Antigen-binding molecules that specifically bind to GUCY2C and CD3 and their pharmaceutical uses

Abstract

An antigen-binding molecule that specifically binds to GUCY2C and CD3 and its pharmaceutical use. The antigen-binding molecule can be used to treat tumors.

Description

[Technical Field]

[0001] The present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to an antigen-binding molecule that specifically binds to GUCY2C and CD3 and its pharmaceutical use. [Background technology]

[0002] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0003] GUCY2C (Guanylyl Cyclase C) belongs to the transmembrane guanylate cyclase family and consists of an extracellular domain, a single transmembrane domain, and an intracellular catalytic domain. It is anchored to the plasma membrane as a homodimer or trimer. Its primary function is to catalyze the formation of cGMP from GTP to maintain normal physiological functions, such as intestinal homeostasis (Michaela Kuhn, Physiological Reviews: (2016), 96(2):751-804). In normal tissues, GUCY2C is expressed exclusively in intestinal epithelial cells, with no or very low expression in other tissues. GUCY2C is expressed in gastrointestinal malignancies, including over 90% of colorectal cancers at all stages and over 50% of gastric or gastroesophageal border cancers (Ruth Birbe MD., Human Pathology, (2005), 36(2):170-179; Hadi Danaee, PLoS One, (2017), 12(12):e0189953). Furthermore, there are clear differences in cellular expression polarity and vascular enrichment between normal intestinal tissue and intestinal cancer, making GUCY2C in intestinal cancer more likely to contact blood vessels than in normal tissue (Divya Mathur, Clinical Cancer Research, (2020), 26(9):2188-2202). Therefore, GUCY2C is a relatively specific and highly promising target for gastrointestinal cancer, especially intestinal cancer.

[0004] Bispecific antibodies targeting CD3 and TAA are a novel immunotherapy that simultaneously bind to T cells and tumor cells, mimicking the interaction between MHC and TCR, and induce T cells to form lytic synapses, releasing perforin and granzymes to specifically kill tumor cells. Activated T cells then release cytokines and activate other immune cells to expand the immune response against tumors, ultimately triggering a cascade reaction that leads to T cell proliferation and tumor cell killing. Summary of the Invention

[0005] The present disclosure provides antigen-binding molecules that specifically bind to GUCY2C and CD3, and antibodies that specifically bind to GUCY2C.

[0006] In one aspect, the present disclosure provides an antigen binding molecule that specifically binds to GUCY2C and CD3, comprising at least one antigen binding module that specifically binds to GUCY2C and at least one antigen binding module that specifically binds to CD3, wherein the antigen binding module that specifically binds to GUCY2C comprises a heavy chain variable region GUCY2C-VH and a light chain variable region GUCY2C-VL, and the antigen binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.

[0007] In some embodiments, the antigen-binding molecule is (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 25, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 54, 26, 50, 51, 52 or 53, respectively; or (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 37 or 15, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 16, 34, 35 or 36, respectively; or (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 42 or 17, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41 or 18, respectively; or iv-1) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 273, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 274, respectively; or iv-2) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 275, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 276, respectively; or iv-3) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 277, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 278, respectively; or iv-4) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 279, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 280, respectively; or iv-5) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 330, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 331, respectively; or iv-6) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 332, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 333, respectively; or iv-7) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 334, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 335, respectively; or iv-8) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 336, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 337, respectively; or iv-9) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 338, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 339, respectively; or iv-10) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 340, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 341, respectively; or iv-11) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 342, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 343, respectively; or iv-12) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 344, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 345, respectively; or iv-13) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 346, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 347, respectively; or iv-14) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 348, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 349, respectively; or iv-15) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 350, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 351, respectively; or iv-16) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 352, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 353.

[0008] In some embodiments, the antigen-binding molecule is (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 25, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 54, respectively; or (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 37, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 16, respectively; or (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 42, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41.

[0009] In some embodiments, the GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0010] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45, or 46, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27 or 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 28, 29 or 30, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 40 or 12, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 39 or 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or iv-1) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 250, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 251, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 252, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 253, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 254, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 255; or iv-2) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 256, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 257, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 258, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 259, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 260; or iv-3) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 262, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 263, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 266, or iv-4) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 267, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 268, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 269, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 270, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 271, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 272; or iv-5) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 281, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 282, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 283, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 284, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 285, or iv-6) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 288, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 289, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 290; or iv-7) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 291, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 292, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 293, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 294, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 295, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 296, or iv-8) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 297, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 298, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-9) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 301, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 303, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 304, or iv-10) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 305, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 306, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or iv-11) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 128, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 307, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 308, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 309, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 310, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 311, or iv-12) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 312, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 313, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 314, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 315, or iv-13) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 316, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 317, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-14) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 318, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 319, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 320, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 321, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 322, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 323, or iv-15) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 324, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 325, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 326, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 327, or iv-16) The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 328, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 302, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 329, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 300.

[0011] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 40, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 39, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0012] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 12, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0013] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or The GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 44, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or The GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 45, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or The above GUCY2C-VH comprises GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 4, GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 5, and GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 6, and the above GUCY2C-VL comprises GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 7, GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 30, and GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 9.

[0014] In some embodiments, the antigen binding molecule of any one of the above, wherein GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 are defined according to the Kabat numbering convention.

[0015] In some embodiments, in any one of the above antigen-binding molecules, the GUCY2C-VH and GUCY2C-VL are humanized and contain the FR region of a human antibody.

[0016] In some embodiments, the antigen-binding molecule according to any one of the above, (i) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 21, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 39L, 43K, 69L, 71V, and 73K; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45, or 46, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 24, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R, and 74K; or (ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 27 or 6, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K, and 67A; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 8, 28, 29, or 30, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 9, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 15P, 16G, 42S, 43S, 45K, 46S, 47W, 49Y, 58V, 71Y, and 76S; or (iii) The GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 40 or 12, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 2F, 28S, 69L, 71V, 73Q and 75S, and the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 39 or 8, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 42S, 43S, 46P, 47W, 58V and 71Y.

[0017] In some embodiments, the antigen-binding molecule according to any one of the above, (i) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 21, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 39L, 43K, 69L, 71V, and 73K; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 24, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R, and 74K; or (ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 27, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K, and 67A; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 9, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 15P, 16G, 42S, 43S, 45K, 46S, 47W, 49Y, 58V, 71Y, and 76S; or (iii) The GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 40, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 2F, 28S, 69L, 71V, 73Q, and 75S, and the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 39, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 42S, 43S, 46P, 47W, 58V, and 71Y.

[0018] In some embodiments, the antigen-binding molecule according to any one of the above, The CD3-VH comprises a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 71, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 73 or 88, and the CD3-VL comprises a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0019] In some embodiments, the antigen-binding molecule according to any one of the above, The CD3-VH comprises CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 71, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and the CD3-VL comprises CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0020] In some embodiments, the antigen-binding molecule according to any one of the above, The CD3-VH comprises CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 71, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 88, and the CD3-VL comprises CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0021] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, 25 or 57, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 26, 48, 49, 50, 51, 52 or 53; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, 15 or 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 16, 31, 32, 34, 35 or 36; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42 or 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41 or 18; or iv-1) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 273, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 274, or iv-2) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 275, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 276; or iv-3) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 277, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 278; or iv-4) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 279, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 280; or iv-5) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 330, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 331; or iv-6) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 332, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 333; or iv-7) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 334, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 335; or iv-8) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 336, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 337; or iv-9) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 338, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 339; or iv-10) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 340, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 341; or iv-11) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 342, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 343; or iv-12) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 344, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 345; or iv-13) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 346, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 347; or iv-14) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 348, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 349; or iv-15) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 350, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 351; or iv-16) The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 352, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 353.

[0022] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 48, 49, 50, 51, or 52; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 25, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 26; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 32, 34, or 36; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO:17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO:18.

[0023] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or iii) The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41.

[0024] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 56, and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 54.

[0025] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 56, and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 55.

[0026] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 37, and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 33.

[0027] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 42 and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 41.

[0028] In some embodiments, the antigen-binding molecule according to any one of the above, The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77 or 89, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78.

[0029] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the CD3-VH is shown in SEQ ID NO:77, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO:78.

[0030] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the CD3-VH is shown in SEQ ID NO:89, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO:78.

[0031] In some embodiments, in any one of the antigen-binding molecules described above, the antigen-binding module that specifically binds to GUCY2C or the antigen-binding module that specifically binds to CD3 comprises a titin chain and an obscurin chain that can form a dimer.

[0032] In some embodiments, in any one of the antigen-binding molecules described above, the antigen-binding module that specifically binds to GUCY2C comprises a Titin chain and an Obscurin chain that can form a dimer.

[0033] In some embodiments, in any one of the antigen-binding molecules described above, the antigen-binding module that specifically binds to CD3 comprises a Titin chain and an Obscurin chain that can form a dimer.

[0034] In some embodiments, in any one of the antigen-binding molecules described above, the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 182 to 200, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 201 to 241.

[0035] In some embodiments, the antigen binding molecule according to any one of the above, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 198.

[0036] In some embodiments, the antigen-binding molecule of any one of the above, wherein the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 236.

[0037] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises an Fc region.

[0038] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises an IgG Fc region.

[0039] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises an IgG1 Fc region.

[0040] In some embodiments, the antigen-binding molecule according to any one of the above aspects comprises an Fc region, and the Fc region comprises one or more amino acid substitutions capable of reducing binding of the Fc region to an Fcγ receptor.

[0041] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises an Fc region, and the Fc region is human IgG1 It is an Fc region and the amino acids at positions 234 and 235 are A, numbered according to the EU index.

[0042] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, and the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce Fc region homodimerization.

[0043] In some embodiments, the antigen-binding molecule of any one of the above is characterized in that the antigen-binding molecule comprises an Fc domain, the Fc domain comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, the Fc1 having a knob structure based on knob-in-hole technology, and the Fc2 having a hole structure based on knob-in-hole technology.

[0044] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the Fc1 has a knob structure according to knobs-in-hole technology and the Fc2 has a hole structure according to knobs-in-hole technology, wherein the amino acid at position 366 of Fc1 is W, and the amino acid at position 366, the amino acid at position 368, the amino acid at position 368, and the amino acid at position 407 of Fc2 are S, A, and V, respectively, and are numbered according to the EU index.

[0045] In some embodiments, the antigen-binding molecule of any one of the above is characterized in that the antigen-binding molecule comprises an Fc region, and the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 79 or 85, and Fc2 comprises the amino acid sequence of SEQ ID NO: 80 or 86.

[0046] In some embodiments, the antigen-binding molecule of any one of the preceding claims, wherein the antigen-binding molecule comprises an Fc region, wherein the Fc region comprises a first subunit, Fc1, and a second subunit, Fc2, which are capable of associating with each other, wherein the Fc1 has a knob structure according to knobs-in-hole technology, and the Fc2 has a hole structure according to knobs-in-hole technology, wherein the Fc1 has a C amino acid at position 354 and a W amino acid at position 366, and the Fc2 has a C amino acid at position 349, an S amino acid at position 366, an A amino acid at position 368, and a V amino acid at position 407, and are numbered according to the EU index.

[0047] In some embodiments, the antigen-binding molecule of any one of the above is characterized in that the antigen-binding molecule comprises an Fc region, and the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 85, and Fc2 comprises the amino acid sequence of SEQ ID NO: 86.

[0048] In some embodiments, the antigen-binding molecule of any one of the preceding embodiments comprises an Fc region, the Fc region comprising a first subunit, Fc1, and a second subunit, Fc2, which are capable of associating with each other, wherein Fc1 has a knob structure according to knobs-in-hole technology, and Fc2 has a hole structure according to knobs-in-hole technology, wherein Fc1 has A at amino acid position 237, C at amino acid position 349, and W at amino acid position 366, and Fc2 has A at amino acid position 237, C at amino acid position 354, S at amino acid position 366, A at amino acid position 368, and V at amino acid position 407, and are numbered according to the EU index.

[0049] In some embodiments, the antigen-binding molecule of any one of the above is characterized in that the antigen-binding molecule comprises an Fc region, and the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 79 and Fc2 comprises the amino acid sequence of SEQ ID NO: 80.

[0050] In some embodiments, the antigen-binding molecule described in any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to GUCY2C and one antigen-binding module that specifically binds to CD3.

[0051] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to GUCY2C and one antigen-binding module that specifically binds to CD3, and both of the antigen-binding modules that specifically bind to GUCY2C and CD3 are scFvs.

[0052] In some embodiments, the antigen-binding molecule according to any one of the above, It comprises one first chain having the structure shown in formula (a) and one second chain having the structure shown in formula (b), (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1], (b) [CD3-VL]-[linker 3]-[GUCY2C-VH]-[linker 2]-[Fc2]; wherein the linker 1, linker 2 and linker 3 are the same or different peptide linkers, or the linker 1, linker 2 and linker 3 do not exist; The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.

[0053] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, or iii) The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 18, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78.

[0054] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 56, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 54, and the amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78.

[0055] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 56, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 55, and the amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78.

[0056] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 15, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 16, and the amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78.

[0057] In some embodiments, in any one of the antigen-binding molecules described above, Linker 1 and Linker 2 are both peptide linkers known in the art, so long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide having 1 to 50 or 3 to 20 amino acid residues. In some embodiments, the peptide linkers each independently have the structure L1-(GGGGS)n-L2, where L1 is a bond, A, G (SEQ ID NO: 82), GS, GGG (SEQ ID NO: 96), GGS (SEQ ID NO: 245), GGGS (SEQ ID NO: 246), or GGGG (SEQ ID NO: 247), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L2 is a bond, G (SEQ ID NO: 82), GG, GGG (SEQ ID NO: 96), or GGGG (SEQ ID NO: 247), and the peptide linker is not a bond. In some embodiments, the amino acid sequence of linker 1 is set forth in SEQ ID NO:81, the amino acid sequence of linker 2 is set forth in SEQ ID NO:82, and the amino acid sequence of linker 3 is set forth in SEQ ID NO:83.

[0058] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the amino acid sequence of Fc1 is represented by SEQ ID NO: 79 and the amino acid sequence of Fc2 is represented by SEQ ID NO: 80.

[0059] In some embodiments, the antigen-binding molecule according to any one of the above is i) one first chain comprising the amino acid sequence of SEQ ID NO: 99 and one second chain comprising the amino acid sequence of SEQ ID NO: 100, or one first chain comprising the amino acid sequence of SEQ ID NO: 101 and one second chain comprising the amino acid sequence of SEQ ID NO: 100; or ii) one first chain comprising the amino acid sequence of SEQ ID NO: 102 and one second chain comprising the amino acid sequence of SEQ ID NO: 103; or iii) one first chain comprising the amino acid sequence of SEQ ID NO: 104 and one second chain comprising the amino acid sequence of SEQ ID NO: 105; Includes:

[0060] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 99 and one second chain whose amino acid sequence is set forth in SEQ ID NO: 100.

[0061] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 101 and one second chain whose amino acid sequence is set forth in SEQ ID NO: 100.

[0062] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 102 and one second chain whose amino acid sequence is set forth in SEQ ID NO: 103.

[0063] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to CD3 and one antigen-binding module that specifically binds to GUCY2C, wherein the antigen-binding module that specifically binds to CD3 is a Fab, and the antigen-binding module that specifically binds to GUCY2C is a substituted Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer.

[0064] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises one first chain having a structure represented by formula (c), one second chain having a structure represented by formula (d), one third chain having a structure represented by formula (e), and one fourth chain having a structure represented by formula (f); (c) [CD3-VH]-[CH1]-[Fc1]; (d) [CD3-VL]-[CL]; (e) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc2], (f) [GUCY2C-VL]-[linker 4]-[Titin chain], wherein the linker 4 is a peptide linker, or the linker 4 is absent; The structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.

[0065] In some embodiments, the antigen-binding molecule according to any one of the above, i) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 89, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 31; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 32; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 34; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 35; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 36; or ii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, or iii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 18.

[0066] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78, and the amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 33.

[0067] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 89, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78, and the amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 33.

[0068] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78, and the amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 42, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 41.

[0069] In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain capable of forming a dimer in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain comprises an amino acid sequence such as SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 236.

[0070] In some embodiments, in any one of the antigen-binding molecules described above, linker 4 is any peptide linker known in the art, so long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide comprising 1 to 50 or 3 to 20 amino acid residues. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have a (GGGGS)n structure, where n is 1, 2, or 3. In some embodiments, the sequence of linker 4 is GGGGS (SEQ ID NO: 90).

[0071] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CH1 is a CH1 sequence of IgG. In some embodiments, the CH1 is a CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.

[0072] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 87.

[0073] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the amino acid sequence of Fc1 is represented by SEQ ID NO: 85 and the amino acid sequence of Fc2 is represented by SEQ ID NO: 86.

[0074] In some embodiments, the antigen-binding molecule according to any one of the above is i) one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or one first chain comprising the amino acid sequence of SEQ ID NO: 242, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 108, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 109; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 111; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 112; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 114; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 115; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 116; or ii) one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 106, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 107; or iii) one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 119, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 120; or one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 117, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 118; Includes:

[0075] In some embodiments, the antigen-binding molecule of any one of the above describes, wherein the antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 91, one second chain whose amino acid sequence is set forth in SEQ ID NO: 92, one third chain whose amino acid sequence is set forth in SEQ ID NO: 110, and one fourth chain whose amino acid sequence is set forth in SEQ ID NO: 113.

[0076] In some embodiments, the antigen-binding molecule of any one of the above embodiments comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 242, one second chain whose amino acid sequence is set forth in SEQ ID NO: 92, one third chain whose amino acid sequence is set forth in SEQ ID NO: 110, and one fourth chain whose amino acid sequence is set forth in SEQ ID NO: 113.

[0077] In some embodiments, the antigen-binding molecule of any one of the above describes, wherein the antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 91, one second chain whose amino acid sequence is set forth in SEQ ID NO: 92, one third chain whose amino acid sequence is set forth in SEQ ID NO: 119, and one fourth chain whose amino acid sequence is set forth in SEQ ID NO: 120.

[0078] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to GUCY2C and one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GUCY2C is a substituted Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer, and the antigen-binding module that specifically binds to CD3 is a Fab.

[0079] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises one first chain having a structure represented by formula (g), one second chain having a structure represented by formula (f), one third chain having a structure represented by formula (h), and one fourth chain having a structure represented by formula (d); (g) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc1], (f) [GUCY2C-VL]-[linker 4]-[Titin chain], (h) [CD3-VH]-[CH1]-[Fc2]; (d) [CD3-VL]-[CL]; wherein the linker 4 is a peptide linker, or the linker 4 is absent; The structures shown in formulas (g), (f), (h) and (d) are arranged from the N-terminus to the C-terminus.

[0080] In some embodiments, the antigen-binding molecule according to any one of the above, i) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, or ii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 31; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 32; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 34; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 35; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 36; or iii) The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41.

[0081] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78, and the amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 56, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 54.

[0082] In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain capable of forming a dimer in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain comprises an amino acid sequence such as SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 236.

[0083] In some embodiments, in any one of the antigen-binding molecules described above, linker 4 is any peptide linker known in the art, so long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide comprising 1 to 50 or 3 to 20 amino acid residues. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have a (GGGGS)n structure, where n is 1, 2, or 3. In some embodiments, the sequence of linker 4 is GGGGS (SEQ ID NO: 90).

[0084] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CH1 is a CH1 sequence of IgG. In some embodiments, the CH1 is a CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.

[0085] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 87.

[0086] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the amino acid sequence of Fc1 is represented by SEQ ID NO: 85 and the amino acid sequence of Fc2 is represented by SEQ ID NO: 86.

[0087] In some embodiments, the antigen-binding molecule according to any one of the above is i) one first chain comprising the amino acid sequence of SEQ ID NO: 121, one second chain comprising the amino acid sequence of SEQ ID NO: 107, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or ii) one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 111, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 112, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 113, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 114, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 115, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 116, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or iii) one first chain comprising the amino acid sequence of SEQ ID NO: 123, one second chain comprising the amino acid sequence of SEQ ID NO: 120, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; Includes:

[0088] In some embodiments, the antigen-binding molecule of any one of the above describes, wherein the antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 121, one second chain whose amino acid sequence is set forth in SEQ ID NO: 107, one third chain whose amino acid sequence is set forth in SEQ ID NO: 93, and one fourth chain whose amino acid sequence is set forth in SEQ ID NO: 92.

[0089] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GUCY2C and two antigen-binding modules that specifically bind to CD3, and both of the antigen-binding modules that specifically bind to GUCY2C and CD3 are scFvs.

[0090] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises two first chains having a structure represented by formula (i) and two second chains having a structure represented by formula (j), (i) [GUCY2C-VH]-[CH1]-[CD3-VH]-[linker 5]-[CD3-VL]-[linker 6]-[one subunit of the Fc region]; (j) [GUCY2C-VL]-[CL], wherein the linker 5 and the linker 6 are peptide linkers, or the linker 5 and the linker 6 do not exist; The structures shown in formulas (i) and (j) are arranged from the N-terminus to the C-terminus.

