Anti-cdh17 antibodies and conjugates thereof

Abstract

Antibodies or antigen-binding fragments thereof having binding specificity for human Cadherin 17 (CDH17) protein and antibody drug conjugates (ADCs) thereof are provided. These antibodies and ADCs are capable of binding to CDH17 with high selectivity, bystander effect, and anti-tumor effect. Methods and uses for treating cancer are also provided.

Description

Background Technology

[0001] cadherin 17 (CDH17) is a transmembrane protein highly expressed in digestive system cancers. Under physiological conditions, CDH17 expression in humans and mice is primarily limited to epithelial cells of the small intestine and colon, rather than in vital organs such as the liver, stomach, heart, lungs, and brain. Under pathophysiological conditions, CDH17 expression has been extensively studied in various cancers originating from the digestive system, such as gastric cancer (GC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), pancreatic cancer (PC), and neuroendocrine carcinoma. Knockdown of CDH17 inhibits tumor development and metastasis in GC, HCC, CRC, and PC.

[0002] Antibody-drug conjugates (ADCs) are among the fastest-growing anticancer drugs. This approach involves a mAb conjugated to a cytotoxic payload via a chemical linker. This mAb targets a target antigen expressed on the surface of cancer cells, thereby reducing systemic exposure and thus decreasing toxicity. ADCs are complex molecules requiring careful attention to their various components. Selecting appropriate targets, mAbs, cytotoxic payloads, and the way the antibody is linked to the payload are key determinants of the safety and efficacy of ADCs.

[0003] There is a need to develop more effective and safer clinical candidates and new designs (such as ADCs with effective cytotoxicity against tumor cells) that target this protein. Summary of the Invention

[0004] This study discovered that anti-CDH17 antibodies and their ADCs exhibit efficient and specific binding to the human CDH17 protein. Compared to benchmark antibodies BMK-BI-905711 and BMK-ARB202, the newly discovered antibodies and ADCs demonstrated comparable binding affinity and specificity. The anti-CDH17 antibody ADCs also exhibited potent in vitro cytotoxic effects against CDH17-expressing tumor cells. In particular, they demonstrated a potent bystander effect on adjacent CDH17-negative cells and significant in vivo antitumor effects.

[0005] Therefore, these antibodies can be used to treat diseases such as cancer.

[0006] An antibody or antigen-binding fragment thereof with binding specificity to human cadherin-17 (CDH17) protein, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions (CDRs) VH CDR1, VH CDR2, and VH CDR3, and the VL comprises VL CDR1, VL CDR2, and VL CDR3, and wherein wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 respectively comprise (a) the amino acid sequences of SEQ ID NO: 11-SEQ ID NO: 16; (b) the amino acid sequences of SEQ ID NO: 17-SEQ ID NO: 22; (c) the amino acid sequences of SEQ ID NO: 23-SEQ ID NO: 28; (d) the amino acid sequences of SEQ ID NO: 29-SEQ ID NO: 34; (e) the amino acid sequences of SEQ ID NO: 35-SEQ ID NO: 16. The amino acid sequence of SEQ ID NO: 40; or (f) the amino acid sequences of SEQ ID NO: 41-SEQ ID NO: 46; or (g) the amino acid sequences of SEQ ID NO: 1-SEQ ID NO: 6.

[0007] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 1, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 2, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 3, the VLCDR1 contains the amino acid sequence of SEQ ID NO: 4, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 5, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0008] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 7, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0009] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 11, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 12, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 13, the VLCDR1 contains the amino acid sequence of SEQ ID NO: 14, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 15, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 16.

[0010] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 47, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 47. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 48, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 48.

[0011] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 17, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 18, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 19, the VLCDR1 contains the amino acid sequence of SEQ ID NO: 20, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 21, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 22.

[0012] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 49, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 49. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 50, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 50.

[0013] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 23, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 24, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 25, the VLCDR1 contains the amino acid sequence of SEQ ID NO: 26, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 27, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 28.

[0014] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 51, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 51. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 52, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 52.

[0015] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 30, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 31, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 32, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 33, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 34.

[0016] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 53, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 53. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 54, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 54.

[0017] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO: 35, the VH CDR2 contains the amino acid sequence of SEQ ID NO: 36, the VH CDR3 contains the amino acid sequence of SEQ ID NO: 37, the VLCDR1 contains the amino acid sequence of SEQ ID NO: 38, the VL CDR2 contains the amino acid sequence of SEQ ID NO: 39, and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 40.

[0018] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 55, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 55. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 56, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 56.

[0019] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43, the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 44, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 46.

[0020] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 57, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 57. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0021] In one embodiment, an antibody or antigen-binding fragment thereof specific to human CDH17 protein is also provided, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of the preceding claims for binding to CDH17 protein.

[0022] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a κ or λ chain constant region. In some embodiments, the antibody or its antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD.

[0023] In some implementations, the antibodies or antigen-binding fragments provided herein are humanized.

[0024] In some implementations, the antibody or its antigen-binding fragment provided herein is F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv.

[0025] In some implementations, the antibody or antigen-binding fragment thereof provided herein is an isotype of IgG1, IgG2, IgG3 or IgG4.

[0026] In some implementations, the antibody or antigen-binding fragment thereof provided herein is an isotype of human IgG1.

[0027] In some implementations, the antibodies or antigen-binding fragments provided herein are conjugated.

[0028] A bifunctional molecule is also provided, comprising a first antigen-binding portion specific to human CDH17 protein and a second portion specific to a second antigen, wherein the first antigen-binding portion comprises an antibody or an antigen-binding fragment thereof disclosed herein.

[0029] Another embodiment provides an antibody-drug conjugate comprising a drug portion conjugated to an antibody or its antigen-binding fragment or bifunctional molecule disclosed herein.

[0030] In some embodiments, the pharmaceutical portion is a cytotoxic agent or cell inhibitor, an immunosuppressant, a radioactive isotope, or a toxin. In some embodiments, the pharmaceutical portion is maytansinoid, auristatin, a macrolide analog, or a topoisomerase inhibitor. In some embodiments, the pharmaceutical portion includes monomethylaurestatin E (MMAE) or monomethylaurestatin F (MMAF).

[0031] In some embodiments, the pharmaceutical ingredient is a cytotoxic agent selected from the group consisting of: auristatin, maytansine alkaloids, macrocyclic ketone analogs, topoisomerase inhibitors, benzodiazepines, tubulolysin, pyroxine, camptothecin, chalcogenine, eczemaconazole, irinotecan (SN38), doxorubicin, anthracycline, pyrrolobenzodiazepines (PBD), TLR agonists, STING agonists, Pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, Staphylococcus aureus enterotoxin A / E-120, antimicrobial antibiotics, Shiga toxin, ricin, and urease.

[0032] In some embodiments, the pharmaceutical part is a cytotoxic agent selected from the group consisting of: monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), maytansine, metansine (DM1), lavtansine (DM4), tubulolysin A, DXd, 7-ethyl-10-hydroxycamptothecin (SN-38), DGN462, ambestatin 269, atrazomycin, SG3199 / SCX, IRDye®700DX, TLR7 / 8 agonists, diABZI STING agonist-2, or any derivative thereof.

[0033] In some embodiments, the pharmaceutical component includes MMAE, MMAF, or DXd.

[0034] In some embodiments, the pharmaceutical portion comprises eczemab or DXd (eczemab derivative targeting ADCs). In some embodiments, the pharmaceutical portion comprises a compound selected from the group consisting of: (LM-D01) (LM-D02) and (LM-D03).

[0035] In some embodiments, the drug portion is linked to the antibody or a fragment thereof via a linker. In some embodiments, the linker is hydrolyzable under acidic conditions.

[0036] In some embodiments, the connector comprises succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (SMCC), sulfonyl-SMCC, p-carboxycyclohexylmethylmaleimide, maleimide-hexanoyl (MC)-valine-citrulline (VC)-p-aminobenzyloxycarbamoyl (PABC), CL2A, maleimide-hexanoyl (MC), MC-glycine-glycine-phenylalanine-glycine (GGFG), MC-PEG8-GGFG, MC-PEG8-GGFG-PAB, or maleimide-propionyl (MP)-PEG8-valine-alanine (VA)-PABC.

[0037] In some embodiments, the antibody-drug conjugate includes ozogamicin, vedotin, mafodotin, emtansine, deruxtecan, govitecan, or tesirine.

[0038] In some embodiments, the antibody-drug conjugate has a drug-to-antibody ratio (DAR) of 1 to 20, 2 to 16, or 4 to 8.

[0039] A chimeric antigen receptor (CAR) specific to human CDH17 protein is also provided, which contains the antigen-binding fragment provided herein or the bifunctional molecule, transmembrane domain and intracellular activation domain provided herein.

[0040] In some implementations, the antigen-binding fragment is a single-stranded fragment (scFv).

[0041] In some embodiments, the transmembrane domain is a transmembrane domain of 4-1BB, the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, or CD154.

[0042] In some embodiments, the chimeric antigen receptor includes an intracellular activation domain that is a signaling domain derived from CD3ζ, CD3ε, CD3δ, and CD3γ.

[0043] In some embodiments, the chimeric antigen receptor further comprises one or more co-stimulatory domains selected from the following signal transduction domains (or other suitable portions): CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory agents (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class 1 molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0044] In some embodiments, the chimeric antigen receptor further includes a hinge domain selected from the hinge domains of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α, any truncated form thereof, or any combination thereof.

[0045] A nucleic acid is also provided that encodes an antibody or its antigen-binding fragment provided herein, a bifunctional molecule provided herein, or a CAR provided herein.

[0046] Vectors containing the nucleic acids provided herein are also provided. Isolated cells containing either the nucleic acids or vectors provided herein are also provided.

[0047] A pharmaceutical composition is also provided comprising an antibody or antigen-binding fragment thereof, a bifunctional molecule or antibody-drug conjugate or CAR disclosed herein, and a pharmaceutically acceptable carrier.

[0048] Another embodiment provides a method for treating cancer in patients in need, the method comprising administering to the patient the antibody or its antigen-binding fragment, the bifunctional molecule, the antibody-drug conjugate, or the CAR, or the use of the antigen-binding fragment, the bifunctional molecule, the antibody-drug conjugate, or the CAR in the preparation of a medicament for treating cancer.

[0049] In some implementations, the cancer is selected from the group consisting of: gastric cancer, colorectal cancer, hepatocellular carcinoma, bladder cancer, liver cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, neuroendocrine carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. Attached Figure Description

[0050] Figures 1A-1B show the specific binding of 8G5G9.p1.z12 to cell lines overexpressing human CDH17.

[0051] Figure 2 The nonspecific binding assay of 8G5G9.p1.z12 with blank HEK-293 cell line is shown.

[0052] Figures 3A-3B show the specific binding of 8G5G9.p1.z12 to endogenous human CDH17 on tumor cells.

[0053] Figures 4A-4D show the cross-reactivity of 8G5G9.p1.z12 to cell lines overexpressing monkey and rodent CDH17.

[0054] Figure 5Epitope binning assays of 8G5G9.p1.z12 against BMK-BI-905711 (anti-CDH17) in HEK-293 / H_CDH17 cell line are shown.

[0055] Figures 6A-6B show the internalization potency of 8G5G9.p1.z12 in cells overexpressing human CDH17.

[0056] Figure 7A-B The specific binding of the anti-CDH17 ADC to cell lines overexpressing human CDH17 was demonstrated.

[0057] Figure 8A-B This demonstrates the specific binding of the anti-CDH17 ADC to endogenous human CDH17 on tumor cells.

[0058] Figure 9A-B The cytotoxic effect of anti-CDH17 ADC on tumor cells expressing human CDH17 was demonstrated.

[0059] Figures 10A-F illustrate the bystander effect of anti-CDH17 ADC on human CDH17 negative cell lines.

[0060] Figure 11A-B The anti-CDH17 ADC was demonstrated to have antitumor activity in the CRC CDX (cell line-derived xenograft) model.

[0061] Figures 12A-D show the cell-based binding of anti-CDH17 antibodies 296G7E10.p4.Z9 (Figure A), 312E10A9.p2.Z7 (Figure B), 35E7D6.p1.z9, 135B2E11.p1.z1, 153A3D7.p3.z5, and 155B9A5.p3.V2.z2 (Figures C-D) in cell lines overexpressing human CDH17.

[0062] Figure 13A-B Cell-based binding of anti-CDH17 antibodies 296G7E10.p4.Z9 (Fig. A) and 312E10A9.p2.Z7 (Fig. B) in HEK293 cell lines overexpressing rhesus monkey CDH17 is shown.

[0063] Figures 14A-D illustrate the cell-based binding of anti-CDH17 antibodies 312E10A9.p2.Z7 (Figure A, SNU16), 296G7E10.p4.Z9 (Figure B, LoVo), 35E7D6.p1.z9, 135B2E11.p1.z1, 153A3D7.p3.z5, and 155B9A5.p3.V2.z2 (Figure C, SNU16), and 35E7D6.p1.z9, 135B2E11.p1.z1, 153A3D7.p3.z5, and 155B9A5.p3.V2.z2 (Figure D, LoVo) in cancer cell lines endogenously expressing CDH17.

[0064] Figure 15A-B The internalization of anti-CDH17 antibody in cancer cell lines SNU16 (Fig. A) and LoVo (Fig. B) that endogenously express CDH17 is shown, respectively.

[0065] Figure 16A-B The internalization of anti-CDH17 antibody in the CDH17-overexpressing cancer cell lines HEK293 (Fig. A) and DLD-1 (Fig. B) is shown, respectively.

[0066] Figure 17A-C The cytotoxic effects of CDH17-targeting ADCs on human CDH17-expressing cells HEK293 (Fig. A), DLD-1 (Fig. B), and LoVo (Fig. C) are shown respectively.

[0067] Figure 18A-C The effective cytotoxic effects of CDH17-targeting ADCs on human CDH17-expressing cells SNU-16 (Fig. A), HCT116 (Fig. B), and DLD-1 / H (Fig. C) are shown respectively.

[0068] Figure 19A-B The antitumor activity of the CDH17-targeting ADC in the CRC CDX (cell line-derived xenograft) model was demonstrated.

[0069] Figure 20A-B The antitumor activity of the CDH17-targeting ADC in the SNU-16 (GC) xenograft model was demonstrated. Detailed Implementation

[0070] definition It should be noted that the term "a" or "an" entity refers to one or more entities within that entity; for example, "antibody" should be understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this document.

[0071] As used herein, the term "polypeptide" is intended to include both the singular and plural forms of "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, lysosomal cleavage, or modification by non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or generated through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be generated in any manner, including through chemical synthesis.

[0072] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be compared for comparative purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences varies with the number of matching or homologous positions shared by the sequences. An "irrelevant" or "non-homologous" sequence has less than 40% identity with one of the sequences in this disclosure, but preferably less than 25% identity.

[0073] "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence at a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that when the two sequences are compared, that percentage of bases (or amino acids) are the same.

[0074] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that shares a degree of homology or sequence identity with the nucleotide sequence of that nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that shares a degree of homology with it or its complement. In one respect, a homolog of a nucleic acid is capable of hybridizing with a nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide that shares a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some respects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some respects, the equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some respects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0075] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing a molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Such examples include, but are not limited to, the complementarity-determining region (CDR) or ligand-binding portion of the heavy or light chain, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region, or any portion thereof, or at least a portion of the binding protein.

