Antigen binding molecule specifically binding to GUCY2C and CD3 and medical use thereof
Patent Information
- Application Number
- CN202380051735.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-05-16
- Filing Date
- 2023-08-04
- Publication Date
- 2025-02-28
AI Technical Summary
Existing technology is difficult to effectively treat gastrointestinal malignant tumors, especially intestinal cancer, and targeted therapy targets are limited.
Develop antigen-binding molecules that specifically bind GUCY2C and CD3, including an antibody module that specifically binds GUCY2C and an antibody module that specifically binds CD3, to simulate the interaction between T cells and tumor cells, and activate T cells to specifically kill tumor cells.
Efficient targeted therapy for gastrointestinal malignant tumors, especially intestinal cancer, has been achieved by activating T cells for specific killing and enhancing the immune response to attack tumors.
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Abstract
Description
Antigen binding molecules specifically binding to GUCY2C and CD3 and their medical uses Technical Field
[0001] The present disclosure belongs to the field of biotechnology, and more specifically, relates to antigen-binding molecules that specifically bind to GUCY2C and CD3 and their medical uses. Background Art
[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.
[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 cell membrane as a homodimer or trimer. Its main function is to catalyze GTP to form cGMP to maintain normal physiological functions such as intestinal homeostasis (Michaela Kuhn, Physiological Reviews: (2016), 96 (2): 751-804). In normal tissues, GUCY2C is only expressed in intestinal epithelial cells and is not expressed or expressed at very low levels in other tissues. At the same time, GUCY2C is expressed in gastrointestinal malignancies, including more than 90% of colorectal cancers at all stages and more than 50% of gastric or gastroesophageal junction cancers (Ruth Birbe MD., Human Pathology, (2005), 36(2):170-179; Hadi Danaee, PLoS One, (2017), 12(12):e0189953). In addition, due to the significant differences in expression polarity and vascular enrichment at the cellular level between normal intestinal tissue and intestinal cancer, the probability of GUCY2C contacting blood vessels in intestinal cancer is higher than that 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 new type of immunotherapy that can simultaneously bind to T cells and tumor cells, mimicking the interaction between MHC and TCR. This allows T cells to form lytic synapses, releasing perforins and granzymes to specifically kill tumor cells. Activated T cells can release cytokines, priming other immune cells and expanding the immune response against tumors, ultimately leading to T cell proliferation and a cascade reaction that kills tumor cells.
[0005] Summary of the Invention
[0006] The present disclosure provides an antigen-binding molecule that specifically binds to GUCY2C and CD3, and an antibody that specifically binds to GUCY2C.
[0007] In one aspect, the present disclosure provides an antigen binding molecule that specifically binds to GUCY2C and CD3, comprising at least one antigen binding moiety that specifically binds to GUCY2C and at least one antigen binding moiety that specifically binds to CD3, wherein the antigen binding moiety 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 moiety that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.
[0008] In some embodiments, the antigen binding molecule as described above, wherein:
[0009] (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 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
[0010] (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 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
[0011] (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41 or 18, respectively; or
[0012] iv-1) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 273, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 274; or
[0013] iv-2) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 275, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 276; or
[0014] iv-3) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 277, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 278; or
[0015] iv-4) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 279, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 280; or
[0016] iv-5) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 330, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 331; or
[0017] iv-6) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 332, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 333; or
[0018] iv-7) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 334, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 335; or
[0019] iv-8) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 336, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 337; or
[0020] iv-9) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 338, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 339; or
[0021] iv-10) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 340, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 341; or
[0022] iv-11) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 342, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 343; or
[0023] iv-12) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 344, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 345; or
[0024] iv-13) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 346, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 347; or
[0025] iv-14) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 348, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 349; or
[0026] iv-15) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 350, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 351; or
[0027] iv-16) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 353.
[0028] In some embodiments, the antigen binding molecule as described above, wherein:
[0029] (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 54, respectively, or
[0030] (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 16, respectively, or
[0031] (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41.
[0032] In some embodiments, the GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering conventions.
[0033] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0034] 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
[0035] ii) the GUCY2C-VH has: 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 has: 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
[0036] 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
[0037] 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
[0038] 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
[0039] 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
[0040] 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
[0041] 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
[0042] iv-6) the GUCY2C-VH has: 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 has: 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
[0043] 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
[0044] 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
[0045] 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
[0046] 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
[0047] 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
[0048] 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
[0049] 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
[0050] 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
[0051] 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
[0052] iv-16) The GUCY2C-VH has: GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 261, GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 328, and GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 302; and the GUCY2C-VL has: GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 264, GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 329, and GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 300.
[0053] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0054] 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
[0055] 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
[0056] 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.
[0057] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0058] 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
[0059] 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
[0060] iii) the GUCY2C-VH has: 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: 12; and the GUCY2C-VL has: 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: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14.
[0061] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0062] 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
[0063] The GUCY2C-VH has: 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 has: 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
[0064] The GUCY2C-VH has: 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 has: 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
[0065] ii) the GUCY2C-VH has: 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 has: 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
[0066] The GUCY2C-VH has: GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and the GUCY2C-VL has: GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.
[0067] In some embodiments, in the antigen binding molecule of any of the preceding items, the GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 are defined according to the Kabat numbering convention.
[0068] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the GUCY2C-VH and GUCY2C-VL are humanized and both comprise the FR regions of human antibodies.
[0069] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0070] (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 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; 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. NO: GUCY2C-LCDR3 having the amino acid sequence of 24, and the FR region of GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R and 74K; or
[0071] (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 FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K and 67A; 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. NO: 9, and the FR region of 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
[0072] (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 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 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: 40 or 12. NO: 14, wherein 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.
[0073] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0074] (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 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; 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, 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
[0075] (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 FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K, and 67A; 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. NO: 9, and the FR region of 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
[0076] (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 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 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, 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.
[0077] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0078] The CD3-VH has: 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 or 88; and the CD3-VL has: 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.
[0079] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0080] The CD3-VH has: 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 has: 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.
[0081] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0082] The CD3-VH has: 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 has: 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.
[0083] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0084] 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
[0085] 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
[0086] 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
[0087] 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
[0088] 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
[0089] 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
[0090] 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
[0091] 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
[0092] 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
[0093] 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
[0094] 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
[0095] 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
[0096] 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
[0097] 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
[0098] 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
[0099] 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
[0100] 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
[0101] 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
[0102] 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.
[0103] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0104] 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
[0105] 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
[0106] 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
[0107] 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
[0108] 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
[0109] 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
[0110] 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.
[0111] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0112] 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
[0113] 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
[0114] 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
[0115] 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.
[0116] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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.
[0117] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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: 55.
[0118] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 33.
[0119] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 42, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 41.
[0120] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0121] 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.
[0122] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0123] 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.
[0124] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0125] 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.
[0126] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding moiety that specifically binds to GUCY2C or the antigen binding moiety that specifically binds to CD3 comprises a titin chain and an obscurin chain that can form a dimer.
[0127] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding moiety that specifically binds to GUCY2C comprises a titin chain and an obscurin chain that can form a dimer.
[0128] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding moiety that specifically binds to CD3 comprises a titin chain and an obscurin chain that can form a dimer.
[0129] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 182 to SEQ ID NO: 200, and the obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 201 to SEQ ID NO: 241.
[0130] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 198.
[0131] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 236.
[0132] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region.
[0133] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an IgG Fc region.
[0134] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an IgG1 Fc region.
[0135] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, and the Fc region comprises one or more amino acid substitutions that can reduce the binding of the Fc region to an Fcγ receptor.
[0136] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, and the numbering is according to the EU index.
[0137] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, the Fc region comprises 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 homodimerization of the Fc region.
[0138] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, the Fc1 has a protrusion structure according to the knob-and-mortar technique, and the Fc2 has a hole structure according to the knob-and-mortar technique.
[0139] In some embodiments, an antigen binding molecule as described in any of the preceding items, 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, the Fc1 having a protrusion structure according to the knob-and-mortar technique, and the Fc2 having a hole structure according to the knob-and-mortar technique, the amino acid at position 366 of the Fc1 is W; and the amino acid at position 366 of the Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, and the numbering is according to the EU index.
[0140] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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, wherein the Fc1 comprises the amino acid sequence of SEQ ID NO: 79 or 85; and the Fc2 comprises the amino acid sequence of SEQ ID NO: 80 or 86.
[0141] In some embodiments, an antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein the Fc1 has a protrusion structure according to the knob-and-mortar technique, and wherein the Fc2 has a hole structure according to the knob-and-mortar technique, wherein the amino acid at position 354 of the Fc1 is C and the amino acid at position 366 is W; and the amino acid at position 349 of the Fc2 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, and the numbering is according to the EU index.
[0142] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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, wherein the Fc1 comprises the amino acid sequence of SEQ ID NO: 85; and the Fc2 comprises the amino acid sequence of SEQ ID NO: 86.
[0143] In some embodiments, an antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein the Fc1 has a protrusion structure according to the knob-and-mortar technique, and the Fc2 has a hole structure according to the knob-and-mortar technique, wherein the amino acid at position 237 of the Fc1 is A, the amino acid at position 349 is C, and the amino acid at position 366 is W; and the amino acid at position 237 of the Fc2 is A, the amino acid at position 354 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, and the numbering is according to the EU index.
[0144] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an Fc region, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein the Fc1 comprises the amino acid sequence of SEQ ID NO: 79; and the Fc2 comprises the amino acid sequence of SEQ ID NO: 80.
[0145] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an antigen binding moiety that specifically binds to GUCY2C and an antigen binding moiety that specifically binds to CD3.
[0146] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an antigen binding moiety that specifically binds to GUCY2C and an antigen binding moiety that specifically binds to CD3, and the antigen binding moieties that specifically bind to GUCY2C and CD3 are both scFv.
[0147] In some embodiments, the antigen binding molecule as described in any of the preceding items comprises
[0148] a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b),
[0149] (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1],
[0150] (b) [CD3-VL]-[Linker 3]-[GUCY2C-VH]-[Linker 2]-[Fc2],
[0151] Wherein: the linker 1, linker 2 and linker 3 are the same or different peptide linkers; or linker 1, linker 2 and linker 3 do not exist;
[0152] The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.
[0153] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0154] 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
[0155] 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
[0156] 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
[0157] 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.
[0158] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0159] 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; and 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.
[0160] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0161] 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; and 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.
[0162] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0163] The amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 15, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 16; and 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.
[0164] In some embodiments, the antigen binding molecules as described in any of the preceding items, wherein the linker 1 and linker 2 are peptide linkers known in the art, as long as the antigen binding molecules can exhibit desired antigen binding activity. For example, the peptide linker can be a flexible peptide having 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of L1-(GGGGS)n-L2, wherein 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 amino acid sequence of linker 1 is shown in SEQ ID NO: 81, the amino acid sequence of linker 2 is shown in SEQ ID NO: 82, and the amino acid sequence of linker 3 is shown in SEQ ID NO: 83.
[0165] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of Fc1 is shown as SEQ ID NO: 79, and the amino acid sequence of Fc2 is shown as SEQ ID NO: 80.
[0166] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has:
[0167] i) a first chain comprising the amino acid sequence of SEQ ID NO: 99 and a second chain comprising the amino acid sequence of SEQ ID NO: 100; or
[0168] a first chain comprising the amino acid sequence of SEQ ID NO: 101 and a second chain comprising the amino acid sequence of SEQ ID NO: 100; or
[0169] ii) a first chain comprising the amino acid sequence of SEQ ID NO: 102 and a second chain comprising the amino acid sequence of SEQ ID NO: 103; or
[0170] iii) a first chain comprising the amino acid sequence of SEQ ID NO: 104 and a second chain comprising the amino acid sequence of SEQ ID NO: 105.
[0171] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 99 and a second chain with an amino acid sequence as shown in SEQ ID NO: 100.
[0172] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 101 and a second chain with an amino acid sequence as shown in SEQ ID NO: 100.
[0173] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 102 and a second chain with an amino acid sequence as shown in SEQ ID NO: 103.
[0174] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an antigen binding moiety that specifically binds to CD3 and an antigen binding moiety that specifically binds to GUCY2C, and the antigen binding moiety that specifically binds to CD3 is Fab; the antigen binding moiety that specifically binds to GUCY2C is a replaced Fab, which comprises a titin chain and an obscurin chain that can form a dimer.
[0175] In some embodiments, the antigen-binding molecule as described in any of the preceding items, wherein the antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (e), and a fourth chain having a structure represented by formula (f),
[0176] (c) [CD3-VH]-[CH1]-[Fc1],
[0177] (d) [CD3-VL]-[CL],
[0178] (e) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc2],
[0179] (f) [GUCY2C-VL]-[Linker 4]-[Titin chain],
[0180] Wherein: the linker 4 is a peptide linker; or the linker 4 does not exist;
[0181] The structures represented by formulae (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.
[0182] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0183] 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
[0184] 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
[0185] 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
[0186] 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
[0187] 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
[0188] 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
[0189] 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
[0190] 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
[0191] 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
[0192] 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
[0193] 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.
[0194] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0195] 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; and the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 33.
[0196] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0197] 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; and the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 33.
[0198] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0199] 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; and the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 42, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 41.
[0200] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain and obscurin chain are any of the titin chains and obscurin chains that can form dimers as described in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the obscurin chain comprises the amino acid sequence of SEQ ID NO: 236.
[0201] In some embodiments, the antigen binding molecules as described in any of the preceding items, wherein the linkers 4 are peptide linkers known in the art, as long as the antigen binding molecules can exhibit the desired antigen binding activity. For example, the peptide linker can be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2 or 3. In some embodiments, the sequence of the linker 4 is GGGGS (SEQ ID NO: 90).
[0202] In some embodiments, the antigen-binding molecule of any of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.
[0203] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 87.
[0204] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of Fc1 is shown as SEQ ID NO: 85, and the amino acid sequence of Fc2 is shown as SEQ ID NO: 86.
[0205] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has:
[0206] i) a first chain comprising the amino acid sequence of SEQ ID NO: 91, a second chain comprising the amino acid sequence of SEQ ID NO: 92, a third chain comprising the amino acid sequence of SEQ ID NO: 110, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or
[0207] a first chain comprising the amino acid sequence of SEQ ID NO: 242, a second chain comprising the amino acid sequence of SEQ ID NO: 92, a third chain comprising the amino acid sequence of SEQ ID NO: 110, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or
[0208] a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:108, and a fourth chain comprising the amino acid sequence of SEQ ID NO:109; or
[0209] a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:111; or
[0210] a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:112; or
[0211] a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:114; or
[0212] a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:115; or
[0213] a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:116; or
[0214] ii) a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:106, and a fourth chain comprising the amino acid sequence of SEQ ID NO:107; or
[0215] iii) a first chain comprising the amino acid sequence of SEQ ID NO: 91, a second chain comprising the amino acid sequence of SEQ ID NO: 92, a third chain comprising the amino acid sequence of SEQ ID NO: 119, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 120; or
[0216] A first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:117, and a fourth chain comprising the amino acid sequence of SEQ ID NO:118.
[0217] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 91, a second chain with an amino acid sequence as shown in SEQ ID NO: 92, a third chain with an amino acid sequence as shown in SEQ ID NO: 110, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 113.
[0218] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 242, a second chain with an amino acid sequence as shown in SEQ ID NO: 92, a third chain with an amino acid sequence as shown in SEQ ID NO: 110, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 113.
[0219] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 91, a second chain with an amino acid sequence as shown in SEQ ID NO: 92, a third chain with an amino acid sequence as shown in SEQ ID NO: 119, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 120.
[0220] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises an antigen binding moiety that specifically binds to GUCY2C and an antigen binding moiety that specifically binds to CD3, the antigen binding moiety that specifically binds to GUCY2C is a replaced Fab, which comprises a titin chain and an obscurin chain that can form a dimer; the antigen binding moiety that specifically binds to CD3 is a Fab.
[0221] In some embodiments, the antigen-binding molecule as described in any of the preceding items, wherein the antigen-binding molecule comprises a first chain having a structure represented by formula (g), a second chain having a structure represented by formula (f), a third chain having a structure represented by formula (h), and a fourth chain having a structure represented by formula (d),
[0222] (g) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc1],
[0223] (f) [GUCY2C-VL]-[Linker 4]-[Titin chain],
[0224] (h) [CD3-VH]-[CH1]-[Fc2],
[0225] (d) [CD3-VL]-[CL],
[0226] Wherein: the linker 4 is a peptide linker; or the linker 4 does not exist;
[0227] The structures represented by formulas (g), (f), (h) and (d) are arranged from the N-terminus to the C-terminus.
[0228] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0229] 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
[0230] 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
[0231] 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
[0232] 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
[0233] 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
[0234] 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
[0235] 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
[0236] 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.
[0237] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0238] 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; and 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.
[0239] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain and obscurin chain are any of the titin chains and obscurin chains that can form dimers as described in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the obscurin chain comprises the amino acid sequence of SEQ ID NO: 236.
[0240] In some embodiments, the antigen binding molecules as described in any of the preceding items, wherein the linkers 4 are peptide linkers known in the art, as long as the antigen binding molecules can exhibit the desired antigen binding activity. For example, the peptide linker can be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2 or 3. In some embodiments, the sequence of the linker 4 is GGGGS (SEQ ID NO: 90).
[0241] In some embodiments, the antigen-binding molecule of any of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.
[0242] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 87.
[0243] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of Fc1 is shown as SEQ ID NO: 85, and the amino acid sequence of Fc2 is shown as SEQ ID NO: 86.
[0244] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has:
[0245] i) a first chain comprising the amino acid sequence of SEQ ID NO: 121, a second chain comprising the amino acid sequence of SEQ ID NO: 107, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0246] ii) a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 111, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0247] a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 112, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0248] a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 113, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0249] a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 114, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0250] a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 115, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0251] a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 116, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or
[0252] iii) a first chain comprising the amino acid sequence of SEQ ID NO: 123, a second chain comprising the amino acid sequence of SEQ ID NO: 120, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92.
