WO2023143444A1 - 抗dkk1抗体、其药物组合物及用途 - Google Patents

抗dkk1抗体、其药物组合物及用途 Download PDF

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WO2023143444A1
WO2023143444A1 PCT/CN2023/073335 CN2023073335W WO2023143444A1 WO 2023143444 A1 WO2023143444 A1 WO 2023143444A1 CN 2023073335 W CN2023073335 W CN 2023073335W WO 2023143444 A1 WO2023143444 A1 WO 2023143444A1
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seq
amino acid
acid sequence
nos
antibody
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何志娟
王亚红
李胜胜
杨灵芝
周岳华
赵强
姚盛
冯辉
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Priority to EP23746323.7A priority Critical patent/EP4471056A4/en
Priority to US18/833,372 priority patent/US20250122271A1/en
Publication of WO2023143444A1 publication Critical patent/WO2023143444A1/zh
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    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • the invention belongs to the field of biomedicine, and relates to an anti-DKK1 antibody, its pharmaceutical composition and application.
  • DKK1 is a secreted inhibitor of the Wnt/ ⁇ -catenin signal transduction pathway, which belongs to the DKK (dickkopf) family, one of the protein families that inhibit Wnt signal transduction, and has the ability to inhibit Wnt-induced axis replication.
  • the DKK family currently has 4 family members, namely DKK1, DKK2, DKK3 and DKK4.
  • the Wnt signaling pathway is involved in the control of embryonic development and tumorigenic processes. Extracellular Wnt proteins are responsible for the growth and differentiation of various cell types during embryonic development and contribute to the development of various cancers. Genetic analysis revealed that DKK1 inhibits Wnt signaling upstream and that DKK1 interacts antagonistically with LRP6, blocking Wnt-mediated signaling activation.
  • DKK1 protein In the absence of DKK1 protein, Wnt, Frizzled, and LRP6 combine to form heterotrimers, activate downstream signaling pathways, maintain the stability of ⁇ -catenin, accumulate after entering the nucleus, and promote the transcription of osteoblast-related genes and tumor suppressor genes and translation.
  • DKK1 protein exists, DKK1 competes with Wnt to bind LRP6 molecules, and a series of downstream signaling reactions promote the phosphorylation of ⁇ -catenin and then be degraded. Thus blocking ⁇ -catenin into the nucleus, thereby blocking the transcription and translation of osteoblast-related genes and tumor suppressor genes.
  • DKK1 can promote the formation of the tumor microenvironment by increasing the inhibitory activity of MDSC and Treg cells, down-regulating the ligands that activate NK cells on the surface of tumor cells, promoting the polarization of Th2 cells and reducing the production of IFN ⁇ .
  • DKK1 can also promote tumor growth and migration, promote the generation of CSC-like cells, and promote tumor angiogenesis, thereby directly promoting the occurrence and development of tumors (Yu et al., Signal Transduct Target Therapy, 2021, 6(1); Nikolai et al., Cancer Letters, 2020, 482).
  • LeapTherapeutics announced that its DKN-01 monoclonal antibody targeting DKK1 had achieved positive results in Phase 2 clinical treatment of advanced gynecological cancers. Clinical results showed that DKN-01 was more effective in patients with high expression of DKK1, with an objective response rate of 14% and a disease control rate of 57%.
  • DKK1 is an important regulator of tumors and deserves more attention as a key therapeutic target; and in fact, strategies to develop DKK1 inhibitors have produced encouraging clinical results in different pathological models (Hewen Jiang et al., Drug Discovery of DKK1 Inhibitors, Frontiers in Pharmacology, March 2022, Volume 13, p1-17).
  • the present inventors prepared a hybridoma cell line expressing anti-DDK1 antibody and obtained the expressed anti-DDK1 antibody. Furthermore, the inventors designed a chimeric antibody on this basis and carried out humanization transformation. The present inventors surprisingly found that the anti-DDK1 antibody of the present invention has high affinity and/or specificity for DDK1, can effectively inhibit tumor growth, and has good anti-tumor potential.
  • the following inventions are thus provided:
  • One aspect of the invention relates to an anti-DKK1 antibody or antigen-binding fragment thereof,
  • the anti-DKK1 antibody comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises HCDR1 to HCDR3
  • the light chain variable region comprises LCDR1 to LCDR3
  • amino acid sequence of HCDR1 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25 and SEQ ID NO:31;
  • amino acid sequence of HCDR2 is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26 and SEQ ID NO:32;
  • amino acid sequence of HCDR3 is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33 and SEQ ID NO:78;
  • amino acid sequence of LCDR1 is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28 and SEQ ID NO:34;
  • amino acid sequence of LCDR2 is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29 and SEQ ID NO:35;
  • the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30 and SEQ ID NO:36.
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein:
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:1
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:2
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:3
  • HCDR1 The amino acid sequence of HCDR1 is shown in SEQ ID NO:1
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:2
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:78
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:7
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:8
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:9
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:13
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:14
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:15
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:19
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:20
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:21
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:25
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:26
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:27, or
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:31
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:32
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:33;
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:6,
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:10
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:12
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:16
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:17
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:18
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:22, the amino acid sequence of LCDR1 is shown in SEQ ID NO:23, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:24,
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:28, the amino acid sequence of LCDR1 is shown in SEQ ID NO:29, the amino acid sequence of LCDR1 is shown in SEQ ID NO:30, or
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:34, the amino acid sequence of LCDR1 is shown in SEQ ID NO:35, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:36.
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein,
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:4
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:6;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:78, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:4
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR1 is shown in SEQ ID NO: as shown in 6;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 7
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:12;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:13
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:14
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:15
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:16
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:17
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:18;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:19
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:20
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:21
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:22
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:23
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:24;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:25
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:26
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:27
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:28
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:29
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:30;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:31
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:32
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:33
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:34
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:35
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:36.
  • variable regions of the light and heavy chains determine the binding of antigens; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3 , the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; which are named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242.
  • CDRs complementarity determining regions
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NOs: 49-53, SEQ ID NOs: 58-62, and SEQ ID NOs: 68-73; and
  • amino acid sequence of the light chain variable region of the antibody is selected from the group consisting of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs: 54-57, SEQ ID NOs: 63-67 and SEQ ID NOs: 74-77.
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 47, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:50, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:58, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 59, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:60, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 62, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:68, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 69, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 70, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 72, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs:63-67 and SEQ ID NOs:74-77;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 73, and the amino acid sequence of the light chain variable region is Selected from the group consisting of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NOs:54-57, SEQ ID NOs :63-67 and SEQ ID NOs:74-77.
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain of the antibody is selected from SEQ ID NOs: 79-83, SEQ ID NOs: 88-92 and SEQ ID NOs: 98-103;
  • the amino acid sequence of the light chain of the antibody is selected from SEQ ID NOs: 84-87, SEQ ID NOs: 93-97 and SEQ ID NOs: 104-107.
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:79, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:80, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:81, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:82, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:83, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:88, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:89, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:90, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:91, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:92, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:98, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:99, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 100, and the amino acid sequence of the light chain is selected from SEQ ID NO: 100 ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:101, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:102, and the amino acid sequence of the light chain is selected from SEQ ID NOs:84-87, SEQ ID NOs:93-97 and SEQ ID NOs:104-107;
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 103, and the amino acid sequence of the light chain is selected from SEQ ID NOs: 84-87, SEQ ID NOs: 93-97 and SEQ ID NOs: 104-107.
  • the anti-DKK1 antibody or its antigen-binding fragment wherein, the anti-DKK1 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv , dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody.
  • Another aspect of the present invention relates to an anti-DKK1 antibody or an antigen-binding fragment thereof, which has the following characteristics:
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein,
  • the antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, eg, from a human antibody.
  • the anti-DKK1 antibody or antigen-binding fragment thereof wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region (such as NCBI ACCESSION: P01857) or Iggamma-4 chain C region (such as NCBI ACCESSION: P01861.1); the light chain constant region is Ig kappa chain C region (such as NCBI ACCESSION: P01834);
  • amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO: 112
  • amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO: 113.
  • Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-DKK1 antibody or antigen-binding fragment thereof according to any one of the present invention.
  • Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.
  • Yet another aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a recombinant vector of the invention.
  • a conjugate which includes an antibody and a coupling part, wherein the antibody is the anti-DKK1 antibody or an antigen-binding fragment thereof according to any one of the present invention, and the coupling part is is a detectable label; preferably, the coupling moiety is a cytotoxin, an immunomodulator, a radioactive isotope, a fluorescent substance, a luminescent substance, an organic color substances or enzymes.
  • the coupling moiety is a cytotoxin, an immunomodulator, a radioactive isotope, a fluorescent substance, a luminescent substance, an organic color substances or enzymes.
  • kits which includes the anti-DKK1 antibody or antigen-binding fragment thereof according to any one of the present invention, or the conjugate of the present invention;
  • the kit also includes a second antibody that specifically recognizes the antibody; optionally, the second antibody also includes a detectable label, such as cytotoxin, immunomodulator, radioactive isotope, fluorescent substance , luminescent substances, colored substances or enzymes.
  • a detectable label such as cytotoxin, immunomodulator, radioactive isotope, fluorescent substance , luminescent substances, colored substances or enzymes.
  • Yet another aspect of the present invention relates to a bispecific antibody comprising a first protein domain and a second protein domain, wherein:
  • the first protein domain targets DKK1,
  • said second protein domain targets a target other than DKK1,
  • the first protein functional domain is the antibody or antigen-binding fragment described in any one of the present invention.
  • the bispecific antibody wherein the first protein functional domain and the second protein functional domain are directly connected or connected through a linking fragment.
  • the first protein functional domain and the second protein functional domain are independently 1, 2 or more than 2.
  • the single-chain antibody is respectively linked to the C-terminals of the two heavy chains of the antibody in the form of immunoglobulin.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises the anti-DKK1 antibody or antigen-binding fragment thereof according to any one of the present invention, or comprises the conjugate of the present invention;
  • the pharmaceutical composition also includes one or more other anti-tumor drugs; preferably, the other anti-tumor drugs are chemotherapy drugs or antibody drugs; preferably, the chemotherapy drugs are tubulin inhibitors ;
  • the antibody drug is an immune checkpoint inhibitor;
  • the chemotherapy drug is paclitaxel;
  • the pharmaceutical composition further includes one or more pharmaceutically acceptable adjuvants (such as carriers and/or excipients).
  • pharmaceutically acceptable adjuvants such as carriers and/or excipients.
  • the pharmaceutical composition wherein, calculated according to the mass of the antibody, the mass ratio of the anti-DKK1 antibody or its antigen-binding fragment to paclitaxel is (1:5)-(5: 1), for example: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.
  • a further aspect of the present invention relates to a combination product comprising a first product and a second product separately packaged, wherein,
  • the first product comprises an anti-DKK1 antibody or an antigen-binding fragment thereof according to any one of the present invention, or a conjugate of the present invention;
  • the second product comprises one or more other anti-tumor drugs; preferably, the other anti-tumor drugs are chemotherapy drugs or antibody drugs; preferably, the chemotherapy drugs are tubulin inhibitors; preferably, the The antibody drug is an immune checkpoint inhibitor; preferably, the chemotherapy drug is paclitaxel;
  • the first product and the second product also independently comprise one or more pharmaceutically acceptable adjuvants (such as carriers and/or excipients);
  • pharmaceutically acceptable adjuvants such as carriers and/or excipients
  • the combined product further includes product instructions.
  • the combination product wherein, calculated according to the mass of the antibody, the mass ratio of the anti-DKK1 antibody or its antigen-binding fragment to paclitaxel is (1:5)-(5:1 ), for example: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.
  • Another aspect of the present invention relates to the antibody or antigen-binding fragment thereof according to any one of the present invention, the conjugate of the present invention, the bispecific antibody according to any one of the present invention or any one of the present invention
  • the antibody or antigen-binding fragment thereof according to any one of the present invention, the conjugate of the present invention, the bispecific antibody according to any one of the present invention or the pharmaceutical composition according to any one of the present invention which is used for the treatment and/or prevention of DKK1-mediated diseases; preferably, the DKK1-mediated diseases are tumors; preferably, the tumors are selected from gallbladder cancer, bladder cancer, osteosarcoma, esophageal cancer, bile duct cancer One or more of endometrial cancer, ovarian cancer, gastric cancer, gastroesophageal junction cancer, colon cancer, rectal cancer, hepatocellular carcinoma, prostate cancer, lung cancer and myeloma.
  • Another aspect of the present invention relates to a method for treating or preventing DKK1-mediated diseases, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in any one of the present invention, the present invention
  • the disease mediated by DKK1 is a tumor;
  • the tumor is selected from gallbladder cancer, bladder cancer, osteosarcoma, esophageal cancer, cholangiocarcinoma, endometrial cancer, ovarian cancer, gastric cancer, gastroesophageal junction cancer, colon cancer, rectal cancer, hepatocellular carcinoma, prostate cancer, One or more of lung cancer and myeloma.
  • the administration method is intravenous drip or intravenous injection.
  • the method for treating or preventing a disease mediated by DKK1 is a method for treating or preventing tumors.
  • the method for treating or preventing tumors further includes the step of administering one or more therapies to the subject in combination, and the therapies include surgical treatment and/or radiation therapy and/or Administration of one or more other antineoplastic drugs;
  • the other anti-tumor drugs are chemotherapy drugs or antibody drugs; preferably, the chemotherapy drugs are tubulin inhibitors; preferably, the antibody drugs are immune checkpoint inhibitors; preferably, the chemotherapy drug for Paclitaxel.
  • Figure 1 ELISA detection of the blocking effect of hybridoma antibody on the binding of DDK1 and LRP6.
  • Figure 2A The antagonism effect of the hybridoma antibody on the inhibitory effect of DDK1 on Wnt signaling was detected by the luciferase reporter gene method.
  • Figure 2B The antagonism effect of the hybridoma antibody on the inhibitory effect of DDK1 on Wnt signaling was detected by the luciferase reporter gene method.
  • Figure 3 ELISA detection of the blocking effect of chimeric antibodies on the binding of DDK1 and LRP6.
  • Figure 4A ELISA detection of chimeric antibody binding to DKK1.
  • Figure 4B ELISA detection of chimeric antibody binding to DKK1.
  • Figure 5A The luciferase reporter gene assay detects the recovery effect of the chimeric antibody on the Wnt signaling pathway.
  • Figure 5B The luciferase reporter gene assay detects the recovery effect of the chimeric antibody on the Wnt signaling pathway.
  • Figure 5C The luciferase reporter gene assay detects the recovery effect of the chimeric antibody on the Wnt signaling pathway.
  • Figure 6 ELISA detection of the binding effect of humanized antibody to DDK1.
  • Figure 7 ELISA detection of the blocking effect of humanized antibodies on the binding of DDK1 and LRP6.
  • Figure 8 Detection of the antagonism effect of humanized antibody on the inhibitory effect of DKK1 on Wnt signaling by luciferase reporter gene method.
  • Figure 9 Inhibitory effect of humanized antibody hu3 on tumor growth.
  • Figure 10 Inhibitory effect of humanized antibody hu3 on tumor growth.
  • the present invention provides an anti-DKK1 antibody or an antigen-binding fragment thereof, which is characterized in that it has a unique CDR sequence composition and has high affinity and high specificity for binding to human DKK1.
  • the anti-DKK1 antibody or antigen-binding fragment thereof provided by the present invention can be used as an independent therapy or in combination with other therapies and/or other anti-cancer agents for the treatment of, for example, cancer.
  • DKK1 herein is a secreted inhibitor of the Wnt/ ⁇ -catenin signal transduction pathway, which belongs to the DKK (dickkopf) family, one of the protein families that inhibit Wnt signal transduction, and refers to any Any native DKK1 of long species (e.g., humans) and rodents (e.g., mice and rats), unless otherwise There are instructions.
  • the term encompasses "full length” unprocessed DKK1 as well as any form of DKK1 or any fragment thereof produced by intracellular processing.
  • the term also includes naturally occurring variants of DKK1, eg, splice variants or allelic variants.
  • DKK1 refers to full length DKK1 or a fragment thereof (such as its mature fragment lacking a signal peptide) from human and mouse. In some embodiments, DKK1 refers to mature human DKK1 (amino acid residues 1-31 is the leader peptide) identical to the sequence of amino acid residues 32-266 of Genbank Accession No. NP_036374.1 (SEQ ID NO: 31).
  • Percent (%) amino acid sequence identity is defined after aligning the sequences (and introducing gaps where necessary) for maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity , the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Immuno response refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines, and complement) by the above cells or the liver, which results in selective damage from the body , destroy or eliminate invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • lymphocytes for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines, and complement) by the above cells or the liver, which results in selective damage from the body , destroy or eliminate invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • Signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, usually initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another .
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a cascade of reactions resulting in signal transduction.
  • the penultimate process often involves nuclear events, resulting in changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, in vivo or in vitro neutralization or Ability to antagonize DKK1 activity, IC50, in vivo stability of antibody, and immunogenic properties of antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., often with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention The ability to express proteins at high levels in mammalian cells.
  • Antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of a binding compound, and in this context means that the molecule is substantially free of other Biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media.