[0076] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a part of an antibody (e.g., F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc.). Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and biantibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0077] "Single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of immunoglobulins. H ) and light chain variable region (V L These are fusion proteins. In some respects, these regions are linked to short linker peptides consisting of 10 to approximately 25 amino acids. The linker can be enriched with glycine for flexibility and with serine or threonine for solubility, and can carry V... H N-terminus and V LThe C-terminus is linked, and vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

[0078] The term antibody encompasses major categories of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as γ-, , , or The immunoglobulin chain, along with some subclasses (e.g., γ1-γ4), determines the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc.) have been well characterized and are known to confer functional specialization. According to this disclosure, modifications of each of these classes and isotypes are readily identifiable to those skilled in the art and therefore fall within the scope of this disclosure. All immunoglobulin classes clearly fall within the scope of this disclosure, and the following discussion will generally pertain to the IgG class of immunoglobulin molecules. In the case of IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons, and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 Daltons. These four chains are typically linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the opening of the "Y" and extends through the variable region to surround the heavy chain.

[0079] The antibodies, antigen-binding peptides, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-derived antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries), and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0080] Light chains are classified as or Each heavy chain class can bind to either a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked, and the "tail" portions of the two heavy chains are linked together via covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration to the C-terminus at the bottom of each chain.

[0081] Both light and heavy chains are divided into structural homologous regions and functional homologous regions. The terms "constant" and "variable" are used according to function. In this regard, it should be understood that the variable domains (VK) of the light chain and the variable domains (VH) of the heavy chain determine antigen recognition and specificity. Conversely, the constant domains (CK) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties (e.g., secretion, transplacental migration, Fc receptor binding, complement binding, etc.). By convention, the numbering of constant domains increases with their distance from the antibody's antigen-binding site or N-terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0082] As shown above, the variable region allows antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, subsets of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), combine to form a variable region defining a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y. More specifically, this antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VK chains. In some cases (e.g., for certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins), the complete immunoglobulin molecule may consist only of the heavy chain, without the light chain. See, for example, Hamers-Casterman et al. Nature 363:446-448 (1993).

[0083] In naturally occurring antibodies, the six complementarity-determining regions (CDRs) present in each antigen-binding domain are short, discontinuous amino acid sequences that are specifically localized to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (called the "framework" region) exhibit less intermolecular variability. The framework region primarily utilizes... - Folded conformation, while CDRs form rings, these rings connect - Folded structure, and in some cases formed - Part of the folded structure. Therefore, the framework region acts as a backbone that positions the CDR in the correct orientation via interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDR defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids containing the CDR and framework region, respectively, as they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI.Biol ., 196:901-917 (1987)).

[0084] Where two or more definitions exist for terms used and / or accepted in this field, the definitions of terms as used herein are intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. This specific region has been described in Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al. J. MoI.Biol References , 196:901-917 (1987), the full text of which are incorporated herein by reference. The CDR definition according to Kabat and Chothia includes overlap or subsets of amino acid residues when compared with each other. However, the application of any definition of a CDR relating to antibodies or variants thereof is intended to fall within the scope of the terminology as defined and used herein. The appropriate amino acid residues containing a CDR as defined in each of the foregoing references are listed in the table below for comparison. The exact number of residues containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues contain a particular CDR based on the amino acid sequence of the variable region of an antibody.

[0085] Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96 Kabat et al. also defined a numbering system applicable to the variable domain sequences of any antibody. Those skilled in the art can explicitly assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data outside of the sequence itself. As used herein, “Kabat numbering” refers to the numbering system proposed by Kabat et al., US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0086] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately the 31st amino acid (i.e., approximately the 9th residue after the first cysteine ​​residue), consists of approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, consists of approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; consists of 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately the 24th residue (i.e., after the cysteine ​​residue); consists of approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and consists of approximately 7 residues. CDR-L3 begins at approximately the 33rd residue after the end of CDR-L2 (i.e., after the cysteine ​​residue); it consists of approximately 7–11 residues and ends at sequence F or WGXG, where X is any amino acid.

[0087] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are from humans, mice, donkeys, rabbits, goats, guinea pigs, camels, llamas, horses, or chickens. In another embodiment, the variable region can be derived from condricthoids (e.g., from sharks).

[0088] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise: a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified to differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0089] The heavy chain constant regions of antibodies disclosed in this paper can be derived from different immunoglobulin molecules. For example, the heavy chain constant regions of peptides can contain components derived from IgG. l The molecule contains the CH1 domain and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region may contain a portion derived from IgG. l The heavy chain portion is derived from the hinge region of the IgG3 molecule. In another example, the heavy chain portion may contain components derived from IgG. l Molecules and chimeric hinges derived from IgG4 molecules.

[0090] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

[0091] A "light chain-heavy chain pair" refers to a combination of light and heavy chains that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0092] As previously described, the subunit structures and three-dimensional conformations of constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0093] As used herein, the term "CH2 domain" encompasses the heavy chain portion of an antibody, extending from approximately residue 244 to residue 360 ​​using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231–340, EU numbering system; see Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it does not pair tightly with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. The CH3 domain is also well-documented, extending from the CH2 domain of the IgG molecule to the C-terminus and containing approximately 108 residues.

[0094] As used herein, the term "hinge region" refers to the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0095] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0096] As used herein, the term "chimeric antibody" will be understood to refer to any antibody in which the immune-reactive region or site is derived from or originates from a first species and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target-binding region or site will be derived from a non-human source (e.g., mouse or primate), while the constant region is human.

[0097] As used in this paper, the "percentage of humanization" is calculated by subtracting the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain from the total number of amino acids, then dividing by the total number of amino acids and multiplying by 100.

[0098] "Specific binding" or "specific to" generally means that an antibody binds to an epitope via its antigen-binding domain, and that this binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is considered to "specifically bind" to an epitope when it binds to that epitope more readily than to a random, unrelated epitope. The term "specific" is used herein to define the relative affinity of an antibody for a given epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than for antibody "B," or antibody "A" may be considered to bind to epitope "C" with higher specificity than to its related epitope "D."

[0099] As used herein, the term “treatment” refers to both therapeutic treatment and preventative or defensive measures aimed at preventing or mitigating (alleviating) undesirable physiological changes or conditions (such as cancer progression). Beneficial or desired clinical outcomes include, but are not limited to, reduction of detectable or undetectable symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and (partial or complete) remission. “Treatment” can also mean extended survival compared to expected survival without treatment. Conditions requiring treatment include those already having a condition or symptom, those susceptible to a condition or symptom, or those requiring prevention of a condition or symptom.

[0100] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject on whom a diagnosis, prognosis or treatment is expected, particularly a mammalian subject. Mammal subjects include humans, livestock, farm animals, as well as zoo animals, sporting animals or pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.).

[0101] As used herein, phrases (e.g., “patient in need of treatment” or “subject in need of treatment”) include subjects (e.g., mammalian subjects) who will benefit from administration of the antibody or composition disclosed herein for use in, for example, detection, diagnostic procedures and / or treatment.

[0102] Anti-CDH17 antibody The anti-CDH17 antibody and its antigen-binding fragment presented in this paper exhibit comparable binding affinity to human CDH17 protein compared to two reference antibodies: BMK-BI-905711 (a monospecific anti-CDH17 Ig molecule synthesized from Boehringer Ingelheim's bispecific antibody BI 905711) and BMK-ARB202 (a monospecific anti-CDH17 Ig molecule synthesized from Arbele's bispecific antibody ARB202). Despite having a different epitope than BMK-BI-905711, the anti-CDH17 antibody and its antigen-binding fragment presented in this paper did not show nonspecific binding to human CDH17 protein and exhibited selective binding to monkey CDH17 protein rather than rodent CDH17 protein.

[0103] Importantly, as shown in Figures 10 and 11, the antibody-drug conjugates (ADCs) based on the antibodies and fragments of this disclosure not only exhibited effective cytotoxicity against CDH17-positive tumor cells but also against adjacent CDH17-negative cells. Subsequently, in vivo tests demonstrated that these ADCs possess excellent antitumor efficacy and superior safety.

[0104] Therefore, according to one embodiment of this disclosure, antibodies capable of binding CDH17 and their antigen-binding fragments are provided. Exemplary antibodies are humanized anti-CDH17 antibodies 8G5G9.p1.z12, 296G7E10.p4.Z9, 312E10A9.p2.Z7, 135B2E11.p1.z1, 153A3D7.p3.z5, 35E7D6.p1.z9, and 155B9A5.p3.V2.z2 having the CDRs described herein. Antibodies containing the same CDRs as described herein are also included. In some embodiments, the disclosed antibodies and fragments include antibodies and fragments binding to the same epitopes as those shown herein, as well as antibodies and fragments competing with the antibodies and fragments of this disclosure for binding to CDH17.

[0105] According to one embodiment of this disclosure, an antibody or antigen-binding fragment thereof is provided, comprising heavy chain and light chain variable domains having the CDR regions disclosed herein, and their bioequivalents.

[0106] Table A1. CDR sequence of 8G5G9.p1.z12 district sequence SEQ ID NO: VH CDR1 TSGMGVS 1 VH CDR2 HIYWDDDKRYNPSLKS 2 VH CDR3 RALITSPFEY 3 VL CDR1 RASQTIGTSIH 4 VL CDR2 YASESIY 5 VL CDR3 QQSNAWPLT 6 Table A2. Variable and constant region sequences of 8G5G9.p1.z12 antibody chain sequence SEQ ID NO: VH <![CDATA[EVTLKESGPALVKPTQTLTLTCTFSGFSLS TSGMGVS WIRQPPGKALEWLA HIYWDDDKRYNPSLKS RLTISKDTSKNQVFLTMTNMDPVDTATYYCAR RALITSPFEY WGQGTTVTVSS]]> 7 VL <![CDATA[EIVMTQSPATLSVSPGERATLSC RASQTIGTSIH WYQQKPGQAPRLLIK YASESIY GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQSNAWPLT FGQGTKLEIK]]> 8 CH ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9 CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10 Table A3. 296G7E10.p4.Z9, 312E10A9.p2.Z7, 135B2E11.p1.z1, 153A3D7.p3.z5, CDR sequences of 35E7D6.p1.z9 and 155B9A5.p3.V2.z2

[0107] Table A4. Antibodies 296G7E10.p4.Z9, 312E10A9.p2.Z7, 135B2E11.p1.z1, 153A3D7.p3.z5 Variable region sequences of 35E7D6.p1.z9 and 155B9A5.p3.V2.z2 Antibody sequence SEQ ID NO: 296G7E10.p4.Z9 VH <![CDATA[EVQLVQSGAEVKKPGASVKVSCKASGYTFI SNWMH WVRQAPGQGLEWMG AIYPGRSETSYN QNFKD RVTMTADTSTSTAYMELRSLRSDDMAVYYCSR HSGFDEFDY WGQGTTVTVSS]]> 47 296G7E10.p4.Z9 VL <![CDATA[DVLMTQSPLSLPVTPGEPASISC RSSQTIVHSSGNAYLE WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPFT FGSGTKLEIK]]> 48 312E10A9.p2.Z7 VH <![CDATA[EVQLVQSGAEVKKPGSSVKVSCKASGYTFT SYTMH WVRQAPGQGLEWMG YIYPTRDYTNYN QKFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCGR QGVYGNYDWYFDV WGQGTTVTVSS]]> 49 312E10A9.p2.Z7 VL <![CDATA[DIVMTQSPLSLPVTPGEPASISC TSSKSLVHSNAKTYLY WYLQKPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQHLEYPFT FGGGTKVEIK]]> 50 135B2E11.p1.z1 VH <![CDATA[EVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWIN WVRQAPGQGLEWMG NIYPSNAYTNYN QKFKD RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR YYGSSYWYFDV WGQGTTVTVSS]]> 51 135B2E11.p1.z1 VL <![CDATA[DIVMTQSPDSLAVSLGERATINC KASENVGPYVS WYQQKPGQPPKLLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC GQSYTYPYT FGGGTKVEIK]]> 52 153A3D7.p3.z5 VH <![CDATA[EVQLQESGPGLVKPSQTLSLTCTVSGDSIS SGYWN WIRQPPGKGLEYIG YISYSGSTYYNP SLRS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR RGYDVYWFFDV WGQGTTVTVSS]]> 53 153A3D7.p3.z5 VL <![CDATA[DVVMTQTPLSLSVTPGQPASISC RSSQSLVLSNANTYLH WYLQKPGQSPQLLIY KISNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQSTHIPFT FGQGTKLEIK]]> 54 35E7D6.p1.z9 VH <![CDATA[EIQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQRLEWMG YIDPFNAGTDYN QQFKG KATITVDKSASTAYMELSSLRSEDMAVYYCTS GYRYPAWFVY WGQGTLVTVSS]]> 55 35E7D6.p1.z9 VL <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASENIYSYLV WYQQKPGKSPQLLVY NVKTLAE GVPSRFSGSGSGTQFTLTISSLQPEDFATYYC QHNYDIPLT FGGGTKVEIK]]> 56 155B9A5.p3.V2.z2 VH <![CDATA[EVQLVQSGAEVKKPGASVKVSCKASGYTFT DYNMH WVRQAPGQRLEWMG YIYPYNAGTDYS QKFKS RVTITRDTSASTAYMELSSLRSEDTAVYYCAR EEIWYGNYGFDY WGQGTTVTVSS]]> 57 155B9A5.p3.V2.z2 VL <![CDATA[DVVMTQTPLSLSVTPGQPASISC RSSQSLVHSNANTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQSTHVPPT FGGGTKVEIK]]> 58 It should be understood that CDRs can be modified to include CDRs with one, two, or three amino acid additions, deletions, and / or substitutions. In some embodiments, substitutions can be conservative substitutions.

[0108] "Conservative amino acid substitution" is the substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin peptides are preferably replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced by a structurally similar string that differs in sequence and / or composition from members of the side chain family.

[0109] The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates that there is a conserved substitution between the two amino acids.

[0110] Table B. Amino Acid Similarity Matrix

[0111] Table C. Conserved Amino Acid Substitutions Replaced amino acids Amino acids used for substitution alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Glun, D-Gln Aspartic acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp glutamic acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp proline D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met Those skilled in the art will also understand that the antibodies disclosed herein can be modified to differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from the specified protein can be similar, for example, having a certain percentage of identity with the starting sequence, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0112] Depending on the specific implementation, the antibody is a humanized antibody. The humanized form of a non-human (e.g., mouse) antibody is a chimeric molecule of an immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibodies) in which residues forming the complementarity-determining region (CDR) of the receptor are replaced by residues from a CDR of a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of a human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are not present in the receptor antibody or in the introduced CDR or framework sequence. Typically, humanized antibodies will contain substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of the human immunoglobulin common sequence. Humanized antibodies preferably also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).

[0113] In one embodiment, the CDRs are those of 296G7E10.p4.Z9, as shown in Table A3. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0114] In some embodiments, those containing the same CDR as 296G7E10.p4.Z9 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 296G7E10.p4.Z9, as well as antibodies or fragments that compete with any of them for binding to CDH17.

[0115] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 47, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 47.