[0253] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 121, a second chain with an amino acid sequence as shown in SEQ ID NO: 107, a third chain with an amino acid sequence as shown in SEQ ID NO: 93, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 92.
[0254] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises two antigen binding moieties that specifically bind to GUCY2C and two antigen binding moieties that specifically bind to CD3, and the antigen binding moieties that specifically bind to GUCY2C and CD3 are both scFv.
[0255] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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),
[0256] (i) [GUCY2C-VH]-[CH1]-[CD3-VH]-[Linker 5]-[CD3-VL]-[Linker 6]-[one subunit of the Fc region],
[0257] (j)[GUCY2C-VL]-[CL],
[0258] Wherein: the linker 5 and the linker 6 are peptide linkers; or the linker 5 and the linker 6 do not exist;
[0259] The structures shown in formulas (i) and (j) are arranged from the N-terminus to the C-terminus.
[0260] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0261] 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
[0262] 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.
[0263] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0264] 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; and 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.
[0265] In some embodiments, the antigen binding molecules as described in any of the preceding items, wherein the linker 5 and linker 6 are peptide linkers known in the art, as long as the antigen binding molecules can exhibit the desired antigen binding activity. For example, the peptide linker can be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of L1-(GGGGS)n-L2, wherein 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 3-15 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2, or 3. In some embodiments, the amino acid sequence of linker 5 is shown in SEQ ID NO: 95, and the amino acid sequence of linker 6 is shown in SEQ ID NO: 96.
[0266] In some embodiments, the antigen-binding molecule of any of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.
[0267] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 59.
[0268] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of one subunit of the Fc region is as shown in SEQ ID NO: 94.
[0269] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has:
[0270] 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
[0271] 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.
[0272] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with two amino acid sequences as shown in SEQ ID NO: 124 and a second chain with two amino acid sequences as shown in SEQ ID NO: 65.
[0273] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule comprises two antigen binding moieties that specifically bind to GUCY2C and two antigen binding moieties that specifically bind to CD3, and the antigen binding moiety that specifically binds to GUCY2C is Fab; the antigen binding moiety that specifically binds to CD3 is a replaced Fab, which comprises a titin chain and an obscurin chain that can form a dimer.
[0274] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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),
[0275] (k) [GUCY2C-VH]-[CH1]-[Linker 7]-[CD3-VH]-[Linker 4]-[Obscurin chain]-[one subunit of the Fc region],
[0276] (j)[GUCY2C-VL]-[CL],
[0277] (l) [CD3-VL]-[Linker 4]-[Titin chain],
[0278] Wherein: the linker 4 and the linker 7 are peptide linkers; or the linker 4 and the linker 7 do not exist;
[0279] The structures shown in formulas (k), (j) and (l) are arranged from the N-terminus to the C-terminus.
[0280] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0281] 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
[0282] 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.
[0283] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0284] 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; and 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.
[0285] In some embodiments, the antigen binding molecules as described in any of the preceding items, wherein the linker 4 and linker 7 are peptide linkers known in the art, as long as the antigen binding molecules can exhibit the desired antigen binding activity. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2 or 3. In some embodiments, the amino acid sequence of linker 4 is as shown in SEQ ID NO: 90, and the amino acid sequence of linker 7 is as shown in SEQ ID NO: 97.
[0286] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain and obscurin chain are any of the titin chains and obscurin chains that can form dimers as described in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the obscurin chain comprises the amino acid sequence of SEQ ID NO: 236.
[0287] In some embodiments, the antigen-binding molecule of any of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.
[0288] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 59.
[0289] In some embodiments, the amino acid sequence of a subunit of the Fc region is shown in SEQ ID NO: 86.
[0290] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has:
[0291] 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
[0292] Two first chains comprise the amino acid sequence of SEQ ID NO: 125, two second chains comprise the amino acid sequence of SEQ ID NO: 66, and two third chains comprise the amino acid sequence of SEQ ID NO: 98.
[0293] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the antigen binding molecule has: a first chain with two amino acid sequences as shown in SEQ ID NO: 125, a second chain with two amino acid sequences as shown in SEQ ID NO: 65, and a third chain with two amino acid sequences as shown in SEQ ID NO: 98.
[0294] In another aspect, the present disclosure further provides an anti-GUCY2C antibody that can specifically bind to GUCY2C, wherein the antibody comprises a heavy chain variable region GUCY2C-VH and a light chain variable region GUCY2C-VL, wherein:
[0295] (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 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
[0296] (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 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
[0297] (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41 or 18, respectively; or
[0298] iv-1) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 273, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 274; or
[0299] iv-2) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 275, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 276; or
[0300] iv-3) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 277, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 278; or
[0301] iv-4) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 279, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 280; or
[0302] iv-5) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 330, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 331; or
[0303] iv-6) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 332, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 333; or
[0304] iv-7) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 334, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 335; or
[0305] iv-8) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 336, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 337; or
[0306] iv-9) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 338, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 339; or
[0307] iv-10) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 340, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 341; or
[0308] iv-11) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 342, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 343; or
[0309] iv-12) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 344, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 345; or
[0310] iv-13) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 346, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 347; or
[0311] iv-14) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 348, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 349; or
[0312] iv-15) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the GUCY2C-VH respectively comprise the amino acid sequences of GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in SEQ ID NO: 350, and GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in the GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 351; or
[0313] iv-16) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 353.
[0314] In some embodiments, the anti-GUCY2C antibody as described above, wherein:
[0315] (i) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 54, respectively, or
[0316] (ii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 16, respectively, or
[0317] (iii) GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 in the 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 GUCY2C-VL respectively comprise the amino acid sequences of GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 in SEQ ID NO: 41.
[0318] In some embodiments, the GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering conventions.
[0319] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0320] 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
[0321] ii) the GUCY2C-VH has: 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 has: 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
[0322] 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
[0323] 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
[0324] 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
[0325] 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
[0326] 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
[0327] 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
[0328] iv-6) the GUCY2C-VH has: 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 has: 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
[0329] 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
[0330] 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
[0331] 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
[0332] 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
[0333] 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
[0334] 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
[0335] 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
[0336] 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
[0337] 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
[0338] iv-16) The GUCY2C-VH has: 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 has: 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.
[0339] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0340] 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
[0341] 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
[0342] 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.
[0343] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0344] 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
[0345] 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
[0346] iii) the GUCY2C-VH has: 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: 12; and the GUCY2C-VL has: 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: 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14.
[0347] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0348] 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
[0349] The GUCY2C-VH has: 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 has: 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
[0350] The GUCY2C-VH has: 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 has: 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
[0351] ii) the GUCY2C-VH has: 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 has: 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
[0352] The GUCY2C-VH has: GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and the GUCY2C-VL has: GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO: 9.
[0353] In some embodiments, in the anti-GUCY2C antibody of any of the preceding items, the GUCY2C-HCDR1, GUCY2C-HCDR2, GUCY2C-HCDR3, GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3 are defined according to the Kabat numbering convention.
[0354] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, wherein the anti-GUCY2C antibody is a murine antibody, a chimeric antibody, or a humanized antibody; preferably a humanized antibody.
[0355] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, 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 κ and λ 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.
[0356] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, wherein the GUCY2C-VH and GUCY2C-VL are humanized and both comprise the FR regions of human antibodies.
[0357] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0358] (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 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; 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. NO: GUCY2C-LCDR3 having the amino acid sequence of 24, and the FR region of GUCY2C-VL comprises one or more amino acid substitutions selected from the group consisting of 2V, 3V, 68R and 74K; or
[0359] (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 FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K and 67A; 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. NO: 9, and the FR region of 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
[0360] (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 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 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: 40 or 12. NO: 14, wherein 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.
[0361] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0362] (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 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; 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, 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
[0363] (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 FR region of the GUCY2C-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 38K, 66K, and 67A; 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. NO: 9, and the FR region of 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
[0364] (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 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 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, 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.
[0365] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein:
[0366] 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
[0367] 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
[0368] 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
[0369] 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
[0370] 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
[0371] 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
[0372] 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
[0373] 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
[0374] 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
[0375] 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
[0376] 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
[0377] 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
[0378] 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
[0379] 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
[0380] 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
[0381] 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
[0382] 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
[0383] 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
[0384] 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.
[0385] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0386] 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
[0387] 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
[0388] 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
[0389] 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
[0390] 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
[0391] 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
[0392] 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.
[0393] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein:
[0394] 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
[0395] 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
[0396] 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
[0397] 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.
[0398] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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.
[0399] In some embodiments, the antigen binding molecule as described in any of the preceding items, 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: 55.
[0400] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 37, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 33.
[0401] In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the amino acid sequence of the GUCY2C-VH is shown in SEQ ID NO: 42, and the amino acid sequence of the GUCY2C-VL is shown in SEQ ID NO: 41.
[0402] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein:
[0403] the heavy chain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 64, and the light chain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 65 or 66; or
[0404] the heavy chain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 60, and the light chain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 61; or
[0405] 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.
[0406] In some embodiments, the anti-GUCY2C antibody of any of the preceding items, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein:
[0407] 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
[0408] 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
[0409] 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.
[0410] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, 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.
[0411] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, 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.
[0412] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, 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.
[0413] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, 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.
[0414] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, wherein the anti-GUCY2C antibody is an antibody fragment, wherein the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv or dAb.
[0415] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, wherein the anti-GUCY2C antibody is a bispecific antibody.
[0416] In some embodiments, the anti-GUCY2C antibody as described in any of the preceding items, wherein the anti-GUCY2C antibody is a bispecific antibody that specifically binds to GUCY2C and CD3.
[0417] In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of any of the aforementioned antigen-binding molecules or any of the aforementioned anti-GUCY2C antibodies, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
[0418] In another aspect, the present disclosure also provides an isolated nucleic acid encoding any of the aforementioned antigen-binding molecules or any of the aforementioned anti-GUCY2C antibodies.
[0419] In another aspect, the present disclosure also provides a vector comprising the aforementioned isolated nucleic acid.
[0420] In another aspect, the present disclosure also provides a host cell comprising the aforementioned isolated nucleic acid.
[0421] In another aspect, the present disclosure also provides a method for treating a disease, comprising administering to a subject a therapeutically effective amount of any of the aforementioned antigen-binding molecules or any of the aforementioned anti-GUCY2C antibodies or any of the aforementioned compositions.
[0422] In another aspect, the present disclosure also provides use of any of the aforementioned antigen-binding molecules or any of the aforementioned antibodies or compositions thereof in the preparation of a medicament for treating a disease.
[0423] In another aspect, the present disclosure further provides any of the aforementioned antigen-binding molecules or any of the aforementioned anti-GUCY2C antibodies or any of the aforementioned compositions for use as a medicament. In some embodiments, the medicament is used to treat a disease.
[0424] In some embodiments, the disease as described in any of the preceding items is a proliferative disease or a tumor. In some embodiments, the disease as described in any of the preceding items is a tumor, and the tumor is gastrointestinal cancer (stomach 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. , endometrial cancer, lung cancer (non-small cell lung cancer (squamous cell carcinoma, adenocarcinoma) and small cell lung cancer), skin cancer, head and neck cancer (head and neck squamous cell carcinoma), brain cancer, glioblastoma multiforme, gastroesophageal cancer (gastroesophageal adenocarcinoma), metastatic adenocarcinoma of the liver, multiple myeloma, lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma or B-cell lymphoma) or leukemia (such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL)).
[0425] In some embodiments, the cells of the tumor as described above or the vascular endothelial cells adjacent to the tumor express GUCY2C.
[0426] In some embodiments, the tumor as described above is a solid tumor.
[0427] In some embodiments, the tumor as described above is a solid tumor, which is gastrointestinal cancer (stomach 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 cancer, 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 (lung squamous cell carcinoma, lung adenocarcinoma) and small cell lung cancer), skin cancer, head and neck cancer (head and neck squamous cell carcinoma), brain cancer, glioblastoma multiforme, gastroesophageal cancer (gastroesophageal adenocarcinoma) or liver metastatic adenocarcinoma.
[0428] In some embodiments, the tumor as described above is gastrointestinal cancer; preferably, gastric cancer or intestinal cancer; more preferably, gastric adenocarcinoma, small intestinal cancer or colorectal cancer.
[0429] In some embodiments, the tumor as described above is a non-solid tumor.
[0430] In some embodiments, the tumor as described above is a non-solid tumor, and the non-solid tumor is multiple myeloma, lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma) or leukemia (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL)).
[0431] In some embodiments, the aforementioned disease is a disease associated with GUCY2C; in some embodiments, the aforementioned disease is a disease expressing GUCY2C.
[0432] In some embodiments, the method for treating a disease as described above further comprises the use of a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an anti-tumor agent, radiotherapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an anti-tumor agent, an immune checkpoint inhibitor, or a combination thereof.
[0433] In some embodiments, in the method of treating a disease as described above, the second therapeutic agent is administered simultaneously, sequentially, or separately with the antigen binding molecule described in any one of the disclosures.
[0434] 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.
[0435] In some embodiments, as described above in the method for treating a disease, the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR, comprising at least one antigen binding moiety that specifically binds to CD28 and at least one antigen binding moiety that specifically binds to EGFR.
[0436] In some embodiments, as described above in the method for treating a disease, the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR, which comprises at least one antigen binding moiety that specifically binds to CD28 and at least one antigen binding moiety that specifically binds to EGFR, wherein the antigen binding moiety 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 moiety that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL.
[0437] In some embodiments, the method of treating a disease as described above, wherein:
[0438] 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
[0439] 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
[0440] 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.
[0441] In some embodiments, the method of treating a disease as described above, wherein:
[0442] 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
[0443] 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
[0444] 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.
[0445] In some embodiments, the method for treating a disease as described above, wherein the antigen binding moiety that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, wherein:
[0446] The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 140, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 141, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 142; and the EGFR-VL has: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 143, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 144, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 145.
[0447] In some embodiments, the method for treating a disease as described above, 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.
[0448] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds 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 that reduce binding of the Fc region to Fcγ receptors. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, numbering according to the EU index.
[0449] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 that can associate with each other, and wherein the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, the Fc1 has a protrusion structure according to the knob-in-hole technique, and the Fc2 has a hole structure according to the knob-in-hole technique. In some embodiments, the amino acid at position 366 of the Fc1 is W; and the amino acid at position 366 of the Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering according to the EU index. In some embodiments, the amino acid at position 354 of Fc1 is C and the amino acid at position 366 is W; and the amino acid at position 349 of Fc2 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering 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.
[0450] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain and obscurin chain are any of the titin chains and obscurin chains that can form dimers as described in Tables 3-1 and 3-2 of the present disclosure. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the obscurin chain comprises the amino acid sequence of SEQ ID NO: 236.
[0451] In some embodiments, the antigen binding molecules as described in any of the preceding items, wherein the linkers 8 are peptide linkers known in the art, as long as the antigen binding molecules can exhibit the desired antigen binding activity. For example, the peptide linker can be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2 or 3. In some embodiments, the sequence of the linker 8 is GGGGS (SEQ ID NO: 90).
[0452] In some embodiments, the antigen-binding molecule of any of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 84.
[0453] In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is an antibody light chain constant region. In some embodiments, the antigen-binding molecule described in any of the preceding items, wherein the CL is a kappa or lamada light chain constant region. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 59.
[0454] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an antigen binding moiety that specifically binds to CD28 and an antigen binding moiety that specifically binds to EGFR, the antigen binding moiety that specifically binds to CD28 is a replaced Fab that comprises a titin chain and an obscurin chain that can form a dimer; the antigen binding moiety that specifically binds to EGFR is a Fab.
[0455] In some embodiments, the method for treating a disease as described above, wherein
[0456] The bispecific antibody that specifically binds to CD28 and EGFR comprises a first chain having a structure represented by formula (m), a second chain having a structure represented by formula (n), a third chain having a structure represented by formula (o), and a fourth chain having a structure represented by formula (p).
[0457] (m) [CD28-VH]-[Linker 8]-[Titin chain]-[Fc1],
[0458] (n) [CD28-VL]-[Linker 8]-[Obscurin chain],
[0459] (o) [EGFR-VH]-[CH1]-[Fc2],
[0460] (p)[EGFR-VL]-[CL],
[0461] Wherein: the linker 8 is a peptide linker; or the linker 8 does not exist;
[0462] The structures represented by formulae (m), (n), (o), and (p) are arranged from the N-terminus to the C-terminus.
[0463] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR has: a first chain comprising the amino acid sequence of SEQ ID NO: 156, a second chain comprising the amino acid sequence of SEQ ID NO: 157, a third chain comprising the amino acid sequence of SEQ ID NO: 158, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 159; or
[0464] The bispecific antibody that specifically binds to CD28 and EGFR has: a first chain comprising the amino acid sequence of SEQ ID NO: 166, a second chain comprising the amino acid sequence of SEQ ID NO: 167, a third chain comprising the amino acid sequence of SEQ ID NO: 158, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 159.
[0465] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR has: a first chain with an amino acid sequence as shown in SEQ ID NO: 156, a second chain with an amino acid sequence as shown in SEQ ID NO: 157, a third chain with an amino acid sequence as shown in SEQ ID NO: 158, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 159.
[0466] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR has: a first chain with an amino acid sequence as shown in SEQ ID NO: 166, a second chain with an amino acid sequence as shown in SEQ ID NO: 167, a third chain with an amino acid sequence as shown in SEQ ID NO: 158, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 159.
[0467] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an antigen binding moiety that specifically binds to CD28 and an antigen binding moiety that specifically binds to EGFR, and the antigen binding moiety that specifically binds to CD28 is a Fab; the antigen binding moiety that specifically binds to EGFR is a replaced Fab that comprises a titin chain and an obscurin chain that can form a dimer.
