Anti-TF antibodies and anti-TF antibody-drug conjugates and their pharmaceutical uses

Abstract

This invention relates to anti-TF antibodies, anti-TF antibody-drug conjugates, and their pharmaceutical uses. Specifically, it relates to anti-TF antibodies, for example, anti-TF antibody-drug conjugates represented by the general formula (Pc-LYD), where Pc is an anti-TF antibody and L, Y, and n are as described in the specification. [Formula 1] JPEG2026518847000143.jpg53138

Description

[Technical Field]

[0001] This disclosure relates to anti-TF antibodies, anti-TF antibody-drug conjugates, and their pharmaceutical uses. [Background technology]

[0002] The descriptions herein are for the sole purpose of providing background information related to this disclosure and do not necessarily constitute prior art.

[0003] Tissue Factor, also known as TF / tissue thromboplastin / FIII / CD142, is a transmembrane glycoprotein with a total length of 47 kDa. It has 219 amino acids in the extracellular domain, 23 amino acids in the transmembrane domain, and 21 amino acids in the intracellular domain. The gene name for human Tissue Factor is F3.

[0004] TF is primarily expressed in perivascular cells (e.g., outer fibroblasts and pericytes) and on the body surface (e.g., skin keratinocytes), and is also expressed in monocytes, cerebral astrocytes, lung epithelial cells, cardiac fibroblasts, and cardiomyocytes. This broad expression is advantageous for the formation of a systemic hemostatic barrier.

[0005] TF is the promoter of exogenous coagulation under normal physiological conditions and is the cell receptor for FVII (coagulation factor VII) and its activated form FVIIa in the blood. When a blood vessel is damaged, TF forms a complex with FVII in the blood, activating FVII to FVIIa and producing a TF / FVIIa complex. TF / FVIIa binds to FX (coagulation factor X) in the blood, activating it to FXa, forming a TF / FVIIa / FXa complex, which further triggers a coagulation cascade reaction, activating prothrombin (FII) to thrombin (FIIa), and thrombin converts coagulation fibrinogen into insoluble coagulation fibers, ultimately completing coagulation.

[0006] The TF / FVIIa complex can activate the PAR2 (proteinase-activated receptor 2) signaling pathway, and it can also form a TF / FVIIa / Integrinβ1 complex with Integrin β1. The downstream signaling pathways of PAR2 and Integrin β1 can promote tumor growth. The downstream products of the TF / FVIIa coagulation gasket reaction, thrombin and fibrin, are associated with tumor metastasis and thrombus formation.

[0007] TF is highly expressed under pathological conditions in various solid tumors, such as pancreatic cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer, ovarian cancer, and bladder cancer. Various growth factors and cytokines induce TF expression, and various transcription factors and microRNAs regulate TF gene expression in cancer cells. For example, the hepatocyte growth factor (HGF) / c-Met and epidermal growth factor receptor (EGFR) signaling pathways can upregulate TF expression by activating various kinase pathways, including JNK, Src, PI3k / Akt / mTOR, and KRAS / Raf / MEK / ERK. Transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) can also increase TF expression. During inflammation, TF can be upregulated by various cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). TF expression may be downregulated by certain microRNAs, such as miR-181b and miR19, or its activity may be suppressed by TFPIs (tissue factor pathway inhibitors) in monocytes.

[0008] High expression of TF under pathological conditions can promote tumor growth, metastasis, and thrombus formation, and is associated with a poor prognosis. Therefore, the development of ADCs against TF can either inhibit tumor growth through the signal blocking effect of the antibody, or achieve specific killing of the toxin against tumors through the targeting properties of the TF antibody. [Overview of the project]

[0009] This disclosure relates to anti-TF antibodies, anti-TF antibody-drug conjugates (particularly anti-TF antibody-exatecan analog conjugates), and their pharmaceutical uses.

[0010] This disclosure provides an anti-TF antibody comprising a heavy chain variable region and a light chain variable region, a. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 40 in HCDR1, an amino acid sequence of SEQ ID NO: 64 or 41 in HCDR2, and an amino acid sequence of SEQ ID NO: 65 or 42 in HCDR3; the light chain variable region includes an amino acid sequence of SEQ ID NO: 66 or 43 in LCDR1, an amino acid sequence of SEQ ID NO: 67 or 44 in LCDR2, and an amino acid sequence of SEQ ID NO: 68, 83, or 45 in LCDR3; b. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 46 in HCDR1, an amino acid sequence of SEQ ID NO: 47 in HCDR2, and an amino acid sequence of SEQ ID NO: 48 in HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 49, 111, 112, or 113 in LCDR1, an amino acid sequence of SEQ ID NO: 50 in LCDR2, and an amino acid sequence of SEQ ID NO: 51 in LCDR3. c. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 73 or 144 for HCDR1, an amino acid sequence of SEQ ID NO: 74, 142, 145 or 146 for HCDR2, an amino acid sequence of SEQ ID NO: 75 or 143 for HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 76, 151, 147 or 150 for LCDR1, an amino acid sequence of SEQ ID NO: 77 or 148 for LCDR2, and an amino acid sequence of SEQ ID NO: 78 or 149 for LCDR3. Preferably, a. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 40 in HCDR1, an amino acid sequence of SEQ ID NO: 64 in HCDR2, an amino acid sequence of SEQ ID NO: 65 in HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 66 in LCDR1, an amino acid sequence of SEQ ID NO: 67 in LCDR2, an amino acid sequence of SEQ ID NO: 68 or 83 in LCDR3, or The heavy chain variable region includes an amino acid sequence of SEQ ID NO:40 in HCDR1, an amino acid sequence of SEQ ID NO:41 in HCDR2, and an amino acid sequence of SEQ ID NO:42 in HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO:43 in LCDR1, an amino acid sequence of SEQ ID NO:44 in LCDR2, and an amino acid sequence of SEQ ID NO:45 in LCDR3. b. The heavy chain variable region includes an amino acid sequence of SEQ ID NO:46 in HCDR1, an amino acid sequence of SEQ ID NO:47 in HCDR2, and an amino acid sequence of SEQ ID NO:48 in HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO:49 in LCDR1, an amino acid sequence of SEQ ID NO:50 in LCDR2, and an amino acid sequence of SEQ ID NO:51 in LCDR3. c. The heavy chain variable region includes an amino acid sequence of SEQ ID NO:73 in HCDR1, an amino acid sequence of SEQ ID NO:74 in HCDR2, and an amino acid sequence of SEQ ID NO:75 in HCDR3. The light chain variable region includes an amino acid sequence of SEQ ID NO:76 or 151 in LCDR1, an amino acid sequence of SEQ ID NO:77 or 148 in LCDR2, and an amino acid sequence of SEQ ID NO:78 in LCDR3.

[0011] In some embodiments, the anti-TF antibodies described above, HCDR1-3 and LCDR1-3 described above, are determined according to the Kabat numbering rules. In some embodiments, the anti-TF antibodies described above, HCDR1-3 and LCDR1-3 described above, are determined according to the IMGT numbering rules. In some embodiments, the anti-TF antibodies described above, HCDR1-3 and LCDR1-3 described above, are determined according to the Chothia numbering rules. In some embodiments, the anti-TF antibodies described above, HCDR1-3 and LCDR1-3 described above, are determined according to the AbM numbering rules. In some embodiments, the anti-TF antibodies described above, HCDR1-3 and LCDR1-3 described above, are determined according to the Contact numbering rules.

[0012] This disclosure provides an anti-TF antibody comprising a heavy chain variable region and a light chain variable region, a. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, or The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 83, respectively, or The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively. b. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. c. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, respectively, or The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:151, SEQ ID NO:148, and SEQ ID NO:78, respectively. The HCDR1-3 and LCDR1-3 described above are determined according to the Kabat numbering rules.

[0013] This disclosure provides an anti-TF antibody comprising a heavy chain variable region and a light chain variable region, a. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively. b. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. c. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, respectively.

[0014] In some embodiments, the anti-TF antibody described above is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0015] In some embodiments, the anti-TF antibody described above is a chimeric antibody or a humanized antibody.

[0016] In some embodiments, the anti-TF antibody described above is a humanized antibody.

[0017] In some embodiments, the anti-TF antibody described in any one of the above clauses comprises a heavy chain variable region and a light chain variable region, where, a. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62, 58, 130, 131, or 132, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 63, 81, 59, 133, 134, or 135, or the heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 37, b. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52 or 114, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 53, 115, 116, 117, 118, 119, 120 or 121, or the heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 38, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 39, c. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 71, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 72, 97, 102, 98, 99, 100, or 101. Preferably, a. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 62, 58, 130, 131, or 132, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 63, 81, 59, 133, 134, or 135, or the heavy chain variable region includes an amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 37. b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 52 or 114, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 53, 115, 116, 117, 118, 119, 120 or 121, or the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 38, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39. c. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 71, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 72, 97, 102, 98, 99, 100, or 101. more, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 62, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 63 or 81, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:36, and the light chain variable region includes the amino acid sequence of SEQ ID NO:37, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:130, and the light chain variable region includes the amino acid sequence of SEQ ID NO:133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:131, and the light chain variable region includes the amino acid sequence of SEQ ID NO:133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:132, and the light chain variable region includes the amino acid sequence of SEQ ID NO:133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:130, and the light chain variable region includes the amino acid sequence of SEQ ID NO:134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:131, and the light chain variable region includes the amino acid sequence of SEQ ID NO:134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:132, and the light chain variable region includes the amino acid sequence of SEQ ID NO:134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:130, and the light chain variable region includes the amino acid sequence of SEQ ID NO:59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:131, and the light chain variable region includes the amino acid sequence of SEQ ID NO:59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:132, and the light chain variable region includes the amino acid sequence of SEQ ID NO:59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:130, and the light chain variable region includes the amino acid sequence of SEQ ID NO:135, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:131, and the light chain variable region includes the amino acid sequence of SEQ ID NO:135, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 135, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:132, and the light chain variable region includes the amino acid sequence of SEQ ID NO:135. b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 52, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 53, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO:38, and the light chain variable region includes the amino acid sequence of SEQ ID NO:39. c. The heavy chain variable region includes the amino acid sequence of SEQ ID NO:71, and the light chain variable region includes the amino acid sequence of SEQ ID NO:72, 97, or 102.

[0018] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region, wherein a. The heavy chain variable region is as shown in SEQ ID NO:36, and the light chain variable region is as shown in SEQ ID NO:37, or, b. The heavy chain variable region is as shown in SEQ ID NO:38, and the light chain variable region is as shown in SEQ ID NO:39.

[0019] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region, wherein a. The heavy chain variable region is as shown in SEQ ID NO:62, and the light chain variable region is as shown in SEQ ID NO:63, or, The heavy chain variable region is as shown in SEQ ID NO:62, and the light chain variable region is as shown in SEQ ID NO:81, or, The heavy chain variable region is as shown in SEQ ID NO:58, and the light chain variable region is as shown in SEQ ID NO:59, or, The heavy chain variable region is as shown in SEQ ID NO:36, and the light chain variable region is as shown in SEQ ID NO:37, or, The heavy chain variable region is as shown in SEQ ID NO:130, and the light chain variable region is as shown in SEQ ID NO:133, or, The heavy chain variable region is as shown in SEQ ID NO:131, and the light chain variable region is as shown in SEQ ID NO:133, or, The heavy chain variable region is as shown in SEQ ID NO: 58, and the light chain variable region is as shown in SEQ ID NO: 133, or, The heavy chain variable region is as shown in SEQ ID NO:132, and the light chain variable region is as shown in SEQ ID NO:133, or, The heavy chain variable region is as shown in SEQ ID NO:130, and the light chain variable region is as shown in SEQ ID NO:134, or, The heavy chain variable region is as shown in SEQ ID NO:131, and the light chain variable region is as shown in SEQ ID NO:134, or, The heavy chain variable region is as shown in SEQ ID NO: 58, and the light chain variable region is as shown in SEQ ID NO: 134, or, The heavy chain variable region is as shown in SEQ ID NO:132, and the light chain variable region is as shown in SEQ ID NO:134, or, The heavy chain variable region is as shown in SEQ ID NO:130, and the light chain variable region is as shown in SEQ ID NO:59, or, The heavy chain variable region is as shown in SEQ ID NO:131, and the light chain variable region is as shown in SEQ ID NO:59, or, The heavy chain variable region is as shown in SEQ ID NO:132, and the light chain variable region is as shown in SEQ ID NO:59, or, The heavy chain variable region is as shown in SEQ ID NO:130, and the light chain variable region is as shown in SEQ ID NO:135, or, The heavy chain variable region is as shown in SEQ ID NO:131, and the light chain variable region is as shown in SEQ ID NO:135, or, The heavy chain variable region is as shown in SEQ ID NO: 58, and the light chain variable region is as shown in SEQ ID NO: 135, or, The heavy chain variable region is as shown in SEQ ID NO:132, and the light chain variable region is as shown in SEQ ID NO:135. b. The heavy chain variable region is as shown in SEQ ID NO: 52, and the light chain variable region is as shown in SEQ ID NO: 53, or, The heavy chain variable region is as shown in SEQ ID NO:38, and the light chain variable region is as shown in SEQ ID NO:39. c. The heavy chain variable region is as shown in SEQ ID NO:71, and the light chain variable region is as shown in SEQ ID NO:72, or, The heavy chain variable region is as shown in SEQ ID NO:71, and the light chain variable region is as shown in SEQ ID NO:97, or, The heavy chain variable region is as shown in SEQ ID NO:71, and the light chain variable region is as shown in SEQ ID NO:102.

[0020] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region, wherein a. The heavy chain variable region is as shown in SEQ ID NO:62, and the light chain variable region is as shown in SEQ ID NO:63, or, b. The heavy chain variable region is as shown in SEQ ID NO: 52, and the light chain variable region is as shown in SEQ ID NO: 53, or, c. The heavy chain variable region is as shown in SEQ ID NO:71, and the light chain variable region is as shown in SEQ ID NO:72.

[0021] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region is selected from those shown in SEQ ID NO: 130, 131, and 132, and the light chain variable region is Select from those shown in SEQ ID NO: 133, 134, and 135.

[0022] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region is selected from those shown in SEQ ID NO:114, and the light chain variable region is selected from those shown in SEQ ID NO:115, 116, 117, 118, 119, 120, and 121.

[0023] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region is selected from those shown in SEQ ID NO: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 71, 94, 95, and 96, and the light chain variable region is selected from those shown in SEQ ID NO: 97, 98, 99, 100, 101, and 102.

[0024] In some embodiments, the anti-TF antibody described in any one of the above clauses is an antibody fragment, preferably selected from Fab, Fab′, F(ab′)2, Fd, Fv, scFv, dsFv, and dAb.

[0025] In some embodiments, the anti-TF antibody described in any one of the above clauses further comprises an antibody heavy chain constant region and a light chain constant region, preferably the heavy chain constant region being selected from human IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region being a human κ or λ chain constant region, more preferably the heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 54, and the light chain constant region comprising the amino acid sequence of SEQ ID NO: 55.

[0026] In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:54, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:55.

[0027] In some embodiments, the anti-TF antibody described in any one of the above clauses is, a. A heavy chain having at least 80% sequence identity with SEQ ID NO: 69, 60, 136, 137, or 138, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 70, 82, 61, 139, 140, or 141, or A heavy chain having at least 80% sequence identity with SEQ ID NO:107, and / or a light chain having at least 80% sequence identity with SEQ ID NO:108. b. A heavy chain having at least 80% sequence identity with SEQ ID NO: 56 or 122, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 57, 123, 124, 125, 126, 127, 128 or 129, or A heavy chain having at least 80% sequence identity with SEQ ID NO:109, and / or a light chain having at least 80% sequence identity with SEQ ID NO:110. c. Selected from an antibody comprising a heavy chain and a light chain, which are described in any one of the following: a heavy chain having at least 80% sequence identity with SEQ ID NO: 79, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 or 164, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 80, 165, 170, 166, 167, 168 or 169. Preferably, a. A heavy chain having at least 80% sequence identity with SEQ ID NO: 69, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 70 or 82, or A heavy chain having at least 80% sequence identity with SEQ ID NO:60, and / or a light chain having at least 80% sequence identity with SEQ ID NO:61, or A heavy chain having at least 80% sequence identity with SEQ ID NO:107, and / or a light chain having at least 80% sequence identity with SEQ ID NO:108, or A heavy chain having at least 80% sequence identity with SEQ ID NO:136, and / or a light chain having at least 80% sequence identity with SEQ ID NO:139, or A heavy chain having at least 80% sequence identity with SEQ ID NO:137, and / or a light chain having at least 80% sequence identity with SEQ ID NO:139, or A heavy chain having at least 80% sequence identity with SEQ ID NO:60, and / or a light chain having at least 80% sequence identity with SEQ ID NO:139, or A heavy chain having at least 80% sequence identity with SEQ ID NO:138, and / or a light chain having at least 80% sequence identity with SEQ ID NO:139, or A heavy chain having at least 80% sequence identity with SEQ ID NO:136, and / or a light chain having at least 80% sequence identity with SEQ ID NO:140, or A heavy chain having at least 80% sequence identity with SEQ ID NO:137, and / or a light chain having at least 80% sequence identity with SEQ ID NO:140, or A heavy chain having at least 80% sequence identity with SEQ ID NO:60, and / or a light chain having at least 80% sequence identity with SEQ ID NO:140, or A heavy chain having at least 80% sequence identity with SEQ ID NO:138, and / or a light chain having at least 80% sequence identity with SEQ ID NO:140, or A heavy chain having at least 80% sequence identity with SEQ ID NO:136, and / or a light chain having at least 80% sequence identity with SEQ ID NO:61, or A heavy chain having at least 80% sequence identity with SEQ ID NO:137, and / or a light chain having at least 80% sequence identity with SEQ ID NO:61, or A heavy chain having at least 80% sequence identity with SEQ ID NO:138, and / or a light chain having at least 80% sequence identity with SEQ ID NO:61, or A heavy chain having at least 80% sequence identity with SEQ ID NO:136, and / or a light chain having at least 80% sequence identity with SEQ ID NO:141, or A heavy chain having at least 80% sequence identity with SEQ ID NO:137, and / or a light chain having at least 80% sequence identity with SEQ ID NO:141, or A heavy chain having at least 80% sequence identity with SEQ ID NO:60, and / or a light chain having at least 80% sequence identity with SEQ ID NO:141, or A heavy chain having at least 80% sequence identity with SEQ ID NO:138, and / or a light chain having at least 80% sequence identity with SEQ ID NO:141. b. A heavy chain having at least 80% sequence identity with SEQ ID NO: 56, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 57, or A heavy chain having at least 80% sequence identity with SEQ ID NO:109, and / or a light chain having at least 80% sequence identity with SEQ ID NO:110. c. A heavy chain having at least 80% sequence identity with SEQ ID NO:79, and / or a light chain having at least 80% sequence identity with SEQ ID NO:80, 165, or 170. more, a. The heavy chain contains the amino acid sequence of SEQ ID NO:69, and the light chain contains the amino acid sequence of SEQ ID NO:70, or The heavy chain contains the amino acid sequence of SEQ ID NO:69, and the light chain contains the amino acid sequence of SEQ ID NO:82, or The heavy chain contains the amino acid sequence of SEQ ID NO:60, and the light chain contains the amino acid sequence of SEQ ID NO:61, or The heavy chain contains the amino acid sequence of SEQ ID NO:107, and the light chain contains the amino acid sequence of SEQ ID NO:108, or The heavy chain contains the amino acid sequence of SEQ ID NO:136, and the light chain contains the amino acid sequence of SEQ ID NO:139, or The heavy chain contains the amino acid sequence of SEQ ID NO:137, and the light chain contains the amino acid sequence of SEQ ID NO:139, or The heavy chain contains the amino acid sequence of SEQ ID NO:60, and the light chain contains the amino acid sequence of SEQ ID NO:139, or The heavy chain contains the amino acid sequence of SEQ ID NO:138, and the light chain contains the amino acid sequence of SEQ ID NO:139, or The heavy chain contains the amino acid sequence of SEQ ID NO:136, and the light chain contains the amino acid sequence of SEQ ID NO:140, or The heavy chain contains the amino acid sequence of SEQ ID NO:137, and the light chain contains the amino acid sequence of SEQ ID NO:140, or The heavy chain contains the amino acid sequence of SEQ ID NO: 60, and the light chain contains the amino acid sequence of SEQ ID NO: 140, or The heavy chain contains the amino acid sequence of SEQ ID NO:138, and the light chain contains the amino acid sequence of SEQ ID NO:140, or The heavy chain contains the amino acid sequence of SEQ ID NO:136, and the light chain contains the amino acid sequence of SEQ ID NO:61, or The heavy chain contains the amino acid sequence of SEQ ID NO:137, and the light chain contains the amino acid sequence of SEQ ID NO:61, or The heavy chain contains the amino acid sequence of SEQ ID NO:138, and the light chain contains the amino acid sequence of SEQ ID NO:61, or The heavy chain contains the amino acid sequence of SEQ ID NO:136, and the light chain contains the amino acid sequence of SEQ ID NO:141, or The heavy chain contains the amino acid sequence of SEQ ID NO:137, and the light chain contains the amino acid sequence of SEQ ID NO:141, or The heavy chain contains the amino acid sequence of SEQ ID NO:60, and the light chain contains the amino acid sequence of SEQ ID NO:141, or The heavy chain comprises the amino acid sequence of SEQ ID NO:138, and the light chain comprises the amino acid sequence of SEQ ID NO:141. b. The heavy chain contains the amino acid sequence of SEQ ID NO: 56, and the light chain contains the amino acid sequence of SEQ ID NO: 57, or The heavy chain comprises the amino acid sequence of SEQ ID NO:109, and the light chain comprises the amino acid sequence of SEQ ID NO:110. c. The heavy chain contains the amino acid sequence of SEQ ID NO:79, and the light chain contains the amino acid sequence of SEQ ID NO:80, or The heavy chain contains the amino acid sequence of SEQ ID NO:79, and the light chain contains the amino acid sequence of SEQ ID NO:165, or The heavy chain contains the amino acid sequence of SEQ ID NO:79, and the light chain contains the amino acid sequence of SEQ ID NO:170.