[0116] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 48, or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 48.

[0117] In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 47, and the light chain variable region contains the amino acid sequence of any of SEQ ID NO: 48.

[0118] In one embodiment, the CDRs are those of 312E10A9.p2.Z7, as shown in Table A3. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0119] In some embodiments, those containing the same CDR as 312E10A9.p2.Z7 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 312E10A9.p2.Z7, as well as antibodies or fragments that compete with any of them for binding to CDH17.

[0120] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 49.

[0121] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 50.

[0122] In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 49, and the light chain variable region contains the amino acid sequence of any of SEQ ID NO: 50.

[0123] In one embodiment, the CDRs are those of 135B2E11.p1.z1, as shown in Table A3. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0124] In some embodiments, those containing the same CDR as 135B2E11.p1.z1 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 135B2E11.p1.z1, as well as antibodies or fragments that compete with any of them for binding to CDH17.

[0125] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 51, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 51.

[0126] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52, or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 52.

[0127] In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 51, and the light chain variable region contains the amino acid sequence of any of SEQ ID NO: 52.

[0128] In one embodiment, the CDRs are those of 153A3D7.p3.z5, as shown in Table A3. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0129] In some embodiments, those containing the same CDR as 153A3D7.p3.z5 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 153A3D7.p3.z5, as well as antibodies or fragments that compete with any of them for binding to CDH17.

[0130] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 53, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 53.

[0131] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54, or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 54.

[0132] In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 53, and the light chain variable region contains the amino acid sequence of any of SEQ ID NO: 54.

[0133] In one embodiment, the CDRs are those of 35E7D6.p1.z9, as shown in Table A3. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0134] In some embodiments, those containing the same CDR as 35E7D6.p1.z9 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 35E7D6.p1.z9, and antibodies or fragments that compete with any of them for binding to CDH17.

[0135] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 55.

[0136] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 56, or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 56.

[0137] In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 55, and the light chain variable region contains the amino acid sequence of any of SEQ ID NO: 56.

[0138] In one embodiment, the CDRs are those of 155B9A5.p3.V2.z2, as shown in Table A3. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0139] In some embodiments, those containing the same CDR as 155B9A5.p3.V2.z2 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 155B9A5.p3.V2.z2, as well as antibodies or fragments that compete with any of them for binding to CDH17.

[0140] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 57.

[0141] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 58, or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 58.

[0142] In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 57, and the light chain variable region contains the amino acid sequence of any of SEQ ID NO: 58.

[0143] In one embodiment, the CDR is one of the CDRs of 8G5G9.p1.z12, as shown in A2. In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof having one, two, or three deletions, additions, substitutions, or combinations thereof.

[0144] In some embodiments, those containing the same CDR as 8G5G9.p1.z12 are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 8G5G9.p1.z12, as well as antibodies or fragments that compete with any of them for binding to CDH17.

[0145] In some embodiments, the antibody and its antigen-binding fragment are humanized and contain a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7.

[0146] In some embodiments, the antibody and its antigen-binding fragment are humanized and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 8.

[0147] In some implementations, the antibodies provided herein also include a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0148] In some embodiments, the antibody provided herein also comprises the heavy chain constant region of SEQ ID NO: 9, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 9.

[0149] In some embodiments, the antibody provided herein also comprises the light chain constant region of SEQ ID NO: 10, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 10.

[0150] The Fc region can be engineered to enhance effector function. IgG antibodies can induce direct antitumor effects by generating indirect antitumor effects through Fc-mediated effector functions that bind to other immune cells or killing mechanisms. As used herein, "effector function" or "antibody effector function" refers to the biological activity resulting from the binding of an antibody's Fc region to its effector (e.g., the C1 complex and Fc receptor (FcγRIIa or FcγRIIIa)). Exemplary effector functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody's Fc region to Fc receptors on effector cells; and antibody-dependent cell-mediated phagocytosis (ADCP), in which non-specific cytotoxic cells expressing FcγR recognize antibodies bound to target cells, subsequently causing phagocytosis of the target cells. Among the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc effector functions. However, because IgG1 has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated functions are IgG1 isotypes.

[0151] In some embodiments, the Fc region of the antibody provided herein is engineered to enhance effector function (e.g., ADCC or CDC). Various methods (primarily Fc mutations) can be found to enhance Fc-mediated effector function, such as those described in PCT disclosures WO2007024249A2 and WO2011044368A1, the entire contents of which are incorporated herein by reference. In some embodiments, the Fc region is engineered to include the S298A / E333A / K334A mutation in human IgG1. Such combinations of Fc mutations have been reported to enhance binding to FcγRIIIa, thereby enhancing ADCC (see Shields RL et al., J. Biol. Chem 276:6591-6604 (2001)).

[0152] In some embodiments, the antibody comprises an amino acid sequence or one or more portions that are not normally associated with the antibody. Exemplary modifications are described in more detail below. For example, the antibodies of this disclosure may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., PEG, a drug, a toxin, or a label).

[0153] The antibodies, variants, or derivatives disclosed herein include modified derivatives, i.e., derivatives obtained by covalently linking any type of molecule to the antibody such that the covalent link does not prevent the antibody from binding to the epitope. For example, but not as a limitation, antibodies can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linking to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Additionally, antibodies may contain one or more non-classical amino acids.

[0154] Antibodies can be detectably labeled by conjugation to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding peptide is then determined by detecting the presence of light emitted during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, and oxalates.

[0155] Fluorescent metals (e.g.) can also be used. 152Antibodies can be detectably labeled using eu or other lanthanides. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The technique of conjugating various parts with antibodies is well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” cited in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd edition), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled "Antibody in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-16 (1985).

[0156] Bifunctional molecules and combination therapies CDH17 is overexpressed on tumor cells. As a tumor antigen-targeting molecule, antibodies or antigen-binding fragments specific to CDH17 can be combined with second antigen-binding fragments specific to immune cells or antigen-binding fragments specific to immune checkpoints to generate combination therapies or bispecific antibodies.

[0157] In some implementations, immune cells are selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells. Molecules that can be targeted on immune cells include, for example, CCL1, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1, PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptor (KIR), and CD47.

[0158] Different forms of bispecific antibodies are also provided. In some embodiments, each anti-CDH17 fragment and the second fragment are independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody also includes an Fc fragment.

[0159] Bifunctional molecules that include not only antibodies or antigen-binding fragments are also provided. As tumor antigen-targeting molecules, antibodies or antigen-binding fragments specific to CDH17 (such as those described herein) can optionally be combined with immune cytokines or ligands via peptide linkers. The linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine immune checkpoint blockade effects with local immunomodulation at the tumor site.

[0160] Chimeric antigen receptor (CAR) This disclosure provides a chimeric antigen receptor (CAR) or engineered T-cell receptor (TCR) comprising the anti-CDH17 antigen-binding fragment provided herein.

[0161] CARs and TCRs are genetically engineered receptors. These engineered receptors can be easily inserted into and expressed by immune cells, such as T cells. CARs can recognize specific antigens, and when they bind to those antigens, they activate immune cells to attack and destroy cells carrying those antigens. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

[0162] CARs can be engineered to bind antigens (e.g., cell surface antigens) by incorporating antigen-binding molecules that interact with the target antigen. Preferably, the antigen-binding molecule is an antibody fragment, and more preferably, one or more single-chain antibody fragments (“scFv”). scFv is a single-chain antibody fragment having variable regions of an antibody heavy chain and a light chain linked together. It should be understood that the antigen-binding molecule is typically contained within the extracellular portion of the CAR, enabling it to recognize and bind to the target antigen. Bispecific and multispecific CARs are covered within the scope of this invention, exhibiting specificity for more than one target.

[0163] In addition to the antigen-binding molecule, the exemplary CAR also includes a transmembrane domain and an activation domain. In some embodiments, the CAR may further include an extracellular domain between the antigen-binding molecule and the transmembrane domain. The extracellular domain may include a hinge domain. In some embodiments, the CAR may further include a co-stimulatory domain.

[0164] A. Antigen-binding molecules The antigen-binding portion of the CAR disclosed herein may be an antibody or antigen-binding fragment (especially scFv) targeting human CDH17, as disclosed herein.

[0165] B. Extracellular domains and transmembrane domains extracellular domain Chimeric antigen receptors may include extracellular domains. Extracellular domains particularly useful in this invention may be derived from (i.e., include) all or some of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory agents (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC. Class 1 molecules, TNF receptor protein, immunoglobulin protein, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 1a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof. Extracellular domains can be derived from natural or synthetic sources.

[0166] Extracellular domains typically contain hinge portions, sometimes referred to as "spacer regions." Various hinges can be used according to the invention, including portions or derivatives of the molecules listed above. In some embodiments, the hinge portion is a hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8α, any truncated form thereof, or any combination thereof.

[0167] Transmembrane domain CARs can be designed to have a transmembrane domain fused to the extracellular domain of the CAR. The transmembrane domain can similarly fuse to the intracellular domain of the CAR. In some cases, the transmembrane domain can be selectively chosen or modified via amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. The transmembrane domain can be derived from natural or synthetic sources. When the source is natural, the domain can originate from any membrane-binding or transmembrane protein.

[0168] Non-limiting examples of such proteins include: CD28, CD8α, CD8β, 4-1BB, B7-H3, BAFFR, BLAME, BTLA, CD100, CD103, CD11a, CD11b, CD11c, CD11d, CD160, CD18, CD19, CD19a, CD2, CD247, CD27, CD276, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD79a, CD84, CD96, CDS, CEACAM1, CRT AM, DAP-10, DNAM1, Fcγ receptor, GADS, GITR, HVEM, IA4, ICAM-1, ICOS, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, ​​Ly9, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, OX-40, PAG, PD-1, PSGL1, SELPLG, SLAM, SLAMF4, SLAMF6, SLAMF7, SLP-76, TNFR2, TNFSF14, TRANCE, VLA1, VLA-6, cytokine receptor, MHC Class 1 molecules, SLAM protein, TNF receptor protein, Toll ligand receptor, activated NK cell receptor, immunoglobulin protein, and integrins.

[0169] C. Intracellular domains – co-stimulatory domains and activation domains The intracellular (cytoplasmic) domain of the engineered T cells of the present invention can activate at least one normal effector function of immune cells. Effector functions of T cells may, for example, refer to cytolytic activity or helper activity, including cytokine secretion. The intracellular domain may include at least an activation domain. The intracellular domain may also include one or more co-stimulatory domains.

[0170] Co-stimulatory domain As used herein, a “co-stimulatory domain” refers to a molecule that provides signals that mediate T cell responses, including but not limited to proliferation, activation, and differentiation. Co-stimulatory domains can provide signals in addition to the primary signals provided by activating molecules as described herein.

[0171] Chimeric antigen receptors can incorporate costimulatory (signaling) domains to enhance their potency. See U.S. Patents 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (ibid.), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59–83 (2016). For example, CD28 is a naturally occurring costimulatory protein on T cells. While various costimulatory molecules have been described herein, it should be understood that other costimulatory molecules are also included within the scope of this invention.

[0172] It should be understood that suitable co-stimulatory domains within the scope of this invention include the following signal transduction domains (or other suitable portions): CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or fragments thereof, or any combination thereof. It should be understood that other co-stimulatory molecules or fragments thereof not listed above are within the scope of this invention.

[0173] Activate the structural domain "Activation domain" refers to molecules on T cells, such as the TCR / CD3 complex, which specifically binds to homologous stimulating ligands present on antigen-presenting cells. This article describes suitable activation molecules. The activation domain is part of the intracellular (cytoplasmic) portion of the CAR. The intracellular domain of the CAR can activate at least one normal effector function of immune cells. T cell effector functions can refer to, for example, cytolytic activity or helper activity, including cytokine secretion.

[0174] CD3 is a T-cell receptor element on natural T cells and has been shown to be an important intracellular activating element in CARs. In a preferred embodiment, CD3 is CD3ζ, CD3ε, CD3δ, or CD3γ.

[0175] CAR immune cells It also provides immune cells containing the CAR of this disclosure or one or more polynucleotides encoding the CAR.

[0176] As described, engineered receptors can be inserted into and expressed by immune cells (such as T cells), which can recognize specific antigens and activate immune cells when they bind to those antigens, thereby attacking and destroying cells carrying those antigens.

[0177] Several types of immune cells have been tested for CAR expression. Non-limiting examples include T cells, NK cells, macrophages, and monocytes. In some embodiments, the T cells may be αβ T cells. In some embodiments, the T cells are γδ T cells.

[0178] Polynucleotides encoding antibodies and methods for preparing antibodies This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives thereof. The polynucleotides of this disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on different polynucleotide molecules. Additionally, the polynucleotides of this disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on different polynucleotide molecules.

[0179] Methods for humanizing nonhuman antibodies are well known in the art. Typically, one or more amino acid residues from a nonhuman source are introduced into the humanized antibody. These nonhuman amino acid residues are generally referred to as importing residues, and these residues are typically derived from the importing variable domain. Humanization can be performed essentially as Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing the corresponding sequence of the human antibody with a rodent CDR or CDR sequence. Thus, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than the complete human variable domain is replaced by a corresponding sequence from a nonhuman species. In practice, humanized antibodies are often human antibodies in which some CDR residues and possibly some FR residues are replaced with residues from similar sites in rodent antibodies.

[0180] Antibody drug conjugates This disclosure provides antibody-drug conjugates (ADCs) of anti-CDH17 antibodies. Compared to antibodies, anti-CDH17 ADCs exhibit binding affinity comparable to CDH17 and demonstrate significant cytotoxic effects and significant in vivo antitumor effects against CDH17-expressing tumor cells. Furthermore, anti-CDH17 ADCs show a potent bystander effect against human CDH17-negative cell lines.

[0181] The "bystander effect" is often used to describe the ability of antibody-targeted cytotoxic agents to eradicate not only antigen-positive cells but also neighboring antigen-negative cells. The efficacy of antibody-drug conjugates is often impaired by the heterogeneous expression of target antigens in certain tumors, as targeted delivery of conjugates via antibody binding to antigen-positive tumor cells avoids any antigen-negative tumor cells that the antibody cannot bind to. Furthermore, the tumor vascular system is an increasingly important target, as shrinking tumors by cutting off their blood supply is an attractive strategy. The tumor vascular system is typically tumor antigen-negative.

[0182] Antibody-drug conjugates (ADCs) comprise an antibody or its antigen-binding fragment conjugated to a drug (or payload) via a linker. The drug can be linked to the antibody via cysteine ​​or lysine residues on the antibody. ADCs can be constructed chemically, for example, by utilizing pre-existing lysine or cysteine ​​residues and randomly conjugating them via appropriate coupling reactions (e.g., amide conjugation and thiol conjugation of cysteine). Active carboxylic acid esters (when available in the linker) are used to link the payload to lysine residues on the antibody. The primary amine in Lys readily reacts with the N-hydroxysuccinimide (NHS) ester introduced into the linker to form a stable amide. A typical IgG1 antibody molecule has approximately 90 Lys residues, of which approximately 30 can be modified for conjugation, meaning that 1 to 30 payloads can be covalently conjugated to the antibody. As for cysteine, after reduction, the disulfide bond can be transformed, exposing free cysteine ​​residues that can be used for coupling reactions (e.g., Michael addition, disulfide bond formation, and α-halocarbonyl alkylation).

