WO2018064964A1 - 半胱氨酸改造的抗体-毒素偶联物及其制备方法 - Google Patents

半胱氨酸改造的抗体-毒素偶联物及其制备方法 Download PDF

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WO2018064964A1
WO2018064964A1 PCT/CN2017/104706 CN2017104706W WO2018064964A1 WO 2018064964 A1 WO2018064964 A1 WO 2018064964A1 CN 2017104706 W CN2017104706 W CN 2017104706W WO 2018064964 A1 WO2018064964 A1 WO 2018064964A1
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antibody
cysteine
pab
mmae
tdc
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PCT/CN2017/104706
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English (en)
French (fr)
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朱义
王一茜
卓识
李�杰
陈澜
万维李
余永国
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四川百利药业有限责任公司
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Priority to CA3039559A priority Critical patent/CA3039559A1/en
Priority to US16/339,719 priority patent/US11484605B2/en
Priority to IL265838A priority patent/IL265838B2/en
Priority to KR1020227044769A priority patent/KR102579211B1/ko
Priority to AU2017340314A priority patent/AU2017340314B2/en
Priority to SG11201903084UA priority patent/SG11201903084UA/en
Priority to EP17857845.6A priority patent/EP3524273A4/en
Priority to JP2019540482A priority patent/JP6998386B2/ja
Priority to RU2019113758A priority patent/RU2762594C2/ru
Priority to KR1020197013122A priority patent/KR102480873B1/ko
Publication of WO2018064964A1 publication Critical patent/WO2018064964A1/zh

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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
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    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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Definitions

  • the present invention relates to a compound and a process for the preparation thereof, and in particular to a cysteine engineered antibody-toxin conjugate and a process for the preparation thereof.
  • ADC Antibody-conjugates
  • the cysteine insertion site comprises one or more sites selected from the following three insertion sites: light chain position 205 ( Kabat number, its amino acid sequence is GLSSP C VTKSF), light chain position 206 (Kabat number, its amino acid sequence is GLSSPV C TKSF), heavy chain position 439 (Kabat number, its amino acid sequence is YTQKSLS C LSPGK).
  • An antibody comprising one or more of the above cysteine insertion mutations retains the ability (affinity) of binding of the parent antibody to the antigen.
  • the present invention performs site-directed coupling of antibody-toxin conjugate (TDC) with a linker-drug by a cysteine thiol group inserted at position 205 or/and position 206 of the light chain or/and position 439 of the heavy chain.
  • TDC antibody-toxin conjugate
  • the antibody is a cysteine site-incorporated antibody
  • the cysteine insertion site comprises one or more sites selected from the following three insertion sites: kappa/ ⁇ light chain
  • the constant region was at position 205 (Kabat numbering)
  • the light chain constant region was at position 206 (Kabat numbering)
  • the IgG antibody heavy chain constant region was at position 439 (Kabat numbering).
  • the cysteine insertion site amino acid sequence comprises one or more of the following three sequences: LC-205ins: GLSSP C VTKSF; LC-206ins: GLSSPV C TKSF or HC-439ins: TQKSLS C LSPGK.
  • the highly active cytotoxin is site-coupled to the inserted cysteine free thiol (-SH) via a linker.
  • the linker is coupled to the inserted cysteine.
  • the light chain amino acid sequence of the antibody is: GLSSP C VTKSF and GLSSPV C TKSF
  • the amino acid sequence of the linker and the inserted cysteine-conjugated antibody heavy chain is: TQKSLS C LSPGK, wherein C is the cysteine inserted at position 205 or position 206 of the target antibody light chain or position 439 of the heavy chain .
  • said antibody light chain comprises a kappa or lambda isoform.
  • the antibody heavy chain comprises an IgGl, IgG2, IgG3 or IgG4 isotype.
  • cysteine comprises a thiol group (-SH).
  • the thiol group (-SH) is capable of chemical coupling.
  • the small molecule highly active cytotoxins that are site-coupled by a linker with a cysteine insertion mutant free sulfhydryl group include, but are not limited to, MMAE, MMAF, PBD, SN-38, Dox, and the like, MMAE, MMAF , PBD, SN-38 and Dox molecular formula are:
  • the main site-directed coupling step is: first reducing the antibody with a reducing agent (such as DTT, TCEP, etc.), removing the shielding on the modified cysteine residue on the antibody, and removing it by cation exchange chromatography or ultrafiltration.
  • a reducing agent such as DTT, TCEP, etc.
  • the DTT is shielded and then oxidized with an oxidizing agent (such as DHAA, CuSO4, etc.) to religate the interchain disulfide bonds of the antibody.
  • Linker-drug is coupled to the free sulfhydryl group on the cystine residue, and the unconjugated antibody molecule Linker- is removed by cation exchange chromatography or ultrafiltration exchange. Drug.
  • GLSSP C VTKSFN sequence to mark the point C of conjugation sites, site-directed coupling with mc-vc-PAB-payload.
  • GLSSPV C TKSFN sequence to mark the point C of conjugation sites, site-directed coupling with mc-vc-PAB-payload.
  • TQKSLS C LSPGK sequence to mark the point C of conjugation sites, site-directed coupling with mc-vc-PAB-payload.
  • GLSSP C TKSFN sequence to mark the point C of conjugation sites, site-directed coupling with mc-vc-PAB-payload.
  • TDC novel cysteine-modified antibody-toxin conjugate
  • Figure 1 is a graph showing the results of HIC-HPLC detection of 2A1-LC-Cys205ins-mc-vc-PAB-MMAE in Example 25.
  • FIG. 2 is a graph showing the results of HIC-HPLC detection of 2A1-LC-Cys206ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 3 is a graph showing the results of HIC-HPLC detection of 2A1-HC-Cys439ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 4 is a graph showing the results of HIC-HPLC detection of 4E1-LC-Cys205ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 5 is a graph showing the results of HIC-HPLC detection of 4E1-LC-Cys206ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 6 is a graph showing the results of HIC-HPLC detection of 4E1-HC-Cys439ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 7 is a graph showing the results of HIC-HPLC detection of 4D3-LC-Cys205ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 8 is a graph showing the results of HIC-HPLC detection of 4D3-LC-Cys206ins-mc-vc-PAB-MMAE in Example 25.
  • Figure 9 is a graph showing the results of RP-HPLC detection of the ratio of 4D3-HC-Cys439ins-mc-vc-PAB-MMAE toxin to antibody in Example 26.
  • Fig. 10 is a graph showing the results of aggregation of TDC antibody skeleton 4D3 by SEC-HPLC in Example 27.
  • Figure 11 is a graph showing the results of aggregation of TDC antibody backbone 4D3-LC-Cys205ins by SEC-HPLC in Example 27.
  • Figure 12 is a graph showing the results of aggregation of TDC antibody backbone 4D3-LC-Cys206ins by SEC-HPLC in Example 27.
  • Figure 13 is a graph showing the results of aggregation of TDC antibody backbone 4D3-HC-Cys439ins by SEC-HPLC in Example 27.
  • Figure 14 is a graph showing the results of the experiment in Example 28.
  • Figure 15 is a graph showing the results of affinity of 4E1-LC-Cys205ins-MVPM, 4E1-LC-Cys206ins-MVPM, 4E1-HC-Cys439ins-MVPM antibody and antigen c-met in Example 29.
  • Figure 16 is a graph showing the results of affinity of TDC 4D3-LC-Cys205ins-MVPM, 4D3-LC-Cys206ins-MVPM, 4D3-HC-Cys439ins-MVPM antibody and antigen Trop2 in Example 29.
  • Figure 17 is 2A1-LC-V205C-mc-vc-PAB-MMAE, 2A1-LC-Cys205ins-mc-vc-PAB-MMAE, 2A1-LC-Cys206ins-mc-vc-PAB-MMAE, 2A1-HC-Cys439ins -mc-vc-PAB-MMA
  • Figure 18 is 2A1-LC-V205C-mc-vc-PAB-MMAE, 2A1-LC-Cys205ins-mc-vc-PAB-MMAE, 2A1-LC-Cys206ins-mc-vc-PAB-MMAE, 2A1-HC-Cys439ins -mc-vc-PAB-MMAE
  • IC50 detection of human glioma cell line U87-EGFRvIII overexpressing FGRvIII The results of IC50 detection of human glioma cell line U87-EGFRvIII overexpressing FGRvIII.