  • isolated does not imply the complete absence of such substances or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.
  • “Monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” characterizes an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • Full-length antibody an immunoglobulin molecule comprising four peptide chains as it occurs in nature, two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains (approximately 25 kDa in full length) interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDRs) of high variability separated by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • an "antigen-binding fragment" of an antibody (“parent antibody”) includes a fragment or derivative of an antibody, typically including at least a fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains the parent antibody at least some of the binding specificities.
  • antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • the binding fragment or derivative typically retains at least 10% of its antigen binding activity when the antigen binding activity is expressed on a molar concentration basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not appreciably alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • a “single-chain Fv” or “scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides generally also comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • Domain antibodies are immunologically functional immunoglobulin fragments that contain only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • a “bivalent antibody” comprises two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • “Diabody” refers to a small antibody fragment with two antigen binding sites comprising a heavy chain variable domain (VL) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). Variable domain (VH). By using a linker that is too short to allow pairing between the two domains of the same chain, this domain is forced to pair with the complementary domain of another chain and creates two antigen-binding sites.
  • a “chimeric antibody” is an antibody that has the variable domains of a first antibody and the constant domains of a second antibody, where the first and second antibodies are from different species.
  • the variable domains are derived from antibodies of experimental animals such as rodents ("parental antibodies”), while the constant domain sequences are derived from human antibodies, such that the resulting chimeric antibody is more effective in human subjects than the parental rodent antibody. Less likely to induce an adverse immune response.
  • Humanized antibody refers to a form of an antibody that contains sequences from both human and non-human (eg, murine, rat) antibodies.
  • a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin.
  • Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • a humanized antibody can comprise, optionally, at least a portion of a human immunoglobulin constant region (Fc).
  • a “fully human antibody” refers to an antibody that contains only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains as produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the antibody class (eg, IgM, IgE, IgG such as IgG1, IgG2 or IgG4) that is contributed by the heavy chain constant region genes.
  • Isotypes also include modified forms of one of these classes, wherein modifications have been made to alter Fc function, for example to enhance or decrease effector function or binding to Fc receptors.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Al's US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage or linear or circular single- or double-stranded DNA or RNA polynucleotide molecules), derived from any source, capable of integrating into the genome or replicating autonomously, constitute polynucleotide molecules which have been linked in a functionally operative manner (i.e. , operably linked to) one or more polynucleotide molecules.
  • a functionally operative manner i.e. , operably linked to
  • Recombinant constructs will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell.
  • transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell.
  • Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of the invention.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • vectors After introduction into a host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate along with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors.”
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest upon transformation, transfection or transduction into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if desired.
  • Activation can have the same meaning, eg, the cell or receptor is activated, stimulated or treated with a ligand, unless the context dictates otherwise or clearly.
  • Ligand includes natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • treatment means, in one embodiment, ameliorating the disease or condition (ie, slowing or arresting or reducing at least one of the progression of the disease or its clinical symptoms).
  • treating refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient.
  • treating or “curing” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiological (eg, stabilization of physical parameters), or both.
  • methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.
  • Administration "in conjunction with” one or more other antineoplastic drugs includes simultaneous (co)administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to the DKK1 antibody of the present invention or its antigen-binding tablet An amount effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the development of the disease or condition when administered to a cell, tissue or subject, alone or in combination with other therapeutic agents.
  • a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates an associated medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • a therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of the active ingredients which produces the therapeutic effect, whether in combination, sequentially or simultaneously.
  • a therapeutically effective amount will result in an increase in diagnostic criteria or parameters of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, most preferably at least 50%.
  • Cancer and “cancerous” refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases.
  • anti-DKK1 antibody capable of binding a DKK1 protein or a fragment thereof with sufficient affinity such that said antibody can be used as a diagnostic agent targeting DKK1 and/or or therapeutic agents.
  • Antibodies of the invention can be produced using any suitable method for producing antibodies. Any suitable form of DKK1 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any DKK1 variant or fragment thereof can be used as an immunogen.
  • hybridoma cells producing murine monoclonal anti-human DKK1 antibodies can be produced by methods known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler et al. (1975) (Nature 256:495-497). Mouse splenocytes are isolated and fused with a mouse myeloma cell line using PEG or by electrofusion, preferably according to standard protocols. Hybridoma cells secreting antibodies that have DKK1 inhibitory activity are then screened. The DNA sequence of the hybridoma immunoglobulin variable region of the present invention can be determined by a method based on degenerate primer PCR.
  • Antibodies derived from rodents can cause unwanted antibody immunogenicity when used in vivo as therapeutic agents, with repeated use leading to an immune response in the body against the therapeutic antibody that results in at least a loss of therapeutic efficacy , and severe, potentially fatal allergic reactions.
  • One approach to reducing the immunogenicity of rodent antibodies involves the generation of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439- 43). However, retention of intact rodent variable regions in chimeric antibodies can still cause deleterious immunogenicity in patients.
  • CDR complementarity-determining region
  • chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the prepared murine monoclonal hybridoma antibody.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric DKK1 antibody of the present invention can be hybridized using methods known in the art Tumor-derived immunoglobulin heavy and light chain variable regions are operably linked to human IgG constant regions (see, eg, US Patent No. 4,816,567 to Cabilly et al.), resulting in chimeric heavy and light chains.
  • the constant region contained in the chimeric antibody of the present invention can be selected from any subtype of human IgG, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric DKK1 antibody of the present invention can be obtained by "mixing and matching" a chimeric light chain and a chimeric heavy chain expression plasmid to transfect expressing cells, such "mixed and matched" DKK1 binding of antibodies can be tested using the binding assays described above and other conventional binding assays (eg, ELISA).
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment systems or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.
  • Antibodies with different specificities have different binding sites for different antigens.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.
  • the murine CDR regions can be inserted into the human germline framework regions using methods known in the art. See US Patent No. 5,225,539 to Winter et al. and US Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 to Queen et al. In short, the inventors searched the human immunoglobulin gene database in the NCBI (http://www.ncbi.nlm.nih.gov/igblast/) website and found that the murine antibody The cDNA sequence of the variable region is homologous to the human germline IgG gene, which can in principle be humanized by grafting selected CDRs.
  • CDR loop exchange still does not uniformly generate antibodies with the same binding properties as the starting antibody.
  • changes in framework residues (FRs) are often also required in order to preserve antigen-binding affinity.
  • the humanization process involves the following steps: A. Compare the gene sequence of each candidate antibody with the human embryonic antibody gene sequence to find out the sequence with high homology; B. Analyze and investigate the HLA-DR parent compatibility, select the human embryonic frame sequence with low affinity; C, use computer simulation technology, apply molecular docking to analyze the variable region and its surrounding framework amino acid sequence, and investigate its spatial three-dimensional binding mode.
  • Fc region variants may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • a human Fc region sequence eg, a human IgGl, IgG2, IgG3 or IgG4 Fc region
  • amino acid modifications eg, substitutions
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are substituted with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art.
  • Moieties suitable for antibody derivatization include, but are not limited to, water soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerin), polyvinyl alcohol, and mixture
  • the anti-DKK1 antibody or antigen-binding fragment thereof of the present invention has one or more of the following properties: (1) specifically binds to human DKK1 protein; (2) cross-reacts with cynomolgus monkey DKK1 ; (3) inhibit the combination of DKK1 and LRP5 or LRP6; (4) inhibit the active signal transduction mediated by DKK1; (5) significantly inhibit the growth of tumors.
  • the anti-DKK1 antibody or antigen-binding fragment thereof of the present invention has at least one of the following properties:
  • (1) binds to human DKK1 with a K of at least about 5 nM, at least about 1 nM, at least about 0.1 nM, at least about 0.01 nM, at least about 0.001 nM;
  • the invention also relates to methods for producing the antibodies or antigen-binding fragments thereof of the invention.
  • Such methods include providing an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment of the invention, or an expression vector comprising such nucleic acid, In particular, vectors for the recombinant production of the antibodies or antigen-binding fragments thereof of the present invention in host cells.
  • the present invention also relates to host cells comprising one or more of the aforementioned recombinant vectors or expression vectors and methods for producing the antibodies of the present invention or comprising antigen-binding fragments thereof, comprising culturing said host cells, purifying and recovering The antibody or antigen-binding fragment thereof.
  • the present invention also provides a method for producing the above-mentioned antibody or antigen-binding fragment thereof, comprising culturing the above-mentioned host cell, and recovering the antibody or antigen-binding fragment thereof from the culture.
  • the present invention also relates to methods of treating cancer in a subject, the methods comprising administering to the subject an effective amount of any of the anti-DKK1 antibodies or antigen-binding fragments thereof described herein, or pharmaceutical compositions thereof.
  • it also relates to the use of any anti-DKK1 antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament for treating cancer in a subject.
  • the present invention also provides the use of the above-mentioned antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell or pharmaceutical composition in the preparation of medicines for treating and/or preventing DKK1-mediated diseases or disorders; preferably, The disease or condition is cancer.
  • the invention also relates to the co-administration of one or more therapies (eg, treatment modalities and/or other antineoplastic agents) to a cancer subject.
  • the aforementioned therapy comprises administering to the subject an effective amount of an anti-DKK1 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof according to any embodiment of the invention.
  • treatment modalities include surgery and radiation therapy.
  • the above-mentioned other anti-tumor drugs include chemotherapeutic drugs and/or immune checkpoint inhibitors, wherein the chemotherapeutic drugs are selected from tubulin inhibitors/or anti-angiogenic agents; wherein the chemotherapeutic drugs are preferably tubulin inhibitors.
  • the invention also relates to the use of any of the anti-DKK1 antibodies or antigen-binding fragments thereof described herein in combination with a tubulin inhibitor for the manufacture of a medicament for the treatment of cancer in a subject.
  • the present invention also relates to a method of detecting DKK1 in a sample comprising: a) contacting the sample with any anti-DKK1 antibody or fragment thereof described herein; and b) detecting a complex between the anti-DKK1 antibody or fragment thereof and DKK1 Formation.
  • the anti-DKK1 antibody is detectably labeled.
  • the invention relates to a kit or article of manufacture comprising any of the anti-DKK1 antibodies or fragments thereof described herein.
  • the kit or article of manufacture comprises an anti-DKK1 antibody or fragment thereof described herein and optionally a pharmaceutically acceptable excipient, and optionally one or more other antineoplastic agents (e.g., immune checkpoint inhibitors, tubulin inhibitors, antiangiogenic agents, or other chemotherapy drugs).
  • antineoplastic agents e.g., immune checkpoint inhibitors, tubulin inhibitors, antiangiogenic agents, or other chemotherapy drugs.
  • the present invention also covers any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof applies to any and all anti-DKK1 antibodies or fragments thereof, methods and uses of the invention described herein.
  • the anti-DKK1 antibody or antigen-binding fragment thereof of the present invention specifically binds DKK1 with a KD of about 1nM-2pM or higher affinity, preferably with a KD or affinity of 1nM, 100pM, 10pM or 2pM Specifically binds DKK1.
  • the antibody of the present invention that specifically binds to human DKK1 also binds to Cynomolgus DKK1 cross-reactivity. "Cross-reactivity" as used herein refers to the ability of an antibody to react with homologous proteins from other species. Whether an antibody specifically binds human DKK1 can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (eg, BIACORE) or similar techniques (eg, ForteBio).
  • the DKK1 antibody or antigen-binding fragment thereof of the present invention has inhibitory activity, such as inhibiting the expression, activity and/or signaling of DKK1 (such as inhibiting the expression of DKK1 on the cell surface), or interfering with DKK1 and LRP5 or Interactions between LRP6s.
  • the DKK1 antibody provided by the invention completely or partially reduces or regulates the expression or activity of DKK1 after binding to or interacting with DKK1 (such as human DKK1).
  • the reduction or modulation of the biological function of DKK1 is complete, substantial or partial following the interaction between the antibody and the human DKK1 polypeptide and/or peptide.
  • the expression or activity level of DKK1 is reduced by at least 95% (e.g., 96%, 97%, 98%, 99% or 100%), the antibody is considered to be able to completely inhibit the expression or activity of DKK1.
  • the expression or activity level of DKK1 is reduced by at least 50% (e.g., by 55%, 60%, 75%, 80% in the presence of a DKK1 antibody) compared to the expression or activity level of DKK1 in the absence of binding to a DKK1 antibody described herein.
  • the DKK1 antibody is considered to be able to significantly inhibit the expression or activity of DKK1.
  • the expression or activity level of DKK1 in the presence of an antibody is reduced by less than 95% (e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%), at this time, the antibody is considered to be able to partially inhibit the expression or activity of DKK1.
  • the invention relates to host cells comprising one or more expression vectors and methods for producing any of the antibodies or fragments thereof of the invention comprising culturing said host cells, purifying and recovering said antibodies or Antigen-binding fragments.
  • the invention provides a nucleic acid encoding any of the above anti-DKK1 antibodies or fragments thereof.
  • the nucleic acid may comprise a nucleic acid encoding an amino acid sequence of a light chain variable region and/or a heavy chain variable region of an antibody, or a nucleic acid comprising an amino acid sequence encoding a light chain and/or a heavy chain of an antibody.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including the many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Select specific cell lines by determining which cell lines have high expression levels Preferred cell lines.
  • ATCC American Type Culture Collection
  • the present invention provides a method for preparing an anti-DKK1 antibody, wherein the method comprises introducing an expression vector into a mammalian host cell, culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, Or more preferably, the antibody is produced by allowing secretion of the antibody into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • non-fucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo and are less likely to be immunogenic , because their sugar structure is a normal component of natural human serum IgG.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more antibodies or antigen-binding fragments thereof that bind to DKK1, which comprises the antibody or antigen-binding fragment thereof as described in any one of the foregoing and a pharmaceutically acceptable carrier or excipient composition of agents.
  • anti-DKK1 antibodies or antigen-binding fragments thereof or pharmaceutical compositions thereof provided herein can be administered in combination with suitable carriers, excipients and other agents in formulations, thereby providing improved transfer, delivery, tolerance wait.
  • composition refers to a preparation that permits the biological activity of the active ingredients contained therein to be present in an effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered. .
  • the pharmaceutical formulation is in the form of an aqueous solution or a lyophilized formulation.
  • compositions or formulations of the invention may also contain one or more other active ingredients as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • other active ingredients are chemotherapeutic drugs, tubulin inhibitors, immune checkpoint inhibitors, growth inhibitors, antibiotics or various known anti-tumor or anti-cancer agents, the active ingredients are for Amounts effective for the intended use are suitably present in combination.
  • the pharmaceutical composition of the present invention further comprises a composition of a polynucleotide encoding an anti-DKK1 antibody or an antigen-binding fragment thereof.
  • the present invention relates to administering to a subject an effective amount of any anti-DKK1 antibody or antigen-binding fragment thereof described herein, or an immunoconjugate comprising said antibody or antigen-binding fragment thereof, or a pharmaceutical composition.
  • method with It is used to induce anti-tumor activity mediated by T cells or NK cells, promote tumor suppressor activity related to the canonical Wnt pathway, or enhance the body's immune response.
  • the present invention relates to administering to a subject an effective amount of any anti-DKK1 antibody or antigen-binding fragment thereof described herein, or an immunoconjugate comprising said antibody or antigen-binding fragment, or a pharmaceutical composition.
  • a method for treating or delaying various cancers, immune-related diseases and T cell dysfunction diseases are also contemplated.
  • the present invention provides a method for treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof as described in any one of the foregoing, or as described in the foregoing The steps of any one of the pharmaceutical compositions.
  • the method as described above further comprising the step of co-administering one or more therapies to said subject, said therapies including surgical treatment and/or radiation therapy and/or administering a or multiple other anti-tumor drugs, and the other anti-tumor drugs are selected from chemotherapy drugs or immune checkpoint inhibitors.
  • the chemotherapeutic drugs are selected from the group consisting of alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylmelamines ), acetogenin, ⁇ -9-tetrahydrocannabinol, ⁇ -lapachone, lapachol, colchicines, betulinic acid (betulinic acid), camptothecin, bryostatin, pemetrexed, podophyllotoxin, podophyllinic acid, teniposide, Cryptophycins, dolastatin, eleutherobin, spongistatin, nitrogen mustards, cholophosphamide, estralimus Estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenyl mustard sterols, prednimustine, trofosfamide, uracil mustard, nitro
  • the chemotherapeutic drug includes paclitaxel.
  • the present invention provides the aforementioned method for treating or preventing cancer, the method comprising administering to the subject an effective amount of the anti-DKK1 antibody or its antigen as described in any one of the foregoing A binding fragment, or a pharmaceutical composition as described above.
  • the method as described above further comprising co-administering to said subject one or more therapies, said therapies comprising administering paclitaxel.
  • the anti-DKK1 antibody or its antigen-binding fragment in the method for treating or preventing cancer as described above, can be administered simultaneously or not simultaneously with paclitaxel; in some embodiments of the present invention, as described above In the above method for treating or preventing cancer, the anti-DKK1 antibody or its antigen-binding fragment can be administered simultaneously or not simultaneously with paclitaxel; in some embodiments of the present invention, as described above in the method for treating or preventing cancer, the anti-DKK1 antibody or an antigen-binding fragment thereof is administered once a week or twice a week; in some embodiments of the present invention, in the method for treating or preventing cancer as described above, paclitaxel is administered once a week or twice a week .