[0183] ADCs can also be constructed via enzymatic methods, such as site-specific conjugation. Site-specific conjugation includes the introduction of engineered reactive cysteine ​​residues, disulfide bond rebridging, non-natural amino acids, enzyme-assisted ligation or glycan reconstruction, sugar conjugation, or click chemistry. Details of conjugation methods can be found in the art, for example, Fu et al., Signal Transduction and Targeted Therapy 7:93 (2022).

[0184] The drug-antibody ratio (DAR) of an ADC can be greater than 2, greater than 4, greater than 6, greater than 8, greater than 10, greater than 16, greater than 20, or greater than 30. In some embodiments, the drug-antibody ratio (DAR) can be 1 to 30, 1 to 20, 1 to 10, 1 to 8, for example, 4 to 8 or 2 to 4. In some embodiments, the drug-antibody ratio (DAR) can be 2, 4, 6, 8, 10, 16, 20, or 30. In some embodiments, the DAR is homogeneous.

[0185] Drug linker compounds can contain linkers that are either uncuttable or cuttable. Cuttable linkers mainly include enzyme-cuttable linkers and chemically sensitive linkers.

[0186] Uncleavable linkers consist of stable bonds that resist protein hydrolysis and ensure greater plasma stability. The mechanism of action of uncleavable linkers is based on the internalization of the ADC complex, followed by the degradation of mAb components in lysosomes, thereby releasing cytotoxic drugs capable of killing tumor cells. They do not release cytotoxic agents at off-target sites and therefore do not harm healthy cells. Uncleavable linkers are classified into two groups: thioether or maleimide hexanoyl (MC). Examples of uncleavable linkers include, but are not limited to, 4-maleimide methylcyclohexane-1-carboxylate (MCC), succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), maleimide hexanoyl (MC), and p-carboxycyclohexylmethylmaleimide.

[0187] Cleavable adapters mainly include chemically sensitive adapters and enzyme-cleavable adapters. Chemically sensitive adapters are usually cleaved by environmental differences (such as redox potential and pH), while enzyme-cleavable adapters are cleaved by specific enzymes in response to extracellular and intracellular environments.

[0188] Chemically sensitive connectors include, but are not limited to, pH-sensitive connectors and glutathione-sensitive disulfide connectors. (Khongorzul et al., Mol Cancer Res; 18(1) (2020)).

[0189] pH-sensitive adapters are a group of adapters that are sensitive to acidic environments but stable in alkaline environments (e.g., systemic circulation), such as hydrazone-based adapters. A successful example of an ADC design using pH-sensitive adapters is the IMMU-110, which consists of a humanized anti-CD74 mAb conjugated with doxorubicin via an acid-labile hydrazone.

[0190] Glutathione-sensitive disulfide linkers utilize the difference in reduction potential between the cytoplasm and plasma. High concentrations of glutathione can be found in cancer cells compared to normal cells. Glutathione-sensitive linkers are stable in the bloodstream and can be specifically cleaved by the elevated intracellular concentration of glutathione in tumor cells, thereby releasing the active drug from a non-toxic prodrug at the tumor site (see above).

[0191] Enzyme-cleavable adapters include, but are not limited to, peptide-based adapters, β-glucuronide-based adapters, and phosphate-based adapters.

[0192] Peptide-based linkers (also known as protease-sensitive linkers) are the most commonly used ADC linkers. These linkers can be cleaved extracellularly and / or intracellularly by specific proteases. For intracellular cleavage, because tumor cells exhibit high expression of lysosomal proteases (such as cathepsin B) compared to normal cells, protease-sensitive peptide linker ADCs selectively bind and transform into cancer cells via receptor-mediated endocytosis. Peptide linkers are stable in systemic circulation and release drugs only in target cells (see above). Examples are valine-citrulline (VC or Val-Cit), valine-alanine (VA), phenylalanine-lysine (PL), and glycine-glycine-phenylalanine-glycine (GGFG).

[0193] The β-glucuronide-based linker is recognized and hydrolyzed by β-glucuronidase or β-galactosidase to release the drug, and both β-glucuronidase and β-galactosidase are enriched in lysosomes and tumor necrosis zones. β-glucuronidase is inactive at physiological pH (circulatory) but active at lysosomal pH. This selective site of action allows for the cleavage of the glycosidic bond in the β-glucuronidase-sensitive β-glucuronide linker, thereby enabling the selective release of cytotoxic payloads (see above).

[0194] Phosphate-based linkers are a class of enzyme-cleavable linkers that are expressed to specifically target target enzymes in lysosomal compartments. These linkers target pyrophosphatases and acid phosphatases, which hydrolyze pyrophosphates and terminal monophosphates into their respective alcohols.

[0195] Parts that can be introduced as part of a linker to facilitate drug linker or antibody linker bonding include maleimide hexanoyl, maleimide-hexanoyl (MC) moiety, maleimide-methylene-cyclohexylcarbonyl moiety spacer, maleimide propionyl (MP), p-aminobenzylcarbamate (PAB) spacer, p-aminobenzyloxycarbamate (PABC) spacer, aminomethoxymethylenecarbonyl (-NH-CH2-O-CH2-CO-) spacer, 4-(4'-acetylphenoxy)butyric acid moiety, acylhydrazide (-CO-NH-NH2) and thiol (-SH) moiety, Lys-PABC, PEG chain, and PEG8.

[0196] Examples of connectors include, but are not limited to, succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), sulfonyl-SMCC, MC-VC-PABC, CL2A, MC-GGFG, MC, MC-PEG8-GGFG, MC-PEG8-GGFG-PAB, or MP-PEG8-VA-PABC.

[0197] The drug or payload can be a cytotoxin, a therapeutic peptide, or a polypeptide.

[0198] The peptide or polypeptide can be any peptide or polypeptide having therapeutic properties (e.g., analgesic, antidiabetic, antitumor, or antiviral activity). Additionally or alternatively, the drug preferably contains an amine group, a thiol group, or a carboxylic acid group, as these types of groups provide ideal sites for the conjugation of the drug with the connectors of this disclosure. Examples of biological agents are Pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, Staphylococcus aureus enterotoxin A / E-120, Shiga toxin, ricin, and urease.

[0199] Cytotoxins, once released from ADCs, are activated in the cytoplasm of tumor cells, thereby destroying them. There are two main types of cytotoxins that can be used in ADC design: microtubule disruptors (e.g., auristatin and maytanyl alkaloids) and DNA damage agents (e.g., chalcogenide, benzodiazepines, and doxorubicin). Examples include, but are not limited to: auristatin, maytanyl alkaloids, benzodiazepines, microtubule-lysin, benzodiazepines, chalcogenide, ixenotac, irinotecan (SN38), doxorubicin, anthracycline, pyrrolobenzodiazepines (PBD), TLR agonists, and STING agonists.

[0200] Auristatins are synthetic antitumor agents derived from the natural product saliton toxin 10. They block the microtubule polymerization process, leading to cell cycle arrest and apoptosis. Examples include monomethylaurestatin E (MMAE) and monomethylaurestatin F (MMAF).

[0201] Maytansine alkaloids are isolated from maytansine (a benzoans acrolide). These drugs inhibit microtubule polymerization. Examples include DM1 and DM4.

[0202] Kazimidox is a type of fungus derived from Micromonospora echinococcosis (…). Micromonospora echinospora Kazimidic acid is an enediyne-based antitumor antibiotic. It recognizes the minor groove of DNA, preventing DNA replication and thus causing mitotic arrest and cell death. An example is N-acetylkazimidic acid, a derivative of kazimidic acid.

[0203] Betamicil is derived from bacteria of the genus Streptomyces (Streptomyces spp.) Streptomyces (This refers to a natural product derivative extracted from a strain of bacteria.) Betainecin is another type of DNA minor groove binding alkylating agent. These drugs exert their effect by binding to the minor groove of DNA, subsequently causing irreparable alkylation of the DNA, thus disrupting the architecture and structural integrity of nucleic acids.

[0204] Doxorubicin works by inserting into DNA to inhibit DNA synthesis. One example is IMMU-110.

[0205] Iciticon is derived from the Chinese tree Camptotheca acuminata (Camptotheca sinensis). Camptotheca acuminata Ecinotecan is a synthetic derivative of the natural cytotoxin camptothecin, isolated from [a specific organism / organism]. Like camptothecin, eccinotecan binds to the topoisomerase 1-DNA complex, preventing DNA rejoining, leading to the accumulation of DNA strand breaks and ultimately cell death. Examples of eccinotecan are DX-8951f and DXd. 7-Ethyl-10-hydroxycamptothecin (SN-38) is also a derivative of camptothecin.

[0206] Other examples of cytotoxins include, but are not limited to, tubulolysin A, camptothecin, DGN462, amberstatin 269, atropine, SG3199 / SCX, IRDye® 700DX, TLR7 / 8 agonists, and diABZI STING agonist-2.

[0207] Cytotoxins can be chemotherapeutic agents, which can be classified according to their mechanism of action into groups such as the following: -Antimetabolites / anticancer agents (e.g., pyrimidine analogues fluorouridine, capecitabine, and cytarabine); -Purine analogues, folic acid antagonists and related inhibitors; - Antiproliferative / antimitotic agents, including natural products (e.g., vinblastine, vincristine) and microtubules (e.g., taxanes (paclitaxel, docetaxel), vinblastin, nocodazole, epothilone, vinorelbine). ® And epipodophyllotoxin (etoposide, teniposide); DNA damaging agents (e.g., actinomycin, acridine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide) ® Actinomycin D, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosourea, procarbazine, paclitaxel, docetaxel, teniposide, etoposide, and triethylene thiophosphoramide). - Antibiotics (such as actinomycin D, daunorubicin, doxorubicin, idarubicin, anthracycline, mitoxantrone, bleomycin, procainox (scintillans), and mitomycin); -Enzymes (such as L-asparaginase, which systemically metabolizes L-asparagine and causes cells that cannot synthesize asparagine to lose it). -Antibacterial agents; - Antiproliferative / antimitotic alkylating agents (e.g., nitrogen mustard cyclophosphamides and their analogues (melphalan, chlorambucil, hexamethylmelamine and thiotepa), alkylnitrosoureas (carmustine) and their analogues, streptozotocin and triazine (dacarbazine)); -Anti-proliferative / anti-mitotic antimetabolites (e.g., folic acid analogues (methotrexate)); - Platinum coordination complexes (cisplatin, oxaliplatin and carboplatin), procarbazine, hydroxyurea, mitotane and ammonia glutathione; - Hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilumid), aromatase inhibitors (letrozole, anastrozole); - Anticoagulants (such as heparin, synthetic heparin salts and other thrombin inhibitors); - Fibrinolytic agents (such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel); -Anti-migration agent; - Antisecretory agents (breveldin); -Immunosuppressants tacrolimus, sirolimus, azathioprine, and mycophenolate mofetil; - Compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitor and fibroblast growth factor inhibitor). -Angiotensin receptor blockers, nitric oxide donors; -Antisense oligonucleotides; - Antibodies (such as trastuzumab and rituximab); - Cell cycle inhibitors and differentiation inducers (e.g., retinoic acid); - Inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, actinomycin D, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan and irinotecan) and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone); - Growth factor signal transduction kinase inhibitors; - Dysfunction inducers; -Toxins (such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and caspase activator). - and chromatin.

[0208] Other examples of chemotherapy agents include: - Alkylating agents (e.g., thiotepa and cyclophosphamide) ® )); -Alkyl sulfonates (e.g., busulfan, indomethacin, and piperazine); -Zalaziridines (e.g., benzodopa, carboquinone, meturedopa, and uredopa); - Emylerumine and memylamelamine (including alfretamine, triemylenemelamine, triemylphosphonamide, triemylthiophosphonamide, and triemylolomelamine); - Acetylginine (especially blatazine and blatazine ketone); - Camptothecin (including its synthetic analogue topotecan); - Lichenin; -Carlistatin; -CC-1065 (including its synthetic analogues of adoresin, cazoresin and pyresin); - Cryptophytic substances (especially cryptophytic 1 and cryptophytic 8); -Sea slug toxin; - Betamethasone (including synthetic analogues KW-2189 and CBI-TMI); - Acanthopanax senticosin; - Pancratistatin; -Cynocoral alcohol; -Spongistatin; - Nitrogen mustards (e.g., chlorambucil, naphthylambucil, cyclophosphamide, estradiol, ifosfamide, dichloromethyldiethylamine, dichloromethyldiethylamine hydrochloride, melphalan, neonitrogen mustard, benzylmustine, prednimustine, trazophosphatide, and uracil mustard); - Nitrosoureas (e.g., carmustine, chloramphenicol, foremustine, lomustine, nimustine, and ramustine); - Antibiotics (e.g., enediyne antibiotics (e.g., chachiin, especially chachiin γII and chachiin φI1), danendomycin (including danendomycin A), bisphosphonates (e.g., clodronate), esperamycin, neomycin chromophores and related pigment proteins, enediyne antibiotic chromophores, aclarubicin, actinomycin, atrazoserine, bleomycin, actinomycin C, carabin, carrninomycin, cymosin, chromomycin, actinomycin D, daunorubicin, detoxin, 6-diazo- 5-O-L-leucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogamycin, olivomycin, pepromycin, pofibromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, tuberculin, ubenmexicin, fenestrated statin, zorubicin); -Antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); - Folic acid analogues (such as demopterin, methotrexate, pteroxate, and trimethoprim); -Purine analogues (e.g., fludarabine, 6-mercaptopurine, nitrothioguanine, and thioguanine); -Pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine); - Androgens (such as capetorone, drotalbutone propionate, cyclothiosterol, meandrolone, and testosterone); - Anti-adrenergic drugs (such as aminoglutide, mitotane and triplostertan). - Folic acid replacement therapy (e.g., folinic acid); - Trichothecene (especially T-2 toxin, wartsporin A, loridin A and serpentin); - Taxanes (e.g., paclitaxel (TAXOL) ® ) and TAXOTERE ® )); - Platinum analogues (e.g., cisplatin, carboplatin); - Acetaldehyde lactone; Aldolphosphatidylglycoside; Aminolevulinic acid; Eniluracil; Acridine; Hestrabucil; Bisaccharide; Edatraxate; Defofamine; Colchicine; Diacinone; Elformthine; Elimethicone; Epotoxin; Etoglobulin; Gallium nitrate; Hydroxyurea; Lentinan; Leucovorin; Clonidamine; Maytansine alkaloids (e.g., Maytansine and Ansericin); Mitoguanidine hydrazone; Mitoantrone; Mopiperol; Nitracrid; Pentostatin; Methionine Mustard; Pirarubicin; Loxoanthraquinone; Fluoropyrimidine; Leucovorin; Podophyllin; 2-Ethylhydrazine; Procarbazine; Polysaccharide-K (PSK); Razosen; Rhizomycin; Cizonan; Germanium Spiroamine; Alternaria solanilic acid; Triaminoquinone; 2,2',2''-Trichlorotriethylamine; Urethane; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeugenol; Piperobromide; Gacytosine; Cytarabine (“Ara-C”); Cyclophosphamide; Thiotepa; Chlorobacillin; Gemcitabine ® ); 6-Thioguanine; mercaptopurine; methotrexate; vincristine; platinum-based drugs; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE) ® ); Norfloxacin; Teniposide; Idatraxa; Daunorubicin; Aminopterin; Xeloda; Ibandronic acid; CPT-11; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DFMO); Retinoids (e.g., retinoic acid); Capecitabine; FOLFIRI (fluorouracil, folinic acid, and irinotecan); - and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

[0209] In some embodiments, the drug and the connector form a drug connector compound of formula I: I Or its stereoisomers or pharmaceutically acceptable salts, wherein: A 1 -NHR 1 , where R 1 For hydrogen, -C(O)R 5 -C6 aryl-C 1-6 Alkyl-NH2 or -C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 2 and A 3 All are hydrogen; or A2 -NHR 2 , where R 2 For hydrogen, -C(O)R 5 -C6 aryl-C 1-6 Alkyl-NH2 or -C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 1 and A 3 All are hydrogen; or A 3 -NHR 3 , where R 3 For hydrogen, -C(O)R 5 -C6 aryl-C 1-6 Alkyl-NH2 or -C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 1 and A 2 All are hydrogen; R 4 -C 1-6 Alkyl; condition is when A 1 -NHR 1 When, then R 4 Not methyl; R 5 For -LR 6 ; L represents the connector portion; and R 6 The group is hydrogen or a heterocyclic group, wherein the heterocyclic group is optionally covalently linked to an antibody or antigen-binding fragment. Further details of these compounds and their synthesis can be found in PCT patent application number: PCT / CN2023 / 123581.