[0091] In some embodiments, the antigen-binding molecule according to any one of the above, The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55.

[0092] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78, and the amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 56, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 54.

[0093] In some embodiments, in any one of the antigen-binding molecules described above, linker 5 and linker 6 are both peptide linkers known in the art, so long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide containing 1 to 50 or 3 to 20 amino acid residues. In some embodiments, the peptide linkers each independently have the structure L1-(GGGGS)n-L2, where L1 is a bond, A, G, GS, GGG (SEQ ID NO: 96), GGS (SEQ ID NO: 245), GGGS (SEQ ID NO: 246), or GGGG (SEQ ID NO: 247), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG (SEQ ID NO: 96), or GGGG (SEQ ID NO: 247), and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3. In some embodiments, the amino acid sequence of linker 5 is set forth in SEQ ID NO:95, and the amino acid sequence of linker 6 is set forth in SEQ ID NO:96.

[0094] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CH1 is a CH1 sequence of IgG. In some embodiments, the CH1 is a CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.

[0095] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 59.

[0096] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the amino acid sequence of one subunit of the Fc region is set forth in SEQ ID NO:94.

[0097] In some embodiments, the antigen-binding molecule according to any one of the above is two first chains comprising the amino acid sequence of SEQ ID NO: 124 and two second chains comprising the amino acid sequence of SEQ ID NO: 65; or two first chains comprising the amino acid sequence of SEQ ID NO: 124 and two second chains comprising the amino acid sequence of SEQ ID NO: 66; Includes:

[0098] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises two first chains whose amino acid sequence is set forth in SEQ ID NO: 124 and two second chains whose amino acid sequence is set forth in SEQ ID NO: 65.

[0099] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GUCY2C and two antigen-binding modules that specifically bind to CD3, wherein the antigen-binding modules that specifically bind to GUCY2C are Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer.

[0100] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises two first chains having a structure represented by formula (k), two second chains having a structure represented by formula (j), and two third chains having a structure represented by formula (l), (k) [GUCY2C-VH]-[CH1]-[linker 7]-[CD3-VH]-[linker 4]-[Obscurin chain]-[one subunit of the Fc region]; (j) [GUCY2C-VL]-[CL], (l) [CD3-VL]-[linker 4]-[Titin chain], wherein the linker 4 and the linker 7 are peptide linkers, or the linker 4 and the linker 7 do not exist; The structures represented by formulae (k), (j) and (l) are arranged from the N-terminus to the C-terminus.

[0101] In some embodiments, the antigen-binding molecule according to any one of the above, The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or The CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55.

[0102] In some embodiments, the antigen-binding molecule according to any one of the above, The amino acid sequence of the above CD3-VH is shown in SEQ ID NO: 77, and the amino acid sequence of the above CD3-VL is shown in SEQ ID NO: 78, and the amino acid sequence of the above GUCY2C-VH is shown in SEQ ID NO: 56, and the amino acid sequence of the above GUCY2C-VL is shown in SEQ ID NO: 54.

[0103] In some embodiments, in any one of the antigen-binding molecules described above, linker 4 and linker 7 are both peptide linkers known in the art, so long as the antigen-binding molecule can exhibit the desired antigen-binding activity. In some embodiments, the length of the peptide linkers is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have a (GGGGS)n structure, where n is 1, 2, or 3. In some embodiments, the amino acid sequence of linker 4 is set forth in SEQ ID NO: 90, and the amino acid sequence of linker 7 is set forth in SEQ ID NO: 97.

[0104] In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain capable of forming a dimer in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain comprises an amino acid sequence such as SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 236.

[0105] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CH1 is a CH1 sequence of IgG. In some embodiments, the CH1 is a CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.

[0106] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 59.

[0107] In some embodiments, the amino acid sequence of one subunit of the Fc region is set forth in SEQ ID NO:86.

[0108] In some embodiments, the antigen-binding molecule according to any one of the above is two first chains comprising the amino acid sequence of SEQ ID NO: 125, two second chains comprising the amino acid sequence of SEQ ID NO: 65, and two third chains comprising the amino acid sequence of SEQ ID NO: 98; or two first chains comprising the amino acid sequence of SEQ ID NO: 125, two second chains comprising the amino acid sequence of SEQ ID NO: 66, and two third chains comprising the amino acid sequence of SEQ ID NO: 98; Includes:

[0109] In some embodiments, the antigen-binding molecule of any one of the above embodiments comprises two first chains whose amino acid sequence is set forth in SEQ ID NO: 125, two second chains whose amino acid sequence is set forth in SEQ ID NO: 65, and two third chains whose amino acid sequence is set forth in SEQ ID NO: 98.

[0110] In another aspect, the present disclosure further provides an anti-GUCY2C antibody capable of specifically binding to GUCY2C, the antibody comprising a heavy chain variable region GUCY2C-VH and a light chain variable region GUCY2C-VL, wherein: (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 25, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 54, 26, 50, 51, 52 or 53, respectively; or (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 37 or 15, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 16, 34, 35 or 36, respectively; or (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 42 or 17, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41 or 18, respectively; or iv-1) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 273, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 274, respectively; or iv-2) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 275, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 276, respectively; or iv-3) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 277, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 278, respectively; or iv-4) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 279, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 280, respectively; or iv-5) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 330, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 331, respectively; or iv-6) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 332, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 333, respectively; or iv-7) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 334, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 335, respectively; or iv-8) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 336, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 337, respectively; or iv-9) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 338, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 339, respectively; or iv-10) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 340, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 341, respectively; or iv-11) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 342, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 343, respectively; or iv-12) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 344, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 345, respectively; or iv-13) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 346, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 347, respectively; or iv-14) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 348, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 349, respectively; or iv-15) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 350, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 351, respectively; or iv-16) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 352, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 353.

[0111] In some embodiments, the anti-GUCY2C antibody is (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 25, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 54, respectively; or (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 37, respectively, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 16, respectively; or (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the above GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 42, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the above GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41.

[0112] In some embodiments, the GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0113] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45, or 46, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27 or 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 28, 29 or 30, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 40 or 12, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 39 or 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or iv-1) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 250, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 251, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 252, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 253, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 254, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 255; or iv-2) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 256, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 257, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 258, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 259, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 260; or iv-3) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 262, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 263, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 266, or iv-4) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 267, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 268, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 269, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 270, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 271, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 272; or iv-5) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 281, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 282, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 283, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 284, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 285, or iv-6) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 288, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 289, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 290; or iv-7) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 291, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 292, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 293, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 294, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 295, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 296, or iv-8) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 297, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 298, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-9) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 301, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 303, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 304, or iv-10) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 305, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 306, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or iv-11) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 128, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 307, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 308, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 309, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 310, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 311, or iv-12) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 312, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 313, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 314, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 315, or iv-13) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 316, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 317, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-14) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 318, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 319, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 320, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 321, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 322, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 323, or iv-15) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 324, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 325, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 326, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 327, or iv-16) The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 328, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 302, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 329, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 300.

[0114] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 40, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 39, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0115] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 12, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0116] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or The GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 44, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or The GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 45, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or The above GUCY2C-VH comprises GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 4, GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 5, and GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 6, and the above GUCY2C-VL comprises GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 7, GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 30, and GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 9.

[0117] In some embodiments, in any one of the above anti-GUCY2C antibodies, GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 are defined according to the Kabat numbering convention.

[0118] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody is a murine antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody.

[0119] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain constant region and a light chain constant region, preferably, the heavy chain constant region is a human IgG1 constant region, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions, more preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 58, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 59.

[0120] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the GUCY2C-VH and GUCY2C-VL are humanized and contain the FR region of a human antibody.

[0121] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: (i) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 21, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 39L, 43K, 69L, 71V, and 73K; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45, or 46, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 24, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R, and 74K; or (ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 27 or 6, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K, and 67A; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 8, 28, 29, or 30, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 9, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 15P, 16G, 42S, 43S, 45K, 46S, 47W, 49Y, 58V, 71Y, and 76S; or (iii) The GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 40 or 12, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 2F, 28S, 69L, 71V, 73Q and 75S, and the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 39 or 8, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 42S, 43S, 46P, 47W, 58V and 71Y.

[0122] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: (i) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 21, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 39L, 43K, 69L, 71V, and 73K; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 24, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R, and 74K; or (ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 27, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K, and 67A; the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 9, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 15P, 16G, 42S, 43S, 45K, 46S, 47W, 49Y, 58V, 71Y, and 76S; or (iii) The GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 40, and the FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 2F, 28S, 69L, 71V, 73Q, and 75S, and the GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 39, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 14, and the FR region of the GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 42S, 43S, 46P, 47W, 58V, and 71Y.

[0123] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein: i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, 25 or 57, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 26, 48, 49, 50, 51, 52 or 53; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, 15 or 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 16, 31, 32, 34, 35 or 36; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42 or 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41 or 18; or iv-1) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 273, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 274, or iv-2) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 275, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 276; or iv-3) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 277, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 278; or iv-4) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 279, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 280; or iv-5) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 330, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 331; or iv-6) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 332, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 333; or iv-7) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 334, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 335; or iv-8) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 336, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 337; or iv-9) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 338, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 339; or iv-10) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 340, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 341; or iv-11) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 342, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 343; or iv-12) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 344, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 345; or iv-13) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 346, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 347; or iv-14) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 348, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 349; or iv-15) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 350, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 351; or iv-16) The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 352, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 353.

[0124] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 48, 49, 50, 51, or 52; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 25, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 26; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 32, 34, or 36; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO:17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO:18.

[0125] In some embodiments, the antigen-binding molecule according to any one of the above, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or iii) The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41.

[0126] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 56, and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 54.

[0127] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 56, and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 55.

[0128] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 37, and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 33.

[0129] In some embodiments, the antigen-binding molecule is any one of the above, wherein the amino acid sequence of the GUCY2C-VH is represented by SEQ ID NO: 42 and the amino acid sequence of the GUCY2C-VL is represented by SEQ ID NO: 41.

[0130] In some embodiments, the anti-GUCY2C antibody according to any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 64, and the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 65 or 66; or the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 60, and the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 61; or The heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:62, and the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:63.

[0131] In some embodiments, the anti-GUCY2C antibody according to any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 64, and the light chain comprises the amino acid sequence of SEQ ID NO: 65 or 66; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 60 and the light chain comprises the amino acid sequence of SEQ ID NO: 61; or The heavy chain comprises the amino acid sequence of SEQ ID NO:62, and the light chain comprises the amino acid sequence of SEQ ID NO:63.

[0132] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 64 and the amino acid sequence of the light chain is shown in SEQ ID NO: 65.

[0133] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 64 and the amino acid sequence of the light chain is shown in SEQ ID NO: 66.

[0134] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 60 and the amino acid sequence of the light chain is shown in SEQ ID NO: 61.

[0135] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 62 and the amino acid sequence of the light chain is shown in SEQ ID NO: 63.

[0136] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody is an antibody fragment, and wherein the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv or dAb.

[0137] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody is a bispecific antibody.

[0138] In some embodiments, the anti-GUCY2C antibody is any one of the above, wherein the anti-GUCY2C antibody is a bispecific antibody that specifically binds to GUCY2C and CD3.

[0139] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the antigen-binding molecules described above or any one of the anti-GUCY2C antibodies described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0140] In another aspect, the present disclosure further provides an isolated nucleic acid encoding any one of the antigen-binding molecules described above or any one of the anti-GUCY2C antibodies described above.

[0141] In another aspect, the present disclosure further provides a vector comprising the isolated nucleic acid described above.

[0142] In another aspect, the present disclosure further provides a host cell comprising the isolated nucleic acid described above.

[0143] In another aspect, the present disclosure further provides a method for treating a disease, the method comprising administering to a subject a therapeutically effective amount of any one of the antigen-binding molecules described above, or any one of the anti-GUCY2C antibodies described above, or any one of the compositions described above.

[0144] In another aspect, the present disclosure further provides use of any one of the above antigen-binding molecules or any one of the above antibodies or compositions thereof in the preparation of a medicament for treating a disease.

[0145] In another aspect, the present disclosure further provides any one of the antigen-binding molecules described above, any one of the anti-GUCY2C antibodies described above, or any one of the compositions described above for use as a medicament. In some embodiments, the medicament is used for the treatment of a disease.

[0146] In some embodiments, the disease described in any one of the above is a proliferative disease or a tumor. In some embodiments, the disease described in any one of the above is a tumor, and the tumor is selected from the group consisting of gastrointestinal cancer (gastric cancer (gastric adenocarcinoma) and intestinal cancer (small intestine cancer, colorectal cancer (colon cancer and rectal cancer))), esophageal cancer (i.e., esophageal cancer), anal cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, kidney cancer (renal cell carcinoma), urothelial carcinoma, breast cancer, bladder cancer, thymic cancer, melanoma, glioma (glioblastoma), sarcoma (osteosarcoma), ovarian cancer, thyroid cancer, cervical squamous cell carcinoma, uterine cancer, and ovarian cancer. endometrial cancer, lung cancer (non-small cell lung cancer (squamous cell carcinoma of the lung, adenocarcinoma of the lung) and small cell lung cancer), skin cancer, head and neck cancer (squamous cell carcinoma of the head and neck), brain cancer, glioblastoma multiforme, gastroesophageal cancer (gastroesophageal adenocarcinoma), metastatic adenocarcinoma of the liver, multiple myeloma, lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, or B-cell lymphoma), or leukemia (e.g., acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL)).

[0147] In some embodiments, cells of the tumor or vascular endothelial cells adjacent to the tumor express GUCY2C.

[0148] In some embodiments, the tumor is a solid tumor.

[0149] In some embodiments, the tumor is a solid tumor, and the solid tumor is selected from the group consisting of gastrointestinal cancer (gastric cancer (gastric adenocarcinoma) and intestinal cancer (small intestine cancer and colorectal cancer (colon cancer and rectal cancer))), esophageal cancer (i.e., esophageal cancer), anal cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, kidney cancer (renal cell carcinoma), urothelial carcinoma, breast cancer, bladder cancer, thymic cancer, melanoma, glioma (glioblastoma), sarcoma (osteosarcoma), ovarian cancer, thyroid cancer, cervical squamous cell carcinoma, endometrial adenocarcinoma, lung cancer (non-small cell lung cancer (squamous cell carcinoma of the lung, adenocarcinoma of the lung) and small cell lung carcinoma), skin cancer, head and neck cancer (squamous cell carcinoma of the head and neck), brain cancer, glioblastoma multiforme, gastroesophageal cancer (gastroesophageal adenocarcinoma), or liver metastatic adenocarcinoma.

[0150] In some embodiments, the tumor is a gastrointestinal cancer, preferably a gastric cancer or intestinal cancer, more preferably a gastric adenocarcinoma, small intestine cancer or colorectal cancer.

[0151] In some embodiments, the tumor is a non-solid tumor.

[0152] In some embodiments, the tumor is a non-solid tumor, and the non-solid tumor is multiple myeloma, lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma), or leukemia (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL)).

[0153] In some embodiments, the disease is a GUCY2C-associated disease, and in some embodiments, the disease is a GUCY2C-expressing disease.

[0154] In some embodiments, the method of treating the disease further comprises using a second therapeutic agent, in some embodiments, the second therapeutic agent comprises an anti-tumor drug, radiation therapy, an antibody-drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an anti-tumor drug, an immune checkpoint inhibitor, or a combination thereof.

[0155] In some embodiments, in the method of treating the above-mentioned disease, the second therapeutic agent is administered simultaneously, sequentially, or separately from the antigen-binding molecule of any one of the present disclosures.

[0156] In some embodiments, in the method of treating the above disease, the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR.

[0157] In some embodiments, in the method of treating a disease as described above, the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR, which comprises at least one antigen-binding module that specifically binds to CD28 and at least one antigen-binding module that specifically binds to EGFR.

[0158] In some embodiments, the method for treating the disease described above, wherein the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR, comprising at least one antigen-binding module that specifically binds to CD28 and at least one antigen-binding module that specifically binds to EGFR, wherein the antigen-binding module that specifically binds to CD28 comprises the heavy chain variable region CD28-VH and the light chain variable region CD28-VL, and wherein the antigen-binding module that specifically binds to EGFR comprises the heavy chain variable region EGFR-VH and the light chain variable region EGFR-VL.

[0159] In some embodiments, a method for treating the above diseases, comprising: i) the CD28-VH comprises CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 146, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 147, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 148, and the CD28-VL comprises CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 149, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 150, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 151; or ii) the CD28-VH comprises CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162, and the CD28-VL comprises CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 149, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 150, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 163; or iii) The CD28-VH comprises CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 168, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 169, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 170, and the CD28-VL comprises CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 171, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 172, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 173.

[0160] In some embodiments, a method for treating the above diseases, comprising: i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 154, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 155; or ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 164, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 165; or iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 174, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 175.

[0161] In some embodiments, the method for treating the above-mentioned disease, wherein the antigen binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, The EGFR-VH comprises an EGFR-HCDR1 having the amino acid sequence of SEQ ID NO: 140, an EGFR-HCDR2 having the amino acid sequence of SEQ ID NO: 141, and an EGFR-HCDR3 having the amino acid sequence of SEQ ID NO: 142, and the EGFR-VL comprises an EGFR-LCDR1 having the amino acid sequence of SEQ ID NO: 143, an EGFR-LCDR2 having the amino acid sequence of SEQ ID NO: 144, and an EGFR-LCDR3 having the amino acid sequence of SEQ ID NO: 145.

[0162] In some embodiments, the method of treating the disease is as described above, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 152, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 153.

[0163] In some embodiments, the method for treating a disease described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an Fc region. In some embodiments, the Fc region is an IgG Fc region. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions capable of reducing binding of the Fc region to an Fcγ receptor. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.

[0164] In some embodiments, a method for treating a disease is provided, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an Fc region, the Fc region comprising a first subunit, Fc1, and a second subunit, Fc2, that can associate with each other, and the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce Fc region homodimerization. In some embodiments, the Fc1 has a knob structure conforming to knob-in-hole technology, and the Fc2 has a hole structure conforming to knob-in-hole technology. In some embodiments, the Fc1 has a W at amino acid position 366, and the Fc2 has an S at amino acid position 366, an A at amino acid position 368, and a V at amino acid position 407, numbered according to the EU index. In some embodiments, the Fc1 has a C at amino acid position 354 and a W at amino acid position 366, and the Fc2 has a C at amino acid position 349, an S at amino acid position 366, an A at amino acid position 368, and a V at amino acid position 407, numbered according to the EU index. In some embodiments, the Fc1 comprises the amino acid sequence of SEQ ID NO:248, and the Fc2 comprises the amino acid sequence of SEQ ID NO:249.

[0165] In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain capable of forming a dimer in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Titin chain comprises an amino acid sequence such as SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 236.

[0166] In some embodiments, in any one of the antigen-binding molecules described above, linker 8 is any peptide linker known in the art, so long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide comprising 1 to 50 or 3 to 20 amino acid residues. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have a (GGGGS)n structure, where n is 1, 2, or 3. In some embodiments, the sequence of linker 8 is GGGGS (SEQ ID NO: 90).

[0167] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CH1 is a CH1 sequence of IgG. In some embodiments, the CH1 is a CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.

[0168] In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule of any one of the above, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 59.

[0169] In some embodiments, there is provided a method for treating a disease as described above, wherein the bispecific antibody that specifically binds to both CD28 and EGFR comprises one antigen-binding module that specifically binds to CD28 and one antigen-binding module that specifically binds to EGFR, the antigen-binding module that specifically binds to CD28 is a substituted Fab comprising a dimerizable titin chain and an obscurin chain, and the antigen-binding module that specifically binds to EGFR is a Fab.

[0170] In some embodiments, a method for treating the above diseases, comprising: The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain having the structure shown in formula (m), one second chain having the structure shown in formula (n), one third chain having the structure shown in formula (o), and one fourth chain having the structure shown in formula (p); (m) [CD28-VH]-[linker 8]-[Titin chain]-[Fc1], (n) [CD28-VL]-[linker 8]-[Obscurin chain], (o) [EGFR-VH]-[CH1]-[Fc2]; (p)[EGFR-VL]-[CL], wherein the linker 8 is a peptide linker, or the linker 8 is absent; The structures represented by formulae (m), (n), (o) and (p) are arranged from the N-terminus to the C-terminus.

[0171] In some embodiments, a method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain comprising the amino acid sequence of SEQ ID NO: 156, one second chain comprising the amino acid sequence of SEQ ID NO: 157, one third chain comprising the amino acid sequence of SEQ ID NO: 158, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 159; or The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain comprising the amino acid sequence of SEQ ID NO: 166, one second chain comprising the amino acid sequence of SEQ ID NO: 167, one third chain comprising the amino acid sequence of SEQ ID NO: 158, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 159.

[0172] In some embodiments, there is provided a method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain having the amino acid sequence set forth in SEQ ID NO: 156, one second chain having the amino acid sequence set forth in SEQ ID NO: 157, one third chain having the amino acid sequence set forth in SEQ ID NO: 158, and one fourth chain having the amino acid sequence set forth in SEQ ID NO: 159.