  • Figure 19 is 4E1-LC-Cys205ins-mc-vc-PAB-MMAE, 4E1-LC-Cys206ins-mc-vc-PAB-MMAE, 4E1-HC-Cys439ins-mc-vc-PAB-MMAE, 4E1 vs. c-Met
  • Figure 20 is a high expression of trop2 by D3-LC-Cys205ins-mc-vc-PAB-MMAE, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE, 4D3-HC-Cys439ins-mc-vc-PAB-MMAE, 4D3 Figure 50 shows the results of IC50 detection of pancreatic cancer cell line BXPC-3.
  • Figure 21 is a graph showing the results of investigation of the stability of 4D3-LC-Cys205ins-mc-vc-PAB-MMAE in human plasma.
  • Figure 22 is a graph showing the results of investigation of the stability of 4D3-LC-Cys206ins-mc-vc-PAB-MMAE in human plasma.
  • Figure 23 is a graph showing the results of investigation of the stability of 4D3-HC-Cys439ins-mc-vc-PAB-MMAE in human plasma.
  • Figure 24 is a graph showing the results of investigation of the stability of 4E1-LC-Cys205ins-mc-vc-PAB-MMAE in human plasma.
  • Figure 25 is a graph showing the results of investigation of the stability of 4E1-LC-Cys206ins-mc-vc-PAB-MMAE in human plasma.
  • Figure 26 is a graph showing the results of investigation of the stability of 4E1-HC-Cys439ins-mc-vc-PAB-MMAE in human plasma.
  • Figure 27 is a 4D3-LC-Cys205ins-mc-vc-PAB-MMAE, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE, 4D3-HC-Cys439ins-mc-vc-PAB-MMAE, 4D3 parent antibody The results of the test results of tumor mice.
  • Figure 28 is a 4D3-LC-Cys205ins-mc-vc-PAB-MMAE, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE, 4D3-HC-Cys439ins-mc-vc-PAB-MMAE, 4D3 parent antibody The results of the test results of tumor mice.
  • Fmoc-vc-PABOH 490 mg (0.815 mmol) was added to 10 ml of NMP and stirred to dissolve, and then 2 ml of diethylamine was added. The reaction was stirred at room temperature for 24 h. After completion of the reaction, the mixture was concentrated under reduced pressure at 40 ° C. EtOAc (EtOAc) m/z: 380.2 (M+H)+.
  • VP-PABOH 205 mg (0.54 mmol) and MC-OSu 184 mg (1.1 eq) were added to 10 ml of NMP, and the reaction was stirred at room temperature for 24 h. After completion of the reaction, the mixture was concentrated under reduced pressure at 40 ° C. Filtration and washing of the cake with methyl tert-butyl ether afforded 310 mg of product in 100% yield. m/z: 573.3 (M+H)+.
  • Example 9 According to the operation of Example 9, about 12.5 mg of mc-vc-PAB-MMAF was obtained, yield 45.2%, m/z: 1345.7 (M+H)+.
  • Example 9 According to the operation of Example 9, 9.5 mg of mc-vc-PAB-PBD was obtained in a yield of 32.5%, m/z: 1325.4 (M+H)+.
  • Example 9 According to the operation of Example 9, about 11.2 mg of mc-vc-PAB-DOX was obtained, yield 38.9%, m/z: 1143.2 (M+H)+.
  • Use Freestyle TM 293-F was suspended cells expressing the antibody.
  • cells were seeded at a density of 6 ⁇ 105 cells/mL in a 1 L shake flask containing 300 mL of F17 complete medium (FreestyleTM F17 expression medium, Gibco), shaken at 37 ° C, 5% CO 2 , 120 rpm. The bed was incubated overnight.
  • transfection of the antibody expression plasmid was carried out with PEI, wherein the ratio of plasmid: PEI was 2:1.
  • the TN1 feed medium was added at 2.5% (v/v), and the culture was continued for 4 days, and the supernatant was collected by centrifugation.
  • the collected cell expression supernatant was eluted by a Protein A affinity chromatography column (Mabselect Sure LX, GE) eluting with 0.1 M citric acid (pH 3.0), and the captured antibody was treated with 1 M Tris-HCl (pH 9.0). Adjust to pH 7.0 at 1/10 (v/v), and remove impurities such as multimers and endotoxin by gel filtration column SEC (Superdex 200, GE), and replace the antibody buffer with PBS. (pH 7.4), a sample of the target peak of UV280 nm was collected and concentrated to 2 mg/ml through an ultrafiltration centrifuge tube (30 KD, Pall Corporation). The target antibody monomer (POI%) obtained by this method was greater than 90% for subsequent experiments.
  • the 2A1-HC-Cys439ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after the low pH elution, it was neutralized by adding Tris solution, and the solution was changed to a pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 2A1-HC-Cys439ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 2A1-LC-Cys205ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after the low pH elution, it was neutralized by adding Tris solution, and the solution was changed to a pH 7.5 Tris-HCl buffer.
  • a 1 M aqueous solution of DTT was added to the 2A1-LC-Cys205ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 2A1-LC-Cys206ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after the low pH elution, it was neutralized by adding Tris solution, and the solution was changed to a pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 2A1-LC-Cys206ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 4D3-HC-Cys439ins antibody expressed by the cells was purified by Mabselect Sure, immediately after low pH elution, and added to Tris solution for neutralization, and the solution was changed to pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 4D3-HC-Cys439ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0 and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography. Subsequently, the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds. Then, mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 4D3-LC-Cys205ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after neutralization with low pH, neutralized with Tris solution, and changed to a pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 4D3-LC-Cys205ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 4D3-LC-Cys206ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after the low pH elution, the Tris solution was neutralized and the solution was changed to a pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 4D3-LC-Cys206ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution so that mc-vc-PAB-MMAE was coupled with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 4E1-HC-Cys439ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after the low pH elution, it was neutralized by adding Tris solution, and the solution was changed to a pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 4E1-HC-Cys439ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 4E1-LC-Cys205ins antibody expressed by the cells was purified by Mabselect Sure, immediately after the low pH elution, and added to the Tris solution for neutralization, and the solution was changed to a pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 4E1-LC-Cys205ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the 4E1-LC-Cys206ins antibody expressed by the cells was purified by Mabselect Sure, and immediately after low pH elution, it was added to Tris solution for neutralization, and the solution was changed to pH 7.5 Tris-HCl buffer.
  • Compound mc-vc-PAB-MMAE, white powder, was dissolved in DMA for use. In order to remove the shield on the mutant cysteine residue, the antibody needs to be reduced first.
  • a 1 M aqueous solution of DTT was added to the 4E1-LC-Cys206ins antibody solution at a molecular ratio of 40 times, and the mixture was reacted at 20 ° C for 2 hours.
  • the pH of the sample was adjusted to 5.0, and the DTT and the shield in the sample were removed by SP Sepharose F.F. cation exchange chromatography.
  • the DHAA solution was added to the sample at a molecular ratio of 20 times, and reacted at 25 ° C for 4 hours in the dark to recombine the interchain disulfide bonds.
  • mc-vc-PAB-MMAE solution was added to couple mc-vc-PAB-MMAE with the antibody mutant cysteine, and the mixture was thoroughly mixed and reacted at 25 ° C for 2 hours.
  • the TDC sample was analyzed by high performance liquid chromatography with hydrophobic chromatography and DAR was calculated from the corresponding peak area.
  • the specific method is as follows:
  • Mobile phase A: 2M ammonium sulfate, 0.025 M, pH 7 phosphate buffer; B: 0.025 M, pH 7 phosphate buffer; C: 100% isopropanol;
  • Buffer A equilibrated, buffer B and buffer C gradient elution, detection at 25 ° C, 214 nm and 280 wavelengths.
  • the fixed-point coupled DAR is calculated to be between 1.6 and 1.7, and the compound uniformity is good.
  • the fixed-point coupled DAR is calculated to be between 1.60-1.95, and the compound uniformity is very good.
  • the fixed-point coupled DAR is calculated between 1.60-1.90, and the compound uniformity is very good.
