CN112402616A - 一种含环丙沙星和抗体药物偶联物的药物组合物及其应用 - Google Patents

一种含环丙沙星和抗体药物偶联物的药物组合物及其应用 Download PDF

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CN112402616A
CN112402616A CN202011321000.3A CN202011321000A CN112402616A CN 112402616 A CN112402616 A CN 112402616A CN 202011321000 A CN202011321000 A CN 202011321000A CN 112402616 A CN112402616 A CN 112402616A
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antibody
ciprofloxacin
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张信玲
罗文婷
陈虎
黄长江
林倩
王阅
周杰
朱梅英
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Rongchang Biopharmaceutical Yantai Co Ltd
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Abstract

本发明提供了一种含抗体药物偶联物和环丙沙星的联合用药组合物，该联合用药组合物能有效抑制肿瘤细胞的增殖，对肿瘤治疗具有显著疗效，其抗肿瘤作用显著优于单一用药，具有显著的增效作用。另外，环丙沙星与一、二线化疗药或靶向药相较，价格低廉，减轻了患者的经济负担，是一种高效低毒的肿瘤治疗药物组合物。

Description

一种含环丙沙星和抗体药物偶联物的药物组合物及其应用
技术领域
本发明涉及生物医药技术领域，尤其涉及一种抗肿瘤的联合用药组合物及其应用。
背景技术
抗体药物偶联物(Antibody Drug Conjuate，ADC)，由三部分组成：抗体、细胞毒素与连接两者的连接头。将单克隆抗体与细胞毒素偶联后，抗体药物偶联物利用单克隆抗体的靶向性，特异性地识别癌细胞表面的受体，并与之结合，然后经内吞作用进入到细胞内部，利用细胞内的蛋白酶释放细胞毒素阻止癌细胞繁殖与杀灭癌细胞。现有技术一般采用哺乳动物细胞培养生产表达抗体，经过高度纯化后的抗体与细胞毒素MMAE通过连接子偶联，获得抗体药物偶联物。抗体药物偶联技术使小分子毒素药物与生物蛋白融为一体，兼具二者之长，成为新一代治疗产品，在极大增强药效的同时减少毒副作用。
环丙沙星(Ciprofloxacin)为合成的第三代喹诺酮类抗菌药物，具广谱抗菌活性，杀菌效果好，常用于预防恶性肿瘤并发症之一——感染(感染是各种癌症死亡的主要原因)。根据文献报导，环丙沙星可提高溶酶体膜通透性(LMP)，而溶酶体通透性的改变可能会导致抗体药物偶联物酶解的速率及MMAE释放速度变慢，进而导致抗瘤效果下降；但是，溶酶体通透性的改变也可能会加快游离MMAE穿透溶酶体膜进入细胞质并与tubulin结合，加快肿瘤细胞的凋亡。
联合用药(Drug combination)是指为了达到治疗目的而采用的两种或两种以上药物同时或先后应用。联合用药往往会发生体内或体外药物的相互影响。对于某些药物组合，联合治疗还允许产生最佳组合剂量来使副作用最小化；两种化合物的联合治疗可获得未预料到的协同作用和引发非由单一化合物诱导的效应。
衡量联合用药作用的方法有多种，其中最简单的为代数和相加法，即EA+B＝EA+EB，EA为药物A在某剂量下单用时产生的效应，EB是药物B在某剂量下单用时产生的效应，EA+B为A药和B药分别以各自单用时的剂量合并使用时产生的效应。但此方法只适用于极其明显的增强现象(强协同)，会使许多独立相加的现象达不到要求水平而被判断为药物的拮抗作用(郭建友,霍海如,姜廷良.衡量联合用药作用研究方法评价[J].中药药理与临床,2005,21(3):60-64.)。最常用的为金正均法及Chou-Talalay合并指数CI(combination index)法。金正均法公式为EA+B＝EA+EB-EA·EB，此法常用于评价A药和B药在体内合并使用时的效果。合并指数法是从通用的质量作用定律概念衍化出来，CI值可由CompuSyn软件根据A、B药物的单药IC50及联用药效计算得出，多用于评价A药和B药在体外合并使用时的效果，此法已被西方医药界所广泛应用。