  • the present invention provides the use of the anti-DKK1 antibody or antigen-binding fragment thereof described herein in the production or preparation of a medicament for treating the above-mentioned related diseases or conditions.
  • the present invention provides the use of an anti-DKK1 antibody or antigen-binding fragment thereof described herein in combination with a tubulin inhibitor for the manufacture of a medicament for treating cancer in a subject.
  • any of the anti-DKK1 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of DKK1 in a biological sample.
  • detection includes quantitative or qualitative detection.
  • the biological sample is blood, serum, or other liquid sample of biological origin.
  • a biological sample comprises cells or tissues.
  • first and second are for the purpose of distinction in reference or clarity of expression, and do not have the meaning of typical sequence.
  • the term "other anti-tumor drugs” refers to anti-tumor drugs other than the anti-DKK1 antibody or its antigen-binding fragment of the present invention and the conjugate of the present invention; preferably, the other anti-tumor drugs are chemotherapy A drug or an immune checkpoint inhibitor; preferably, the chemotherapy drug is a tubulin inhibitor; preferably, the chemotherapy drug is paclitaxel.
  • CD CHO medium (chemically defined medium for China hamster ovary cell) represents a chemically defined medium for CHO;
  • PEI Polyethyleneimine
  • BSA Bovine serum albumin
  • amino acid sequence distribution of the heavy chain and light chain of DKN-01 is shown in SEQ ID NO:108 and SEQ ID NO:109;
  • amino acid sequence distribution of the heavy chain and light chain of BHQ-880 is shown in SEQ ID NO:110 and SEQ ID NO:111.
  • the antigen for animal immunization is the fusion protein of human DKK1 and human IgG1 Fc.
  • the designed sequence is handed over to Jinweizhi Biotechnology Co., Ltd. for gene synthesis and the synthesized plasmid is transformed into top10 strains.
  • the strains were cultured in LB medium at a ratio of 1:100, collected for 18 hours, the supernatant was discarded, and the plasmid was extracted with a plasmid extraction kit for controlling endotoxin (Tiangen).
  • CD CHO medium to subculture and expand CHO-K1 cells (the cells have been modified at the genome level to make the cells suitable for transient expression).
  • the transfection mixture was slowly added to the cells, and the cells were cultured on a shaker under the culture conditions of 36.5° C., 120 rpm, and 7% CO 2 .
  • the culture period is 6-10 days after transfection, with feeding every two days.
  • the antigen DKK1-hFc fusion protein prepared in Example 1 was used to immunize 5 BALB/c female mice by means of immunization with foot pads on both sides.
  • the dose of initial immunization was 50 ⁇ g/rat
  • the dose of 6 times of impulse immunization was 25 ⁇ g/raw
  • the dose of impulse immunization was carried out every other week.
  • the inguinal lymph nodes, popliteal lymph nodes and spleens of the mice were taken, and the suspension rich in lymphocytes was taken after grinding in normal saline, and were mixed with mouse myeloma cells Sp2/ 0 fusion.
  • the fusion products were cultured in DMEM complete medium containing 1:50 HAT (Hypoxanthine, Aminopterin and Thymidine) for 5 days to screen for successfully fused cells, and then replaced with 1:50 HT in DMEM complete medium until the end of the selection.
  • the ratio of DMEM complete medium is: 15% FBS+1:50L-glutamine+100U/mL penicillin+1:100OPI (Oxaloacetate, Pyruvate, and Insulin), the incubator parameter condition is 8% CO 2 , 37°C.
  • polyclonal secreted antibodies could compete with LPR6 for binding to DKK1 by competition ELISA, among which 3 strains had weak blocking effect and 12 strains had strong blocking effect. Fifteen antibodies were subcloned and screened by competitive ELISA to obtain 7 monoclonal antibodies ( Figure 1).
  • DKK1 was detected by target cells overexpressing LRP5/6 and luciferase reporter gene system 293 TOP and recombinant Wnt3a (R&D systems, cat#5036-WN) and human DKK1 (Shenzhou, cat#10170-H08H) protein Antibody cell biological activity.
  • 293 TOP target cells were inoculated into 96-well flat-bottomed white plate (corning, cat#3917) at 50,000 cells per well and cultured overnight, then the supernatant was discarded, and the antibody to be tested was added in a gradient dilution and pre-incubated at 37°C for 0.5h; After incubation, dilute working concentrations of 0.05 ⁇ g/ml and 0.1 ⁇ g/ml Wnt3a (R&D systems, cat#5036-WN) and human DKK1 proteins were added to the mixture containing cells and antibodies, and incubated at 37°C. co-incubation 6-8h.
  • the luciferase substrate One-Lite (Vazyme, cat#DD1203-03-AB) was added and the chemiluminescence value corresponding to each hybridoma antibody was detected.
  • the luciferase reporter gene method it was detected that 7 of the antibodies had different degrees of recovery effects on the Wnt signaling pathway ( Figure 2A and Figure 2B).
  • the DNA sequence of the variable region of the mouse antibody expressed by the candidate hybridoma cells was determined using a degenerate primer PCR-based method. Briefly, the hybridoma cell lines were expanded and cultured separately, the cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. Using this as a template, after the first-strand cDNA is synthesized, the corresponding variable region DNA sequence is amplified by PCR using the first-strand cDNA as a subsequent template, and the PCR primers used are based on the Ig-primer set.
  • the germline and rearranged Ig variable region sequence databases were searched for consensus sequences using NCBI Ig-Blast (http://www.ncbi.nlm.nih.gov/projects/igblast/). Based on Kabat (Wu, T.T and Kabat, E.A.1970 J.Exp.Med., 132:211-250) and IMGT system (Lefranc M.-P.
  • Example 3 Design, construction and expression of chimeric antibodies
  • the light chain variable region and the heavy chain variable region of the anti-huDKK1 murine antibodies 7M2.6, 11I18.4, 17F14.3, 18E24.6, 18J21.1 and 19O15.6 were selected ( The sequence is shown in Table 3 below), the heavy chain variable region is constructed into the hIgG4 heavy chain constant region (the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 112), and the light chain variable region is constructed into the hIg ⁇ chain (wherein the light chain is constant The amino acid sequence of the region is shown in SEQ ID NO: 113), and the chimeric heavy and light chain variable regions were randomly combined into 36 strains of chimeric antibodies, as shown in Table 3 below.
  • CD CHO medium to subculture and expand CHO-K1 cells (the cells have been modified at the genome level to make the cells suitable for transient expression).
  • the transfection mixture was slowly added to the cells, and the cells were cultured on a shaker under the culture conditions of 36.5° C., 120 rpm, and 7% CO 2 .
  • the culture period is 6-10 days after transfection, with feeding every two days.
  • ELISA method was used to detect the binding activity of chimeric antibody to human DKK1: the plate was coated with human DKK1 antigen 2.0 ⁇ g/mL (Sino Biological, 10170-H08H) for 90 minutes, and after blocking with 2% BSA for 90 minutes, serially diluted anti-DKK1 chimeric antibody was added and positive Control antibody DKN-01 (starting at 1 ⁇ g/ml, 2.5-fold serial dilution to 1.64ng/mL, 8 concentrations in total), incubated for 60 min, washed the plate and incubated 5000-fold diluted HRP-coupled goat anti-human IgG (Fc-specific nature) (Sigma, A0170) secondary antibody, then use 0.1mg/ml TMB (Sigma, T2885) to develop color, and finally stop the reaction with 2M hydrochloric acid solution, use Thermo Scientific microplate reader, read the plate at 450nm/620nm, use The four-parameter logarithmic regression (4PL)
  • ELISA method was used to detect the inhibition of the binding of LRP6(629-1244) and DKK1 by the chimeric antibody: the plate was coated with human DKK1 antigen 5.0 ⁇ g/mL (Sino Biological, 10170-H08H) for 90 minutes, blocked with 2% BSA for 90 minutes, and human LRP6 mFc(629-1244 ) (Suzhou Junmeng) diluted anti-DKK1 chimeric antibody and positive control antibody DKN-01 (starting at 10 ⁇ g/ml, 2.5-fold gradient dilution to 16.38ng/mL, a total of 8 concentrations), incubated for 60min after adding the sample, and washed the plate Then add 5000-fold diluted HRP-coupled goat anti-mouse IgG (Fc-specific) (Sigma, A2554) and incubate for 30 min.
  • human DKK1 antigen 5.0 ⁇ g/mL Seo Biological, 10170-H08H
  • each chimeric antibody was fitted using a four-parameter logarithmic regression (4PL) model ( Figure 4A to 4B ), and compare the relative competitive inhibitory activity of each chimeric antibody.
  • 4PL logarithmic regression
  • chi1 supernatant and chi15 supernatant are fermentation broth supernatants without purification; chi1, chi8, chi15 and chi21 are protein samples obtained after purification, and the preparation of fermentation broth supernatant and protein samples refers to the previous Example 3.
  • hDKK1 by overexpressing LRP5/6 and luciferase reporter gene system target cells 293 TOP and recombinant Wnt3a (R&D systems, cat#5036-WN) and human DKK1 (Shenzhou, cat#10170-H08H) protein Cell Biological Activity of Chimeric Antibodies.
  • Example 4 Through the screening of chimeric antibodies in Example 4, three antibodies chi1, chi8 and chi15 with the best in vitro activity were obtained, which were used for subsequent humanization.
  • human germline IgG genes homologous to the DNA sequence of the murine antibody variable region were searched in the human immunoglobulin gene database on the NCBI (http://www.ncbi.nlm.nih.gov/igblast/) website.
  • the amino acid sequences of the CDRs in the variable region are then defined by the Kabat numbering system or the IMGT numbering system.
  • human IGHV (immunoglobulin heavy chain constant region) and IGKV (immunoglobulin light chain constant region) which have high homology with the murine antibody variable region are selected as humanized templates, and the antibody variable region is implemented by CDR grafting humanization.
  • chimeric antibody humanization The specific steps of chimeric antibody humanization are as follows: 1. Compare the gene sequence of the antibody secreted by each hybridoma cell with the human embryonic antibody gene sequence to find out the sequence with high homology; 2. Analyze and investigate the HLA- DR affinity, select the human embryonic frame region sequence with low affinity; 3. Using computer simulation technology, apply molecular docking to analyze the amino acid sequence of the variable region and its surrounding framework region, and investigate its three-dimensional binding mode.
  • No. 1 chimeric antibody molecule (chi-1) was humanized and transformed to obtain 5 heavy chains and 4 light chains (HCDR3 was mutated from SEQ ID NO:78 to SEQ ID NO:3 after humanization), and No. 2 Chimeric antibody molecule (chi-8) humanized With 5 heavy chains and 5 light chains, chimeric antibody molecule No. 3 (chi-15) was humanized and transformed to obtain 6 heavy chains and 4 light chains.
  • CDR sequences are defined by Kabat scheme
  • Table 4a Numbering of chi-1-based humanized antibodies and their combinations of heavy and light chain variable regions
  • Table 4b Numbering of chi-8-based humanized antibodies and their combinations of heavy and light chain variable regions
  • Table 4c Numbering of chi-15-based humanized antibodies and their combinations of heavy and light chain variable regions
  • Table 5a Numbering of chi-1-based humanized antibodies and their heavy and light chain combinations
  • Table 5b Numbering of chi-8-based humanized antibodies and their heavy and light chain combinations
  • Table 5c Numbering of chi-15-based humanized antibodies and their combinations of heavy and light chains
  • the plasmids encoding the heavy and light chains of 69 humanized antibodies hu1-hu69 were entrusted to Nanjing GenScript Biotechnology Co., Ltd. for gene synthesis. After receiving the synthesized plasmids and strains, the strains were transferred to LB medium After culturing overnight, the plasmid was extracted with the Tiangen Plasmid Extraction Kit.
  • CD CHO medium to subculture and expand CHO-K1 cells (the cells have been modified at the genome level to make the cells suitable for transient expression).
  • the cells were cultured on a shaker, and the culture conditions were 36.5° C., 120 rpm, and 7% CO 2 .
  • the culture period is 6-10 days after transfection, with feeding every two days.
  • the 69 strains of humanized antibodies were purified using the same purification method for hybridoma antibodies in Example 1. After purification, the size and purity of the antibody protein were identified by SDS-PAGE, and the purity of the protein was detected by HPLC-SEC. used in subsequent experiments.
  • Detection of the binding of humanized antibody to DKK1 by ELISA coat the plate with human DKK1 antigen 2.0 ⁇ g/mL (Sino Biological, 10170-H08H) for 90 min, then block with 2% BSA for 90 min, wash the plate and add serially diluted anti-DKK1 humanized Antibody (starting at 1 ⁇ g/ml, 10-fold serial dilution to 1ng/mL, 4 concentrations in total), incubated for 60 min, washed and incubated with 5000-fold diluted HRP-coupled goat anti-human IgG (Fc specific) (Sigma, A0170) secondary antibody for 30min, then add 100 ⁇ L 0.1mg/mL TMB (Sigma, T2885) to each well to develop color for 10min, and finally add 50 ⁇ L 2M hydrochloric acid solution to each well to stop the reaction, use Thermo Scientific microplate reader, read at 450nm/620nm plate. Using a four-parameter logarith
  • Detection of humanized antibody inhibiting the binding of LRP6(629-1244) to DKK1 by ELISA method using human DKK1 antigen 5.0 ⁇ g/mL (Sino Biological, 10170-H08H) coated plate for 90min, then blocked with 2% BSA for 90min, after washing the plate, dilute DKK1 antibody humanized antibody with 10 ⁇ g/mL human LRP6 mFc (629-1244) (Suzhou Junmeng) And anti-DKK1 chimeric antibody (starting at 10 ⁇ g/ml, 10-fold serial dilution to 10ng/mL, a total of 4 concentrations), and incubated for 60min after adding the sample.
  • the obtained partially humanized antibody has an improved ability to inhibit the binding of DKK1 and LRP6 ( FIG. 7 ).
  • DKK1 was detected by using target cells overexpressing LRP5/6 and luciferase reporter gene system 293 TOP and recombinant Wnt3a (R&D systems, cat#5036-WN) and human DKK1 (Shenzhou, cat#10170-H08H) protein Antibody cell biological activity.
  • the luciferase substrate One-Lite (Vazyme, cat#DD1203-03-AB) was added and the chemiluminescence value corresponding to each humanized antibody was detected.
  • the obtained partially humanized Antibodies Antibodies increased the degree of restoration of expression of the Wnt3a pathway ( FIG. 8 ).
  • the experimental animals were CB17/SCID female mice, and the mice were subcutaneously inoculated with A549 cells (5 ⁇ 10 6 ) to establish a subcutaneous transplanted tumor model.
  • the test was divided into anti-KLH IgG4 (5mg/kg) group, DKN-01 (5mg/kg) group, hu3 (5mg/kg) group, paclitaxel (20mg/kg) group, DKN-01 (5mg/kg) + paclitaxel ( 20mg/kg) group, DKN-01(5mg/kg)+hu3(5mg/kg) group, DKN-01 is the positive control antibody, and anti-KLH IgG4 is the negative control antibody.
  • TGI tumor inhibition rate
  • the experimental results showed that on the 35th day after tumor inoculation, according to the descending order of TGI effect, they were DKN-01 (5mg/kg)+hu3 (5mg/kg) group, paclitaxel (20mg/kg) group, DKN-01 (5mg/kg) kg)+paclitaxel (20mg/kg) group, hu3 group, DKN-01 group and anti-KLH IgG4 group, the values were 74.48%, 60.48%, respectively, 60.04%, 35.29%, 23.27% and 0%.
  • the experimental animals were CB17/SCID male mice, and PC-3 cells (10 ⁇ 10 6 ) were subcutaneously inoculated into the right back of the mice to establish a subcutaneous transplanted tumor model.
  • the test was divided into anti-KLH IgG4 (10mg/kg) group, hu3 (5mg/kg) group, hu3 (10mg/kg) group, hu3 (20mg/kg) group, paclitaxel (7.5mg/kg) group, hu3 (5mg/kg) group, kg)+paclitaxel (7.5mg/kg) group, hu3(10mg/kg)+paclitaxel (7.5mg/kg) group, hu3(20mg/kg)+paclitaxel (7.5mg/kg) group, anti-KLH IgG4 as negative control Antibody.
  • the experimental results show that on the 21st day after tumor inoculation, according to the descending order of TGI effect, they are hu3 (20mg/kg) + paclitaxel (7.5mg/kg) group, hu3 (10mg/kg) + paclitaxel (7.5mg/kg) group , hu3(5mg/kg)+paclitaxel(7.5mg/kg) group, paclitaxel(7.5mg/kg) group, hu3(20mg/kg) group, hu3(10mg/kg) group, hu3(5mg/kg) group, Anti-KLH IgG4 (10mg/kg) group, the values were 88.4%, 88.3%, 81.5%, 71.8%, 56.4%, 48.8%, 30.7% and 0%.