  • the ratio of toxin to antibody was measured by RP-HPLC.
  • the sample treated with DTT was analyzed by reversed-phase hydrophobic high performance liquid chromatography, and DAR was calculated from the corresponding peak area.
  • the specific method is as follows:
  • Mobile phase A and mobile phase B were eluted in a proportional gradient at 80 ° C, 214 nm and 280 wavelengths.
  • the fixed-point coupled DAR was calculated to be 1.82, and the compound uniformity was excellent.
  • Table 1 shows that the coupling efficiency of site-directed TDC compounds by cysteine insertion mutation modification is high (theoretical maximum is 2.0), DAR ⁇ 1.6.
  • TDC antibody skeleton samples were stored at 37 ° C, and their aggregation was analyzed by SEC-HPLC on days 0, 7, 21, and 29, respectively.
  • SEC-HPLC SEC-HPLC on days 0, 7, 21, and 29, respectively.
  • Mobile phase 0.1 M sodium sulfate, 0.1 M, pH 6.7 phosphate buffer.
  • SEC-HPLC was used to detect the aggregation of TDC antibody backbones 4D3, 4D3-LC-Cys205ins, 4D3-LC-Cys206ins and 4D3-HC-Cys439ins.
  • the samples were stored at 37 ° C for 4 weeks, and the aggregate content remained basically unchanged. .
  • Table 2 shows that the target monomer content of the TDC compound coupled by cysteine is above 90%.
  • Example 28 Skeletal antibody and parental antibody 2A1 modified by cysteine site-directed mutagenesis and insertional mutagenesis Affinity for EGFRvIII, affinity of 4E1 for c-met, affinity of 4D3 for Trop2.
  • Recombinant EGFRvIII-His*6 antigen-coated plate fish skin gelatin blocked; diluted 2A1, 2A1-LC-V205C, 2A1-LC-Cys205ins, 2A1-LC-Cys206ins and 2A1-HC-Cys439ins, respectively, the highest concentration 10ug/ml, 4 Dilute gradient dilution, a total of 11 concentrations; HRP-labeled secondary antibody incubation; TMB coloration, detection of absorption at 450 nm.
  • 2A1-LC-V205C, 2A1-LC-Cys205ins, 2A1-LC-Cys206ins, 2A1-HC-Cys439ins antibodies retained the affinity of 2A1 to the antigen EGFRvIII.
  • Example 29 Affinity of a backbone antibody engineered with a cysteine site-directed mutagenesis and an insertional mutation and TDC for the corresponding antigen. 4E1 affinity for c-met, 4D3 affinity for Trop2.
  • Recombinant c-met-His*10 antigen-coated plate fish skin gelatin closed; diluted 4E1-LC-Cys205ins-MVPM, 4E1-LC-Cys206ins-MVPM, 4E1-HC-Cys439ins-MVPM and 4E1, respectively, the highest concentration 10ug/ml , 4 times gradient dilution, a total of 11 concentrations; HRP-labeled secondary antibody incubation; TMB coloration, detection of absorption at 450 nm.
  • 4E1-LC-Cys205ins-MVPM, 4E1-LC-Cys206ins-MVPM, 4E1-HC-Cys439ins-MVPM antibodies retained the affinity of 4E1 for antigen c-met.
  • 4D3-LC-Cys205ins-MVPM, 4D3-LC-Cys206ins-MVPM, 4D3-HC-Cys439ins-MVPM antibodies maintained the affinity of 4D3 for the antigen Trop2.
  • TDC cytotoxic activity was determined by the following experimental procedure: TDC was separately added to human tumor cell culture medium in which EGFR was overexpressed or EGFRVIII was expressed, and cell viability was measured after 72 hours of cell culture. Cell-based in vitro assays were used to determine cell viability, cytotoxicity, and TDC-induced apoptosis in the present invention.
  • CellTiter Solution Cell Proliferation Assay is commercially available (Promega Corp., Madison, WI).
  • CellTiter AQueous One Solution Cell Proliferation Assay (a) is a detection reagent for colorimetric detection of the number of viable cells in cell proliferation and cytotoxicity experiments.
  • This reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and An electronic coupling agent (phenazine ethosulfate; PES).
  • PES has enhanced chemical stability which allows it to be mixed with MTS to form a stable solution.
  • This convenient "single solution" mode is in the first generation of CellTiter Improvements based on Assay, CellTiter
  • the electron coupling agent PMS used in Assay is supplied separately from the MTS solution.
  • MTS (Owen's reagent) is biologically reduced to a colored formazan product that is directly soluble in the medium ( Figure 1). This transformation is most likely accomplished by the action of NADPH or NADH produced by dehydrogenase in metabolically active cells.
  • NADPH NADPH
  • NADH NADH
  • the amount of formazan product detected at 490 nm is directly proportional to the number of viable cells in the culture. Since the MTS hyperthyroid product is soluble in tissue culture medium, CellTiter The AQueous One Solution Assay has fewer steps than the MTT or INT method.
  • A431 EGFR overexpressing cells
  • U87-EGFRVIII EGFR mutant stable cell line
  • U87-MG highly expressed glioblastoma cell line in c-Met
  • BXPC-3 high expression of trop2
  • the pancreatic cancer cell line is used as a research system for in vitro drug efficacy testing.
  • cell plating was performed at 6000/well, and after 24 hours, antibody administration was performed.
  • the initial concentrations of various TDCs corresponding to A431, U87-EGFRVIII cell lines were 10 uM, which were sequentially diluted according to a 5-fold gradient.
  • the initial concentrations of various TDCs corresponding to U87-MG and BXPC-3 cell lines were 1 uM, which were sequentially diluted according to a 5-fold gradient.
  • MTS assayed for cell activity after 72 hours of treatment.
  • ADC sample Take a certain amount of ADC sample, add it to human plasma with human IgG removed, repeat 2 tubes of each ADC, incubate in 37 °C water bath, incubate for 0h, 72h, take ADC samples, add ProteinA (MabSelect) SuReTM LX Lot: #10221479GE, washed with PBS 100 ul, shaken by a vertical mixer for 2 h, and subjected to a washing and elution step to obtain an ADC after incubation.
  • the ADC samples of the specific time of incubation were subjected to HIC-HPLC and RP-HPLC to determine the plasma stability of the samples.
  • TDC was relatively stable after being incubated at 37 ° C for 72 hours in human plasma samples, and had good drug-forming properties.
  • TDC with 439 insertion mutations had the best stability, followed by TDC with 205 and 206 insertion mutations.
  • a BXPC-3 tumor-bearing mouse model was established to evaluate the in vivo efficacy of TDC and parental antibodies. That is, 3 ⁇ 106 BXPC-3 cells were subcutaneously injected into the back side of 4-8 weeks old BALB/c nude mice, and the average tumor size of the mice was grown to 400-500 mm3, randomly grouped, 5 rats in each group.
  • 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC and 4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC was administered in a single intravenous dose at a dose of 5 mg/kg, and the parent antibody 4D3 was administered at a dose of 5 mg/kg.
  • Data A is shown as the mean tumor volume ⁇ SE at the time of measurement
  • data B is shown as the average body weight of the mouse at the time of measurement ⁇ SE.

