ig sequences, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and associated with human germline VH and VL sequences, may not be naturally present in the in vivo repertoire of human antibodies.

[0074] The term "CDR" refers to one of the six hypervariable regions within the antibody's variable domain, primarily facilitating antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991), Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of the light chain variable domain, and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3) of the heavy chain variable domain.

[0075] Methods and techniques for identifying CDRs within amino acid sequences of HCVR and LCVR are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883) based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics Database (IMGT) (imgt.cines.fr / on the World Wide Web), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art can readily identify CDRs defined by each numbering system.

[0076] The following are useful comparisons of CDR numbers: Note 1: Some of these definitions (particularly for the Chothia ring) vary depending on the individual publication examined; Note 2: Any numbering scheme can be used for these CDR definitions, except for the contact definition using the Chothia or Martin (enhanced Chothia) definition; Note 3: When numbering using the Kabat numbering convention, the end of the ChothiaHCDR1 ring varies between H32 and H34 depending on the ring length. This is because the Kabat numbering scheme places the insertion at H35A and H35B.

[0077] The term "Fab fragment" includes both the variable domains of the heavy chain and the variable domains of the light chain, and also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. A "Fab' fragment" differs from a Fab fragment by the addition of several residues (including one or more cysteine ​​residues from the antibody hinge region) to the carboxyl terminus of the CH1 domain of the heavy chain. "Fab'-SH" refers to a Fab' fragment in which the cysteine ​​residues of the constant domain carry a free thiol group. The F(ab')2 antibody fragment is initially generated as a pair of Fab' fragments with hinge cysteine ​​residues between them. Other chemical conjugations of antibody fragments are also known.

[0078] The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes the Fc region of the native sequence and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered based on the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0079] As will be understood by those skilled in the art, the precise numbering and placement of heavy chain constant region structural domains may differ between different numbering systems. A useful comparison of the heavy chain constant region numbering according to the EU and Kabat is provided below, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, U.S. Public Health Service, National Institutes of Health, Bethesda, incorporated in its entirety by reference. "Conservative modification," "conservative substitution," or "conservative variant" refers to the replacement of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent substitutions without altering the protein's biological activity. Those skilled in the art will understand that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to affect biological activity.

[0080] As used herein, the term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. A nucleic acid is "effectively linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

[0081] The preparation method of nucleic acid is a conventional method in the art. Preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full-length sequence synthesis.

[0082] Those skilled in the art will recognize that the base sequence encoding the amino acid sequence of a protein can be appropriately substituted, deleted, altered, inserted, or added to provide polynucleotide homologs. The polynucleotide homologs of the present invention can be prepared by substituting, deleting, or adding one or more bases of a gene encoding a protein sequence within a range that maintains antibody activity.

[0083] As used herein, the term "linker unit" refers to the portion that links an antibody to a drug in an antibody-drug conjugate (i.e., an ADC), and this linker may be cleavable or non-cleavable. Cleavable linkers (i.e., breakable or biodegradable linkers) can cleave within or on the surface of target cells, thereby releasing the drug. In some embodiments, the linker units or linkers of the present invention exhibit excellent stability, significantly reducing drug release during delivery to the target (e.g., in the bloodstream), thereby reducing side effects and toxicity. In some specific embodiments, the linker units or linkers of the present invention are selected from cleavable linkers, such as disulfide-based linkers (which selectively cleave in tumor cells at higher thiol concentrations), peptide linkers (which are enzymatically cleaved in tumor cells), and hydrazone linkers.

[0084] The “connector unit” may comprise one or more connector elements. Exemplary connector elements include 6-maleimide hexanoyl (“MC”), maleimide propionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimide-4-(2-pyridinylthio)valerate (“SPP”), N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), and N-succinimide-4-iodoacetyl)aminobenzoate (“SIAB™”). The connector may comprise one or more or a combination of the following elements: extension units, spacer units, and amino acid units, synthesized by methods known in the art. The connector may be a “cleavable connector” that facilitates drug release into cells. For example, acid-labile adapters (e.g., hydrazones), protease-sensitive adapters (e.g., peptidase-sensitive adapters), photolabile adapters, dimethyl adapters, or adapters containing disulfide bonds can be used (Chari et al., Cancer Research, 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0085] The terms "toxin drug" or "cytotoxic drug" refer to chemical molecules that can strongly disrupt the normal growth of tumor cells. In principle, toxin drugs can kill tumor cells at sufficiently high concentrations, but due to their lack of specificity, they also induce apoptosis in normal cells, leading to serious side effects. Cytotoxic drugs can be selected from any agent that is harmful (e.g., kills) cells.Suitable cytotoxic agents for forming the immunoconjugates of the present invention include, but are not limited to, paclitaxel, microtubule inhibitors, duostatin, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, 48haracteri, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, maytansin or its analogues or derivatives, mitoxantrone, styraxin D, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine. Propranolol and puromycin; galicillin or its analogues or derivatives; antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 48haracteriz, 5-fluorouracil, hydroxyurea, asparaginase, gemcitabine, cladribine); alkylating agents (such as nitrogen mustard, thiamethoxam, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DT) IC), procarbazine, mitomycin C, cisplatin and other platinum derivatives (such as carboplatin), and docalamycin A, docalamycin SA, CC-1065 (also known as rachelin); or analogues or derivatives of CC-1065), dolalastatin, olprestatin, pyrrolo[2,1-c][1,4]benzodiazepines (PDB), indoline benzodiazepines (IGN) or their analogues, antibiotics (such as dactinomycin) (proactinomycin) n)), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, scintillan, mitomycin, mitoxantrone, procainox, anthramycin (AMC), antimitotic agents (e.g., tubulin-targeting agents), such as diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules); ricin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, shiga-like toxins (SLTI, SLT II, ​​SLT III), LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alarin, saporin, modeccin, gelanin, abrutin A chain, modeccin A chain, α-salinmycin, tung oil (). Aleurites fordiiProteins including dianthin, phytolacca 48-haracter proteins (PAPI, PAPII, and PAPS), 48-haracter bitter melon inhibitor, curcumin, crotonin, and saponins. Sapaonaria officinalis Inhibitors, gelonin, mitomycin, restriction bacteria, phenylmycin, and 48haracte toxin. Other suitable conjugates include antimicrobial / anti-cleavage peptides such as CLIP, macaine 2, melittin, cephalosporin, and P18; ribonucleases (RNases), DNase I, staphylococcal enterotoxin A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin.

[0086] Specifically, the toxin drug can be selected from mitosis inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors, and DNA synthesis inhibitors, with microtubule inhibitors and topoisomerase inhibitors being preferred.

