WO2018184558A1 - 一种人源化抗tpbg抗体及其制备方法、其偶联物和应用 - Google Patents

一种人源化抗tpbg抗体及其制备方法、其偶联物和应用 Download PDF

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WO2018184558A1
WO2018184558A1 PCT/CN2018/081853 CN2018081853W WO2018184558A1 WO 2018184558 A1 WO2018184558 A1 WO 2018184558A1 CN 2018081853 W CN2018081853 W CN 2018081853W WO 2018184558 A1 WO2018184558 A1 WO 2018184558A1
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seq
sequence
variable region
chain variable
amino acid
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French (fr)
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张莹
宫世勇
孙晓岚
徐腾蛟
彭菲
陈雨竹
刘礼乐
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凯惠科技发展(上海)有限公司
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Priority to US16/499,995 priority Critical patent/US11608384B2/en
Priority to EP18781311.8A priority patent/EP3608335A4/en
Priority to JP2019554774A priority patent/JP7305549B2/ja
Publication of WO2018184558A1 publication Critical patent/WO2018184558A1/zh

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Definitions

  • TPBG trophoblast-specific glycoprotein
  • 5T4 trophoblast-specific glycoprotein
  • the human TPBG protein has a molecular weight of about 72 kDa and contains 420 amino acids. Its N-terminal oligosaccharide structure is diverse, prevents protein hydrolysis, and interacts with other molecules during cell membrane signaling.
  • the TPBG protein contains a total of seven repeat leucine domains (LRRs) that are involved in protein-protein interactions.
  • the trophoblast is a special layer of embryonic stem cells between the placenta and the fetus.
  • TPBG is widely expressed in various trophoblast cells during embryonic development. For normal adult tissues, TPBG is only expressed in a limited number of epithelial cells. However, TPBG is expressed in many cancer cells, such as uterine cancer, colon cancer, gastric cancer, ovarian cancer, oral cancer, prostate cancer, lung cancer or kidney cancer tissues, and is detected in colon cancer, gastric cancer or ovarian cancer. There is evidence that the expression of TPBG is associated with a low cure rate for cancer. In tissues of non-small cell lung cancer, kidney cancer or pancreatic cancer, the expression of TPBG is as high as 95% or more.
  • TPBG epithelial cell cadherin
  • EMT epithelial mesenchymal transition
  • TPBG and CXCR4 colocalize on the surface of the cell membrane, which can induce the binding of its ligand CXCL12 chemokine and promote the spread of inflammation and tumor.
  • CXCL12 binds to another receptor, CXCR7, which inhibits chemotactic responses and facilitates cell growth and survival.
  • the Wnt/b-catenin signaling pathway plays a very important role in development and cell regeneration.
  • TPBG inhibits Wnt signaling pathway by inhibiting endocytosis of LRP6 and Wnt receptors, thereby inhibiting cell adhesion and cytoskeletal formation, and promoting tumorigenesis. Migration and diffusion.
  • TPBG is involved in non-canonical Wnt pathways in breast and gastric cancer cells, as well as promoting cancer cell migration and infiltration.
  • Monoclonal antibodies are developing into new diagnostic and therapeutic drugs due to their advantages of targeting, specificity, specificity and high affinity.
  • early clinical trials revealed that the use of non-human monoclonal antibodies in humans often results in severe immune responses due to human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, and antibodies are rapidly cleared.
  • HAMA human anti-mouse antibody
  • HAA human anti-rat antibody
  • antibodies with less immunogenicity were developed, including chimeric antibodies, humanized antibodies, and fully human antibodies.
  • therapeutic monoclonal antibody drugs can be divided into four types: murine antibody (unmanned amino acid sequence), chimeric antibody (60% to 70% humanized amino acid sequence), CDR graft antibody (90% to 95% humanized amino acid sequence) and fully human antibody (100% human amino acid sequence).
  • non-rat monoclonal antibodies can alleviate human anti-mouse antibody responses (HAMA and HARA responses) during human therapy, gradually eliminating the immunogenicity of heterologous antibodies, and maintaining high affinity for antigens.
  • HAMA and HARA responses human anti-mouse antibody responses
  • the pharmacokinetics of the antibodies have been improved, and these antibody drugs have been used in a large amount in the clinic for targeted therapy.
  • the antibody drug-conjugated drug is an antibody drug conjugate formed by coupling an antibody and a high-efficiency small molecule drug through a linker, and enables a highly toxic small molecule drug to specifically recognize a target protein on a cancer cell, thereby specifically killing the cancer. cell.
  • antibody-based immunotherapy and chemical-based chemotherapy have been the two major treatment strategies for clinical cancer treatment.
  • Antibodies target antigens overexpressed by tumor cells, and a variety of therapeutic monoclonal antibodies have achieved great clinical success.
  • therapeutic antibodies have good targeting properties, the killing effect is limited.
  • small molecule chemical drugs have an effective killing effect on cancer cells, they also cause the same damage to non-cancer cells.
  • ADC drugs involves: screening of drug targets, preparation of recombinant antibodies, development of linker technology, and screening optimization of high cytotoxic compounds.
  • TPBG is a candidate for ADC drugs.
  • the technical problem to be solved by the present invention is to provide a humanized anti-TPBG antibody with high affinity and high specificity, a preparation method and application thereof, and a humanized anti-TPBG antibody and the like, in order to overcome the deficiency of the current lack of TPBG antibody.
  • TPBG protein has a high affinity, and with the increase of humanization, it can reduce the human anti-mouse antibody response (HAMA reaction) during human treatment, gradually eliminate the immunogenicity of heterologous antibodies, and maintain high affinity for antigens. At the same time, the pharmacokinetics of the antibodies are improved.
  • the present invention also provides a conjugate of a pharmaceutically active ingredient comprising the humanized anti-TPBG antibody and a small molecule compound having an antitumor function coupled thereto, the conjugate being capable of entering a cell
  • a conjugate of a pharmaceutically active ingredient comprising the humanized anti-TPBG antibody and a small molecule compound having an antitumor function coupled thereto, the conjugate being capable of entering a cell
  • the cytotoxic killing effect on TPBG-positive cells can be used in the preparation of drugs for treating tumors and the like.
  • the present inventors used a human TBPG protein or a recombinant cell strain overexpressing a human TBPG protein as an immunogen, using a conventional hybridoma preparation technique (Kohler and Milstein, Nature, 1975, 256:495), through a series of adjustments. Improved to obtain a lead antibody for the TPBG antibody. Further, by preliminary production, purification and identification of the lead antibody, a TPBG antibody having a high affinity with a protein such as a human TBPG protein is obtained. The amino acid sequence of the heavy chain variable region of the obtained mouse-derived TPBG antibody and the light chain variable region of the TPBG antibody was obtained by sequencing by molecular biological methods.
  • the human antibody heavy chain framework region residues may include germline DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11), DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68 and DP69, especially FR1, FR2, FR3 of these germ lines; and JH fragments JH1, JH2, JH3, JH4, JH4b, JH5 and JH6,
  • the FR4 encoded sequences of these lines, or the consensus sequences of the heavy chain framework regions may include germline DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11), DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP
  • Such framework region sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be obtained from the "VBase” human germline sequence database (www.mrcco8.com.ac.uk/vbase), as well as in Kabat, EA et al., 1991 Sequences. Of Proteins of Immunological Interest, found in the 5th edition.
  • the amino acid sequence of the CDR2 of the murine antibody heavy chain variable region is shown in positions 50 to 66 of SEQ ID No. 2; more preferably, the murine antibody heavy chain variable region is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR2 is shown in positions 148 to 198 of SEQ ID No. 1 of the Sequence Listing;
  • amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 24 to 38 of SEQ ID No. 4; more preferably, the murine antibody light chain is variable
  • the nucleotide sequence of the amino acid sequence of CDR1 of the region is shown in positions 70 to 114 of SEQ ID No. 3 of the Sequence Listing;
  • the amino acid sequence of the CDR3 of the murine antibody light chain variable region is shown in positions 93 to 101 of SEQ ID No. 4; more preferably, the murine antibody heavy chain variable region is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR3 is shown in positions 277 to 303 of SEQ ID No. 3 of the Sequence Listing;
  • the amino acid sequence of CDR1 of the murine antibody heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 6 of the Sequence Listing; more preferably, the murine antibody heavy chain is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR1 of the variable region is shown in positions 91 to 105 of SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the CDR2 of the murine antibody heavy chain variable region is shown in positions 50 to 66 of SEQ ID No. 6; more preferably, the murine antibody heavy chain variable region is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR2 is shown in positions 148 to 198 of SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the CDR3 of the murine antibody heavy chain variable region is shown in positions 99 to 109 of SEQ ID No. 6; more preferably, the murine antibody heavy chain variable region is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR3 is shown in positions 295 to 327 of SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the CDR2 of the murine antibody light chain variable region is shown in positions 50 to 56 of SEQ ID No. 8; more preferably, the murine antibody light chain variable region is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR2 is shown in positions 148 to 168 of SEQ ID No. 7 of the Sequence Listing;
  • the amino acid sequence of the CDR3 of the murine antibody light chain variable region is shown in positions 89 to 97 of SEQ ID No. 8; more preferably, the murine antibody light chain variable region is encoded.
  • the nucleotide sequence of the amino acid sequence of CDR3 is shown in positions 265 to 291 of SEQ ID No. 7 of the Sequence Listing.
  • the humanized anti-TPBG antibody comprises at least one heavy chain variable region and/or at least one light chain variable region, wherein the amino acid sequence of the heavy chain variable region is SEQ ID in the sequence listing NO.2, SEQ ID NO. 6, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 36. SEQ ID NO. 38 or SEQ ID NO. 40; the light chain variable region sequence is SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO in the sequence listing. .28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 42, SEQ ID NO. 44 or SEQ ID NO.
  • nucleotide sequences are respectively SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. in the Sequence Listing. 23. SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37 or SEQ ID No. 39; the nucleotide sequence encoding the amino acid sequence of the light chain variable region is as shown in the sequence listing, respectively. SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 25, SEQ ID No.27, SEQ ID No.29, SEQ ID No.31, SEQ ID No.41, SEQ ID No.43 or SEQ ID shown a No. 45;
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO in the sequence listing. .22, the amino acid sequence of SEQ ID NO. 24, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38 or SEQ ID NO. 40 is represented by an amino acid sequence having at least 80% sequence homology;
  • the light chain variable region sequence is SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ in the Sequence Listing.
  • the amino acid sequence encoded by the nucleotide sequence represented by No. 33, SEQ ID No. 35, SEQ ID No. 37 or SEQ ID No. 39 has an amino acid sequence of 80% sequence homology; the light chain variable region
  • the amino acid sequence is SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 41, SEQ ID No. 43 or
  • the amino acid sequence encoded by the nucleotide sequence shown by SEQ ID No. 45 has an amino acid sequence of 80% sequence homology.
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 26 of the Sequence Listing. More preferably, the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is SEQ ID No. The sequence shown in 25;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 26 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 25 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 26 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 25 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 16 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 15 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 27 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 27 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 27 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 22 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 21 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 27 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 27 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 30 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 29 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 30 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 30 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 29 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 16 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 15 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 31 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 31 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 31 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 22 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 21 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 31 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 31 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 41 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 41 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 42 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 41 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 36 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 36 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing. The sequence shown.
  • the full-length antibody protein is a conventional full-length antibody of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.
  • the preparation method of the humanized anti-TPBG antibody is a conventional preparation method in the art.
  • the preparation method is preferably obtained by isolating the expression transformant recombinantly expressing the humanized anti-TPBG antibody or by artificially synthesizing the protein sequence.
  • the method for isolating the expression transformant recombinantly expressing the humanized anti-TPBG antibody preferably obtains a method of cloning the nucleic acid molecule encoding the humanized anti-TPBG antibody into a recombinant vector, and transforming the resulting recombinant vector into a transformation.
  • a recombinant expression transformant is obtained, and the humanized anti-TPBG antibody can be obtained by isolation and purification by culturing the resulting recombinant expression transformant.
  • the preparation of representative humanized anti-TPBG antibodies of the invention is described in Example 1.
  • Amino acids 89-97 (CDR3-L). Due to the change in the length of the variable region, in the different central or different subgroups, the 27th position may have 1-6 amino acids, and the 95th position may have 1-6 amino acids, which are added to the original number. English letters are numbered, such as: 27A, 27B, 95A, 95B, etc. According to the Kabat definition, the CDRs of the heavy chain variable region are defined by residues at positions 31 and 35B (CDR1-H), positions 50 and 65 (CDR2-H), and positions 95 and 102 (CDR3-H). (according to Kabat numbering).
  • the CDRs of the heavy chain variable region are defined by residues at positions 26 and 32 (CDR1-H), positions 52 and 56 (CDR2-H), and positions 95 and 102 (CDR3-H) (according to the Chothia number).
  • the CDRs of the heavy chain variable region are defined by the residues at positions 26 and 35B (CDR1-H), at positions 50 and 58 (CDR2-H), and at positions 95 and 102 (CDR3-H) (according to Kabat number).
  • a plurality of amino acids may be present at positions 35, 52, 82, and 100, and are numbered A, B, C, and the like. See Martin et al.
  • a framework residue is a portion of a light chain variable region or a heavy chain variable region, and is an antibody variable region residue other than a hypervariable residue (a hypervariable residue multi-finger complementarity determining region or CDR) or a CDR residue, It serves as a scaffold for the antigen binding loop (CDR) of this variable domain.
  • the framework residues can be derived from naturally occurring human antibodies, such as the framework regions of human antibodies that are substantially similar to the framework regions of murine anti-TPBG antibodies 12B12 or 28D4. Artificial framework region sequences representing consensus sequences between individual sequences can also be used. When framework regions for humanization are selected, sequences that are widely present in humans may be superior to less common sequences.
  • the humanized antibodies can be prepared using any of a variety of methods, including inlays of complementarity determining regions (CDRs), transplantation, transplantation of shortened CDRs, transplantation of specificity determining regions (SDRs), and Frankenstein assembly.
  • CDRs complementarity determining regions
  • SDRs specificity determining regions
  • Frankenstein assembly a variety of methods, including inlays of complementarity determining regions (CDRs), transplantation, transplantation of shortened CDRs, transplantation of specificity determining regions (SDRs), and Frankenstein assembly.
  • the humanized antibody also includes a superhumanized antibody, which is a method for preparing a humanized antibody, which does not rely on the human framework sequence as an analysis point, but relies on comparing the normative CDRs of the non-human antibody.
  • the structural type and the CDR structure type of the human antibody particularly the human antibody encoded by the human germline sequence, from which a candidate human antibody sequence from which a suitable human framework sequence can be obtained is identified.
  • a human residue can replace a non-human residue in a CDR, wherein one or more changes have been introduced into the CDRs.
  • the inlay is based on the concept of reducing the amino acid sequence of potential immunogenicity in rodents or other non-human antibodies by reconstituting the solvent-accessible surface of the antibody with a human amino acid sequence.
  • a human amino acid sequence See Padlan (1991) Mol. Immunol. 28: 489-980. Identification of residues of the outer framework regions of solvent accessible residues exposed to the surface in non-human antibodies (the residues are different from the framework regions of human antibodies) Those residues at the same position) and replacing the identified residues with amino acids occupying the same position in the human antibody for inlaying, ie, the embedded antibody, the surface residues of which are predominantly human sequences, and The internal residues are primarily the original murine sequences.
  • Transplantation of CDRs is performed by replacing one or more CDRs of a receptor antibody (eg, a human antibody or other antibody comprising the desired framework residues) with CDRs of a donor antibody (eg, a non-human antibody).
  • the receptor antibody can be selected based on the similarity of the framework residues between the candidate receptor antibody and the donor antibody. For example, according to the Frankenstein method, a human framework region having substantial sequence homology to each framework region of a related non-human antibody is identified, and CDRs of non-human antibodies are grafted onto the complex of these different human framework regions.
  • the above methods can be combined to produce an anti-TPBG antibody of any desired sequence.
  • the invention also provides a nucleic acid encoding the above-described humanized anti-TPBG antibody, comprising a nucleic acid encoding the heavy chain variable region, and/or a nucleic acid encoding the light chain variable region.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 3 of the Sequence Listing; SEQ ID No. 7, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 26 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 17 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 25;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 26 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .25;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 26 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .25;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 16 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 15 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .27;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .27;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 22 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 21 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .27;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 28 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .27;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 30 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .29;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 30 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .29;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 30 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .29;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 18 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 17 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. As shown in .31;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 20 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 19 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. As shown in .31;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 22 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 21 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. As shown in .31;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 24 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 23 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. As shown in .31;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 36 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 42 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 35 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .41;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 42 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 37 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .41;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 42 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 39 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .41;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 33 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .43;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 36 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 35 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .43;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 37 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .43;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 39 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .43;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 36 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 35 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .45;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 37 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .45;
  • amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 39 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. .45 is shown.
  • nucleotide sequence numbers are shown in Table 1-2.
  • nucleic acids encoding the above-described humanized anti-TPBG antibodies can be suitably introduced with substitutions, deletions, alterations, insertions or additions to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more nucleotides of a nucleic acid encoding the humanized anti-TPBG antibody while maintaining antibody activity.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors.
  • the expression vector is a variety of vectors conventional in the art as long as it can accommodate the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell is an E. coli TG1 or BL21 cell (expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (expressing a full-length IgG antibody).
  • the recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.
  • the invention further provides a cell or cell line comprising the recombinant expression vector described above.
  • the cells are mammalian or human cells, more preferably CHO cells, HEK-293 cells, HeLa cells, CV-1 cells or COS cells. Methods for producing stable cell lines after transformation of a heterologous construct into said cells are known in the art.
  • the non-mammalian host cell is preferably an insect cell (Potter et al. (1993) Int. Rev. Immunol. 10(2-3): 103-112).
  • the antibodies can also be used in transgenic animals (Houdebine (2002) Curr. Opin. Biotechno J. 13(6): 625-629) or transgenic plants (Schillberg et al. (2003) Cell Mol. Life Sci. 60(3): Production is carried out in 433-45).
  • the present invention also provides a method for producing a humanized anti-TPBG antibody, which comprises the steps of culturing the above recombinant expression transformant, or a cell, or a cell line, and obtaining a humanized anti-TPBG antibody from the culture.
  • the invention also provides an immunoconjugate comprising the above-described humanized anti-TPBG antibody covalently attached to a cytotoxic agent.
  • the above 1 equivalent of the protein is linked to the y equivalent of the cytotoxic agent via the x equivalent linker, and has the structure of the formula 1.
  • Ab is the above-mentioned humanized anti-TPBG antibody; L is a linker; D is a cytotoxic agent; the x is a conventional degree of crosslinking in the art, x is a natural number, preferably an integer of 1-20; y is 0 or a natural number Preferably, an integer of from 0 to 20; x and y are each independently preferably from 1 to 2, or from 2 to 4, or from 3 to 5, or from 4 to 8, or from 8 to 20; the ratio of x to y is preferably 1 :1.
  • Ar is composed of (C 1 -C 6 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', 1, 2 or 3 groups of -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' Substituted 1,2-, 1,3- or 1,4-phenylene, wherein n and R' are as defined above, or Ar is each from C 1 -C 6 )alkyl, (C 1 -C 5 ) Alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -2 , 3, or
  • the cytotoxin is a cytotoxin conventional in the art and generally refers to an active agent that inhibits or prevents cellular function and/or causes cell destruction.
  • an active agent that inhibits or prevents cellular function and/or causes cell destruction.
  • Amine chemotherapeutic agents nitrogen mustard, nitrourea chemotherapeutics, antibiotics, antimetabolites, folic acid chemotherapeutics, purine analogs, pyrimidine analogs, androgens, anti-adrenalin, folic acid supplements, and beauty
  • the alkylating agent is a conventional alkylating agent in the art, preferably selected from the group consisting of thiotepa or cyclophosphamide.
  • the alkyl sulfonate chemotherapeutic agent is a conventional alkyl sulfonate chemotherapeutic agent, preferably selected from the group consisting of busulfan, propylene bromide or pipersulfuron.
  • the aziridine chemotherapeutic agent is a conventional aziridine chemotherapeutic agent in the art, preferably selected from the group consisting of aziridine, carbazone, metopril or uridine.
  • the antimetabolite is a conventional antimetabolite in the art, preferably selected from methotrexate or 5-fluorouracil (5-FU).
  • the folic acid chemotherapeutic agent is a conventional folic acid chemotherapeutic agent in the art, preferably selected from the group consisting of dimethyl folic acid, pterostilbose or trimethoate.
  • the purine analogs are conventional anthraquinone analogs in the art, preferably selected from fludarabine, 6-oxime, thioxime or thioguanine.
  • the folic acid supplement is a conventional folic acid supplement in the art, preferably selected from the group consisting of folinic acid, acetolactone, aldophosphamide glucoside, amino keto valeric acid, amsacrine, amoxicilin, specific life.
  • the maytansinol is a conventional maytansinol in the art, preferably selected from the group consisting of maytansin, anspirin, mitoxantrone, mitoxantrone, moppeldol, diamine nitidine, Pentastatin, egg ammonia, pirarubicin, loxoprost, podophyllin, 2-ethyl hydrazide or procarbazine.
  • the polysaccharide complex is a conventional polysaccharide complex in the art, preferably selected from the group consisting of razuxon, rhizomycin, cilostazol, quercetin, fine-chain oxysporic acid, and triammine oxime 2, 2' , 2"-trichlorotriethylamine, trichothecenes, urethane, vindesine, dacarbazine, mannimostatin, dibromomannitol, dibromodusol, piperobbrane, Gacytosine, cytarabine, cyclophosphamide or thiotepa.
  • T-2 toxin More preferably selected from the group consisting of T-2 toxin, fusarium A, porphyrin A or anguidine.
  • the taxane is a conventional purple in the art.