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Abstract

将目标抗体的重链和轻链定点***半胱氨酸(C)，通过定点***的半胱氨酸的游离巯基(-SH)与偶联了小分子高活性细胞毒素的连接子进行定点偶联，形成均一性优良的半胱氨酸改造的抗体-毒素偶联物。所述半胱氨酸***位点为抗体轻链第205位和/或第206位(Kabat编号)，和/或重链第439位(Kabat编号)。

Description

半胱氨酸改造的抗体-毒素偶联物及其制备方法 技术领域
本发明涉及一种化合物及其制备方法，特别涉及一类半胱氨酸改造的抗体-毒素偶联物及其制备方法。
技术背景
抗体-毒素偶联物(Antibody drug conjugate,ADC)是靶向治疗的热点领域，已在美国获批上市的两个药物Adcetris和Kadcyla表现出良好的临床疗效，并且有超过50个ADC药物在进行临床阶段研究。
发明内容
本发明的化合物半胱氨酸改造的抗体-毒素偶联物(TDC)，半胱氨酸***位点包含选自以下3个***位点的一个或多个位点：轻链第205位(Kabat编号，其氨基酸序列为GLSSPCVTKSF)，轻链第206位(Kabat编号，其氨基酸序列为GLSSPVCTKSF)，重链第439位(Kabat编号，其氨基酸序列为YTQKSLSCLSPGK)。包含一个或多个上述半胱氨酸***突变的抗体保持了其亲本抗体与抗原结合的能力(亲和力)。本发明通过其轻链第205位或/和第206位或/和重链第439位***的半胱氨酸巯基与linker-drug进行抗体-毒素偶联物(TDC)定点偶联。
氨酸改造的抗体-毒素偶联物，抗体为半胱氨酸定点***抗体，半胱氨酸***位点包含选自以下3个***位点的一个或多个位点：kappa/λ轻链恒定区第205位(Kabat编号)，轻链恒定区第206位(Kabat编号)或IgG抗体重链恒定区第439位(Kabat编号)。
半胱氨酸***位点氨基酸序列包含以下3个序列的一个或多个：LC-205ins:GLSSPCVTKSF；LC-206ins:GLSSPVCTKSF或HC-439ins:TQKSLSCLSPGK。
高活性细胞毒素通过连接子与***的半胱氨酸游离巯基(-SH)进行定点偶联，连接子与***的半胱氨酸偶联的抗体轻链氨基酸序列为：GLSSPCVTKSF和GLSSPVCTKSF，连接子与***的半胱氨酸偶联的抗体重链氨基酸序列为：TQKSLSCLSPGK，其中，C为目标抗体轻链205位或206位或重链第439位***的半胱氨酸。
作为优选，其中所述抗体轻链包括kappa或λ同种型。
作为优选，其中所述抗体重链包括IgG1、IgG2、IgG3或IgG4同种型。
作为优选，其中所述半胱氨酸包含巯基(-SH)。
作为优选，其中巯基(-SH)能够进行化学偶联。
作为优选，通过连接子与半胱氨酸***突变的游离巯基进行定点偶联的小分子高活性细胞毒素包括但不限于MMAE、MMAF、PBD、SN-38、Dox及其类似物，MMAE、MMAF、PBD、SN-38及Dox分子式为：
Figure PCTCN2017104706-appb-000001
主要定点偶联步骤为：首先使用还原剂(如DTT、TCEP等)还原抗体，解除抗体上改造的半胱氨酸残基上的屏蔽，并通过阳离子交换层析或超滤换液等方式去除DTT与屏蔽物，然后使用氧化剂(如DHAA、CuSO4等)氧化抗体，使抗体的链间二硫键重新连接。最后加入Linker-drug与胱氨酸残基上的游离巯基偶联，并通过阳离子交换层析或超滤换液等方式去除未偶联上抗体分子Linker- drug。
本发明氨基酸简表：
中文名称 英文名称 符号与缩写
丙氨酸 Alanine A或Ala
精氨酸 Arginine R或Arg
天冬酰胺 Asparagine N或Asn
天冬氨酸 Aspartic acid D或Asp
半胱氨酸 Cysteine C或Cys
谷氨酰胺 Glutamine Q或Gln
谷氨酸 Glutamic acid E或Glu
甘氨酸 Glycine G或Gly
组氨酸 Histidine H或His
异亮氨酸 Isoleucine I或Ile
亮氨酸 Leucine L或Leu
赖氨酸 Lysine K或Lys
蛋氨酸 Methionine M或Met
苯丙氨酸 Phenylalanine F或Phe
脯氨酸 Proline P或Pro
丝氨酸 Serine S或Ser
苏氨酸 Threonine T或Thr
色氨酸 Tryptophan W或Trp
酪氨酸 Tyrosine Y或Tyr
缬氨酸 Valine V或Val
SEQ ID NO:6 LC-Cys205ins轻链恒定区(Kappa)氨基酸序列
>LC-Cys205ins-Kappa
Figure PCTCN2017104706-appb-000002
其中，GLSSPCVTKSFN序列中标示出的C为定点偶联位点，与mc-vc-PAB-payload进行定点偶联。
SEQ ID NO:8 LC-Cys206ins轻链恒定区(Kappa)氨基酸序列
>LC-Cys206ins-Kappa
Figure PCTCN2017104706-appb-000003
其中，GLSSPVCTKSFN序列中标示出的C为定点偶联位点，与mc-vc-PAB-payload进行定点偶联。
SEQ ID NO:10 IgG1-Fc-Cys439ins重链恒定区(Fc)氨基酸序列
>IgG1-Fc-Cys439ins
Figure PCTCN2017104706-appb-000004
其中，TQKSLSCLSPGK序列中标示出的C为定点偶联位点，与mc-vc-PAB-payload进行定点偶联。
SEQ ID NO:12 LC-V205C轻链恒定区(Kappa)氨基酸序列
>LC-V205C-Kappa
Figure PCTCN2017104706-appb-000005
其中，GLSSPCTKSFN序列中标示出的C为定点偶联位点，与mc-vc-PAB-payload进行定点偶联。
本发明披露的全新半胱氨酸改造的抗体-毒素偶联物(TDC)相较非定点偶联的ADC具有药物均一性好，副作用小等优点，临床前研究结果显示其显著优于非定点偶联ADC。
附图说明
图1为实施例25中HIC-HPLC检测2A1-LC-Cys205ins-mc-vc-PAB-MMAE的结果图。
图2为实施例25中HIC-HPLC检测2A1-LC-Cys206ins-mc-vc-PAB-MMAE的结果图。
图3为实施例25中HIC-HPLC检测2A1-HC-Cys439ins-mc-vc-PAB-MMAE的结果图。
图4为实施例25中HIC-HPLC检测4E1-LC-Cys205ins-mc-vc-PAB-MMAE的结果图。
图5为实施例25中HIC-HPLC检测4E1-LC-Cys206ins-mc-vc-PAB-MMAE的结果图。
图6为实施例25中HIC-HPLC检测4E1-HC-Cys439ins-mc-vc-PAB-MMAE的结果图。
图7为实施例25中HIC-HPLC检测4D3-LC-Cys205ins-mc-vc-PAB-MMAE的结果图。
图8为实施例25中HIC-HPLC检测4D3-LC-Cys206ins-mc-vc-PAB-MMAE的结果图。
图9为实施例26中RP-HPLC检测4D3-HC-Cys439ins-mc-vc-PAB-MMAE毒素比抗体比例的结果图。
图10为实施例27中SEC-HPLC检测TDC抗体骨架4D3的聚集情况结果图。
图11为实施例27中SEC-HPLC检测TDC抗体骨架4D3-LC-Cys205ins的聚集情况结果图。
图12为实施例27中SEC-HPLC检测TDC抗体骨架4D3-LC-Cys206ins的聚集情况结果图。
图13为实施例27中SEC-HPLC检测TDC抗体骨架4D3-HC-Cys439ins的聚集情况结果图。
图14为实施例28中实验结果图。
图15为实施例29中4E1-LC-Cys205ins-MVPM、4E1-LC-Cys206ins-MVPM、4E1-HC-Cys439ins-MVPM抗体与抗原c-met的亲和力结果图。
图16为实施例29中TDC 4D3-LC-Cys205ins-MVPM、4D3-LC-Cys206ins-MVPM、4D3-HC-Cys439ins-MVPM抗体与抗原Trop2的亲和力结果图。
图17为2A1-LC-V205C-mc-vc-PAB-MMAE、2A1-LC-Cys205ins-mc-vc-PAB-MMAE、2A1-LC-Cys206ins-mc-vc-PAB-MMAE、2A1-HC-Cys439ins-mc-vc-PAB-MMA对EGFRwt过表达的人皮肤鳞癌细胞A431的IC50检测结果图。
图18为2A1-LC-V205C-mc-vc-PAB-MMAE、2A1-LC-Cys205ins-mc-vc-PAB-MMAE、2A1-LC-Cys206ins-mc-vc-PAB-MMAE、2A1-HC-Cys439ins-mc-vc-PAB-MMAE对FGRvIII过表达的人脑胶质瘤细胞株U87-EGFRvIII的IC50检测结果图。
图19为4E1-LC-Cys205ins-mc-vc-PAB-MMAE、4E1-LC-Cys206ins-mc-vc-PAB-MMAE、4E1-HC-Cys439ins-mc-vc-PAB-MMAE、4E1对c-Met中高度表达的恶性胶质瘤细胞株U87-MG的IC50检测结果图。
图20为D3-LC-Cys205ins-mc-vc-PAB-MMAE、4D3-LC-Cys206ins-mc-vc-PAB-MMAE、4D3-HC-Cys439ins-mc-vc-PAB-MMAE、4D3对trop2高度表达的胰腺癌细胞株BXPC-3的IC50检测结果图。
图21为4D3-LC-Cys205ins-mc-vc-PAB-MMAE在人血浆中稳定性的考察结果图。
图22为4D3-LC-Cys206ins-mc-vc-PAB-MMAE在人血浆中稳定性的考察结果图。
图23为4D3-HC-Cys439ins-mc-vc-PAB-MMAE在人血浆中稳定性的考察结果图。
图24为4E1-LC-Cys205ins-mc-vc-PAB-MMAE在人血浆中稳定性的考察结果图。
