CN110269943A - 一种靶向于egfr的抗体-药物偶联物及其制备方法与应用 - Google Patents
一种靶向于egfr的抗体-药物偶联物及其制备方法与应用 Download PDFInfo
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- CN110269943A CN110269943A CN201910687951.3A CN201910687951A CN110269943A CN 110269943 A CN110269943 A CN 110269943A CN 201910687951 A CN201910687951 A CN 201910687951A CN 110269943 A CN110269943 A CN 110269943A
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Abstract
本发明提供一种靶向于EGFR的抗体‑药物偶联物,其特征在于,包含式Ⅰ结构的抗体药物偶联物或其药学上可接受的盐或溶剂合物,其结构式为:其中,n表示药物‑抗体比例,n为3~5;R为O,CONH或NHCO;R为对位或间位;p为1~6;m为0~20。本发明是基于我们发明的双乙烯磺酰胺连接子技术,首次合成靶向于EGFR的抗体‑药物偶联物。本发明基于抗体内在的硫‑硫键断开后再桥联,可得到DAR为4左右的均一性好的偶联产物,并且对抗体的适用性广。
Description
技术领域
本发明属于生物制药及生物技术领域,具体的涉及一类靶向于表皮生长因子受体(EGFR)的抗体-药物偶联物(ADCs)以及其制备方法与用途。
背景技术
表皮生长因子受体(EGFR)与肿瘤细胞的增殖、肿瘤侵袭、细胞凋亡的抑制等密切相关。针对EGFR进行抑制和干预,是控制肿瘤的重要措施。
抗体-药物偶联物(ADCs)是通过一个化学链接将具高活性药物分子连接到抗体上,抗体作为载体靶向运输药物分子到目标细胞。高活性药物分子虽然具备高度杀伤效力,但选择性差,常常引起严重的副作用;而作为载体的抗体,虽然靶向性强,但治疗效果有限。ADCs能够协同发挥化学药物和抗体药物各自的优点,利用抗体的特异专一性,有效输送高活性药物分子到特定靶标。
ADCs的研究中不仅要优化药物分子、抗体,链接技术的发展和优化也至关重要。早期的ADCs多基于抗体中的氨基、巯基等氨基酸残基进行修饰。例如上市ADCs药物Kadcyla、Mylotarg和Besponsa均是通过抗体上赖氨酸(lysine)的侧链胺基进行修饰连接的。然而,由于抗体上有近90个赖氨酸残基,当其与高活性药物偶联时,化学选择性差,药物的结合位点及结合数目非常复杂。另一上市药物Adcetris是通过同抗体中硫-硫键还原后得到的巯基偶联修饰所得。单抗中一般含有4对易于接近的硫-硫键,还原后可得到8个可以反应的巯基,当药物-抗体比列为2-4时(DAR=2-4),偶联产物同样比较复杂。这类均一性差的ADCs不仅会对药代动力学、药效和药物安全性产生很大的影响,同时对产品质量控制造成极大的挑战。因此近些年,定位偶联受到越来越多的重视。目前最为常见的定位偶联方式是基于抗体的定点突变技术,该技术虽然可以很好的解决前期ADCs研究中链接的非特异性问题,但存在突变抗体程序繁琐,成本高等问题。
发明内容
本发明的目的是提供一种以双乙烯磺酰胺连接子制备靶向于EGFR的抗体-药物偶联物(ADCs)以及所制备的ADCs在制备治疗非小细胞肺癌等疾病的药物中的用途。
为达到上述发明目的,本发明采用的技术方案:
本发明提供一种靶向于EGFR的抗体-药物偶联物,其特征在于,包含式Ⅰ结构的抗体药物偶联物或其药学上可接受的盐或溶剂合物,其结构式为:
其中,n表示药物-抗体比例,n为3~5;R为O,CONH或NHCO;R为对位或间位;p为1~6;m为0~20。
优选地,所述Anti-EGFR为抗EGFR抗体,Anti-EGFR为尼妥珠单抗(Nimotuzumab)或西妥昔单抗(Cetuximab)。
优选地,所述靶向于EGFR的抗体-药物偶联物为抗体-药物偶联物22或抗体-药物偶联物23;
所述抗体-药物偶联物22的结构式为:
所述抗体-药物偶联物23的结构式为:
本发明还提供了上述靶向于EGFR的抗体-药物偶联物的制备方法,其特征在于,包括以下步骤:
步骤1:制备化合物13:
将MMAE、11-叠氮基-3,6,9-三氧代十一酸、N-甲基吗啉NMM和HOAt溶于DMF中,室温下反应12h,用HPLC分离得到化合物13;
步骤2:制备药物-链接子:
将步骤1所得的化合物13、链接子、抗坏血酸钠和硫酸铜溶于tBuOH/H2O/DMF中,室温下反应1h后,用HPLC分离得到药物-链接子;
步骤3:制备抗体-药物偶联物:
将步骤2所得的药物-链接子溶于DMSO配制成药物-链接子溶液,将抗EGFR抗体溶于PBS缓冲溶液配制成抗EGFR抗体溶液,将TCEP溶于纯水配制成TCEP溶液;
在微孔板的孔中加入抗EGFR抗体溶液,TCEP溶液,然后放在微孔板振荡器上室温反应2h,随后加入药物-链接子溶液,然后放在微孔板振荡器上室温下反应12h后,通过Zeba脱盐柱除去过量的小分子化合物,得到抗体抗体-药物偶联物。
优选地,所述步骤2中,化合物13、链接子、抗坏血酸钠和硫酸铜的用量比例为1equiv:1.2equiv:1.2equiv:1.2equiv。
优选地,所述步骤2中,连接子为双乙烯磺酰胺连接子,其结构式为
其中,R为O,CONH或NHCO;R为对位或间位。
更优选地,所述双乙烯磺酰胺连接子为化合物3,化合物5,化合物8或化合物12中的一种;
所述化合物3的结构式为:
所述化合物5的结构式为:
所述化合物8的结构式为:
所述化合物12的结构式为:
进一步地,所述化合物3的制备方法包括:
步骤1:将4-氨基苯酚、Boc酸酐和Et3N溶于THF中,室温反应12h,减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/10作洗脱剂,快速柱层析,得到化合物1;
步骤2:将步骤1所得的化合物1、3-溴丙炔和K2CO3溶于DMF中,室温下反应12h,然后用水和乙酸乙酯萃取产物,合并后的有机相依次用饱和NaHCO3、NH4Cl和NaCl洗,再用无水Na2SO4干燥,减压旋蒸除去溶剂后得到的中间体用DCM溶解,在冰浴下冷却至0℃后加入三氟乙酸,冰浴下反应1h后减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/8作洗脱剂,快速柱层析,得到化合物2;
步骤3:将步骤2所得的化合物2和Et3N溶于DCM中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯,反应液45℃下冷凝回流2h,冷却至室温后加入水,产物用DCM萃取,合并后的有机相用饱和NaCl洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/6作洗脱剂,快速柱层析得到化合物3。
进一步地,所述化合物5的制备方法包括:
步骤1:将4-氨基苯甲酸,炔丙胺,HOBt,EDCl,N’N-二异丙基乙胺溶于四氢呋喃中,在室温下搅拌反应12h,减压旋转蒸除溶剂四氢呋喃后得粗品,将固体溶于二氯甲烷中,用水稀释反应液后,二氯甲烷萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入CH2Cl2和60~100目硅胶,拌匀旋干,粗品经硅胶柱层析得到化合物4;
步骤2:将步骤1所得的化合物4溶于二氯甲烷中,冰浴下,加入三乙胺,充分冷却后缓慢滴加2-氯乙烷磺酰氯,冰浴下反应2h,用水稀释反应液后,二氯甲烷萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入CH2Cl2和60~100目硅胶,拌匀旋干,粗品经硅胶柱层析得到化合物5。
进一步地,所述化合物8的制备方法包括:
步骤1:将(4-氨基苯基)氨基甲酸叔丁酯,1-叠氮丁酸,HOBt,EDCl,N’N-二异丙基乙胺溶于二氯甲烷中,在室温下搅拌反应12h,用水稀释反应液后,二氯甲烷萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入CH2Cl2和60~100目硅胶,拌匀旋干,粗品经硅胶柱层析得到化合物6;
步骤2:将步骤1所得的化合物6溶于二氯甲烷中,冰浴下加入三氟乙酸,冰浴下反应4h,减压旋转蒸除溶液后得到化合物7;
步骤3:将步骤2所得的化合物7溶于二氯甲烷中,冰浴下,加入三乙胺,充分冷却后缓慢滴加2-氯乙烷磺酰氯,冰浴下反应2h,用水稀释反应液后,二氯甲烷萃取,合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入CH2Cl2和60~100目硅胶,拌匀旋干,粗品经硅胶柱层析得到化合物8。
进一步地,所述化合物8的制备方法包括:
步骤1:将3-氨基苯酚和Boc酸酐溶于THF中,室温反应12h,减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/10作洗脱剂,快速柱层析,得到化合物9;
步骤2:将步骤1所得的化合物9,3-溴丙炔和K2CO3溶于DMF中,室温下反应12h,然后用水和乙酸乙酯萃取产物,合并后的有机相依次用饱和NaHCO3、NH4Cl和NaCl洗,再用无水Na2SO4干燥,减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/6作洗脱剂,快速柱层析得到化合物10;
步骤3:将步骤2所得的化合物10用DCM溶解,在冰浴下冷却至0℃后加入三氟乙酸,冰浴下反应3h后用水和CH2Cl2萃取产物,合并后的有机相用饱和NaHCO3洗,再用无水Na2SO4干燥,减压旋蒸除去溶剂,得到化合物11;
步骤4:将步骤3所得的化合物11和Et3N溶于DCM中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯,反应液45℃下冷凝回流1h,冷却至室温后加入水,产物用DCM萃取,合并后的有机相用饱和NaCl洗后用无水Na2SO4干燥,减压旋蒸除去溶剂,加入CH2Cl2和60~100目硅胶,拌匀旋干,采用乙酸乙酯/石油醚=1/5作洗脱剂,快速柱层析得到化合物12。
优选地,所述步骤3中,抗EGFR抗体溶液、TCEP溶液和药物-链接子溶液的用量比为1equiv:5equiv:10equiv。
本发明还提供了上述靶向于EGFR的抗体-药物偶联物在制备治疗增殖性疾病或病灶的药物中的用途。
优选地,所述增殖性疾病或病灶的特征为细胞生成一种抗原或靶点,该抗原或靶点能与EGFR的抗体特异性结合。
优选地,所述增殖性疾病为非小细胞肺癌。
与现有技术相比,本发明的有益效果在于:
(1)本发明是基于我们发明的双乙烯磺酰胺连接子技术,首次合成靶向于EGFR的抗体-药物偶联物。
(2)本发明基于抗体内在的硫-硫键断开后再桥联,可得到DAR为4左右的均一性好的偶联产物,并且对抗体的适用性广;
(3)本发明提供的ADCs,在体内外效果良好。
附图说明
图1显示了本发明中涉及的典型的药物-连接子的制备路线及结构;
图2显示了本发明中涉及的药物-连接子14、15、16、17的制备路线及结构;
图3显示了本发明中涉及的药物-链接子与抗体硫-硫键的选择性桥联;
其中,反应条件为:a:三(2-羧乙基)膦(TCEP),磷酸盐缓冲液(PBS),室温,2h;b:化合物14或者15或者16或者17,PBS/二甲亚砜,室温,12h;