  • Cedarane preferably selected from paclitaxel, non-hydrogenated castor oil, paclitaxel, albumin engineered nanoparticle preparation (American Pharmaceutical Partners, Schaumberg, Illinois), docetaxel, chlorambucil, gemcitabine, 6-sulfur A guanine, guanidine or methotrexate.
  • the platinum analog is a conventional platinum analog, preferably selected from the group consisting of cisplatin, carboplatin, vinblastine, etoposide, ifosfamide, Mitoxantrone, vincristine, nooto, teniposide, edarshasha, daunorubicin, aminopterin, Peitabin ibandronate, CPT-11, topoisomerase inhibitor RFS 2000 or difluoromethylornithine.
  • the retinoid is a retinoid in the field, preferably Retinoic acid.
  • the radioisotope is a conventional radioisotope in the art, preferably, it is directly bound to the above humanized anti-TPBG antibody, or is bound to the above humanized anti-TPBG antibody by a chelating agent. More preferably, it binds directly to the cysteine residue of the humanized anti-TPBG antibody.
  • the radioisotope is selected from the group consisting of alpha-emitters, beta-emitters and Auger electrons suitable for radiation therapy and positron emitters or gamma-emitters suitable for diagnosis. More preferably, the radioisotope is selected from the group consisting of 18 fluoro, 64 copper, and 65 copper.
  • the immunomodulatory agent is a conventional immunomodulator in the art, ie, an agent that elicits an immune response, including a humoral immune response (eg, production of an antigen-specific antibody) and a cell-mediated immune response (eg, lymphocyte proliferation).
  • a humoral immune response eg, production of an antigen-specific antibody
  • a cell-mediated immune response eg, lymphocyte proliferation
  • cytokine a cytokine
  • a growth factor eg, a hormone, an anti-hormonal agent, an immunosuppressive agent or a corticosteroid.
  • the cytokine is a cytokine conventional in the art, preferably selected from the group consisting of xanthine, interleukin or interferon.
  • the growth factor is a conventional growth factor in the art, preferably selected from the group consisting of TNF, CSF, GM-CSF or G-CSF.
  • the hormone is a conventional hormone in the art, preferably selected from the group consisting of estrogen, androgen or progestin. More preferably, the estrogen is diethylstilbestrol or estradiol. More preferably, the androgen is testosterone or fluorotestosterone. More preferably, the progestin is megestrol acetate or medroxyprogesterone acetate.
  • the corticosteroids are conventional corticosteroids in the art, preferably selected from prednisone, dexamethasone or cortisone.
  • the antihormonal agent is a conventional antihormonal agent in the art, which can block the action of a hormone on a tumor, inhibit the production of a cytokine, down-regulate the expression of an autoantigen, or mask an immunosuppressant of an MHC antigen. It is preferably selected from the group consisting of an antiestrogenic drug, an antiandrogen or an antinephrine. More preferably, the antiestrogens are selected from the group consisting of tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, travoxifene or toremi Fen.
  • the anti-angiogenic agent is a conventional anti-angiogenic agent in the art, preferably selected from the group consisting of a farnesyl transferase inhibitor, a COX-2 inhibitor, a VEGF inhibitor, a bFGF inhibitor, a steroid sulfatase Inhibitor, interleukin-24, thrombospondin, metallospondin protein, class I interferon, interleukin 12, protamine, angiostatin, laminin, endostatin or prolactin fragment. More preferably, it is 2-methoxyestradiol diaminosulphonate (2-MeOE2bisMATE).
  • the anti-proliferative pro-apoptotic agent is a conventional anti-proliferative pro-apoptotic agent in the art, preferably selected from the group consisting of a PPAR- ⁇ activator, a retinoid, a triterpenoid, an EGF receptor inhibitor, and an end.
  • the PPAR-gamma activators are conventional PPAR-gamma activators in the art, preferably cyclopentenone prostaglandins (cyPGs).
  • the triterpenoids are conventional triterpenoids in the art, preferably selected from the group consisting of cyclobungane, lupin, ursane, zitherane, xylan, dammarane, cucurbitacin, lemon A bitter analog or a triterpenoid.
  • the EGF receptor inhibitor is a conventional EGF receptor inhibitor in the art, preferably selected from the group consisting of HER4, rapamycin or 1,25-dihydroxycholecalciferol (vitamin D).
  • the iron chelate is a conventional iron chelate compound, preferably 3-aminopyridine-2-formaldehyde thiosemicarbazone.
  • the apoptotic protein is a conventional apoptotic protein in the art, preferably the viral protein 3-VP3 of chicken anemia virus.
  • the PI3K-Akt survival pathway signaling inhibitor is a conventional PI3K-Akt survival pathway signaling inhibitor in the art, preferably UCN-01 or geldanamycin.
  • the cytolytic enzyme is a cytolytic enzyme conventional in the art, preferably an RNase.
  • m is from 1 to 10, preferably m is 5, that is, maleimidohexanoyl (MC).
  • the preparation method comprises the steps of: dialyzing the above humanized anti-TPBG antibody through sodium borate buffer having a pH of 6.5 to 8.5, and adding tris(2-carboxyethyl) Phosphine (TCEP), wherein the molar ratio of TCEP to the above humanized anti-TPBG antibody is 2 to 10, and reduction is carried out at room temperature for 1 to 4 hours to obtain a reaction liquid A.
  • the reaction solution A was eluted to remove excess humanized anti-TPBG antibody to obtain a reaction solution B.
  • MC-MMAF was added to the reaction solution B, wherein the molar ratio of the MC-MMAF to the purified humanized TPBG antibody was 5 to 20, and the reaction was carried out at 10 to 37 ° C for 4 hours.
  • the immunoconjugate can be present in any physical form known in the art, preferably a clear solution.
  • the pharmaceutically acceptable carrier is a carrier conventional in the art and may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above humanized anti-TPBG antibody and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being the mass percentage of the pharmaceutical composition.
  • the dosage level of the pharmaceutical composition of the present invention can be adjusted depending on the amount of the composition that achieves the desired diagnosis or treatment result.
  • the administration regimen can also be a single injection or multiple injections, or adjusted.
  • the selected dosage level and regimen will depend on the activity and stability (ie, half-life) of the pharmaceutical composition, the formulation, the route of administration, the combination with other drugs or treatments, the disease or condition to be detected and/or treated, Reasonable adjustments are made to various factors such as the health status of the subject to be treated and the prior medical history.
  • the above-described humanized anti-TPBG antibodies, immunoconjugates and/or additional therapeutic or diagnostic agents described above, each as a single agent can be used in any time range suitable for performing the intended treatment or diagnosis
  • the therapeutic or diagnostic agent can be another anti-tumor antibody, such as an antibody against a target during the cancer immune cycle, preferably an anti-PD-1 antibody.
  • these single agents can be administered substantially simultaneously (i.e., as a single formulation or over a few minutes or hours) or sequentially. For example, these single agents can be administered within one year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or 5, 4, 3, 2, or 1 day.
  • the present invention provides the use of the above pharmaceutical composition for the preparation of an antitumor drug.
  • the tumor is a conventional tumor, preferably a tumor overexpressing TPBG protein, more preferably squamous/adenoma lung cancer (non-small cell lung cancer), invasive breast cancer, colon cancer, rectal cancer, gastric cancer Squamous cervical cancer, invasive endometrial adenocarcinoma, invasive pancreatic cancer, ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial carcinoma, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
  • squamous/adenoma lung cancer non-small cell lung cancer
  • invasive breast cancer colon cancer
  • rectal cancer gastric cancer Squamous cervical cancer
  • invasive endometrial adenocarcinoma invasive pancreatic cancer
  • ovarian cancer squamous bladder cancer
  • choriocarcinoma bronchial carcinoma
  • breast cancer cervical cancer
  • pancreatic cancer pancreatic cancer or seminal vesicle
  • the present invention also provides a method for detecting a cell overexpressing a TPBG protein, comprising the steps of: contacting the humanized anti-TPBG antibody with a sample to be tested in vitro, and detecting the humanized anti-TPBG antibody and the Check the combination of samples.
  • the meaning of the overexpression is conventional in the art, preferably in the sample to be tested, the cells are subjected to flow detection, and the average fluorescence density (MFI) value of the above humanized anti-TPBG antibody is the MFI value of the subtype IgG. 3 times and above.
  • MFI average fluorescence density
  • the detection mode of the combination is a conventional detection method in the art, preferably FACS detection.
  • Figure 4 Sequence comparison of humanized anti-TPBG antibody 12B12 light chain variable region h12B12.Vk2 and its variants with 12B12 chimeric antibody Vk and human germline Vk exon A2/Jk-2.
  • the box is the CDR.
  • Figure 5 Sequence comparison of humanized anti-TPBG antibody 28D4 heavy chain variable region h28D4.VH1 and its variants with 28D4 chimeric antibody VH and human germline VH exon hVH3-11/JH-6.
  • the box is the CDR.
  • Figure 10C FACS detection of the binding reaction of the humanized 28D4 variant to the surface-expressing mouse TPBG protein stable cell line CHOK1-cTPBG.
  • Figure 11A Detection of the cytotoxic activity of the humanized antibody 12B12 variant and its antibody drug conjugate against the TPBG positive tumor cell line NCI-H1568.
  • Figure 11B Detection of the cytotoxic activity of the humanized antibody 28D4 variant and its antibody drug conjugate against the TPBG positive tumor cell line NCI-H1568.
  • Figure 12A Detection of the cytotoxic activity of the humanized TPBG antibody drug conjugate against the TPBG positive tumor cell line NCI-H1568.
  • Figure 12B Detection of the cytotoxic activity of the humanized TPBG antibody drug conjugate against the TPBG weakly positive tumor cell line NCI-H1975.
  • Figure 12C Detection of the cytotoxic activity of the humanized TPBG antibody drug conjugate against the TPBG positive tumor cell line MDA-MB-468.
  • Figure 13A is a graph showing the volume change of tumors in different doses of humanized 28D4-3-MMAF antibody drug conjugate in an in vivo pharmacodynamic experiment of a NCI-H1975 mouse xenograft model.
  • Figure 13B is a graph showing changes in body weight of mice in different doses of humanized 28D4-3-MMAF antibody drug conjugate in a NCI-H1975 mouse xenograft model.
  • Figure 14A Humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody drug conjugates coupled to different linker-toxins at the same dose in NCI-H1975 mouse xenograft model A graph of the volume change of a tumor in an in vivo pharmacodynamic experiment.
  • Figure 14B Humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody drug conjugates coupled to different linker-toxins at the same dose in NCI-H1975 mouse xenograft model Changes in body weight of mice in an in vivo pharmacodynamic experiment in an in vivo pharmacodynamic experiment of NCI-H1975 mouse xenograft tumor model.
  • FIG. 15A Different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins in NCI-H1568 mouse xenograft tumors The volume change map of the tumor in the in vivo efficacy experiment of the model.
  • Figure 15B Different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins in NCI-H1568 mouse xenograft tumors A graph of changes in body weight in mice in a model of in vivo pharmacodynamic experiments.
  • FIG 16A Different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins in MDA-MB-468 mouse xenogeneic
  • Figure 16B Different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins in MDA-MB-468 mouse xenogeneic A graph of changes in body weight in mice in an in vivo pharmacodynamic experiment of a transplanted tumor model.
  • FIG. 18A Staining of humanized anti-TPBG antibody 12B12-3 on PDX tumor tissue sections.
  • Figure 18B Staining of negative control antibody hIgG on PDX tumor tissue sections.
  • FIG. 20D Mice in which the humanized 28D4-3-MMAE antibody drug conjugate or/and anti-PD-1 combination therapy was completely re-vaccinated with CT26-TPBG.
  • the pCpC vector purchased from Invitrogen, V044-50 harboring the human IgG Fc fragment (hFc) was cloned and the plasmid was prepared according to established standard molecular biology methods. For a specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • HEK293 cells purchased from ATCC
  • polyetherimide PEI purchased from Polysciences
  • FreeStyleTM 293 purchased from Invitrogen
  • the culture supernatant was applied to a Protein A affinity chromatography column (Mabselect Sure, available from GE Healthcare) while monitoring the change in ultraviolet absorption value (A280 nm) with an ultraviolet (UV) detector.
  • A280 nm ultraviolet absorption value
  • a 30 ⁇ L PCR system was configured using the primers M13F and M13R on the T vector, colony PCR was performed, and the colony was pipetted in a PCR reaction system, and 0.5 ⁇ L of the LB containing 100 nM ampicillin was aspirated. The strain was preserved on a solid petri dish. After the end of the PCR reaction, 5 ⁇ L was taken for agarose gel electrophoresis detection, and the positive samples were sequenced and analyzed [see Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)].
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 12B12C7C3 is 148th to 198th in SEQ ID No. 1 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 12B12C7C3 is 295th to 327th in SEQ ID No. 1 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 12B12C7C3 is shown in positions 160 to 180 of SEQ ID No. 3 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 12B12C7C3 is shown in positions 277 to 303 of SEQ ID No. 3 of the Sequence Listing;
  • nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 28D4E6A9 is shown in positions 295 to 327 of SEQ ID No. 5 of the Sequence Listing;
  • the recombinant plasmid (the above experimental principle and steps of the plasmid recombination can be found in "Molecular Cloning Experimental Guide (Third Edition)", (US) J. Sambrook et al.) and verified by sequencing.
  • the recombinant plasmid having a purity increased by mass spectrometry using an alkaline lysis kit (purchased from MACHEREY-NAGEL) was used at a mass of 500 ⁇ g or more, and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • Peptone was added the next day to a final concentration of 0.5% (w/v).
  • the antibody titer of the culture solution was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 ⁇ m filter to obtain a filtered cell supernatant for purification.
  • Human germline antibody heavy and light chain variable region templates that best match the non-CDR regions of chimeric antibody 12B12 or 28D4 described above were selected in the Germline database.
  • TPBG humanized antibody human germline sequence selected receiving exon V H, J H, V k and J k sequences.
  • Figure 2 is a comparison of the sequence of humanized anti-TPBG antibody 12B12 heavy chain variable region h12B12.VH2 and its variants with 12B12 chimeric antibody VH and human germline VH exon hVH3-30/JH-6
  • Figure 3 is a human Comparison of the sequence of the sourced anti-TPBG antibody 12B12 light chain variable region h12B12.Vk1 and its variant with 12B12 chimeric antibody Vk and human germline Vk exon B3/Jk-2
  • Figure 4 is humanized anti-TPBG antibody 12B12 Comparison of the sequence of the light chain variable region h12B12.Vk2 and its variants with the 12B12 chimeric antibody Vk and the human germline Vk exon A2/Jk-2
  • Figure 5 is the humanized anti-TPBG antibody 28D4 heavy chain variable region h28D4 Comparison of the sequence of VH1 and its variants with the 28D4 chimeric antibody VH and the human germline VH exon hVH3-11/JH
  • JK2 FGQGTKLEIK.
  • JK5 FGQGTRLEIK.
  • c begins with a chimeric antibody
  • h begins with a humanized antibody
  • each humanized anti-TPBG antibody 12B12 heavy chain variable region h12B12 "A93S" in .VH1 and its variants indicates that the 93rd amino acid shown in Figure 1 is mutated from "A" alanine to "S" serine, and the site of the back mutation is located in the framework region, for example, Not one by one.
  • the cDNA was synthesized based on the amino acid sequences of the light chain variable region and the heavy chain variable region of each humanized antibody (ie, SEQ ID NO. 15, 17, 19, 21, 23, 25, 27, 29 in the sequence listing, respectively. The sequences shown in 31, 33, 35, 37, 39, 41, 43 and 45), the heavy chain cDNA was digested with FspAI and AfeI, and the light chain cDNA was subjected to FspAI/AfeI or FspAI/BsiwI enzyme after FspAI and BsiwI.
  • the cleavage sites were inserted into an expression vector containing the signal peptide and the human IgG1 constant region of the human heavy chain antibody and an expression vector comprising the signal peptide and the human antibody light chain kappa constant region (wherein the expression vector was purchased from Invitrogen, and the recombination step was also In Shanghai Ruizhi Chemical, the recombinant plasmid was verified by sequencing, and the recombinant plasmid with a purity of 500 ⁇ g or more was obtained by using an alkali lysis kit (purchased from MACHEREY-NAGEL). The mass was 500 ⁇ g or more (purified from Millopore). ) Filtered for transfection.
  • 293E cells Prior to transfection, 293E cells (purchased from Invitrogen) were cultured in medium Freestyle 293 expression medium (purchased from Invitrogen). At the time of transfection, 10% (v/v) F68 (purchased from Invitrogen) was added to Freestyle 293 expression medium to a final concentration of 0.1% (v/v) of F68 to obtain Freestyle 293 expression culture containing 0.1% (v/v) F68.
  • Base medium A. 5 mL of Medium A and 200 ⁇ g/mL of PEI (purchased from Sigma) were mixed to obtain Medium B.
  • TPBG antibody was characterized (the procedure is as described in Example 2, Example 3, and Example 4 below).
  • Example 2 Enzyme-linked immunosorbent assay (ELISA) for detection of binding of humanized TPBG antibody to TPBG protein
  • the purified humanized TPBG antibody obtained in Example 1 was reacted with a human TBPG-hFc protein.
  • the method is as shown in the first (5) lead antibody assay in Example 1, and the results are shown in Figures 7-8 and 11-12.
  • Table 11 and Table 12 show the EC50 values calculated from the OD 450 nm values for the h12B12 variant and the h28D4 variant, respectively, indicating that the purified humanized TPBG antibody variant binds well to the TPBG recombinant protein at the ELISA level.
  • 7A and 7B are the binding reactions of the purified humanized h12B12 variant to the human TBPG-hFc protein
  • FIGS. 8A and 8B are the binding reactions of the purified humanized h28D4 variant to the human TBPG-hFc protein.
  • Biacore analysis was performed using human TPBG antigen immobilized on a CM5 chip.
  • the Biacore technique utilizes a change in the refractive index of the surface layer of the antibody after binding to the TPBG antigen immobilized on the surface layer.
  • the detection is performed in combination with surface plasmon resonance (SPR) of a laser refracted from the surface. Analysis of signal kinetics binding rate and dissociation rate allows for the discrimination of non-specific and specific interactions.
  • SPR surface plasmon resonance
  • CHO-k1 stable cell line of human TPBG (CHOk1-hTPBG stable cell line), CHO-k1 stable cell line of monkey-derived TPBG (CHOk1-cTPBG stable cell line), and CHO-k1 stable cell line of mouse-derived TPBG (for the preparation method of the CHOk1-mTPBG stable cell strain, the detection method of the flow cytometer, and the reading of the results, see Example 1 (5) "Test of the lead antibody”.
  • the analysis results are shown in Tables 14, 15 and Figs. 10 and 10.
  • the data of Figures 9A to D and 10A to D are the average fluorescence density (MFI) of the cells.
  • the data in Tables 14 and 15 are EC50 values calculated from MFI.
  • Tables 14 and 15 show that humanized TPBG antibody variants specifically bind to cells expressing human TPBG protein on the surface and cells expressing cynomolgus TPBG protein, and cells expressing mouse TPBG protein and CHO- negative for TBPG expression. K1 has no combination.
  • the purified humanized TPBG antibody variant obtained in Example 1 was coupled to MC-MMAF. After the antibody was dialyzed against sodium borate buffer of pH 6.5-8.5, tris(2-carboxyethyl)phosphine (TCEP) was added, wherein the molar ratio of TCEP to purified TPBG antibody was 3, and the reaction was carried out at room temperature for 1 hour to obtain a reaction. Liquid A. The reaction solution A was desalted (purchased from GE) through a G25 column to remove excess TCEP to obtain a reaction liquid B.
  • TCEP tris(2-carboxyethyl)phosphine
  • MC-MMAF (purchased from Nanjing Lianning) was added to the reaction solution B, wherein the molar ratio of MC-MMAF to purified humanized TPBG antibody was 10, and the reaction was carried out for 4 hours at room temperature. Cysteine was added to neutralize excess MC-MMAF and desalted through a G25 column to remove excess small molecules. A purified humanized TPBG antibody drug conjugate was obtained (for coupling methods, see Doronina, 2006, Bioconjugate Chem. 17, 114-124). In vitro cytotoxic activity assays were performed after analyzing the cross-linking rate and purity parameters of the drug by HPLC-HIC and HPLC-SEC, respectively.
  • the drug cross-linking rate (DAR) of all humanized TPBG antibody conjugates ranged from 3.0 to 5.0. Wherein, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on one antibody molecule after antibody coupling.
  • Humanized TPBG antibody drug conjugates were serially diluted with complete medium, and 96-well cell culture plates were added to 100 ⁇ l of TPCG-positive non-small cell lung cancer cell line NCI-H1568 at 2000 cells/well (purchased from ATCC, article number #CRL-5876) After overnight incubation of the cell suspension, add 10 ⁇ l of each dilution of purified TPBG antibody drug conjugate to each well and continue to culture for 5 days, using CellTiter-Glo kit (purchased from Promega, The method of use refers to the product specification) to detect cell viability.
  • IC50 of Table 16 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • 11A shows the cytotoxic activity of the humanized antibody 12B12 variant and its antibody drug conjugate against the TPBG-positive tumor cell line NCI-H1568
  • FIG. 11B shows the humanized antibody 28D4 variant and its antibody drug conjugate. The cytotoxic activity of the TPBG-positive tumor cell line NCI-H1568 was detected.
  • the purified humanized antibodies 12B12-12 and 28D4-3 obtained in Example 1 were coupled to MC-MMAF or MC-VC-PAB-MMAE, respectively.