图25为4E1-LC-Cys206ins-mc-vc-PAB-MMAE在人血浆中稳定性的考察结果图。
图26为4E1-HC-Cys439ins-mc-vc-PAB-MMAE在人血浆中稳定性的考察结果图。
图27为4D3-LC-Cys205ins-mc-vc-PAB-MMAE、4D3-LC-Cys206ins-mc-vc-PAB-MMAE、4D3-HC-Cys439ins-mc-vc-PAB-MMAE、4D3亲本抗体在荷瘤小鼠药效上测试结果图。
图28为4D3-LC-Cys205ins-mc-vc-PAB-MMAE、4D3-LC-Cys206ins-mc-vc-PAB-MMAE、4D3-HC-Cys439ins-mc-vc-PAB-MMAE、4D3亲本抗体在荷瘤小鼠药效上测试结果图。
具体实施方式
实施例1 mc的合成
Figure PCTCN2017104706-appb-000006
于30ml冰醋酸中加入6-氨基己酸3.9g(0.03mol)和1.2eq的马来酸酐3.5g(0.036mol)。反应液于120℃搅拌反应4～6h。反应完毕后，停止加热，自 然冷却到室温。60℃减压浓缩除去大部分醋酸。所得棕黄色粘稠液倒入水中，再加入乙酸乙酯20ml×3萃取，合并有机层。有机层依次用水、饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到棕黄色油状物，加入50ml水搅拌，有类白色固体析出，过滤，50℃减压干燥的目标产品5.08g，收率80％。mp：89-92℃。m/z：212.2[M+H]+。1HNMR(400Mz,DMSO)：13.21(br,1H,COOH)、6.75(s,2H,COCH＝CHCO)、3.63(t,2H,J＝7.2Hz,NCH2CH2)、2.42(t,2H,J＝7.4Hz,CH2COOH)、1.52-1.68(m,4H,NCH2CH2CH2CH2)、1.30-1.42(m,2H,NCH2CH2CH2CH2)。
实施例2 Mc-OSu的合成
Figure PCTCN2017104706-appb-000007
在氮气保护下于50ml乙腈中加入4.7g(22mmol)MC和25g(22mmol)HOSu。另取4.5g(22mmol)DCC溶于25ml乙腈中，保持内温在0℃左右，将其缓慢滴入反应液中。反应液于0℃反应2小时后再室温反应过夜。过滤，滤饼用乙腈10ml×3洗涤，滤液减压浓缩至干。所得油状物于室温减压干燥6h得到浅棕色固体6.4g，收率95％。(不纯化直接投下一步反应)m/z：309.2[M+H]+。1HNMR(400Mz,CDCl3)：1～2(m,6H,CCH2CH2CH2C)、2.68(t,2H,CH2CO)，2.95(s,4H,COCH2CH2CO)、3.68(t,2H,CH2N)、6.81(s,2H,CH＝CH)。
实施例3 Fmoc-Val-OSu的合成
Figure PCTCN2017104706-appb-000008
于100mlTHF中加入Fmoc-Val 10g和HOSu 3.4g。另取DCC6g溶于50ml乙腈 中，保持内温在0℃左右，将其缓慢滴入反应液中。反应液于室温搅拌反应24小时。过滤，滤饼用THF洗涤，滤液减压浓缩得到透明油状物。油状物未经纯化直接投下一步反应。m/z：437.4[M+H]+。
实施例4.Fmoc-vc的合成
Figure PCTCN2017104706-appb-000009
于20mlTHF中加入Cit 4.0g(1.05eq)和碳酸氢钠的水溶液60ml(NaHCO32g,1.05eq)。另取22.35mmolFmoc-Val-OSu溶于60mlDME中，再将其加入反应液中。反应液于室温下搅拌反应24小时。反应完毕后，向体系中加入15％柠檬酸水溶液110ml，然后再用EA萃取两次，合并有机层，减压浓缩得到白色固体。并向白色固体中加入甲基叔丁基醚100ml搅洗，过滤，滤饼于40℃减压干燥4h得产品4.83g，收率65％。m/z：497.6(M+H)+。1HNMR(400Mz,DMSO):0.92(6H,m)、1.35～1.65(4H,m)、2.10(1H,m)、3.01(2H,q)、3.99(1H,t)、4.01-4.45(2H,m)、4.45(2H,t)、5.46(2H,br)、6.03(1H,t)、7.20-8.02(8H,m)、8.25(1H,d)。
实施例5.Fmoc-vc-PABOH的合成
Figure PCTCN2017104706-appb-000010
于反应瓶中加入DCM/MeOH＝2/1混合溶剂60ml，再加入Fmoc-vc 2g(4.2mmol)和PABOH 1.04g(2eq)，搅拌溶解部分后再加入EEDQ 2.0g(2eq)。反应体系在室温条件下避光搅拌反应2.0d。反应完毕后，40℃减压浓缩得到白色固体。收集白色固体，加入甲基叔丁基醚100ml搅洗，过滤，滤饼用甲基叔丁基醚洗涤，所得白色固体40℃减压干燥得到2.2g，收率约88％。m/z：602.6 (M+H)+。1HNMR(400Mz,DMSO):0.95(6H,m)、1.45～1.69(4H,m)、2.10(1H,m)、3.11(2H,m)、3.99(1H,m)、4.30(2H,d)、4.05～-4.66(2H,m)、4.55(2H,d)、5.21(1H,t)、5.51(2H,br)、6.11(1H,t)、7.09-8.10(12H,m)、8.21(1H,d)、10.51(1H,br)。
实施例6.vc-PABOH的合成
Figure PCTCN2017104706-appb-000011
于10mlNMP中加入Fmoc-vc-PABOH 490mg(0.815mmol)搅拌溶解，再加入二乙胺2ml。于室温下搅拌反应24h。反应完毕后，于40℃减压浓缩，所得油状物中加入20mlDCM搅拌析晶，过滤，滤饼用DCM洗涤，所得固体减压干燥得到277mg,收率90％。m/z：380.2(M+H)+。1HNMR(400Mz,DMSO):0.89(6H,m),1.31～1.61(4H,m),1.82(1H,m),2.86(1H,m),2.89(2H,d),4.38(2H,d),4.44(1H,m),5.01(1H,br),5.35(2H,br),5.84(1H,br),7.14(2H,d),7.42(2H,d),8.08(1H,br),9.88(1H,br)。
实施例7.mc-vc-PABOH的合成
Figure PCTCN2017104706-appb-000012
于10mlNMP中加入vc-PABOH 205mg(0.54mmol)和MC-OSu 184mg(1.1eq)，加毕于室温下搅拌反应24h。反应完毕，于40℃减压浓缩，所得油状物中加入20ml甲基叔丁基醚搅拌析晶。过滤，滤饼用甲基叔丁基醚洗涤，得到310mg产品，收率100％。m/z:573.3(M+H)+。1HNMR(400Mz,DMSO):0.89(6H,m)、1.15-1.99(10H,m)、2.11(1H,m)、2.31(2H,t)、3.21(2H,m)、3.53(2H,t)、4.32(1H,t)、4.51(1H,m)、4.59(2H,br)、5.24(1H,br)、5.56 (2H,br)、6.20(1H,br)、7.12(2H,s)、7.23(2H,d),7.58(2H,d)、7.94(1H,d),8.17(1H,d)、10.21(1H,br)。
实施例8.mc-vc-PAB-PNP的合成
Figure PCTCN2017104706-appb-000013
在氮气保护下取mc-vc-PABOH 168.6mg(0.294mmol)溶于5ml无水吡啶中，反应体系冷却到0℃左右。另取PNP179mg(3eq)溶于5mlDCM中，再将其缓慢加入到反应体系中。并于0℃左右保持10min后除去冰浴，再于室温搅拌反应3h。反应完毕，加入70mlEA和100ml 15％柠檬酸水溶液，分取有机层。有机层依次用柠檬酸，水，饱和食盐水洗涤，再无水硫酸钠干燥，过滤，滤液减压浓缩至干得到浅黄色油状物，加入甲基叔丁基醚析晶得到类白色固体86mg,收率40％。m/z:738(M+H)+。1HNMR(400Mz,CDCl3/CD3OD):0.84(6H,m)、1.11-1.84(10H,m)、2.05(1H,m)、2.15(2H,t)、3.09(2H,m)、3.32(2H,t)、4.12(1H,m)、4.38(1H,m)、5.15(2H,s)、6.61(2H,s)、6.84(1H,d),7.61(1H,d)、7.21(2H,d),7.50(2H,d)、7.61(2H,d),8.18(2H,d)、9.59(1H,br)。
实施例9.mc-vc-PAB-MMAE的合成
Figure PCTCN2017104706-appb-000014
于2mlDMF中加入20mg mc-vc-PAB-PNP(1.5eq)和3mgHOBT。室温搅拌片刻后， 加入13mgMMAE，0.5ml吡啶，25ulDIEA。反应液于室温下搅拌反应2d。反应完毕后，反应液直接用制备柱纯化，收集所需成分浓缩后冻干，得到约10mg产品，收率约42％。m/z:1317.1(M+H)+。
实施例10.mc-vc-PAB-MMAF的合成
Figure PCTCN2017104706-appb-000015
按照实施例9的操作，得到mc-vc-PAB-MMAF约12.5mg，收率45.2％，m/z:1345.7(M+H)+。
实施例11mc-vc-PAB-PBD的合成
Figure PCTCN2017104706-appb-000016
按照实施例9的操作，得到mc-vc-PAB-PBD约9.5mg，收率32.5％，m/z:1325.4(M+H)+。
实施例12mc-vc-PAB-DOX的合成
Figure PCTCN2017104706-appb-000017
按照实施例9的操作，得到mc-vc-PAB-DOX约11.2mg，收率38.9％，m/z:1143.2(M+H)+。
实施例14mc-vc-PAB-SN-38的合成
Figure PCTCN2017104706-appb-000018