图4显示了本发明中涉及的抗体-药物偶联物的SDS-PAGE跑胶图;
其中,1为曲妥珠单抗;2为还原后的曲妥珠单抗;3为抗体-药物偶联物18;4为抗体-药物偶联物19;5为抗体-药物偶联物20;6为抗体-药物偶联物21;
图5显示了本发明中涉及的抗体-药物偶联物的HR-ESI-MS谱图;
图6显示了本发明中涉及的抗体药物偶联物的SEC-HPLC谱图;
图7显示了涉及10nM的抗体-药物偶联物及西妥昔单抗在HCC827细胞的亲和力;
图8显示了本发明中涉及的不同浓度梯度的抗体-药物偶联物及西妥昔单抗在HCC827细胞的亲和力;
图9显示了本发明中涉及10nM的抗体-药物偶联物及西妥昔单抗在NCI-H2228细胞的亲和力;
图10显示了本发明中涉及的抗体-药物偶联物及西妥昔单抗在HCC827细胞的内吞效应;
图11显示了本发明中涉及的抗体-药物偶联物、西妥昔单抗和MMAE对HCC827细胞的体外增殖抑制作用;
图12显示了本发明中涉及的抗体-药物偶联物、西妥昔单抗和MMAE对NCI-H2228细胞的体外增殖抑制作用;
图13显示如下给药后对人非小细胞肺癌HCC827裸小鼠移植瘤的肿瘤体积均值随时间变化的图:(1)溶剂对照PBS;(2)22;(3)23;
图14显示如下给药后对人非小细胞肺癌HCC827裸小鼠移植瘤后小鼠体重均值随时间变化的图:(1)溶剂对照PBS;(2)22;(3)23。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
以下实验中所用的试剂及耗材来源于:所有化学试剂均为分析级,从商业来源获得,除非另有说明,否则无需进一步纯化即可使用。曲妥珠单抗购于(Trastuzumab)(上海环耀生物科技有限公司,货号:180288-69-1),未进一步纯化。
实施例1:药物-链接子的制备
如图1~2所示,本实施例提供了药物-链接子14、15、16、17的制备,具体如下:
化合物1
将4-氨基苯酚(2.0g,18.3mmol)、Boc酸酐(5mL,22mmol)和Et3N(36.6mmol,5mL)溶于THF(50mL)中,室温反应12h。减压旋蒸除去溶剂,加入30mL CH2Cl2和8g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/10作洗脱剂,快速柱层析,得到产物白色固体1(3.27g,15.6mmol),产率96%。1H NMR(500MHz,DMSO)δ9.04(s,1H),8.99(br,1H),7.22(d,J=7.1Hz,2H),6.71–6.58(m,2H),1.45(s,9H)ppm.13C NMR(126MHz,DMSO)δ153.06,152.56,131.08,119.99,115.07,78.45,28.24ppm.ESI-HRMS calcd for C11H16NO3[(M+H)+]:210.1130,found:210.1154.
化合物2
将化合物1(1.7g,8.1mmol)、3-溴丙炔(0.84ml,9.7mmol)和K2CO3(3.3g,24.3mmol)溶于DMF(30mL)中,室温下反应12h。然后用水(600mL)和乙酸乙酯(3x 50mL)萃取产物,合并后的有机相依次用饱和NaHCO3(20mL)、NH4Cl(20mL)和NaCl(20mL)洗,再用无水Na2SO4干燥。减压旋蒸除去溶剂后得到的中间体用DCM(30mL)溶解,在冰浴下冷却至0℃后加入三氟乙酸(12mL),冰浴下反应1h后减压旋蒸除去溶剂,加入20mL CH2Cl2和3g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/8作洗脱剂,快速柱层析,得到产物红色油状物2(1.0g,6.8mmol),两步总产率84%。1H NMR(500MHz,CDCl3)δ6.80(dd,J=8.7,1.6Hz,2H),6.60(dd,J=8.8,3.1Hz,2H),4.58(t,J=2.4Hz,2H),3.49(s,2H),2.51(d,J=2.3Hz,1H)ppm.13C NMR(126MHz,CDCl3)δ150.38,140.99,116.23,116.09,79.15,75.27,56.57ppm.ESI-HRMScalcd for C9H10NO[(M+H)+]:148.0762,found:148.0815.
化合物3
将化合物2(180mg,1.22mmol)和Et3N(7.32mmol,1.0mL)溶于DCM(15mL)中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯(0.288ml,2.69mmol),反应液45℃下冷凝回流2h。冷却至室温后加入水(5mL),产物用DCM(3x 15mL)萃取,合并后的有机相用饱和NaCl(10mL)洗后用无水Na2SO4干燥。减压旋蒸除去溶剂,加入10mL CH2Cl2和0.4g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/6作洗脱剂,快速柱层析得到产物淡黄色固体3(252mg,0.77mmol),产率64%。1H NMR(500MHz,CDCl3)δ7.23–7.17(m,2H),7.09–6.96(m,4H),6.28(d,J=16.6Hz,2H),6.14(d,J=9.9Hz,2H),4.70(d,J=2.4Hz,2H),2.56(t,J=2.4Hz,1H)ppm.13C NMR(126MHz,CDCl3)δ159.16,136.18,132.24,129.79,126.81,115.76,77.97,76.37,56.17ppm.ESI-HRMS calcd for C13H14NO5S2[(M+H)+]:328.0313,found:328.1552.