  • TCEP tris(2-carboxyethyl)phosphine
  • MC-MMAF or MC-VC-PAB-MMAE purchased from Nanjing Lianning
  • the reaction solution B wherein the molar ratio of the MC-MMAF or MC-VC-PAB-MMAE to the purified humanized TPBG antibody was 10, react at room temperature for 4 hours. Additional cysteine was added to neutralize excess MC-MMAF or MC-VC-PAB-MMAE and desalted through a G25 column to remove excess small molecules.
  • a purified humanized TPBG antibody drug conjugate was obtained (for coupling methods, see Doronina, 2006, Bioconjugate Chem. 17, 114-124).
  • the drug cross-linking rate (DAR) of all humanized TPBG antibody conjugates ranged from 3.0 to 5.0.
  • DAR drug antibody ratio refers to the average number of small molecule drugs carried on one antibody molecule after antibody coupling.
  • the purified humanized antibodies 12B12-12 and 28D4-3 obtained in Example 1 were respectively mixed with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC). ) Coupling. After the antibody was dialyzed against a phosphate buffer solution having a pH of 6.5 to 7.4, SMCC was added in the presence of a volume ratio of 30% DMA, wherein the molar ratio of SMCC to the purified TPBG chimeric antibody was 8, and the reaction was carried out at room temperature for 1 hour to obtain a reaction. Liquid A. The reaction solution A was desalted (purchased from GE) through a G25 column to remove excess small molecules to obtain a reaction liquid B.
  • SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester
  • reaction solution B A final volume of 10% DMA (N,N-dimethylacetamide) was added to the reaction solution B, followed by the addition of DM1 (chemical name N2'-deacetyl-N2'-3-mercapto-1 oxopropyl) - maytansine), wherein the molar ratio of DM1 to purified TPBG antibody is 9, and the reaction is carried out for 3.5 hours at room temperature to obtain a reaction liquid C.
  • the reaction solution C was desalted (purchased from GE) through a G25 column to remove excess small molecules to obtain a purified humanized TPBG antibody drug conjugate (see US Pat. No. 5,208,020 for the coupling method).
  • the in vitro cytotoxic activity assay and in vivo pharmacodynamic analysis were performed by analyzing the cross-linking rate of the drug by LC-MS, analyzing the purity of the antibody drug conjugate by SEC, and the like.
  • the drug cross-linking rate (DAR) of all humanized TPBG antibody conjugates ranged from 3.0 to 5.0.
  • DAR drug antibody ratio
  • DAR drug antibody ratio
  • the purified humanized TPBG antibody drug conjugate obtained in Example 6 was separately diluted with complete medium, and 100 ⁇ l of TPCG-positive non-small cell lung cancer cell line NCI was added to the 96-well cell culture plate at 2000 cells/well. -H1568 (purchased from ATCC, Cat. #CRL-5876) After cell culture overnight, add 10 ⁇ l of each dilution of purified TPBG antibody drug conjugate to each well, continue to culture for 5 days, and use CellTiter- Cell viability was measured using the Glo kit (purchased from Promega, using the method reference).
  • the non-small cell lung cancer cell line NCI-H1975 (purchased from ATCC, item #CRL-5908) and the breast cancer cell line MDA-MB-468 (purchased from ATCC, item #HTB-132) with weak expression of TPBG were used for cell killing.
  • Activity detection the method is the same as above.
  • IC50 of Table 18 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • 12A shows the cytotoxic activity of the humanized TBPG antibody drug conjugate against the TPBG-positive tumor cell line NCI-H1568
  • FIG. 12B shows the humanized TPBG antibody drug conjugate against the TPBG-positive tumor cell line NCI- The cell killing activity of H1975 was detected.
  • Fig. 12C shows the cytotoxic activity of the humanized TPBG antibody drug conjugate against the TPBG-positive tumor cell line MDA-MB-468.
  • Purified humanized TPBG antibody drug conjugates coupled with different small molecule toxins have different degrees of killing effect on TPBG-positive cells, and TPBG chimeric antibody drug conjugates coupled with MC-MMAF have higher The low IC50 indicates that the cell killing ability is the strongest.
  • NCI-H1975 non-small cell lung cancer cell line, ATCC, CRL-5908
  • mice 200 ⁇ l of NCI-H1975 (non-small cell lung cancer cell line, ATCC, CRL-5908) (2 ⁇ 10 6 ) was inoculated subcutaneously with the right rib of Balb/c nude mice, and after 7-10 days, the tumor grew to 200 mm 3 . The body weight was removed, the tumor was too large and too small, and the mice were randomly divided into several groups according to the tumor volume, with 7 rats in each group. Grouped as Table 19, for the therapeutic grouping of humanized 28D4-3-MMAF antibody drug conjugates prepared in different doses of Example 6, and the same dose of humanized 12B12-12-MMAF and humanized 28D4- 3-MMAF antibody drug conjugate treatment group.
  • the same dose treatment component group of humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody drug conjugates coupled to different linker-toxins D0 began to inject the antibody into the tail vein, once every 4 days, a total of 4 times, the tumor volume was measured twice a week, the rats were weighed, and the data were recorded.
  • FIG. 13A Volumetric changes in tumors after treatment with different doses of humanized 28D4-3-MMAF antibody drug conjugate
  • Figure 13B Different doses of humanized 28D4-3-MMAF antibody drug conjugate treatment After the mouse weight change graph.
  • the results showed that the tumor volume of all treated mice was significantly reduced compared with untreated mice, and the tumor volume decreased more significantly with increasing dose, and the tumor volume of the 10 mg/kg dose of the treated group was after administration.
  • the tumor gradually subsided in the 35 days, and the tumor began to slowly resume growth in subsequent continuous observation.
  • MC-MMAF antibody drug conjugates conjugated to humanized antibodies 12B12-12 and 28D4-3 were comparable in efficacy at the 10 mg/kg dose of the treatment group.
  • Figure 14A Volumetric changes in tumors after treatment with the same dose of humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody drug conjugates coupled to different linker-toxins
  • Figure 14B Figures of body weight change after treatment of humanized 28D4-3-MMAF, 28D4-3-MMAE and 28D4-3-DM1 antibody drug conjugates coupled to different linker-toxins at the same dose.
  • the results showed that the tumor volume of the humanized 28D4-3 antibody drug conjugate treatment group coupled to MC-MMAF and MC-VC-PAB-MMAE was significantly higher than that of untreated mice at the same dose.
  • the tumors of the humanized 28D4-3-MMAF treatment group gradually regained growth after the regression, and the tumors of the humanized 28D4-3-MMAE treatment group basically subsided.
  • NCI-H1568 non-small cell lung cancer cell line, ATCC, CRL-5876
  • V 1/2 ⁇ a ⁇ b2; where a and b represent length and width, respectively.
  • Figure 15A Volumetric changes in tumors after treatment with different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins
  • Figure 15B Changes in body weight of mice after treatment with different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins.
  • the results showed that the tumor volume of all treated mice was significantly reduced compared to untreated mice.
  • the high-dose 10 mg/kg treatment group had a stronger inhibitory effect on NCI-H1568 tumors than the low-dose 2 mg/kg treatment group.
  • MDA-MB-468 (purchased from ATCC, item #HTB-132) cells were suspended in L-15 basal medium and 50% Matrigel (1 ⁇ 10 7 ) 200 ⁇ l was added to inoculate subcutaneously with CB17SCID mice. After 7-10 days of tumor growth to 200 mm 3 , the body weight was removed, the tumor was too large and too small, and the mice were randomly divided into several groups according to the tumor volume, 6 in each group, grouped as shown in Table 22, which was coupled to different linkers. - Different dose treatment groups of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates of toxin.
  • V 1/2 ⁇ a ⁇ b2; where a and b represent length and width, respectively.
  • Figure 16A Volumetric changes in tumors after treatment with different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins
  • Figure 16B Changes in body weight of mice after treatment with different doses of humanized 12B12-12-MMAF, 12B12-12-MMAE and 12B12-12-DM1 antibody drug conjugates coupled to different linker-toxins.
  • the results showed a significant reduction in tumor volume in all treatment groups compared to untreated mice.
  • the humanized 12B12-12 antibody drug conjugates conjugated to MC-MMAF and MC-VC-PAB-MMAE had tumor regression, but were coupled.
  • the humanized 12B12-12 antibody drug conjugate in the SMCC-DM1 treatment group slowly grew after the tumor became smaller. It is shown that antibody drug conjugates coupled to different linker-toxins have different inhibitory capacities for NCI-H1568 tumor growth.
  • the total humanized anti-TPBG antibody concentration in the serum was detected by ELISA, captured using an anti-human Fc antibody and detected using a horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody.
  • the concentration of the antibody drug conjugate carrying at least one cytotoxic drug in the serum is detected by ELISA, captured using an anti-MMAE antibody, and detected using a horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody,
  • anti-MMAE antibody (trade name anti-MMAF-mIgG1, purchased from Shanghai Ruizhi Chemical Research Co., Ltd.) was prepared by using hybridoma technology.
  • Anti-human Fc antibody and anti-MMAE antibody were separately diluted with PBS to a certain concentration (2-5 ug/ml), coated on a 96-well ELISA plate, and coated at 4 ° C overnight.
  • the liquid in the well was discarded and dried, and the blocking solution (PBS, 1% BSA, 0.05% Tween-20) was added for 1 hour at room temperature, and then washed 3 times with PBST (PBS, 0.05% Tween-20) for use. Dilute the standard to a certain concentration with PBST, and prepare the serum sample as appropriate. Add 200 ul/well of the diluted serum sample or standard to the coated 96-well plate and incubate for 1-3 hours at room temperature.
  • HRP horseradish peroxidase
  • CL represents the total clearance rate, the higher the CL value represents the faster metabolism or clearance
  • Vss represents the apparent volume of distribution in steady state, the higher the Vss value, the wider the tissue distribution
  • V1 represents the volume of distribution of the central chamber, The value should be close to the serum volume per kilogram of the experimental animal
  • Alpha t1/2 represents the distribution phase half-life, which is related to the distribution rate
  • Beta t1/2 represents the elimination phase half-life, which is related to the elimination rate
  • AUC represents the time of administration.
  • the area under concentration-time curve represents the exposure of the test substance in serum, and the exposure amount and the efficacy are directly related to the general time.
  • the coupled antibody and the total antibody have similar pharmacokinetic characteristics, such as longer half-life and nonlinear distribution and elimination.
  • the difference is that the conjugated antibody shows a higher clearance rate (CL) and a lower elimination half-life (Beta t 1/2 ) and exposure (AUC) compared to the total antibody, which is in contrast to classical ADC drug metabolism.
  • the analysis may be because the ADC drug is a mixture of various molecular components, the heterogeneity is high, some of the conjugates degrade or induce an immune reaction during the blood circulation, so that the antibody against the ADC drug of the body product causes the ADC drug to be cleared.
  • the ADC drug is a mixture of various molecular components, the heterogeneity is high, some of the conjugates degrade or induce an immune reaction during the blood circulation, so that the antibody against the ADC drug of the body product causes the ADC drug to be cleared.
  • FIG. 18A-B The expression level of TPBG in the PDX model is shown in Figures 18A-B, wherein Figure 18A is the staining of the humanized anti-TPBG antibody 12B12-3 on the PDX model tumor tissue sections, and Figure 18B is the negative control antibody hIgG in the PDX model tumor tissue. Stain on the slice. Based on the results shown in Figure 18, the PDX model can be used for subsequent pharmacodynamic experiments.
  • the humanized 28D4-3-MMAE antibody drug conjugate significantly enhanced the anti-tumor activity of the anti-PD-1 antibody against the CT26-hTPBG mouse model.
  • the complete response rate of the combination of the two drugs reached 37.5%.
  • the combination of humanized 28D4-3-MMAE antibody drug conjugate and anti-PD-1 antibody can be significantly extended compared to humanized 28D4-3-MMAE antibody drug conjugate or anti-PD-1 alone.
  • the survival time of the mice, and the mice with complete tumor remission after combined treatment will form immunological memory.

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Abstract

本发明提供了一种人源化抗TPBG抗体及其制备方法、其偶联物和应用。其包含：(a)包含人抗体构架区残基的构架区；和(b)SEQ ID NO.4或8所示的轻链可变区的一个或多个CDRs，或SEQ ID NO.2或6所示的重链可变区的一个或多个CDRs。

Description

一种人源化抗TPBG抗体及其制备方法、其偶联物和应用
本申请要求申请日为2017年04月05日的中国专利申请CN201710218524.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及抗体领域，具体涉及一种人源化抗TPBG抗体及其制备方法、其偶联物和应用。
背景技术
在对胚胎干细胞滋养层和癌细胞进行比较时发现一种细胞表面分子，滋养层特异性糖蛋白(TPBG，又称为5T4)，是胚胎滋养层表达的特异性蛋白。人类TPBG蛋白的分子量约为72kDa，包含420个氨基酸，其N端低聚糖结构具有多样性，能够防止蛋白质水解，并且在细胞膜信号传导过程中与其他分子有交互作用。TPBG蛋白共包含7个重复的亮氨酸结构域(LRR)，可参与蛋白质与蛋白质的相互作用。
滋养层是胎盘和胎儿之间的一层特殊的胚胎干细胞，TPBG广泛表达于胚胎发育期的各种滋养层细胞。对于正常成人组织中，TPBG只在有限的几种上皮细胞中有表达。但是，TPBG在很多癌细胞中表达，如子宫癌、结肠癌、胃癌、卵巢癌、口腔癌、前列腺癌、肺癌或肾癌组织中都检测到TPBG的表达，在结肠癌、胃癌或卵巢癌中有证据表明TPBG的表达量与癌症的低治愈率相关。而在非小细胞肺癌、肾癌或胰腺癌的组织中，TPBG的表达高达95％以上。
很多研究显示，过表达该TPBG能够促进细胞迁移，同时还能够避免免疫监控。在小鼠纤维原细胞中过表达TPBG能够诱导细胞呈现纺锤体形态，降低细胞粘附。在小鼠正常上皮细胞中，同样发现TPBG能够抑制上皮细胞钙粘蛋白(E-cadherin)，促进细胞迁移。而TPBG在细胞内的部分有抑制细胞骨架形成的功能。同时，TPBG与上皮细胞间质转移(EMT)相关，TPBG作为胚胎干细胞发育的早期标志物，增加细胞间质蛋白酶的活性，干扰肌动蛋白细胞骨架的排列，下调E-cadherin的表达。还有研究发现，TPBG与CXCR4共定位于细胞膜表面，能够诱导其配体CXCL12趋化因子的结合，促进炎症和肿瘤的扩散。在TPBG阴性细胞中，CXCL12结合另一个受体CXCR7，抑制趋化反应，有利于细胞的生长和存活。Wnt/b-catenin信号通路对发育和细胞再生起到非常重要的作用，TPBG通过抑制LRP6与Wnt受体的内吞，抑制Wnt信号通路，从而抑制细胞粘附 和细胞骨架的形成，促进肿瘤的迁移和扩散。还有证据表明TPBG在乳腺癌和胃癌细胞中参与非经典的Wnt通路，同样促进癌细胞的迁移和浸润。
单克隆抗体由于具有靶向性、特异性、专一性、高亲和力等优势，正发展成为新型诊断和治疗药物。然而，早期的临床试验揭示，在人体中使用非人源单克隆抗体，常常因为人抗小鼠抗体(HAMA)和人抗大鼠抗体(HARA)应答，导致严重的免疫反应，抗体被快速清除。随后开发出免疫原性较小的抗体，包括嵌合抗体、人源化抗体和全人源抗体。根据人源化程度不同,治疗性单克隆抗体药物可分为4种：鼠源性抗体(无人源氨基酸序列)、嵌合抗体(60％～70％人源化氨基酸序列)、CDR移植抗体(90％～95％人源化氨基酸序列)以及全人源抗体(100％人源氨基酸序列)。随着人源化程度增加，非鼠源单克隆抗体可以减轻人体治疗过程中人抗鼠抗体反应(HAMA和HARA反应)，逐步消除异源性抗体的免疫原性问题，在保持对抗原高亲和力的同时，改善了抗体的药代动力学，临床上已大量使用这些抗体药物进行靶向治疗。
抗体药物偶联药物，是由抗体与高效小分子药物通过连接物偶联形成的抗体药物偶联物，能够使高毒性小分子药物特异识别癌细胞上的靶点蛋白，从而特异性杀死癌细胞。近一百年间，基于抗体的免疫疗法与基于化学药物的化学疗法，一直是临床上癌症治疗的两大治疗策略。抗体以肿瘤细胞过度表达的抗原为靶点，多种治疗性单抗已经在临床上取得了巨大成功。在临床实践中，治疗性抗体虽然具有很好的靶向性，但是杀伤作用存在局限性。小分子化学药物虽然具备对癌细胞的高效杀伤作用，但是对非癌细胞也造成同样的伤害。因此临床上抗体药物以及小分子药物各自的局限性，对药物研发提出了新的要求。新一代抗体药物偶联物，利用抗体对靶细胞的特异结合能力，输送高细胞毒的化学药物，实现对癌细胞的靶向高效杀伤。随着新型化学连接技术的出现，抗体药物偶联药在八十年代末开始进入临床研究，目前已经有2个ADC药物经FDA批准上市。
ADC药物的开发涉及：药物靶点的筛选、重组抗体的制备、连接物技术开发以及高细胞毒性化合物的筛选优化等几个方面。TPBG作为癌细胞特异表达的蛋白，是ADC药物的候选靶点。
发明内容
本发明所要解决的技术问题是为了克服目前缺少TPBG抗体的不足，提供一种亲和力高、特异性强的人源化抗TPBG抗体及其制备方法和应用，所述的人源化抗TPBG抗体与TPBG蛋白具有高度亲和力，并且随着人源化程度增加，可减轻人体治疗过程中人抗鼠抗体反应(HAMA反应)，逐步消除异源性抗体的免疫原性问题，在保持对抗原高亲 和力的同时，改善了抗体的药代动力学。本发明还提供一种药物活性成分的偶联物，其包括所述的人源化抗TPBG抗体和与其偶联的、具有抗肿瘤的功能的小分子化合物，所述的偶联物能够进入细胞，对TPBG阳性的细胞进行细胞毒杀伤作用，能够运用于治疗肿瘤等药物的制备中。
本发明人以人源TPBG蛋白或者过表达人源TPBG蛋白的重组细胞株作为免疫原，采用传统的杂交瘤制备技术(Kohler and Milstein，Nature，1975，256:495)，通过一系列的调整和改进，获得TPBG抗体的先导抗体。再通过对先导抗体的初步生产、纯化和鉴定，获得具备与人源TPBG蛋白等蛋白具有高度亲和力的TPBG抗体。通过分子生物学方法测序获知所得的小鼠源TPBG抗体的重链可变区和TPBG抗体的轻链可变区的氨基酸序列。利用经过详细分析的小鼠抗体，取其与抗原结合的CDR区与人的抗体框架嫁接，经亲和力重塑，形成CDR移植抗体，维持其特异性和大部分的亲和力，同时几乎完全去除免疫原性和毒副作用。该人源化TPBG抗体与小分子化合物如MMAF偶联得偶联物，所述偶联物能够进入细胞，对TPBG阳性细胞有优异的细胞毒杀伤作用。
本发明提供一种人源化抗TPBG抗体，其包含：
(a)包含人抗体构架区残基的构架区；和
(b)如序列表SEQ ID NO.4或8所示的轻链可变区的一个或多个CDRs，或SEQ ID NO.2或6所示的重链可变区的一个或多个CDRs。
所述的人抗体构架区包括重链构架区和轻链构架区，人抗体轻链构架区残基可以包含种系O2、O12、DPK1(O18)、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、DPK12(A2)、DPK13、DPK15、DPK16、DPKI8、DPK19、DPK20、DPK21、DPK22、DPK23、DPK24(B3)、DPK25、DPK26和DPK28，特别是这些种系的FR1、FR2、FR3；以及由Jk片段Jk1、Jk2、Jk3、Jk4和JK5，特别是这些种系的FR4编码的序列。人抗体重链构架区残基可以包含种系DP4、DP7、DP8、DP9、DP10、DP31、DP33、DP35(VH3-11)、DP45、DP46、DP47、DP48、DP49(VH3-30)、DP50、DP51(VH3-48)、DP53、DP54、DP65、DP66、DP67、DP68和DP69，特别是这些种系的FR1、FR2、FR3；以及JH片段JH1、JH2、JH3、JH4、JH4b、JH5和JH6，特别是这些种系的FR4编码的序列，或重链构架区的共有序列。此类构架区序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在"VBase"人种系序列数据库(www.mrcco8.com.ac.uk/vbase)获得，以及在Kabat，E.A等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。
在本发明一个优选的实施方案中，所述的人源化抗TPBG抗体中，CDR是小鼠的CDR，选自SEQ ID NO.4或8所示的轻链可变区的一个或多个CDRs，或SEQ ID NO.2或6所示的重链可变区的一个或多个CDRs。较佳地，所述鼠源抗体重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.2中的第31位至第35位所示；更佳地，编码所述鼠源抗体重链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.1中的第91位至第105位所示；
所述鼠源抗体重链可变区的CDR2的氨基酸序列如序列表SEQ ID No.2中的第50位至第66位所示；更佳地，编码所述鼠源抗体重链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.1中的第148位至第198位所示；
所述鼠源抗体重链可变区的CDR3的氨基酸序列如序列表SEQ ID No.2中的第99位至第109位所示；更佳地，编码所述鼠源抗体重链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.1中的第295位至第327位所示；
和所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.4中的第24位至第38位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.3中的第70位至第114位所示；
所述鼠源抗体轻链可变区的CDR2的氨基酸序列如序列表SEQ ID No.4中的第54位至第60位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.3中的第160位至第180位所示；
所述鼠源抗体轻链可变区的CDR3的氨基酸序列如序列表SEQ ID No.4中的第93位至第101位所示；更佳地，编码所述鼠源抗体重链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.3中的第277位至第303位所示；
或者，所述鼠源抗体重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.6中的第31位至第35位所示；更佳地，编码所述鼠源抗体重链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.5中的第91位至第105位所示；
所述鼠源抗体重链可变区的CDR2的氨基酸序列如序列表SEQ ID No.6中的第50位至第66位所示；更佳地，编码所述鼠源抗体重链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.5中的第148位至第198位所示；
所述鼠源抗体重链可变区的CDR3的氨基酸序列如序列表SEQ ID No.6中的第99位至第109位所示；更佳地，编码所述鼠源抗体重链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.5中的第295位至第327位所示；
和/或，所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.8中的 第24位至第34位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.7中的第70位至第102位所示；
所述鼠源抗体轻链可变区的CDR2的氨基酸序列如序列表SEQ ID No.8中的第50位至第56位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.7中的第148位至第168位所示；
所述鼠源抗体轻链可变区的CDR3的氨基酸序列如序列表SEQ ID No.8中的第89位至第97位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.7中的第265位至第291位所示。
人的抗体可变区框架经过选择，其中所述抗体轻链可变区上的轻链FR序列，来源于人种系轻链包含1)A2、B3或O18的FR1、FR2、FR3区，和2)JK2或JK5的FR4区的人抗体轻链构架区的组合；所述抗体的重链可变区上的重链FR序列，来源于人种系重链序列包含1)VH3-48、VH3-30或VH3-11的FR1、FR2、FR3区，和2)JH6的FR4区的人抗体重链构架区的组合。一般而言，人受体构架区的选择应类似于供体抗体的构架区，或最类似于可变区亚家族的共有序列进行选择。移植后，可以在供体和/或受体序列中进行序列突变，以优化抗原结合、功能性、密码子使用、表达水平等，包括将非人残基引入构架区内。
较佳地，所述的人源化抗TPBG抗体包含至少一个重链可变区和/或至少一个轻链可变区，其中，所述重链可变区的氨基酸序列如序列表中SEQ ID NO.2、SEQ ID NO.6、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.34、SEQ ID NO.36、SEQ ID NO.38或SEQ ID NO.40所示；所述的轻链可变区序列如序列表中SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.26、SEQ ID NO.28、SEQ ID NO.30、SEQ ID NO.32、SEQ ID NO.42、SEQ ID NO.44或SEQ ID NO.46所示；更佳地，编码所述重链可变区的氨基酸序列的核苷酸序列分别如序列表中SEQ ID No.1、SEQ ID No.5、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.33、SEQ ID No.35、SEQ ID No.37或SEQ ID No.39所示；编码所述的轻链可变区的氨基酸序列的核苷酸序列分别如序列表中SEQ ID No.3、SEQ ID No.7、SEQ ID No.25、SEQ ID No.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.41、SEQ ID No.43或SEQ ID No.45所示；
或者，所述的重链可变区的氨基酸序列如序列表中SEQ ID NO.2、SEQ ID NO.6、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.34、SEQ ID NO.36、SEQ ID NO.38或SEQ ID NO.40所示的氨基酸序列至少有80％ 序列同源的氨基酸序列所示；所述的轻链可变区序列如序列表中SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.26、SEQ ID NO.28、SEQ ID NO.30、SEQ ID NO.32、SEQ ID NO.42、SEQ ID NO.44或SEQ ID NO.46所示的氨基酸序列至少有80％序列同源的氨基酸序列所示；较佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.1、SEQ ID No.5、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.33、SEQ ID No.35、SEQ ID No.37或SEQ ID No.39所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.3、SEQ ID No.7、SEQ ID No.25、SEQ ID No.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.41、SEQ ID No.43或SEQ ID No.45所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列。
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.25所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.25所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.25所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.16所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.15所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻 链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.22所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.21所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.29所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.29所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.29所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.16所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.15所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.22所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.21所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.33所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.36所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.35所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.33所示的序列，且所述轻链可变区的核苷酸序列如序列 表SEQ ID No.43所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.36所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.35所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.33所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.36所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.35所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.37所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列；
或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；更佳地，所述重链可变区的核苷酸序列如序列表SEQ ID No.39所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列。
综上所述，上述氨基酸序列的编号如表1-1所示。
表1-1.人源化TPBG抗体蛋白序列编号
抗体编号 重链可变区SEQ ID NO. 轻链可变区SEQ ID NO.