[0028] The above "at least 80% sequence identity" includes at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0029] In some embodiments, an anti-TF antibody according to any one of the above claims, comprising a heavy chain and a light chain, wherein a. A heavy chain having at least 85% sequence identity with SEQ ID NO:69, and / or a light chain having at least 85% sequence identity with SEQ ID NO:70, or A heavy chain having at least 85% sequence identity with SEQ ID NO:69, and / or a light chain having at least 85% sequence identity with SEQ ID NO:82, or A heavy chain having at least 85% sequence identity with SEQ ID NO:60, and / or a light chain having at least 85% sequence identity with SEQ ID NO:61, or A heavy chain having at least 85% sequence identity with SEQ ID NO:107, and / or a light chain having at least 85% sequence identity with SEQ ID NO:108, or A heavy chain having at least 85% sequence identity with SEQ ID NO:136, and / or a light chain having at least 85% sequence identity with SEQ ID NO:139, or A heavy chain having at least 85% sequence identity with SEQ ID NO:137, and / or a light chain having at least 85% sequence identity with SEQ ID NO:139, or A heavy chain having at least 85% sequence identity with SEQ ID NO:60, and / or a light chain having at least 85% sequence identity with SEQ ID NO:139, or A heavy chain having at least 85% sequence identity with SEQ ID NO:138, and / or a light chain having at least 85% sequence identity with SEQ ID NO:139, or A heavy chain having at least 85% sequence identity with SEQ ID NO:136, and / or a light chain having at least 85% sequence identity with SEQ ID NO:140, or A heavy chain having at least 85% sequence identity with SEQ ID NO:137, and / or a light chain having at least 85% sequence identity with SEQ ID NO:140, or A heavy chain having at least 85% sequence identity with SEQ ID NO:60, and / or a light chain having at least 85% sequence identity with SEQ ID NO:140, or A heavy chain having at least 85% sequence identity with SEQ ID NO:138, and / or a light chain having at least 85% sequence identity with SEQ ID NO:140, or A heavy chain having at least 85% sequence identity with SEQ ID NO:136, and / or a light chain having at least 85% sequence identity with SEQ ID NO:61, or A heavy chain having at least 85% sequence identity with SEQ ID NO:137, and / or a light chain having at least 85% sequence identity with SEQ ID NO:61, or A heavy chain having at least 85% sequence identity with SEQ ID NO:138, and / or a light chain having at least 85% sequence identity with SEQ ID NO:61, or A heavy chain having at least 85% sequence identity with SEQ ID NO:136, and / or a light chain having at least 85% sequence identity with SEQ ID NO:141, or A heavy chain having at least 85% sequence identity with SEQ ID NO:137, and / or a light chain having at least 85% sequence identity with SEQ ID NO:141, or A heavy chain having at least 85% sequence identity with SEQ ID NO:60, and / or a light chain having at least 85% sequence identity with SEQ ID NO:141, or A heavy chain having at least 85% sequence identity with SEQ ID NO:138, and / or a light chain having at least 85% sequence identity with SEQ ID NO:141. b. A heavy chain having at least 85% sequence identity with SEQ ID NO: 56, and / or a light chain having at least 85% sequence identity with SEQ ID NO: 57, or A heavy chain having at least 85% sequence identity with SEQ ID NO:109, and / or a light chain having at least 85% sequence identity with SEQ ID NO:110, c. A heavy chain having at least 85% sequence identity with SEQ ID NO:79, and / or a light chain having at least 85% sequence identity with SEQ ID NO:80, or A heavy chain having at least 85% sequence identity with SEQ ID NO:79, and / or a light chain having at least 85% sequence identity with SEQ ID NO:165, or A heavy chain having at least 85% sequence identity with SEQ ID NO:79, and / or a light chain having at least 85% sequence identity with SEQ ID NO:170.

[0030] The above "at least 85% sequence identity" includes at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0031] In some embodiments, an anti-TF antibody according to any one of the above descriptions is selected from an antibody comprising a heavy chain and a light chain as described in any one of the following: a. The heavy chain is as shown in SEQ ID NO:69, and the light chain is as shown in SEQ ID NO:70, or The heavy chain is as shown in SEQ ID NO:69, and the light chain is as shown in SEQ ID NO:82, or, The heavy chain is as shown in SEQ ID NO:60, and the light chain is as shown in SEQ ID NO:61, or The heavy chain is as shown in SEQ ID NO:107, and the light chain is as shown in SEQ ID NO:108, or, The heavy chain is as shown in SEQ ID NO:136, and the light chain is as shown in SEQ ID NO:139, or, The heavy chain is as shown in SEQ ID NO:137, and the light chain is as shown in SEQ ID NO:139, or, The heavy chain is as shown in SEQ ID NO:60, and the light chain is as shown in SEQ ID NO:139, or, The heavy chain is as shown in SEQ ID NO:138, and the light chain is as shown in SEQ ID NO:139, or, The heavy chain is as shown in SEQ ID NO:136, and the light chain is as shown in SEQ ID NO:140, or, The heavy chain is as shown in SEQ ID NO:137, and the light chain is as shown in SEQ ID NO:140, or, The heavy chain is as shown in SEQ ID NO:60, and the light chain is as shown in SEQ ID NO:140, or, The heavy chain is as shown in SEQ ID NO:138, and the light chain is as shown in SEQ ID NO:140, or, The heavy chain is as shown in SEQ ID NO:136, and the light chain is as shown in SEQ ID NO:61, or The heavy chain is as shown in SEQ ID NO:137, and the light chain is as shown in SEQ ID NO:61, or, The heavy chain is as shown in SEQ ID NO:138 and / or as shown in SEQ ID NO:61, or The heavy chain is as shown in SEQ ID NO:136, and the light chain is as shown in SEQ ID NO:141, or, The heavy chain is as shown in SEQ ID NO:137, and the light chain is as shown in SEQ ID NO:141, or, The heavy chain is as shown in SEQ ID NO:60, and the light chain is as shown in SEQ ID NO:141, or, The heavy chain is as shown in SEQ ID NO:138, and the light chain is as shown in SEQ ID NO:141. b. The heavy chain is as shown in SEQ ID NO: 56, the light chain is as shown in SEQ ID NO: 57, or, The heavy chain is as shown in SEQ ID NO:109, and the light chain is as shown in SEQ ID NO:110. c. The heavy chain is as shown in SEQ ID NO:79, and the light chain is as shown in SEQ ID NO:80, or, The heavy chain is as shown in SEQ ID NO:79, and the light chain is as shown in SEQ ID NO:165, or, The heavy chain is as shown in SEQ ID NO:79, and the light chain is as shown in SEQ ID NO:170.

[0032] In some embodiments, an anti-TF antibody according to any one of the above claims comprises a heavy chain and a light chain, a. The heavy chain is as shown in SEQ ID NO:69, and the light chain is as shown in SEQ ID NO:70. b. The heavy chain is as shown in SEQ ID NO: 56, and the light chain is as shown in SEQ ID NO: 57. c. The heavy chain is as shown in SEQ ID NO:79, and the light chain is as shown in SEQ ID NO:80.

[0033] In some embodiments, the disclosure further provides nucleic acid molecules encoding the anti-TF antibody described in any one of the preceding paragraphs.

[0034] In some embodiments, the present disclosure further provides host cells comprising the nucleic acid molecules described above.

[0035] In some embodiments, the Disclosure further provides an immunoconjugate comprising an anti-TF antibody and an effector molecule as described in any one of the preceding paragraphs, wherein the effector molecule is conjugated to the anti-TF antibody as described in any one of the preceding paragraphs, and preferably the effector molecule is selected from antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0036] In some embodiments, the present disclosure further provides methods for in vivo and / or in vitro immunodetection or measurement of TF, the methods comprising the step of contacting a subject or a sample from a subject with an anti-TF antibody as described in any one of the preceding paragraphs.

[0037] In some embodiments, the disclosure further provides antibody-drug conjugates represented by the general formula (Pc-LYD) or pharmaceutically acceptable salts thereof. [ka] Here, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, Here, R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, and a C 1-6 alkyl group, R 1 is a C 3-6 cycloalkyl-C 1-6 alkyl group or a C 3-6 cycloalkyl group, R 2 is selected from a hydrogen atom, a halo C 1-6 alkyl group, and a C 3-6 cycloalkyl group, or, R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group, m is an integer from 0 to 4, L is a linker unit, n is from 1 to 10, preferably n is from 1 to 8, more preferably n is from 2 to 8, and most preferably n is from 4 to 8, Pc is the anti-TF antibody according to any one of the above.

[0038] In some embodiments, n is from 0 to 10 (including decimals and integers), preferably 1 to 10, more preferably 1 to 8, or 2 to 8, or 2 to 7, 2 to 6, or 2 to 5, or 2 to 4, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, and in some embodiments, n is an average value, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1,0.

[0039] In some embodiments, R a and R b are the same or different and are each independently selected from a hydrogen atom, a halogen, and a C 1-6 alkyl group, preferably, R a and R b are the same or different and are each independently a hydrogen atom or a C1-6 It is an alkyl group.

[0040] In some embodiments, R 1 C 3-6 It is a cycloalkyl group, preferably R 1 This is a cyclopropyl group.

[0041] In some embodiments, R 2 is a hydrogen atom or halo C 1-6 It is an alkyl group, preferably R 2 This is a hydrogen atom.

[0042] In some embodiments, R 1 and R 2 These, together with the carbon atoms linked to them, form a cyclopropyl group.

[0043] In some embodiments, R 1 C 3-6 It is a cycloalkyl group, R 2 is a hydrogen atom or halo C 1-6 It is an alkyl group, preferably R 1 It is a cyclopropyl group, and R 2 This is a hydrogen atom.

[0044] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) described above, or a pharmaceutically acceptable salt thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -and, Here, L 1 is -(succinimido-3-yl-N)-WC(O)-,-CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)- are selected, where W is C 1-6 Alkylene group and C 1-6 Alkylene-C 3-6 Selected from cycloalkyl groups, where C 1-6 Alkylene group or C1-6 Alkylene-C 3-6 Each cycloalkyl group can be independently and optionally selected as a halogen, hydroxyl group, cyano group, amino group, or C 1-6 Alkyl, halo C 1-6 Alkyl groups, deuterated C 1-6 Alkyl alkyl group, C 1-6 Alkoxy groups and C 3-6 Substituted with one or more substituents selected from cycloalkyl groups, L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, where p 1 These are integers from 1 to 20, L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, where the amino acid residues are amino acids selected from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 4 -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - Selected from chemical bonds, where t is an integer from 1 to 6, R 3 , R 4 and R 5 They are either the same or different, and each is independently a hydrogen atom, C 1-6 Alkyl, halo C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and C 1-6selected from a hydroxyalkyl group, R 6 and R 7 are the same or different and each independently is a hydrogen atom, a halogen, a C 1-6 alkyl group, a haloC 1-6 alkyl group, a deuterated C 1-6 alkyl group, and a C 1-6 hydroxyalkyl group.

[0045] In some embodiments, L 1 is selected from -(succinimid-3-yl-N)-W-C(O)-, where W is a C 1-6 alkylene group and a C​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ In some embodiments, L 4 is selected from -NR 5 (CR 6 R 7 ) t -, where t is an integer from 1 to 6, and R 5 , R 6 and R 7 are the same or different and each independently is a hydrogen atom or a C 1-6 alkyl group. Preferably, L 4 is -NR 5 (CR 6 R 7 )t-, where R 5 , R 6 or R 7 are the same or different and each independently is a hydrogen atom or a C 1-6 alkyl group, and t is 1 or 2.

[0049] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-L-Y-D) described above or a pharmaceutically acceptable salt thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, where L 1 is

Chemical formula

[0050] In some embodiments, an antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above paragraphs, or a pharmaceutically acceptable salt thereof, wherein it is represented by the general formula (Pc-LYD). a -YD) is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof. [ka] Here, Pc is an anti-TF antibody as described in any one of the above items. m is an integer between 0 and 4. n is between 1 and 10. R 1 C 3-6 Cycloalkyl-C 1-6 Alkyl alkyl group or C 3-6 It is a cycloalkyl group, R 2 This is a hydrogen atom, halo C 1-6 Alkyl and C 3-6 Selected from cycloalkyl groups, Or, R 1 and R 2 C 3-6 Forms a cycloalkyl group, W is C 1-6 Alkylene group and C 1-6 Alkylene-C 3-6 Selected from cycloalkyl groups, where C 1-6 Alkylene group and C 1-6 Alkylene-C 3-6Each cycloalkyl group is independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, where p 1 These are integers from 1 to 20, L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, where the amino acid residues are amino acids selected from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R 4 and R 5 This is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups. R 6 and R 7 These elements are either the same or different, and each is independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0051] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, is the antibody-drug conjugate represented by the general formula (Pc-9-A), [ka] Here, n is between 1 and 8. Pc is an anti-TF antibody as described in any one of the above items. Preferably, the Pc is selected from an anti-TF antibody comprising a heavy chain variable region and a light chain variable region as described in any one of the following: a. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, or The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 83, respectively, or The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively. b. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. c. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, respectively. More preferably, the Pc is selected from an anti-TF antibody comprising a heavy chain variable region and a light chain variable region as described in any one of the following: a. The heavy chain variable region is as shown in SEQ ID NO:62, and the light chain variable region is as shown in SEQ ID NO:63, or, The heavy chain variable region is as shown in SEQ ID NO:62, and the light chain variable region is as shown in SEQ ID NO:81, or, The heavy chain variable region is as shown in SEQ ID NO:58, and the light chain variable region is as shown in SEQ ID NO:59, or, The heavy chain variable region is as shown in SEQ ID NO:36, and the light chain variable region is as shown in SEQ ID NO:37. b. The heavy chain variable region is as shown in SEQ ID NO: 52, and the light chain variable region is as shown in SEQ ID NO: 53, or, The heavy chain variable region is as shown in SEQ ID NO:38, and the light chain variable region is as shown in SEQ ID NO:39. c. The heavy chain variable region is as shown in SEQ ID NO:71, and the light chain variable region is as shown in SEQ ID NO:72. Most preferably, the Pc is selected from an antibody comprising a heavy chain and a light chain as described in any one of the following: a. The heavy chain is as shown in SEQ ID NO:69, and the light chain is as shown in SEQ ID NO:70, or The heavy chain is as shown in SEQ ID NO:69, and the light chain is as shown in SEQ ID NO:82, or, The heavy chain is as shown in SEQ ID NO:60, and the light chain is as shown in SEQ ID NO:61. b. The heavy chain is as shown in SEQ ID NO: 56, and the light chain is as shown in SEQ ID NO: 57. c. The heavy chain is as shown in SEQ ID NO:79, and the light chain is as shown in SEQ ID NO:80.

[0052] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, is the antibody-drug conjugate represented by the general formula (Pc-9-A), [ka] Here, n is 1 to 8, preferably 2 to 8, and more preferably 4 to 8. Pc is an anti-TF antibody as described in any one of the above items. Preferably, the Pc is selected from an anti-TF antibody comprising a heavy chain variable region and a light chain variable region as described in any one of the following: a. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, or b. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively, or c. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, respectively, or More preferably, the Pc is selected from an anti-TF antibody comprising a heavy chain variable region and a light chain variable region as described in any one of the following: a. The heavy chain variable region is as shown in SEQ ID NO:62, and the light chain variable region is as shown in SEQ ID NO:63. b. The heavy chain variable region is as shown in SEQ ID NO: 52, and the light chain variable region is as shown in SEQ ID NO: 53. c. The heavy chain variable region is as shown in SEQ ID NO:71, and the light chain variable region is as shown in SEQ ID NO:72. Most preferably, the Pc is selected from an antibody comprising a heavy chain and a light chain as described in any one of the following: a. The heavy chain is as shown in SEQ ID NO:69, and the light chain is as shown in SEQ ID NO:70, or b. The heavy chain is as shown in SEQ ID NO: 56, the light chain is as shown in SEQ ID NO: 57, or, c. The heavy chain is as shown in SEQ ID NO:79, and the light chain is as shown in SEQ ID NO:80.

[0053] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, is the antibody-drug conjugate represented by the general formula (Pc-9-A), [ka] Here, n is 1 to 8, preferably 2 to 8, and more preferably 4 to 8. Pc is an anti-TF antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:40, SEQ ID NO:64, and SEQ ID NO:65, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively.

[0054] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, is the antibody-drug conjugate represented by the general formula (Pc-9-A), [ka] Here, n is 1 to 8, preferably 2 to 8, and more preferably 4 to 8. Pc is an anti-TF antibody containing a heavy chain variable region and a light chain variable region, the heavy chain variable region being as shown in SEQ ID NO:62 and the light chain variable region being as shown in SEQ ID NO:63.

[0055] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, is the antibody-drug conjugate represented by the general formula (Pc-9-A), [ka] Here, n is 1 to 8, preferably 2 to 8, and more preferably 4 to 8. Pc is an anti-TF antibody comprising a heavy chain and a light chain, the heavy chain being as shown in SEQ ID NO:69 and the light chain being as shown in SEQ ID NO:70.

[0056] In some embodiments, the general formula (Pc-L a The present invention further provides a method for producing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in -YD), which, [ka] After Pc reduction, the general formula (L a The compound shown in -YD) is conjugated and reacted with the general formula (Pc-L a The step of obtaining an antibody-drug conjugate shown in -YD) or a pharmaceutically acceptable salt thereof, Here, Pc is an anti-TF antibody as described in any one of the above items. m is an integer between 0 and 4. n is between 1 and 10. R 1 C 3-6 Cycloalkyl-C 1-6 Alkyl alkyl group or C 3-6 It is a cycloalkyl group, R 2 This is a hydrogen atom, halo C 1-6 Alkyl and C 3-6 Selected from cycloalkyl groups, Or, R 1 and R 2 C 3-6 Forms a cycloalkyl group, W is C 1-6 Alkylene group and C 1-6 Alkylene-C 3-6 Selected from cycloalkyl groups, where C 1-6 Alkylene group and C 1-6 Alkylene-C 3-6 Each cycloalkyl group is independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 Selected from C(O)- and chemical bonds, where p 1 These are integers from 1 to 20, L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, where the amino acid residues are amino acids selected from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R 4 and R 5 This is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups. R 6 and R 7 These elements are either the same or different, and each is independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0057] In some embodiments, the present disclosure further provides a method for producing an antibody-drug conjugate represented by general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof, which, [ka] The process includes the step of reducing Pc and then conjugating it with compound 9-A to obtain an antibody-drug conjugate represented by the general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof. Here, n is 1 to 8, preferably 2 to 8, and more preferably 4 to 8. Pc is an anti-TF antibody as described in any one of the above items.

[0058] In some embodiments, the Disclosure further provides a pharmaceutical composition comprising an anti-TF antibody as described in any one of the preceding paragraphs, or a nucleic acid molecule as described above, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding paragraphs, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0059] In some embodiments, the disclosure further provides applications of the anti-TF antibody described in any of the preceding paragraphs, or the nucleic acid molecule described above, or the antibody-drug conjugate described in any of the preceding paragraphs or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above, in the manufacture of drugs for treating TF-mediated diseases or disorders.

[0060] In some embodiments, the disclosure further provides applications of the anti-TF antibody described in any of the preceding paragraphs, or the nucleic acid molecule described above, or the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above, in the manufacture of drugs for treating tumors or cancer.

[0061] In some embodiments, the Disclosure further provides uses of the anti-TF antibodies described in any of the preceding paragraphs, or the nucleic acid molecules described above, or the antibody-drug conjugates or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described above, in the manufacture of drugs for treating tumors or cancer, preferably, said tumors or cancer being selected from breast cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer (including colon cancer and rectal cancer), head and neck cancer and squamous cell carcinoma.

[0062] In some embodiments, the Disclosure further provides a kit comprising an anti-TF antibody as described in any one of the preceding paragraphs, or a nucleic acid molecule as described above, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding paragraphs, or a pharmaceutical composition as described above.

[0063] In some embodiments, an antibody-drug conjugate represented by the general formula (Pc-LYD) described in any one of the preceding paragraphs, or a pharmaceutically acceptable salt thereof, wherein m is not limited to being selected from 0, 1, 2, 3, and 4.

[0064] In some embodiments, the Disclosure further provides a method for preventing or treating a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of an anti-TF antibody as described in any of the preceding paragraphs, or a nucleic acid molecule as described in any of the preceding paragraphs, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding paragraphs, or a pharmaceutical composition as described in any of the preceding paragraphs. In some embodiments, the disease or disorder is a tumor or cancer, preferably a disease or disorder related to TF. In some embodiments, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer (including colon cancer and rectal cancer), head and neck cancer, and squamous cell carcinoma.

[0065] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-TF antibody, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding paragraphs, and one or more pharmaceutically acceptable excipients, diluents or carriers. In some embodiments, the unit dose of the pharmaceutical composition contains 0.1 mg to 3000 mg or 1 mg to 1000 mg of the aforementioned anti-TF antibody or antibody-drug conjugate.

[0066] In another aspect, the Disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, as a drug.

[0067] In another embodiment, the Disclosure provides applications of the antibody-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising them, as drugs for treating TF-mediated diseases or disorders, in some embodiments, the TF-mediated diseases or disorders being TF-high-expression cancers, TF-moderate-expression cancers, or TF-low-expression cancers. In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising them, are used to treat tumors or cancers.

[0068] In another embodiment, the Disclosure further relates to a method for treating and / or preventing a tumor or cancer, the method comprising administering to a subject in need a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, wherein in some embodiments, the tumor or cancer is a cancer associated with high TF expression, a cancer associated with moderate TF expression, or a cancer associated with low TF expression.

[0069] The active compound may be formulated in a form suitable for administration via any appropriate route, and may be in the form of a unit dose or in a form that can be self-administered by a patient in a single dose. The unit dose form of the compound or composition of this disclosure may be a tablet, capsule, cachet, ampoule, powder, granule, lozenge, suppository, regenerated powder or liquid formulation.

[0070] The dose of the compound or composition used in the therapeutic method disclosed herein usually varies depending on the severity of the disease, the subject's body weight, and the relative efficacy of the compound. However, as a general guideline, an appropriate unit dose may range from 0.1 mg to 1000 mg.

[0071] The pharmaceutical compositions of this disclosure may contain one or more excipients in addition to the active compound, the excipients being selected from fillers, diluents, adhesives, wetting agents, disintegrants, and other similar agents. Depending on the administration method, the composition may contain 0.1 to 99% by weight of the active compound.

[0072] The anti-TF antibody according to this disclosure has good affinity for cell surface antigens and good cell endocytosis efficiency, and has little effect on coagulation. The anti-TF antibody-drug conjugate according to this disclosure has a potent tumor suppressor effect and exhibits superior efficacy and lower toxicity and side effects in animals.