因此，本发明的目的在于：通过对肿瘤细胞增殖的抑制作用及对小鼠模型肿瘤组织生长抑制作用的研究，提供了一种含抗体药物偶联物和环丙沙星的联合用药组合物；实现了以下技术效果：抗体药物偶联物和环丙沙星联合用药能有效抑制肿瘤细胞的增殖，对肿瘤治疗具有显著疗效，其抗肿瘤作用显著优于单一用药，具有显著的增效作用，是一种高效低毒的肿瘤治疗药物组合物。
发明内容
本发明提供了一种含环丙沙星和抗体药物偶联物的联合用药组合物，联用药物之一为临床上应用的高效广谱抗菌药物—环丙沙星，且采用的临床常规剂量，安全性高。环丙沙星与一、二线化疗药或靶向药相较，价格低廉，减轻了患者的经济负担，大幅度降低了靶向药生产公司的时间成本和经济成本。环丙沙星和抗体药物偶联物的联合用药组合物能有效抑制肿瘤细胞的增殖，对肿瘤治疗具有显著疗效，其抗肿瘤作用显著优于单一用药，具有显著的增效作用，是一种高效低毒的肿瘤治疗药物组合物。
具体的，本发明提供了药物组合物，其包含：
(1)靶向Her2的抗体药物偶联物，和
(2)环丙沙星，
其中，所述的抗体药物偶联物的抗体部分包含重链和轻链，其中(i)所述重链包含三个CDR区，其中所述CDR区分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；和/或(ii)所述轻链包含三个CDR区，其中所述CDR区分别具有如SEQ ID NO：4、5和6所示的氨基酸序列。
进一步的，所述抗体的重链可变区具有如SEQ ID NO：7所示的氨基酸序列，和/或所述的抗体的轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
进一步的，所述抗体的重链可变区具有如SEQ ID NO：7所示的氨基酸序列，和/或所述的抗体的轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
进一步的，所述抗体的重链具有如SEQ ID NO：9所示的氨基酸序列，和/或所述的抗体的轻链具有如SEQ ID NO：10所示的氨基酸序列。
进一步的，所述抗体药物偶联物中所述抗体为抗Her2单克隆抗体，其中所述抗体与一个或多个细胞毒素偶联，所述细胞毒素为MMAE、MMAF及其洐生物或DM1、DM4及其洐生物。
进一步的，所述抗体与细胞毒素接头通过巯基连接；其中所述接头选自mc-vc-pAB和或者mc共价连接。
进一步的，所述的抗体药物偶联物和环丙沙星分别、同时或顺序给药，优选先给药抗体药物偶联物再给药环丙沙星。
进一步的，其中所述的抗体药物偶联物与环丙沙星的物质的量比为：0.0327:1270-20390。
本发明进一步提供了抗体药物偶联物和环丙沙星联合用药组合物在制备用于治疗或预防癌症的药物中的用途，其中所述癌症为Her2阳性癌症，优选癌症为乳腺癌、卵巢癌或胃癌。
附图说明
图1AAE ADC和环丙沙星的单药抗瘤药效
图2AAE ADC与环丙沙星给药间隔时间对联用效果的影响
图3环丙沙星给药剂量对联用效果的影响
具体实施方式
下面将通过实施例对本发明进行进一步地阐述，需要说明的是，以下实施例是对本发明进行进一步地阐述和解释，而不应被看作是对本发明的限制。
实施例1：抗体纯化
基于专利申请(CN105008398A或WO2015074528A1)实施例公开的相关方法获得了鼠源单克隆抗体mAAE以及相关人源化抗体AAE，其包含人IgG1γ重链恒定区和重链可变区AAE-VH，以及人IgG1κ轻链恒定区和轻链可变区AAE-VL，将上述扩增到的各片段分别亚克隆到表达载体pcDNA3.0上。将构建的不同质粒转染到悬浮CHO细胞中。在标准条件下培养，待培养基中的营养物质耗尽、细胞不再进一步生长时放罐，采用离心或过滤的方法将细胞分离出去，取上清液，抗体蛋白存在于上清液中，上ProteinA亲和层析柱进行第一步纯化，
洗脱下来的目的蛋白，上阳离子填料层析柱进行第二步纯化，收集目的蛋白峰，然后上第三步柱以目的蛋白穿透模式进行第三步纯化，纯化后的蛋白经过各项指标检测合格后，然后进行超滤浓缩，蛋白浓度达到约20-30mg/ml，此即为抗体蛋白原液，可在-80℃长期保存。
实施例2：人源化抗体AAE和细胞毒素偶联
将TCEP(Tris-2-carboxyethyl-phosphine)母液溶于偶联缓冲液中，先用偶联缓冲液稀释，与人源化抗体AAE按照体积比1：1(v:v＝1：1)混合，TCEP与抗体的终浓度摩尔比1.9：1，于25℃搅拌反应2.5h。TCEP还原可重复性好，还原后自由巯基数可以达到3.5-4.5。