[0468] In some embodiments, the method for treating a disease as described above, wherein
[0469] The bispecific antibody that specifically binds to CD28 and EGFR comprises a first chain having a structure represented by formula (q), a second chain having a structure represented by formula (r), a third chain having a structure represented by formula (s), and a fourth chain having a structure represented by formula (t).
[0470] (q) [CD28-VH]-[CH1]-[Fc1],
[0471] (r)[CD28-VL]-[CL],
[0472] (s) [EGFR-VH]-[Linker 8]-[Obscurin chain]-[Fc2],
[0473] (t) [EGFR-VL]-[Linker 8]-[Titin chain],
[0474] Wherein: the linker 8 is a peptide linker; or the linker 8 does not exist;
[0475] The structures represented by formulas (q), (r), (s) and (t) are arranged from the N-terminus to the C-terminus.
[0476] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR has: a first chain comprising the amino acid sequence of SEQ ID NO: 176, a second chain comprising the amino acid sequence of SEQ ID NO: 177, a third chain comprising the amino acid sequence of SEQ ID NO: 178, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 179.
[0477] In some embodiments, the method for treating a disease as described above, wherein the bispecific antibody that specifically binds to CD28 and EGFR has: a first chain with an amino acid sequence as shown in SEQ ID NO: 176, a second chain with an amino acid sequence as shown in SEQ ID NO: 177, a third chain with an amino acid sequence as shown in SEQ ID NO: 178, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 179.
[0478] The antigen-binding molecules provided by the present disclosure have the characteristics of good therapeutic activity, safety, pharmacokinetic properties and drugability (such as stability). BRIEF DESCRIPTION OF THE DRAWINGS
[0479] Figure 1A: Schematic diagram of the structure of Format 1 of the GUCY2C-CD3 bispecific antibody; Figure 1B: Schematic diagram of the structure of Format 2 of the GUCY2C-CD3 bispecific antibody, wherein Ob represents Obscurin; Figure 1C: Schematic diagram of the structure of Format 3 of the GUCY2C-CD3 bispecific antibody, wherein Ob represents Obscurin; Figure 1D: Schematic diagram of the structure of Format 4 of the GUCY2C-CD3 bispecific antibody; Figure 1E: Schematic diagram of the structure of Format 5 of the GUCY2C-CD3 bispecific antibody, wherein Ob represents Obscurin.
[0480] Figures 2A-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.
[0481] 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.
[0482] FIG4A : HCT116 / hGUCY2C were used as target cells to test the ability of antibodies to activate Jurkat / NFAT cells; FIG4B : LS1034 was used as target cells to test the ability of antibodies to activate Jurkat / NFAT cells.
[0483] Figure 5A shows the results of IFN-γ release by the co-action of GUCY2C-CD3 bispecific antibody and LS1034 cells; Figure 5B and Figure 5C show the results of IFN-γ release by the co-action of GUCY2C-CD3 bispecific antibody and LS174T cells.
[0484] Figure 6A shows the results of IL-6 release from the GUCY2C-CD3 bispecific antibody and LS1034 cells; Figure 6B and Figure 6C show the results of IL-6 release from the GUCY2C-CD3 bispecific antibody and LS174T cells. DETAILED DESCRIPTION
[0485] the term
[0486] The terms used herein are for the purpose of describing the embodiments only and are 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 to which this disclosure belongs.
[0487] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," and the like should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, to mean "including but not limited to." Unless otherwise indicated, "comprising" includes "consisting of." For example, a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 specifically encompasses a GUCY2C-HCDR1 having the amino acid sequence of SEQ ID NO: 1.
[0488] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).
[0489] The term "and / or," such as "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.
[0490] The term "CD28" (cluster of differentiation 28, Tp44) refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells by CD28 can also provide effective signals for the production of various interleukins. CD28 is a receptor for CD80 (B7.1) protein and CD86 (B7.2) protein, and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747.
[0491] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as 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 naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, a carboxyl 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 naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.
[0492] The term "amino acid mutation" includes amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as 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 carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. 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 with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, are also useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has mutated from T to W.
[0493] The term "antigen binding molecule" is used in the broadest sense to encompass various molecules that specifically bind to antigens, including but not limited to antibodies, other polypeptides with antigen binding activity, and antibody fusion proteins formed by fusion of the two, as long as they exhibit the desired antigen binding activity. The antigen binding molecules herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen binding domain. Exemplary, the antigen binding molecules herein are bispecific antigen binding molecules (e.g., bispecific antibodies).
[0494] The term "antibody" is used in the broadest sense and encompasses various 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 antigen-binding portions), as long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL).
[0495] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, they can be divided into bispecific antibodies with different valencies, such as bispecific antibodies with different valencies. Based on whether the structure is symmetrical, they can be divided into bispecific antibodies with different symmetric structures and bispecific antibodies with different asymmetric structures. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, are formed by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragments) have a relatively large molecular weight. The Fc fragment helps to purify the antibody and improve its solubility and stability. The Fc part may also bind to the receptor FcRn to increase the antibody serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 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. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).
[0496] The term "variable region" or "variable domain" refers to the domain of an antigen-binding molecule that binds to an antigen. Herein, the heavy chain variable region in an antigen-binding moiety that specifically binds to GUCY2C is designated GUCY2C-VH, and the light chain variable region is designated GUCY2C-VL; the heavy chain variable region in an antigen-binding moiety that specifically binds to CD3 is designated CD3-VH, and the light chain variable region is designated CD3-VL. VH and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining regions" or "CDRs" refers to the regions within the variable domain that primarily contribute to antigen binding; "framework" or "FRs" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; 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; the three CDR regions in GUCY2C-VL are designated GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3; the three CDR regions in CD3-VH are designated CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3; and the three CDR regions in CD3-VL are designated CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3. From N-terminus to C-terminus, each VH and VL consists of the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.
[0497] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "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), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art. The numbering convention of the present disclosure is shown in Table 1 below.
[0498] Table 1. Relationships between CDR numbering systems
[0499] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are all numbered using the "Kabat" convention.
[0500] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. 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) formed from antibody fragments.
[0501] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise indicated, the numbering convention for the Fc region is the EU index.
[0502] The term "Titin chain" refers to a peptide segment of 78-118 amino acids in length containing the Titin Ig-like 152 domain or a functional variant thereof in the Titin protein, wherein the Titin chain is capable of binding 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 segment of 87-117 amino acids in length containing the Obscurin Ig-like 1 domain or a functional variant thereof on the Obscurin protein, or a peptide segment of 78-118 amino acids in length containing the Obscurin-like Ig-like 1 domain or a functional variant thereof on the Obscurin-like 1 protein, wherein the Obscurin chain is capable of binding to the Titin Ig-like 152 domain to form a dimerization complex. The Titin chain and the Obscurin chain disclosed herein can be used to replace the CH1 and CL in Fab to form a replaced Fab (Fab-S), and this replacement does not affect the binding of the antigen-binding molecule to the antigen.
[0503] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.
[0504] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant region and the framework region portion of the variable region).
[0505] The term "affinity" refers to the overall intensity of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its part Y can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art (including those described herein). The terms "kassoc" or "ka" refer to the on-rate of a specific antibody-antigen interaction, and the terms "kdis" or "kd" as used herein are intended to refer to the off-rate of a specific antibody-antigen interaction. As used herein, the term "KD" refers to an equilibrium dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).
[0506] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for the natural mutations that may be present in small amounts. In contrast, polyclonal antibody preparations are typically comprised of a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" represents the characteristic of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any ad hoc method. In some embodiments, the antibody provided by the present disclosure is a monoclonal antibody.
[0507] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent of an antigen binding protein (eg, an antibody). An antigen may have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).
[0508] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody or its antigen-binding fragment. An epitope can be formed by contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, when the non-contiguous amino acids are brought into spatial proximity due to the folding of the antigen (i.e., by the tertiary folding of the antigen in the case of proteinaceous properties). Conformational epitopes differ from linear epitopes in that antibody binding to the conformational epitope 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 particular epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, 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)).
[0509] The terms "capable of specifically binding", "specific binding" or "binding" refer to the ability of an antibody to bind to an antigen or epitope with a higher affinity than to other antigens or epitopes. - 7 M or less (e.g., about 1×10 -8In some embodiments, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of 10% or less (e.g., 1%) of the KD of the antibody bound to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as those measured by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzees (Pan troglodytes (chimpanzee, chimp), or marmosets (Callithrix jacchus (common marmoset, marmoset).
[0510] The term "no binding" means that the antibody is unable to bind to an antigen or an epitope within the antigen in the manner described above. For example, when the antibody is about 1×10 -6 The antigen binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of M or greater.
[0511] The term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to a target antigen. Specific antigen binding moieties include the antigen binding domain of an antibody, for example, comprising a heavy chain variable region and a light chain variable region. The term "antigen binding moiety 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 moiety that specifically binds to GUCY2C has the following equilibrium dissociation constant (KD): <about 10 nM, which is measured by surface plasmon resonance assay. Antigen binding moieties include antibody fragments as defined herein, such as Fab, replaced Fab or scFv.
[0512] The term "linker" refers to a connecting unit that connects 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 comprising one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in a structural formula, it indicates that the units on either side are directly connected by a covalent bond.
[0513] "Tm" is the melting denaturation temperature (intrinsic fluorescence). When a protein denatures (by heat or denaturants), the tertiary structure opens up, the microenvironment of the aromatic amino acids changes, and the emitted fluorescence spectrum shifts. For purposes of this disclosure, Tm1 refers to the temperature at which fluorescence reaches half its maximum value.
[0514] "Tonset" is the denaturation onset temperature. It refers to the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.
[0515] "Tagg" stands for aggregation onset temperature. Aggregates are detected by static light scattering at two wavelengths, 266 nm and 473 nm, to measure the temperature at which the sample begins to aggregate. Tagg 266 refers to the aggregation onset temperature measured at 266 nm.
[0516] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this article and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0517] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from the components of its natural environment.Isolated nucleic acid includes the nucleic acid molecule contained in the following cells, and the cell usually contains the nucleic acid molecule, but the nucleic acid molecule is present in extrachromosomal or in a chromosomal position different from its natural chromosomal position. The isolated nucleic acid encoding the antigen-binding molecules refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such one or more nucleic acid molecules in a single vector or a separate vector, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise indicated, specific nucleic acid sequences also implicitly encompass conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences and sequences explicitly indicated. Specifically, as described in detail below, degenerate codon substitutions can be obtained by producing sequences in which the third position of one or more selected (or all) codons is replaced by degenerate bases and / or deoxyinosine residues.
[0518] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
[0519] The term sequence "identity" refers to the degree (percentage) at which the amino acid / nucleic acid of the two sequences are identical in the equivalent position when two sequences are optimally aligned. In the alignment process, it may be allowed, if necessary, to introduce a gap to obtain maximum sequence identity percentage, but any conservative substitution is not considered to constitute a part for sequence identity. For measuring sequence identity percentage, alignment can be achieved by techniques known in the art, for example, using 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 applicable to measuring alignment, including any algorithm required for maximum alignment, which is included in the full length of the sequence being compared.
[0520] The term "fused" or "linked" means that the components (eg, an antigen binding moiety and an Fc domain) are covalently linked directly or via a linker.
[0521] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence for guiding and / or controlling (together with the host cell) the expression of one or more heterologous coding regions operably connected thereto. An expression construct can include but is not limited to affecting or controlling transcription, translation and affecting the sequence of RNA splicing of the coding region operably connected thereto when introns are present.
[0522] The terms "host cell", "host cell line", and "host cell culture" are 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", which include the primary transformed cell and the progeny derived therefrom, without regard to the number of passages. The progeny may not be completely 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 the cell screened or selected in the primary transformed cell. Host cells include prokaryotic and eukaryotic host cells, wherein 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, cow, 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, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia truncatula, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha, polymorpha), Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa. The host cells of this patent do not include subject matter not authorized under patent law.
[0523] As used in this application, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cell from which they were selected are included.
[0524] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
[0525] The term "pharmaceutical composition" refers to a mixture containing one or more antigen binding molecules or antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.
[0526] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
[0527] 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 explicitly stated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cynomolgus monkey" or "cynomolgus monkey" refers to cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the individual or subject is a human.
[0528] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.
[0529] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.
[0530] "Treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention attempted to be applied to the individual being treated, and can be performed for preventive purposes or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. In some embodiments, the molecules of the present disclosure are used to delay the development of the disease or slow the progression of the disease.
[0531] The terms "recurrence," "relapse," and "relapsed" refer to the return of a cancer or disease after clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence may be considered a relapse.
[0532] The term "refractory" or "resistant" refers to a cancer or disease that does not respond to treatment.
[0533] "Effective amount" is generally enough to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the damage (e.g., lung disease) caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is enough to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete treatment or prophylactic effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. A "therapeutically effective amount" and a "prophylactically effective amount" may vary depending on factors such as 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.
[0534] 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 act as "brakes" to downregulate or inhibit anti-tumor 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.
[0535] The immune checkpoint inhibitors for the present disclosure are substances that inhibit the function of immune checkpoint molecules. There are no particular restrictions on immune checkpoint inhibitors, as long as the inhibitor is a substance that can inhibit the function (signal) of immune checkpoint molecules. The immune checkpoint inhibitors that can be used in the present disclosure method include but are not limited to 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 inhibitors. The inhibition of inhibitory molecules can be implemented by suppressing at the DNA, RNA or protein levels. In embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA or shRNA) can be used to suppress the expression of inhibitory molecules. In other embodiments, the inhibitor of inhibitory signals is a polypeptide bound to inhibitory molecules, for example, a soluble ligand or antibody. Examples of the immune checkpoint inhibitor used in the present disclosure may include, but are not particularly limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and anti-PD-1 antibodies and anti-PD-L1 antibodies may be preferred.
[0536] Antigen-binding molecules of the present disclosure
[0537] 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 drugability (such as yield, purity, and stability).
[0538] Exemplary Antigen Binding Molecules
[0539] The antigen-binding molecules disclosed herein include antigen-binding molecules (e.g., bispecific antibodies) and anti-GUCY2C antibodies that specifically bind to GUCY2C and CD3. In particular, the antigen-binding molecules disclosed herein have any of the following properties:
[0540] a. 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.
[0541] b. Has good specific binding to GUCY2C on the cell surface. In some embodiments, the GUCY2C monoclonal antibody has better binding activity than 1608. In some embodiments, the disclosed GUCY2C-CD3 bispecific antibody has good specific binding activity to cells expressing different levels of GUCY2C, and the binding activity is better than 1608-BsAb. The disclosed GUCY2C-CD3 bispecific antibody has good specific binding activity to cells expressing Jurkat, and the binding activity is better than 1608-BsAb.
[0542] c. Nonspecific binding to non-target cells (e.g., T cells). In some embodiments, the GUCY2C monoclonal antibody does not nonspecifically bind to Jurkat cells without GUCY2C expression; 1608 significantly binds to Jurkat cells. In some embodiments, the disclosed GUCY2C monoclonal antibody does not nonspecifically bind to PBMCs and T cells without GUCY2C expression, while the control antibody 1608 significantly nonspecifically binds to PBMCs and T cells. The disclosed GUCY2C monoclonal antibody has superior target specificity to 1608.
[0543] d. The antigen epitope bound by GUCY2C monoclonal antibody is quite different from that of 1608 and cannot competitively bind to GUCY2C antigen.
[0544] e. has good binding to human GUCY2C protein. In some embodiments, the GUCY2C-CD3 bispecific antibody 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 were measured by ELISA.
[0545] f.GUCY2C-CD3 bispecific antibody has good cytotoxic activity against target cells with different expression levels.
[0546] g.GUCY2C-CD3 bispecific antibody can effectively induce the release of IFN-γ associated with cytotoxic activity, and the release level of IL-6, a cytokine associated with inflammation, is relatively low.
[0547] h. Enhanced in vivo therapeutic activity. In some embodiments, a GUCY2C-CD3 bispecific antibody, or a GUCY2C-CD3 bispecific antibody in combination with an EGFR-CD28 bispecific antibody, exhibits excellent in vivo therapeutic activity. Combining with an EGFR-CD28 bispecific antibody can effectively enhance the efficacy of each agent alone.
[0548] The present disclosure provides an antigen binding molecule comprising at least one antigen binding moiety that specifically binds to GUCY2C and at least one antigen binding moiety that specifically binds to CD3, wherein the antigen binding moiety 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 moiety that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.
[0549] Specifically, the examples herein disclose antibody series P3-C7, 81, and 146. The antibodies herein are described below using antibody P3-C7 as an example.
[0550] An antigen-binding molecule or anti-GUCY2C antibody that specifically binds to GUCY2C and CD3, wherein:
[0551] The GUCY2C-VH has: 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 has: 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.
[0552] An antigen-binding molecule or anti-GUCY2C antibody that specifically binds to GUCY2C and CD3, wherein:
[0553] The GUCY2C-VH has: GUCY2C-HCDR1 as shown in SEQ ID NO: 19, GUCY2C-HCDR2 as shown in SEQ ID NO: 20, and GUCY2C-HCDR3 as shown in SEQ ID NO: 21; and the GUCY2C-VL has: GUCY2C-LCDR1 as shown in SEQ ID NO: 47, GUCY2C-LCDR2 as shown in SEQ ID NO: 23, and GUCY2C-LCDR3 as shown in SEQ ID NO: 24.
[0554] In some embodiments, in the antigen binding molecules or anti-GUCY2C antibodies described above, 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.
[0555] In some embodiments, the humanized GUCY2C-VH has FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and 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 has FR1, FR2, FR3 derived from IGKV2-40*01 or IGKV1-39*01 and FR4 derived from IGKJ4*01, and 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.
[0556] In some embodiments, the antigen binding molecule or anti-GUCY2C antibody as described in any of the preceding items, 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.
[0557] In some embodiments, the antigen binding molecule or anti-GUCY2C antibody as described in any of the preceding items, wherein:
[0558] 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
[0559] 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.
