WO2024109944A1 - Anticorps anti-liv-1, conjugué médicamenteux associé et utilisation médicale associée - Google Patents

Anticorps anti-liv-1, conjugué médicamenteux associé et utilisation médicale associée Download PDF

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WO2024109944A1
WO2024109944A1 PCT/CN2023/134161 CN2023134161W WO2024109944A1 WO 2024109944 A1 WO2024109944 A1 WO 2024109944A1 CN 2023134161 W CN2023134161 W CN 2023134161W WO 2024109944 A1 WO2024109944 A1 WO 2024109944A1
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seq
amino acid
acid sequence
liv
cancer
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Chinese (zh)
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董丽伟
张伟
陈思萌
王利利
廖成
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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    • A—HUMAN NECESSITIES
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Definitions

  • the present invention discloses LIV-1 binding molecules, their immunoconjugates, medical uses, and preparation methods.
  • the present invention also relates to pharmaceutical compositions comprising the LIV-1 binding molecules or their immunoconjugates, methods for treating and preventing diseases, and methods for detecting LIV-1.
  • Human LIV-1 (also known as ZIP6, zinc ion transporter, encoding gene SLC39A6, hereinafter referred to as hLIV-1) is a member of the LIZ subfamily of the zinc ion transporter ZIP family. It is an 8-transmembrane cell membrane protein with a full length of 755 amino acid residues, including a long extracellular N-terminus and a short extracellular C-terminus.
  • the fourth transmembrane region of hLIV-1 contains a conserved metalloproteinase motif (HEXPHE) unique to the LIV-1 superfamily, and the N-terminus of the extracellular region contains 4 predicted N-glycosylation sites and histidine-rich repeats (Kathryn M. TAYLOR1, Helen E. Biochem J 2003; 375: 51–9).
  • HEXPHE conserved metalloproteinase motif
  • LIV-1 As a zinc ion transporter, LIV-1 has the function of transferring zinc ions from outside the cell into the cell, and plays an important role in maintaining the homeostasis of zinc ions in the cell. LIV-1 also participates in regulating the mesenchymal transformation of cells by regulating the Snail and E-cadherin signaling pathways. In the zebrafish model, mesenchymal transformation disorders occurred after LIV-1 knockout (Yamashita S. et al, Nature 2004; 429: 298-302). In addition, cellular level studies have shown that LIV-1 on the cell surface can promote the survival and migration of ovarian cancer cells (Zhao L. et al, BBRC 2007; 363: 82-8). In animal models, it was also observed that in two LIV-1 overexpressing prostate cell line mouse models, LIV-1 can promote the growth of cell lines in mice (Lue HW. et al, PLoS ONE 2011; 6: e27720).
  • LIV-1 is an estrogen-induced gene (Manning DL. et al, Mol Cell Endocrinol 1988; 59: 205-12), which is specifically expressed in breast, prostate, and testicular tissues. Immunohistochemical analysis of clinical samples showed that hLIV-1 is highly expressed in a variety of epithelial cell cancers such as breast cancer, prostate cancer, ovarian cancer, lung cancer, and digestive tract tumors, and its expression level is much higher than that in normal tissues (Mol Cancer Ther. 2014 Dec; 13 (12): 2991-3000; Seagen Phase 2 trial, ESMO 2020). In triple-negative breast cancer, tumor tissue sections of 20 patients showed that 65% of the patients' tumors were positive for LIV-1 (CN103533957B).
  • SGN-LIV-1A Ladiratuzumab Vedotin
  • ADC drug targeting LIV-1 which uses the anti-LIV-1 antibody hLIV22 and MMAE as a toxin (DAR value of 4).
  • the hLIV22 antibody is connected to MMAE with a protease-degradable mc-val-cit-PABC linker.
  • SGN-LIV-1A shows good anti-tumor activity in breast cancer and has no target-mediated toxic side effects.
  • the present disclosure provides LIV-1 binding molecules, immunoconjugates, and medical uses and preparation methods, as well as pharmaceutical compositions comprising LIV-1 binding molecules and immunoconjugates, methods for treating and preventing diseases, and methods for detecting LIV-1.
  • the present disclosure provides LIV-1 binding molecules comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein:
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 20 or 27, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO: 21 or 22;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:4, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:5;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:6, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:7; or,
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in any one of SEQ ID NO:23-25, 28, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:26.
  • CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact definition schemes. In some specific embodiments, the CDRs are defined according to the Kabat definition scheme.
  • the LIV-1 binding molecule comprises a VH and a VL as shown in any of the following:
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO: 4, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO: 5.
  • VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:20
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:21;
  • the VH comprises the same HCDR1, HCDR2 and HCDR3 as those in the amino acid sequence shown in SEQ ID NO:20
  • the VL comprises LCDR1, LCDR2 and LCDR3 as those in the amino acid sequence shown in SEQ ID NO:22;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:27
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:21;
  • the VH comprises the same HCDR1, HCDR2 and HCDR3 as those in the amino acid sequence shown in SEQ ID NO:27
  • the VL comprises LCDR1, LCDR2 and LCDR3 as those in the amino acid sequence shown in SEQ ID NO:22;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:6
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:7;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:23
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:26;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:24
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:26;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:25
  • the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:26;
  • the VH comprises the same HCDR1, HCDR2 and HCDR3 as in the amino acid sequence shown in SEQ ID NO:28, and the VL comprises LCDR1, LCDR2 and LCDR3 as in the amino acid sequence shown in SEQ ID NO:26.
  • the LIV-1 binding molecule comprising VH and/or VL, comprises:
  • the VH comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or 14;
  • the VH comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9 or 15;
  • the VH comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10 or 16;
  • the VL comprises LCDR1, and the LCDR1 comprises the amino acid set forth in SEQ ID NO: 42 or 17 sequence;
  • the VL comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or 18;
  • the VL comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:13 or 19.
  • the amino acid sequence shown in SEQ ID NO:42 is RSSQSLERSXGNTFLN, and X is selected from T or N.
  • X is T
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:41 (RSSQSLERSTGNTFLN);
  • X is N
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:11 (RSSQSLERSNGNTFLN).
  • the LIV-1 binding molecule comprises a VH and a VL, wherein:
  • the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 8-10
  • the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 41, 12, and 13;
  • the VH comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NO:8-10
  • the VL comprises LCDR1, LCDR2, LCDR3 shown in SEQ ID NO:11-13;
  • the VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 14-16
  • the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 17-19;
  • the LIV-1 binding molecule comprises a VH and a VL, wherein:
  • Any HCDR contained in the VH shown has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any HCDR in the VH shown in any of a-1), a-2), and b); and/or,
  • Any LCDR contained in the VL has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any LCDR in the VL shown in any of a-1), a-2), and b).
  • the amino acid mutations in the HCDR or LCDR are conservative substitutions.
  • the CDR in the present disclosure may also be a CDR sequence defined by other numbering systems.
  • the following examples provide HCDR1, HCDR2, HCDR3 in the VH shown in SEQ ID NO:27, and LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO:21, as defined by the IMGT, Chothia, AbM, or Contact numbering systems.
  • the CDR sequences defined by the IMGT numbering system are as follows:
  • HCDR1 DYTFSSYW (SEQ ID NO: 47)
  • HCDR2 IHPDSGRT (SEQ ID NO: 48)
  • HCDR3 ARSSVYVGFDY (SEQ ID NO: 49)
  • LCDR3 LQLTHVPLT (SEQ ID NO: 51)
  • CDR sequences defined by the Chothia numbering system are as follows:
  • HCDR1 DYTFSSY (SEQ ID NO: 52)
  • HCDR2 HPDSGR (SEQ ID NO: 53)
  • LCDR1 RSSQSLERSTGNTFLN (SEQ ID NO:55)
  • LCDR3 LQLTHVPLT (SEQ ID NO: 51)
  • the CDR sequences defined by the AbM numbering system are as follows:
  • HCDR1 DYTFSSYWMH (SEQ ID NO: 57)
  • HCDR2 MIHPDSGRTN (SEQ ID NO: 58)
  • LCDR1 RSSQSLERSTGNTFLN (SEQ ID NO:55)
  • LCDR3 LQLTHVPLT (SEQ ID NO: 51)
  • the CDR sequence defined by the Contact numbering system is as follows:
  • HCDR2 WIGMIHPDSGRTN (SEQ ID NO: 60)
  • HCDR3 ARSSVYVGFD (SEQ ID NO: 61)
  • LCDR1 ERSTGNTFLNWY (SEQ ID NO: 62)
  • LCDR2 LLIYRVSTRF (SEQ ID NO: 63)
  • LCDR3 LQLTHVPL (SEQ ID NO: 64)
  • the present disclosure relates to a LIV-1 binding molecule comprising any of the aforementioned VH and/or VL that specifically binds to LIV-1.
  • the LIV-1 binding molecule specifically binds to human LIV-1.
  • the LIV-1 binding molecule specifically binds to the extracellular domain of human LIV-1.
  • the LIV-1 binding molecule has cell internalization activity. In some specific embodiments, the LIV-1 binding molecule specifically binds to human LIV-1 and has internalization activity of tumor cells. In some specific embodiments, the LIV-1 binding molecule specifically binds to the extracellular domain of human LIV-1 and has internalization activity of tumor cells.
  • the LIV-1 binding molecule is an anti-LIV-1 antibody or an antigen-binding fragment thereof.
  • the antibody is selected from a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, or a fragment thereof. In some specific embodiments, the antibody is a humanized antibody, a human antibody, or a fragment thereof.
  • the LIV-1 binding molecule is a humanized antibody
  • the humanized light chain template can be at least one of IGKV2-29*02, IGKV2-30*01, IGKV2-28*01, IGKJ4*01
  • the humanized heavy chain template can be at least one of IGHV1-2*02, IGHV3-21*01, IGHJ4*01.
  • the LIV-1 binding molecule comprises a VH and a VL, wherein:
  • the VH comprises FR1 to FR3 derived from IGHV1-2*02, and FR4 derived from IGHJ4*01;
  • the VL comprises FR1 to FR3 derived from IGKV2-29*02, and FR4 derived from IGKJ4*01;
  • the VH comprises FR1 to FR3 derived from IGHV3-21*01, and FR4 derived from IGHJ4*01; the VL comprises FR1 to FR3 derived from IGKV2-28*01, and FR4 derived from IGKJ4*01.
  • the humanized antibody or fragment thereof further comprises a back mutation to VH, VL.
  • the back mutation is located in the framework region (FR) of VH and/or VL.
  • the back mutation is located in the CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) of VH and/or VL.
  • the LIV-1 binding molecule comprises a VH and a VL, wherein:
  • the VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4, 6, 20, 27, 23-25 and 28, or an amino acid sequence having at least 80% sequence identity thereto; and/or,
  • the VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 5, 7, 21-22 and 26, or an amino acid sequence having at least 80% sequence identity therewith.
  • the LIV-1 binding molecule comprises the VH and/or VL of any of the following:
  • the VH comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:20, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VL comprises the amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises the amino acid sequence as shown in SEQ ID NO:23, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VL comprises the amino acid sequence as shown in SEQ ID NO:26, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises the amino acid sequence as shown in SEQ ID NO: 24, or has at least 90%
  • the VL comprises the amino acid sequence shown in SEQ ID NO: 26, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises the amino acid sequence as shown in SEQ ID NO:25, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VL comprises the amino acid sequence as shown in SEQ ID NO:26, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 90% sequence identity thereto.
