CN113181373B - 一种抗体药物偶联制剂及其制备方法和应用 - Google Patents

一种抗体药物偶联制剂及其制备方法和应用 Download PDF

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CN113181373B
CN113181373B CN202110506892.2A CN202110506892A CN113181373B CN 113181373 B CN113181373 B CN 113181373B CN 202110506892 A CN202110506892 A CN 202110506892A CN 113181373 B CN113181373 B CN 113181373B
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antibody
antibody drug
drug conjugate
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CN113181373A (zh
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李卓玲
李晓彤
倪大禹
李宝山
林植平
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Jilin Baohe Ante Biomedical Technology Co ltd
Shenzhen Ante Biomedical Technology Co ltd
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Shenzhen Ante Biomedical Technology Co ltd
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Abstract

本发明公开了一种抗体药物偶联物制剂及其制备方法和应用，属于生物医药技术领域。所述抗体药物偶联物由DAR2和DAR4组成，DAR2的重量百分比为12‑20%，DAR4的重量百分比为80‑88%。本发明通过对抗体药物Glembatumumab vedotin偶联纯化工艺的改进，得到了只含有DAR2和DAR4两种有效成分的抗体药物偶联物制剂，并通过药效学实验证明其抗癌效果远优于抗体药物Glembatumumab vedotin。

Description

一种抗体药物偶联制剂及其制备方法和应用
技术领域
本发明属于生物医药技术领域，具体涉及一种抗体药物偶联物制剂及其制备方法和应用。
背景技术
抗体偶联药物(Antibody-Drug Conjugates，ADC)是由靶向特异性抗原的单克隆抗体药物和小分子细胞毒药物通过连接子偶联而成，兼具传统小分子化疗的强大杀伤效应及抗体药物的肿瘤靶向性。
非转移性黑色素瘤糖蛋白B(glycoprotein nonmetastatic melanoma proteinB，GPNMB)是一种Ⅰ型跨膜糖蛋白，研究证实其在多种肿瘤组织中表达，并且与肿瘤浸润和转移有关。由于GPNMB在多种肿瘤细胞中表达，故其可能成为肿瘤免疫治疗的分子靶点。
抗体药物Glembatumumab vedotin(CDX-011)可用于靶向治疗有GPNMB表达的肿瘤细胞，在美国已经完成临床I、II、III期试验，对三阴乳腺癌，黑色素癌，头颈癌，肺鳞癌等肿瘤有效。
目前，CDX-011的制备工艺得到的制剂是含有7个组分的混合物，而具有抑制癌细胞的有效成分只是混合物中的2个组分。因此，为了得到高纯度的抗体药物偶联物制剂，需要对CDX-011的制备工艺进行改进。
发明内容
本发明的目的是提供一种抗体药物偶联物制剂其制备方法和应用，该抗体药物偶联物制剂对GPNMB阳性的肿瘤组织具有高抗肿瘤活性。
为了实现上述发明目的，本发明采用以下技术方案：