[0173] In some embodiments, there is provided a method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain having the amino acid sequence set forth in SEQ ID NO: 166, one second chain having the amino acid sequence set forth in SEQ ID NO: 167, one third chain having the amino acid sequence set forth in SEQ ID NO: 158, and one fourth chain having the amino acid sequence set forth in SEQ ID NO: 159.

[0174] In some embodiments, there is provided a method for treating a disease described above, wherein the bispecific antibody that specifically binds to both CD28 and EGFR comprises one antigen-binding module that specifically binds to CD28 and one antigen-binding module that specifically binds to EGFR, the antigen-binding module that specifically binds to CD28 is a Fab, and the antigen-binding module that specifically binds to EGFR is a substituted Fab that includes a Titin chain and an Obscurin chain that can form a dimer.

[0175] In some embodiments, a method for treating the above diseases, comprising: The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain having the structure represented by formula (q), one second chain having the structure represented by formula (r), one third chain having the structure represented by formula (s), and one fourth chain having the structure represented by formula (t), (q) [CD28-VH]-[CH1]-[Fc1]; (r)[CD28-VL]-[CL]; (s) [EGFR-VH]-[linker 8]-[Obscurin chain]-[Fc2], (t) [EGFR-VL]-[linker 8]-[Titin chain], wherein the linker 8 is a peptide linker, or the linker 8 is absent; The structures represented by formulas (q), (r), (s) and (t) are arranged from the N-terminus to the C-terminus.

[0176] In some embodiments, there is provided a method of treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain comprising the amino acid sequence of SEQ ID NO: 176, one second chain comprising the amino acid sequence of SEQ ID NO: 177, one third chain comprising the amino acid sequence of SEQ ID NO: 178, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 179.

[0177] In some embodiments, there is provided a method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 176, one second chain whose amino acid sequence is set forth in SEQ ID NO: 177, one third chain whose amino acid sequence is set forth in SEQ ID NO: 178, and one fourth chain whose amino acid sequence is set forth in SEQ ID NO: 179.

[0178] The antigen-binding molecules provided in the present disclosure have favorable characteristics in terms of therapeutic activity, safety, pharmacokinetic properties, and druggability (e.g., stability). [Brief explanation of the drawings]

[0179] [Figure 1A] FIG. 1 is a schematic diagram of the structure of Format 1 of the GUCY2C-CD3 bispecific antibody. [Figure 1B] 1 is a schematic diagram of the structure of Format 2 of the GUCY2C-CD3 bispecific antibody, in which Ob stands for Obscurin. [Figure 1C] 1 is a schematic diagram of the structure of GUCY2C-CD3 bispecific antibody Format 3, in which Ob stands for Obscurin. [Figure 1D] FIG. 1 is a schematic diagram of the structure of Format 4 of the GUCY2C-CD3 bispecific antibody. [Figure 1E] 1 is a schematic diagram of the structure of GUCY2C-CD3 bispecific antibody Format 5, in which Ob stands for Obscurin. [Figure 2A] Binding ability of GUCY2C monoclonal antibody to HCT116 / hGUCY2C cells. [Figure 2B] Binding ability of GUCY2C monoclonal antibody to HCT116 / hGUCY2C cells. [Figure 2C] Binding ability of GUCY2C monoclonal antibody to HCT116 / hGUCY2C cells. [Figure 2D] Binding ability of GUCY2C monoclonal antibody to HT-55 cells. [Figure 2E] Binding ability of GUCY2C monoclonal antibody to T84 cells. [Figure 3A] Binding ability of GUCY2C-CD3 bispecific antibody to HCT116 / hGUCY2C cells. [Figure 3B] Binding ability of GUCY2C-CD3 bispecific antibody to HT55 cells. [Figure 3C] Binding ability of GUCY2C-CD3 bispecific antibody to Jurkat cells. [Figure 4A] The objective of this study was to test the activating ability of the antibodies against Jurkat / NFAT cells using HCT116 / hGUCY2C as target cells. [Figure 4B]The purpose of this study was to test the activation ability of antibodies against Jurkat / NFAT cells using LS1034 as target cells. [Figure 5A] This shows the results of IFN-γ release due to the cooperative action of GUCY2C-CD3 bispecific antibody and LS1034 cells. [Figure 5B] This shows the results of IFN-γ release due to the cooperative action of GUCY2C-CD3 bispecific antibody and LS174T cells. [Figure 5C] This shows the results of IFN-γ release due to the cooperative action of GUCY2C-CD3 bispecific antibody and LS174T cells. [Figure 6A] This shows the results of IL-6 release due to the cooperative action of GUCY2C-CD3 bispecific antibody and LS1034 cells. [Figure 6B] This shows the results of IL-6 release due to the cooperative action of GUCY2C-CD3 bispecific antibody and LS174T cells. [Figure 6C] This shows the results of IL-6 release due to the cooperative action of GUCY2C-CD3 bispecific antibody and LS174T cells. DETAILED DESCRIPTION OF THE INVENTION

[0180] term The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0181] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "comprise," "have," "contain," and the like should be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning. Unless otherwise specified, "comprise" includes "consisting of." For example, in the case of GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, it specifically covers GUCY2C-HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 1.

[0182] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0183] The term "and / or," e.g., "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be used to provide clear support for both meanings or either meaning.

[0184] The term "CD28" (cluster of differentiation 28, Tp44) refers to any CD28 protein from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides costimulatory signals necessary for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells by CD28 can also signal effectively for the production of various interleukins. CD28 is the receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is listed in UniProt (www.uniprot.org) accession number P10747.

[0185] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by codons and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in a manner similar to a naturally occurring amino acid.

[0186] The term "amino acid mutation" includes amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be used to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or derivatives of the 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods for modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be utilized. As used herein, various names may be used to refer to the same amino acid mutation. As used herein, the amino acid residue at a particular site may be designated by the position + amino acid residue, e.g., 366W indicates that the amino acid residue at position 366 is W. T366W indicates that the amino acid residue at position 366 has been mutated from the original T to W.

[0187] The term "antigen-binding molecule" is used in the broadest sense to encompass a variety of molecules that specifically bind to antigens as long as they exhibit the desired antigen-binding activity, including, but not limited to, antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins formed by fusing both. The antigen-binding molecule herein comprises a variable region (VH) and a variable region (VL), which together constitute an antigen-binding domain. Illustratively, the antigen-binding molecule herein is a bispecific antigen-binding molecule (e.g., a bispecific antibody).

[0188] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or portions thereof), provided they exhibit the desired antigen-binding activity. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain contains one variable region (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains one variable region (VL), also called a variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL).

[0189] The term "bispecific antibody" refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that can specifically bind to two different antigens or at least two different antigen epitopes on the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art; they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies based on the integrity of the IgG molecule, bispecific antibodies can be divided into bivalent, trivalent, tetravalent or higher valent bispecific antibodies based on the number of antigen-binding regions, and bispecific antibodies with symmetrical structures can be divided into bispecific antibodies with symmetrical structures and bispecific antibodies with asymmetrical structures. Here, bispecific antibodies based on antibody fragments are, for example, Fab fragments lacking Fc fragments. Bispecific antibodies are formed by binding two or more Fab fragments into a single molecule, which have relatively low immunogenicity, a small molecular weight, and relatively high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-like bispecific antibodies (e.g., having Fc fragments) have a relatively large molecular weight, and the Fc fragment contributes to antibody purification and improves its solubility and stability. The Fc portion can also bind to the receptor FcRn to increase the serum half-life of the antibody. Typical structural models of bispecific antibodies include, for example, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (Aran See F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages585~608 (2019), Chen S1 et al., J Immunol Res.2019 Feb 11, 2019:4516041).

[0190] The term "variable region" or "variable domain" refers to the domain that binds to an antigen in an antigen-binding molecule. Herein, the heavy chain variable region of an antigen-binding module that specifically binds to GUCY2C is designated GUCY2C-VH, and the light chain variable region thereof is designated GUCY2C-VL. The heavy chain variable region of an antigen-binding module that specifically binds to CD3 is designated CD3-VH, and the light chain variable region thereof is designated CD3-VL. VH and VL each contain four conserved framework regions (FR) and three complementarity-determining regions (CDR). The term "complementarity-determining region" or "CDR" refers to the region that primarily promotes antigen binding in the variable domain, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, while VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Herein, the three CDR regions in GUCY2C-VH are designated GUCY2C-HCDR1, GUCY2C-HCDR2, and GUCY2C-HCDR3, respectively; the three CDR regions in GUCY2C-VL are designated GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3, respectively; the three CDR regions in CD3-VH are designated CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3, respectively; and the three CDR regions in CD3-VL are designated CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3, respectively. Each VH and VL has, from N-terminus to C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A single VH or VL can be sufficient to confer antigen-binding specificity.

[0191] The amino acid sequence boundaries of CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278), and the correspondence between various numbering systems is well known to those of skill in the art. The numbering conventions of the present disclosure are as shown in Table 1 below.

[0192] [Table 1]

[0193] Unless otherwise stated, all variable region and CDR sequences in the examples of this disclosure follow the "Kabat" numbering convention.

[0194] The term "antibody fragment" refers to a molecule distinct from an intact antibody, including a portion of the intact antibody that retains the intact antibody's antigen-binding ability. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies (e.g., bispecific antibodies) composed of antibody fragments.

[0195] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, and includes native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226, or from Pro230, to its carboxy terminus. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise specified, the numbering convention for the Fc region is the EU index.

[0196] The term "Titin chain" refers to a peptide fragment of the Titin protein that is 78 to 118 amino acids in length and contains the Titin Ig-Like 152 domain, or a functional variant thereof; the Titin chain can bind to the Obscurin Ig-Like 1 or Obscurin-Like Ig-Like 1 domain to form a dimerization complex. The term "Obscurin chain" refers to a peptide fragment of the Obscurin protein that is 87 to 117 amino acids in length and contains the Obscurin Ig-Like 1 domain, or a functional variant thereof; or a peptide fragment of the Obscurin-Like 1 protein that is 78 to 118 amino acids in length and contains the Obscurin-Like Ig-Like 1 domain, or a functional variant thereof; the Obscurin chain can bind to the Titin Ig-Like 152 domain to form a dimerization complex. The Titin chain and Obscurin chain of the present disclosure can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S), and the substitution does not affect the binding of the antigen-binding molecule to an antigen.

[0197] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular origin or species, while the remainder of the heavy and / or light chain is derived from a different origin or species.

[0198] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while having relatively low immunogenicity in humans, which may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (i.e., the constant and variable region framework region portions).

[0199] The term "affinity" refers to the overall strength of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to internal binding affinity and reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can typically be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art (such as those described herein). The terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, while the terms "kdis" or "kd" as used herein refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured by methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

[0200] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules within the population are the same except for possible minor natural mutations. In contrast, a polyclonal antibody preparation typically contains a plurality of different antibodies with different amino acid sequences in their variable domains, which are generally specific for different epitopes. "Monoclonal" describes the character of an antibody obtained from an essentially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

[0201] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent of an antigen-binding protein (e.g., an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0202] The term "epitope" refers to an area or region on an antigen capable of specific binding by an antibody or antigen-binding fragment thereof. An epitope may be formed from contiguous amino acids (linear epitope) or may contain non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity by antigen folding (i.e., tertiary folding of a proteinaceous antigen). Conformational and linear epitopes differ in that antibody binding to conformational epitopes is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by conventional methods in the art, including, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies," Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0203] The terms "capable of specifically binding," "specifically binds," or "binding" refer to the ability of an antibody to bind to an antigen or epitope with greater affinity than to other antigens or epitopes. Generally, antibodies bind to antigens with a greater affinity than other antigens or epitopes. -7 M or less (e.g., about 1 × 10 -8An antibody binds to an antigen or epitope with an equilibrium dissociation constant (KD) of 1 M or less. In some embodiments, the KD of an antibody for binding to an antigen is 10% or less (e.g., 1%) of the KD for binding to a non-specific antigen (e.g., BSA, casein) of the antibody. KD can be measured by known methods, for example, FACS or surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or epitope within an antigen may have cross-reactivity to other related antigens, for example, cross-reactivity to corresponding antigens from other species (homologs) (e.g., humans, or monkeys such as cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes) (chimpanzee, chimp)) or common marmosets (Callithrix jacchus) (marmoset).

[0204] The term "does not bind" means that the antibody is unable to bind to an antigen or an epitope within the antigen in the manner of specific binding described above. For example, the antibody may bind to an antigen in a concentration of about 1 x 10 -6 It binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of M or greater.

[0205] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to a target antigen. Specific antigen-binding modules include, for example, the antigen-binding domain of an antibody, including a heavy chain variable region and a light chain variable region. The term "antigen-binding module that specifically binds to GUCY2C" refers to a module that can bind to GUCY2C with sufficient affinity so that a molecule containing the module can be used as a diagnostic and / or therapeutic agent targeting GUCY2C. For example, an antigen-binding module that specifically binds to GUCY2C has an equilibrium dissociation constant (KD) of < about 10 nM, as measured by a surface plasmon resonance assay. Antigen-binding modules include antibody fragments, such as Fab, substituted Fab, or scFv, as defined herein.

[0206] The term "linker" refers to a linking unit that links two polypeptide fragments. As used herein, linkers appearing in the same structural formula may be the same or different. A linker may be a peptide linker and contain one or more amino acids, typically about 1 to 30, 2 to 24, or 3 to 15 amino acids. Linkers used herein may be the same or different. When "-" appears in a structural formula, it indicates that the units on both sides are directly linked by a covalent bond.

[0207] "Tm" is the melting denaturation temperature (intrinsic fluorescence). When a protein is denatured (by heating or the action of a denaturing agent), the tertiary structure opens and the microenvironment of aromatic amino acids is altered, resulting in a change in the emission fluorescence spectrum. In this disclosure, Tm1 refers to the temperature at which the fluorescence is changed to half of its maximum value.

[0208] "Tonset" is the denaturation onset temperature. It refers to the temperature at which the protein begins to denature, i.e., the temperature at which the fluorescence value begins to change.

[0209] "Tagg" is the aggregation onset temperature. Aggregates were detected by static light scattering at two wavelengths, 266 nm and 473 nm, to monitor the temperature at which sample aggregation began. Tagg 266 indicates that the aggregation onset temperature was monitored at 266 nm.

[0210] The term "nucleic acid" may be used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term covers nucleic acids containing known nucleoside analogs or modified backbone residues or linkers, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleoside. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0211] An "isolated nucleic acid" refers to a nucleic acid molecule separated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells, as defined below. These cells typically contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. The isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including one or more such nucleic acid molecules contained in a single vector or separate vectors, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. Specifically, as described in more detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a degenerate base and / or deoxyinosine residue.

[0212] The terms "polypeptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and apply to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0213] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. Gaps can be introduced as needed during the alignment process to maximize the percentage of sequence identity, and any conservative substitutions are not considered part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved using techniques known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to the measured alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0214] The terms "fused" or "linked" refer to covalent linkage of elements (e.g., an antigen-binding module and an Fc domain) either directly or via a linker.

[0215] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop that can be ligated to an additional DNA segment. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which another DNA segment can be ligated to the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after introduction and thereby replicate along with the host genome. The term "expression vector" or "expression construct" refers to a vector that is applicable to transformation of a host cell and contains (in conjunction with the host cell) nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or regulate transcription, translation, and RNA splicing of coding regions operably linked to introns, if any.

[0216] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. As used herein, the term includes mutant progeny that have the same function or biological activity as cells screened or selected from the original transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells, including, but not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, and Pichia stiptis. stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.Host cells of this patent do not include those not permitted by patent law.

[0217] As used herein, the expressions "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is further understood that not all progeny will have exactly the same DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as the initially transformed cell for which they were screened are included.

[0218] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but need not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0219] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules or antibodies described herein and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0220] The term "pharmaceutically acceptable carrier" refers to an ingredient in a drug formulation that is different from the active ingredient and that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0221] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0222] "Administration" or "giving," as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means contacting an exogenous agent, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0223] The term "sample" refers to fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secreted tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ media (including cell or organ conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically resected tissues, organ cultures, or cell cultures.

[0224] "Treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention intended to be administered to the individual being treated, and can be performed prophylactically or during a clinical pathological process. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction / reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, amelioration or alleviation of the disease state, and regression or improvement of prognosis. In some embodiments, the molecules of the present disclosure are used to delay the formation of disease or slow the progression of disease.

[0225] The terms "recurrence," "relapse," and "relapsed" mean the reappearance of cancer or disease after it has been clinically determined that the disease has disappeared. A diagnosis of distant metastasis or local recurrence of cancer may be considered a recurrence.

[0226] The terms "refractory" or "resistant" refer to a cancer or disease that does not respond to treatment.

[0227] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate those symptoms and / or their underlying causes, prevent the onset of symptoms and / or their underlying causes, and / or improve or ameliorate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or to otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any manner. A "prophylactically effective amount" is an amount that, when administered to a subject, confers a desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state or a reduced likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or prophylactic effect does not necessarily occur with a single dose, but may occur after a series of doses have been administered. Thus, a therapeutically or prophylactically effective amount can be administered in one or multiple doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on various factors, such as, for example, the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0228] The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively function as "brakes" that downregulate or inhibit antitumor immune responses. Immune checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG-3, which directly inhibit immune cells.

[0229] The immune checkpoint inhibitor used in the present disclosure is a substance that inhibits the function of an immune checkpoint molecule. Immune checkpoint inhibitors are not particularly limited, as long as they are capable of inhibiting the function (signal) of an immune checkpoint molecule. Immune checkpoint inhibitors that can be used in the methods of the present disclosure include, but are not limited to, inhibitors of PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), and / or TGFRβ. Inhibition of inhibitory molecules can be achieved by inhibition at the DNA, RNA, or protein level. In embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit the expression of inhibitory molecules. In other embodiments, inhibitors of inhibitory signals are polypeptides that bind to inhibitory molecules, such as soluble ligands or antibodies. Examples of immune checkpoint inhibitors used in the present disclosure include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and may preferably be anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0230] Antigen-binding molecules according to the present disclosure The present disclosure provides antigen-binding molecules that have many advantageous properties, such as good in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties, and druggability (e.g., yield, purity, stability, etc.).

[0231] Exemplary antigen-binding molecules The antigen-binding molecules of the present disclosure include antigen-binding molecules that specifically bind to GUCY2C and CD3 (e.g., bispecific antibodies) and anti-GUCY2C antibodies. In particular, the antigen-binding molecules of the present disclosure have any one of the following properties: High affinity for GUCY2C. In some embodiments, the GUCY2C monoclonal antibody binds to human GUCY2C with a KD value of less than 100 nM (e.g., less than 80 nM, less than 50 nM, less than 10 nM, less than 8 nM, less than 1 nM, less than 0.5 nM), as measured by Biacore.

[0232] b. Relatively good specific binding to cell surface GUCY2C. In some embodiments, the GUCY2C monoclonal antibody has better binding activity than 1608. In some embodiments, the GUCY2C-CD3 bispecific antibody of the present disclosure has relatively good specific binding activity to cells expressing GUCY2C at different levels and has better binding activity than 1608-BsAb, while the GUCY2C-CD3 bispecific antibody of the present disclosure has relatively good specific binding activity to cells expressing Jurkat and has better binding activity than 1608-BsAb.

[0233] c. No non-specific binding to non-target cells (e.g., T cells). In some embodiments, the GUCY2C monoclonal antibody does not non-specifically bind to Jurkat cells that do not express GUCY2C, whereas 1608 clearly binds to Jurkat cells. In some embodiments, the GUCY2C monoclonal antibody of the present disclosure does not non-specifically bind to PBMCs and T cells that do not express GUCY2C, whereas control antibody 1608 clearly binds to PBMCs and T cells non-specifically. The GUCY2C monoclonal antibody of the present disclosure has superior target specificity to 1608.

[0234] d. The antigen epitope bound by the GUCY2C monoclonal antibody is relatively different from that of 1608, and the antibody is unable to competitively bind to the GUCY2C antigen.

[0235] e. Good binding activity to human GUCY2C protein. In some embodiments, the GUCY2C-CD3 biantibody binds to human GUCY2C with a KD value of less than 1 nM (e.g., less than 0.5 nM, less than 0.2 nM, less than 0.1 nM), and the EC 50 Values are measured by ELISA.

[0236] f. The GUCY2C-CD3 bispecific antibody has relatively good cytotoxic activity against target cells expressing different amounts of GUCY2C.

[0237] g. The GUCY2C-CD3 bispecific antibody can effectively induce the release of IFN-γ, which is associated with cytotoxic activity, while releasing a relatively low level of the inflammation-related cytokine IL-6.

[0238] h. Stronger in vivo therapeutic activity. In some embodiments, the GUCY2C-CD3 bispecific antibody, or a combination of the GUCY2C-CD3 bispecific antibody and an EGFR-CD28 bispecific antibody, has superior in vivo therapeutic activity. Combination with the EGFR-CD28 bispecific antibody can effectively improve the efficacy of each agent alone.