[0073] Epitope analysis was performed by FACS binding experiments, and the antibodies in this disclosure bind to TF-specific epitopes. In particular, antibody hu67Y116 binds to different TF epitopes than antibodies in the prior art. [Modes for carrying out the invention]

[0074] term Unless otherwise noted, all technical and scientific terms used herein are consistent with the general understanding of those skilled in the art of the field to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to carry out or test the disclosure, preferred methods and materials are described herein. In describing and asserting this disclosure, the following terms are used based on their definitions.

[0075] When a trade name is used in this disclosure, it is intended to include formulations of the product of the trade name, the pharmaceutical product and the active drug portion of the product of the trade name.

[0076] Unless otherwise stated, terms used in the specification and claims have the following meanings:

[0077] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0078] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and mimetic compounds that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, later modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-serine phosphate. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and α-carbon bonded to an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetic compounds refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0079] In this specification, the term "antibody" is used in its broadest sense and covers a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding moieties), but is not limited to these, as long as they exhibit the desired antigen-binding activity.

[0080] "Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From N to the C-terminus, each heavy chain has one variable region (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant regions (CH1, CH2, and CH3). Similarly, from N to the C-terminus, each light chain has one variable region (VL), also called a variable light chain domain or light chain variable region, followed by one constant light chain domain (also called a light chain constant region or CL).

[0081] The terms “full-length antibody,” “complete antibody,” and “whole antibody” are used synonymously herein and refer to antibodies that have a structure substantially similar to that of a natural antibody, or whose heavy chain contains an Fc region as defined herein.

[0082] "Isolated" antibodies refer to antibodies that have already been isolated from components of their natural environment. In some embodiments of this disclosure, antibodies may be purified to a purity of 90% or more than 99%. In some embodiments, antibodies are purified and measured by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0083] The terms "variable region," "variable area," or "variable domain" refer to regions in the antibody heavy and / or light chain that relate to the antibody-binding antigen. Natural IgG antibodies VH and VL each contain four conservative framework regions (FRs) and three complementary determining regions (CDRs). Here, the terms "complementary determining region" or "CDR" refer to regions in the variable domain that primarily contribute to antigen binding, and "framework" or "FR" refer to variable domain residues other than CDR residues in the variable domain. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino group terminus to the carboxyl group terminus. A single VH or VL is sufficient to confer antigen-binding specificity.

[0084] The amino acid sequence boundaries of CDRs can be determined by various known schemes, such as the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system, the "ABM" numbering system, the "contact" numbering system (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), Front Immunol. 2018 Oct 16;9:2278), and the correspondences between various numbering systems are well known to those skilled in the art, and are exemplified in Table 1 below. [Table 1]

[0085] The term "light chain" includes both a variable region domain (VL) and a constant region domain (CL). The VL is located at the amino group terminus of the light chain. The light chain includes both a κ chain and a λ chain.

[0086] The term "heavy chain" includes a variable region domain VH and three constant region domains CH1, CH2, and CH3. VH is located at the amino group terminus of the heavy chain, and the CH domains are located at the carboxyl group terminus, where CH3 is closest to the carboxyl group terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0087] The term "antibody fragment" refers to a molecule distinct from the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. 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 consisting of antibody fragments.

[0088] The terms “Fc region” or “fragment crystallizable region” are used to define the C-terminal region of an antibody heavy chain, and include both the natural sequence Fc region and modified Fc regions. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226 or from Pro230 to its carboxyl group terminus. The boundaries of the Fc region of an antibody heavy chain can also vary, for example, by the deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or by the deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Therefore, in some embodiments, a complete antibody composition may include an antibody population from which all K447 residues and / or G446+K447 residues have been removed. In some embodiments, a complete antibody composition may include an antibody population from which the K447 residues and / or G446+K447 residues have not been removed. In some embodiments, the complete antibody composition comprises an antibody population consisting of a mixture of antibodies with and without the K447 residue and / or G446+K447 residue. Suitable native sequence Fc regions used in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise specified herein, the numbering scheme for amino acid residues in the Fc region or constant region is based on the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0089] The term "chimeric antibody" refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, but the remaining parts of the heavy chain and / or light chain originate from a different source or species.

[0090] The term "humanized antibody" refers to an antibody that retains the reactivity of a non-human antibody while exhibiting low immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the framework region portion of the constant and variable regions).

[0091] The term "human antibody" is intended to refer to antibodies whose variable and constant regions are human sequences. The term covers modified antibodies derived from human genes but which have sequences that remove, for example, cysteine ​​or glycosylation sites that may reduce possible immunogenicity, increase affinity, or cause undesirable folding. The term covers these antibodies produced recombinantly in non-human cells (which may confer glycosylation that does not possess human cell characteristics). The term also covers antibodies produced in genetically modified mice containing some or all human immunoglobulin heavy and light chain loci. The definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0092] The term "affinity-matured" antibody refers to an antibody whose affinity for an antigen is improved compared to the parent antibody due to one or more modifications in one or more CDRs of the antibody. In some embodiments, affinity-matured antibodies have nanomolar or picomolar levels of affinity for the target antigen. Affinity-matured antibodies can be produced by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. The following literature describes random mutagenesis of CDRs and / or framework residues: Barbas et al., PNAS, 91:3809-3813 (1994), Schier et al., Gene 169:147-155 (1995), Yelton et al., J.Immunol. 155:1994-2004 (1995), Jackson et al., J.Immunol. 154(7):3310-9 (1995), and Hawkins et al., J.Mol.Biol. 226:889-896 (1992).

[0093] The term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, meaning that, apart from naturally occurring mutations that may be present in small amounts, the amino acid sequences of the antibody molecules within that population are identical. In contrast, polyclonal antibody preparations typically contain multiple different antibodies, each having a different amino acid sequence in its variable domain, and they typically exhibit specificity for different epitopes. "Monoclonal" describes a characteristic of antibodies obtained from a substantially homogeneous population and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies may be produced by hybridoma methods as described by Kohler et al., (1975) Nature 256:495, or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Alternatively, monoclonal antibodies may be isolated from a phage antibody library using the techniques described in Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J.Mol.Biol.222:581-597. Alternatively, monoclonal antibodies may be produced by referring to Presta (2005) J.Allergy Clin.Immunol.116:731, or by genetically modified animal methods containing all or some human immunoglobulin loci.

[0094] The term "antigen" refers to a molecule or part of a molecule that can be selectively bound by an antibody and, when used in an animal, can produce an antibody capable of binding to that antigen. An antigen may have one or more epitopes that can interact with different antibodies.

[0095] The term "epitope" refers to a region in an antigen that can specifically bind to an antibody. Epitopes may be formed by a continuous amino acid sequence (linear epitope) or by a discontinuous sequence of amino acids (conformational epitope), and may be spatially close due to, for example, antigen folding (i.e., tertiary folding of the antigen due to protein properties). The difference between conformational epitopes and linear epitopes is that in the presence of a denaturing solvent, the binding of the antibody to the conformational epitope is lost. Epitopes contain 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.

[0096] Screening for antibodies that bind to a specific epitope (i.e., those antibodies that bind to the same epitope) may be carried out by methods well known in this field, including, but not limited to, alanine scanning, peptide blotting (see Meth.Mol.Biol.248(2004)443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot.Sci.9(2000)487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0097] An antibody that binds to the same epitope as a reference antibody or an antibody that binds competitively with a reference antibody refers to an antibody that blocks the binding of the reference antibody to an antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by 50% or more compared to the reference antibody. Further, for example, to measure whether a test antibody binds to the same epitope as a reference antibody, under saturation conditions, the binding of the reference antibody to the antigen is allowed. After removing the excess reference antibody, the antigen-binding ability of the test antibody is evaluated. To confirm whether the test antibody binds to the same epitope or its binding is hindered due to steric reasons, common experiments (e.g., binding analysis using peptide mutations and ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art) can be used. Such assays need to be performed in two setups, i.e., in a state where both antibodies are saturated antibodies. If in both setups only the first (saturated) antibody can bind to the antigen, it can be concluded that the test antibody and the reference antibody compete for binding to the antigen.

[0098] In some embodiments, as measured by a competition binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495 - 1502), if an excess of one antibody by 1-fold, 5-fold, 10-fold, 20-fold or 100-fold inhibits the binding of another antibody by at least 50%, at least 75%, at least 90% or even 99% or more, the two antibodies are considered to bind to the same or overlapping epitopes.

[0099] In some embodiments, for those amino acid mutations in an antigen that can reduce or eliminate the binding of one antibody, if substantially all of them can reduce or eliminate the binding of another antibody, the two antibodies are considered to bind to the "same epitope", and if only some of them can reduce or eliminate the binding of another antibody, the two antibodies are considered to have "overlapping epitopes".

[0100] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y may typically be expressed as the equilibrium dissociation constant (KD). Affinity may be measured by conventional methods known in the art, including those described herein.

[0101] As used herein, the terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured by methods well known in the art. For example, a biosensing system can be used to systematically measure surface plasmon resonance or to measure affinity in solution by solution equilibrium titration assay (SET).

[0102] The terms "specifically binds", "specific binding" or "binds" refer to an antibody binding to an antigen or an epitope within the antigen with a higher affinity than to other antigens or epitopes. Typically, an antibody binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 1×10 -7 M or less (e.g., about 1×10 -8 M or less, about 1×10 -9 M or less). In some embodiments, the KD of the binding between an antibody and an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). KD can be measured by methods known in the art, such as BIACORE <~ (登録商標)Measurement is performed by surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or an epitope within an antigen may cross-react to other relevant antigens, for example, to relevant antigens from other species (homologs) (e.g., humans or monkeys, e.g., cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp)) or marmosets (Callithrix jacchus) (commonmarmoset, marmoset).

[0103] The terms "anti-TF antibody" and "antibody that binds to TF" refer to antibodies that can bind to TF with sufficient affinity. In some examples, the antibodies that bind to the anti-TF antibody are <approximately 1 μM, <approximately 100 nM, <approximately 10 nM, <approximately 1 nM, <approximately 0.1 nM, <approximately 0.01 nM, or <approximately 0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -9 M, for example, 10 -9 It has an equilibrium dissociation constant (KD) of M or less. In some examples, the anti-TF antibody binds to a conserved TF epitope in TF from different species.

[0104] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages). These cytotoxic effector cells then specifically bind to antigen-carrying target cells, subsequently killing them with cytotoxins. Antibodies "arm" cytotoxic cells and are necessary for this killing action. NK cells, the main cells mediating ADCC, express only FcγRIII, while mononuclear cells express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, *Annual Commentary on Immunology* (Annu. Rev. Immunol.) 9:457-92 (1991). To evaluate the ADCC activity of the target molecule, in vitro ADCC analysis may be performed, and the analytical method is described, for example, in U.S. Patent No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Selectively, the ADCC activity of the target antibody can also be evaluated in vivo, for example, in animal models (disclosed, for example, in Clynes et al., (USA) 95:652-656 (1998)).

[0105] "Antibody-dependent cell phagocytosis" ("ADCP") refers to the mechanism by which phagocytic cells (e.g., macrophages or dendritic cells) remove antibody-coated target cells through internalization.

[0106] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death, where the Fc effector domain of a target-binding antibody binds to and activates complement component C1q, subsequently activating the complement gasket and causing target cell death. Complement activation can also lead to the deposition of complement components on the target cell surface, and these complement components promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0107] The terms “nucleic acid” and “polynucleotide” are used synonymously herein and refer to single-stranded or double-stranded deoxyribonucleotides or ribonucleotides and polymers thereof. The terms cover nucleic acids containing or linked to known nucleotide analogs or modified skeletal residues, which are synthetic, natural, and unnatural, possess similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated” nucleic acids refer to nucleic acid molecules isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which typically contain such nucleic acid molecules, but which are extrachromosomal or located at chromosomal positions different from their natural chromosomal locations. "Nucleic acid encoding an anti-TF antibody" refers to one or more nucleic acid molecules that encode the antibody heavy chain and light chain (or fragments thereof).

[0108] Unless otherwise specified, a given nucleic acid sequence implicitly includes its complementary sequences and explicitly indicated sequences, as well as its conservatively modified variants (e.g., degenerate codon substitutions). Specifically, as described in detail below, degenerate codon substitutions can be obtained by producing the following sequences, in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0109] The terms “polypeptide” and “protein” are used synonymously herein and refer to polymers of amino acid residues. The terms are applicable to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, and are applicable to both natural and non-natural amino acid polymers.

[0110] The term "naked antibody" refers to an antibody that is not conjugated with a heterogeneous module (e.g., a cytotoxic module) or a radioactive marker. Naked antibodies can be present in pharmaceutical formulations.

[0111] The terms "identity," "sequence identity," or "amino acid sequence identity" refer to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when optimal alignment is performed on two sequences (by introducing gaps as necessary to obtain the maximum sequence identity percentage, and by not considering any conservative substitutions as part of sequence identity). To measure the sequence identity percentage, alignment can be performed by various methods well known in the art, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for the measurement alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0112] The terms “conservatively modified variant” or “conservative substitution” refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, skeletal configuration and rigidity) that can usually bring about such a change without altering the protein’s biological activity. Those skilled in the art generally know that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224 (4th Ed.)). When the term “conservatively modified variant” is applied to a nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or substantially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for substantially the same sequence. Due to the degeneracy of the genetic code, any given protein can be coded by multiple nucleic acids that have the same function. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at each position of alanine where a codon is specified, the codon can be replaced with any of the corresponding codons without changing the polypeptide it encodes. Such nucleic acid mutations are “silencing mutations,” and they are one of the conservatively modified mutations. In this specification, each nucleic acid sequence encoding a polypeptide also describes each possible silencing mutation of the nucleic acid. Those skilled in the art will recognize that each codon in the nucleic acid (except AUG—usually the sole codon of methionine and TGG—usually the sole codon of tryptophan) can be modified to produce molecules with the same function. Therefore, each of the sequences implicitly contains all the silencing mutations of the nucleic acid encoding the polypeptide.

[0113] The terms “expression vector” or “expression construct” refer to a vector containing nucleic acid sequences suitable for transformation into host cells and which dictate and / or control the expression of one or more heterogeneous coding regions that are manipulatively ligated to it (in conjunction with the host cell). An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, where introns are present, affect the RNA splicing of the manipulatively ligated coding region.

[0114] As used herein, “operably ligated” means a relationship that enables the components to which the term applies to perform their inherent function under appropriate conditions. For example, in an expression vector, a regulatory sequence that is “operably ligated” to a protein-coding sequence is ligated thereto to achieve the expression of the protein-coding sequence under conditions compatible with the transcriptional activity of the regulatory sequence.

[0115] The terms “host cell,” “host cell line,” and “host cell culture” are used synonymously and refer to cells into which exogenous nucleic acids have been introduced, and the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which, without regard to passage number, include primary transformed cells and their offspring. Offspring may not be exactly the same as the parent cells in terms of nucleic acid material and may contain mutations. This specification includes mutant offspring having the same function or biological activity as those screened or selected from the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells include 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, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei.

[0116] As used in this disclosure, the terms “cells,” “cell lines,” and “cell cultures” are used synonymously and include the offspring of such cells. Therefore, “transformed organisms” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of passages. Furthermore, it should be understood that not all offspring will have exactly the same DNA content as a result of sense or nonsense mutations, including mutant offspring that have the same function or biological activity as the original transformed cells from which they were screened.

[0117] Conventional techniques for producing and purifying antibodies and antigen-binding fragments are well known, for example, in Chapters 5-8 and 15 of the Cold Spring Harbor Guidelines for Antibody Experimental Techniques. The antibodies or antigen-binding fragments described in the disclosure are obtained by adding one or more human FR regions to a non-human CDR region using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 012441351, by matching them with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software.

[0118] The engineered antibodies or antigen-binding fragments of this disclosure can be manufactured and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned into expression vectors and recombinant. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred conventional technique, mammalian expression systems induce antibody glycosylation, particularly at the N-terminal region of the Fc domain. Stable clones are obtained by expressing antibodies that specifically bind to human TF. Positive clones are expanded and cultured in bioreactor culture medium to produce antibodies. The culture medium secreted by the antibodies can be purified by conventional techniques, for example, by purification using an A or G Sepharose FF column. Nonspecific binding components are washed away. The bound antibodies are then eluted by pH gradient elution, and antibody fragments are detected and collected by SDS-PAGE. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, e.g., molecular sieves, ion exchange. The resulting product must be immediately frozen, for example, at -70°C, or freeze-dried.

[0119] "Isolated" refers to a purified state, and in this case, the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Usually, the term "isolated" does not imply the complete absence of these materials, or the absence of water, buffer, or salt, as long as they do not significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0120] The term "drug" refers to a chemical substance that can change or specify the physiological function or pathological state of a living body and can be used for the prevention, diagnosis, and treatment of diseases. Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons. Poisons refer to chemical substances that can cause harmful effects on the living body even in small amounts and damage health. Any drug can cause toxic reactions if overdosed.

[0121] The term "cytotoxic drug" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic drugs can, in principle, kill tumor cells at a sufficiently high concentration, but due to lack of specificity, they kill tumor cells and at the same time induce apoptosis of normal cells, causing serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, radioisotopes (e.g., radioisotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0122] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group, which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), and more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, and 5-methylhexyl group. This includes 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof.More preferably, the lower alkyl group contains 1 to 6 carbon atoms. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, and 2,3-dimethylbutyl group. The alkyl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linking point, and the substituent is preferably one or more substituents independently selected optionally from a D atom, halogen, alkyl group, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0123] The term "alkoxy group" refers to -O-(alkyl), where the definition of alkyl group is as described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy groups. Alkoxy groups may be optionally substituted or unsubstituted, and if substituted, the substituent is preferably one or more of the following groups, which are independently selected from a D atom, halogen, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0124] The term "alkylene group" refers to a saturated linear or branched aliphatic hydrocarbon group, which is a residue derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, which is a linear or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12), and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-restrictive examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), and 1,4-butylene (-CH2CH2CH2CH2-). The alkylene group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linkage point, and the substituent is preferably one or more substituents independently selected from alkenyl groups, alkynyl groups, alkoxy groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0125] The term "alkenyl group" refers to an alkyl group compound in which the molecule contains at least one carbon-carbon double bond, where the definition of alkyl group is as described above. Preferably, it is an alkenyl group containing 2 to 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12), and more preferably, an alkenyl group containing 2 to 6 carbon atoms. The alkenyl group may be substituted or unsubstituted, and if substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0126] The term "alkynyl group" refers to an alkyl group compound in which the molecule contains at least one carbon-carbon triple bond, where the definition of alkyl group is as described above. Preferably, it is an alkynyl group containing 2 to 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12), and more preferably, an alkynyl group containing 2 to 6 carbon atoms. The alkynyl group may be substituted or unsubstituted, and if substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl groups, alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0127] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, where the ring of the cycloalkyl group contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms (e.g., C 3-6 (Cycloalkyl groups). Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups, while polycyclic cycloalkyl groups include cycloalkyl groups of spiro rings, fused rings, and crosslinked rings.

[0128] The term "spirocycloalkyl group" refers to a 5-20 membered polycyclic group in which monocyclic rings share one carbon atom (called a spiro atom), and which may contain one or more double bonds. Preferably, it is 6-14 membered (e.g., 6, 7, 8, 9, 10, 11, 12, 13, and 14), and more preferably 7-10 membered (e.g., 7, 8, 9, or 10). Depending on the number of shared spiro atoms between the rings, spirocycloalkyl groups are classified into monospirocycloalkyl groups, bisspirocycloalkyl groups, or polyspirocycloalkyl groups, preferably monospirocycloalkyl groups and bisspirocycloalkyl groups. More preferably, they are 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, and 6-membered / 6-membered monospirocycloalkyl groups. Non-limiting examples of spirocycloalkyl groups are: [ka] Includes.

[0129] The term "condensed cycloalkyl group" refers to a 5-20 member all-carbon polycyclic group in which each ring in the system shares one adjacent pair of carbon atoms with the other rings in the system, where one or more rings may contain one or more double bonds. Preferably, they are 6-14 member (e.g., 6, 7, 8, 9, 10, 11, 12, 13, and 14), and more preferably 7-10 member (e.g., 7, 8, 9, or 10). Depending on the number of rings, they may be classified as bicyclic, tricyclic, tetracyclic, or polycyclic condensed cycloalkyl groups, preferably bicyclic or tricyclic, and more preferably 3-member / 4-member, 3-member / 5-member, 3-member / 6-member, 4-member / 4-member, 4-member / 5-member, 4-member / 6-member, 5-member / 4-member, 5-member / 5-member, 5-member / 6-member, 6-member / 3-member, 6-member / 4-member, 6-member / 5-member, and 6-member / 6-member bicyclic cycloalkyl groups. Non-limiting examples of condensed cycloalkyl groups are: [ka] Includes.

[0130] A "crosslinked cycloalkyl group" refers to a 5-20 membered all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly bonded, and which may contain one or more double bonds. Preferably, it is 6-14 membered (e.g., 6, 7, 8, 9, 10, 11, 12, 13, and 14), and more preferably 7-10 membered (e.g., 7, 8, 9, or 10). Depending on the number of rings, crosslinked cycloalkyl groups may be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of crosslinked cycloalkyl groups include: [ka]

[0131] The cycloalkyl ring mentioned above includes cycloalkyl groups (including monocyclic, spirocyclic, fused, and crosslinked rings) that are fused to an aryl group, a heteroaryl group, or a heterocycloalkyl ring, where the ring linked to the basic skeleton is a cycloalkyl group, and non-limiting examples include an indanyl group, a tetrahydronaphthyl group, and a benzocycloheptyl group, preferably an indanyl group and a tetrahydronaphthyl group.

[0132] The cycloalkyl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linking point, and the substituent is preferably one or more substituents independently selected optionally from halogen, alkyl, alkoxy group, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0133] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent that contains 3 to 20 ring atoms, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O)2, but do not contain -OO-, -OS- or -SS- ring portions, and the other ring atoms are carbon. Preferably, the ring group contains 3 to 12 ring atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), where 1 to 4 ring atoms (e.g., 1, 2, 3, and 4) are heteroatoms; more preferably, it contains 3 to 8 ring atoms (e.g., 3, 4, 5, 6, 7, and 8), where 1 to 3 ring atoms are heteroatoms (e.g., 1, 2, and 3); more preferably, it contains 3 to 6 ring atoms, where 1 to 3 ring atoms are heteroatoms; most preferably, it contains 5 or 6 ring atoms, where 1 to 3 ring atoms are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl groups, tetrahydropyranyl groups, 1,2,3,6-tetrahydropyridyl groups, piperidine groups, piperazinyl groups, morpholinyl groups, thiomorpholinyl groups, and homopiperazinyl groups. Polycyclic heterocyclyl groups include spiro rings, fused rings, and bridging ring heterocyclyl groups.