[0087] The term "alkyl" refers to a saturated aliphatic hydrocarbon group containing 1 to 20 carbon atoms in a straight or branched chain, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (containing 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups having 1 to 6 carbon atoms are preferred. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group may be substituted or unsubstituted. When substituted, the substituent can be substituted at any available connection point, wherein the substituent is preferably one or more groups independently selected from the following: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0088] In some respects, anti-CDH17 antibodies can be conjugated with one or more topoisomerase inhibitors to form ADCs for cancer treatment. Topoisomerases are enzymes capable of altering the topology of DNA in eukaryotic cells. They are crucial for cell function and cell proliferation. In some respects, topoisomerase inhibitors are camptothecin or camptothecin analogs. Camptothecin is a water-insoluble cytotoxic alkaloid derived from the tree *Camptotheca acuminata* (native to the Chinese camptotheca tree). Camptotheca accuminata ) and the smelly false dragon tree native to India ( Nothapodytes foetida Camptothecin exhibits inhibitory activity against the growth of various tumor cells. Camptothecin analogues are typically specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" refers to any tumor cell growth inhibitory compound structurally related to camptothecin.

[0089] In some embodiments, the camptothecin analogue is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analogue is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by the hydrolysis of irinotecan by a carboxylesterase. In some embodiments, the camptothecin analogue is eciletecan mesylate. Ecinotecan mesylate is a water-soluble camptothecin (CPT) that exhibits more potent topoisomerase I inhibitory activity and antitumor activity compared to other CPT analogues. Furthermore, eciletecan is effective against p-glycoprotein (P-gp)-mediated multidrug-resistant cells. In some embodiments, the camptothecin analogue is deruxtecan (Dxd), a potent derivative of eciletecan that is 10 times more potent in inhibiting topoisomerase I than SN-38.

[0090] The term "ligand-cytotoxic drug conjugate" refers to a ligand linked to a biologically active drug via a linker unit. In some specific embodiments, "ligand-cytotoxic drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a linker unit.

[0091] The term "drug-to-antibody ratio (DAR)" refers to the average amount of cytotoxic drug loaded on each ligand, and can also be expressed as the ratio of drug amount to antibody amount. The drug loading per ligand (Ab) can range from 1 to 20 cytotoxic drugs (D). In embodiments of the invention, the drug-to-antibody ratio is represented by y. The average amount of drug in each ADC molecule after the conjugation reaction can be identified by conventional methods, such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0092] As used herein, the term “transfected tumor” includes recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS / O cells, HEK293 cells, plant cells, or fungi (including yeast cells).

[0093] The sequence of the DNA molecule of the antibody or fragment thereof according to the invention can be obtained using conventional techniques (e.g., PCR amplification or genomic library screening). Furthermore, sequences encoding the light and heavy chains can be fused together to form a single-chain antibody.

[0094] Once the relevant sequence is obtained, it can be obtained in batches using recombination methods. This is typically done by cloning the sequence into a vector using conventional methods, transforming cells with the vector, and then separating the relevant sequence from the proliferating host cells.

[0095] In addition, related sequences can be synthesized artificially, especially when the fragment length is short. Typically, several small fragments are synthesized first, and then they are joined together to obtain a long sequence fragment.

[0096] Currently, the DNA sequence encoding the antibody (or a fragment thereof or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0097] Generally, host cells obtained are cultured under conditions suitable for expressing the antibodies according to the invention. The antibodies of the invention are then purified using conventional immunoglobulin purification steps, such as conventional separation and purification methods known to those skilled in the art, including protein A-agarose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0098] Monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined by, for example, Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

[0099] The antibodies according to the invention can be expressed in cells or on cell membranes, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and any other liquid chromatography, and combinations thereof.

[0100] The term "identity" in sequence refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment. Generally, identity refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences, taking into account the number of vacancies and the length of each vacancy, which need to be introduced to achieve optimal alignment of the two sequences. Typically, sequence alignment is performed and vacancies (if any) are introduced before calculating the percentage of identity between two amino acid or nucleotide sequences. If amino acid residues or bases in two sequences are the same at an alignment position, the two sequences are considered identical or matched at that position. If amino acid residues or bases in two sequences are different, they are considered inconsistent or mismatched at that position. In some algorithms, the number of matched positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number and / or length of notches are also considered. For the purposes of this disclosure, the known alignment software BLAST (available at ncbi.nlm.nih.gov) can be used, with default settings to obtain optimal sequence alignment for calculating sequence identity between two amino acid or nucleotide sequences. When an amino acid sequence is described as being at least 85%, at least 90%, or at least 95% identical to another amino acid sequence, the difference in the amino acid sequence may lie in conserved substitutions (including all of which are conserved substitutions).

[0101] The term "variant" of a polypeptide (such as an antigen-binding fragment, protein, or antibody) refers to a polypeptide in which one or more amino acid residues are inserted, deleted, added, and / or substituted compared to another polypeptide sequence, and includes fusion polypeptides. Furthermore, protein variants include protein variants that are modified by protease cleavage, phosphorylation, or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding to CDH17 and specificity. Variants may be approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of the antibodies or antigen-binding fragments disclosed herein. The percentage of identity or homology (%) can be calculated with reference to the following description.

[0102] In one implementation, the homology or identity percentage can be calculated as 100 × [(identical position) / min(TGA, TGB)], where TGA and TGB are the sum of the number of residues in the compared sequences A and B and the internal vacancy positions (Russell et al., J. Mol Biol., 244: 332-350 (1994)).

[0103] In this invention, the antibodies of this invention also include conserved variants thereof, meaning that, compared to the amino acid sequence of the antibodies of this invention, up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are substituted with amino acids having similar or similar properties to form a polypeptide. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table A.

[0104] Table A As used in this article, the term "K" D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. "K" D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a The Kc of an antibody is obtained and expressed as a molar concentration (M). In view of this disclosure, methods in the art can be used to determine the Kc of an antibody. D Value. For example, the K value of an antibody. D It can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g., the system) or by using biolayer interferometry (e.g., the Octet RED96 system).

[0105] The term "affinity" refers to the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, determined by the properties of the antigen (such as its size, shape, and / or charge) and the CDR sequence of the antibody or antigen-binding fragment. Methods for determining affinity are known in the art and can be found below.

[0106] When the dissociation constant (K) D )< l0 -6 When M occurs, the antibody or its antigen-binding fragment is said to have "specifically bound" to its target (such as an antigen). When K occurs... D It is < l0 -9 When M occurs, the antibody binds specifically to its target with "high affinity".

[0107] As used herein, the term "pharmaceutical composition" is intended to refer to a mixture containing one or more compounds described herein, or physiologically / pharmaceutically acceptable salts thereof, or prodrugs thereof, along with other chemical components such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, which is beneficial for the absorption of the active ingredient and the exertion of its biological activity.