[0239] The present disclosure provides an antigen-binding molecule, which comprises at least one antigen-binding module that specifically binds to GUCY2C and at least one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GUCY2C comprises a heavy chain variable region GUCY2C-VH and a light chain variable region GUCY2C-VL, and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.

[0240] Specifically, the Examples herein disclose the antibody series P3-C7, 81, and 146. Hereinafter, the antibodies of the present specification will be described using antibody P3-C7 as an example.

[0241] An antigen-binding molecule or anti-GUCY2C antibody that specifically binds to GUCY2C and CD3, The above GUCY2C-VH comprises a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 having the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 having the amino acid sequence of SEQ ID NO: 21, and the above GUCY2C-VL comprises a GUCY2C-LCDR1 having the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45 or 46, a GUCY2C-LCDR2 having the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 having the amino acid sequence of SEQ ID NO: 24.

[0242] An antigen-binding molecule or anti-GUCY2C antibody that specifically binds to GUCY2C and CD3, The above GUCY2C-VH comprises GUCY2C-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 19, GUCY2C-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 20, and GUCY2C-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 21, and the above GUCY2C-VL comprises GUCY2C-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 47, GUCY2C-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 23, and GUCY2C-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 24.

[0243] In some embodiments, in the antigen-binding molecule or anti-GUCY2C antibody, the GUCY2C-VH and / or the GUCY2C-VL are murine or humanized. In some embodiments, the GUCY2C-VH and / or the GUCY2C-VL are humanized.

[0244] In some embodiments, the humanized GUCY2C-VH comprises FR1, FR2, and FR3 from IGHV1-46*01 and FR4 from IGHJ6*01, which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 39L, 43K, 69L, 71V, and 73K, and / or the humanized GUCY2C-VL comprises FR1, FR2, and FR3 from IGKV2-40*01 or IGKV1-39*01 and FR4 from IGKJ4*01, which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R, and 74K. In some embodiments, the amino acid positions in the variable regions are defined according to the Kabat numbering convention.

[0245] In some embodiments, the antigen binding molecule or anti-GUCY2C antibody described in any one of the above, wherein the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, 25 or 57, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 26, 48, 49, 50, 51, 52 or 53.

[0246] In some embodiments, the antigen-binding molecule or anti-GUCY2C antibody described above is an antigen-binding molecule or anti-GUCY2C antibody, wherein: The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 48, 49, 50, 51, or 52; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO:25, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO:26.

[0247] In some embodiments, the antigen-binding molecule or anti-GUCY2C antibody described above is an antigen-binding molecule or anti-GUCY2C antibody, wherein: The amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 56, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 54; or The amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO:56, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO:55.

[0248] In some embodiments, the antigen binding molecule described above, wherein the CD3-VH comprises a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 71, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 73 or 88; and the CD3-VL comprises a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 76.

[0249] In some embodiments, in any one of the antigen-binding molecules described above, the CD3-VH and / or the CD3-VL are murine or humanized.

[0250] In some embodiments, the CD3-VH and / or the CD3-VL are humanized.

[0251] In some embodiments, the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77 or 89, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78.

[0252] In some embodiments, the amino acid sequence of the CD3-VH is set forth in SEQ ID NO:77, and the amino acid sequence of the CD3-VL is set forth in SEQ ID NO:78.

[0253] In some embodiments, the variable regions and CDRs are defined according to the Kabat numbering convention.

[0254] The antibody series 81 and 146 disclosed in the Examples herein have a similar scope of technical solutions to the antibody series P3-C7 described above.

[0255] Structure of antigen-binding molecules The bispecific antigen-binding molecule of the present disclosure is not limited to a specific molecular structure as long as it has the desired antigen-binding function. For example, the bispecific antigen-binding molecule of the present disclosure may be bivalent (1+1), trivalent (2+1), or tetravalent (2+2). The antigen-binding module in the antigen-binding molecule may be any antibody fragment with antigen-binding activity fused via a peptide linker. The peptide linker of the present disclosure (e.g., linkers 1 to 11) may be any appropriate peptide chain as long as the antigen-binding molecule exhibits the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide comprising 1 to 50 or 3 to 20 amino acid residues. In some embodiments, the peptide linkers each independently have the structure L1-(GGGGS)n-L2, where L1 is a bond, A, G (SEQ ID NO: 82), GS, GGG (SEQ ID NO: 96), GGS (SEQ ID NO: 245), GGGS (SEQ ID NO: 246), or GGGG (SEQ ID NO: 247), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G (SEQ ID NO: 82), GG, GGG (SEQ ID NO: 96), or GGGG (SEQ ID NO: 247), and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3. In some embodiments, the amino acid sequence of linker 1 is set forth in SEQ ID NO: 81, the amino acid sequence of linker 2 is set forth in SEQ ID NO: 82, the amino acid sequence of linker 3 is set forth in SEQ ID NO: 83, the amino acid sequence of linker 4 is set forth in SEQ ID NO: 90, the amino acid sequence of linker 5 is set forth in SEQ ID NO: 95, the amino acid sequence of linker 6 is set forth in SEQ ID NO: 96, and the amino acid sequence of linker 7 is set forth in SEQ ID NO: 97.

[0256] Exemplarily, the antigen-binding molecule of the present disclosure has one first chain having a structure shown in formula (a) and one second chain having a structure shown in formula (b), (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1], (b) [CD3-VL]-[linker 3]-[GUCY2C-VH]-[linker 2]-[Fc2]; wherein the linker 1, linker 2 and linker 3 are the same or different peptide linkers, or the linker 1, linker 2 and linker 3 do not exist; The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.

[0257] Exemplarily, the antigen-binding molecule of the present disclosure has one first chain having a structure shown in formula (a) and one second chain having a structure shown in formula (b), (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1], (b) [CD3-VL]-[linker 3]-[GUCY2C-VH]-[linker 2]-[Fc2]; Among them, The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, wherein linker 1, linker 2 and linker 3 are the same or different peptide linkers, or linker 1, linker 2 and linker 3 are absent; The amino acid sequence of Fc1 is shown in SEQ ID NO: 79, and the amino acid sequence of Fc2 is shown in SEQ ID NO: 80. The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.

[0258] Exemplarily, the antigen-binding molecule of the present disclosure has one first chain having a structure shown in formula (a) and one second chain having a structure shown in formula (b), (a) [GUCY2C-VL]-[GGGSGGGG]-[CD3-VH]-[G]-[Fc1], (b) [CD3-VL]-[GGGGGSGGGG]-[GUCY2C-VH]-[G]-[Fc2], Among them, the structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.

[0259] Exemplarily, the antigen-binding molecule of the present disclosure has one first chain having a structure shown in formula (a) and one second chain having a structure shown in formula (b), (a) [GUCY2C-VL]-[GGGSGGGG]-[CD3-VH]-[G]-[Fc1], (b) [CD3-VL]-[GGGGGSGGGG]-[GUCY2C-VH]-[G]-[Fc2], Among them, The GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, The CD3-VH comprises CD3-HCDR1 represented by SEQ ID NO: 71, CD3-HCDR2 represented by SEQ ID NO: 72, and CD3-HCDR3 represented by SEQ ID NO: 73, and the CD3-VL comprises CD3-LCDR1 represented by SEQ ID NO: 74, CD3-LCDR2 represented by SEQ ID NO: 75, and CD3-LCDR3 represented by SEQ ID NO: 76, The amino acid sequence of Fc1 is shown in SEQ ID NO: 79, and the amino acid sequence of Fc2 is shown in SEQ ID NO: 80. The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.

[0260] Exemplary antigen binding molecules include: one first chain comprising the amino acid sequence of SEQ ID NO: 99 and one second chain comprising the amino acid sequence of SEQ ID NO: 100; or one first chain comprising the amino acid sequence of SEQ ID NO: 101 and one second chain comprising the amino acid sequence of SEQ ID NO: 100; Includes:

[0261] Exemplarily, the antigen-binding molecule comprises one first chain having the structure shown in formula (c), one second chain having the structure shown in formula (d), one third chain having the structure shown in formula (e), and one fourth chain having the structure shown in formula (f), (c) [CD3-VH]-[CH1]-[Fc1]; (d) [CD3-VL]-[CL]; (e) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc2], (f) [GUCY2C-VL]-[linker 4]-[Titin chain], wherein the linker 4 is a peptide linker, or the linker 4 is absent; The structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.

[0262] Exemplarily, the antigen-binding molecule comprises one first chain having the structure shown in formula (c), one second chain having the structure shown in formula (d), one third chain having the structure shown in formula (e), and one fourth chain having the structure shown in formula (f), (c) [CD3-VH]-[CH1]-[Fc1]; (d) [CD3-VL]-[CL]; (e) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc2], (f) [GUCY2C-VL]-[linker 4]-[Titin chain], Among them, The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, wherein linker 4 is a peptide linker or linker 4 is absent; The amino acid sequence of the Obscurin chain is shown in SEQ ID NO: 236, and the amino acid sequence of the Titin chain is shown in SEQ ID NO: 198. The amino acid sequence of Fc1 is shown in SEQ ID NO: 85, and the amino acid sequence of Fc2 is shown in SEQ ID NO: 86. The amino acid sequence of the CH1 is represented by SEQ ID NO: 84, and the amino acid sequence of the CL is represented by SEQ ID NO: 87; The structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.

[0263] Exemplarily, the antigen-binding molecule comprises one first chain having the structure shown in formula (c), one second chain having the structure shown in formula (d), one third chain having the structure shown in formula (e), and one fourth chain having the structure shown in formula (f), (c) [CD3-VH]-[CH1]-[Fc1]; (d) [CD3-VL]-[CL]; (e) [GUCY2C-VH]-[GGGGS]-[Obscurin chain]-[Fc2], (f) [GUCY2C-VL]-[GGGGS]-[Titin chain], Among them, the structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.

[0264] Exemplarily, the antigen-binding molecule comprises one first chain having the structure shown in formula (c), one second chain having the structure shown in formula (d), one third chain having the structure shown in formula (e), and one fourth chain having the structure shown in formula (f), (c) [CD3-VH]-[CH1]-[Fc1]; (d) [CD3-VL]-[CL]; (e) [GUCY2C-VH]-[GGGGS]-[Obscurin chain]-[Fc2], (f) [GUCY2C-VL]-[GGGGS]-[Titin chain], Among them, The GUCY2C-VH comprises a GUCY2C-HCDR1 represented by SEQ ID NO: 4, a GUCY2C-HCDR2 represented by SEQ ID NO: 5, and a GUCY2C-HCDR3 represented by SEQ ID NO: 27, and the GUCY2C-VL comprises a GUCY2C-LCDR1 represented by SEQ ID NO: 7, a GUCY2C-LCDR2 represented by SEQ ID NO: 8, and a GUCY2C-LCDR3 represented by SEQ ID NO: 9, The CD3-VH comprises CD3-HCDR1 represented by SEQ ID NO: 71, CD3-HCDR2 represented by SEQ ID NO: 72, and CD3-HCDR3 represented by SEQ ID NO: 73, and the CD3-VL comprises CD3-LCDR1 represented by SEQ ID NO: 74, CD3-LCDR2 represented by SEQ ID NO: 75, and CD3-LCDR3 represented by SEQ ID NO: 76, The amino acid sequence of the Obscurin chain is shown in SEQ ID NO: 236, and the amino acid sequence of the Titin chain is shown in SEQ ID NO: 198. The amino acid sequence of Fc1 is shown in SEQ ID NO: 85, and the amino acid sequence of Fc2 is shown in SEQ ID NO: 86. The amino acid sequence of the CH1 is represented by SEQ ID NO: 84, and the amino acid sequence of the CL is represented by SEQ ID NO: 87; The structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.

[0265] Exemplary antigen binding molecules include: one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 113; Includes:

[0266] Antigen-binding molecule variants In certain embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are covered. For example, these variants are expected to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antigen-binding molecule. The final construct can be obtained by any combination of deletion, insertion, and substitution, provided that the final construct possesses the desired properties, such as antigen-binding properties.

[0267] Mutant substitutions, insertions and deletions In one embodiment, variants of antigen-binding molecules are provided that have one or more amino acid substitutions. Substitutions can be made in the CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classifications. Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0268] [Table 2]

[0269] Amino acids can be grouped according to common side chain properties as follows: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0270] Non-conservative substitutions refer to the replacement of a member of one class with a member of another class.

[0271] One type of substitutional variant involves substituting one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study has altered (e.g., improved) biological properties relative to the parent antibody (e.g., improved affinity, decreased immunogenicity) and / or essentially retains certain biological properties of the parent antibody. One exemplary substitutional variant is an affinity-matured antibody, which can be readily produced, for example, by phage display-based affinity maturation techniques (e.g., techniques such as those described herein). Briefly, one or more CDR residues are mutated, the mutated antibodies are displayed on phage, and then screened for a particular biological activity (e.g., binding affinity). For example, changes (e.g., substitutions) can be made in the CDRs to improve antibody affinity. Such changes can be made to CDR "hot spots," i.e., residues encoded by codons frequently mutated during somatic maturation and / or residues that contact the antigen, and the resulting variant VH or VL can be tested for binding affinity. In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-targeting, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding.

[0272] In certain embodiments, substitutions, insertions, or deletions may be made in one or more CDRs, so long as such changes do not substantially reduce the antibody's ability to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to a CDR that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0273] A method that can be used to identify potential target sites for mutagenesis in antibodies is called "alanine scanning mutagenesis." In this method, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Additionally, contact points between the antibody and antigen can be identified by examining crystal structures of antigen-antibody complexes. These contact residues and neighboring residues can be targeted or removed as substitution candidates. Mutants can be screened to determine whether they contain desired properties.

[0274] Amino acid sequence insertions include the fusion of polypeptides of one residue or more than 100 residues in length at the amino and / or carboxyl termini, and intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of an enzyme (or a polypeptide which extends the serum half-life of the antibody) to the N- or C-terminus of the antibody.

[0275] Fab modification In one aspect, in an antigen-binding molecule of the present disclosure, one of the antigen-binding module that specifically binds to GUCY2C and the antigen-binding module that specifically binds to CD3 is a substituted Fab comprising a heavy chain variable region, a light chain variable region, a Titin chain, and an Obscurin chain. In the substituted Fab, the original CH1 and CL in the Fab are replaced with the Titin chain and the Obscurin chain. Exemplary sequences of the Titin chain and the Obscurin chain are shown in Tables 3-1 and 3-2.

[0276] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0277] Fc region modification In one aspect, the Fc region of an antigen-binding molecule of the present disclosure comprises one or more amino acid substitutions that reduce binding to its Fc receptor, e.g., binding to its Fcγ receptor, and reduce or eliminate effector function. Native IgG Fc regions are specifically IgG1 Fc regions or IgG4 Fc regions, which may allow antigen-binding molecules of the present disclosure to target Fc receptor-expressing cells rather than antigen-expressing cells. Modified Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region exhibits 50%, 80%, 90%, or 95% or more reduced binding affinity to Fc receptors compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the altered Fc region also has reduced binding affinity to complement, such as C1q, relative to the native Fc region. In some embodiments, the altered Fc region does not have reduced binding affinity to the neonatal Fc receptor (FcRn) relative to the native Fc region. In some examples, the altered Fc region has reduced effector functions, which may include, but are not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen-presenting cell uptake, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced cell apoptosis, reduced dendritic cell maturation, or reduced T cell triggering. For the IgG1 Fc region, substitution of amino acid residues at positions such as 238, 265, 269, 270, 297, 327, and 329 can reduce effector function.In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For IgG4 Fc regions, substitution of amino acid residues at positions such as 228 can reduce effector function.

[0278] The antigen-binding molecule may further comprise disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of the antigen-binding molecule, the following mutations can be introduced: 252Y, 254T, and 256E.

[0279] When an antigen-binding molecule contains different binding modules fused to two subunits in the Fc region, undesired homodimerization may occur. To improve yield and purity, it is advantageous to introduce modifications that promote heterodimerization into the Fc region of an antigen-binding molecule of the present disclosure. In some embodiments, the Fc region of the present disclosure includes modifications using the knob-into-hole (KIH) technique, which involves introducing a knob structure at the interface of the first subunit and a hole structure at the interface of the second subunit. This positions the knob structure at the hole structure, promoting heterodimer formation and inhibiting homodimer formation. The knob structure is constructed by replacing a small amino acid side chain from the interface of the first subunit with a relatively large side chain (e.g., tyrosine or tryptophan). Meanwhile, the hole structure is constructed at the interface of the second subunit by replacing a large amino acid side chain with a relatively small amino acid side chain (e.g., alanine or threonine). The knob and hole structures are prepared by altering the nucleic acid encoding the polypeptide, with selectable amino acid substitutions as shown in Table 4 below.

[0280] [Table 5]

[0281] In addition to the knobs-in-holes technique, other techniques for modifying the CH3 domain of the heavy chain to achieve heterodimerization are also known in the art, e.g., WO1996027011A1, WO1998050431A2, EP1870459A1, WO2007110205A2, WO2009089004A1, WO2010129304A2, WO2011143545A1, WO2012058768A1, WO2013157954A1 and WO2013096291A2.

[0282] The C-terminus of the Fc region may be a complete C-terminus ending with amino acid residues PGK, or may be a truncated C-terminus, e.g., the truncated C-terminus has one or two C-terminal amino acid residues removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, a composition of whole antibodies may comprise a population of antibodies from which any K447 residue and / or G446+K447 residues have been removed. In some embodiments, a composition of whole antibodies may comprise a population of antibodies from which K447 residue and / or G446+K447 residues have not been removed. In some embodiments, a composition of whole antibodies comprises a population of antibodies that are a mixture of antibodies with and without K447 residue and / or G446+K447 residues.

[0283] Recombinant methods Antigen-binding molecules can be produced by recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecule are provided.

[0284] In the case of a natural antibody, a natural antibody fragment, or a bispecific antibody having a homodimeric heavy chain, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. Such nucleic acids encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the light and / or heavy chains of the antibody). The nucleic acids can be present in the same or different expression vectors.

[0285] In the case of a bispecific antibody having a heterodimeric heavy chain, for example, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising the first heterologous monomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heterologous monomeric Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors; typically, the nucleic acids are located in two or three expression vectors, i.e., one vector may contain one or more of these nucleic acids.

[0286] In one embodiment, the present disclosure provides isolated nucleic acids encoding the above-described antibodies. Such nucleic acids can each independently encode any one of the above-described polypeptide chains. In another aspect, the present disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, a method for preparing an antigen-binding molecule is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0287] To produce an antigen-binding molecule recombinantly, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced by conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an antibody), or produced by recombinant methods or obtained by chemical synthesis.

[0288] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0289] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. Suitable host cells for antibody expression (glycosylation) can be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plants and insect cells. Many baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures, such as those described in US Pat. No. 5,959,177, US Pat. No. 6,040,498, US Pat. No. 6,420,548, US Pat. No. 7,125,978, and US Pat. No. 6,417,429, can also be used as hosts. Vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines are SV40-transformed monkey kidney CV1 (COS-7), human embryonic kidney (293 or 293T) cells, baby hamster kidney (BHK) cells, mouse Sertoli (TM4) cells, monkey kidney (CV1) cells, African green monkey kidney (VERO-76) cells, human cervical carcinoma (HELA) cells, canine kidney (MDCK) cells, buffalo rat hepatocytes (BRL3A), human lung (W138) cells, human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of several mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0290] immune complex The present disclosure further provides immunoconjugates comprising antigen-binding molecules conjugated to one or more cytotoxic agents, wherein the one or more cytotoxic agents are, for example, a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioisotope.

[0291] Diagnostic and Therapeutic Compositions In some embodiments, the antigen-binding molecules provided herein can be used to detect the presence of GUCY2C and / or CD3 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In some embodiments, the biological sample includes cells or tissues, such as tumor tissue.

[0292] In one embodiment, an antigen-binding molecule is provided for use in a diagnostic or detection method. In another aspect, a method for detecting the presence of GUCY2C and / or CD3 in a biological sample is provided. In one embodiment, the method comprises contacting a biological sample with the antigen-binding molecule under appropriate conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such a method may be an in vitro method or an in vivo method. In one embodiment, the antigen-binding molecule is used to select subjects suitable for treatment, for example, GUCY2C and / or CD3 are biomarkers for patient selection.

[0293] Exemplary diseases that can be diagnosed using the antigen binding molecules of the present disclosure are, for example, proliferative diseases or tumors (e.g., cancer).

[0294] In some embodiments, a labeled antigen-binding molecule is provided. Labels include, but are not limited to, directly detected labels or modules (e.g., fluorescent, colorimetric, electron-dense, chemiluminescent, and radioactive labels) and indirectly detected modules (e.g., modules indirectly detected via enzymatic reactions or molecular interactions, such as enzymes or ligands).

[0295] In another aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule described above, for example, for use in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and at least one other therapeutic agent.

[0296] Pharmaceutical compositions of the antigen-binding molecules described in the present disclosure are prepared by mixing such antigen-binding molecules having the required purity with one or more pharmaceutically acceptable carriers, and the pharmaceutical compositions are in the form of a lyophilized composition or an aqueous solution. Formulations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0297] Treatment methods and routes of administration Any of the antigen-binding molecules provided herein can be applied in therapeutic methods.