  • hu3 showed obvious tumor inhibitory effect at the doses of 5mg/kg, 10mg/kg, and 20mg/kg, and the higher the dose, the higher the TGI.
  • the tumor inhibitory effect of hu3 combined with 7.5 mg/kg paclitaxel was significantly better than that of the corresponding concentrations of hu3 and paclitaxel alone, indicating that the combination of anti-DKK1 antibody hu3 and paclitaxel can play a synergistic antitumor effect.

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Abstract

本发明属于生物医药领域，涉及一种抗DKK1抗体、其药物组合物及用途。具体地，本发明涉及一种抗DKK1抗体或其抗原结合片段，其中，所述抗DKK1抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中，HCDR1的氨基酸序列选自SEQ ID NO:1等；HCDR2的氨基酸序列选自SEQ ID NO:2等；HCDR3的氨基酸序列选自SEQ ID NO:3等；LCDR1的氨基酸序列选自SEQ ID NO:4等；LCDR2的氨基酸序列选自SEQ ID NO:5等；LCDR3的氨基酸序列选自SEQ ID NO:6等。本发明的抗DKK1抗体能够与DKK1高特异性地结合，有效地阻断Wnt信号通路，从而抑制DKK1的对肿瘤微环境形成的促进作用和对肿瘤发生发展的促进作用，具有良好的抗肿瘤效果。

Description

抗DKK1抗体、其药物组合物及用途 技术领域
本发明属于生物医药领域，涉及一种抗DKK1抗体、其药物组合物及用途。
背景技术
DKK1是Wnt/β-catenin信号转导通路的分泌性抑制剂，属于抑制Wnt信号传导的蛋白质家族之一的DKK(dickkopf)家族，具有抑制Wnt诱导的轴复制的能力。DKK家族目前有4个家族成员，即DKK1，DKK2，DKK3和DKK4。
Wnt信号通路涉及对胚胎发育和致瘤过程的控制。胞外Wnt蛋白负责胚胎发育过程中的多种细胞类型的生长和分化，并对多种癌症的发展有促进作用。遗传分析表明DKK1在上游抑制Wnt信号转导，DKK1与LRP6拮抗性的相互作用，阻断Wnt介导的信号激活。
当DKK1蛋白不存在时，Wnt和Frizzled，LRP6结合形成异源三聚体，激活下游信号通路，维持β-catenin的稳定，其入核后聚集，促进成骨细胞相关基因和抑癌基因的转录和翻译。而当DKK1蛋白存在时，DKK1与Wnt竞争结合LRP6分子，下游一系列的信号反应促使β-catenin磷酸化然后被降解。从而阻断了β-catenin入核，进而阻断了成骨细胞相关基因和抑癌基因的转录翻译。当抗DKK1抗体存在时，抗体竞争LRP6与DKK1分子的结合，有利于Wnt和Frizzled，LRP6重新形成异源三聚体。下游信号通路回复，使得成骨细胞相关基因和抑癌基因的转录翻译恢复正常，起到促进成骨细胞稳定和增殖的作用以及抑制癌细胞发生和增殖的作用。DKK1可以通过增加MDSC和Treg细胞的抑制活性，下调肿瘤细胞表面激活NK细胞的配体，促进Th2细胞极化以及降低IFNγ的产生，从而促进了肿瘤微环境的形成。此外，DKK1还可以促进肿瘤的生长和迁移，促进CSC样细胞的产生，促进肿瘤血管生成，从而直接促进肿瘤的发生发展(Yu等，Signal Transduct Target Therapy，2021，6(1)；Nikolai等，Cancer Letters，2020，482)。2021年3月22日，LeapTherapeutics宣布其靶向DKK1靶点的DKN-01单克隆抗体在2期临床治疗晚期妇科癌症取得积极结果。临床结果表面，DKN-01在高表达DKK1的患者中更有效，其客观缓解率为14％，疾病控制率为57％。
DKK1是肿瘤的重要调节因子，作为一个关键的治疗靶点值得更多的关注；并且事实上，开发DKK1抑制剂的策略已经在不同的病理模型产生了令人鼓舞的临床结果(Hewen Jiang等，Drug Discovery of DKK1 Inhibitors，Frontiers in Pharmacology，March 2022,Volume 13,p1-17)。然而迄今为止，针对该靶点的抗体药物仍未上市，其中在癌症治疗方面进展最快的两个分子正处于单药或联合用药的临床研究阶段(Jiang等，Front Pharmacol，2022， 13)，分别为正处于治疗多发性骨髓瘤、胆囊癌、食管癌、肝癌、肝细胞癌、膀胱癌、前列腺癌、肺癌、妇科癌症、结肠癌、直肠癌的研究中的DKN-01(CN110114087A)，以及处于治疗多发性骨髓瘤和骨肉瘤研究中的BHQ-880(WO2007084344)。然而对于提供能够单独地或与其它药剂相组合，通过增强对人类DKK1的抑制活性，从而促进Wnt通路介导的抑癌基因表达的其他更有效、特异、安全和/或稳定的药剂，仍存在着未满足的需求。
发明内容
本发明人经过深入的研究和创造性的劳动，制得表达抗DDK1抗体的杂交瘤细胞株并获得了表达的抗DDK1抗体。进一步地，本发明人在此基础上设计了嵌合抗体并进行了人源化改造。本发明人惊奇地发现，本发明的抗DDK1抗体对DDK1具有较高的亲和力和/或特异性，能够有效地抑制肿瘤生长，具有良好的抗肿瘤潜力。由此提供了下述发明：
本发明的一个方面涉及一种抗DKK1抗体或其抗原结合片段，
其中，所述抗DKK1抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，
其中，
HCDR1的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:13、SEQ ID NO:19、SEQ ID NO:25和SEQ ID NO:31；
HCDR2的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:8、SEQ ID NO:14、SEQ ID NO:20、SEQ ID NO:26和SEQ ID NO:32；
HCDR3的氨基酸序列选自SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:15、SEQ ID NO:21、SEQ ID NO:27、SEQ ID NO:33和SEQ ID NO:78；
LCDR1的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:10、SEQ ID NO:16、SEQ ID NO:22、SEQ ID NO:28和SEQ ID NO:34；
LCDR2的氨基酸序列选自SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:17、SEQ ID NO:23、SEQ ID NO:29和SEQ ID NO:35；
LCDR3的氨基酸序列选自SEQ ID NO:6、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:24、SEQ ID NO:30和SEQ ID NO:36。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中：
所述抗体的重链可变区，其中，
HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示，
HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2 所示、HCDR3的氨基酸序列如SEQ ID NO:78所示，
HCDR1的氨基酸序列如SEQ ID NO:7所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:9所示，
HCDR1的氨基酸序列如SEQ ID NO:13所示、HCDR2的氨基酸序列如SEQ ID NO:14所示、HCDR3的氨基酸序列如SEQ ID NO:15所示，
HCDR1的氨基酸序列如SEQ ID NO:19所示、HCDR2的氨基酸序列如SEQ ID NO:20所示、HCDR3的氨基酸序列如SEQ ID NO:21所示，
HCDR1的氨基酸序列如SEQ ID NO:25所示、HCDR2的氨基酸序列如SEQ ID NO:26所示、HCDR3的氨基酸序列如SEQ ID NO:27所示，或者
HCDR1的氨基酸序列如SEQ ID NO:31所示、HCDR2的氨基酸序列如SEQ ID NO:32所示、HCDR3的氨基酸序列如SEQ ID NO:33所示；
并且
所述抗体的轻链可变区，其中，
LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR1的氨基酸序列如SEQ ID NO:5所示、LCDR1的氨基酸序列如SEQ ID NO:6所示，
LCDR1的氨基酸序列如SEQ ID NO:10所示、LCDR1的氨基酸序列如SEQ ID NO:11所示、LCDR1的氨基酸序列如SEQ ID NO:12所示，
LCDR1的氨基酸序列如SEQ ID NO:16所示、LCDR1的氨基酸序列如SEQ ID NO:17所示、LCDR1的氨基酸序列如SEQ ID NO:18所示，
LCDR1的氨基酸序列如SEQ ID NO:22所示、LCDR1的氨基酸序列如SEQ ID NO:23所示、LCDR1的氨基酸序列如SEQ ID NO:24所示，
LCDR1的氨基酸序列如SEQ ID NO:28所示、LCDR1的氨基酸序列如SEQ ID NO:29所示、LCDR1的氨基酸序列如SEQ ID NO:30所示，或者
LCDR1的氨基酸序列如SEQ ID NO:34所示、LCDR1的氨基酸序列如SEQ ID NO:35所示、LCDR1的氨基酸序列如SEQ ID NO:36所示。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，
HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR1的氨基酸序列如SEQ ID NO:5所示、LCDR1的氨基酸序列如SEQ ID NO:6所示；
HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:78所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR1的氨基酸序列如SEQ ID NO:5所示、LCDR1的氨基酸序列如SEQ ID NO: 6所示；
HCDR1的氨基酸序列如SEQ ID NO:7所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:9所示、LCDR1的氨基酸序列如SEQ ID NO:10所示、LCDR1的氨基酸序列如SEQ ID NO:11所示、LCDR1的氨基酸序列如SEQ ID NO:12所示；
HCDR1的氨基酸序列如SEQ ID NO:13所示、HCDR2的氨基酸序列如SEQ ID NO:14所示、HCDR3的氨基酸序列如SEQ ID NO:15所示、LCDR1的氨基酸序列如SEQ ID NO:16所示、LCDR1的氨基酸序列如SEQ ID NO:17所示、LCDR1的氨基酸序列如SEQ ID NO:18所示；
HCDR1的氨基酸序列如SEQ ID NO:19所示、HCDR2的氨基酸序列如SEQ ID NO:20所示、HCDR3的氨基酸序列如SEQ ID NO:21所示、LCDR1的氨基酸序列如SEQ ID NO:22所示、LCDR1的氨基酸序列如SEQ ID NO:23所示、LCDR1的氨基酸序列如SEQ ID NO:24所示；
HCDR1的氨基酸序列如SEQ ID NO:25所示、HCDR2的氨基酸序列如SEQ ID NO:26所示、HCDR3的氨基酸序列如SEQ ID NO:27所示、LCDR1的氨基酸序列如SEQ ID NO:28所示、LCDR1的氨基酸序列如SEQ ID NO:29所示、LCDR1的氨基酸序列如SEQ ID NO:30所示；
或者
HCDR1的氨基酸序列如SEQ ID NO:31所示、HCDR2的氨基酸序列如SEQ ID NO:32所示、HCDR3的氨基酸序列如SEQ ID NO:33所示、LCDR1的氨基酸序列如SEQ ID NO:34所示、LCDR1的氨基酸序列如SEQ ID NO:35所示、LCDR1的氨基酸序列如SEQ ID NO:36所示。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Bethesda M.d.,Sequences of Proteins of Immunological Interest,Fifth Edition,NIH Publication 1991；1-3:91-3242。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，
所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NOs:49-53、SEQ ID NOs:58-62和SEQ ID NOs:68-73；并且
所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:37所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:39所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:41所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:45所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:47所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:49所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:50所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:51所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:52所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:53所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、 SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:58所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:59所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:60所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:61所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:62所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:68所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:69所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:70所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:71所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:72所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:73所示，轻链可变区的氨基酸序 列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，
所述抗体的重链的氨基酸序列选自SEQ ID NOs:79-83、SEQ ID NOs:88-92和SEQ ID NOs:98-103；并且
所述抗体的轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，
所述抗体的重链的氨基酸序列如SEQ ID NO:79所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:80所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:81所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:82所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:83所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:88所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:89所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:90所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:91所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:92所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:98所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:99所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:100所示，轻链的氨基酸序列选自SEQ  ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:101所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
所述抗体的重链的氨基酸序列如SEQ ID NO:102所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
或者
所述抗体的重链可的氨基酸序列如SEQ ID NO:103所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，所述抗DKK1抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
本发明的另一方面涉及一种抗DKK1的抗体或其抗原结合片段，其具有如下特征：
(a)与本文任一项所述的抗体或抗原结合片段结合相同的、或完全重叠的、或部分重叠的人DKK1蛋白的表位；或
(b)与本文任一项所述的抗体或抗原结合片段竞争性结合人DKK1蛋白的表位。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方式中，所述的抗DKK1抗体或其抗原结合片段，其中，所述抗体的重链恒定区为Ig gamma-1 chain C region(例如NCBI ACCESSION:P01857)或Iggamma-4 chain C region(例如NCBI ACCESSION:P01861.1)；轻链恒定区为Ig kappa chain C region(例如NCBI ACCESSION:P01834)；
优选地，所述抗体的重链恒定区的氨基酸序列如SEQ ID NO:112所示，所述抗体的轻链恒定区的氨基酸序列如SEQ ID NO:113所示。
本发明的另一方面涉及一种分离的核酸分子，其编码本发明中任一项所述的抗DKK1抗体或其抗原结合片段。
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的重组载体。
本发明的再一方面涉及一种偶联物，其包括抗体以及偶联部分，其中，所述抗体为本发明中任一项所述的抗DKK1抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为细胞毒素、免疫调节剂、放射性同位素、荧光物质、发光物质、有 色物质或酶。
本发明的再一方面涉及一种试剂盒，其包括本发明中任一项所述的抗DKK1抗体或其抗原结合片段，或者包括本发明的偶联物；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如细胞毒素、免疫调节剂、放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中：
所述第一蛋白功能区靶向DKK1，
所述第二蛋白功能区靶向不同于DKK1的靶点，
其中，所述第一蛋白功能区为本发明中任一项所述的抗体或抗原结合片段。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述单链抗体分别连接在免疫球蛋白形式的抗体的两条重链的C末端。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗DKK1抗体或其抗原结合片段，或者包含本发明的偶联物；
可选地，所述药物组合物还包含一种或多种其它抗肿瘤药物；优选地，所述其它抗肿瘤药物为化疗药物或抗体药物；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述抗体药物为免疫检查点抑制剂；优选地，所述化疗药物为紫杉醇；
可选地，所述药物组合物还包括一种或多种药学上可接受的辅料(例如载体和/或赋形剂)。
在本发明的一个或多个实施方式中，所述的药物组合物，其中，按照抗体的质量计算，抗DKK1抗体或其抗原结合片段与紫杉醇的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
本发明的再一方面涉及一种组合产品，其包含独立包装的第一产品和第二产品，其中，
所述第一产品包含其包含本发明中任一项所述的抗DKK1抗体或其抗原结合片段，或者包含本发明的偶联物；
所述第二产品包含一种或多种其它抗肿瘤药物；优选地，所述其它抗肿瘤药物为化疗药物或抗体药物；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述抗体药物为免疫检查点抑制剂；优选地，所述化疗药物为紫杉醇；
优选地，所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料(例如载体和/或赋形剂)；
优选地，所述组合产品还包含产品说明书。
在本发明的一个或多个实施方式中，所述的组合产品，其中，按照抗体的质量计算，抗DKK1抗体或其抗原结合片段与紫杉醇的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
本发明的再一方面涉及本发明中任一项所述的抗体或其抗原结合片段、本发明的偶联物、本发明中任一项所述的双特异性抗体或者本发明中任一项所述的药物组合物在制备治疗和/或预防DKK1介导的疾病的用途；优选地，所述DKK1介导的疾病为肿瘤；优选地，所述肿瘤选自胆囊癌、膀胱癌、骨肉瘤、食管癌、胆管癌、子宫内膜癌、卵巢癌、胃癌、胃食管交界处癌、结肠癌、直肠癌、肝细胞癌、前列腺癌、肺癌和骨髓瘤中的一种或多种。
