Anti-CDH17 antibody and conjugate comprising same

Abstract

Provided are an antibody specifically binding to CDH17 or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or the antigen-binding fragment thereof, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector. Also provided are a conjugate, CAR, and pharmaceutical composition comprising the antibody, and a treatment, diagnosis and detection method using the antibody.

Description

Anti-CDH17 antibodies and their conjugates

[0001] This application claims priority to Chinese Patent Application No. 2024119579791, filed on December 27, 2024, and Chinese Patent Application No. 2025104873503, filed on April 17, 2025, the entire contents of each of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the fields of biomedicine or biopharmaceutical technology, and more specifically to anti-CDH17 antibodies and their applications. Background Technology

[0003] Cadherin-17 (CDH17), also known as liver-intestine cadherin (LI-cadherin), is a calcium-dependent transmembrane glycoprotein. Belonging to the 7D cadherin family, CDH17 differs from most of its family members in that its structure includes seven extracellular cadherin domains, a transmembrane region, and approximately 20 intracellular amino acid sequences. The physiological function of CDH17 is to mediate peptide uptake as a proton-dependent peptide transporter. Furthermore, CDH17 maintains epithelial cell integrity through interactions with integrins at extracellular tight junctions. Studies have reported that aberrant expression of CDH17 is closely related to tumor metastasis and growth. Research has found that the main mechanism by which CDH17 regulates tumor growth and metastasis is that the RGD domain of CDH17 binds to integrin α2β1, inducing the activation of β1 integrin, thereby increasing tumor cell proliferation and adhesion. Furthermore, CDH17 can regulate tumor metastasis and invasion through the Wnt / β-catenin and NFκB signaling pathways. A growing body of research reports on CDH17 expression in tumor tissues, and this specific expression in tumors makes it a promising therapeutic target. Summary of the Invention

[0004] This invention aims to develop antibodies that specifically bind to CDH17 for the treatment of various cancer diseases. The inventors also humanized the obtained antibody clones and obtained humanized antibody sequences, which exhibit high affinity.

[0005] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CDH17.

[0006] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0007] (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:106, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:107; or

[0008] (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:153, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:154; or

[0009] (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:155, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:154; or

[0010] (4) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:155, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:156.

[0011] In some embodiments, the VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:9, 11 and 13, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:62, 64 and 66, respectively.

[0012] In some embodiments, (1) the VH comprises 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%, or 100% sequence identity with SEQ ID NO:106, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:107; or

[0013] (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:153, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:154; or

[0014] (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:155, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:154; or

[0015] (4) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:155, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:156.

[0016] In a preferred embodiment, (1) the VH comprises the amino acid sequence shown in SEQ ID NO:106, and the VL comprises the amino acid sequence shown in SEQ ID NO:107; or

[0017] (2) The VH contains the amino acid sequence shown in SEQ ID NO:153, and the VL contains the amino acid sequence shown in SEQ ID NO:154; or

[0018] (3) The VH contains the amino acid sequence shown in SEQ ID NO:155, and the VL contains the amino acid sequence shown in SEQ ID NO:154; or

[0019] (4) The VH contains the amino acid sequence shown in SEQ ID NO:155, and the VL contains the amino acid sequence shown in SEQ ID NO:156.

[0020] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0021] (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:104, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:105; or

[0022] (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:148, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149; or

[0023] (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:150, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149; or

[0024] (4) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:151, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149; or

[0025] (5) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:152, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149.

[0026] In some embodiments, (1) the VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:2, 4 and 6, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:55, 57 and 59, respectively; or

[0027] (2) The VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:2, 4 and 166, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:55, 57 and 59, respectively.

[0028] In some embodiments, (1) the VH comprises 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%, or 100% sequence identity with SEQ ID NO:104, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:105; or

[0029] (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:148, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149; or

[0030] (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:150, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149; or

[0031] (4) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:151, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149; or

[0032] (5) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:152, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:149.

[0033] In a preferred embodiment, (1) the VH comprises the amino acid sequence shown in SEQ ID NO:104, and the VL comprises the amino acid sequence shown in SEQ ID NO:105; or

[0034] (2) The VH contains the amino acid sequence shown in SEQ ID NO:148, and the VL contains the amino acid sequence shown in SEQ ID NO:149; or

[0035] (3) The VH contains the amino acid sequence shown in SEQ ID NO:150, and the VL contains the amino acid sequence shown in SEQ ID NO:149; or

[0036] (4) The VH contains the amino acid sequence shown in SEQ ID NO:151, and the VL contains the amino acid sequence shown in SEQ ID NO:149; or

[0037] (5) The VH contains an amino acid sequence as shown in SEQ ID NO:152, and the VL contains an amino acid sequence as shown in SEQ ID NO:149.

[0038] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0039] (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:114, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:115; or

[0040] (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:157, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:158; or

[0041] (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:159, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:160; or

[0042] (4) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:159, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:161.

[0043] In some embodiments, the VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:27, 29 and 31, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:80, 64 and 83, respectively.

[0044] In some embodiments, (1) the VH comprises 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%, or 100% sequence identity with SEQ ID NO:114, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:115; or

[0045] (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:157, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:158; or

[0046] (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:159, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:160; or

[0047] (4) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:159, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:161.

[0048] In a preferred embodiment, (1) the VH comprises the amino acid sequence shown in SEQ ID NO:114, and the VL comprises the amino acid sequence shown in SEQ ID NO:115; or

[0049] (2) The VH contains the amino acid sequence shown in SEQ ID NO:157, and the VL contains the amino acid sequence shown in SEQ ID NO:158; or

[0050] (3) The VH contains the amino acid sequence shown in SEQ ID NO:159, and the VL contains the amino acid sequence shown in SEQ ID NO:160; or

[0051] (4) The VH contains an amino acid sequence as shown in SEQ ID NO:159, and the VL contains an amino acid sequence as shown in SEQ ID NO:161.

[0052] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0053] (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:118, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:119; or

[0054] (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:162, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163; or

[0055] (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:164, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163; or

[0056] (4) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:165, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163.

[0057] In some embodiments, (1) the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:40, 42, and 44, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:62, 94, and 96, respectively; or

[0058] (2) The VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:40, 167 and 44 respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:62, 94 and 96 respectively.

[0059] In some embodiments, (1) the VH comprises 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%, or 100% sequence identity with SEQ ID NO:118, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:119; or

[0060] (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:162, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163; or

[0061] (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:164, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163; or

[0062] (4) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:165, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:163.

[0063] In a preferred embodiment, (1) the VH comprises the amino acid sequence shown in SEQ ID NO:118, and the VL comprises the amino acid sequence shown in SEQ ID NO:119; or

[0064] (2) The VH contains the amino acid sequence shown in SEQ ID NO:162, and the VL contains the amino acid sequence shown in SEQ ID NO:163; or

[0065] (3) The VH contains the amino acid sequence shown in SEQ ID NO:164, and the VL contains the amino acid sequence shown in SEQ ID NO:163; or

[0066] (4) The VH contains an amino acid sequence as shown in SEQ ID NO:165, and the VL contains an amino acid sequence as shown in SEQ ID NO:163.

[0067] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0068] (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:108, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:109; or

[0069] (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:110, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:111; or

[0070] (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:112, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:113; or

[0071] (4) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:116, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:117; or

[0072] (5) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:120, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:121; or

[0073] (6) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:122, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:123; or

[0074] (7) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:124, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:125.

[0075] In some implementations, the VH and VL comprise a CDR sequence as shown below:

[0076] (1) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 16, and 6, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:69, 57, and 59, respectively; or

[0077] (2) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:20, 22, and 24, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:74, 76, and 78, respectively; or

[0078] (3) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:33, 35, and 37, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:86, 88, and 90, respectively; or

[0079] (4) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO: 9, 11, and 13, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO: 62, 64, and 97, respectively; or

[0080] (5) The VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:2, 16, and 47, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:55, 57, and 59, respectively; or

[0081] (6) The VH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:49, 51 and 53, respectively, and the VL comprises light chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:101, 57 and 59, respectively.

[0082] In some implementation schemes,

[0083] (1) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:108, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:109; or

[0084] (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:110, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:111; or

[0085] (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:112, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:113; or

[0086] (4) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:116, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:117; or

[0087] (5) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:120, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:121; or

[0088] (6) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:122, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:123; or

[0089] (7) The VH contains 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%, or 100% sequence identity with SEQ ID NO:124, and the VL contains 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%, or 100% sequence identity with SEQ ID NO:125.

[0090] In a preferred embodiment,

[0091] (1) The VH contains the amino acid sequence shown in SEQ ID NO:108, and the VL contains the amino acid sequence shown in SEQ ID NO:109; or

[0092] (2) The VH contains the amino acid sequence shown in SEQ ID NO:110, and the VL contains the amino acid sequence shown in SEQ ID NO:111; or

[0093] (3) The VH contains the amino acid sequence shown in SEQ ID NO:112, and the VL contains the amino acid sequence shown in SEQ ID NO:113; or

[0094] (4) The VH contains the amino acid sequence shown in SEQ ID NO:116, and the VL contains the amino acid sequence shown in SEQ ID NO:117; or

[0095] (5) The VH contains the amino acid sequence shown in SEQ ID NO:120, and the VL contains the amino acid sequence shown in SEQ ID NO:121; or

[0096] (6) The VH contains the amino acid sequence shown in SEQ ID NO:122, and the VL contains the amino acid sequence shown in SEQ ID NO:123; or

[0097] (7) The VH contains the amino acid sequence shown in SEQ ID NO:124, and the VL contains the amino acid sequence shown in SEQ ID NO:125.

[0098] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to CDH17 disclosed in this invention, the antibodies are selected from mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0099] In some embodiments, the antibody is an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is a subtype selected from IgG1, IgG2, IgG3, and IgG4.

[0100] In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to CDH17 disclosed in this invention, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv and ds-scFv.

[0101] In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to CDH17 disclosed in this invention, the antibody is a monovalent, bivalent, or multivalent antibody.

[0102] In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to CDH17 disclosed in this invention, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0103] In a second aspect, the present invention provides a nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0104] In some implementations, the nucleic acid comprises:

[0105] (1) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:126, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:127;

[0106] (2) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:128, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:129;

[0107] (3) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:130, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:131;

[0108] (4) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:132, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:133;

[0109] (5) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:134, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:135;

[0110] (6) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:136, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:137;

[0111] (7) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:138, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:139;

[0112] (8) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:140, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:141;

[0113] (9) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:142, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:143;

[0114] (10) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:144, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:145; or

[0115] (11) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:146, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:147.

[0116] In a third aspect, the present invention provides a carrier comprising nucleic acids as described in the second aspect of the present invention.

[0117] In a fourth aspect, the present invention provides a host cell comprising a nucleic acid as described in the second aspect of the present invention or a vector as described in the third aspect of the present invention.

[0118] In a fifth aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising:

[0119] a) Culture host cells as described in the fourth aspect of the invention under conditions suitable for the expression of the antibody or its antigen-binding fragment; and

[0120] b) Isolate the antibody or its antigen-binding fragment from the culture and / or culture supernatant of the host cells.

[0121] In a sixth aspect, the present invention provides a conjugate comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the invention and a chemical portion conjugated to said antibody or antigen-binding fragment thereof via a linker, preferably said chemical portion being selected from therapeutic agents, detectable portions and immunostimulatory molecules.