[0134] The term "spiroheterocyclyl group" refers to a 5-20 membered polycyclic heterocyclyl group in which two rings share one atom (called a spiro atom), where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O), or S(O)2, and the remaining ring atoms are carbon. It may also contain one or more double bonds. Preferably, it is 6-14 membered, and more preferably 7-10 membered (e.g., 7, 8, 9, or 10 membered). Depending on the number of shared spiro atoms between the rings, spiroheterocyclyl groups are classified into monospiroheterocyclyl groups, bisspiroheterocyclyl groups, or polyspiroheterocyclyl groups, preferably monospiroheterocyclyl groups and bisspiroheterocyclyl groups. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, 5-membered / 6-membered, or 6-membered / 6-membered monospiroheterocyclyl group. Non-restrictive examples of spiroheterocyryl groups are: [ka] Includes.

[0135] The term "condensed heterocyclyl group" refers to a 5- to 20-membered polycyclic heterocyclyl group in which each ring in the system shares one pair of adjacent atoms with the other rings in the system, and one or more rings may contain one or more double bonds, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O)2, and the remaining ring atoms are carbon. Preferably, it is 6- to 14 members, more preferably 7- to 10 members (e.g., 7, 8, 9 or 10 members). Depending on the number of rings that make up the group, it may be classified into bicyclic, tricyclic, tetracyclic, or polycyclic condensed heterocyclyl groups, preferably bicyclic or tricyclic, and more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, and 6-membered / 6-membered bicyclic condensed heterocyclyl groups. Non-limiting examples of condensed heterocyclyl groups are: [ka] Includes.

[0136] The term "bridged heterocyclyl group" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two atoms that are not directly linked, and which may contain one or more double bonds, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O)2, and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of rings that make up the group, it may be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclyl group, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl groups are: [ka] Includes.

[0137] The heterocyclyl ring described above includes heterocyclyl groups (including monocyclic, spiroheterocyclic, fused heterocyclic, and crosslinked heterocyclic groups) that are fused to an aryl group, heteroaryl group, or cycloalkyl group ring, where the ring linked to the basic skeleton is a heterocyclyl group, and non-limiting examples thereof are: [ka] This includes, among others.

[0138] The heterocyclyl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linkage point, and the substituent is preferably one or more substituents independently and optionally selected from halogen, alkyl, alkoxy group, haloalkyl, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0139] The term "aryl group" refers to a 6-14 membered all-carbon monocyclic or fused polycyclic (a fused polycyclic is a ring that shares adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6-10 membered, such as a phenyl group and a naphthyl group. The aryl group ring includes those formed by the condensation of the aryl group ring described above with a heteroaryl group, a heterocyclyl group, or a cycloalkyl group, where the ring linked to the basic skeleton is an aryl group ring, and non-limiting examples include: [ka] Includes.

[0140] The aryl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linkage point, and the substituent is preferably one or more substituents independently selected optionally from halogen, alkyl, alkoxy group, haloalkyl, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl, heterocyclyl group, aryl group, and heteroaryl group.

[0141] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms (e.g., 1, 2, 3, and 4) and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10 membered (e.g., 5, 6, 7, 8, 9, or 10 membered), more preferably 5 or 6 membered, and includes, for example, a furyl group, thienyl group, pyridyl group, pyrrolyl group, N-alkylpyrrolyl group, pyrimidinyl group, pyrazinyl group, pyridadinyl group, imidazolyl group, pyrazolyl group, triazolyl group, and tetraazolyl group. The ring of the heteroaryl group includes those in which the heteroaryl group described above is condensed with an aryl group, heterocyclyl group, or cycloalkyl group, where the ring linked to the basic skeleton is the ring of the heteroaryl group, and non-limiting examples are: [ka] Includes.

[0142] The heteroaryl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linkage point, and the substituent is preferably one or more substituents independently and optionally selected from halogen, alkyl, alkoxy group, haloalkyl, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group and heteroaryl group.

[0143] The above-mentioned cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups include residues derived by removing one hydrogen atom from a parent ring atom, or residues derived by removing two hydrogen atoms from the same ring atom or two different ring atoms of the parent, i.e., "divalent cycloalkyl groups," "divalent heterocyclyl groups," "arylene groups," or "heteroarylene groups."

[0144] The term "amino protecting group" refers to a group that can be easily removed to protect an amino group so that it does not change even if reactions occur at other parts of the molecule. Non-limiting examples include (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tert-butoxycarbonyl (Boc), acetyl, benzyl, allyl, p-methoxybenzyl, and tert-butyldimethylsilyl (TBS). These groups can be optionally substituted with 1 to 3 substituents selected from halogens, alkoxy groups, or nitro groups.

[0145] The term "hydroxy protecting group" usually refers to a hydroxy derivative on which the reaction proceeds on another functional group of the compound, used to block or protect the hydroxyl group. For example, preferably, the hydroxy protecting group is (C 1-10 It may also be an alkyl or aryl)3-silyl group, for example, a triethylsilyl group, a triisopropylsilyl group, a tert-butyldimethylsilyl group (TBS), and a tert-butyldiphenylsilyl group, C 1-10 They may be alkyl groups or substituted alkyl groups, preferably alkyl groups substituted with alkoxy or aryl groups, and more preferably C 1-6 C substituted with an alkoxy group 1-6 C substituted with alkyl or phenyl groups 1-6 It is an alkyl group, most preferably C 1-4 C substituted with an alkoxy group 1-4 These are alkyl groups, such as methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, and 2-tetrahydropyranyl (THP), (C1-10 It may be an alkyl or aryl)acyl group, for example, a formyl group, an acetyl group, a benzoyl group and a p-nitrobenzoyl group, (C 1-6 It may also be an alkyl or 6-10 membered aryl)sulfonyl group, (C 1-6 It may also be an alkoxy or a 6-10 membered aryloxy)carbonyl group.

[0146] The term "cycloalkyloxy group" refers to a cycloalkyl-O- group, where cycloalkyl is defined as described above.

[0147] The term "heterocyclyloxy group" refers to a heterocyclyl-O-, where the heterocyclyl group is as described above.

[0148] The term "alkylthio group" refers to alkyl-S-, where alkyl is as described above.

[0149] The term "haloalkyl group" refers to a group in which an alkyl group is substituted with one or more halogens, where the alkyl group is as described above.

[0150] The term "haloalkoxy group" refers to a group in which an alkoxy group is substituted with one or more halogens, where the alkoxy group is as described above.

[0151] The term "deuterated alkyl group" refers to a group in which an alkyl group is substituted with one or more deuterium atoms, where the alkyl group is as described above.

[0152] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxyl groups, where the alkyl group is as defined above.

[0153] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0154] The term "hydroxyl group" refers to the -OH group.

[0155] The term "mercapto group" refers to the -SH group.

[0156] The term "amino group" refers to -NH2.

[0157] The term "cyano group" refers to -CN.

[0158] The term "nitro group" refers to -NO2.

[0159] The term "oxo group" refers to the group "=O".

[0160] The term "carbonyl group" refers to C=O.

[0161] The term "carboxyl group" refers to -C(O)OH.

[0162] The term "carboxylic acid ester group" refers to -C(O)O(alkyl), -C(O)O(cycloalkyl), (alkyl)C(O)O-, or (cycloalkyl)C(O)O-, where alkyl groups and cycloalkyl groups are as described above.

[0163] The term "antibody-drug conjugate (ADC)" refers to a conjugate unit that links an antibody to a bioactive cytotoxin or a small molecule drug with cytotoxic activity.

[0164] The antibodies or antibody fragments described herein can be conjugated to effector molecules in any manner. For example, the antibodies or antibody fragments can be conjugated to cytotoxic drugs chemically or recombinantly. Chemical methods for producing fusions or conjugates are known in the art and can be used for the production of immunoconjugates. Methods for conjugating antibodies or antibody fragments with drugs must be able to link the antibody and the cytotoxic drug without inhibiting the antibody's ability to bind to the target molecule.

[0165] In one embodiment, both the antibody and the cytotoxic drug are proteins and can be conjugated using techniques well known in the art. Hundreds of crosslinking agents are disclosed in the art, and they can conjugate two proteins. The crosslinking agent is selected based on whether it is usable with the antibody or cytotoxic drug or based on the inserted reactive functional group. If a reactive group is not present, a photoactivatable crosslinking agent can be used. In some cases, a spacer is required between the antibody and the cytotoxic drug. Crosslinking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyladipineimide, and bis(diazonium benzidine), and heterobifunctional agents: m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.

[0166] Crosslinking agents that can be used to conjugate effector molecules to antibody fragments include, for example, TPCH (S-(2-thiopyridyl)-L-cysteine ​​hydrazide) and TPMPH (S-(2-thiopyridyl)mercaptopropionyl hydrazide). TPCH and TPMPH react with the carbohydrate portion of the glycoprotein, which has been previously oxidized by mild periodate treatment, thereby forming a hydrazone bond between the hydrazide portion of the crosslinking agent and the aldehyde from the periodate. Heterobifunctional crosslinking agents GMBS (N-(γ-maleimidobutyryloxy)succinimidile) and SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane) react with primary amines, thereby introducing maleimide groups into the components. These maleimide groups can then react with sulfhydryl groups, which may be introduced by crosslinking agents, in other components, thereby forming stable thioether bonds between the components. If steric hindrance between components interferes with the activity of any component, a crosslinking agent may be used to introduce a long spacer arm between the components, such as n-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Therefore, many suitable crosslinking agents exist, and they are selected based on their availability and their impact on the optimal immunoconjugate yield.

[0167] The term “drug loading capacity” is also known as the drug-to-antibody ratio (DAR), and is the average number of drugs conjugated to each antibody in an ADC. It may be, for example, a range in which about 1 to about 10 drugs are conjugated per antibody, and in some examples, a range in which about 1 to about 8 drugs are conjugated per antibody, preferably selected from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 4-8, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug loading capacity may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula for the ADC of this disclosure includes the set of antibody-drug conjugates within the aforementioned ranges. In embodiments of this disclosure, the drug loading capacity may be represented as n, and is a decimal or an integer. Drug loading capacity can be measured using conventional methods such as UV / visible spectrophotometric analysis, mass spectrometry, ELISA, and HPLC.

[0168] The terms "linker unit," "linking fragment," or "linking unit" refer to a chemical structural fragment or linkage in which one end is linked to an antibody or its antigen-binding fragment, and the other end is linked to a drug, and which may be linked to another linker before being linked to the drug.

[0169] The term “linker” may include one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those derived from conjugates with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodoacetyl)aminobenzoate ("SIAB"). The linker may include a stretcher, a spacer, and an amino acid unit, and can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates the release of the drug in the cell. For example, an acid-unstable linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photo-unstable linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020).

[0170] The linker assembly includes, but is not limited to, the following: MC=6-maleimidocaproyl has the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in protease-cleavable linkers), Citrulline = 2-amino-5-ureidopentanoic acid, The PAB group is a p-aminobenzyloxycarbonyl group (an example of a "self-sacrificing" linker assembly), and its structure is as follows: [ka] Me-Val-Cit=N-methyl-valine-citrulline (where the linker peptide bond is modified so as not to be cleaved by cathepsin B), MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (adheres to antibody cysteine), SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate, SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, IT = Iminothiolan, In one embodiment of the present disclosure, a cytotoxic drug is conjugated to a mercapto group of an antibody via a linking unit.

[0171] The ligand-drug conjugate load can be controlled using the following non-limiting methods: (1) Controlling the molar ratio of the compound binding portion to the monoclonal antibody, (2) Controlling the reaction time and temperature, (3) Includes selecting different reaction reagents.

[0172] The terms “optionally” or “optionally” mean that the events or environments described later may occur, but are not required to occur, and the description includes cases where the events or environments occur or do not occur. For example, “C where optional halogen or cyano group substitution is required.” 1-6 The term "alkyl group" means that a halogen or cyano group may be present, but is not necessarily present, and this description includes cases where the alkyl group is substituted with a halogen or cyano group, and cases where the alkyl group is not substituted with a halogen or cyano group.

[0173] The term "substituted" refers to the substitution of one or more hydrogen atoms in a group, preferably 1 to 6, and more preferably 1 to 3, with a corresponding number of substituents, independently of each other. Those skilled in the art can determine possible or impossible substitutions with little effort (experimentally or theoretically). For example, an amino or hydroxyl group with free hydrogen can become unstable if bonded to a carbon atom having an unsaturated (e.g., olefin) bond.

[0174] In this disclosure, the antibody-drug conjugate concentration is calculated as the protein concentration, i.e., the concentration of the antibody portion in the antibody-drug conjugate.

[0175] With respect to drugs or pharmacological activators, the "therapeutic effective dose" refers to the amount of drug or agent that is non-toxic but sufficient to achieve the desired therapeutic effect. Determining the effective dose varies from person to person and depends on the age and general condition of the receptor, as well as on the specific active substance. The appropriate effective dose in each case can be determined by a person skilled in the art based on conventional experiments.

[0176] The compounds and intermediates of this disclosure may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms “tautomer” or “tautomer form” refer to structural isomers of different energies that can be converted to one another by a low-energy barrier. For example, proton tautomers (also called proton-transfer tautomers) include tautomerization by proton transfer, e.g., keto-enol and imine-enamine, lactam-lactim isomerization. An example of lactam-lactim equilibrium is shown between A and B below. [ka]

[0177] All compounds in this disclosure may be depicted as either type A or type B. All tautomer forms are within the scope of this disclosure. The nomenclature of the compounds does not exclude any tautomers.

[0178] As used herein, the term “pharmaceutically acceptable” means that these compounds, materials, compositions and / or dosage forms are, to the extent of reasonable medical judgment, suitable for contact with the tissues of a subject, have a reasonable benefit-risk ratio, and are effective for the desired use without excessive toxicity, irritation, allergic reactions or other problems or complications.

[0179] The manufacture of conventional pharmaceutical compositions is based on the Chinese Pharmacopoeia.

[0180] As used herein, the singular forms “one,” “one kind,” and “the” include multiple references unless explicitly stated in the context, and vice versa.

[0181] "Approximately" means within the acceptable margin of error of a particular value as determined by those skilled in the art, which is in part determined by how the value is measured or determined, i.e., limited by the measuring system. In the context of a particular measurement, result, or embodiment, unless otherwise specified elsewhere in the example or specification, "approximately" means within one of the standard deviations according to the convention of the art.

[0182] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to a component in a drug formulation that is different from the active ingredient and is non-toxic to the subject. Pharmacochemically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0183] The terms “subject” or “individual” include humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), e.g., non-human primates (e.g., cynomolgus macaques), sheep, dogs, cattle, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms “patient” or “subject” can be used synonymously herein. As used herein, the terms “cynomolgus macaque (cyno)” ​​or “cynomolgus macaque (cynomolgus)” refer to the cynomolgus macaque (Macaca fascicularis). In some embodiments, the individual or subject is human.

[0184] The term "excipient" refers to additives in drug formulations other than the active ingredient, and is also called an adjuvant. For example, adhesives, fillers, disintegrants, and lubricants in tablets, the matrix components in semi-solid ointments and creams, and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid formulations can all be called excipients.

[0185] The term "diluent," also known as a filler, primarily serves to increase the weight and volume of tablets. Adding diluents not only ensures a consistent volume but also reduces variations in the dosage of the main component and improves the compressibility of the drug. If the drug in the tablet contains oily components, an absorbent is needed to absorb the oil and dry it, making it easier to form into a tablet. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0186] The term "pharmaceutical composition" means a mixture of one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components, and other components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to exert biological activity by facilitating administration to a living organism and facilitating the absorption of the active ingredient.

[0187] The pharmaceutical composition may be in the form of a sterile aqueous solution for injection. Acceptable vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injection formulation may also be a sterile oil-in-water microemulsion for injection in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and treated to form a microemulsion. The injection solution or microemulsion can be injected into the bloodstream of the subject by local bolus injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of this disclosure. A continuous intravenous delivery device can be used to maintain such a constant concentration. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.

[0188] The pharmaceutical composition may be in the form of a sterile aqueous or oily suspension for intramuscular and subcutaneous administration. The suspension can be prepared using the above-mentioned suitable dispersants or wetting agents and suspending agents according to known techniques. The sterile injection formulation may also be a sterile injection solution or suspension prepared in a non-toxic, parenterally permissible diluent or solvent, such as a solution prepared in 1,3-butanediol. Sterile non-volatile oils can be conveniently used as solvents or suspension media. Any compounded non-volatile oil, including synthetic monoglycerides or diglycerides, can be used for this purpose. Fatty acids, such as oleic acid, can also be used in the preparation of injections.

[0189] When applied to animals, humans, experimental subjects, cells, tissues, organs, or bodily fluids, "administration" or "giving" refers to contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or bodily fluid.

[0190] The term "sample" refers to a collection of bodily fluids, cells, or tissues similar to those isolated from a subject, and to bodily fluids, cells, or tissues present in the body of a subject. Exemplary samples include biological fluids, such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions from secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, bodily fluids from the abdomen and other body cavities, bodily fluids collected by bronchoalveolar perfusion, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ culture media (including conditioned media for cells or organs), perfusion fluids, tissue biopsy specimens, needle punctures, surgically excised tissues, organ cultures, or cell cultures.

[0191] "Pharmacologically acceptable salt" or "pharmaceutically acceptable salt" means a salt of the antibody-drug conjugate of the present disclosure, or a salt of a compound described herein, such salts are safe and effective when used in a mammalian body and have the desired biological activity, and the antibody-drug conjugate of the present disclosure contains at least one amino group and can therefore form salts with an acid.

[0192] "Treatment" (or "treat") (and its grammatical variations) refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for preventive purposes or during the course of a clinical condition. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, symptom reduction, reduction / decreasation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of this disclosure are used to delay disease development or slow disease progression.

[0193] "Effective dose" generally refers to an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or their potential causes, prevent the onset of symptoms and / or their potential causes, and / or improve or repair damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, the effective dose is a therapeutic effective dose or a preventive effective dose. "Therapeutic effective dose" refers to an amount sufficient to treat a disease state or symptoms, in particular a condition or symptoms associated with the disease state, or to prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptoms associated with the disease in any way. "Preventive effective dose" refers to an amount that, when administered to a subject, has a predetermined preventive effect, e.g., prevention or delay of the onset (or recurrence) of the disease state, or reduction of the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete treatment or prevention does not necessarily occur after a single dose, and in some cases, after a series of doses. Therefore, a therapeutic or preventive effective dose may be administered in one or more doses. The "therapeutic dose" and "preventive dose" may vary depending, for example, on the individual's disease state, age, sex and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, the improved health status of the subject.

[0194] The above specification describes in detail one or more embodiments of the present disclosure. Other features, purposes and advantages of the present disclosure will become apparent throughout the specification and claims. In the specification and claims, unless otherwise clearly indicated in the context, the singular form includes the case of the plural referent. Unless otherwise noted, all technical and scientific terms used herein have the meaning generally understood by those skilled in the art to which the present disclosure belongs. All patents and publications incorporated herein by reference are incorporated by reference. The following examples are provided to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed in any way as limiting the scope of the present disclosure, which is limited by the claims. [Brief explanation of the drawing]

[0195] [Figure 1] This figure shows the results of binding the antibody disclosed herein to the mouse TF antigen protein. [Figure 2] This figure shows the results of binding the antibody of this disclosure to rat TF antigen protein. [Figure 3] This figure shows the results of PT detection in pharmacokinetic experiments of ADC-1 in cynomolgus monkeys. [Figure 4] This figure shows the results of APTT detection in pharmacokinetic experiments of ADC-1 in cynomolgus monkeys.

[0196] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure.

[0197] Experimental methods in the examples or test cases of this disclosure that do not specify concrete conditions generally follow conventional conditions or conditions suggested by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, and Contemporary Molecular Biology Methods, edited by Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents whose specific source is not specified are common reagents purchased on the market. This disclosure incorporates all the contents of application WO2020063676A1. [Examples]

[0198] Example 1: Clonal construction of antigen and antibody Molecular cloning of antibodies (including light and heavy chains) and antigens was performed using overlap extension PCR or gene synthesis methods, which are well-known in this field. The antibody and protein antigen sequences were each constructed in a pTT5 vector, and then Expi293F was used. TM The cells (A14528, Gibco) were transfected to obtain the corresponding antibodies and antigen proteins. Antigen protein sequence

[0199] The human TF (hTF) protein sequence is derived from NCBI(AAA61151.1), and its extracellular region (ECD) contains 219 amino acids (Ser33-Glu251). The monkey TF (cyno TF, cTF) protein sequence is derived from NCBI(XP_005542723.1), and its extracellular region (ECD) contains 220 amino acids (Ser33-Glu252). The rat TF (rTF) protein sequence is derived from NCBI(AAA16966.1), and its extracellular region (ECD) contains 224 amino acids (Ala29-Glu252). The mouse TF (mTF) protein sequence is derived from UniProtKB(P20352), and its extracellular region (ECD) contains 223 amino acids (Ala29-Glu251). The TF extracellular region sequence may be tagged with His, human Fc region (hFc), or mouse Fc region (mFc).

[0200] For TF immunization, plasmids involved in the proteins used for detection are prepared using the pTT5 vector, or ExpiFectamine. TM Using the 293 transfect kit (A14525, Gibco), the plasmid was transfected to Expi293F TM The target protein was obtained by transfecting cells.