c12B12 2 4
h12B12-1 18 26
h12B12-2 20 26
h12B12-3 24 26
h12B12-4 16 28
h12B12-5 18 28
h12B12-6 20 28
h12B12-7 22 28
h12B12-8 24 28
h12B12-9 18 30
h12B12-10 20 30
h12B12-11 24 30
h12B12-12 16 32
h12B12-13 18 32
h12B12-14 20 32
h12B12-15 22 32
h12B12-16 24 32
c28D4 6 8
h28D4-1 34 42
h28D4-2 36 42
h28D4-3 38 42
h28D4-4 40 42
h28D4-5 34 44
h28D4-6 36 44
h28D4-7 38 44
h28D4-8 40 44
h28D4-9 34 46
h28D4-10 36 46
h28D4-11 38 46
h28D4-12 40 46
其中，表1-1中的数字即为序列表中序列号，如h12B12-1的重链蛋白可变区的氨基酸序列为SEQ ID No.18，而h12B12-1的轻链蛋白可变区的氨基酸序列为SEQ ID No.26。
所述的人源化抗TPBG抗体，较佳地为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(Single chain antibody fragment，scFv)、单域抗体(Single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。本发明还包括超人源化抗体、双抗体(diabody)等。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。
较佳地，所述的人源化抗TPBG抗体还包括人源抗体重链恒定区和/或人源抗体轻链恒定区。所述的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成人源化抗体全长蛋白。其中所述的人源化抗体重链恒定区为本领域常规，可以包含衍生自人恒定区的恒定区，其进一步包含人源IgG1，IgG2，IgG3，IgG4或其变体的重链恒定区；所述的人源化抗体轻链恒定区为本领域常规，可以包含衍生自人恒定区的恒定区，其进一步包含人源κ、λ链或其变体的轻链恒定区。
所述的单链抗体为本领域常规的单链抗体，其包括所述的重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述人源化抗TPBG抗体的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该人源化抗TPBG抗体的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该人源化抗TPBG抗体的表达转化体中分离获得优选如下方法：将编码所述人源化抗TPBG抗体的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述人源化抗TPBG抗体。本发明的代表性人源化抗TPBG抗体的制 备在实施例1中得到描述。
人源化抗体广义上也是一类嵌合抗体，其中负责抗原结合的可变区残基，包括衍生自非人物种的互补决定区、缩短的互补决定区、或参与抗原结合的任何其它残基；而其余可变区残基如，构架区的残基和恒定区至少部分衍生自人抗体序列。人源化抗体的构架区残基和恒定区残基的一个子集可以衍生自非人来源。人源化抗体的可变区也描述为人源化的轻链可变区和/或重链可变区。非人物种一般是用于由抗原免疫接种的物种，例如小鼠、大鼠、兔、非人灵长类、或其他非人哺乳动物物种。人源化抗体一般比传统嵌合抗体的免疫原性小，并且在给人施用后显示出改善的稳定性。
互补决定区(CDRs)是参与抗原结合的抗体可变区的残基。用于标识CDRs的几种编号系统是常用的，包括例如Kabat定义、Chothia定义和AbM定义。概括地说，Kabat定义是基于序列的变异性、Chothia定义是基于结构环区的位置，AbM定义是Kabat和Chothia方法之间的折中。根据Kabat、Chothia或AbM算法，轻链可变区有三个CDR区，其CDR1位于由第24-34位氨基酸(CDR1-L)、CDR2位于第50-56位氨基酸(CDR2-L)、CDR3位于第89-97位氨基酸(CDR3-L)。由于可变区的长度变化，在不同的中枢或不同的亚群中，第27位可有1-6个氨基酸，第95位也可有1-6个氨基酸，它们在原编号的基础上加上英文字母进行编位，如：27A、27B、95A、95B等。根据Kabat定义，重链可变区的CDRs由在第31和35B位(CDR1-H)、第50和65位(CDR2-H)、以及第95和102位(CDR3-H)的残基界定(根据Kabat编号)。根据Chothia定义，重链可变区的CDRs由第26和32位(CDR1-H)、第52和56位(CDR2-H)、以及第95和102位(CDR3-H)上的残基界定(根据Chothia编号)。根据AbM定义，重链可变区的CDRs由26和35B位(CDR1-H)、第50和58位(CDR2-H)、以及第95和102位(CDR3-H)的残基界定(根据Kabat编号)。与轻链可变区类似，在第35位、52位、82位及100位也可有多个氨基酸，以A、B、C……等编号。参见Martin等人(1989)Proc.Nat l.Acad.Sci.USA 86:9268-9272；Martin等人(1991)Methods Enzymol.203:121-153；Pedersen等人(1992)lmmunomethods 1:126；Protein Structure Prediction,Oxford University Press,Oxford,第141-172页。
特异性决定区(SDRs)是与抗原直接相互作用的CDRs内的残基。SDRs对应高变残基。参见Padlan等人(1995)FASEB J.9:133-139)。
构架残基是轻链可变区或重链可变区的一部分，是除高变残基(高变残基多指互补决定区或CDR)或CDR残基外的抗体可变区残基，其用作该可变结构域的抗原结合环(CDR)的支架。构架残基可以衍生自天然存在的人的抗体，例如基本上类似于鼠源抗 TPBG抗体12B12或28D4的构架区的人抗体的构架区。还可以使用代表个体序列之间的共有序列的人工构架区序列。当选择用于人源化的构架区时，在人类中广泛呈现的序列可能优于较不常见的序列。可以制备人构架受体序列的另外突变，以恢复被认为涉及抗原接触的鼠类残基和/或涉及抗原结合位点的结构完整性的残基，或改善抗体表达。肽结构预测可以用于分析人源化重链可变区和轻链可变区序列，以鉴定和避免由人源化设计引入的翻译后蛋白质修饰位点。
所述的人源化抗体可以使用各种方法中的任何一种进行制备，包括互补决定区(CDRs)的镶饰、移植、缩短的CDRs的移植、特异性决定区(SDRs)的移植、和Frankenstein装配。
所述的人源化抗体还包括超人源化抗体，它是一种人源化抗体的制备方法，此方法不依赖于将人构架序列作为分析点，而是依赖于比较非人抗体的规范CDR结构类型和人抗体的CDR结构类型，尤其是人胚系序列所编码的人抗体，从中识别出可以得到适宜的人构架序列的候选的人抗体序列。例如，人残基可以置换CDRs中的非人残基，其中一个或多个变化已引入CDRs中。镶饰(veneering)的一个前提是鼠源抗体可变区的免疫原性起源于它的表面残基，且残基的运动性和溶剂的可及性是其成为抗原决定簇的基本条件。根据对现有的抗体晶体结构数据的分析结果统计，在序列配对位置上，人和鼠的抗体可变区残基的相对溶剂可及性分布的保真度达98％，这说明在异种间诱导免疫反应的残基是由其余的种特异性溶剂可及表面残基引起的。因此将鼠特异性表面残基换成人源性的，就可以模拟人源抗体的表面轮廓，逃避人体免疫系统的识别，达到人源化的目的。简单来说，镶饰基于通过用人氨基酸序列重建抗体的溶剂可及的表面来减少在啮齿类动物或其他非人抗体中的潜在免疫原性的氨基酸序列的概念。参见Padlan(1991)Mol.Immunol.28:489-980.通过鉴定非人抗体中暴露在表面的溶剂可及性残基的外部构架区残基(所述残基不同于人抗体的构架区中相同位置上的那些残基)，并用占据人抗体中的那些相同位置的氨基酸替换所鉴定的残基，以进行镶饰，即镶饰的抗体，其表面残基主要是人源序列，而包裹在内部的残基主要是最初的鼠源序列。CDRs的移植通过用供体抗体(如，非人抗体)的CDRs替换受体抗体(例如，包含所需构架残基的人抗体或其他抗体)的一个或多个CDRs来进行。受体抗体可以基于在候选受体抗体和供体抗体之间的构架残基的相似性进行选择。例如，根据Frankenstein方法，鉴定与相关的非人抗体的各构架区具有实质上的序列同源性的人构架区，并且将非人抗体的CDRs移植到这些不同的人构架区的复合物上。可以结合上述方法，以产生任何所需序列的抗TPBG抗体。
本发明还提供一种核酸，其编码上述的人源化抗TPBG抗体，包括编码所述重链可 变区的核酸，和/或编码所述轻链链可变区的核酸。
较佳地，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID NO.2、SEQ ID NO.6、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.34、SEQ ID NO.36、SEQ ID NO.38或SEQ ID NO.40所示；更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.1、SEQ ID No.5、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.33、SEQ ID No.35、SEQ ID No.37或SEQ ID No.39；编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.3、SEQ ID No.7、SEQ ID No.25、SEQ ID No.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.41、SEQ ID No.43或SEQ ID No.45所示。
进一步更佳地，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.25所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.25所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.25所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.16所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.15所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区 的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.22所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.21所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.29所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.29所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.29所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.16所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.15所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所 述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.22所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.21所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.33所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.35所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.37所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.39所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.33所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.35所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.37所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.39所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.33所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.35所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.37所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示；
或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.39所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示。
综上所述，上述核苷酸序列的编号如表1-2所示。
表1-2.TPBG抗体基因序列编号
Figure PCTCN2018081853-appb-000001
Figure PCTCN2018081853-appb-000002
Figure PCTCN2018081853-appb-000003
其中，表1-2中的数字即为序列表中序列号，如h12B12-1的重链蛋白可变区的核苷酸序列为SEQ ID No.17，而h12B12-1的轻链蛋白可变区的核苷酸序列为SEQ ID No.25。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述人源化抗TPBG抗体的核酸分子，或者通过人工全序列合成的方法得到编码上述人源化抗TPBG抗体的核酸分子。
本领域技术人员知晓，编码上述人源化抗TPBG抗体的核酸可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该人源化抗TPBG抗体的核酸的一个或多个核苷酸在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明进一步提供了包含上述重组表达载体的细胞或细胞系。较佳的，所述的细胞为哺乳动物或人细胞，更佳的为CHO细胞、HEK-293细胞、HeLa细胞、CV-l细胞或COS细胞。在将异源构建体转化到所述的细胞内后用于产生稳定细胞系的方法是本领域已知的。所述的非哺乳动物宿主细胞较佳的为昆虫细胞(Potter等人(1993)Int.Rev.Immunol.10(2-3):103-112)。所述的抗体还可以在转基因动物(Houdebine(2002)Curr.Opin.BiotechnoJ.13(6):625-629)或转基因植物(Schillberg等人(2003)Cell Mol.Life Sci.60(3):433-45)中进行生产。
本发明还提供一种人源化抗TPBG抗体的制备方法，其包括如下步骤：培养上述重 组表达转化体、或者细胞、或者细胞系，从培养物中获得人源化抗TPBG抗体。
本发明还提供一种免疫偶联物，其包括共价附着至细胞毒剂的上述人源化抗TPBG抗体。
较佳地，所述的免疫偶联物中，上述的1当量的蛋白质通过x当量接头与y当量的细胞毒剂相连，具有式1的结构，
Ab-(L) x-(D) y
式1
其中，Ab为上述的人源化抗TPBG抗体；L为接头；D为细胞毒剂；所述x为本领域常规的交联度，x为自然数，优选1-20的整数；y为0或自然数，优选0-20的整数；x和y各自独立地优选为1～2，或2～4，或3～5，或4～8，或8～20的整数；x和y的比例优选为1:1。
所述L是本领域常规的接头(或称交联剂或偶联剂)。所述L包含2个官能团，即与抗体反应的基团，和与药物反应的基团(例如，醛或酮)。
药物经由接头分子与上述的人源化抗TPBG抗体偶联。所述L进入细胞后释放，其包括但不限于如下的官能团，活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段。
较佳地，所述L主要含有式2的结构，其为L中离去基团离去后对应的剩余部分：
(CO-Alk 1-Sp 1-Ar-Sp 2-Alk 2-C(Z 1)＝Q-Sp)
式2
其中，Alk 1和Alk 2独立地是键或分支的或不分支的(C 1-C 10)亚烷基链；Sp 1是-S-、-O-、-CONH-、-NHCO-、-NR’-、-N(CH 2CH 2) 2N-、或-X-Ar’-Y-(CH 2) n-Z，其中X、Y和Z是独立的键、-NR’-、-S-或-O-，条件是当n＝0时，Y和Z中的至少一个必须是键，且Ar’是由(C 1-C 5)烷基、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-(CH 2) nCOOR’、S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1，2-、1，3-或1，4-亚苯基，n是0-5的整数，条件是当Alk 1是键时，Sp 1是键；R’是由-OH、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、(C 1-C 3)二烷基氨基、或(C 1-C 3)三烷基铵-A的一个或2个基团任选取代的分支的或不分支的(C 1-C 5)链，其中A是完成盐的药学上可接受的阴离子；Ar是由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR”或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1,2-、1,3-或1,4-亚苯基，其中n和R’如上述的定义，或Ar是1,2-、 1,3-、1,4-、1,5-、1,6-、1,7-、1,8-、2,3-、2,6-或2,7-亚萘基，其中亚萘基或吩噻嗪各任选地由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、或-S(CH 2) nCONHR’的1、2、3或4个基团取代，其中n和R’如上文定义，条件是当Ar是吩噻嗪时，Sp 1是仅与氮连接的键；
Sp 2是键、-S-或-O-，条件是当Alk 2是键时，Sp 2是键；
Z 1是H、(C 1-C 5)烷基、或由(C 1-C 5)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、或3个基团任选取代的苯基，其中n和R’如上文定义；
Sp是直链或支链二价或三价(C 1-C 18)基团，二价或三价芳基或杂芳基基团，二价或三价(C 3-C 18)环烷基或杂环烷基基团，二价或三价芳基或杂芳基-芳基(C 1-C 18)基团，二价或三价环烷基或杂环烷基-烷基(C 1-C 18)基团，或二价或三价(C 2-C 18)不饱和的烷基基团，其中杂芳基优选是呋喃基、噻吩基，N-甲基吡咯基、吡啶基、N-甲基咪唑基、噁唑基、嘧啶基。喹啉基、异喹啉基、N-甲基咔唑基、氨基豆素基、或吩嗪基、并且其中如果Sp是三价基团，那么Sp还可以由低级(C 1-C 5)二烷基氨基、低级(C 1-C 5)烷氧基、羟基、或低级(C 1-C 5)烷硫基任选取代；且，Q是＝NHNCO-、＝NHNCS-、＝NHNCONH-、＝NHNCSNH-或＝NHO-。
优选地，Alk 1是分支或不分支的(C 1-C 5)亚烷基链，Sp 1是键、-S-、-O-、-CONH-、-NHCO-或-NR’,其中R’如上文定义，条件是当Alk 1是键时，Sp 1是键；
Ar是由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1,2-、1,3-或1,4-亚苯基，其中n和R’如上文定义，或Ar是各自由C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、3或4个基团任选取代的1,2-、1,3-、1,4-、1,5-、1,6-、1,7-、1,8-、2,3-、2,6-或2,7-亚萘基。
Z 1是(C 1-C 5)烷基、或由(C 1-C 5)烷基、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、或3个基团任选取代的苯基；Alk 2和Sp 2均为键；且Sp和Q如仅在上文中所定义的。上述的键的含义为共价键。
所述L优选为马来酰亚胺基己酰(maleimidocaproyl，MC)、马来酰亚胺基己酰-L- 缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)。
所述D为本领域常规的细胞毒剂，较佳地选自细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶。
其中，所述细胞毒素为本领域常规的细胞毒素，一般指抑制或阻止细胞功能和/或导致细胞破坏的活性剂。较佳地选自抗生素、微管蛋白聚合的抑制剂、烷化剂、蛋白合成抑制剂、蛋白激酶抑制剂、磷酸酶抑制剂、拓扑异构酶抑制剂、蛋白激酶、磷酸酶、拓扑异构酶或细胞周期蛋白。更佳地选自多柔比星、柔红霉素、依达比星、阿柔比星、佐柔比星、米托蒽醌、表柔比星、卡柔比星、诺加霉素、美诺立尔、吡柔比星、戊柔比星、阿糖胞苷、吉西他滨、曲氟尿苷、安西他滨、依诺他滨、阿扎胞苷、去氧氟尿苷、喷司他丁、溴尿苷、卡培他滨、克拉屈滨、地西他滨、氟尿苷、氟达拉滨、谷氏菌素、嘌呤霉素、替加氟、噻唑羧胺核苷、阿霉素、顺铂、卡铂、环磷酰胺、达卡巴嗪、长春碱、长春新碱、博来霉素、氮芥、强的松、甲基苄肼、氨甲喋呤、氟尿嘧啶、依托泊苷、泰素、泰素类似物、铂类(如顺铂和卡铂)、丝裂霉素、噻替派、紫杉烷、道诺红菌素、放线菌素、安曲霉素、氮丝氨酸、它莫西芬、多拉司他汀、奥瑞他汀及其衍生物、哈米特林、埃斯波霉素或美登素类化合物，最佳地选自甲基奥瑞他汀E(MMAE)、甲基奥瑞他汀F(MMAF)或N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素(DM1)。
其中，所述化学治疗剂为本领域常规的化学治疗剂，较佳地选自烷化剂、烷基磺酸酯类化学治疗剂、氮丙啶类化学治疗剂、乙烯酰胺类和甲基密胺类化学治疗剂、氮芥、硝基脲类化学治疗剂、抗生素、抗代谢物、叶酸类化学治疗剂、嘌呤类似物、嘧啶类似物、雄激素、抗肾上腺素、叶酸补充剂、美登醇、多糖复合物、紫杉烷、铂类似物或类视黄醇，或者，其在药学上可接受的盐、酸和衍生物。
所述的烷化剂为本领域常规的烷化剂，较佳地选自噻替派或环磷酰胺。所述的烷基磺酸酯类化学治疗剂为本领域常规的烷基磺酸酯类化学治疗剂，较佳地选自白消安、英丙舒凡或哌泊舒凡。所述的氮丙啶类化学治疗剂为本领域常规的氮丙啶类化学治疗剂，较佳地选自氮丙唳如、卡巴醌、美妥替哌或乌瑞替派。所述乙烯酰胺类和甲基密胺类化学治疗剂为本领域常规的乙烯酰胺类和甲基密胺类化学治疗剂，较佳地选自六甲蜜胺、三乙撑蜜胺、三亚乙基磷酰胺，三亚乙基硫代磷酰胺或三羟甲蜜胺。所述的氮芥为本领域常规的氮芥，较佳地选自苯丁酸氮芥、萘氮芥、雌氮芥(estramustine)、异环磷酰胺、氮芥、氧氮芥盐酸盐、苯丙氨酸氮芥、新氮芥、苯芥胆甾醇、泼尼氮芥、曲磷胺或尿嘧啶氮芥。所述硝基脲类化学治疗剂为本领域常规的硝基脲类化学治疗剂，较佳地选自卡 莫司汀、氯脲菌素、福莫司汀、洛莫司汀、尼莫司汀或雷莫司汀。所述抗生素为本领域常规的抗生素，较佳地选自阿克拉霉素、放线菌素、安曲霉素、氮丝氨酸、博来霉素、放线菌素c、加力车霉素、卡柔比星、洋红霉素、嗜癌素、色霉素、更生霉素、柔红霉素、地托比星、6-重氮基-5-氧代-L-正亮氨酸、多柔比星、表柔比星、依索比星、依达比星、发波霉素、丝裂霉素、霉酚酸、诺加霉素、橄榄霉素、培洛霉素、紫菜霉素、嘌呤霉素、三铁阿霉素、罗多比星、链黑菌素、链脲菌素、杀结核菌素、乌苯美司、静司他丁或佐柔比星。所述的抗代谢物为本领域常规的抗代谢物，较佳地选自氨甲喋呤或5-氟尿嘧啶(5-FU)。所述的叶酸类化学治疗剂为本领域常规的叶酸类化学治疗剂，较佳地选自二甲叶酸、蝶罗呤或三甲曲沙。所述的嘌呤类似物为本领域常规的嘌呤类似物，较佳地选自氟达拉滨、6-巯嘌呤、硫咪嘌呤或硫鸟嘌呤。所述的嘧啶类似物为本领域常规的嘧啶类似物，较佳地选自安西他滨、阿扎胞苷、6-阿扎尿苷、卡莫氟、阿糖胞苷、二脱氧尿苷、去氧氟尿苷、依诺他滨、氟尿苷或5-EU。所述的雄激素为本领域常规的雄激素，较佳地选自卡普睾酮、丙酸甲雄烷酮、环硫雄醇、美雄烷或睾内酯。所述的抗肾上腺素为本领域常规的抗肾上腺素，较佳地选自安鲁米特、米托坦或曲洛司坦。所述的叶酸补充剂为本领域常规的叶酸补充剂，较佳地选自亚叶酸、醋葡全内酯、醛磷酰胺糖苷、氨基酮戊酸、安吖啶、阿莫司汀、比生群、依达曲沙、地磷酰胺、秋水仙胺、地吖醌、依氟鸟氨酸、依利醋铵、埃坡西龙、依托格鲁、硝酸镓、羟基脲、香菇多糖或氯尼达明。所述的美登醇为本领域常规的美登醇，较佳地选自美登素、安丝菌素、米托胍腙、米托蒽醌、莫哌达醇、二胺硝吖啶、喷司他丁、蛋氨氮芥、吡柔比星、洛索蒽醌、鬼臼酸、2-乙基酰肼或丙卡巴肼。所述的多糖复合物为本领域常规的多糖复合物，较佳地选自雷佐生、根霉素、西佐喃、锗螺胺、细交链孢菌酮酸、三亚胺醌2，2′，2″-三氯三乙胺、单端孢霉烯族毒素、乌拉坦、长春地辛、达卡巴嗪、甘露莫司汀、二溴甘露醇、二溴卫矛醇、哌泊溴烷、gacytosine、阿糖胞苷、环磷酰胺或噻替派。更佳地选自T-2毒素、疣孢菌素A、杆孢菌素A或anguidine。所述紫杉烷为本领域常规的紫杉烷，较佳地选自紫杉醇、无氢化蓖麻油、紫杉醇的白蛋白工程化纳米颗粒制剂(American Pharmaceutical Partners，Schaumberg，Illinois)、多西他赛、苯丁酸氮芥、吉西他滨、6-硫代鸟嘌呤、巯嘌呤或甲氨蝶呤。所述的铂类似物为本领域常规的铂类似物，较佳地选自顺铂、卡铂、长春碱、依托泊苷、异环磷酰胺、米托蒽醌、长春新碱、诺安托、替尼泊苷、依达曲沙、道诺霉素、氨基蝶呤、卡培他滨伊班膦酸盐、CPT-11、拓扑异构酶抑制剂RFS 2000或二氟甲基鸟氨酸。所述的类视黄醇为本领域的类视黄醇，较佳地为视黄酸。
其中，所述放射性同位素为本领域常规的放射性同位素，较佳地，其与上述人源化 抗TPBG抗体直接结合，或者通过螯合剂与上述人源化抗TPBG抗体结合。更佳地，其与所述人源化抗TPBG抗体的半胱氨酸残基直接结合。较佳地，所述放射性同位素选自适于放射治疗的α-发射体、β-发射体和俄歇电子以及适于诊断的正电子发射体或γ-发射体。更佳地，所述放射性同位素选自 18氟、 64铜、 65铜。 67镓、 68镓、 77溴、 80m溴、 95钌、 97钌、 103钌、 105钌、 99m锝、 107汞、 203汞、 123碘、 124碘、 125碘、 126碘、 131碘、 133碘、 111铟、 113铟、 99m铼、 105铼、 101铼、 186铼、 188铼、 121m碲、 99锝、 122m碲、 125m碲、 165铥、 167铥、 168铥、 90钇、 213铋、 213铅或 225锕，或者其衍生的氮化物或氧化物。