将100mg购买的10-O-Boc-SN-38用10ml干燥的二氯甲烷溶解后，加入25.6mg(1eq)DMAP，于0℃下滴加三光气的二氯甲烷溶液(62mg三光气用2ml二氯甲烷溶解)，滴毕，继续于0℃下反应12h，减压除去二氯甲烷，用10ml干燥的DMF溶解后，加入144mg mc-vc-PABOH，转至室温搅拌24h，经过制备液相分离得到mc-vc-PAB-SN-38 41mg，两步收率总的为19.7％，m/z:1063.2(M+H)+。实施例15、目标抗体的表达与纯化
使用FreestyleTM 293-F(Invitrogen)悬浮细胞表达目标抗体。转染前一 天，以6×105个/mL密度将细胞接种于含300mL F17完全培养基(FreestyleTM F17表达培养基，Gibco公司)的1L摇瓶中，37℃，5％CO2，120rpm细胞培养摇床过夜培养。次日，用PEI进行抗体表达质粒的转染，其中质粒:PEI比例为2:1。转染后一天，按2.5％(v/v)加入TN1补料培养基，继续培养4天后离心收集上清。
收集得到的细胞表达上清，经Protein A亲和层析柱(Mabselect Sure LX，GE公司)，以0.1M柠檬酸(pH3.0)洗脱，捕获的抗体用1M Tris-HCl(pH9.0)按1/10(v/v)调节至pH7.0，再通过凝胶过滤层析柱SEC(Superdex 200，GE公司)去除多聚体和内毒素等杂质，同时将抗体缓冲液置换成PBS(pH7.4)，收集UV280nm目标峰样品经超滤离心管(30KD，Pall公司)浓缩至2mg/ml。通过此方法获得的目标抗体单体(POI％)大于90％，用于后续试验。
实施例16、通过偶联2A1-HC-Cys439ins抗体和mc-vc-PAB-MMAE制备2A1-HC-Cys439ins-mc-vc-PAB-MMAE TDC样品
细胞表达的2A1-HC-Cys439ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入2A1-HC-Cys439ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到2A1-HC-Cys439ins-mc-vc-PAB-MMAE TDC样品。
实施例17、通过偶联2A1-LC-Cys205ins抗体和mc-vc-PAB-MMAE制备2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC样品
细胞表达的2A1-LC-Cys205ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物 mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入2A1-LC-Cys205ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC样品。
实施例18、通过偶联2A1-LC-Cys206ins抗体和mc-vc-PAB-MMAE制备2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC样品
细胞表达的2A1-LC-Cys206ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入2A1-LC-Cys206ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC样品。
实施例19、通过偶联4D3-HC-Cys439ins抗体和mc-vc-PAB-MMAE制备4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC样品
细胞表达的4D3-HC-Cys439ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入4D3-HC-Cys439ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将 样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC样品。
实施例20、通过偶联4D3-LC-Cys205ins抗体和mc-vc-PAB-MMAE制备4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC样品
细胞表达的4D3-LC-Cys205ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入4D3-LC-Cys205ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC样品。
实施例21、通过偶联4D3-LC-Cys206ins抗体和mc-vc-PAB-MMAE制备4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC样品
细胞表达的4D3-LC-Cys206ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入4D3-LC-Cys206ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得 mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC样品。
实施例22、通过偶联4E1-HC-Cys439ins抗体和mc-vc-PAB-MMAE制备4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC样品
细胞表达的4E1-HC-Cys439ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入4E1-HC-Cys439ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC样品。
实施例23、通过偶联4E1-LC-Cys205ins抗体和mc-vc-PAB-MMAE制备4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC样品
细胞表达的4E1-LC-Cys205ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入4E1-LC-Cys205ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC样品。
实施例24、通过偶联4E1-LC-Cys206ins抗体和mc-vc-PAB-MMAE制备4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC样品
细胞表达的4E1-LC-Cys206ins抗体，经过Mabselect Sure纯化，低pH洗脱后马上加入Tris溶液中和，并换液为pH7.5的Tris-HCl缓冲液。化合物mc-vc-PAB-MMAE，白色粉末，将其溶解于DMA中备用。为了去除突变半胱氨酸残基上的屏蔽物，需要先将抗体还原。按照40倍分子比将1M的DTT水溶液加入4E1-LC-Cys206ins抗体溶液中，混匀后20℃反应2小时。反应时间到后将样品的pH调整至5.0，并通过SP Sepharose F.F.阳离子交换层析去除样品中的DTT和屏蔽物。随后按照20倍分子比将DHAA溶液加入样品中，25℃避光反应4小时，使抗体链间二硫键重新连接。然后加入mc-vc-PAB-MMAE溶液，使得mc-vc-PAB-MMAE与抗体突变半胱氨酸进行偶联，充分混匀后25℃反应2小时。反应结束后使用SP Sepharose F.F.阳离子交换层析去除未偶联上抗体分子的mc-vc-PAB-MMAE,得到4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC样品。
实施例25、HIC-HPLC检测毒素比抗体比例DAR
用高效液相色谱疏水层析方法分析TDC样品，根据对应峰面积计算DAR。具体方法如下：
色谱柱：
Figure PCTCN2017104706-appb-000019
HICBu‐NP5(5μm,4.6x 35mm)；
流动相：A:2M硫酸铵，0.025M、pH7的磷酸盐缓冲液；B：0.025M、pH7的磷酸盐缓冲液；C：100％异丙醇；
缓冲液A平衡，缓冲液B与缓冲液C梯度洗脱，25℃，214nm和280波长下检测。
根据附图1-3计算得到定点偶联DAR在1.6-1.7之间，其化合物均一性很好。
根据附图4-6计算得到定点偶联DAR在1.60-1.95之间，其化合物均一性很好。
根据附图7-8计算得到定点偶联DAR在1.60-1.90之间，其化合物均一性很好。
实施例26、RP-HPLC检测毒素比抗体比例DAR
RP-HPLC检测毒素比抗体比例DAR，采用反相疏水高效液相色谱法分析用DTT处理的样品，根据对应峰面积计算DAR。具体方法如下：
色谱柱：Proteomix RP-1000(5μm,4.6×100mm)
流动相：A：0.1％TFA水溶液；B：0.1％乙腈溶液
流动相A与流动相B按比例梯度洗脱，80℃，214nm和280波长下检测。
根据附图9计算得到定点偶联DAR为1.82，其化合物均一性很好。
附表1.2A1-LC-V205C-mc-vc-PAB-MMAE TDC、2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys206ins--mc-vc-PAB-MMAE TDC、2A1-HC-Cys439ins-mc-vc-PAB-MMAE、4E1-LC-Cys205ins-mc-vc-PAB-MMAE、4E1-LC-Cys206ins-mc-vc-PAB-MMAE、4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys205ins-mc-vc-PAB-MMAE、4D3-LC-Cys206ins-mc-vc-PAB-MMAE、4D3-HC-Cys439ins-mc-vc-PAB-MMAE的偶联效率DAR表.