[0201] The sequence of the antigen protein is as follows: Human TF complete sequence: METPAWPRVPRPETAVARTLLLGWVFAQVAGASGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNSESTGSAGEPLYENSPEFTPYLETNLGQPTIQS FEQVGTKVNTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREIFYIIGAVAFVVIILVIILAISLHKCRKAGVGQSWKENSPLNVS SEQ ID NO:1 Cyno TF full array: METPAWPRVPRPETAVARTLLLGWVFAQVAGASGTTNTVAAYNLTWKSTNFKTILEWEPKPINQVYTVQISTKSGDWKSKCFYTADTECDLTDEIVKDVKQTYLARVFSYPAGHVESTGSTEEPPYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVQDEWTLVRRNDTFSLSRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRRTANRKSTDSPVECMGHEKGESREIFYIIGAVVFVVIILVIILAISLHKCKKARVGRSWKENSPLNVA SEQ ID NO:2 Rat TF full array: MAIPMRPLLAALAPTFLGFLLLQVAVGAGTPPGKAFNLTWISTDFKTILEWQPKPTNYTYTVQISDRSRNWKYKCTGTTDTECDLTDEIVKDVNWTYEARVLSVPWRNSTHGKETLFGTHGEEPFTNARKFLPYRDTKIGQPVIQKYEQGGTKLKVTVKDSFTLVRKNGTFLTLRQVFGNDLGYILTYRKDSSTGRKTNTTHTNEFLIDVEKGVSYCFFAQAVIFSRKTNHKSPESITKCTEQWKSVLGETLIIVGAVVFLVTVFIILLTISLCKRRKNRAGQKRKNTPSRLA SEQ ID NO:3 Mouse TF full array: MAILVRPRLLAALAPTFLGCLLLQVIAGAGIPEKAFNLTWISTDFKTILEWQPKPTNYTYTVQISDRSRNWKNKCFSTTDTECDLTDEIVKDVTWAYEAKVLSVPRRNSVHGDGDQLVIHGEEPPFTNAPKFLPYRDTNLGQPVIQQ FEQDGRKNLVVVKDSLTLVRKNGTFLTLRQVFGKDLGYIITYRKGSSTGKKTNITNTNEFSIDVEEGVSYCFFVQAMIFSRKTNQNSPGSSTVCTEQWKSFLGETLIIVGAVVLLATIFIILLSISLCKRRKNRAGQKGKNTPSRLA SEQ ID NO:4 Human TF ECD-His sequence (hTF-his): SGTTNTVAAYNLTWKSTNFKTILEWPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRETGHHHHH SEQ ID NO:5 Human TF ECD-hFc sequence (hTF-hFc): SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREEPKSSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK SEQ ID NO:6 cyno TF ECD-hFc sequence (cTF-hFc): SGTTNTVAAYNLTWKSTNFKTILEWEPKPINQVYTVQISTKSGDWKSKCFYTADTECDLTDEIVKDVKQTYLARVFSYPAGHVESTGSTEEPPYENSPEFTPYLETNLQPTIQSFEQVGTKVNVTVQDEWTLVRRNDTFSLSRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRRTANRKSTDSPVECMGHEKGESREEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:7 Cyno TF ECD-His sequence (cTF-his): SGTTNTVAAYNLTWKSTNFKTILEWEPKPINQVYTVQISTKSGDWKSKCFYTADTECDLTDEIVKDVKQTYLARVFSYPAGHVESTGSTEEPPYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVQDEWTLVRRNDTFSLSRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRRTANRKSTDSPVECMGHEKGESRETGHHHHHH SEQ ID NO:8 cyno TF ECD-mFc sequence (cTF-mFc): SGTTNTVAAYNLTWKSTNFKTILEWEPKPINQVYTVQISTKSGDWKSKCFYTADTECDLTDEIVKDVKQTYLARVFSYPAGHVESTGSTEEPPYENSPEFTPYLETNLQPTIQSFEQVGTKVNVTVQDEWTLVRRNDTFSLSRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRRTANRKSTDSPVECMGHEKGESREEPRGPTIKPCPPCKCPAPNLGGPSVFIFPPKIKDVLMISLSPIVTCVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO:106 Rat TF ECD-His sequence: AGTPPGKAFNLTWISTDFKTILEWQPKPTNYTYTVQISDRSRNWKYKCTGTTDTECDLTDEIVKDVNWTYEARVLSVPWRNSTHGKETLFGTHGEEPFTNARKFLPYRDTKIGQPVIQKYEQGGTKLKVTVKDSFTLVRKNGTFLTLRQVFGNDLGYILTYRKDSSTGRKTNTTHTNEFLIDVEKGVSYCFFAQAVIFSRKTNHKSPESITKCTEQWKSVLGETGHHHHH SEQ ID NO:9 Mouse TF ECD-His sequence: AGIPEKAFNLTWISTDFKTILEWQPKPTNYTYTVQISDRSRNWKNKCFSTTDTECDLTDEIVKDVTWAYEAKVLSVPRRNSVHGDGDQLVIHGEEPPFTNAPKFLPYRDTNLGQP VIQQFEQDGRKLNVVVKDSLTLVRKNGTFLTLRQVFGKDLGYIITYRKGSSTGKKTNITNTNEFSIDVEEGVSYCFFVQAMIFSRKTNQNSPGSSTVCTEQWKSFLGETGHHHHHH SEQ ID NO:10

[0202] A hybrid human-rat antigen, HuRatTF, was constructed by combining amino acids 1-130 of rat TF (Met1-Ala130) and amino acids 130-245 of human TF (Pro130-Glu245), and adding a His tag. Here, individual amino acids affecting coagulation were replaced with the amino acid Ala, and individual sites in the rat TF sequence were mutated to the corresponding sequences in human TF. The resulting protein sequence (including the signal peptide) is as follows: JPEG2026518847000027.jpg43166SEQ ID NO:11 Note: Italics represent human TF sequences, dashed lines represent signal peptides, double underlines indicate mutations in rat TF amino acids to their corresponding human TF amino acids, underlines indicate mutations in the coagulation-affecting site to alanine (Ala), and wavy lines represent His tags. Sequences used in the construction of antigen cell lines

[0203] The plasmids involved in the antigen cell lines used for TF immunization and detection were prepared with reference to the Cell, 01 Aug 2005, 122(3):473-48 vector. Using Lipofectamine 3000 transfect reagent (Invitrogen, L3000015) and the transposase hyPBase plasmid, the piggyBac transposon plasmid containing the antigen sequence was transfected into NIH / 3T3 (ATCC, CRL-1658) and / or CHO-K1 (ATCC, CCL-61) cells, followed by flow cytometry sorting (BD FACSAria). TM The target cell line was obtained using a Fusion Cell Sorter. The antigen sequence used to construct the above cell line is composed of the full-length TF antigen, the self-cleaving protein (2A), and the green fluorescent protein (EGFP) sequence. Specifically, it is as follows: piggyBac transposon plasmid constituent elements (PB 3' LTR and PB 5' LTR, NCBI-KF658273.1): PB 3' LTR-CMV promoter-antigen sequence-beta-globin poly(A)-PB 5' LTR -Ampicillin resisitance gene-bla promoter Transposase hyPBase plasmid constituent elements (hyPBase, NCBI-OL519599.1): EF1a promoter-hyPBase-BGH polyA-f1 origin-SV40 early promoter and origin-Neomycin resistance gene-SV40 polyA-pUC origin-Ampicillin resisitance gene-bla promoter Human TF-2A-EGFP sequence (hTF): JPEG2026518847000028.jpg85166SEQ ID NO:12 Cyno TF-2A-EGFP sequence (cTF): JPEG2026518847000029.jpg85166SEQ ID NO:13 Note: Italics indicate TF sequences, double underlines indicate 2A sequences, and wavy lines indicate EGFP sequences.

[0204] Referring to patent WO2010066803A2, a total of seven human-mouse chimeric antigens were produced by replacing some sequences in human TF with corresponding sequences in mouse TF, namely hTF1 (1-41 mm), hTF2 (42-84 mm), hTF3 (85-122 mm), hTF4 (123-137 mm), hTF7 (138-184 mm), hTF5 (185-225 mm), and hTF6 (226-250 mm). These seven antigens are abbreviated as hTF1, hTF2, hTF3, hTF4, hTF7, hTF5, and hTF6. Here, hTF7 is not mentioned in patent WO2010066803A2 and is newly added in this disclosure. The corresponding human TF antigen epitopes for these seven antigens in the test examples are shown in Table 2. [Table 2] The seven chimeric antigen sequences are as follows: hTF1 (1-41mm) array: JPEG2026518847000031.jpg79166SEQ ID NO:21 hTF2 (42-84mm) array: JPEG2026518847000032.jpg84166SEQ ID NO:22 hTF3 (85-122mm): JPEG2026518847000033.jpg87166SEQ ID NO:23 hTF4 (123-137mm): JPEG2026518847000034.jpg84167SEQ ID NO:24 hTF7 (138-184mm): JPEG2026518847000035.jpg86167SEQ ID NO:25 hTF5 (185-225mm): JPEG2026518847000036.jpg86167SEQ ID NO:26 hTF6 (226-250mm): JPEG2026518847000037.jpg82167SEQ ID NO:27 Note: Italicized text indicates human TF sequences, underlined text indicates mouse TF sequences, double underlined text indicates 2A sequences, and wavy text indicates EGFP sequences.

[0205] Epitope analysis was performed by FACS binding experiments, and the antibody of this disclosure binds to specific epitopes of TF. Positive control antibody sequence

[0206] The positive control TF011 antibody sequence is derived from the monoclonal antibody sequence of tisotumab vedotin, the H39 antibody variable region sequence is derived from WO2018036117A1, and the 10H10 antibody variable region sequence is derived from WO2012125559A1. TF011 antibody heavy chain sequence (monoclonal antibody sequence of tisotumab vedotin): EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISGSGDYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPWGYYLDSWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:28 TF011 antibody light chain sequence (monoclonal antibody sequence of tisotumab vedotin): DIQMTQSPPSLSASAGDRVTITCRASQGISSRLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:29 H39 antibody heavy chain sequence (variable region + human IgG1 constant region): EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMNWVRQMPGKGLEWMGMIYPADSETRLNQKFKDQATLSVDKSISTAYLQWSSLKASDTAMYYCAREDYGSSDYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:30 H39 antibody light chain sequence (variable region + human κ constant region): DIQLTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKSPKIWIYGISNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKSSFPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:31 10H10 antibody heavy chain sequence (variable region + human IgG1 constant region): QVHLQQSGAELMKPGASVKISCKASGYTFITYWIEWVKQRPGHGLEWIGDILPGSGSTNYNENFKGKATFTADSSSNTAYMQLSSLTSEDSAVYYCARSGYYGNSGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:32 10H10 antibody light chain sequence (variable region + human κ constant region): DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLSSGNQKNYLTWYQQIPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:33 TF011-mIgG2a heavy chain sequence (variable region + mouse mIgG2a constant region): EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISGSGDYTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPWGYYLDSWGQGTLVTVSS AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO:34 TF011-mκ light chain sequence (variable region + mouse κ constant region): DIQMTQSPPSLSASAGDRVTITCRASQGISSRLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:35

[0207] Negative control antibody hIgG1 (where VH / VL is derived from the C25 antibody in patent US6114143A): Amino acid sequence of the hIgG1 antibody heavy chain (C25 heavy chain variable region + human IgG1 constant region): QVQLVQSGAEVKKPGASVKVSCKASGYTFTNSWIGWFRQAPGQGLEWIGDIYPGGGYTNYNEIFKGKATMTADTSTNTAYMELSSLRSEDTAVYYCSRGIPGYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:103 Amino acid sequence of the hIgG1 antibody light chain (C25 light chain variable region + human κ constant region): DIQMTQSPSSLSASVGDRVTMSCKSSQSLLNSGDQKNYLTWYQQKPGKAPKLLIYWASTGESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNDYSYPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:104 Note: The underlined portion is a variable region array. Example 2: Purification of antigen protein and antibody protein

[0208] Antigens with His tags were purified by affinity using a nickel column Ni Sepharose excel 25 mL (GE Healthcare, 17-3712-01), and antigens or antibodies with Fc tags were purified by affinity using ProteinA-MabSelect SuRe 25 mL (GE Healthcare, 17-5438-01). The affinity-purified proteins were then subjected to molecular exclusion chromatography to remove polymers or lysed fragments. The specific methods are as follows:

[0209] Purification using a nickel column: The nickel column was washed and equilibrated with PBS solution, and the culture supernatant after cell transfection was rapidly centrifuged to remove impurities before loading. The nickel column was washed with PBS solution, and A 280 After the readings decreased to baseline, the column was washed with a 10 mM imidazole-containing PBS solution to remove nonspecifically bound impurity proteins. Then, the target protein was eluted with a 300 mM imidazole-containing PBS solution, the eluted peak liquid was collected, and the buffer was changed to PBS for storage.

[0210] Purification by exclusion chromatography: An SEC column (GE, superdex75) was washed with PBS solution and equilibrated. The purified protein was loaded onto a nickel column or Protein A column (loading volume ≤ 3% column volume), eluted using PBS solution as the mobile phase, and each elution peak was collected. The components of the target protein were identified by SDS-PAGE.

[0211] Affinity chromatography of antibodies or Fc fusion proteins: The culture supernatant after cell transfection was rapidly centrifuged to remove impurities and then loaded onto a Protein A column. The Protein A column was washed with PBS solution and then A 280After the readings decreased to baseline, the target protein was eluted with acetic acid at pH 3.5, 100 mM. The eluate was neutralized with Tris-HCl at pH 8.0, 1 M Tris-HCl, and the buffer was changed to PBS. If necessary, further removal of polymers may be performed using gel chromatography (Superdex200, GE). Example 3: Production of anti-human TF monoclonal antibody 3.1 Immunity

[0212] Anti-human TF monoclonal antibodies were obtained using hybridoma technology. Experimental SJL white mice, female, 6-8 weeks old (Shanghai SJL Laboratory Animals Co., Ltd.) Animal production license number: (JPEG2026518847000038.jpg766). Rearing environment: SPF grade. After purchasing the mice, they were reared in a laboratory environment for one week, with a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. Mice adapted to the environment were immunized according to the following regimen. 3.2 Immunology Scheme

[0213] The immune antigens for anti-human TF antibody 67 are hTF-NIH / 3T3 cells (SEQ ID NO: 12), cTF-NIH / 3T3 cells (SEQ ID NO: 13), hTF-his protein (SEQ ID NO: 5), and cTF-mFc protein (SEQ ID NO: 106), and the immunization method is cell-protein immunization. In the initial immunization, hTF-NIH / 3T3 cells were used, and before immunizing the cells, TiterMax (登録商標) Gold Adjuvant (Sigma, T2684) was pre-injected into the peritoneum of mice at a dose of 0.1 mL / mice, and 30 minutes later, 0.1 mL of physiological saline was added. 8 A cell suspension diluted to 10 cells / mL was intraperitoneally injected into each mouse. A second immunization was performed on day 14, using cTF-NIH / 3T3 cells, and 10 immunizations were performed with 0.1 mL of physiological saline. 8Cell suspensions diluted to 100 cells / mL were intraperitoneally injected into each mouse. A third immunization was performed on day 35 using hTF-his protein, 50 μg / mouse / dose, along with antigen and adjuvant (TiterMax). (登録商標) The ratio of the antigen to the adjuvant (Gold Adjuvant) was 1:1, and the antigen and adjuvant were thoroughly emulsified before inoculation. On day 49, the fourth immunization was performed using hTF-his protein, with 50 μg / animal / dose, and the antigen and adjuvant (TiterMax (登録商標) The ratio with Gold Adjuvant was 1:1, and the antigen and adjuvant were thoroughly emulsified before inoculation. Blood was collected on days 29, 47, and 61, and antibody titers in mouse serum were determined by ELISA and FACS. After the fourth immunization, mice with high serum antibody titers and a tendency for the titer to plateau were selected, and spleen cell fusion was performed. Three days before spleen cell fusion, an additional immunization was performed, and an antigen protein solution of hTF-his and cTF-mFc mixed in physiological saline in a 1:1 mass ratio was injected intraperitoneally (ip) at a dose of 50 μg / mifacial.

[0214] The anti-human TF antibody 310 immune antigens are hTF-NIH / 3T3 cells, cTF-NIH / 3T3 cells, hTF-CHO-K1 cells, and cTF-CHO-K1 cells, and the immunization method is cellular immunity. In the initial immunization, hTF-NIH / 3T3 cells are used, and before immunizing the cells, TiterMax (登録商標) Gold Adjuvant was pre-injected into the peritoneum of mice at a dose of 0.1 mL / mice, and 30 minutes later, 0.1 mL of physiological saline was added. 8 A cell suspension diluted to 10 cells / mL was injected intraperitoneally into each mouse. A second immunization was performed on day 59, using cTF-NIH / 3T3 cells and 10 immunizations with 0.1 mL of physiological saline. 8 A cell suspension diluted to 10 cells / mL was injected intraperitoneally into each mouse. A third immunization was performed on day 74, using hTF-CHO-K1 cells and 10 immunizations with 0.1 mL of physiological saline. 8 A cell suspension diluted to 10 cells / mL was intraperitoneally injected into each mouse. On day 90, a fourth immunization was performed, using cTF-CHO-K1 cells and 10 immunizations with 0.1 mL of physiological saline. 8A cell suspension diluted to 10 cells / mL was injected intraperitoneally into each mouse. On day 104, a fifth immunization was performed, using hTF-CHO-K1 cells and 100 ml of physiological saline. 8 Cell suspensions diluted to 100 cells / mL were intraperitoneally injected into each mouse. Blood was collected on days 68, 85, and 118, and antibody titers in mouse serum were determined by ELISA and FACS. After the fifth immunization, mice with high serum antibody titers and a tendency for the titer to plateau were selected for spleen cell fusion. Three days prior to spleen cell fusion, an additional immunization was performed, and an antigen protein solution consisting of hTF-his (SEQ ID NO: 5) and cTF-his (SEQ ID NO: 8) mixed in physiological saline in a 1:1 mass ratio was injected intraperitoneally (ip) at a dose of 50 μg / mouse. 3.3 Splenocyte Fusion

[0215] By electrofusion, splenic lymphocytes and myeloma cells Sp2 / 0-Ag14 cells (ATCC, CRL-8287) are fused. TM ) were fused to obtain hybridoma cells. 3-4 × 10⁻¹⁶ hybridoma cells were obtained. 5 The cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI) at a density of / mL and inoculated into 96-well cell culture plates at 150μL / well. After incubation at 37°C and 5%CO2 for 3-4 days, the supernatant was removed, and 200μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. The cells were incubated at 37°C and 5%CO2 for 3-5 days before detection. 3.4 Screening of Hybridoma Cells

[0216] When the hybridoma cell density reached 90%, the cell supernatant was aspirated, and a preliminary screening of the 96-well plate was performed using a cell-based ELISA method. Subsequently, the screened positive wells were transferred to a 24-well plate, and after 2-3 days, when the cell density reached 90%, the cell supernatant was aspirated, and the 24-well plate was re-screened by experiments such as FACS and / or competition and / or coagulation. Cells were selected from a single well and subcloned to obtain single-cell clones. The supernatant of the subcloned cells was collected and re-detected. Hybridoma clones were obtained by screening using the above experiments and used for subsequent tests. Example 4: Determination of the variable region amino acid sequence of a mouse monoclonal antibody

[0217] The variable region amino acid sequence was determined for the hybridoma monoclonal cell line obtained in Example 3. The mouse hybridoma variable region sequence was combined with the human constant region sequence to obtain a human mouse chimeric antibody (CHAb) sequence, and expression and identification of the chimeric antibody were performed. The specific method is as follows.

[0218] First, the total RNA of the hybridoma monoclonal cell line is obtained using conventional, well-known methods, and then the reverse transcription kit PrimeScript is used. TM IV. cDNA was prepared using 1st strand cDNA Synthesis Mix (TAKARA, 6215A). PCR was performed using the cDNA as a template, and the resulting fragments containing the light and heavy chain variable regions were amplified to obtain PCR fragments. The corresponding variable region sequences were then obtained by PCR fragment sequencing. The obtained light and heavy chain sequences were cloned into pTT5 vectors using conventional, well-known methods, and recombinant monoclonal antibodies were expressed to verify their activity.

[0219] The amino acid residues of the VH / VL CDR of the anti-human TF antibody were determined and annotated using the Kabat numbering system.

[0220] Sequence of mouse hybridoma cell monoclonal antibody 67#: 67# Heavy chain variable region: QVQLKQSGPGLVQPSQSLSITCIVSGFSLTNYGVNWVRQSPRKGLEWLGVIWSRGNTDYNAAFISRLIISKDNSKSQVFLKMNRLQADDTAIYYCARKDAVVATDALDYWGQGTSVTVSS SEQ ID NO:36 67# Light chain variable region: DIVMTQSHKFMSTSLGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGGTKLEIK SEQ ID NO:37

[0221] Sequence of mouse hybridoma cell monoclonal antibody 310#: 310# Heavy chain variable region: EVQLQQSGPELVKPGASLKISCKASGYSFTGYYMNWVKQSPEKSLEWIGEINPSTGGSTYNQKFKAKATLTVDQSSNTAYMQLKSLTSEDSAVYYCARRELGGPFGYWGQGTPVTVSA SEQ ID NO:38 310#Light chain variable region: SIVMTQTPKFLLVSSGDRVTITCKASQSVSNGIAWYQQKPGQSPKLLIYFASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGGTKLEIK SEQ ID NO:39 [Table 3] [Table 4] Human IgG1 heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:54 Human κ-light chain constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:55

[0222] The heavy chain and light chain sequences of the chimeric antibody CH67 are as follows: CH67 heavy chain: QVQLKQSGPGLVQPSQSLSITCIVSGFSLTNYGVNWVRQSPRKGLEWLGVIWSRGNTDYNAAFISRLIISKDNSKSQVFLKMNRLQADDTAIYYCARKDAVVATDALDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:107 CH67 Light Chain: DIVMTQSHKFMSTSLGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:108

[0223] The heavy chain and light chain sequences of the chimeric antibody CH310 are as follows: CH310 heavy chain: EVQLQQSGPELVKPGASLKISCKASGYSFTGYYMNWVKQSPEKSLEWIGEINPSTGGSTYNQKFKAKATLTVDQSSNTAYMQLKSLTSEDSAVYYCARRELGGPFGYWGQGTPVTVSA ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:109 CH310 Light Chain: SIVMTQTPKFLLVSSGDRVTITCKASQSVSNGIAWYQQKPGQSPKLLIYFASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:110 Example 5: Humanization of anti-human TF antibody 310#

[0224] The MOE software was used to query the IMGT human antibody heavy chain and light chain variable region germline gene database. Germline genes with high homology to 310# were selected as templates, and the CDRs of mouse-derived antibodies were transplanted into the corresponding human-derived templates to form the variable region sequences FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order. Exemplarily, the CDR amino acid residues in the following specific examples were determined and annotated using the Kabat numbering system.