[0298] In a further aspect, the present disclosure provides a use of an antigen-binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a proliferative disease or tumor. The medicament is present in an amount effective for the disease. In some embodiments, the effective amount is a daily dose or a weekly dose. In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents). The "subject" according to any of the above embodiments may be a human.

[0299] In a further aspect, there is provided a pharmaceutical composition comprising the antigen-binding molecule, for example, for use in any of the pharmaceutical uses or therapeutic methods described above. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0300] The antigen-binding molecules of the present disclosure can be used alone or in combination with other reagents for treatment. For example, the antigen-binding molecules of the present disclosure can be administered together with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a bispecific antibody that specifically binds to EGFR and CD28.

[0301] The antigen-binding molecules of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and, when localized treatment is required, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0302] The antigen-binding molecules of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disease being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the delivery site of the reagent, the administration method, the administration schedule, and other factors known to medical professionals. The antigen-binding molecules may not be formulated together with one or more reagents currently used to prevent or treat the disease, but they may optionally be. The effective amount of such other reagents depends on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. They are generally used in the same dosages and by the same administration routes as described herein, or at about 1% to about 99% of the dosages described herein, or at any dosage and by any route empirically / clinically confirmed to be appropriate.

[0303] The appropriate dose of an antigen-binding molecule according to the present disclosure (used alone or in combination with one or more additional therapeutic agents) for preventing or treating a disease depends on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is for prophylaxis or treatment, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient as appropriate in one or a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to about 15 mg / kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dose may range from about 1 μg / kg to about 100 mg / kg or more, depending on the factors discussed above. Thus, for example, for a 50 kg body weight, an exemplary daily dose would be 50 μg to 5 g.

[0304] product Another aspect of the present disclosure provides an article of manufacture containing materials applicable to the treatment, prevention, and / or diagnosis of the above-mentioned conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition that, alone or in combination with another composition, effectively treats, prevents, and / or diagnoses a disease and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a plug pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen-binding molecule of the present disclosure. The label or package insert indicates that the composition is used to treat a selected condition. The article of manufacture may also include (a) a first container containing a composition comprising an antigen-binding molecule of the present disclosure and (b) a second container containing a composition comprising another cytotoxic or other therapeutic agent. The product in this embodiment of the present disclosure may further include a package insert indicating that the composition is applicable to the treatment of a particular medical condition. Alternatively, or in addition, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer. From a business and user perspective, it may further include other necessary materials, including other buffers, diluents, filters, needles, and syringes.

[0305] Examples and Test Examples The present disclosure will be further explained below with reference to examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples and test examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual, or conditions recommended by manufacturers of raw materials or products. Reagents for which specific sources are not specified are standard commercially available reagents.

[0306] Example 1 Antigen-binding molecule containing Titin chain / Obscurin chain The Titin chain / Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including those disclosed in WO2021139758A1 (incorporated herein by reference in their entireties) and CN202110527339.7, and patents claiming priority thereto (incorporated herein by reference in their entireties). A bispecific antibody was constructed, where CL is the kappa light chain constant region in WO2021139758A1, the amino acid sequences of the Titin chain and Obscurin chain are shown in Tables 3-1 and 3-2, and the linker sequence comprises GGGGS (SEQ ID NO: 90), ASTKG (SEQ ID NO: 243), or RTVAS (SEQ ID NO: 244). The amino acid sequences of Fc1, Fc2, and CH1 in this example are as follows:

[0307] Example 1 - Fc1 (knob, SEQ ID NO: 85) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 1 - Fc2 (hole, SEQ ID NO: 86) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 1-CH1 (SEQ ID NO: 84) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0308] 1.1 DI bispecific antibodies With reference to Example 5 of WO2021139758A1, anti-hNGF and hRANKL DI bispecific antibodies: DI-2 to DI-20 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain and second light chain: First heavy chain: from the N-terminus to the C-terminus, [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2]; First light chain: [VL1-I]-[Linker 2]-[Titin chain] from the N-terminus to the C-terminus; A second heavy chain: from the N-terminus to the C-terminus, [VH2-D]-[CH1]-[Fc1]; and Second light chain: [VL2-D]-[CL] from the N-terminus to the C-terminus; VH1-I and VL1-I are the heavy chain variable region and light chain variable region of I0 in WO2021139758A1, respectively, and VH2-D and VL2-D are the heavy chain variable region and light chain variable region of D0 in WO2021139758A1, respectively. In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.

[0309] [Table 6]

[0310] The binding activity of the DI-2 to DI-20 bispecific antibodies to their antigens was determined using the method described in Test Example 4 of WO2021139758A1. The thermal stability of the antibodies was also investigated. Research method: The antibodies were diluted to 5 mg / mL with PBS, and their thermal stability was measured using a high-throughput differential scanning fluorometer (UNCHAINED, model number: Unit). Experimental results showed that the modified bispecific antibodies had no significant change in antigen-binding activity, and compared to DI-2, DI-4 to DI-8, DI-10 to DI-16, and DI-20 had significantly improved Tm1 (°C) and Tonset (°C), indicating superior thermal stability of the bispecific antibodies.

[0311] [Table 7] [Table 8]

[0312] A solution containing DI bispecific antibodies was prepared in a buffer solution of 10 mM acetic acid, pH 5.5, and 9% sucrose. The solution was incubated in an incubator at 40°C for 4 weeks, at which point the antibody concentration was concentrated to the initial concentration, and the solution was observed for precipitation. The experimental results showed that precipitation appeared in the solution of the DI-2 bispecific antibody group, while DI-3 to DI-7 had better stability than DI-2.

[0313] [Table 9]

[0314] 1.2 PL bispecific antibodies Anti-hPDL1 and hCTLA4 PL bispecific antibodies: PL-1 to PL-19 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain, and second light chain: First heavy chain: from the N-terminus to the C-terminus, [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1]; First light chain: [VL1-P]-[Linker 2]-[Titin chain] from the N-terminus to the C-terminus; A second heavy chain: from the N-terminus to the C-terminus, [VH2-L]-[CH1]-[Fc2]; and Second light chain: [VL2-L]-[CL] from the N-terminus to the C-terminus; Among them, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1, respectively, and the amino acid sequences of VH2-L and VL2-L are as follows:

[0315] >VH2-L (SEQ ID NO: 180) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS >VL2-L (SEQ ID NO: 181) EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0316] In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the PL bispecific antibody are shown in the table below.

[0317] [Table 10]

[0318] The binding activity of the PL bispecific antibodies was determined using the ELISA method in Test Example 4 of WO2021139758A1. The hPDL1 and hCTLA4 antigens were purchased from Sino Biology. The thermal stability of the antibodies was investigated. Methods: The antibodies were diluted with PBS to a concentration of 1.4-3 mg / mL, and their thermal stability was measured using a high-throughput differential scanning fluorometer (UNCHAINED, model number: Unit). Experimental results showed that the PL bispecific antibodies still maintained good binding activity to the antigens. Furthermore, compared with PL-1, PL-2 to PL-19 exhibited significantly improved Tm1 (°C), Tagg 266 (°C), and Tonset (°C), demonstrating superior thermal stability of the bispecific antibodies.

[0319] [Table 11] [Table 12]

[0320] 1.3 HJ bispecific antibody Anti-hIL5 and hTSLP HJ bispecific antibodies: HJ-3 to HJ11 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain, and second light chain: First heavy chain: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1] from the N-terminus to the C-terminus; First light chain: from the N-terminus to the C-terminus, [VL1-H]-[Linker 2]-[Obscurin chain]; A second heavy chain: from the N-terminus to the C-terminus, [VH2-J]-[CH1]-[Fc2]; and Second light chain: [VL2-J]-[CL] from the N-terminus to the C-terminus; Here, VH1-H and VL1-H are the heavy chain variable region and light chain variable region of H0 in WO2021139758A1, respectively, and VH2-J and VL2-J are the heavy chain variable region and light chain variable region of J1 in WO2021139758A1, respectively. In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody are shown in the table below.

[0321] [Table 13]

[0322] The antigen-binding activity of the HJ bispecific antibody was detected by following the method in Test Example 4 of WO2021139758A1. The thermal stability of the antibody was investigated as follows. A diluted solution of the HJ bispecific antibody was prepared in a buffer solution containing 10 mM acetic acid, pH 5.5, and 9% sucrose. The bispecific antibody was then concentrated by ultrafiltration to obtain HJ bispecific antibody solutions of different concentrations (the concentrations of the HJ bispecific antibody are shown in Table 13-2). The concentrated solutions were then incubated in an incubator at 40°C. The SEC purity of the samples was determined on day 0 (i.e., before the start of incubation at 40°C, D0), day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21), and day 28 (day 28 of incubation at 40°C, D28). After 28 days of incubation at 40°C, the samples were immediately sampled for CE-SDS purity determination. Experimental results showed that the HJ bispecific antibodies constructed in this disclosure did not show significant changes in antigen-binding activity, and compared to HJ-3, the HJ-5 to HJ-11 bispecific antibodies had superior thermal stability.

[0323] [Table 14] [Table 15]

[0324] Example 2. Screening and identification of anti-human GUCY2C mouse antibodies 2.1 Cell line construction CHO-K1 / hGUCY2C and HCT116 / hGUCY2C cell lines were constructed, and the sequences of the relevant proteins are as follows:

[0325] Human GUCY2C protein (UniProtKB:P25092): SEQ ID NO: 1 The full-length gene encoding human GUCY2C was cloned into the mammalian cell expression vector pCDH. Three plasmids, pVSV-G, pCMV-dR8.91, and pCDH-human GUCY2C, were cotransfected into HEK293T cells (ATCC, CRL-11268) to package the virus. After 48 h of transfection, the virus was collected and used to infect CHO-K1 (ATCC, CCL-61) or HCT116 cells (ATCC, CCL-247). After 2 weeks of pressure screening, the cells were subcloned and detected by FACS to obtain CHO-K1 / hGUCY2C and HCT116 / hGUCY2C cell lines with high GUCY2C expression.

[0326] 2.2 Construction of fusion proteins for immunization or screening The extracellular domain of human GUCY2C was selected and linked to a His or Fc tag at the C-terminus to construct a fusion protein for immunization and screening detection. The sequences of the relevant proteins are as follows:

[0327] hGUCY2C ECD-hFc: SQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYSWSTSSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLEN GENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFWTWKNSKLPNDITGRGPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK sequence number 2 hGUCY2C ECD-His: SQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTY STFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQD KEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQHHHHHH SEQ ID NO: 3

[0328] 2.3 Screening and identification of anti-GUCY2C mouse antibodies The present disclosure prepares a monoclonal antibody against human GUCY2C by hybridoma fusion or Single B technology, and the obtained antibody specifically binds to human GUCY2C with high affinity and can cross-link with cynomolgus monkey GUCY2C, and has relatively good binding activity to human GUCY2C on the cell surface.

[0329] hGUCY2C ECD-hFc, hGUCY2C ECD-His, CHO-K1 / hGUCY2C cells, or HCT116 / hGUCY2C cells were used as immunogens. 50 μg of protein was used for the primary immunization, followed by a single boost of 25 μg. Cross-immunization was performed using TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and Thermo Imject® Alum (Thermo Cat No. 77161) as adjuvants. Cell immunization was performed at 1 × 10^7 per immunization per animal. After the primary immunization and four to seven booster immunizations, mice with high serum antibody titers were selected and screened for splenocyte fusion or single B cell screening.

[0330] After fusion, the hybridoma culture supernatant was detected based on the growth density of the hybridoma cells. Hybridomas with relatively good binding activity to human GUCY2C on the cell surface and the antigen were screened. Monoclonal hybridoma cells were harvested, and RNA was extracted using NucleoZol (MN) (following the steps in the reagent kit's instruction manual) and reverse transcribed (PrimeScript® Reverse Transcriptase, Takara, cat #2680A). The reverse-transcribed cDNA was PCR amplified using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced to obtain the amino acid sequences of the antibody CDRs and variable regions.

[0331] [Table 16] >M81 VH: EFQLQQSGPELVKPGASVKISCKASGYSFT DHNMN WVKQSNGKSLEWIG VMNPKFGTINYNQKFKD KATLTVDQSSSTAYMQLNSLTSEDSAVYYCVR DDYPNYFDY WGQGTTLTVSS SEQ ID NO: 15 >M81 VL: QIVLTQSPAIMSASPGEKVTISC SASSSVNYMY WYQQKPGSSPKSWIY RTSNLAS GVSARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHDYPPT FGGGTKLEIK SEQ ID NO: 16 >M146 VH: EFQLQQSGPELVKPGASVKISCKASGYSFT DYNMN WVKQSNGKSLEWIG LINPKYGTTNYNQKFKG KATLTVDQSSSTAYMQLNSLTSEDSAVYYCAI DDYPAWFAY WGQGTLVTVSA SEQ ID NO: 17 >M146 VL: QIVLTQSPAIMSASPGEKVTISC SASSSLNYMY WYQQKPGSSPKSWIY RTSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHSYPPT FGAGTKLELK SEQ ID NO: 18 >M42VH: QIQLKQSGPGLVQPSQSLSITCTVSGFSLT SYAVH WVRQSPGKGLEWLG VIWTGGNTRSNAAFMS RLTITKDNSKSQVFFKMNSLQADDTAIYFCAK NSLLDYAMDY WGQGTSVTVSS SEQ ID NO: 273 >M42VL: ENVLTQSPVIMSASPGEKVTMTC RASSFVGSGYLH WYQQKSGASPKLWIY STSKLAS GVPARFSGSGSGTSFLTISRVEIEDAAAYYC QQSSGYPLT FGGGSKLEIK SEQ ID NO: 274 >M61VH: EVQLQQSGPELVRPGASVKISCTASGYTIT DYYMS WVKQSHGKSPEWIG DINPNNGGTSDNQKFKG KATLTVDKSSSTAYMEYRSLTSEDSAVYYCAR GRGMVARYFDI WGTGTTVTVSS SEQ ID NO: 275 >M61VL: EILLTQSPTTMAASPGEKITITC SASSSISSNFLH WYQEKPGFSPKLLIY RTSNLAS GVPARFSGSGSGTSYSLTIGSMEAEDVATYYC QQGSGLPLT FGAGTKLELK SEQ ID NO: 276 >M64VH: EVQLVESGGGLVKPGGSLKLSCAASGFTFS DYGMH WVRQAPEKGLEWVA YISSGSSTIYYADTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCAR TRVYFYGSYWYFDV WGTGTTVTVSS SEQ ID NO: 277 >M64VL: QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMY WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSLEAEDAATYYC QQWSSDPPYT FGGGTKLEIK SEQ ID NO: 278 >M93VH: QVQLQQPGAELVRPGTSVKLSCKASGYTFT RYWMH GVKRRPGQGLEWIG MIDPSDSYTKYNQKFMG KAILTVDTSSSTAYMQFSSLTSEDSAVYYCAR SIKVAGWYFDV WGTGTTVTVSS SEQ ID NO: 279 >M93VL: DIVMTQSHKFMSTSVGDRVSITC KASQDVSSAVA WYQQKPGQSPKLLIY SASYRST GVPDRFTGSGSGTTDFTFTISSVQAEDLAIYYC QQHFSTTLT FGAGTKLELK SEQ ID NO: 280 Note: The single underlined parts are CDRs based on the Kabat rules, and the remaining parts are FRs.

[0332] Single B cells were screened primarily using positive and negative screening, 10*genomics, and bioinformatics techniques to obtain a series of antibody variable region sequences. The resulting sequences were then subjected to CDR typing, and the mouse variable region sequences were selected and linked to human antibody constant region sequences to express chimeric antibodies. These were then screened using methods such as binding to hGUCY2C ECD-His and cynoGUCY2C ECD-His (ACRO# GUC-C52H6) proteins, surface plasmon resonance, and cell binding, to select antibody sequences that specifically bind to hGUCY2C ECD and cynoGUCY2C ECD. The heavy and light chain variable region sequences of the antibodies obtained through screening are as follows:

[0333] [Table 17-1] [Table 17-2] >P3-C7 VH: EVQLQQSGPVLLKPGASVKMSCKASGYTFT DHYIH WVKLSHGKSLEWIG VINPYNDGATYNQKFKG KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR PPRAYGNYAGY WGQGTTLTVSS SEQ ID NO: 25 >P3-C7 VL: DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFIGSGSRTDFTLKISRVEAEDLGVYFC SQSTHVPYT FGGGTRLEIK SEQ ID NO: 26 >P1-D2 VH EFQLQQSGPELVKPGASVKISCKASGYSFT DHNMN WVKQSNGKSLEWIG VINPKYGTISNNQKFKG KATLTVDQSSSTAYMQLNSLTAEDSAVYYCAR DDYPNYFDY WGQGTTLTVSS SEQ ID NO: 330 >P1-D2 VL QIVLTQSPAIMSASPGEKVTISC SANSSVNYIY WYQQKPGSSPKPWIY RTSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHSFPPT FGGGTKLEIK SEQ ID NO: 331 >P1-D3 VH QIQLIQSGPELKKPGETVKISCKASGYTFT TYGMS WVKQAPGKGLKWMG WINTYSGVPTYADDFKG RFAFSLETSANTAYLQINNLKNEDTATYFCAR RGYSNLYYYALDY WGQGASVTVSS SEQ ID NO: 332 >P1-D3 VL QIVLTQSPAIMSASPGEKVTMTC SASSSVNYMH WYQQKSGTSPKRWIY DTSKLAS GVPTRFSGSGSGTSYSLTISSMEAEDAATYFC QQWSSNPST FGGGTKLEIK SEQ ID NO: 333 >P1-C6 VH QVQLQQSGAELARPGASVKLSCKASGYTFT SYGIT WVKRRTGQGLGWIG EIYPRSGNTYYNEKFKG KATLTADKSSSTAYMELRSLTSEDSAVYFCAR AITTVVGDWYFDV WGTGTTVTVSS SEQ ID NO: 334 >P1-C6 VL DIVLTQSPASLAMSLGKRATISC RASESVSLIGTNLIH WYQQKPGQPPKLLIH HASNLEA GVPARFSGSGSRTDFTLTIDPVEEDDVAIYYC LQSRKIRT FGGGTKLEIK SEQ ID NO: 335 >P1-D9 VH EVQLVESGGGLVKPGGSLKLSCAASGFSFS DYGMH WVRQAPEKGLEWIA HISSGSSTIYYADTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCAR SRNYYGSRIFDY WGQGTTLTVSS SEQ ID NO: 336 >P1-D9 VL QIVLTQSPARMSASPGEKVTMTC SASSSVSYMY WYQQKPRSSPKPWIY LTSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPT FGSGTKLEIK SEQ ID NO: 337 >P1-F10 VH EVQLVESGGGLVKPGGSLKLSCAASGFAFS DYGMH WVRQAPEKGLEWVA HISSGSSTIYYADTVKG RFTISRDNVKNTLFLQMTSLRSEDTAMYYCAR SRNYYGSRVFDY WGQGTPLTVSS SEQ ID NO: 338 >P1-F10 VL QIVLTQSPALMSVSPGEKVTMTCSASSSVNYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPPTFGGSGTKLEIK SEQ ID NO: 339 >P2-C8 VH EVQLQQSGPVLVKPGASVKMSCKVSGYTFT DYYMH WVKQSHGKSLEWIG LINPYNGVTSYNQKFKG KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR PPRAYGNYAGY WGQGTTLTVSS SEQ ID NO: 340 >P2-C8 VL DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPYT FGGGTKLEIK SEQ ID NO: 341 >P2-B9 VH QVQLQQPGAELVKPGASVKLSCKASGYTFS SYWMH WVKQRPGQGLEWIGMIHPKSDSVNNNEKFKNKATLTVDKSSSTVYLQLSSLTSEDSAVYSCAR GGDSGYSMDY WGQGTSVTVSS SEQ ID NO: 342 >P2-B9 VL DIVLTQSPASLAVSLGQKATISC KASKKVTIFGSISALH WYQQKPGQPPKLIY NGAKLES GVSARFSDSGSQNRSPFGNQLSFTLTIDPVEADDAATYYC LQNKEVPYT FGGGTKLEIK SEQ ID NO: 343 >P3-D2 VH EVQLQQSGPVLVKPGASVKMSCKASGYTFT DYFMH WVKQSHGKSLEWIG LINPYSGGTTYNQKFKG KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR PPRAYGDYAGY WGQGTTLTVSS SEQ ID NO: 344 >P3-D2 VL DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPLT FGTGTKLEIK SEQ ID NO: 345 >P3-B4 VH QIQLVQSGPELKKPGEIVKISCKASGYTFT TYGMS WVKQAPGKGLKWMG WINTYSGVPTYADDFKG RFAFSLETSASTAYLQINNLKNEDTATYFCAR RAGSSLYYYAMDY WGQGTSVTVSS SEQ ID NO: 346 >P3-B4 VL QIVLTQSPAIMSASPGEKVTMTC SASSSVSYML WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPT FGGGTKLEIK SEQ ID NO: 347 >P3-E4 VH QVQLQQPGAELVMPGAAVKLSCKASGYTFI NYWMH WLKQRPGQGLEWIG EIDPSDGYTDYNQKFKG KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAI SLRGPITFGPFPY WGQGTLVTVSS SEQ ID NO: 348 >P3-E4 VL DIVLTQSPASLAVSLGQRATISC RASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLES GVPARFSGSGSETDFTLNIHPVAEEDAATYFC QHSIEDPRT FGGGTKLEIK SEQ ID NO: 349 >P3-F6 VH EVQLQQSGPELVKPGASVKISKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNNGGTSYNQKFKG KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR KTWLPYASVY WGQGTSVTVSS SEQ ID NO: 350 >P3-F6 VL DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPPYT FGGGTKLEIK SEQ ID NO: 351 >P3-D9 VH EVQLVESGGGLVKPGGSLKLSCAASGFAFS DYGMH WVRQAPEKGLEWVA HISSGSSTIYYGDTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCTR SRNYYGSRVFDY WGQGTTLTVSS SEQ ID NO: 352 >P3-D9 VL QIVLTQSPALMSVSPGEKVTMTC SASSSVSYMYWYQQKPRSSPKPWIY LTSSLAS GVPARFSGSGSGTSYSLTINNVEAEDAATYYC QQWSSNPPT FGSGTKVEIK SEQ ID NO: 353 Note: The single underlined parts are CDRs based on the Kabat rules, and the remaining parts are FRs.