Figure PCTCN2017104706-appb-000020
附表1显示，通过半胱氨酸***突变改造进行定点偶联的TDC化合物偶联效率均较高(理论最高值为2.0)，DAR≥1.6。
实施例27、SEC-HPLC检测TDC抗体骨架聚集情况
将TDC抗体骨架样品保存于37℃，第0、7、21、29天分别用SEC-HPLC分析其聚集情况，具体方法如下：
色谱柱：TSKgel SuperSW mAb HR(7.8mm×30cm)
流动相：0.1M硫酸钠、0.1M，pH6.7的磷酸盐缓冲液。
25℃，280nm检测
附图10-13，SEC-HPLC检测TDC抗体骨架4D3、4D3-LC-Cys205ins、4D3-LC-Cys206ins和4D3-HC-Cys439ins的聚集情况，样品于37℃存放4周，聚集体含量基本没有变化。
同上述抗体的检测方法，将2A1-LC-V205C-mc-vc-PAB-MMAE TDC、2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys206ins--mc-vc-PAB-MMAE TDC、2A1-HC-Cys439ins-mc-vc-PAB-MMAE、4E1-LC-Cys205ins-mc-vc-PAB-MMAE、4E1-LC-Cys206ins-mc-vc-PAB-MMAE 4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys205ins-mc-vc-PAB-MMAE、4D3-LC-Cys206ins-mc-vc-PAB-MMAE、4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC进行SEC检测。
附表2.2A1-LC-V205C-mc-vc-PAB-MMAE TDC、2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys206ins--mc-vc-PAB-MMAE TDC和2A1-HC-Cys439ins-mc-vc-PAB-MMAE、4E1-LC-Cys205ins-mc-vc-PAB-MMAE、4E1-LC-Cys206ins-mc-vc-PAB-MMAE、4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys205ins-mc-vc-PAB-MMAE、4D3-LC-Cys206ins-mc-vc-PAB-MMAE、4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC目标单体含量列表
Figure PCTCN2017104706-appb-000021
附表2显示，通过半胱氨酸定点偶联的TDC化合物目标单体含量在90％以上。
实施例28、经半胱氨酸定点突变和***突变改造后的骨架抗体与亲本抗体2A1 对于EGFRvIII的亲和力,4E1对于c-met的亲和力，4D3对于Trop2的亲和力。
用间接ELISA法对比2A1-LC-V205C、2A1-LC-Cys205ins、2A1-LC-Cys206ins、2A1-HC-Cys439ins及2A1对于EGFRvIII的相对亲和力。具体步骤如下：
重组EGFRvIII-His*6抗原包板；鱼皮明胶封闭；分别稀释2A1、2A1-LC-V205C、2A1-LC-Cys205ins、2A1-LC-Cys206ins和2A1-HC-Cys439ins，最高浓度10ug/ml，4倍梯度稀释，共11个浓度；HRP标记的二抗孵育；TMB显色，检测450nm处吸收。检测结果以A450对浓度作图，半胱氨酸定点突变或***突变后的抗体2A1-LC-V205C、2A1-LC-Cys205ins、2A1-LC-Cys206ins和2A1-HC-Cys439ins保持了与2A1相似的亲和力，EC50值很接近；说明2A1上轻链V205C的定点突变、第205位***突变或第206位***突变，或重链第439位***突变不影响其与EGFRvIII抗原的亲和力。
如附图可见，2A1-LC-V205C、2A1-LC-Cys205ins、2A1-LC-Cys206ins、2A1-HC-Cys439ins抗体保持了2A1与抗原EGFRvIII的亲和力。
实施例29、经半胱氨酸定点突变和***突变改造后的骨架抗体与TDC对于相应抗原的亲和力。4E1对于c-met的亲和力，4D3对于Trop2的亲和力。
用间接ELISA法对比4E1-LC-Cys205ins-MVPM、4E1-LC-Cys206ins-MVPM、4E1-HC-Cys439ins-MVPM及4E1对于EGFRvIII的相对亲和力。具体步骤如下：
重组c-met-His*10抗原包板；鱼皮明胶封闭；分别稀释4E1-LC-Cys205ins-MVPM、4E1-LC-Cys206ins-MVPM、4E1-HC-Cys439ins-MVPM和4E1，最高浓度10ug/ml，4倍梯度稀释，共11个浓度；HRP标记的二抗孵育；TMB显色，检测450nm处吸收。检测结果以A450对浓度作图，半胱氨酸定点***突变后偶联得到的TDC4E1-LC-Cys205ins-MVPM、4E1-LC-Cys206ins-MVPM、4E1-HC-Cys439ins-MVPM保持了与亲本抗体4E1相似的亲和力，EC50值很接近；说明4E1轻链第205位***突变或第206位***突变，或重链第439位***突变所得到的TDC不影响其与c-met抗原的亲和力。
用间接ELISA法对比4D3-LC-Cys205ins-MVPM、4D3-LC-Cys206ins-MVPM、4D3-HC-Cys439ins-MVPM及4D3对于EGFRvIII的相对亲和力。具体步骤如下：
重组Trop2-His*10抗原包板；鱼皮明胶封闭；分别稀释4D3-LC- Cys205ins-MVPM、4D3-LC-Cys206ins-MVPM、4D3-HC-Cys439ins-MVPM和4D3，最高浓度10ug/ml，4倍梯度稀释，共11个浓度；HRP标记的二抗孵育；TMB显色，检测450nm处吸收。检测结果以A450对浓度作图，半胱氨酸定点***突变后偶联得到的TDC4D3-LC-Cys205ins-MVPM、4D3-LC-Cys206ins-MVPM、4D3-HC-Cys439ins-MVPM保持了与亲本抗体4D3相似的亲和力，EC50值很接近；说明4D3轻链第205位***突变或第206位***突变，或重链第439位***突变所得到的TDC不影响其与Trop2抗原的亲和力。
如图15，4E1-LC-Cys205ins-MVPM、4E1-LC-Cys206ins-MVPM、4E1-HC-Cys439ins-MVPM抗体保持了4E1与抗原c-met的亲和力。
如图16，4D3-LC-Cys205ins-MVPM、4D3-LC-Cys206ins-MVPM、4D3-HC-Cys439ins-MVPM抗体保持了4D3与抗原Trop2的亲和力。
实施例30、细胞毒性药效检测
通过下列实验过程测定TDC细胞毒性活性：将TDC分别加入到EGFR过量表达或EGFRVIII表达的人的肿瘤细胞培养基中，细胞培养72小时后测定细胞存活率。基于细胞的体外实验用于测定细胞存活率、细胞毒性和本发明TDC诱导的细胞程序性死亡。
通过细胞增殖试验测定抗体-毒素偶联物的体外药效。CellTiter 
Figure PCTCN2017104706-appb-000022
Solution Cell Proliferation Assay为商购的(Promega Corp.，Madison,WI)。CellTiter
Figure PCTCN2017104706-appb-000023
AQueous One Solution Cell Proliferation Assay(a)是一种用比色法来检测细胞增殖和细胞毒性实验中的活细胞数量的检测试剂。此试剂含有一个新型的四唑化合物[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt；MTS]和一种电子偶联剂(phenazine ethosulfate；PES)。PES具有增强的化学稳定性，这使它可与MTS混合形成稳定的溶液。这种方便的“单溶液”模式，是在第一代CellTiter
Figure PCTCN2017104706-appb-000024
Assay的基础上的改进，CellTiter
Figure PCTCN2017104706-appb-000025
Assay中使用的电子偶联剂PMS与MTS溶液是分开提供的。MTS(Owen’s reagent)被细胞生物还原成为一种有色的甲臜产物，可直接溶解于培养基中(图1)。这种转化很可能是在代谢活跃的细胞中的脱氢酶产生的NADPH或NADH的作用下完成的。检测时，只需 将少量的CellTiter
Figure PCTCN2017104706-appb-000026
