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WO2025242100A1 - Anticorps dirigé contre cdh17 et son utilisation - Google Patents

Anticorps dirigé contre cdh17 et son utilisation

Info

Publication number
WO2025242100A1
WO2025242100A1 PCT/CN2025/096099 CN2025096099W WO2025242100A1 WO 2025242100 A1 WO2025242100 A1 WO 2025242100A1 CN 2025096099 W CN2025096099 W CN 2025096099W WO 2025242100 A1 WO2025242100 A1 WO 2025242100A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
antibody
sequence identity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/096099
Other languages
English (en)
Chinese (zh)
Inventor
陈博
王常玉
徐刚
郭步静
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Kangnuoxing Biopharma Inc
Keymed Biosciences Chengdu Co Ltd
Original Assignee
Chengdu Kangnuoxing Biopharma Inc
Keymed Biosciences Chengdu Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chengdu Kangnuoxing Biopharma Inc, Keymed Biosciences Chengdu Co Ltd filed Critical Chengdu Kangnuoxing Biopharma Inc
Publication of WO2025242100A1 publication Critical patent/WO2025242100A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • This invention relates to antibodies that bind to CDH17 and their uses, particularly in the treatment of cancer.
  • Cadherin-17 (CDH17) is a new member of the cadherin family. It was first identified from a mouse hepatocyte cDNA library. Because it is expressed only in the liver and small intestine in mice, it was also named liver-intestine cadherin (LI-cadherin) (Berndorff, D. et al., 1994, Journal of Cell Biology, 125, 1353-1369). Recent studies have shown that it plays an important role in the invasion and metastasis of various tumors.
  • LI-cadherin liver-intestine cadherin
  • the human CDH17 gene is located on chromosome 8q22.1, and its encoded protein belongs to the 7D-cadherin family. CDH17 is commonly expressed in embryonic and adult intestinal epithelial cells, as well as some pancreatic duct epithelial cells, but it is almost not significantly expressed in hepatocytes, esophageal epithelial cells, and gastric mucosa in healthy individuals (Gul, I.S. et al., 2017, Experimental Cell Research, 358, 3-9).
  • CDH17 While its structure shares some homology with classical cadherins, CDH17 also has unique structural features: firstly, the extracellular region of CDH17 consists of seven repeat sequences, distinct from the five repeat sequences found in non-classical cadherins; secondly, within the extracellular EC1 cell adhesion recognition region, CDH17 contains an AAL sequence that replaces the corresponding HAV sequence in classical cadherins; and thirdly, the cytoplasmic tail region of CDH17 contains only 20 amino acid residues, while the cytoplasmic tail region of classical cadherins contains 150–160 amino acid residues.
  • CDH17 Due to its short cytoplasmic tail domain, CDH17 cannot interact with chain protein networks or the actin cytoskeleton (Kreft, B. et al., 1997, Journal of Cell Biology, 136, 1109-1121). Therefore, CDH17 is classified as a variant of classical cadherins (Nollet, F. et al., 2000, Journal of Molecular Biology, 299, 551-572).
  • Cathecins exert their biological functions by mediating calcium-dependent intercellular connections.
  • the ligands of classic cadherins are calcatenins, including ⁇ -chain protein, ⁇ -chain protein, ⁇ -chain protein, and P120 protein.
  • Cathecins regulate cell adhesion by interacting with the intracellular cytoskeleton.
  • CDH17 functions as a functional Ca2 + -dependent cell adhesion molecule; however, CDH17 does not achieve tight binding through intracellular calcatenin to actin in the cellular scaffold, but rather directly binds to the cellular scaffold for cell adhesion (Marshall, JF, 2018, Clinical Cancer Research, 24, 253-255).
  • CDH17 expression is an independent prognostic predictor of patient survival (Lee, HJet.al., 2010, Gastroenterology, 139, 213-25.e3). CDH17 expression affects the prognosis of patients with lymph node-negative gastric cancer, which may reflect the role of this protein in maintaining polarity and normal intercellular adhesion.
  • Gastric cancer is one of the most common malignant tumors of the digestive system, with a high incidence and mortality rate (Zuo Tingting et al., Current Status of Gastric Cancer Epidemiology in China [J]. Chinese Journal of Clinical Oncology, 2017, 44(1):52-58). Invasion and metastasis of tumor cells are one of the main reasons for poor prognosis in patients. Decreased cell adhesion mediated by some adhesion molecules in gastric cancer is a crucial factor in invasion and metastasis.
  • CDH17 Aberrant splice variants in gastric cancer, especially the expression of E-cadherin (CDH17) with exon 8 or exon 9 deletion, which is dominant in gastric cancer
  • CDH17 exhibits the ability to regulate homocellular adhesion in a Ca2 + -dependent manner, independent of cytoskeleton interactions, suggesting its important role in tumor metastasis (Ito, R. et al., 2005, Virchows Archiv, 447, 717-722).
  • CDH17 can serve as a relatively specific and sensitive marker for gastrointestinal tumors, and can provide relevant diagnostic evidence for identifying primary gastrointestinal tumors (Wong, B. Wet. al., 2003, Biochemical and Biophysical Research Communications, 311, 618-624). Meanwhile, high expression of CDH17 is closely related to unfavorable clinicopathological features of gastric cancer, such as a positive correlation with histological stage, tumor invasion, and lymph node metastasis.
  • CDH17 may be an important indicator for predicting the progression and prognosis of gastric cancer (Long, ZWet.al., 2015, World Journal of Gastroenterology, 21, 3694-3705; Park, SSet.al., 2007, Annals of Surgical Oncology, 14, 94-99; Li, R.et.al., 2017, International Journal of Oncology, 50, 15-22).
  • Cellular and animal experiments have shown that knockdown of CDH17 can lead to inactivation of the Wnt signaling pathway, thereby inhibiting the invasive activity of cancer cells (Liu, LXet.al., Hepatology, 2010, 51, 358).
  • CDH17 can regulate the Wnt/ ⁇ -catenin signaling pathway, thereby affecting downstream effector factors and influencing the proliferation, invasion, and apoptosis of gastric cancer cells (Qu, LP et al., 2017, European Review for Medical and Pharmacological Sciences, 21, 1234-1241). Furthermore, in vitro studies have shown that inhibition of CDH17 reduces the proliferation and increases apoptosis of the MKN28 gastric cancer cell line in vitro, and significantly reduces its tumorigenicity in vivo. In conclusion, CDH17 is abnormally expressed in gastric cancer tissues and is associated with the occurrence, development, invasion, metastasis, and poor prognosis of gastric cancer.
  • HCC Hepatocellular carcinoma
  • CDH17 is present in the fetal liver and gastrointestinal tract during embryonic development, but its expression is silenced in the liver and stomach tissues of healthy adults (Lee, N.P. et al., 2010, Biochimica et Biophysica Acta, 1806, 138-145).
  • Overexpression of CDH17 can be detected in approximately 80% of patients in human HCC cancer cell lines (Wang, X.Q. et al., 2005, Clinical Cancer Research, 11, 483-489).
  • CDH17 can inactivate the Wnt signaling pathway and activate tumor suppressor genes, thereby inhibiting hepatocellular carcinoma (HCC) tumor growth (Wang, X.Q. et al., 2005, Clinical Cancer Research, 11, 483-489).
  • HCC hepatocellular carcinoma
  • shRNA short hairpin RNA
  • CDH17-targeting antibodies can specifically inhibit CDH17, thereby inhibiting tumor growth by inactivating the Wnt/ ⁇ -catenin pathway. This confirms that CDH17 can inhibit hepatocellular carcinoma by targeting the Wnt/ ⁇ -catenin pathway (Qiu, H.B. et al., 2019, PLoS ONE, 14, Article ID: e0217124).
  • CDH17 expression plays a pro-tumor role in hepatocellular carcinoma and could serve as a therapeutic target for the disease.
  • CDH17 is highly expressed in colorectal cancer, and downregulating CDH17 gene expression inhibits the invasion and metastasis of colon cancer cells. Meanwhile, other studies have shown that CDH17 expression level is an important prognostic predictor affecting the survival of colorectal cancer patients, and CDH17 has certain clinical application value, such as as a molecular marker for disease stage classification and assessment of treatment outcomes in colorectal cancer (Kwak, J. M. et al., 2007, Diseases of the Colon & Rectum, 50, 1873-1880).
  • pancreas as a component of the gastrointestinal tract, plays a role in the morphology and organization of the liver and intestines. Disruption of CDH17 expression or function can lead to increased tumor cell migration and excessive proliferation during pancreatic tumor development (Ivanov, D.B. et al., 2001, Biochemistry (Moscow), 66, 1174-1186). Takamura et al. (Takamura, M. et al., 2003, Cancer Science, 94, 425-430) detected strong CDH17 expression in well-differentiated pancreatic cancer, but not in differentiated regions or poorly differentiated cancers.
  • CDH17 is closely related to the development of various tumors, and developing antibodies targeting CDH17 to kill tumor cells is a possible approach to treating cancer.
  • the inventors immunized mice with recombinant CDH17 protein, a gene gun, or a combination thereof, obtaining multiple antibodies that recognize recombinant CDH17 protein in humans and cynomolgus monkeys. These antibodies were then humanized to obtain humanized antibodies.
  • the antibodies of this invention possess high binding affinity, good endocytic activity, and tumor-killing effects, making them particularly suitable for antibody-drug conjugates (ADCs).
  • ADCs antibody-drug conjugates formed by conjugating the antibodies of this invention with toxins (such as MMAE or DXD and their analogues) can effectively kill tumor cells and inhibit tumor growth in mice.
  • this disclosure provides an antibody or antigen-binding fragment thereof that binds to CDH17, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 5, 6, and 7, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 10, 11, and 12, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 15, 16, and 17, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 20, 21, and 22, respectively; or
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:25 or 107, HCDR2 having the amino acid sequence shown in SEQ ID NO:26 or 125, and HCDR3 having the amino acid sequence shown in SEQ ID NO:27, and the VL comprises LCDR1 having the amino acid sequence shown in SEQ ID NO:30 or 120, LCDR2 having the amino acid sequence shown in SEQ ID NO:31, and LCDR3 having the amino acid sequence shown in SEQ ID NO:32; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:35, 36, and 37, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:40, 41, and 42, respectively; or
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1 having the amino acid sequence shown in SEQ ID NO:50 or 90, LCDR2 having the amino acid sequence shown in SEQ ID NO:51, 93, or 96, and LCDR3 having the amino acid sequence shown in SEQ ID NO:52; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 55, 56, and 57, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 60, 61, and 62, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:65, 66, and 67, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:70, 71, and 72, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 5, 6, and 7, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 10, 11, and 12, respectively;
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 15, 16, and 17, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 20, 21, and 22, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:25, 26, and 27, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:30, 31, and 32, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:35, 36, and 37, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:40, 41, and 42, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:45, 46, and 47, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:50, 51, and 52, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 55, 56, and 57, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 60, 61, and 62, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 65, 66, and 67, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 70, 71, and 72, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, 125, and 27, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 120, 31, and 32, respectively; or
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:45, 46, and 47, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:90, 51, and 52, respectively.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:13
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 23, 99, 101, 103, 105, 108, 110, 112, 114, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 28, 116, 118, 121, 123; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:33
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 43, 73, 75, 77, 79, 82, 84, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 48, 86, 88, 91, 94, 97; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:53
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:58; or
  • the VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:63
  • the VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:68.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:13
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:23
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:33
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:48; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:53
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:58; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:63
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:68; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:101
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:118; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:101
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:123; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:103
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:118; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:103
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:123; or
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:77
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:86; or
  • the VH contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:82
  • the VL contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:88.