一种抗体药物偶联物制剂，包括抗体药物偶联物和药学上允许的佐剂；
所述抗体药物偶联物由DAR2和DAR4组成，DAR2的重量百分比为12-20％，DAR4的重量百分比为80-88％；
所述DAR2具有下式所示结构：
所述DAR4具有下式所示结构：
上式中，所述抗体为人IgG2单克隆抗体。
上述抗体药物偶联物制剂的制备方法，包括以下步骤：
步骤1，将解冻后的抗体置于偶联缓冲液中，进行超滤透析；
步骤2，将超滤透析后的抗体溶液和偶联缓冲液混合，然后先加入二乙烯三胺五乙酸溶液，再加入还原剂三(2-羧乙基)膦盐酸盐溶液，进行还原反应；
步骤3，向还原反应液中加入MC-Val-Cit-PAB-MMAE母液，制备抗体药物偶联物，之后加入L-Cys溶液终止反应，得到偶联反应液；
步骤4，向偶联反应液中加入疏水层析洗脱液进行第一次稀释，得到第一稀释液，然后向第一稀释液中再加入疏水层析稀释液进行第二次稀释；
步骤5，对稀释后的样品进行深层过滤，深层过滤后的样品除菌过滤；
步骤6，将步骤5得到的样品进行疏水层析，将疏水样品合并后进行除菌过滤；
步骤7，将步骤6得到的样品进行超滤透析，超滤透析后的样品除菌过滤；
步骤8，将步骤7得到的样品用制剂缓冲液进行稀释，即可得到所述抗体药物偶联物制剂。
进一步地，步骤2中二乙烯三胺五乙酸溶液加入后的终浓度为1-2mM，还原剂三(2-羧乙基)膦盐酸与抗体的摩尔比为5.0:1-7:1，还原反应体系中抗体的浓度为6.0-15mg/mL，还原反应的温度为28-39℃、反应时间为110-190min。
进一步地，步骤3中MC-Val-Cit-PAB-MMAE与抗体的摩尔比为8:1-16:1，偶联反应的温度为18-27℃、反应时间为1-6h。
进一步地，步骤4中偶联反应液和疏水层析洗脱液的重量比为1-5:0.25，第一稀释液和疏水层析稀释液的重量比为1:1.069。
上述抗体药物偶联物制剂在制备用于肿瘤靶向治疗的药物中的用途，所述的肿瘤为三阴乳腺癌、黑色素癌、头颈癌或肺鳞癌。
本发明通过对抗体药物Glembatumumab vedotin偶联纯化工艺的改进，得到了只含有DAR2和DAR4两种有效成分的抗体药物偶联物制剂，并通过药效学实验证明其抗癌效果远优于抗体药物Glembatumumab vedotin。
附图说明
图1为抗体药物偶联物制剂的液相结果。其中第一幅为抗体药物Glembatumumabvedotin(CDX-011)的液相色谱，第二幅和第三幅为实施例1制得的抗体药物偶联物制剂的液相色谱。
图2和图3为治疗组和对照组给药后肿瘤生长情况的结果。
图4为治疗组和对照组给药后的体重变化。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
本发明中超滤均采用Pellicon 3超滤膜。
本发明中采用的抗体中间体为C19399抗体中间体，购自烟台迈百瑞国际生物医药有限公司；采用的MC-Val-Cit-PAB-MMAE，购自烟台迈百瑞国际生物医药有限公司。
以下实施例中，采用的相关试剂配方如下：
50mM PB缓冲液：每1L含量：Na2HPO4·2H2O 6.86g，NaH2PO4·H2O 1.58g，WFI定容至1000g，用0.50M NaOH或0.50M HCl调节pH至7.30-7.50。
10mM二乙烯三胺五乙酸母液：每1L含量：DTPA 3.90g，NaOH 1.20g，WFI定容至1000g。
10mM三(2-羧乙基)膦盐酸盐母液：每1L含量：TCEP·HCl 2866mg，WFI定容至1000g。
6mg/mL MC-Val-Cit-PAB-MMAE母液：每1L含量：MC-Val-Cit-PAB-MMAE 6000mg，DMSO定容至1100g。
30mM L-Cys溶液：每1L含量：L-Cys 3.63g，WFI定容至1000g。
疏水层析洗脱液：每1L含量：Na2HPO4·2H2O 6.18g，NaH2PO4·H2O 2.11g，DMSO66.00g，WFI定容至1000g。