[0108] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "application" and "treatment" mean contacting said animal, human, subject, cell, tissue, organ, or biological fluid with an exogenous pharmaceutical reagent, therapeutic reagent, diagnostic reagent, or composition. "Application" and "treatment" can, for example, refer to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Cell treatment encompasses contacting cells with a reagent, as well as contacting a liquid with a reagent, wherein the liquid contacts the cells. "Application" and "treatment" also refer to in vitro and ex vivo treatments, such as in vitro and ex vivo treatments of cells with a reagent, diagnostic agent, conjugate composition, or with another cell. When applied to humans, veterinarians, or research subjects, "treatment" means therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0109] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. Therapeutic effective doses can vary due to factors such as an individual's disease state, age, sex, weight, and the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indications for an effective treatment or combination of treatments include, for example, improvement in the patient's health condition.

[0110] This disclosure also includes various deuterated forms of compounds of formulas (I) and (A). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formulas (I) and (A) with reference to relevant literature. When preparing the deuterated forms of compounds of formulas (I) and (A), commercially available deuterated starting materials can be used, or conventional techniques can be used, employing deuterating agents including but not limited to deuterated boranes, trideuterated boranes in tetrahydrofurans, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0111] In addition, this disclosure includes medicaments for treating diseases associated with CDH17, which contain antibodies, antigen-binding fragments thereof, or antibody-drug conjugates of this disclosure as active ingredients.

[0112] There are no restrictions on the diseases associated with CDH17, as long as they are CDH17-related diseases. For example, the therapeutic response induced by the molecules disclosed in this disclosure can be reduced by binding to human CDH17. Therefore, the molecules of this disclosure are very useful for people suffering from tumors, cancers, or infectious diseases when in formulations and formulations suitable for therapeutic applications.

[0113] Furthermore, this disclosure relates to methods for the immune detection or measurement of CDH17, reagents for the immune detection or measurement of CDH17, methods for the immune detection or measurement of CDH17-expressing cells, and diagnostic reagents for diagnosing diseases associated with CDH17-positive cells, comprising an antibody or antigen-binding fragment of the present disclosure that specifically recognizes human CDH17 as an active ingredient.

[0114] In this disclosure, the method for detecting or determining the amount of CDH17 can be any known method. For example, it includes immunoassay or assay.

[0115] Immunoassay or assay is a method that detects or measures the amount of an antibody or antigen by using labeled antigens or antibodies. Examples of immunoassays or assays include radiolabeled antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0116] The aforementioned diseases associated with CDH17-positive cells can be diagnosed by detecting or measuring cells expressing CDH17 using the antibodies or antibody fragments of the present invention.

[0117] To detect cells expressing peptides, known immunoassays can be used, with immunoprecipitation, fluorescent cell staining, or immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining methods can be employed using the FMAT8100HTS (Applied Bio system).

[0118] Example The present invention is further illustrated by the following specific embodiments. It will be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not detailed in the following embodiments generally follow the conditions described in conventional conditions, such as those in Sambrook J. et al., *Molecular Cloning: A Laboratory Manual* (translated by Huang Peitang et al., Beijing: Science Press, 2002), or the manufacturer's recommended conditions (such as the product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0119] The room temperature described in the examples is a conventional room temperature in the art, typically 10-30°C.

[0120] Example 1: Mouse immune and hybridoma fusion Immunization regimen: Anti-CDH17 antibodies were obtained by immunizing genetically modified mice encoding the variable regions of the human immunoglobulin heavy and κ light chains with the recombinant protein antigen human CDH17 His tag (Sino Biological, catalog number: 11360-H08H), followed by two booster doses with the same antigen. Antibody immune responses were monitored using a CDH17-specific immunoassay. When the desired immune response was achieved, spleen cells were harvested from each mouse and fused with mouse myeloma cells to maintain their viability and form hybridoma cells, which were then screened for CDH17 specificity.

[0121] Spleen cell fusion Hybridoma cells are obtained by fusing spleen lymphocytes and myeloma cells Sp2 / 0 (ATCC® CRL-158) via electrofusion or PEG fusion. Clonacells are used according to the manufacturer's instructions. TM HY (STEMCELL technologies) are used for PEG fusion. For electrofusion, the ratio of primary cells to mouse myeloma cell lines is 1:1, while for PEG fusion it is 10:1.

[0122] Example 2: Hybridoma Screening Hybridoma clones that specifically bind to human CDH17 protein were screened using ELISA. ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). The ELISA plate was coated overnight with 1 µg / ml human CDH17 His-tagged plate (Sino Biological, catalog number: 11360-H08H) or BSA. Excess unbound protein was washed three times with washing buffer, followed by blocking at room temperature for 1 hour. 100 μl of CDH17 hybridoma supernatant was added to each well and incubated at room temperature for 1 hour. Excess unbound antibody was washed away, and 100 μl of a 1:30000 dilution of goat anti-mouse IgG Fc-HRP (ab5870) was added to each well, followed by incubation for another 1 hour. Following the manufacturer's protocol, the plate was washed, followed by the addition of 50 μL of chemiluminescent reagent (color A and color B). The reaction was terminated with 25 μL of stop solution. The optical density of the samples at 450 nm was measured using a microplate reader (PerkinElmer). All clones tested selectively bound to human CDH17 but not to BSA.

[0123] Table 1. Characterization of the binding of hybridoma clones to human CDH17 protein by ELISA (OD450) Hybridoma clones that specifically bind to CDH17-expressing cancer cells were screened by flow cytometry. Flow cytometry analysis was used to assess the binding of hybridoma supernatant to CDH17-positive cell lines AsPC1 (ATCC, CRL-1682), GP2d (Creative bioarray, CSC-J9456), and CDH17-negative cell line SW480 (ATCC, CCL-228). Briefly, 50 μL of cells (2 × 10⁶ cells / mL) in cell staining buffer were used. 6 Mix cells (1 cell / mL) with 50 µL of undiluted supernatant. Incubate the mixture on ice for 1 hour, then wash twice with ice-cold staining buffer. Subsequently, stain cells for 20 minutes with 50 µL of PE-labeled secondary antibody (1:250 dilution, Biolegend, catalog number: 405307). After washing with staining buffer and fixing with 4% PFA, analyze the cells by flow cytometry.

[0124] Purified anti-human CDH17 antibody was used as a positive control (Sino Biological, catalog number: 11360-MM02). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog number: 400102). Figure 1Examples of selected cell binding signals measured by flow cytometry are shown. Hybridoma clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as having enhanced binding profiles to AsPC1 and GP2d cells compared to SW480 cells.