  • VH comprises the amino acid sequence shown in SEQ ID NO:3
  • VL comprises the amino acid sequence shown in SEQ ID NO:8;
  • VH contains the amino acid sequence shown in SEQ ID NO:13
  • VL contains the amino acid sequence shown in SEQ ID NO:18;
  • VH contains the amino acid sequence shown in SEQ ID NO:23
  • VL contains the amino acid sequence shown in SEQ ID NO:28;
  • VH contains the amino acid sequence shown in SEQ ID NO:33
  • VL contains the amino acid sequence shown in SEQ ID NO:38
  • VH contains the amino acid sequence shown in SEQ ID NO:43
  • VL contains the amino acid sequence shown in SEQ ID NO:48
  • VH comprises the amino acid sequence shown in SEQ ID NO:53
  • VL comprises the amino acid sequence shown in SEQ ID NO:58;
  • VH comprises the amino acid sequence shown in SEQ ID NO:63
  • VL comprises the amino acid sequence shown in SEQ ID NO:68
  • VH comprises the amino acid sequence shown in SEQ ID NO:101
  • VL comprises the amino acid sequence shown in SEQ ID NO:118;
  • VH comprises the amino acid sequence shown in SEQ ID NO:101
  • VL comprises the amino acid sequence shown in SEQ ID NO:123;
  • VH comprises the amino acid sequence shown in SEQ ID NO:103
  • VL comprises the amino acid sequence shown in SEQ ID NO:118;
  • VH comprises the amino acid sequence shown in SEQ ID NO:103
  • VL comprises the amino acid sequence shown in SEQ ID NO:123;
  • VH comprises the amino acid sequence shown in SEQ ID NO:77
  • VL comprises the amino acid sequence shown in SEQ ID NO:86
  • VH contains the amino acid sequence shown in SEQ ID NO:82
  • VL contains the amino acid sequence shown in SEQ ID NO:88.
  • the antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.
  • the antibody is an isotype selected from IgG, IgA, IgM, IgE and IgD.
  • the antibody belongs to a subtype selected from IgG1, IgG2, IgG3 and IgG4.
  • the antibody comprises a heavy chain (HC) and a light chain (LC), wherein:
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:126 or SEQ ID NO:130
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:128 or SEQ ID NO:132; or
  • the HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:134 or SEQ ID NO:140, and the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:136 or SEQ ID NO:138.
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:126
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:128; or
  • the HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:130, and the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:132; or
  • the HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:134
  • the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:136; or
  • the HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:134
  • the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:138; or
  • the HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:140, and the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:136; or
  • the HC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:140, and the LC contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:138.
  • the HC contains the amino acid sequence shown in SEQ ID NO:126, and the LC contains the amino acid sequence shown in SEQ ID NO:128; or
  • the HC contains the amino acid sequence shown in SEQ ID NO:130, and the LC contains the amino acid sequence shown in SEQ ID NO:132; or
  • the HC contains the amino acid sequence shown in SEQ ID NO:134, and the LC contains the amino acid sequence shown in SEQ ID NO:136; or
  • the HC contains the amino acid sequence shown in SEQ ID NO:134, and the LC contains the amino acid sequence shown in SEQ ID NO:138; or
  • the HC contains the amino acid sequence shown in SEQ ID NO:140, and the LC contains the amino acid sequence shown in SEQ ID NO:136; or
  • the HC contains the amino acid sequence shown in SEQ ID NO:140, and the LC contains the amino acid sequence shown in SEQ ID NO:138.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , Fv, scFv, and ds-scFv.
  • the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.
  • the antibody is a bispecific antibody that further includes a second antigen-binding region that binds to a second antigen.
  • nucleic acids comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof disclosed herein.
  • this disclosure provides a carrier containing the nucleic acid disclosed herein.
  • this disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.
  • compositions comprising (i) an antibody or an antigen-binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition further comprises a second therapeutic agent.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.
  • conjugates comprising the antibody or antigen-binding fragment thereof disclosed herein, and a chemical portion conjugated thereto.
  • the chemical component is selected from therapeutic agents, detectable components, and immunostimulatory molecules.
  • this disclosure provides a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof disclosed herein.
  • CAR chimeric antigen receptor
  • this disclosure provides a method for treating a subject's disease, comprising administering to the subject an effective amount of the antibody disclosed herein or an antigen-binding fragment thereof, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a CAR disclosed herein.
  • the disease is cancer, such as cancer associated with CDH17 expression.
  • the cancer is selected from neuroendocrine tumors, gastric cancer, colon cancer, rectal cancer, small bowel cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hidradenoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial cancer, esophageal cancer, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial cancer;
  • the disease is selected from gastric cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, pancreatic cancer, esophageal cancer, neuroendocrine tumors, and breast cancer.
  • the method further includes administering a second therapeutic agent to the subject.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.
  • Figure 1 Western blot analysis of recombinant human and cynomolgus monkey CDH17 protein expressed in eukaryotes.
  • Figure 2 FACS detection of stable transgenic cell lines HEK293-hCDH17 and HEK293-cyCDH17.
  • Figure 3 FACS detection of stable cell line CHO-hCDH17.
  • Figure 4 BLI pattern of anti-CDH17 chimeric antibody binding to hCDH17.
  • Figure 5 Binding of anti-CDH17 chimeric antibody to tumor cells.
  • Figure 6 Endocytosis of anti-CDH17 chimeric antibody.
  • Figure 7 BLI map of anti-CDH17 humanized antibody binding to hCDH17.
  • Figures 8A-8B Binding of anti-CDH17 humanized antibody to tumor cells.
  • Figures 9A-9B Endocytosis of anti-CDH17 humanized antibody.
  • Figure 10 Killing of SNU-16 tumor cells by anti-CDH17 chimeric antibody ADC.
  • FIGS 11A-11C Killing of SNU-16 tumor cells by anti-CDH17 humanized antibody ADC.
  • Figure 11D Killing of ASPC-1 tumor cells by anti-CDH17 humanized antibody ADC.
  • Figure 12 Bystander effect of anti-CDH17 humanized antibody ADC.
  • Figure 13 Inhibition of tumor growth in mice by anti-CDH17 humanized antibody ADC (using ASPC-1 as the target cell).
  • Figure 14 Inhibition of tumor growth in mice by anti-CDH17 humanized antibody ADC (using SNU-16 as the target cell).
  • references to “an antibody” include a plural number of antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.
  • the term "antibody” refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Such molecules typically consist of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (or domain) (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The variable regions of both the antibody heavy and light chains contain binding domains that interact with the antigen. The antibody constant regions mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical complement activation pathway).
  • the heavy chain of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment).
  • the Fd region contains the VH and CH1 domains and binds to the light chain to form the Fab (antigen-binding fragment).
  • the Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement binding and binding to homologous Fc receptors on effector cells.
  • the hinge region found in the IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region.
  • the hinge domain is structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.
  • the immunoglobulin hinge region can be further subdivided into three regions structurally and functionally: the upper hinge, the core hinge, and the lower hinge.
  • the upper hinge includes the amino acid from the carboxyl terminus of CH1 to the first motility-restricting residue in the hinge, typically the first cysteine residue forming an interchain disulfide bond between the two heavy chains.
  • the length of the upper hinge region is related to the fragmental flexibility of the antibody.
  • the core hinge region contains the interchain disulfide bonds.
  • the lower hinge region connects to the amino terminus of the CH2 domain and includes residues within the CH2 domain.
  • the light chain variable region (VL) or heavy chain variable region (VH) consists of a "framework" region separated by three “complementarity-determining regions” or "CDRs".
  • the framework regions are used to align with the CDRs that specifically bind to the antigenic epitopes.
  • the CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding.
  • Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper.
  • each VL and VH domain is consistent with any conventional definition of CDR.
  • Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of Chothia and Kabat CDR, where CDR-H1 is a complex of Chothia and Kabat CDR; the AbM definition used by the Oxford Molecular antibody modeling software; and the CONTACT definition by Martin et al. (worldwide web bioinfo.org.uk/abs).
  • Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, but the preferred embodiment uses the CDRs defined by Kabat.
  • immunoglobulin molecules Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the antibody light chain Based on the amino acid sequence of the light chain, the antibody light chain can be divided into lambda ( ⁇ ) chains and kappa ( ⁇ ) chains.
  • antibody should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.
  • the term "monoclonal antibody” refers to an antibody derived from a substantially homogeneous population of antibodies. That is, each antibody that makes up the population is identical, except for a small number of mutations that may be naturally present. Monoclonal antibodies are highly specific and target a single antigen.
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies to be produced by any particular method.
  • bispecific antibody should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target.
  • the term “bispecific antibody” should be understood in its broadest sense, including full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.
  • chimeric antibody typically refers to an antibody in which the variable region originates from one species and the constant region originates from another species.
  • the variable region is derived from antibodies from laboratory animals such as rodents ("parental antibodies”), and the constant region is derived from human antibodies, making the resulting chimeric antibody less likely to elicit an adverse immune response in human individuals compared to parental (e.g., mouse-derived) antibodies.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids within the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen.
  • Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulins. "Humanized antibodies” retain antigen specificity similar to the original antibody.
  • the "humanized" form of a non-human (e.g., mouse) antibody may contain, to a minimum, chimeric antibody with a sequence derived from a non-human immunoglobulin.
  • CDR region residues in a human immunoglobulin may be replaced with CDR region residues from a non-human species (donor antibody) (such as a mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and/or capabilities.
  • donor antibody such as a mouse, rat, rabbit, or non-human primate
  • FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications not present in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.
  • antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind antigens. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • antigen-binding fragments covered in the term "antigen-binding portion" of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') 2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a partially hinge region; (iv) Fd fragments, consisting of VH and CH1 domains; (v) Fd' fragments, having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) Fv fragments, consisting of VL and VH domains in a single arm of the antibody; (vii) dAb fragments, consisting of a VH domain; (viii) separate complementarity-determining regions (CDRs); and (ix) nanobodies, heavy chain variable regions containing a single
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via synthetic linkers using recombinant methods, enabling them to be made into a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)).
  • single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibody.
  • the term also includes "straight-chain antibodies” comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1) that together form the antigen-binding region with a complementary light-chain polypeptide, as well as any modified forms of the aforementioned fragments that retain antigen-binding activity.
  • antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as for intact antibodies.
  • binding refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen.
  • the binding specificity of an antibody can be determined based on affinity and/or cohesion.
  • Affinity represents the equilibrium constant (KD) for the dissociation of the antigen and antibody, and is a measure of the strength of binding between the antigenic determinant and the antigen-binding site of the antibody: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody.
  • affinity can also be expressed as the affinity constant (KA), which is 1/KD.
  • Affinity is a measure of the strength of binding between an antibody and an associated antigen. Affinity involves the affinity between an antigenic determinant and an antigen-binding site on the antibody, as well as the number of associated binding sites present on the antibody.
  • antibody binding to an antigen will result in a dissociation constant (KD) of 10 ⁇ 5 M to 10 ⁇ 12 M or less, preferably 10 ⁇ 7 M to 10 ⁇ 12 M or less, and more preferably 10 ⁇ 8 M to 10 ⁇ 12 M, and/or a binding affinity of at least 107 M ⁇ 1 , preferably at least 108 M ⁇ 1 , more preferably at least 109 M ⁇ 1 , such as at least 1012 M ⁇ 1 .
  • KD dissociation constant
  • Any KD value greater than 10 ⁇ 4 M is generally considered to indicate nonspecific binding.
  • Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, as well as various variations thereof known in the art.
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competitive assays as well as various variations thereof known in the art.
  • epitopes refers to the site on an antigen where an antibody binds.
  • Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed within the ternary folds of one or more proteins.
  • Epitopes formed from consecutive amino acids also known as linear epitopes
  • epitopes formed from ternary folds also known as conformational epitopes
  • Epitopes typically comprise at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimum binding site for antibodies and are therefore specific targets for antibodies or their antigen-binding fragments.
  • sequence identity refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned.
  • amino acid sequence is X% identical to SEQ ID NO:Y
  • amino acid sequence has X% identity with SEQ ID NO:Y, and is described as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO:Y.
  • Exemplary programs for comparing and aligning sequence pairs include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.
  • conserved amino acid substitutions can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.
  • Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.
  • the particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.