[0225] For the 310# humanized antibody, FR1, FR2, and FR3 of IGKV4-1*01 or IGKV1-33*01, and FR4 of IGKJ4*01 were selected as light chain framework region templates, and FR1, FR2, and FR3 of IGHV1-46*01 and FR4 of IGHJ6*01 were selected as heavy chain framework region templates. Optionally, amino acid residues at positions 1, 9, 10, 31, 43, 67, and / or 73 in the light chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 1, 28, 69, 71, 73, 76, and / or 78 in the heavy chain variable region of the humanized antibody were substituted. [Table 5] [Table 6]

[0226] The heavy chain variable region sequence and light chain variable region sequence of the 310# humanized antibody are as follows: >hu310-VH1: JPEG2026518847000043.jpg21167SEQ ID NO:52 >hu310-VH2: JPEG2026518847000044.jpg26167SEQ ID NO:114 >hu310-VL1: DIVMTQSPDSLAVSLGERATINC KASQSVSNGIA WYQQKPGQPPKLLIY FASNRYT GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC QQDYSSPWT FGGGTKVEIK SEQ ID NO:115 >hu310-VL2: JPEG2026518847000045.jpg20164SEQ ID NO:53 >hu310-VL3: JPEG2026518847000046.jpg21164SEQ ID NO:116 >hu310-VL4: DIQMTQSPSSLSASVGDRVTITC KASQSVSNGIA WYQQKPGKAPKLLIY FASNRYT GVPSRFSGSGSGTTDFTFTISSLQPEDIATYYC QQDYSSPWT FGGGTKVEIK SEQ ID NO:117 >hu310-VL5: JPEG2026518847000047.jpg20164SEQ ID NO:118 >hu310-VL6: JPEG2026518847000048.jpg18164SEQ ID NO:119 >hu310-VL7: JPEG2026518847000049.jpg21159SEQ ID NO:120 >hu310-VL8: JPEG2026518847000050.jpg22159SEQ ID NO:121 Note: In the above sequence, the sequences are FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order. The underlined parts are CDR sequences determined according to the Kabat numbering system, the non-underlined parts are FR sequences, and the double-underlined bold parts are amino acid point mutations.

[0227] The heavy chain variable region and light chain variable region of the 310# humanized antibody were rearranged with the human heavy chain IgG1 constant region (SEQ ID NO: 54) and the human light chain Kappa constant region (SEQ ID NO: 55), respectively, to obtain the humanized antibodies shown in Table 7 below. [Table 7]

[0228] The heavy chain and light chain sequences of the 310# humanized antibody are as follows: hu310-1 / hu310-2 / hu310-3 / hu310-4 / hu310-5 / hu310-6 / hu310-7 / hu310-8 double chain: EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMNWVRQAPGQGLEWMGEINPSTGGSTYNQKFKARVTMTVDQSTSTVYMELSSLRSEDTAVYYCARRELGGPFGYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:56 hu310-9 / hu310-10 / hu310-11 / hu310-12 / hu310-13 / hu310-14 / hu310-15 / hu310-16 heavy chain: JPEG2026518847000052.jpg66165SEQ ID NO:122 hu310-1 / hu310-9 light chain: DIVMTQSPDSLAVSLGERATINC KASQSVSNGIA WYQQKPGQPPKLLIY FASNRYT GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC QQDYSSPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:123 hu310-2 / hu310-10 light chain: SIVMTQSPDSLAVSLGERATINCKASQSVSNGIAWYQQKPGQSPKLLIYFASNRYTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYCQQDYSSPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:57 hu310-3 / hu310-11 light chain: JPEG2026518847000053.jpg36165SEQ ID NO:124 hu310-4 / hu310-12 light chain: DIQMTQSPSSLSASVGDRVTITC KASQSVSNGIA WYQQKPGKAPKLLIY PHASE SURFACES GVPSRFSGSGSGTTDFTFTISSLQPEDIATYYC QQDYSSPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:125 hu310-5 / hu310-13 light chain: JPEG2026518847000054.jpg36165SEQ ID NO:126 hu310-6 / hu310-14 light chain: JPEG2026518847000055.jpg35165SEQ ID NO:127 hu310-7 / hu310-15 light chain: JPEG2026518847000056.jpg35165SEQ ID NO:128 hu310-8 / hu310-16 light chain: JPEG2026518847000057.jpg35166SEQ ID NO:129 Example 6: Humanized anti-human TF antibody 67#

[0229] For the light and heavy chain sequences of the mouse anti-TF monoclonal antibody 67# obtained in Example 4, homology comparisons were performed using MOE software with those in the IMGT human antibody heavy chain and light chain variable region germline gene databank. Heavy chain and light chain variable region germline genes with high homology to 67# were used as templates, and the CDRs of this mouse antibody were transplanted into the corresponding human templates to form the variable region sequences FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order. Exemplarily, the CDR amino acid residues in the following specific examples were determined and annotated using the Kabat numbering system.

[0230] For the 67# antibody, the human germline light chain template was selected from FR1, FR2, and FR3 of IGKV4-1*01 or IGKV1-33*01, and FR4 of IGKJ4*01. For the human germline heavy chain template, the FR1, FR2, and FR3 of IGHV2-26*01 or IGHV4-30-4*01, and FR4 of IGHJ6*01 were selected. Optionally, amino acid residues at positions 8, 9, 10, 43, and / or 73 in the light chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 1, 6, 16, 27, 29, 30, 42, 44, 49, 71, 73, 76, 78, and / or 81 in the heavy chain variable region of the humanized antibody were substituted. [Table 8]

[0231] The heavy chain variable region and light chain variable region sequences of the 67# humanized antibody are as follows: >hu67-VH1: JPEG2026518847000059.jpg23166SEQ ID NO:130 >hu67-VH2: JPEG2026518847000060.jpg24166SEQ ID NO:131 >hu67-VH3: JPEG2026518847000061.jpg23166SEQ ID NO:58 >hu67-VH4: JPEG2026518847000062.jpg23166SEQ ID NO:132 >67VL-VL1: JPEG2026518847000063.jpg19166SEQ ID NO:133 >67VL-VL2: JPEG2026518847000064.jpg20166SEQ ID NO:134 >67VL-VL3: DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLIY STSYRYT GVPSRFSGSGSGTTDFTFTISSLQPEDIATYYC QQHYSTPYT FGGGTKVEIK SEQ ID NO:59 >67VL-VL4: JPEG2026518847000065.jpg22165SEQ ID NO:135 Note: In the above sequence, the sequences are FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order. The underlined parts are CDR sequences determined according to the Kabat numbering system, the non-underlined parts are FR sequences, and the double-underlined bold parts are amino acid point mutations.

[0232] The heavy chain variable region and light chain variable region of the 67# humanized antibody were rearranged with the human heavy chain IgG1 constant region (SEQ ID NO: 54) and the human light chain Kappa constant region (SEQ ID NO: 55), respectively, to obtain the humanized antibodies shown in Table 9 below. [Table 9]

[0233] The heavy chain and light chain sequences of the humanized antibody are as follows: hu67-1 / hu67-5 / hu67-9 / hu67-13 heavy chain sequence: JPEG2026518847000067.jpg66168SEQ ID NO:136 hu67-2 / hu67-6 / hu67-10 / hu67-14 heavy chain sequence: JPEG2026518847000068.jpg67168SEQ ID NO:137 hu67-3 / hu67-7 / hu67-11 / hu67-15 heavy chain sequence: EVQLQESGPGLVKPSQTLSLTCTVSGFSLTNYGVNWIRQPPGKGLEWIGVIWSRGNTDYNAAFISRVTISKDTSKNQVSLKLSSVTAADTAVYCARKDAVVADTALDYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:60 hu67-4 / hu67-8 / hu67-12 / hu67-16 heavy chain sequence: JPEG2026518847000069.jpg67165SEQ ID NO:138 hu67-1 / hu67-2 / hu67-3 / hu67-4 light chain sequence: JPEG2026518847000070.jpg36165SEQ ID NO:139 hu67-5 / hu67-6 / hu67-7 / hu67-8 light chain sequence: JPEG2026518847000071.jpg37165SEQ ID NO:140 hu67-9 / hu67-10 / hu67-11 / hu67-12 light chain sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:61 hu67-13 / hu67-14 / hu67-15 / hu67-16 light chain sequence: JPEG2026518847000072.jpg36167SEQ ID NO:141 Example 7: Affinity maturation of hu67-11

[0234] For the human anti-TF monoclonal antibody hu67-11 obtained in Example 6, an antibody yeast library was constructed using hTF-hFc (SEQ ID NO: 6) and cTF-hFc (SEQ ID NO: 7) as antigens, and affinity maturation was performed to improve the cell binding ability of the target antibody. The optimal humanized antibody was selected by cloning, expressing, purifying, and detecting the above antibody. 7.1 Construction of an affinity-mature yeast library and library screening

[0235] To obtain better anti-TF hu67-11 related antibodies, affinity maturation against hu67-11 was performed using yeast display platform technology. Based on hu67-11-scFv (SEQ ID NO: 171), an affinity-matured yeast library for human TF and monkey TF-binding CDRs was designed and created, and novel mutants were screened from it.

[0236] Yeast library construction: Degenerate primers were designed, and the designed mutant amino acids were introduced into the hu67-11-scFv mutant library by PCR, with each library being approximately 10 units in size. 9 The diversity of the constructed yeast library was then verified using next-generation sequencing methods.

[0237] In the first round of screening, yeast and biotinylated hTF-hFc proteins from the hu67-11-scFv mutant library were incubated in 50 mL of 0.1% bovine serum albumin (BSA)-phosphate buffer (called PBSA) at room temperature for 1 hour. The mixture was then washed three times with PBSA to remove unbound antibody fragments. Streptabidin microbeads (Mi1envi Biotec, Auburn, CA) were added to the mutant library bound to biotinylated hTF-hFc protein, and this was loaded into an AutoMACS system for sorting. Antibody libraries with high affinity for human TF were collected, amplified, and cultured. The cultures were then induced and expressed. The resulting enriched libraries were screened in the second round for binding to biotinylated cTF-hFc (SEQ ID NO: 7).

[0238] For the third and fourth rounds of screening, library cells from the previous round, along with biotinylated hTF-hFc and Mouse Anti-cMyc (9E10, sigma) antibody, were incubated in PBSA at room temperature for 1 hour. The mixture was washed three times with PBSA to remove unbound antibody fragments. Goat anti-mouse-Alexa488 (Life Technologies, A-11001) and Strepavidin-PE (Life Technologies, S-866) were added and incubated at 4°C for 1 hour. The mixture was washed three times with PBSA to remove unbound antibody fragments. Finally, antibodies with high affinity were screened using FACS (BD FACSAria™ FUSION).

[0239] A mutant library was subjected to 2-3 rounds of MACS screening (streptavidin microbeads, Invitrogen) and 2-3 rounds of FACS screening (BD FACSAria™ FUSION) using biotinylated TF-hFc antigen. Next, approximately 600 yeast monoclonals were selected, cultured, induced, and expressed. Binding of the yeast monoclonals to the aforementioned hTF-hFc (SEQ ID NO:6) and cTF-hFc (SEQ ID NO:7) was detected using FACS (BD FACSCanto II). Yeast monoclonals with higher affinity than the wild type (hu67-11-scfv) were selected, sequenced for validation, and alignment analysis was performed on the sequenced clones. After removing redundant sequences, non-redundant sequences were converted to full-length human IgG and then expressed in mammalian cells. hu67-11-scFv array: EVQLQESGPGLVKPSQTLSLTCTVSGFSLTNYGVNWIRQPPGKGLEWIGVIWSRGNTDYNAAFISRVTISKDTSKNQVSLKLSSVTAADTAVYCARKDAVVADTALDYWGQGTTVTVSS GGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPYTFGGGTKVEIK SEQ ID NO:171 Example 8: Modification of the hu67-11 affinity mature molecule

[0240] The humanized antibody hu67-11 obtained in Example 7 was subjected to a CDR point mutation (single point and / or combination) and further enhanced with a P40S point mutation in the FR region, thereby improving its cell binding ability. The antibody was cloned, expressed, purified, and detected. Screening revealed the variable region sequences of antibodies hu67Y116 and hu67Y118, which exhibited even greater cell binding ability. Antibody hu67Y116 / hu67Y118 heavy chain variable region sequence: EVQLQESGPGLVKPSQTLSLTCTVSGFSLT NYGVN WIRQSPGKGLEWIG VIWSRGNTDYNAAFKS RVTISKDTSKNQVSLKLSSVTAADTAVYYCAR KDAIVATDALDY WGQGTTVTVSS SEQ ID NO:62 antibody hu67Y116 light chain variable region sequence: DIQMTQSPSSLSASVGDRVTITC KASQDVQTAVA WYQQKPGKAPKLLIY STORYTY GVPSRFSGSGSGTTDFTFTISSLQPEDIATYYC LQSYTLPFS FGGGTKVEIK SEQ ID NO:63 antibody hu67Y118 light chain variable region sequence: DIQMTQSPSSLSASVGDRVTITC KASQDVQTAVA WYQQKPGKAPKLLIY STORY GVPSRFSGSGSGTTDFTFTISSLQPEDIATYYC LQLIGVPHT FGGGTKVEIK SEQ ID NO:81 [Table 10] [Table 11] hu67Y116 / hu67Y118 heavy chain sequence: EVQLQESGPGLVKPSQTLSLTCTVSGFSLTNYGVNWIRQSPGKGLEWIGVIWSRGNTDYNAAFKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKDAIVATDALDYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:69 hu67Y116 light chain sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVQTAVAWYQQKPGKAPKLLIYSTHYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQSYTLPFSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:70 hu67Y118 light chain sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVQTAVAWYQQKPGKAPCLLIYSTHYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQLIGVPHTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:82 Example 9: Modification of the molecular sequence of 10H10

[0241] Crystal structure analysis demonstrated that antibody 10H10 does not affect coagulation (Cellular Signalling 36(2017)139-144). After homology comparison using an antibody database, a humanized antibody model was constructed. Based on the model, reverse mutations were selected and the CDR region was optimized. The above antibody was cloned, expressed, purified, and detected.

[0242] For antibody 10H10, the human germline light chain templates were selected from FR1, FR2, FR3 of IGKV1-39*01 and FR4 of IGKJ2*01, and the human germline heavy chain templates were selected from FR1, FR2, FR3 of IGHV1-46*01 or IGHV7-4-1*02 and FR4 of IGHJ1*01. Optionally, amino acid residues at positions 24, 25, 27b, 27d, 27f, 56 and / or 97 in the light chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 1, 26, 27, 30, 31, 38, 40, 53, 54, 56, 57, 67, 68, 69, 71, 81 and / or 101 in the heavy chain variable region of the humanized antibody were substituted. [Table 12] JPEG2026518847000076.jpg37170

[0243] The sequence of the variable region of the obtained hu10H10 humanized antibody is as follows: hu10H10 VH1 (SEQ ID NO:84) JPEG2026518847000077.jpg28167hu10H10 VH2(SEQ ID NO:85) JPEG2026518847000078.jpg23168hu10H10 VH3(SEQ ID NO:86) JPEG2026518847000079.jpg26165hu10H10 VH4(SEQ ID NO:87) JPEG2026518847000080.jpg28165hu10H10 VH5(SEQ ID NO:88) JPEG2026518847000081.jpg27165hu10H10 VH6(SEQ ID NO:89) JPEG2026518847000082.jpg28165hu10H10 VH7(SEQ ID NO:90) JPEG2026518847000083.jpg26165hu10H10 VH8(SEQ ID NO:91) JPEG2026518847000084.jpg26165hu10H10 VH9(SEQ ID NO:92) JPEG2026518847000085.jpg25165hu10H10 VH10(SEQ ID NO:93) JPEG2026518847000086.jpg26165hu10H10 VH11(SEQ ID NO:71) JPEG2026518847000087.jpg27165hu10H10 VH12(SEQ ID NO:94) JPEG2026518847000088.jpg27165hu10H10 VH13(SEQ ID NO:95) JPEG2026518847000089.jpg25166hu10H10 VH14(SEQ ID NO:96) JPEG2026518847000090.jpg25166hu10H10 VL1(SEQ ID NO:97) DIQMTQSPSSLSASVGDRVTITC KSSQSLLSSGNQKNYLT WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QNDYTYPLT FGQGTKLEIK hu10H10 VL2 (SEQ ID NO:98) JPEG2026518847000091.jpg18166hu10H10 VL3(SEQ ID NO:99) JPEG2026518847000092.jpg20166hu10H10 VL4(SEQ ID NO:100) JPEG2026518847000093.jpg22166hu10H10 VL5(SEQ ID NO:101) JPEG2026518847000094.jpg21166hu10H10 VL6(SEQ ID NO:102) JPEG2026518847000095.jpg18166hu10H10 VL9(SEQ ID NO:72) JPEG2026518847000096.jpg18166 Note: The single underlined portion is the CDR, the single underlined and bold portion is the CDR amino acid substitution site, the double underlined and bold portion is the FR amino acid substitution site, the remaining portion is the FR region, and so on. [Table 13] [Table 14] [Table 15]

[0244] The heavy chain and light chain sequences of the 10H10 humanized antibody are as follows: hu10H10-1 / hu10H10-7 / hu10H10-13 / hu10H10-19 heavy chain: JPEG2026518847000100.jpg66166SEQ ID NO:152 hu10H10-2 / hu10H10-8 / hu10H10-14 / hu10H10-20 / hu10H10-57 / hu10H10-58 heavy chain: JPEG2026518847000101.jpg67169SEQ ID NO:153 hu10H10-3 / hu10H10-9 / hu10H10-15 / hu10H10-21 heavy chain: JPEG2026518847000102.jpg69169SEQ ID NO:154 hu10H10-4 / hu10H10-10 / hu10H10-16 / hu10H10-22 double chain: JPEG2026518847000103.jpg69169SEQ ID NO:155 hu10H10-5 / hu10H10-11 / hu10H10-17 / hu10H10-23 double chain: JPEG2026518847000104.jpg68166SEQ ID NO:156 hu10H10-6 / hu10H10-12 / hu10H10-18 / hu10H10-24 double chain: JPEG2026518847000105.jpg68166SEQ ID NO:157 hu10H10-25 / hu10H10-33 / hu10H10-41 / hu10H10-49 heavy chain: JPEG2026518847000106.jpg69166SEQ ID NO:158 hu10H10-26 / hu10H10-34 / hu10H10-42 / hu10H10-50 heavy chain: JPEG2026518847000107.jpg67165SEQ ID NO:159 hu10H10-27 / hu10H10-35 / hu10H10-43 / hu10H10-51 heavy chain: JPEG2026518847000108.jpg72165SEQ ID NO:160 hu10H10-28 / hu10H10-36 / hu10H10-44 / hu10H10-52 double chain: JPEG2026518847000109.jpg69165SEQ ID NO:161 hu10H10-29 / hu10H10-37 / hu10H10-45 / / hu10H10-53 / hu10H10-77 Heavy Chain: EVQLVQSGSELKKPGASVKVSCKASGYTFITYWIEWVKQRPGQGLEWMGDILPYSGSTNYNENFKGRATFSADTSVSTAYLQISSLKAEDTAVYYCARSGYYGNSGFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:79 hu10H10-30 / hu10H10-38 / hu10H10-46 / hu10H10-54 heavy chain: JPEG2026518847000110.jpg67168SEQ ID NO:162 hu10H10-31 / hu10H10-39 / hu10H10-47 / hu10H10-55 heavy chain: JPEG2026518847000111.jpg68168SEQ ID NO:163 hu10H10-32 / hu10H10-40 / hu10H10-48 / hu10H10-56 heavy chain: JPEG2026518847000112.jpg69165SEQ ID NO:164 hu10H10-1 / hu10H10-2 / hu10H10-3 / hu10H10-4 / hu10H10-5 / hu10H10-6 / hu10H10-25 / hu10H10-26 / hu10H10-27 / hu10H10-28 / hu10H10-29 / hu10H10-30 / hu10H10-31 / hu10H10-32 Light chain: DIQMTQSPSSLSASVGDRVTITC KSSQSLLSSGNQKNYLT WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QNDYTYPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:165 hu10H10-7 / hu10H10-8 / hu10H10-9 / hu10H10-10 / hu10H10-11 / hu10H10-12 Light Chain: JPEG2026518847000113.jpg38165SEQ ID NO:166 hu10H10-13 / hu10H10-14 / hu10H10-15 / hu10H10-16 / hu10H10-17 / hu10H10-18 / hu10H10-33 / hu10H10-34 / hu10H10-35 / hu10H10-36 / hu10H10-37 / hu10H10-38 / hu10H10-39 / hu10H10-40 Light Chain: JPEG2026518847000114.jpg37165SEQ ID NO:167 hu10H10-19 / hu10H10-20 / hu10H10-21 / hu10H10-22 / hu10H10-23 / hu10H10-24 Light Chain: JPEG2026518847000115.jpg38165SEQ ID NO:168 hu10H10-41 / hu10H10-42 / hu10H10-43 / hu10H10-44 / hu10H10-45 / hu10H10-46 / hu10H10-47 / hu10H10-48 / hu10H10-57 Light Chain: JPEG2026518847000116.jpg32165SEQ ID NO:169 hu10H10-49 / hu10H10-50 / hu10H10-51 / hu10H10-52 / hu10H10-53 / hu10H10-54 / hu10H10-55 / hu10H10-56 / hu10H10-58 Light Chain: JPEG2026518847000117.jpg38165SEQ ID NO:170 hu10H10-77 Light Chain: DIQMTQSPSSLSASVGDRVTITCQSSQSLLSSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNDYTYPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:80 Example 10: Manufacturing of ADC ADC DAR value measurement

[0245] The method for calculating the DAR value of the ADC in this disclosure was performed using RP-HPLC (reverse-phase high-performance liquid chromatography).