  • the LIV-1 binding molecule wherein:
  • VH is shown in SEQ ID NO:4, and the VL is shown in SEQ ID NO:5;
  • VH is shown in SEQ ID NO: 6
  • VL is shown in SEQ ID NO: 7;
  • VH is shown in SEQ ID NO: 20
  • VL is shown in SEQ ID NO: 21;
  • VH is shown in SEQ ID NO: 20
  • VL is shown in SEQ ID NO: 22;
  • VH is shown in SEQ ID NO: 27, and the VL is shown in SEQ ID NO: 21;
  • VH is shown in SEQ ID NO: 27, and the VL is shown in SEQ ID NO: 22;
  • VH is shown in SEQ ID NO: 23
  • VL is shown in SEQ ID NO: 26;
  • VH is shown in SEQ ID NO: 24, and the VL is shown in SEQ ID NO: 26;
  • VH is shown in SEQ ID NO: 25
  • VL is shown in SEQ ID NO: 26; or,
  • the VH is shown in SEQ ID NO:28, and the VL is shown in SEQ ID NO:26.
  • a LIV-1 binding molecule comprises a VH and a VL, wherein:
  • the VH has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the VH shown in any one of 1) to 10); and/or,
  • the VL has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the VL shown in any one of 1)-10).
  • amino acid mutations in VH or VL are conservative substitutions.
  • the LIV-1 binding molecule further comprises a constant region.
  • the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4 or a variant thereof
  • the light chain constant region can be derived from a ⁇ , ⁇ chain or a variant thereof.
  • the LIV-1 binding molecule is a murine antibody or a fragment thereof, whose heavy chain further comprises a murine IgG1, IgG2, IgG3, IgG4 or a variant thereof heavy chain constant region, and whose light chain further comprises a murine ⁇ , ⁇ chain or a variant thereof light chain constant region.
  • the LIV-1 binding molecule is a human antibody or a fragment thereof, whose heavy chain further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and whose light chain further comprises a light chain constant region of human ⁇ , ⁇ chain or a variant thereof.
  • the LIV-1 binding molecule is a chimeric antibody, which comprises a heavy chain further comprising a rabbit IgG1, IgG2, IgG3, IgG4 or a variant thereof heavy chain constant region, and a light chain further comprising a rabbit ⁇ , The light chain constant region of a lambda chain or a variant thereof.
  • the Fc region of the constant region of the LIV-1 binding molecule is derived from IgG1, IgG2, IgG3 or IgG4.
  • the Fc of IgG1 comprises an amino acid sequence as shown in SEQ ID NO: 29 or 43 or an amino acid sequence having at least 80% sequence identity thereto.
  • the LIV-1 binding molecule comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 30, 33-34, 36-38 and 45, or an amino acid sequence having at least 80% sequence identity thereto; and/or,
  • the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:31-32, 35 and 46, or an amino acid sequence having at least 80% sequence identity thereto.
  • the LIV-1 binding molecule comprises a heavy chain and/or a light chain as described in any one of the following:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% sequence identity thereto; or,
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 45 or has at least 80% sequence homology thereto.
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 46 or an amino acid sequence having at least 80% sequence identity thereto.
  • the LIV-1 binding molecule comprises a heavy chain and a light chain, wherein:
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain is shown in SEQ ID NO: 31;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain is shown in SEQ ID NO: 32;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain is shown in SEQ ID NO: 31;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain is shown in SEQ ID NO: 32;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:34, and the amino acid sequence of the light chain is shown in SEQ ID NO:35;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:36, and the amino acid sequence of the light chain is shown in SEQ ID NO:35;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain is shown in SEQ ID NO: 35;
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38, and the amino acid sequence of the light chain is shown in SEQ ID NO:35; or,
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:45
  • amino acid sequence of the light chain is shown in SEQ ID NO:46.
  • the LIV-1 binding molecule comprises a heavy chain and a light chain, wherein:
  • the heavy chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the heavy chain of any of the preceding items; and/or,
  • the light chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the light chain described in any of the above.
  • amino acid mutations in the heavy or light chain are conservative substitutions.
  • the LIV-1 binding molecule is an antigen-binding fragment of an anti-LIV-1 antibody, including but not limited to any of the following: Fab, Fv, sFv, Fab', F(ab') 2 , linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv), for example, specifically scFv, Fv, Fab or Fab' fragments.
  • the LIV-1 binding molecule is a fusion protein comprising any of the aforementioned anti-LIV-1 antibodies or antigen-binding fragments thereof.
  • At least 80% encompasses 80% and above, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
  • any of the aforementioned LIV-1 binding molecules has at least one of the following properties:
  • the LIV-1 binding molecule binds to LIV-1 on the cell surface and is internalized (or, endocytosed) into the cell. In some specific embodiments, the LIV-1 binding molecule binds to the extracellular domain of LIV-1 and is internalized into the cell. In some specific embodiments, the LIV-1 binding molecule in the present disclosure has enhanced cellular internalization activity compared to the hLIV22 antibody (sequence see Publication No.: US 2016/0185858 A1, which is incorporated herein by reference in its entirety).
  • the LIV-1 binding molecules have tumor cell internalization activity. In some specific embodiments, the LIV-1 binding molecules of the present disclosure have enhanced tumor cell internalization activity compared to hLIV22 antibodies.
  • the LIV-1 binding molecule and the hLIV22 antibody have different antigenic epitopes. In some embodiments, the LIV-1 binding molecule and the hLIV22 antibody have partially overlapping antigenic epitopes.
  • the LIV-1 binding molecule binds to human LIV-1 with a K value of less than or equal to 100 nM, such as less than or equal to about 75 nM, about 50 nM, about 25 nM, about 50 nM, about 10 nM, about 5 nM, etc.
  • the present disclosure provides a LIV-1 binding molecule that binds to the same epitope as the aforementioned LIV-1 binding molecule.
  • LIV-1 binding molecules are provided that cross-block the binding of the aforementioned LIV-1 binding molecules to human LIV-1.
  • LIV-1 binding molecules whose binding to human LIV-1 is cross-blocked by the aforementioned LIV-1 binding molecules.
  • the present disclosure provides an immunoconjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL) of any of the aforementioned LIV-1 binding molecules.
  • the immunoconjugate comprises a LIV-1 binding molecule, and an effector molecule conjugated to the LIV-1 binding molecule.
  • the LIV-1 binding molecule is any one of the aforementioned LIV-1 binding molecules.
  • effector molecules include, but are not limited to, cytotoxic drugs, immunomodulators, and cytostatic agents.
  • cytotoxic drugs such as small molecules of bacterial, fungal, plant, or animal origin
  • toxins such as small molecules of bacterial, fungal, plant, or animal origin
  • toxins or enzymatically active toxins such as small molecules of bacterial, fungal, plant, or animal origin
  • radioactive isotopes such as small molecules of bacterial, fungal, plant, or animal origin
  • chemotherapeutic drugs such as antibiotics and nucleolytic enzymes, etc.
  • the immunoconjugate disclosed in the present invention can bind to the extracellular domain of LIV-1 and then undergo endocytosis, and the effector molecules are released inside the cells, which can not only effectively exert cell killing and inhibitory effects, but also have a good bystander effect.
  • the effector molecule is selected from MMAF or a derivative thereof, MMAE or a derivative thereof, exitecan or a derivative thereof, eribulin or a derivative thereof. In some specific embodiments, the effector molecule is exitecan or a derivative thereof.
  • the immunoconjugate is an antibody or fragment thereof-exitecan or a derivative thereof conjugate, which is represented by the general formula (Pc-LYD) of formula (I):
  • Y is selected from -O-( CRaRb ) m - CR1R2- C ( O )- , -O- CR1R2- ( CRaRb ) m- , -O-CR1R2-, -NH-(CRaRb)m-CR1R2-C(O)- and -S-(CRaRb)m - CR1R2 - C ( O ) -;
  • Ra and Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group and a heterocyclic group; or, Ra and Rb together with the carbon atom to which they are attached form a cycloalkyl group and a heterocyclic group;
  • R1 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl
  • R2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, R1 and R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;
  • Ra and R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group
  • n is an integer from 0 to 4.
  • n 1 to 10
  • n is a decimal or an integer, preferably, n is 2 to 8 or 5 to 9;
  • Pc is any one of the LIV-1 binding molecules disclosed herein.
  • Ra and Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;
  • R1 is C3-6 cycloalkylalkyl or C3-6 cycloalkyl, hydrogen atom;
  • R2 is selected from hydrogen atom, haloalkyl or C3-6 cycloalkyl; preferably hydrogen atom;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • m 0 or 1.
  • the conjugate of the antibody or fragment thereof-exitecan or a derivative thereof of the present disclosure wherein the structural unit -Y- is -O-(CH 2 )m-CR 1 R 2 -C(O)-;
  • R1 is C3-6 cycloalkylalkyl or C3-6 cycloalkyl, hydrogen atom;
  • R2 is selected from a hydrogen atom, a haloalkyl group or a C3-6 cycloalkyl group
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • m 0 or 1.
  • the conjugate of the antibody or fragment thereof-exitecan or a derivative thereof of the present disclosure wherein the structural unit -Y- is -O-(CH 2 )m-CR 1 R 2 -C(O)-;
  • R1 is C3-6 cycloalkylalkyl or C3-6 cycloalkyl, hydrogen atom;
  • R2 is a hydrogen atom
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • m 0 or 1.
  • the conjugate of the antibody or fragment thereof-exitecan or a derivative thereof of the present disclosure wherein the structural unit -Y- is -O-(CH 2 )m-CR 1 R 2 -C(O)-;
  • R1 is C3-6 cycloalkylalkyl or C3-6 cycloalkyl, hydrogen atom;
  • R2 is a hydrogen atom
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • the conjugate of the antibody or fragment thereof-exitecan or a derivative thereof of the present disclosure wherein the structural unit -Y- is -O-(CH 2 )m-CR 1 R 2 -C(O)-;
  • R1 is a hydrogen atom
  • R2 is a hydrogen atom
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • Y is selected from:
  • the O end of Y is connected to the linker unit L.
  • the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, which is shown in the general formula (Pc-L-D1) of formula (II):
  • R1 is a hydrogen atom, a C3-6 cycloalkylalkyl group or a C3-6 cycloalkyl group;
  • R2 is selected from hydrogen atom, haloalkyl or C3-6 cycloalkyl; preferably hydrogen atom;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • n 0 or 1
  • n is 1 to 10, and can be an integer or a decimal.
  • n is a decimal or an integer from 1 to 8 or from 1 to 6. More preferably, n is a decimal or an integer from 1 to 5 or from 2 to 4.
  • the present disclosure provides a conjugate of an antibody or a fragment thereof-ixitecan or a derivative thereof, wherein n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and can be an integer or a decimal; preferably 1 to 6, and can be an integer or a decimal.
  • n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and can be an integer or a decimal; preferably 1 to 6, and can be an integer or a decimal.