  • carrier is intended to refer to a nucleic acid molecule that is capable of transporting another nucleic acid to which it is attached.
  • the term "host cell” refers to a cell into which an expression vector has been introduced.
  • pharmaceutical acceptable means that the carrier or excipient is compatible with the other components of the composition and does not cause substantial toxicity to the recipient, and/or that such carrier or excipient is approved or may be used in pharmaceutical compositions for parenteral administration to humans.
  • treatment refers to the administration of a drug or the conduct of a procedure for the purpose of achieving an effect. These effects may be preventative in the complete or partial prevention of a disease or its symptoms, and/or therapeutic in the partial or complete cure of a disease and/or its symptoms.
  • treatment can include treating a disease or condition (e.g., an inflammatory disease) in mammals, particularly humans, and includes: (a) preventing the onset of a disease or its symptoms in subjects who may be susceptible to the disease (e.g., including diseases that may be related to or caused by the primary disease) but have not yet been diagnosed with the disease; (b) suppressing the disease, i.e., halting its progression; and (c) alleviating the disease, i.e., causing its regression.
  • a disease or condition e.g., an inflammatory disease
  • Treatment can refer to any indicator of success in the treatment, improvement, or prevention of cancer, including any objective or subjective parameter such as a reduction in symptoms; remission; elimination of disease symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or weakening the final stage of deterioration.
  • Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term “treatment” includes the administration of the antibodies, compositions, or conjugates disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms or conditions associated with a disease (e.g., an inflammatory disease).
  • therapeutic effect refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.
  • the term "effective dose” refers to a dose administered to a subject that is sufficient to treat the disease.
  • the term “subject” refers to any mammalian subject for whom a diagnosis, treatment, or therapy is intended.
  • “Mammalian” for therapeutic purposes means any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sport, or pet animals such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
  • This disclosure provides an antibody or antigen-binding fragment thereof that binds to CDH17, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:5, 6, and 7, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:10, 11, and 12, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 15, 16, and 17, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 20, 21, and 22, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:25 or 107, HCDR2 having the amino acid sequence shown in SEQ ID NO:26 or 125, and HCDR3 having the amino acid sequence shown in SEQ ID NO:27
  • the VL comprises LCDR1 having the amino acid sequence shown in SEQ ID NO:30 or 120, LCDR2 having the amino acid sequence shown in SEQ ID NO:31, and LCDR3 having the amino acid sequence shown in SEQ ID NO:32.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:25 or 107, HCDR2 having the amino acid sequence shown in SEQ ID NO:26, and HCDR3 having the amino acid sequence shown in SEQ ID NO:27, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:30, 31, and 32, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 25 or 107, HCDR2 having the amino acid sequence shown in SEQ ID NO: 125, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 30, 31, and 32, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 25 or 107, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 120, 31, and 32, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 25 or 107, HCDR2 having the amino acid sequence shown in SEQ ID NO: 125, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 120, 31, and 32, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 25, 26, and 27, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 30, 31, and 32, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, 125, and 27, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 120, 31, and 32, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:35, 36, and 37, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:40, 41, and 42, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1 having the amino acid sequence shown in SEQ ID NO:50 or 90, LCDR2 having the amino acid sequence shown in SEQ ID NO:51, 93, or 96, and LCDR3 having the amino acid sequence shown in SEQ ID NO:52.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:50, 51, and 52, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:90, 51, and 52, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:50, 93, and 52, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:50, 96, and 52, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:90, 93, and 52, respectively.
  • the VH comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:45, HCDR2 having the amino acid sequence shown in SEQ ID NO:46 or 81, and HCDR3 having the amino acid sequence shown in SEQ ID NO:47, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:90, 96, and 52, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO:45, 46, and 47, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO:50, 51, and 52, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:45, 46, and 47, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:90, 51, and 52, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 55, 56, and 57, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 60, 61, and 62, respectively.
  • the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 65, 66, and 67, respectively
  • the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 70, 71, and 72, respectively.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:8.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:13
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:18.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 23, 99, 101, 103, 105, 108, 110, 112, 114, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 28, 116, 118, 121, 123.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:23, 101, 103
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:28, 118, 123.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:23
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:28.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:101
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:118.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:101
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:123.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:103
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:118.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:103
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:123.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:33
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:38.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:43, 73, 75, 77, 79, 82, 84, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:48, 86, 88, 91, 94, 97.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:43, 77, and 82
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:48, 86, and 88.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:43
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:48.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:77
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:86.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:82
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:88.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:53
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:58.
  • the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:63
  • the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:68.
  • VH comprises a functional variant of the amino acid sequence disclosed herein by means of insertion, deletion, and/or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to CDH17.
  • VL comprises a functional variant of the amino acid sequence disclosed herein by means of insertion, deletion, and/or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to CDH17.
  • the functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.
  • the number of inserted, deleted, and/or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, even more preferably 1% to 33%, more preferably 5% to 30%, even more preferably 10% to 25%, and even more preferably 15% to 20%.
  • the number of inserted, deleted, and/or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2.
  • the number of inserted, deleted, and/or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.
  • insertions, deletions, and/or substitutions can be performed in frame (FR) regions, such as FR1, FR2, FR3, and/or FR4.
  • the substitution of one or more amino acids may be a conservative substitution of one or more amino acids.
  • a conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.
  • the particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:3
  • the VL comprises the amino acid sequence shown in SEQ ID NO:8.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:13
  • the VL comprises the amino acid sequence shown in SEQ ID NO:18.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:23
  • the VL comprises the amino acid sequence shown in SEQ ID NO:28.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:33
  • the VL comprises the amino acid sequence shown in SEQ ID NO:38.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:43
  • the VL comprises the amino acid sequence shown in SEQ ID NO:48.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:53
  • the VL comprises the amino acid sequence shown in SEQ ID NO:58.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:63
  • the VL comprises the amino acid sequence shown in SEQ ID NO:68.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:101
  • the VL comprises the amino acid sequence shown in SEQ ID NO:118.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:101
  • the VL comprises the amino acid sequence shown in SEQ ID NO:123.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:103
  • the VL comprises the amino acid sequence shown in SEQ ID NO:118.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:103
  • the VL comprises the amino acid sequence shown in SEQ ID NO:123.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:77
  • the VL comprises the amino acid sequence shown in SEQ ID NO:86.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:82
  • the VL comprises the amino acid sequence shown in SEQ ID NO:88.
  • the antibody is a mouse antibody, a chimeric antibody, or a humanized antibody. In a preferred embodiment, the antibody is a humanized antibody.
  • immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the antibody light chain can be divided into lambda ( ⁇ ) chains and kappa ( ⁇ ) chains.
  • the antibodies disclosed herein can be any of the above classes or subtypes.
  • the antibody belongs to an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody belongs to a subtype selected from IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.
  • the antibodies disclosed herein may be complete antibodies or antigen-binding fragments thereof.
  • the antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), wherein the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:126 or SEQ ID NO:130, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:128 or SEQ ID NO:132.
  • HC immunoglobulin heavy chain
  • LC immunoglobulin light chain
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:126
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:128.
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:130
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:132.
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:134
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:136.
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:134
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:138.
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:140
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:136.
  • the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:140
  • the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:138.
  • the HC comprises the amino acid sequence shown in SEQ ID NO:126
  • the LC comprises the amino acid sequence shown in SEQ ID NO:128.
  • the HC comprises the amino acid sequence shown in SEQ ID NO:130
  • the LC comprises the amino acid sequence shown in SEQ ID NO:132.
  • the HC comprises the amino acid sequence shown in SEQ ID NO:134
  • the LC comprises the amino acid sequence shown in SEQ ID NO:136.
  • the HC comprises the amino acid sequence shown in SEQ ID NO:134
  • the LC comprises the amino acid sequence shown in SEQ ID NO:138.
  • the HC comprises the amino acid sequence shown in SEQ ID NO:140
  • the LC comprises the amino acid sequence shown in SEQ ID NO:136.
  • the HC comprises the amino acid sequence shown in SEQ ID NO:140
  • the LC comprises the amino acid sequence shown in SEQ ID NO:138.
  • the antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to IL-13.
  • antigen-binding fragments include, but are not limited to: Fab fragments; F(ab’)2 fragments; Fab’ fragments; Fd fragments; Fd’ fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity-determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd fragments (VH-CH1-VH-CH1); and any modified forms of the aforementioned fragments that retain antigen-binding activity.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , Fv, scFv, and ds-scFv.
  • the antigen-binding fragment is Fab.
  • the antigen-binding fragment is Fv.
  • the antigen-binding fragment is scFv.
  • the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody that further includes a second antigen-binding region for binding to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen.
  • TAs tumor-associated antigens
  • TAs are antigens that can elicit a significant tumor-specific immune response. Some of these antigens are encoded by normal cells but are not necessarily expressed by them. These antigens can be characterized as antigens that are normally silenced (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed over time, such as embryonic and fetal antigens.
  • cancer cell antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogenes), repressor genes (e.g., p53 mutants), and fusion proteins resulting from internal deletions or chromosomal translocations.
  • oncogenes e.g., activated ras oncogenes
  • repressor genes e.g., p53 mutants
  • fusion proteins resulting from internal deletions or chromosomal translocations e.g., p53 mutants
  • Other cancer antigens can be encoded by viral genes such as those carried by RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and/or commercially available and can also be prepared by those skilled in the art.
  • tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate-binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, melanoma-associated antigen, MUC-1, mutant p53, mutant ras, ROR1, GPC3, VEGFR2, and combinations thereof.
  • the second antigen is a T-cell antigen.
  • the T-cell antigen is selected from T-cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof.
  • the T-cell antigen is CD3, and the second antigen binding region binds to any one of the ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains of CD3.
  • the antibody of the present invention binds to CDH17 in humans and cynomolgus monkeys.
  • the EC50 of the antibody of the present invention binding to CDH17-positive tumor cells is in the nM range, for example, about 0.5-2.0 nM.
  • the IC50 of the antibody of the present invention being internalized by CDH17-positive tumor cells is less than 1 nM, for example, 0.01-0.30 nM.
  • the antibody of the present invention is capable of inhibiting the Wnt/ ⁇ -catenin signaling pathway.
  • the antibodies disclosed herein may contain an Fc region.
  • the Fc region may be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications.
  • the Fc region is an IgG1 or IgG4 isotype or derived therefrom, optionally having one or more mutations or modifications.
  • the Fc region is human IgG1 Fc.
  • the Fc region has reduced effector function, such as reduced binding to ADCC, ADCP, CDC and/or Clq, Fc ⁇ RI, Fc ⁇ RII, or Fc ⁇ RIIIA.
  • the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector function.
  • Such mutations have been described, for example, in Dall’Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).
  • the Fc region may contain an amino acid sequence with one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G.
  • the Fc region contains L234A and L235A (LA mutation).
  • the Fc region contains a mutation that removes the Asn-linked glycosylation receptor site or is otherwise manipulated to alter the glycosylation properties.
  • the N297Q mutation can be used to remove the Asn-linked glycosylation site. Therefore, in a specific embodiment, the Fc region contains an IgG1 sequence with the N297Q mutation.
  • the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, it can be used... The method described by Natsume et al. (1999) Nature Biotech 17:176 optimizes ADCC.
  • the Fc region is engineered to enhance complement activation, as described by Natsume et al. (2009) Cancer Sci. 100:2411.
  • the Fc region has an extended serum half-life.
  • altering e.g., decreasing or increasing
  • the in vivo half-life of an antibody can be found, for example, International Publications WO 02/060919, WO 98/23289, and WO 97/34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety.
  • one, two, or more amino acid mutations are introduced into the constant domain of IgG or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to reduce the in vivo half-life of the antibody.
  • one, two, or more amino acid mutations are introduced into the constant domain of IgG or its FcRn binding fragment (preferably an Fc or hinge-Fc domain fragment) to increase the in vivo half-life of the antibody.
  • the antibody may have one or more amino acid mutations (e.g., substitutions) (according to EU numbering system) in the second constant (CH2) domain (residues 231-340 of human IgG1) and/or the third constant (CH3) domain (residues 341-447 of human IgG1).