根据本发明中任一项所述的抗体或其抗原结合片段、本发明的偶联物、本发明中任一项所述的双特异性抗体或者本发明中任一项所述的药物组合物，其用于治疗和/或预防DKK1介导的疾病；优选地，所述DKK1介导的疾病为肿瘤；优选地，所述肿瘤选自胆囊癌、膀胱癌、骨肉瘤、食管癌、胆管癌、子宫内膜癌、卵巢癌、胃癌、胃食管交界处癌、结肠癌、直肠癌、肝细胞癌、前列腺癌、肺癌和骨髓瘤中的一种或多种。
本发明的再一方面涉及一种治疗或预防DKK1介导的疾病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的偶联物、本发明中任一项所述的双特异性抗体或者本发明中任一项所述的药物组合物的步骤；；优选地，所述DKK1介导的疾病为肿瘤；优选地，所述肿瘤选自胆囊癌、膀胱癌、骨肉瘤、食管癌、胆管癌、子宫内膜癌、卵巢癌、胃癌、胃食管交界处癌、结肠癌、直肠癌、肝细胞癌、前列腺癌、肺癌和骨髓瘤中的一种或多种。
优选地，给药方式为静脉滴注或静脉注射。
在本发明的一些实施方式中，所述治疗或预防DKK1介导的疾病的方法为疗或预防肿瘤的方法。在本发明的一些实施方式中，所述治疗或预防肿瘤的方法还包括向所述受试者联合施用一种或多种疗法的步骤，所述疗法包括手术治疗和/或放射疗法和/或施用一种或多种其它抗肿瘤药物；
优选地，所述其它抗肿瘤药物为化疗药物或抗体药物；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述抗体药物为免疫检查点抑制剂；优选地，所述化疗药物为 紫杉醇。
附图简述
图1：ELISA检测杂交瘤抗体对DDK1与LRP6结合的阻断作用。
图2A：荧光素酶报告基因法检测杂交瘤抗体对DDK1对Wnt信号抑制作用的拮抗效果。
图2B：荧光素酶报告基因法检测杂交瘤抗体对DDK1对Wnt信号抑制作用的拮抗效果。
图3：ELISA检测嵌合抗体对DDK1与LRP6结合的阻断作用。
图4A：ELISA检测嵌合抗体与DKK1的结合作用。
图4B：ELISA检测嵌合抗体与DKK1的结合作用。
图5A：荧光素酶报告基因法检测嵌合抗体对Wnt信号通路的回复作用。
图5B：荧光素酶报告基因法检测嵌合抗体对Wnt信号通路的回复作用。
图5C：荧光素酶报告基因法检测嵌合抗体对Wnt信号通路的回复作用。
图6：ELISA检测人源化抗体与DDK1的结合作用。
图7：ELISA检测人源化抗体对DDK1与LRP6结合的阻断作用。
图8：荧光素酶报告基因法检测人源化抗体对DKK1对Wnt信号抑制作用的拮抗效果。
图9：人源化抗体hu3对肿瘤生长的抑制作用。
图10：人源化抗体hu3对肿瘤生长的抑制作用。
本发明涉及的部分序列(下划线标出部分为CDR)
SEQ ID NO:1
EYTMH
SEQ ID NO:2
VIDPSNGDTSYNQKFKG
SEQ ID NO:3
TTGPWFAS
SEQ ID NO:4
RASQDISNYLN
SEQ ID NO:5
YKSRLHS
SEQ ID NO:6
QQVHTLPPT
SEQ ID NO:7
ENTMH
SEQ ID NO:8
GINPHKGGTSYNQKFKG
SEQ ID NO:9
DWLGAMDY
SEQ ID NO:10
ITSTDIDDDMN
SEQ ID NO:11
EGNTLRP
SEQ ID NO:12
LQSDNLPYT
SEQ ID NO:13
SYNML
SEQ ID NO:14
VIFPGNGDTSYNPKFKG
SEQ ID NO:15
DGYPSMDY
SEQ ID NO:16
ITSTDIDDDMN
SEQ ID NO:17
EGNSLRP
SEQ ID NO:18
LQFNNMPLT
SEQ ID NO:19
TSGMGVG
SEQ ID NO:20
HIWWDDVKRYNPALKS
SEQ ID NO:21
IARSPTGFAY
SEQ ID NO:22
SASSSVSYMY
SEQ ID NO:23
DTSNLAS
SEQ ID NO:24
QQWSSYPPALT
SEQ ID NO:25
AFDVH
SEQ ID NO:26
VIWIGGTTDYNGAFIS
SEQ ID NO:27
KRGNYYNMDY
SEQ ID NO:28
RSSTGTVTTTNYAN
SEQ ID NO:29
GTNNRAP
SEQ ID NO:30
ALWYSNHWV
SEQ ID NO:31
NHGMN
SEQ ID NO:32
WINTNTGEPTYADDFKG
SEQ ID NO:33
RGYGFPYYYTMDY
SEQ ID NO:34
RSSTGAVTTRNYAN
SEQ ID NO:35
GTKNRAP
SEQ ID NO:36
ALWYSNRLV
SEQ ID NO:37
MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVRISCKTSGYTFTEYTMHWVKQSHGKSLEWIGVIDPSNGDTSYNQKFKGRATLTVDKSSSTAYMDLRSLTSEDSALYYCSSTTGPWFACWGQGTLVTVSA
SEQ ID NO:38
MSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTINCRASQDISNYLNWYQQKPDGTVKLLIYYKSRLHSGVPTRFSGSGSGTDFSLTISNLVQEDIATYFCQQVHTLPPTFGGGTKLEIK
SEQ ID NO:39
MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKIFCKTSGYTFTENTMHWVKQTHGKSLEWIGGINPHKGGTSYNQKFKGKATLTVDKSSSTACMELRSLTSEDSTVYYCARDWLGAMDYWGQGTSVTVSP
SEQ ID NO:40
MFSLALLLSLLLLCVSDSRAETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK
SEQ ID NO:41
MGWNCFILFLVATATGVHSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMLWVKQTPGQGLEWIGVIFPGNGDTSYNPKFKGKAALTADKSSSTAYMQLSSLTSEDSAVYYCARDGYPSMDYWGQGTSVTVSS
SEQ ID NO:42
MLSLAPLLSLLLLCVSDSRAETIVTQSPASLSVATGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNSLRPGVPSRFSSSGYGTDFVFTIENTLSEDVADYYCLQFNNMPLTFGAGTKLELK
SEQ ID NO:43
MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSSSQVFLKIASVDTTDTASYYCSRIARSPTGFAYWGQGTL
SEQ ID NO:44
MDFQVQIFSFLLISASVILSRGQIVLTQSPVIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPP ALTFGAGTKLELK
SEQ ID NO:45
MAVLGLLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTAFDVHWVRQSPGKGLEWLGVIWIGGTTDYNGAFISRLSISKDNSKSQVFFKMNSLQAKDTAIYYCARKRGNYYNMDYWGQGTSVTVAS
SEQ ID NO:46
MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGTVTTTNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH WVFGGGTKLTVL
SEQ ID NO:47
MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTNHGMNWVKQAPGKGLKWMGWINTNTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRGYGFPYYYTMDYWGQGTSVTVSS
SEQ ID NO:48
MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTRNYANWVQEKADHLLTGLIGGTKNRAPGVPARFSGSLIGDKAALTITGAQTEDEAKYFCALWYSN RLVFGGGTKLTVL
SEQ ID NO:49
EVQLLQSAAEVKRPGESLRISCKTSGYTFTEYTMHWVRQMPGKELEWMGVIDPSN GDTSYNQKFKGHVTISADKSSSTAYLQWSSLKASDAAMYYCSSTTGPWFASWGQGTLVTVSS
SEQ ID NO:50
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQGLEWMGVIDP SNGDTSYNQKFKGRVTLTVDKSTSTAYMELRSLRSDDTAVYYCARTTGPWFASWGQGTLVTVSS
SEQ ID NO:51
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYTMHWVRQAPGQGLEWMGVIDP SNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCSSTTGPWFASWGQGTLVTVSS
SEQ ID NO:52
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQRLEWMGVIDP SNGDTSYNQKFKGRVTITVDKSSSTAYMELSSLRSEDTAVYYCSSTTGPWFASWGQGTLVTVSS
SEQ ID NO:53
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQRLEWMGVIDP SNGDTSYNQKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARTTGPWFASWGQGTLVTVSS
SEQ ID NO:54
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYKSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQVHTLPPTFGQGTKLEIK
SEQ ID NO:55
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYKSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVHTLPPTFGQGTKLEIK
SEQ ID NO:56
EIVMTQSPPTLSLSPGERVTLSCRASQDISNYLNWYQQKPGQAPRLLIYYKSRLHSGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQVHTLPPTFGQGTKLEIK
SEQ ID NO:57
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYKSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQVHTLPPTFGQGTKLEIK
SEQ ID NO:58
EVQLLQSAAEVKRPGESLRISCKTSGYSFTENTMHWVRQMPGKELEWMGGINPH KGGTSYNQKFKGHVTISVDKSSSTAYLQWSSLKASDAAMYYCARDWLGAMDYWGQGTLVTVSS
SEQ ID NO:59
EVQLLQSAAEVKRPGESLRISCKTSGYTFTENTMHWVRQMPGKELEWMGGINPH KGGTSYNQKFKGHVTISADKSSSTAYLQWSSLKASDAAMYYCARDWLGAMDYWGQGTLVTVSS
SEQ ID NO:60
EVQLLQSAAEVKRPGESLRISCKTSGYTFTENTMHWVRQMPGKELEWMGGINPH KGGTSYNQKFKGHVTLSVDKSSSTAYLQWSSLKASDAAMYYCARDWLGAMDYWGQGTLVTVSS
SEQ ID NO:61
QVQLVQSGAEVKKPGASVKVSCKASGYTFTENTMHWVRQAPGQGLEWMGGINP HKGGTSYNQKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYYCARDWLGAMDYWGQGTLVTVSS
SEQ ID NO:62
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTENTMHWVRQAPGQGLEWMGGINP HKGGTSYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARDWLGAMDYWGQGTLVTVSS
SEQ ID NO:63
DIQLTQSPSSLSASVGDRVTITCITSTDIDDDMNWYRQKPGKVPKLLISEGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDNLPYTFGQGTKLEIK
SEQ ID NO:64
DIQVTQSPSSLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNTLRPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQSDNLPYTFGQGTKLEIK
SEQ ID NO:65
DIVLTQSPASLAVSPGQRATITCITSTDIDDDMNWYQQKPGQPPKLLISEGNTLRPGVPARFSSSGSGTDFTLTINPVEANDTANYYCLQSDNLPYTFGQGTKLEIK
SEQ ID NO:66
DTVLTQSPASLAVSPGQRATITCITSTDIDDDMNWYQQKPGQPPKLLISEGNTLRPGVPARFSSSGSGTDFTLTINPVEANDTANYYCLQSDNLPYTFGQGTKLEIK
SEQ ID NO:67
DIVLTQSPASLAVSPGQRATITCITSTDIDDDMNWYQQKPGQPPKLLISEGNTLRPGVPARFSSSGSGTDFTLTINPVEANDVANYYCLQSDNLPYTFGQGTKLEIK
SEQ ID NO:68
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMLWVRQAPGQRLEWMGVIFP GNGDTSYNPKFKGRVTITADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSS
SEQ ID NO:69
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMLWVRQAPGQRLEWMGVIFP GNGDTSYNPKFKGRVTLTADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSS
SEQ ID NO:70
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYNMLWVRQAPGQGLEWMGVIFPG NGDTSYNPKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSS
SEQ ID NO:71
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMLWVRQAPGQGLEWMGVIFP GNGDTSYNPKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSS
SEQ ID NO:72
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMLWVRQAPGQGLEWMGVIFP GNGDTSYNPKFKGRVTLTADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSS
SEQ ID NO:73
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMLWVRQAPGQRLEWMGVIFP GNADTSYNPKFKGRVTLTADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSS
SEQ ID NO:74
DIQMTQSPSSVSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNSLRPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFNNMPLTFGQGTKLEIK
SEQ ID NO:75
DIQLTQSPSFLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNSLRPGVPSRFSSSGSGTEFTLTISSLQPEDFATYYCLQFNNMPLTFGQGTKLEIK
SEQ ID NO:76
DTQLTQSPSFLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNSLRPGVPSRFSSSGSGTEFTLTISSLQPEDFATYYCLQFNNMPLTFGQGTKLEIK
SEQ ID NO:77
EIVLTQSPDFQSVTPKEKVTITCITSTDIDDDMNWYQQKPDQSPKLLISEGNSLRPGVPSRFSSSGSGTDFTLTINSLEAEDAATYYCLQFNNMPLTFGQGTKLEIK
SEQ ID NO:78
TTGPWFAC
SEQ ID NO:79
EVQLLQSAAEVKRPGESLRISCKTSGYTFTEYTMHWVRQMPGKELEWMGVIDPSN GDTSYNQKFKGHVTISADKSSSTAYLQWSSLKASDAAMYYCSSTTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:80
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQGLEWMGVIDP SNGDTSYNQKFKGRVTLTVDKSTSTAYMELRSLRSDDTAVYYCARTTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:81
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYTMHWVRQAPGQGLEWMGVIDP SNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCSSTTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:82
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQRLEWMGVIDP SNGDTSYNQKFKGRVTITVDKSSSTAYMELSSLRSEDTAVYYCSSTTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:83
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQRLEWMGVIDP SNGDTSYNQKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARTTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:84
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYKSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQVHTLPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:85
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYKSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVHTLPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:86
EIVMTQSPPTLSLSPGERVTLSCRASQDISNYLNWYQQKPGQAPRLLIYYKSRLHSGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQVHTLPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:87
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYKSRLHS GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQVHTLPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:88
EVQLLQSAAEVKRPGESLRISCKTSGYSFTENTMHWVRQMPGKELEWMGGINPH KGGTSYNQKFKGHVTISVDKSSSTAYLQWSSLKASDAAMYYCARDWLGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:89
EVQLLQSAAEVKRPGESLRISCKTSGYTFTENTMHWVRQMPGKELEWMGGINPH KGGTSYNQKFKGHVTISADKSSSTAYLQWSSLKASDAAMYYCARDWLGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:90
EVQLLQSAAEVKRPGESLRISCKTSGYTFTENTMHWVRQMPGKELEWMGGINPH KGGTSYNQKFKGHVTLSVDKSSSTAYLQWSSLKASDAAMYYCARDWLGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:91
QVQLVQSGAEVKKPGASVKVSCKASGYTFTENTMHWVRQAPGQGLEWMGGINP HKGGTSYNQKFKGRVTLTVDKSSSTAYMELSRLRSDDTAVYYCARDWLGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:92
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTENTMHWVRQAPGQGLEWMGGINP HKGGTSYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARDWLGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:93
DIQLTQSPSSLSASVGDRVTITCITSTDIDDDMNWYRQKPGKVPKLLISEGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQSDNLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:94
DIQVTQSPSSLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNTLRPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQSDNLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:95
DIVLTQSPASLAVSPGQRATITCITSTDIDDDMNWYQQKPGQPPKLLISEGNTLRPGVPARFSSSGSGTDFTLTINPVEANDTANYYCLQSDNLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:96
DTVLTQSPASLAVSPGQRATITCITSTDIDDDMNWYQQKPGQPPKLLISEGNTLRPGVPARFSSSGSGTDFTLTINPVEANDTANYYCLQSDNLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:97
DIVLTQSPASLAVSPGQRATITCITSTDIDDDMNWYQQKPGQPPKLLISEGNTLRPGVPARFSSSGSGTDFTLTINPVEANDVANYYCLQSDNLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:98
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMLWVRQAPGQRLEWMGVIFP GNGDTSYNPKFKGRVTITADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:99
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMLWVRQAPGQRLEWMGVIFP GNGDTSYNPKFKGRVTLTADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:100
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYNMLWVRQAPGQGLEWMGVIFPG NGDTSYNPKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:101
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMLWVRQAPGQGLEWMGVIFP GNGDTSYNPKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:102
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMLWVRQAPGQGLEWMGVIFP GNGDTSYNPKFKGRVTLTADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:103
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMLWVRQAPGQRLEWMGVIFP GNADTSYNPKFKGRVTLTADKSSSTAYMELSSLRSEDTAVYYCARDGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:104
DIQMTQSPSSVSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNSLRPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQFNNMPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:105
DIQLTQSPSFLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNSLRPGVPSRFSSSGSGTEFTLTISSLQPEDFATYYCLQFNNMPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:106
DTQLTQSPSFLSASVGDRVTITCITSTDIDDDMNWYQQKPGKAPKLLISEGNSLRPGVPSRFSSSGSGTEFTLTISSLQPEDFATYYCLQFNNMPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:107
EIVLTQSPDFQSVTPKEKVTITCITSTDIDDDMNWYQQKPDQSPKLLISEGNSLRPGVPSRFSSSGSGTDFTLTINSLEAEDAATYYCLQFNNMPLTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:108
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISGGGFGTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYNNYYFDIWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
SEQ ID NO:109
EIVLTQSPATLSLSPGERATLSCHASDSISNSLHWYQQKPGQAPRLLIYYARQSIQGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSESWPLHFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:110
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISYSGSNTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGIDLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:111
DIALTQPASVSGSPGQSITISCTGTSSDVGGFNYVSWYQQHPGKAPKLMIHDGSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCQSWDVSPITAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO:112
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:113
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
发明详述
本发明提供了抗DKK1抗体或其抗原结合片段，其特征在于具有独特的CDR序列组成，与人类DKK1结合具有高亲和力和高特异性。本发明提供的抗DKK1抗体或其抗原结合片段可作为独立的疗法或与其它疗法/或其他抗癌药剂联合，用于诸如癌症的治疗。
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
本文术语“DKK1”，是Wnt/β-catenin信号转导通路的分泌性抑制剂，属于抑制Wnt信号传导的蛋白质家族之一的DKK(dickkopf)家族，指来自任何脊椎动物(包括哺乳动物如灵长类动物(例如人)和啮齿类动物(例如，小鼠和大鼠)的任何天然DKK1，除非另 有说明。该术语涵盖“全长”未加工的DKK1以及由细胞内加工产生的任何形式的DKK1或其任何片段。该术语还包括天然存在的DKK1的变体，例如，剪接变体或等位变体。在一些实施方案中，DKK1是指来自人和小鼠DKK1全长或其片段(诸如其缺乏信号肽的成熟片段)。在一些实施方案中，DKK1是指与Genbank登录号NP_036374.1(SEQ ID NO:31)氨基酸残基32-266序列一致的成熟人DKK1(氨基酸残基1-31为前导肽)。
“百分比(％)氨基酸序列同一性”定义为在将所述序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选序列中的氨基酸残基与参比多肽序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，并包括但不局限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗DKK1活性的能力、IC50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人抗体、嵌合抗体和骆驼源化单结构域抗体。
“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它 生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可能少量天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
“全长抗体”，天然存在时包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常可变结构域获自啮齿动物等实验动物的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
“人源化抗体”是指含有来自人和非人(例如鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体可包含任选的至少一部分的人免疫球蛋白恒定区(Fc)。
“完全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则完全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则完全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、 噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方案中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方案中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方案中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。
“联合”一种或多种其它抗肿瘤药物的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的DKK1抗体或其抗原结合片 段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。
抗DKK1抗体及其产生
术语“抗DKK1抗体”、“抗DKK1”、“DKK1抗体”或“结合DKK1的抗体”能够以足够的亲合力结合DKK1蛋白或其片段以致所述抗体可以用作靶向DKK1的诊断剂和/或治疗剂。
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的DKK1都可用作产生抗体的免疫原(抗原)。通过举例而非限制，任何DKK1变体或其片段都可用作免疫原。在一些实施方式中，产生鼠源的单克隆抗人DKK1抗体的杂交瘤细胞可通过本领域公知的方法产生。这些方法包括但不限于最初由Kohler等(1975)(Nature 256:495-497)研发的杂交瘤技术。优选根据标准方案，分离出小鼠脾细胞，用PEG或通过电融合与小鼠骨髓瘤细胞系融合。然后通过筛选分泌抗体具有DKK1抑制活性的杂交瘤细胞。本发明的杂交瘤细胞免疫球蛋白可变区的DNA序列可利用基于简并引物PCR的方法测定。
来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84:3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321:522；Verhoeyen等(1988)Science 239:1534)。
在一些实施方案中，本发明的嵌合或人源化抗体可基于所述制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方案中，本发明所述的嵌合DKK1抗体，可使用本领域已知的方法将杂交 瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链。在一些实施方案中，本发明的嵌合抗体包含的恒定区可选自人IgG任何亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG4。
在一些实施方案中，本发明的嵌合DKK1抗体可由一种嵌合型轻链与一种嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的DKK1结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglob μLins”,Journal of Molec μLar Biology,273,927-948(1997))基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat、Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101、5,585,089、5,693,762和6,180,370。简言之，发明人藉由NCBI(http://www.ncbi.nlm.nih.gov/igblast/)网站中的人类免疫球蛋白基因数据库搜寻与鼠源抗体 可变区的cDNA序列同源的人类种系IgG基因，原则上藉由选定的CDR嫁接实现人源化。然而，CDR环交换仍不能均匀产生具有与起始抗体相同的结合性质的抗体。在人源化抗体中，常常还需要构架残基(FR)(参与CDR环支持的残基)改变以保持抗原结合亲和力。简言之，人源化改造过程涉及以下步骤：A、把各候选抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析各候选的抗体基因序列中可与DKK1作用以及维护空间构架的关键氨基酸个体，将其嫁接回已经选择的人胚胎系基因框架，并在此基础上标配出必须保留的框架区氨基酸位点，合成人源化抗体。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如取代)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4 Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基取代。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
在一些实施方案中，本发明所述的抗DKK1抗体或其抗原结合片段具有以下特性中的一种或多种：(1)特异性结合人DKK1蛋白；(2)与食蟹猴DKK1交叉反应；(3)抑制DKK1与LRP5或LRP6结合；(4)抑制由DKK1介导的活性信号转导；(5)能显著抑制肿瘤的生长。
在一些实施方案中，本发明所述的抗DKK1抗体或其抗原结合片段具有下列性质中的至少一种：
(1)以至少约5nM、至少约1nM、至少约0.1nM、至少约0.01nM、至少约0.001nM的KD与人DKK1结合；
(2)与食蟹猴DKK1交叉反应。
本发明还涉及用于产生本发明所述抗体或其抗原结合片段的方法。此类方法包括提供一种编码本发明的抗体或抗原结合片段的分离的核酸分子，或包含此类核酸的表达载体， 特别是用于在宿主细胞中重组产生本发明所述的抗体或其抗原结合片段的载体。
本发明还涉及包含一种或多种上述重组载体或表达载体的宿主细胞以及用于产生本发明所述抗体或包含其抗原结合片段的方法，所述方法包括培养所述宿主细胞、纯化和回收所述抗体或其抗原结合片段。
本发明还提供一种产生上述抗体或其抗原结合片段的方法，包括培养上述的宿主细胞，并从培养物中回收所述抗体或其抗原结合片段。
本发明还涉及治疗受试者癌症的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗DKK1抗体或其抗原结合片段或其药物组合物。
在一种实施方式中，还涉及本发明所述的任何抗DKK1抗体或其抗原结合片段在制备用于在受试者中治疗癌症的药物的用途。
本发明还提供了上述的抗体或其抗原结合片段、核酸分子、载体、宿主细胞或药物组合物在制备用于治疗和/或预防DKK1介导的疾病或病症的药物中的用途；优选地，所述疾病或病症是癌症。
本发明还涉及向癌症受试者联合施用一种或多种疗法(例如治疗方式和/或其它抗肿瘤药物)。在一些实施方案中，上述疗法包括向所述受试者施用有效量的本发明任一实施方案所述的抗DKK1抗体或其抗原结合片段或其药物组合物。在一些实施方案中，治疗方式包括手术治疗和放射疗法。在一些实施方案中，上述其它抗肿瘤药物包含化疗药物和/或免疫检查点抑制剂，其中化疗药物选自微管蛋白抑制剂/或抗血管生成剂；其中化疗药物优选微管蛋白抑制剂。
本发明还涉及本文所述的任何抗DKK1抗体或其抗原结合片段与微管蛋白抑制剂组合在制备用于在受试者中治疗癌症的药物中的用途。
本发明还涉及检测样品中DKK1的方法，所述方法包括：a)将样品与本文所述的任何抗DKK1抗体或其片段接触；和b)检测抗DKK1抗体或其片段和DKK1间的复合物的形成。在一个实施方案中，抗DKK1抗体是被可检测地标记的。
在一些实施方案中，本发明涉及试剂盒或制品，其包含本文所述的任何抗DKK1抗体或其片段。在一些实施方案中，所述试剂盒或制品包含本文所述的抗DKK1抗体或其片段与任选的可药用辅料，以及任选地一种或多种其它抗肿瘤药物(例如免疫检查点抑制剂、微管蛋白抑制剂、抗血管生成剂或其他化疗药物)。
本发明还涵盖本文所述的任何实施方案的任意组合。本文所述的任何实施方案或其任何组合适用于本文所述的发明的任何和所有抗DKK1抗体或其片段、方法和用途。
在一些实施方案中，本发明所述的抗DKK1抗体或其抗原结合片段以约lnM-2pM的KD或更高亲和力特异性结合DKK1，优选地其以1nM、100pM、10pM或2pM的KD或亲和力特异性结合DKK1。在一个实施方案中，特异性结合人DKK1的本发明抗体还与食 蟹猴DKK1交叉反应。本文所述的“交叉反应性”是指抗体与来自其它物种的同源蛋白反应的能力。抗体是否特异性结合人DKK1可使用本领域中已知的任何测定法确定。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(例如，BIACORE)或类似技术(例如，ForteBio)。
在一些实施方案中，本发明所述的DKK1抗体或其抗原结合片段具有抑制活性，例如抑制DKK1的表达(如抑制细胞表面DKK1的表达)、活性和/或信号传递，或干扰DKK1和LRP5或LRP6之间的相互作用。本发明提供的DKK1抗体在结合DKK1(如人DKK1)或与其相互作用后完全或部分地降低或调节DKK1的表达或活性。在抗体与人DKK1多肽和/或肽之间相互作用后，DKK1生物学功能的降低或调节是完全、显著或部分的。当与不存在同本文所述的抗体相互作用(如结合)时DKK1的表达或活性水平相比，存在抗体时DKK1的表达或活性水平降低了至少95％(例如降低了96％、97％、98％、99％或100％)时，所述抗体被认为能够完全抑制DKK1的表达或活性。与不存在同本文所述的DKK1抗体结合时DKK1的表达或活性水平相比，存在DKK1抗体时DKK1的表达或活性水平降低了至少50％(例如降低了55％、60％、75％、80％、85％或90％)，此时所述DKK1抗体被认为能够显著抑制DKK1的表达或活性。与不存在同本文所述的抗体相互作用(如结合)时DKK1的表达或活性水平相比，存在抗体时DKK1的表达或活性水平降低了少于95％(例如降低了10％、20％、25％、30％、40％、50％、60％、75％、80％、85％或90％)，此时所述抗体被认为能够部分抑制DKK1的表达或活性。
抗体表达
在一个方面，本发明涉及包含一种或多种表达载体的宿主细胞以及用于产生本发明的任何抗体或其片段的方法，所述方法包括培养所述宿主细胞、纯化和回收所述抗体或抗原结合片段。
在一个方面，本发明提供了编码以上任何抗DKK1抗体或其片段的核酸。所述核酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的核酸，或包含编码抗体的轻链和/或重链的氨基酸序列的核酸。
在一些实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别 优选的细胞系。
在一个实施方案中，本发明提供制备抗DKK1抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中，将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选允许抗体分泌到宿主细胞生长的培养基中，从而产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
药物组合物和药物制剂
本发明提供了包含一种或多种结合DKK1的抗体或其抗原结合片段的药物组合物，其包含如前文任一项所述的抗体或其抗原结合片段和药学上可接受的载体或赋形剂的组合物。
应理解，本发明提供的抗DKK1抗体或其抗原结合片段或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性以有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗DKK1抗体或其抗原结合片段与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编(1980))混合来制备包含本文所述的抗DKK1抗体或其抗原结合片段的药物制剂，优选地，所述药物制剂为水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗药物、微管蛋白抑制剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗DKK1抗体或其抗原结合片段的多核苷酸的组合物。
抗体的医药用途
在一个方面，本发明涉及向受试者施用有效量的本文所述的任何抗DKK1抗体或其抗原结合片段，或包含所述抗体或其抗原结合片段的免疫辍合物，或药物组合物的方法，用 于诱导T细胞或NK细胞介导的抗肿瘤活性、促进经典Wnt通路相关的抑癌活性或增强机体的免疫应答。
在又一个方面，本发明涉及向受试者施用有效量的本文所述的任何抗DKK1抗体或其抗原结合片段，或包含所述抗体或抗原结合片段的免疫辍合物，或药物组合物的方法，用于治疗或延缓各种癌症、免疫相关疾病和T细胞功能障碍性疾病。