[0246] The drug portion of the conjugate of this disclosure may be any suitable drug. Particularly suitable drugs are described, for example, in WO2020063676A1 (which is incorporated herein by reference in its entirety). Compound 9A of this disclosure (i.e., Compound 9-A of Example 9 of WO2020063676A1) has the following structure: [ka]

[0247] This disclosure describes the production of an antibody-drug conjugate represented by the ADC general formula (TF-9A) by adjusting the reaction parameters using the following method. [ka] Example 10-1 ADC-1

[0248] Under conditions of 37°C, an aqueous solution of PBS buffer containing antibody hu67Y116 (pH=6.5, 0.05M) (10.0 mg / mL, 7.4 mL, 511 nmol) was mixed with an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 131.8 μL, 1.318 μmol). The mixture was placed in a constant-temperature oscillator and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0249] Compound 9A (5.49 mg, 5.11 μmol) was dissolved in 370 mL of DMSO and added dropwise to the reaction mixture. The mixture was placed in a constant-temperature oscillator and shaken for 3 hours at 25°C to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: His buffer aqueous solution with pH 5.0) to obtain His buffer (3.8 mg / mL, 17.6 mL) of ADC-1 (hu67Y116-9A), an exemplary product of conjugate TF-9A, which was stored at 4°C. Average value calculated by RP-HPLC: DAR = 4.14. Example 10-2 ADC-2

[0250] Under conditions of 37°C, an aqueous solution of PBS buffer containing antibody hu67Y116 (pH=6.5, 0.05M) (10.0 mg / mL, 7.4 mL, 511 nmol) was mixed with an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 178.9 μL, 1.789 μmol). The mixture was placed in a water bath oscillator and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0251] Compound 9A (6.586 mg, 6.132 μmol) was dissolved in 370 μL of DMSO and added to the above reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: His buffer aqueous solution with pH 5.0) to obtain PBS buffer (3.52 mg / mL, 17.9 mL) of ADC-2 (hu67Y116-9A), an exemplary product of conjugate TF-9A, which was stored at 4°C. Average value calculation by RP-HPLC: DAR = 5.91. Example 10-3 ADC-3

[0252] Under conditions of 37°C, an aqueous solution of antibody hu67Y116 buffered in PBS (pH=6.5, 0.05M) (10.0 mg / mL, 1 mL, 69 nmol) was added to an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 48.3 μL, 483 nmol). The mixture was placed in a water bath oscillator and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0253] Compound 9A (1.1 mg, 1.035 μmol) was dissolved in 50 μL of DMSO and added to the above reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: His buffer aqueous solution with pH 5.0) to obtain PBS buffer (0.8 mg / mL, 11.5 mL) of ADC-3 (hu67Y116-9A), an exemplary product of conjugate TF-9A, which was stored at 4°C. Average value calculation by RP-HPLC: DAR = 7.23. Example 10-4 ADC-4

[0254] Under conditions of 37°C, an aqueous solution of the antibody hu10H10-77 buffered in PBS (pH=6.5, 0.05M) (10.0 mg / mL, 8 mL, 549 nmol) was mixed with an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 145.5 μL, 1.455 μmol). The mixture was placed in a water bath oscillator and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0255] Compound 9A (5.9 mg, 5.49 μmol) was dissolved in 400 μL of DMSO and added to the above reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: His buffer aqueous solution with pH 5.0) to obtain PBS buffer (3.85 mg / mL, 19 mL) of ADC-4 (hu10H10-77-9A), an exemplary product of conjugate TF-9A, which was stored at 4°C. Average value calculation by RP-HPLC: DAR = 4.2. Example 10-5 ADC-5

[0256] Under conditions of 37°C, an aqueous solution of the antibody hu10H10-77 buffered in PBS (pH=6.5, 0.05M) (10.0 mg / mL, 8 mL, 549 nmol) was mixed with an aqueous solution of the prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 175.7 μL, 1.757 μmol). The mixture was placed in a water bath oscillator and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0257] Compound 9A (7.08 mg, 6.59 μmol) was dissolved in 400 μL of DMSO and added to the above reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: His buffer aqueous solution with pH 5.0) to obtain PBS buffer (3.57 mg / mL, 19.5 mL) of ADC-5 (hu10H10-77-9A), an exemplary product of conjugate TF-9A, which was stored at 4°C. Average value calculation by RP-HPLC: DAR = 5.87. Example 10-6 ADC-6

[0258] Under conditions of 37°C, an aqueous solution of antibody hu310-2 buffered in PBS (pH=6.5, 0.05M) (10.0 mg / mL, 8 mL, 553 nmol) was mixed with an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 124.4 μL, 1.244 μmol). The mixture was placed in a water bath oscillator and shaken for 3 hours at 37°C to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0259] Compound 9A (5.94 mg, 5.53 μmol) was dissolved in 400 μL of DMSO and added to the above reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: His buffer aqueous solution with pH 5.0) to obtain PBS buffer (3.77 mg / mL, 19.2 mL) of ADC-6 (hu310-2-9A), an exemplary product of conjugate TF-9A, which was stored at 4°C. Average value calculation by RP-HPLC: DAR = 4.05. Example 10-7 ADC-7 [ka]

[0260] Under conditions of 37°C, an aqueous solution of prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 16.6 μL, 166 nmol) was added to a PBS-buffered aqueous solution of the antibody hIgG1 (0.05 M PBS-buffered aqueous solution with pH=6.5, 10.0 mg / mL, 1 mL, 67.6 nmol). The mixture was placed in a water bath oscillator and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0261] Compound 9A (0.73 mg, 676 nmol) was dissolved in 60 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath oscillator and shaken at 25°C for 3 hours to stop the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-7 in PBS buffer (0.91 mg / mL, 9.8 mL), which was stored at 4°C.

[0262] Calculation of average values ​​using RP-HPLC: n=4.73. Test example: Test Example 1: Binding experiment between antibody and free human and monkey TF proteins

[0263] PBS with a pH of 7.4 AffiniPure Goat Anti-Human IgG (Jackon, 109-005-008) protein was diluted to 2 μg / mL in buffer (JPEG2026518847000121.jpg757, B320KJ) and added to a 96-well microplate at a volume of 100 μL / well, and incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added per well, the wells were blocked, and incubated at 37°C for 2 hours. After blocking, the mounting medium was discarded, the plate was washed three times with PBST buffer (pH 7.4, containing 0.1% tween-20 PBS), and then 50 μL of the target antibody solution diluted to 2 μg / mL was added per well, and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with PBST, and 50 μL of biotinylated human or monkey TF-his protein (SEQ ID No. 5 / 8) (biotin labeling kit: DO JINDO, LK03-10), which had been gradient diluted (10 μg / mL, 5-fold gradient dilution), was added per well and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with PBST, and Streptavidin-Peroxidase Polymer (sigma, S2438-250UG) was added, diluted 1:8000, and incubated at 37°C for 1 hour. After washing the plate three times with PBST, add 50 μL of TMB chromogenic substrate (KPL, 5120-0077) per well, incubate at room temperature for 5 minutes, add 50 μL of 1 M H2SO4 per well to terminate the reaction, read the absorption value at 450 nm using a microplate reader, and fit the antibody-antigen binding curve using GraphPad Prism. 50 The value was calculated. [Table 16] The antibodies disclosed herein have good binding activity to the TF antigen. Experiment Example 2: Antibody-cell binding experiment

[0264] Using MDA-MB-231 cells, NCI-H358 (abbreviated as H358 cells), and HCT116 cells, 1 × 10⁶ cells were sampled in FACS buffer (1% BSA, pH 7.4 PBS). 6 Cell suspensions of 100 μL / mL were prepared and added to 96-well round-bottom plates at 100 μL / well. After centrifugation and removal of supernatant, 100 μL / well of the target antibody, diluted to different concentrations in FACS buffer (60 μg / mL, 3-fold gradient dilution), was added and incubated at 4°C in the dark for 1 hour. After centrifugation with 300 g of FACS buffer and washing three times, working concentration APC Rat Anti-human IgG Fc (BioLegend, 410712) was added and incubated at 4°C in the dark for 40 minutes. After centrifugation with 300 g of FACS buffer and washing three times, the geometric mean of fluorescence intensity was detected by Thermo Attune Nxt flow cytometry, and EC 50 The value was calculated. [Table 17] JPEG2026518847000124.jpg26158 The antibody disclosed herein has good binding activity to cells expressing the TF antigen. Test Example 3, Affinity Binding Experiment

[0265] Using antibody samples, a 4 μg / mL solution was prepared in 1 × HBS-EP buffer solution (pH 7.4) (Cytiva, BR-1006-69). The antibody was affinity-captured using a Protein A biosensing chip (Cytiva, 29127556), and a constant concentration of the antigen molecule, human TF-His protein (SEQ ID NO: 5) or monkey TF-His protein (SEQ ID NO: 8), was flowed over the chip surface. The reaction signal was detected in real time using a Biacore T200 (Cytiva) instrument to obtain binding and dissociation curves. After dissociation was completed in each experimental cycle, the biosensing chip was washed with 10 mM glycine-HCl (pH 1.5) (Cytiva, BR-1003-54) and regenerated. A 1:1 binding model was used for data fitting. The detection results are shown in Table 18. [Table 18] The antibody disclosed herein has high affinity for the TF antigen. Test Example 4: Binding experiment between antibody and mouse and rat TF protein

[0266] Mouse TF-His protein (SEQ ID NO: 10) or rat TF-His protein (SEQ ID NO: 9) was diluted to 4 μg / mL in pH 7.4 PBS buffer and added to a 96-well microplate at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the supernatant, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added per well, the plate was blocked, and incubated at 37°C for 2 hours. After blocking, the mounting medium was discarded, the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20), and then gradient-diluted antibody solution was added at 100 μL per well. The plate was incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of Peroxidase AffiniPure Goat Anti-Human IgG (H+L) (Jackson, 109-035-003), diluted 1:4000, was added per well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 50 μL of TMB chromogenic substrate (KPL, 5120-0077) was added per well and incubated at room temperature for 5 minutes. Then, 50 μL of 1 M H2SO4 was added per well to terminate the reaction, and the absorption values ​​at 450 nm were read using a microplate reader. The antibody-antigen binding curves were fitted using GraphPad Prism (Figures 1 and 2).

[0267] The results showed that none of the proteins hu10H10-77, hu310-2, or hu67Y116 bound to mouse TF-His protein (Figure 1) or rat TF-His protein (Figure 2). Experiment Example 5: Factor-VII-Human Chromogenic-Activity Coagulation Pathway Experiment

[0268] In the TF-FVII-FX pathway, TF ultimately affects coagulation by influencing the production of FX substrates. This test is used to detect the effects of different antibodies on the coagulation pathway.

[0269] The 96-well plate from the Factor-VII-Human Chromogenic-Activity kit (Abcam, ab 108830) was left at room temperature for approximately 10 minutes, then 100 μL / well of 0.5 μg / mL FVIIa (recombinant human clotting factor VIIa for injection, i.e., NovoSeven) was added and incubated at 37°C for 2 hours. After incubation, the washing solution (included in the kit) was used. The cells were washed five times with buffer, diluted antibody was added at 40 μL / well, then a mixture of MDA-MB-231 cells and FX (included in the kit) was added at 40 μL / well, and incubated at 37°C for 30 minutes. 20 μL / well of substrate (included in the kit) was added to start the reaction. Using a microplate reader, the absorption values ​​at 405 nm were read at 3-minute intervals for a total of 30 minutes. The value in the well without antibody was taken as the maximum value (MAX), and the value in the well without FVIIa was taken as the minimum value (Min). Data within the linear range were selected, and the ratio of coagulation pathway influence was calculated. The coagulation ratios are shown in Table 19. [Table 19] The results showed that none of the three antibodies in this disclosure affected the coagulation pathway, while the positive controls TF011 and H39 did affect the coagulation pathway. Test Example 6: DT3C Endocytosis Experiment

[0270] After the DT3C protein entered the cell, activated diphtheria toxin (DT) killed the cell, indirectly reflecting the endocytosis status of the antibody. 50The in vitro endocytosis activity of the antibody was evaluated using Imax.

[0271] DT3C is a recombinant fusion protein composed of the fusion of diphtheria toxin Fragment A (toxin portion only) and the 3C fragment (IgG binding portion) of Group G Streptococcus. This protein has high affinity for the antibody's Fc structure and was able to enter cells together with the antibody when endocytosis occurred. Under the action of intracellular frin proteases, toxic DT was released. DT was able to inhibit EF2-ADP ribosylation activity, block the protein translation process, and induce cell death. DT3C that is not inside a cell does not have cell-killing activity. The endocytic activity of the antibody was evaluated based on the cell-killing status.

[0272] MDA-MB-231 cell suspension was prepared using fresh cell medium containing 20% ​​low IgG FBS (Gibco, 1921005PJ) with a cell density of 4 × 10⁶. 4 The cells were divided into 1000 cells / mL and added to a cell culture plate at 50 μL / well, meaning each well contained 2000 cells. They were then cultured in 5% carbon dioxide at 37°C for 16 hours.

[0273] A 4× concentration of DT3C (120 μg / mL) was prepared in serum-free RPMI 1640 medium and sterilized by filtration through a 0.22 μm filter. A 4× concentration of antibody (40 μg / mL) was prepared in serum-free RPMI 1640 medium. 80 μL of DT3C (approximately 70 KD) and 80 μL of antibody (approximately 145 KD) were uniformly mixed in a 1:1 ratio, incubated at room temperature for 30 minutes, and then 40 μL of the mixture was taken and added to 120 μL of serum-free RPMI 1640 medium. 160 μL of serum-free RPMI 1640 medium alone was added to the cell wells until the final antibody concentration was 10 μg / mL. The well to which only the medium was added was designated as the zero point for endocytosis rate calculation. 50 μL of diluted antibody was added to 50 μL of cells and incubated at 37°C and 5% CO2 for 3 days. Add 50 μL of CellTiter-Glo to each well. TM(CTG)(Promega, G7573) was added, incubated at room temperature in the dark for 10 minutes, and chemiluminescence was read using ENVISION. The results are shown in Table 20. [Table 20] The results indicate that the antibodies of this disclosure possess endocytotic activity. Experiment Example 7: Prothrombin Time (PT) Experiment

[0274] The effect of anti-TF antibodies on in vitro PT in human plasma was evaluated by measuring the PT values ​​of human plasma to which the antibodies were added. Venous blood was collected from the upper limb veins of volunteers, placed in 15 ml centrifuge tubes, and 3.8% trisodium citrate anticoagulant (Guoyao Group Chemical Reagents Co., Ltd., 6132-04-3) (anticoagulant:whole blood = 1:9) was pre-added to the tubes. The centrifuge was performed at room temperature at 2500 rpm for 10 minutes to collect the plasma. Working solutions were prepared using PBS (Meilen Bio, PWL050) with concentrations of the target antibody at 16000 nM, 8000 nM, 4000 nM, 2000 nM, 1000 nM, and 500 nM (final concentrations were 1600 nM, 800 nM, 400 nM, 200 nM, 100 nM, and 50 nM). 4 mL of purified water was added to the PT reagent (Shanghai Dacheng Medical Equipment Co., Ltd., OUHP29) and incubated at 37°C for 30 minutes. Following the operating manual for the CA600 coagulation analyzer (sysmex), human plasma, PT reagent, target antibody, blank control PBS, and washing agent Clean I (Shanghai Dacheng Medical Equipment Co., Ltd., GSA-500A) were placed in the designated positions on the instrument, the program was started, and the PT value was measured. The results were obtained, curve fitting was performed using GraphPad Prism, and the values ​​(Table 21) were analyzed and calculated to evaluate the effect of the antibody on PT, mainly based on the reading of the maximum antibody concentration. [Table 21] The results showed that none of the three antibodies in this disclosure affected coagulation, and their effect on PT was smaller than that of TF011 and H39. TF011 showed clear inhibition of coagulation in PT experiments. Test Example 8: Binding experiment between antibody and human rat hybrid TF protein HuRatTF

[0275] To screen for antibodies against specific epitopes that do not affect relevant human coagulation function after binding to human TF, the human-rat hybrid antigen HuRatTF protein (SEQ ID No. 11) from Example 1 was constructed and used in antibody binding experiments. The HuRatTF protein already contains partial human and rat sequences, with the coagulation-affecting site mutated to alanine (Ala).

[0276] The HuRatTF protein was diluted to 4 μg / mL in pH 7.4 PBS buffer and added to a 96-well microplate at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the supernatant, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added per well, the plate was blocked, and the plate was incubated at 37°C for 2 hours. After blocking, the mounting medium was discarded, the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20), and then 100 μL of gradient-diluted antibody solution (starting at 20 μg / mL, 5-fold gradient dilution) was added per well. The plate was incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of Peroxidase AffiniPure Goat Anti-Human IgG(H+L) (Jackson, 109-035-003), diluted 1:4000, was added per well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 50 μL of TMB chromogenic substrate (KPL, 5120-0077) was added per well and incubated at room temperature for 5 minutes. 50 μL of 1 M H2SO4 was added per well to terminate the reaction, and the absorption values ​​at 450 nm were read using a microplate reader. Antibody-antigen binding curves were fitted using GraphPad Prism, and the data are shown in Table 22. [Table 22] The results showed that the antibody TF011, which affects coagulation function, did not bind to the HuRatTF protein, while the antibody of this disclosure, which does not affect coagulation function, was able to bind to the HuRatTF protein. Test Example 9, IL-8 Cytokine Test Experiment

[0277] Using MDA-MB-231 cells, 3 × 10⁶ cells were cultured in serum-free medium. 5 A cell suspension of 100 μL / mL was prepared and added to a 96-well plate at 100 μL / well. It was incubated at 37°C under starvation conditions for 2 hours. The supernatant was discarded, and 100 μL / well of a gradient-diluted antibody (starting at 10 μg / mL, 6-fold gradient dilution) prepared in serum-free medium was added. The mixture was incubated at 37°C for 0.5 hours. The supernatant was discarded, and 120 μL / well of FVIIa (recombinant human coagulation factor VIIa for injection, NovoSeven) prepared in serum-free medium was added. The mixture was incubated at 37°C for 5 hours. After incubation, the mixture was centrifuged at 300 g for 5 minutes, and 100 μL of the supernatant was transferred to a 96-well plate in an IL-8 kit (Xinbo Sheng Bio, EHC008). The IL-8 test was performed according to the instructions. Absorption values ​​at 450 nm were read using a microplate reader, and antibody-antigen binding curves were fitted using GraphPad Prism. EC 50 The values ​​were calculated, and the results are shown in Table 23. [Table 23] The results showed that the antibody described herein exerted a potent inhibitory effect on IL8. Experiment Example 10: Cellular Activity Experiment of ADC Molecules

[0278] The purpose of this experiment is to detect the cell-killing effect of ADC samples and IC 50 The goal is to evaluate the in vitro activity of ADCs based on Imax.

[0279] HPAFII cells (human pancreatic cancer cells), MDA-MB-231 cells (human breast cancer cells), H358 cells (human non-small cell lung cancer cells), and CHO-K1 cells (hamster ovary cells) were digested with trypsin, neutralized with fresh medium, centrifuged at 1000 rpm, resuspended in culture medium, and counted. The cell suspension densities were then determined as follows: HPAFII cells 3700 cells / mL, MDA-MB-231 cells 7400 cells / mL, and H358 cells 7400 cells / mL, respectively. The cell suspension was adjusted to 3700 cells / mL of CHO-K1 cells, and 135 μL of the suspension was taken and added to a 96-well cell culture plate, with 500 cells per HPAFII cell well, 1000 cells per MDA-MB-231 cell well, 1000 cells per H358 cell well, and 500 cells per CHO-K1 cell well. Only 135 μL of medium was added to the 11th row without plating the cells, and the cells were cultured at 37°C in 5% carbon dioxide for 16 hours.

[0280] The ADC sample was diluted with PBS to 3.333 μM (10× concentration), which was used as the initial concentration. A sequential 5-fold gradient dilution was then performed using PBS to obtain a total of 9 concentrations. 15 μL of the 10× gradient dilution solution was added to each well, with the 10th and 11th wells being culture medium. Pre-set wells were placed at each concentration point, and the samples were incubated in 5% carbon dioxide at 37°C for 6 days.

[0281] After culturing was complete, 50 μL of CTG was added to each well, incubated at room temperature in the dark for 10 minutes, a white basement membrane was attached to the bottom of the cell culture plate, and the chemiluminescence was read using ENVISION. The results of the in vitro cell-killing activity are shown in Table 24. [Table 24] The results show that the above ADC has a strong killing effect in cell lines HPAFII, MDA-MB-231, and H358. Test Example 11: In vivo efficacy experiment of HPAFII using a CDX model.

[0282] This study evaluated and compared the therapeutic effects of each ADC drug on subcutaneous tumors in NUNU mice.

[0283] Each Nunu mouse (4 - 6 weeks old, ♀ SPF grade, Beijing Vital River Laboratory Animal Technology Co., Ltd.) was subcutaneously inoculated with 4×10 6 / 200 μL (containing 50% matrigel) of HPAF-II cells (human pancreatic cancer), and after the tumor grew to about 250 - 300 mm 3 , the animals were grouped based on tumor volume (D0). The mice were administered by intraperitoneal injection (IP) once a week for a total of 3 times. The administration volume once a week was 10 mL / kg, and the specific dosage and administration scheme are as shown in Table 25. The tumor volume was measured twice a week, the mouse body weight was weighed, and the data were recorded.

[0284] The experimental index is to consider the effect of the drug on tumor growth. The specific index is T / C% or tumor growth inhibition rate TGI%. The tumor diameter was measured twice a week using Vernier calipers, and the calculation formula for tumor volume (V) is V = 1 / 2 × a × b 2 , where a and b represent the length and width respectively. T / C% = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Here, T is the tumor volume of the ADC administration group, and C is the tumor volume of the control group. The tumor growth inhibition rate % (TGI%) = 100 - T / C%. When tumor regression occurs, the tumor growth inhibition rate % (TGI%) = 100 - (T - T0) / T0 × 100. When the tumor shrinks compared to the starting volume, i.e., T < T0 or C < C0, it is defined as partial regression (PR) of the tumor. When the tumor completely disappears, it is defined as complete regression (CR) of the tumor. Unless otherwise specified, for the tumor volumes of the two groups, a two-way ANOVA assay was used, and P < 0.05 is defined as having a statistically significant difference. See Table 25 for the results.

Table 25

[0285] After three administrations, antibodies in the ADC-4 (2 mg / kg) and ADC-1 (2 mg / kg) groups both showed a clear therapeutic effect against HPAF-II nude mouse subcutaneous graft tumors, with a significant difference compared to the blank control group, indicating a clear dose-dependent effect. Tumor-bearing mice showed good resistance to all of the above drugs. Test Example 12: In vivo efficacy experiment of the CDX model H358

[0286] This study evaluated and compared the therapeutic effects of each ADC drug on subcutaneous tumors in nude mice.