[0122] In some embodiments, the therapeutic agent is selected from microtubule inhibitors, minor groove binders, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors. In some embodiments, the therapeutic agent is selected from MMAE, MMAF, eczemab (DX8951), duocarmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin, and PBDs (benzodiazepines). In a preferred embodiment, the therapeutic agent is MMAE or MMAF.

[0123] In some embodiments, the connector comprises a cleavable connector or a non-cleavable connector. In some embodiments, the connector is selected from acid-unstable connectors, light-unstable connectors, protease-sensitive connectors, hydrazone connectors, esterase-cleavable connectors, dimethyl connectors, disulfide-containing connectors, hydrophilic connectors, charged connectors, and acid-based connectors. In some embodiments, the connector is selected from mc(6-maleimide-hexanoyl), valine-citrulline (val-cit), PABC (p-amino-benzyloxycarbonyl), DMEA (dimethylethylamine), val-cit-PABC, mc-val-cit-PABC, mc-val-cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG, AcBut (4-(4-acetylphenoxy)-butyric acid), and AcBut-dimethylhydrazine, preferably val-cit or mc-val-cit-PABC.

[0124] In a seventh aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0125] In an eighth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as in the first aspect of the present invention, a nucleic acid as in the second aspect of the present invention, a vector as in the third aspect of the present invention, a host cell as in the fourth aspect of the present invention, a conjugate as in the sixth aspect of the present invention, or a CAR as in the seventh aspect of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0126] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, optionally selected from antibodies, chemotherapeutic agents, oligonucleotides (such as siRNA), peptides, small molecule drugs, immune checkpoint inhibitors, or cytokines.

[0127] In a ninth aspect, the present invention provides a method for treating cancers associated with CDH17 expression, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a nucleic acid as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, a conjugate as described in the sixth aspect of the present invention, a CAR as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention.

[0128] In some embodiments, the cancer associated with CDH17 expression is a solid tumor. In a preferred embodiment, the solid tumor is gastric cancer or colorectal cancer.

[0129] In some embodiments, the method further includes administering a second therapeutic agent to the subject, optionally selected from antibodies, chemotherapeutic agents, oligonucleotides (such as siRNA), peptides, small molecule drugs, immune checkpoint inhibitors, or cytokines.

[0130] In a tenth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a nucleic acid as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, a conjugate as described in the sixth aspect of the present invention, a CAR as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a medicament for treating cancers associated with CDH17 expression.

[0131] In some embodiments, the cancer associated with CDH17 expression is a solid tumor. In a preferred embodiment, the solid tumor is gastric cancer or colorectal cancer.

[0132] In some embodiments, the drug is used in combination with a second therapeutic agent, optionally selected from antibodies, chemotherapeutic agents, oligonucleotides (such as siRNA), peptides, small molecule drugs, immune checkpoint inhibitors, or cytokines.

[0133] In an eleventh aspect, the present invention provides an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a nucleic acid as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, a conjugate as described in the sixth aspect of the present invention, a CAR as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention for treating cancers associated with CDH17 expression.

[0134] In some embodiments, the cancer associated with CDH17 expression is a solid tumor. In a preferred embodiment, the solid tumor is gastric cancer or colorectal cancer.

[0135] In some embodiments, the antibody or its antigen-binding fragment, nucleic acid, vector, host cell, conjugate, CAR, or pharmaceutical composition is used in combination with a second therapeutic agent, optionally selected from antibodies, chemotherapeutic agents, oligonucleotides (such as siRNA), peptides, small molecule drugs, immune checkpoint inhibitors, or cytokines.

[0136] In a twelfth aspect, the present invention provides a method for detecting the presence or level of CDH17 in a sample, comprising:

[0137] (1) Contacting the sample with an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0138] (2) The presence or level of CDH17 in the sample is determined by detecting the binding of the antibody to the sample. Detailed Implementation

[0139] The above-described features and advantages of the present invention, as well as their additional features and advantages, will become more clearly understood below in conjunction with the detailed description of the following embodiments.

[0140] The embodiments described herein are illustrative and exemplary, and are intended for a general understanding of the invention. The embodiments should not be construed as limiting the scope of the invention. Identical or similar elements and elements having the same or similar functions are represented by the same reference numerals throughout the specification.

[0141] Terminology Definition

[0142] Unless otherwise stated or defined, all terms used have the common meaning in the art as is clear to a person skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology" (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York. York (2005), and the general background technology cited above.

[0143] In this document, CDH17, cadherin-17, and hepato-gut cadherin are used interchangeably and include any variant, isotype, and species homolog of human CDH17 expressed naturally in cells or on cells transfected with the CDH17 gene. The term “CDH17” as a target should be interpreted broadly in this document, intended to encompass various forms of the CDH17 molecule at different stages in mammals (e.g., humans), such as, but not limited to, molecules produced during the amplification, replication, transcription, splicing, translation, and modification of the CDH17 gene (e.g., precursor CDH17, mature CDH17, membrane-expressed CDH17, CDH17 splice variants, modified CDH17, or fragments thereof). The term also covers CDH17 prepared artificially or expressed in vitro.

[0144] As used herein, 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).

[0145] 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.

[0146] 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.

[0147] The amino acid arrangement of each VL and VH domain is consistent with any conventional definition of CDR. Common 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 combination of Chothia and Kabat CDRs, where CDR-H1 is a combination of Chothia and Kabat CDRs; the AbM definition used by the Oxford Molecular antibody modeling software; the CONTACT definition by Martin et al. (world wide web bioinfo.org.uk / abs); and the IMGT definition proposed by Lefranc based on a comparison of the V domains of immunoglobulins and T cell receptors (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0148] In the technical solution of this invention, the amino acid residues in the variable domain sequence can be determined using Kabat definitions, Chothia definitions, Combined definitions (which include both Kabat and Chothia definitions), AbM definitions, CONTACT definitions, IMGT definitions, etc., as detailed in the table below. In a preferred embodiment, the CDR of the antibody in this application is defined according to the IMGT rule.

[0149] Those skilled in the art will understand that, unless otherwise specified, the “CDR” and “complementarity-determining region” of an antibody or its region (e.g., a variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes described herein. While in the preferred embodiment, the amino acid sequence of the CDR is based on the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also be within the scope of protection of this invention.

[0150] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal 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 at 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.

[0151] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the CDH17 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0152] Examples of “antigen-binding fragments” of the term 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 of an antibody single arm; (vii) dAb fragments, consisting of a VH domain; (viii) a single complementarity-determining region (CDR); and (ix) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via synthetic linkers using recombination methods, enabling them to form a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibody. Additionally, the term also includes "straight-chain antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1), which, together with a complementary light-chain polypeptide and any modified form of the aforementioned fragment that retains antigen-binding activity, form the antigen-binding region.

[0153] These 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.

[0154] As used herein, the term "binding" or "specific 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, expressed as the equilibrium constant (KD) for antigen-antibody dissociation, is a measure of the strength of binding between an 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. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Cohesion is a measure of the strength of binding between an antibody and its associated antigen. Cohesion involves the affinity between the antigenic determinant and the antigen-binding site of the antibody, as well as the number of associated binding sites present on the antibody. Typically, antibodies will bind antigens with the following dissociation constant (KD): 10 -5 M to 10 -10 M or smaller, and preferably 10 -7 M to 10 -10 M or smaller, and more preferably 10 -8 M to 10 -10 M, and / or binds to the antigen with the following binding affinity: at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 such as at least 10 10 M -1 It is generally considered that anything greater than 10... -4 The KD value of M indicates 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 variants known in the art.

[0155] In this application, the term "KD" generally refers to the equilibrium dissociation constant, which is the ratio of the dissociation rate constant (kdis, also known as "off-rate" (koff) or "kd") to the binding rate constant (kon, also known as "on-rate" or "ka"). The binding rate constant (kon), dissociation rate constant (kdis), and equilibrium dissociation constant (KD) can be used to represent the binding affinity of an antigen-binding protein (e.g., an antibody) to an antigen. Methods for determining the binding and dissociation rate constants are well known in the art, including but not limited to biomembrane interference (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction may vary depending on the conditions measured (e.g., salt concentration, pH).

[0156] The term "epitope" 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 in the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally preserved upon exposure to denaturing solvents, while epitopes formed from ternary folding (also known as conformational epitopes) are generally lost during treatment with denaturing solvents. Epitopes typically consist of 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.

[0157] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence is % identical to SEQ ID NO: Y and is described as having X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y.

[0158] Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0159] As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies in the cluster are identical, except for a small number of possible natural mutations. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody formulations (which usually have different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to the invention can be prepared in hybridoma cells or by recombinant DNA methods.

[0160] In the context of this invention, the term "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. As used herein, 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.

[0161] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to elicit an adverse immune response in human individuals compared to parental (e.g., mouse-derived) antibodies.

[0162] In this application, the term "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 in 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 minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) 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. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0163] In this application, the term "fully human antibody" generally refers to an antibody expressed by transferring a human antibody-encoding gene into a genetically engineered animal lacking an antibody gene. All parts of the antibody (including the variable and constant regions) are encoded by human-derived genes. Fully human antibodies can significantly reduce the immune side effects caused by heterologous antibodies in humans. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0164] In this application, the term "subject" generally refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). The term "primate" generally refers to monkey and ape species, and includes monkey species such as those from the genera *Macaca* (e.g., cynomolgus monkeys and rhesus monkeys) and baboons (e.g., pig-tailed baboons), as well as marmosets (species from the genus *Callithrix*), squirrel monkeys (species from the genus *Saimiri*) and tamarins (species from the genus *Saguinus*), and ape species such as chimpanzees (*Pan troglodytes*), and also includes *Homo sapiens*.

[0165] In this application, the term "nucleic acid" generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue thereof, isolated from its natural environment or synthesized artificially.

[0166] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules to host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0167] In this application, the term "host cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the antibodies or antigen-binding fragments described in this application. The cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the antibodies or antigen-binding fragments described in this application. The cell can be obtained by in vitro transfection of cells using the vector described in this application. The cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell. In this application, the term "recombinant cell" generally refers to a cell in which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific type of cell but also the progeny of these cells.

[0168] In this application, the term "pharmaceutical composition" generally refers to a formulation which is present in a form that allows the biological activity of the active ingredient to be effective and does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered.

[0169] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations employed. Typically, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0170] As used herein, the terms “treatment,” “therapy,” “treatment,” etc., refer to the administration of a drug or the performance 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. As used herein, “treatment” can include the treatment of a disease or condition (e.g., cancer) 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. Treatment can refer to any indicator of success in the treatment or improvement or prevention of cancer, including any objective or subjective parameter such as a reduction in symptoms; remission; elimination or making the disease or its symptoms 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 or compositions or conjugates disclosed herein to prevent or delay, alleviate, or prevent or inhibit the development of symptoms associated with a disease or condition (e.g., cancer).

[0171] As used herein, the term “diagnosis” typically includes determining a subject’s susceptibility to a disease or condition, determining whether a subject is currently infected with a disease or condition, the prognosis of a subject infected with a disease or condition (e.g., identifying pre- or metastatic cancer status, cancer stage, or cancer’s responsiveness to treatment), and therapeutic dosages (e.g., monitoring a subject’s condition to provide information about the treatment’s effect or efficacy).