化合物4
将4-氨基苯甲酸(1.508g,11mmol),炔丙胺(0.686mL,10mmol),HOBt(0.817g,6.0mmol),EDCl(2.30g,12.0mmol),N’N-二异丙基乙胺(3.636mL,12mmol)溶于四氢呋喃(30mL)中,在室温下搅拌反应12h。减压旋转蒸除溶剂四氢呋喃后得粗品,将固体溶于二氯甲烷(30mL)中,用100mL水稀释反应液后,二氯甲烷萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入15mL CH2Cl2和8g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=1:1)得白色固体4(1g,5.74mmol,57.40%)。1H NMR(500MHz,DMSO)δ8.42(t,J=5.1Hz,1H),7.58(d,J=8.5Hz,2H),6.54(d,J=8.5Hz,2H),5.65(s,2H),3.99(dd,J=5.4,2.2Hz,2H),3.04(s,1H).13C NMR(126MHz,DMSO)δ166.42,152.31,129.32,120.93,113.03,82.45,72.83,28.70.ESI-HRMS Calculated for C10H11N2O[M+H]+:175.0871,Found:175.0952.
化合物5
将上一步所得化合物4(1g,5.74mmol)溶于二氯甲烷(20mL)中,冰浴下,加入三乙胺(2.386mL,17.22mmol),充分冷却后缓慢滴加2-氯乙烷磺酰氯(1.86mL,17.22mmol),冰浴下反应2h。用100mL水稀释反应液后,二氯甲烷萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入10mL CH2Cl2和5g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=1:1)得黄色固体5(1.508g,4.25mmol,74.16%)。1H NMR(500MHz,DMSO)δ9.08(t,J=5.5Hz,1H),7.96–7.88(m,2H),7.51–7.41(m,2H),7.27(dd,J=16.3,9.8Hz,2H),6.40(dd,J=9.8,1.0Hz,2H),6.27(dd,J=16.3,1.0Hz,2H),4.07(dd,J=5.5,2.5Hz,2H),3.14(t,J=2.5Hz,1H).13C NMR(126MHz,DMSO)δ165.51,136.54,136.31,136.02,132.01,131.53,129.00,81.50,73.50,29.09.ESI-HRMS Calculated forC14H15N2O5S2[M+H]+:355.0422Found:355.0351.
化合物6
将(4-氨基苯基)氨基甲酸叔丁酯(2.291g,11mmol),1-叠氮丁酸(1.1213g,10mmol),HOBt(1.633g,12mmol),EDCl(2.30g,12mmol),N’N-二异丙基乙胺(4.958mL,30mmol)溶于二氯甲烷(30mL)中,在室温下搅拌反应12h。用100mL水稀释反应液后,二氯甲烷萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入15mLCH2Cl2和8g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=1:1)得黄色固体6(2.117g,7mmol,70%)。1H NMR(500MHz,MeOD)δ7.46–7.41(m,2H),7.33(d,J=8.8Hz,2H),2.50–2.45(m,2H),2.30–2.24(m,3H),1.92–1.83(m,2H),1.51(s,9H).13C NMR(126MHz,MeOD)δ172.15,153.99,135.41,133.37,120.48,118.81,82.77,68.90,35.15,27.35,24.38,17.30.ESI-HRMS Calculated for C17H23N2O3[M+H]+:303.1709,Found:303.1800.
化合物7
将上一步所得化合物6(2.117g,7mmol)溶于二氯甲烷(20mL)中,冰浴下加入三氟乙酸(3.12mL,42mmol),冰浴下反应4h,减压旋转蒸除溶液后得黄色固体7(1.2306mg,6.08mmol,86.92%)。1H NMR(500MHz,MeOD)δ7.28–7.22(m,2H),6.72–6.63(m,2H),2.48–2.38(m,2H),2.29–2.20(m,3H),1.91–1.81(m,2H).13C NMR(126MHz,MeOD)δ172.04,144.16,129.26,122.10,121.99,115.33,83.00,69.05,35.15,24.54,17.40.ESI-HRMS Calculatedfor C12H15N2O[M+H]+:203.1184,Found:203.1351.
化合物8
将上一步所得化合物7(1.2306mg,6.08mmol)溶于二氯甲烷(20mL)中,冰浴下,加入三乙胺(2.53mL,18.25mmol),充分冷却后缓慢滴加2-氯乙烷磺酰氯(1.97mL,18.25mmol),冰浴下反应2h。用100mL水稀释反应液后,二氯甲烷萃取(3*20mL),合并有机相,无水硫酸钠干燥,减压旋转蒸除溶剂后得粗品,加入10mL CH2Cl2和5g 60-100目硅胶,拌匀旋干。粗品经硅胶柱层析(石油醚:乙酸乙酯=1:2)得金黄色固体8(1.912g,5mmol,82.23%)。1H NMR(500MHz,MeOD)δ7.68–7.61(m,2H),7.25–7.19(m,2H),7.11(dd,J=16.3,10.0Hz,2H),6.25(dd,J=7.8,0.6Hz,2H),6.22(d,J=0.6Hz,2H),2.53(t,J=7.5Hz,2H),2.32–2.24(m,3H),1.93–1.84(m,2H).13C NMR(126MHz,MeOD)δ172.54,140.50,136.23,131.36,129.24,119.89,82.71,68.93,35.20,24.11,17.23.ESI-HRMS Calculated forC16H19N2O5S2[M+H]+:383.0735,Found:383.1048.