[0125] Hybridoma clones with internalization activity were screened using an indirect kill assay. In AsPC1 cells, the intracellular activity of hybridoma supernatant was measured using an indirect killing assay. AsPC1 cells were seeded at 8,000 cells / well in 96-well plates, and 500 ng / mL propidium iodide (abcam, catalog number: ab14083) and a 1:2000 dilution of SPY650-DNA (Cytoskeleton, Inc., catalog number: CYSC501) were added. Cells were incubated overnight at 37°C with 5% CO2. Hybridoma supernatant from each hybridoma clone was diluted with hybridoma medium containing 500 ng / mL propidium iodide and a 2000-fold dilution of SPY650-DNA, and mixed with Fab anti-mouse IgG Fc-MMAF conjugate with a cleavable adapter (Moradec, AM-202AF), then added to each well. The final concentrations of mouse IgG were approximately 10 nM, 3.33 nM, and 1.11 nM. The final concentration of the Fab anti-mouse IgG Fc-MMAF conjugate in each well was 20 nM. Due to the presence of secondary Fab-vc-MMAF, the internalized antibody / Fab-vc-MMAF conjugate complex will release a cytotoxic payload and kill cells. The plate was imaged every 8 hours using a Cytation5 (Agilent) to determine the viability of cells in each well.

[0126] Purified anti-human CDH17 antibody was used as a positive control (Invitrogen, catalog number: MA5-29135). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, catalog number: 400102). Cells treated with selected hybridoma supernatant showed reduced viability, such as... Figure 2 As shown, this indicates antibody internalization. Using an indirect killing assay, clones 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 were identified as capable of internalization into CDH17-positive cell lines.

[0127] Example 3: Sequencing of positive hybridoma clones The sequencing process from positive hybridoma clones is as follows: Hybridoma cells in logarithmic growth phase are collected, RNA is extracted, reverse transcribed, and then the VDJ region is amplified. Next-generation sequencing is performed on the cDNA library amplified from each clone. The amino acid sequences of the heavy and light chain variable regions (CDRs) of antibodies 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 15A4, 17F3, 19C7, and 20B4 are obtained. The amino acid sequences of the heavy and light chain variable regions and CDRs of each antibody are shown in the following tables (Tables 2 to 4). The amino acid residues of the CDRs in VH / VL are numbered and annotated according to the Kabat numbering system.

[0128] Table 2. CDR sequences of heavy chain variable domains in CDH17 hybridoma clones Table 3. CDR sequences of the light chain variable domain in CDH17 hybridoma clones Table 4. Sequences of the heavy and light chain variable domains of CDH17 hybridoma clones Example 4: Expression, purification, and binding characterization of recombinant antibodies Molecular cloning of recombinant antibodies The cDNA sequences encoding the VH and VL regions of the selected clones were directly synthesized into DNA fragments with a 5' end leader sequence (MGWSCIILFLVATATGVHS). These DNA fragments were cloned into selected vectors using the NEBuilder DNA Assembly Cloning Kit (New England Biolabs). The VH region was cloned into the pFUSE-CHIg_hG1 vector (InvivoGen, catalog number: pfuse-hchg1), in the same frame as the constant region of the hIgG1 heavy chain in the vector. The VL region was cloned into the pFUSE2-CLIg_hk vector (InvivoGen, catalog number: pfuse2-hclk), in the same frame as the constant region of the hIgκ light chain in the vector. The amino acid sequences of the hIgG1 heavy chain constant region and the hIgκ light chain constant region are as follows: Heavy chain constant region (SEQ ID NO: 99): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain constant region (SEQ ID NO: 100): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The full lengths of the heavy and light chains in the form of antibody IgG are disclosed in Table 5 below. 1H10: SEQ ID NO: 77 (heavy chain) and 78 (light chain); 2F9: SEQ ID NO: 79 (heavy chain) and 80 (light chain); 4F9: SEQ ID NO: 81 (heavy chain) and 82 (light chain); 9E2: SEQ ID NO: 83 (heavy chain) and 84 (light chain); 10E11: SEQ ID NO: 85 (heavy chain) and 86 (light chain); 13C7: SEQ ID NO: 87 (heavy chain) and 88 (light chain); 14B12: SEQ ID NO: 89 (heavy chain) and 90 (light chain); 15A4: SEQ ID NO: 91 (heavy chain) and 92 (light chain); 17F3: SEQ ID NO: 93 (heavy chain) and 94 (light chain); 19C7: SEQ ID NO: 95 (heavy chain) and 96 (light chain); 20B4: SEQ ID NO: 97 (heavy chain) and 98 (light chain).

[0129] Table 5. Full-length heavy and light chain sequences of the anti-CDH17 recombinant antibody Expression and purification of recombinant antibodies Heavy chain expression plasmids and light chain plasmids were co-transfected into CHO cells (ATCC, catalog number: CCL-61) using the ExpiFectamine 293 transfection kit (ThermoFisher, A14524), or co-transfected into ExpiCHO-S cells (ThermoFisher, catalog number: A29127) using the ExpiFectamine CHO transfection kit (ThermoFisher, A29129). Following the manufacturer's instructions, the plasmid DNA concentration was 1.0 μg per ml of suspended cells, with an LC:HC vector ratio of 1:1. Transfected cells were cultured for 5 to 7 days on an orbital oscillator at 37°C and 8% CO2. Conditioned medium was collected, and antibodies were purified using a HiTrap MabSelect SuRe column (Cytiva, catalog number: 17549112) on an AKTA Pure 25 instrument (Cytiva). The eluted antibodies were neutralized with Tris buffer (pH 9.0) and exchanged with PBS buffer. The concentration of the product was measured by UV absorbance, and the mass was determined by SDS-PAGE and HPLC.

[0130] The binding of the anti-CDH17 recombinant antibody to CDH17-positive and CDH17-negative cell lines was characterized by flow cytometry. Using cancer cell lines comprising CDH17-positive AsPC1 cells and CDH17-negative SW480 cells, the binding of recombinant antibody (human IgG1) to CDH17 on the cell surface was determined by FACS analysis.

[0131] AsPC1 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. SW480 cells were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin and streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0132] To determine the binding of the recombinant antibody to the CDH17 receptor on the cell surface, cells were first harvested and stored at 1.3 × 10⁻⁶. 6 -1.5×10 6Cells / mL were resuspended in cell staining buffer (BioLegend, catalog number: 420201). Then, the cells were treated with human Fc receptor blocking reagent (BioLegend, catalog number: 422302) on ice for 10 minutes. The resulting cell suspension was divided into 50 µL aliquots. 25 µL of recombinant antibody at different concentrations was mixed with each aliquot, with the final concentration of recombinant antibody ranging from 1.1 pM to 200 nM. The cells were incubated on ice for 1 hour, followed by washing twice with cell staining buffer. 50 µL of secondary antibody (PE conjugated to goat anti-human Fc, eBioscience) was added... TM Add (1:250 dilution) to each sample to resuspend cells. Incubate cells on ice for 20 min. Then wash cells twice with cell staining buffer and resuspend in 4% PFA to fix cells. Analyze samples using iQue3 to measure median fluorescence intensity using the corresponding channel.