[0334] The variable region sequences of the mouse anti-GUCY2C antibody were combined with the constant regions shown in SEQ ID NO: 58 and SEQ ID NO: 59 to obtain a chimeric antibody. For example, M81CHI or P3-C7 CHI refers to a chimeric antibody comprising the M81 or P3-C7 mouse heavy chain variable region, light chain variable region, and the above-mentioned constant region, with the specific sequences as follows: The remaining chimeric antibodies, M146 CHI, M42 CHI, M61 CHI, M64 CHI, M93 CHI, P1-D2 CHI, P1-D3 CHI, P1-C6 CHI, P1-D9 CHI, P1-F10 CHI, P2-C8 CHI, P2-B9 CHI, P3-D2 CHI, P3-B4 CHI, P3-E4 CHI, P3-F6 CHI, and P3-D9 CHI, are derived in this manner. For space reasons, their full-length sequences can be determined without any doubt, and therefore are not listed here.

[0335] Exemplary, the full-length sequence of an anti-GUCY2C chimeric antibody is as follows:

[0336] M81CHI heavy chain: EFQLQQSGPELVKPGASVKISCKASGYSFT DHNMN WVKQSNGKSLEWIG VMNPKFGTINYNQKFKD KATLTVDQSSSTAYMQLNSLTSEDSAVYYCVR DDYPNYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 354 M81CHI light chain: QIVLTQSPAIMSASPGEKVTISC SASSSVNYMY WYQQKPGSSPKSWIY RTSNLAS GVSARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHDYPPT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 355 P3-C7 CHI heavy chain: EVQLQQSGPVLLKPGASVKMSCKASGYTFT DHYIH WVKLSHGKSLEWIG VINPYNDGATYNQKFKG KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR PPRAYGNYAGYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 356 P3-C7 CHI light chain: DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFIGSGSRTDFTLKISRVEAEDLGVYFC SQSTHVPYT FGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 357

[0337] Example 3. Humanization of anti-GUCY2C monoclonal antibody The humanization of mouse antibodies was carried out according to the methods disclosed in many publications in this field. Briefly, based on the typical structure of the VH / VL CDR of the obtained mouse antibody, homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched for in a human germline database and arranged in descending order of FR homology. The germline with the highest FR homology was used as a template, and the CDR region of the mouse antibody was grafted onto the human template, certain amino acids in the variable region were mutated, and the constant region of the mouse antibody was replaced with a human constant region to obtain the final humanized antibody.

[0338] 3-1. Humanization of M81 antibody For the humanized M81 antibody, IGKV3-11*01, IGKV6-21*02, or IGKV1-9*01 and IGKJ4*01 were selected as light chain framework region templates, and IGHV1-3*01 and IGHJ6*01 were selected as heavy chain framework region templates. The CDR regions of a mouse antibody were grafted onto the selected humanized template, and certain amino acids in the variable regions were substituted to obtain the light and heavy chain variable regions of the humanized antibody. The specific framework substitutions and amino acid substitutions are as follows:

[0339] [Table 18]

[0340] The CDRs of the M81 humanized antibody are as follows:

[0341] [Table 19]

[0342] The variable region sequences of the M81 humanized antibody are as follows:

[0343] > h81 L1: EIVLTQSPATLSLSPGERATLSC SASSSVNYMY WYQQKPGQAP KSW IY RTSNLAS G V PARFSGSGSGTD Y TLTISSLEPEDFAVYYC QQYHDYPPT FGGGTKVEIK SEQ ID NO: 31 >h81 L2: EIVLTQSPDFQSVTP G EKVTITC SASSSVNYMY WYQQKPDQSPK SW I YRTSNLAS GVPSRFSGSGSGTDFTLTI S SLEAEDAATYYC QQYHDYPPTFGGGTKVEIK SEQ ID NO: 32 >h81 L3: DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNLAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK SEQ ID NO: 33 >h81 L4: DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNDAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK SEQ ID NO: 34 >h81 L5: DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNLAQ GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK SEQ ID NO: 35 >h81 L6: DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTQNLAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK SEQ ID NO: 36 >h81 H1: E VQLVQSGAEVKKPGASVKVSCKASGYTFT DHNMN WVK QAPGQRLEWMG VMNPKFGTINYNQKFKDKA TITRDTSASTAYMELSSLRSEDTAVYYCAR DNYPNYFDY WGQGTTVTVSS SEQ ID NO: 37 >h81 H2: E VQLVQSGAEVKKPGASVKVSCKASGYTFT DHNMN WV K QAPGQRLEWMG VMNPKFGTINYNQKFKDKA TITRDTSASTAYMELSSLRSEDTAVYYCAR DDYPNYFDY SEQ ID NO: 38 Note: The single underlined parts are CDRs, the bolded parts under dotted lines are amino acid substitution sites, and the remaining parts are FRs.

[0344] 3-2. Humanization of M146 antibody For the humanized M146 antibody, IGKV3-11*01 and IGKJ4*01 were selected as light chain framework region templates, and IGHV1-3*01 and IGHJ6*01 were selected as heavy chain framework region templates. The CDR regions of a mouse antibody were grafted onto the selected humanized template, and certain amino acids in the variable regions were substituted to obtain the light and heavy chain variable regions of the humanized antibody. The specific framework substitutions and amino acid substitutions are as follows:

[0345] [Table 20]

[0346] The CDRs of the humanized antibody M146 are as follows: [Table 21]

[0347] The variable region sequences of humanized antibody M146 are as follows: >h146 L1: EIVLTQSPATLSLSPGERATLSC SASSSLNYMYWYQQKPG SS PR PW IY RTSQLAS G V PARFSGSGSGTD Y TLTISSLEPEDFAVYYC QQYHSYPPT FGGGTKVEIK SEQ ID NO: 41 >h146 H1: EF QLVQSGAEVKKPGASVKVSCKASGY S FT DYNMN WVRQAPGQRLEWMG LINPKYGTTNYNQKFKG RVT L T V D Q S S STAYMELSSLRSEDTAVYYCAR DNYPAWFAY WGQGTTVTVSS SEQ ID NO: 42 Note: The single underlined parts are CDRs, the bolded parts under dotted lines are amino acid substitution sites, and the remaining parts are FRs.

[0348] 3-3. Humanization of P3-C7 antibody For the humanized P3-C7 antibody, IGKV2-40*01 or IGKV1-39*01 and IGKJ4*01 were selected as the light chain framework region templates, and IGHV1-46*01 and IGHJ6*01 were selected as the heavy chain framework region templates. The CDR regions of a mouse antibody were grafted onto the selected humanized template, and certain amino acids in the variable regions were substituted to obtain the light and heavy chain variable regions of the humanized antibody. The specific framework substitutions and amino acid substitutions are as follows:

[0349] [Table 22]

[0350] The CDRs of the humanized antibody P3-C7 are as follows:

[0351] [Table 23]

[0352] The variable region sequences of humanized antibody P3-C7 are as follows: >hP3-C7 L1: DIVMTQTPLSLPVTPGEPASISC RSSQSLVHSNGNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO: 48 >hP3-C7 L2: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSNGNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO: 49 >hP3-C7 L3: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSQGNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO:50 >hP3-C7 L4: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSSGNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO:51 >hP3-C7 L5: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSTGNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS RTDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO:52 >hP3-C7 L6: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSNVNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO:53 >hP3-C7 L7: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSNQNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO:54 >hP3-C7 L8: D VV MTQSPSSLSASVGDRVTITC RSSQSLVHSNQNTYLH WYQQKPGKAPKLLIY KVSNRFS GVPSRFSGSGS R TDFTL K ISSLQPEDFATYYC SQSTHVPYT FGGGTKVEIK SEQ ID NO: 55 >hP3-C7 H1: E VQLVQSGAEVKKPGASVKVSCKASGYTFT DHYIH WVRQAPGQGLEWMG VINPYNDGATYNQKFKG RVTMT V D K STSTVYMELSSLRSEDTAVYYCAR PPRAYGNYAGY WGQGTTVTVSS SEQ ID NO:56 >hP3-C7 H2: E VQLVQSGAEVKKPGASVKVSCKASGYTFT DHYIH WVR L APGK GLEWMG VINPYNDGATYNQKFKG RVT L T V D K STSTVYMELSSLRSEDTAVYYCAR PPRAYGNYAGY WGQGTTVTVSS SEQ ID NO:57 Note: The single underlined parts are CDRs, the dotted underlined parts are amino acid substitution sites, and the remaining parts are FRs.

[0353] The heavy chain variable region and light chain variable region of the anti-GUCY2C humanized antibody were recombined with the heavy chain constant region hIgG1:CH1-Fc and the light chain constant region CL, respectively, to obtain a full-length humanized antibody.

[0354] >hIgG1:CH1-Fc: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:58 >CL: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:59 The anti-GUCY2C humanized antibodies obtained in this disclosure are as follows:

[0355] [Table 24] [Table 25] [Table 26]

[0356] Exemplary, the full-length sequence of an anti-GUCY2C humanized antibody is as follows: 81 L3H1 heavy chain: EVQLVQSGAEVKKPGASVKVSCKASGYTFT DHNMN WV K QAPGQRLEWMG VMNPKFGTINYNQKFKDKA TITRDTSASTAYMELSSLRSEDTAVYYCAR DNYPNYFDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 60 81 L3H1 light chain: DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNLAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 61 146 L1H1 heavy chain: E F QLVQSGAEVKKPGASVKVSCKASGY S FT DYNMN WVRQAPGQRLEWMG LINPKYGTTNYNQKFKG RVT L T V D Q S S STAYMELSSLRSEDTAVYYCAR DNYPAWFAY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 62 146 L1H1 light chain: EIVLTQSPATLSLSPGERATLSC SASSSLNYMY WYQQKPG SS PR PW IY RTSQLAS G V PARFSGSGSGTD Y TLTISSLEPEDFAVYYC QQYHSYPPT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 63 P3-C7 L7H1 heavy chain: EVQLVQSGAEVKKPGASVKVSCKASGYTFT DHYIH WVRQAPGQGLEWMG VINPYNDGATYNQKFKG RVTMT V D K STSTVYMELSSLRSEDTAVYYCAR PPRAYGNYAGY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 64 P3-C7 L7H1 light chain: D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSNQNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 65 P3-C7 L8H1 heavy chain (similar to P3-C7 L7H1 heavy chain, SEQ ID NO: 64) P3-C7 L8H1 light chain: D VV MTQSPSSLSASVGDRVTITC RSSQSLVHSNQNTYLH WYQQKPGKAPKLLIY KVSNRFS GVPSRFSGSGS R TDFTL K ISSLQPEDFATYYC SQSTHVPYT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 66 Note: The single underlined parts are CDRs, the bolded parts under dotted lines are amino acid substitution sites, and the italicized parts are constant regions.

[0357] The control antibodies for the anti-GUCY2C antibodies used in the present disclosure are as follows:

[0358] 1608 (the variable region sequence was constructed with reference to the GUCY2c end of the GUCY2c-1608 bispecific antibody in patent WO2019224716A2, and the constant region sequence is shown in SEQ ID NO: 58 and SEQ ID NO: 59), and its full-length sequence is as follows:

[0359] > 1608 Heavy Chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGLEWIG EIKPSNELTNVHEKFKD RFTISVDKAKNSAYLQMNSLRAEDTAVYYCTR TITTTEGYWFFDV WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 67 > 1608 light chain: DIQLTQSPSSLSASVGDRVTITC RASESVDYYGSSLLQ WYQQKPGKAPKLLIY AASKLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQTRKAYT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 68 The antibody 5F9 (the variable region sequences were prepared with reference to patent WO2014134311A1, and the constant region sequences are shown in SEQ ID NO: 58 and SEQ ID NO: 59) has the following specific sequences:

[0360] >5F9 heavy chain: QVQLQQWGAGLLKPSETLSLTCAVFGGSFS GYYWS WIRQPPGKGLEWIG EINHRGNTNDNPSLKS RVTISVDTSKNQFALKLSSVTAADTAVYYCAR ERGYTYGNFDH WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 69 >5F9 light chain: EIVMTQSPATLSVSPGERATLSC RASQSVSRNLA WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTEFTLTIGSLQSEDFAVYYC QQYKTWPRT FGQGTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 70

[0361] Example 4. Preparation of anti-GUCY2C-CD3 bispecific antibody The GUCY2C arm of the GUCY2C-CD3 bispecific antibody of the present disclosure is an anti-GUCY2C antibody of the present disclosure, and the CD3 arm may be derived from any suitable antibody. Specifically, the CD3 arm used in the present disclosure is S107E or N103QN106Q.

[0362] [Table 27]

[0363] The variable region sequence of S107E is as follows: >S107E-VH: EVQLVESGGGLVQPGGSLKLSCAASGFTFN KYAMN WVRQAPGKGLEWVA RIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNEYISYWAY WGQGTLVTVSS SEQ ID NO:77 >S107E-VL: QTVVTQEPSLTVSPGGTVTLTC GSSTGAVTSGNYPN WVQQKPGQAPRGLIG GTKFLAP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC VLWYSNRWV FGGGTKLTVL SEQ ID NO:78 [Table 28]

[0364] The variable region sequences of N103QN106Q are as follows:

[0365] >N103QN106Q-VH: EVQLVESGGGLVQPGGSLKLSCAASGFTFN KYAMN WVRQAPGKGLEWVA RIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGQFGQSYISYWAY WGQGTLVTVSS SEQ ID NO:89 >N103QN106Q-VL (similar to S107E-VL, SEQ ID NO: 78) The GUCY2C-CD3 bispecific antibodies disclosed herein have the following molecular structures of Format 1 to Format 5: Format 1 is an asymmetric molecule, and contains two chains as a complete molecule, and the two chains are different from each other, as follows:

[0366] Chain 1: [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[IgG1Fc(Knob-DI)], Chain 2: [CD3-VL]-[Linker 3]-[GUCY2C-VH]-[Linker 2]-[IgG1Fc(Hole-DI)], The schematic diagram is shown in Figure 1A.

[0367] The relevant sequences are as follows:

[0368] >IgG1Fc(Knob-DI, L234A / L235A / G237A / Y349C / T366W): CPPCPAPE AA G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:79 >IgG1Fc(Hole-DI, L234A / L235A / G237A / S354C / T366S / L368A / Y407V): CPPCPAPE AA G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 80 >Linker 1: GGGSGGGG SEQ ID NO: 81 >Linker 2: G SEQ ID NO:82 >Linker 3: GGGGSGGGG SEQ ID NO: 83 Format 2 is an asymmetric molecule, and the complete molecule contains a total of four chains, all of which are different. Specifically, the chains are as follows:

[0369] Chain 1: [CD3-VH]-[IgG1(CH1)]-[IgG1Fc(Knob)], Chain 2: [CD3-VL]-[CL(Lambda)]; Chain 3: [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[IgG1Fc(Hole)], Chain 4: [GUCY2C-VL]-[Linker 4]-[Titin chain], The schematic diagram is shown in Figure 1B (where Ob stands for Obscurin).

[0370] The relevant sequences are as follows:

[0371] >IgG1(CH1): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:84 >IgG1Fc(Knob, L234A / L235A / S354C / T366W): DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 85 >IgG1Fc(Hole, L234A / L235A / Y349C / T366S / L368A / Y407V): DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:86 >CL(Lambda): GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO:87 >Titin(T.16): GIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDD S TTL T IKDVQKQDGGLYTLTLRNEFGSDSATATVNIHIRSI SEQ ID NO: 198 >Obscurin(O.28): SGAPRFLTRPKA S VVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNA H GEAFACLGLQVDAEA SEQ ID NO: 236 >Linker 4: GGGGS SEQ ID NO: 90 Format 3 is an asymmetric molecule, and the complete molecule contains a total of four chains, all of which are different. Specifically, the chains are as follows:

[0372] Chain 1: [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[IgG1Fc(Knob)], Chain 2: [GUCY2C-VL]-[Linker 4]-[Titin chain], Chain 3: [CD3-VH]-[IgG1(CH1)]-[IgG1Fc(Hole)], Chain 4: [CD3-VL]-[CL(Lambda)]; A schematic diagram of this is shown in Figure 1C (where Ob stands for Obscurin).

[0373] The relevant sequences are as follows:

[0374] >IgG1(CH1) (SEQ ID NO: 84) >IgG1Fc(Knob) (SEQ ID NO: 85) >IgG1Fc(Hole)(SEQ ID NO:86) >CL(Lambda) (SEQ ID NO: 87) >Titin (T.16) (SEQ ID NO: 198) >Obscurin (O.28) (SEQ ID NO: 236) >Linker 4 (SEQ ID NO: 90) >Chain 3 (Format 3-3, CD3-VH-IgG1(CH1)-IgG1Fc(Hole)): EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 93 >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92) Format 4 is a symmetrical molecule, and the complete molecule consists of a total of four chains, each consisting of two identical chains 1 and two identical chains 2, as follows:

[0375] Chain 1: [GUCY2C-VH]-[IgG1(CH1)]-[CD3-VH]-[Linker 5]-[CD3-VL]-[Linker 6]-[IgG1Fc]; Chain 2: [GUCY2C-VL]-[CL], The schematic diagram is shown in FIG. 1D.

[0376] The relevant sequences are as follows:

[0377] >IgG1(CH1) (SEQ ID NO: 84) >CL (SEQ ID NO: 59) >IgG1Fc(L234A / L235A): DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:94 >Linker 5: GGGGSGGGGSGGGGS SEQ ID NO: 95 Linker 6 GGG SEQ ID NO:96 Format 5 is a symmetric molecule, and the complete molecule consists of a total of six chains, which are two identical chains 1, two identical chains 2, and two identical chains 3, as follows:

[0378] Chain 1: [GUCY2C-VH]-[IgG1(CH1)]-[Linker 7]-[CD3-VH]-[Linker 4]-[Obscurin chain]-[IgG1Fc], Chain 2: [GUCY2C-VL]-[CL], Chain 3: [CD3-VL]-[Linker 4]-[Titin chain], A schematic diagram of this is shown in Figure 1E (where Ob stands for Obscurin).

[0379] The relevant sequences are as follows:

[0380] >IgG1(CH1) (SEQ ID NO: 84) >IgG1Fc (SEQ ID NO: 86) >CL (SEQ ID NO: 59) >Titin (T.16) (SEQ ID NO: 198) >Obscurin (O.28) (SEQ ID NO: 236) >Linker 4 (SEQ ID NO: 90) >Linker 7: GGGGSGGGGS SEQ ID NO:97 >Chain 3 (Format 5-3, CD3-VL-Linker 4-Titin) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVLGGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 98 Antigen-binding molecules were constructed according to Table 25, and all bibody molecules employed the variable region of S107E or N103QN106Q as the CD3 arm. In the numbers, the variable region of the anti-GUCY2C antibody refers to the antibody name, and the number following indicates the format used. When "(N103QN106Q)" is written after "Format," it indicates that the CD3 arm used in the bibody is N103QN106Q. When no notation is written after "Format," it indicates that the CD3 arm used in the bibody is S107E. For example, P3-C7 L7H1-1 indicates that the molecule employs the variable region of the P3-C7 L7H1 antibody as the GUCY2C-binding domain, Format 1 is used as the molecular structure, and the CD3 arm is S107E. Others are deduced similarly.