[0560] In some embodiments, the antigen binding molecule or anti-GUCY2C antibody as described in any of the preceding items, wherein:
[0561] 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
[0562] 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.
[0563] In some embodiments, in the antigen binding molecule as described above, the CD3-VH has: 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 has: 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.
[0564] In some embodiments, according to any of the preceding antigen binding molecules, the CD3-VH and / or the CD3-VL are murine or humanized.
[0565] In some embodiments, the CD3-VH and / or the CD3-VL are humanized.
[0566] 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.
[0567] In some embodiments, 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.
[0568] In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.
[0569] 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.
[0570] Structure of antigen-binding molecules
[0571] The bispecific antigen binding molecules disclosed herein are not limited to specific molecular structure, as long as they have desired antigen binding function.For example, the bispecific antigen binding molecules herein can be divalent (1+1), trivalent (2+1) or quadrivalent (2+2).The antigen binding module in the antigen binding molecules can be any antibody fragment with antigen binding activity, which is fused by a peptide connexon.The peptide connexons (such as connexons 1 to 11) disclosed herein can be any suitable peptide chain, as long as the antigen binding molecules can show desired antigen binding activity.For example, the peptide connexons can be flexible peptides comprising 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of L1-(GGGGS)n-L2, wherein 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 peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2, or 3. In some embodiments, the amino acid sequence of linker 1 is shown in SEQ ID NO: 81, the amino acid sequence of linker 2 is shown in SEQ ID NO: 82, the amino acid sequence of linker 3 is shown in SEQ ID NO: 83, the amino acid sequence of linker 4 is shown in SEQ ID NO: 90, the amino acid sequence of linker 5 is shown in SEQ ID NO: 95, the amino acid sequence of linker 6 is shown in SEQ ID NO: 96, and the amino acid sequence of linker 7 is shown in SEQ ID NO: 97.
[0572] Exemplarily, the antigen-binding molecule disclosed herein has a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b),
[0573] (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1],
[0574] (b) [CD3-VL]-[Linker 3]-[GUCY2C-VH]-[Linker 2]-[Fc2],
[0575] Wherein: the linker 1, linker 2 and linker 3 are the same or different peptide linkers; or linker 1, linker 2 and linker 3 do not exist;
[0576] The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.
[0577] Exemplarily, the antigen-binding molecule disclosed herein has a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b),
[0578] (a) [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1],
[0579] (b) [CD3-VL]-[Linker 3]-[GUCY2C-VH]-[Linker 2]-[Fc2],
[0580] in:
[0581] 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
[0582] 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;
[0583] The linker 1, linker 2 and linker 3 are the same or different peptide linkers; or linker 1, linker 2 and linker 3 do not exist;
[0584] 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;
[0585] The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.
[0586] Exemplarily, the antigen-binding molecule disclosed herein has a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b),
[0587] (a)[GUCY2C-VL]-[GGGSGGGG]-[CD3-VH]-[G]-[Fc1],
[0588] (b)[CD3-VL]-[GGGGSGGGG]-[GUCY2C-VH]-[G]-[Fc2],
[0589] Wherein: the structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.
[0590] Exemplarily, the antigen-binding molecule disclosed herein has a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b),
[0591] (a)[GUCY2C-VL]-[GGGSGGGG]-[CD3-VH]-[G]-[Fc1],
[0592] (b)[CD3-VL]-[GGGGSGGGG]-[GUCY2C-VH]-[G]-[Fc2],
[0593] in:
[0594] 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;
[0595] The CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 71, CD3-HCDR2 as shown in SEQ ID NO: 72, and CD3-HCDR3 as shown in SEQ ID NO: 73; and the CD3-VL has: CD3-LCDR1 as shown in SEQ ID NO: 74, CD3-LCDR2 as shown in SEQ ID NO: 75, and CD3-LCDR3 as shown in SEQ ID NO: 76;
[0596] 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;
[0597] The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus.
[0598] Exemplary antigen binding molecules have:
[0599] a first chain comprising the amino acid sequence of SEQ ID NO: 99 and a second chain comprising the amino acid sequence of SEQ ID NO: 100; or
[0600] A first chain comprising the amino acid sequence of SEQ ID NO: 101 and a second chain comprising the amino acid sequence of SEQ ID NO: 100.
[0601] Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (e), and a fourth chain having a structure represented by formula (f).
[0602] (c) [CD3-VH]-[CH1]-[Fc1],
[0603] (d) [CD3-VL]-[CL],
[0604] (e) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc2],
[0605] (f) [GUCY2C-VL]-[Linker 4]-[Titin chain],
[0606] Wherein: the linker 4 is a peptide linker; or the linker 4 does not exist;
[0607] The structures represented by formulae (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.
[0608] Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (e), and a fourth chain having a structure represented by formula (f).
[0609] (c) [CD3-VH]-[CH1]-[Fc1],
[0610] (d) [CD3-VL]-[CL],
[0611] (e) [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[Fc2],
[0612] (f) [GUCY2C-VL]-[Linker 4]-[Titin chain],
[0613] in:
[0614] 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;
[0615] The linker 4 is a peptide linker; or the linker 4 does not exist;
[0616] 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;
[0617] 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;
[0618] The amino acid sequence of CH1 is shown in SEQ ID NO: 84, and the amino acid sequence of CL is shown in SEQ ID NO: 87;
[0619] The structures represented by formulae (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.
[0620] Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (e), and a fourth chain having a structure represented by formula (f).
[0621] (c) [CD3-VH]-[CH1]-[Fc1],
[0622] (d) [CD3-VL]-[CL],
[0623] (e) [GUCY2C-VH]-[GGGGS]-[Obscurin chain]-[Fc2],
[0624] (f) [GUCY2C-VL]-[GGGGS]-[Titin chain],
[0625] Wherein: the structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.
[0626] Exemplarily, the antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (e), and a fourth chain having a structure represented by formula (f).
[0627] (c) [CD3-VH]-[CH1]-[Fc1],
[0628] (d) [CD3-VL]-[CL],
[0629] (e) [GUCY2C-VH]-[GGGGS]-[Obscurin chain]-[Fc2],
[0630] (f) [GUCY2C-VL]-[GGGGS]-[Titin chain],
[0631] in:
[0632] The GUCY2C-VH has: GUCY2C-HCDR1 as shown in SEQ ID NO: 4, GUCY2C-HCDR2 as shown in SEQ ID NO: 5, and GUCY2C-HCDR3 as shown in SEQ ID NO: 27; and the GUCY2C-VL has: GUCY2C-LCDR1 as shown in SEQ ID NO: 7, GUCY2C-LCDR2 as shown in SEQ ID NO: 8, and GUCY2C-LCDR3 as shown in SEQ ID NO: 9;
[0633] The CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 71, CD3-HCDR2 as shown in SEQ ID NO: 72, and CD3-HCDR3 as shown in SEQ ID NO: 73; and the CD3-VL has: CD3-LCDR1 as shown in SEQ ID NO: 74, CD3-LCDR2 as shown in SEQ ID NO: 75, and CD3-LCDR3 as shown in SEQ ID NO: 76;
[0634] 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;
[0635] 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;
[0636] The amino acid sequence of CH1 is shown in SEQ ID NO: 84, and the amino acid sequence of CL is shown in SEQ ID NO: 87;
[0637] The structures represented by formulae (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus.
[0638] Exemplary antigen binding molecules have:
[0639] A first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:113.
[0640] Variants of antigen-binding molecules
[0641] In certain embodiments, the amino acid sequence variants of the antigen binding molecules provided herein are encompassed.For example, it is desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications can be introduced into the nucleotide sequence encoding the antibody, or the amino acid sequence variants of the antibody can be prepared by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or replacement of the residues in the amino acid sequence of the antigen binding molecules. Any combination of deletion, insertion and replacement can be performed to obtain final construct, as long as final construct has desired features, for example, antigen binding properties.
[0642] Substitution, insertion, and deletion variants
[0643] In certain embodiments, antigen binding molecule variants with one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 2. More substantial variations are provided under the heading of "exemplary substitutions" in Table 2, and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the desired activity of the product screening is determined, such as the antigen binding to which retention / improvement is sought, the immunogenicity to which reduction is sought, or the ADCC or CDC to which improvement is sought.
[0644] Table 2. Amino acid substitutions
[0645] Based on common side chain properties, amino acids can be grouped as follows:
[0646] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0647] (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0648] (3) Acidic: Asp, Glu;
[0649] (4) Basic: His, Lys, Arg;
[0650] (5) Residues that affect chain orientation: Gly, Pro;
[0651] (6) Aromatic: Trp, Tyr, Phe.
[0652] Non-conservative substitutions would involve exchanging a member from one class for a member from another class.
[0653] A class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., humanized or human antibody). Generally, the variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be readily produced using, for example, affinity maturation techniques based on phage display (e.g., those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on phage, and screened for specific biological activity (e.g., binding affinity). Changes (e.g., substitutions) can be made to CDR, for example, to improve antibody affinity. Such changes can be made to CDR "hot spots," i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process, and / or residues contacting antigens, while binding affinity is tested for the variant VH or VL obtained. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., fallibility PCR, chain shuffling, or oligonucleotide-guided mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variant with desired affinity. Another method of introducing diversity relates to a CDR-directed method in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues that specifically identify antigen binding can be, for example, identified using alanine scanning mutagenesis or modeling.
[0654] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to the CDRs 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 1, 2, or 3 amino acid substitutions.
[0655] A method that can be used to identify residues or regions that can be used as mutagenesis targets in antibodies is called "alanine scanning mutagenesis". In this method, a residue or residue group (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. In addition, the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain desired properties.
[0656] Amino acid sequence insertions include: fusions of one residue or polypeptides of 100 or more residues in length to the amino and / or carboxyl termini; and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of enzymes (or polypeptides that extend the serum half-life of the antibody) to the N- or C-termini of the antibody.
[0657] Fab Transformation
[0658] In one aspect, in the antigen-binding molecules disclosed herein, one of the antigen-binding moiety that specifically binds to GUCY2C and the antigen-binding moiety that specifically binds to CD3 is a replaced Fab, wherein the replaced Fab comprises a heavy chain variable region, a light chain variable region, a titin chain, and an obscurin chain. In the replaced Fab, the original CH1 and CL of the Fab are replaced by 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.
[0659] Table 3-1. Amino acid sequence of titin chain
[0660] Table 3-2. Amino acid sequence of obscurin chain
[0661] Renovation of the Fc region
[0662] In one aspect, the Fc region of the antigen-binding molecule of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fcγ receptor, and reduce or eliminate effector function. A native IgG Fc region, specifically an IgG1 Fc region or an IgG4 Fc region, may cause the antigen-binding molecule of the present disclosure to target cells expressing Fc receptors rather than cells expressing antigens. The modified Fc region of the present disclosure exhibits reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region has a binding affinity to Fc receptors that is reduced by 50%, 80%, 90% or more than 95% 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, such as FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the Fc region of the transformation has a reduced binding affinity for complement, such as C1q, compared to the native Fc region. In some embodiments, the Fc region of the transformation has a reduced binding affinity for neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the Fc region of the transformation has reduced effector functions, which can include but are not limited to one or more of the following: 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 uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming. For the IgG1 Fc region, substitutions of amino acid residues at positions 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, numbering according to the EU index. For the IgG4 Fc region, substitutions of amino acid residues at positions 228, etc. can reduce effector function.
[0663] 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, mutations 252Y, 254T, and 256E may be introduced.
[0664] When the antigen binding molecules include different binding modules fused to the two subunits in the Fc region, undesirable homodimerization may result. In order to improve yield and purity, it is therefore advantageous to introduce a modification that promotes heterodimerization in the Fc region of the antigen binding molecules of the present disclosure. In some embodiments, the Fc region of the present disclosure includes the transformation according to knob-into-hole (KIH) technology, which involves introducing a protrusion structure (knob) at the interface of the first subunit and introducing a hole structure (hole) at the interface of the second subunit. The protrusion structure is positioned in the hole structure, promoting the formation of heterodimers and suppressing the generation of homodimers. The protrusion structure is constructed by replacing the small amino acid side chains from the interface of the first subunit with larger side chains (such as tyrosine or tryptophan). And the hole structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine). Protrusion structure and hole structure are prepared by changing the nucleic acid encoding the polypeptide, and optional amino acid substitutions are as shown in Table 4:
[0665] Table 4. KIH mutation combinations
[0666] In addition to the knob-in-hole technique, other techniques for modifying the CH3 domain of the heavy chain to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431A2, EP1870459A1, WO2007110205A2, WO2009089004A1, WO2010129304A2, WO2011143545A1, WO2012058768A1, WO2013157954A1 and WO2013096291A2.
[0667] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues are removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Therefore, in some embodiments, a composition of intact antibodies can include a population of antibodies in which all K447 residues and / or G446+K447 residues have been removed. In some embodiments, a composition of intact antibodies can include a population of antibodies in which the K447 residue and / or G446+K447 residues have not been removed. In some embodiments, a composition of intact antibodies has a population of antibodies that are a mixture of antibodies with and without the K447 residue and / or G446+K447 residues.
[0668] Recombination methods
[0669] Antigen binding molecules can be produced using recombinant methods.For these methods, one or more isolated nucleic acids encoding the antigen binding molecules are provided.
[0670] In the case of a natural antibody, a natural antibody fragment or a bispecific antibody with a homodimer heavy chain, two nucleic acids are required, one for a light chain or its fragment and one for a heavy chain or its fragment. Such nucleic acid encoding comprises the amino acid sequence of antibody VL and / or comprises the amino acid sequence of antibody VH (e.g., light chain and / or heavy chain of an antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
[0671] In the case of a bispecific antibody with a heterodimeric heavy chain, for example, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising a first heterologous monomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, typically these nucleic acids are located on two or three expression vectors, i.e., one vector can contain more than one of these nucleic acids.
[0672] In one embodiment, present disclosure provides the nucleic acid encoding the separation of an antibody as described above. Such nucleic acid can independently encode any of the aforementioned polypeptide chains. On the other hand, present disclosure provides one or more vectors (such as expression vectors) comprising such nucleic acid. On the other hand, present disclosure provides a host cell comprising such nucleic acid. In one embodiment, there is provided a method for preparing an antigen binding molecule, wherein the method comprises, under conditions suitable for antibody expression, cultivating a host cell comprising the nucleic acid encoding the antibody, as provided above, and optionally reclaiming the antibody from a host cell (or host cell culture medium).
[0673] In order to recombinantly produce antigen binding molecules, the nucleic acid of coded protein is separated and inserted into one or more vectors, for further cloning and / or expressing in host cell.Such nucleic acid can be easily separated and sequenced (for example, by using oligonucleotide probes that can be combined with the gene specificity of encoding antibody heavy chain and light chain) using conventional procedures, or produced by recombinant method or obtained by chemical synthesis.
[0674] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required for the antibodies. Following expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
[0675] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or 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 expressing (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, for example, US5959177, US6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted for growth in suspension. Other examples of suitable mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (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 review of certain 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.
[0676] Immunoconjugates
[0677] The present disclosure also provides immunoconjugates comprising an antigen binding molecule conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes.
[0678] Diagnostic and therapeutic compositions
[0679] In certain 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 certain embodiments, the biological sample comprises cells or tissues, such as tumor tissue.
[0680] In one embodiment, an antigen-binding molecule for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of GUCY2C and / or CD3 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an antigen-binding molecule under suitable conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods can be in vitro or in vivo. In one embodiment, the antigen-binding molecule is used to select subjects suitable for treatment, for example, GUCY2C and / or CD3 are biomarkers for selecting patients.
[0681] Exemplary disorders that can be diagnosed using the antigen binding molecules of the disclosure include, for example, proliferative diseases or tumors (eg, cancer).
[0682] In certain embodiments, labeled antigen binding molecules are provided. Labels include, but are not limited to, directly detected labels or modules (such as fluorescence, chromogenic, electron-dense, chemiluminescence, and radiolabels), and indirectly detected modules (e.g., modules indirectly detected via enzyme reactions or molecular interactions, such as enzymes or ligands).
[0683] In other aspects, a pharmaceutical composition comprising the antigen binding molecules is provided, for example, for any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises any antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any antigen binding molecules provided herein and at least one other therapeutic agent.
[0684] The pharmaceutical composition of the antigen binding molecules described in this disclosure is prepared by mixing such antigen binding molecules with the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized composition or an aqueous solution. The preparation for in vivo administration is generally sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.
[0685] Treatment methods and administration routes
[0686] Any of the antigen binding molecules provided herein can be used in therapeutic methods.
[0687] In yet another aspect, the disclosure provides the purposes of antigen binding molecules in the manufacture or preparation of medicine. In one embodiment, the medicine is used to treat proliferative diseases or tumors. And the medicine is in the form of an effective amount for the above-mentioned diseases. In some embodiments, the effective amount is a unit daily dose or a unit weekly dose. In one such embodiment, the purposes further includes administering an effective amount of at least one other therapeutic agent (such as one, two, three, four, five or six other therapeutic agents) to the subject. According to any of the above embodiments, "subject" can be a person.
[0688] In yet another aspect, a pharmaceutical composition comprising the antigen binding molecule is provided, for example, for use in any of the above pharmaceutical uses or treatment methods. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.
[0689] The antigen binding molecules of the present disclosure can be used alone or in combination with other agents for treatment. For example, the antigen binding molecules of the present disclosure can be co-administered 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.
[0690] The antigen binding molecules of the present disclosure (and any other therapeutic agent) can be applied by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is needed, then intralesional application. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether administration is short-term or long-term. A variety of administration time schemes are contemplated herein, including but not limited to, single or multiple administrations at multiple time points, push administration and pulse infusion.