疏水层析稀释液：每1L含量：Na2HPO4·2H2O 8.90g，(NH4)2SO4 105.71g，DMSO66.00g，WFI定容至1069g。
疏水层析平衡液：每1L含量：Na2HPO4·2H2O 8.01g，NaH2PO4·H2O 0.69g，(NH4)2SO452.86g，DMSO 66.00g，WFI定容至1041g。
20mM组氨酸缓冲液：每1L含量：组氨酸1.46g，一水盐酸组氨酸2.22g，WFI定容至1000g。
4X制剂缓冲液：每1L含量：蔗糖400g，聚山梨酯20 6.80g，20mM His/His-HCl缓冲液定容至1151g。
1X制剂缓冲液：每1L含量：4X制剂缓冲液287.75g，20mM His/His-HCl缓冲液定容至1038g。
实施例1
1、抗体解冻
将抗体中间体从冰箱中取出，置室温(20-26℃)存放，至完全解冻，控制解冻时间≤48h。
2、抗体中间体超滤透析
膜包载量100-300g/m2，超滤前用≥3L/m2的50mM PB缓冲液冲膜。然后将解冻后的抗体中间体置换到50mM PB缓冲液中，进行超滤透析。
控制进口流速5-6L/min/m2，透析浓度8.0-12.0mg/mL，TMP 12-16psi，透析体积为6-8DV。超滤透析后抗体浓度在7.0-10.0mg/mL。将超滤透析后的裸抗用除菌滤器过滤。
3、还原反应
将抗体溶液、50mM PB缓冲液加入反应釜，然后加入10mM二乙烯三胺五乙酸母液至终浓度为1-2mM，将反应釜保温至35-39℃。保温至所需温度后，按照三(2-羧乙基)膦盐酸盐与抗体的摩尔比5.0:1-7.0:1加入10mM三(2-羧乙基)膦盐酸盐母液，进行还原反应，还原反应温度为28-39℃，还原反应时间为110-190min。还原反应体系抗体终浓度为6.0mg/mL，终pH为7.30-7.50。
还原反应结束之后将反应体系温度降低至20-23℃，从还原反应开始到降温至目标温度不超过230min。
4、偶联反应
按照小分子毒素与抗体摩尔比8:1-16：1计算添加6mg/mL MC-Val-Cit-PAB-MMAE母液的体积。然后在10-20min之内添加完所需体积的MC-Val-Cit-PAB-MMAE母液。偶联温度为18-27℃，偶联时间为1-6h。
按照L-Cys:MC-Val-Cit-PAB-MMAE摩尔比为2:1加入30mM L-Cys溶液终止反应，反应温度为23-27℃，反应时间为15-30min。终止反应结束后，从反应釜中放出反应液。
5、疏水层析样品稀释
首先按照偶联反应液：疏水层析洗脱液(g/g)＝1:0.25，对样品进行第一步稀释。
然后稀释后得到的偶联样品，再用疏水层析稀释液稀释，稀释比为稀释后偶联样品1：疏水层析稀释液(g/g)＝1:1.069，稀释时间10-30min。
6、深层过滤
对稀释后的样品进行深层过滤(深层过滤膜包，MD0HC05FS1)，控制入口压力≤1bar，入口通量150-250LMH，体积载量≤600L/m2。
深层过滤后，用疏水层析平衡液进行膜包顶洗，控制入口通量150-250LMH，顶洗体积为40-60L/m2。
深层过滤后样品除菌过滤。
7、疏水层析
采用丁基柱(Butyl Sepharose 4FF填料，粒径90μm)进行疏水层析，层析柱消毒(0.50M NaOH)6-8CV，层析柱预平衡(注射水)4-5CV，层析柱平衡(疏水层析平衡液)保留时间6-10min，4-6CV。
上样：上样载量12-22g/L填料，保留时间6-10min。
层析柱再平衡：疏水层析平衡液，保留时间6-10min，10-12CV。
洗脱：疏水层析洗脱液，保留时间6-10min，收集UV起始值40-60mAU/mm，收集UV结束值40-60mAU/mm。
疏水样品收集液合并后，取样送检ADC浓度。随后，将疏水样品合并液进行除菌过滤。
8、ADC超滤透析
冲膜：超滤前用≥3L/m2的20mM组氨酸缓冲液冲膜。
浓缩：目标浓度5.0mg/mL(4.0-6.0mg/mL)，进口流速5-6L/min/m2，TMP 12-16psi。