[0381] [Table 29]

[0382] Exemplarily, the specific amino acid sequences of bispecific antibodies constructed using Format 1 in the present disclosure are as follows:

[0383] 1. Full-length sequence of P3-C7 L7H1-1 >Chain 1 (P3-C7 L7H1-1-1): DVVMTQTPLSLPVTPGEPASISCRSSQSLVHSNQNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSRTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKGGGSGGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence number 99 > Chain 2 (P3-C7 L7H1-1-2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSGGGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYIHWVRQAPGQGLEWMGVINPYNDGATYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARPPRAYGNYAGYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 100 2. Full-length sequence of P3-C7 L8H1-1 >Strand 1 (P3-C7 L8H1-1-1): DVVMTQSPSSLSASVGDRVTITCRSSQSLVHSNQNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSRTDFTLKISSLQPEDFATYYCSQSTHVPYTFGGGTKVEIKGGGS GGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWA YWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVCTLPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 101 >Strand 2 (P3-C7 L8H1-1-2, similar to P3-C7 L7H1-1-2, SEQ ID NO: 100)

[0384] 3. Full-length sequence of M81-1 >Chain 1 (M81-1-1): QIVLTQSPAIMSASPGEKVTISCSASSSVNYMYWYQQKPGSSPKSWIYRTSNLASGVSARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHDYPPTFGGGTKLEIK GGGSGGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHHGNFGNEYISYWAYWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 102 >Chain 2 (M81-1-2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSGGGG EFQLQQSGPELVKPGASVKISCKASGYSFTDHNMNWVKQSNGKSLEWIGVMNPKFGTINYNQKFKDKATLTVDQSSSTAYMQLNSLTSEDSAVYYCVRDDYPNYFDYWGQGTTLTVSS GCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 103

[0385] 4. Full-length sequence of M146-1 >Chain 1 (M146-1-1): QIVLTQSPAIMSASPGEKVTISC SASSSLNYMY WYQQKPGSSPKSWIY RTSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHSYPPT FGAGTKLELKGGGSGGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 104 >Chain 2 (M146-1-2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSGGGGEFQLQQSGPELVKPGASVKISCKASGYSFT DYNMN WVKQSNGKSLEWIG LINPKYGTTNYNQKFKG KATLTVDQSSSTAYMQLNSLTSEDSAVYYCAI DDYPAWFAY WGQGTLVTVSAGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 105 Exemplary specific amino acid sequences of bispecific antibodies constructed using Format 2 in the present disclosure are as follows:

[0386] 1. Full-length sequence of P3-C7 L7H1-2 >Chain 1 (Format 2-1): EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 91 >Chain 2 (Format 2-2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC Sequence number 92 >Chain 3 (P3-C7 L7H1-2-3): EVQLVQSGAEVKKPGASVKVSCKASGYTFT DHYIH WVRQAPGQGLEWMG VINPYNDGATYNQKFKG RVTMT V D KSTSTVYMELSSLRSEDTAVYYCAR PPRAYGNYAGY WGQGTTVTVSS GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 106 >Chain 4 (P3-C7 L7H1-2-4): D V VMTQTPLSLPVTPGEPASISC RSSQSLVHSNQNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGS R TDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 107

[0387] 2. Full-length sequence of M81-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Chain 3 (M81-2-3): EFQLQQSGPELVKPGASVKISCKASGYSFT DHNMN WVKQSNGKSLEWIG VMNPKFGTINYNQKFKD KATLTVDQSSSTAYMQLNSLTSEDSAVYYCVR DDYPNYFDY WGQGTTLTVSS GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 108 >Chain 4 (M81-2-4): QIVLTQSPAIMSASPGEKVTISC SASSSVNYMY WYQQKPGSSPKSWIY RTSNLAS GVSARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHDYPPT FGGGTKLEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 109

[0388] 3.81 Full-length sequence of L1H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Chain 3 (81 L1H1-2-3): EVQLVQSGAEVKKPGASVKVSCKASGYTFT DHNMN WV K QAPGQRLEWMG VMNPKFGTINYNQKFKDKA TITRDTSASTAYMELSSLRSEDTAVYYCAR DNYPNYFDY WGQGTTVTVSS GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 110 >Chain 4 (81 L1H1-2-4): EIVLTQSPATLSLSPGERATLSC SASSSVNYMY WYQQKPGQAP KSW IY RTSNLAS G V PARFSGSGSGTD Y TLTISSLEPEDFAVYYC QQYHDYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 111

[0389] 4.81 Full-length sequence of L2H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Strand 3 (81 L2H1-2-3, similar to 81 L1H1-2-3, SEQ ID NO: 110) >Chain 4 (81 L2H1-2-4): EIVLTQSPDFQSVTP G EKVTITC SASSSVNYMY WYQQKPDQSPK SW I YRTSNLAS GVPSRFSGSGSGTDFTLTI S SLEAEDAATYYC QQYHDYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 112

[0390] 5.81 Full-length sequence of L3H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Strand 3 (81 L3H1-2-3, similar to 81 L1H1-2-3, SEQ ID NO: 110) >Chain 4 (81 L3H1-2-4): DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNLAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 113

[0391] 6.81 Full-length sequence of L3H1-2 (N103QN106Q) >Chain 1(Format 2-1(N103QN106Q)) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGQFGQSYISYWA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 242 >Strand 2 (Format 2-2, SEQ ID NO: 92) >Strand 3 (81 L3H1-2-3, similar to 81 L1H1-2-3, SEQ ID NO: 110) >Strand 4 (81 L3H1-2-4, SEQ ID NO: 113):

[0392] 6.81 Full-length sequence of L4H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Strand 3 (81 L4H1-2-3, similar to 81 L1H1-2-3, SEQ ID NO: 110) >Chain 4 (81 L4H1-2-4): DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNDAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 114

[0393] 7.81 Full-length sequence of L5H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Strand 3 (81 L5H1-2-3, similar to 81 L1H1-2-3, SEQ ID NO: 110) >Chain 4 (81 L5H1-2-4): DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTSNLAQ GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 115

[0394] 8.81 Full-length sequence of L6H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Strand 3 (81 L6H1-2-3, similar to 81 L1H1-2-3, SEQ ID NO: 110) >Chain 4 (81 L6H1-2-4) DIQLTQSPSFLSAS P GDRVTITC SASSSVNYMY WYQQKPG SS Penalty kick SW IY RTQNLAS GVPSRFSGSGSGTE Y TLTISSLQPEDFATYYC QQYHDYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 116

[0395] 9. Full-length sequence of M146-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Chain 3 (M146-2-3): EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGLINPKYGTTNYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCAIDDYPAWFAYWGQGTLVTVSAGGGGS SGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 117 >Chain 4 (M146-2-4): QIVLTQSPAIMSASPGEKVTISC SASSSLNYMY WYQQKPGSSPKSWIY RTSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHSYPPT FGAGTKLELK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 118

[0396] 10.146 Full-length sequence of L1H1-2 >Strand 1 (Format 2-1, SEQ ID NO: 91) >Strand 2 (Format 2-2, SEQ ID NO: 92) >Chain 3 (146 L1H1-2-3): E F QLVQSGAEVKKPGASVKVSCKASGY S FT DYNMN WVRQAPGQRLEWMG LINPKYGTTNYNQKFKG RVT L T V D Q S S STAYMELSSLRSEDTAVYYCAR DNYPAWFAY WGQGTTVTVSS GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 119 >Chain 4 (146 L1H1-2-4): EIVLTQSPATLSLSPGERATLSC SASSSLNYMY WYQQKPG SS PR PW IY RTSQLAS G V PARFSGSGSGTD Y TLTISSLEPEDFAVYYC QQYHSYPPT FGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 120 Exemplarily, the specific amino acid sequences of bispecific antibodies constructed using Format 3 in the present disclosure are as follows:

[0397] 1. Full-length sequence of P3-C7 L7H1-3 >Chain 1 (P3-C7 L7H1-3-1): EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYIHWVRQAPGQGLEWMGVINPYNDGATYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARPPRAYGNYAGYWGQ GTTVTVSSGGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEADK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 121 >Strand 2 (similar to P3-C7 L7H1-3-2, P3-C7 L7H1-2-4, SEQ ID NO: 107) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0398] 2.81 Full-length sequence of L1H1-3 >Chain 1 (81 L1H1-3-1): EVQLVQSGAEVKKPGASVKVSCKASGYTFT DHNMN WV K QAPGQRLEWMG VMNPKFGTINYNQKFKDKA TITRDTSASTAYMELSSLRSEDTAVYYCAR DNYPNYFDY WGQGTTVTVSSGGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 122 >Strand 2 (81 L1H1-3-2, similar to 81 L1H1-2-4, SEQ ID NO: 111) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0399] 3.81 Full-length sequence of L2H1-3 >Strand 1 (81 L2H1-3-1, similar to 81 L1H1-3-1, SEQ ID NO: 122) >Strand 2 (81 L2H1-3-2, similar to 81 L2H1-2-4, SEQ ID NO: 112) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0400] 4.81 Full-length sequence of L3H1-3 >Strand 1 (81 L3H1-3-1, similar to 81 L1H1-3-1, SEQ ID NO: 122) >Strand 2 (81 L3H1-3-2, similar to 81 L3H1-2-4, SEQ ID NO: 113) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0401] 5.81 Full-length sequence of L4H1-3 >Strand 1 (81 L4H1-3-1, similar to 81 L1H1-3-1, SEQ ID NO: 122) >Strand 2 (81 L4H1-3-2, similar to 81 L4H1-2-4, SEQ ID NO: 114) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0402] 6.81 Full-length sequence of L5H1-3 >Strand 1 (81 L5H1-3-1, similar to 81 L1H1-3-1, SEQ ID NO: 122) >Strand 2 (81 L5H1-3-2, similar to 81 L5H1-2-4, SEQ ID NO: 115) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0403] 7.81 Full-length sequence of L6H1-3 >Strand 1 (81 L6H1-3-1, similar to 81 L1H1-3-1, SEQ ID NO: 122) >Strand 2 (81 L6H1-3-2, similar to 81 L6H1-2-4, SEQ ID NO: 116) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92)

[0404] 8.146 Full-length sequence of L1H1-3 >Chain 1 (146 L1H1-3-1): E F QLVQSGAEVKKPGASVKVSCKASGY S FT DYNMN WVRQAPGQRLEWMG LINPKYGTTNYNQKFKG RVT L T V D Q S S STAYMELSSLRSEDTAVYYCAR DNYPAWFAYWGQGTTVTVSSGGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 123 >Strand 2 (146 L1H1-3-2, similar to 146 L1H1-2-4, SEQ ID NO: 120) >Strand 3 (Format 3-3, SEQ ID NO: 93) >Strand 4 (Format 3-4, similar to Format 2-2, SEQ ID NO: 92) Exemplarily, the specific amino acid sequences of bispecific antibodies constructed using Format 4 in the present disclosure are as follows:

[0405] 1. Full-length sequence of P3-C7 L7H1-4 >Chain 1 (P3-C7 L7H1-4-1): EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYIHWVRQAPGQGLEWMGVINPYNDGATYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARPPRAYGNYAGYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 124 > Chain 2 (P3-C7 L7H1-4-2, similar to P3-C7 L7H1 light chain, SEQ ID NO: 65)

[0406] 2. Full-length sequence of P3-C7 L8H1-4 > Chain 1 (P3-C7 L8H1-4-1, similar to P3-C7 L7H1-4-1, SEQ ID NO: 124) > Chain 2 (P3-C7 L8H1-4-2, similar to P3-C7 L8H1 light chain, SEQ ID NO: 66) The specific amino acid sequences of bispecific antibodies constructed using Format 5 as an example in the present disclosure are as follows:

[0407] 1. Full-length sequence of P3-C7 L7H1-5 >Chain 1 (P3-C7 L7H1-5-1): EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYIHWVRQAPGQGLEWMGVINPYNDGATYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARPPRAYGNYAGYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC GGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSGGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 125 > Chain 2 (P3-C7 L7H1-5-2, similar to P3-C7 L7H1 light chain, SEQ ID NO: 65) >Strand 3 (Format 5-3, SEQ ID NO: 98)

[0408] 2. Full-length sequence of P3-C7 L8H1-5 >Strand 1 (P3-C7 L8H1-5-1, similar to P3-C7 L7H1-5-1, SEQ ID NO: 125) > Chain 2 (P3-C7 L8H1-5-2, similar to P3-C7 L8H1 light chain, SEQ ID NO: 66) >Strand 3 (Format 5-3, SEQ ID NO: 98) Furthermore, the present disclosure also uses the GUCY2C-CD3 bispecific antibody GUCY2c-1608 (designated 1608-BsAb in the present disclosure), which was prepared with reference to Patent WO2019224716A2, and which has a DIABODY structure, and the complete molecule contains two chains, specifically as follows:

[0409] >Strand 1 (GUCY2c-1608-Knob sequence): DIQLTQSPSSLSASVGDRVTITCRASESVDYYGSSLLQWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKAYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSG CPPCPAPE AA G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 126 >Strand 2 (GUCY2c-1608-Hole sequence): DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIKGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWIGEIKPSNELTNVHEKFKDRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSG CPPCPAPE AA G APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 127 [Table 30]

[0410] In addition, the present disclosure also uses the EGFR-CD28 bispecific antibody 97H2L3OT-ZAL prepared with reference to Patent CN202210371538.8, and its sequence is specifically as follows:

[0411] [Table 31] EGFR-VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS SEQ ID NO: 152 EGFR-VL: DIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK SEQ ID NO: 153 CD28-VH: EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYWITWVRQAPGQGLEWMGDIYPGSGRTNYNEMFKNRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRHYGTGYESFDVWGQGTTVTVSS SEQ ID NO: 154 CD28-VL: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK SEQ ID NO: 155 Full-length sequence of EGFR-CD28 bispecific antibody 97H2L3OT-ZAL: >Chain 1: EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYWITWVRQAPGQGLEWMGDIYPGSGRTNYNEMFKNRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRHYGTGYESFDVWGQGTTVTVSS GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSIDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 156 >Chain 2: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA SEQ ID NO: 157 >Chain 3: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 158 >Chain 4: DIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 159 In addition, the present disclosure also uses the EGFR-CD28 bispecific antibodies 129H4L1OT-ZAL and 94H6L12-ZALOT, both of which were prepared with reference to Patent CN202210371538.8, and their sequences are specifically as follows:

[0412] [Table 32] EGFR-VH (similar to EGFR-VH of 129H4L1OT-ZAL, SEQ ID NO: 152) EGFR-VL (similar to EGFR-VL in 129H4L1OT-ZAL, SEQ ID NO: 153) CD28-VH: EVQLVQSGAEVKKPGASVKVSCKAPGYTFTSHWIQWVRQAPGQGLEWMGEIFPGSGSTHYNEKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARLHYGTSYDAMDYWGQGTTVTVSS SEQ ID NO: 164 CD28-VL: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGYTLPRTFGGGTKVEIK SEQ ID NO: 165 Full-length sequence of EGFR-CD28 bispecific antibody 129H4L1OT-ZAL: >Strand 1 (SEQ ID NO: 166): EVQLVQSGAEVKKPGASVKVSCKAPGYTFTSHWIQWVRQAPGQGLEWMGEIFPGSGSTHYNEKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARLHYGTSYDAMDYWGQGTTVTVSS GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSIDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Strand 2 (SEQ ID NO: 167): DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGYTLPRTFGGGTKVEIK GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA >Strand 3 (similar to strand 3 of 97H2L3OT-ZAL, SEQ ID NO: 158) >Strand 4 (similar to strand 4 of 97H2L3OT-ZAL, SEQ ID NO: 159) [Table 33] EGFR-VH (similar to EGFR-VH of 129H4L1OT-ZAL, SEQ ID NO: 152) EGFR-VL (similar to EGFR-VL in 129H4L1OT-ZAL, SEQ ID NO: 153) CD28-VH: EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMNWVRQAPGQRLEWMGEINPSTGDTTYNQKFKAKVTLTVDKSASTAYMELSSLRSEDTAVYYCASPYYDLTPFLYWGQGTLVTVSS SEQ ID NO: 174 CD28-VL: DIQMTQSPSSVSASVGDRVTITCLASQTIGTWLAWYQQKPGKSPKLLIYAATTGADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSTPYTFGGGTKVEIK SEQ ID NO: 175 Full-length sequence of EGFR-CD28 bispecific antibody 94H6L12-ZALOT: >Chain 1: EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMNWVRQAPGQRLEWMGEINPSTGDTTYNQKFKAKVTLTVDKSASTAYMELSSLRSEDTAVYCASPYYDLTPFLYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 176 >Chain 2: DIQMTQSPSSVSASVGDRVTITCLASQTIGTWLAWYQQKPGKSPKLLIYAATTGADGVPSRFSGSGSGTKFTLTISSLQPEDFATYYCQQLYSTPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 177 >Chain 3: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS GGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 178 >Chain 4: DIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPCLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK GGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI SEQ ID NO: 179 >IgG1Fc-Knob(L234A / L235A / S354C / T366W) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 248 >IgG1Fc-Hole(L234A / L235A / Y349C / T366S / L368A / Y407V) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 249 >Linker 8 (SEQ ID NO: 90)

[0413] Test Example Test Example 1. Affinity of the GUCY2C monoclonal antibody of the present disclosure to the antigen To test the affinity of the GUCY2C monoclonal antibody of the present disclosure for GUCY2C, this test example used Biacore 8K and Biacore T200 instruments to detect the antibody's binding ability to hGUCY2C-ECD-His (SEQ ID NO: 3). The specific method is as follows: After affinity capturing the antibody using a Protein A biosensor chip coupled with an anti-human antibody, a solution containing GUCY2C protein was flowed over the chip surface, and the instrument detected the reaction signal in real time to obtain an association-dissociation curve. After the dissociation of each experimental cycle was completed, the biosensor chip was regenerated by washing with 10 mM glycine-HCl (pH 1.5). A 1:1 model was used for data fitting. The affinity data for the GUCY2C monoclonal antibody of the present disclosure binding to its corresponding antigen are shown in the table below.

[0414] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38]

[0415] As a result, it was shown that the GUCY2C monoclonal antibody according to the present disclosure has relatively good binding affinity to any of the human GUCY2Cs.

[0416] Test Example 2. Binding activity of anti-GUCY2C monoclonal antibody to cell surface antigens In this test example, the binding activity of GUCY2C monoclonal antibodies to human GUCY2C on the surface of the stable transformed cell line HCT116 / hGUCY2C, which overexpresses human GUCY2C, and the intestinal cancer cells HT-55 and T84 was detected by flow cytometry.

[0417] 1E5 cells resuspended in 1% BSA were added to the reaction wells, centrifuged, and the supernatant was removed. The corresponding diluted antibody was added and incubated at room temperature for 30 minutes. The cells were then washed once with 1% BSA. Subsequently, F(ab')2-goat anti-human IgG Fc secondary antibody, PE (Cat. #H10104, Invitrogen) secondary antibody was added, resuspended, and incubated in the dark at room temperature for 30 minutes. After washing twice with 1% BSA, the fluorescent signal was read using a flow cytometer. Exemplary FACS results are shown in Figures 2A-2E.

[0418] As a result, it was shown that the GUCY2C monoclonal antibody according to the present disclosure binds relatively well and specifically to GUCY2C on the cell surface, and has a binding activity superior to that of 1608.

[0419] Test Example 3. Non-specific binding activity of the GUCY2C humanized antibody according to the present disclosure This test example detected the non-specific binding activity of the GUCY2C humanized antibody to non-target cells (e.g., T cells), as follows.

[0420] Experiment 1: Flow cytometry was performed. 1E5 Jurkat cells (ATCC #TIB-152) resuspended in 1% BSA were added to each well, centrifuged, and the supernatant removed. Diluted antibodies were added at the corresponding concentrations, incubated at room temperature for 30 minutes, and washed once with 1% BSA. F(ab')2-goat anti-human IgG Fc secondary antibody, PE (Cat. #H10104, Invitrogen) secondary antibody was then added, resuspended, and incubated in the dark at room temperature for 30 minutes. After two washes with 1% BSA, the fluorescence signal was read using a flow cytometer. The relative mean fluorescence intensity was calculated by subtracting the background fluorescence intensity of unincubated cells from the mean fluorescence intensity of antibody binding. The results are shown in Table 31 below.

[0421] [Table 39]

[0422] As a result, it was shown that the GUCY2C humanized monoclonal antibody according to the present disclosure does not bind nonspecifically to Jurkat cells that do not express GUCY2C, whereas 1608 clearly binds to Jurkat cells.

[0423] Experiment 2: Flow cytometry was used to detect the nonspecific binding of the GUCY2C humanized monoclonal antibody of the present disclosure to human PBMCs and CD3+ T cells in PBMCs. 3E5 human peripheral blood cells resuspended in 1% BSA were added to each well, centrifuged, and the supernatant removed. The corresponding diluted antibody was added, incubated at room temperature for 30 minutes, and washed once with 1% BSA. A secondary antibody working solution of PE-Cy7 anti-human CD3 (BD #557851) and APC anti-human IgG Fc (BioLegend #409306) was then added, resuspended, and incubated in the dark at room temperature for 30 minutes. After washing twice with 1% BSA, the fluorescence signal was read using a flow cytometer. The relative mean fluorescence intensity was calculated by subtracting the background fluorescence intensity of cells not incubated with antibody from the mean fluorescence ...