[0691] The antigen binding molecules of the present disclosure will be prepared, administered and used in a manner that meets good medical practice. The factors considered in this context include the specific disease for treatment, the specific mammal for treatment, the clinical condition of the individual patient, the cause of the disease, the delivery site of the reagent, the method of administration, the administration time arrangement and other factors known to medical practitioners. Antigen binding molecules do not need to but are optionally prepared together with one or more reagents currently used to prevent or treat the disease. The effective amount of such other reagents depends on the amount of the antigen binding molecules present in the pharmaceutical composition, the type of disease or treatment and other factors discussed above. These are usually used with the same dosage and administration route as described herein, or with about 1 to 99% of dosage described herein, or with any dosage, and are determined to be suitable any approach use by experience / clinical practice.
[0692] In order to prevent or treat disease, the appropriate dosage of the antigen binding molecules of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether to use for prevention or treatment purposes, previous treatment, the patient's clinical history and the response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient once or through a series of treatments. Depending on the type and severity of the disease, the antigen binding molecules of about 1 μg / kg to 15mg / kg can be the initial candidate dose for administration to the patient, no matter whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose may be in the range of about 1 μg / kg to 100mg / kg or more, depending on the factors mentioned above. Accordingly, taking 50kg body weight as an example, an exemplary unit daily dose is 50 μg-5g.
[0693] Products
[0694] In another aspect of the present disclosure, a product is provided, the product comprising materials that can be used to treat, prevent and / or diagnose the above-mentioned conditions. The product comprises a container and a label or package insert on or in combination with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container is filled with a composition that is effective for treating, preventing and / or diagnosing the disease, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic 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 the selected condition. In addition, the product may comprise: (a) a first container containing a composition, wherein the composition comprises an antigen-binding molecule of the present disclosure; and (b) a second container containing a composition, wherein the composition comprises another cytotoxic agent or a therapeutic agent of other aspects. The product in this embodiment of the present disclosure may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the product may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. From a commercial and user perspective, it may further comprise other materials required, including other buffers, diluents, filters, needles, and syringes.
[0695] Examples and test cases
[0696] The present disclosure is further described below with reference to the following examples and test cases, but these examples and test cases are not intended to limit the scope of this disclosure. Experimental methods in the examples and test cases disclosed herein, where specific conditions are not specified, generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents whose sources are not specified are commercially available reagents.
[0697] Example 1. Antigen-binding molecules containing titin chains / obscurin chains
[0698] The titin chain / obscurin chain disclosed herein can be derived from any suitable polypeptide, including polypeptides from WO2021139758A1 (incorporated herein by reference in its entirety) and CN202110527339.7, and patents to which they are priority documents (incorporated herein by reference in their entirety). A bispecific antibody was constructed, wherein 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, the linker sequence includes GGGGS (SEQ ID NO: 90), ASTKG (SEQ ID NO: 243), or RTVAS (SEQ ID NO: 244), and the amino acid sequences of Fc1, Fc2, and CH1 in this example are shown below.
[0699] >Example 1-Fc1 (knob, SEQ ID NO: 85)
[0700] >Example 1-Fc2 (hole, SEQ ID NO: 86)
[0701] >Example 1-CH1 (SEQ ID NO: 84)
[0702] 1.1 DI bispecific antibodies
[0703] With reference to Example 5 of WO2021139758A1, DI bispecific antibodies against hNGF and hRANKL were constructed: DI-2 to DI-20, which comprised the first heavy chain, the second heavy chain, the first light chain, and the second light chain as described below:
[0704] First heavy chain: from N-terminus to C-terminus: [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2],
[0705] First light chain: from N-terminus to C-terminus: [VL1-I]-[Linker 2]-[Titin chain],
[0706] The second heavy chain: from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and
[0707] Second light chain: from N-terminus to C-terminus: [VL2-D]-[CL];
[0708] Among them, 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. The structures of the obscurin chain, titin chain, linker 1, and linker 2 in the DI bispecific antibody in this example are shown in the table below.
[0709] Table 5. Correspondence between Obscurin Chain / Titin Chain and Linker in DI Bispecific Antibodies
[0710] Note: The numbers of titin chains and obscurin chains are shown in Table 3-1 and Table 3-2.
[0711] The binding activity of DI-2 to DI-20 bispecific antibodies to their antigens was detected using the method in Test Example 4 of WO2021139758A1. The thermal stability of the antibodies was studied. Research method: The concentration of the antibody was diluted to 5 mg / mL with PBS, and its thermal stability was determined using a high-throughput differential scanning fluorimeter (UNCHAINED, specification model: Unit). The experimental results showed that the binding activity of the modified bispecific antibody to the antigen did not change significantly; and compared with DI-2, the Tm1 (℃) and Tonset (℃) of DI-4 to DI-8, DI-10 to DI-16, and DI-20 were significantly improved, and the bispecific antibody had better thermal stability.
[0712] Table 6. Binding activity detection of DI bispecific antibodies
[0713] Table 7. Thermal stability test results of DI bispecific antibodies
[0714] A solution containing the DI bispecific antibody was prepared in a buffer solution of 10 mM acetic acid, pH 5.5, and 9% sucrose. The solution was incubated in a 40°C incubator for four weeks. After the incubation period, the antibody concentration was concentrated to the initial concentration, and the solution was observed for precipitation. The experimental results showed that the DI-2 bispecific antibody solution exhibited precipitation, while DI-3 through DI-7 exhibited greater stability than DI-2.
[0715] Table 8. Precipitation of DI bispecific antibodies
[0716] 1.2 PL bispecific antibodies
[0717] PL bispecific antibodies against hPDL1 and hCTLA4 were constructed: PL-1 to PL-19, which comprise the first heavy chain, the second heavy chain, the first light chain, and the second light chain as described below:
[0718] First heavy chain: from N-terminus to C-terminus: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],
[0719] First light chain: from N-terminus to C-terminus: [VL1-P]-[Linker 2]-[Titin chain],
[0720] The second heavy chain: from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and
[0721] Second light chain: from N-terminus to C-terminus: [VL2-L]-[CL];
[0722] 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 shown below.
[0723] >VH2-L (SEQ ID NO: 180)
[0724] >VL2-L (SEQ ID NO: 181)
[0725] The structures of the obscurin chain, titin chain, linker 1, and linker 2 in the PL bispecific antibody of this embodiment are shown in the table below.
[0726] Table 9. Correspondence between Obscurin Chain / Titin Chain and Linker in PL Bispecific Antibodies
[0727] Note: The numbers of titin chains and obscurin chains are shown in Table 3-1 and Table 3-2.
[0728] The binding activity of the PL bispecific antibody was tested using the ELISA method described in Test Example 4 of WO2021139758A1. The hPDL1 and hCTLA4 antigens were purchased from Sino Biology. The thermal stability of the antibody was investigated. Methods: The antibody was diluted to a concentration of 1.4-3 mg / mL in PBS, and its thermal stability was measured using a high-throughput differential scanning fluorimeter (UNCHAINED, specification model: Unit). The experimental results demonstrated that the PL bispecific antibody maintained good binding activity to the antigen. Furthermore, compared to PL-1, the Tm1 (°C), Tagg 266 (°C), and Tonset (°C) values of PL-2 to PL-19 were significantly improved, indicating that the bispecific antibody exhibited superior thermal stability.
[0729] Table 10. Binding activity detection of PL bispecific antibodies
[0730] Table 11. Thermal stability test results of PL bispecific antibodies
[0731] 1.3 HJ bispecific antibodies
[0732] HJ bispecific antibodies against hIL5 and hTSLP were constructed: HJ-3 to HJ11, which comprise the first heavy chain, the second heavy chain, the first light chain, and the second light chain as described below:
[0733] First heavy chain: from N-terminus to C-terminus: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],
[0734] First light chain: from N-terminus to C-terminus: [VL1-H]-[Linker 2]-[Obscurin chain],
[0735] The second heavy chain: from N-terminus to C-terminus: [VH2-J]-[CH1]-[Fc2], and
[0736] Second light chain: from N-terminus to C-terminus: [VL2-J]-[CL];
[0737] Among them, 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. The structures of the obscurin chain, titin chain, linker 1, and linker 2 in the HJ bispecific antibody in this example are shown in the table below.
[0738] Table 12. Correspondence between Obscurin Chain / Titin Chain and Linker in HJ Bispecific Antibodies
[0739] The antigen binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 of WO2021139758A1. The thermal stability of the antibody was studied as follows: a dilute HJ bispecific antibody solution was prepared with a buffer solution of 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 (see Table 13-2 for the concentration of the HJ bispecific antibody). The concentrated solution was then incubated in a 40°C incubator. The SEC purity of the samples was tested on day 0 (i.e., before the start of incubation at 40°C, D0), day 7 (7th day of incubation at 40°C, D7), day 14 (14th day of incubation at 40°C, D14), day 21 (21st day of incubation at 40°C, D21), and day 28 (28th day of incubation at 40°C, D28). After incubation at 40°C for 28 days, the samples were immediately sampled for CE-SDS purity. The experimental results showed that the antigen binding activity of the HJ bispecific antibodies constructed in the present disclosure did not change significantly; and compared with HJ-3, the thermal stability of the HJ-5 to HJ-11 bispecific antibodies was better.
[0740] Table 13-1. Binding activity detection of HJ bispecific antibodies
[0741] Table 13-2. Results of accelerated stability test of HJ bispecific antibody
[0742] Example 2. Screening and identification of anti-human GUCY2C mouse antibodies
[0743] 2.1 Cell line construction
[0744] CHO-K1 / hGUCY2C and HCT116 / hGUCY2C cell lines were constructed. The relevant protein sequences are as follows:
[0745] Human GUCY2C protein (UniProtKB: P25092):
[0746] The full-length gene encoding human GUCY2C was cloned into the mammalian cell expression vector pCDH. HEK293T cells (ATCC, CRL-11268) were co-transfected with three plasmids, pVSV-G, pCMV-dR8.91, and pCDH-human GUCY2C, to package the virus. Forty-eight hours after transfection, the virus was harvested and infected with CHO-K1 (ATCC, CCL-61) or HCT116 cells (ATCC, CCL-247). After two weeks of pressure selection, the cells were subcloned, and high-GUCY2C-expressing CHO-K1 / hGUCY2C and HCT116 / hGUCY2C cell lines were obtained by FACS analysis.
[0747] 2.2 Construction of fusion proteins for immunization or screening
[0748] The extracellular region of human GUCY2C was selected and a His or Fc tag was connected to the C-terminus to construct a fusion protein for immunization and screening detection. The relevant protein sequence is as follows:
[0749] hGUCY2C ECD-hFc:
[0750] hGUCY2C ECD-His:
[0751] 2.3 Screening and identification of anti-GUCY2C mouse antibodies
[0752] The present disclosure discloses a monoclonal antibody against human GUCY2C prepared by hybridoma fusion or Single B technology. The resulting antibody specifically binds to human GUCY2C with high affinity, can cross-bind to cynomolgus monkey GUCY2C, and has good binding activity to human GUCY2C on the cell surface.
[0753] hGUCY2C ECD-hFc, hGUCY2C ECD-His, CHO-K1 / hGUCY2C cells or HCT116 / hGUCY2C cells were used as immunogens. Protein was used for the first immunization at 50 μg and for the booster immunization at 25 μg each time. Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Fisher Scientific Cat. No. 77161) was used as an adjuvant for cross-immunization. Cellular immunization was performed at a dose of 10^7 per mouse. After the initial immunization and 4 to 7 booster immunizations, mice with high serum antibody titers were selected for spleen cell fusion or single B cell screening.
[0754] After fusion, the hybridoma culture supernatant was tested according to the growth density of the hybridoma cells. Hybridomas with good binding activity to human GUCY2C and antigens on the cell surface were screened. Monoclonal hybridoma cells were collected, RNA was extracted using NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript TM The cDNA obtained by reverse transcription was amplified by PCR using Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced to obtain the amino acid sequences of the CDR and variable regions of the antibody.
[0755] Table 14. CDRs of anti-GUCY2C antibodies obtained by hybridoma fusion screening
[0756] >M81 VH:
[0757] >M81 VL:
[0758] >M146 VH:
[0759] >M146 VL:
[0760] >M42VH:
[0761] >M42VL:
[0762] >M61VH:
[0763] >M61VL:
[0764] >M64VH:
[0765] >M64VL:
[0766] >M93VH:
[0767] >M93VL:
[0768] Note: The single underlined part is CDR according to Kabat rule, and the rest are FR.
[0769] Single B cell screening primarily involves positive and negative screening, using 10*genomic and bioinformatics techniques to generate a series of antibody variable region sequences. The resulting sequences were then subjected to CDR classification, and the mouse variable region sequences were selected for ligation with human antibody constant region sequences to express chimeric antibodies. These antibodies were then screened using binding to hGUCY2C ECD-His and cynoGUCY2C ECD-His (ACRO#GUC-C52H6) proteins, surface plasmon resonance, and cell binding assays to identify antibody sequences that specifically bind to hGUCY2C ECD and cynoGUCY2C ECD. The heavy and light chain variable region sequences of the resulting antibodies are as follows:
[0770] Table 15. CDRs of anti-GUCY2C antibodies obtained by Single B screening
[0771] >P3-C7 VH:
[0772] >P3-C7 VL:
[0773] >P1-D2 VH
[0774] >P1-D2 VL
[0775] >P1-D3 VH
[0776] >P1-D3 VL
[0777] >P1-C6 VH
[0778] >P1-C6 VL
[0779] >P1-D9 VH
[0780] >P1-D9 VL
[0781] >P1-F10 VH
[0782] >P1-F10 VL
[0783] >P2-C8 VH
[0784] >P2-C8 VL
[0785] >P2-B9 VH
[0786] >P2-B9 VL
[0787] >P3-D2 VH
[0788] >P3-D2 VL
[0789] >P3-B4 VH
[0790] >P3-B4 VL
[0791] >P3-E4 VH
[0792] >P3-E4 VL
[0793] >P3-F6 VH
[0794] >P3-F6 VL
[0795] >P3-D9 VH
[0796] >P3-D9 VL
[0797] Note: The single underlined part is CDR according to Kabat rule, and the rest are FR.
[0798] Chimeric antibodies were obtained by combining the variable region sequences of the murine anti-GUCY2C antibody with the constant regions set forth in SEQ ID NO: 58 and SEQ ID NO: 59. Exemplary chimeric antibodies are M81CHI or P3-C7CHI, which comprise the murine heavy chain variable region, light chain variable region, and constant regions of M81 or P3-C7, respectively. Their specific sequences are shown below. The remaining chimeric antibodies are M146CHI, M42CHI, M61CHI, M64CHI, M93CHI, P1-D2CHI, P1-D3CHI, P1-C6CHI, P1-D9CHI, P1-F10CHI, P2-C8CHI, P2-B9CHI, P3-D2CHI, P3-B4CHI, P3-E4CHI, P3-F6CHI, and P3-D9CHI, and so on. To conserve space, their full-length sequences can be unambiguously determined and are therefore not listed in full.
[0799] Exemplary, the full-length sequence of the anti-GUCY2C chimeric antibody is as follows:
[0800] M81CHI heavy chain:
[0801] M81CHI light chain:
[0802] P3-C7 CHI Heavy Chain:
[0803] P3-C7 CHI Light Chain:
[0804] Example 3. Humanization of anti-GUCY2C monoclonal antibody
[0805] Humanization of murine antibodies is performed using methods published in numerous literature in the field. Briefly, based on the representative VH / VL CDR structures of the murine antibody, homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) are searched from a human germline database. These sequences are ranked from highest to lowest based on FR homology, and the germline with the highest FR homology is selected as the template. The CDR regions of the murine antibody are then transplanted onto the human template, and certain amino acids in the variable regions are mutated. The constant regions of the murine antibody are then replaced with human constant regions to yield the final humanized antibody.
[0806] 3-1. Humanization of M81 Antibody
[0807] The humanized M81 antibody selected IGKV3-11*01, IGKV6-21*02, or IGKV1-9*01 and IGKJ4*01 as light chain framework region templates; IGHV1-3*01 and IGHJ6*01 were selected as heavy chain framework region templates. The CDR regions of the murine antibody were transplanted onto the selected humanized templates, and certain amino acid substitutions were made in the variable regions to obtain the light and heavy chain variable regions of the humanized antibody. The specific framework replacements and amino acid substitutions are as follows:
[0808] Table 16. Framework replacement and amino acid substitution of humanized antibody M81
[0809] Note: Amino acid numbering follows the Kabat numbering system.
[0810] The CDRs of the humanized antibody M81 are as follows:
[0811] Table 17. CDRs of M81 humanized antibody
[0812] The variable region sequence of the M81 humanized antibody is as follows:
[0813] >h81 L1:
[0814] >h81 L2:
[0815] >h81 L3:
[0816] >h81 L4:
[0817] >h81 L5:
[0818] >h81 L6:
[0819] >h81 H1:
[0820] >h81 H2:
[0821] Note: The single underlined part is CDR, the dotted underlined bold part is the amino acid substitution site, and the rest is FR.
[0822] 3-2. Humanization of M146 Antibody
[0823] The humanized M146 antibody selected IGKV3-11*01 and IGKJ4*01 as light chain framework region templates; IGHV1-3*01 and IGHJ6*01 as heavy chain framework region templates. The CDR regions of the murine antibody were transplanted onto the selected humanized templates, 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 replacements and amino acid substitutions are as follows:
[0824] Table 18. Framework replacement and amino acid substitution of humanized antibody M146
[0825] Note: Amino acid numbering follows the Kabat numbering system.
[0826] The CDRs of the humanized antibody M146 are as follows:
[0827] Table 19. CDRs of M146 humanized antibody
[0828] The variable region sequence of the humanized antibody M146 is as follows:
[0829] >h146 L1:
[0830] >h146 H1:
[0831] Note: The single underlined part is CDR, the dotted underlined bold part is the amino acid substitution site, and the rest is FR.