其中，所述治疗性核酸为本领域常规的核酸，较佳地为编码免疫调节剂、抗血管生成剂、抗增殖剂或促凋亡剂的基因。所述治疗剂包括所述治疗剂、其衍生物和所述治疗剂在药学上可接受的盐、酸及衍生物。
其中，所述的免疫调节剂为本领域常规的免疫调节剂，即引发免疫应答，包括体液免疫应答(例如抗原特异性抗体的产生)和细胞介导的免疫应答(例如淋巴细胞增殖)的试剂。较佳地选自细胞因子、生长因子、激素、抗激素药、免疫抑制剂或皮质类固醇。所述细胞因子为本领域常规的细胞因子，较佳地选自黄嘌呤、白介素或干扰素。所述的生长因子为本领域常规的生长因子，较佳地选自TNF、CSF、GM-CSF或G-CSF。所述的激素为本领域常规的激素，较佳地选自雌激素、雄激素或孕激素。更佳地，所述的雌激素为已烯雌酚或雌二醇。更佳地，所述的雄激素为睾酮或氟甲睾酮。更佳地，所述的孕激素为乙酸甲地孕酮或乙酸甲羟孕酮。所述的皮质类固醇为本领域常规的皮质类固醇，较佳地选自强的松、地塞米松或化可的松。所述抗激素药为本领域常规的抗激素药，其能阻断激素对肿瘤的作用，抑制细胞因子生产，下调自身抗原表达、或掩蔽MHC抗原的免疫抑制剂。较佳地选自抗雌激素药、抗雄激素药或抗肾上腺素药。更佳地，所述抗雌激素药选自它莫西芬、雷洛昔芬、芳香酶抑制性4(5)-咪唑类、4-羟基它莫西芬、曲沃昔芬或托瑞米芬。所述抗雄激素药选自氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林或戈舍瑞林。所述免疫抑制剂为本领域常规的免疫抑制剂，较佳地选自2-氨基-6芳基-5取代的嘧啶类、硫唑嘌呤、环磷酰胺、溴隐亭、达那唑、氨苯砜、戊二醛、针对MHC抗原和MHC片段的抗独特型抗体、环孢菌素A、类固醇例如糖皮质类固醇、链激酶、TGFb、雷帕霉素、T细胞受体、T细胞受体片段、细胞因子受体拮抗剂或T细胞受体抗体。更佳地，所述细胞因子受体拮抗剂选自抗干扰素抗体、抗IL10抗体、抗TNFa抗体或抗IL2抗体。
其中，所述的抗血管生成剂为本领域常规的抗血管生成剂，较佳地选自法尼基转移酶抑制剂、COX-2抑制剂、VEGF抑制剂、bFGF抑制剂、类固醇硫酸酯酶抑制剂、白介 素-24、凝血栓蛋白、metallospondin蛋白质、I类干扰素、白介素12、鱼精蛋白、血管他丁、层粘连蛋白、内皮他丁或催乳激素片段。更佳地为2-甲氧基雌二醇二氨基磺酸酯(2-MeOE2bisMATE)。
其中，所述抗增殖促凋亡剂为本领域常规的抗增殖促凋亡剂，较佳地选自PPAR-γ激活剂、类视黄醇、三萜类化合物、EGF受体抑制剂、端粒末端转移酶抑制剂、铁螯合剂、凋亡蛋白、Bcl-2和Bcl-X(L)的抑制剂、TNF-α/FAS配体/TNF相关的凋亡诱导配体及其信号传导的激活物或PI3K-Akt存活途径信号抑制剂。所述PPAR-γ激活剂为本领域常规的PPAR-γ激活剂，较佳地为环戊烯酮前列腺素(cyPGs)。所述三萜类化合物为本领域常规的三萜类化合物，较佳地选自环菠萝蜜烷、羽扇豆烷、乌苏烷、齐敦果烷、木栓烷、达玛烷、葫芦素、柠檬苦素类似物或三萜类化合物。所述EGF受体抑制剂为本领域常规的EGF受体抑制剂，较佳地选自HER4、雷帕霉素或1，25-二羟基胆钙化醇(维生素D)。所述的铁螯合物为本领域常规的铁螯合物，较佳地为3-氨基吡啶-2-甲醛硫代缩氨基脲。所述的凋亡蛋白为本领域常规的凋亡蛋白，较佳地为鸡贫血病病毒的病毒蛋白质3-VP3。所述PI3K-Akt存活途径信号抑制剂为本领域常规的PI3K-Akt存活途径信号抑制剂，较佳地为UCN-01或格尔德霉素。
其中，所述细胞溶解酶为本领域常规的细胞溶解酶，较佳地为RNA酶。
本发明优选地，式1中x＝y＝n；由此
在一个优选实施例中，-(L) x-(D) y为：
Figure PCTCN2018081853-appb-000004
其中m为1～10，优选m为5，即为马来酰亚胺基己酰(MC)。
在一个优选实施例中，-(L) x-(D) y为：
Figure PCTCN2018081853-appb-000005
在一个优选实施例中，-(L) x-(D) y为：
Figure PCTCN2018081853-appb-000006
最佳地，所述D为微管蛋白合成酶抑制剂——甲基奥瑞他汀F，(MMAF)，且所述接头L为马来酰亚胺基己酰(maleimidocaproyl，MC)，所述免疫偶联物的结构如式3所示，
Figure PCTCN2018081853-appb-000007
其中，mAb为上述的人源化抗TPBG抗体。
或者，所述L为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；D为N2’-脱乙酰-N2’-3-巯基-1氧代丙基)-美登素(DM1)，所述免疫偶联物的结构如式4所示，
Figure PCTCN2018081853-appb-000008
或者，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇，D为甲基奥瑞他汀E(MMAE)，所述免疫偶联物的结构如式5所示，
Figure PCTCN2018081853-appb-000009
其中，n为自然数，优选为1～20的整数，更优选为1～2，或2～4，或3～5，或4～8，或8～20的整数。
所述的免疫偶联物的制备方法为本领域常规，较佳地采用Doronina，2006，Bioconjugate Chem.17,114-124所记载的制备方法。较佳地，所述的制备方法产生具有最低限度的低偶联级分(LCF)小于10％的免疫偶联物。
在本发明一较佳实施例中，所述的制备方法包括以下的步骤：将上述人源化抗TPBG抗体经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与上述人源化抗TPBG抗体的摩尔比比率为2～10，室温下还原1～4小时，得反应液A。将反应液A洗脱去除多余的上述人源化抗TPBG抗体得反应液B。向反应液B中加入MC-MMAF，其中MC-MMAF与纯化的人源化TPBG抗体的摩尔比比率为5～20，10～37℃下反应4小时。
所述的免疫偶联物能够以本领域所知的任何物理形态而存在，较佳地为澄清溶液。
本发明还提供一种药物组合物，其包括上述的免疫偶联物和药学可接受的载体；较佳地，所述的药物组合物还包括其他抗肿瘤抗体例如针对癌症免疫循环过程中靶点的其他抗体作为活性成分，所述的其他抗体优选抗PD-1抗体。其中，癌症免疫循环具体包括(死亡)癌细胞释放抗原、肿瘤抗原提呈、启动和激活、T细胞运输至肿瘤部位、T细胞浸润肿瘤、T细胞识别癌细胞以及癌细胞被杀死(详见Chen D S,Mellman I.Oncology Meets Immunology:The Cancer-Immunity Cycle[J].Immunity,2013,39(1):1-10.)；本发明中的免疫偶联ADC在癌症免疫循环中的第1阶段[(死亡)癌细胞释放抗原]发挥作用，抗PD-1抗体在癌症免疫循环中的第7阶段(癌细胞被杀死中发挥作用)。
所述的药学可接受的载体为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳地包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述人源化抗TPBG抗体和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明所述的药物组合物的给药途径较佳地为肠胃外施用、注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，即可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。更佳地为经由血管内、皮下、腹膜内或肌内施用。较佳地，所述药物组合物还可以作为气雾剂或粗喷雾剂施用，即经鼻施用；或者，鞘内、髓内或心室内施用。更佳地，所述的药物组合物还可以透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。
本发明所述的药物组合物的给药剂量水平可以根据达到所需诊断或治疗结果的组合物量而调整。施用方案也可以为单次注射或多次注射，或进行调整。所选择的剂量水平和方案依赖于包括所述药物组合物的活性和稳定性(即，半衰期)、制剂、施用途径、与其他药物或治疗的组合、待检测和/或治疗的疾病或病症、以及待治疗的受试者的健康状况和先前医疗史等各种因素而进行合理地调整。
对于本发明的所述药物组合物的治疗有效剂量可以最初在细胞培养实验或动物模型例如啮齿类动物、兔、犬、猪和/或灵长类动物中进行估计。动物模型也可以用于测定合适的施用浓度范围和途径。随后可以用于确定在人中施用的有用剂量和途径。一般地，施用有效量或剂量的确定和调整以及何时和如何进行此类调整的评估为本领域技术人员已知。
对于组合疗法，上述人源化抗TPBG抗体、上述免疫偶联物和/或另外的治疗或诊断 剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行使用，所述的治疗或诊断剂可以为其他抗肿瘤抗体例如针对癌症免疫循环过程中靶点的抗体，所述的抗体优选抗PD-1抗体。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。例如，这些单一药剂可以在一年内，或10、8、6、4或2个月内，或4、3、2、或1周内，或5、4、3、2或1天内施用。
关于制剂、剂量、施用方案和可测量的治疗结果的另外指导，参见Berkow等人(2000)The Merck Manual of Medical Information(Merck医学信息手册)和Merck&Co.Inc.,Whitehouse Station,New Jersey；Ebadi(1998)CRC Desk Reference of Clinical Pharmacology(临床药理学手册)等著作。
本发明提供一种上述的人源化抗TPBG抗体在制备抗肿瘤药物中的应用。
本发明提供一种上述的免疫偶联物在制备抗肿瘤药物中的应用。
本发明提供一种上述的药物组合物在制备抗肿瘤药物中的应用。
以上所述的应用当中，所述的人源化TPBG抗体、所述的免疫偶联物或者所述的药物组合物优选与针对癌症免疫循环过程中靶点的抗体联合施用，所述的抗体优选抗PD-1抗体。
所述的肿瘤为常规的肿瘤，较佳的是过表达TPBG蛋白的肿瘤，更佳的为鳞状/腺瘤性肺癌(非小细胞肺癌)、浸润性乳腺癌、结肠癌、直肠癌、胃癌、鳞状宫颈癌、浸润性子宫内膜腺癌、浸润性胰腺癌、卵巢癌、鳞状膀胱癌、绒毛膜癌、支气管癌、乳腺癌、子宫颈癌、胰腺癌或精囊癌。
本发明还提供一种检测过表达TPBG蛋白的细胞的方法，包括如下的步骤：上述的人源化抗TPBG抗体与待检样品在体外接触，检测上述的人源化抗TPBG抗体与所述待检样品的结合即可。
所述的过表达的含义为本领域常规，较佳地为待检样品中，细胞经过流式检测，上述的人源化抗TPBG抗体的平均荧光密度(MFI)值是亚型IgG的MFI值的3倍及以上。
所述结合的检测方式是本领域常规的检测方式，较佳地为FACS检测。
本发明所述的“TPBG阳性”的细胞即为过表达TPBG蛋白的细胞，如NCI-H1568细胞株；反之，则称为“TPBG阴性”的细胞，如肿瘤细胞系NCI-H1770。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明所述的TPBG抗体是一人源化抗体，具有对于 人TPBG抗原至少约1×10 -7M至约1×10 -12M的结合亲和力，所述的人源化抗TPBG抗体及其偶联物还可以显示在体内靶向TPBG表达细胞的特异性结合。本发明的人源化抗TPBG抗体能够在蛋白水平和细胞水平结合TPBG蛋白受体的胞外区。所述的人源化TPBG抗体与如MC-MMAF的小分子化合物偶联后得到偶联物，所述偶联物能够有效地对TPBG阳性细胞进行细胞毒杀伤作用。此外，人源化TPBG抗体能把小分子化合物，如MMAF，通过内吞作用带入细胞，并在细胞内降解释放小分子化合物，从而起到细胞毒杀伤作用。因此所述的人源化TPBG抗体用于制备抗体交联药，能够有效杀伤肿瘤细胞，用于治疗肿瘤；例如与抗PD-1抗体联合治疗可以显著延长小鼠的生存期，且组合治疗后肿瘤完全缓解的小鼠体内会形成免疫记忆。偶联抗体还显示出较高的清除速率、较低的消除半衰期以及暴露量，对非小细胞肺癌患者衍生的异种移植瘤PDX的小鼠体内生长具有显著抑制作用。
附图说明
图1.人源化抗TPBG抗体12B12重链可变区h12B12.VH1及其变体与12B12嵌合抗体VH及人种系VH外显子hVH3-48/JH-6的序列比较。方框处为CDR。
图2.人源化抗TPBG抗体12B12重链可变区h12B12.VH2及其变体与12B12嵌合抗体VH及人种系VH外显子hVH3-30/JH-6的序列比较。方框处为CDR。
图3.人源化抗TPBG抗体12B12轻链可变区h12B12.Vk1及其变体与12B12嵌合抗体Vk及人种系Vk外显子B3/Jk-2的序列比较。方框处为CDR。
图4.人源化抗TPBG抗体12B12轻链可变区h12B12.Vk2及其变体与12B12嵌合抗体Vk及人种系Vk外显子A2/Jk-2的序列比较。方框处为CDR。
图5.人源化抗TPBG抗体28D4重链可变区h28D4.VH1及其变体与28D4嵌合抗体VH及人种系VH外显子hVH3-11/JH-6的序列比较。方框处为CDR。
图6.人源化抗TPBG抗体28D4轻链可变区h28D4.Vk1及其变体与28D4嵌合抗体Vk及人种系Vk外显子O18/Jk-5的序列比较。方框处为CDR。
图7A和7B.ELISA检测人源化抗体12B12变体与人TPBG-hFc蛋白的结合反应。
图8A和8B.ELISA检测人源化抗体28D4变体与人TPBG-hFc蛋白的结合反应。
图9A.FACS检测人源化12B12变体与表面表达人TPBG蛋白的稳转细胞系CHOK1-hTPBG的结合反应。
图9B.FACS检测人源化12B12变体与表面表达食蟹猴TPBG蛋白的稳转细胞系CHOK1-cTPBG的结合反应。
图9C.FACS检测人源化12B12变体与表面表达小鼠TPBG蛋白的稳转细胞系CHOK1-cTPBG的结合反应。
图9D.FACS检测人源化12B12变体与TPBG阴性的细胞系CHO-k1的结合反应。
图10A.FACS检测人源化28D4变体与表面表达人TPBG蛋白的稳转细胞系CHOK1-hTPBG的结合反应。
图10B.FACS检测人源化28D4变体与表面表达食蟹猴TPBG蛋白的稳转细胞系CHOK1-cTPBG的结合反应。
图10C.FACS检测人源化28D4变体与表面表达小鼠TPBG蛋白的稳转细胞系CHOK1-cTPBG的结合反应。
图10D FACS检测人源化28D4变体与TPBG阴性的细胞系CHO-k1的结合反应。
图11A.人源化抗体12B12变体及其抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测。
图11B.人源化抗体28D4变体及其抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测。
图12A.人源化TPBG抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测。
图12B.人源化TPBG抗体药物偶联物对TPBG弱阳性的肿瘤细胞系NCI-H1975的细胞杀伤活性检测。
图12C.人源化TPBG抗体药物偶联物对TPBG阳性的肿瘤细胞系MDA-MB-468的细胞杀伤活性检测。
图13A.为不同剂量的人源化28D4-3-MMAF抗体药物偶联物在NCI-H1975小鼠异种移植瘤模型体内药效实验中肿瘤的体积变化图。
图13B.为不同剂量的人源化28D4-3-MMAF抗体药物偶联物在NCI-H1975小鼠异种移植瘤模型体内药效实验中的小鼠体重变化图。
图14A.为偶联于不同连接子-毒素的人源化28D4-3-MMAF、28D4-3-MMAE和28D4-3-DM1抗体药物偶联物同一剂量在NCI-H1975小鼠异种移植瘤模型体内药效实验中肿瘤的体积变化图。
图14B.为偶联于不同连接子-毒素的人源化28D4-3-MMAF、28D4-3-MMAE和28D4-3-DM1抗体药物偶联物同一剂量在NCI-H1975小鼠异种移植瘤模型体内药效实验中在NCI-H1975小鼠异种移植瘤模型体内药效实验中的小鼠体重变化图。
图15A.为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和 12B12-12-DM1抗体药物偶联物的不同剂量在NCI-H1568小鼠异种移植瘤模型体内药效实验中肿瘤的体积变化图。
图15B.为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量在NCI-H1568小鼠异种移植瘤模型体内药效实验中的小鼠体重变化图。
图16A.为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量在MDA-MB-468小鼠异种移植瘤模型体内药效实验中肿瘤的体积变化图。
图16B.为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量在MDA-MB-468小鼠异种移植瘤模型体内药效实验中的小鼠体重变化图。
图17.人源化28D4-3-MMAE偶联物在大鼠中的药代动力学分析。
图18A.人源化抗TPBG抗体12B12-3在PDX肿瘤组织切片上的染色。
图18B.阴性对照抗体hIgG在PDX肿瘤组织切片上的染色。
图19A.人源化28D4-3-MMAE偶联物的不同剂量在非小细胞肺癌患者衍生的异种移植瘤模型(PDX)中的肿瘤体积变化图。
图19B.人源化28D4-3-MMAE偶联物的不同剂量在非小细胞肺癌患者衍生的异种移植瘤模型(PDX)中的小鼠体重变化图。
图20A.人源化28D4-3-MMAE抗体药物偶联物或/和抗-PD-1组合治疗后肿瘤的体积变化图
图20B.人源化28D4-3-MMAE抗体药物偶联物或/和抗-PD-1组合治疗对小鼠存活影响的寿命图
图20C.人源化28D4-3-MMAE抗体药物偶联物或/和抗-PD-1组合治疗后个体小鼠肿瘤的体积变化图。
图20D.人源化28D4-3-MMAE抗体药物偶联物或/和抗-PD-1组合治疗后肿瘤完全缓解的小鼠再次接种CT26-TPBG。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照 商品说明书选择。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
若无特别说明，实施例中所述的PBS为PBS磷酸缓冲液，pH7.2。
实施例1 嵌合抗体12B12和28D4以及人源化TPBG抗体的制备
一、鼠源抗体12B12和28D4的制备
(一)免疫原A(人源TPBG-hFc蛋白)的制备
将含有编码人源TPBG蛋白胞外区氨基酸序列32-355(Ser32-Ser355)(其中，编码人源TPBG蛋白的核苷酸序列在Genebank的编号为Genebank ID:AAH37161.1)的核苷酸序列克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒。具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293细胞(购自ATCC)进行瞬时转染(聚醚酰亚胺PEI，购自Polysciences)并使用FreeStyle TM 293(购自Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含TPBG蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的TPBG蛋白(即人源TPBG-hFc)。用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的人源TPBG-hFc蛋白。
人源TPBG-hFc蛋白在使用前需要进行一系列质控检测，如检测其蛋白浓度、纯度、分子量、生物活性等，结果发现人源TPBG-hFc蛋白各项指标良好，能够作为抗原进行后续制备TPBG抗体的试验。
(二)免疫原B的制备
编码人源TPBG全长氨基酸序列的核苷酸序列(其中，编码人源TPBG蛋白的核苷酸序列在Genebank的编号为Genebank ID:AAH37161.1)被克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系(购自ATCC)进行质粒转染(PEI，购自Polysciences)后，在含0.5μg/ml的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃、5％(v/v)CO 2培养，大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的TPBG抗体(购 自Sigma，货号#SAB1404485)经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B。具体选择结果如表2所示，IgG亚型对照为小鼠IgG对照。表2说明，已经制得一系列TPBG阳性表达的HEK293细胞系。表2的结果说明，293F-hTPBG 5E5为TPBG高水平表达细胞株，其中抗TPBG抗体标记的细胞平均细胞荧光密度为280.5，迁移率为98.5％。
表2 人源TPBG蛋白转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2018081853-appb-000010
(三)杂交瘤细胞的制备和抗体筛选
A、免疫原A免疫
采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(均购自上海斯莱克公司)，小鼠在SPF条件下饲养。初次免疫时，将上述第(一)部分制得的免疫原A用弗氏完全 佐剂乳化后腹腔注射0.25mL，即每只小鼠注射50μg免疫原A蛋白。加强免疫时，免疫原A用弗氏不完全佐剂乳化后腹腔注射0.25mL，即每只小鼠注射50微克免疫原A。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性，结果如表3所示。表3说明，经免疫原A免疫的小鼠的免疫后血清对免疫原A均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一百万左右。其中空白对照为1％(w/w)BSA，其中批次指第二次加强免疫后第七天的小鼠血清，表中的数据为OD 450nm值。
表3 ELISA检测TPBG蛋白免疫后Balb/c小鼠血清抗体效价
Figure PCTCN2018081853-appb-000011
B、免疫原B免疫
采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(均购自上海斯莱克公司)，小鼠在SPF条件下饲养。根据上述第(二)部分的，含有编码人源TPBG全长氨基酸序列的核苷酸序列的pIRES质粒转染HEK293细胞系，得含有人源TPBG的HEK293稳定细胞系(293F-hTPBG 5E5)(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)。在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液稀释至2╳10 7细胞每mL。每只小鼠每次免疫时腹腔注射0.5mL细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在第二次加强免疫后，FACS检测血清抗体效价达到1:1000以上。
A～B步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克纯化的免疫原A(针对免疫原A进行免疫反应的小鼠)或含有人源TPBG的HEK293稳定细胞系(针对免疫原B进行免疫反应的小鼠)，5天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(w/w)，裂解脾细胞中参杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000 转每分钟离心清洗细胞3次，然后按照活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％(w/w)胎牛血清、1╳HAT的DMEM培养基中。然后按1╳10 5/200微升每孔加入到96孔细胞培养板中，放入5％(v/v)CO 2、37℃培养箱中培养。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD 450nm>1.0和Acumen中MFI值>100的阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清的DMEM(invitrogen)中，于37℃、5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析，用ELISA、FACS确定对TPBG蛋白和TPBG阳性细胞的结合活性。
根据24孔板筛选结果，挑选ELISA实验中OD 450nm>1.0、FACS实验中MFI值>50和间接细胞毒杀伤实验中杂交瘤细胞培养上清对TPBG阳性细胞杀伤率达到50％的杂交瘤细胞为符合条件的阳性克隆，选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自invitrogen)37℃、5％(v/v)CO 2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性评估生物活性，评估标准为ELISA实验中OD 450nm>1.0和FACS实验中MFI值>50。
根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(w/w)FBS的DMEM培养基中(购自invitrogen)在37℃、5％(v/v)CO 2条件下将该最优的克隆进行扩大培养，液氮冻存即得本发明杂交瘤细胞，并可用于后续的获得先导抗体、生产和纯化抗体。
(四)先导抗体的生产和纯化
杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/mL，浓度变化较大。且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5mg)抗体生产纯化。
将上述第(三)部分所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入200mL生产培养基，接种细胞密度为1.0╳10 5/mL。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45μm的滤膜过滤至培养上清液澄清，得澄清的杂交瘤细胞的培养上清液。澄清的杂交瘤细胞的培养上清液可立即进行纯化或于-30℃冻存。
将获得的的培养上清液(200mL)中的TPBG抗体用2mL蛋白A柱(购自GE  Healthcare)纯化。蛋白G柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.4)平衡，然后将培养上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白G柱，平衡缓冲液的体积为蛋白A柱柱床体积的4倍。用洗脱液(0.1M柠檬酸钠缓冲液，pH3.5)洗脱结合在蛋白A柱上的TPBG抗体，用紫外检测器监测洗脱情况(A 280nm紫外吸收峰)。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的TPBG抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的TPBG抗体。
