WO2018233572A1 - 半胱氨酸改造的抗体-毒素偶联物(tdc)定点偶联位点筛选 - Google Patents

半胱氨酸改造的抗体-毒素偶联物(tdc)定点偶联位点筛选 Download PDF

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WO2018233572A1
WO2018233572A1 PCT/CN2018/091623 CN2018091623W WO2018233572A1 WO 2018233572 A1 WO2018233572 A1 WO 2018233572A1 CN 2018091623 W CN2018091623 W CN 2018091623W WO 2018233572 A1 WO2018233572 A1 WO 2018233572A1
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antibody
cysteine
pab
tdc
mmae
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PCT/CN2018/091623
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English (en)
French (fr)
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朱义
王一茜
卓识
李�杰
余永国
万维李
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四川百利药业有限责任公司
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Priority to JP2020551558A priority Critical patent/JP7153082B2/ja
Priority to CA3098491A priority patent/CA3098491A1/en
Priority to NZ766990A priority patent/NZ766990A/en
Priority to US16/976,475 priority patent/US20210393794A1/en
Priority to EP18821450.6A priority patent/EP3770170A4/en
Priority to AU2018288030A priority patent/AU2018288030B2/en
Priority to SG11202008821PA priority patent/SG11202008821PA/en
Publication of WO2018233572A1 publication Critical patent/WO2018233572A1/zh
Priority to JP2022158036A priority patent/JP2023011567A/ja

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Definitions

  • the present invention relates to a compound, a preparation method and use thereof, and in particular to a cysteine engineered antibody-toxin conjugate (TDC), a preparation method thereof and use thereof.
  • TDC cysteine engineered antibody-toxin conjugate
  • ADC Antibody-conjugates
  • Adcetris and Kadcyla which have been approved for marketing in the United States, have shown good clinical efficacy, and more than 50 ADC drugs are in progress.
  • the novel cysteine-modified antibody-toxin conjugate (TDC) disclosed in this patent has better drug uniformity and less side effects than non-site-coupled ADC. Preclinical studies have shown that it is significantly better than non-fixed point. Coupled with an ADC.
  • the cysteine insertion site comprises one or more sites selected from the following 15 insertion sites: light chain position 110 ( Kabat number, its amino acid sequence EIKRTCVAAPS), light chain position 111 (Kabat number, amino acid sequence IKRTVCAAPSV), light chain position 142 (Kabat number, amino acid sequence NNFYPCREAKV), heavy chain position 254 (its amino acid sequence is ISRTPCEVTCV) ), heavy chain 255 (the amino acid sequence is SRTPECVTCVV), heavy chain 258 (the amino acid sequence is PEVTCCVVVDV), heavy chain 259 (the amino acid sequence is EVTCVCVVDVS), heavy chain 354 (the amino acid sequence) PSRDECLTKNQ), heavy chain 355 (its amino acid sequence is SRDELCTKNQV), heavy chain 357 (amino acid sequence DELTKCNQVSL), heavy chain 378 (amino acid sequence IAVEWCESNGQ),
  • 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 cysteine thiol group inserted into the light chain or the heavy cyst inserted into the cysteine thiol group and the payload are subjected to antibody-toxin conjugate (TDC) site-directed coupling, and the toxin ratio antibody ratio DAR is 1.6-2.0 or 3.2. -4.0.
  • 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 9.5 mg of mc-vc-PAB-PBD was obtained, the yield was 32.5%, m/z: 1325.4 (M+H)+
  • Use Freestyle TM 293-F was suspended cells expressing antibody 2A1-HC-Cys254ins.
  • 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 2A1-HC-Cys254ins antibody obtained by this method had a concentration of 2 mg/ml and a target antibody monomer (POI%) of more than 90%, which was used for subsequent experiments.
  • Example 15 prepared by coupling 2A1-HC-Cys357ins antibody and mc-vc-PAB-MMAE
  • the 2A1-HC-Cys357ins 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-Cys357ins 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 then 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 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, 25 ° C, 214 nm detection.
  • HIC-HPLC detects 2A1-HC-Cys357ins-mc-vc-PAB-MMAE,
  • the fixed-point coupled DAR was calculated to be between 1.6 and 1.7, and the compound uniformity was good.
  • 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, 14, and 21, respectively.
  • SEC-HPLC SEC-HPLC on days 0, 7, 14, and 21, respectively.
  • Mobile phase 0.1 M sodium sulfate, 0.1 M, pH 6.7 phosphate buffer.
  • FIGS. 2A-2D SEC results for 2A1-HC-Cys378ins on days 0, 7, 14, and 21. The data shows that the sample is stored at 37 ° C for 3 weeks and is very stable.
  • Table 3 shows that the target monomer content of the TDC compound coupled by cysteine is above 90%.
  • Example 18 Affinity of skeletal antibody engineered with cysteine insertion mutation and parent antibody 2A1 for EGFRvIII
  • 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 Aqueous One 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 tetrazole compound
  • PES [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron 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 AQueous Assay, CellTiter The electronic coupling agent PMS used in the AQueous 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 EGFRVIII mutant stable cell line
  • A431 EGFR overexpressing cells
  • U87-EGFRVIII EGFRVIII mutant stable cell line
  • FIG. 4A-4B 2A1-HC-Cys357ins-mc-vc-PAB-MMAE,
  • TDC sample Take a certain amount of TDC sample, add it to human plasma from which human IgG has been removed, repeat three tubes of each TDC, incubate in a 37 °C water bath, incubate for 0h, 24h, 96h, remove TDC samples, and add ProteinA to each tube.
  • 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 TDC after incubation.
  • the TDC samples incubated for a specific time were subjected to HIC-HPLC and RP-HPLC detection to determine the plasma stability of the samples.
  • the present invention establishes a subcutaneous xenograft model of human pancreatic cancer cell BxPC3 nude mice to evaluate the in vivo efficacy of TDC-conjugated drugs such as 2A1-HC-Cys357ins-mc-vc-PAB-MMAE.
  • TDC-conjugated drugs such as 2A1-HC-Cys357ins-mc-vc-PAB-MMAE.
  • 5 ⁇ 10 6 A431 cells were injected subcutaneously into the back of BALB/c nude mice of 4-6 weeks old mice.
  • the average tumor size of the mice was grown to about 150-200 mm 3 and randomly divided into groups of 6 rats.