  • the constant region of the antibody IgG1 described herein includes a substitution of methionine (M) at position 252 to tyrosine (Y), a substitution of serine (S) at position 254 to threonine (T), and a substitution of threonine (T) at position 256 to glutamate (E) (according to EU numbering system). See U.S. Patent No.
  • the Fc region contains M252Y, S254T, and T256E (YTE mutation).
  • nucleic acids comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof disclosed herein.
  • nucleic acid includes single-stranded and double-stranded nucleotide polymers.
  • Nucleic acids can be ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide. These modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and nucleotide bond modifications such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.
  • the present invention provides nucleic acid molecules encoding any of the heavy chain variable region sequences disclosed herein.
  • the present invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.
  • the present invention provides nucleic acid molecules encoding any of the light chain variable region sequences disclosed herein.
  • the present invention also provides nucleic acid molecules that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding any of the light chain variable region sequences disclosed herein.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 19.
  • the nucleic acids disclosed herein comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 24, 100, 102, 104, 106, 109, 111, 113, 115, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 29, 117, 119, 122, 124.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29.
  • nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 102 and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 119.
  • nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 104 and the nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 119.
  • nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 104 and the nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 124.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 34, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 39.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 44, 74, 76, 78, 80, 83, 85, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 49, 87, 89, 92, 95, 98.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 44, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 49.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 78, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 87.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 83, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 89.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 54, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 59.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 64, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 69.
  • the present invention provides nucleic acid molecules encoding the heavy chain sequences disclosed herein.
  • the present invention also provides nucleic acid molecules that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding the heavy chain sequences disclosed herein.
  • the present invention provides nucleic acid molecules encoding the light chain sequences disclosed herein.
  • the present invention also provides nucleic acid molecules that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding the light chain sequences disclosed herein.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 127, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 129.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 131, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 133.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 135, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 137.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 135, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 139.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 141, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 137.
  • the nucleic acid disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 141, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 139.
  • the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • deoxyribonucleic acid can be introduced into human cells in vivo.
  • the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent.
  • the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.
  • ribonucleic acid can be introduced into human cells in vivo.
  • the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.
  • This disclosure provides vectors containing the nucleic acids disclosed herein.
  • the vector is an expression vector capable of expressing a polypeptide containing a variable region of the heavy or light chain of an antibody.
  • the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.
  • the vector is a viral vector.
  • the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.
  • AAV adenovirus-associated vector
  • Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.
  • the expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses.
  • vectors can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.).
  • Phage vectors such as ⁇ GT10, ⁇ GT11, ⁇ ZapII (Stratagene), ⁇ EMBL4, and ⁇ NM1149 can also be used.
  • plant expression vectors that can be used in this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech).
  • animal expression vectors that can be used in this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd ed., *Cold Spring Harbor Press*, *Cold Spring Harbor*, N.Y., 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, *Greene Publishing Associates and John Wiley & Sons*, NY, 1994.
  • Circular or linear expression vector constructs can be prepared to contain the functionality of a replication system in prokaryotic or eukaryotic host cells.
  • the replication system can be derived from, for example, COLEL, 2 ⁇ plasmid, ⁇ , SV40, bovine papillomavirus, etc.
  • the vector could be an adenoviral vector containing a nucleotide sequence encoding an antibody disclosed herein.
  • the vector could be administered to a subject, then enter the subject's cells, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the cell's genome, and subsequently the cells express the antibody disclosed herein.
  • This disclosure provides host cells containing the nucleic acids or vectors disclosed herein.
  • the cell may be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cell such as a mammalian cell.
  • Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium.
  • the genera *Serratia* include *Serratia typhimurium*; *Serratia* species, such as *Serratia marcescans* and *Shigella*; *Bacilli* species, such as *B. subtilis* and *B. licheniformis*; *Pseudomonas* species, such as *P. aeruginosa*; and *Streptomyces*.
  • the cells are human cells.
  • the cells are immune cells.
  • the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.
  • the host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or ex vivo.
  • the host cells of the present invention can be administered to a subject, and the host cells express the antibodies disclosed herein in vivo.
  • This invention provides host cells in which any of the above-described vectors have been introduced.
  • This invention also provides a method for preparing the antibodies of this invention, the method comprising a) culturing the host cells disclosed herein under conditions suitable for antibody production; and b) obtaining the antibodies from the culture.
  • compositions comprising an antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.
  • compositions suitable for administration typically comprise an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration.
  • Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin.
  • Liposomes and non-aqueous media such as non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Additional active compounds may also be incorporated into the composition.
  • the pharmaceutical composition further comprises a second therapeutic agent.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.
  • compositions of the present invention can be formulated to be compatible with their intended routes of administration.
  • routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), mucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as tetraethylamine oxalate (EDTA); buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or dextran.
  • the pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide.
  • Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.
  • Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, antibacterial water, Cremophor EL TM (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS).
  • the composition must be sterile and should be a fluid present in a manner easily injectable. It must be stable under preparation and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.
  • Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.
  • isotonic agents such as sugars, polyols like mannitol, sorbitol, and sodium chloride.
  • the absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by filtration and sterilization.
  • dispersions are prepared by introducing the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above.
  • the preparation method is vacuum drying and freeze-drying to produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.
  • Oral compositions typically include inert diluents or edible carriers. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be mixed with excipients and administered in tablet, lozenge, or capsule form. Oral compositions may also be prepared using fluid carriers used as mouthwashes, wherein the compound in the fluid carrier is administered orally and rinsed and spat out or swallowed. Pharmaceutically compatible binders and/or adjuvants may be included as part of the composition.
  • Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint oil, methyl salicylate, or orange flavoring.
  • binders such as microcrystalline cellulose, astragalus gum, or gelatin
  • excipients such as starch or lactose
  • disintegrants such as alginate, Primogel, or corn starch
  • lubricants such as magnesium stearate or sterotes
  • gliding agents such as colloidal silica
  • sweeteners such as sucrose or saccharin
  • the compound is delivered as an aerosol spray from a pressure vessel or dispenser or sprayer containing a suitable propellant (e.g., a gas such as carbon dioxide).
  • a suitable propellant e.g., a gas such as carbon dioxide.
  • Systemic application can also be performed via transmucosal or transdermal routes.
  • a penetrant suitable for the target penetration barrier is used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for transmucosal application.
  • Transmucosal application can be accomplished using nasal sprays or suppositories.
  • the active compound is formulated as an ointment, cream, gel, or lotion known in the art.
  • Active compounds can also be formulated as suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or as retention enemas for rectal delivery.
  • suppositories e.g., using conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compound is prepared together with a carrier (such as a controlled-release formulation, including implants and microencapsulated delivery systems) that protects the compound from rapid elimination from the body.
  • a carrier such as a controlled-release formulation, including implants and microencapsulated delivery systems
  • Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.
  • This invention provides therapeutic compositions comprising the antibodies of the invention or antigen-binding fragments thereof.
  • the therapeutic compositions according to the invention will be administered together with suitable carriers, excipients, and other pharmaceutical agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc.
  • suitable formulations are available in all formulations known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J Pharm Sci Technol 52:238-311.
  • conjugates comprising the antibody or antigen-binding fragment thereof disclosed herein, and the chemical portion conjugated thereto.
  • a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety.
  • the chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus.
  • Antibody conjugates are commonly referred to as “immunoconjugates.”
  • a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent)
  • the conjugate is commonly referred to as an “antibody-drug conjugate” or “ADC.”
  • conjugation can refer to the process by which two polypeptides become a single, continuous polypeptide molecule.
  • an antibody is linked to a chemical moiety.
  • the antibody linked to the chemical moiety is further linked to a lipid or other molecule to a protein or peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly.
  • the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety produces a covalent bond formed between the two molecules to form a single molecule.
  • a peptide linker short peptide sequence
  • the chemical moiety can be attached to the antibody of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent attachment methods can be used.
  • the procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety.
  • Peptides typically contain multiple functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with suitable functional groups on the antibody to result in the binding of the chemical moiety.
  • the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attaching any of a number of known linker molecules.
  • the linker can be any molecule used to attach the antibody to the chemical moiety.
  • the linker is capable of forming a covalent bond with both the antibody and the chemical moiety.
  • Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linker can be attached via their side groups to the constituent amino acids (e.g., via disulfide bonds to cysteine) or to the ⁇ -carbon amino and carboxyl groups of the terminal amino acid.
  • the immunoconjugate when an immunoconjugate reaches its target site, it is desirable to release a chemical moiety from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site.
  • Conditions experienced by enzymatically active or immunoconjugates within or near target cells may induce linker cleavage to release the chemical portion from the antibody.
  • antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein).
  • antibodies or portions thereof are derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling.
  • antibodies can be functionally linked (through chemical coupling, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a divalent antibody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association between the antibody or an antibody portion and another molecule (such as the streptavidin core region or a multihistidine tag).
  • One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same or different types).
  • Suitable crosslinking agents include heterobifunctional crosslinkers (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester) or homobifunctional crosslinkers (e.g., disuccinimide octanoate ester) having two distinct reactive moieties separated by a suitable spacer.
  • heterobifunctional crosslinkers e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester
  • homobifunctional crosslinkers e.g., disuccinimide octanoate ester
  • the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.
  • the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins.
  • the therapeutic agent may be, for example, maytansine, galdromycin, microtubule inhibitors such as microtubule binding agents (e.g., olistatins), or small groove binding agents such as galicarmycin.
  • the therapeutic agent may be, for example, MMAE, Dxd, or derivatives thereof.
  • the detectable portion may be selected from biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, or fluorescent, phosphorescent, or chemiluminescent molecules.
  • Detectable portions for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.
  • Antibodies can be conjugated to detectable markers; for example, detectable markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy.
  • detectable markers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection).
  • useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc.
  • Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), can also be used.
  • Antibody or antigen-binding fragments can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase, and glucose oxidase.
  • enzymes that can be used for detection
  • detection can be achieved by adding additional reagents to generate a recognizable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually.
  • Antibody or antigen-binding fragments can also be conjugated to biotin and detected indirectly by binding to avidin or streptavidin. It should be noted that avidin itself can be conjugated to enzymes or fluorescent labels.
  • Antibodies can be fused with self-labeled protein tags, such as HaloTag.
  • protein tags can be cloned to the ends of constant regions.
  • HaloTag is a self-labeled protein tag derived from bacterial enzymes (haloalkyl dehalogenases) and designed to be covalently bound to synthetic ligands.
  • the synthetic ligands contain chloroalkane linkers attached to fluorophores such as near-infrared fluorophores (Los et al. (2008) ACS Chem Biol. 3(6):373-82).
  • Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.
  • Paramagnetic particles such as superparamagnetic iron oxide
  • Antibodies can also be labeled with predetermined polypeptide epitopes recognized by a second reporter gene (such as leucine zipper pairs, binding sites of the second antibody, metal-binding domains, or epitope tags).
  • the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.
  • Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes or radionucleotides: 3H , 14C , 15N , 35S , 90Y , 99Tc , 111In , 125I , and 131I .
  • immunostimulatory molecules are immune effector molecules that stimulate an immune response.
  • immunostimulatory molecules can be cytokines such as IL-2 and IFN- ⁇ , chemokines such as IL-8, platelet-4, melanoma growth-stimulating proteins, complement activators; viral/bacterial protein domains, or viral/bacterial peptides.
  • This disclosure provides a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof disclosed herein.
  • CAR chimeric antigen receptor
  • This disclosure also provides genetically modified cells comprising the chimeric antigen receptors disclosed herein.
  • chimeric antigen receptor refers to a molecule engineered to contain an antigen-binding domain that targets a specific antigen, and upon binding to that antigen, activates immune cells (such as T cells or NK cells, including naive T cells, central memory T cells, effector memory T cells, or combinations thereof) to attack and destroy cells carrying that antigen.