在又一个方面，本发明提供治疗或预防受试者癌症的方法，所述方法包括向所述受试者施用有效量的如前文任一项所述的抗体或其抗原结合片段、或如前文任一项所述的药物组合物的步骤。在一些实施方式中，如前文所述的方法，其还包括向所述受试者联合施用一种或多种疗法的步骤，所述疗法包括手术治疗和/或放射疗法和/或施用一种或多种其它抗肿瘤药物，所述的其它抗肿瘤药物选自化疗药物或免疫检查点抑制剂。所述化疗药物选自烷化剂类(alkylating agents)、磺酸烷基酯类(alkyl sulfonates)、氮丙啶类(aziridines)、乙撑亚胺类(ethylenimines)和甲基蜜胺类(methylamelamines)、番荔枝内酯类(acetogenin)、δ-9-四氢大麻酚(tetrahydrocannabinol)、β-拉帕醌(lapachone)、拉帕醇(lapachol)、秋水仙素类(colchicines)、白桦脂酸(betulinic acid)、喜树碱(camptothecin)、苔藓抑素(bryostatin)、培美曲塞(pemetrexed)、鬼臼毒素(podophyllotoxin)、鬼臼酸(podophyllinic acid)、替尼泊苷(teniposide)、隐藻素类(cryptophycins)、多拉司他汀(dolastatin)、艾榴塞洛素(eleutherobin)、海绵抑素(spongistatin)、氮芥类(nitrogen mustards)、胆磷酰胺(cholophosphamide)、雌莫司汀(estramustine)、异环磷酰胺(ifosfamide)、双氯乙基甲胺(mechlorethamine)、盐酸氧氮芥(mechlorethamine oxide hydrochloride)、美法仑(melphalan)、新氮芥(novembichin)、苯芥胆甾醇(phenesterine)、泼尼莫司汀(prednimustine)、曲磷胺(trofosfamide)、尿嘧啶氮芥(uracil mustard)、亚硝脲类(nitrosoureas)、抗生素类、地托比星(detorubicin)、6-二氮-5-氧-L-正亮氨酸、多柔比星(doxorubicin)、表柔比星(epirubicin)、依索比星(esorubicin)、伊达比星(idarubicin)、麻西罗霉素(marcellomycin)、丝裂霉素类(mitomycins)、抗代谢物类、叶酸类似物、嘌呤类似物、嘧啶类似物、c-Kit抑制剂、抗肾上腺类、醋葡醛内酯(aceglatone)、醛磷酰胺糖苷(aldophosphamide glycoside)、氨基乙酰丙酸(aminolevulinic acid)、恩尿嘧啶(eniluracil)、安吖啶(amsacrine)、bestrabucil、比生群(bisantrene)、依达曲沙(edatraxate)、地磷酰胺(defofamine)、地美可辛(demecolcine)、地吖醌(diaziquone)、elfornithine、依利醋铵(elliptinium acetate)、依托格鲁(etoglucid)、硝酸镓、羟脲(hydroxyurea)、香菇多糖(lentinan)、氯尼达明(lonidainine)、美登木素生物碱类(maytansinoids)、莫哌达醇(mopidanmol)、二胺硝吖啶(nitraerine)、喷司他丁(pentostatin)、蛋氨氮芥(phenamet)、吡柔比星(pirarubicin)、洛索蒽醌(losoxantrone)、2-乙基酰肼(ethylhydrazide)、丙卡巴肼(procarbazine)、雷佐生(razoxane)、根霉素(rhizoxin)、西索菲兰(sizofiran)、螺旋锗(spirogermanium)、细交链孢菌酮酸(tenuazonic acid)、三亚胺醌(triaziquone)、2,2',2″-三氯三乙胺、单端孢菌素类(trichothecenes)、乌拉坦(urethan)、长春地 辛(vindesine)、达卡巴嗪(dacarbazine)、甘露醇氮芥(mannomustine)、二溴甘露醇(mitobronitol)、二溴卫矛醇(mitolactol)、哌泊溴烷(pipobroman)、gacytosine、阿糖胞苷(arabinoside)、塞替派(thiotepa)、类紫杉醇(taxoids)、苯丁酸氮芥(chloranbucil)、6-硫鸟嘌呤(thioguanine)、巯基嘌呤(mercaptopurine)、甲氨蝶呤(methotrexate)、铂类似物、依托泊苷(etoposide)(VP-16)、异环磷酰胺(ifosfamide)、米托蒽醌(mitoxantrone)、长春新碱(vincristine)、奥沙利铂(oxaliplatin)、亚叶酸(leucovovin)、长春瑞滨(vinorelbine)、能灭瘤(novantrone)、依达曲沙(edatrexate)、道诺霉素(daunomycin)、氨基蝶呤(aminopterin)、伊本膦酸盐(ibandronate)、二氟甲基鸟氨酸(DMFO)、类维A酸(retinoids)、任何上述物质的药学可接受盐、酸或衍生物、以及两种或更多种上述物质的组合，诸如CHOP(环磷酰胺、多柔比星、长春新碱和泼尼松龙联合疗法的缩写)和FOLFOX(奥沙利铂(ELOXATINTM)联合5-FU和亚叶酸的治疗方案的缩写)。优选地，所述化疗药物包括紫杉醇。在本发明的一些实施方式中，本发明提供的如前述治疗或预防癌症的方法，所述方法包括向所述受试者施用有效量的如前文任一项所述的抗DKK1抗体或其抗原结合片段、或如前文任一项所述的药物组合物。在一些实施方式中，如前文所述的方法，其还包括向所述受试者联合施用一种或多种疗法，所述疗法包括施用紫杉醇。在本发明的一些实施方式中，如前文所述治疗或预防癌症的方法中，抗DKK1抗体或其抗原结合片段可与紫杉醇同时或不同时施用；在本发明的一些实施方式中，如前文所述治疗或预防癌症的方法中，抗DKK1抗体或其抗原结合片段可与紫杉醇同时或不同时施用；在本发明的一些实施方式中，如前文所述治疗或预防癌症的方法中，抗DKK1抗体或其抗原结合片段为每周1次或每周2次施用；在本发明的一些实施方式中，如前文所述治疗或预防癌症的方法中，紫杉醇为每周1次或每周2次施用。
在又一个方面，本发明提供本文所述的抗DKK1抗体或其抗原结合片段在生产或制备药物中的用途，所述药物用于治疗上文提及的相关疾病或病症。
在又一个方面，本发明提供本文所述的抗DKK1抗体或其抗原结合片段与微管蛋白抑制剂的组合在制备用于在受试者中治疗癌症的药物中的用途。
用于诊断和检测的方法
在某些实施方案中，本文中提供的任何抗DKK1抗体或其抗原结合片段可以用于检测DKK1在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定 实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
在本发明中，如果没有特别说明，所述“第一”(例如，第一产品、第一抗体、第一蛋白功能区等)和“第二”(例如，第二产品、第二抗体、第二蛋白功能区等))是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
本发明中，术语“其它抗肿瘤药物”是指除了本发明的抗DKK1抗体或其抗原结合片段以及本发明的偶联物之外的抗肿瘤药物；优选地，所述其它抗肿瘤药物为化疗药物或免疫检查点抑制剂；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述化疗药物为紫杉醇。
本发明采用下述缩略词：
CD CHO培养基(chemically defined medium for China hamster ovary cell)代表化学成分确定的CHO培养基；
PEI(Polyethylenimine)代表聚乙烯亚胺；
BSA(Bovine serum albumin)代表牛血清白蛋白。
具体实施方式
DKN-01的重链和轻链的氨基酸序列分布如SEQ ID NO:108和SEQ ID NO:109所示；
BHQ-880的重链和轻链的氨基酸序列分布如SEQ ID NO:110和SEQ ID NO:111所示。
实施例1：重组抗原蛋白DKK1-hFC的制备和检测
进行动物免疫的抗原为human DKK1和human IgG1 Fc的融合蛋白，将设计好的序列交给金唯智生物科技有限公司进行基因合成并将合成的质粒转化至top10菌种中。将菌种按照1：100的比例接到LB培养基中进行培养，18h收集，弃上清，用控内毒质粒抽提试剂盒(天根)提取质粒。
用CD CHO培养基对CHO-K1细胞(经过基因组水平的改造使细胞适用于瞬转表达)进行传代扩增，转染前一天将细胞密度稀释至1.5-2.0×106/mL，次日当细胞密度达到约 3.5×106/mL时用于转染。先加入十分之一转染体积的培养基，再加入1-2μg/mL转染体积的上述合成质粒，最后加入3-14μg/mL转染体积的PEI，混匀后室温孵育不超过30分钟后，将转染混合物缓慢加入细胞中，将细胞放入摇床培养，培养条件为36.5℃，120rpm，7％CO2。培养周期为转染后6-10天，每两天补料一次。
培养结束后，1000g离心10min弃去沉淀，然后12000g离心30min收集细胞上清。细胞上清进行无菌过滤后，用AKTA Avant纯化仪进行纯化。先用0.1M NaOH对装填mabselect sure LX柱子进行CIP 15-20min，然后用3-5柱体积的PBS缓冲液平衡后再上样，上样完成后用亲和层析淋洗缓冲液进行淋洗，最后用pH 3.6的醋酸-醋酸钠缓冲液洗脱目的蛋白，并用1M Tris缓冲液中和样品。经SDS-PAGE方法鉴定蛋白的大小和纯度，所制备的抗原质量检测合格。
实施例2：杂交瘤细胞的制备、筛选及测序
2.1动物免疫
采用两侧脚垫免疫的方式，将实施例1中制得的抗原DKK1-hFc融合蛋白用于免疫5只BALB/c雌性小鼠。初次免疫的剂量为50μg/只，6次冲击免疫的剂量为25μg/只，每隔一周进行一次冲击免疫。
2.2细胞融合
在最后一针加强免疫后，取小鼠腹股沟淋巴结,腘窝淋巴结和脾脏，在生理盐水中碾磨后取富含淋巴细胞的悬浮液，按常规电转方法将其与小鼠骨髓瘤细胞Sp2/0融合。将融合产物在含1:50 HAT(Hypoxanthine,Aminopterin and Thymidine)的DMEM完全培养基中培养5天以筛选成功融合的细胞，然后换成含1:50 HT的DMEM完全培养基培养直至筛选结束。DMEM完全培养基配比是：15％FBS+1:50L-谷氨酰胺+100U/mL青链霉素+1:100OPI(Oxaloacetate,Pyruvate,and Insulin)，培养箱参数条件是8％CO2，37℃。
2.3杂交瘤细胞的筛选
在11520株不同的多克隆杂交瘤细胞中，通过竞争ELISA，筛选出15株多克隆分泌抗体可与LPR6竞争结合DKK1，其中3株有弱的阻断效果，12株有强阻断效果。将15株抗体进行亚克隆，并通过竞争ELISA筛选，得到了7株单克隆抗体(图1)。
用过表达LRP5/6以及荧光素酶报告基因系统的靶细胞293 TOP和重组Wnt3a(R&D systems,cat#5036-WN)以及人DKK1(义翘神州，cat#10170-H08H)蛋白，来检测DKK1抗体细胞生物学活性。即将靶细胞293 TOP按每孔50,000个细胞接种到96孔平底白板(corning,cat#3917)中培养过夜，然后弃上清并且加入梯度稀释的待测抗体并于37℃预孵育0.5h；预孵育结束后，向含细胞和抗体的混合物中加入稀释的工作浓度分别为0.05μg/ml和为0.1μg/ml的Wnt3a(R&D systems,cat#5036-WN)和人DKK1蛋白，并于37℃共孵育 6-8h。最后，加入荧光素酶底物One-Lite(Vazyme,cat#DD1203-03-AB)并检测各杂交瘤抗体对应的化学发光值。通过荧光素酶报告基因法，检测到其中7株抗体对Wnt信号通路均具有不同程度的回复作用(图2A和图2B)。
将7株杂交瘤细胞送至南京金斯瑞生物科技有限公司进行测序，其中两株细胞序列完全一致，最终得到6株杂交瘤抗体，分别命名为7M2.6，11I18.4，17F14.3，18E24.6，18J21.1和19O15.6。
2.4候选抗体可变区序列的获得(Kabat定义方案)
用基于简并引物PCR的方法，测定由候选杂交瘤细胞表达的小鼠抗体可变区的DNA序列。简言之，将杂交瘤细胞株分别扩大培养，1000rpm离心收集细胞，并以Trizol提取总RNA。以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板PCR扩增对应的可变区DNA序列，所用PCR引物基于Ig-引物组。回收纯化PCR产物，将扩增产物测序后，得到候选杂交瘤抗体重链可变区和轻链可变区的编码序列，由此得到杂交瘤抗体重链可变区和轻链可变区的氨基酸序列。如下面的表1所示。
表1：杂交瘤抗体可变区的氨基酸序列
用NCBI Ig-Blast(http://www.ncbi.nlm.nih.gov/projects/igblast/)在种系和重排Ig可变区序列数据库中搜索共有序列。基于Kabat(Wu,T.T及Kabat,E.A.1970 J.Exp.Med.，132:211-250)及IMGT系统(Lefranc M.-P.等人，1999 Nucleic Acids Research,27,209-212)，藉由序列批注及藉由基于因特网的序列分析(http://www.Imgt.org/IMGT_vquest/share/textes/index.html与http://www.ncbi.nlm.nih.gov/igblast/)鉴定互补决定区(CDR)。如下面的表2所示。
表2：杂交瘤抗体CDR区的氨基酸序列(Kabat定义方案)

实施例3：嵌合抗体的设计、构建与表达
根据实施例2的测序结果，选取抗huDKK1鼠源抗体7M2.6，11I18.4，17F14.3，18E24.6，18J21.1和19O15.6的轻链可变区和重链可变区(序列如下表3)，重链可变区构建到hIgG4重链恒定区(重链恒定区的氨基酸序列如SEQ ID NO:112所示)，轻链可变区构建到hIgκ链(其中轻链恒定区的氨基酸序列如SEQ ID NO:113所示)，嵌合重轻链可变区再随机组合成36株嵌合抗体，如下面的表3所示。
表3：嵌合抗体的编号(chi1-chi36)及其重链可变区和轻链可变区的组合

用CD CHO培养基对CHO-K1细胞(经过基因组水平的改造使细胞适用于瞬转表达)进行传代扩增，转染前一天将细胞密度稀释至1.5-2.0×106/mL，次日当细胞密度达到约3.5×106/mL时用于转染。先加入十分之一转染体积的培养基，再分别加入1-2μg/mL转染体积的编码嵌合抗体的质粒，最后加入3-14μg/mL转染体积的PEI，混匀后室温孵育不超过30分钟后，将转染混合物缓慢加入细胞中，将细胞放入摇床培养，培养条件为36.5℃，120rpm，7％CO2。培养周期为转染后6-10天，每两天补料一次。
培养结束后，1000g离心10min弃去沉淀，然后12000g离心30min收集细胞上清。细胞上清进行无菌过滤后，用AKTA Avant纯化仪进行纯化。先用0.1M NaOH对装填mabselect sure LX柱子进行CIP 15-20min，然后用3-5柱体积的PBS缓冲液平衡后再上样，上样完成后用亲和层析淋洗缓冲液进行淋洗，最后用pH 3.6的醋酸-醋酸钠缓冲液洗脱目的蛋白，并用1M Tris缓冲液中和样品。经SDS-PAGE方法鉴定蛋白的大小和纯度，所制备的嵌合抗体质量检测合格。
实施例4：嵌合抗体的筛选
4.1蛋白水平的筛选
ELISA法检测嵌合抗体与人DKK1的结合活性：用人DKK1抗原2.0μg/mL(Sino Biological，10170-H08H)包板90min，2％BSA封闭90min后，加入梯度稀释的抗DKK1嵌合抗体及阳性对照抗体DKN-01(1μg/ml起始，2.5倍梯度稀释至1.64ng/mL，共8个浓度)，孵育60min，洗板后孵育5000倍稀释的HRP偶联的羊抗人IgG(Fc特异性)(Sigma，A0170)二抗，然后用0.1mg/ml TMB(Sigma，T2885)显色，最后用2M盐酸溶液终止反应，使用Thermo Scientific的酶标仪，在450nm/620nm下读板，使用四参数对数回归(4PL)模型拟合得到各嵌合抗体的EC50(图3)，并比较各嵌合抗体的相对结合活性，选取活性较优的chi-1，chi-8，chi-15分子的VH和VL用于后续人源化抗体的构建。
ELISA法检测嵌合抗体抑制LRP6(629-1244)与DKK1的结合：用人DKK1抗原5.0μg/mL(Sino Biological,10170-H08H)包板90min，2％BSA封闭90min，用人LRP6 mFc(629-1244)(苏州君盟)稀释抗DKK1嵌合抗体及阳性对照抗体DKN-01(10μg/ml起始，2.5倍梯度稀释至16.38ng/mL，共8个浓度)，加样后孵育60min，洗板后加入5000倍稀释的HRP偶联的羊抗鼠IgG(Fc特异性)(Sigma，A2554)孵育30min，洗板后每孔加入100μL 0.1mg/ml TMB(Sigma，T2885)显色10min，最后每孔加入2M盐酸溶液终止反应，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合各嵌合抗体的IC50(图4A至 图4B)，并比较各个嵌合抗体的相对竞争抑制活性。
4.2细胞水平筛选
chi1上清和chi15上清为发酵液上清，未经纯化；chi1、chi8、chi15和chi21为纯化后得到的蛋白样品，发酵液上清和蛋白样品的制备参照前面的实施例3。
利用过表达LRP5/6以及荧光素酶报告基因系统的靶细胞293 TOP和重组Wnt3a(R&D systems,cat#5036-WN)以及人DKK1(义翘神州，cat#10170-H08H)蛋白，来检测hDKK1嵌合抗体的细胞生物学活性。
将靶细胞293 TOP按每孔50,000个细胞接种到96孔平底白板(corning,cat#3917)中培养过夜，然后弃上清并分别加入梯度稀释的待测抗体，于37℃预孵育0.5h；预孵育结束后，向含细胞和抗体的混合物中加入稀释的工作浓度分别为0.05μg/ml和为0.1μg/ml的Wnt3a(R&D systems,cat#5036-WN)和人DKK1蛋白，并于37℃共孵育6-8h。最后，加入荧光素酶底物One-Lite(Vazyme,cat#DD1203-03-AB)并检测各嵌合抗体对应的化学发光值(图5A至图5C)。
通过实施例4嵌合抗体的筛选得到了3个体外活性最好的抗体chi1、chi8和chi15，用于后续的人源化改造。
实施例5：嵌合抗体的人源化
首先在NCBI(http://www.ncbi.nlm.nih.gov/igblast/)网站中的人类免疫球蛋白基因数据库搜寻与鼠源抗体可变区的DNA序列同源的人类种系IgG基因。再通过Kabat编号系统或IMGT编号系统定义可变区中的CDR的氨基酸序列。原则上将与鼠源抗体可变区具有高同源性的人类IGHV(免疫球蛋白重链恒定区)及IGKV(免疫球蛋白轻链恒定区)选为人源化模板，通过CDR嫁接实施抗体可变区的人源化。
嵌合抗体人源化的具体步骤如下：1、将各杂交瘤细胞分泌的抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；2、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架区序列；3、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架区氨基酸序列，考察其空间立体结合方式。通过计算静电力、范德华力、亲疏水性和熵值，分析各杂交瘤细胞分泌的抗体的基因序列中与huDKK1作用以及维护空间架构的关键氨基酸，将其嫁接到所选择的人胚胎系基因框架区中，并在此基础上标示必须保留的框架区氨基酸位点；4、以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并优化导致抗体CDR区化学不稳定的氨基酸残基。
最终1号嵌合抗体分子(chi-1)人源化改造得到了5条重链和4条轻链(人源化后HCDR3由SEQ ID NO:78突变为SEQ ID NO:3)，2号嵌合抗体分子(chi-8)人源化改造得 到了5条重链和5条轻链，3号嵌合抗体分子(chi-15)人源化改造得到了6条重链和4条轻链。3个分子内重轻链随机组合一共得到69株人源化分子(CDR序列为Kabat定义方案)，其组合分别见下面的表4a至4c和5a至5c。
表4a：基于chi-1的人源化抗体的编号及其重链可变区和轻链可变区的组合
表4b：基于chi-8的人源化抗体的编号及其重链可变区和轻链可变区的组合
表4c：基于chi-15的人源化抗体的编号及其重链可变区和轻链可变区的组合

表5a：基于chi-1的人源化抗体的编号及其重链和轻链的组合
表5b：基于chi-8的人源化抗体的编号及其重链和轻链的组合
表5c：基于chi-15的人源化抗体的编号及其重链和轻链的组合

实施例6：人源化抗体的制备
将编码69个人源化抗体hu1-hu69的重链和轻链的质粒委托南京金斯瑞生物科技有限公司进行基因合成，收到合成的质粒和菌种后，将菌种转接到LB培养基进行过夜培养，用天根质粒大抽试剂盒进行质粒抽提。
用CD CHO培养基对CHO-K1细胞(经过基因组水平的改造使细胞适用于瞬转表达)进行传代扩增，转染前一天将细胞密度稀释至1.5-2.0×106/mL，次日当细胞密度达到约3.5×106/mL时用于转染。先加入十分之一转染体积的培养基，再分别加入1-2μg/mL转染体积的编码嵌合抗体的质粒，最后加入3-14μg/mL转染体积的PEI，混匀后室温孵育不超过30分钟后，将转染混合物缓慢加入细胞中，终体积约为40mL。将细胞放入摇床培养，培养条件为36.5℃，120rpm，7％CO2。培养周期为转染后6-10天，每两天补料一次。
培养结束后，1000g离心10min弃去沉淀，然后12000g离心30min收集细胞上清并进行无菌过滤。采用同实施例1中对杂交瘤抗体的纯化方法，对69株人源化抗体进行纯化。纯化后采用SDS-PAGE鉴定抗体蛋白的大小和纯度，并采用HPLC-SEC检测蛋白的纯度。用于后面的实验。
实施例7：人源化抗体的筛选
7.1蛋白水平的筛选
ELISA法检测人源化抗体与DKK1的结合：用人DKK1抗原2.0μg/mL(Sino Biological,10170-H08H)包板90min，然后2％BSA封闭90min，洗板后加入梯度稀释的抗DKK1人源化抗体(1μg/ml起始，10倍梯度稀释至1ng/mL，共4个浓度)，孵育60min，洗板后孵育5000倍稀释的HRP偶联的羊抗人IgG(Fc特异性)(Sigma，A0170)二抗30min，然后每孔加入100μL 0.1mg/mL TMB(Sigma，T2885)显色10min，最后每孔加入50μL 2M盐酸溶液终止反应，使用Thermo Scientific的酶标仪，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合各人源化抗体的EC50，相比嵌合抗体chi-15，得到的部分人源化抗体与人DKK1结合能力提高(图6)。
ELISA法检测人源化抗体抑制LRP6(629-1244)与DKK1的结合：用人DKK1抗原 5.0μg/mL(Sino Biological,10170-H08H)包板90min，然后2％BSA封闭90min，洗板后用10μg/mL人LRP6 mFc(629-1244)(苏州君盟)稀释DKK1抗体人源化抗体及抗DKK1嵌合抗体(10μg/ml起始，10倍梯度稀释至10ng/mL，共4个浓度),加样后孵育60min。洗板加入5000倍稀释的HRP偶联的羊抗鼠IgG(Fc特异性)(Sigma，A2554)每孔加入100μL,孵育30min，洗板后每孔加入100μL 0.1mg/ml TMB(Sigma，T2885)显色10min，最后每孔加入50μL 2M盐酸溶液终止反应，使用Thermo Scientific的酶标仪，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合各人源化抗体的IC50，相比嵌合抗体chi-15，得到的部分人源化抗体抑制DKK1与LRP6结合的能力提高(图7)。
7.2细胞水平的筛选
利用过表达LRP5/6以及荧光素酶报告基因系统的靶细胞293 TOP和重组Wnt3a(R&D systems,cat#5036-WN)以及人DKK1(义翘神州，cat#10170-H08H)蛋白，来检测DKK1抗体细胞生物学活性。将靶细胞293 TOP按每孔50,000个细胞接种到96孔平底白板(corning,cat#3917)中培养过夜，然后弃上清并且加入梯度稀释的待测抗体并于37℃预孵育0.5h；预孵育结束后，向含细胞和抗体的混合物中加入稀释的工作浓度分别为0.05μg/ml和为0.1μg/ml的Wnt3a(R&D systems,cat#5036-WN)和人DKK1蛋白，并于37℃共孵育6-8h。最后，加入荧光素酶底物One-Lite(Vazyme,cat#DD1203-03-AB)并检测各人源化抗体对应的化学发光值，相比嵌合抗体chi-15，得到的部分人源化抗体抗体使Wnt3a通路的表达的恢复程度提高(图8)。
细胞水平的筛选结果显示，活性较优的分子为hu3、hu2、hu64、hu52和hu50。