化合物9
将3-氨基苯酚(5.45g,50mmol)和Boc酸酐(12.4g,57.5mmol)溶于THF(50mL)中,室温反应12h。减压旋蒸除去溶剂,加入30mL CH2Cl2和8g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/10作洗脱剂,快速柱层析,得到产物白色固体9(9.927g,47.5mmol),产率95%。ESI-HRMS calcd for C11H16NO3[(M+H)+]:210.1130,found:210.1148.
化合物10
将化合物9(0.93g,4.45mmol)、3-溴丙炔(0.582g,4.9mmol)和K2CO3(0.737g,5.34mmol)溶于DMF(30mL)中,室温下反应12h。然后用水(600mL)和乙酸乙酯(3x 50mL)萃取产物,合并后的有机相依次用饱和NaHCO3(20mL)、NH4Cl(20mL)和NaCl(20mL)洗,再用无水Na2SO4干燥。减压旋蒸除去溶剂,加入10mL CH2Cl2和1g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/6作洗脱剂,快速柱层析得到产物淡黄色固体10(880mg,3.56mmol),产率80%。ESI-HRMS calcd for C14H18NO3[(M+H)+]:248.1287,found:248.1295.
化合物11
将化合物10(0.37g,1.5mmol)用DCM(4mL)溶解,在冰浴下冷却至0℃后加入三氟乙酸(1mL),冰浴下反应3h后用水(20mL)和CH2Cl2(3x 20mL)萃取产物,合并后的有机相用饱和NaHCO3(20mL)洗,再用无水Na2SO4干燥。减压旋蒸除去溶剂,得到产物无色油状物11(0.18g,1.22mmol),产率82%。ESI-HRMS calcd for C9H10NO[(M+H)+]:148.0762,found:148.0783.
化合物12
化合物11(180mg,1.22mmol)和Et3N(7.32mmol,1.0mL)溶于DCM(15mL)中,冰浴下冷却至0℃后缓慢加入2-氯乙烷磺酰氯(0.288ml,2.69mmol),反应液45℃下冷凝回流1h。冷却至室温后加入水(5mL),产物用DCM(3x 15mL)萃取,合并后的有机相用饱和NaCl(10mL)洗后用无水Na2SO4干燥。减压旋蒸除去溶剂,加入10mL CH2Cl2和0.4g 60-100目硅胶,拌匀旋干。采用乙酸乙酯/石油醚=1/5作洗脱剂,快速柱层析得到产物淡黄色固体12(280mg,0.85mmol),产率70%。ESI-HRMS calcd for C13H14NO5S2[(M+H)+]:328.0313,found:328.0325.
化合物13的合成
将MMAE(100mg,0.139mmol)、11-叠氮基-3,6,9-三氧代十一酸(35.58mg,0.153mmol)、N-甲基吗啉NMM(140.6mg,1.39mmol)、EDCl(53.48mg,0.279mmol)和HOAt(37.92mg,0.279mmol)溶于DMF(5mL)中,室温下反应12h。用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体13(111mg,0.1189mmol),产率85.57%。ESI-HRMSCalculated for C47H81N8O11[M+H]+:993.6025,Found:993.5729.
药物-连接子14的合成
将化合物13(37mg,0.0397mmol)、化合物3(15.58mg,0.0476mmol)、抗坏血酸钠(8.38mg,0.0476mmol)和硫酸铜(7.57mg,0.0476mmol)溶于6mLtBuOH/H2O/DMF(1/1/1)中。室温下反应1h后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体的药物-连接子14(14.6mg,0.01158mmol),产率29.17%。ESI-HRMSCalculated for C60H94N9O16S2[M+H]+:1260.6260,Found:1260.5866。用HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,10分钟)分析产物的纯度,纯度为98.6%。
药物-连接子15的合成
将化合物13(37mg,0.0397mmol)、化合物8(18.21mg,0.0476mmol)、抗坏血酸钠(8.38mg,0.0476mmol)和硫酸铜(7.57mg,0.0476mmol)溶于6mLtBuOH/H2O/DMF(1/1/1)中。室温下反应1h后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体的药物连接子15(6.1mg,0.004636mmol),产率11.67%。ESI-HRMSCalculated for C63H99N10O16S2[M+H]+:1315.6682,Found:1315.6358。用HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,10分钟)分析产物的纯度,纯度为98.2%。
药物-连接子16的合成
将化合物13(30mg,0.032mmol)、化合物5(13.82mg,0.039mmol)、抗坏血酸钠(6.87mg,0.039mmol)和硫酸铜(6.2mg,0.039mmol)溶于6mLtBuOH/H2O/DMF(1/1/1)中。室温下反应1h后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体的药物-连接子16(17.5mg,0.0136mmol),产率42.47%。ESI-HRMSCalculated for C63H99N10O16S2[M+H]+:1287.6369,Found:1287.6890。用HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,10分钟)分析产物的纯度,纯度为97.9%。