将纯化的TPBG抗体进行蛋白浓度(A 280nm/1.4)、纯度、内毒(Lonza试剂盒)等检测分析，结果如表4所示，表4说明，抗体最终产品内毒素浓度在1.0EU/mg以内。
表4 纯化的TPBG抗体检测分析
Figure PCTCN2018081853-appb-000012
(五)先导抗体的检定
A、酶联免疫吸附实验(ELISA)检测TPBG抗体与TPBG蛋白的结合
对所得的纯化的TPBG抗体与人源TPBG-hFc蛋白进行反应。
将上述第(一)部分制得的人源TPBG-hFc蛋白用PBS稀释到终浓度1.0μg/mL，然后以100μL每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[含0.01％(v/v)Tween20的PBS]洗板2次，加入封闭液[含0.01％(v/v)Tween20和1％(w/w)BSA的PBS]室温封闭2小时。倒掉封闭液，加入纯化的TPBG抗体100μL每孔。37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A 450nm数值，结果如表5所示，表5说明，纯化的TPBG抗体与TPBG重组蛋白在ELISA水平结合。表5中IgG对照为对照小鼠IgG，表中的数据为OD 450nm值，Blank的含义为板中只有PBS缓冲液时的OD 450nm值。
表5 ELISA检测TPBG抗体与人TPBG-hFc蛋白的结合反应
Figure PCTCN2018081853-appb-000013
Figure PCTCN2018081853-appb-000014
B、流式细胞实验(FACS)检测TPBG抗体与TPBG表达细胞的结合
将编码人源TPBG全长氨基酸序列的核苷酸序列(其中，编码人源TPBG蛋白的氨基酸序列和核苷酸序列在Genebank中的编号分别为Genebank ID:AAH37161.1和Gene ID:7162)克隆到pIRES载体(购自Clontech)并制备质粒。然后将编码人源TPBG全长氨基酸序列的核苷酸序列的pIRES质粒转染(PEI，购自Polysciences)CHO-k1细胞株(购自ATCC)得含人源TPBG的CHO-k1稳定细胞株(此处称为CHOk1-hTPBG稳定细胞株)。类似地，将带有猴源TPBG全长氨基酸序列的核苷酸序列(其中，编码猴源TPBG蛋白的氨基酸序列和核苷酸序列在Genebank中的编号分别为Genebank ID:BAE00432.1和Gene ID:102132149)克隆到pIRES载体(购自Clontech)并制备质粒。然后将编码猴源TPBG全长氨基酸序列的核苷酸序列的pIRES质粒转染(PEI，购自Polysciences)CHO-k1细胞株(购自ATCC)得含猴源TPBG的CHO-k1稳定细胞株(此处称为CHOk1-cTPBG稳定细胞株)。同理，将带有小鼠源TPBG全长氨基酸序列的核苷酸序列(其中，编码小鼠TPBG蛋白的氨基酸序列和核苷酸序列在Genebank中的编号分别为Genebank ID:CAA09931.1和Gene ID:21983)克隆到pIRES载体(购自Clontech)并制备质粒。然后将编码小鼠源TPBG全长氨基酸序列的核苷酸序列的pIRES质粒转染(PEI，购自Polysciences)CHO-k1细胞株(购自ATCC)得含小鼠源TPBG的CHO-k1稳定细胞株(此处称为CHOk1-mTPBG稳定细胞株)。
用FACS检测血清中TPBG抗体的效价和特异性，检测方法参见上述第(二)部分“免疫原B的制备”中鉴定HEK293-hTPBG稳定细胞株的方法。检测结果如表6所示。表6的结果说明，CHOk1-hTPBG稳定细胞株、CHOk1-cTPBG稳定细胞株和CHOk1-mTPBG稳定细胞株的细胞膜上分别过表达人、猴或小鼠的TPBG蛋白，其可以用于筛选TPBG抗体。
表6 人/猴/小鼠TPBG转染的CHO-k1细胞FACS筛选检测结果
Figure PCTCN2018081853-appb-000015
将CHOk1-hTPBG稳定细胞株、CHOk1-cTPBG稳定细胞株、CHOk1-mTPBG稳定细胞株(即表6所示的CHOk1-hTPBG 3A2F1、CHOk1-cTPBG 3F13G4和CHOk1-mTPBG 3A3)以及CHO-k1细胞分别在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution)(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution:购自Life technology公司)处理和收集细胞。用HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2╳10 6个细胞/mL，加入10％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA，所述百分比为质量百分比)悬浮至2╳10 6个细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入上述第(四)部分所得的纯化的TPBG抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]重悬细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism5)分析，进行数据拟合，计算EC50值。分析结果如表7所示。表7中的数据为根据MFI计算得到的EC50值。表7说明，TPBG抗体可结合细胞表面的TPBG蛋白。
表7 FACS分析TPBG抗体与人/猴/小鼠TPBG表达细胞株结合活性
Figure PCTCN2018081853-appb-000016
(六)竞争性ELISA检测分析TPBG抗体与抗原的表位分布
为了鉴定抗体对抗原的结合位点，采用竞争ELISA的方法对上述TPBG抗体进行分组。
纯化的待测抗体用PBS稀释至1μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250微升封闭液[含有0.01％(v/v)Tween20和1％(w/w)BSA的PBS]进行室温一小时封闭，每孔加入0.05μg/mL的生物素标记的重组TPBG蛋白。同时加入5μg/mL的竞争抗体，即上述所得的纯化的TPBG抗体，其克隆号分别为12B12C7C3和28D4E6A9，并于25-37℃孵育1-2小时。用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次，加入HRP(辣根过氧化物酶)标记的链亲和素(购自Sigma)。37℃孵育0.5小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus， 购自Molecular Device)读取A 450nm数值。根据A 450nm数值，计算出抗体相互之间的竞争率，结果如表8所示。竞争率的数值越高，表示两个抗体的抗原表面越是接近。
表8.12B12C7C3抗体与28D4E6A9抗体相互之间的竞争率
Figure PCTCN2018081853-appb-000017
结果说明，12B12C7C3和28D4E6A9的表位不同。
(七)轻重链可变区氨基酸序列测定
总RNA分离：通过离心搜集上述第(三)部分所得的杂交瘤细胞5×10 7个，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置10分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％(v/v)乙醇，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促进溶解10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μL体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μL PCR体系，包括1μL cDNA、每种引物25pmol、1μL DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，95℃预变性3分钟，95℃变性30秒，55℃退火30秒，72℃延伸35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒包括Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2018081853-appb-000018
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μL，缓冲液1μL，反应体系10μL，于16℃反应半小时得连接产物。其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟，而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μL无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟。取出200μL涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μLPCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含 100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat，“Sequences of Proteins of Immunological Interest，”National Institutes of Health，Bethesda，Md.(1991)]。
结果显示，所述12B12C7C3重链可变区的氨基酸序列如序列表中SEQ ID NO.2所示，轻链可变区序列如序列表中SEQ ID NO.4所示。
其中重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.2中的第31位至第35位所示，CDR2的氨基酸序列如序列表SEQ ID No.2中的第50位至第66位所示，CDR3的氨基酸序列如序列表SEQ ID No.2中的第99位至第109位所示；
所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.4中的第24位至第38位所示，CDR2的氨基酸序列如序列表SEQ ID No.4中的第54位至第60位所示，CDR3的氨基酸序列如序列表SEQ ID No.4中的第93位至第101位所示。
所述28D4E6A9重链可变区的氨基酸序列如序列表中SEQ ID NO.6所示，轻链可变区序列如序列表中SEQ ID NO.8所示。
其中重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.6中的第31位至第35位所示，CDR2的氨基酸序列如序列表SEQ ID No.6中的第50位至第66位所示，CDR3的氨基酸序列如序列表SEQ ID No.6中的第99位至第109位所示；
所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.8中的第24位至第34位所示，CDR2的氨基酸序列如序列表SEQ ID No.8中的第50位至第56位所示，CDR3的氨基酸序列如序列表SEQ ID No.8中的第89位至第97位所示。
核苷酸测序结果：
12B12C7C3的重链可变区的核苷酸序列如SEQ ID NO.1所示，轻链可变区的核苷酸序列如SEQ ID NO.3所示；
28D4E6A9的重链可变区的核苷酸序列如SEQ ID NO.5所示，轻链可变区的核苷酸序列如SEQ ID NO.7所示；
其中，编码12B12C7C3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.1中的第91位至第105位所示；
编码12B12C7C3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.1中的第148位至第198位；
编码12B12C7C3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.1中的第295位至第327位；
编码12B12C7C3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.3 中的第70位至第114位所示；
编码12B12C7C3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.3中的第160位至第180位所示；
编码12B12C7C3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.3中的第277位至第303位所示；
编码28D4E6A9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.5中的第91位至第105位所示；
编码28D4E6A9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.5中的第148位至第198位所示；
编码28D4E6A9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.5中的第295位至第327位所示；
编码28D4E6A9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.7中的第70位至第102位所示；
编码28D4E6A9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.7中的第148位至第168位所示；
编码28D4E6A9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.7中的第265位至第291位所示。
二、鼠-人嵌合抗体12B12和28D4的制备
SEQ ID NO：2、4分别列举了抗TPBG鼠单克隆抗体12B12的重链可变区和轻链可变区的氨基酸序列。SEQ ID NO：6、8分别列举了抗TPBG鼠单克隆抗体28D4的重链可变区和轻链可变区的氨基酸序列。
1)质粒构建与准备：将上述鼠源先导抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)中，将TPBG抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)当中，得重组质粒(上述质粒重组的实验原理及步骤的出处见《分子克隆实验指南(第三版)》，(美)J.萨姆布鲁克等著)并经测序验证。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
2)细胞转染：在培养基Freestyle 293expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM和8％CO 2(v/v)。Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg步骤1)所得的重组质粒(此处为上述重链重组质粒和轻链重组质粒按常规等比例混合的混合重组质粒)混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293expression medium中至293E的细胞密度为1.5×10 6个/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
3)抗体纯化：对于连续生产的无内毒素的层析柱和蛋白A填料，使用0.1M NaOH处理30min或者5个柱体积0.5M NaOH冲洗；对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％Triton X100对柱料清洗。使用5个柱体积的PBS进行平衡，将过滤好的细胞上清上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用1/10体积的pH8.5的1M Tris-HCl(1.5M NaCl)中和。收获抗体后，在1×PBS中透析过夜，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。
分别获得纯化的TPBG嵌合抗体12B12及28D4。
三、人源化TPBG抗体的制备
在Germline数据库中选取与上述嵌合抗体12B12或28D4的非CDR区匹配最好的人种系抗体重链和轻链可变区模板。人源化TPBG抗体的人接受序列选自人种系外显子V H、J H、V k和J k序列。其中12B12抗体重链可变区的模板为人种系抗体重链V H外显子的VH3-48和VH3-30，J H外显子的J H-6，轻链可变区的模板为人种系抗体轻链V K外显子的B3和A2，J K外显子的/J K-2。其中28D4抗体重链可变区的模板为人种系抗体重链V H外显子的VH3-11，J H外显子的J H-6，轻链可变区的模板为人种系抗体轻链V K外显子的O18和A2，J K外显子的J K-5。
根据Kabat定义确定的嵌合抗体12B12或28D4的重链和轻链CDR分别移植到所选人种系模板中，替换人种系模板的CDR区，得到人源化的抗体。然后，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 构象有重要影响的构架区的残基进行回复突变，得到人源化之后的抗体。简要说，产生跨越人源化V H或V L结构域的合成重叠寡核苷酸、并利用PCR重叠延伸来组装各结构域。利用掺入PCR产物的限制性位点将V H结构域定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体，将V L结构域定向克隆到包含信号肽和人源抗体轻链kappa恒定区的表达载体中，得到的重组质粒经过测序验证，使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
人源化抗TPBG抗体变体的重链和轻链可变区与人种系重链和轻链可变区及嵌合抗体的重链和轻链可变区的序列比对如图1-6所示，其中图1为人源化抗TPBG抗体12B12重链可变区h12B12.VH1及其变体与12B12嵌合抗体VH及人种系VH外显子hVH3-48/JH-6的序列比较，图2为人源化抗TPBG抗体12B12重链可变区h12B12.VH2及其变体与12B12嵌合抗体VH及人种系VH外显子hVH3-30/JH-6的序列比较，图3为人源化抗TPBG抗体12B12轻链可变区h12B12.Vk1及其变体与12B12嵌合抗体Vk及人种系Vk外显子B3/Jk-2的序列比较，图4为人源化抗TPBG抗体12B12轻链可变区h12B12.Vk2及其变体与12B12嵌合抗体Vk及人种系Vk外显子A2/Jk-2的序列比较，图5为人源化抗TPBG抗体28D4重链可变区h28D4.VH1及其变体与28D4嵌合抗体VH及人种系VH外显子hVH3-11/JH-6的序列比较，图6.为人源化抗TPBG抗体28D4轻链可变区h28D4.Vk1及其变体与28D4嵌合抗体Vk及人种系Vk外显子O18/Jk-5的序列比较。
人种系重链可变区模板VH3-48/JH6(SEQ ID NO.9)
VH3-48：
Figure PCTCN2018081853-appb-000019
JH6：WGQGTTVTVSS。
人种系重链可变区模板VH3-30/JH6(SEQ ID NO.10)
VH3-30：
Figure PCTCN2018081853-appb-000020
JH6：WGQGTTVTVSS。
人种系轻链可变区模板B3/Jk2(SEQ ID NO.11)
B3：
Figure PCTCN2018081853-appb-000021
Figure PCTCN2018081853-appb-000022
JK2：FGQGTKLEIK。
人种系轻链可变区模板A2/Jk2(SEQ ID NO.12)
A2：
Figure PCTCN2018081853-appb-000023
JK2：FGQGTKLEIK。
人种系重链可变区模板VH3-11/JH6(SEQ ID NO.13)
VH3-11：
Figure PCTCN2018081853-appb-000024
JH6：WGQGTTVTVSS。
人种系轻链可变区模板O18/Jk5(SEQ ID NO.14)
O18：
Figure PCTCN2018081853-appb-000025
JK5：FGQGTRLEIK。
选择数个框架位置以重新引入小鼠供体残基。可通过在VH结构域中掺入小鼠框架供体残基或在VL结构域中掺入人CDR残基的不同组合产生数种人源化12B12和28D4变体。下表9和表10小结了这些变体。其中，表9和表10显示的是这些变体的可变区，不包括恒定区。表9、10中，c开头表示嵌合抗体，h开头表示人源化抗体；其中供体构架残基及回复突变栏中显示的、例如各人源化抗TPBG抗体12B12重链可变区h12B12.VH1及其变体中“A93S”表明如图1中所示的第93位氨基酸由“A”丙氨酸突变为“S”丝氨酸，回复突变的位点位于构架区，以此为例，不一一说明。
表9
Figure PCTCN2018081853-appb-000026
Figure PCTCN2018081853-appb-000027
Figure PCTCN2018081853-appb-000028
表10
Figure PCTCN2018081853-appb-000029
Figure PCTCN2018081853-appb-000030
注：表中的“/”代表的含义是“和”，为并列关系。
根据各人源化抗体轻链可变区和重链可变区的氨基酸序列合成cDNA(即分别为序列表中的SEQ ID NO.15，17，19，21，23，25，27，29，31，33，35，37，39，41，43，45所示序列)，重链cDNA用FspAI和AfeI消化，轻链cDNA用FspAI和BsiwI后，将cDNA片段通过FspAI/AfeI或FspAI/BsiwI酶切位点分别插入到包含信号肽和人源重链抗体 IgG1恒定区的表达载体及包含信号肽和人源抗体轻链kappa恒定区的表达载体当中(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)中，重组质粒并经测序验证，使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
转染前，用培养基Freestyle 293expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。转染时在Freestyle 293expression medium中添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg重链和轻链重组质粒(重链重组质粒与轻链重组质粒的质量比为1:1-1:3范围)混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle293expression medium中至293E的细胞密度为1.5×10 6个/mL，边加边振荡，避免PEI过度集中，放入摇床培养，摇床设置为37℃、130RPM和8％CO 2(v/v)。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
抗体纯化时，对于连续生产的无内毒素的层析柱和蛋白A填料(购自GE)，使用5个柱体积的0.5M NaOH冲洗。然后用5个柱体积的PBS(PBS缓冲液，pH7.4)进行平衡至中性后，将过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH 3.0的Glycine-HCl进行洗脱，收集洗脱液，并立刻在洗脱液中加入0.1倍体积的pH 8.5的1MTris-HCl(1.5M NaCl)中和TPBG抗体。上述所用溶液均需要新鲜配制。收获TPBG抗体后，在1×PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。并对所获TPBG抗体进行特性鉴定(操作步骤如下述实施例2，实施例3，实施例4所述)。
实施例2 酶联免疫吸附实验(ELISA)检测人源化TPBG抗体与TPBG蛋白的结合
对实施例1所得的纯化的人源化TPBG抗体与人源TPBG-hFc蛋白进行反应，方法详见实施例1中(五)先导抗体的检定，结果如图7-8和表11-12所示，表11和表12分别为h12B12变体和h28D4变体的根据OD 450nm值计算出来的EC50值，说明纯化的人源化TPBG抗体变体与TPBG重组蛋白在ELISA水平有较好的结合。其中，图7A和7B为纯化的人源化h12B12变体与人源TPBG-hFc蛋白的结合反应，图8A和8B为纯化的 人源化h28D4变体与人源TPBG-hFc蛋白的结合反应。
表11.ELISA检测人源化h12B12抗体变体与人TPBG-hFc蛋白的结合反应
抗体名称 EC50(nM)
嵌合抗体12B12(对照) 0.137
人源化抗体12B12-1 0.248
人源化抗体12B12-2 0.274
人源化抗体12B12-3 0.333
人源化抗体12B12-4 0.227
人源化抗体12B12-5 0.253
人源化抗体12B12-6 0.362
人源化抗体12B12-7 0.367
人源化抗体12B12-8 0.287
人源化抗体12B12-9 0.223
人源化抗体12B12-10 0.25
人源化抗体12B12-11 0.239
人源化抗体12B12-12 0.206
人源化抗体12B12-13 0.289
人源化抗体12B12-14 0.257
人源化抗体12B12-15 0.193
人源化抗体12B12-16 0.2
表12.ELISA检测人源化h28D4抗体变体与人TPBG-hFc蛋白的结合反应
Figure PCTCN2018081853-appb-000031
Figure PCTCN2018081853-appb-000032
实施例3 人源化TPBG抗体的表征分析(Biacore)
为了评估人源化抗TPBG抗体的结合特异性和亲和力，使用固定在CM5芯片上的人TPBG抗原进行Biacore分析。Biacore技术利用抗体与固定在表层上的TPBG抗原结合后表层上的折射率改变。结合通过从表面折射的激光的表面等离子体共振(SPR)进行检测。信号动力学结合速率和解离速率的分析允许区分非特异性和特异性相互作用。嵌合抗体12B12和28D4用作对照。
表13.Biacore试验结果
抗体 ka(1/Ms) kd(1/s) KD(M)
嵌合抗体12B12 1.61E+06 1.64E-05 1.02E-11
人源化抗体12B12-4 1.58E+06 3.46E-05 2.20E-11
人源化抗体12B12-5 1.17E+06 2.10E-05 1.79E-11
人源化抗体12B12-12 1.23E+06 2.29E-05 1.86E-11
人源化抗体12B12-15 1.77E+06 <1e-5 <5.66E-12
人源化抗体12B12-16 1.59E+06 3.65E-05 2.29E-11