[0172] As used herein, the term "detection" includes the determination or confirmation of the presence or absence of a target CDH17 (the CDH17 gene product (peptide) encoding a nucleic acid sequence), its subunits, or a combination of targets of a binding reagent, or a determination used to inquire, probe, confirm, or determine one or more actual characteristics of cancer, metastasis, stage, or similar condition. The term includes diagnostic, predictive, and surveillance applications of CDH17 and other cancer biomarkers. The term includes quantitative, semi-quantitative, and qualitative detection methods. In embodiments involving the detection of the CDH17 protein (rather than the nucleic acid molecule encoding the CDH17 protein), the detection method is preferably based on an ELISA method. Preferably, the detection method provides an output (i.e., a readout or signal) of information regarding the presence, absence, or amount of CDH17 in a subject's sample. For example, the output may be qualitative (e.g., "positive" or "negative") or quantitative (e.g., concentration, e.g., nanograms per milliliter).

[0173] As used in this article, the term "effective dose" refers to a dose administered to a subject that is sufficient to treat the disease.

[0174] As used herein, the term "conjugate" generally refers to a substance formed by linking an antibody to another chemical moiety. The chemical moiety can be a cytotoxic drug, an immunostimulatory molecule, or a detectable marker. Drugs can be, for example, microtubule inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors.

[0175] As used herein, the term "chimeric antigen receptor" or "CAR" 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. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

[0176] As used herein, the singular forms “a,” “an,” and “the” include plural objects unless the context clearly indicates otherwise. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

[0177] Unless otherwise stated or defined, the terms “comprising,” “including,” “containing,” and “having” should be understood to mean including the said element or step or group of elements or steps, but not excluding any other element or step or group of elements or steps.

[0178] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0179] Anti-CDH17 antibody

[0180] A first aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CDH17, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0181] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:106, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:107.

[0182] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:153, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:154.

[0183] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:155, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:154.

[0184] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:155, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:156.

[0185] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:104, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:105.

[0186] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:148, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149.

[0187] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:150, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149.

[0188] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:151, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149.

[0189] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:152, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149.

[0190] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:114, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:115.

[0191] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:157, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:158.

[0192] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:159, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:160.

[0193] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:159, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:161.

[0194] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:118, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:119.

[0195] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:162, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163.

[0196] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:164, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163.

[0197] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:165, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163.

[0198] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:108, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:109.

[0199] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:110, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:111.

[0200] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:112, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:113.

[0201] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:116, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:117.

[0202] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:120, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:121.

[0203] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:122, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:123.

[0204] In some embodiments, the VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:124, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:125.

[0205] In some implementations, the CDR sequence is defined according to the Kabat definition rule. In some implementations, the CDR sequence is defined according to the Chothia definition rule. In some implementations, the CDR sequence is defined according to the Combined definition rule. In some implementations, the CDR sequence is defined according to the AbM definition rule. In some implementations, the CDR sequence is defined according to the CONTACT definition rule. In some implementations, the CDR sequence is defined according to the IMGT definition rule.

[0206] In a preferred embodiment, the CDR sequence is defined according to the IMGT definition rules.

[0207] In some embodiments, the VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:9, 11 and 13, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:62, 64 and 66, respectively.

[0208] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 4 and 6, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:55, 57 and 59, respectively.

[0209] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 4, and 166, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:55, 57, and 59, respectively.

[0210] In some embodiments, the VH comprises heavy chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:27, 29 and 31, respectively, and the VL comprises light chain CDR1-3 having amino acid sequences as shown in SEQ ID NO:80, 64 and 83, respectively.

[0211] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:40, 42 and 44, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:62, 94 and 96, respectively.

[0212] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:40, 167, and 44, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:62, 94, and 96, respectively.

[0213] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 16 and 6, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:69, 57 and 59, respectively.

[0214] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:20, 22 and 24, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:74, 76 and 78, respectively.

[0215] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:33, 35 and 37, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:86, 88 and 90, respectively.

[0216] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:9, 11 and 13, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:62, 64 and 97, respectively.

[0217] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 16 and 47, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:55, 57 and 59, respectively.

[0218] In some embodiments, the VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:49, 51 and 53, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:101, 57 and 59, respectively.

[0219] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:106, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:107.

[0220] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:153, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:154.

[0221] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:155, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:154.

[0222] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:155, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:156.

[0223] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:104, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:105.

[0224] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:148, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149.

[0225] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:150, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149.

[0226] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:151, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149.

[0227] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:152, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149.

[0228] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:114, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:115.

[0229] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:157, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:158.

[0230] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:159, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:160.

[0231] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:159, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:161.

[0232] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:118, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:119.

[0233] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:162, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163.

[0234] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:164, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163.

[0235] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:165, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163. In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:108, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:109.

[0236] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:110, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:111.

[0237] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:112, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:113.

[0238] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:116, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:117.

[0239] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:120, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:121.

[0240] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:122, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:123.

[0241] In some embodiments, the VH comprises 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%, or 100% sequence identity with SEQ ID NO:124, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:125.

[0242] In some embodiments, VH comprises a functional variant formed by inserting, deleting, and / or substituting one or more amino acids in the amino acid sequences shown in SEQ ID NO: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124, provided that the functional variant retains the ability to bind CDH17. In some embodiments, VL comprises a functional variant formed by inserting, deleting, and / or substituting one or more amino acids in the amino acid sequences shown in SEQ ID NO: 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125, provided that the functional variant retains the ability to bind CDH17.

[0243] 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.

[0244] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parent amino acid sequence, more preferably not more than 35%, more preferably 1% to 33%, and even more preferably 5% to 30%, more preferably 10% to 25%, and even more preferably 15% to 20%. For example, 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.

[0245] In some implementations, insertions, deletions, and / or substitutions can be performed in frame (FR) regions, such as FR1, FR2, FR3, and / or FR4.

[0246] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such 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.

[0247] 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.

[0248] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:106, and the VL comprises an amino acid sequence as shown in SEQ ID NO:107.

[0249] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:153, and the VL comprises an amino acid sequence as shown in SEQ ID NO:154.

[0250] In a preferred embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO:155, and the VL comprises the amino acid sequence shown in SEQ ID NO:154.

[0251] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:155, and the VL comprises an amino acid sequence as shown in SEQ ID NO:156.

[0252] In a preferred embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO:104, and the VL comprises the amino acid sequence shown in SEQ ID NO:105.

[0253] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:148, and the VL comprises an amino acid sequence as shown in SEQ ID NO:149.

[0254] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:150, and the VL comprises an amino acid sequence as shown in SEQ ID NO:149.

[0255] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:151, and the VL comprises an amino acid sequence as shown in SEQ ID NO:149.

[0256] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:152, and the VL comprises an amino acid sequence as shown in SEQ ID NO:149.

[0257] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:114, and the VL comprises an amino acid sequence as shown in SEQ ID NO:115.

[0258] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:157, and the VL comprises an amino acid sequence as shown in SEQ ID NO:158.

[0259] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:159, and the VL comprises an amino acid sequence as shown in SEQ ID NO:160.

[0260] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:159, and the VL comprises an amino acid sequence as shown in SEQ ID NO:161.

[0261] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:118, and the VL comprises an amino acid sequence as shown in SEQ ID NO:119.

[0262] In a preferred embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO:162, and the VL comprises the amino acid sequence shown in SEQ ID NO:163.

[0263] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:164, and the VL comprises an amino acid sequence as shown in SEQ ID NO:163.

[0264] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:165, and the VL comprises an amino acid sequence as shown in SEQ ID NO:163.

[0265] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:108, and the VL comprises an amino acid sequence as shown in SEQ ID NO:109.

[0266] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:110, and the VL comprises an amino acid sequence as shown in SEQ ID NO:111.

[0267] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:112, and the VL comprises an amino acid sequence as shown in SEQ ID NO:113.

[0268] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:116, and the VL comprises an amino acid sequence as shown in SEQ ID NO:117.

[0269] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:120, and the VL comprises an amino acid sequence as shown in SEQ ID NO:121.

[0270] In a preferred embodiment, the VH comprises an amino acid sequence as shown in SEQ ID NO:122, and the VL comprises an amino acid sequence as shown in SEQ ID NO:123.

[0271] In a preferred embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO:124, and the VL comprises the amino acid sequence shown in SEQ ID NO:125.

[0272] In some implementations, the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0273] 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. Based on the amino acid sequence of the light chain, the antibody light chain can be divided into lambda (λ) chains and kappa (κ) chains. The antibodies disclosed herein can be any of the above classes or subtypes.

[0274] In some embodiments, the antibody is an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is a subtype selected from IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

[0275] The antibodies disclosed herein can be complete antibodies or antigen-binding fragments thereof. An antigen-binding fragment can be any fragment of an antibody that retains its ability to specifically bind to CDH17. Examples of 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; individual complementarity-determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd fragments (VH-CH1-VH-CH1); and modified forms of any of the aforementioned fragments that retain antigen-binding activity.

[0276] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In yet another preferred embodiment, the antigen-binding fragment is scFv.

[0277] In some implementations, the antibody is a monovalent, bivalent, or multivalent antibody.

[0278] In some implementations, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0279] In some embodiments, the antibody is a bispecific antibody. A bispecific antibody comprises an antibody that binds to CDH17 as described herein, or an antigen-binding fragment thereof, and a second antigen-binding domain that binds to a second antigen. The second antigen may be selected from tumor-associated antigens, immune cell antigens, and immune checkpoint molecules.

[0280] Numerous tumor-associated antigens (TAAs) associated with specific cancers have been identified in the art. In some embodiments, 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. Other 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. 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.

[0281] Examples of 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.

[0282] In some embodiments, the second antigen is an immune cell antigen, such as a T-cell antigen. In some embodiments, the T-cell antigen is selected from T-cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 4-1BB (CD137), and NKG2D, or any combination thereof. In some embodiments, the T-cell antigen is CD3, and the second antigen binding region binds to any one of the γ, δ, ε, ζ, and η chains of CD3.

[0283] In some implementations, the second antigen is an immune checkpoint molecule. In some implementations, the immune checkpoint molecule may be selected from PD-1, PD-L1, CTLA-4, CD4, CD40, CD80, CD86, B7-H3, LAG3, TIM-3, IDO1, etc.

[0284] In some embodiments, the antibody of the present invention has a binding affinity of 10⁻⁶ to CDH17. 9 M to 10-10 M. In some embodiments, the KD value of the antibody of the present invention binding to CDH17 is 10- 9 M to 10- 10 M. In some embodiments, the antibody of the present invention binds to integrin α2β1. In some embodiments, the antibody of the present invention induces activation of β1 integrin. In some embodiments, the antibody of the present invention regulates the Wnt / β-catenin and NFκB signaling pathways.

[0285] In some embodiments, the antibody of the present invention may comprise 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. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG Fc. In one embodiment, the Fc region is human IgG1 Fc. In one embodiment, the Fc region is mouse IgG Fc. In one embodiment, the Fc region is mouse IgG1 Fc.

[0286] In one implementation, the Fc region has reduced effector function, such as reduced binding to ADCC, ADCP, CDC and / or Clq, FcγRI, FcγRII, or FcγRIIIA. For example, 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). For example, compared to the wild-type sequence, the Fc region may contain an amino acid sequence with one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G.

[0287] In a further embodiment, 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. In another implementation, the Fc region is engineered to enhance complement activation, as described by Natsume et al. (2009) Cancer Sci. 100:2411.

[0288] Nucleic acid

[0289] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof disclosed herein.

[0290] The term "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.

[0291] For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.