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Abstract

本发明公开了一种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，半胱氨酸***位点包含选自以下位点的一个或多个：kappa/λ轻链恒定区轻链第110位、第111位、第142位，IgG抗体重链恒定区重链第254位、第255位、第258位、第259位、第354位、第355位、第357位、第378位、第379位、第386位、第387位或第410位。

Description

半胱氨酸改造的抗体-毒素偶联物(TDC)定点偶联位点筛选 技术领域
本发明涉及一种化合物及其制备方法和用途，特别涉及一类半胱氨酸改造的抗体-毒素偶联物(TDC)及其制备方法和用途。
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。此专利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。
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15个***位点的一个或多个位点：轻链第110位(Kabat编号，其氨基酸序列EIKRTCVAAPS)、轻链第111位(Kabat编号，其氨基酸序列IKRTVCAAPSV)、轻链第142位(Kabat编号，其氨基酸序列NNFYPCREAKV)，重链第254位(其氨基酸序列为ISRTPCEVTCV)、重链第255位(其氨基酸序列为SRTPECVTCVV)、重链第258位(其氨基酸序列为PEVTCCVVVDV)、重链第259位(其氨基酸序列为EVTCVCVVDVS)、重链第354位(其氨基酸序列为PSRDECLTKNQ)、重链第355位(其氨基酸序列为SRDELCTKNQV)、重链第357位(其氨基酸序列为DELTKCNQVSL)、重链第378位(其氨基酸序列为IAVEWCESNGQ)、重链第379位(其氨基酸序列为AVEWECSNGQP)、重链第386位(其氨基酸序列为GQPENCNYKTT)、重链第387位(其氨基酸序列为QPENNCYKTTP)、重链第410位(其氨基酸序列为KLTVDCKSRWQ)。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)。通过其轻链***的半胱氨酸巯基或/和重链***的半胱氨酸巯基与payload进行抗体-毒素偶联物(TDC)定点偶联，毒素比抗体比例DAR为1.6-2.0或3.2-4.0。
实施例
实施例1 mc的合成
Figure PCTCN2018091623-appb-000001
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 PCTCN2018091623-appb-000002
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 PCTCN2018091623-appb-000003
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 PCTCN2018091623-appb-000004
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 PCTCN2018091623-appb-000005
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 PCTCN2018091623-appb-000006
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 PCTCN2018091623-appb-000007
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),8.1 7(1H,d)、10.21(1H,br)。
实施例8.mc-vc-PAB-PNP的合成
Figure PCTCN2018091623-appb-000008
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 PCTCN2018091623-appb-000009
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 PCTCN2018091623-appb-000010
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11 mc-vc-PAB-PBD的合成
Figure PCTCN2018091623-appb-000011
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12 mc-vc-PAB-DOX的合成
Figure PCTCN2018091623-appb-000012
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13 mc-vc-PAB-SN-38的合成
Figure PCTCN2018091623-appb-000013
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-3841mg，两步收率总的为19.7％，m/z:1063.2(M+H)+
实施例14、2A1-HC-Cys254ins抗体的表达与纯化
使用Freestyle TM 293-F(Invitrogen)悬浮细胞表达2A1-HC-Cys254ins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2A1-HC-Cys254ins抗体，浓度为2mg/ml，目标抗体单体(POI％)大于90％，用于后续试验。
实施例15、通过偶联2A1-HC-Cys357ins抗体和mc-vc-PAB-MMAE制备
2A1-HC-Cys357ins-mc-vc-PAB-MMAE TDC样品
细胞表达的2A1-HC-Cys357ins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-Cys357ins抗体溶液中，混匀后20℃反应2小时。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-Cys357ins-mc-vc-PAB-MMAE TDC样品。
实施例16、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 PCTCN2018091623-appb-000014
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检测。
Figure PCTCN2018091623-appb-000015
Figure PCTCN2018091623-appb-000016
Figure PCTCN2018091623-appb-000017
Figure PCTCN2018091623-appb-000018
附图1A-1D.HIC-HPLC分别检测2A1-HC-Cys357ins-mc-vc-PAB-MMAE，
2A1-HC-Cys378ins-mc-vc-PAB-MMAE,2A1-HC-Cys387ins-mc-vc-PAB-MMAE、
2A1-HC-Cys410ins-mc-vc-PAB-MMAE TDC DAR。
根据附图1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。
附表1.2A1-LC-Cys110ins-mc-vc-PAB-MMAE TDC、