药物-连接子17的合成
化合物13(40mg,0.0428mmol)、化合物12(16.84mg,0.0514mmol)、抗坏血酸钠(9.05mg,0.0514mmol)和硫酸铜(8.18mg,0.0514mmol)溶于6mLtBuOH/H2O/DMF(1/1/1)中。室温下反应1h后用制备HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,30分钟)分离得到产物白色固体的药物-连接子17(24.3mg,0.01927mmol),产率45.03%。ESI-HRMSCalculated for C60H94N9O16S2[M+H]+:1260.6260,Found:1260.6067。用HPLC(固定相为C-18硅胶柱,流动相为CH3CN/H2O=10~100%,10分钟)分析产物的纯度,纯度为99.3%。
实施例2:药物-链接子与抗体硫-硫键的选择性桥联
如图3所示,将药物-连接子14、15、16和17溶于DMSO配成10mM的溶液,将曲妥珠单抗(Trastuzumab)(上海环耀生物科技有限公司,货号:180288-69-1)溶于PBS(pH=7.4)缓冲溶液配成2.5mg/mL的溶液,TCEP溶于纯水配成10mM的溶液(用NaOH/H3PO4调节PH为7.0)。
在微孔板(330uL LABTIDE 96 Round Well)的四个孔中分别加入100uL的曲妥珠单抗溶液,0.83uL的TCEP溶液,然后放在微孔板振荡器上室温反应2h。随后分别加入1.67uL(10eq.)含有药物分子的双乙烯磺酰胺化合物,然后放在微孔板振荡器上室温下反应12h后,通过Zeba脱盐柱(ZebaTM Spin Desalting Columns,7K MWCO,0.5mL)除去过量的小分子化合物。得到抗体抗体-药物偶联物,分别命名为抗体-药物偶联物18、抗体-药物偶联物19、抗体-药物偶联物20、抗体-药物偶联物21。
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶分析所得抗体药物偶联物。按照标准实验室程序进行4-12%丙烯酰胺凝胶的非还原性甘氨酸-SDS-PAGE。使用4-12%堆积凝胶并共同运行宽范围MW标记物(10-250kDa,Prestained PagerulerPlus蛋白质标准品,Bio-Rad)以估计蛋白质重量。将得到的抗体药物偶联物18、19、20或21(10μL)转移到管中,将NuPAGE LDS样品缓冲液(4X,10μL)和磷酸盐缓冲液((pH=7.4),20μL)加入管中。将溶液(10μL)加载到具有4-12%梯度聚丙烯酰胺浓度的NuPAGE Bis-Tris微型凝胶中,并通过电泳(120V)分析缀合反应。使用的缓冲系统是1X SDS运行缓冲液(NuPAGE MOPS SDS运行缓冲液,20X,pH 7.3,50至950mL去离子水)。然后,用考马斯染料(0.5%)染色凝胶,并在室温下混合后2小时读取凝胶。图4说明本发明中所制备得到的抗体-药物偶联物18、19和21正确形成桥接抗体并且未发现“半抗体”。
用UPLC-MS(型号ABsciex 4600)分析所得抗体药物偶联物。流动相为:0.1%HCOOH水溶液(溶剂A)和乙腈(溶剂B)。LC条件:GL Sciences C4柱:2.1×150mm,5μm,柱温:70℃,λ=254nm。梯度编程如下:0-2.5分钟15%B,2.5-5分钟15-95%B,5-6.5分钟95%B,6.51-8.0分钟5%B。电喷雾源用毛细管电压操作使用2.0kV,锥电压为40V的氮气作为去溶剂化气体,总流量为850L/h。在LC-MS分析之前,使用Endo S酶将曲妥珠单抗样品去糖基化。图5说明本发明中所制备得到的抗体-药物偶联物18、19和21的Drug Antibody Ratio(DAR)约为4。
使用Agilent technologies 1260Infinity进行排阻色谱(SEC)分析所得抗体药物偶联物。流动相是磷酸盐缓冲盐水(PBS(pH 7.4))。LC条件:TSKgel G3000SWXL柱:7.8×300mm,5μm,柱温:40℃,λ=280nm,梯度:0-20分钟100%PBS(pH7.4),流速:1mL/min。图6说明本发明中所制备得到的抗体-药物偶联物18、19、20、21没有明显的聚集。
实施例3:抗体-药物偶联物的亲和力测定
将含有药物分子的双乙烯基磺酰胺化合物14和15溶于DMSO配成10mM的溶液,将西妥昔单抗(Cetuximab)(上海环耀生物科技有限公司,货号:205923-56-4)溶于PBS(pH=7.4)缓冲溶液配成2.5mg/mL的溶液,TCEP溶于纯水配成10mM的溶液(用NaOH/H3PO4调节PH为7.0)。
在微孔板(330uL LABTIDE 96Round Well)的四个孔中分别加入100uL的西妥昔单抗溶液,0.83uL的TCEP溶液,然后放在微孔板振荡器上室温反应2h。随后分别加入1.67uL(10eq.)含有药物分子的双乙烯磺酰胺化合物14或15,然后放在微孔板振荡器上室温下反应12h后,通过Zeba脱盐柱(ZebaTM Spin Desalting Columns,7K MWCO,0.5mL)除去过量的小分子化合物。得到抗体抗体-药物偶联物,分别命名为抗体-药物偶联物22、抗体-药物偶联物23。
在本实施例中,研究了Cetuximab、抗体-药物偶联物22、抗体-药物偶联物23对肿瘤细胞系的亲和作用。
以下实验中所用的试剂及耗材来源于:Goat Anti-Human IgG H&L(FITC)购置于Abcam公司,DAPI购置于Cell Signaling公司,face清洗液(PBS+1%BSA),人非小细胞肺癌细胞系HCC827和NCI-H2228来自美国模式培养物集存库ATCC。