  • the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,
  • L 1 is selected from -(succinimid-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -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 C(O)-, or a chemical bond, wherein p 1 is an integer from 1 to 20;
  • L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH 2 ) t -, or a chemical bond, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;
  • R6 and R7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.
  • the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit L1 is selected from -(succinimidyl-3-yl- N )-( CH2 ) s1 -C(O)-, -(succinimidyl-3-yl-N)-CH2 - cyclohexyl-C(O)-, -(succinimidyl -3 - yl-N)-(CH2CH2O)s2 - CH2CH2- C(O)-, -CH2 -C(O)-NR3-( CH2 ) s3 - C (O)-, or -C(O)-( CH2 ) s4C (O)-, wherein s1 is an integer from 2 to 8, s2 is an integer from 1 to 3, s3 is an integer from 1 to 8, and s4 is an integer from 1 to 8; s1 is an integer from 2 to
  • the present disclosure provides a conjugate of an antibody or fragment thereof- exitecan or a derivative thereof, wherein the linker unit L2 is selected from -NR4 ( CH2CH2O ) p1CH2C (O)- or a chemical bond, and p1 is an integer of 6-12.
  • the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, wherein L 4 is selected from -NR 5 (CR 6 R 7 ) t-, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, t is 1 or 2, preferably 2; L 4 is preferably -NR 5 CR 6 R 7 -; L 4 is more preferably -NHCH 2 -.
  • the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,
  • L1 is s1 is an integer from 2 to 8;
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.
  • the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,
  • L 1 is -(succinimidyl-3-yl-N)-CH 2 -cyclohexyl-C(O)-;
  • L 2 is -NR 4 (CH 2 CH 2 O) 9 CH 2 C(O)-;
  • L 3 is a tetrapeptide residue
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.
  • the present disclosure provides a conjugate of an antibody or a fragment thereof-ixitecan or a derivative thereof, wherein the peptide residue of L3 is an amino acid residue formed by one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (N); preferably, it is an amino acid residue formed by one, two or more amino acids selected from phenylalanine and glycine; more preferably, it is a tetrapeptide residue; and most preferably, it is a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).
  • the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- has its L1 end connected to the antibody and its L4 end connected to Y.
  • the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, wherein the -LY- is:
  • L 1 is selected from -(succinimid-3-yl-N)-(CH 2 )s 1 -C(O)- or -(succinimid-3-yl-N)-CH 2 -cyclohexyl-C(O)-;
  • L 2 is -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)- or a chemical bond, p 1 is an integer from 6 to 12;
  • L 3 is a tetrapeptide residue of GGFG
  • R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a C 3-6 cycloalkylalkyl group or a C 3-6 cycloalkyl group;
  • R2 is selected from hydrogen atom, haloalkyl or C3-6 cycloalkyl; preferably hydrogen atom;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • R 5 is selected from a hydrogen atom or an alkyl group
  • R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group
  • s 1 is an integer from 2 to 8; preferably 5;
  • n is an integer of 0-4.
  • the conjugate of antibody or fragment thereof-exitecan or its derivative is conjugate of antibody or fragment thereof-exitecan or its derivative
  • L 2 is -NR 4 (CH 2 CH 2 O) 9 CH 2 C(O)-;
  • L 3 is a tetrapeptide residue of GGFG
  • R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a C 3-6 cycloalkylalkyl group or a C 3-6 cycloalkyl group;
  • R2 is selected from hydrogen atom, haloalkyl or C3-6 cycloalkyl; preferably hydrogen atom;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • R 5 is selected from a hydrogen atom or an alkyl group
  • R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group
  • n is an integer of 0-4.
  • the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by formula (III):
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue of GGFG
  • R1 is a hydrogen atom, a C3-6 cycloalkylalkyl group or a C3-6 cycloalkyl group;
  • R2 is selected from a hydrogen atom, a haloalkyl group or a C3-6 cycloalkyl group
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • R 5 is selected from a hydrogen atom or an alkyl group
  • R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group
  • s1 is an integer from 2 to 8;
  • n is an integer of 0-4.
  • the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-):
  • L 1 is selected from -(succinimid-3-yl-N)-(CH 2 )s 1 -C(O)- or -(succinimid-3-yl-N)-CH 2 -cyclohexyl-C(O)-;
  • L 2 is -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)- or a chemical bond, p 1 is an integer from 1 to 20;
  • L 3 is a tetrapeptide residue of GGFG
  • R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a C 3-6 cycloalkylalkyl group or a C 3-6 cycloalkyl group;
  • R2 is selected from hydrogen atom, haloalkyl or C3-6 cycloalkyl; preferably hydrogen atom;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • R 5 , R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group
  • s1 is an integer from 2 to 8;
  • n is an integer of 0-4.
  • the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-):
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue of GGFG
  • R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a C 3-6 cycloalkylalkyl group or a C 3-6 cycloalkyl group;
  • R2 is selected from hydrogen atom, haloalkyl or C3-6 cycloalkyl; preferably hydrogen atom;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • R 5 is selected from a hydrogen atom or an alkyl group
  • R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group
  • s1 is an integer from 2 to 8;
  • n is an integer of 0-4.
  • the antibody or fragment thereof-exitecan or a derivative thereof conjugate is a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof represented by the general formula (Pc- La -Y-Dr) of formula (IV):
  • W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -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 C(O)- or a chemical bond, and p 1 is an integer from 1 to 20;
  • L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • R1 is selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;
  • R2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;
  • R1 and R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group
  • R4 and R5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;
  • R6 and R7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;
  • n is an integer from 0 to 4.
  • n 1 to 10 and can be an integer or a decimal
  • Pc is a LIV-1 binding molecule provided by the present disclosure.
  • the antibody or fragment thereof-exitecan or a derivative thereof conjugate is a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof represented by the general formula (Pc-L b -Y-Dr) of formula (V):
  • s 1 is an integer from 2 to 8; preferably 5;
  • the linker unit -LY- of the antibody or fragment thereof-exitecan or derivative thereof conjugate includes, but is not limited to:
  • the present disclosure provides antibody or fragment thereof-exitecan or derivative thereof conjugates including, but not limited to:
  • n 1 to 10 and can be an integer or a decimal
  • Pc is the aforementioned LIV-1 binding molecule of the present disclosure.
  • n is ⁇ 0.4, for example, n is 4.
  • the LIV-1 binding molecule comprises VH and VL, wherein:
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 8-10
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 41, 12, 13;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:8-10
  • the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:11-13; or
  • VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:14-16
  • VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:17-19.
  • the LIV-1 binding molecule comprises VH and VL, wherein:
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 20 or 27, or at least 80% identical thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 21 or 22, or at least 80% identical thereto
  • the VH comprises an amino acid sequence as shown in SEQ ID NO:4 or having at least 80% identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO:5 or having at least 80% identity thereto;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO:6 or having at least 80% identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO:7 or having at least 80% identity thereto;
  • the VH comprises an amino acid sequence as shown in any one of SEQ ID NO:23-25, 28, or a sequence that is at least 80% identical thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO:26, or a sequence that is at least 80% identical thereto.
  • the VH comprises an amino acid sequence as shown in SEQ ID NO:27 or has at least 80% identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO:21 or has at least 80% identity thereto.
  • the VH comprises an amino acid sequence as shown in SEQ ID NO:28 or has at least 80% identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO:26 or has at least 80% identity thereto.
  • the LIV-1 binding molecule comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31 or 32 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31 or 32 or an amino acid sequence having at least 80% sequence identity thereto; or,
  • the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:34, 36-38, or an amino acid sequence having at least 80% sequence identity thereto
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO:35, or an amino acid sequence having at least 80% sequence identity thereto.
  • the LIV-1 binding molecule comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% sequence identity thereto; or,
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% sequence identity thereto.
  • the disclosure provides polynucleotides encoding any of the LIV-1 binding molecules of the disclosure, or encoding an antibody or antigen-binding fragment thereof in any of the immunoconjugates.
  • the polynucleotides of the present disclosure may be RNA, DNA or cDNA. In some embodiments, the polynucleotides may be isolated polynucleotides.
  • the aforementioned polynucleotides may also be in the form of vectors, may be present in vectors and/or may be part of vectors.
  • the vectors comprising the polynucleotides may be eukaryotic vectors, prokaryotic vectors, viral vectors, such as plasmids, cosmids, bacteriophages, etc.
  • the vector may be an expression vector in particular, i.e., a vector that provides expression of the LIV-1 binding molecule in vitro and/or in vivo (i.e., in a suitable host cell, host organism, and/or expression system).
  • the expression vector generally comprises at least one nucleic acid of the present disclosure, which may be operably connected to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host.
  • suitable expression control elements e.g., promoters, enhancers, terminators, etc.
  • polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA technology) based on the information of the amino acid sequence of the antibodies or fragments thereof of the present disclosure, and/or can be isolated from suitable natural sources.
  • the polynucleotides and vectors of the present disclosure can be used to prepare LIV-1 binding molecules. In some embodiments, the polynucleotides and vectors of the present disclosure are used to express LIV-1 binding molecules in vitro or in vivo, and the LIV-1 binding molecules bind to LIV-1 protein for different purposes such as detection, diagnosis, treatment, and regulation.
  • the present disclosure provides a host cell comprising any of the aforementioned vectors; or expressing any of the aforementioned LIV-1 binding molecules, or antibodies or antigen-binding fragments thereof in immunoconjugates.
  • the host cell is a prokaryotic or eukaryotic cell.
  • the host cell includes a prokaryotic microorganism (such as Escherichia coli), or various eukaryotic cells (such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells or lymphocytes (such as Y0, NS0, Sp20 cells), yeast cells (such as Pichia pastoris), insect cells, etc.)
  • a prokaryotic microorganism such as Escherichia coli
  • various eukaryotic cells such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) cells or lymphocytes (such as Y0, NS0, Sp20 cells), yeast cells (such as Pichia pastoris), insect cells, etc.
  • CHO Chinese hamster ovary cells
  • HEK human embryonic kidney
  • lymphocytes such as Y0, NS0, Sp20 cells
  • yeast cells such as Pichia pastoris
  • the host cell may be derived from a multicellular organism (including, for example, invertebrates and vertebrates).
  • Vertebrate cells can also be used as host cells, for example, mammalian cell lines grown in suspension, monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse breast tumor cells (MMT060562), human breast cancer cells (MCF-7), human prostate cancer cells (PC3), TRI cells (as described, for example, in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells, Chinese hamster ovary (CHO) cells, myel, my
  • the present disclosure provides a pharmaceutical composition comprising any one of the aforementioned LIV-1 binding molecules, immunoconjugates, polynucleotides or vectors.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the pharmaceutical composition comprises a therapeutically or prophylactically effective amount of a LIV-1 binding molecule or immunoconjugate.
  • the LIV-1 binding molecules, immunoconjugates or pharmaceutical compositions of the present disclosure can be formulated into any dosage form known in the medical field, such as lyophilized preparations, liquid preparations, etc., and the dosage form selection depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical compositions of the present disclosure should be sterile and stable under production and storage conditions.
  • the LIV-1 binding molecules and immunoconjugates of the present disclosure can be present in a pharmaceutical composition in a unit dosage form for ease of administration.
  • the unit dosage can be 0.1-2000 mg (e.g., 1-1000 mg).