Claims

1. An antigen-binding molecule that specifically binds to GUCY2C and CD3, comprising at least one antigen-binding module that specifically binds to GUCY2C and at least one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GUCY2C comprises a heavy chain variable region GUCY2C-VH and a light chain variable region GUCY2C-VL, and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein: i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45, or 46, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27 or 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 28, 29, or 30, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 40 or 12, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 39 or 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or iv-1) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 250, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 251, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 252, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 253, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 254, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 255; or iv-2) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 256, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 257, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 258, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 259, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 260; or iv-3) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 262, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 263, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 266; or iv-4) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 267, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 268, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 269, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 270, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 271, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 272; or iv-5) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 281, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 282, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 283, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 284, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 285; or iv-6) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 288; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 289, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 290; or iv-7) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 291, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 292, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 293; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 294, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 295, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 296; or iv-8) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 297, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 298; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-9) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 301, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 303, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 304; or iv-10) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 305, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 306, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or iv-11) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 128, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 307, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 308; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 309, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 310, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 311; or iv-12) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 312, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 313, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 314, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 315; or iv-13) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 316, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 317, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-14) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 318, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 319, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 320, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 321, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 322, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 323; or iv-15) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 324, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 325, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 326, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 327; or iv-16) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 328, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 329, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; Preferably, i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 40, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; Antigen-binding molecules.

2. the CD3-VH comprises a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 71, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 73 or 88; and the CD3-VL comprises a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; Preferably, the CD3-VH comprises a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 71, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and the CD3-VL comprises a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:

76. The antigen-binding molecule of claim 1.

3. i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, 25 or 57, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 26, 48, 49, 50, 51, 52 or 53; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, 15, or 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 16, 31, 32, 34, 35, or 36; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42 or 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41 or 18; Preferably, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 48, 49, 50, 51, or 52; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 25, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 26; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 32, 34, or 36; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 18; or More preferably, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; The antigen-binding molecule of claim 1 or 2.

4. the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77 or 89, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; Preferably, the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; The antigen-binding molecule according to any one of claims 1 to 3.

5. the antigen-binding module that specifically binds to GUCY2C or the antigen-binding module that specifically binds to CD3 comprises a titin chain and an obscurin chain that can form a dimer; Preferably, The Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 182-200, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-241; More preferably, The Titin chain comprises the amino acid sequence of SEQ ID NO: 198, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO:

236. The antigen-binding molecule according to any one of claims 1 to 4.

6. the antigen-binding molecule comprises an Fc region, and the Fc region is preferably an IgG Fc region, more preferably an IgG1 Fc region; More preferably, the Fc region comprises one or more amino acid substitutions capable of reducing binding of the Fc region to an Fcγ receptor; Most preferably, the Fc region is human IgG1 an Fc region and the amino acids at positions 234 and 235 are A, numbered according to the EU index; The antigen-binding molecule according to any one of claims 1 to 5.

7. the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 and Fc2 each independently having one or more amino acid substitutions that reduce Fc region homodimerization; Preferably, the Fc1 has a knob structure based on knob-in-hole technology, and the Fc2 has a hole structure based on knob-in-hole technology; More preferably, said Fc1 has an amino acid at position 366 that is W and said Fc2 has an amino acid at position 366 that is S, an amino acid at position 368 that is A, and an amino acid at position 407 that is V, numbered according to the EU index; Most preferably, said Fc1 comprises the amino acid sequence of SEQ ID NO: 79 or 85, and said Fc2 comprises the amino acid sequence of SEQ ID NO: 80 or 86. The antigen-binding molecule according to any one of claims 1 to 6.

8. the antigen-binding molecule comprises one antigen-binding module that specifically binds to GUCY2C and one antigen-binding module that specifically binds to CD3, and both of the antigen-binding modules that specifically bind to GUCY2C and CD3 are scFvs; Preferably, The antigen-binding molecule comprises one first chain having a structure represented by formula (a) and one second chain having a structure represented by formula (b), (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1]; (b) [CD3-VL]-[linker 3]-[GUCY2C-VH]-[linker 2]-[Fc2]; wherein the linker 1, linker 2 and linker 3 are the same or different peptide linkers, or the linker 1, linker 2 and linker 3 do not exist; The structures represented by formulas (a) and (b) are arranged from the N-terminus to the C-terminus, More preferably, among them: i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 18, and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78; Most preferably, i) the antigen-binding molecule one first chain comprising the amino acid sequence of SEQ ID NO: 99 and one second chain comprising the amino acid sequence of SEQ ID NO: 100; or one first chain comprising the amino acid sequence of SEQ ID NO: 101 and one second chain comprising the amino acid sequence of SEQ ID NO: 100; or ii) the antigen-binding molecule is one first chain comprising the amino acid sequence of SEQ ID NO: 102 and one second chain comprising the amino acid sequence of SEQ ID NO: 103; or iii) the antigen-binding molecule is one first chain comprising the amino acid sequence of SEQ ID NO: 104 and one second chain comprising the amino acid sequence of SEQ ID NO: 105; The antigen-binding molecule according to any one of claims 1 to 4.

9. the antigen-binding molecule comprises one antigen-binding module that specifically binds to CD3 and one antigen-binding module that specifically binds to GUCY2C, wherein the antigen-binding module that specifically binds to CD3 is a Fab, and the antigen-binding module that specifically binds to GUCY2C is a substituted Fab comprising a titin chain and an obscurin chain capable of forming a dimer; Preferably, The antigen-binding molecule comprises one first chain having the structure represented by formula (c), one second chain having the structure represented by formula (d), one third chain having the structure represented by formula (e), and one fourth chain having the structure represented by formula (f); (c) [CD3-VH]-[CH1]-[Fc1], (d) [CD3-VL]-[CL], (e) [GUCY2C-VH]-[linker 4]-[Obscurin chain]-[Fc2], (f) [GUCY2C-VL]-[linker 4]-[Titin chain], wherein the linker 4 is a peptide linker or the linker 4 is absent; The structures represented by formulas (c), (d), (e), and (f) are arranged from the N-terminus to the C-terminus, More preferably, among them: i) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 89, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 31; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 32; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 34; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 35; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 36; or ii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or iii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 18; Most preferably, The antigen-binding molecule i) one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 110, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or one first chain comprising the amino acid sequence of SEQ ID NO:242, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:110, and one fourth chain comprising the amino acid sequence of SEQ ID NO:113; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:108, and one fourth chain comprising the amino acid sequence of SEQ ID NO:109; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:110, and one fourth chain comprising the amino acid sequence of SEQ ID NO:111; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:110, and one fourth chain comprising the amino acid sequence of SEQ ID NO:112; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:110, and one fourth chain comprising the amino acid sequence of SEQ ID NO:114; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:110, and one fourth chain comprising the amino acid sequence of SEQ ID NO:115; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:110, and one fourth chain comprising the amino acid sequence of SEQ ID NO:116; or ii) one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 106, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 107; or iii) one first chain comprising the amino acid sequence of SEQ ID NO: 91, one second chain comprising the amino acid sequence of SEQ ID NO: 92, one third chain comprising the amino acid sequence of SEQ ID NO: 119, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 120; or one first chain comprising the amino acid sequence of SEQ ID NO:91, one second chain comprising the amino acid sequence of SEQ ID NO:92, one third chain comprising the amino acid sequence of SEQ ID NO:117, and one fourth chain comprising the amino acid sequence of SEQ ID NO:118; The antigen-binding molecule of claim 7 .

10. the antigen-binding molecule comprises one antigen-binding module that specifically binds to GUCY2C and one antigen-binding module that specifically binds to CD3, the antigen-binding module that specifically binds to GUCY2C is a substituted Fab comprising a titin chain and an obscurin chain capable of forming a dimer, and the antigen-binding module that specifically binds to CD3 is a Fab; Preferably, The antigen-binding molecule has one first chain having the structure represented by formula (g), one second chain having the structure represented by formula (f), one third chain having the structure represented by formula (h), and one fourth chain having the structure represented by formula (d), (g) [GUCY2C-VH]-[linker 4]-[Obscurin chain]-[Fc1], (f) [GUCY2C-VL]-[linker 4]-[Titin chain], (h) [CD3-VH]-[CH1]-[Fc2], (d) [CD3-VL]-[CL], wherein the linker 4 is a peptide linker or the linker 4 is absent; The structures represented by formulae (g), (f), (h) and (d) are arranged from the N-terminus to the C-terminus, More preferably, among them: i) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or ii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 31; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 32; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 34; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 35; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 36; or iii) the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; Most preferably, The antigen-binding molecule i) one first chain comprising the amino acid sequence of SEQ ID NO: 121, one second chain comprising the amino acid sequence of SEQ ID NO: 107, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or ii) one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 111, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 112, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 113, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 114, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 115, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 116, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or iii) one first chain comprising the amino acid sequence of SEQ ID NO: 123, one second chain comprising the amino acid sequence of SEQ ID NO: 120, one third chain comprising the amino acid sequence of SEQ ID NO: 93, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 92; The antigen-binding molecule of claim 7 .

11. the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GUCY2C and two antigen-binding modules that specifically bind to CD3, and the antigen-binding modules that specifically bind to GUCY2C and CD3 are both scFvs; Preferably, The antigen-binding molecule has two first chains having a structure represented by formula (i) and two second chains having a structure represented by formula (j), (i) [GUCY2C-VH]-[CH1]-[CD3-VH]-[linker 5]-[CD3-VL]-[linker 6]-[one subunit of the Fc region]; (j) [GUCY2C-VL]-[CL], wherein the linker 5 and the linker 6 are peptide linkers, or the linker 5 and the linker 6 are absent; The structures represented by formulas (i) and (j) are arranged from the N-terminus to the C-terminus, More preferably, among them: the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55; Most preferably, The antigen-binding molecule two first chains comprising the amino acid sequence of SEQ ID NO: 124 and two second chains comprising the amino acid sequence of SEQ ID NO: 65; or two first chains comprising the amino acid sequence of SEQ ID NO: 124 and two second chains comprising the amino acid sequence of SEQ ID NO: 66; The antigen-binding molecule of any one of claims 1 to 4, comprising:

12. the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GUCY2C and two antigen-binding modules that specifically bind to CD3, the antigen-binding modules that specifically bind to GUCY2C are Fabs, and the antigen-binding module that specifically binds to CD3 is a substituted Fab that includes a titin chain and an obscurin chain capable of forming a dimer; Preferably, The antigen-binding molecule comprises two first chains having a structure represented by formula (k), two second chains having a structure represented by formula (j), and two third chains having a structure represented by formula (l), (k) [GUCY2C-VH]-[CH1]-[linker 7]-[CD3-VH]-[linker 4]-[Obscurin chain]-[one subunit of the Fc region]; (j) [GUCY2C-VL]-[CL], (l) [CD3-VL]-[linker 4]-[Titin chain], wherein the linker 4 and the linker 7 are peptide linkers, or the linker 4 and the linker 7 are absent; The structures represented by formulae (k), (j) and (l) are arranged from the N-terminus to the C-terminus, More preferably, among them: the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or the CD3-VH comprises the amino acid sequence of SEQ ID NO: 77, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 78, and the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55; Most preferably, The antigen-binding molecule two first chains comprising the amino acid sequence of SEQ ID NO: 125, two second chains comprising the amino acid sequence of SEQ ID NO: 65, and two third chains comprising the amino acid sequence of SEQ ID NO: 98; or two first chains comprising the amino acid sequence of SEQ ID NO: 125, two second chains comprising the amino acid sequence of SEQ ID NO: 66, and two third chains comprising the amino acid sequence of SEQ ID NO: 98; The antigen-binding molecule of claim 7 .

13. The anti-GUCY2C antibody can specifically bind to GUCY2C, and the anti-GUCY2C antibody comprises a heavy chain variable region GUCY2C-VH and a light chain variable region GUCY2C-VL, wherein: (i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, 22, 43, 44, 45, or 46, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27 or 6, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 28, 29, or 30, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 40 or 12, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 39 or 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or iv-1) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 250, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 251, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 252, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 253, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 254, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 255; or iv-2) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 256, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 257, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 258, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 259, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 260; or iv-3) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 262, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 263, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 266; or iv-4) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 267, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 268, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 269, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 270, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 271, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 272; or iv-5) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 281, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 282, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 283, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 284, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 285; or iv-6) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 288; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 289, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 290; or iv-7) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 291, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 292, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 293; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 294, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 295, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 296; or iv-8) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 297, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 298; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-9) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 301, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 303, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 304; or iv-10) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 305, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 306, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or iv-11) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 128, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 307, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 308; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 309, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 310, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 311; or iv-12) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 312, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 313, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 314, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 315; or iv-13) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 286, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 316, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 317, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 265, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; or iv-14) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 318, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 319, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 320, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 321, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 322, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 323; or iv-15) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 324, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 325, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 326, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 327; or iv-16) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 328, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302; and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 329, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300; Preferably, i) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or ii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9; or iii) the GUCY2C-VH comprises a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 40, and the GUCY2C-VL comprises a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; Anti-GUCY2C antibody.

14. i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, 25 or 57, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 26, 48, 49, 50, 51, 52 or 53; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, 15, or 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 16, 31, 32, 34, 35, or 36; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42 or 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41 or 18; or iv-1) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 273, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 274; or iv-2) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 275, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 276; or iv-3) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 277, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 278; or iv-4) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 279, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 280; or iv-5) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 330, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 331; or iv-6) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 332, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 333; or iv-7) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 334, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 335; or iv-8) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 336, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 337; or iv-9) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 338, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 339; or iv-10) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 340, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 341; or iv-11) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 342, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 343; or iv-12) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 344, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 345; or iv-13) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 346, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 347; or iv-14) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 348, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 349; or iv-15) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 350, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 351; or iv-16) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 352, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 353; Preferably, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54, 55, 48, 49, 50, 51, or 52; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 25, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 26; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 38, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33, 32, 34, or 36; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 15, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 16; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; or The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 17, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 18; More preferably, i) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 54; or the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 56, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 55; or ii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 37, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 33; or iii) the GUCY2C-VH comprises the amino acid sequence of SEQ ID NO: 42, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO: 41; The anti-GUCY2C antibody described in claim 13.

15. The anti-GUCY2C antibody according to claim 13 or 14, which is a murine antibody, a chimeric antibody or a humanized antibody.

16. The anti-GUCY2C antibody according to any one of claims 13 to 15, wherein the anti-GUCY2C antibody comprises a heavy chain constant region and a light chain constant region, preferably wherein the heavy chain constant region is a human IgG1 constant region and the light chain constant region is selected from human antibody κ and λ chain constant regions, more preferably wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 58 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:

59.

17. The anti-GUCY2C antibody comprises a heavy chain and a light chain, the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 64, and the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 65 or 66; or the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 60, and the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 61; or the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 62, and the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 63; Preferably, the heavy chain comprises the amino acid sequence of SEQ ID NO: 64 and the light chain comprises the amino acid sequence of SEQ ID NO: 65 or 66; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 62, and the light chain comprises the amino acid sequence of SEQ ID NO: 63; An anti-GUCY2C antibody according to any one of claims 13 to 16.

18. The anti-GUCY2C antibody according to any one of claims 13 to 17, wherein the anti-GUCY2C antibody is an antibody fragment, and preferably the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv or dAb.

19. A pharmaceutical composition comprising a therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 12 or the anti-GUCY2C antibody of any one of claims 13 to 18, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

20. An isolated nucleic acid encoding the antigen-binding molecule of any one of claims 1 to 12 or the anti-GUCY2C antibody of any one of claims 13 to 18.

21. 21. A host cell comprising the isolated nucleic acid of claim 20.

22. A method for treating a disease, comprising administering to a subject a therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 12, the anti-GUCY2C antibody of any one of claims 13 to 18, or the pharmaceutical composition of claim 19; Preferably, the disease is a tumor, more preferably, the tumor is gastric cancer, gastrointestinal cancer, intestinal cancer, gastric adenocarcinoma, small intestine cancer, colorectal cancer, esophageal cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, kidney cancer, urothelial cancer, breast cancer, bladder cancer, thymic cancer, melanoma, glioma, sarcoma, osteosarcoma, ovarian cancer, thyroid cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial cancer, lung cancer, skin cancer, head and neck cancer, brain cancer, glioblastoma multiforme, gastroesophageal cancer, gastroesophageal adenocarcinoma, liver metastatic adenocarcinoma, multiple myeloma, lymphoma, leukemia, or B-cell lymphoma, and most preferably, cells of the tumor or vascular endothelial cells adjacent to the tumor express GUCY2C. method.

23. 23. The method of treating a disease according to claim 22, further comprising using a second therapeutic agent, preferably wherein said second therapeutic agent comprises an anti-tumor drug, radiation therapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated with an anti-tumor drug, an immune checkpoint inhibitor or a combination thereof.

24. the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR, and comprises at least one antigen-binding module that specifically binds to CD28 and at least one antigen-binding module that specifically binds to EGFR, the antigen-binding module that specifically binds to CD28 comprises a heavy chain variable region CD28-VH and a light chain variable region CD28-VL, and the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL; Preferably, i) the CD28-VH comprises a CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 146, a CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 147, and a CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 148, and the CD28-VL comprises a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 149, a CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 150, and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 151; or ii) the CD28-VH comprises a CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, a CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and a CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162, and the CD28-VL comprises a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 149, a CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 150, and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 163; or iii) the CD28-VH comprises a CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 168, a CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 169, and a CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 170; and the CD28-VL comprises a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 171, a CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 172, and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 173; More preferably, i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 154, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 155; or ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 164, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 165; or iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 174, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 175; 24. A method for treating the disease of claim 23.

25. The antigen binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, The EGFR-VH comprises an EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 140, an EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 141, and an EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 142; and the EGFR-VL comprises an EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 143, an EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 144, and an EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 145; More preferably, the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 152, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 153; 25. A method for treating the disease of claim 24.

26. The bispecific antibody that specifically binds to CD28 and EGFR comprises an Fc region, Preferably, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, the Fc1 having a knob structure in a knob-in-hole technology, and the Fc2 having a hole structure in a knob-in-hole technology; More preferably, said Fc1 has an amino acid at position 366 that is W and said Fc2 has an amino acid at position 366 that is S, an amino acid at position 368 that is A, and an amino acid at position 407 that is V, numbered according to the EU index; Most preferably, said Fc1 comprises the amino acid sequence of SEQ ID NO: 248 and said Fc2 comprises the amino acid sequence of SEQ ID NO:

249.

26. A method for treating the disease of claim 25.

27. The bispecific antibody that specifically binds to CD28 and EGFR comprises one antigen-binding module that specifically binds to CD28 and one antigen-binding module that specifically binds to EGFR, wherein the antigen-binding module that specifically binds to CD28 is a substituted Fab that includes a titin chain and an obscurin chain capable of forming a dimer, and the antigen-binding module that specifically binds to EGFR is a Fab; Preferably, The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain having the structure shown in formula (m), one second chain having the structure shown in formula (n), one third chain having the structure shown in formula (o), and one fourth chain having the structure shown in formula (p); (m) [CD28-VH]-[linker 8]-[Titin chain]-[Fc1], (n) [CD28-VL]-[linker 8]-[Obscurin chain]; (o) [EGFR-VH]-[CH1]-[Fc2], (p) [EGFR-VL]-[CL], wherein the linker 8 is a peptide linker or the linker 8 is absent; The structures represented by formulae (m), (n), (o) and (p) are arranged from the N-terminus to the C-terminus, Most preferably, the bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain comprising the amino acid sequence of SEQ ID NO: 156, one second chain comprising the amino acid sequence of SEQ ID NO: 157, one third chain comprising the amino acid sequence of SEQ ID NO: 158, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 159; or The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain comprising the amino acid sequence of SEQ ID NO: 166, one second chain comprising the amino acid sequence of SEQ ID NO: 167, one third chain comprising the amino acid sequence of SEQ ID NO: 158, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

159.

27. A method for treating the disease of claim 26.

28. The bispecific antibody that specifically binds to CD28 and EGFR comprises one antigen-binding module that specifically binds to CD28 and one antigen-binding module that specifically binds to EGFR, wherein the antigen-binding module that specifically binds to CD28 is a Fab, and the antigen-binding module that specifically binds to EGFR is a substituted Fab that includes a titin chain and an obscurin chain capable of forming a dimer; Preferably, The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain having the structure shown in formula (q), one second chain having the structure shown in formula (r), one third chain having the structure shown in formula (s), and one fourth chain having the structure shown in formula (t), (q) [CD28-VH]-[CH1]-[Fc1], (r) [CD28-VL]-[CL], (s) [EGFR-VH]-[linker 8]-[Obscurin chain]-[Fc2], (t) [EGFR-VL]-[linker 8]-[Titin chain], wherein the linker 8 is a peptide linker or the linker 8 is absent; The structures represented by formulae (q), (r), (s) and (t) are arranged from the N-terminus to the C-terminus, More preferably, The bispecific antibody that specifically binds to CD28 and EGFR comprises one first chain comprising the amino acid sequence of SEQ ID NO: 176, one second chain comprising the amino acid sequence of SEQ ID NO: 177, one third chain comprising the amino acid sequence of SEQ ID NO: 178, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

179.

27. A method for treating the disease of claim 26.

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