本实施例中使用流式细胞术(flow cytometry,FCM)来分析抗体及ADC对肿瘤细胞系的亲和作用。FCM是利用流式细胞仪在细胞分子水平上通过单克隆抗体对单个细胞进行多参数、快速的定量分析、分选的技术。Cetuximab可以特异性结合在靶细胞的Her-2抗原位点,然后用特异的荧光二抗标记Cetuximab,利用FCM检测荧光二抗的荧光强度可以间接指示Cetuximab与靶细胞的抗原位点的结合能力。
分别收集HCC827和NCI-H2228细胞至15mL离心管,在4℃下1200rpm离心3min,弃上清;用face洗液洗涤细胞1次,分装到1.5mL离心管(每个管大于1×105个细胞)中,在4℃下1200rpm离心3min,去上清。
每个样品管内加入200μL Cetuximab、抗体-药物偶联物22、抗体-药物偶联物23(浓度分别设置为:0.01、0.03、0.1、0.3、1、3、10、50、200nM),在冰上孵育30min。
在4℃下1200rpm离心3min,去上清,用face洗液洗涤细胞3次;每个样品管内加入200μL Goat Anti-Human IgG H&L(FITC)抗体(1:200),在冰上孵育30min。
在4℃下1200rpm离心3min,去上清,用face洗液洗涤细胞3次;每个样品管内加入200μL DAPI(0.1μg/mL),冰上孵育15min后在CytoFLEX(Beckman Coulter)流式细胞仪上进行检测分析。
图7显示了本发明中涉及抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23及西妥昔单抗在EGFR高表达细胞株HCC827细胞中的亲和力。在10nM浓度下,抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23及西妥昔单抗与HCC827细胞亲和力相同。
图8显示了本发明中涉及的不同浓度梯度的抗体-药物偶联物及西妥昔单抗在HCC827细胞的亲和力。抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23及西妥昔单抗与HCC827细胞亲和力拟合曲线具有高度一致性。
图9显示了本发明中涉及抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23及西妥昔单抗在EGFR低表达细胞株NCI-H2228细胞中的亲和力,在10nM浓度下,抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23和西妥昔单抗与NCI-H2228细胞具有相同的亲和力,并且显著低于HCC827细胞。因此对西妥昔单抗的上述改造没有影响其在靶细胞中的亲和力。
实施例4:抗体-药物偶联物的内吞作用测定
在本实施例中,研究了人非小细胞肺癌细胞系HCC827对Cetuximab、抗体-药物偶联物22、抗体-药物偶联物23的内吞作用。
以下实验中所用的试剂及耗材来源于:Goat Anti-Human IgG H&L(FITC)购置于Abcam公司,DAPI购置于Cell Signaling公司,face清洗液(PBS+1%BSA),人非小细胞肺癌细胞系HCC827来自美国模式培养物集存库ATCC。
本实施例中使用流式细胞术(flow cytometry,FCM)来分析肿瘤细胞系对抗体及ADC的内吞作用。Cetuximab抗体结合在靶细胞HCC827的EGFR抗原位点后会发生內吞作用进入细胞内。细胞在37℃可以正常內吞,在4℃细胞绝大多数生理活动受到抑制,视为不內吞。Cetuximab抗体与HCC827细胞分别在4℃和37℃孵育一段时间,该时间段内的內吞量=(4℃细胞表面抗体量-37℃细胞表面抗体量)/4℃细胞表面抗体量×100%。
收集HCC827细胞至15mL离心管,在4℃下1200rpm离心3min,弃上清;用face洗液洗涤细胞1次,分装到1.5mL离心管(每个管大于1×105个细胞)中,在4℃下1200rpm离心3min,去上清。
每个样品管内加入200μL Cetuximab、抗体-药物偶联物22、抗体-药物偶联物23(浓度为10nM),在4℃孵育30min。
在4℃下1200rpm离心3min,去上清,用face洗液洗涤细胞3次;将其中每个样品管内加入200μL Goat Anti-Human IgG H&L(FITC)抗体(1:200),在冰上孵育30min。
在4℃下1200rpm离心3min,去上清,用face洗液洗涤细胞3次;每个样品管内加入200μL DAPI(0.1μg/mL),冰上孵育15min后在CytoFLEX(Beckman Coulter)流式细胞仪上进行检测分析。
图10显示了本发明中涉及的抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23及西妥昔单抗在EGFR高表达细胞株HCC827细胞的内吞作用。10nM的抗体-药物偶联物抗体-药物偶联物22、抗体-药物偶联物23及西妥昔单抗在HCC827细胞中经过3h的內吞效率分别为42.16%、43.85%及46.45%。因此,对西妥昔单抗的上述改造没有影响其在靶细胞中的內吞效率。
实施例5:抗体-药物偶联物的体外细胞增殖生物活性测定
在本实施例中,研究了Cetuximab、抗体-药物偶联物22、抗体-药物偶联物23、MMAE对肿瘤细胞系增殖的作用。
以下实验中所用的试剂及耗材来源于:RPMI1640培养基、0.25%胰蛋白酶-EDTA、胎牛血清、重组人胰岛素、100×青链霉素、1×PBS(pH 7.4)、CCK8显色试剂购置于Gibco公司;人非小细胞肺癌细胞系HCC827和NCI-H2228来自美国模式培养物集存库ATCC。10cm培养皿(Corning)及96孔细胞培养板(Corning);多功能酶标仪(SpectraMax i3)。