[0133] HBMAB-81 is a reformulated mAb with a CDH17 binding arm from the bispecific antibody BI 905771 and a human IgG1 backbone (see WO 2018115231A2; SEQ ID:116, SEQ ID:117). B12 is an internal isotype control antibody disclosed in patent US005652138A. Results disclosed in Figure 3 And in Table 6. As a result, it was confirmed that the anti-CDH17 antibody of this disclosure specifically binds to human CDH17 originally expressed in cells in a concentration-dependent manner, while HBMAB81 nonspecifically binds to the CDH17-negative cell line SW480.

[0134] Table 6. K+ binding of anti-CDH17 recombinant antibody to CDH17-positive AsPC1 cells D value Example 5: Characterization of the internalization of recombinant anti-CDH17 antibody in CDH17-expressing cells by indirect killing assay. To evaluate antibody endocytosis induced by anti-CDH17 antibody binding, an indirect killing assay was used to characterize antibody internalization. A Fab anti-human IgG Fc-MMAF conjugate with a cleavable linker (Moradec, AH-202AF) was incubated with and conjugated to the recombinant anti-CDH17 antibody. The resulting complex was then incubated with CDH17-expressing cells. Upon binding to the CDH17 receptor on the cell surface, the complex was internalized, and the conjugated MMAF was released after lysosomal cleavage of the linker. The released MMAF subsequently inhibited cell division by blocking microtubule polymerization. In summary, CDH17-expressing AsPC1 cells were seeded at 5,000 cells / well in 96-well plates and incubated overnight. The recombinant anti-CDH17 antibody was mixed with the Fab anti-human IgG Fc-MMAF conjugate with a cleavable linker at a ratio of 1:6 (mol / mol) and incubated for 10 minutes to form the complex. Then, serial dilutions of the complex (from 4.5 pM to 30 nM) were added to each well, and cell viability was measured for 48 hours using a Cytation 5 imaging system. Dose-response curves were plotted and fitted using a GraphPad Prism 9. Figure 4 As shown in Table 7, all antibody clones exhibited cytotoxic activity in the AsPC1 cell line compared to the negative control clone (B12), indicating effective delivery of MMAF into the cells.

[0135] Table 7. IC50 assay used to evaluate the indirect killing effect of recombinant anti-CDH17 antibody internalization in AsPC1 cells. 50 . Example 6: Characterization of the binding activity of anti-CDH17 clones to cross-species CDH17 and homologs using Octet.

[0136] The affinity between CDH17 antibody and CDH17 antigen and homolog was determined using an Octet (Octet Red 384) instrument. An anti-hIgG Fc capture (AHC) biosensor was selected and equilibrated with buffer solution for 10 minutes. Subsequently, the sensor was immersed in a well containing 1 μg / ml of CDH17 lead antibody for antibody loading onto the probe. Excess unbound antibody was washed away. Antigen binding was performed in wells containing serially diluted human CDH17-His tags (Sino Biological, catalog number: 11360-H08H), cynomolgus monkey CDH17-His tags (Acro Biosystems, catalog number: CA7-C52H4), homologous human CDH6-His tags (Acro Biosystems, catalog number: CA6-H5229), and human CDH16-His tags (Sinobiological, catalog number: 10915-H08H) at concentrations ranging from 200 nM to 3.1 nM for 5 minutes. The probes were then immersed in wells containing fresh buffer to initiate an additional 15 minutes of dissociation.

[0137] The results are shown in Table 8. 10E11, 14B12 and 20B4 all bind to the CDH17 protein in humans and cynomolgus monkeys, while HBMAB81 binds only to the human CDH17 protein.

[0138] Table 8. Binding activity of anti-CDH17 clones to cross-species CDH17 and its homologs. Note: N / A: Not bound.

[0139] Example 7 Competitive Combination Following the manufacturer's instructions (ThermoFisher, catalog number: A20186), 20B4 and HBMAB81 mAb were labeled using Alexa Flour 647. In the competitive binding assay, 20B4-AF647 and HBMAB81-AF647 were immobilized at EC80 concentrations of 1.51 nM and 0.97 nM, respectively. First, 20B4 and HBMAB81 in the range of 0.03 nM to 1800 nM were mixed with 1.51 nM 20B4-AF647 or 0.97 nM HBMAB81-647 and then incubated with AsPC1 cells on ice for 1 h. Cells were washed twice with cell staining buffer. Samples were analyzed using iQue3 to measure median fluorescence intensity using the corresponding channel.

[0140] The results are as follows Figure 5The results show that neither the “20B4-AF647 + HBMAB81” group nor the “HBMAB-AF647 + 20B4” group exhibited binding inhibition. This indicates that 20B4 and HBMAB81 do not bind to the same or similar epitopes.

[0141] Example 8: Generation of Anti-CDH17 Antibody Drug Conjugate (ADC) The antibodies of the present invention possess cell affinity and endocytic activity, making them suitable for conjugation with drugs to form antibody-drug conjugates for the treatment of CDH17-mediated diseases.

[0142] Purification of monoclonal antibodies Refer to the method for preparing the anti-CDH17 antibody described in Example 4. Mota, 1H10, 2F9, 4F9, 9E2, 10E11, 13C7, 14B12, 17F3, 19C7, 20B4, and HBMAB-81 were purified for drug conjugation. Mota is an internal isotype antibody obtained from patent US008568726B2. Other antibodies used for conjugation may include any antibodies described herein (see Example 4).

[0143] Preparation of drug intermediates The following intermediate compounds are used to generate antibody-drug conjugates (ADCs) against CDH17 antibodies. The mc-vc-MMAE drug linker can be generated using a method similar to that described in US 2005 / 0238649. Compound C is prepared using the method disclosed in PCT patent application (see WO2020063673, filed September 25, 2019), and compound D is prepared using the method disclosed in PCT patent application (see WO2022161385, filed January 26, 2022). Compound C Compound D Conjugation of monoclonal antibodies to drug molecules Compound D Anti-CDH17 antibody (5 mg / mL in PBS, pH 7.4) was treated with an excess equivalent of 10 mM (tris(2-carboxyethyl)phosphine (TCEP)) at 37 °C for 2 h at 37 °C. Sufficient molar equivalents (6–12 eq) of the drug linker (compound D in DMSO) were added to the reducing antibody in PBS. The sample was then incubated with compound D overnight at room temperature. The drug-antibody ratio (DAR) of the ADC was determined using a 6230 LC / MS-TOF system (Agilent), and the mean values ​​are summarized in Table 9.