还原后抗体可直接进行后续偶联。配制10mmol/L细胞毒素(vc-MMAE、vc-MMAF、mc-MMAF)溶于DMSO(dimethyl sulfoxide，二甲亚砜)，按照细胞毒素与巯基的摩尔比1.1:1缓慢加药，于25℃搅拌反应2h。用DTNB法于412nm检测自由巯基浓度(接近0)，纯化除去残余未反应细胞毒素以及DMSO等游离小分子，SDS-PAGE电泳及SEC、HPLC法检测偶联情况。偶联反应可重复性好，打开的自由巯基可以偶联完全，得到AAE ADC，AAE ADC偶联度在3.5-4.5。
实施例3：AAE ADC和环丙沙星单药的体外抗癌活性
(1)药物处理
汇合度为80％左右的SK-BR-3细胞，经胰酶消化后，新鲜培养基重悬细胞并计数，将细胞浓度稀释为5×104/ml，100μl/孔接种到96孔板中，37℃、5％CO2培养箱中培养24h，加入梯度浓度的AAE ADC或环丙沙星处理72h，每个浓度梯度设3-6个复孔，同时设置空白对照孔(无药物处理细胞对照孔)。
环丙沙星的临床给药剂量因适应症的不同有所差异，常规剂量为0.5g～1.5g/天，分2～3次给药。根据文献报导，0.5g/天、1.0g/天及1.5g/天给药方案下，环丙沙星的最大血药浓度(Cmax)分别为2.06±0.51μg/ml、4.65±0.68μg/ml及6.25μg/ml(李慈.环丙沙星不同给药方案血药浓度与疗效[J].中国中医药现代远程教育,2012,010(008):158-159.)。因此，本试验检测了环丙沙星在≤7.5μg/ml剂量下的单药抗瘤药效及与AAE ADC的联合抗瘤药效。
(2)细胞增殖能力测定(CCK8法)
弃掉培养基，用无血清培养基配置10％CCK8稀释液，每孔加100ul，培养箱内孵育1-4小时。酶标仪检测450nm条件下各孔的吸光度(OD值)并计算增殖抑制率。细胞增殖抑制率(％)＝(1-药物组平均OD值/空白对照组平均OD值)×100。
(3)结果分析
如图1所示，AAE ADC单药对SK-BR-3细胞增殖有显著的抑制作用，半抑制浓度(IC50)为4.91ng/ml(32.7pM)，最大抑制率(Imax)为89.2％。环丙沙星在468～7500ng/ml(1.27～20.39μM)剂量范围内无显著的抗瘤活性。
实施例4：AAE ADC和环丙沙星同时用药的体外抗瘤效果评价
(1)药物处理汇合度为80％左右的SK-BR-3细胞，经胰酶消化后，新鲜培养基重悬细胞并计数，将细胞浓度稀释为5×104/ml，100μl/孔接种到96孔板中，37℃、5％CO2培养箱中培养24h后，恒定浓度的AAE ADC(IC50,4.91ng/ml[32.7pM])分别与不同浓度的环丙沙星(7500ng/ml[20.39μM]、3750ng/ml[10.20μM]、1875ng/ml[5.10μM]、937.5ng/ml[2.55μM]、468.75ng/ml[1.27μM])配比共组成5个联用组(分别为联用组1、2、3、4、5)并同时处理细胞，每个联用组设置3-6个重复，同时设置空白对照孔，72h后检测细胞活力。
(2)细胞增殖能力测定(CCK8法)
弃掉培养基，用无血清培养基配置10％CCK8稀释液，每孔加100ul，培养箱内孵育1-4小时。酶标仪检测450nm条件下各孔的吸光度(OD值)并计算增殖抑制率，即为实际测得的两种药物联合使用的抑瘤率，用Effect表示。另外，根据两种药物单药的药效，计算两种药物联用的理论抑瘤率，即两种药物的单药药效之和，用Effect’表示，该数据可辅助评价两种药物的联用效果。
(3)联合效果评价
利用Chou-Talalay法，即联合指数(combination index，CI)法评价AAE ADC与环丙沙星联合给药在不同配比下作用72h对SK-BR-3细胞增殖抑制的协同/拮抗作用。利用CompuSyn软件可计算不同配比条件下两种药物联用的CI值，CI值可评价两种药物的联用效果，评价标准如表1所示。
表1 CI值与联用效果
Range of Combination Index Description
<0.1 极强协同(Very strong synergism)
0.1-0.3 强协同(Strong synergism)
0.3-0.7 协同(Synergism)
0.7-0.85 中度协同(Moderate synergism)
0.85-0.90 轻微协同(Slight synergism)