2A1-LC-Cys111ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys142ins-mc-vc-PAB-MMAE TDC和2A1-HC-Cys254ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys255ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys258ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys259ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys354ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys355ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys357ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys378ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys379ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys386ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys410ins-mc-vc-PAB-MMAE
附表1 TDC的偶联效率DAR表
Figure PCTCN2018091623-appb-000019
Figure PCTCN2018091623-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17、SEC-HPLC检测TDC抗体骨架稳定性情况以及TDC聚集情况
将TDC抗体骨架样品保存于37℃，第0、7、14、21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。
Figure PCTCN2018091623-appb-000021
Figure PCTCN2018091623-appb-000022
Figure PCTCN2018091623-appb-000023
Figure PCTCN2018091623-appb-000024
附表2
Figure PCTCN2018091623-appb-000025
附图2A-2D 2A1-HC-Cys378ins在第0、7、14、21天的SEC结果。数据显示样品在于37℃存放3周，非常稳定。
附表2.SEC-HPLC检测TDC抗体骨架2A1-HC-Cys357ins、2A1-HC-Cys387ins、2A1-HC-Cys378ins、和2A1-HC-Cys410ins的聚集情况，样品于37℃存放3周，单体POI含量基本没有变化；
2A1-LC-Cys110ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys111ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys142ins-mc-vc-PAB-MMAE TDC和2A1-HC-Cys254ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys255ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys258ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys259ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys354ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys355ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys357ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys378ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys379ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys386ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys410ins-mc-vc-PAB-MMAE
附表3 TDC目标单体含量列表
Figure PCTCN2018091623-appb-000026
Figure PCTCN2018091623-appb-000027
附表3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18、经半胱氨酸***突变改造后的骨架抗体与亲本抗体2A1对于EGFRvIII的亲和力
用间接ELISA法对比2A1-LC-Cys110ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys111ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys142ins-mc-vc-PAB-MMAE TDC和2A1-HC-Cys254ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys255ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys258ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys259ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys354ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys355ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys357ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys378ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys379ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys386ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys410ins-mc-vc-PAB-MMAE TDC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；分别稀释各个***突变体抗体，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，如图3和附表3所示，半胱氨酸***突变后的抗体保持了与2A1相似的亲和力，EC50值很接近；说明2A1上轻链或重链***突变不影响其与EGFRvIII抗原的亲和力。
Figure PCTCN2018091623-appb-000028
Figure PCTCN2018091623-appb-000029
Figure PCTCN2018091623-appb-000030
Figure PCTCN2018091623-appb-000031
Figure PCTCN2018091623-appb-000032
附表4 突变抗体的亲和力测定
Figure PCTCN2018091623-appb-000033
Figure PCTCN2018091623-appb-000034
附图3及附表4.2A1-LC-Cys110ins、2A1-LC-Cys111ins、2A1-LC-Cys142ins、2A1-HC-Cys254ins、2A1-HC-Cys255ins、2A1-HC-Cys258ins、2A1-HC-Cys259ins、2A1-HC-Cys354ins、2A1-HC-Cys355ins、2A1-HC-Cys357ins、2A1-HC-Cys378ins、2A1-HC-Cys379ins、2A1-HC-Cys386ins、2A1-HC-Cys387ins、2A1-HC-Cys410ins抗体保持了2A1与抗原EGFRvIII的亲和力。