[0832] 3-3. Humanization of P3-C7 Antibody
[0833] The humanized P3-C7 antibody selected IGKV2-40*01 or IGKV1-39*01 and IGKJ4*01 as the light chain framework region template; IGHV1-46*01 and IGHJ6*01 were selected as the heavy chain framework region template. The CDR regions of the murine antibody were transplanted onto the selected humanized templates, 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 replacements and amino acid substitutions are as follows:
[0834] Table 20. Framework replacements and amino acid substitutions of humanized antibody P3-C7
[0835] Note: Amino acid numbering follows the Kabat numbering system.
[0836] The CDRs of the humanized antibody P3-C7 are as follows:
[0837] Table 21. CDRs of P3-C7 humanized antibody
[0838] The variable region sequence of the humanized antibody P3-C7 is as follows:
[0839] >hP3-C7 L1:
[0840] >hP3-C7 L2:
[0841] >hP3-C7 L3:
[0842] >hP3-C7 L4:
[0843] >hP3-C7 L5:
[0844] >hP3-C7 L6:
[0845] >hP3-C7 L7:
[0846] >hP3-C7 L8:
[0847] >hP3-C7 H1:
[0848] >hP3-C7 H2:
[0849] Note: The single underlined part is CDR, the dotted underlined part is the amino acid substitution site, and the rest is FR.
[0850] 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.
[0851] >hIgG1:CH1-Fc:
[0852] >CL:
[0853] The humanized anti-GUCY2C antibodies obtained in this disclosure are as follows:
[0854] Table 22-1. 81 series of humanized antibodies
[0855] Table 22-2. 146 series of humanized antibodies
[0856] Table 22-3. Humanized antibodies of the P3-C7 series
[0857] Exemplary, the full-length sequence of the anti-GUCY2C humanized antibody is as follows:
[0858] 81 L3H1 heavy chain:
[0859] 81 L3H1 light chain:
[0860] 146 L1H1 heavy chain:
[0861] 146 L1H1 light chain:
[0862] P3-C7 L7H1 heavy chain:
[0863] P3-C7 L7H1 light chain:
[0864] P3-C7 L8H1 heavy chain (same as P3-C7 L7H1 heavy chain, SEQ ID NO: 64)
[0865] P3-C7 L8H1 light chain:
[0866] Note: The single underlined part is CDR, the dotted underlined bold part is the amino acid substitution site, and the italic part is the constant region.
[0867] The control antibodies for the anti-GUCY2C antibodies used in this disclosure are as follows:
[0868] 1608 (the variable region sequence is 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). The full-length sequence is as follows:
[0869] >1608 heavy chain:
[0870] >1608 Light Chain:
[0871] Antibody 5F9 (variable region sequence prepared with reference to patent WO2014134311A1, constant region sequence shown in SEQ ID NO: 58 and SEQ ID NO: 59), the specific sequence is as follows:
[0872] >5F9 heavy chain:
[0873] >5F9 light chain:
[0874] Example 4. Preparation of anti-GUCY2C-CD3 bispecific antibody
[0875] The GUCY2C arm of the GUCY2C-CD3 bispecific antibody disclosed herein is the anti-GUCY2C antibody disclosed herein, and the CD3 arm can be derived from any suitable antibody. Specifically, the CD3 arm used in the disclosure is S107E or N103QN106Q.
[0876] Table 23. CDRs of S107E
[0877] The variable region sequence of S107E is as follows:
[0878] >S107E-VH:
[0879] >S107E-VL:
[0880] Table 24. CDRs of N103Q and N106Q
[0881] The variable region sequence of N103QN106Q is as follows:
[0882] >N103QN106Q-VH:
[0883] >N103QN106Q-VL (same as S107E-VL, SEQ ID NO: 78)
[0884] The GUCY2C-CD3 bispecific antibodies disclosed herein have the following molecular structures of Format 1 to Format 5:
[0885] Format 1 is an asymmetric structure molecule, containing two chains in the complete molecule, and the two chains are different, as follows:
[0886] Chain 1: [GUCY2C-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[IgG1Fc (Knob-DI)]
[0887] Chain 2: [CD3-VL]-[Linker 3]-[GUCY2C-VH]-[Linker 2]-[IgG1Fc(Hole-DI)];
[0888] The schematic diagram is shown in FIG1A .
[0889] The relevant sequences are as follows:
[0890] >IgG1Fc (Knob-DI, L234A / L235A / G237A / Y349C / T366W):
[0891] >IgG1Fc (Hole-DI, L234A / L235A / G237A / S354C / T366S / L368A / Y407V):
[0892] >Linker 1:
[0893] >Linker 2:
[0894] >Linker 3:
[0895] Format 2 is an asymmetric structure molecule. The complete molecule has four chains, and the four chains are different. The details are as follows:
[0896] Chain 1: [CD3-VH]-[IgG1(CH1)]-[IgG1Fc(Knob)];
[0897] Chain 2: [CD3-VL]-[CL(Lambda)];
[0898] Chain 3: [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[IgG1Fc (Hole)];
[0899] Chain 4: [GUCY2C-VL]-[Linker 4]-[Titin chain];
[0900] Its schematic diagram is shown in FIG1B (wherein: Ob represents Obscurin).
[0901] The relevant sequences are as follows:
[0902] >IgG1(CH1):
[0903] >IgG1Fc (Knob, L234A / L235A / S354C / T366W):
[0904] >IgG1Fc (Hole, L234A / L235A / Y349C / T366S / L368A / Y407V):
[0905] >CL(Lambda):
[0906] >Titin(T.16):
[0907] >Obscurin(O.28):
[0908] >Linker 4:
[0909] Format 3 is an asymmetric structure molecule. The complete molecule has four chains, and the four chains are different. The details are as follows:
[0910] Chain 1: [GUCY2C-VH]-[Linker 4]-[Obscurin chain]-[IgG1Fc (Knob)];
[0911] Chain 2: [GUCY2C-VL]-[Linker 4]-[Titin chain];
[0912] Chain 3: [CD3-VH]-[IgG1(CH1)]-[IgG1Fc(Hole)];
[0913] Chain 4: [CD3-VL]-[CL(Lambda)];
[0914] The schematic diagram is shown in FIG1C (wherein: Ob represents Obscurin).
[0915] The relevant sequences are as follows:
[0916] >IgG1 (CH1) (SEQ ID NO: 84)
[0917] >IgG1 Fc (Knob) (SEQ ID NO: 85)
[0918] >IgG1 Fc (Hole) (SEQ ID NO: 86)
[0919] >CL (Lambda) (SEQ ID NO: 87)
[0920] >Titin (T.16) (SEQ ID NO: 198)
[0921] >Obscurin(O.28)(SEQ ID NO: 236)
[0922] >Linker 4 (SEQ ID NO: 90)
[0923] >Chain 3 (Format 3-3, CD3-VH-IgG1(CH1)-IgG1Fc(Hole)):
[0924] >Chain 4 (Format 3-4, same as Format 2-2, SEQ ID NO: 92)
[0925] Format 4 is a symmetrical structure molecule. The complete molecule has four chains, which are composed of two identical chains 1 and two identical chains 2, as follows:
[0926] Chain 1: [GUCY2C-VH]-[IgG1(CH1)]-[CD3-VH]-[Linker 5]-[CD3-VL]-[Linker 6]-[IgG1Fc];
[0927] Chain 2: [GUCY2C-VL]-[CL];
[0928] The schematic diagram is shown in FIG1D .
[0929] The relevant sequences are as follows:
[0930] >IgG1 (CH1) (SEQ ID NO: 84)
[0931] >CL (SEQ ID NO: 59)
[0932] >IgG1Fc(L234A / L235A):
[0933] >Linker 5:
[0934] >Linker 6
[0935] Format 5 is a symmetrical structure molecule. The complete molecule has six chains, which are composed of two identical chains 1, two identical chains 2, and two identical chains 3. The details are as follows:
[0936] Chain 1: [GUCY2C-VH]-[IgG1(CH1)]-[Linker 7]-[CD3-VH]-[Linker 4]-[Obscurin chain]-[IgG1Fc];
[0937] Chain 2: [GUCY2C-VL]-[CL];
[0938] Chain 3: [CD3-VL]-[Linker 4]-[Titin chain];
[0939] The schematic diagram is shown in FIG1E (wherein: Ob represents Obscurin).
[0940] The relevant sequences are as follows:
[0941] >IgG1 (CH1) (SEQ ID NO: 84)
[0942] >IgG1 Fc (SEQ ID NO: 86)
[0943] >CL (SEQ ID NO: 59)
[0944] >Titin (T.16) (SEQ ID NO: 198)
[0945] >Obscurin(O.28)(SEQ ID NO: 236)
[0946] >Linker 4 (SEQ ID NO: 90)
[0947] >Linker 7:
[0948] > Chain 3 (Format 5-3, CD3-VL-Linker 4-Titin)
[0949] Antigen-binding molecules were constructed according to Table 25. All bispecific antibodies used the variable regions of S107E or N103QN106Q as the CD3 arm. In the numbering, the variable regions of the anti-GUCY2C antibodies are listed in the antibody name, followed by the number representing the format used. A format followed by (N103QN106Q) indicates that the CD3 arm used in the bispecific antibody is N103QN106Q. No format designation indicates that the CD3 arm used in the bispecific antibody is S107E. For example, P3-C7 L7H1-1 indicates that the molecule uses the variable region of antibody P3-C7 L7H1 as the GUCY2C binding domain, uses format 1 as the molecular structure, and has a CD3 arm of S107E. This applies to other bispecific antibodies.
[0950] Table 25. GUCY2C-CD3 bispecific antibodies of the present disclosure
[0951] For example, the specific amino acid sequence of the bispecific antibody constructed using Format 1 disclosed herein is as follows:
[0952] 1. Full-length sequence of P3-C7 L7H1-1
[0953] >Chain 1 (P3-C7 L7H1-1-1):
[0954] >Chain 2 (P3-C7 L7H1-1-2):
[0955] 2. Full-length sequence of P3-C7 L8H1-1
[0956] >Chain 1 (P3-C7 L8H1-1-1):
[0957] >Chain 2 (P3-C7 L8H1-1-2, same as P3-C7 L7H1-1-2, SEQ ID NO: 100)
[0958] 3. Full-length sequence of M81-1
[0959] >Chain 1 (M81-1-1):
[0960] >Chain 2 (M81-1-2):
[0961] 4. Full-length sequence of M146-1
[0962] >Chain 1 (M146-1-1):
[0963] >Chain 2 (M146-1-2):
[0964] For example, the specific amino acid sequence of the bispecific antibody constructed using Format 2 disclosed herein is as follows:
[0965] 1. Full-length sequence of P3-C7 L7H1-2
[0966] >Chain 1 (Format 2-1):
[0967] >Chain 2 (Format 2-2):
[0968] >Chain 3 (P3-C7 L7H1-2-3):
[0969] >Chain 4 (P3-C7 L7H1-2-4):
[0970] 2. Full-length sequence of M81-2
[0971] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[0972] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[0973] >Chain 3 (M81-2-3):
[0974] >Chain 4 (M81-2-4):
[0975] 3. Full-length sequence of 81 L1H1-2
[0976] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[0977] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[0978] >Chain 3 (81 L1H1-2-3):
[0979] >Chain 4 (81 L1H1-2-4):
[0980] 4.81 Full-length sequence of L2H1-2
[0981] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[0982] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[0983] >Chain 3 (81 L2H1-2-3, same as 81 L1H1-2-3, SEQ ID NO: 110)
[0984] >Chain 4 (81 L2H1-2-4):
[0985] 5.81 Full-length sequence of L3H1-2
[0986] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[0987] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[0988] >Chain 3 (81 L3H1-2-3, same as 81 L1H1-2-3, SEQ ID NO: 110)
[0989] >Chain 4 (81 L3H1-2-4):
[0990] 6.81 Full-length sequence of L3H1-2(N103QN106Q)
[0991] >Chain 1 (Format 2-1 (N103QN106Q))
[0992] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[0993] >Chain 3 (81 L3H1-2-3, same as 81 L1H1-2-3, SEQ ID NO: 110)
[0994] >Chain 4 (81 L3H1-2-4, SEQ ID NO: 113):
[0995] 6.81 Full-length sequence of L4H1-2
[0996] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[0997] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[0998] >Chain 3 (81 L4H1-2-3, same as 81 L1H1-2-3, SEQ ID NO: 110)
[0999] >Chain 4 (81 L4H1-2-4):
[1000] 7.81 Full-length sequence of L5H1-2
[1001] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[1002] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[1003] >Chain 3 (81 L5H1-2-3, same as 81 L1H1-2-3, SEQ ID NO: 110)
[1004] >Chain 4 (81 L5H1-2-4):
[1005] 8.81 Full-length sequence of L6H1-2
[1006] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[1007] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[1008] >Chain 3 (81 L6H1-2-3, same as 81 L1H1-2-3, SEQ ID NO: 110)
[1009] >Chain 4 (81 L6H1-2-4)
[1010] 9.Full-length sequence of M146-2
[1011] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[1012] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[1013] >Chain 3 (M146-2-3):
[1014] >Chain 4 (M146-2-4):
[1015] 10.146 Full-length sequence of L1H1-2
[1016] >Chain 1 (Format 2-1, SEQ ID NO: 91)
[1017] >Chain 2 (Format 2-2, SEQ ID NO: 92)
[1018] >Chain 3 (146 L1H1-2-3):
[1019] >Chain 4 (146 L1H1-2-4):
[1020] For example, the specific amino acid sequence of the bispecific antibody constructed using Format 3 disclosed herein is as follows:
[1021] 1. Full-length sequence of P3-C7 L7H1-3
[1022] >Chain 1 (P3-C7 L7H1-3-1):
[1023] >Chain 2 (P3-C7 L7H1-3-2, same as P3-C7 L7H1-2-4, SEQ ID NO: 107)
[1024] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1025] >Chain 4 (Format 3-4, same as Format 2-2; SEQ ID NO: 92)
[1026] 2. Full-length sequence of 81 L1H1-3
[1027] >Chain 1 (81 L1H1-3-1):
[1028] >Chain 2 (81 L1H1-3-2, same as 81 L1H1-2-4, SEQ ID NO: 111)
[1029] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1030] >Chain 4 (Format 3-4, same as Format 2-2, SEQ ID NO: 92)
[1031] 3. Full-length sequence of 81 L2H1-3
[1032] >Chain 1 (81 L2H1-3-1, same as 81 L1H1-3-1, SEQ ID NO: 122)
[1033] >Chain 2 (81 L2H1-3-2, same as 81 L2H1-2-4, SEQ ID NO: 112)
[1034] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1035] >Chain 4 (Format 3-4, same as Format 2-2, SEQ ID NO: 92)
[1036] 4.81 Full-length sequence of L3H1-3
[1037] >Chain 1 (81 L3H1-3-1, same as 81 L1H1-3-1, SEQ ID NO: 122)
[1038] >Chain 2 (81 L3H1-3-2, same as 81 L3H1-2-4, SEQ ID NO: 113)
[1039] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1040] >Chain 4 (Format 3-4, same as Format 2-2, SEQ ID NO: 92)
[1041] 5.81 Full-length sequence of L4H1-3
[1042] >Chain 1 (81 L4H1-3-1, same as 81 L1H1-3-1, SEQ ID NO: 122)
[1043] >Chain 2 (81 L4H1-3-2, same as 81 L4H1-2-4, SEQ ID NO: 114)
[1044] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1045] >Chain 4 (Format 3-4, same as Format 2-2; SEQ ID NO: 92)
[1046] 6.81 Full-length sequence of L5H1-3
[1047] >Chain 1 (81 L5H1-3-1, same as 81 L1H1-3-1, SEQ ID NO: 122)
[1048] >Chain 2 (81 L5H1-3-2, same as 81 L5H1-2-4, SEQ ID NO: 115)
[1049] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1050] >Chain 4 (Format 3-4, same as Format 2-2; SEQ ID NO: 92)
[1051] 7.81 Full-length sequence of L6H1-3
[1052] >Chain 1 (81 L6H1-3-1, same as 81 L1H1-3-1, SEQ ID NO: 122)
[1053] >Chain 2 (81 L6H1-3-2, same as 81 L6H1-2-4, SEQ ID NO: 116)
[1054] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1055] >Chain 4 (Format 3-4, same as Format 2-2, SEQ ID NO: 92)
[1056] 8.146 Full-length sequence of L1H1-3
[1057] >Chain 1 (146 L1H1-3-1):
[1058] >Chain 2 (146 L1H1-3-2, same as 146 L1H1-2-4, SEQ ID NO: 120)
[1059] >Chain 3 (Format 3-3, SEQ ID NO: 93)
[1060] >Chain 4 (Format 3-4, same as Format 2-2, SEQ ID NO: 92)
[1061] For example, the specific amino acid sequence of the bispecific antibody constructed using Format 4 disclosed herein is as follows:
[1062] 1. Full-length sequence of P3-C7 L7H1-4
[1063] >Chain 1 (P3-C7 L7H1-4-1):
[1064] >Chain 2 (P3-C7 L7H1-4-2, same as P3-C7 L7H1 light chain, SEQ ID NO: 65)
[1065] 2. Full-length sequence of P3-C7 L8H1-4
[1066] >Chain 1 (P3-C7 L8H1-4-1, same as P3-C7 L7H1-4-1, SEQ ID NO: 124)
[1067] >Chain 2 (P3-C7 L8H1-4-2, same as P3-C7 L8H1 light chain, SEQ ID NO: 66)
[1068] The specific amino acid sequence of the bispecific antibody constructed using Format 5 is as follows:
[1069] 1. Full-length sequence of P3-C7 L7H1-5
[1070] >Chain 1 (P3-C7 L7H1-5-1):
[1071] >Chain 2 (P3-C7 L7H1-5-2, same as P3-C7 L7H1 light chain, SEQ ID NO: 65)
[1072] >Chain 3 (Format 5-3, SEQ ID NO: 98)
[1073] 2. Full-length sequence of P3-C7 L8H1-5
[1074] >Chain 1 (P3-C7 L8H1-5-1, same as P3-C7 L7H1-5-1, SEQ ID NO: 125)
[1075] >Chain 2 (P3-C7 L8H1-5-2, same as P3-C7 L8H1 light chain, SEQ ID NO: 66)
[1076] >Chain 3 (Format 5-3, SEQ ID NO: 98)
[1077] In addition, this disclosure also uses the GUCY2C-CD3 bispecific antibody GUCY2c-1608 (named 1608-BsAb in this disclosure), which was prepared with reference to patent WO2019224716A2. It has a DIABODY structure, and the complete molecule contains two chains, as follows:
[1078] >Chain 1 (GUCY2c-1608-Knob sequence):
[1079] >Chain 2 (GUCY2c-1608-Hole sequence):
[1080] Table 26. CDR sequences of 1608-BsAb
[1081] In addition, this disclosure also uses the EGFR-CD28 bispecific antibody 97H2L3OT-ZAL, which was prepared with reference to patent CN202210371538.8, and its sequence is as follows:
[1082] Table 27. CDRs of EGFR-CD28 bispecific antibody 97H2L3OT-ZAL
[1083] EGFR-VH:
[1084] EGFR-VL:
[1085] CD28-VH:
[1086] CD28-VL:
[1087] Full-length sequence of the EGFR-CD28 bispecific antibody 97H2L3OT-ZAL:
[1088] Chain 1:
[1089] Chain 2:
[1090] >Chain 3:
[1091] >Chain 4:
[1092] In addition, this disclosure also uses the EGFR-CD28 bispecific antibodies 129H4L1OT-ZAL and 94H6L12-ZALOT, both prepared with reference to patent CN202210371538.8, and their sequences are as follows:
[1093] Table 28. CDRs of EGFR-CD28 bispecific antibody 129H4L1OT-ZAL
[1094] EGFR-VH (same as EGFR-VH of 129H4L1OT-ZAL, SEQ ID NO: 152)
[1095] EGFR-VL (same as EGFR-VL of 129H4L1OT-ZAL, SEQ ID NO: 153)
[1096] CD28-VH:
[1097] CD28-VL:
[1098] Full-length sequence of the EGFR-CD28 bispecific antibody 129H4L1OT-ZAL:
[1099] >Chain 1 (SEQ ID NO: 166):
[1100] >Chain 2 (SEQ ID NO: 167):
[1101] >Chain 3 (same as chain 3 of 97H2L3OT-ZAL, SEQ ID NO: 158)
[1102] >Chain 4 (same as chain 4 of 97H2L3OT-ZAL, SEQ ID NO: 159)
[1103] Table 29. CDRs of EGFR-CD28 bispecific antibody 94H6L12-ZALOT
[1104] EGFR-VH (same as EGFR-VH of 129H4L1OT-ZAL, SEQ ID NO: 152)
[1105] EGFR-VL (same as EGFR-VL of 129H4L1OT-ZAL, SEQ ID NO: 153)
[1106] CD28-VH:
[1107] CD28-VL:
[1108] Full-length sequence of the EGFR-CD28 bispecific antibody 94H6L12-ZALOT:
[1109] Chain 1:
[1110] Chain 2:
[1111] >Chain 3:
[1112] >Chain 4:
[1113] >IgG1Fc-Knob(L234A / L235A / S354C / T366W)
[1114] >IgG1Fc-Hole(L234A / L235A / Y349C / T366S / L368A / Y407V)
[1115] >Linker 8 (SEQ ID NO: 90)
[1116] Test Case
[1117] Test Example 1. Affinity of the disclosed GUCY2C monoclonal antibody for antigen
[1118] In order to test the affinity of the GUCY2C monoclonal antibody disclosed herein for GUCY2C, this test example used instruments Biacore 8K and Biacore T200 to detect the binding ability of the antibody to hGUCY2C-ECD-His (SEQ ID NO: 3). The specific method is as follows: a Protein A biosensor chip coupled with an anti-human antibody is used to affinity capture the antibody, and then a solution containing GUCY2C protein is passed over the chip surface, and the reaction signal is detected in real time by the instrument to obtain the binding and dissociation curves. After each experimental cycle of dissociation is completed, the biosensor chip is washed and regenerated with 10mM glycine-hydrochloric acid (pH 1.5). The data fitting model uses a 1:1 model. The affinity data of the GUCY2C monoclonal antibody disclosed herein binding to the corresponding antigen are shown in the following table.