0.90-1.10 近似相加(Nearly additive)
1.10-1.20 轻微拮抗(Slight antagonism)
1.20-1.45 中度拮抗(Moderate antagonism)
1.45-3.3 拮抗(Antagonism)
3.3-10 强拮抗(Strong antagonism)
>10 极强拮抗(Very strong antagonism)
(4)结果分析
结果如表2、图2所示，4.91ng/ml(32.7pM)AAE ADC(IC50)单药对SK-BR-3细胞的增殖抑制率为49.27％，AAE ADC在该剂量条件分别与7500ng/ml(20.39μM)、3750ng/ml(10.20μM)、1875ng/ml(5.10μM)、937.5ng/ml(2.55μM)、468.75ng/ml(1.27μM)环丙沙星同时给药测得的联合抑制率分别为34.68％、32.63％、32.67％、31.33％及31.34％，均显著低于AAEADC单药药效，经计算两种药物联合使用时的CI值≥1.41，显示为中度拮抗或拮抗，说明两种药物同时给药时环丙沙星对AAE ADC的抗瘤活性具有拮抗作用。但拮抗强度没有显著的环丙沙星剂量依赖性，随着环丙沙星浓度的增加/降低，拮抗程度变化不大(图2)。
表2 SK-BR-3细胞中不同剂量环丙沙星对AAE ADC抑瘤活性的影响(同时给药)
Figure BDA0002792902300000071
实施例5：AAE ADC和环丙沙星顺序用药的体外抗瘤效果评价
(1)试验方法
环丙沙星与AAE ADC同时使用对AAE ADC的抗瘤能力产生的拮抗作用，可能是由于环丙沙星在AAE ADC进入溶酶体前或已进入溶酶体但未完全酶解释放出MMAE分子时破坏了溶酶体膜，导致AAE ADC药效下降。因此，我们尝试先用AAE ADC处理细胞，待AAE ADC进入溶酶体且完全酶解释放出MMAE小分子后，适时加入环丙沙星增加溶酶体的通透性，加快MMAE分子穿过溶酶体膜作用于靶蛋白tubulin，从而增强AAE ADC药效。因此，接下来我们分别在AAE ADC给药后2h、6h、24h、30h及48h后加入环丙沙星，然后检测两者的联用效果。
(2)细胞增殖能力测定(CCK8法)
(3)联合效果评价联合指数(CI)法
(4)结果分析
为了检测AAE ADC和环丙沙星顺序用药的联合抗瘤药效，先用4.91ng/ml(32.7pM)AAE ADC(IC50)处理SK-BR-3细胞，2h、6h、24h、30h或48h后再加入不同剂量的环丙沙星(7500ng/ml[20.39μM]、3750ng/ml[10.20μM]、1875ng/ml[5.10μM]、937.5ng/ml[2.55μM]、468.75ng/ml[1.27μM]),待AAE ADC处理72h后检测细胞增殖抑制率。两种药物间隔2h给药，联合药效低于AAE ADC单药药效，不同剂量环丙沙星与AAE ADC联用的CI值为1.19～1.51，表现为拮抗作用(表3)，但给药间隔延长到6h、24h、30h或48h时，联合药效不仅显著高于AAEADC单药药效，还显著高于两种药物的单药药效之和，且CI值均≤0.70，表现为显著的协同作用(表4-7)。此外，与同时给药相似的是，顺序给药的联用效果依然没有环丙沙星剂量相关性(图2、表3-7)。但两种药物给药的间隔时间显著影响联合药效，AAE ADC给药后0-2h给环丙沙星，AAE ADC抗瘤药效被显著抑制；AAE ADC给药后6-48h给环丙沙星，AAE ADC抗瘤药效显著提高，且给药间隔为6h时，协同效果最优(图3，表2-7)。
表3 SK-BR-3细胞中不同剂量环丙沙星对AAE ADC抑瘤能力的影响(间隔2h给药)
Figure BDA0002792902300000081
表4 SK-BR-3细胞中不同剂量环丙沙星对AAE ADC抑瘤能力的影响(间隔6h给药)
Figure BDA0002792902300000082
Figure BDA0002792902300000091
表5 SK-BR-3细胞中不同剂量环丙沙星对AAE ADC抑瘤能力的影响(间隔24h给药)
Figure BDA0002792902300000092
表6 SK-BR-3细胞中不同剂量环丙沙星对AAE ADC抑瘤能力的影响(间隔30h给药)
Figure BDA0002792902300000093
Figure BDA0002792902300000101
表7 SK-BR-3细胞中不同剂量环丙沙星对AAE ADC抑瘤能力的影响(间隔48h给药)