[0292] For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0293] In some embodiments, the nucleic acid molecules of the present invention encode the heavy chain variable region as shown in SEQ ID NO: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124. In some embodiments, the nucleic acid molecules of the present invention encode at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124. In some embodiments, the nucleic acid molecules of the present invention encode the light chain variable region as shown in SEQ ID NO: 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125. In some embodiments, the nucleic acid molecules of the present invention encode at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125.

[0294] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide sequence as shown in any one of SEQ ID NO:126-147, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:126-147.

[0295] In some embodiments, the nucleic acid molecules encoding the heavy strand variable region sequence of the present invention are as shown in SEQ ID NO: 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, or 146. In some embodiments, the nucleic acid molecules encoding the heavy strand variable region sequence of the present invention are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid molecules shown in SEQ ID NO: 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, or 146.

[0296] In some embodiments, the nucleic acid molecules encoding the light chain variable region sequence of the present invention are as shown in SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, or 147. In some embodiments, the nucleic acid molecules encoding the light chain variable region sequence of the present invention are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid molecules shown in SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, or 147.

[0297] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:126, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:127.

[0298] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:128, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:129.

[0299] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:130, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:131.

[0300] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:132, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:133.

[0301] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:134, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:135.

[0302] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:136, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:137.

[0303] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:138, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:139.

[0304] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:140, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:141.

[0305] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:142, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:143.

[0306] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:144, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:145.

[0307] In some embodiments, the nucleic acid molecule of the present invention comprises a nucleotide 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%, or 100% sequence identity with SEQ ID NO:146, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:147.

[0308] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed in this invention is provided. In some embodiments, this invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed in this invention.

[0309] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

[0310] In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0311] carrier

[0312] In another aspect, the present invention provides a vector comprising a nucleic acid disclosed herein, the nucleic acid comprising encoding a CDH17 antibody or an antigen-binding fragment thereof disclosed herein.

[0313] In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a variable region of the heavy or light chain of an antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

[0314] Any vector may be used in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, 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 adeno-associated virus (AAV) vector, a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pBY, 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.

[0315] Recombinant expression vectors 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. For example, vectors can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript sequences (Stratagene, LaJolla, Calif.), pET sequences (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0316] 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, NY 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 containing a replication system functional in prokaryotic or eukaryotic host cells can be prepared. The replication system can be derived from, for example, COLEL, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0317] For example, 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.

[0318] host cells

[0319] In another aspect, the present invention provides a host cell comprising the nucleic acid or vector disclosed herein.

[0320] Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, 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; Serratia, for example, Serratia marcescens. The bacteria include *Bacillus subtilis* and *Shigella*; *Bacillus* species, such as *B. subtilis* and *B. licheniformis*; *Pseudomonas* species, such as *P. aeruginosa*; and *Streptomyces*. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, 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.

[0321] 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.

[0322] Preparation method

[0323] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof disclosed herein, comprising culturing the host cell provided herein under conditions that cause the antibody or antigen-binding fragment thereof disclosed herein to be expressed; and separating the antibody or antigen-binding fragment thereof from the culture and / or culture supernatant of the host cell.

[0324] Any suitable method for producing antibodies can be used to produce the antibodies of this application. For example, antibodies can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0325] Immunostimulants typically comprise protein antigens, fragments thereof, or fusion proteins thereof. In this document, any suitable form of CDH17 can be used as an immunostimulant (antigen) to generate nonhuman antibodies specific to CDH17 and to screen for the biological activity of said antibodies. Immunostimulants can be full-length mature human CDH17, including natural homodimers, or peptides containing one or more epitopes. Immunostimulants can be used alone or in combination with one or more immunogenicity enhancers known in the art. Peripheral blood lymphocytes are typically used if human cells are desired, and spleen cells or lymph node cells are used if nonhuman mammalian sources are desired. The lymphocytes are then fused with immortalized cell lines using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevent the growth of HGPRT-deficient cells.

[0326] Preferred immortalized cell lines are those that can fuse efficiently, support stable high-level antibody expression in cells producing the selected antibody, and are sensitive to culture media such as HAT medium. More preferred immortalized cell lines are mouse myeloma cell lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center in San Diego, California, and the American Collection of Type Cultures in Manassas, Virginia. Human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies are also described (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

[0327] The presence of monoclonal antibodies against the antigen can then be determined in the culture medium for hybridoma cells. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[0328] After identifying the desired hybridoma cells, the clone can be subcloned using a limiting dilution procedure and cultured using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells can be grown in vivo as ascites in mammals.

[0329] Subclonal secreted monoclonal antibodies can be isolated or purified from culture medium or ascites using routine immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0330] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of this invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells of this invention are used as a preferred source of such DNA. The isolated DNA can be placed in an expression vector and then transfected into host cells, such as non-immunoglobulin-producing cells like simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain antibodies in recombinant host cells for the synthesis of monoclonal antibodies. The DNA can also be modified, for example, by replacing the homologous mouse sequence with the coding sequence of the human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368,812-13 (1994)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. This non-immunoglobulin polypeptide can replace the constant domain of the antibody of the present invention, or can replace the variable domain of an antigen-binding site of the antibody of the present invention to produce a chimeric bivalent antibody.

[0331] The antibody sequences of this application can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single-chain antibody.

[0332] Once the relevant sequence is obtained, it can be mass-produced using recombination. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. Alternatively, the sequence can be synthesized artificially, especially for shorter fragments. Longer fragments are usually obtained by first synthesizing multiple small fragments and then ligating them. This nucleic acid molecule can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0333] Conjugate

[0334] In another aspect, the present invention provides conjugates comprising an antibody or antigen-binding fragment thereof disclosed herein and a chemical moiety conjugated to said antibody or antigen-binding fragment thereof.

[0335] In the context of this disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety may be selected from cytotoxic drugs, immunostimulatory molecules, and detectable markers, such as drugs, toxins, therapeutic agents, detectable markers, proteins, nucleic acids, lipids, nanoparticles, carbohydrates, or recombinant viruses. When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is generally referred to as an “antibody-drug conjugate” or “ADC.” Therefore, this disclosure provides antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof disclosed herein and a chemical moiety conjugated to said antibody or antigen-binding fragment thereof via a linker, wherein said chemical moiety is a therapeutic agent.

[0336] The terms "conjugation" or "linkage" can refer to the process by which two polypeptides become a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, 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. In one embodiment, 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) may optionally be included between the antibody and the chemical moiety.

[0337] 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. Alternatively, 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. In the case where the antibody and chemical moiety are peptides, the linker can be attached via their side groups to the constituent amino acids (e.g., via disulfide bonds to cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acid.

[0338] Linkers can be readily cleaved (cleavable linkers), for example, by acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under which conditions the compound or antibody retains its activity. Alternatively, linkers can be substantially cleavable (e.g., stable linkers or cleavable linkers).

[0339] In some embodiments, the connector comprises a cleavable connector or a non-cleavable connector. For example, the connector may be selected from, for example, acid-labile connectors, light-labile connectors, protease-sensitive connectors, hydrazone connectors, esterase-cleavable connectors, dimethyl connectors, disulfide-containing connectors, hydrophilic connectors, charged connectors, and acid-based connectors.

[0340] In some embodiments, the linker may include an amino acid unit. In one such embodiment, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of a drug from the antibody-drug conjugate upon exposure to an intracellular protease (e.g., a lysosomal enzyme). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-gly (gly-gly-gly). In some embodiments, the linker comprises a vc (valine-citrulline) unit. The amino acid unit can be designed and optimized to be selectively cleaved by specific enzymes (e.g., tumor-associated proteases, cathepsins B, C, and D, or plasminogen activator).

[0341] In some embodiments, the connector is selected from mc (6-maleimide hexanoyl), val-cit, PABC (p-amino-benzyloxycarbonyl), DMEA (dimethylethylamine), val-cit-PABC, mc-val-cit-PABC, mc-val-cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG, AcBut (4-(4-acetylphenoxy)-butyric acid), and AcBut-dimethylhydrazine, preferably val-cit or mc-val-cit-PABC.

[0342] In some cases, when a conjugate reaches its target site, it is desirable to release a chemical moiety from the antibody. Therefore, in these cases, the conjugate will contain a cleavable linker near the target site.

[0343] The conditions that the enzymatic activity or conjugate undergoes in or near the target cell may induce linker cleavage to release the chemical portion from the antibody.

[0344] Given the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapy compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, those skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other peptide.

[0345] The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Typically, antibodies or portions thereof are derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, 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).

[0346] 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. Such linkers are commercially available.

[0347] In some embodiments of the conjugates disclosed in this invention, the chemical portion is selected from therapeutic agents, detectable portions, and immunostimulatory molecules.

[0348] In some embodiments, the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, the therapeutic agent may be selected from microtubule inhibitors, minor groove binders, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors. In a preferred embodiment, the therapeutic agent is a microtubule inhibitor. The microtubule inhibitor may be, for example, monomethylolpropionate E (MMAE) or monomethylolpropionate F (MMAF). Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals capable of cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include drug prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, lactammycin, cazithromycin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photosensitizer II, temozolomide, topotecan, metformin, olistatin E, vincristine, and doxorubicin; peptide cytotoxins, which are proteins or fragments thereof capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNases, and RNases; radionuclides, which are unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; chelating agents can be used to promote the binding of these radionuclides to molecules or polymers thereof.

[0349] In some implementations, the therapeutic agent is selected from MMAE, MMAF, ixotecan (DX8951), duocarmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin, and PBD (benzodiazepines).

[0350] In some implementations, the linker-load portion conjugated with the antibody includes an SPDB-DM4 portion, a vc-MMAD portion, a vc-MMAE portion, a vc-carcinomamycin portion, or a PEG2-vc-MMAD portion.

[0351] In some embodiments, the antibody-drug conjugates of this disclosure have the following structures:

[0352] In some implementations, 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.

[0353] In some implementations, immunostimulatory molecules are immune effector molecules that stimulate an immune response. For example, immunostimulatory molecules can be cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet-4, melanoma growth-stimulating proteins, complement activators; viral / bacterial protein domains, or viral / bacterial peptides.

[0354] Chimeric antigen receptor

[0355] This disclosure provides a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof disclosed herein. This disclosure also provides genetically modified cells comprising the chimeric antigen receptor disclosed herein.

[0356] A classic chimeric antigen receptor (CAR) 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. 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. Depending on the antigen, more compact spacer regions may be suitable, such as the stem from CD8α, 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).

[0357] According to at least one non-limiting viewpoint, at least three “generations” of CAR molecules have existed. In first-generation CARs, they are designed with an intracellular domain having an intracellular portion of the γ chain derived from FcεR1 or the intracellular portion of CD3ζ. 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. To overcome this limitation, second-generation CARs have been constructed with a complex intracellular domain resulting from the fusion of the intracellular portion of a T cell co-stimulatory molecule with the intracellular portion of CD3ζ, thus enabling the simultaneous delivery of activation and co-stimulatory signals after antigen recognition. The most commonly used co-stimulatory domain is the CD28 co-stimulatory domain. This provides the most potent co-stimulatory signal, i.e., an immune signal², which triggers T cell proliferation. Several 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.

[0358] Therefore, 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.

[0359] An "antigen-binding domain" refers to the portion of a chimeric antigen receptor that recognizes an antigen. In a classic CAR, 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. In this application, the antigen-binding domain may be the antibody or its antigen-binding fragment described herein.

[0360] 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 a modified immunoglobulin hinge region.