  • the pharmaceutical composition unit dosage can contain 0.01 to 99% by weight of the LIV-1 binding molecules or immunoconjugates of the present disclosure.
  • the present disclosure provides a method for preparing an LIV-1 binding molecule comprising: expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment thereof from the host cell.
  • the preparation method may further include a purification step, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away nonspecifically bound components, eluting the bound antibodies using a pH gradient method, detecting using SDS-PAGE, and collecting.
  • a purification step for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away nonspecifically bound components, eluting the bound antibodies using a pH gradient method, detecting using SDS-PAGE, and collecting.
  • filtration and concentration are performed using conventional methods. Soluble mixtures and polymers may also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or freeze-dried.
  • mice can be immunized with human FcRn or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods.
  • Antigen-binding fragments can also be prepared using conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells. Mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected using conventional techniques. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves, ion exchange, etc.
  • the present disclosure provides a method for preparing an immunoconjugate, comprising: coupling any one of the aforementioned LIV-1 binding molecules with an effector molecule to obtain the immunoconjugate. Further, the method further comprises: purifying the immunoconjugate.
  • the method for preparing a conjugate of a ligand-exitecan or a derivative thereof as represented by the general formula (Pc-L a -YD) comprises the following steps:
  • Pc is the LIV-1 binding molecule of the present disclosure
  • W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in formula (IV).
  • the present disclosure provides at least one of the following uses of an LIV-1 binding molecule, immunoconjugate, pharmaceutical composition, polynucleotide, or vector:
  • the diseases associated with LIV-1 include but are not limited to cancer or tumor.
  • the cancer or tumor is selected from the group consisting of breast cancer, endometrial cancer, ovarian cancer, prostate cancer, Prostate cancer, melanoma, pancreatic cancer, kidney cancer, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), liver cancer, gastric cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer, leukemia, multiple myeloma, non-Hodgkin lymphoma.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • liver cancer gastric cancer
  • colorectal cancer colorectal cancer
  • bladder cancer bladder cancer
  • head and neck cancer thyroid cancer
  • esophageal cancer cervical cancer
  • leukemia multiple myeloma
  • non-Hodgkin lymphoma non-Hodgkin lymphom
  • the immunoconjugate disclosed in the present invention has tumor targeting, tumor cell toxicity, and bystander killing, and can be used to inhibit the activity of tumor cells in vitro or in vivo in a subject, kill tumor cells, and treat diseases associated with LIV-1.
  • the present disclosure provides methods of treating a subject having cancer or a tumor, or a subject at risk of having cancer, comprising: administering to the subject a therapeutically effective amount of a LIV-1 binding molecule, immunoconjugate, pharmaceutical composition, polynucleotide, or vector.
  • the present disclosure provides a method of treating a subject having cancer or a tumor, or a subject at risk of having cancer, comprising: administering to the subject a therapeutically effective amount of an immunoconjugate or a pharmaceutical composition.
  • the present disclosure provides a method of killing or inhibiting cells expressing LIV-1 in vitro or in vivo in a subject, comprising:
  • An effective amount of an LIV-1 binding molecule, immunoconjugate, pharmaceutical composition, polynucleotide or vector of the present disclosure is administered to a subject.
  • the LIV-1 binding molecules, immunoconjugates or pharmaceutical compositions of the present disclosure can be administered by any suitable method known in the art, including, but not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), intratumoral administration.
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • intratumoral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • the LIV-1 binding molecules, immunoconjugates or pharmaceutical compositions can be administered by any suitable route, such as by infusion or rapid injection. Administration can be systemic or local.
  • the dosage regimen can be adjusted to obtain the best intended response (e.g., a therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.
  • the best intended response e.g., a therapeutic or preventive response
  • the present disclosure provides a method for detecting LIV-1 in vivo or in vitro, comprising: contacting a sample to be tested with a LIV-1 binding molecule, immunoconjugate, polynucleotide or carrier of the present disclosure.
  • the method can be used to detect the presence or content of LIV-1 in a sample to be tested.
  • the LIV-1 binding molecules and immunopharmaceuticals disclosed herein are also detectably labeled.
  • the method further comprises detecting the LIV-1 binding molecules and immunoconjugates disclosed herein using a reagent with a detectable label.
  • the method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient).
  • the present disclosure provides a method for detecting LIV-1 in vivo or in vitro, comprising:
  • the method further comprises:
  • a reference sample e.g., a control sample
  • an LIV-1 binding molecule, polynucleotide, or vector of the present disclosure
  • test sample contains LIV-1 by comparing it with a reference sample; or, determine the content of LIV-1 in the test sample by comparing it with a reference sample.
  • sample to be tested refers to any type of sample in which it is necessary to determine whether LIV-1 is contained or the content of LIV-1 needs to be determined.
  • the sample to be tested is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to unicellular organisms, such as bacteria, yeast, protozoa and amoeba, etc., multicellular organisms (such as plants or animals, including samples from healthy or apparently healthy human subjects or human patients affected by the illness or disease to be diagnosed or investigated, such as infection by pathogenic microorganisms such as pathogenic bacteria or viruses).
  • the sample to be tested can be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any body secretions, exudates, exudates or fluids obtained from joints, or swabs from skin or mucosal surfaces.
  • the sample to be tested can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsy) or can contain cells (primary cells or cultured cells) or a medium conditioned by any cell, tissue or organ.
  • Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cell centrifugation preparations, cytological smears, body fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).
  • body fluids e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.
  • tissue biopsies e.g., tumor biopsies
  • fine needle aspirates e.g., cryostat tissue sections and/or paraffin-embedded tissue sections.
  • the antibodies or antigen-binding fragments disclosed herein can specifically bind to LIV-1, and thus can be used to detect the presence or level of LIV-1 in a sample, and to diagnose whether a subject suffers from a disease associated with LIV-1.
  • LIV-1 binding molecules can be used for the diagnosis, prognosis, efficacy monitoring, etc. of cancer or tumors by detecting the expression level of LIV-1 protein in the subject.
  • the present disclosure provides a method for diagnosing, prognosing or detecting the therapeutic effect of a disease related to LIV-1, comprising: using the LIV-1 binding molecule of the present disclosure to detect the presence or level of LIV-1 in a sample from the subject.
  • the antibody or immunoconjugate of the present disclosure also carries a detectable label, and by detecting the label, the presence or level of LIV-1 specifically bound to the LIV-1 binding molecule or immunoconjugate of the present disclosure is detected.
  • the method further comprises the step of using a reagent with a detectable label to detect the LIV-1 binding molecule or immunoconjugate of the present disclosure.
  • the LIV-1 binding molecules provided by the present disclosure can be used to detect the expression level of LIV-1 antigen in cancer tissues to determine whether the cancer can be treated with immunoconjugates.
  • the LIV-1 binding molecule is selected from any one of the following:
  • VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 8-10
  • VL comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 11-13;
  • VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 8-10
  • VL comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 41, 12-13;
  • VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 14-16
  • VL comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 17-19;
  • VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO:4, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO:5;
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 20 or 27, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO: 21 or 22;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:6, and the VL comprises LCDR1, LCDR2 and LCDR in the amino acid sequence shown in SEQ ID NO:7;
  • the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in any one of SEQ ID NO:23-25, 28, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:26.
  • the LIV-1 binding molecule is selected from any one of the following:
  • the VH comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence of SEQ ID NO: 21 or 22, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence of SEQ ID NO:27, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence of SEQ ID NO:21 or 22, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NO:23-25, 28, or an amino acid sequence having at least 80% sequence identity thereto;
  • the VL comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 80% sequence identity thereto.
  • the LIV-1 binding molecule is selected from any one of the following:
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 30 or has at least 80% sequence identity thereto.
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 31 or 32 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% sequence identity thereto
  • the light chain comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 34, 36-38, or an amino acid sequence having at least 80% sequence identity thereto
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80% sequence identity thereto;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:45 or an amino acid sequence having at least 80% sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO:46 or an amino acid sequence having at least 80% sequence identity thereto.
  • the heavy chain of the LIV-1 binding molecule comprises the amino acid sequence shown in SEQ ID NO:45, and the light chain comprises the amino acid sequence shown in SEQ ID NO:46.
  • the above antibodies are used for immunohistochemistry (Immunohistochemistry, IHC) detection.
  • IHC technology uses antibodies to detect and display cell antigens in situ, usually by colorimetric or fluorescent methods.
  • LIV-1 binding molecules are applied to IHC detection of FFPE tissues, or IHC detection of frozen tissues.
  • the above antibodies are used for IHC detection in formalin fixed and embedded (FFPE) tissue samples.
  • the IHC detection method is known in the art, for example, refer to (Development of a LAG-3 immunohistochemistry assay for melanoma. J Clin Pathol. 2022 May 9; jclinpath-2022-208254), or the IHC staining method disclosed in CN111051345A.
  • the IHC detection methods in the above-mentioned documents and patents are incorporated into the present disclosure by reference.
  • the above LIV-1 binding molecules are used for IHC detection, which can clearly show the localization of LIV-1 on cancer tissue samples and have high binding specificity and sensitivity.
  • the LIV-1 binding molecules in the present disclosure are used for LIV-1 protein detection, and have better binding specificity and sensitivity than LIV-1/ZIP6 Antibody (14236-1-AP).
  • the present disclosure provides a kit, a detection system or a detection device, which includes the LIV-1 binding molecule, immunoconjugate, polynucleotide or vector of the present disclosure.
  • the LIV-1 binding molecule or immunoconjugate carries a detectable label.
  • the kit, detection system or detection device further comprises a second antibody that specifically recognizes the first antibody or scFv of the LIV-1 antibody of the present disclosure.
  • the second antibody further comprises a detectable label.
  • the detectable label can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • labels can be suitable for use in immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.).
  • immunological assays e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.
  • enzymes e.g., horseradish peroxidase, alkali metal ions.
  • radionuclides e.g., 3H, 125I, 35S, 14C or 32P
  • fluorescent dyes e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)
  • acridinium ester compounds such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels.
  • the labels encompassed in the present disclosure can be detected by methods known in the art.
  • radiolabels can be detected using photographic film or a scintillation counter
  • fluorescent labels can be detected using a photodetector to detect the emitted light.
  • Enzymatic labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label.
  • detectable labels as described above can be attached to the bispecific antibodies of the present disclosure via linkers of varying lengths to reduce potential steric hindrance.
  • Figure 1 shows the results of the endocytosis ability test of different anti-LIV-1 antibodies in CHO-hLIV-1 cells.
  • the endocytosis and killing effects of PR0082 and hLIV22 in CHOK1-hLIV-1 cells were determined by DT3C.
  • FIG2 shows the results of the endocytosis ability test of different anti-LIV-1 antibodies in SK-OV-3 cells.
  • the internalization of antibodies in SK-OV-3 cells was measured by pHrodo.
  • LIV-1 also known as Zip6, refers to a zinc transporter belonging to the subfamily of ZIP zinc transporters known as LZT, encoding the gene SLC39A6.
  • LIV-1 is also known as SLC39A6 (solute carrier protein family 39 (zinc transporter), member 6).
  • the LIV-1 sequence can be obtained from GenBank, UniProt, etc.