[0210] In some implementation schemes, A 1 -NHR 1 , where R 1 For hydrogen, -C(O)R 5 -C6 aryl-C 1-6 Alkyl-NH2 or -C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 2 and A 3 All are hydrogen. Therefore, in some embodiments, compounds of formula II are provided: II Or its stereoisomer or its pharmaceutically acceptable salt, wherein R 4 -C 2-6 Alkyl, and A 1 Independently as defined in this article.

[0211] In some implementation schemes, A 1 It is -NH2; and A 2 and A 3 Both are hydrogen.

[0212] In some implementation schemes, A 1 -NH-C(O)R 5 And A 2 and A 3 Both are hydrogen.

[0213] In some implementation schemes, A 1 -NH-C6 aryl-C 1-6 Alkyl-NH2; and A 2 and A 3 Both are hydrogen. In some implementations, A 1 It is -NH-C6 aryl-CH2-NH2; and A 2 and A 3 Both are hydrogen.

[0214] In some implementation schemes, A 1 -NH-C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 2 and A 3 Both are hydrogen. In some implementations, A 1 -NH-C6 aryl-CH2-NHC(O)R 5 And A 2 and A 3 Both are hydrogen.

[0215] In some implementation schemes, A 2 -NHR 2 , where R 2 For hydrogen, -C(O)R 5 -C6 aryl-C 1-6 Alkyl-NH2 or -C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 1 and A 3 All are hydrogen. Therefore, in some embodiments, compounds of formula III are provided: III Or its stereoisomer or its pharmaceutically acceptable salt, wherein A 2 and R 4 Each is defined independently as described in this article.

[0216] In some implementation schemes, A 2It is -NH2; and A 1 and A 3 Both are hydrogen.

[0217] In some implementation schemes, A 2 -NH-C(O)R 5 And A 1 and A 3 Both are hydrogen.

[0218] In some implementation schemes, A 2 -NH-C6 aryl-C 1-6 Alkyl-NH2; and A 1 and A 3 Both are hydrogen. In some implementations, A 2 It is -NH-C6 aryl-CH2-NH2; and A 1 and A 3 Both are hydrogen.

[0219] In some implementation schemes, A 2 -NH-C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 1 and A 3 Both are hydrogen. In some implementations, A 2 -NH-C6 aryl-CH2-NHC(O)R 5 And A 1 and A 3 Both are hydrogen.

[0220] In some implementation schemes, A 3 -NHR 3 , where R 3 For hydrogen, -C(O)R 5 -C6 aryl-C 1-6 Alkyl-NH2 or -C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 1 and A 2 All are hydrogen. Therefore, in some embodiments, compounds of formula IV are provided: IV Or its stereoisomer or its pharmaceutically acceptable salt, wherein A 3 and R 4 Each is defined independently as described in this article.

[0221] In some implementation schemes, A 3 It is -NH2; and A 1 and A 2 Both are hydrogen.

[0222] In some implementation schemes, A 3 -NH-C(O)R 5 And A 1 and A 2 Both are hydrogen.

[0223] In some implementation schemes, A 3 -NH-C6 aryl-C 1-6 Alkyl-NH2; and A 1 and A 2 Both are hydrogen. In some implementations, A 3 It is -NH-C6 aryl-CH2-NH2; and A 1 and A 2 Both are hydrogen.

[0224] In some implementation schemes, A 3 -NH-C6 aryl-C 1-6 Alkyl-NHC(O)R 5 And A 1 and A 2 Both are hydrogen. In some implementations, A 3 -NH-C6 aryl-CH2-NHC(O)R 5 And A 1 and A 2 Both are hydrogen.

[0225] In some implementation schemes, R 4 It is methyl, ethyl, or isopropyl. In some embodiments, R 4 It is ethyl or isopropyl. In some embodiments, R 4 It is methyl. In some embodiments, R 4 It is an ethyl group.

[0226] In some implementation schemes, R 4 It is isopropyl.

[0227] In some implementations, compounds of formula IVA are provided: IVA Or its stereoisomer or its pharmaceutically acceptable salt, wherein A 3 Independently as defined in this article.

[0228] In some implementations, compounds of formula IVB are provided: IVB Or its stereoisomer or its pharmaceutically acceptable salt, wherein R 5 Independently as defined in this article.

[0229] In some implementations, when R 1 R 2 Or R 3 When it is not hydrogen, the compound contains a connecting portion L, which connects R. 6 It is covalently attached to the rest of the compound.

[0230] In some embodiments, L comprises one or more chain heteroatoms and one or more alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkyl moieties; wherein each alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkyl moieties may independently and optionally be selected from one to five independently selected from oxo, halogen, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Substitution of alkyl halogens and benzyl groups.

[0231] In some embodiments, L is an alkylene linker that optionally contains one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thiosuccinimide, or ketone functional groups.

[0232] In some embodiments, L comprises one or more amino acids. In some embodiments, L comprises one or more straight-chain or branched natural or non-natural amino acids. In some embodiments, L comprises a polypeptide.

[0233] In some embodiments, L comprises one or more polyethylene glycol units (e.g., PEG with an average molecular weight of 300 g / mol to 10,000 g / mol).

[0234] In some implementation schemes, R 5 -L 1 -(AA) n -L 2 -R 6 , where each L 1 AA, n, L 2 and R 6 Independently as defined in this article.

[0235] In some implementations, L 1 C 1-20 Alkylene or C 2-20 Heteroalkylene; n is 0, 1, 2, 3, 4, 5, or 6; Each AA is an amino acid independently; and L 2 C 1-40 Alkylene or C 2-40 Heteroalkylene, wherein C1-40 Alkylene or C 2-40 Heteroalkylene groups are optionally located at C 1-40 Alkylene or C 2-40 The heteroalkylene chain contains phenylene, and C 1-40 Alkylene or C 2-40 The heteroalkylene group is optionally substituted with one or more (e.g., one or two) oxo groups.

[0236] In some implementations, L 1 C 1-20 Alkylene or C 2-20 Heteroalkylene; n is 0, 1, 2, 3, 4, 5, or 6; Each AA is an amino acid independently; and L 2 C 1-40 Alkylene or C 2-40 Heteroalkylene, wherein C 1-40 Alkylene or C 2-40 The heteroalkylene group may optionally be substituted with one or more (e.g., one or two) oxo groups.

[0237] In some implementations, n is 0. In some implementations, n is 1, 2, 3, 4, 5, 6, or 7. In some implementations, n is 2, 3, 4, 5, 6, or 7. In some implementations, n is 1. In some implementations, n is 2. In some implementations, n is 3. In some implementations, n is 4. In some implementations, n is 5. In some implementations, n is 6. In some implementations, n is 7.

[0238] In some implementations, L 1 C 1-20 Alkylene. In some embodiments, L 1 C 1-10 Alkylene. In some embodiments, L 1 C 2-6 Alkylene.

[0239] In some implementations, L 1 C 2-20 Heteroalkylene compounds. In some embodiments, L 1 C 2-10 Heteroalkylene compounds. In some embodiments, L 1 C 2-6 Heteroalkylene.

[0240] In some implementations, L 1 -(CH2) p -X 1 -(CH2) q-X 2 - or -X 1 -(CH2) p -phenylene-(CH2) q -X 2 - ;in Key and -(AA) n -L 2 -R 6 Connect; and X 1 For bonds, -O-, -S-, or -NH-; X 2 For bonds, -O-, -S-, or -NH-; p is 1, 2, 3, or 4; and q can be 1, 2, 3 or 4.

[0241] In some implementations, L 1 -CH2-X 1 -CH2-X 2 - In some implementations, L 1 -(CH2)2-X 1 -CH2-X 2 - In some implementations, L 1 -(CH2)3-X 1 -CH2-X 2 - In some implementations, L 1 -(CH2)4-X 1 -CH2-X 2 - .

[0242] In some implementations, L 1 -CH2-X 1 -(CH2)2-X 2 - In some implementations, L 1 -(CH2)2-X 1 -(CH2)2-X 2 - In some implementations, L 1 -(CH2)3-X 1 -(CH2)2-X 2 - In some implementations, L 1 -(CH2)4-X 1 -(CH2)2-X2 - .

[0243] In some implementations, L 1 -CH2-X 1 -(CH2)3-X 2 - In some implementations, L 1 -(CH2)2-X 1 -(CH2)3-X 2 - In some implementations, L 1 -(CH2)3-X 1 -(CH2)3-X 2 - In some implementations, L 1 -(CH2)4-X 1 -(CH2)3-X 2 - .

[0244] In some implementations, L 1 -CH2-X 1 -(CH2)4-X 2 - In some implementations, L 1 -(CH2)2-X 1 -(CH2)4-X 2 - In some implementations, L 1 -(CH2)3-X 1 -(CH2)4-X 2 - In some implementations, L 1 -(CH2)4-X 1 -(CH2)4-X 2 - .

[0245] In some implementation schemes, X 1 As a key. In some implementations, X 1 For -O-. In some implementations, X 1 For -S-. In some implementations, X 1 It is -NH-.

[0246] In some implementation schemes, X 2 As a key. In some implementations, X 2 For -O-. In some implementations, X 2 For -S-. In some implementations, X2 It is -NH-.

[0247] In some implementations, L 1 -CH2-X 1 -CH2-NH- In some implementations, L 1 -(CH2)2-X 1 -CH2-NH- In some implementations, L 1 -(CH2)3-X 1 -CH2-NH- In some implementations, L 1 -(CH2)4-X 1 -CH2-NH- .

[0248] In some implementations, L 1 -CH2-X 1 -(CH2)2-NH- In some implementations, L 1 -(CH2)2-X 1 -(CH2)2-NH- In some implementations, L 1 -(CH2)3-X 1 -(CH2)2-NH- In some implementations, L 1 -(CH2)4-X 1 -(CH2)2-NH- .

[0249] In some implementations, L 1 -CH2-X 1 -(CH2)3-NH- In some implementations, L 1 -(CH2)2-X 1 -(CH2)3-NH- In some implementations, L 1 -(CH2)3-X 1 -(CH2)3-NH- In some implementations, L 1 -(CH2)4-X 1 -(CH2)3-NH- .

[0250] In some implementations, L 1 -CH2-X 1-(CH2)4-NH- In some implementations, L 1 -(CH2)2-X 1 -(CH2)4-NH- In some implementations, L 1 -(CH2)3-X 1 -(CH2)4-NH- In some implementations, L 1 -(CH2)4-X 1 -(CH2)4-NH- .

[0251] In some implementations, L 1 -CH2-O-CH2-X 2 - In some implementations, L 1 -(CH2)2-O-CH2-X 2 - In some implementations, L 1 -(CH2)3-O-CH2-X 2 - In some implementations, L 1 -(CH2)4-O-CH2-X 2 - .

[0252] In some implementations, L 1 -CH2-O-(CH2)2-X 2 - In some implementations, L 1 -(CH2)2-O-(CH2)2-X 2 - In some implementations, L 1 -(CH2)3-O-(CH2)2-X 2 - In some implementations, L 1 -(CH2)4-O-(CH2)2-X 2 - .

[0253] In some implementations, L 1 -CH2-O-(CH2)3-X 2 - In some implementations, L 1 -(CH2)2-O-(CH2)3-X 2 - In some implementations, L 1 -(CH2)3-O-(CH2)3-X 2 - In some implementations, L 1 -(CH2)4-O-(CH2)3-X 2 - .

[0254] In some implementations, L 1 -CH2-O-(CH2)4-X 2 - In some implementations, L 1 -(CH2)2-O-(CH2)4-X 2 - In some implementations, L 1 -(CH2)3-O-(CH2)4-X 2 - In some implementations, L 1 -(CH2)4-O-(CH2)4-X 2 - .

[0255] In some implementations, L 1 for , or ,in Key and -(AA) n -L 2 -R 6 Connected.

[0256] In some implementations, L 1 -(CH2) p -O-(CH2) q -NH- .

[0257] In some implementations, L 1 -CH2-O-CH2-NH- In some implementations, L 1 -(CH2)2-O-CH2-NH- In some implementations, L 1 -(CH2)3-O-CH2-NH- In some implementations, L 1 -(CH2)4-O-CH2-NH- .

[0258] In some implementations, L1 -CH2-O-(CH2)2-NH- In some implementations, L 1 -(CH2)2-O-(CH2)2-NH- In some implementations, L 1 -(CH2)3-O-(CH2)2-NH- In some implementations, L 1 -(CH2)4-O-(CH2)2-NH- .

[0259] In some implementations, L 1 -CH2-O-(CH2)3-NH- In some implementations, L 1 -(CH2)2-O-(CH2)3-NH- In some implementations, L 1 -(CH2)3-O-(CH2)3-NH- In some implementations, L 1 -(CH2)4-O-(CH2)3-NH- .

[0260] In some implementations, L 1 -CH2-O-(CH2)4-NH- In some implementations, L 1 -(CH2)2-O-(CH2)4-NH- In some implementations, L 1 -(CH2)3-O-(CH2)4-NH- In some implementations, L 1 -(CH2)4-O-(CH2)4-NH- .

[0261] In some embodiments, L contains more than one consecutive amino acid to form the formula -(AA). n - peptide units. In some embodiments, formula -(AA) n The peptide unit of - consists of 2 to 7 amino acids linked by peptide (i.e., amide) bonds. In some embodiments, formula -(AA) n - peptide units, at their C-terminal linker L 1 It is bonded to AA and to -L at its N-terminus. 2 -R 6 Partial bonding.

[0262] Peptide unit-(AA)n The amino acid sequence of - is not particularly limited, but examples include L-amino acids or D-amino acids, and may include amino acids or non-natural amino acids (e.g., N-methylated amino acids) having structures such as β-alanine, ε-aminohexanoic acid, γ-aminobutyric acid, α-amino acids, etc. Specific examples include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), and aspartic acid (Asp; D). In some embodiments, the peptide unit - (AA) n - Contains one or more (e.g., 2 to 7, or 2 to 5, or 3 to 5, or 4) amino acid residues, which are independently selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid.