  • immune cells such as T cells or NK cells, including naive T cells, central memory T cells, effector memory T cells, or combinations thereof
  • CAR-expressing immune cells can target and kill the tumor cells.
  • a classic chimeric antigen receptor is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain.
  • the antigen-binding domain is typically an antigen-binding fragment derived from a monoclonal antibody (mAb) (e.g., scFv), but it can be based on other forms containing antibody-like antigen-binding sites or on a native ligand derived from the antigen.
  • mAb monoclonal antibody
  • a hinge domain is usually required to separate the antigen-binding domain from the membrane and allow for its proper orientation.
  • a common hinge domain used is the Fc of IgG1.
  • more compact spacer regions may be suitable, such as the stem from CD8 ⁇ , and even just the IgG1 hinge alone.
  • the transmembrane domain anchors the protein within the cell membrane and connects the hinge domain to the intracellular domain (intramocyte domain).
  • At least three “generations” of CAR molecules have existed.
  • first-generation CARs they are designed with an intracellular domain having an intracellular portion of the ⁇ chain derived from Fc ⁇ R1 or the intracellular portion of CD3 ⁇ . Therefore, these first-generation CARs deliver an immune signal1 sufficient to trigger T cell killing of homologous target cells, but cannot fully activate T cell proliferation and survival.
  • second-generation CARs have been constructed with a complex intracellular domain resulting from the fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3 ⁇ , thus enabling the simultaneous delivery of activation and costimulatory signals after antigen recognition.
  • the most commonly used costimulatory domain is the CD28 costimulatory domain. This provides the most potent costimulatory signal, i.e., an immune signal2, which triggers T cell proliferation.
  • costimulatory signal i.e., an immune signal2
  • CARs have also been described that include intracellular domains of the TNF receptor family, such as the closely associated OX40 and 41BB, which deliver survival signals. Even more potent third-generation CARs have now been described, possessing intracellular domains capable of delivering activation, proliferation, and survival signals.
  • a CAR typically comprises: (i) an antigen-binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain that includes a signal transduction domain and one or more co-stimulatory domains.
  • an "antigen-binding domain” refers to the portion of a chimeric antigen receptor that recognizes an antigen.
  • the antigen-binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody.
  • CARs have also been generated using domain-specific antibodies (dAbs), VHH antigen-binding domains, or antigen-binding domains derived from natural ligands of the antigen.
  • the antigen-binding domain may be the antibody or its antigen-binding fragment described herein.
  • the "hinge domain” positions the antigen-binding domain away from the effector cell surface to facilitate proper cell/cell contact, antigen binding, and activation.
  • CARs optionally include one or more hinge domains between the binding domain and the transmembrane domain (TM).
  • the hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources.
  • the hinge domain can contain the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • hinge domains suitable for use in CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8 ⁇ ), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be altered.
  • type 1 membrane proteins such as CD8 (e.g., CD8 ⁇ ), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be altered.
  • a “transmembrane domain” refers to a portion of a CAR that optionally fuses an extracellular binding portion with an intracellular portion (e.g., a co-stimulatory domain and an intracellular signaling domain) via a hinge domain, and anchors the CAR to the plasma membrane of an immune effector cell.
  • a transmembrane domain is typically a hydrophobic region of the CAR that crosses the cell membrane.
  • a TM domain can be a transmembrane region or fragment of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof.
  • the intracellular domain is the signal transduction portion of a chimeric antigen receptor. It contains a signal transduction domain and one or more co-stimulatory domains. Upon antigen recognition, the receptor clusters native CD45 and CD148 are expelled from the synapse, and signals are transduced into the cell, thereby activating one or more immune cell effector functions (e.g., innate immune cell effector functions).
  • the most commonly used intracellular domain component is the CD3 ⁇ intracellular domain component containing three ITAMs. After antigen binding, it transmits an activation signal to T cells. CD3 ⁇ may not provide a completely sufficient activation signal, and additional co-stimulatory signal transduction may be required.
  • Co-stimulatory signals promote T cell proliferation and survival. There are two main types of co-stimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).
  • “Intracellular signal transduction domain” refers to a portion of a CAR polypeptide that participates in transducing information about the binding of an effective CAR to a target antigen into immune effector cells to trigger effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into CAR-bound target cells, or other cellular responses triggered upon antigen binding to the extracellular CAR domain.
  • Non-limiting examples of intracellular signal transduction domains containing immune receptor tyrosine-based activation motifs include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • a "co-stimulatory domain” refers to the intracellular signal transduction domain of a co-stimulatory molecule.
  • Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for effective activation and function of T lymphocytes when binding to antigens.
  • Co-stimulatory domains can be, for example, 4-1BB, CD27, CD28, or OX40.
  • This disclosure provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of an antibody disclosed herein or an antigen-binding fragment thereof, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein.
  • This disclosure also provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the conjugates disclosed herein, or the chimeric antigen receptors disclosed herein in the preparation of medicaments for treating a disease in a subject.
  • This disclosure also provides the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the conjugates disclosed herein, or the chimeric antigen receptors disclosed herein for the treatment of a subject's disease.
  • the disease is cancer, such as cancer associated with CDH17 expression or CDH17-positive cancer.
  • the cancer is selected from neuroendocrine tumors, gastric cancer, colon cancer, rectal cancer, small bowel cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hidradenoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial cancer, esophageal cancer, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, embryonal carcinosarcoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, or epithelial cancer.
  • the disease is selected from gastric cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, pancreatic cancer, esophageal cancer, neuroendocrine tumors, and breast cancer.
  • the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site of treatment and the subject. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as antibody activity and the route of administration.
  • the dosage of the antibodies, compositions, conjugates, or chimeric antigen receptors described herein can be administered to mammals in a single dose or in a series of sub-dose over an appropriate period of time, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed.
  • Dosage units containing an effective amount of the antibody, composition, conjugate, or chimeric antigen receptor can be administered as a single daily dose, or the total daily dose can be administered as needed in two, three, four, or more daily sub-dose administrations.
  • Administration routes may include parenteral administration, such as by injection, nasal administration, pulmonary administration, or percutaneous administration.
  • Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.
  • antibodies, compositions, conjugates, or chimeric antigen receptors are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration.
  • the dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.
  • the method further includes administering a second therapeutic agent to the subject.
  • the antibody or antigen-binding fragment thereof disclosed herein, pharmaceutical composition, conjugate, or chimeric antigen receptor is used in combination with the second therapeutic agent.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.
  • the antibody, composition, conjugate, or chimeric antigen receptor disclosed herein is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent.
  • kits or delivery devices comprising the antibodies or antigen-binding fragments thereof disclosed herein, the nucleic acid molecules disclosed herein, the vectors disclosed herein, the host cells disclosed herein, the conjugates disclosed herein, the pharmaceutical compositions disclosed herein, or the chimeric antigen receptors disclosed herein.
  • the kit or delivery device includes one or more containers containing one or more components of the pharmaceutical composition described herein, such as antibodies or antigen-binding fragments disclosed herein.
  • the kit includes a first container containing the antibody disclosed herein.
  • the kit includes a first container which is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit also includes a second container containing a pharmaceutically acceptable fluid.
  • this document provides an injection device containing antibodies.
  • the injection device contains antibodies in a sterile solution.
  • the injection device is a syringe.
  • the kit includes instructional material disclosing how to use the antibodies disclosed herein.
  • the instructional material may be in written, electronic (e.g., computer floppy disk or optical disc), or visual (e.g., video file).
  • the kit may also include additional components to facilitate the application for which the kit is designed.
  • the kit may additionally contain tools for detecting labeled substances (e.g., enzyme substrates for enzymatic labeling, filter sets for detecting fluorescent labels, suitable secondary labels such as secondary antibodies, etc.).
  • the kit may also include buffers and other reagents conventionally used to perform a particular method. Such kits and suitable contents are well known to those skilled in the art.
  • the nucleic acid sequence encoding the antigen protein with his and Fc tags was integrated into a mammalian cell expression vector. After bacterial infection, lysis, plasmid extraction, and washing, the plasmid expressing the antigen protein was obtained. HEK293F cells (passages up to P11) were passaged to an appropriate number. In a clean bench, the complex formed by PEI and the plasmid was added dropwise to the 293 cells passaged the previous day at a density of 1.5 ⁇ 106 cells/ml. The complex was added while shaking the container, or a portion of the complex was added, mixed with the cells, and then repeated to avoid excessively high concentrations of the complex in localized areas of the cells.
  • the 293 cells were cultured in a cell culture incubator at 37°C, 120 rpm, and 5% CO2 .
  • Day 0 was the day of transient transfection.
  • days 1 and 3 of transient transfection 5% OPM-293Profeed feed (the same volume as the cells on the day of transfection) was added.
  • day 4 after transfection cell viability was assessed using Vi-cell. When cell viability dropped to approximately 70%, the fermentation broth was collected, filtered, and purified to obtain the antigen protein, which was then analyzed by Western blot.
  • the results are shown in Figure 1.
  • Recombinant human CDH17 (hCDH17) and cynomolgus monkey CDH17 (cyCDH17) proteins were obtained after purification.
  • the amino acid sequences of the human and cynomolgus monkey CDH17 proteins are shown below:
  • the full-length human or cynomolgus monkey CDH17 gene was cloned into an expression vector containing a puromycin resistance gene using molecular cloning technology, resulting in plasmids plenti-hCDH17-IRES-p or plenti-cyCDH17-IRES-p.
  • HEK293 cells were then transfected with these expression vectors. After entering the host cells, the target gene underwent reverse transcription and integrated into the genome, thus expressing the human or cynomolgus monkey CDH17 gene.
  • Cells were continuously screened for 30 days using a puromycin-containing selection medium to obtain cell populations containing HEK293-hCDH17 or HEK293-cyCDH17 positive cell lines (HEK293-hCDH17 mix pool or HEK293-cyCDH17 mix pool). Next, subcloning was performed using either the HEK293-hCDH17 mix pool or the HEK293-cyCDH17 mix pool. Cells were seeded at 1 cell/well in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 2 weeks. Single-clone cells were then expanded to 24-well plates.
  • the full-length hCDH17 gene was cloned into an expression vector containing a puromycin resistance gene using molecular cloning technology, resulting in... Plasmids were transfected into CHO cells using electroporation. After the target gene entered the host cell, it underwent reverse transcription and integrated into the genome, thereby expressing the hCDH17 gene. Cell lines expressing the hCDH17 gene were obtained through cell line selection with the addition of puromycin. Cells were continuously screened for 2 weeks with 8 ⁇ g/mL puromycin selection medium to obtain a cell population containing CHO-hCDH17 positive cells (CHO-hCDH17 mix pool).
  • the CHO-hCDH17 mix pool was subcloned, and cells were seeded at 1 cell/well in a 96-well cell culture plate. After culturing at 37°C in a 5% CO2 incubator for 10–15 days, the single clones were expanded to 24-well plates. Once the cells had grown sufficiently, the single clones were screened and validated using an anti-hCDH17 antibody. As shown in Figure 3, the hCDH17 single clone cell line was successfully constructed.
  • mice Female Balb/C mice aged 4-6 weeks (from Beijing Vital River) were selected and immunized using the following four immunization strategies.
  • Group 1 Immunization was performed using a combination of gene gun and antigen.
  • mice were immunized with the mFlt3L plasmid, with each mouse receiving 3 ⁇ g of plasmid via gene gun immunization per dose, for a total of one dose.
  • each mouse received 3 ⁇ g of plasmid via gene gun immunization per dose, once a week, for a total of four immunizations.
  • mice were immunized with hCDH17 protein, with each mouse receiving 15 ⁇ g of the protein, for a total of one immunization.
  • Group 2 Cellular immunization was used.
  • each mouse was immunized subcutaneously and in the footpads with CHO-hCDH17 cells, using the Sigma adjuvant system.
  • Each mouse was immunized with 1 ⁇ 106 cells, for a total of one immunization.
  • the footpads were immunized with CHO-hCDH17 cells, using the Sigma adjuvant system, every two weeks.