本实施例使用CCK8比色法来评价待测物的抗增殖作用。Cell Counting Kit-8(简称CCK-8)试剂中含有WST-8【化学名:2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐】。它在电子载体1-甲氧基-5-甲基吩嗪鎓硫酸二甲酯(1-Methoxy PMS)的作用下被细胞中的脱氢酶还原为具有高度水溶性的黄色甲瓒产物(Formazan dye)。生成的甲瓒物的数量与活细胞的数量成正比,在450nm波长下产生最大吸收峰。因此可利用这一特性直接进行细胞增殖和毒性分析。
本实例选用的细胞系有:HCC827和NCI-H2228细胞在含有10%胎牛血清和1%青链霉素的RPMI1640培养基中,所有细胞于37℃、5%二氧化碳培养箱中培养至对数生长期,将处于对数生长期的上述细胞以5×103个细胞每孔的密度接种至96孔培养板,每孔100μL,培养24h后加入不同浓度的药物处理72h,药物原始浓度为500nM,以5倍稀释制备10个浓度,每个浓度设置3个复孔,并设相应浓度的溶媒对照及无细胞培养基孔。作用结束后,每孔加入10μL CCK8试剂,在于37℃,5%二氧化碳培养箱中继续培养一段1~4h。然后在450nm波长下测定吸光度(OD值)。
抑制率(%)=(OD对照-OD给药)/(OD对照-OD空白)×100%
图11和图12显示了本发明中涉及的抗体-药物偶联物、西妥昔单抗和MMAE对在HCC827和NCI-H2228细胞的体外增殖抑制作用。
表1本发明中涉及的抗体-药物偶联物、西妥昔单抗和MMAE对在HCC827和NCI-H2228细胞中的IC50(nM)
结果表明,本发明中涉及的抗体-药物偶联物22、23对EGFR高表达细胞HCC827有较强的抑制作用,IC50分别为5.84nM、6.376nM,抑制作用强于原抗Cetuximab,低于小分子毒性药物MMAE;抗体-药物偶联物22、23对EGFR低表达细胞NCI-H2228没有显著抑制作用。
实施例6:抗体-药物偶联物的体内抗肿瘤生物活性测定
实验动物:NOD-Scid免疫缺陷小鼠,6±0.5周龄,雌性,购自江苏集萃药康生物科技有限公司。许可证号:SCXK(苏)-0008。饲养环境:国家蛋白质科学中心实验动物房(SPF级)。
实验步骤:实验小鼠在SPF级别封闭环境中适应5天后,选取健康小鼠在其右侧皮下接种人非小细胞肺癌HCC827细胞(5×106个细胞/只),待肿瘤生长至150mm3左右后,将动物随机分组。给药剂量和给药方案见表1。每4天监测肿瘤体积,称量体重,记录数据。肿瘤体积(V)计算公式为:V=1/2×a×b2(其中a、b分别表示肿瘤长、宽)。
表2给药剂量和给药方案
图13显示,溶剂对照PBS组人非小细胞肺癌HCC827异种移植小鼠皮下肿瘤模型肿瘤体积均值持续生长,在分组处理后第40d肿瘤体积均值超过2000mm3;而20mg/kg抗体-药物偶联物22及抗体-药物偶联物23处理组在给药后肿瘤体积显著减小,在分组处理后第12d肿瘤完全消失。
图14显示,抗体-药物偶联物22及抗体-药物偶联物23处理组小鼠体重持续增长,而溶剂对照PBS组小鼠体重增长受到抑制。
Claims (9)
1.一种靶向于EGFR的抗体-药物偶联物,其特征在于,包含式Ⅰ结构的抗体药物偶联物或其药学上可接受的盐或溶剂合物,其结构式为:
其中,n表示药物-抗体比例,n为3~5;R为O,CONH或NHCO;R为对位或间位;p为1~6;m为0~20。
2.如权利要求1所述的靶向于EGFR的抗体-药物偶联物,其特征在于,所述Anti-EGFR为抗EGFR抗体,Anti-EGFR为尼妥珠单抗或西妥昔单抗。
3.如权利要求1所述的靶向于EGFR的抗体-药物偶联物,其特征在于,所述靶向于EGFR的抗体-药物偶联物为抗体-药物偶联物22或抗体-药物偶联物23;
所述抗体-药物偶联物22的结构式为:
所述抗体-药物偶联物23的结构式为:
4.权利要求1~3任一项所述的靶向于EGFR的抗体-药物偶联物的制备方法,其特征在于,包括以下步骤:
步骤1:制备化合物13:
将MMAE、11-叠氮基-3,6,9-三氧代十一酸、N-甲基吗啉NMM和HOAt溶于DMF中,室温下反应12h,用HPLC分离得到化合物13;
步骤2:制备药物-链接子:
将步骤1所得的化合物13、链接子、抗坏血酸钠和硫酸铜溶于tBuOH/H2O/DMF中,室温下反应1h后,用HPLC分离得到药物-链接子;
步骤3:制备抗体-药物偶联物:
将步骤2所得的药物-链接子溶于DMSO配制成药物-链接子溶液,将抗EGFR抗体溶于PBS缓冲溶液配制成抗EGFR抗体溶液,将TCEP溶于纯水配制成TCEP溶液;
在微孔板的孔中加入抗EGFR抗体溶液,TCEP溶液,然后放在微孔板振荡器上室温反应2h,随后加入药物-链接子溶液,然后放在微孔板振荡器上室温下反应12h后,通过Zeba脱盐柱除去过量的小分子化合物,得到抗体抗体-药物偶联物。
5.如权利要求4所述的靶向于EGFR的抗体-药物偶联物的制备方法,其特征在于,所述步骤2中,连接子为双乙烯磺酰胺连接子,其结构式为
其中,R为O,CONH或NHCO;R为对位或间位。
6.如权利要求5所述的靶向于EGFR的抗体-药物偶联物的制备方法,其特征在于,所述双乙烯磺酰胺连接子为化合物3,化合物5,化合物8或化合物12中的一种;
所述化合物3的结构式为:
所述化合物5的结构式为:
所述化合物8的结构式为:
所述化合物12的结构式为:
7.权利要求1~3任一项所述的靶向于EGFR的抗体-药物偶联物在制备治疗增殖性疾病或病灶的药物中的应用。
8.如权利要7所述的应用,其特征在于,所述增殖性疾病或病灶的特征为细胞生成抗原或靶点,该抗原或靶点能与EGFR的抗体特异性结合。
9.如权利要7所述的应用,其特征在于,所述增殖性疾病为非小细胞肺癌。
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