[1119] Table 30-1. Binding of monoclonal antibodies to human GUCY2C antigen
[1120] Table 30-2. Binding of monoclonal antibodies to human GUCY2C antigen
[1121] Table 30-3. Binding of monoclonal antibodies to human GUCY2C antigen
[1122] Table 30-4. Binding of monoclonal antibodies to human GUCY2C antigen
[1123] Table 30-5. Binding of monoclonal antibodies to human GUCY2C antigen
[1124] The results showed that the GUCY2C monoclonal antibody disclosed herein had good binding affinity to human GUCY2C.
[1125] Test Example 2. Binding activity of anti-GUCY2C monoclonal antibody to cell surface antigens
[1126] This test example uses flow cytometry to detect the binding activity of GUCY2C monoclonal antibody to human GUCY2C on the surface of HCT116 / hGUCY2C, a stably transfected cell line overexpressing human GUCY2C, and colorectal cancer cells HT-55 and T84 cells. The details are as follows:
[1127] 1E5 cells resuspended in 1% BSA were added to the reaction wells, centrifuged, and the supernatant discarded. Antibodies at the corresponding dilution concentrations were added, 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), was then added, resuspended, and incubated at room temperature for 30 minutes in the dark. After washing twice with 1% BSA, fluorescence signals were read using a flow cytometer. Exemplary FACS results are shown in Figures 2A to 2E.
[1128] The results showed that the disclosed GUCY2C monoclonal antibody had good specific binding to GUCY2C on the cell surface, and the binding activity was better than that of 1608.
[1129] Test Example 3. Non-specific binding of the GUCY2C humanized antibody disclosed herein
[1130] This test example detects the non-specific binding of the GUCY2C humanized antibody to non-target cells (e.g., T cells). The details are as follows:
[1131] Experiment 1: Using flow cytometry, 1E5 Jurkat cells (ATCC #TIB-152) resuspended in 1% BSA were added to each well. The cells were centrifuged and the supernatant discarded. Antibodies of the appropriate dilution concentration were added, and the cells were incubated at room temperature for 30 minutes. The cells were then washed once with 1% BSA. F(ab')2-goat anti-human IgG Fc secondary antibody, PE (Cat. #H10104, Invitrogen), was then added, the cells were resuspended, and the cells were incubated at room temperature for 30 minutes in the dark. After washing twice with 1% BSA, the fluorescence signal was read using a flow cytometer. The relative mean fluorescence intensity (RMFI) was calculated by subtracting the background fluorescence intensity of cells not incubated with antibody from the mean fluorescence intensity of antibody-bound cells. The results are shown in Table 31 below.
[1132] Table 31. Binding of GUCY2C humanized mAb to Jurkat
[1133] The results showed that the disclosed GUCY2C humanized monoclonal antibody had no non-specific binding to Jurkat cells without GUCY2C expression; while 1608 had significant binding to Jurkat cells.
[1134] Experiment 2: Flow cytometry was used to examine whether the disclosed GUCY2C humanized monoclonal antibody nonspecifically binds to human PBMCs and CD3+ T cells within the PBMCs. 3E5 human peripheral blood cells resuspended in 1% BSA were added to each well and the supernatant was removed after centrifugation. Diluted antibodies of the corresponding concentration were added, incubated at room temperature for 30 minutes, and washed once with 1% BSA. Secondary antibodies containing PE-Cy7 anti-human CD3 (BD#557851) and APC anti-human IgG Fc (BioLegend#409306) were 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 (RMFI) was calculated by subtracting the background fluorescence intensity of cells not incubated with the antibody from the mean fluorescence intensity of the antibody-bound cells. The results are shown in Table 32 below.
[1135] Table 32. Binding of GUCY2C humanized mAb to human PBMC
[1136] The results showed that the disclosed humanized GUCY2C monoclonal antibody showed no nonspecific binding to PBMCs and T cells without GUCY2C expression, while the control antibody 1608 showed significant nonspecific binding to PBMCs and T cells. The disclosed humanized GUCY2C monoclonal antibody had superior target specificity to 1608.
[1137] Test Example 4. Epitope Analysis of the GUCY2C Monoclonal Antibody Disclosed
[1138] To test the epitope to which the disclosed GUCY2C monoclonal antibody binds to the antigen, an ELISA method was used to detect the difference in the epitope binding of the antibody to the antigen. The details are as follows:
[1139] 100 ng of the tested monoclonal antibody was coated on a plate and incubated overnight at 4°C. The plate was washed and blocked with 1% BSA. Then, 50 ng of hGUCY2C ECD-His antigen was added and incubated at 37°C for 1 hour. The plate was washed and 100 ng of each biotinylated antibody was added and incubated at 37°C for 1 hour. Finally, the signal was detected using HRP-streptavidin. Relative competition coefficient = (antibody self-competition signal value / other antibody competition signal value) * 100. Two antibodies with identical epitopes have a competition coefficient of 100 in competitive binding. Antibodies with greater epitope differences have smaller competition coefficients. This is shown in Table 33 below:
[1140] Table 33. Competitive binding of GUCY2C mAb to antigen
[1141] The results showed that the antigen epitope bound by the disclosed GUCY2C monoclonal antibody was quite different from that of 1608 and could not competitively bind to the GUCY2C antigen.
[1142] Test Example 5. Protein Binding Activity of GUCY2C-CD3 Bispecific Antibody to Antigen
[1143] This test example uses ELISA to detect the binding activity of the GUCY2C-CD3 bispecific antibody to human GUCY2C. The details are as follows:
[1144] Add 100 ng of streptavidin (ACRO #STN-N5116) to the reaction wells and coat the plates at 4°C overnight. After blocking with 1% BSA (PBS), add 100 ng of hGUCY2C ECD-His and incubate at 37°C for 1 hour. After washing the plates, add the diluted gradient antibodies and incubate at 37°C for 1 hour. Then add Peroxidase AffiniPure goat anti-human IgG (H+L) (Cat. #109-035-003, Jackson ImmunoResearch) and incubate at 37°C for 1 hour. After washing the plates, add the colorimetric solution. After sufficient color development, add the stop solution to terminate the reaction. Read the signal value at 450 nM on a microplate reader. The binding activity of the antibodies is shown in Table 34 below.
[1145] Table 34. Binding activity of GUCY2C-CD3 bispecific antibodies to human GUCY2C protein (absorbance values)
[1146] The results showed that the GUCY2C-CD3 bispecific antibody disclosed herein has a good binding effect with the human GUCY2C protein.
[1147] Test Example 6. Binding activity of GUCY2C-CD3 bispecific antibody to cell surface antigens
[1148] This test example used flow cytometry to detect the binding activity of the GUCY2C-CD3 bispecific antibody against the stably transfected human GUCY2C-overexpressing cell line HCT116 / hGUCY2C and colorectal cancer cell line HT55 (Nanjing Kebai #CBP60012), as well as CD3-expressing Jurkat cells (ATCC #TIB-152). The expression levels of GUCY2C in HCT116 / hGUCY2C cells and colorectal cancer cell line HT55 were high (++++) and moderate (++), respectively. The details are as follows:
[1149] Experiment 1: Add 1E5 cells resuspended in 1% BSA to the reaction wells, centrifuge, and discard the supernatant. Add the corresponding diluted antibody concentration, incubate at room temperature for 30 minutes, and wash twice with 1% BSA. Then, add a 1:400 dilution of goat anti-human IgG(γ) R-PE conjugate F(ab') secondary antibody (Life Technologies #H10104), resuspend, and incubate at room temperature for 30 minutes in the dark. After washing twice with 1% BSA, read the fluorescence signal using a flow cytometer. Example FACS results are shown in Figures 3A-3B.
[1150] The results showed that the GUCY2C-CD3 bispecific antibody disclosed in this batch had good specific binding activity to cells expressing different levels of GUCY2C, and the binding activity was better than that of 1608-BsAb.
[1151] Experiment 2: The same method as Experiment 1 was used. The binding activity of the antibody to Jurkat cells is shown in Figure 3C.
[1152] The results showed that the GUCY2C-CD3 bispecific antibody disclosed in this batch had good specific binding activity to cells expressing Jurkat, and its binding activity was better than that of 1608-BsAb.
[1153] Test Example 7. Activation of T cells by GUCY2C-CD3 bispecific antibody
[1154] To test the activation of T cells by the GUCY2C-CD3 bispecific antibody disclosed in this disclosure under the combined action of target cells HCT116 / GUCY2C and LS1034 cells. TM NFAT (InvivoGen, jktl-nfat) was used for cell detection. The method is as follows:
[1155] Jurkat Lucia TMNFAT cells and target cells were subcultured in complete medium and corresponding culture medium, respectively. Jurkat-NFAT lum cell collection was collected by centrifugation and resuspended and counted in 1640+10% FBS culture medium to adjust the cell number to 1E6 cells / mL. Target cells HCT116 / GUCY2C and LS1034 were resuspended and counted to adjust the cell number to 2E5 cells / mL and 4E5 cells / mL, respectively. TM NFAT and target cells were mixed in equal volumes and 100 μL was added to each well, ensuring that the E:T ratio of HCT116 / GUCY2C and LS1034 cells was 5:1 and 2.5:1 respectively. The antibody was diluted in 1640+10% FBS medium and 20 μL was added to the cell wells. After incubation at 37°C for 5 hours, QUANTI-Luc was dissolved in 25 mL of ddH2O according to the instructions. TM Gold reagent. Add 50μL QUANTI-Luc TM Gold, incubated at room temperature for 5 minutes, and the luminescence was detected by Vendor, and the antibody activity against Jurkat Lucia at different concentrations was calculated. TM Activation of NFAT cells. The relative multiple of Lumin value after antibody action divided by background Lumin value is plotted. The relative multiple value and EC 50 The results are shown in Figures 4A-4B.
[1156] The results showed that the GUCY2C-CD3 bispecific antibody disclosed in this batch acted together with target cells and had a good activation effect on T cells, and its activation effect was better than that of 1608-BsAb.
[1157] Test Example 8. Cytotoxic activity of the GUCY2C-CD3 bispecific antibody disclosed herein
[1158] This test case studies the cytotoxicity of the disclosed GUCY2C-CD3 bispecific antibody as a T cell engager molecule against tumor cells. The details are as follows:
[1159] LucG stably transfected cell lines constructed with GUCY2C-expressing intestinal cancer cell lines LS1034, HT-55 and LS174T (ATCC#CL-188) were used as high-expressing, moderately-expressing and low-expressing target cells to detect the target-specific cytotoxic activity of the bispecific antibodies disclosed herein. After resuscitation, frozen PBMCs (purchased from Miaoshun Biological Company) were resuspended in 1640+10% FBS complete medium and placed in a T75 culture flask for 4 hours (density of 2E6 cells / mL). After collecting and centrifuging, PBMCs were resuspended and counted with 1640+10% FBS, and the cell number was adjusted to 1.5E6 cells / mL. Target cells were collected, centrifuged at 1000rpm for 3 minutes, resuspended, counted, and the cell concentration was adjusted to 1.5E5 cells / mL. The above-mentioned PBMC suspension and target cell suspension were mixed in equal volumes, and 100 μL was added to each well to ensure that the E:T Ratio was 10:1. In addition, a PBMC ONLY group was set up, and the above PBMC suspension and 1640+10% FBS complete medium were mixed in equal volumes. The antibody was diluted with 1640+10% FBS medium, with a starting concentration of 600nM (6x final concentration), 5-fold dilution, 9 dose points, and 20μL was added to each well. The treated cells were cultured in a 37°C, 5% CO2 incubator for 48 hours and then luminescence detection was performed. Graphpad Prism8.0 software was used to draw a dose-effect curve based on the logarithmic concentration and signal value of the antibody, and the maximum inhibition rate (Imax) and IC of antibody-mediated killing were obtained by fitting. 50 Wells containing only target cells and PBMC without antibody were set as 0% inhibition, and the maximum inhibition rate of the antibody was calculated. The results are shown in Tables 35-1, 35-2, and 35-3 below.
[1160] Table 35-1. Cytotoxic activity of GUCY2C-CD3 bispecific antibody against target cells Note: + indicates the expression level of GUCY2C, where the higher the +, the higher the expression level of GUCY2C.
[1161] Table 35-2. Cytotoxic activity of GUCY2C-CD3 bispecific antibody against target cells
[1162] Table 35-3. Cytotoxic activity of GUCY2C-CD3 bispecific antibody against target cells
[1163] The results showed that the GUCY2C-CD3 bispecific antibody disclosed in this batch had good cytotoxic activity against target cells with different expression levels.
[1164] Test Example 9. Cytokine Release of the GUCY2C-CD3 Bispecific Antibody Disclosed
[1165] This test example detected the changes in the secretion of cytokines IFN-γ and IL-6 in PBMC under the combined action of GUCY2C-CD3 dual antibody and target cells (LS1034 cells and LS174T cells in Test Example 8).
[1166] The supernatant collected in the cell killing experiment (Test Example 8) and stored in a -20 refrigerator was thawed at room temperature, shaken and mixed, and the sample was diluted 20 times with 1640+4% FBS medium for standby use. Sterile water was used to dissolve the IL-6 and IFN-γ standard powders, and the standards were diluted to the required concentrations using 1640+4% FBS medium. Take out the kits for the cytokines to be tested, including human IL6 ELISA kit (Xinbosheng Biotechnology Co., Ltd., EHC007.96) and human IFN-γ kit (CISBIO, 62HIFNGPEG), balance the kit to room temperature, and detect according to the instructions. The results are shown in Figures 5A-5C and 6A-6C.
[1167] The results showed that the GUCY2C-CD3 bispecific antibody disclosed herein can effectively induce the release of IFN-γ associated with cytotoxic activity, and the release level of the cytokine IL-6 associated with inflammation is relatively low.