透析：20mM组氨酸缓冲液，进口流速5-6L/min/m2，TMP 12-16psi，透析倍数8-10DV。
再浓缩：目标浓度14.0-16.0mg/mL，进口流速5-6L/min/m2，TMP 12-16psi。
ADC回收：目标浓度11.0-15.0mg/mL。
将超滤透析后的ADC用除菌滤器过滤。
9、原液制备
第一步稀释：向超滤透析后的ADC收集液中加入4X制剂缓冲液及1X制剂缓冲液，目标浓度8.0mg/mL(7.0-9.0mg/mL)。
第二步稀释：根据第一步稀释后ADC浓度，加入1X制剂缓冲液，目标浓度5.0mg/mL(4.5-5.5mg/mL)。
除菌过滤后，即可得到ADC原液，即所述抗体药物偶联物制剂，其中：DAR2＝12-20％，DAR4＝80-88％。
如图1所示，采用上述工艺制得的抗体药物偶联物制剂中，仅有DAR2和DAR4，单体含量之和在99.5％以上，说明上述工艺可制得只含有DAR2和DAR4两种有效成分的抗体药物偶联物制剂。
实施例2
抗体药物偶联物制剂在人源黑色素瘤SK-MEL-5细胞株皮下异种移植雌性BALB/c裸鼠模型中的药效学评价
1.供试品
C19399-1为抗体药物Glembatumumab vedotin(CDX-011)，DAR＝4.56。
C19399-2为实施例1所制备的抗体药物偶联物制剂，DAR＝3.31。
2.给药方案
按下表所示给药途径、剂量及方案给药。
其中，s.c.表示皮下注射，Q5D×6表示每五天给药一次、连续六次。
3.实验方法
3.1细胞培养
SK-MEL-5细胞(购自ATCC；细胞编号CL-00154)培养在含10％胎牛血清的MEM+0.01mMNEAA培养液中。收集指数生长期的SK-MEL-5细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
3.2动物造模和随机分组
53只雌性小鼠右侧皮下接种1×107SK-MEL-5细胞，细胞重悬在1：1的PBS与基质胶中(0.2mL/只)定期观察肿瘤生长情况。待肿瘤平均体积146mm3左右时，根据肿瘤大小随机分组，分组当天定义为第0天。给药起始于第0天。
3.3实验过程中动物体重下降的处理
当单只动物的体重下降超过15％时(BWL≥15％)，给予相应单只动物/该组动物停药期，直到其体重下降恢复至15％以内(BWL≤15％)。
当单只小鼠体重下降>15％超过72小时/下降>20％，按照动物福利对其实施安乐死。
3.4疗效评价标准
相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
或T/C％＝TTW/CTW×100％(TTW：治疗组实验终结时平均瘤重；CTW：溶媒对照组实验终结时平均瘤重)。
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW))。
3.5实验终点
开始给药后第35天称瘤重，肿瘤、荷瘤鼠拍照以及福尔马林固定瘤块后石蜡包埋。
4.实验结果
4.1测试药C19399在SK-MEL-5人黑色素瘤模型中的抗肿瘤作用
溶媒对照组小鼠在开始给药后第三十五天平均肿瘤体积为682.24mm3。测试药C19399-1(2.5mg/kg)治疗组在开始给药后第三十五天平均肿瘤体积为365.87mm3，相较溶媒对照组统计学上没有显著性差异(p＝0.249)，相对肿瘤抑制率TGI(％)为48％。测试药C19399-1(5mg/kg)治疗组在开始给药后第三十五天平均肿瘤体积为215.01mm3，相较溶媒对照组统计学上没有显著性差异(p＝0.136)，相对肿瘤抑制率TGI(％)为64％。C19399-1(10mg/kg)治疗组在开始给药后第三十五天平均肿瘤体积为0.00mm3，相较溶媒对照组统计学上有显著性差异(p＜0.001)，相对肿瘤抑制率TGI(％)为100％。C19399-2(3.14mg/kg)治疗组在开始给药后第三十五天平均肿瘤体积为127.47mm3，相较溶媒对照组统计学上有显著性差异(p＝0.0446)，相对肿瘤抑制率TGI(％)为78％。C19399-2(6.28mg/kg)治疗组在开始给药后第三十五天平均肿瘤体积为3.22mm3，相较溶媒对照组统计学上有显著性差异(p＜0.001)，相对肿瘤抑制率TGI(％)为99％。C19399-2(12.56mg/kg)治疗组在开始给药后第三十五天平均肿瘤体积为0.00mm3，相较溶媒对照组统计学上有显著性差异(p＜0.001)，相对肿瘤抑制率TGI(％)为100％。瘤重分析结果与相对瘤体积分析结果基本吻合。