实施例19、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 PCTCN2018091623-appb-000035
AqueousOne Solution Cell Proliferation Assay为商购的(Promega Corp.，Madison,WI)。CellTiter
Figure PCTCN2018091623-appb-000036
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 PCTCN2018091623-appb-000037
AQueous Assay的基础上的改进，CellTiter
Figure PCTCN2018091623-appb-000038
AQueous 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 PCTCN2018091623-appb-000039
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 PCTCN2018091623-appb-000040
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 PCTCN2018091623-appb-000041
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)和U87-EGFRVIII(EGFRVIII突变体稳定细胞系)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。对A431和U87-EGFRvIII的用药浓度为200nM-10pM，四倍稀释，对U87-EGFRVIII的用药浓度为500nM-30pM。处理72小时后MTS检测细胞活性。
Figure PCTCN2018091623-appb-000042
Figure PCTCN2018091623-appb-000043
附图4A-4B.2A1-HC-Cys357ins-mc-vc-PAB-MMAE，
2A1-HC-Cys378ins-mc-vc-PAB-MMAE,2A1-HC-Cys387ins-mc-vc-PAB-MMAE、2A1-HC-Cys410ins-mc-vc-PAB-MMAE对EGFRwt过表达的人皮肤鳞癌细胞A431和EFGRvIII过表达的人脑胶质瘤细胞株U87-EGFRvIII的IC50检测结果。
附表5.TDC对EGFRwt过表达细胞系A431及EGFRvIII表达稳定性株U87-EGFRVIII细胞毒性IC 50检测结果
Figure PCTCN2018091623-appb-000044
Figure PCTCN2018091623-appb-000045
附表5结果显示2A1-LC-Cys110ins-mc-vc-PAB-MMAE TDC、
2A1-LC-Cys111ins-mc-vc-PAB-MMAE TDC、2A1-LC-Cys142ins-mc-vc-PAB-MMAE TDC和2A1-HC-Cys254ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys255ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys258ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys259ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys354ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys355ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys357ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys378ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys379ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys386ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys410ins-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。
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。
实施例20、血浆稳定性检测
取取一定量的TDC样品，加入到已去除人IgG的人血浆中，每种TDC重复三管，放置37℃水浴中孵育,分别孵育0h,24h、96h后，取出TDC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TDC。对孵育特定时间的TDC样品进行HIC-HPLC和RP-HPLC检测.判定样品的血浆稳定性。
Figure PCTCN2018091623-appb-000046
Figure PCTCN2018091623-appb-000047
Figure PCTCN2018091623-appb-000048
Figure PCTCN2018091623-appb-000049
Figure PCTCN2018091623-appb-000050
附图5A-5D.2A1-HC-Cys357ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys410ins-mc-vc-PAB-MMAE TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE TDC 2A1-HC-Cys378ins-mc-vc-PAB-MMAE TDC HIC-HPLC血浆稳定性检测结果图示，三个分子均具有良好的血浆稳定性。
附表6.TDC血浆稳定性检测结果
Figure PCTCN2018091623-appb-000051
Figure PCTCN2018091623-appb-000052
实施例21、荷瘤小鼠药效检测
本发明建立了人胰腺癌细胞BxPC3裸小鼠皮下移植瘤模型，以评价2A1-HC-Cys357ins-mc-vc-PAB-MMAE等TDC偶联药物的体内药效。以5×10 6A431细胞通过皮下注射到4～6周鼠龄的BALB/c裸鼠背部，待小鼠肿瘤平均大小生长至150～200mm 3左右，随机分组，每组6只。在第1天，2A1-HC-Cys357ins-mc-vc-PAB-MMAE(2A1-HC-Cys357ins TDC)、2A1-HC-Cys410ins-mc-vc-PAB-MMAE(2A1-HC-Cys410ins TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE(2A1-HC-Cys387insTDC)、2A1-HC-Cys378ins-mc-vc-PAB-MMAE(2A1-HC-Cys378insTDC)以5mg/kg剂量进行单次尾静脉给药(附图6)数据显示为测量时肿瘤平均体积±SE。
Figure PCTCN2018091623-appb-000053
附图6、2A1-HC-Cys357ins-mc-vc-PAB-MMAE(2A1-HC-Cys357ins TDC)、2A1-HC-Cys410ins-mc-vc-PAB-MMAE(2A1-HC-Cys410ins TDC、2A1-HC-Cys387ins-mc-vc-PAB-MMAE(2A1-HC-Cys387insTDC)、2A1-HC-Cys378ins-mc-vc-PAB-MMAE(2A1-HC-Cys378insTDC)以5mg/kg剂量进行单 次静脉给药,抑瘤效果显著。