  • Each mouse was immunized with 1 ⁇ 106 cells, for a total of three immunizations.
  • Group 3 Immunization using antigens.
  • each mouse was immunized subcutaneously and in the footpads with hCDH17 protein, using TiterMax as an adjuvant, at a dose of 50 ⁇ g per mouse, for a total of one immunization.
  • the footpads were immunized with hCDH17 protein, using the Sigma adjuvant system as an adjuvant, every two weeks, at a dose of 15 ⁇ g per mouse, for a total of three immunizations.
  • Group 4 Immunization using antigens.
  • each mouse was immunized subcutaneously and in the footpads with hCDH17 protein, using CFA as an adjuvant, at a dose of 50 ⁇ g per mouse, for a total of one immunization.
  • the footpads were immunized with hCDH17 protein, using the Sigma adjuvant system as an adjuvant, every two weeks, at a dose of 15 ⁇ g per mouse, for a total of three immunizations.
  • the serum antibody titers of each group were detected by ELISA and FACS.
  • ELISA detected antibody binding at the antigen level
  • FACS detected antibody binding at the cellular level. Once the antibody titers met the requirements, fusion and screening were performed.
  • the first group underwent two fusions. Initial screening yielded 845 positive clones, and retesting revealed 739 positive clones. The first fusion screened 53 384-well plates, identifying 1210 clones that bound to hCDH17 via ELISA, followed by FACS screening of 257 clones that simultaneously bound to both HEK293-hCDH17 and HEK293-cyCDH17. Finally, 217 positive clones were obtained at the cellular level, and endocytosis and killing were detected using the supernatant from these 217 clones.
  • the second fusion screened 63 384-well plates, identifying 1749 clones that bound to hCDH17 via ELISA, followed by FACS screening of 588 clones that simultaneously bound to both HEK293-hCDH17 and HEK293-cyCDH17. Finally, 522 positive clones were obtained at the cellular level.
  • the second group consisted of 7 96-well plates selected from Clonepix. ELISA screening yielded 10 clones that bound to hCDH17, followed by FACS screening to find 7 clones that bound both HEK293-hCDH17 and HEK293-cyCDH17. Finally, 7 positive clones were obtained at the cellular level.
  • the third group screened 62 384-well plates.
  • ELISA identified 939 clones that bound to hCDH17, and FACS further screened 139 clones that bound both HEK293-hCDH17 and HEK293-cyCDH17. Finally, 33 positive clones were obtained at the cellular level.
  • Table 1 ELISA binding activity of supernatant culture medium from different hybridoma clones for human or cynomolgus monkey CDH17 protein.
  • RNA was extracted with Trizol.
  • first-strand cDNA was synthesized, and the variable region DNA sequence corresponding to the hybridoma cells was amplified using the first-strand cDNA as a subsequent template.
  • 1 ⁇ L of cDNA, 5 ⁇ L of 10 ⁇ PCR buffer, 1 ⁇ L each of forward and reverse primers, 1 ⁇ L of dNTP, 1 ⁇ L of 25 mmol MgCl2, 39 ⁇ L of H2O , and 1 ⁇ L of Taq enzyme were added.
  • the mixture was pre-denatured at 95°C for 10 minutes, followed by temperature cycling for PCR amplification.
  • the reaction conditions were: denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 15 seconds, for a total of 30 cycles, followed by incubation at 72°C for 10 minutes.
  • the variable region sequences of positive clones were amplified. After sequencing, the heavy and light chain variable region sequences of the candidate positive clones were obtained.
  • the heavy/light chain variable region sequences of the anti-CDH17 murine antibody are as follows (wherein, the CDR sequence is defined according to the Kabat definition rules):
  • amino acid sequence of the mAb1 heavy chain VH is shown in SEQ ID NO.3, the encoded nucleic acid is shown in SEQ ID NO.4, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.5, 6 and 7, respectively.
  • amino acid sequence of the mAb1 light chain VL is shown in SEQ ID NO.8, the encoded nucleic acid is shown in SEQ ID NO.9, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.10, 11 and 12, respectively.
  • the amino acid sequence of the mAb2 heavy chain VH is shown in SEQ ID NO.13
  • the encoded nucleic acid is shown in SEQ ID NO.14
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.15, 16 and 17, respectively.
  • the amino acid sequence of the mAb2 light chain VL is shown in SEQ ID NO.18, and its encoded nucleic acid is shown in SEQ ID NO.19. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.20, 21 and 22, respectively.
  • the amino acid sequence of the mAb3 heavy chain VH is shown in SEQ ID NO.23, and its encoded nucleic acid is shown in SEQ ID NO.24. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, 26 and 27, respectively.
  • the amino acid sequence of the mAb3 light chain VL is shown in SEQ ID NO.28, and its encoded nucleic acid is shown in SEQ ID NO.29. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.30, 31 and 32, respectively.
  • the amino acid sequence of the mAb4 heavy chain VH is shown in SEQ ID NO.33, and its encoded nucleic acid is shown in SEQ ID NO.34. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.35, 36 and 37, respectively.
  • the amino acid sequence of the mAb4 light chain VL is shown in SEQ ID NO.38, and its encoded nucleic acid is shown in SEQ ID NO.39. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.40, 41 and 42, respectively.
  • amino acid sequence of mAb5 heavy chain VH is shown in SEQ ID NO.43, and its encoded nucleic acid is shown in SEQ ID NO.44. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, 46 and 47, respectively.
  • the amino acid sequence of the mAb5 light chain VL is shown in SEQ ID NO.48, and its encoded nucleic acid is shown in SEQ ID NO.49. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.50, 51 and 52, respectively.
  • amino acid sequence of mAb6 heavy chain VH is shown in SEQ ID NO.53, and its encoded nucleic acid is shown in SEQ ID NO.54. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.55, 56 and 57, respectively.
  • the amino acid sequence of the mAb6 light chain VL is shown in SEQ ID NO.58, and its encoded nucleic acid is shown in SEQ ID NO.59. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.60, 61 and 62, respectively.
  • amino acid sequence of mAb7 heavy chain VH is shown in SEQ ID NO.63, and its encoded nucleic acid is shown in SEQ ID NO.64. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.65, 66 and 67, respectively.
  • amino acid sequence of the mAb7 light chain VL is shown in SEQ ID NO.68, and its encoded nucleic acid is shown in SEQ ID NO.69. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.70, 71 and 72, respectively.
  • variable region sequence fragments of the heavy and light chains were amplified by PCR.
  • the variable region of the heavy chain was cloned into a vector containing the constant region of the human heavy chain to express the complete IgG1 heavy chain in mammalian cells.
  • the variable region of the light chain was cloned into a vector containing the constant region of the human light chain to express the complete kappa light chain in mammalian cells.
  • the cells were transfected into HEK293.6E mammalian cells. IgG was expressed and secreted into the culture medium. The supernatant was collected, filtered, and purified. IgG was purified by Protein A chromatography. The eluted protein was concentrated by ultrafiltration, and the concentration of IgG was determined by spectrophotometry. The purity of IgG was analyzed by size exclusion chromatography.
  • Biological membrane interference (BLI/gator prime) was used to immobilize candidate chimeric antibodies with Protein A probes. The probes were then transferred to serially diluted hCDH17 avi cHis antigen. The affinity between the antibodies and antigens was obtained by detecting signal changes on the probes and through kinetic fitting, as shown in Figure 4 and Table 4. The results indicate that all chimeric antibodies can bind to hCDH17.
  • the cell density was adjusted to 1 ⁇ 107 cells/ml with blocking buffer and incubated on ice for 30 minutes, during which time samples were prepared.
  • the antibody was diluted to 30 ⁇ g/ml with flow cytometry buffer and serially diluted 5-fold.
  • the cell density was then diluted to 5 ⁇ 105 cells/ml with flow cytometry buffer, and 100 ⁇ l was seeded into 96-well U-shaped plates. After centrifugation and discarding the supernatant, 100 ⁇ l of the diluted antibody was added to each well, and the plates were incubated on ice for 60 minutes.
  • the cells were washed three times with 200 ⁇ l of flow cytometry buffer. 50 ⁇ l of AF647-Goat Anti-human IgG,Fc (1:300 dilution) labeled on the cell surface was added to each well. After incubation on ice for 25 minutes, 50 ⁇ l of PI staining solution (1:100 dilution) was added to each well, and incubation continued for 5 minutes. After one wash, the cells were resuspended in PBS. The mean fluorescence intensity of AF647 in single viable SNU-16 cells was detected by flow cytometry.
  • the cell density was adjusted to 1 ⁇ 105 to 5 ⁇ 105 cells per ml using RPMI-1640 complete medium containing 20% FBS (low IgG), and 100 ⁇ l was seeded into 96-well flat plates.
  • the purified chimeric antibody and DT3C were diluted to a concentration of 3.33 ⁇ g/ml using RPMI-1640 basal medium. An equal volume of antibody and DT3C were mixed and incubated at 37°C for 30 min. A four-fold serial dilution was then performed, with 100 ⁇ l added to each well (at this point, the actual working antibody concentration was 2.5 ⁇ g/ml, a four-fold dilution, an antibody-to-DT3C molar ratio of 1:2, and an FBS concentration of 10%). Cells were cultured for another 72 h. After staining with 10% CCK-8 reagent, absorbance was read at 450 nm, and relative activity was calculated.
  • the IC50 of the samples was obtained using a four-parameter regression analysis with antibody concentration on the x-axis and relative activity on the y-axis, as shown in Figure 6 and Table 6.
  • the unrelated anti-KLH antibody was used as a control.
  • the results showed that all antibody clones exhibited high endocytic activity, with IC50 values all below 1 nM.
  • the mouse antibody mAb3 sequence was compared with the human antibody germline sequence to identify human germline light chain genes IMGT_Hvk1-39 or IMGT_Hvk3-11, IGKJ2*01 and human germline heavy chain genes IMGT_hVH1-46 or IMGT_hVH3-23, IGHJ4*01 with good homology, maintaining the key amino acid sequence of the antibody upper hydrophobic core, and occurring frequently in humans. These genes were then used for mouse antibody CDR transplantation. Computer-aided homology modeling was used to analyze the CDR region and its surrounding framework amino acid sequences to avoid concentrated distribution of molecular surface charges or hydrophobic regions. A total of eight heavy chain variants hmAb3-H1 ⁇ H8 and four light chain variants hmAb3-K1 ⁇ K4 were designed.
  • the mouse antibody mAb5 sequence was aligned with the human antibody germline sequence to identify the human germline light chain genes IMGT_hVK1-39 and IGKJ2*01, and the human germline heavy chain genes IMGT_Hvh1-46 and IGHJ4*01, which were then used for mouse antibody CDR transplantation.
  • a total of 6 heavy chain variants mAb5-H1 ⁇ H6 and 5 light chain variants mAb5-K1 ⁇ K5 were designed.
  • the heavy chain variable region and light chain variable region sequences of the anti-CDH17 humanized antibody are shown in Tables 7 and 8, respectively.
  • the heavy/light chain variable region sequence of the anti-CDH17 humanized antibody is as follows:
  • amino acid sequence of mAb5 H1 is shown in SEQ ID NO.73
  • the encoded nucleic acid is shown in SEQ ID NO.74
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, 46 and 47, respectively.
  • amino acid sequence of mAb5 H2 is shown in SEQ ID NO.75, and the nucleic acid it encodes is shown in SEQ ID NO.76. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, 46 and 47, respectively.
  • amino acid sequence of mAb5 H3 is shown in SEQ ID NO.77, and the nucleic acid it encodes is shown in SEQ ID NO.78. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, 46 and 47, respectively.
  • amino acid sequence of mAb5 H4 is shown in SEQ ID NO.79, and the nucleic acid it encodes is shown in SEQ ID NO.80. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, SEQ ID NO.81 (YIYPGSGNLKYNEKFKG) and SEQ ID NO.47, respectively.
  • amino acid sequence of mAb5 H5 is shown in SEQ ID NO.82, and the nucleic acid it encodes is shown in SEQ ID NO.83. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, 46 and 47, respectively.