  • the sequence of human LIV-1 (hereinafter referred to as hLIV-1) can be referenced by accession numbers such as UniProtKB-Q13433 and NP_036451.4.
  • the crab-eating monkey LIV-1 protein can be referenced (GenBank, accession number: XP_005586923.1), and the mouse LIV-1 protein can be referenced (GenBank, accession number: NP_631882.2).
  • LIV-1 should be understood in the broadest sense. The term covers LIV-1 in its native form, naturally occurring variants, and artificially expressed forms. When involved in antigen-antibody interactions, LIV-1 covers the scope of the entire protein, the extracellular domain and its epitopes.
  • Binding molecule encompasses any protein, polypeptide, or any molecule comprising the protein or polypeptide that is capable of specifically binding to an antigen (eg, LIV-1), including but not limited to antibodies as defined in the present disclosure.
  • an antigen eg, LIV-1
  • Antibody is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibodies may refer to immunoglobulins, which are tetrapeptide chains formed by two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulins are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, respectively.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into ⁇ chains or ⁇ chains according to the differences in the constant region.
  • Each of the five Ig classes can have either kappa or lambda chains.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes three hypervariable regions (CDRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • CDR refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • CDR the deterministic depiction of CDR and the identification of residues comprising the binding site of the antibody can be completed by resolving the structure of the antibody and/or resolving the structure of the antibody-antibody complex. This can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR definition systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
  • the antibody light chain may further comprise a light chain constant region.
  • the light chain constant region comprises a human or murine ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain may further comprise a heavy chain constant region.
  • the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • murine antibody in the present disclosure refers to a monoclonal antibody against human LIV-1 or an epitope thereof prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with LIV-1 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.
  • the murine LIV-1 antibody or its antigen-binding fragment may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • human antibody includes antibodies with variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term “human antibody” does not include “humanized antibodies”.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. In order to avoid a decrease in activity while reducing immunogenicity, the variable region of the fully human antibody can be subjected to minimal reverse mutations to maintain activity.
  • chimeric antibody refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can reduce the immune response induced by the antibody of the first species.
  • a hybridoma that secretes mouse-specific monoclonal antibodies is selected, and then the variable region gene is cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody is cloned as needed, and the mouse variable region gene and the human constant region gene are connected into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system.
  • the constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, such as a heavy chain constant region containing human IgG2 or IgG4, or an IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
  • human IgG1, IgG2, IgG3 or IgG4 or a variant thereof such as a heavy chain constant region containing human IgG2 or IgG4, or an IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
  • antigen-binding fragment includes single-chain antibodies (i.e., heavy or light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, divalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • Fab-Fv format was first disclosed in WO2009/040562, and its disulfide-stabilized form Fab-dsFv First disclosed in WO2010/035012.
  • Antigen binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multispecificity, such as bispecificity or monospecificity (see, for example, WO92/22583 and WO05/113605).
  • single-chain antibody means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • binding to LIV-1 refers to the ability to interact with LIV-1 or an epitope thereof, and the LIV-1 or the epitope thereof may be of human origin.
  • antigen binding site refers to a discrete, three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present disclosure.
  • epitope refers to a site on an antigen that binds to an immunoglobulin or antibody.
  • An epitope can be formed by adjacent amino acids, or non-adjacent amino acids (non-adjacent amino acids are brought into close proximity in space by tertiary folding of the protein). Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes usually exist in a unique spatial conformation, which includes at least 3-15 amino acids. Methods for determining epitopes are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody binds to the predetermined antigen or its epitope with an equilibrium dissociation constant ( KD ) of approximately less than 10-7 M or even less when measured in an instrument by surface plasmon resonance (SPR) technology, and the affinity of the antibody to the predetermined antigen or its epitope is at least twice the affinity of the antibody to the predetermined antigen or its epitope (or a non-specific antigen other than a closely related antigen, such as BSA, etc.).
  • KD equilibrium dissociation constant
  • SPR surface plasmon resonance
  • Binding affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding antibody (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity” refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its antibody Y can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.
  • kassoc or "ka” refers to the association rate of a specific antibody-antigen interaction
  • kdis or “kd” as used herein refers to the association rate of a specific antibody-antigen interaction. It is intended to refer to the dissociation rate of a specific antibody-antigen interaction.
  • KD refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M).
  • the KD value of an antibody can be determined using methods known in the art, for example: the method for determining the KD of an antibody includes using a biosensor system such as a system measuring surface plasmon resonance, or measuring the affinity in a solution by a solution equilibrium titration method (SET).
  • SET solution equilibrium titration method
  • Cross-reactivity refers to the ability of an antibody (or fragment thereof) of the present invention to bind to LIV-1 from different species.
  • an antibody of the present invention that binds to human LIV-1 can also bind to LIV-1 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in a binding assay (e.g., SPR or ELISA), or by binding or functional interaction with cells that physiologically express LIV-1.
  • the method for determining cross-reactivity includes standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.
  • amino acid sequence identity refers to the percentage of amino acid residues in the first sequence that are identical to those in the second sequence, after aligning the amino acid sequences and introducing gaps when necessary to achieve the maximum sequence identity percentage (and not considering conservative substitutions as part of the sequence identity).
  • alignment can be achieved in a variety of ways within the scope of the art, for example, using computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.
  • cross-reactivity refers to the ability of an antibody of the present disclosure to bind to LIV-1 from a different species.
  • an antibody of the present disclosure that binds to human LIV-1 may also bind to LIV-1 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells expressing LIV-1.
  • binding assays e.g., SPR and ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.
  • internalization refers to the transport of a portion from the outside of a cell to the inside.
  • the internalized portion can be located in an intracellular compartment.
  • An "internalized” or “internalized” antigen or antibody refers to an antigen or antibody that can be transported from the outside of a target cell to the inside. It is generally understood by those skilled in the art that the process of cellular internalization generally refers to the movement of cell surface molecules from the cell surface to the inside of the cell across the plasma membrane. After internalization, the endosome can be transported to the lysosome for degradation or recycled to the cell surface.
  • the cellular internalization rate of a given cell surface molecule provides a measurement of the kinetics of the molecule moving from the cell surface through the plasma membrane to the inside of the cell.
  • the internalization activity or internalization rate of antigens and antibodies can be monitored and/or measured by a variety of techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305–17, 2008) and toxin killing assays (Pahara J. et al. Exp Cell Res., 316:2237–50, 2010; and Mazor et al., J. Immunol. Methods, 321:41–59, 2007).
  • Many antibody labeling techniques, dyes, and kits for antibody labeling that can be used to quantify and monitor internalization are commercially available (e.g., pHrodo iFL antibody labeling methods, reagents, and kits sold by Thermo Fisher Scientific).
  • antibody-drug conjugates bind to tumor cell surface antigens through the antibody in the ADC, which then undergoes endocytosis into endosomes.
  • the ADC is transformed into lysosomes, and then the bioactive molecules (e.g., toxins or payloads) are dissociated from the ADC under the action of hydrolases in the lysosomes.
  • the dissociated bioactive molecules enter the cytoplasm from the lysosomes and kill tumor cells.
  • the bioactive molecules that escape from the killed tumor cells can further kill the surrounding tumor cells that do not express or express low antigens (the so-called bystander effect).
  • “Mutant”, “variant” refers to a polypeptide containing at least one amino acid mutation (such as substitution, deletion or insertion) compared to the "parent" amino acid sequence, as long as the variant is still able to bind to LIV-1, in particular human LIV-1 as shown in any one of SEQ ID NO: 1-3.
  • Variants of the binding molecules (such as antibodies or antigen-binding fragments thereof) disclosed herein are generally prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or antibody fragment or by peptide synthesis.
  • the above-mentioned amino acid modifications can be introduced into the variable region or the constant region.
  • amino acid mutations can be introduced to adjust antibody properties that affect drug development, such as thermodynamic stability, solubility or viscosity ("sequence optimization").
  • Amino acid mutations include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of a binding molecule (preferably an antibody or antigen-binding fragment). Any combination of deletions, insertions and substitutions can be introduced into the "parent" amino acid sequence to obtain the final variant. Amino acid mutations also include post-translational processes that can change the binding molecule, such as changing the number or position of glycosylation sites.
  • Insertion variants of binding molecules (particularly antibodies or antibody fragments) of the present disclosure include fusion products of antibodies or antibody fragments with enzymes or another functional polypeptide (e.g., which improves the serum half-life of the binding molecule (e.g., antibody or antibody fragment)).
  • amino acid substitutions may be introduced into the CDR, VH or FR regions of the heavy and/or light chains. Conservative substitutions are preferred, for example, and may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved.
  • Constant substitution refers to replacement with another amino acid residue having a property similar to the original amino acid residue.
  • lysine, arginine and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains.
  • tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when replacing an amino acid residue in a group showing similar properties as described above, it will not show a specific change in properties.
  • modifications may also be introduced into the Fc portion of a binding molecule (preferably an antibody or antigen-binding fragment thereof). Such modifications may be used to modulate the functional properties of the antibody, for example, interactions with complement proteins on other immune cells (such as C1q and/or Fc receptors), or to modulate serum half-life or antigen-dependent cellular cytotoxicity (ADCC). Mutations that alter effector function may be introduced using conventional methods known in the art.
  • Exemplary modifications include: Asn297 ⁇ Ala297 and Asn297 ⁇ Gln297, or Lys322 ⁇ Ala322 and optionally Leu234 ⁇ Ala234 and Leu235 ⁇ Ala234, which result in IgG1 glycosylation, which are reported to reduce or eliminate antibody-derived cell-mediated cytotoxicity (ADCC) and/or complement-derived cytotoxicity (CDC).
  • ADCC antibody-derived cell-mediated cytotoxicity
  • CDC complement-derived cytotoxicity
  • linker refers to a fragment or bond that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers and then connected to a ligand or drug.
  • the joint may include one or more joint components.
  • exemplary joint components include 6-maleimidocaproyl ("MC”), maleimidopropionyl ("MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), and N-succinimidyl 4-(2-pyridylthio) valerate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC”, also referred to herein as "MCC”) and N-succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • val-cit valine-citrulline
  • alanine-phenylalanine ala-p
  • the joint may include a stretching unit, a spacer unit, an amino acid unit and an extension unit, which may be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the joint may be a "cleavable joint" that is convenient for releasing the drug in the cell.
  • an acid-labile linker e.g., a hydrazone
  • a protease-sensitive e.g., a peptidase-sensitive
  • a photolabile linker e.g., a dimethyl linker, or a disulfide-containing linker
  • a disulfide-containing linker can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020).
  • amino acid unit refers to an amino acid to which the carbonyl group in the following structural formula Y R can be connected to the stretching unit if a stretching unit is present, or to which Y R can be directly connected to the cytotoxic drug if no stretching unit is present.
  • the amino acid unit is represented by -K k -:
  • -K k - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide, and the -K- units each independently have the following structural formula Ka or Kb , and k is an integer between 0 and 10:
  • R 23 in the above amino acid unit is -H or methyl
  • R25 is -aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl-, -heterocyclyl-, -alkyl-heterocyclyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-alkyl-, -aryl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-aryl-alkyl-, -heteroaryl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-, -alkyl-heteroaryl-, -alkyl-heteroaryl-alkyl-, -alkyl-heteroaryl-alkyl-, -alkyl
  • -K k - is a dipeptide, preferably -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline-, and more preferably -valine-citrulline-.