[0361] A "transmembrane domain" (TM 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, anchoring the CAR to the plasma membrane of an immune effector cell. The transmembrane domain is typically a hydrophobic region of the CAR that crosses the cell membrane. The 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.

[0362] 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 released from the synapse, and signals are transmitted to the cell, thereby activating one or more immune cell effector functions (such as innate immune cell effector functions). The most commonly used intracellular domain component is the CD3ζ intracellular domain component containing three ITAMs. After antigen binding, it transmits activation signals to T cells.

[0363] "Intracellular signal transduction domain" refers to a portion of the 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 after the antigen binds to the extracellular CAR domain.

[0364] The "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 the second signal required for effective activation and function of T lymphocytes when binding to antigens.

[0365] Pharmaceutical Composition

[0366] In another aspect, the present invention provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, a conjugate disclosed herein, and / or a CAR disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0367] The antibodies or antigen-binding fragments or agents thereof (also referred to herein as “active compounds”) of the present invention may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise antibodies or antigen-binding fragments or agents thereof, along with a pharmaceutically acceptable carrier. As used herein, the term “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. Their use in compositions is contemplated unless any conventional media or agent is incompatible with the active compound. Pharmaceutical compositions of this application may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such pharmaceuticals depend, for example, the amount and type of antagonists present in the formulation, and the clinical parameters of the subject.

[0368] In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, peptides, small molecule drugs, immune checkpoint inhibitors, or cytokines.

[0369] The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended routes of administration. The routes of administration for the pharmaceutical compositions of the present invention are preferably parenteral, injectable, or oral. Injectable administration preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The pharmaceutical compositions are in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, i.e., aqueous, non-aqueous, or suspension forms, more preferably tablets, capsules, granules, injections, or infusions. More preferably, they are administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the pharmaceutical compositions can also be administered as aerosols or coarse sprays, i.e., nasal administration; or intrathecal, intramedullary, or intraventricular administration. More preferably, the pharmaceutical compositions can also be administered transdermally, percutaneously, topically, intraenterically, intravaginally, sublingually, or rectally. The pharmaceutical compositions of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a physician based on factors such as patient type, age, weight, general disease condition, and route of administration. Administration methods may include injection or other treatment methods.

[0370] The dosage level of the pharmaceutical composition described in this invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are subject to reasonable adjustment based on various factors, including the activity and stability (i.e., half-life) of the pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0371] Treatment methods and uses

[0372] This disclosure provides a method for treating cancer in a subject, 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 CAR disclosed herein, or a conjugate disclosed herein.

[0373] This disclosure provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the CARs disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for treating a subject's cancer.

[0374] This disclosure also provides the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the CARs disclosed herein, or the conjugates disclosed herein for the treatment of cancer in a subject.

[0375] In some embodiments of the methods and uses disclosed herein, the cancer is cancer associated with CDH17 expression. In some embodiments, the cancer is a solid tumor. In a preferred embodiment, the solid tumor is gastric cancer or colorectal cancer.

[0376] In some implementation methods, 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.

[0377] The dosage of the antibodies, compositions, CARs, or conjugates described herein may 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, CAR, or conjugate may be administered as a single daily dose, or the total daily dose may be administered as needed in two, three, four, or more daily fractions.

[0378] The appropriate route of administration can be chosen by the physician. 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. In some embodiments, antibodies, compositions, CARs, or conjugates 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.

[0379] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or forms. As used herein, the terms “combined” or “in combination” administration should be understood as the delivery of two (or more) different treatments to a subject during the course of the subject’s illness, such that the effects of the treatments on the patient overlap at some point in time. In some embodiments, the delivery of one treatment is still in progress when the second treatment begins, thus creating an overlap in administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. For example, fewer doses of the second treatment may be observed when combined with the first treatment compared to when the second treatment is administered alone without the first treatment, or the second treatment may alleviate symptoms to a greater extent; or a similar situation may exist with the first treatment. In some embodiments, combined administration results in a reduction in symptoms or other parameters related to the illness that is greater than that observed in the absence of the other treatment. The effects of the two treatments may be partially additive, completely additive, or greater than additive. Combination therapy allows the effect of the first treatment to still be detectable when the second treatment is administered.

[0380] This disclosure provides a method for treating a subject by co-administering a second therapeutic agent with an anti-CDH17 antibody or an antigen-binding fragment thereof disclosed herein. Therefore, in some embodiments, the method herein further includes administering the second therapeutic agent to the subject.

[0381] This disclosure provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the CARs disclosed herein, or the conjugates and second therapeutic agents disclosed herein in the preparation of a medicament for treating a subject's cancer.

[0382] This disclosure provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the CARs disclosed herein, or the conjugates disclosed herein in the preparation of a medicament for administration in combination with a second therapeutic agent to treat a subject’s cancer.

[0383] This disclosure provides the use of a second therapeutic agent in the preparation of a medicament for administration in combination with an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a CAR disclosed herein, or a conjugate disclosed herein to treat a subject's cancer.

[0384] This disclosure also provides the antibodies or antigen-binding fragments thereof disclosed herein, the pharmaceutical compositions disclosed herein, the CARs disclosed herein, or the conjugates disclosed herein for use in combination with a second therapeutic agent to treat a subject's cancer.

[0385] The amount of the antibody or its antigen-binding fragment and the relative timing of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering combination therapy to a patient requiring such administration, the therapeutic agents in the combination, or one or more pharmaceutical compositions containing therapeutic agents, can be administered in any order, such as sequentially, jointly, together, simultaneously, etc. Furthermore, for example, the antibody or its antigen-binding fragment can be administered while the second therapeutic agent exerts its preventive or therapeutic effect, and vice versa.

[0386] In some embodiments, the antibodies, compositions, CARs, or conjugates disclosed herein are administered before, substantially simultaneously with, or after the administration of a second therapeutic agent.

[0387] In some implementations, the second therapeutic agent is an immune checkpoint inhibitor. Examples of immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, adenosine A2A receptor inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, KIR inhibitors, LAG3 inhibitors, TIM-3 inhibitors, VISTA inhibitors, galactagogue-9 inhibitors, or TIGIT inhibitors.

[0388] In some implementations, the second therapeutic agent is a cytokine. Examples of cytokines include, but are not limited to, interleukins (e.g., IL-2, IL-7, IL-10, IL-12, IL-15) and interferons (e.g., IFNα, IFNγ).

[0389] In some implementations, the second therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthraquinones, anthracyclines, epipodophyllotoxin, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., holothurin, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, aziridines, nitrogen mustard, nitrosoureas, platinum derivatives, triazine, etc.), alkaloids, terpenoids, and kinase inhibitors.

[0390] Diagnostic / Detection Methods and Uses

[0391] In another aspect, this disclosure provides a method for detecting CDH17 protein in vitro or in vivo, which includes contacting a sample with the CDH17 antibody disclosed herein to detect the presence of CDH17 protein.

[0392] This article provides methods for detecting the presence or level of CDH17 in samples, including:

[0393] (1) Contact the sample with the antibody or its antigen-binding fragment disclosed herein; and

[0394] (2) The presence or level of CDH17 in the sample is determined by detecting the binding of the antibody to the sample.

[0395] This disclosure also provides a method for diagnosing cancers associated with CDH17 expression in subjects, comprising:

[0396] (1) Obtain samples from the subjects;

[0397] (2) Contact the sample with the antibody or its antigen-binding fragment disclosed herein; and

[0398] (3) Detect the binding of antibodies to samples.

[0399] Compared to the binding of the antibodies or their antigen-binding fragments disclosed herein to control samples, the binding of the antibodies or their antigen-binding fragments disclosed herein to samples increases the indication that the subject has CDH17-related cancer.

[0400] This disclosure also provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, or the conjugates disclosed herein, in the preparation of kits for diagnosing cancer in subjects.

[0401] This disclosure also provides antibodies or antigen-binding fragments thereof disclosed herein, or conjugates disclosed herein, for the diagnosis of cancer in a subject.

[0402] In some embodiments, the sample can be any sample, including but not limited to blood samples, tissue from biopsies, autopsies, and pathological specimens. Samples also include bodily fluids such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. In some embodiments, the control sample is a sample from a subject without cancer. In certain embodiments, the sample is a blood or tissue sample.

[0403] In some embodiments of the methods and uses disclosed herein, the cancer is cancer associated with CDH17 expression. In some embodiments, the cancer is a solid tumor. In a preferred embodiment, the solid tumor is gastric cancer or colorectal cancer. In some embodiments of the diagnostic and detection methods, the anti-CDH17 antibody is directly labeled with a detectable marker. In another embodiment, the anti-CDH17 antibody (the first antibody) is unlabeled, while the second antibody or other molecules that can bind to the first antibody are labeled. As is well known to those skilled in the art, the second antibody is selected to specifically bind to a particular species and class of the first antibody. For example, if the first antibody is human IgG, the second antibody may be anti-human IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0404] Suitable labeling for antibodies or secondary antibodies includes various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium; and a non-limiting exemplary radioactive labeling includes… 125 I, 131 I, 35 S or 3 H.

[0405] In an alternative implementation, CDH17 can be determined in biological samples using a competitive immunoassay that utilizes a CDH17 protein standard labeled with a detectable substance and an unlabeled anti-CDH17 antibody. In this assay, the biological sample, the labeled CDH17 protein standard, and the anti-CDH17 antibody are combined, and the amount of labeled CDH17 protein standard binding to the unlabeled antibody is determined. The amount of CDH17 in the biological sample is inversely proportional to the amount of labeled CDH17 protein standard binding to the anti-CDH17 antibody.

[0406] The immunoassay and detection methods disclosed herein can be used for a variety of purposes. In one embodiment, the anti-CDH17 antibody can be used to detect the production of CDH17 in cells within a cell culture. In another embodiment, the antibody can be used to detect the amount of CDH17 in a biological sample (such as a tissue sample or a blood or serum sample). In some instances, CDH17 is cell surface CDH17.

[0407] On the other hand, this disclosure provides a method for imaging cancers associated with CDH17 expression in subjects, comprising:

[0408] (1) Administering to a subject the antibody or antigen-binding fragment thereof disclosed herein, wherein the antibody is conjugated to a detectable marker, and

[0409] (2) Detect the presence of markers.

[0410] In some implementations, the detectable marker is 111 In some preferred embodiments, the marker is detected by single-photon emission computed tomography (SPECT).

[0411] In some implementations, the detectable marker is 89 Zr. In some preferred embodiments, the marker is detected by positron emission tomography (PET).

[0412] Reagent kit / drug delivery device

[0413] In another aspect, the present invention provides a kit or delivery device comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a host cell disclosed herein, a conjugate disclosed herein, a CAR disclosed herein, and / or a pharmaceutical composition disclosed herein.

[0414] In some embodiments, the kit may further include (i) means for administering the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned host cell, the aforementioned conjugate, the aforementioned CAR and / or the aforementioned pharmaceutical composition; and / or (ii) instructions for use.