AQueous One Solution Reagent直接加入培养板孔的培养基中，孵育1–4小时，然后以酶标仪读取490nm的吸光度值。
Figure PCTCN2017104706-appb-000027
在490nm处检测到的甲臜产物的量与培养中的活细胞数成正比。由于MTS的甲臜产物在组织培养基中可溶，CellTiter
Figure PCTCN2017104706-appb-000028
AQueous One Solution Assay与MTT或INT法相比操作步骤更少。
本发明中采用A431(EGFR过表达细胞)、U87-EGFRVIII(EGFR突变体稳定细胞系)、U87-MG(c-Met中高度表达的恶性胶质瘤细胞株)和BXPC-3(trop2高度表达的胰腺癌细胞株)作为体外药效检测的研究体系。在96孔板中，进行细胞铺板6000/孔，24小时后，进行抗体加药。A431,U87-EGFRVIII细胞株对应的各种TDC始浓度为10uM,依次按照5倍梯度稀释,U87-MG,BXPC-3细胞株对应的各种TDC始浓度为1uM,依次按照5倍梯度稀释。处理72小时后MTS检测细胞活性。
附表3、TDC、ADC对EGFRwt过表达细胞系A431及EGFRvIII表达稳定性株U87-EGFRVIII细胞毒性IC50检测结果
Figure PCTCN2017104706-appb-000029
Figure PCTCN2017104706-appb-000030
附表3结果显示，2A1-LC-V205C-mc-vc-PAB-MMAE TDC、2A1-LC-Cys205Cins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys439ins-mc-vc-PAB-MMAE TDC对EGFRwt过表达细胞系A431及EGFRvIII表达稳定性株U87-EGFRVIII细胞毒性活性相当,439位***的突变的TDC活性略好于205位和206位***突变的TDC,
4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC在U87-MG细胞毒性活性和偶联位置有一定的关联,439位***的突变的TDC活性略好于205位和206位***突变的TDC,TDC的活性要显著好于亲本抗体的活性。
4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC在胰腺癌细胞株BXPC-3上细胞 毒性活性上相当,439位***的突变的TDC活性略好于205位和206位***突变的TDC，TDC的活性要显著好于亲本抗体的活性。
实施例31、血浆稳定性检测
取取一定量的ADC样品，加入到已去除人IgG的人血浆中,每种ADC重复2管，放置37℃水浴中孵育,分别孵育0h,72h后，取出ADC样品，每管加入ProteinA(MabSelect SuReTM LX Lot:#10221479GE，用取PBS洗涤过的)100ul，垂直混合仪晃动吸附2h,经过洗涤洗脱步骤,获得孵育后的ADC。对孵育特定时间的ADC样品进行HIC-HPLC和RP-HPLC检测，判定样品的血浆稳定性。
附图21-23，4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC在人血浆中稳定性的考察结果，其中4D3-HC-Cys439ins-mc-vc-PAB-MMAE的检测方法是RP-HPLC,4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys206ins-mc-vc-PAB-MMAE的检测方法是HIC-HPLC。
附图24-26,4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC、在人血浆中稳定性的考察结果，检测方法为HIC-HPLC。
附表4.TDC血浆稳定性检测结果表(以DAR的变化情况计算)
Figure PCTCN2017104706-appb-000031
上述TDC加入人血浆样品中37℃孵育72小时后均相对稳定，具备良好的成药性。比较而言，439位***突变的TDC稳定性最佳，205位、206位***突变的TDC次之。
实施例32、荷瘤小鼠药效检测
本发明中建立了BXPC-3荷瘤小鼠模型，以评价TDC和亲本抗体的体内药效。即以3×106BXPC-3细胞通过皮下注射到4～6周鼠龄的BALB/c裸鼠背部一侧，待小鼠肿瘤平均大小生长至400～500mm3，随机分组，每组5只，在第0天和第7天，4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC分别以5mg/kg剂量进行单次静脉给药，亲本抗体4D3以5mg/kg剂量给药。数据A显示为测量时肿瘤平均体积±SE,数据B显示为测量时小鼠平均体重±SE。
附图27、4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC以及4D3亲本抗体在荷瘤小鼠药效上测试结果，相对于亲本抗体，TDC能显著体现出体内抑瘤效果。
附图28、4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC、4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC和4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC以及4D3亲本抗体在荷瘤小鼠药效上测试，小鼠体重没有明显的改变，证明TDC体内毒性较小或无体内毒性。
本发明并不限于由实施例中披露的具体实施方案的范围，这些实施例用来例示本发明的几个方面，在功能上等效的任意实施方案均属于本发明的范围。实际上，除本文所示和所述的以外，本发明的各种变型对本领域技术人员而言也是显而易见的并且属于本文所附权利要求的范围。

Claims (9)

  1. 半胱氨酸改造的抗体-毒素偶联物，其特征在于：抗体为半胱氨酸定点***抗体，所述半胱氨酸***位点包含选自以下3个***位点的一个或多个位点：kappa/λ轻链恒定区Kabat编号第205位、轻链恒定区Kabat编号第206或IgG抗体重链恒定区Kabat编号第439位。
  2. 如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述半胱氨酸***位点氨基酸序列包含以下3个序列的一个或多个：
    LC-205ins:GLSSPCVTKSF、LC-206ins:GLSSPVCTKSF或HC-439ins:TQKSLSCLSPGK，其中，C为目标抗体轻链205位或206位或重链第439位***的半胱氨酸。
  3. 如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：高活性细胞毒素通过连接子与***的半胱氨酸游离巯基进行定点偶联，连接子与***的半胱氨酸偶联的抗体轻链氨基酸序列为：GLSSPCVTKSF和GLSSPVCTKSF；连接子与***的半胱氨酸偶联的抗体重链氨基酸序列为：TQKSLSCLSPGK；
    其中，C为目标抗体轻链205位或206位或重链第439位***的半胱氨酸。
  4. 如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述抗体轻链包括kappa或λ同种型。
  5. 如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述抗体重链包括IgG1、IgG2、IgG3或IgG4同种型。
  6. 如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述半胱氨酸包含巯基。
  7. 如权利要求5所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述巯基能够进行化学偶联。
  8. 如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物，其特征在于：通过连接子与半胱氨酸***突变的游离巯基进行定点偶联的高活性细胞毒素选自：MMAE、MMAF、PBD、SN-38或Dox及其类似物。
  9. 一种如权利要求1所述的半胱氨酸改造的抗体-毒素偶联物的制备方法，其特征在于：定点偶联步骤为：首先使用还原剂还原抗体，解除抗体上改 造的半胱氨酸残基上的屏蔽，并通过阳离子交换层析或超滤换液等方式去除DTT与屏蔽物；然后使用氧化剂氧化抗体，使抗体的链间二硫键重新连接；最后加入Linker-drug与胱氨酸残基上的游离巯基偶联，并通过阳离子交换层析或超滤换液等方式去除未偶联上抗体分子Linker-drug。
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