  • stretcher refers to a chemical structure fragment that is covalently linked to a ligand via a carbon atom at one end and to a cytotoxic drug via a sulfur atom at the other end.
  • spacer unit refers to a bifunctional chemical structural fragment that can be used to couple a linker unit and a cytotoxic drug to ultimately form a ligand-cytotoxic drug conjugate. This coupling method can selectively connect the cytotoxic drug to the linker unit.
  • stretching unit refers to a chemical structure that can couple the amino acid unit to a cytotoxic drug when the amino acid unit is present, or can couple the cytotoxic drug via the carbonyl group on YR when the amino acid unit is absent.
  • the stretching unit is represented by -Q q -, where q is selected from 0, 1, and 2.
  • the stretching unit in the present disclosure is PAB, the structure of which is a 4-iminobenzylcarbamoyl fragment, the structure of which is shown in the following formula, connected to D,
  • Joint components include but are not limited to:
  • MC 6-maleimidocaproyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker)
  • PAB p-aminobenzyloxycarbonyl (an example of a "self-immolative” linker component)
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B)
  • MC(PEG)6-OH Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine)
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • PBS Phosphate buffered saline
  • immunoconjugate also known as “antibody-drug conjugate” (ADC) or "ligand-drug conjugate” refers to an antibody or a fragment thereof connected to a drug via a linker (or a connecting unit).
  • ADC antibody-drug conjugate
  • ligand-drug conjugate refers to an antibody or a fragment thereof connected to a drug via a linker (or a connecting unit).
  • linker or a connecting unit
  • immunoconjugate refers to an anti-LIV-1 antibody or an antigen-binding fragment thereof connected to a toxic drug via a connecting unit.
  • drug loading refers to the average number of cytotoxic drugs loaded on each ligand in ADC, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) connected to each antibody (Pc).
  • drug loading is expressed as n or k, and exemplary is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or the mean of any two numerical values.
  • Preferably 1-10, more preferably 1-8, or 1-8, or 1-7, or 2-8, or 2-7, or 2-6, or 2-5, or 2-3, or 1-2, or 2-4, or 1-4, or 1-5, or 1-6, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean.
  • the average amount of drug substance per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
  • the monoclonal antibody molecular size variant determination method (CE-SDS) disclosed in the present invention can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method to quantitatively determine the purity of the recombinant monoclonal antibody product according to the capillary electrophoresis method (2015 edition of the "Chinese Pharmacopoeia" 0542) based on the molecular weight under reducing and non-reducing conditions.
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through a linker.
  • the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.
  • the loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:
  • the drug-to-antibody ratio has a specific value for a particular conjugate molecule (e.g., n in Formula (I)), it will be appreciated that when used to describe a sample containing many molecules, the value will often be an average value due to some degree of heterogeneity typically associated with the conjugation step.
  • the average loading of an immunoconjugate sample is referred to herein as the drug-to-antibody ratio or "DAR".
  • the DAR is between about 1 and about 6, and is typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 8.5, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0 7.5, 8.0.
  • At least 50% of the sample by weight is a compound having an average DAR plus or minus 2, and preferably at least 50% of the sample is a conjugate containing an average DAR plus or minus 1.
  • Examples include where the DAR is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,.
  • a DAR of 'about x' means that the measured value of the DAR is within 20% of x.
  • the detection method of DAR extrapolates the DAR value from the LC-MS data of reduced and deglycosylated samples.
  • LC/MS allows the average number of effective load (drug moiety) molecules connected to the antibody in quantitative ADC.
  • HPLC separates the antibody into light chain and heavy chain, and also separates the heavy chain (HC) and light chain (LC) according to the number of linker-effective load groups of each chain.
  • Mass spectrometry data can identify the types of components in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc.
  • the average DAR of ADC can be calculated.
  • the DAR of a given immunoconjugate sample represents the average number of drug (effective load) molecules connected to a tetrameric antibody containing two light chains and two heavy chains.
  • the DAR detection method in WO2018142322.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,
  • lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available attachment point, and the substituents are preferably one or more of the following groups, which Independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane radical by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms.
  • alkylene groups include, but are not limited to, methylene (—CH 2 —), 1,1-ethylene (—CH(CH 3 )—), 1,2-ethylene (—CH 2 CH 2 )—, 1,1-propylene (—CH(CH 2 CH 3 )—), 1,2-propylene (—CH 2 CH(CH 3 )—), 1,3-propylene (—CH 2 CH 2 CH 2 —), 1,4-butylene (—CH 2 CH 2 CH 2 CH 2 —), and 1,5-butylene (—CH 2 CH 2 CH 2 CH 2 CH 2 —), and the like.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • alkoxy refers to-O-(alkyl) and-O-(non-substituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or non-substituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.
  • ring atoms preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.
  • Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.
  • spiro heterocyclic group refers to a polycyclic heterocyclic group in which the monocyclic rings of 5 to 20 members share one atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a completely conjugated ⁇ electron system. For example, 6 to 14 members, and for example, 7 to 10 members.
  • spiro heterocyclic groups are divided into monospiro heterocyclic groups, bispiro heterocyclic groups or polyspiro heterocyclic groups, preferably monospiro heterocyclic groups and bispiro heterocyclic groups.
  • monospiro heterocyclic groups preferably monospiro heterocyclic groups and bispiro heterocyclic groups.
  • spiro heterocyclic groups include:
  • fused heterocyclic group refers to a polycyclic heterocyclic group of 5 to 20 members, each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated ⁇ electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. For example, 6 to 14 members, and for example, 7 to 10 members.
  • the number of constituent rings it can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, for example, a bicyclic or tricyclic group, and for example, a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
  • fused heterocyclic groups include:
  • bridged heterocyclic group refers to a polycyclic heterocyclic group of 5 to 14 members, any two rings sharing two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings has a completely conjugated ⁇ electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon.
  • m is an integer from 0 to 2
  • 6 to 14 members and another example is 7 to 10 members.
  • bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups for example, bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
  • bridged heterocyclic groups include:
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated ⁇ electron system, for example, 6- to 10-membered, such as phenyl and naphthyl, specifically phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • the heteroaryl is preferably 5 to 10 members, more preferably 5 or 6 members, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc.
  • the heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • amino protecting group is used to keep the amino group unchanged when other parts of the molecule are reacted.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl. These groups may be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • aminoheterocyclyl refers to a heterocyclyl group substituted by one or more amino groups, preferably by one amino group, wherein the heterocyclyl group is as defined above, and wherein “amino” refers to -NH 2 .
  • aminoheterocyclyl refers to a heterocyclyl group substituted by one or more amino groups, preferably by one amino group, wherein the heterocyclyl group is as defined above, and wherein “amino” refers to -NH 2 .
  • heterocyclylamino refers to an amino group substituted by one or more heterocyclyl groups, preferably substituted by one heterocyclyl group, wherein the amino group is as defined above, and wherein the heterocyclyl group is as defined above.
  • Representative embodiments of the present disclosure are as follows:
  • cycloalkylamino refers to an amino group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the amino group is as defined above, and wherein the cycloalkyl group is as defined above.
  • Representative embodiments of the present disclosure are as follows:
  • cycloalkylalkyl refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein alkyl is as defined above and wherein cycloalkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxy refers to an -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • Substituted means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (such as olefinic) bonds.
  • Optionally or “optionally” means that the subsequently described event or circumstance may but need not occur.
  • the description includes situations where the event or circumstance occurs or does not occur.
  • C1-C6 alkyl optionally substituted by halogen or cyano means that halogen or cyano may but need not be present, and the description includes situations where the alkyl is substituted by halogen or cyano and situations where the alkyl is not substituted by halogen and cyano.
  • administering when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell.
  • Treatment when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, to a subject who has, is suspected of having, or is prone to having one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree.
  • the amount of a therapeutic agent that is effective to alleviate any specific disease symptom may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of the target disease in a subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • any statistical test known in the art such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • an "effective amount” includes an amount sufficient to improve or prevent the symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.
  • drug carrier refers to a drug that can change the way the drug enters the human body and
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, such as micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.
  • excipient refers to the additives in pharmaceutical preparations other than the main drug, which can also be called excipients.
  • the binders, fillers, disintegrants, and lubricants in tablets can also be called excipients.
  • the matrix part in semi-solid preparations such as ointments and creams
  • the preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.
  • diluent is also called filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients, improves the compression molding of the drug, etc.
  • an absorbent needs to be added to absorb the oily substance and keep it in a "dry" state to facilitate tableting.
  • the amino acid sequence (extracellular domain) of 1-323 of human LIV-1 protein (GenBank, accession number: NP_036451.4) was selected, and a polyhistidine (His) tag and a human immunoglobulin Fc tag were added to the C-terminus, respectively.
  • the sequence is shown in SEQ ID NO: 1-3.
  • Recombinant monkey LIV-1-His protein (purchased from ACRO Biosystems) is a His tag fused to the C-terminus of amino acids 1-309 of cynomolgus monkey LIV-1 protein (GenBank, accession number: XP_005586923.1).
  • Recombinant mouse LIV-1-His protein (purchased from ACRO Biosystems) is a His tag fused to the C-terminus of amino acids 1-335 of mouse LIV-1 protein (GenBank, accession number: NP_631882.2).
  • Plasmids carrying target protein (SEQ ID NO: 1-3) coding genes were synthesized separately, and expression vectors containing target protein coding genes were obtained by enzyme digestion, ligation, and transformation. After DNA sequencing screening, plasmids were extracted in large quantities for clones with correct insertion sequences. The extracted expression vectors were transiently expressed in Expi293 cells (purchased from ADCC) for 7 days. After ELISA detection, the expression supernatant was separated and purified, and stored at -70°C for later use.
  • the plasmid encoding the recombinant human LIV-1 protein (SEQ ID NO: 1) or the plasmid encoding the cynomolgus monkey LIV-1 protein After CHO-K1 cells (purchased from ADCC) were transfected with a plasmid containing the recombinant human or cynomolgus monkey LIV-1 protein (GenBank, accession number: XP_005586923.1), cell lines stably expressing recombinant human or cynomolgus monkey LIV-1 protein were obtained by screening.
  • nucleotide sequence encoding human or crab-eating macaque LIV-1 protein was cloned and connected to a lentiviral expression vector, CHO-K1 cells were transfected using a lentiviral transfection method well known in the industry, and selectively cultured using F-12 medium containing puromycin (purchased from Gibco), subcloned in a 96-well culture plate using the limiting dilution method, and cultured in a cell culture incubator.
  • LIV-1 on the surface of lentivirus-infected CHO-K1 cells was detected by flow cytometry (FACS), and the monoclonal cell line CHO-hLIV-1 with high expression of human LIV-1 and the monoclonal cell line CHO-cynoLIV-1 with high expression of monkey LIV-1 were selected, expanded for culture, and frozen for storage.
  • LIV-1 The expression level of LIV-1 on the surface of tumor cell lines was detected by FACS, and the antibody used was hLIV122 antibody (sequence see published patent number: US 2016/0185858A1, which is incorporated into the present disclosure by reference).