[0415] In some embodiments, this application provides a kit that may contain an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule, a vector, a host cell, a conjugate, a CAR, and / or a pharmaceutical composition. It may include the antibody, nucleic acid molecule, vector, host cell, antibody-drug conjugate, or CAR described herein in a single, commonly used container. In some cases, the kit may include instructions for use, including information about the antibody, pharmaceutical composition, and dosage form in the kit. Typically, such information helps patients and physicians use the packaged antibody, pharmaceutical composition, and dosage form effectively and safely. Containers used in such kits typically contain at least one vial, test tube, flask, bottle, syringe, or other suitable container in which one or more of the detection and / or therapeutic compositions may be placed, preferably appropriately aliquoted. Where a second therapeutic agent is also provided, the kit may also contain a second, different container in which the second detection and / or therapeutic composition may be placed. Alternatively, multiple compounds may be prepared into a single pharmaceutical composition and packaged in a single-container device such as a vial, flask, syringe, bottle, or other suitable single-container.

[0416] In one embodiment, the kit is used to detect CDH17 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a subject's cancer diagnosis, a biopsy may be performed to obtain a tissue sample for histological examination. Kits for detecting peptides typically contain an anti-CDH17 antibody, such as any monoclonal antibody disclosed herein. In a further embodiment, the antibody is labeled (e.g., with fluorescent, radioactive, or enzyme-catalyzed labeling).

[0417] In some embodiments, the kit may also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit may additionally contain tools for detecting labeled substances (e.g., enzyme substrates for enzymatic labeling, filter banks 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.

[0418] In some embodiments, this application provides a drug delivery device (e.g., a plastic or vial, such as a hollow pin or syringe cylinder) that can be used to administer the disclosed antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, conjugate, CAR, and / or pharmaceutical composition. The device can introduce a substance into a patient via a parenteral route (e.g., intramuscular, subcutaneous, or intravenous). For example, the injection device can be a syringe (e.g., a pre-filled syringe containing the antibody or pharmaceutical composition described in this application, such as an auto-injector), which may include a syringe and needle (which can be used to pierce the skin and / or blood vessels) for containing the fluid to be injected (e.g., the antibody or pharmaceutical composition described in this application). The route of administration can be varied. Routes of administration may include oral, intramuscular, subcutaneous, rectal, etc.

[0419] Example

[0420] Example 1. Production of anti-human CDH17 monoclonal antibody

[0421] 1.1 Mouse Immunization

[0422] Four 6-8 week old female Balb / c mice (SPF grade) housed in an SPF barrier system were immunized subcutaneously at multiple sites using a fusion protein of human CDH17 protein (Gln23-Met787, UniProtKB: Q12864) and Fc (Pro100-Lys330, UniProtKB: P01857) as the antigen. The initial immunization consisted of 20 μg of antigen per mouse, followed by 10 μg per mouse. Serum titers were measured after every two immunizations. Another four 6-8 week old female Balb / c mice (SPF grade) were immunized with DNA using a full-length human CDH17 plasmid (WuXi Biologics) and adjuvant via intramuscular and intradermal injections. The initial immunization consisted of 200 μg of antigen per mouse, followed by 100 μg per mouse. Serum titers were measured after every four immunizations.

[0423] 1.2 Cell Fusion

[0424] Two protein-immunized mice and one DNA-immunized mouse with good immunogenicity were used for shock immunization. Three days later, the mice were euthanized by cervical dislocation, and their spleens were obtained under sterile conditions. B-cell suspensions were prepared and mixed with non-secreting SP2 / 0 myeloma cells at a ratio of 1:5. Cell fusion was performed using an electrofusion instrument. After electrofusion, all cells were immediately suspended in complete culture medium (DMEM, 20% FBS and HAT (a mixture of hypoxanthine, aminopterin, and thymine deoxyribonucleoside)) and seeded into 96-well plates, which were then cultured in a CO2 incubator.

[0425] 1.3 Hybridoma Screening

[0426] Twelve days after fusion, hybridoma supernatant was collected from 96-well plates and subjected to FACS analysis with CHO-K1 cells (WuXi Biologics) stably expressing human CDH17 to preliminarily screen hybridomas with cell affinity. The selected hybridoma supernatant underwent further testing, including FACS assays to determine cell affinity, SNU-16 cell endocytosis assays, and reverse ELISA assays with other CDH family members. Specific experimental procedures are described below:

[0427] 1.3.1 FACS Screening

[0428] SNU-16 cell line (WuXi Biologics) was plated in wells, with 1x10 cells per well. 5 100 μl of hybridoma supernatant was incubated with SNU-16 cells, and after thorough mixing, incubated at 4°C for 1 hour. Cells were washed three times with PBS, and goat anti-mouse IgG Fc A647 was added, mixed thoroughly, and incubated at 4°C in the dark for 30 minutes. Finally, cells were washed three more times with PBS, and flow cytometry was used for analysis. The results are shown in Table 1.

[0429] Table 1. FACS data of hybridoma clones and SNU-16 cells

[0430] 1.3.2 Internalization Experiment

[0431] SNU-16 cell lines were seeded into plates, 1 x 10⁶ cells per well. 5 SNU-16 cells were incubated with 100 μl of hybridoma supernatant at 4°C for 1 hour after thorough mixing. Cells were washed three times with PBS, and goat anti-mouse IgG Fc A647 was added and thoroughly mixed before incubation at 4°C in the dark for 30 minutes. Cells were then washed three more times with PBS and incubated at 37°C for 2 hours. Finally, cells were washed three times with PBS, incubated at 4°C for 5 minutes, and analyzed by flow cytometry. The results are shown in Table 2.

[0432] Table 2. Data on the endocytic activity of hybridoma clones in SNU-16 cells.

[0433] 1.3.3 ELISA Screening

[0434] 96-well plates were coated with recombinant CDH17-His protein from cynomolgus monkeys (Acro-CA7-C52H4, Bipsys) (final concentration 0.25 μg / ml) and incubated overnight at 4°C. After washing three times with PBST (containing 0.5% Tween), blocking buffer (2% BSA) was added, and the plates were incubated at 25°C for 1 hour. After washing three times with PBST, 30 μl of serially diluted hybridoma supernatant (initial concentration 20 nM) was added, and the plates were incubated at 25°C for 1.5 hours. After washing three times with PBST, 30 μl of goat anti-mouse IgG Fc A647-HRP was added, and the plates were incubated at 25°C for 1 hour. After washing three times with PBST, TMB chromogenic solution was added, and the plates were incubated at room temperature in the dark for 6 minutes. The reaction was terminated by adding stop solution, and the OD values ​​in the wells were read using a microplate reader. The results are shown in Table 3.

[0435] Table 3. Hybridoma clones and OD values ​​of CDH17 protein from cynomolgus monkeys determined by ELISA.

[0436] Example 2. Sequencing of anti-human CDH17 monoclonal antibody

[0437] Based on the results of ELISA, FACS, and endocytosis assays, positive monoclonal hybridoma cells meeting the requirements were selected for expanded culture. Cells were lysed using Trizol, and total RNA was extracted from hybridoma cells according to standard methods. The total RNA was reverse transcribed into cDNA samples using a reverse transcription kit. Then, using hybridoma sequencing primers, PCR was used to amplify the cDNA of the antibody's heavy chain variable region and light chain variable region. After purification and recovery, the PCR product fragments were subcloned into a T vector, and single clones were selected for sequencing. Sequencing yielded the antibody heavy chain variable region (VH) and light chain variable region (VL) sequences, with amino acid sequences shown in Table 4 and nucleotide sequences shown in Table 5. The complementarity-determining regions (CDRs) (according to IMGT definition) and framework regions (FRs) of VH and VL are shown in Tables 6 and 7, respectively.

[0438] Table 4. Amino acid sequences of the antibody heavy chain variable region (VH) and light chain variable region (VL).

[0439] Table 5. Nucleotide sequences of antibody heavy chain variable region (VH) and light chain variable region (VL) sequences.

[0440] Example 3. Generation of chimeric antibodies

[0441] The mouse antibody heavy chain variable region gene fragment was constructed into the multiple cloning site binding region of the eukaryotic expression plasmid pCDNA3.4-IgG1, thus constructing the heavy chain expression plasmid. pCDNA3.4-IgG1 was obtained by ligating the coding nucleic acid of the human IgG1 antibody heavy chain constant region fragment into the backbone plasmid pCDNA3.4. The mouse antibody light chain variable region coding nucleic acid was constructed into the multiple cloning site binding region of the eukaryotic expression plasmid pCDNA3.4-IgKc, thus constructing the light chain expression plasmid. pCDNA3.4-IgKc was obtained by ligating the coding nucleic acid of the human κ light chain constant region fragment into the backbone plasmid pCDNA3.4. The constructed light chain and heavy chain expression vectors were transformed into Ecoli. DH5α competent cells. Single colonies were picked, sequenced, and plasmids were extracted in large quantities to obtain chimeric antibody light chain and chimeric antibody heavy chain expression plasmids.

[0442] Plasmids containing the heavy chain gene and plasmids containing the light chain gene were transiently co-transfected into Expi293F cells. After transfection, the cells were cultured for 4-6 days, centrifuged at 4000 rpm for 10 minutes, and the cell culture supernatant containing the target protein was collected. After filtration through a 0.22 μm filter membrane, the supernatant was loaded onto a Protein A affinity chromatography column to obtain purified antibody.

[0443] Example 4. Characterization of chimeric antibodies

[0444] 4.1 Affinity of chimeric antibodies

[0445] Carterra LSA was used for detection: anti-mouse Fc IgG was immobilized on the surface of an HC200M chip. The buffer was 1xHBS-EP+, and biotinylated human CDH17-His protein was diluted to 800 nM, 200 nM, 50 nM, 12.5 nM, and 3.13 nM. After the chimeric antibody was captured onto the chip by the anti-mouse Fc IgG, different concentrations of biotinylated human CDH17-His protein were sequentially injected, with binding for 240 seconds and dissociation for 1200 seconds. The detection temperature was 25℃. The kinetic parameters of binding between each sample and biotinylated human CDH17-His (Ka: binding rate; Kd: dissociation rate; KD: binding-dissociation equilibrium constant) were obtained using Carterra LSA Kinetics software. The control antibody BMK is the CDH17 sequence from the TRAILR2 / DH17 bispecific antibody, linked to the human IgG1 backbone (WO 2018 / 115231). The results are shown in Table 8.

[0446] Table 8. Affinity data of chimeric antibodies

[0447] 4.2 Verification of binding and species specificity of chimeric antibodies

[0448] 4.2.1 FACS

[0449] SNU-16 cell lines were seeded into plates, 1 x 10⁶ cells per well. 5 SNU-16 cells were incubated with 100 μl of chimeric antibody (starting at 200 nM, serially diluted 4 times). After thorough mixing, the cells were incubated at 4°C for 1 hour. Cells were washed three times with PBS, and anti-human IgG FcγA647 was added. After thorough mixing, the cells were incubated at 4°C in the dark for 30 minutes. Finally, the cells were washed three more times with PBS, and flow cytometry was used for analysis. The results are shown in Table 9.

[0450] Table 9. Affinity data between chimeric antibodies and SNU-16

[0451] 4.2.2 Internalization Experiment

[0452] SNU-16 cell lines were seeded into plates, 1 x 10⁶ cells per well. 5 SNU-16 cells were incubated with 100 μl of chimeric antibody (starting at 200 nM, serially diluted 4 times). After thorough mixing, the cells were incubated at 4°C for 1 hour. Cells were washed three times with PBS, and anti-human IgG FcγA647 was added. After thorough mixing, the cells were incubated at 4°C in the dark for 30 minutes. Cells were then washed three more times with PBS and incubated at 37°C for 2 hours. Finally, the cells were washed three times with PBS, incubated at 4°C for 5 minutes, and analyzed by flow cytometry. The results are shown in Table 10.