Claims (10)

  1. 一种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，半胱氨酸***位点包含选自以下位点的一个或多个：kappa/λ轻链恒定区轻链第110位、第111位、第142位，IgG抗体重链恒定区重链第254位、第255位、第258位、第259位、第354位、第355位、第357位、第378位、第379位、第386位、第387位或第410位。
  2. 根据权利要求1所述的一种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***位点的氨基酸序列包含以下序列的一个或多个：
    LC-110ins：EIKRTCVAAPS；
    LC-111ins：IKRTVCAAPSV；
    LC-142ins：NNFYPCREAKV；
    HC-254ins：ISRTPCEVTCV；
    HC-255ins：SRTPECVTCVV；
    HC-258ins：PEVTCCVVVDV；
    HC-259ins：EVTCVCVVDVS；
    HC-354ins：PSRDECLTKNQ；
    HC-355ins：SRDELCTKNQV；
    HC-357ins：DELTKCNQVSL；
    HC-378ins：IAVEWCESNGQ；
    HC-379ins：AVEWECSNGQP；
    HC-386ins：GQPENCNYKTT；
    HC-387ins：QPENNCYKTTP；
    HC-410ins：KLTVDCKSRWQ。
  3. 根据权利要求1所述的一种半胱氨酸改造的抗体-毒素偶联物，其特征 在于：所述***的半胱氨酸游离巯基(-SH)通过连接子与高活性细胞毒素进行定点偶联；连接子与***的半胱氨酸偶联的抗体轻链氨基酸序列为：EIKRT CVAAPS、IKRTV CAAPSV和NNFYP CREAKV；连接子与***的半胱氨酸偶联的抗体重链氨基酸序列为：ISRTP CEVTCV、SRTPE CVTCVV、PEVTC CVVVDV、EVTCV CVVDVS、PSRDE CLTKNQ、SRDEL CTKNQV、DELTK CNQVSL、IAVEW CESNGQ、AVEWE CSNGQP、GQPEN CNYKTT、QPENN CYKTTP、KLTVD CKSRWQ；其中， C为目标抗体轻链或重链***的半胱氨酸。
  4. 根据权利要求1所述的一种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. 根据权利要求1所述的一种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. 根据权利要求1所述的一种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. 根据权利要求6所述的一种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. 根据权利要求3所述的一种半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述高活性细胞毒素选自：MMAE、MMAF、PBD、SN-38、Dox或Amanitin及其类似物，分子式如下为：
    Figure PCTCN2018091623-appb-100001
    Figure PCTCN2018091623-appb-100002
  9. 根据权利要求1所述的一种半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述毒素与抗体比例为1.6-2.0或3.2-4.0。
  10. 根据权利要求1所述的一种半胱氨酸改造的抗体-毒素偶联物，其特征在于：所述定点偶联步骤为：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payload与抗体改造的半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的payload。
PCT/CN2018/091623 2017-06-20 2018-06-15 半胱氨酸改造的抗体-毒素偶联物(tdc)定点偶联位点筛选 WO2018233572A1 (zh)

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