[0263] In some implementations, -(AA) n - Selected from -FGG-, -FGGD-, -FGG-(D-)D-, -FGGE-, -GFGG-, -FGGS-, -FGGK-, -GFGGD-, -GGFGG-, -GFGGDD-, -GFGGDK-, -FGGGFGG-, where (D-)D refers to D-aspartic acid.

[0264] In some implementations, n is 2, 3, 4, 5, 6 or 7, and each AA is independently selected from Gly and Phe.

[0265] In some implementations, -(AA) n - is -Gly-Phe-Gly-Gly-(-GFGG-).

[0266] In some implementations, -(AA) n -for or The key aa and L 2 Connected.

[0267] In some implementations, L 2 C is a C that is optionally substituted with one or more oxygen groups. 1-40 Alkylene. In some embodiments, L 2 C is a C that is optionally substituted with one or more oxygen groups. 1-30 Alkylene. In some embodiments, L 2 C is a C that is optionally substituted with one or more oxygen groups. 1-20 Alkylene. In some embodiments, L 2C is a C that is optionally substituted with one or more oxygen groups. 1-10 Alkylene. In some embodiments, L 2 C is a C that is optionally substituted with one or more oxygen groups. 5-40 Alkylene. In some embodiments, L 2 C is a C that is optionally substituted with one or more oxygen groups. 5-30 Alkylene. In some embodiments, L 2 C is a C that is optionally substituted with one or more oxygen groups. 5-20 Alkylene. In some embodiments, L 2 C is a C that is optionally substituted with one or more oxygen groups. 5-10 Alkylene.

[0268] In some implementations, L 2 -C(O)-C 0-39 Alkylene. In some embodiments, L 2 -C(O)-C 1-15 Alkylene. In some embodiments, L 2 -C(O)-C 1-10 Alkylene. In some embodiments, L 2 -C(O)-C 5-10 Alkylene.

[0269] In some implementations, L 2 C is a C that is optionally substituted with one or more oxygen groups. 2-40 Heteroalkylene compounds. In some embodiments, L 2 -C(O)-C 1-39 Heteroalkylene.

[0270] In some implementations, L 2 It is -C(O)-(CH2CH2O) 1-10 -CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O) 1-8 -CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O) 1-5 -CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O) 2-10 -CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O) 2-8 -CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O) 2-5 -CH2CH2-.

[0271] In some implementations, L 2 It is -C(O)-CH2CH2O-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)2-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)3-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)4-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)5-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)6-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)7-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)8-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O)9-CH2CH2-. In some implementations, L 2 It is -C(O)-(CH2CH2O) 10 -CH2CH2-.

[0272] In some implementations, L 2 for or Among them, the key ab and R 6 connect.

[0273] In some implementation schemes, R 5 -(CH2) p -O-(CH2) q -NH-(AA) n -C(O)-C 1-10 Alkylene-R 6 In some implementations, R 5 -(CH2) p -O-(CH2) q -NH-(AA) n -C(O)-(CH2) 1-10 -CH2CH2-R 6 .

[0274] In some implementation schemes, R 5 -(CH2) p -O-(CH2) q-NH-GFGG-C(O)-C 1-10 Alkylene-R 6 In some implementations, R 5 -(CH2) p -O-(CH2) q -NH-GFGG-C(O)-(CH2) 1-10 -CH2CH2-R 6 .

[0275] In some implementation schemes, R 5 -(CH2) p -O-(CH2) q -NH-(AA) n -C(O)-C 1-30 Heteroalkylene-R 6 In some implementations, R 5 -(CH2) p -O-(CH2) q -NH-(AA) n -C(O)-(CH2CH2O) 1-10 -CH2CH2-R 6 .

[0276] In some implementation schemes, R 5 -(CH2) p -O-(CH2) q -NH-GFGG-C(O)-C 1-35 Heteroalkylene-R 6 In some implementations, R 5 -(CH2) p -O-(CH2) q -NH-GFGG-C(O)-C 1-30 Heteroalkylene-R 6 In some implementations, R 5 -(CH2) p -O-(CH2) q -NH-GFGG-C(O)-(CH2CH2O) 1-10 -CH2CH2-R 6 .

[0277] In some implementation schemes, R 5 -(CH2) p -O-(CH2) q -NH-GFGG-C(O)-C 1-35 Heteroalkylene-R 6 In some implementations, R 5 -(CH2)p -O-(CH2) q -NH-GFGG-C(O)-C 1-30 Heteroalkylene-R 6 In some implementations, R 5 -(CH2) p -O-(CH2) q -NH-GFGG-C(O)-(CH2CH2O) 1-10 -CH2CH2-R 6 .

[0278] In some implementation schemes, R 5 for: , Where R 6 It is a heterocyclic group, wherein the heterocyclic group is optionally covalently linked to an antibody or antigen-binding fragment.

[0279] In some implementation schemes, R 5 for: ; Where R 6 It is a heterocyclic group, wherein the heterocyclic group is optionally covalently linked to an antibody or antigen-binding fragment.

[0280] In some implementations, p is 1. In some implementations, p is 2. In some implementations, p is 3. In some implementations, p is 4.

[0281] In some implementations, q is 1. In some implementations, q is 2. In some implementations, q is 3. In some implementations, q is 4.

[0282] In some embodiments, p is 1 and q is 1. In some embodiments, p is 2 and q is 1. In some embodiments, p is 3 and q is 1. In some embodiments, p is 4 and q is 1. In some embodiments, p is 1 and q is 2. In some embodiments, p is 2 and q is 2. In some embodiments, p is 3 and q is 2. In some embodiments, p is 4 and q is 2. In some embodiments, p is 1 and q is 3. In some embodiments, p is 2 and q is 3. In some embodiments, p is 3 and q is 3. In some embodiments, p is 4 and q is 3. In some embodiments, p is 1 and q is 4. In some embodiments, p is 2 and q is 4. In some embodiments, p is 3 and q is 4. In some embodiments, p is 4 and q is 4.

[0283] In some implementation schemes, A 3 -NH-C(O)R 5And A 1 and A 2 Both are hydrogen.

[0284] In some implementation schemes, A 3 -NH-C(O)-L 1 -(AA) n -L 2 -R 6 And A 1 and A 2 Both are hydrogen.

[0285] In some implementation schemes, A 3 -NH-C(O)-L 1 -(AA) n -L 2 -R 6 And A 1 and A 2 All are hydrogen; L 1 -(CH2) p -X 1 -(CH2) q -X 2 - or -X 1 -(CH2) p -phenylene-(CH2) q -X 2 - ;in Key and -(AA) n -L 2 -R 6 connect; n is 0, 1, 2, 3, 4, 5, or 6; Each AA is an amino acid independently; and L 2 C 1-40 Alkylene or C 2-40 Heteroalkylene, wherein C 1-40 Alkylene or C 2-40 Heteroalkylene groups are optionally located at C 1-40 Alkylene or C 2-40 The heteroalkylene chain contains phenylene, and C 1-40 Alkylene or C 2-40 The heteroalkylene group is optionally substituted with one or more (e.g., one or two) oxo groups.

[0286] In some implementation schemes, R 1 R 2 Or R 3 for:

[0287] Where R 6 It is a heterocyclic group, wherein the heterocyclic group is optionally covalently linked to an antibody or antigen-binding fragment.

[0288] In some implementation schemes, R 1 R 2 Or R 3 for:

[0289] Where R 6 It is a heterocyclic group, wherein the heterocyclic group is optionally covalently linked to an antibody or antigen-binding fragment.

[0290] In some implementation schemes, R 6 It is a heterocyclic group, which is covalently linked to an antibody or antigen-binding fragment.

[0291] In some implementation schemes, R 6 The heterocyclic group is optionally covalently linked to the antibody or antigen-binding fragment via a cystine or lysine residue on the antibody or antigen-binding fragment.

[0292] In some implementation schemes, R 6 for .

[0293] In some implementation schemes, R 6 for Where Ab is the antibody or antigen-binding fragment provided in this article.

[0294] In some implementations, a drug connector compound selected from the following is provided: (a) Formula VIII (LM-D01) , (b) Equation IX (LM-D02) ,and (c) Formula X (LM-D03) .

[0295] In some implementations, an ADC selected from the following is provided: , and

[0296] Wherein, Ab represents the antibody or antigen-binding fragment provided in this article.

[0297] In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, x is 6. In some embodiments, x is 7. In some embodiments, x is 8. In some embodiments, x is 4, 6, or 8. In some embodiments, x is 4 to 8.

[0298] In certain embodiments of this disclosure, the drug forms a drug connector compound with the connector, such as oxazolidin (a derivative of chachiomycin, see U.S. Patent No. 5,773,001), vedotin (MC-VC-PABC-MMAE, see U.S. Patent No. 7,659,241), maltodine (MC-MMAF, see U.S. Patent No. 7,498,298), emmetine (SMCC-DM1, see U.S. Patent No. 5,208,020), delutecan (MC-GGFG-DXd, see U.S. Patent No. 10,195,288), goxatecan (CL2A-SN38, see U.S. Patent No. 8,420,086), or tercilin (MP-PEG8-VA-PABC-SG3199 / SCX, see U.S. Patent No. 9,889,207), the full text of which is incorporated herein by reference.

[0299] Other examples of drug linker compounds include parsudotox (PE38), solatanine (DM4), lavtanine (DM4), and metanine (DM1).

[0300] In some embodiments of this disclosure, the drug connector compound is vitoline having formula VI or a derivative thereof: (Mc-vc-PAB-MMAE).

[0301] In some embodiments of this disclosure, the drug connector compound is a derutin having formula VII or a derivative thereof: (Mc-GGFG-DXd).

[0302] Treatment As described herein, the antibodies, ADCs, variants, or derivatives disclosed herein can be used in certain therapeutic and diagnostic approaches.

[0303] This disclosure also relates to antibody-based therapies involving the administration of an antibody or ADC of this disclosure to a patient (e.g., an animal, mammal, or human) for the treatment of one or more of the conditions or illnesses described herein. Therapeutic compounds of this disclosure include, but are not limited to, antibodies or ADCs of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of this disclosure (including variants and derivatives thereof as described herein).

[0304] The antibodies or ADCs disclosed herein can also be used to treat or inhibit cancer. In some embodiments, cancer cells in a patient express or overexpress CDH17. As mentioned above, CDH17 can be overexpressed in tumor cells, particularly in gastric, pancreatic, colorectal, hepatocellular carcinoma, and neuroendocrine tumors. Inhibition of CDH17 has been shown to be useful in the treatment of tumors.

[0305] Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the disclosed antibody or ADC to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses CDH17.

[0306] This disclosure also provides cell therapies (e.g., chimeric antigen receptor (CAR) T-cell therapy). Suitable cells can be used, contacted with the anti-CDH17 antibody of this disclosure (or alternatively, engineered to express the anti-CDH17 antibody of this disclosure). In some embodiments, the antibody is present in the chimeric antigen receptor (CAR). Following such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient can have any type of cancer disclosed herein. The cells (e.g., T cells) can be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

[0307] In some implementations, cells are isolated from the cancer patient themselves. In other implementations, cells are provided by a donor or from a cell bank. When cells are isolated from cancer patients, unwanted immune responses can be minimized.

[0308] Non-limiting examples of cancer include stomach cancer, colorectal cancer, hepatocellular carcinoma, bladder cancer, liver cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, neuroendocrine carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

[0309] The antibodies or variants or derivatives thereof disclosed herein can be used to treat, prevent, diagnose, and / or prognose other diseases or conditions associated with increased cell survival, including but not limited to the progression and / or metastasis of malignancies and related conditions such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, and liposarcoma. Sarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangioma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0310] The specific dosage and treatment regimen for any particular patient will depend on a number of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex and diet, timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The healthcare professional's judgment of these factors is within the realm of ordinary technical skill in this art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using pharmacological and pharmacokinetic principles known in the art.

[0311] Methods of administration for antibodies, ADCs, variants, or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides, ADCs, or compositions can be administered via any convenient route (e.g., by infusion or bolus, absorption through the epithelial or mucosal inner layer of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.)) and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides or ADCs of this disclosure can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), sublingually, or as oral or nasal sprays.

[0312] As used in this article, the term "parenteral" refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0313] Administration can be systemic or local. Furthermore, it is desirable to introduce the antibodies or ADCs of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter connected to a reservoir (e.g., an Ommaya reservoir). Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and formulating it with a nebulizing agent.

[0314] It is desirable to apply the antigen-binding peptides, ADCs, or compositions of this disclosure topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, local application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implants that are porous, non-porous, or gel-like materials, including membranes (e.g., sialic acid membranes) or fibers. Preferably, when applying the proteins of this disclosure (including antibodies or ADCs), care must be taken to use materials that do not absorb the proteins.

[0315] The amount at which the antibody or ADC of this disclosure will effectively treat, inhibit, and prevent immune or malignant diseases, symptoms, or conditions can be determined using standard clinical techniques. Additionally, in vitro assays may optionally be used to help identify the optimal dose range. The precise dose used in the formulation will also depend on the route of administration and the severity of the disease, symptom, or condition, and should be determined based on the physician's judgment and the individual patient's circumstances. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0316] As a general recommendation, the dose of the antigen-binding peptide or ADC of this disclosure administered to patients is typically 0.1 mg / kg to 100 mg / kg patient body weight, 0.1 mg / kg to 20 mg / kg patient body weight, or 1 mg / kg to 10 mg / kg patient body weight. Generally, due to the immune response to exogenous peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and less frequent administration are generally possible. Furthermore, the dosage and frequency of administration of the antibodies or ADCs of this disclosure can be reduced by modifications (e.g., lipolysis) that enhance antibody uptake and tissue penetration (e.g., into the brain).

[0317] In another embodiment, the compositions of this disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of this disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0318] In another embodiment, the compositions disclosed herein are administered in combination with other treatment or preventative measures (e.g., radiotherapy).

[0319] Diagnostic methods CDH17 overexpression has been observed in certain tumor samples, and patients with CDH17-overexpressing cells may respond to treatment with the anti-CDH17 antibody of this disclosure. Therefore, the antibody of this disclosure can also be used for diagnostic and prognostic purposes.

[0320] The preferred sample, including cells, can be obtained from a patient, who may be a cancer patient or a patient requiring diagnosis. The cells are cells from tumor tissue or tumor masses, blood samples, urine samples, or any sample from the patient. After optional pretreatment of the sample, it can be incubated with the antibody of this disclosure under conditions that allow the antibody to interact with the CDH17 protein that may be present in the sample. The presence of the CDH17 protein in the sample can be detected using, for example, an ELISA method utilizing an anti-CDH17 antibody.

[0321] The presence of CDH17 protein in a sample (optionally in a specific amount or concentration) can be used for cancer diagnosis, as an indicator of a patient's suitability for antibody therapy, or as an indicator of whether a patient has (or has not) responded to cancer treatment. For prognostic methods, one, two, or more tests can be performed at certain stages after the initiation of cancer treatment to indicate the progress of treatment.