  • amino acid sequence of mAb5 H6 is shown in SEQ ID NO.84, and the encoded nucleic acid is shown in SEQ ID NO.85. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.45, 81 and 47, respectively.
  • amino acid sequence of mAb5 K1 is shown in SEQ ID NO.86, and the nucleic acid it encodes is shown in SEQ ID NO.87. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.50, 51 and 52, respectively.
  • amino acid sequence of mAb5 K2 is shown in SEQ ID NO.88, and the nucleic acid it encodes is shown in SEQ ID NO.89. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.90 (RASQDVGTAVA), SEQ ID NO.51 and SEQ ID NO.52, respectively.
  • amino acid sequence of mAb5 K3 is shown in SEQ ID NO.91, and the nucleic acid it encodes is shown in SEQ ID NO.92. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.90, SEQ ID NO.93 (WASTRHS) and SEQ ID NO.52, respectively.
  • amino acid sequence of mAb5 K4 is shown in SEQ ID NO.94, and the nucleic acid it encodes is shown in SEQ ID NO.95. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.90, SEQ ID NO.96 (YASTRHS) and SEQ ID NO.52, respectively.
  • amino acid sequence of mAb5 K5 is shown in SEQ ID NO.97, and the nucleic acid it encodes is shown in SEQ ID NO.98. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.90, 96 and 52, respectively.
  • amino acid sequence of mAb3 H1 is shown in SEQ ID NO.99, and its encoded nucleic acid is shown in SEQ ID NO.100. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, 26 and 27, respectively.
  • amino acid sequence of mAb3 H2 is shown in SEQ ID NO.101, and the encoded nucleic acid is shown in SEQ ID NO.102. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, SEQ ID NO.125(AIYPGDGVTRYSQKFKD) and SEQ ID NO.27, respectively.
  • amino acid sequence of mAb3 H3 is shown in SEQ ID NO.103, and the nucleic acid it encodes is shown in SEQ ID NO.104. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, 125 and 27, respectively.
  • amino acid sequence of mAb3 H4 is shown in SEQ ID NO.105
  • the encoded nucleic acid is shown in SEQ ID NO.106
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.107 (SYYMQ), SEQ ID NO.125 and SEQ ID NO.27, respectively.
  • amino acid sequence of mAb3 H5 is shown in SEQ ID NO.108
  • the encoded nucleic acid is shown in SEQ ID NO.109
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, 26 and 27, respectively.
  • amino acid sequence of mAb3 H6 is shown in SEQ ID NO.110, and its encoded nucleic acid is shown in SEQ ID NO.111. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, 26 and 27, respectively.
  • amino acid sequence of mAb3 H7 is shown in SEQ ID NO.112, and the encoded nucleic acid is shown in SEQ ID NO.113. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.25, 26 and 27, respectively.
  • amino acid sequence of mAb3 H8 is shown in SEQ ID NO.114
  • the encoded nucleic acid is shown in SEQ ID NO.115
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.107, 26 and 27, respectively.
  • amino acid sequence of mAb3 K1 is shown in SEQ ID NO.116
  • the encoded nucleic acid is shown in SEQ ID NO.117
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.30, 31 and 32, respectively.
  • amino acid sequence of mAb3 K2 is shown in SEQ ID NO.118
  • the encoded nucleic acid is shown in SEQ ID NO.119
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.120(RASESVDDYGFSFLN), SEQ ID NO.31 and SEQ ID NO.32, respectively.
  • amino acid sequence of mAb3 K3 is shown in SEQ ID NO.121, and its encoded nucleic acid is shown in SEQ ID NO.122. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.30, 31 and 32, respectively.
  • amino acid sequence of mAb3 K4 is shown in SEQ ID NO.123, and the nucleic acid it encodes is shown in SEQ ID NO.124. Its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.120, 31 and 32, respectively.
  • the heavy chain amino acid sequence of the anti-CDH17 human antibody MAB5 H3K1 is shown in SEQ ID NO.126, and its encoded nucleic acid is shown in SEQ ID NO.127; the light chain amino acid sequence is shown in SEQ ID NO.128, and its encoded nucleic acid is shown in SEQ ID NO.129.
  • the heavy chain amino acid sequence of the anti-CDH17 human antibody MAB5 H5K2 is shown in SEQ ID NO.130, and its encoded nucleic acid is shown in SEQ ID NO.131; the light chain amino acid sequence is shown in SEQ ID NO.132, and its encoded nucleic acid is shown in SEQ ID NO.133.
  • the heavy chain amino acid sequence of the anti-CDH17 human antibody MAB3 H2K2 is shown in SEQ ID NO.134, and its encoded nucleic acid is shown in SEQ ID NO.135; the light chain amino acid sequence is shown in SEQ ID NO.136, and its encoded nucleic acid is shown in SEQ ID NO.137.
  • the heavy chain amino acid sequence of the anti-CDH17 human antibody MAB3 H2K4 is shown in SEQ ID NO.134, and its encoded nucleic acid is shown in SEQ ID NO.135; the light chain amino acid sequence is shown in SEQ ID NO.138, and its encoded nucleic acid is shown in SEQ ID NO.139.
  • the heavy chain amino acid sequence of the anti-CDH17 human antibody MAB3 H3K2 is shown in SEQ ID NO.140, and its encoded nucleic acid is shown in SEQ ID NO.141; the light chain amino acid sequence is shown in SEQ ID NO.136, and its encoded nucleic acid is shown in SEQ ID NO.137.
  • the heavy chain amino acid sequence of the anti-CDH17 human antibody MAB3 H3K4 is shown in SEQ ID NO.140, and its encoded nucleic acid is shown in SEQ ID NO.141; the light chain amino acid sequence is shown in SEQ ID NO.138, and its encoded nucleic acid is shown in SEQ ID NO.139.
  • the CDH17-targeting antibody TORL-3-600 monoclonal antibody was used as a control antibody. It was prepared according to patent WO2023107558A1.
  • the heavy chain amino acid sequence of this antibody is shown in SEQ ID NO.142, and its encoded nucleic acid is shown in SEQ ID NO.143; the light chain amino acid sequence is shown in SEQ ID NO.144, and its encoded nucleic acid is shown in SEQ ID NO.145.
  • the cell density was adjusted to 1 ⁇ 107 cells/ml with blocking buffer and incubated on ice for 30 minutes, during which time samples were prepared.
  • the antibody was diluted to 30 ⁇ g/ml with flow cytometry buffer and serially diluted 5-fold.
  • the cell density was then diluted to 5 ⁇ 105 cells/ml with flow cytometry buffer, and 100 ⁇ l was seeded into 96-well U-shaped plates. After centrifugation and discarding the supernatant, 100 ⁇ l of the diluted antibody was added to each well, and the plates were incubated on ice for 60 minutes.
  • the binding activities of mAb5 H3K1 and mAb5 H5K2 did not change significantly compared with the parent cell line; the binding activities of four humanized antibodies, including mAb3 H2K4, were slightly higher than those of the parent cell line.
  • the cell density was adjusted to 1 x 105 cells per ml using RPMI-1640 complete medium containing 20% FBS (Low IgG), and 100 ⁇ l was seeded into 96-well flat plates.
  • the purified hybridoma antibody and DT3C were diluted to a concentration of 40 mg/ml using RPMI-1640 basal medium. An equal volume of antibody and DT3C were mixed and incubated at 37°C for 30 min. A four-fold serial dilution was then performed, with 100 cells added to each well (at this point, the actual working concentration of the antibody was 10 ⁇ g/ml, a four-fold dilution, an antibody-to-DT3C molar ratio of 1:2, and an FBS concentration of 10%). Cells were cultured for another 72 h. After staining with 10% CCK-8 reagent, the absorbance was read at 450 nm, and the relative activity was calculated.
  • the IC50 of the samples was determined using a four-parameter regression analysis with antibody concentration on the x-axis and relative activity on the y-axis. The results are shown in Figure 9A and Table 12, respectively.
  • the endocytic activity of the humanized antibodies did not change significantly compared to the parent antibody.
  • SNU-16 cells as the target cell line, the endocytic activity of mAb5 H3K1, the control antibody TORL-3-600, and Anti-KLH was detected.
  • SNU-16 cells were cultured in RPMI 1640 complete medium at 37°C and 5% CO2. Cells were collected by centrifugation at 300 ⁇ g for 10 minutes at 4°C. The cell density was adjusted to 1 ⁇ 105 cells/ml with RPMI 1640 reaction buffer, and 100 ⁇ l was seeded into each well of a 96-well cell culture plate and cultured overnight at 37°C and 5% CO2.
  • the prepared mAb5 H3K1, TORL-3-600, and Anti-KLH were mixed with serially diluted DT3C in equal volumes and incubated at 37°C for 30 minutes. 100 ⁇ l of the mixture was added to each well and cultured at 37°C and 5% CO2 for 3 days. 20 ⁇ l of CCK-8 chromogenic solution was added to each well, and the cells were cultured at 37°C and 5% CO2 for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the results were recorded. Wells containing 100 ⁇ l of reaction buffer served as control wells, and wells containing 200 ⁇ l of reaction buffer served as blank wells. The results were measured using the same method. The results are shown in Figure 9B and Table 13. The endocytic activity of mAb5-H3K1 was superior to that of TORL-3-600.
  • the anti-CDH17 antibody of the present invention is conjugated to the linker-drug conjugate mc-vc-PABC-MMAE (also known as VcMMAE) to prepare an ADC.
  • the specific preparation steps are as follows.
  • the antibody was replaced with an ultrafiltration tube with a molecular weight cutoff of 50 kDa and then transferred to 50 mM PBS/1.0 mM EDTA buffer (pH adjusted to 6.5 with sodium hydroxide solution). 2-4 equivalents of 10 mM TCEP aqueous solution were added, and the mixture was shaken at 25°C for 3 hours.
  • the linker-drug conjugate was dissolved in DMSO, and 8-20 equivalents of the linker-drug conjugate were added dropwise to the reduced antibody solution. The mixture was vortexed to ensure homogeneity and shaken at 25°C for 2 hours.
  • exemplary product ADC-reference 2 in PBS buffer was prepared from the conjugated mixtures FADC-reference 2A, FADC-reference 2B, and FADC-reference 2C.
  • an exemplary product ADC-20 of the conjugated mixture FADC-20 was prepared in PBS buffer.
  • the specific preparation steps are as follows.
  • LD-13 g 500 mg, 1.48 mmol was dissolved in 5 mL of N,N-dimethylformamide, followed by the sequential addition of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (564 mg, 1.48 mmol), N,N-diisopropylethylamine (574 mg, 4.44 mmol), and 3,6,9-trioxa-1-aminodecane (242 mg, 1.48 mmol). The mixture was stirred at 25 °C for 2 hours. 15 mL of water was added, and the mixture was extracted with ethyl acetate (30 mL ⁇ 3).
  • LD-112a 600 mg, 1.24 mmol was dissolved in 6 mL of methanol. Under nitrogen protection, palladium on carbon (60 mg, 10%, moistened with approximately 55% water) was added. The mixture was purged with hydrogen three times and stirred at 25°C for 1 hour. The reaction mixture was then stirred at 25°C for 1 hour under hydrogen (15 Psi) atmosphere. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated by vacuum distillation to obtain the title product LD-112b (350 mg, yield: 81%) as a colorless oil.
  • LD-112b (350 mg, 1.01 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and then 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (384 mg, 1.01 mmol) and N,N-diisopropylethylamine (390 mg, 3.02 mmol) were added sequentially. Then LD-13j (271 mg, 1.01 mmol, prepared by the method disclosed in Example 2 on page 30 of patent application "WO2021228141 A1" was added, and the mixture was stirred at 25 °C for 2 hours.
  • LD-112c (50.0 mg, 0.08 mmol) was dissolved in 1 mL of dichloromethane, and then 0.5 mL of trifluoroacetic acid was slowly added dropwise. The mixture was stirred at 25 °C for 0.5 hours. The reaction solution was concentrated by vacuum distillation to obtain the crude title product LD-112d (45 mg) as a colorless oil. The product was used directly in the next reaction without purification.