经过对人源化抗体蛋白和细胞水平的筛选(图7，图8)，将两轮筛选中效果较好的分子用于体内药效的研究。
实施例8：人源化抗DKK1抗体抑制小鼠体内肿瘤生长
实验动物为CB17/SCID雌性小鼠，向小鼠皮下接种A549细胞(5×106)，建立皮下移植肿瘤模型。试验分为抗KLH IgG4(5mg/kg)组、DKN-01(5mg/kg)组、hu3(5mg/kg)组、紫杉醇(20mg/kg)组、DKN-01(5mg/kg)+紫杉醇(20mg/kg)组、DKN-01(5mg/kg)+hu3(5mg/kg)组，DKN-01为阳性对照抗体，抗KLH IgG4为阴性对照抗体。每组6只动物,各组均为腹腔注射给药，阴性对照、阳性对照、抗DKK1抗体hu3均为每周给药1次，共给药4周；紫杉醇为每周给药2次，共给药4周，根据相对肿瘤抑制率(TGI)进行疗效评价(图9)。
实验结果显示在接种肿瘤后第35天时，按TGI效果递减排序，依次为DKN-01(5mg/kg)+hu3(5mg/kg)组，紫杉醇(20mg/kg)组，DKN-01(5mg/kg)+紫杉醇(20mg/kg)组，hu3组，DKN-01组和抗KLH IgG4组，数值分别为74.48％，60.48％， 60.04％，35.29％，23.27％和0％。结果显示hu3在5mg/kg剂量表现明显的肿瘤抑制作用(p＝0.013)，效果优于阳性对照DKN-01；此外，在前述浓度下，抗DKK1抗体hu3与紫杉醇联用组合的肿瘤抑制效果明显优于hu3和紫杉醇两组分分别单用，同时效果明显优于相同浓度下阳性对照与紫杉醇的联用组合。
实施例9：人源化抗DKK1抗体抑制小鼠体内肿瘤生长
实验动物为CB17/SCID雄性小鼠，向小鼠的右后背皮下接种PC-3细胞(10×106)，建立皮下移植肿瘤模型。试验分为抗KLH IgG4(10mg/kg)组、hu3(5mg/kg)组、hu3(10mg/kg)组、hu3(20mg/kg)组、紫杉醇(7.5mg/kg)组、hu3(5mg/kg)+紫杉醇(7.5mg/kg)组、hu3(10mg/kg)+紫杉醇(7.5mg/kg)组、hu3(20mg/kg)+紫杉醇(7.5mg/kg)组，抗KLH IgG4为阴性对照抗体。每组8只动物，各组均为腹腔注射给药，阴性对照抗KLH IgG4、紫杉醇、抗DKK1抗体hu3，均为每周给药2次，共给药3周，根据相对肿瘤抑制率(TGI)进行疗效评价(图10)。
实验结果显示在接种肿瘤后第21天时，按TGI效果递减排序，依次为hu3(20mg/kg)+紫杉醇(7.5mg/kg)组，hu3(10mg/kg)+紫杉醇(7.5mg/kg)组，hu3(5mg/kg)+紫杉醇(7.5mg/kg)组，紫杉醇(7.5mg/kg)组，hu3(20mg/kg)组，hu3(10mg/kg)组，hu3(5mg/kg)组，抗KLH IgG4(10mg/kg)组，数值分别为88.4％，88.3％，81.5％，71.8％，56.4％，48.8％，30.7％和0％。结果显示，hu3在5mg/kg、10mg/kg、20mg/kg的剂量下均表现出明显的肿瘤抑制作用，且剂量越高TGI越高。此外，在前述三个浓度剂量下，hu3分别与7.5mg/kg紫杉醇联用，肿瘤抑制效果明显优于对应浓度下hu3和紫杉醇两组分分别单用，说明抗DKK1抗体hu3和紫杉醇联合施用能够起到协同的抗肿瘤效果。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (26)

  1. 抗DKK1抗体或其抗原结合片段，
    其中，所述抗DKK1抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，
    其中，
    HCDR1的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:13、SEQ ID NO:19、SEQ ID NO:25和SEQ ID NO:31；
    HCDR2的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:8、SEQ ID NO:14、SEQ ID NO:20、SEQ ID NO:26和SEQ ID NO:32；
    HCDR3的氨基酸序列选自SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:15、SEQ ID NO:21、SEQ ID NO:27、SEQ ID NO:33和SEQ ID NO:78；
    LCDR1的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:10、SEQ ID NO:16、SEQ ID NO:22、SEQ ID NO:28和SEQ ID NO:34；
    LCDR2的氨基酸序列选自SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:17、SEQ ID NO:23、SEQ ID NO:29和SEQ ID NO:35；
    LCDR3的氨基酸序列选自SEQ ID NO:6、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:24、SEQ ID NO:30和SEQ ID NO:36。
  2. 根据权利要求1所述的抗DKK1抗体或其抗原结合片段，其中，所述抗DKK1抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，
    其中，
    所述抗体的重链可变区，其中，
    HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示，
    HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:78所示，
    HCDR1的氨基酸序列如SEQ ID NO:7所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:9所示，
    HCDR1的氨基酸序列如SEQ ID NO:13所示、HCDR2的氨基酸序列如SEQ ID NO:14所示、HCDR3的氨基酸序列如SEQ ID NO:15所示，
    HCDR1的氨基酸序列如SEQ ID NO:19所示、HCDR2的氨基酸序列如SEQ ID NO: 20所示、HCDR3的氨基酸序列如SEQ ID NO:21所示，
    HCDR1的氨基酸序列如SEQ ID NO:25所示、HCDR2的氨基酸序列如SEQ ID NO:26所示、HCDR3的氨基酸序列如SEQ ID NO:27所示，或者
    HCDR1的氨基酸序列如SEQ ID NO:31所示、HCDR2的氨基酸序列如SEQ ID NO:32所示、HCDR3的氨基酸序列如SEQ ID NO:33所示；
    并且
    所述抗体的轻链可变区，其中，
    LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR1的氨基酸序列如SEQ ID NO:5所示、LCDR1的氨基酸序列如SEQ ID NO:6所示，
    LCDR1的氨基酸序列如SEQ ID NO:10所示、LCDR1的氨基酸序列如SEQ ID NO:11所示、LCDR1的氨基酸序列如SEQ ID NO:12所示，
    LCDR1的氨基酸序列如SEQ ID NO:16所示、LCDR1的氨基酸序列如SEQ ID NO:17所示、LCDR1的氨基酸序列如SEQ ID NO:18所示，
    LCDR1的氨基酸序列如SEQ ID NO:22所示、LCDR1的氨基酸序列如SEQ ID NO:23所示、LCDR1的氨基酸序列如SEQ ID NO:24所示，
    LCDR1的氨基酸序列如SEQ ID NO:28所示、LCDR1的氨基酸序列如SEQ ID NO:29所示、LCDR1的氨基酸序列如SEQ ID NO:30所示，或者
    LCDR1的氨基酸序列如SEQ ID NO:34所示、LCDR1的氨基酸序列如SEQ ID NO:35所示、LCDR1的氨基酸序列如SEQ ID NO:36所示。
  3. 根据权利要求1至2中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，
    HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR1的氨基酸序列如SEQ ID NO:5所示、LCDR1的氨基酸序列如SEQ ID NO:6所示；
    HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:78所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR1的氨基酸序列如SEQ ID NO:5所示、LCDR1的氨基酸序列如SEQ ID NO:6所示；
    HCDR1的氨基酸序列如SEQ ID NO:7所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:9所示、LCDR1的氨基酸序列如SEQ ID NO:10所示、LCDR1的氨基酸序列如SEQ ID NO:11所示、LCDR1的氨基酸序列如SEQ ID  NO:12所示；
    HCDR1的氨基酸序列如SEQ ID NO:13所示、HCDR2的氨基酸序列如SEQ ID NO:14所示、HCDR3的氨基酸序列如SEQ ID NO:15所示、LCDR1的氨基酸序列如SEQ ID NO:16所示、LCDR1的氨基酸序列如SEQ ID NO:17所示、LCDR1的氨基酸序列如SEQ ID NO:18所示；
    HCDR1的氨基酸序列如SEQ ID NO:19所示、HCDR2的氨基酸序列如SEQ ID NO:20所示、HCDR3的氨基酸序列如SEQ ID NO:21所示、LCDR1的氨基酸序列如SEQ ID NO:22所示、LCDR1的氨基酸序列如SEQ ID NO:23所示、LCDR1的氨基酸序列如SEQ ID NO:24所示；
    HCDR1的氨基酸序列如SEQ ID NO:25所示、HCDR2的氨基酸序列如SEQ ID NO:26所示、HCDR3的氨基酸序列如SEQ ID NO:27所示、LCDR1的氨基酸序列如SEQ ID NO:28所示、LCDR1的氨基酸序列如SEQ ID NO:29所示、LCDR1的氨基酸序列如SEQ ID NO:30所示；
    或者
    HCDR1的氨基酸序列如SEQ ID NO:31所示、HCDR2的氨基酸序列如SEQ ID NO:32所示、HCDR3的氨基酸序列如SEQ ID NO:33所示、LCDR1的氨基酸序列如SEQ ID NO:34所示、LCDR1的氨基酸序列如SEQ ID NO:35所示、LCDR1的氨基酸序列如SEQ ID NO:36所示。
  4. 根据权利要求1至3中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，
    所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NOs:49-53、SEQ ID NOs:58-62和SEQ ID NOs:68-73；并且
    所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77。
  5. 根据权利要求1至4中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:37所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:39所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:41所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:45所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:47所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:49所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:50所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:51所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:52所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:53所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:58所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:59所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:60所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:61所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:62所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:68所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:69所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:70所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:71所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:72所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77；
    或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:73所示，轻链可变区的氨基酸序列选自SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NOs:54-57、SEQ ID NOs:63-67和SEQ ID NOs:74-77。
  6. 根据权利要求1至5中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其 中，
    所述抗体的重链的氨基酸序列选自SEQ ID NOs:79-83、SEQ ID NOs:88-92和SEQ ID NOs:98-103；并且
    所述抗体的轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107。
  7. 根据权利要求1至6中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，
    所述抗体的重链的氨基酸序列如SEQ ID NO:79所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:80所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:81所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:82所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:83所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:88所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:89所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:90所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:91所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:92所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:98所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:99所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:100所示，轻链的氨基酸序列选自SEQ  ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:101所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    所述抗体的重链的氨基酸序列如SEQ ID NO:102所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107；
    或者
    所述抗体的重链的氨基酸序列如SEQ ID NO:103所示，轻链的氨基酸序列选自SEQ ID NOs:84-87、SEQ ID NOs:93-97和SEQ ID NOs:104-107。
  8. 根据权利要求1至7中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，所述抗DKK1抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
  9. 一种抗DKK1的抗体或其抗原结合片段，其具有如下特征：
    (a)与权利要求1-8中任一项所述的抗体或抗原结合片段结合相同的、或完全重叠的、或部分重叠的人DKK1蛋白的表位；或
    (b)与权利要求1-8任一项所述的抗体或抗原结合片段竞争性结合人DKK1蛋白的表位。
  10. 根据权利要求1至9中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，
    所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  11. 根据权利要求1至10中任一权利要求所述的抗DKK1抗体或其抗原结合片段，其中，所述抗体的重链恒定区为Ig gamma-1 chain C region(例如NCBI ACCESSION:P01857)或Ig gamma-4 chain C region(例如NCBI ACCESSION:P01861.1)；轻链恒定区为Ig kappa chain C region(例如NCBI ACCESSION:P01834)；
    优选地，所述抗体的重链恒定区的氨基酸序列如SEQ ID NO:112所示，所述抗体的轻链恒定区的氨基酸序列如SEQ ID NO:113所示。
  12. 分离的核酸分子，其编码权利要求1至11中任一权利要求所述的抗DKK1抗体或其抗原结合片段。
  13. 一种重组载体，其包含权利要求12所述的分离的核酸分子。
  14. 一种宿主细胞，其包含权利要求12所述的分离的核酸分子，或者权利要求13所述的重组载体。
  15. 偶联物，其包括抗体以及偶联部分，其中，所述抗体为权利要求1至11中任一权利要求所述的抗DKK1抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为细胞毒素、免疫调节剂、放射性同位素、荧光物质、发光物质、有色物质或酶。
  16. 试剂盒，其包括权利要求1至11中任一权利要求所述的抗DKK1抗体或其抗原结合片段，或者包括权利要求15所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如细胞毒素、免疫调节剂、放射性同位素、荧光物质、发光物质、有色物质或酶。
  17. 一种双特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中：
    所述第一蛋白功能区靶向DKK1，
    所述第二蛋白功能区靶向不同于DKK1的靶点，
    其中，所述第一蛋白功能区为权利要求1至11中任一权利要求所述的抗体或抗原结合片段。
  18. 根据权利要求17所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接。
  19. 根据权利要求17至18中任一权利要求所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
  20. 根据权利要求17至19中任一权利要求所述的双特异性抗体，其中，所述单链抗体分别连接在免疫球蛋白形式的抗体的两条重链的C末端。
  21. 一种药物组合物，其包含权利要求1至11中任一权利要求所述的抗DKK1抗体或其抗原结合片段，或者包含权利要求15所述的偶联物，或者包含权利要求17至20中 任一权利要求所述的双特异性抗体；
    可选地，所述药物组合物还包含一种或多种其它抗肿瘤药物；优选地，所述其它抗肿瘤药物为化疗药物或抗体药物；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述抗体药物为免疫检查点抑制剂；优选地，所述化疗药物为紫杉醇；
    可选地，所述药物组合物还包括一种或多种药学上可接受的辅料。
  22. 一种组合产品，其包含独立包装的第一产品和第二产品，其中，
    所述第一产品包含其包含权利要求1至11中任一权利要求所述的抗DKK1抗体或其抗原结合片段，或者包含权利要求15所述的偶联物，或者包含权利要求17至20中任一权利要求所述的双特异性抗体；
    所述第二产品包含一种或多种其它抗肿瘤药物；优选地，所述其它抗肿瘤药物为化疗药物或抗体药物；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述抗体药物为免疫检查点抑制剂；优选地，所述化疗药物为紫杉醇；
    优选地，所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料；
    优选地，所述组合产品还包含产品说明书。
  23. 权利要求1至11中任一权利要求所述的抗体或其抗原结合片段、权利要求15所述的偶联物、权利要求17至20中任一权利要求所述的双特异性抗体或者权利要求21所述的药物组合物在制备治疗和/或预防DKK1介导的疾病的药物中的用途；优选地，所述DKK1介导的疾病为肿瘤。
  24. 根据权利要求1至11中任一权利要求所述的抗体或其抗原结合片段、权利要求15所述的偶联物、权利要求17至20中任一权利要求所述的双特异性抗体或者权利要求21所述的药物组合物，其用于治疗和/或预防DKK1介导的疾病；优选地，所述DKK1介导的疾病为肿瘤。
  25. 一种治疗或预防DKK1介导的疾病的方法，包括给予有需求的受试者以有效量的权利要求1至11中任一权利要求所述的抗体或其抗原结合片段、权利要求15所述的偶联物、权利要求17至20中任一权利要求所述的双特异性抗体或者权利要求21所述的药物组合物的步骤；优选地，所述DKK1介导的疾病为肿瘤。
  26. 根据权利要求25所述的方法，其还包括向所述受试者联合施用一种或多种疗法的步骤，所述疗法包括手术治疗和/或放射疗法和/或施用一种或多种其它抗肿瘤药物；
    优选地，所述其它抗肿瘤药物为化疗药物或抗体药物；优选地，所述化疗药物为微管蛋白抑制剂；优选地，所述抗体药物为免疫检查点抑制剂；优选地，所述化疗药物为紫杉醇。
PCT/CN2023/073335 2022-01-28 2023-01-20 抗dkk1抗体、其药物组合物及用途 Ceased WO2023143444A1 (zh)

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