嵌合抗体28D4 2.50E+06 3.24E-05 1.30E-11
人源化抗体28D4-3 1.12E+06 4.57E-05 4.06E-11
人源化抗体28D4-4 1.55E+06 3.58E-05 2.31E-11
人源化抗体28D4-7 3.68E+06 9.42E-05 2.56E-11
人源化抗体28D4-8 1.02E+06 3.01E-05 2.96E-11
人源化抗体28D4-12 1.03E+06 3.94E-05 3.81E-11
Biacore结果显示，人源化抗TPBG抗体12B12和28D4各变体与嵌合抗体12B12和28D4具有非常类似的KD值，这验证了人源化抗TPBG抗体与嵌合抗体相比，没有显著降低抗原结合活性。
实施例4 流式细胞实验(FACS)检测TPBG抗体与TPBG表达细胞的结合
人源TPBG的CHO-k1稳定细胞株(CHOk1-hTPBG稳定细胞株)、猴源TPBG的CHO-k1稳定细胞株(CHOk1-cTPBG稳定细胞株)以及小鼠源TPBG的CHO-k1稳定细胞株(CHOk1-mTPBG稳定细胞株)的制备方法、流式细胞仪的检测方法以及结果的读取详见实施例1(五)“先导抗体的检定”。
分析结果如表14、15以及图9，10所示，图9A～D、10A～D的数据为细胞的平均荧光密度(MFI)。表14、15中的数据为根据MFI计算得到的EC50值。表14、15说明，人源化TPBG抗体变体与表面表达人TPBG蛋白的细胞和表面表达食蟹猴TPBG蛋白的细胞有特异结合，与表达小鼠TPBG蛋白的细胞及TPBG表达阴性的CHO-k1没有结合。
表14.FACS分析人源化抗体12B12变体与TPBG表达细胞株结合活性
Figure PCTCN2018081853-appb-000033
表15.FACS分析人源化28D4抗体变体与TPBG表达细胞株结合活性
Figure PCTCN2018081853-appb-000034
实施例5 人源化TPBG抗体药物偶联物的体外药效实验
将实施例1获得的纯化的人源化TPBG抗体变体与MC-MMAF进行偶联。抗体经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TPBG抗体的摩尔比比率为3，室温下还原1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF(购 自南京联宁)，其中MC-MMAF与纯化的人源化TPBG抗体的摩尔比比率为10，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF，并通过G25柱脱盐除去多余的小分子。得到纯化的人源化TPBG抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。分别通过HPLC-HIC和HPLC-SEC分析药物的交联率和纯度等参数后，进行体外细胞毒活性分析。所有人源化TPBG抗体偶联物的药物交联率(DAR)为3.0-5.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。
人源化TPBG抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以2000细胞/孔加入100微升TPBG阳性的非小细胞肺癌细胞系NCI-H1568(购自ATCC，货号#CRL-5876)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TPBG抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。
结果如表16，17以及图11所示，其中表16的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图11A为人源化抗体12B12变体及其抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测，图11B为人源化抗体28D4变体及其抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测。结果说明，人源化TPBG抗体变体的抗体药物偶联物对TPBG阳性的NCI-H1568细胞有杀伤作用，但没有偶联的人源化抗体变体对TPBG阳性的NCI-H1568没有杀伤作用。
表16.细胞杀伤实验检测人源化12B12变体及其抗体药物偶联物对TPBG阳性细胞的杀伤作用
克隆号 IC50(nM)
阴性对照抗体 阴性
嵌合抗体12B12 阴性
人源化抗体12B12-12 阴性
人源化抗体12B12-15 阴性
阴性对照抗体-MMAF 阴性
嵌合抗体12B12-MMAF 0.063
人源化抗体12B12-12-MMAF 0.066
人源化抗体12B12-15-MMAF 0.058
表17.细胞杀伤实验检测人源化28D4变体及其抗体药物偶联物对TPBG阳性细胞的杀伤作用
克隆号 IC50(nM)
嵌合抗体28D4 阴性
人源化抗体28D4-3 阴性
人源化抗体28D4-4 阴性
人源化抗体28D4-7 阴性
嵌合抗体28D4-MMAF 0.091
人源化抗体28D4-3-MMAF 0.061
人源化抗体28D4-4-MMAF 0.074
人源化抗体28D4-7-MMAF 0.066
实施例6 偶联不同连接子-毒素的抗体药物偶联物
将实施例1所得的纯化的人源化抗体12B12-12和28D4-3分别与MC-MMAF或MC-VC-PAB-MMAE进行偶联。抗体经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TPBG抗体的摩尔比比率为3，室温下还原1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF或MC-VC-PAB-MMAE(购自南京联宁)，其中MC-MMAF或MC-VC-PAB-MMAE与纯化的人源化TPBG抗体的摩尔比比率为10，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF或MC-VC-PAB-MMAE，并通过G25柱脱盐除去多余的小分子。得到纯化的人源化TPBG抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。分别通过HPLC-HIC和HPLC-SEC分析药物的交联率和纯度等参数后，进行体外细胞毒活性分析和体内药效分析。所有人源化TPBG抗体偶联物的药物交联率(DAR)为3.0-5.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。
将实施例1所得的纯化的人源化抗体12B12-12和28D4-3分别与4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)进行偶联。抗体经过pH 6.5～7.4的磷酸盐缓冲液透析后，在体积比为30％DMA存在下加入SMCC，其中SMCC与纯化的TPBG嵌合抗体的摩尔比比率为8，室温下反应1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的小分子，得反应液B。向反应液B中加入终体积为10％DMA(N，N-二甲基乙酰胺)，然后加入DM1(化学名称为N2’-脱乙酰-N2’-3-巯基-1氧代 丙基)-美登素)，其中DM1与纯化的TPBG抗体的摩尔比比率为9，室温下反应3.5小时，得反应液C。将反应液C经过G25柱脱盐(购自GE)，去除多余的小分子，得到纯化的人源化TPBG抗体药物偶联物(偶联方法参见US5208020)。通过LC-MS分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行体外细胞毒活性分析和体内药效分析。所有人源化TPBG抗体偶联物的药物交联率(DAR)为3.0-5.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。
实施例7 人源化TPBG抗体药物偶联物的体外药效实验
将实施例6获得的纯化的人源化TPBG抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以2000细胞/孔加入100微升TPBG阳性的非小细胞肺癌细胞系NCI-H1568(购自ATCC，货号#CRL-5876)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TPBG抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。同时选用TPBG弱表达的非小细胞肺癌细胞系NCI-H1975(购自ATCC，货号#CRL-5908)和乳腺癌细胞系MDA-MB-468(购自ATCC，货号#HTB-132)进行细胞杀伤活性检测，方法同上。
结果如表18以及图12所示，其中表18的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图12A为人源化TPBG抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测，图12B为人源化TPBG抗体药物偶联物对TPBG弱阳性的肿瘤细胞系NCI-H1975的细胞杀伤活性检测，图12C为人源化TPBG抗体药物偶联物对TPBG阳性的肿瘤细胞系MDA-MB-468的细胞杀伤活性检测。结果说明，人源化TPBG抗体药物偶联物对TPBG阳性的细胞有杀伤作用。偶联不同的小分子毒素的纯化的人源化TPBG抗体药物偶联物对TPBG阳性的细胞均具有不同程度的杀伤作用，并且偶联了MC-MMAF的TPBG嵌合抗体药物偶联物具有较低的IC50，说明其细胞杀伤能力最强。
表18.细胞杀伤实验检测人源化TPBG抗体药物偶联物对TPBG阳性细胞的杀伤作用
Figure PCTCN2018081853-appb-000035
Figure PCTCN2018081853-appb-000036
实施例8 非小细胞肺癌细胞系NCI-H1975的小鼠异种移植瘤模型
将NCI-H1975(非小细胞肺癌细胞株，ATCC，CRL-5908)(2×10 6个)200μl接种与Balb/c nude小鼠右肋皮下，待7-10天肿瘤长至200mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为几组，每组7只。分组如表19，为不同剂量的实施例6制得的人源化28D4-3-MMAF抗体药物偶联物的治疗分组，以及同一剂量的人源化12B12-12-MMAF和人源化28D4-3-MMAF抗体药物偶联物治疗组。分组如表20，为偶联于不同连接子-毒素的人源化28D4-3-MMAF、28D4-3-MMAE和28D4-3-DM1抗体药物偶联物同一剂量治疗组分组。D0开始尾静脉注射抗体，4天一次，共给药4次，每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b2；其中a、b分别表示长、宽。
表19.不同剂量的人源化TPBG抗体药物偶联物在NCI-H1975小鼠异种移植瘤模型中的体内药效实验
Figure PCTCN2018081853-appb-000037
表20.偶联不同连接子-毒素的人源化TPBG抗体药物偶联物在NCI-H1975小鼠异种移植瘤模型中的体内药效实验
Figure PCTCN2018081853-appb-000038
Figure PCTCN2018081853-appb-000039
结果见图13A：不同剂量的人源化28D4-3-MMAF抗体药物偶联物治疗后肿瘤的体积变化图，和图13B：不同剂量的人源化28D4-3-MMAF抗体药物偶联物治疗后的小鼠体重变化图。结果显示，与未治疗的小鼠相比，所有治疗小鼠的肿瘤体积显著减小，且随着剂量的增加肿瘤体积减小越明显，10mg/kg剂量的治疗组的肿瘤体积在给药后的35天中肿瘤基本消退，在后续持续观察中肿瘤开始缓慢恢复生长。另外，偶联于人源化抗体12B12-12和28D4-3的MC-MMAF抗体药物偶联物在10mg/kg剂量的治疗组中的效能相当。
结果见图14A：偶联于不同连接子-毒素的人源化28D4-3-MMAF、28D4-3-MMAE和28D4-3-DM1抗体药物偶联物同一剂量治疗后肿瘤的体积变化图，和图14B：偶联于不同连接子-毒素的人源化28D4-3-MMAF、28D4-3-MMAE和28D4-3-DM1抗体药物偶联物同一剂量治疗后的小鼠体重变化图。结果显示，在同一剂量下与未治疗的小鼠相比，偶联于MC-MMAF和MC-VC-PAB-MMAE的人源化28D4-3抗体药物偶联物治疗组的小鼠肿瘤体积显著减小，且在给药后的50天中，人源化28D4-3-MMAF治疗组的肿瘤先消退后缓慢恢复生长，人源化28D4-3-MMAE治疗组的肿瘤基本消退。与未治疗的小鼠相比，偶联于SMCC-DM1的人源化28D4-3抗体药物偶联物治疗组的小鼠肿瘤体积没有减小。说明，偶联不同连接子-毒素的抗体药物偶联物对NCI-H1975肿瘤生长的抑制能力不同。
实施例9 非小细胞肺癌NCI-H1568的小鼠异种移植瘤模型
将NCI-H1568(非小细胞肺癌细胞株，ATCC，CRL-5876)(1×10 7个)200μl接种与Balb/c nude小鼠右肋皮下，待7-10天肿瘤长至200mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为几组，每组6只，分组如表21，为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量治疗组。D0开始尾静脉注射抗体，4天一次，共给药4次，每周测2次瘤体积， 称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b2；其中a、b分别表示长、宽。
表21.人源化TPBG抗体药物偶联物的NCI-H1568小鼠异种移植瘤模型的体内药效实验
Figure PCTCN2018081853-appb-000040
结果见图15A：偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量治疗后肿瘤的体积变化图，和图15B：偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量治疗后的小鼠体重变化图。结果显示，与未治疗的小鼠相比，所有治疗小鼠的肿瘤体积显著减小。高剂量10mg/kg治疗组比低剂量2mg/kg治疗组对NCI-H1568肿瘤的抑制能力更强。另外，在10mg/kg剂量给药后的35天中，偶联于 MC-MMAF和MC-VC-PAB-MMAE的人源化12B12-12抗体药物偶联物治疗组肿瘤基本消退，但偶联于SMCC-DM1的人源化12B12-12抗体药物偶联物治疗组肿瘤先变小后缓慢恢复生长。说明，偶联不同连接子-毒素的抗体药物偶联物对NCI-H1568肿瘤生长的抑制能力不同。
实施例10 乳腺癌MDA-MB-468的小鼠异种移植瘤模型
将MDA-MB-468(购自ATCC，货号#HTB-132)细胞悬于L-15基础培养基中并加入50％Matrigel(1×10 7个)200μl接种与CB17SCID小鼠右肋皮下，待7-10天肿瘤长至200mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为几组，每组6只，分组如表22，为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量治疗组。D0开始尾静脉注射抗体，4天一次，共给药4次，每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b2；其中a、b分别表示长、宽。
表22.人源化TPBG抗体药物偶联物的MDA-MB-468小鼠异种移植瘤模型的体内药效实验
Figure PCTCN2018081853-appb-000041
结果见图16A：偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量治疗后肿瘤的体积变化图，和图16B：偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗 体药物偶联物的不同剂量治疗后的小鼠体重变化图。结果显示，与未治疗的小鼠相比，所有治疗组小鼠的肿瘤体积显著减小。另外，在2mg/kg剂量给药后的43天中，偶联于MC-MMAF和MC-VC-PAB-MMAE的人源化12B12-12抗体药物偶联物治疗组肿瘤基本消退，但偶联于SMCC-DM1的人源化12B12-12抗体药物偶联物治疗组肿瘤先变小后缓慢恢复生长。说明，偶联不同连接子-毒素的抗体药物偶联物对NCI-H1568肿瘤生长的抑制能力不同。
实施例11 人源化抗TPBG抗体药物偶联物在大鼠血清中的稳定性
通过尾静脉单次内推将3mg/kg的人源化28D4-MMAE偶联物注射入Sprague-Dawley大鼠(购于上海斯莱克实验动物有限公司)后，分别在给药前、注射后10分钟、1小时、4小时、8小时及1、2、4、7、14、21和28天收集200ul全血，14000转离心5分钟后分离血清，用ELISA方法分别测定血清中的抗TPBG总抗体(总抗体包含裸抗和偶联抗体)以及携带至少一个细胞毒性药物的偶联物浓度。血清中总的人源化抗TPBG抗体浓度用ELISA进行检测，使用抗人Fc抗体进行捕捉并使用辣根过氧化物酶(HRP)偶联的抗小鼠Fc抗体进行检测。血清中携带至少含一个细胞毒性药物的抗体药物偶联物的浓度用ELISA进行检测，使用抗MMAE抗体进行捕捉并使用辣根过氧化物酶(HRP)偶联的抗小鼠Fc抗体进行检测，其中抗MMAE抗体(商品名anti-MMAF-mIgG1，购自上海睿智化学研究有限公司)由采用杂交瘤技术生产制备。分别将抗人Fc抗体和抗MMAE抗体用PBS稀释到一定浓度(2-5ug/ml)后包被于96孔ELISA板上，置于4℃包被过夜。弃孔中液体并甩干，加入封闭液(PBS,1％BSA,0.05％Tween-20)室温封闭1小时，然后用PBST(PBS,0.05％Tween-20)清洗3次，备用。用PBST稀释标准品至一定浓度，血清样本根据情况适当稀释，将200ul/孔的稀释后的待测血清样品或标准品加入到上述包被好的96孔板中，室温孵育1-3小时，弃上清后用PBST清洗3次；然后用PBS稀释辣根过氧化物酶(HRP)偶联的抗小鼠Fc抗体至适当浓度，100ul/孔加到96孔板中37℃孵育30分钟，弃上清后用PBST清洗3次；每孔加入100ul TMB显色液，室温下孵育5-10分钟；每孔加入100ul 2N硫酸终止液终止反应，酶标仪测定OD 450nm/630nm的比值。使用具有IV推注输入、一阶消除和宏观速率常数的两隔室模型(Model 8，WinNonlinear Pro v.5.0.1,Pharsight Corporation,Mountain View,CA)分析了来自每只动物的不同采样时间点下的血清中的总抗体和偶联物的浓度数据，分析人源化抗TPBG抗体药物偶联物的体内稳定性。
在大鼠中进行的28天药代动力学分析的结果如图17及表23所示。表23中，CL代表总清除率，CL值越高代表代谢或清除越快；Vss代表稳态下的表观分布容积，Vss值 越高组织分布越广泛；V1代表中央室的分布容积，该值应与实验动物每公斤体积的血清体积接近；Alpha t1/2代表分布相半衰期，该值与分布速率相关；Beta t1/2代表消除相半衰期，该值与消除速度相关；AUC代表给药时曲线下面积(area under concentration-time curve)，代表受试物在血清中的暴露量,一般时间下暴露量和药效直接相关。
表23 抗TPBG抗体MMAE偶联物28D4-3-MMAE在大鼠中的药物代谢
PK参数 CL Vss V1 Alpha t 1/2 Beta t 1/2 AUC
单位 mL/day/kg mL/kg mL/kg day day day*μg/mL
总抗体 17.2±1.8 225±16.1 60.7±0.61 0.39±0.016 10.4±1.5 176±17.8
偶联抗体 51.3±2.7 246±23.4 70.8±0.73 0.52±0.052 6.18±1.0 58.5±3.0
从结果来看，偶联抗体与总抗体均具有相似的药代动力学特征，如较长的半衰期及非线性分布和消除等特征。不同之处在于与总抗体相比，偶联抗体显示出较高的清除速率(CL)以及较低的消除半衰期(Beta t 1/2)和暴露量(AUC)，这与经典的ADC药物代谢相符，分析可能因为ADC药物是多种分子组成的混合物，异质性较高，部分偶联物在血液循环过程中发生降解或诱发免疫反应从而使机体产品针对ADC药物的抗体导致ADC药物被清除所致。
实施例12 人源化抗TPBG抗体药物偶联物在人肿瘤患者衍生的异种移植瘤模型(PDX)中的药效实验
PDX模型为非小细胞肺癌(NSCLC)人源肿瘤模型(是将病人的肿瘤组织接种到小鼠上体内，依靠小鼠提供的环境生长的一种异种移植模型；本发明的PDX模型购自上海睿智化学研究有限公司)。通过免疫组化评估NSCLC PDX模型中TPBG的表达。简而言之，将PDX模型新鲜肿瘤组织液氮速冻后存放于-80℃冰箱。用切片机切片，室温下4％多聚甲醛固定10分钟；室温下3％双氧水室温孵育5分钟以封闭内源性过氧化物酶；室温下5％胎牛血清孵育以封闭15分钟非特异性蛋白结合点；室温下将组织切片与人源化抗TPBG抗体12B12-12或同型对照抗体hIgG室温孵育1小时；室温下与辣根过氧化物酶标记的羊抗人二抗Goat anti-human IgG(H+L)Secondary Antibody,HRP conjugate(购于Thermo)孵育30分钟，DAB显色5分钟后将切片放入盛有自来水的缸中终止反应；用苏木素复染10秒后自来水冲洗3次；盐酸酒精分化1秒后自来水冲洗3分钟；分别用75％酒精脱水2分钟、95％酒精脱水2分钟、100％酒精脱水3次、二甲苯处理3次，每次2分钟。然后中性树胶封片，显微镜观察染色结果并拍照。PDX模型中TPBG的表达量如图18A-B所示，其中图18A是人源化抗TPBG抗体12B12-3在PDX模型肿瘤组织 切片上的染色，图18B为阴性对照抗体hIgG在PDX模型肿瘤组织切片上的染色。根据图18显示的结果，PDX模型可以用于后续的药效实验。
从液氮存储罐中取出已冻存的P1代次(上述PDX模型的第一代肿瘤组织样品)肿瘤样本(FP1)，解冻后皮下接种于SCID小鼠(购于北京维通利华实验动物技术有限公司)体内，命名为FP1+1，每日监测小鼠的健康状态且最初通过目视检查每周两次监测肿瘤生长。每周2～3次记录小鼠体重。每周2～3次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝1/2a×b 2，a和b分别表示肿瘤的长径和短径。当肿瘤生长到1000mm 3时，将小鼠实行安乐死操作，取出新鲜的肿瘤组织，切成小块后皮下接种到雌性裸鼠体内，在以此方法传代3次以上后用于药效试验(FP1+3)。
采购6-8周龄的雌性Nu/Nu小鼠，体重在16-19g(购于北京维通利华实验动物技术有限公司)，动物到达后在SPF级动物房以IVC(独立送风系统)笼具饲养7天后开始实验。因小鼠个体差异可能导致肿瘤组织的生长速率差异，因此需要至少多准备50％的动物数量以允许随机分组是的最小肿瘤体积方差。经上述复苏后的肿瘤组织接种雌性Nu/Nu小鼠，每日监测小鼠的健康状态且最初通过目视检查每周两次监测肿瘤生长，并每周2～3次记录小鼠体重。待肿瘤可触及时，每周2～3次用游标卡尺测量肿瘤直径并计算肿瘤体积。待肿瘤体积长至100mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分组，每组10只，分组如表24，为偶联于不同连接子-毒素的人源化12B12-12-MMAF、12B12-12-MMAE和12B12-12-DM1抗体药物偶联物的不同剂量治疗组。D0开始尾静脉注射抗体，4天一次，共给药4次，每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：1/2a×b 2；其中a、b分别表示长、宽。
表24.人源化TPBG抗体药物偶联物在人肿瘤患者衍生的异种移植瘤模型(PDX)中的药效实验
Figure PCTCN2018081853-appb-000042
Figure PCTCN2018081853-appb-000043
结果见图19A：人源化28D4-MMAE偶联物不同剂量治疗后的肿瘤体积变化图，和图19B：人源化28D4-MMAE偶联物不同剂量治疗后的动物体重变化图。结果显示，未治疗组小鼠肿瘤体积持续生长，在实验进行到第35天时肿瘤体积超过1000mm 3而进行安乐死；与未治疗组的小鼠相比，所有治疗组小鼠的肿瘤体积显著减小，在给药3天后开始抑制肿瘤生长，给药10天后明显抑制肿瘤生长，且在给药28天后剂量1，3，10mg/ml不同剂量组均使肿瘤完全消退，并在之后一个月的持续观察中保持肿瘤消退状态，说明抗TPBG抗体MMAE药物偶联物对非小细胞肺癌患者衍生的异种移植瘤PDX的小鼠体内生长具有显著抑制作用。
实施例13 人源化抗TPBG抗体药物偶联物与抗PD-1抗体组合治疗抑制肿瘤生长在小鼠体内形成免疫记忆
人源TPBG的CT26稳定细胞株(CT26-hTPBG稳定细胞株)(购自上海睿智化学研究有限公司)，制备方法及流式细胞仪检测方法与实施例1(二)“免疫原B的制备”相同，其中小鼠结肠癌细胞CT26购自ATCC。
将CT26-TPBG细胞(1×10 6个)50μl接种于Balb/c小鼠右肋皮下，接种5～7天后选择肿瘤体积在50～100mm 3的32只动物，按肿瘤体积将小鼠随机分为4组，每组8只。分别为溶剂对照组，单独的人源化28D4-3-MMAE抗体药物偶联物治疗组，单独的抗-PD-1治疗组，以及人源化28D4-3-MMAE抗体药物偶联物和抗-PD-1组合治疗组。分组如表25。
人源化28D4-3-MMAE抗体药物偶联物通过尾静脉注射于分组当天(定义为D0)给药一次。抗-PD-1抗体从D0开始通过腹腔注射给药，一周2次，共给药8次，每周2次记录小鼠体重，并用游标卡尺测量肿瘤长短径，通过以下公式计算肿瘤体积V：V＝1/2a×b2；其中a、b分别表示肿瘤长径、短径。肿瘤体积达到2000mm 3的动物视为达到实验终点，进行安乐死，并对动物生存时间进行生存期分析。
表25.人源化TPBG抗体药物偶联物或/和抗-PD-1抗体在肿瘤模型中的体内药效实验
Figure PCTCN2018081853-appb-000044
Figure PCTCN2018081853-appb-000045
各组肿瘤生长曲线显示，在给药后D14天时，相比溶剂对照组，各个给药组的肿瘤生长均得到了显著抑制，且联合治疗组的肿瘤抑制作用显著优于单药治疗组(图20A，p值<0.001，双因素重复测量方差分析)。以肿瘤体积达到2000mm 3作为实验终点所进行的生存期分析显示，与溶剂对照组相比，人源化28D4-3-MMAE抗体药物偶联物与抗-PD-1的单药治疗和联合治疗显著延长了荷瘤鼠的生存期，且联合治疗显著优于单药治疗(图20B，p值<0.05，对数秩检验)。另外对各组单个肿瘤生长情况进行分析，结果显示对照组以及各单药治疗组的肿瘤均在60天内达到2000mm 3，与之相反，联合治疗组在给药D12时出现肿瘤完全消退的现象，至D60时，联合治疗组共有3只动物肿瘤完全消除，其完全缓解率达到37.5％(图20C)。在给药后D106时用相同数量的CT26-hTPBG细胞接种于3只完全缓解小鼠左侧肋部皮下，第二次接种30天研究期内各小鼠均没有肿瘤形成，提示这些小鼠体内已经形成免疫记忆(图20D)。