[0453] Table 10. Data on the endocytic activity of chimeric antibodies on SNU-16

[0454] 4.2.3 ELISA Screening

[0455] Biotinylated human CDH17-His and cynomolgus monkey CDH17-His recombinant protein (final concentration 0.25 μg / ml) were coated onto 96-well plates and incubated overnight at 4°C. After washing three times with PBST (containing 0.5% Tween), blocking buffer (2% BSA) was added, and the plates were incubated at 25°C for 1 hour. After washing three times with PBST, 30 μl of serially diluted hybridoma supernatant (initial concentration 20 nM) was added, and the plates were incubated at 25°C for 1.5 hours. After washing three more times with PBST, 30 μl of goat anti-human IgG Fc-HRP was added, and the plates were incubated at 25°C for 1 hour. After washing three more times with PBST, TMB chromogenic solution was added, and the plates were incubated at room temperature in the dark for 6 minutes. The reaction was terminated by adding stop solution, and the OD values ​​in the wells were read using a microplate reader. The results showed that the antibody of this application had high affinity for both human CDH17 protein and cynomolgus monkey CDH17 protein (Table 11).

[0456] Table 11. ELISA affinity data of antibodies to human CDH17 protein and cynomolgus monkey CDH17 protein

[0457] Example 5. Preparation of antibody-drug conjugates

[0458] 5.1 Fabrication method of DAR4 ADC

[0459] (1) Antibody replacement: Before conjugation, the antibody (IgG) of this invention was desalted using an AKTA system with a G25 dextran gel column, and the protein was replaced in the conjugation buffer (PBS: 20mM, EDTA: 2mM, pH 6.5–8.0; or L-HIs: 20mM, EDTA: 2mM, pH 6.5–8.0). The absorbance of the protein was measured using a UV spectrophotometer, and the antibody concentration after buffer replacement was calculated using the extinction coefficient method. When the antibody concentration was lower than the conjugation concentration, ultrafiltration was performed to concentrate the protein and increase the protein concentration.

[0460] (2) Preparation of conjugation reaction solution: According to the required amount of conjugated antibody (1 eq), use a pipette to accurately transfer the corresponding conjugation system buffer to make the antibody concentration the initial conjugation concentration, usually 1 to 10 mg / mL.

[0461] (3) Antibody reduction: Add 2.0 to 2.5 eq of TCEP·HCl solution (1.0 mg / mL to 5.73 mg / mL) to the reaction vial containing the antibody solution under slow stirring; after the addition is complete, stir slowly at room temperature for 60 to 180 min.

[0462] (4) Antibody conjugation: Calculate the volume of organic solvent (DMAC or DMSO) to be added, making it 5%–15% of the total volume (usually ≤10%); simultaneously calculate the mass of the linker-payload portion mc-vc-PABC-MMAE (Biode Pharmaceuticals, catalog number: BD317613-0.1g, CAS: 646502-53-6, batch: CSC271) to be added for antibody conjugation. The linker-payload portion usually needs to be slightly excessive (usually 8 eq). Then calculate the concentration of the organic solution containing the required load. After accurately preparing the load solution, slowly add it dropwise to the reduced antibody reaction solution. Continue stirring slowly at room temperature for 0.25–1.0 h, depending on the specific conjugation situation. The structure of the prepared ADC is as follows:

[0463] The antibody portions are antibody clones 1.24.15, 1.66.15, 1.107.1, 1.123.15, 1.191.14, 1.228.6, 1.229.13, 1.240.7, 1.268.8, 1.255.1, and 1.256.5 of the present invention, and their corresponding ADCs are ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, ADC-9, ADC-10, and ADC-11, respectively.

[0464] (5) Quenching of the reaction: After the reaction solution reaches the predetermined conjugation time, add an excess of water-soluble small molecule N-acetylcysteine ​​(NAC) solution containing reducing thiol groups (1.0-3.26 mg / mL), and stir slowly to continue the reaction for 15-60 min.

[0465] (6) ADC product purification: After the conjugation reaction is quenched, the reaction solution is first filtered, and then desalted using the AKTA system with a G25 dextran gel column. The first part (about 80%) of the component eluent is collected, concentrated by ultrafiltration again, sterilely filtered, and then sampled. Except for the portion of ADC samples reserved for analysis which is stored at 4°C for a short period of time, the other ADC products are stored at -80°C for later use.

[0466] 5.2 DAR Value Analysis Method of ADC

[0467] ADC-DAR values ​​were analyzed by RP-HPLC. A YMC-Triart BioC4 column (4.6 × 250 mm, 3 μm, 30 nm) was used at 60 °C. Gradient elution was performed with mobile phase A being H₂O (containing 0.1% TFA) and mobile phase B being acetonitrile (containing 0.1% TFA). The reduced ADC components (L0, L1, H0, H1, H2, and H3) were separated. The peak areas corresponding to each component were integrated and weighted by ×2 to calculate the DAR value of the ADC. The control ADC BMK-17 had the antibody terminus of the control antibody BMK and the linker-loading portion being mc-vc-PABC-MMAE. The results are shown in Table 12.

[0468] Table 12. Structure and DAR value of ADC

[0469] Example 6. In vitro killing experiment of ADC

[0470] SNU-16 cells were seeded in 384-well plates. After cell adhesion, the ADC samples to be tested were added at final concentrations of 100 nM, 25 nM, 6.25 nM, 1.56 nM, 0.39 nM, 0.098 nM, 0.024 nM, 0.006 nM, 0.0015 nM, and 0.00038 nM. Cells were cultured for another 5 days, and cell viability was assessed using the Celltiter Glo assay kit (Promega). The results showed that the ADCs proposed in this application effectively killed SNU-16 cells, with IC50 values ​​ranging from 0.28 nM to 0.72 nM (Table 13).

[0471] Table 13. Cell killing assays of ADC on SNU-16

[0472] Example 7. In vivo PDX efficacy experiment of ADC

[0473] STAD-021 is a PDX model of subcutaneous tumors from human gastric cancer xenografts. Tumor tissue was passaged in mice, and the subcutaneous tumors in the tumor-bearing mice grew to 500-1000 mm. 3 Afterwards, the mice were euthanized, the tumors were dissected, necrotic parts were removed, and the tumors were cut into small pieces approximately 2mm*2mm*2mm in size. One tumor piece was then inoculated subcutaneously into the right rib area of ​​each NCG mouse using a cannula. The inoculation continued until the average tumor volume reached 100-120mm. 3 Around 45 days after the start of the experiment, mice were randomly divided into groups of 5 mice each, based on tumor volume and body weight. The administration was intravenous injection of 3 mg / kg, administered once. The carrier group served as a blank control, and the BMK17 group served as a positive control. From grouping until the end of the experiment (D45), tumor volume was measured twice weekly using calipers, measuring both the long and short diameters. The volume was calculated using the formula: Tumor volume = 0.5 × long diameter × short diameter, and the average tumor volume was calculated. The tumor growth inhibition rate (TGI) was calculated based on the average tumor volume: TGI(%) = (1 - T / C) × 100%, where T / C = mean tumor volume in the treatment group / mean tumor volume in the control group. Simultaneously with tumor volume measurement, mouse body weight was measured, and the relationship between body weight changes and administration time was recorded, with the average body weight calculated. The survival and health status of the mice, such as activity and feeding during the administration period, were also observed. The in vivo PDX efficacy results of the ADCs are shown in Table 14. All the ADCs in this application achieved better effects than the control ADCs.

[0474] Table 14. In vivo PDX efficacy results of ADC

[0475] Example 8. Humanization of Antibodies

[0476] 8.1 Humanization design, removal of post-translational modification sites, and plasmid construction

[0477] Based on the performance of the chimeric antibodies, 1.24.15, 1.66.15, 1.228.6, and 1.240.7 were selected for humanization. Using the amino acid sequences of the heavy and light chains of the selected murine antibodies, a homology model was obtained through modeling. Framework amino acids outside the CDR were analyzed, as these sites typically affect the conformation of the CDR or indirectly participate in antigen binding. The human germline with the highest sequence similarity was obtained through IMGT analysis. After splicing the selected human germline framework with the antibody's CDR, the framework region sequences of the designed humanized antibody and the original antibody were compared. By analyzing the homology modeling results of the parental antibody, reversion mutations were performed on framework region sequences that might affect antigen-binding activity. This involved replacing as few murine antibody residues as possible while maintaining antibody activity and reducing heterology. Post-translational modification (PTM) hotspots in variable regions were modified to reduce PTM risk. After verifying the template sequence and codon optimization, the heavy chain variable region sequence was synthesized and constructed into the pCDNA3.4-uIgG1 plasmid containing human heavy chain CH1, hinge, CH2, and CH3 fragments. pCDNA3.4-uIgG1 was obtained by ligating the coding nucleic acid of the heavy chain constant region fragment (UniprotKB / Swiss-Prot: P0DOX5, Seq120-449) into the backbone plasmid pCDNA3.4. The light chain variable region sequence was synthesized and constructed into the pCDNA3.4-IgKc plasmid containing the human light chain constant region fragment. pCDNA3.4-IgKc was obtained by ligating the coding nucleic acid of the light chain constant region fragment (UniprotKB / Swiss-Prot: P01834.2, Seq1-107) into the backbone plasmid pCDNA3.4. The constructed plasmids have the same constant region but contain different site mutations in the heavy chain and light chain variable regions.

[0478] 8.2 Production of humanized antibodies

[0479] The pCDNA3.4 plasmid containing the antibody heavy chain variable region (VH) and light chain variable region (VL) genes was co-transfected into Expi293F cells. After 6 days of cell culture, the supernatant was collected. Using the AKTA sample loading system, the supernatant was automatically loaded onto a protein A (MabSelect SuRe, GE-17543804) affinity chromatography column. After loading, the column was washed with 0.1M Tris-HCl, pH 7.0 equilibration buffer, followed by elution and collection of the target protein with 0.1M glycine, pH 3.5 elution buffer. The variable region sequences of the obtained humanized antibodies are shown in Table 15, and the corresponding CDR sequences are shown in Table 16.

[0480] Table 15. Amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized antibodies.

[0481] Table 16. CDR sequences of humanized antibodies

[0482] Example 9. Characterization of humanized antibodies

[0483] 9.1 Affinity Verification of Humanized Antibodies - SPR

[0484] The affinity of humanized antibodies for human CDH17-His (Acro-CA7-H52H3, Bipsys) antigen was detected using SPR technology on a Biacore 8K microarray. Humanized antibodies were captured by anti-human IgG Fc antibodies coated on a CM5 chip. Seven serially diluted analytes of human CDH17-His (18.75, 37.5, 75, 150, 300, 600, and 1200 nM) and buffer were injected through the chip channels at a rate of 30 μL / min, with a binding time of 180 s and a dissociation time of 3600 s. After each binding and dissociation, the chip surface was regenerated by treatment with 10 mM Glycine-HCl, pH 1.5 buffer. The final binding-dissociation curves are the results after subtracting the reference channel (FcI) and buffer channel. The experimental data were fitted using a 1:1 binding model to obtain kinetic data. The results are shown in Table 17, indicating that all humanized antibodies strongly bind to the human CDH17-His antigen.