[0322] Composition This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody or ADC and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0323] In one specific implementation, the term "pharmaceutical acceptable" means a substance approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more particularly for human use. Furthermore, "pharmaceutical acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0324] The term "carrier" refers to a diluent, adjuvant, excipient, or solvent that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Physiological saline, as well as aqueous glucose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers (e.g., acetates, citrates, or phosphates). Antimicrobial agents (e.g., benzyl alcohol or methylparaben); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., EDTA); and agents for regulating tension (e.g., sodium chloride or glucose) may also be considered. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Compositions can be formulated into suppositories using conventional binders and carriers (e.g., triglycerides). Oral formulations may contain standard carriers (e.g., pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.). Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in a purified form) and a suitable amount of carrier to provide a form suitable for appropriate administration to the patient. The formulation should be suitable for the administration mode. The parent formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0325] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional methods. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also contain a solubilizer and a local anesthetic (e.g., lidocaine) to relieve pain at the injection site. These components are typically provided individually or in combination in unit dosage forms, for example, as lyophilized powder or anhydrous concentrate in a sealed container (e.g., an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or physiological saline. When the composition is administered by injection, a single ampoule of sterile water for injection or physiological saline can be provided so that the components can be mixed prior to administration.

[0326] The compounds disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions (e.g., anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.) and those formed with cations (e.g., cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0327] Example Example 1: Binding with cell lines overexpressing human CDH17 This embodiment examines the specific binding of the newly developed humanized antibody 8G5G9.p1.z12.

[0328] Cell-based binding of 8G5G9.p1.z12 and reference antibodies BMK-BI-905711 (a monospecific form of the bispecific antibody BI-905711, which is specific for TRAILR2 and CDH17) and BMK-ARB202 (a monospecific form of the bispecific antibody ARB202, which is specific for CDH17 and CD3), as well as human IgG1 isotype control and cell lines overexpressing human CDH17, was evaluated using flow cytometry.

[0329] In short, the cell lines HEK-293 / H_CDH17 and DLD-1 / H_CDH17 were incubated with titrated test mAbs (starting from 200 nM, 3-fold dilution, 12 spots) at 4°C for 60 min. The cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. The cells were washed twice more, and the fluorescence signal was analyzed by flow cytometry. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0330] The results in Figures 1A and 1B show that 8G5G9.p1.z12 binds effectively to cells overexpressing human CDH17 in a dose-dependent manner.

[0331] Example 2. Non-specific binding assay with blank HEK-293 cell line This embodiment examined the nonspecific binding of 8G5G9.p1.z12.

[0332] The nonspecific binding of 8G5G9.p1.z12, reference antibodies BMK-BI-905711 and BMK-ARB202, and human IgG1 isotype control to blank HEK-293 cell line was evaluated using flow cytometry.

[0333] In short, HEK-293 cells were incubated with titrated test mAbs (starting from 200 nM, 3-fold dilution, 12 spots) at 4°C for 60 min. Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. Cells were washed twice more, and fluorescence signals were analyzed by flow cytometry. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0334] like Figure 2 As shown, unlike BMK-BI-905711, 8G5G9.p1.z12 did not bind nonspecifically to blank HEK-293 cells.

[0335] Example 3. Specific binding to endogenous human CDH17 on tumor cells This embodiment examined the binding of 8G5G9.p1.z12 to endogenously expressed human CDH17.

[0336] Cell-based binding of 8G5G9.p1.z12, reference antibodies BMK-BI-905711 and BMK-ARB202, and human IgG1 isotype control to tumor cell lines expressing endogenous human CDH17 was assessed using flow cytometry. Briefly, the gastric cancer (GC) cell line SNU-16 and the colorectal cancer (CRC) cell line LoVo were incubated with titrated test mAbs (starting at 200 nM, 3-fold dilution, 12 spots) at 4°C for 60 min. Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. Cells were washed twice again, and fluorescence signals were analyzed by flow cytometry. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0337] As shown in Figure 3, 8G5G9.p1.z12 effectively binds to human tumor cells expressing endogenous CDH17 in a dose-dependent manner.

[0338] Example 4. Cross-reactivity of cell lines overexpressing monkey and rodent CDH17 This embodiment examines the cross-species reactivity of 8G5G9.p1.z12.

[0339] Cell-based binding of 8G5G9.p1.z12, reference antibodies BMK-BI-905711 and BMK-ARB202, and human IgG1 isotype control to cell lines overexpressing CDH17 in cynomolgus monkeys, rhesus monkeys, rats, and mice was assessed using flow cytometry. Briefly, engineered cell lines HEK-293 / cynomolgus monkey_CDH17, HEK-293 / rhesus monkey_CDH17, HEK-293 / rat_CDH17, and HEK-293 / mouse_CDH17 were incubated with titrated test mAbs (starting from 200 nM, 3-fold dilution, 12 spots) at 4°C for 60 min. Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. Cells were washed twice more, and fluorescence signals were analyzed by flow cytometry. Create a graph and calculate EC50 in GraphPadPrism software (LaJolla, CA).

[0340] As shown in Figures 4A to 4D, 8G5G9.p1.z12 cross-reacts with monkey CDH17, but does not bind to rodent CDH17.

[0341] Example 5. Tabletop Sub-box This embodiment performs tabletop binning on 8G5G9.p1.z12.

[0342] Cell-based epitope binning assays were performed on HEK-293 / H_CDH17 cells using flow cytometry to evaluate 8G5G9.p1.z12, BMK-BI-905711, and a human IgG1 isotype control targeting the mouse IgG2a Fc fragment with BMK-BI-905711. Briefly, HEK-293 / H_CDH17 cells were incubated for 60 min at 4°C with a mixture of BMK-BI-905711 (anti-CDH17)-mFc dilution (1 nM) and titrated test mAbs (starting from 200 nM, 3-fold dilution, 12 spots). Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-mouse Fc secondary antibody at 4°C for 30 min. Cells were washed twice again, and fluorescence signals were analyzed by flow cytometry. Graphs were created and IC50 was calculated in GraphPad Prism software (LaJolla, CA).

[0343] result( Figure 5 As shown in Table 1, 8G5G9.p1.z12 does not compete with the reference antibody BMK-BI-905711 for binding to CDH17, therefore they do not bind to the same epitope.

[0344] Table 1. Inhibition Rate Inhibition rate BMK-BI-905711 8G5G9.p1.z12 BMK-BI-905711 96.81% 3.18% Example 6. Internalizing efficacy in cells overexpressing human CDH17 This embodiment examines the internalization effectiveness of 8G5G9.p1.z12.

[0345] The internalization potency of 8G5G9.p1.z12 was assessed using a secondary antibody labeled with a pH-sensitive dye (pHAb dye), which fluoresces strongly under acidic pH conditions. In short, the test mAb 8G5G9.p1.z12, the reference antibody, and a human IgG1 isotype control (100 nM) were incubated with the pHAb-conjugated secondary antibody (100 nM) at room temperature for 30 minutes, followed by a 3-fold serial dilution. The titrated mixture was then added to HEK-293 / H_CDH17 or DLD-1 / H_CDH17 cells seeded the previous day in 96-well plates. After incubation overnight at 37°C, the cells were gently washed twice with DPBS, and fluorescence density was detected using a Tecan microplate reader. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0346] The results (Figures 6A to 6B) showed that 8G5G9.p1.z12 had effective internalization activity against cell lines overexpressing CDH17.

[0347] Example 7. Specific binding of anti-CDH17 ADC to cell lines overexpressing human CDH17 In this embodiment, an antibody-drug conjugate (ADC) conjugated with 8G5G9.p1.z12 was prepared, and its binding to a cell line overexpressing human CDH17 was tested.

[0348] Flow cytometry was used to assess the cell-based binding of 8G5G9.p1.z12, a reference antibody, a human IgG1 isotype control, and corresponding ADCs (designated as DAR conjugated with MMAE, DXd, LM-D01, LM-D02, and LM-D03) to cell lines overexpressing human CDH17. The structures of LM-D01, LM-D02, and LM-D03 are shown in Table 2.

[0349] Table 2. Structure of LM-D01, LM-D02 and LM-D03

[0350] In short, HCT116 / H_CDH17 and DLD-1 / H_CDH17 cells were incubated with titrated test samples (starting from 50 nM, 5-fold dilution, 8 spots) at 4°C for 60 min. The cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. The cells were washed twice again, and fluorescence density was analyzed using an Agilent NovoCyte flow cytometer. Graphs were created and EC50 was calculated in GraphPad Prism software (LaJolla, CA).

[0351] The results (Figures 7A to 7B and Table 3) show that the anti-CDH17 ADC binds effectively to cells overexpressing human CDH17 in a dose-dependent manner, with an affinity comparable to that of the naked antibody.

[0352] Table 3. Binding with cell lines overexpressing CDH17

[0353] Dar: Drug antibody ratio Example 8. Specific binding of anti-CDH17 ADC to endogenous human CDH17 on tumor cells This embodiment measures the binding of anti-CDH17 ADC to endogenous human CDH17 on tumor cells.

[0354] Cell-based binding of 8G5G9.p1.z12, a reference antibody, a human IgG1 isotype control, and corresponding ADCs (conjugated to DAR with MMAE, DXd, LM-D01, LM-D02, or LM-D03) to tumor cell lines expressing endogenous human CDH17 was assessed using flow cytometry. Briefly, human colorectal cancer (CRC) cell line DLD-1 and gastric cancer (GC) cell line SNU-16 were incubated with titrated test samples (starting at 50 nM, 5-fold dilution, 8 spots) at 4°C for 60 min. Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. Cells were washed twice again, and fluorescence density was analyzed using an Agilent NovoCyte flow cytometer. Graphs were created and EC50 was calculated in GraphPad Prism software (LaJolla, CA).

[0355] The results (Figures 8A to 8B and Table 4) show that the anti-CDH17 ADC binds effectively to tumor cells expressing endogenous human CDH17 in a dose-dependent manner, with an affinity comparable to that of the anti-CDH17 mAb.

[0356] Table 4. Binding with endogenous human CDH17 on tumor cells

[0357] Example 9. Cytotoxic effect of anti-CDH17 ADC on tumor cells expressing human CDH17 This embodiment measures the cytotoxic effects of anti-CDH17 ADC.

[0358] HCT116 / H_CDH17 and DLD-1 / H_CDH17 cells overexpressing human CDH17 were seeded into 96-well tissue culture plates and incubated with titrated test ADCs (starting from 50 nM, 5-fold dilution, 8 spots) at 37°C in a 5% CO2 incubator. Cells without ADC treatment served as a control group. After 6 days of treatment, Cell Titer-Glo reagent (Promega) was added, and relative luminescence units (RLU) were measured using EnVision. Cell viability was determined as the percentage of sample well RLU in the mean control RLU. Graphs were created and IC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0359] The results are shown in Figure 9 and Table 5. The tested anti-CDH17 ADCs exhibited significant cytotoxic effects on cells expressing human CDH17.

[0360] Table 5. Cytotoxic effects of anti-CDH17 ADC

[0361] Example 10. Bystander effect of anti-CDH17 ADC on human CDH17 negative cell lines This embodiment measures the bystander effect of the anti-CDH17 ADC.

[0362] CRC cell lines DLD-1 / H_CDH17 and HCT116 / H_CDH17, overexpressing human CDH17, were incubated with ADC dilution buffer at 37°C and 5% CO2 for 6 days to generate conditioned medium (CM) containing free payload. Cells not treated with ADC were used as a control group. On day 6, the conditioned medium was collected and added at a 1:1 volume ratio to CDH17-negative human tumor cells MDA-MB-231 and HCT116 seeded in black 96-well plates. After incubation at 37°C for 6 days, Cell Titer-Glo reagent (Promega) was added, and relative luminescence units (RLU) were measured using EnVision. Cell viability was determined as the percentage of RLU in the sample wells relative to the average RLU in the control group. Graphs were plotted in GraphPad Prism software (LaJolla, CA).

[0363] The results are shown in Figures 10A to 10F, which show that the anti-CDH17 ADC conjugated with DXd and LM-D03 exhibited a significant bystander effect on CDH17-negative MDA-MB-231 and HCT116 cells, while the ADC conjugated with MMAE exhibited a significant bystander effect on MDA-MB-231 cells.

[0364] Example 11. Antitumor activity of anti-CDH17 ADC in a colorectal cancer (CRC) CDX (cell line-derived xenograft) model This embodiment measures the antitumor effect of anti-CDH17 ADC.

[0365] A CRC CDX model was established by subcutaneously injecting 5 million DLD-1 / H_CDH17 cells per mouse into the right flank of Balb / C nude mice. The tumor volume reached 100 mm. 3 Previously, tumor-bearing mice were randomly assigned to treatment and control groups (n=5 per group) and drug administration began on day 0. Each ADC was administered intravenously to mice at a dose of 3 mg / kg or 6 mg / kg once weekly (qw) for 3 weeks. DPBS buffer was used as the solvent and administered at the same volume as the ADC.

[0366] Most of the tested ADCs induced effective tumor regression at the specified doses (Figures 11A to 11B) and were safe.

[0367] Example 12. Characterization of anti-CDH17 antibody The VH / VL sequences of the other six lead humanized antibodies are further provided in Table A3, and the CDR sequences are provided in Table A4. The properties of the anti-CDH17 antibodies were evaluated in cell-based binding assays and antibody internalization assays.

[0368] Cell-based binding assays Cells were incubated at 4°C for 60 minutes with titrated antibodies or antibody-drug conjugates (the highest concentration range for different cell lines was 200 nM to 40 nM, all at 5-fold dilutions). Cells were then washed twice with FACS buffer and treated with a fluorescently conjugated secondary antibody (Alexa Fluor). ® 647 goat anti-human IgG (Jackson, 109-605-098) was stained at 4°C for 60 minutes. The cells were then washed twice and analyzed by flow cytometry.

[0369] Both anti-CDH17 antibodies 296G7E10.p4.Z9 and 312E10A9.p2.Z7 bound to HE293 cells overexpressing human CDH17 (Fig. 12, Fig. A to Fig. B) and HE293 cells overexpressing rhesus monkey CDH17 (Fig. 13, Fig. A to Fig. B).

[0370] Cell-based binding of anti-CDH17 antibodies (e.g., 35E7D6.p1.z9, 135B2E11.p1.z1, 153A3D7.p3.z5, and 155B9A5.p3.V2.z2) and human IgG1 isotype controls to cell lines overexpressing human CDH17 was assessed using flow cytometry. Briefly, HEK-293 / H_CDH17 and DLD-1 / H_CDH17 cells were incubated with titrated test mAbs (starting at 200 nM, 3-fold dilution, 12 spots) at 4°C for 60 min. Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. Cells were washed twice again, and fluorescence signals were analyzed by flow cytometry. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0371] Based on the results shown in Figure 12 (Figures C to D) and Table 6, the anti-CDH17 antibody can bind effectively to cells overexpressing human CDH17 in a dose-dependent manner.

[0372] Table 6. Binding of anti-CDH17 antibody to cell lines overexpressing human CDH17

[0373] Cell-based binding of anti-CDH17 antibody and human IgG1 isotype control to human tumor cell lines expressing endogenous CDH17 was assessed using flow cytometry. Briefly, the gastric cancer (GC) cell line SNU-16 and the colorectal cancer (CRC) cell line LoVo were incubated with titrated test mAbs (starting at 200 nM, 3-fold dilution, 12 spots) at 4°C for 60 min. Cells were then washed twice with 2% FBS / PBS buffer and stained with fluorescently conjugated anti-human Fc secondary antibody at 4°C for 30 min. Cells were washed twice again, and fluorescence signals were analyzed by flow cytometry. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0374] As shown in Figure 14 (Figures A to D) and Table 7, anti-CDH17 antibodies can bind effectively to human tumor cell lines expressing endogenous CDH17 in a dose-dependent manner.