[0144] Compound C Anti-CDH17 antibody (5 mg / mL in PBS, pH 7.4) was treated with an excess equivalent of 10 mM (tris(2-carboxyethyl)phosphine (TCEP)) at 37 °C for 2 h at 37 °C. Sufficient molar equivalents (6–12 eq) of the drug linker (compound C in DMSO) were added to the reduced antibody in PBS. The sample was then incubated with compound C by rotation overnight at 4 °C. The drug-antibody ratio (DAR) of the ADC was determined using a 6230 LC / MS-TOF system (Agilent), and the mean values ​​are summarized in Table 9.

[0145] Vc-MMAE Anti-CDH17 antibody (5 mg / mL, PBS solution, pH 7.4) was treated with an excess equivalent of 10 mM (tris(2-carboxyethyl)phosphine (TCEP)) at 37 °C for 1 h. Sufficient molar equivalents (8 eq) of the drug linker (vc-MMAE in DMSO) were added to the reducing antibody in PBS. The sample was then incubated with vc-MMAE at room temperature for 1 h. The drug-antibody ratio (DAR) of the ADC was determined using a 6230 LC / MS-TOF system (Agilent), and the mean values ​​are summarized in Table 9. The mean DAR value of the anti-CDH17 ADC conjugated with vc-MMAE was approximately 4.0.

[0146] Table 9. Conjugation of monoclonal antibodies to linker / payload motifs Example 9: Inhibitory effect of ADC on tumor cell growth In vitro cytotoxicity of ADCs in multiple cell lines This embodiment analyzes ADC immunoconjugates with different antibodies targeting CDH17 and exhibiting varying degrees of internalization. The CDH17-mediated cytotoxicity of these conjugates was tested in cell cultures to limit the potency of various adaptor cytotoxic agent combinations. Different types of cancer cell lines were tested, including colorectal cancer cell lines GP2d, NCI-H716, SK-CO-1, SNU16, ESO26, and SW480; and pancreatic cancer cell line AsPC1.

[0147] We used two methods (Cell titer glo and Cytation5) to test the cytotoxicity of the immunoconjugates in the aforementioned cell lines. For the Cytation5 method, cells were collected during the exponential growth phase and distributed at 5000 cells / well in 96-well plates. 500 ng / mL propidium iodide (abcam, catalog number: ab14083) and a 1:2000 dilution of SPY650-DNA (Cytoskeleton, Inc., catalog number: CYSC501) were added. Cells were incubated overnight at 37°C and 5% CO2. The immunoconjugates were diluted with cell culture medium containing 500 ng / mL propidium iodide and a 2000-fold dilution of SPY650-DNA and added to each well. The final concentration of the immunoconjugates ranged from 0.003 nM to 200 nM. The plates were imaged every 12 hours for a total of 96 hours using Cytation5 (Agilent) to assess cell viability in each well. For the Celltiter glo method, cells were seeded at 2000 cells / well in 96-well plates. After overnight incubation, an immunoconjugate was added to each well. The final concentration of the immunoconjugate in the wells ranged from 0.0001 nM to 200 nM. After 5 or 7 days of incubation, cell viability in each well was determined using the Cell Titer Glo 2.0 assay (Promega).

[0148] Using S-shaped dose-response nonlinear regression fitting in GraphPad Prism to generate curves and IC 50 Values. The data from these experiments are summarized in Tables 10 and 11. Figure 6 All ADC molecules exhibited good cytotoxic potential against tumor cells. ADC molecules showed CDH17 expression level-dependent cytotoxicity: the higher the CDH17 expression level, the greater the cytotoxicity of the ADC, suggesting that ADC molecules are less cytotoxic to cells that do not express CDH17 and may be safer in vivo. Anti-CDH17 antibodies conjugated with mc-vc-MMAE also exhibited excellent tumor cell cytotoxicity.

[0149] Table 10. In vitro cytotoxicity evaluation of antibody-drug conjugates against tumor cells Note 1: *: Cytogenicity assays were performed using Cytation5 (96 hours). Other assays were performed using Celltiter Glo (7 days).

[0150] Note 2: NA: Not applicable based on the current dose-response curve or IC50. 50 No definitive conclusion yet (indicating no / low toxicity). Note 3: "-": Not tested.

[0151] Table 11. In vitro cytotoxicity evaluation of antibody-drug conjugates against tumor cells Note 1: *: Cytogenicity assay was performed using Cytation5 (96 hours). Other assays were performed using Celltiter Glo for 5 days or #: 7 days.

[0152] Note 2: NA: Not applicable based on the current dose-response curve or IC50 is inconclusive (indicating no / low toxicity).

[0153] Comparison of in vitro cytotoxicity of anti-CDH17 ADCs with different DAR values Cytotoxicity of anti-CDH17 ADCs with different DAR values ​​was tested using the same methods (Cell Titer Glo and Cytation5). Plates were imaged every 12 hours using Cytation5 (Agilent) for a total of 96 hours to assess AsPC1 cell viability in each well. The final concentration of the immunoconjugate ranged from 0.003 nM to 200 nM. Cell viability of GP2d and SKCO1 cell lines after 7 days of incubation was assessed using the Cell Titer Glo 2.0 assay (Promega). The final concentration of the immunoconjugate in the wells ranged from 0.0007 nM to 50 nM.

[0154] Using S-shaped dose-response nonlinear regression fitting in GraphPad Prism to generate curves and IC 50 Values. Data for anti-CDH17 ADCs containing compound D are summarized in Table 12. Figures 7 to 8 In the study, all ADCs with different DAR values ​​exhibited cytotoxicity against tumor cells. Data on anti-CDH17 ADCs containing compound C are summarized in Table 13. Figures 9 to 10 The cytotoxicity of all ADCs was within reasonable limits.

[0155] Table 12. Comparison of in vitro cytotoxicity of antibody-drug conjugates against tumor cells *Note: Cytogenicity assays were performed using Cytation5 (96 hours). Other assays were performed using the Celltiter Glo assay (7 days).

[0156] Table 13. Comparison of in vitro cytotoxicity of antibody-drug conjugates against tumor cells *: Cytogenicity assays were performed using Cytation5 (96 hours). Other assays were performed using Celltiter Glo (7 days).

[0157] Example 10: Tumor suppression experiment of ADC on a subcutaneous transplantation tumor model of CDH17-positive cancer cells in nude mice. The efficacy of anti-CDH17 ADCs in a mouse model of human pancreatic cancer cell xenograft AsPC1 cells (pancreatic cancer) were subcutaneously transplanted into female BALB / c nude mice. When the tumor volume reached approximately 150-200 mm... 3 At the time of transplantation, the transplanted mice were randomly divided into seven groups (n=5 per group). These groups were the load cell control, ADC-02, ADC-05, ADC-06, ADC-07, ADC-10, and ADC-11. Mice were treated with intravenous injection of ADC (6 mg / kg) every 4 days for 3 days. The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0158] The research results are shown in Table 14 and Figure 11 As shown in the figure. Compared with the load / PBS group, tumor growth in mice treated with ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10 was inhibited. ADC-10 showed the highest tumor inhibition rate (80.76%), followed by ADC-02 (79.25%), ADC-07 (77.77%), ADC-05 (76.27%), and ADC-06 (76.26%), all of which were superior to the positive control ADC-11 (67.62%). The anti-CDH17 antibody conjugated with compound C also showed excellent tumor inhibition (Table 15 and 10). Figure 11 ).