[0485] Table 17. Affinity data between humanized antibodies and human CDH17-His

[0486] 9.2 Affinity Verification of Humanized Antibodies - FACS Binding

[0487] SNU-16 cell lines were plated in 384-well plates at a density of 3 x 10⁴ cells per well. 30 μL of humanized antibody (starting at 200 nM, serially diluted 3.16 times) was added to each SNU-16 cell and incubated at 4°C for 1 hour after thorough mixing. Cells were washed twice with 1% BSA, and anti-human IgG FcγA647 was added. After thorough mixing, the cells were incubated at 4°C in the dark for 30 minutes. Finally, the cells were washed twice more with 1% BSA, and flow cytometry was used for analysis. The results are shown in Table 18, indicating that all humanized antibodies strongly bound to SNU-16 cells.

[0488] Table 18. Affinity data between humanized antibodies and SNU-16

Claims

1. An antibody that specifically binds to CDH17 or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:106, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:107; or (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:153, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:154; or (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:155, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:154; or (4) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:155, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:

156.

2. The antibody or its antigen-binding fragment according to claim 1, wherein... The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:9, 11 and 13, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:62, 64 and 66, respectively.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein... (1) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:106, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:107; or (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:153, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:154; or (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:155, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:154; or (4) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:155, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

156.

4. The antibody or its antigen-binding fragment according to claim 3, wherein... (1) The VH contains the amino acid sequence shown in SEQ ID NO:106, and the VL contains the amino acid sequence shown in SEQ ID NO:107; or (2) The VH contains the amino acid sequence shown in SEQ ID NO:153, and the VL contains the amino acid sequence shown in SEQ ID NO:154; or (3) The VH contains the amino acid sequence shown in SEQ ID NO:155, and the VL contains the amino acid sequence shown in SEQ ID NO:154; or (4) The VH contains the amino acid sequence shown in SEQ ID NO:155, and the VL contains the amino acid sequence shown in SEQ ID NO:

156.

5. An antibody that specifically binds to CDH17 or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:104, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:105; or (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:148, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149; or (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:150, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149; or (4) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:151, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:149; or (5) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:152, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:

149.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein... (1) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 4, and 6, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:55, 57, and 59, respectively; or (2) The VH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:2, 4 and 166, respectively, and the VL comprises light chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:55, 57 and 59, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 5 or 6, wherein... (1) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:104, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:105; or (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:148, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149; or (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:150, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149; or (4) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:151, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:149; or (5) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:152, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

149.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein... (1) The VH contains the amino acid sequence shown in SEQ ID NO:104, and the VL contains the amino acid sequence shown in SEQ ID NO:105; or (2) The VH contains the amino acid sequence shown in SEQ ID NO:148, and the VL contains the amino acid sequence shown in SEQ ID NO:149; or (3) The VH contains the amino acid sequence shown in SEQ ID NO:150, and the VL contains the amino acid sequence shown in SEQ ID NO:149; or (4) The VH contains the amino acid sequence shown in SEQ ID NO:151, and the VL contains the amino acid sequence shown in SEQ ID NO:149; or (5) The VH contains an amino acid sequence as shown in SEQ ID NO:152, and the VL contains an amino acid sequence as shown in SEQ ID NO:

149.

9. An antibody that specifically binds to CDH17 or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:114, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:115; or (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:157, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:158; or (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:159, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:160; or (4) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:159, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:

161.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein... The VH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:27, 29 and 31, respectively, and the VL comprises light chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:80, 64 and 83, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein... (1) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:114, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:115; or (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:157, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:158; or (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:159, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:160; or (4) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:159, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

161.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein... (1) The VH contains the amino acid sequence shown in SEQ ID NO:114, and the VL contains the amino acid sequence shown in SEQ ID NO:115; or (2) The VH contains the amino acid sequence shown in SEQ ID NO:157, and the VL contains the amino acid sequence shown in SEQ ID NO:158; or (3) The VH contains the amino acid sequence shown in SEQ ID NO:159, and the VL contains the amino acid sequence shown in SEQ ID NO:160; or (4) The VH contains an amino acid sequence as shown in SEQ ID NO:159, and the VL contains an amino acid sequence as shown in SEQ ID NO:

161.

13. An antibody that specifically binds to CDH17 or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (1) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:118, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:119; or (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:162, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163; or (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:164, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:163; or (4) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:165, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:

163.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein... (1) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:40, 42, and 44, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:62, 94, and 96, respectively; or (2) The VH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:40, 167 and 44, respectively, and the VL comprises light chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:62, 94 and 96, respectively.

15. The antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein... (1) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:118, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:119; or (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:162, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163; or (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:164, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:163; or (4) The VH contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:165, and the VL contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

163.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein... (1) The VH contains the amino acid sequence shown in SEQ ID NO:118, and the VL contains the amino acid sequence shown in SEQ ID NO:119; or (2) The VH contains the amino acid sequence shown in SEQ ID NO:162, and the VL contains the amino acid sequence shown in SEQ ID NO:163; or (3) The VH contains the amino acid sequence shown in SEQ ID NO:164, and the VL contains the amino acid sequence shown in SEQ ID NO:163; or (4) The VH contains an amino acid sequence as shown in SEQ ID NO:165, and the VL contains an amino acid sequence as shown in SEQ ID NO:

163.

17. An antibody or antigen-binding fragment thereof that specifically binds to CDH17, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (1) the VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:108, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:109; or (2) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:110, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:111; or (3) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:112, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:113; or (4) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:116, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:117; or (5) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:120, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:121; or (6) The VH comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:122, and the VL comprises light chain CDR1, light chain CDR2, and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:123; or (7) The VH comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO:124, and the VL comprises light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO:

125.

18. The antibody or antigen-binding fragment thereof according to claim 17, wherein... (1) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 16, and 6, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:69, 57, and 59, respectively; or (2) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:20, 22, and 24, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:74, 76, and 78, respectively; or (3) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:33, 35, and 37, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:86, 88, and 90, respectively; or (4) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO: 9, 11, and 13, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO: 62, 64, and 97, respectively; or (5) The VH comprises heavy chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:2, 16, and 47, respectively, and the VL comprises light chain CDRs 1-3 having amino acid sequences as shown in SEQ ID NO:55, 57, and 59, respectively; or (6) The VH comprises heavy chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:49, 51 and 53, respectively, and the VL comprises light chain CDR 1-3 having amino acid sequences as shown in SEQ ID NO:101, 57 and 59, respectively.

19. The antibody or antigen-binding fragment thereof according to claim 17 or 18, wherein... (1) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:108, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:109; or (2) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:110, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:111; or (3) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:112, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:113; or (4) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:116, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:117; or (5) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:120, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:121; or (6) The VH comprises 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%, or 100% sequence identity with SEQ ID NO:122, and the VL comprises 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%, or 100% sequence identity with SEQ ID NO:123; or (7) The VH contains 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%, or 100% sequence identity with SEQ ID NO:124, and the VL contains 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%, or 100% sequence identity with SEQ ID NO:

125.

20. The antibody or antigen-binding fragment thereof according to claim 19, wherein... (1) The VH contains the amino acid sequence shown in SEQ ID NO:108, and the VL contains the amino acid sequence shown in SEQ ID NO:109; or (2) The VH contains the amino acid sequence shown in SEQ ID NO:110, and the VL contains the amino acid sequence shown in SEQ ID NO:111; or (3) The VH contains the amino acid sequence shown in SEQ ID NO:112, and the VL contains the amino acid sequence shown in SEQ ID NO:113; or (4) The VH contains the amino acid sequence shown in SEQ ID NO:116, and the VL contains the amino acid sequence shown in SEQ ID NO:117; or (5) The VH contains the amino acid sequence shown in SEQ ID NO:120, and the VL contains the amino acid sequence shown in SEQ ID NO:121; or (6) The VH contains the amino acid sequence shown in SEQ ID NO:122, and the VL contains the amino acid sequence shown in SEQ ID NO:123; or (7) The VH contains the amino acid sequence shown in SEQ ID NO:124, and the VL contains the amino acid sequence shown in SEQ ID NO:

125.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1-20, wherein the antibody is selected from mouse antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1-21, wherein the antibody is an isotype selected from IgG, IgA, IgM, IgE and IgD.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody is a subtype selected from IgG1, IgG2, IgG3 and IgG4.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1-23, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv and ds-scFv.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1-24, wherein the antibody is a monovalent, bivalent, or multivalent antibody.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1-25, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

27. A nucleic acid comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-26.

28. The nucleic acid according to claim 27, comprising: (1) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:126, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:127; (2) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:128, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:129; (3) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:130, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:131; (4) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:132, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:133; (5) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:134, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:135; (6) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:136, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:137; (7) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:138, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:139; (8) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:140, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:141; (9) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:142, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:143; (10) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:144, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:145; or (11) A nucleotide 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%, or 100% sequence identity with SEQ ID NO:146, and a nucleotide 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%, or 100% sequence identity with SEQ ID NO:

147.

29. A vector comprising the nucleic acid according to claim 27 or 28.

30. A host cell comprising the nucleic acid according to claim 27 or 28 or the vector according to claim 29.

31. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-26, the method comprising: a) Culture the host cells according to claim 30 under conditions suitable for the expression of the antibody or its antigen-binding fragment; and b) Isolate the antibody or its antigen-binding fragment from the culture and / or culture supernatant of the host cells.

32. A conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-26 and a chemical moiety conjugated to said antibody or antigen-binding fragment thereof via a linker, preferably said chemical moiety being selected from therapeutic agents, detectable moieties, and immunostimulatory molecules.

33. The conjugate of claim 32, wherein the therapeutic agent is selected from microtubule inhibitors, minor groove binders, antibiotics, DNA synthesis inhibitors, topoisomerase inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors, such as MMAE or MMAF.

34. The conjugate of claim 33, wherein the linker comprises a cleavable linker or a non-cleavable linker, preferably, the linker comprises valine-citrulline (val-cit) or mc-val-cit-PABC.

35. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-26.

36. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-26, a nucleic acid according to claim 27 or 28, a vector according to claim 29, a host cell according to claim 30, a conjugate according to any one of claims 32-34, or a CAR according to claim 35, and optionally a pharmaceutically acceptable carrier or excipient.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutical composition further comprises a second therapeutic agent, optionally selected from antibodies, chemotherapeutic agents, antisense oligonucleotides (such as siRNA), peptides, small molecule drugs, immune checkpoint inhibitors, and cytokines.

38. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-26, a nucleic acid according to claim 27 or 28, a vector according to claim 29, a host cell according to claim 30, a conjugate according to any one of claims 32-34, a CAR according to claim 35, or a pharmaceutical composition according to claim 36 or 37.

39. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-26, a nucleic acid according to claim 27 or 28, a vector according to claim 29, a host cell according to claim 30, a conjugate according to any one of claims 32-34, a CAR according to claim 35, or a pharmaceutical composition according to claim 36 or 37.

40. The method of claim 39, wherein the cancer is a cancer associated with CDH17 expression, preferably a solid tumor, and more preferably gastric cancer or colorectal cancer.

41. The method according to claim 39 or 40, the method further comprising administering a second therapeutic agent to the subject, preferably, the second therapeutic agent being selected from antibodies, chemotherapeutic agents, antisense oligonucleotides (such as siRNA), peptides, small molecule drugs, immune checkpoint inhibitors, and cytokines.

42. Methods for detecting the presence or level of CDH17 in a sample, including: (1) Contact the sample with the antibody or antigen-binding fragment thereof as described in any one of claims 1-26; and (2) The presence or level of CDH17 in the sample is determined by detecting the binding of the antibody to the sample.

Images