  • LD-99a (3.00 g, 7.87 mmol, prepared by the method disclosed in Example 1.15 on page 226 of patent application "WO2022135332 A1" was dissolved in 50 mL of dichloromethane, and pyridine 4-methylbenzenesulfonic acid (1.98 g, 7.87 mmol) and benzyl (R)-2-hydroxypropionate (2.84 g, 15.7 mmol) were added sequentially. The mixture was stirred at 50 °C for 10 hours. 40 mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane (50 mL ⁇ 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by vacuum distillation. The residue was purified by silica gel column chromatography using system B, yielding the title product LD-99b (1.55 g, yield: 40%) as a yellow gel.
  • LD-99b (2.63 g, 5.38 mmol) was dissolved in 30 mL of dichloromethane, and 1,8-diazabicyclo[5.4.0]undec-7-ene (491 mg, 3.23 mmol) was added. The mixture was stirred at 25 °C for 1 hour.
  • LD-99c (1.60 g, 1.97 mmol) was dissolved in 15 mL of dichloromethane, and 1,8-diazabicyclo[5.4.0]undec-7-ene (179 mg, 1.18 mmol) was added. The mixture was stirred at 25 °C for 1 hour.
  • LD-99d 500 mg, 602 ⁇ mol
  • palladium on carbon 448 mg, 10%, moistened with approximately 55% water
  • the reaction solution was stirred at 25°C for 2 hours under hydrogen (15 Psi).
  • the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated by vacuum distillation to give the crude title product LD-99e (330 mg, yield: 76%) as a white solid.
  • the product was used directly in the next reaction without purification.
  • LD-99e (30.2 mg, 41.7 ⁇ mol) and 1i-1-1 (15.0 mg, 32.1 ⁇ mol) were dissolved in 2 mL of N,N-dimethylformamide. Then, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (18.3 mg, 48.1 ⁇ mol) and N,N-diisopropylethylamine (12.4 mg, 96.2 ⁇ mol) were added sequentially, and the mixture was stirred at 25 °C for 1 hour. 1 mL of water was added, and the mixture was extracted with dichloromethane (3 mL ⁇ 2).
  • LD-99f (44.0 mg, 39.9 ⁇ mol) was dissolved in 1 mL of acetonitrile, and diethylamine (142 mg, 1.94 mmol) was added. The mixture was stirred at 25 °C for 0.5 hours. The reaction solution was concentrated by vacuum distillation to obtain crude product LD-99 g (30.0 mg) as a yellow solid. The product was used directly in the next reaction without purification.
  • LD-112d (10.2 mg, 18.7 ⁇ mol) and LD-99g (15.0 mg, 17.0 ⁇ mol) were dissolved in 1.0 mL of N,N-dimethylformamide, and then 1-hydroxybenzotriazole (3.5 mg, 25.5 ⁇ mol), N,N-diisopropylethylamine (6.6 mg, 51 ⁇ mol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.9 mg, 25.5 ⁇ mol) were added sequentially. The mixture was stirred at 25 °C for 2 hours.
  • the antibody was replaced with an ultrafiltration tube with a molecular weight cutoff of 50 kDa and placed in 50 mM PBS/1.0 mM EDTA buffer (pH adjusted to 7.4 with sodium hydroxide solution). 5–10 equivalents of 10 mM TCEP aqueous solution were added, and the mixture was shaken at 25°C for 3 hours.
  • the linker-drug conjugate LD-114 was dissolved in DMSO. 10–20 equivalents of the linker-drug conjugate were then added to the reduced antibody solution, vortexed to mix thoroughly, and shaken at 25°C for 2 hours. Once the reaction was complete, 40 equivalents of 100 mM NAC aqueous solution were added, and the reaction was stopped by shaking at 25°C for 20 minutes.
  • the cell density was adjusted to 5 ⁇ 104 cells per ml using RPMI-1640 complete medium containing 20% FBS, and 100 ⁇ l was seeded into 96-well plates.
  • the antibody conjugated with MMAE was diluted to a concentration of 2 ⁇ g/ml using RPMI-1640 basal medium, and serially diluted 5-fold. 100 ⁇ l was added to each well of the cells (at this point, the actual working concentration of the ADC was 1 ⁇ g/ml, 5-fold dilution, and the FBS concentration was 10%).
  • the cells were cultured for 3 days, stained with 10% CCK-8 reagent, and the absorbance was read at 450 nm to calculate the relative viability.
  • ADC concentration as the x-axis and relative viability as the y-axis
  • the IC50 of the samples was calculated using a four-parameter regression method. The results are shown in Figure 10 and Table 12, respectively.
  • ADC reference 2 as the control
  • ADC-7, ADC-8, ADC-9, ADC-10, ADC-11, and ADC-12 can all effectively kill SNU-16 cells, among which ADC-8 has the best killing effect.
  • the cell density was adjusted to 1 ⁇ 105 cells per ml using RPMI-1640 complete medium containing 20% FBS, and 100 ⁇ l was seeded into 96-well plates.
  • the anti-CDH17 humanized antibody ADC was diluted to a concentration of 2 ⁇ g/ml using RPMI-1640 basal medium, and serially diluted 4-fold. 100 ⁇ l of the ADC was added to each well (at this point, the actual working concentration of ADC was 1 ⁇ g/ml, 4-fold dilution, and the FBS concentration was 10%).
  • the cells were cultured for 3 days, stained with 10% CCK-8 reagent, and the absorbance was read at 450 nm to calculate the relative viability.
  • the cell density was adjusted to 3 ⁇ 104 cells per mL with RPMI-1640 complete medium containing 20% FBS, and 100 ⁇ L was seeded into 96-well plates.
  • ADC-20 and ADC-25 were diluted to a concentration of 29 ⁇ g/mL with RPMI-1640 basal medium, and serially diluted 4-fold. 100 ⁇ L was added to each well of the cells (at this point, the actual working concentration of ADC was 14.5 ⁇ g/mL, 4-fold dilution, and the FBS concentration was 10%). After culturing for 7 days, the cells were stained with 10% CCK-8 reagent, and the absorbance was read at 450 nm.
  • the relative viability was calculated.
  • the IC50 of the samples was obtained by four-parameter regression analysis, with ADC concentration as the x-axis and relative viability as the y-axis.
  • the IC50 of ADC-20 was 0.26 nM
  • the IC50 of ADC-25 was 0.31 nM.
  • ADC-20 can effectively kill SNU-16 tumor cells, and the killing is more thorough, with better results than ADC-25.
  • the cell density was adjusted to 1 ⁇ 105 cells per mL with RPMI-1640 complete medium containing 20% FBS, and 100 ⁇ L was seeded into 96-well plates.
  • ADC-20 and ADC-25 were diluted to a concentration of 20 ⁇ g/mL with RPMI-1640 basal medium, performing a 4-fold serial dilution. 100 ⁇ L was added to each well (at this point, the actual working concentration of ADC was 10 ⁇ g/mL, 4-fold dilution, and the FBS concentration was 10%). After 3 days of culture, the cells were stained with 10% CCK-8 reagent, and the absorbance at 450 nm was read to calculate the relative viability.
  • the IC50 of the samples was obtained using a four-parameter regression analysis with ADC concentration on the x-axis and relative viability on the y-axis, as shown in Table 15.
  • the IC50 of ADC-20 was 0.26 nM
  • the IC50 of ADC-25 was 0.52 nM.
  • ADC-20 can effectively kill ASPC-1 tumor cells, and the killing is more thorough, with better results than ADC-25.
  • Cell suspensions were prepared using 1640 complete medium containing 10% FBS. Positive SNU-16 cells and negative KATOIII cells (1 ⁇ 105 and 5 ⁇ 104 respectively) were added to the same well of a 6-well plate at a volume of 2 ml and cultured overnight. The next day, ADC samples were prepared using complete medium at a concentration of 10 ⁇ g/ml, diluted 5-fold (3 concentrations total). 1 ml of the prepared ADC sample was added to each well, and an untreated control was added to the medium. Culture was continued for 3 days to induce cell death. After the corresponding time points, all cells in the wells were collected, and viable cell counts were performed using a cell counter. Flow cytometry analysis was then performed to determine the proportions of positive and negative cells. The actual number of each cell type in the wells was calculated based on the viable cell count results. The results are shown in Figure 12. Compared to the control, ADC-18 exhibited a bystander effect, effectively killing CDH17-negative KATOIII cells.
  • Example 8 In vivo efficacy of anti-CDH17 humanized antibody ADC
  • the treatment groups included ADC-reference 2 (6 mg/kg) and ADC-18 (6 mg/kg), administered once a week for a total of three times. Administration was via tail vein injection.
  • TGI tumor growth inhibition rate
  • CDH17-overexpressing SNU-16 cells were passaged in RPMI-1640 complete medium containing 10% FBS until the total cell count reached 4.5 ⁇ 108 cells.
  • Tumor cells in the logarithmic growth phase (growth density not exceeding 70%) were collected, centrifuged at 300g for 7 min, washed three times with PBS, and counted. The cell density was adjusted to 4 ⁇ 107 cells/ml, and the cells were resuspended in an appropriate volume of PBS.
  • 100 ⁇ L of the cell suspension was injected subcutaneously into each mouse for tumor inoculation, and the day of inoculation was recorded as D0. Tumor size was measured three times a week to create tumor growth curves; mouse body weight changes were also monitored. On day 10, when the tumor grew to 100-200 mm3, drug administration was initiated in groups.
  • the treatment groups included ADC-reference 2 and ADC-18 (1, 0.3, and 0.1 mg/kg), administered as a single dose via tail vein injection.

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Abstract

L'invention concerne un anticorps dirigé contre CDH17 et son utilisation, en particulier un anticorps qui se lie à CDH17 ou un fragment de liaison à l'antigène de celui-ci, un acide nucléique comprenant une séquence nucléotidique codant pour l'anticorps, un vecteur comprenant l'acide nucléique, et une cellule hôte comprenant l'acide nucléique ou le vecteur. L'invention concerne également une composition pharmaceutique comprenant l'anticorps, un conjugué, un récepteur antigénique chimérique et une méthode de traitement utilisant l'anticorps.
PCT/CN2025/096099 2024-05-21 2025-05-20 Anticorps dirigé contre cdh17 et son utilisation Pending WO2025242100A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103534268A (zh) * 2010-10-20 2014-01-22 牛津生物疗法有限公司 抗体
CN113817059A (zh) * 2016-01-09 2021-12-21 嘉立医疗科技(广州)有限公司 用于癌症治疗的钙粘蛋白-17特异性抗体和细胞毒性细胞
CN113999308A (zh) * 2021-12-30 2022-02-01 深圳市人民医院 靶向钙粘蛋白17的纳米抗体及其应用
WO2023015169A1 (fr) * 2021-08-02 2023-02-09 Tavotek Biotech (Suzhou) Ltd Anticorps monoclonaux et bispécifiques anti-cdh17 et leurs utilisations
WO2023107558A1 (fr) * 2021-12-07 2023-06-15 The Regents Of The University Of California Anticorps cdh17 et méthodes de traitement du cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103534268A (zh) * 2010-10-20 2014-01-22 牛津生物疗法有限公司 抗体
CN113817059A (zh) * 2016-01-09 2021-12-21 嘉立医疗科技(广州)有限公司 用于癌症治疗的钙粘蛋白-17特异性抗体和细胞毒性细胞
WO2023015169A1 (fr) * 2021-08-02 2023-02-09 Tavotek Biotech (Suzhou) Ltd Anticorps monoclonaux et bispécifiques anti-cdh17 et leurs utilisations
WO2023107558A1 (fr) * 2021-12-07 2023-06-15 The Regents Of The University Of California Anticorps cdh17 et méthodes de traitement du cancer
CN113999308A (zh) * 2021-12-30 2022-02-01 深圳市人民医院 靶向钙粘蛋白17的纳米抗体及其应用

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