[0287] Each nude mouse (4-6 weeks old, female SPF grade, Shanghai Slice Laboratory Animals Co., Ltd.) was given 5 x 10 6 H358 cells (human non-small cell lung cancer) in 200 μL (containing 50% matrigel) were subcutaneously inoculated, and the tumor was approximately 300 mm. 3 After tumor growth, the animals were grouped based on tumor volume (D0). Mice were administered intraperitoneally (IP) once a week for a total of three times. The weekly dose was 10 mL / kg, and the specific dosage and administration scheme are shown in Table 26. Tumor volume was measured twice a week, mouse body weight was weighed, and the data were recorded.

[0288] The experimental indicator is to examine the effect of the drug on tumor growth, and the specific indicator is T / C% or tumor suppression rate TGI%. Tumor diameter is measured twice a week using a vernier, and the formula for calculating tumor volume (V) is V = 1 / 2 × a × b 2where a and b represent length and width respectively. T / C% = (T - T0) / (C - C0)×100, where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Here, T is the tumor volume with ADC administration, and C is the tumor volume of the control group. Tumor growth inhibition rate % (TGI%) = 100 - T / C%. When tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100 - (T - T0) / T0×100. When the tumor shrinks smaller than the starting volume, i.e., T < T0 or C < C0, it is defined as partial regression (PR) of the tumor. When the tumor completely disappears, it is defined as complete regression (CR) of the tumor. Unless otherwise specified, for the tumor volumes of two groups, a two-way ANOVA assay is used, and P < 0.05 is defined as having a statistically significant difference. See Table 26 for the results.

Table 26

[0289] After three administrations of ADC-1 (3 mg / kg, 1 mg / kg), ADC-4 (3 mg / kg, 1 mg / kg), and ADC-6 (3 mg / kg, 1 mg / kg), all had good tumor suppression effects, caused partial regression of the tumor, and the drugs showed a certain dose-dependence. The tumor-bearing mice showed good tolerance to any of the above drugs. [[ID=X]] Test Example 13, In Vivo Pharmacodynamic Experiment of CDX Model of MDA-MB-231

[0290] This test example evaluated and compared the therapeutic effects of each ADC drug on subcutaneous transplanted tumors in NOD scid mice.

[0291] Each NOD scid mouse (4 - 6 weeks old, ♀ SPF grade, Shanghai Slack Experimental Animal Co., Ltd.) was subcutaneously inoculated with 3×10 6 / 100 μL (containing 50% Matrigel) of MDA-MB-231 cells (human breast cancer), and when the tumor was about 250 - 300 mm 3After growth until, animals were grouped based on tumor volume (D0). They were administered by intraperitoneal injection (IP) once a week for a total of 3 times to mice. The administration volume once a week was 10 mL / kg. The tumor volume was measured twice a week, the body weight of the mice was weighed, and the data were recorded.

[0292] The experimental index is to consider the effect of the drug on tumor growth, and the specific index is T / C% or tumor growth inhibition rate TGI%. The tumor diameter was measured twice a week using Vernier calipers, and the calculation formula for tumor volume (V) is V = 1 / 2 × a × b 2 where a and b represent the length and width respectively. T / C% = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment. Here, T is the tumor volume of the ADC administration group, and C is the tumor volume of the control group. Tumor growth inhibition rate % (TGI%) = 100 - T / C%. When tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100 - (T - T0) / T0 × 100. When the tumor shrinks smaller than the starting volume, i.e., T < T0 or C < C0, it is defined as partial regression (PR) of the tumor. When the tumor completely disappears, it is defined as complete regression (CR) of the tumor. Unless otherwise specified, for the tumor volumes of the two groups, a two-way ANOVA assay is used, and P < 0.05 is defined as having a statistically significant difference. The tumor-bearing mice showed good tolerance to any of the above drugs, and no obvious symptoms such as weight loss occurred. PBS was used as a blank control. See Table 27 for the results.

Table 27

[0293] After three administrations, antibodies in the ADC-1 (3 mg / kg, 1 mg / kg), ADC-2 (1 mg / kg), ADC-3 (1 mg / kg), ADC-6 (3 mg / kg, 1 mg / kg), and hIgG-9A (3 mg / kg) groups all showed clear therapeutic effects against subcutaneous tumors in MDA-MB-231 mice, with significant differences compared to the blank control group, indicating clear dose-dependent efficacy. Tumor-bearing mice showed good resistance to all of the above drugs. Test Example 14: Cynomolgus monkey in vivo PK and toxicity observation experiment

[0294] This study evaluated the pharmacokinetics (PK) of ADC-1 and ADC-2 in vivo using cynomolgus monkeys, and also conducted toxicity observations.

[0295] Each cynomolgus monkey (Suzhou Xishan Zhongke Experimental Animal Co., Ltd., SCXK(Su)2018-0001) was administered intravenously at a dose of 12 mg / kg. Blood samples were collected at 0.083 h (5 min ± 1 min), 1 h (± 5 min), 6 h (± 15 min), 12 h (± 15 min), 24 h (± 30 min), 48 h (± 30 min), 96 h (± 30 min), 168 h (± 30 min), 240 h (± 60 min), 336 h (± 60 min), and 504 h (± 60 min) after administration, and drug blood concentration was detected and other toxicological observations were performed.

[0296] PK detection was performed using the LBA method (Delfia) to quantitatively detect the concentrations of total antibody and complete ADC in cynomolgus monkey serum containing ADC-1. [Table 28] [Table 29]

[0297] The total antibody and complete ADCs of the ADC-1 and ADC-2 molecules were substantially identical in half-life and AUC in cynomolgus monkeys in vivo. The ADC-1 and ADC-2 molecules exhibited good stability in cynomolgus monkeys in vivo. The second dose had lower half-life and AUC than the first dose due to the presence of immunogenicity.

[0298] During the detection of ADC-1 pharmacokinetics, blood samples were collected from all animals before the first dose, on day 2, and on day 8, and coagulation function was detected. Animals were fasted overnight before blood collection, and blood samples were collected. PT and APTT were detected using a Sysmex CS-2000i fully automated coagulation analyzer or a Sysmex CA-1500 automated coagulation analyzer. The PT and APTT detection results are shown in Figures 3 and 4. The results showed no significant prolongation of coagulation time or increased bleeding risk compared to before administration. Test Example 15, TGA Experiment

[0299] This study investigated whether the antibody hu67Y116 affects TF coagulation activity.

[0300] Human plasma contains coagulation factors other than TF from the exogenous coagulation pathway, such as FVII, FX, FV, FII, and FI. If the antibody did not affect the coagulation activity of TF, it did not affect the coagulation time and peak value of human plasma. Human plasma was prepared according to the instructions for the Technothrombin TGA Kit. A 20X antibody was prepared, and the antibody and plasma were mixed in a 1:19 ratio. This mixture was added to a 96-well black plate at a rate of 40 μL / well, followed by 10 μL / well of RCH (TGA reagent C high, included in the kit), and finally 50 μL / well of substrate to start the reaction. Fluorescence was read using a microplate reader (~360 nm / ~460 nm, excitation / emission). The results are shown in Table 30. TF011 showed significant coagulation inhibition compared to the negative control antibody, while hu67Y116 had little effect on coagulation. [Table 30]

Claims

1. An anti-TF antibody comprising a heavy chain variable region and a light chain variable region, a. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 40, HCDR2 containing the amino acid sequence of SEQ ID NO: 64 or 41, and HCDR3 containing the amino acid sequence of SEQ ID NO: 65 or 42; the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 66 or 43, LCDR2 containing the amino acid sequence of SEQ ID NO: 67 or 44, and LCDR3 containing the amino acid sequence of SEQ ID NO: 68, 83 or 45. b. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 46 for HCDR1, an amino acid sequence of SEQ ID NO: 47 for HCDR2, and an amino acid sequence of SEQ ID NO: 48 for HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 49, 111, 112, or 113 for LCDR1, an amino acid sequence of SEQ ID NO: 50 for LCDR2, and an amino acid sequence of SEQ ID NO: 51 for LCDR3. c. The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 73 or 144, HCDR2 containing the amino acid sequence of SEQ ID NO: 74, 142, 145 or 146, and HCDR3 containing the amino acid sequence of SEQ ID NO: 75 or 143; the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 76, 151, 147 or 150, LCDR2 containing the amino acid sequence of SEQ ID NO: 77 or 148, and LCDR3 containing the amino acid sequence of SEQ ID NO: 78 or 149. Preferably, a. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 40 for HCDR1, an amino acid sequence of SEQ ID NO: 64 for HCDR2, an amino acid sequence of SEQ ID NO: 65 for HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 66 for LCDR1, an amino acid sequence of SEQ ID NO: 67 for LCDR2, an amino acid sequence of SEQ ID NO: 68 or 83 for LCDR3, or The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 40 for HCDR1, an amino acid sequence of SEQ ID NO: 41 for HCDR2, and an amino acid sequence of SEQ ID NO: 42 for HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 43 for LCDR1, an amino acid sequence of SEQ ID NO: 44 for LCDR2, and an amino acid sequence of SEQ ID NO: 45 for LCDR3. b. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 46 for HCDR1, an amino acid sequence of SEQ ID NO: 47 for HCDR2, and an amino acid sequence of SEQ ID NO: 48 for HCDR3, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 49 for LCDR1, an amino acid sequence of SEQ ID NO: 50 for LCDR2, and an amino acid sequence of SEQ ID NO: 51 for LCDR3. c. An anti-TF antibody in which the heavy chain variable region comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 73, HCDR2 containing the amino acid sequence of SEQ ID NO: 74, and HCDR3 containing the amino acid sequence of SEQ ID NO: 75, and the light chain variable region comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 76 or 151, LCDR2 containing the amino acid sequence of SEQ ID NO: 77 or 148, and LCDR3 containing the amino acid sequence of SEQ ID NO:

78.

2. The anti-TF antibody according to claim 1, which is a mouse antibody, a chimeric antibody, or a humanized antibody, preferably a chimeric antibody or a humanized antibody, and more preferably a humanized antibody.

3. It includes a heavy chain variable region and a light chain variable region, a. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62, 58, 130, 131, or 132, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 63, 81, 59, 133, 134, or 135, or the heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 37, b. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52 or 114, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 53, 115, 116, 117, 118, 119, 120 or 121, or the heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 38, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 39, c. The heavy chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 71, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, and the light chain variable region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 72, 97, 102, 98, 99, 100, or 101. Preferably, a. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 62, 58, 130, 131, or 132, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 63, 81, 59, 133, 134, or 135, or the heavy chain variable region includes an amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 37, b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 52 or 114, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 53, 115, 116, 117, 118, 119, 120 or 121, or the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 38, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 39, c. The heavy chain variable region includes an amino acid sequence of SEQ ID NO: 71, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, and the light chain variable region includes an amino acid sequence of SEQ ID NO: 72, 97, 102, 98, 99, 100, or 101. more, a. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 62, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 63 or 81, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 36, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 37, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 130, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 131, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 132, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 133, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 130, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 131, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 132, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 134, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 130, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 131, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 132, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 59, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 130, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 135, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 131, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 135, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 58, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 135, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 132, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

135. b. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 52, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 53, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 38, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

39. c. The anti-TF antibody according to claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 72, 97, or 102.

4. An anti-TF antibody according to any one of claims 1 to 3, which is an antibody fragment, preferably selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, and dAb.

5. An anti-TF antibody according to any one of claims 1 to 4, comprising a heavy chain constant region and a light chain constant region, preferably the heavy chain constant region being a human IgG1, IgG2, IgG3, and IgG4 heavy chain constant region, and the light chain constant region being a human κ or λ chain constant region, more preferably the heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 54, and the light chain constant region comprising the amino acid sequence of SEQ ID NO:

55.

6. a. A heavy chain having at least 80% sequence identity with SEQ ID NO: 69, 60, 136, 137, or 138, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 70, 82, 61, 139, 140, or 141, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 107, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 108, b. A heavy chain having at least 80% sequence identity with SEQ ID NO: 56 or 122, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 57, 123, 124, 125, 126, 127, 128 or 129, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 109, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 110, c. An antibody comprising a heavy chain and a light chain, selected from any one of the following: a heavy chain having at least 80% sequence identity with SEQ ID NO: 79, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 or 164, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 80, 165, 170, 166, 167, 168 or 169. Preferably, a. A heavy chain having at least 80% sequence identity with SEQ ID NO: 69, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 70 or 82, A heavy chain having at least 80% sequence identity with SEQ ID NO: 60, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 61, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 107, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 108, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 136, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 139, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 137, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 139, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 60, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 139, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 138, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 139, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 136, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 140, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 137, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 140, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 60, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 140, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 138, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 140, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 136, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 61, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 137, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 61, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 138, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 61, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 136, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 141, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 137, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 141, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 60, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 141, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 138, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 141, b. A heavy chain having at least 80% sequence identity with SEQ ID NO: 56, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 57, or A heavy chain having at least 80% sequence identity with SEQ ID NO: 109, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 110, c. A heavy chain having at least 80% sequence identity with SEQ ID NO: 79, and / or a light chain having at least 80% sequence identity with SEQ ID NO: 80, 165, or 170. more, a. The heavy chain contains the amino acid sequence of SEQ ID NO: 69, and the light chain contains the amino acid sequence of SEQ ID NO: 70, or The heavy chain contains the amino acid sequence of SEQ ID NO: 69, and the light chain contains the amino acid sequence of SEQ ID NO: 82, or The heavy chain contains the amino acid sequence of SEQ ID NO: 60, and the light chain contains the amino acid sequence of SEQ ID NO: 61, or The heavy chain contains the amino acid sequence of SEQ ID NO: 107, and the light chain contains the amino acid sequence of SEQ ID NO: 108, or The heavy chain contains the amino acid sequence of SEQ ID NO: 136, and the light chain contains the amino acid sequence of SEQ ID NO: 139, or The heavy chain contains the amino acid sequence of SEQ ID NO: 137, and the light chain contains the amino acid sequence of SEQ ID NO: 139, or The heavy chain contains the amino acid sequence of SEQ ID NO: 60, and the light chain contains the amino acid sequence of SEQ ID NO: 139, or The heavy chain contains the amino acid sequence of SEQ ID NO: 138, and the light chain contains the amino acid sequence of SEQ ID NO: 139, or The heavy chain contains the amino acid sequence of SEQ ID NO: 136, and the light chain contains the amino acid sequence of SEQ ID NO: 140, or The heavy chain contains the amino acid sequence of SEQ ID NO: 137, and the light chain contains the amino acid sequence of SEQ ID NO: 140, or The heavy chain contains the amino acid sequence of SEQ ID NO: 60, and the light chain contains the amino acid sequence of SEQ ID NO: 140, or The heavy chain contains the amino acid sequence of SEQ ID NO: 138, and the light chain contains the amino acid sequence of SEQ ID NO: 140, or The heavy chain contains the amino acid sequence of SEQ ID NO: 136, and the light chain contains the amino acid sequence of SEQ ID NO: 61, or The heavy chain contains the amino acid sequence of SEQ ID NO: 137, and the light chain contains the amino acid sequence of SEQ ID NO: 61, or The heavy chain contains the amino acid sequence of SEQ ID NO: 138, and the light chain contains the amino acid sequence of SEQ ID NO: 61, or The heavy chain contains the amino acid sequence of SEQ ID NO: 136, and the light chain contains the amino acid sequence of SEQ ID NO: 141, or The heavy chain contains the amino acid sequence of SEQ ID NO: 137, and the light chain contains the amino acid sequence of SEQ ID NO: 141, or The heavy chain contains the amino acid sequence of SEQ ID NO: 60, and the light chain contains the amino acid sequence of SEQ ID NO: 141, or The heavy chain comprises the amino acid sequence of SEQ ID NO: 138, and the light chain comprises the amino acid sequence of SEQ ID NO:

141. b. The heavy chain contains the amino acid sequence of SEQ ID NO: 56, and the light chain contains the amino acid sequence of SEQ ID NO: 57, or The heavy chain comprises the amino acid sequence of SEQ ID NO: 109, and the light chain comprises the amino acid sequence of SEQ ID NO:

110. c. The heavy chain contains the amino acid sequence of SEQ ID NO: 79, and the light chain contains the amino acid sequence of SEQ ID NO: 80, or The heavy chain contains the amino acid sequence of SEQ ID NO: 79, and the light chain contains the amino acid sequence of SEQ ID NO: 165, or The anti-TF antibody according to any one of claims 1 to 5, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 79, and the light chain comprises the amino acid sequence of SEQ ID NO:

170.

7. A nucleic acid molecule encoding an anti-TF antibody according to any one of claims 1 to 6.

8. A host cell comprising the nucleic acid molecule described in claim 7.

9. An immunoconjugate comprising an anti-TF antibody and an effector molecule according to any one of claims 1 to 6, wherein the effector molecule is conjugated to the anti-TF antibody, and preferably the effector molecule is selected from an antitumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

10. An antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 Here, Y is -O-(CR ａ R ｂ ) ｍ -CR １ R ２ -C(O)-, Here, R ａ and R ｂ They are either the same or different, and each is independently a hydrogen atom, a deuterium atom, a halogen, and C １－６ Selected from alkyl groups, R １ is a C ３－６ cycloalkyl-C １－６ alkyl group or a C ３－６ cycloalkyl group, and R ２ This is a hydrogen atom, halo C １－６ Alkyl and C ３－６ Selected from cycloalkyl groups, Or, R １ and R ２ C ３－６ Forms a cycloalkyl group, m is an integer between 0 and 4. L is a linker unit, n is 1 to 10, preferably 1 to 8, and more preferably 2 to 8. Pc is an anti-TF antibody according to any one of claims 1 to 6, an antibody-drug conjugate represented by the general formula (Pc-L-Y-D), or a pharmaceutically acceptable salt thereof.

11. Linker unit -L- is -L １ -L ２ -L ３ -L ４ - and Here, L １ is -(succinimido-3-yl-N)-W-C(O)-,-CH ２ -C(O)-NR ３ Selected from -W-C(O)- and -C(O)-W-C(O)-, where W is C １－６ Alkylene group and C １－６ Alkylene-C ３－６ Selected from cycloalkyl groups, where C １－６ Alkylene group or C １－６ Alkylene-C ３－６ Each cycloalkyl group can be independently and optionally selected as a halogen, hydroxyl group, cyano group, amino group, or C １－６ Alkyl, Halo C １－６ Alkyl alkyl groups, deuterated C １－６ alkyl group, C １－６ Alkoxy groups and C ３－６ Substituted with one or more substituents selected from cycloalkyl groups, L ２ -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ ) p １ Selected from C(O)- and chemical bonds, where p １ These are integers from 1 to 20, L ３ This is a peptide residue consisting of 2 to 7 amino acid residues, where the amino acid residues are amino acids selected from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ４ -NR ５ (CR ６ R ７ ) ｔ -, -C(O)NR ５ -, -C(O)NR ５ (CH ２ ) ｔ - Selected from chemical bonds, where t is an integer from 1 to 6, R ３ , R ４ and R ５ They are either the same or different, and each is independently a hydrogen atom, C １－６ Alkyl, Halo C １－６ Alkyl alkyl groups, deuterated C １－６ Alkyl and C １－６ Selected from hydroxyalkyl groups, R ６ and R ７ They are either the same or different, and each is independently a hydrogen atom, halogen, and C １－６ Alkyl, Halo C １－６ Alkyl alkyl groups, deuterated C １－６ Alkyl and C １－６ Selected from hydroxyalkyl groups, Preferably, the linker unit -L １ -L ２ -L ３ -L ４ - is as follows: L １ teeth, 【Chemistry 2】 And here, s １ These are integers from 2 to 8, L ２ It is a chemical bond, L ３ is a tetrapeptide residue, preferably L ３ This is a tetrapeptide residue shown in GGFG (SEQ ID NO: 105), L ４ is - NR ５ (CR ６ R ７ )t−, where R ５ , R ６ or R ７ is the same or different and each independently is a hydrogen atom or a C １－６ alkyl group, and t is 1 or 2, Here, the L of -L- １ The end is connected to Pc, L ４ The terminal is linked to Y, and the antibody-drug conjugate represented by the general formula (Pc-L-Y-D) as described in claim 10, or a pharmaceutically acceptable salt thereof.

12. General formula (Pc-L ａ The antibody-drug conjugate shown in (-Y-D) or a pharmaceutically acceptable salt thereof, 【Transformation 3】 Here, Pc is an anti-TF antibody according to any one of claims 1 to 6, m is an integer between 0 and 4. n is between 1 and 10. R １ is a C ３－６ cycloalkyl-C １－６ alkyl group or a C ３－６ cycloalkyl group, and R ２ This is a hydrogen atom, halo C １－６ Alkyl and C ３－６ Selected from cycloalkyl groups, Or, R １ and R ２ C ３－６ Forms a cycloalkyl group, W is C １－６ Alkylene group and C １－６ Alkylene-C ３－６ Selected from cycloalkyl groups, where C １－６ Alkylene group and C １－６ Alkylene-C ３－６ Each cycloalkyl group is independently and optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ２ -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ ) p １ Selected from C(O)- and chemical bonds, where p １ These are integers from 1 to 20, L ３ This is a peptide residue consisting of 2 to 7 amino acid residues, where the amino acid residues are amino acids selected from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R ４ and R ５ This is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups. R ６ and R ７ The antibody-drug conjugate represented by the general formula (Pc-L-Y-D) according to claim 10 or 11, or a pharmaceutically acceptable salt thereof, wherein the atoms are the same or different and each is independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

13. An antibody-drug conjugate represented by the general formula (Pc-9-A) or a pharmaceutically acceptable salt thereof, 【Chemistry 4】 Here, n is between 1 and 8. Pc is an anti-TF antibody according to any one of claims 1 to 6, an antibody-drug conjugate represented by the general formula (Pc-L-Y-D) according to any one of claims 10 to 12, or a pharmaceutically acceptable salt thereof.

14. General formula (Pc-L ａ A method for producing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in -Y-D), 【Transformation 5】 After Pc reduction, the general formula (L ａ The compound shown in (-Y-D) is conjugated and reacted with the compound shown in the general formula (Pc-L ａ The process includes the step of obtaining an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in (-Y-D), Here, Pc is an anti-TF antibody according to any one of claims 1 to 6, W, L ２ , L ３ , R １ , R ２ , R ５ ~R ７ A method wherein m and n are as defined in claim 12.

15. A pharmaceutical composition comprising an anti-TF antibody according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 7, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 10 to 13, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

16. An application of an anti-TF antibody according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 7, or an antibody-drug conjugate according to any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 15, in the manufacture of a drug for treating tumors or cancer, Preferably, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, fallopian tube cancer, peritoneal cancer, colorectal cancer, head and neck cancer, and squamous cell carcinoma.

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