Figure BDA0002792902300000102
本发明已通过各具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各具体实施方式，普通技术人员在本发明的范文内可以作出各种改动或变型，并且在本说明书中各处提及的各个技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。
序列表
<110> 荣昌生物制药（烟台）股份有限公司
<120> 一种含环丙沙星和抗体药物偶联物的药物组合物及其应用
<130> 2020
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Claims (8)

1.药物组合物，其包含：
(1)靶向Her2的抗体药物偶联物，和
(2)环丙沙星，
其中，所述的抗体药物偶联物的抗体部分包含重链和轻链，其中(i)所述重链包含三个CDR区，其中所述CDR区分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；和/或(ii)所述轻链包含三个CDR区，其中所述CDR区分别具有如SEQ ID NO：4、5和6所示的氨基酸序列。
2.根据权利要求1所述的组合物，其中所述抗体的重链可变区具有如SEQ ID NO：7所示的氨基酸序列，和/或所述的抗体的轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
3.根据权利要求1所述的组合物，其中所述抗体的重链具有如SEQ ID NO：9所示的氨基酸序列，和/或所述的抗体的轻链具有如SEQ ID NO：10所示的氨基酸序列。
4.根据权利要求1所述的联合用药组合物，其中所述抗体药物偶联物中所述抗体为抗Her2单克隆抗体，其中所述抗体与一个或多个细胞毒素偶联，所述细胞毒素为MMAE、MMAF及其洐生物或DM1、DM4及其洐生物。
5.根据权利要求4所述的联合用药组合物，其中所述抗体与细胞毒素接头通过巯基连接；其中所述接头选自mc-vc-pAB和或者mc共价连接。
6.根据权利要求1-5任一项所述的药物组合物，其中所述的抗体药物偶联物和环丙沙星分别、同时或顺序给药，优选先给药抗体药物偶联物再给药环丙沙星。
7.根据权利要求1-5任一项所述的药物组合物，其中所述的抗体药物偶联物与环丙沙星的物质的量比为:0.0327:1270-20390。
8.根据权利要求1-5任一项所述的靶向Her2抗体药物偶联物和环丙沙星药物组合物在制备用于治疗或预防癌症的药物中的用途，其中所述癌症为Her2阳性癌症，优选癌症为乳腺癌、卵巢癌或胃癌。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5637580A (en) * 1988-10-24 1997-06-10 The Procter & Gamble Company Antimicrobial penem-quinolones
CN1938046A (zh) * 2003-11-06 2007-03-28 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物
CN109320612A (zh) * 2013-11-19 2019-02-12 荣昌生物制药（烟台）有限公司 抗her2抗体及其缀合物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5637580A (en) * 1988-10-24 1997-06-10 The Procter & Gamble Company Antimicrobial penem-quinolones
CN1938046A (zh) * 2003-11-06 2007-03-28 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物
CN107213469A (zh) * 2003-11-06 2017-09-29 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物
CN109320612A (zh) * 2013-11-19 2019-02-12 荣昌生物制药（烟台）有限公司 抗her2抗体及其缀合物

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