[0375] Table 7. Binding of anti-CDH17 antibodies to endogenous human CDH17 on tumor cells

[0376] Antibody internalization assay Internalization based on CDH17 endogenous expression cell lines Internalization of 296G7E10.p4.Z9 and 312E10A9.p2.Z7 in cells endogenously expressing human CDH17 (SNU16 and LoVo) was assessed using flow cytometry. Briefly, a secondary antibody conjugated with the pHAb amine-reactive dye (Promega) (AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific, Jackson) was mixed with 296G7E10.p4.Z9, 312E10A9.p2.Z7, and an isotype control at 37°C for 30 min. The mixture of primary and secondary antibodies was then titrated and incubated with SNU16 or LoVo cells overnight at 37°C. The following day, cells were collected and analyzed by flow cytometry by evaluating fluorescence values.

[0377] The internalization based on cancer cell lines is shown in Figures A to B of Figure 15.

[0378] Internalization based on CDH17 overexpression cell lines The internalizing potency of anti-CDH17 antibodies was assessed using secondary antibodies labeled with a pH-sensitive dye (pHAb dye), which fluoresces strongly under acidic pH conditions. In short, the test mAb anti-CDH17 antibody or human IgG1 isotype control (33.3333 nM) was incubated with a pHAb-conjugated secondary antibody (33.3333 nM) at room temperature for 30 minutes, followed by a 3-fold serial dilution. The titrated mixture was then added to HEK-293 / H_CDH17 or DLD-1 / H_CDH17 cells seeded the previous day in 96-well plates. After overnight incubation at 37°C, cells were gently washed twice with DPBS, and fluorescence density was detected using a Tecan microplate reader. Graphs were created and EC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0379] As shown in Figures 16 (A to B) and Table 8, the anti-CDH17 antibody exhibited effective internalization activity against cell lines overexpressing CDH17.

[0380] Table 8. Internalizing activity of anti-CDH17 antibody against CDH17-overexpressing cell lines

[0381] Example 13. Cytotoxic effects of CDH17-targeting ADCs on cells expressing human CDH17 HEK-293 / H_CDH17 and DLD-1 / H_CDH17 cells overexpressing human CDH17, or LoVo cells endogenously expressing CDH17, were seeded into 96-well tissue culture plates and incubated with titrated test ADCs (starting from 10 nM, 3-fold dilution, 9 spots) at 37°C in a 5% CO2 incubator. Cells without ADC treatment served as a control group. After 6 days of treatment, Cell Titer-Glo reagent (Promega) was added, and relative luminescence units (RLU) were measured using EnVision. Cell viability was determined as the percentage of sample well RLU in the average control RLU. Graphs were created and IC50 was calculated using GraphPad Prism software (LaJolla, CA).

[0382] As shown in Figure 17 (Figures A to C) and Table 9, the anti-CDH17 ADC exhibited a significant cytotoxic effect on cells expressing human CDH17.

[0383] Table 9. Cytotoxic effects of CDH17-targeting ADCs on cells expressing human CDH17

[0384] Example 14. Cytotoxic effects of CDH17-targeting ADCs on cells expressing human CDH17 Six days after ADC treatment, the viability of colorectal cancer (CRC) or gastric cancer (GC) cells expressing human CDH17 was assessed using CellTiter-Glo. As shown in Figures 18 (Figures A to C) and Table 10, the anti-CDH17 ADC exhibited significant cytotoxic effects against SNU-16, HCT116 / H_CDH17, and DLD-1 / H_CDH17 cells, with IC50 values ​​ranging from 0.016 nM to 0.35 nM.

[0385] Table 10. Cytotoxic effects of CDH17-targeting ADCs on cells expressing human CDH17

[0386] Example 15. In vivo antitumor effect of CDH17-targeting ADC 1. Antitumor activity of anti-CDH17 ADC in CRC CDX (cell line-derived xenograft) model A CRC CDX model was established by subcutaneously injecting 5 million DLD-1 / H_CDH17 cells per mouse into the right flank of Balb / C nude mice. The tumor volume reached 100 mm. 3Previously, tumor-bearing mice were randomly assigned to treatment and control groups (n=5 per group) and drug administration began on day 0. Each ADC was administered intravenously to mice at a dose of 3 mg / kg or 6 mg / kg, once weekly (qw) for 3 weeks. DPBS buffer was used as the solvent and administered at the same volume as the ADC.

[0387] Therefore, the 3 mg / kg dose of MMAE-conjugated ADC showed moderate tumor growth inhibitory activity, while 6 mg / kg of 8G5G9.p1.z12-LM-D01 induced effective tumor regression (Figures A and B of Figure 19 and Table 11). TORL is TORL-3-600, produced by TORL BioTherapeutic from a fully humanized CDH17 mAb via MMAE conjugation.

[0388] Table 11. Tumor growth inhibition rate (%) and complete response (CR) Group %TGI CR BMK-MMAE (TORL) 3mg / kg 48.62 / 8G5G9.p1.z12-MMAE_3mg / kg 56.15 / 8G5G9.p1.z12-LM-D01 _6mg / kg 89.08 / 2. Antitumor activity of CDH17-targeting ADCs in the SNU-16 (GC) xenograft model The SNU-16 model was established by subcutaneously injecting 10 million cells suspended in a Matrigel matrix into the right flank of CB17-SCID mice. The tumor volume reached 100 mm. 3 Up to 150 mm 3 At that time, tumor-bearing mice were randomly divided into a treatment group and a control group (n=5 mice / group) and were administered intravenously once with a dose of 3 mg / kg or 6 mg / kg (day 0). DPBS buffer was used as the solvent and administered in the same volume as the ADC.

[0389] As shown in Figure 20 (Figures A and B) and Table 12, a single dose of 3 mg / kg or 6 mg / kg of 8G5G9.p1.z12-LM-D01 (Dar8) induced effective tumor regression in the GC CDX model.

[0390] Table 12. Tumor growth inhibition rate (%) and complete response (CR) Group %TGI CR 8G5G9.p1.z12-LM-D01_3 mg / kg 129 3 / 5 8G5G9.p1.z12-LM-D01_6 mg / kg 129 4 / 5 Human IgG1-LM-D01_6 mg / kg 18 /

[0391] This disclosure is not limited to the specific embodiments described herein, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover such modifications and variations, provided that such modifications and variations are within the scope of the appended claims and their equivalents.

[0392] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated herein by reference.

Claims

1. An antibody or antigen-binding fragment thereof having binding specificity to human cadherin-17 (CDH17) protein, wherein the antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions (CDRs) VH CDR1, VH CDR2, and VH CDR3, and the VL comprises VL CDR1, VL CDR2, and VL CDR3, and wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 respectively comprise: (a) The amino acid sequences of SEQ ID NO: 11-SEQ ID NO: 16; (b) The amino acid sequences of SEQ ID NO: 17-SEQ ID NO: 22; (c) The amino acid sequences of SEQ ID NO: 23-SEQ ID NO: 28; (d) The amino acid sequences of SEQ ID NO: 29-SEQ ID NO: 34; (e) The amino acid sequence of SEQ ID NO: 35-SEQ ID NO: 40; (f) The amino acid sequence of SEQ ID NO: 41-SEQ ID NO: 46; or (g) The amino acid sequences of SEQ ID NO: 1-SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

11. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

12. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

13. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

14. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 15, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises the amino acid sequence of SEQ ID NO: 47, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 47, and the VL comprises the amino acid sequence of SEQ ID NO: 48, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

48.

4. The antibody or its antigen-binding fragment according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

17. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

18. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

19. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

20. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 21, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

22.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the VH comprises the amino acid sequence of SEQ ID NO: 49, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 49, and the VL comprises the amino acid sequence of SEQ ID NO: 50, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

50.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

23. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

24. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

25. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

26. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 27, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

28.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO: 51, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 51, and the VL comprises the amino acid sequence of SEQ ID NO: 52, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

52.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

29. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

30. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

31. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

32. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 33, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

34.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the VH comprises the amino acid sequence of SEQ ID NO: 53, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 53, and the VL comprises the amino acid sequence of SEQ ID NO: 54, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

54.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

35. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

36. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

37. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

38. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 39, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

40.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VH comprises the amino acid sequence of SEQ ID NO: 55, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO: 56, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

56.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

41. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

42. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

43. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

44. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 45, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

46.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 57, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 57, and the VL comprises the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

58.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein... The VH CDR1 contains the amino acid sequence of SEQ ID NO:

1. The VH CDR2 contains the amino acid sequence of SEQ ID NO:

2. The VH CDR3 contains the amino acid sequence of SEQ ID NO:

3. The VL CDR1 contains the amino acid sequence of SEQ ID NO:

4. The VL CDR2 contains the amino acid sequence of SEQ ID NO: 5, and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

6.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

8.

16. An antibody or antigen-binding fragment thereof specific to human CDH17 protein, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of the preceding claims for binding to the CDH17 protein.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof is humanized.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment thereof is F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof is an isotype of IgG1, IgG2, IgG3 or IgG4.

21. The antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody or antigen-binding fragment thereof is an isotype of human IgG1.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof is conjugated.

23. A bifunctional molecule comprising a first antigen-binding portion specific to human CDH17 protein and a second portion specific to a second antigen, wherein the first antigen-binding portion comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22.

24. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22 or a bifunctional molecule according to claim 23, and a pharmaceutical portion.

25. The antibody-drug conjugate of claim 24, wherein the drug portion is a cytotoxin, an immunosuppressant, a radioactive isotope, or a toxin.

26. The antibody-drug conjugate of claim 25, wherein the drug portion is a cytotoxin selected from the group consisting of: auristatin, maytansine alkaloids, macrocyclic ketone analogs, topoisomerase inhibitors, benzodiazepines, tubulolysin, pyroxine, camptothecin, chalcogenine, eczemaconazole, irinotecan (SN38), doxorubicin, anthracycline, pyrrolobenzodiazepines (PBD), TLR agonists, STING agonists, Pseudomonas aeruginosa exotoxin PE38, diphtheria toxin, Staphylococcus aureus enterotoxin A / E-120, antimicrobial antibiotics, Shiga toxin, ricin, and urease.

27. The antibody-drug conjugate of claim 25, wherein the drug moiety is a cytotoxic agent selected from the group consisting of: monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), maytansine, metansine (DM1), lavtansine (DM4), tubulolysin A, DXd, 7-ethyl-10-hydroxycamptothecin (SN-38), DGN462, ambestatin 269, atrazomycin, SG3199 / SCX, IRDye®700DX, TLR7 / 8 agonist, diABZI STING agonist-2, or any derivative thereof.

28. The antibody-drug conjugate according to any one of claims 25 to 27, wherein the drug portion comprises MMAE, MMAF, or DXd.

29. The antibody-drug conjugate of claim 26, wherein the drug portion comprises a compound selected from the group consisting of: (LM-D01) (LM-D02) and (LM-D03).

30. The antibody-drug conjugate according to any one of claims 24 to 29, wherein the antibody or its antigen-binding fragment is conjugated to the drug portion via a linker.

31. The antibody-drug conjugate of claim 30, wherein the linker is hydrolyzable under acidic conditions.

32. The antibody-drug conjugate according to any one of claims 30 to 31, wherein the linker comprises succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (SMCC), sulfonyl-SMCC, p-carboxycyclohexylmethylmaleimide, maleimide-hexanoyl (MC)-valine-citrulline (VC)-p-aminobenzyloxycarbamoyl (PABC), CL2A, maleimide-hexanoyl (MC), MC-glycine-glycine-phenylalanine-glycine (GGFG), MC-PEG8-GGFG, MC-PEG8-GGFG-PAB, or maleimide propionyl (MP)-PEG8-valine-alanine (VA)-PABC.

33. The antibody-drug conjugate according to any one of claims 24 to 32, wherein the antibody-drug conjugate comprises oxazolidin, vedotin, maltodine, emmetantine, delutecan, goxatecan, or tercilin.

34. The antibody-drug conjugate according to any one of claims 24 to 33, wherein the drug-antibody ratio (DAR) is 1 to 20.

35. The antibody-drug conjugate according to any one of claims 24 to 33, wherein the drug-antibody ratio (DAR) is 4 to 8.

36. A chimeric antigen receptor (CAR) specific for human CDH17 protein, said CAR comprising an antigen-binding fragment according to any one of claims 1 to 22 or a bifunctional molecule, a transmembrane domain and an intracellular activation domain according to claim 23.

37. The CAR of claim 36, wherein the antigen-binding fragment is a single-chain fragment (scFv).

38. The CAR according to claim 36 or 37, wherein the transmembrane domain is a transmembrane domain of 4-1BB, the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD3ε, CD4, CD5, CD8α, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, or CD154.

39. The CAR according to any one of claims 36 to 38, wherein the chimeric antigen receptor comprises an intracellular activation domain, the intracellular activation domain being a signal transduction domain derived from CD3ζ, CD3ε, CD3δ and CD3γ.

40. The CAR according to any one of claims 36 to 39, wherein the CAR further comprises one or more co-stimulatory domains selected from the following signal transduction domains (or other suitable portions): CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class 1 molecules, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activating molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

41. The CAR according to any one of claims 36 to 40, wherein the CAR further comprises a hinge domain selected from the hinge domains of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28 or CD8α, any truncated form thereof, or any combination thereof.

42. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, a bifunctional molecule according to claim 23, or a CAR according to any one of claims 34 to 39.

43. A vector comprising the nucleic acid according to claim 42.

44. An isolated cell comprising the nucleic acid according to claim 42 or the vector according to claim 43.

45. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22, a bifunctional molecule according to claim 23, an antibody-drug conjugate according to any one of claims 24 to 35, or a CAR according to any one of claims 36 to 41, and a pharmaceutically acceptable carrier.

46. ​​A method of treating cancer in a patient in need, the method comprising administering to the patient an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, a bifunctional molecule according to claim 23, an antibody-drug conjugate according to any one of claims 24 to 35, a CAR according to any one of claims 36 to 41, or a pharmaceutical composition according to claim 45.

47. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, the bifunctional molecule according to claim 23, the antibody-drug conjugate according to any one of claims 24 to 35, or the CAR according to any one of claims 36 to 41, or the pharmaceutical composition according to claim 45, in the preparation of a medicament for treating cancer.

48. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, a bifunctional molecule according to claim 23, an antibody-drug conjugate according to any one of claims 24 to 35, a CAR according to any one of claims 36 to 41, or a pharmaceutical composition according to claim 45, for treating cancer in patients in need.

49. The method of claim 46 or the use of claim 47, or the antibody or antigen-binding fragment thereof, bispecific antibody or antigen-binding fragment thereof, antibody-drug conjugate, CAR, or pharmaceutical composition of claim 46, wherein the cancer is selected from the group consisting of: gastric cancer, colorectal cancer, hepatocellular carcinoma, bladder cancer, liver cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, neuroendocrine carcinoma, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

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