[1168] In vivo biological evaluation
[1169] Test Example 10. Efficacy of the GUCY2C-CD3 bispecific antibody disclosed herein in the LS1034 subcutaneous transplant tumor model
[1170] This disclosure uses a subcutaneous transplant tumor model of human colon cancer cell LS1034 in mice to evaluate the in vivo efficacy of the GUCY2C-CD3 bispecific antibody.
[1171] Female NOG mice were purchased from Beijing Weitonglihua (Shanghai Branch) at 6-8 weeks of age. Human PBMCs used in this test were purchased commercially from Miaoshun Biotechnology.
[1172] LS1034 cells 1×10 6 Cells / mouse / 200 μL (containing 50% basement membrane matrix) were inoculated subcutaneously on the right rib of NOG mice. The next day after inoculation, human PBMCs in good condition after recovery were added at a concentration of 5×10 6 Cells / mouse / 100 μL were injected into the peritoneal cavity of NOG mice. When the tumor volume of the tumor-bearing mice reached 150 mm 3 When the tumor volume exceeded 1000 mm, the mice were randomly divided into groups of 8. The day of grouping was defined as day 0 of the experiment. The antibodies were injected intraperitoneally in equal molar amounts twice a week for two to three weeks. The tumor volume and animal weight were monitored and recorded twice a week. When the tumor volume of the experimental animal exceeded 1000 mm, the mice were randomly divided into groups of 8. The day of grouping was defined as day 0 of the experiment. The antibodies were injected intraperitoneally in equal molar amounts twice a week for two to three weeks.3 Or when most tumors ruptured or body weight decreased by 20%, the tumor-bearing animals were euthanized as the experimental endpoint.
[1173] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 , where a and b represent length and
[1174] Width.
[1175] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.
[1176] Tumor inhibition rate TGI (%) = 1-T / C (%).
[1177] Table 36-1. Anti-tumor activity of GUCY2C-CD3 bispecific antibody Note: *p<0.05, **p<0.01, ***p<0.001 vs. vehicle control, T test.
[1178] Table 36-2. Anti-tumor activity of GUCY2C-CD3 bispecific antibody Note: *p<0.05, **p<0.01, ***p<0.001 vs. vehicle control, T test.
[1179] The results showed that the GUCY2C-CD3 dual antibody disclosed herein had a significant tumor inhibitory effect on this model.
[1180] Test Example 11. Efficacy of the bispecific antibody disclosed herein in the LS174T subcutaneous transplant tumor model
[1181] The present disclosure uses a subcutaneous transplant tumor model of human colon cancer cells LS174T in mice to evaluate the in vivo efficacy of the GUCY2C-CD3 bispecific antibody.
[1182] Female NOG mice were purchased from Beijing Weitonglihua (Shanghai Branch) at 6-8 weeks of age. Human PBMCs used in this test were purchased commercially from Miaoshun Biotechnology.
[1183] LS174T cells 1×10 6 100 μL of cells / mouse were mixed with frozen and revived PBMC (E:T=1:1) and inoculated subcutaneously in the right rib of NOG mice. 3When the tumor volume was too large, too small, or too light, mice were removed and randomly divided into groups of 8. The day of grouping was defined as day 0 of the experiment, and each antibody was intraperitoneally injected on day 0, day 3, day 7, and day 10 for a total of 4 to 5 doses. The tumor volume and animal weight were monitored and recorded twice a week. When the tumor volume of the experimental animal exceeded 1000 mm 3 Or when most tumors ruptured or body weight decreased by 20%, the tumor-bearing animals were euthanized as the experimental endpoint.
[1184] Table 37-1. Anti-tumor activity of GUCY2C-CD3 bispecific antibody Note: *p<0.05, **p<0.01, ***p<0.001 vs. vehicle control, T test.
[1185] Table 37-2. Anti-tumor activity of GUCY2C-CD3 bispecific antibody Note: *p<0.05, **p<0.01, ***p<0.001 vs. vehicle control, T test.
[1186] The results showed that the disclosed GUCY2C-CD3 dual antibody had a significant tumor inhibitory effect in this model. The tumor weight data during the experiment were generally consistent with the tumor volume trend.
[1187] Test Example 12. Anti-tumor activity of the combined use of GUCY2C-CD3 dual antibody and EGFR-CD28 dual antibody
[1188] This disclosure uses the LS174T subcutaneous tumor model to study the anti-tumor activity of the combination of GUCY2C-CD3 dual antibody and EGFR-CD28 dual antibody (97H2L3OT-ZAL, prepared with reference to patent CN202210371538.8).
[1189] Female NOG mice were purchased from Beijing Weitonglihua (Shanghai Branch) at 6-8 weeks of age. Human PBMCs used in this test were purchased commercially from Miaoshun Biotechnology.
[1190] LS174T cells 1×10 6 100 μL of cells / mouse were mixed with frozen and revived PBMC (E:T=1:1) and inoculated subcutaneously in the right rib of NOG mice. 3 When the mice had tumors that were too large or too small or too light, they were randomly divided into groups of 8. The day of grouping was defined as day 0 of the experiment, and each antibody was intraperitoneally injected on day 0, day 3, day 7, and day 11, for a total of 4 doses. The combination group drugs were prepared separately and administered simultaneously. The tumor volume and animal weight were monitored and recorded twice a week. When the tumor volume of the experimental animals exceeded 1000 mm 3When most tumors ruptured or the body weight decreased by 20%, the tumor-bearing animals were euthanized as the experimental endpoint. The specific results are shown in Table 38 below.
[1191] Table 38. Antitumor activity of GUCY2C-CD3 dual antibody combined with EGFR-CD28 dual antibody Note: *p<0.05, **p<0.01, ***p<0.001 vs. vehicle control, T test.
[1192] The results showed that the combination of the disclosed EGFR-CD28 dual antibody and the GUCY2C-CD3 dual antibody can effectively improve the efficacy of each single drug.
[1193] Although the above invention has been described in detail with the aid of the accompanying drawings and examples for clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.
Claims
1. An antigen-binding molecule that specifically binds to GUCY2C and CD3, comprising at least one antigen-binding moiety that specifically binds to GUCY2C and at least one antigen-binding moiety that specifically binds to CD3, wherein the antigen-binding moiety 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 moiety 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 has: 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 has: 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 sequence of GUCY2C-LCDR3; 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 has: 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 has: 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: 265 GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 299, and 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 has: GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 321, GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 322, and 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 having: 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 having: 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.
2. The antigen-binding molecule according to claim 1, 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; Preferably, the CD3-VH has: 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 has: 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.
3. The antigen-binding molecule according to claim 1 or 2, 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; 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.
4. The antigen-binding molecule according to any one of claims 1 to 3, wherein: 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.
5. The antigen binding molecule according to any one of claims 1 to 4, wherein the antigen binding moiety that specifically binds to GUCY2C or the antigen binding moiety 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 NO: 182 to SEQ ID NO: 200, and the obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 201 to SEQ ID NO: 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.
6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises an Fc region, preferably an IgG Fc region, more preferably an IgG1 Fc region; More preferably, the Fc region comprises one or more amino acid substitutions that can reduce the binding of the Fc region to Fcγ receptors; Most preferably, the Fc region is a human IgG1 Fc region and the amino acids at positions 234 and 235 are A, numbering according to the EU index.
7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region; Preferably, the Fc1 has a protrusion structure according to the pestle-and-mortar technique, and the Fc2 has a hole structure according to the pestle-and-mortar technique; More preferably, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering according to the EU index; Most preferably, the Fc1 comprises the amino acid sequence of SEQ ID NO: 79 or 85; and the Fc2 comprises the amino acid sequence of SEQ ID NO: 80 or 86.
8. The antigen binding molecule according to any one of claims 1 to 4, wherein the antigen binding molecule comprises one antigen binding moiety that specifically binds to GUCY2C and one antigen binding moiety that specifically binds to CD3, and both the antigen binding moieties that specifically bind to GUCY2C and CD3 are scFv; Preferably, The antigen-binding molecule comprises a first chain having a structure represented by formula (a) and a 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], in: The linker 1, linker 2 and linker 3 are the same or different peptide linkers; or 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; More preferably, wherein: 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 has: a first chain comprising the amino acid sequence of SEQ ID NO: 99 and a second chain comprising the amino acid sequence of SEQ ID NO: 100; or a first chain comprising the amino acid sequence of SEQ ID NO: 101 and a second chain comprising the amino acid sequence of SEQ ID NO: 100; or ii) The antigen-binding molecule has: a first chain comprising the amino acid sequence of SEQ ID NO: 102 and a second chain comprising the amino acid sequence of SEQ ID NO: 103; or iii) The antigen-binding molecule has: A first chain comprising the amino acid sequence of SEQ ID NO: 104 and a second chain comprising the amino acid sequence of SEQ ID NO:
105.
9. The antigen binding molecule according to claim 7, wherein the antigen binding molecule comprises an antigen binding moiety that specifically binds to CD3 and an antigen binding moiety that specifically binds to GUCY2C, wherein the antigen binding moiety that specifically binds to CD3 is a Fab; and the antigen binding moiety that specifically binds to GUCY2C is a replaced Fab comprising a titin chain and an obscurin chain that can form a dimer; Preferably, The antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (e), and a 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], in: The linker 4 is a peptide linker; or the linker 4 does not exist; The structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus; More preferably, wherein: 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: 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 has: i) a first chain comprising the amino acid sequence of SEQ ID NO: 91, a second chain comprising the amino acid sequence of SEQ ID NO: 92, a third chain comprising the amino acid sequence of SEQ ID NO: 110, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or a first chain comprising the amino acid sequence of SEQ ID NO: 242, a second chain comprising the amino acid sequence of SEQ ID NO: 92, a third chain comprising the amino acid sequence of SEQ ID NO: 110, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 113; or a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:108, and a fourth chain comprising the amino acid sequence of SEQ ID NO:109; or a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:111; or a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:112; or a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:114; or a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:115; or a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:110, and a fourth chain comprising the amino acid sequence of SEQ ID NO:116; or ii) a first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:106, and a fourth chain comprising the amino acid sequence of SEQ ID NO:107; or iii) a first chain comprising the amino acid sequence of SEQ ID NO: 91, a second chain comprising the amino acid sequence of SEQ ID NO: a second chain comprising the amino acid sequence of SEQ ID NO: 92, a third chain comprising the amino acid sequence of SEQ ID NO: 119, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 120; or A first chain comprising the amino acid sequence of SEQ ID NO:91, a second chain comprising the amino acid sequence of SEQ ID NO:92, a third chain comprising the amino acid sequence of SEQ ID NO:117, and a fourth chain comprising the amino acid sequence of SEQ ID NO:
118.
10. The antigen binding molecule according to claim 7, wherein the antigen binding molecule comprises an antigen binding moiety that specifically binds to GUCY2C and an antigen binding moiety that specifically binds to CD3, wherein the antigen binding moiety that specifically binds to GUCY2C is a replaced Fab comprising a titin chain and an obscurin chain that can form a dimer; and the antigen binding moiety that specifically binds to CD3 is a Fab; Preferably, The antigen-binding molecule comprises a first chain having a structure represented by formula (g), a second chain having a structure represented by formula (f), a third chain having a structure represented by formula (h), and a 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], in: The linker 4 is a peptide linker; or the linker 4 does not exist; The structures shown in formulas (g), (f), (h), and (d) are arranged from the N-terminus to the C-terminus; More preferably, wherein: 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 said 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 has: i) a first chain comprising the amino acid sequence of SEQ ID NO: 121, a second chain comprising the amino acid sequence of SEQ ID NO: 107, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or ii) a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 111, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 112, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 113, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 114, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or A first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: a second chain comprising the amino acid sequence of SEQ ID NO: 115, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or a first chain comprising the amino acid sequence of SEQ ID NO: 122, a second chain comprising the amino acid sequence of SEQ ID NO: 116, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 92; or iii) a first chain comprising the amino acid sequence of SEQ ID NO: 123, a second chain comprising the amino acid sequence of SEQ ID NO: 120, a third chain comprising the amino acid sequence of SEQ ID NO: 93, and a fourth chain comprising the amino acid sequence of SEQ ID NO:
92.
11. The antigen binding molecule according to any one of claims 1 to 4, wherein the antigen binding molecule comprises two antigen binding moieties that specifically bind to GUCY2C and two antigen binding moieties that specifically bind to CD3, and both the antigen binding moieties that specifically bind to GUCY2C and CD3 are scFv; Preferably, 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], in: The linker 5 and linker 6 are peptide linkers; or linker 5 and linker 6 do not exist; The structures shown in formulas (i) and (j) are arranged from the N-terminus to the C-terminus; More preferably, wherein: 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 has: 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.
12. The antigen binding molecule according to claim 7, wherein the antigen binding molecule comprises two antigen binding moieties that specifically bind to GUCY2C and two antigen binding moieties that specifically bind to CD3, wherein the antigen binding moiety that specifically binds to GUCY2C is a Fab; and the antigen binding moiety that specifically binds to CD3 is a replaced Fab comprising a titin chain and an obscurin chain that can form 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], in: The linker 4 and linker 7 are peptide linkers; or linker 4 and linker 7 do not exist; The structures shown in formulas (k), (j), and (l) are arranged from the N-terminus to the C-terminus; More preferably, wherein: 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 has: 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 comprise the amino acid sequence of SEQ ID NO: 125, two second chains comprise the amino acid sequence of SEQ ID NO: 66, and two third chains comprise the amino acid sequence of SEQ ID NO:
98.
13. An anti-GUCY2C antibody capable of specifically binding to GUCY2C, the anti-GUCY2C antibody comprising 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. ID NO: 21; and the GUCY2C-VL has: 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 has: 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 has: 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 has: an amino acid sequence comprising SEQ ID NO: 267 GUCY2C-HCDR1, GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO: 268, and GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO: 269; and the GUCY2C-VL has: GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO: 270, GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO: 271, and 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 has: 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 has: 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. sequence of GUCY2C-LCDR3; or iv-16) the GUCY2C-VH having: 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 having: 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.
14. The anti-GUCY2C antibody according to claim 13, 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; 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 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, 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, 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 according to any one of claims 13 to 16, wherein the anti-GUCY2C antibody comprises a heavy chain and a light chain, wherein: 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. columns; or the heavy chain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 60, and the light chain comprises an amino acid sequence that has 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.
18. The anti-GUCY2C antibody according to any one of claims 13 to 17, wherein the anti-GUCY2C antibody is an antibody fragment; 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. 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, or 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, metastatic liver adenocarcinoma, multiple myeloma, lymphoma, leukemia or B-cell lymphoma; most preferably, the cells of the tumor or the vascular endothelial cells near the tumor express GUCY2C.
23. The method for treating a disease according to claim 22, further comprising using a second therapeutic agent; preferably, the second therapeutic agent comprises an anti-tumor agent, radiotherapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an anti-tumor agent, an immune checkpoint inhibitor, or a combination thereof.
24. The method for treating a disease according to claim 23, wherein the second therapeutic agent is a bispecific antibody that specifically binds to CD28 and EGFR, comprising at least one antigen-binding moiety that specifically binds to CD28 and at least one antigen-binding moiety that specifically binds to EGFR, wherein the antigen-binding moiety that specifically binds to CD28 comprises a heavy chain variable region CD28-VH and a light chain variable region CD28-VL, and wherein the antigen-binding moiety 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.
25. The method for treating a disease according to claim 24, wherein the antigen binding moiety that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, wherein: 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.
26. The method for treating a disease according to claim 25, wherein 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 capable of associating with each other, the Fc1 having a protrusion structure according to the knob-in-hole technique, and the Fc2 having a hole structure according to the knob-in-hole technique; More preferably, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering according to the EU index; Most preferably, 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.
27. The method for treating a disease according to claim 26, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an antigen-binding moiety that specifically binds to CD28 and an antigen-binding moiety that specifically binds to EGFR, wherein the antigen-binding moiety that specifically binds to CD28 is a replaced Fab comprising a titin chain and an obscurin chain that can form a dimer; and the antigen-binding moiety that specifically binds to EGFR is a Fab; Preferably, The bispecific antibody that specifically binds to CD28 and EGFR comprises a first chain having a structure represented by formula (m), a second chain having a structure represented by formula (n), a third chain having a structure represented by formula (o), and a fourth chain having a structure represented by 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], in: The linker 8 is a peptide linker; or the linker 8 does not exist; The structures shown in formulas (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 has: a first chain comprising the amino acid sequence of SEQ ID NO: 156, a second chain comprising the amino acid sequence of SEQ ID NO: 157, a third chain comprising the amino acid sequence of SEQ ID NO: 158, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 159; or The bispecific antibody that specifically binds to CD28 and EGFR has: a first chain comprising the amino acid sequence of SEQ ID NO: 166, a second chain comprising the amino acid sequence of SEQ ID NO: 167, a third chain comprising the amino acid sequence of SEQ ID NO: 158, and a fourth chain comprising the amino acid sequence of SEQ ID NO:
159.
28. The method for treating a disease according to claim 26, wherein the bispecific antibody that specifically binds to CD28 and EGFR comprises an antigen-binding moiety that specifically binds to CD28 and an antigen-binding moiety that specifically binds to EGFR, wherein the antigen-binding moiety that specifically binds to CD28 is a Fab; and the antigen-binding moiety that specifically binds to EGFR is a replaced Fab that comprises a titin chain and an obscurin chain that can form a dimer; Preferably, The bispecific antibody that specifically binds to CD28 and EGFR comprises a first chain having a structure represented by formula (q), a second chain having a structure represented by formula (r), a third chain having a structure represented by formula (s), and a fourth chain having a 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], in: The linker 8 is a peptide linker; or the linker 8 does not exist; The structures shown in formulas (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 has: a first chain comprising the amino acid sequence of SEQ ID NO: 176, a second chain comprising the amino acid sequence of SEQ ID NO: 177, a third chain comprising the amino acid sequence of SEQ ID NO: 178, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 179.