各治疗组和对照组肿瘤生长情况如图2和图3所示。
4.2测试药C19399在SK-MEL-5人黑色素瘤模型中的安全性
测试药C19399-1(2.5mg/kg)、C19399-2(3.14mg/kg)、C19399-1(5mg/kg)组小鼠体重严重下降，但溶媒对照组也出现了小鼠体重下降的现象,应该由SK-MEL-5模型的恶病质特性引起的。测试药各治疗组均无动物死亡，没有表现药物毒性，治疗期间耐受良好。
治疗组和对照组给药后体重变化如图4所示。

Claims (3)

1.一种抗体药物偶联物制剂，其特征在于：包括抗体药物偶联物和药学上允许的佐剂；
所述抗体药物偶联物由DAR2和DAR4组成，DAR2的重量百分比为12-20％，DAR4的重量百分比为80-88％；
所述DAR2具有下式所示结构：
所述DAR4具有下式所示结构：
上式中，所述抗体为人IgG2单克隆抗体；
所述抗体药物偶联物制剂的制备方法，包括以下步骤：
步骤1，将解冻后的抗体置于偶联缓冲液中，进行超滤透析；
步骤2，将超滤透析后的抗体溶液和偶联缓冲液混合，然后先加入二乙烯三胺五乙酸溶液，再加入还原剂三(2-羧乙基)膦盐酸盐，进行还原反应；
步骤3，向还原反应液中加入MC-Val-Cit-PAB-MMAE溶液，制备抗体药物偶联物，之后加入L-Cys溶液终止反应，得到偶联反应液；
步骤4，向偶联反应液中加入疏水层析洗脱液进行第一次稀释，得到第一稀释液，然后向第一稀释液中再加入疏水层析稀释液进行第二次稀释；
步骤5，对稀释后的样品进行深层过滤，深层过滤后的样品除菌过滤；
步骤6，将步骤5得到的样品进行疏水层析，将疏水样品合并后进行除菌过滤；
步骤7，将步骤6得到的样品进行超滤透析，超滤透析后的样品除菌过滤；
步骤8，将步骤7得到的样品用制剂缓冲液进行稀释，即可得到所述抗体药物偶联物制剂；
步骤2中二乙烯三胺五乙酸溶液加入后的终浓度为1-2mM，还原剂三(2-羧乙基)膦盐酸与抗体的摩尔比为5.0:1-7:1，还原反应体系中抗体的浓度为6.0-15mg/mL，还原反应的温度为28-39℃、反应时间为110-190min；
步骤3中MC-Val-Cit-PAB-MMAE与抗体的摩尔比为8:1-16:1，偶联反应的温度为18-27℃、反应时间为1-6h；
步骤4中偶联反应液和疏水层析洗脱液的重量比为1-5:0.25，第一稀释液和疏水层析稀释液的重量比为1:1.069；
所述偶联缓冲液的配方为：Na2HPO4·2H2O 6.86g、NaH2PO4·H2O 1.58g、定容至1000g，pH为7.30-7.50；
所述疏水层析洗脱液的配方为：Na2HPO4·2H2O 6.18g、NaH2PO4·H2O 2.11g、DMSO66.00g，定容至1000g；
所述疏水层析稀释液的配方为：Na2HPO4·2H2O 8.90g、(NH4)2SO4 105.71g、DMSO66.00g，定容至1069g；
步骤6中疏水层析采用的填料为丁基-琼脂糖凝胶；
所述制剂缓冲液是含有蔗糖和聚山梨酯20的His/His-HCl缓冲液。
2.权利要求1所述的抗体药物偶联物制剂在制备用于肿瘤靶向治疗的药物中的用途，所述的肿瘤为三阴乳腺癌、黑色素癌、头颈癌或肺鳞癌。
3.以权利要求1所述的抗体药物偶联物制剂为活性成分的用于肿瘤靶向治疗的药物组合物，其特征在于，含有药学上有效量的权利要求1所述的抗体药物偶联物制剂及药学上允许的佐剂。
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