综上，人源化28D4-3-MMAE抗体药物偶联物显著增强抗-PD-1抗体对CT26-hTPBG小鼠模型的抗肿瘤活性。二者联合用药的完全缓解率达到37.5％。与人源化28D4-3-MMAE抗体药物偶联物或抗-PD-1单独治疗相比，人源化28D4-3-MMAE抗体药物偶联物和抗-PD-1抗体联合治疗可以显著延长小鼠的生存期，且组合治疗后肿瘤完全缓解的小鼠体内会形成免疫记忆。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (20)

  1. 一种人源化抗TPBG抗体，其特征在于，其包含：
    (a)包含人抗体构架区残基的构架区；和
    (b)如SEQ ID NO.4或8所示的鼠源抗体轻链可变区的一个或多个CDRs，或SEQ ID NO.2或6所示的鼠源抗体重链可变区的一个或多个CDRs。
  2. 如权利要求1所述的人源化抗TPBG抗体，其特征在于，所述的人抗体构架区包括人抗体重链构架区和人抗体轻链构架区；所述人抗体轻链构架区包含1)种系O2、O12、O18、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、A2、DPK13、DPK15、DPK16、DPKI8、DPK19、DPK20、DPK21、DPK22、DPK23、B3、DPK25、DPK26或DPK 28中的FR1、FR2和FR3区，以及2)种系Jk片段Jk1、Jk2、Jk3、Jk4或JK5中的FR4区；
    和/或，所述人抗体重链构架区包含1)种系DP4、DP7、DP8、DP9、DP10、DP31、DP33、VH3-11、DP45、DP46、DP47、DP48、VH3-30、DP50、VH3-48、DP53、DP54、DP65、DP66、DP67、DP68或DP69中的FR1、FR2和FR3区，以及2)种系JH片段JH1、JH2、JH3、JH4、JH4b、JH5或JH6中的FR4区；
    较佳地，所述鼠源抗体重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.2中的第31位至第35位所示；更佳地，编码所述鼠源抗体重链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.1中的第91位至第105位所示；
    所述鼠源抗体重链可变区的CDR2的氨基酸序列如序列表SEQ ID No.2中的第50位至第66位所示；更佳地，编码所述鼠源抗体重链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.1中的第148位至第198位所示；
    所述鼠源抗体重链可变区的CDR3的氨基酸序列如序列表SEQ ID No.2中的第99位至第109位所示；更佳地，编码所述鼠源抗体重链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.1中的第295位至第327位所示；
    和/或，所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.4中的第24位至第38位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.3中的第70位至第114位所示；
    所述鼠源抗体轻链可变区的CDR2的氨基酸序列如序列表SEQ ID No.4中的第54位至第60位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.3中的第160位至第180位所示；
    所述鼠源抗体轻链可变区的CDR3的氨基酸序列如序列表SEQ ID No.4中的第93位至第101位所示；更佳地，编码所述鼠源抗体重链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.3中的第277位至第303位所示；
    或者，所述鼠源抗体重链可变区的CDR1的氨基酸序列如序列表SEQ ID No.6中的第31位至第35位所示；更佳地，编码所述鼠源抗体重链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.5中的第91位至第105位所示；
    所述鼠源抗体重链可变区的CDR2的氨基酸序列如序列表SEQ ID No.6中的第50位至第66位所示；更佳地，编码所述鼠源抗体重链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.5中的第148位至第198位所示；
    所述鼠源抗体重链可变区的CDR3的氨基酸序列如序列表SEQ ID No.6中的第99位至第109位所示；更佳地，编码所述鼠源抗体重链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.5中的第295位至第327位所示；
    和/或，所述鼠源抗体轻链可变区的CDR1的氨基酸序列如序列表SEQ ID No.8中的第24位至第34位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR1的氨基酸序列的核苷酸序列如序列表SEQ ID No.7中的第70位至第102位所示；
    所述鼠源抗体轻链可变区的CDR2的氨基酸序列如序列表SEQ ID No.8中的第50位至第56位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR2的氨基酸序列的核苷酸序列如序列表SEQ ID No.7中的第148位至第168位所示；
    所述鼠源抗体轻链可变区的CDR3的氨基酸序列如序列表SEQ ID No.8中的第89位至第97位所示；更佳地，编码所述鼠源抗体轻链可变区的CDR3的氨基酸序列的核苷酸序列如序列表SEQ ID No.7中的第265位至第291位所示。
  3. 如权利要求2所述的人源化抗TPBG抗体，其特征在于，所述的人源化抗TPBG抗体包含至少一个重链可变区和/或至少一个轻链可变区，
    所述重链可变区的氨基酸序列如序列表中SEQ ID NO.2、SEQ ID NO.6、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.34、SEQ ID NO.36、SEQ ID NO.38或SEQ ID NO.40所示；所述的轻链可变区序列如序列表中SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.26、SEQ ID NO.28、SEQ ID NO.30、SEQ ID NO.32、SEQ ID NO.42、SEQ ID NO.44或SEQ ID NO.46所示；
    较佳地，编码所述重链可变区的氨基酸序列的核苷酸序列分别如序列表中SEQ ID No.1、SEQ ID No.5、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.33、SEQ ID No.35、SEQ ID No.37或SEQ ID No.39所示；编码所 述的轻链可变区的氨基酸序列的核苷酸序列分别如序列表中SEQ ID No.3、SEQ ID No.7、SEQ ID No.25、SEQ ID No.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.41、SEQ ID No.43或SEQ ID No.45所示；
    或者，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID NO.2、SEQ ID NO.6、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.34、SEQ ID NO.36、SEQ ID NO.38或SEQ ID NO.40所示的氨基酸序列至少有80％序列同源的氨基酸序列；所述的轻链可变区序列为，与如序列表中SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.26、SEQ ID NO.28、SEQ ID NO.30、SEQ ID NO.32、SEQ ID NO.42、SEQ ID NO.44或SEQ ID NO.46所示的氨基酸序列至少有80％序列同源的氨基酸序列所示；
    较佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.1、SEQ ID No.5、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.33、SEQ ID No.35、SEQ ID No.37或SEQ ID No.39所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.3、SEQ ID No.7、SEQ ID No.25、SEQ ID No.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.41、SEQ ID No.43或SEQ ID No.45所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列。
  4. 如权利要求3所述的人源化抗TPBG抗体，其特征在于，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.25所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.25所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.25所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.16所示的序列，且所述轻 链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.15所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.22所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.21所示的序列，且所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.27所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.29所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.29所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.29所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.16所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.15所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.18所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.17所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.20所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.19所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.22所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.21所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.24所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.23所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.31所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.33所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.36所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.35所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.37所示的序列，且编码所述轻链可变区的核苷酸序 列如序列表SEQ ID No.41所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.39所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.41所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.33所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.36所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.35所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.37所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.39所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.43所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.34所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.33所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.36所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.35所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.38所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；较佳地，编码所述重链可变 区的核苷酸序列如序列表SEQ ID No.37所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列；
    或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.40所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示的序列；较佳地，编码所述重链可变区的核苷酸序列如序列表SEQ ID No.39所示的序列，且编码所述轻链可变区的核苷酸序列如序列表SEQ ID No.45所示的序列。
  5. 如权利要求1所述的人源化抗TPBG抗体，其特征在于，所述的人源化抗TPBG抗体还包括人源抗体重链恒定区和/或人源抗体轻链恒定区。
  6. 如权利要求1所述的人源化抗TPBG抗体，其特征在于，所述的人源化抗TPBG抗体为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体或单区抗体；
    或者，所述的人源化抗TPBG抗体为单克隆抗体或多克隆抗体；
    或者，所述的人源化抗TPBG抗体为超人源化抗体或双抗体。
  7. 一种核酸，其特征在于，其编码如权利要求1-6任一项所述的人源化抗TPBG抗体；
    较佳地，其包括编码所述重链可变区的核酸，和/或，编码所述轻链链可变区的核酸；所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID NO.2、SEQ ID NO.6、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ ID NO.34、SEQ ID NO.36、SEQ ID NO.38或SEQ ID NO.40所示；更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.1、SEQ ID No.5、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.23、SEQ ID No.33、SEQ ID No.35、SEQ ID No.37或SEQ ID No.39；
    所述轻链可变区的核酸编码的氨基酸序列如序列表SEQ ID NO.4、SEQ ID NO.8、SEQ ID NO.26、SEQ ID NO.28、SEQ ID NO.30、SEQ ID NO.32、SEQ ID NO.42、SEQ ID NO.44或SEQ ID NO.46所示；更佳地，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.3、SEQ ID No.7、SEQ ID No.25、SEQ ID No.27、SEQ ID No.29、SEQ ID No.31、SEQ ID No.41、SEQ ID No.43或SEQ ID No.45所示。
  8. 如权利要求7所述的核酸，其特征在于，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.25所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.25所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.26所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.25所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.16所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.15所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.22所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.21所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.28所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.27所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷 酸序列如序列表SEQ ID No.29所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.29所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.30所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.29所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.16所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.15所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.18所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.17所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.19所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.22所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.21所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.32所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.23所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.31所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区 的核酸的核苷酸序列如序列表SEQ ID No.33所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.35所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.37所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.42所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.39所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.41所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.33所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.35所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.37所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.44所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.39所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.43所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.34所示，且所 述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.33所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.35所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.37所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示；
    或者，所述重链可变区的核酸编码的氨基酸序列如序列表SEQ ID No.40所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.46所示；较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.39所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.45所示。
  9. 一种包含如权利要求7或8所述的核酸的重组表达载体。
  10. 一种包含如权利要求9所述的重组表达载体的重组表达转化体。
  11. 一种如权利要求1-6任一项所述的人源化抗TPBG抗体的制备方法，其特征在于，其包括如下步骤：培养如权利要求10所述的重组表达转化体，从培养物中获得人源化抗TPBG抗体。
  12. 一种免疫偶联物，其特征在于，其包括共价附着至细胞毒剂的如权利要求1-6任一项所述的人源化抗TPBG抗体。
  13. 如权利要求12所述的免疫偶联物，其特征在于，1当量如权利要求1-6任一项所述的人源化抗TPBG抗体通过x当量接头与y当量细胞毒剂相连，其具有式1所示结构，
    Ab-(L) x-(D) y
    式1
    其中，Ab为如权利要求1-6中任一项所述的人源化抗TPBG抗体；L为接头；D为细胞毒剂；x为自然数，优选1-20的整数；y为0或自然数，优选0-20的整数；x和y各自独立地更优选为1-2，或2-4，或3～5，或4-8，或8-20的整数；x和y的比例优选为1:1。
  14. 如权利要求13所述的免疫偶联物，其特征在于，所述接头L为活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段。
  15. 如权利要求14所述的免疫偶联物，其特征在于，所述接头L选自马来酰亚胺基己酰(MC)、马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)、4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)；和/或，所述D选自细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶。
  16. 如权利要求12～15任一项所述的免疫偶联物，其特征在于，所述式1中x＝y＝n，所述免疫偶联物的结构如式3或者如式4或者如式5所示，
    Figure PCTCN2018081853-appb-100001
    式3中m为1～10，优选m为5，即马来酰亚胺基己酰；D为甲基奥瑞他汀F；
    Figure PCTCN2018081853-appb-100002
    式4中，L为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；D为N2’-脱乙酰-N2’-3-巯基-1氧代丙基)-美登素(DM1)；
    Figure PCTCN2018081853-appb-100003
    式5中，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇，D为甲基奥瑞他汀E(MMAE)；
    其中，n为自然数，优选为1～20的整数，更优选为1～2，或2～4，或3～5，或4～8，或8～20的整数。
  17. 一种药物组合物，其特征在于，其包括如权利要求12～16任一项所述的免疫偶联物和药学可接受的载体；所述的药物组合物较佳地还包括其他抗肿瘤抗体作为活性成分；所述的抗体优选抗PD-1抗体。
  18. 一种套装药盒，其特征在于，其包含成分A和成分B，所述的成分A为如权利要求1～6所述的人源化抗TPBG抗体、或者如权利要求12～16任一项所述的免疫偶联物、或者如权利要求17所述的药物组合物，所述的成分B为其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物；所述的其他抗体优选抗PD-1抗体。
  19. 一种如权利要求1～6所述的人源化抗TPBG抗体、或者如权利要求12～16任一项所述的免疫偶联物、或者如权利要求17所述的药物组合物或者如权利要求18所述的套装药盒在制备抗肿瘤药物中的应用。
  20. 一种检测过表达TPBG蛋白的细胞的方法，其特征在于，其包括以下的步骤：如权利要求1-6任一项所述的人源化抗TPBG抗体与待检样品在体外接触，检测如权利要求1-6任一项所述的人源化抗TPBG抗体与所述待检样品的结合即可。
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