  • the cell lines with higher expression levels were MCF-7 and SK-OV-3. Hela, PC3 and ZR-75-1 expressed LIV-1 at lower levels.
  • the recombinant human LIV-1-hFc fusion protein or recombinant human LIV-1-His protein in Example 1 was used as an immunogen and for later screening.
  • the CHO-K1 stable cell line or MCF-7 expressing human LIV-1 in Example 2 was used to determine the antiserum titer and screen for antigen-specific antibodies.
  • recombinant human LIV-1-His protein or recombinant human LIV-1-hFc protein was used as an immunogen to immunize 6-8 week old Balb/c mice, SJL/J mice or C57 mice.
  • the initial immunization dose was 50 ⁇ g per mouse.
  • booster immunization was performed with an immunization dose of 25 ⁇ g per mouse, and each subsequent booster immunization was separated by 3 weeks. Serum samples were collected one week after each booster immunization, and the antibody activity in the mouse serum was detected by protein ELISA and FACS.
  • the protein ELISA detection process is as follows: coat the plate with 1 ⁇ g/mL recombinant human LIV-1-His, incubate at 4°C overnight, block with 1% BSA/PBST buffer for 1 hour, and wash the plate 3 times. Mix the plate with 1:100 in the blocking buffer. Initially, mouse serum was diluted 10-fold in series, incubated at 37°C for 1 hour, washed 3 times, and incubated with HRP-labeled anti-mouse IgG Fc secondary antibody for 1 hour. Washed 3 times with PBST, 100 ⁇ L TMB substrate was added to each well, and the reaction was terminated with 2M HCl after 15 minutes, and the absorbance at 450 nm was read using a microplate reader.
  • the FACS detection process is as follows: cells were plated, 1x 10 5 /100 ⁇ L cells per well, centrifuged and supernatant was discarded, and mouse serum was diluted in series. Incubated at 4°C for 1 hour. Washed twice with 1% BSA/PBS buffer and supernatant was discarded, Alexa Flour 647 goat anti-mouse secondary antibody (purchased from Jackson immuno research) was added, and incubated at 4°C in the dark for 30 minutes. Washed twice with 1% BSA/PBS buffer, resuspended in 200 ⁇ L buffer, and FACS detected.
  • the last immunization was intraperitoneal injection of 100 ⁇ g recombinant human LIV-1-hFc or recombinant human LIV-1-His.
  • the mice were killed 5 days later and the spleen was taken out.
  • the spleen cells were collected by grinding.
  • NH 4 OH with a final concentration of 1% (w/w) was added to lyse the mixed red blood cells in the spleen cells to obtain a spleen cell suspension, and the cells were resuspended and washed 3 times by centrifugation at 1000 rpm.
  • Mouse spleen cells were mixed with mouse myeloma cells SP2/0 at a ratio of 5:1 in the number of live cells, and cell fusion was performed using a high-efficiency electrofusion method.
  • the fused cells were diluted into a 96-well cell culture plate using DMEM medium containing 20% fetal bovine serum and 1 ⁇ HAT (w/w), with a total of 1x 10 5 cells per well in 200 ⁇ L, and placed in a 37°C incubator with 5% (v/v) CO 2 . After 14 days, the hybridoma cell supernatant was screened by cell ELISA, and the positive clones with OD450nm>0.2 were expanded to 24-well cell plates.
  • the culture was continued to be expanded at 37°C and 5% (v/v) CO 2 using DMEM medium containing 10% (w/w) HT fetal bovine serum. After 3 days, the culture fluid of the 24-well cell plate was centrifuged to collect the supernatant, and the binding activity to recombinant human LIV-1 positive cells was determined by FACS. Two rounds of monoclonal cloning were performed, and the monoclonal cell lines with excellent binding activity were screened using the above-mentioned flow cytometry method. Hybridoma cells in the logarithmic growth phase were collected, RNA was extracted with Trizol, and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was PCR amplified and sequenced using a mouse Ig-primer set (Novagen, TB326 Rev.B 0503).
  • the mouse variable regions are expressed in mammalian host cells to provide chimeric antibodies.
  • the nucleotide sequence encoding the variable region of the mouse monoclonal antibody is cloned into the pTT5 vector containing the protein sequence of the human heavy and light chain constant regions (Human IgG1, kappa) and transfected into HEK293 cells. After 5 days, the cells were removed by centrifugation, and the cell culture fluid was collected and filtered.
  • the harvested cell culture supernatant was loaded onto a protein A column (MabSelect SuRe, GE), the bound antibody was eluted with glycine, and the eluate was neutralized with 1M Tris. After testing, an anti-LIV-1 chimeric antibody with mouse VH/VL was obtained.
  • the LIV-1 binding activity experiment of the anti-LIV-1 chimeric antibody on cells was conducted by detecting the fluorescence signal of the cell surface-bound antibody, and the binding strength of the antibody was evaluated according to the strength of the fluorescence signal. Specifically, the gradient-diluted antibody molecules and control molecules were incubated with 1x 10 5 cells at 4°C for 1 hour, the excess antibody was washed off, and the anti-human Fc antibody labeled with mouse Alexa Flour 647 was added, incubated at 4°C for 30 minutes, and the excess antibody was washed off and resuspended with 200 ⁇ L 1% BSA/PBS buffer, and the fluorescence signal on the cell surface was read by a Thermo Attune NxT flow cytometer.
  • the cells used were CHO-hLIV-1 expressing human LIV-1, CHO-cynoLIV-1 expressing monkey LIV-1, and MCF-7 prepared in Example 2.
  • Table 3 exemplarily lists the amino acid sequences of the variable regions of the anti-LIV-1 antibodies obtained in the present disclosure, and the underlined complementary determining region CDR sequences (using the Kabat numbering system) are shown in Table 4.
  • the heavy chain variable region and light chain variable region of the above-mentioned antibody are connected to the heavy and light chain constant regions of different animal or human antibodies, a vector is constructed, cells are transiently transfected, antibodies are expressed and purified, and can be used to prepare chimeric antibodies.
  • the present disclosure uses the constant region of rabbit IgG1, whose sequence is shown in SEQ ID NO: 43.
  • the light chain constant region can be selected from the constant region of rabbit ⁇ chain, whose sequence is shown in SEQ ID NO: 44.
  • the heavy and light chain variable regions of the anti-LIV-1 antibody of the present disclosure are recombined with the above constant regions to obtain the full-length sequences of the heavy and light chains.
  • the sequence of the antibody is as follows:
  • the underlined sequences are the constant region sequences of the heavy and light chains.
  • the CDR of the mouse antibody was chimerized into a suitable human GermLine framework (Bioinformation. 2014; 10(4): 180-186; Methods Mol Biol. 2019; 1904: 213-230), and then reverse mutations were introduced at sites that may affect antibody-antigen binding.
  • the nucleotide sequence encoding the variable region of the humanized monoclonal antibody was cloned into the pTT5 vector containing the human heavy and light chain constant region (Human IgG1, kappa) protein sequence and transfected into HEK293 cells to prepare the antibody.
  • the variable region framework selected by the VH/VL of the mouse antibody during the humanization process is shown in Table 5.
  • TCE sites are predicted by software. After predicting the structure of humanized antibodies by homology modeling, mutations are introduced at sites that may not affect antibody-antigen binding to reduce the predicted number of TCE sites.
  • Antibody molecules with TCE removed are produced according to the aforementioned method. After TCE removal, the following sequence is obtained:
  • PR0046 and PR0047 are humanized antibodies of cAb0009
  • PR0098, PR0100, and PR0102 are humanized antibodies of cAb0064.
  • PR0046 and PR0102 were modified by removing TCE to obtain PR0082 and PR0114, respectively.
  • the modified VH and VL were used to construct a full-length antibody, and the human IgG1 Fc sequence used and the obtained full-length sequence are shown below:
  • the underline of the full-length heavy chain sequence above is Fc of IgG1, and the underline of the full-length light chain sequence is C ⁇ of IgG1.
  • SPR detection was performed using the BIAcore 8K (Cityva) system.
  • the sensor chip protein A and related reagents used for detection were purchased from Cytiva.
  • Antibodies PR0082, PR0102 and control antibodies were diluted to 1 ⁇ g/mL with HBS-EP+ buffer, the flow rate was set to 10 ⁇ L/min, and the antibody was captured to a level of 200RU.
  • the His-tagged LIV-1 antigen was diluted with HBS-EP+ buffer in a certain proportion to a concentration gradient of 0nM, 3.125nM, 6.25nM, 12.5nM (two replicates), 25nM, 50nM, 100nM, and 200nM.
  • the flow rate was set to 30 ⁇ L/min for sample analysis.
  • the binding time was 120s and the dissociation time was 900s. Then proceed Regeneration, pH 1.5Gly-HCl buffer was used as the regeneration buffer, the regeneration flow rate was set to 30 ⁇ L/min, and the regeneration was 30s.
  • the response signal was plotted with the analysis time as the abscissa and the response value as the ordinate.
  • the obtained data were fitted by BIAcore 8K analysis software, and the 1:1 Langmuir binding model was used to determine the kinetic constants such as the association rate constant (K a ), dissociation rate constant (K d ) and dissociation equilibrium constant (K D ).
  • the epitopes between antibodies were distinguished by ELISA competitive binding. Specifically, 100 ⁇ L of 0.5 ⁇ g/mL anti-human Fc capture antibody was coated on the ELISA plate and incubated overnight at 4°C. The next day, the corresponding detection antibody was added using the matrix loading method, and the detection antibody and hLIV-1-His were mixed at an appropriate concentration and incubated at room temperature for 1 hour. After the ELISA plate antibody incubation was completed, it was washed three times and a mixture of the detection antibody and LIV-His was added. Incubate at room temperature for 1 hour. Wash three times, add 100 ⁇ L of HRP-labeled anti-His tag secondary antibody to each well, and incubate at room temperature for 1 hour.
  • the synthetic anti-LIV-1 antibody hLIV22 (sequence see patent publication number: US 2016/0185858 A1, which is incorporated herein by reference in its entirety, corresponding to SEQ ID NO: 39 and 40 in the present disclosure) was used as a control.
  • the results in Table 8 show that cAb0009 and hLIV22 belong to different epitopes, and the epitopes of cAb0064 and hLIV22 have a certain overlap.
  • FACS detection of anti-LIV-1 antibodies was performed by the method described in Example 5.
  • DT3C is a recombinantly expressed fusion protein with a molecular weight of 70KD. It is formed by the fusion of fragment A of diphtheria toxin (toxin part only) and fragment 3C of group G streptococcus (IgG binding part). This protein has a high affinity with the IgG part of the antibody and enters the cell together when the antibody is internalized. Under the action of intracellular furin, toxic DT is released. DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and eventually lead to cell death. By using this system, the cell killing effect caused by antibody internalization and immunotoxin can be observed simultaneously (Yamaguchi, M., Hama, H., et al., Biochemical and Biophysical Research Communications 454 (2014) 600-603).