[0159] Table 14. Therapeutic effects of ADCs in AsPC1 tumor-bearing mice Table 15. Therapeutic effects of ADCs in AsPC1 tumor-bearing mice The efficacy of anti-CDH17 ADC in a mouse model of human colorectal cancer cell xenograft GP2d cells (colorectal cancer) were subcutaneously transplanted into female BALB / c nude mice. When the tumor volume reached approximately 100-150 mm... 3 The transplanted mice were randomly divided into seven groups (n=5 mice per group). These groups were the loading control, ADC-02, ADC-05, ADC-06, ADC-07, ADC-10, and ADC-11. Mice were treated with intravenous ADC (6 mg / kg, Q4D×3).

[0160] The research results are shown in Tables 16 and 17. Figure 12 As shown in the figure. In vivo efficacy showed that all ADC molecules effectively inhibited tumor volume increase compared to the control group (PBS). Compared to the load cell / PBS group, tumor growth was inhibited in mice treated with ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10, with TGI values ​​of 104.20%, 104.22%, 92.03%, 104.22%, and 104.10%, respectively. Specifically, tumor growth was inhibited with ADC-02, ADC-05, ADC-07, and ADC-10 compared to the ADC-11 control group. Tumor volume increased in the ADC-11 group approximately 30 days after the first treatment. Our ADCs showed better efficacy compared to the positive control ADC-11.

[0161] Anti-CDH17 antibodies conjugated with compound C also exhibit excellent tumor-suppressive effects (Table 18). Figure 12 ADCs coupled with compound C with different DAR values ​​have been compared in the GP2d CDX model. Tumor inhibition was observed in all treatment groups. When the ADCs had equivalent payloads, they showed comparable in vivo efficacy (Table 19). Figure 12 ).

[0162] Table 16. The therapeutic effects of ADCs in GP2d tumor-bearing mice Table 17. Comparison of the therapeutic effects of anti-CDH17 ADC and positive control in GP2d tumor-bearing mice. Table 18. Therapeutic effects of ADCs in GP2d tumor-bearing mice Table 19. Comparison of the efficacy of anti-CDH17 ADCs with different DAR values ​​in GP2d tumor-bearing mice The efficacy of anti-CDH17 ADC in a mouse model of xenograft of human gastric cancer cells SNU16 cells (gastric cancer) were subcutaneously transplanted into female NU / J mice. When the tumor volume reached approximately 150-200 mm... 3 At that time, the transplanted mice were randomly divided into ten groups (n=5 mice per group). Detailed dosing strategies are described in Table 20. ADCs were conjugated to compound C at different DAR values. In the ADC-24 3 mg / kg group, tumor regrowth began after 17 days of treatment. Figure 13 At lower doses, there was a significant difference in tumor volume between ADC-24 and ADC-21.

[0163] Table 20. Comparison of the efficacy of anti-CDH17 ADCs with different DAR values ​​in SNU16 tumor-bearing mice. Example 11 Pharmacokinetics of ADC To investigate the pharmacokinetics of ADCs, ADC-02, ADC-05, ADC-06, ADC-07, ADC-10, and ADC-11 were administered intravenously to Balb / c mice (n=3 per group) at a concentration of 5 mg / kg. Following injection, serum samples were collected from mice at specified time points (pre-sampling (blank), 1 hour, 4 hours, 24 hours, 2 days, 3 days, 5 days, 7 days, 14 days, and 21 days) and stored at -80°C.

[0164] Detection of total antibodies in mouse serum Human CDH17 His-tagged protein (Sino Biological, catalog number: 11360-H08H) was coated onto a bare MSD standard plate (Meso Scale Discovery, catalog number: L15XA-3) at a concentration of 4 µg / mL and incubated overnight at 4°C. Excess unbound protein was washed away with PBST buffer, and the plate was blocked with 3% BSA in PBS buffer at room temperature for 1 hour with shaking at 700 rpm. After washing three times with PBST, standard samples, QC samples, SC samples, and mouse serum samples were added to the plate and incubated at room temperature for 1 hour with shaking at 700 rpm. The final concentration range of the standard samples was 0.02 ng / mL to 1000 ng / mL. The quality control (QC) and sample control (SC) samples were diluted to four different concentrations (0.06 ng / mL, 0.244 ng / mL, 3.906 ng / mL, and 250 ng / mL). Mouse serum samples were also diluted to the detection range. The plate was then washed three times with PBST and incubated with the sulfonyl-tagged anti-Fc antibody at room temperature with shaking for 1 hour. Finally, 150 µL of MSD GOLD Read buffer B was added to each well, and the plate was read using a MESO QuickPlex SQ 120 MM (Meso Scale Discovery). The raw data were analyzed on the MSDdiscovery workbench software, and the AUC data of total mouse serum antibody were calculated using the pK solver.

[0165] Detection of ADC in mouse serum Human CDH17 His-tagged protein (Sino Biological, catalog number: 11360-H08H) was coated onto MSD naked standard plates (Meso Scale Discovery, catalog number: L15XA-3) at a concentration of 8 µg / mL and incubated overnight at 4°C. Excess unbound protein was washed away with PBST buffer, and the plates were blocked with 3% BSA in PBS buffer at room temperature for 1 hour with shaking at 700 rpm. After washing three times with PBST, standard samples, QC samples, SC samples, and mouse serum samples were added to the plates and incubated at room temperature for 1 hour with shaking at 700 rpm. The final concentration range of the standard samples was 0.02 ng / mL to 1000 ng / mL. The QC and SC samples were diluted to four different concentrations (0.06 ng / mL, 0.244 ng / mL, 3.906 ng / mL, and 250 ng / mL). Mouse serum samples were also diluted to the detection range. The plate was then washed three times with PBST and incubated with the sulfonyl-tagged anti-compound D antibody at room temperature with shaking for 1 hour. Finally, 150 µL of MSD GOLD Read buffer B was added to each well, and the plate was read using a MESO QuickPlex SQ 120 MM (Meso Scale Discovery). The raw data were analyzed on the MSDdiscovery workbench software, and the AUC data of ADC in mouse serum were calculated using the pK solver.