  • DT3C Sterile filtered DT3C and chimeric antibody to be tested (DT3C molar concentration is 6 times the antibody molar concentration) are mixed at a volume ratio of 1:1, incubated at 37°C for 30 minutes, then diluted with complete medium, added to cells (CHO-hLIV-1) plated one day in advance (1000 cells/well), and incubated at 37°C in a 5% carbon dioxide incubator for 6 days. Add CellTiter-Glo, incubate at room temperature in the dark for 10 minutes, and read chemiluminescence on a PerkinElmer.
  • ⁇ HFc-CL-MMAF is an anti-human Fc antibody-conjugated toxin MMAF with a cleavable linker.
  • the antibody portion is a polyclonal antibody that can specifically bind to the Fc portion of human IgG.
  • MMAF is a toxic small molecule that inhibits cell division by blocking the polymerization of tubulin.
  • the linker is stable in the extracellular matrix and can be cleaved by lysosomal tissue proteases after entering the cell through endocytosis, thereby releasing the toxin. The endocytic activity of the antibody is thus evaluated based on the cell-killing activity.
  • ⁇ HFc-CL-MMAF and anti-LIV-1 antibody were mixed at a volume ratio of 1:1, incubated at 37°C for 30 min, then diluted 3-fold with complete medium, added to cells (ATCC, MCF-7/CRL-3435) plated one day in advance (2000 cells/well), and incubated at 37°C in a 5% carbon dioxide incubator for 6 days.
  • CellTiter-Glo purchased from Promega was added, incubated at room temperature in the dark for 10 min, and chemiluminescence was read on a PerkinElmer.
  • Tables 9-1 to 9-2 show the results of cell surface antigen affinity test of anti-LIV-1 antibodies.
  • the results show that the humanized, PTM-modified and TCE-removed antibodies PR0046, PR0047, PR0082, PR0098, PR0100, PR0102, and PR0114 can all retain the binding activity of cAb0009 or cAb0064 to cell surface antigens, and the binding ability is equivalent to that of hLIV22.
  • Figure 1 and Tables 10-1 to 10-4 show the results of the endocytic ability test of the anti-LIV-1 antibodies.
  • Figure 1 shows that the anti-LIV-1 antibody PR0082 has strong internalization and immunotoxin killing activity in the stably transfected cell line CHO-hLIV-1, and its molecular internalization and immunotoxin killing activity are higher than hLIV22, and PR0114 also shows strong molecular internalization and immunotoxin killing activity.
  • This example uses Zenon TM pHrodo TM iFL IgG indicator reagent (Invitrogen, catalog number Z25612) to evaluate the internalization of the antibody.
  • the pHrodo iFL Red-labeled Fab fragment can bind to the Fc portion of the intact IgG antibody to form a labeled complex within 5 minutes.
  • the fluorescence intensity can be recorded in real time by the Incucyte instrument to determine the degree of internalization of the antibody.
  • the cells are plated, 5000 cells per well, and 50 ⁇ L per well.
  • labeled complex After the cells are attached, 50 ⁇ L of labeled complex is added to each well.
  • the labeled complex is configured in the same centrifuge tube: humanized antibody 80nM, pHrodo 240nM, mixed and incubated at room temperature for 5 minutes. Put in the Incucyte instrument (instrument model) and set the plate reading conditions according to the instrument operation steps. After the plate reading is completed, the data is analyzed according to the steps of the instrument's own analysis software.
  • Figure 2 shows the results of pHrodo assay for the internalization of anti-LIV-1 antibodies in SK-OV-3 cells. As shown in Figure 2, PR0082 internalized better than hLIV22, and PR0114 also showed strong cell internalization activity.
  • Compound 9-A (ie, compound 9-A of Example 9 of WO2020063676A1) is N-((2R, 10S)-10-benzyl-2-cyclopropyl-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexa-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide.
  • WO2020063676 is incorporated herein by reference in its entirety.
  • ADC antibody-drug conjugate
  • TCEP-HCl tris(2-carboxyethyl)phosphine hydrochloride
  • Compound 9-A (3.7 mg, 3424.6 nmol) was dissolved in 250 ⁇ L dimethyl sulfoxide, added to the above reaction solution, placed in a water bath oscillator, and oscillated at 25°C for 3 hours to stop the reaction.
  • the reaction solution was desalted with HiPrep 26/10 (buffer: PBS pH 7.2-7.4, flow rate 10 mL/min), concentrated using an ultrafiltration tube to obtain ADC-1 PBS buffer (2.0 mg/mL, 22.5 mL), and stored at 4°C.
  • Example 12 Determination of the activity of ADC in inhibiting the proliferation of tumor cell lines in vitro
  • MCF-7 was cultured in DMEM growth medium containing 10% fetal bovine serum (Gibco).
  • PC3 was cultured in F12-K medium containing 10% fetal bovine serum (Gibco).
  • Cells were grown overnight in 96-well culture plates at a density of 2000 (MCF-7) or 1000 (PC3) per well.
  • An equal volume of serial dilutions of ADC were added the next day.
  • Cell viability was determined 5 days later using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) as described in the manufacturer's protocol. Cell viability was assessed as a percentage of control untreated cells.
  • the ADC drug has nanomolar killing activity on MCF-7 and PC3 tumor cell lines.
  • the expression of LIV-1 on the surface of MCF-7 cells is higher than that of PC3, which indicates that the in vitro cell killing ability of ADC-1 is positively correlated with the abundance of target expression.
  • Example 13 Evaluation of the anti-tumor activity of ADC in a mouse model with high expression of LIV-1
  • the antitumor activity was evaluated in MCF-7 and PC3 mouse xenograft models.
  • MCF-7 xenograft tumor model MCF-7 cells were cultured and 1.5 x 10 7 /0.25 mL (PBS+gel) of MCF-7 cells were inoculated into the third pair of mammary pads of NOD/SCID mice (purchased from Beijing Weitong Lihua). When the mean tumor volume reached about 150 mm 3 , the mice were randomly divided into 6 groups according to tumor volume and body weight, with 8 mice in each group. The mice were intraperitoneally injected with drugs starting from the day of grouping (D0), once a week, for a total of 3 times. The tumor volume and body weight were measured twice a week, and the data were recorded.
  • D0 day of grouping
  • PC3 xenograft tumor model BALB/c nude mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used in the experiment. Male, 6-8 weeks old, 18-22g. Housing environment: SPF grade. 0.2mL (1 ⁇ 10 7 ) PC3 cells (with matrix gel, volume ratio of 1:1) were subcutaneously inoculated on the right back of each mouse. When the average tumor volume reached about 100-150mm 3 , grouping and dosing began. The animals were randomly divided into groups, with 6 animals in each group. On the day of grouping (D0), intraperitoneal injection was started for dosing once a week for a total of 3 times. The tumor volume and body weight were detected twice a week and the data were recorded.
  • Tumor volume V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
  • Relative tumor growth rate T/C (%) (T-T0)/(C-C0) ⁇ 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.
  • Tumor inhibition rate TGI (%) 1-T/C (%). As mentioned above, PBS was used as a negative control. TGI represents the maximum tumor growth inhibition during the experiment.
  • ADC-1 3 mg/kg
  • ADC-1 10 mg/kg
  • PBS P values were 0.0001****, ⁇ 0.0001****, respectively.
  • ADC-1 has dose-dependent antitumor activity.
  • the results of the MCF-7 xenograft tumor model are shown in Tables 12 and 13.
  • the growth inhibition rates of ADC-1 3 mg/kg and ADC-1 10 mg/kg on MCF-7 tumors were higher than those of ADC-2. Since the activities of PR0082 and hLIV22 monoclonal antibodies are comparable, the in vivo results showed that ADC-1 has stronger antitumor activity than ADC-2, indicating that ADC-1 has a stronger bystander effect and is beneficial to anti-tumor growth.
  • the experimental animals were active and ate well during the administration period, and their body weight did not decrease significantly even at the 10 mg/kg dose of ADC-1, indicating that the test drug did not cause obvious toxic side effects on the experimental animals, and had good tolerance and safety to toxins.
  • the experimental animals were active and ate well during the administration period, and their body weight did not decrease significantly even at the 10 mg/kg dose of ADC-1, indicating that the test drug did not cause obvious toxic side effects on the experimental animals, and had good tolerance and safety to toxins.
  • NOD-SCID mice normal grade, animal source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • ADC-1 was injected into the tail vein.
  • the single dose was set at 10 mg/kg, and the administration volume was 10 mL/kg (diluted with 0.9% sodium chloride injection).
  • PBST buffer After washing five times with PBST buffer, the color developing solution was added, and the stop solution was added after the color development was completed, and the plate was read with an enzyme reader. Take OD450. Add mouse-derived IgG Fc antibody linked to compound 9-A to another portion, using a concentration of 1 ⁇ g/mL, 100 ⁇ L per well. Incubate at room temperature for 1 hour. After washing three times with PBST buffer, add HRP-labeled anti-mouse IgG Fc antibody and incubate at room temperature for 1 hour. After washing five times with PBST buffer, add colorimetric solution and stop solution. Read OD450 with an enzyme reader. PK parameters were calculated using the non-compartmental model of Phoenix software.

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Abstract

L'invention concerne une molécule de liaison à LIV-1 et son procédé de préparation, ainsi qu'un immunoconjugué correspondant, une composition pharmaceutique, une utilisation médicale, et une méthode de prévention d'une maladie, en particulier une méthode de traitement du cancer ou d'une tumeur.
PCT/CN2023/134161 2022-11-25 2023-11-24 Anticorps anti-liv-1, conjugué médicamenteux associé et utilisation médicale associée WO2024109944A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040141983A1 (en) * 1999-03-15 2004-07-22 Protein Design Labs, Inc. Compositions against cancer antigen LIV-1 and uses thereof
US20050175619A1 (en) * 2004-02-05 2005-08-11 Robert Duffy Methods of producing antibody conjugates
CN1849337A (zh) * 2003-01-27 2006-10-18 比奥根艾迪克Ma公司 利用igsf9和liv－1的、用于治疗癌症的组合物及方法
CN103533957A (zh) * 2010-12-06 2014-01-22 西雅图遗传学公司 抗liv-1的人源化抗体及其在治疗癌症中的用途
CN106999517A (zh) * 2014-10-07 2017-08-01 免疫医疗公司 抗体‑药物缀合物的新辅助剂用途
CN114206933A (zh) * 2019-06-10 2022-03-18 加的夫大学学院咨询有限公司 包含针对zip6和/或zip10的抗体的抗有丝分裂组合物

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040141983A1 (en) * 1999-03-15 2004-07-22 Protein Design Labs, Inc. Compositions against cancer antigen LIV-1 and uses thereof
CN1849337A (zh) * 2003-01-27 2006-10-18 比奥根艾迪克Ma公司 利用igsf9和liv－1的、用于治疗癌症的组合物及方法
US20050175619A1 (en) * 2004-02-05 2005-08-11 Robert Duffy Methods of producing antibody conjugates
CN103533957A (zh) * 2010-12-06 2014-01-22 西雅图遗传学公司 抗liv-1的人源化抗体及其在治疗癌症中的用途
CN106999517A (zh) * 2014-10-07 2017-08-01 免疫医疗公司 抗体‑药物缀合物的新辅助剂用途
CN114206933A (zh) * 2019-06-10 2022-03-18 加的夫大学学院咨询有限公司 包含针对zip6和/或zip10的抗体的抗有丝分裂组合物

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