[0166] The total antibody here refers to the antibody derived from the ADC (antibody-dependent antibody-drug conjugate). We tested both the total antibody and the ADC simultaneously during the assay. The results are as follows: Figure 14 As shown in Tables 21 and 22, all ADC molecules exhibited favorable pharmacokinetic properties. The AUCs of ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10 were significantly higher than those of the control group ADC-11. ADC-02, ADC-05, ADC-06, ADC-07, and ADC-10 were detectable in mouse serum samples at day 7, while ADC-11 was only detectable in mouse serum samples at day 5.

[0167] Table 21. In vivo pharmacokinetics (PK) of mice after single-dose administration of total antibody Table 22. In vivo pharmacokinetics (PK) of mice after single-dose administration of the conjugate antibody For ADC-21, ADC-24, and ADC-34, similar administration, detection, and analysis methods were used. Balb / c mice (n=3 per group) were administered the drug intravenously at a concentration of 5 mg / kg. Serum samples were collected at 13 time points: pre-collection, 1 hour, 4 hours, 24 hours, 2 days, 3 days, 5 days, 7 days, 9 days, 12 days, 14 days, 17 days, and 21 days. MSD bare standard plates were coated at a concentration of 2 µg / mL for total antibody and ADC pharmacokinetic assays. Quality control (QC) and sample control (SC) samples were diluted to three different concentrations (0.22 ng / mL, 3.52 ng / mL, and 225 ng / mL).

[0168] Sulfonyl-tagged anti-Fc antibodies and anti-payload antibodies were used to detect total antibody and ADC, respectively. Tables 23 and 24 show that ADC-21 and ADC-34 had larger AUCs compared to ADC-24. Figure 15 ).

[0169] Table 23. In vivo pharmacokinetics (PK) of mice after single-dose administration of total antibody Table 24. In vivo pharmacokinetics (PK) of mice after single-dose administration of the conjugated antibody Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the disclosed teachings, and these are also included within the scope of the invention. The scope of protection of the invention is defined by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvate thereof, ； in: L1 and L2 are connection units; where L2 is -La-Lb-Lc-Ld-, La is shown in general formula (II-b): s1 is selected from 4, 5, 6, 7 or 8; Lb is a chemical bond; Lc is a tetrapeptide residue; preferably, Lc is a tetrapeptide residue of glycine-glycine-phenylalanine-glycine (GGFG). Ld is –NR1(CR2R3)s3-, where R1, R2, and R3 are the same or different, and each is independently hydrogen or alkyl, and s3 is 1 or 2; wherein, the La end is connected to Ab, and the Ld end is connected to L1. y is a number selected from 1 to 10, preferably from 2 to 8, and most preferably 2, 4, 6, or 8; Ab is an anti-CDH17 antibody or its antigen-binding fragment, wherein the anti-CDH17 antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region of the antibody, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 21, and SEQ ID NO: 31, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 47, and SEQ ID NO: 54, respectively; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 07, SEQ ID NO: 17, and SEQ ID NO: 28, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 45, and SEQ ID NO: 53, respectively; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 05, SEQ ID NO: 15, and SEQ ID NO: 26, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 43, and SEQ ID NO: 51, respectively.

2. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 1, wherein the anti-CDH17 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a multispecific antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a recombinant antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

3. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 1 or 2, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises: a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 65, 61, 59 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith; and / or a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 76, 72, 70 or a sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith.

4. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 3, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises: Heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 65 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; and / or light chain variable region having an amino acid sequence as shown in SEQ ID NO: 76 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it. or Heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 61 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; and / or light chain variable region having an amino acid sequence as shown in SEQ ID NO: 72 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it. or Heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 59 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it; and / or light chain variable region having an amino acid sequence as shown in SEQ ID NO: 70 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with it.

5. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 4, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises: The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 76; or The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO: 72; or The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59; and / or the light chain variable region having the amino acid sequence shown in SEQ ID NO:

70.

6. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 5, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises: The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 65 and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 76; or The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 61 and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 72; or The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 59 and the light chain variable region having the amino acid sequence shown in SEQ ID NO:

70.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the anti-CDH17 antibody or its antigen-binding fragment further comprises a human antibody constant region; Preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region of the human antibody constant region is selected from the κ chain and λ chain constant regions of human antibodies and their conventional variants; More preferably, the full-length antibody comprises a human antibody heavy chain constant region having at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 99, and a human light chain constant region having at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:

100. More preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 99 and the human light chain constant region of SEQ ID NO:

100.

8. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 7, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises: Heavy chains having the amino acid sequence shown in SEQ ID NO: 97 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it, and light chains having the amino acid sequence shown in SEQ ID NO: 98 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or Heavy chains having the amino acid sequence shown in SEQ ID NO: 89 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it, and light chains having the amino acid sequence shown in SEQ ID NO: 90 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with it; or Heavy chains having an amino acid sequence as shown in SEQ ID NO: 85 or having an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the sequence therein, and light chains having an amino acid sequence as shown in SEQ ID NO: 86 or having an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the sequence therein.

9. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 8, wherein the anti-CDH17 antibody or its antigen-binding fragment comprises: A heavy chain having the amino acid sequence shown in SEQ ID NO: 97 and a light chain having the amino acid sequence shown in SEQ ID NO: 98; or A heavy chain having the amino acid sequence shown in SEQ ID NO: 89 and a light chain having the amino acid sequence shown in SEQ ID NO: 90; or The heavy chain having the amino acid sequence shown in SEQ ID NO: 85 and the light chain having the amino acid sequence shown in SEQ ID NO:

86.

10. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 9, wherein the anti-CDH17 antigen-binding fragment is selected from: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (dimeric antibody), disulfide-stabilized Fv (dsFv), and peptides containing CDR.

11. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 10, wherein L1 is selected from: and .

12. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 11, wherein the antibody-drug conjugate has the following structure: ， ， ， ， ， ， ， ， ， ， ； y is a number selected from 1 to 10, preferably from 2 to 8, and most preferably 2, 4, 5, 6, 7, or 8; Ab is any one of the anti-CDH17 antibodies or antigen-binding fragments thereof as claimed in claims 1 to 10.

13. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 1 to 12, wherein the antibody-drug conjugate is selected from the group consisting of: ， ， ； in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8, further preferably a number selected from 4 to 6 or 6 to 8, and most preferably a number selected from 4, 5, 6, 7 or 8.

14. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers.

15. Use of the antibody-drug conjugate of any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 14, in the preparation of a medicament for the treatment and / or prevention of CDH17-mediated diseases or conditions.

16. The use according to claim 15, wherein the CDH17-mediated disease or condition is a tumor or cancer.

17. The use according to claim 16, wherein the tumor or cancer is associated with moderate or high CDH17 expression.

18. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described in any one of claims 1 to 13, or the pharmaceutical composition of claim 14, in the preparation of a medicament for treating and / or preventing tumors or cancer; preferably, the tumor or cancer is gastrointestinal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, gastric cancer, intestinal cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, or glioblastoma.

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