CN112714648A - 抗癌用组合物 - Google Patents
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Abstract
公开一种包含曲贝德生及IL‑2的抗癌用配合组合物。
Description
技术领域
本发明涉及抗癌用配合组合物。更具体而言,本发明涉及一种包含曲贝德生(trabedersen)和IL-2(interleukin-2:白细胞介素-2)的抗癌用配合组合物及其联合给药。
背景技术
原有的细胞毒性抗癌剂不仅攻击癌细胞,而且存在连正常细胞也攻击的问题,因而为了改善这种问题,靶向治疗抗癌剂成为抗癌治疗剂开发的主流。靶向治疗抗癌剂只选择性地攻击致癌过程中的特定靶向因子,不攻击正常细胞,因而被认为对患者造成的痛苦比原有细胞毒性抗癌剂更小。
但是,这种靶向治疗抗癌剂也发现了在给药后产生耐药性的缺点。为了在克服这种靶向治疗抗癌剂缺点的同时,通过抗癌治疗延长生存期,免疫抗癌剂正在成为抗癌治疗剂开发的核心。
本发明旨在提供一种在治疗时减小副作用并增进抗癌效果的抗癌剂及其联合给药。
[现有技术文献]
[非专利文献]
Schlingensiepen KH et al,Cancer Sci.2011;102:1193-200
发明内容
要解决的技术问题
本发明旨在借助于作为TGF-β2抑制剂的曲贝德生与IL-2联合给药来增进抗癌效果。
解决技术问题的手段
为了解决所述课题,本发明提供一种包含TGF-β抑制剂及细胞因子的抗癌用配合组合物。
具体而言,本发明提供一种包含作为TGF-β抑制剂的曲贝德生(trabedersen)及白细胞介素(IL)-2的抗癌用配合组合物。
TGF-β在肿瘤中的表达执行全面免疫抑制功能。TGF-β是表现出对免疫抗癌剂药物的抵抗性的生物标志物。
PD-1及PD-L1等的免疫检查点(immune checkpoint)担负全面免疫抑制中的一部分作用,因此,在未去除TGF-β的状态下,将对PD-1及PD-L1等免疫检查点的抑制剂用作免疫抗癌剂,使得恢复免疫功能的尝试只能部分地发挥作用,无法完全恢复免疫系统的细胞凋亡功能。
曲贝德生(trabedersen)是以人TGF-β为标靶而特别研发的合成18-mer硫代磷酸酯反义寡核苷酸,基于展现TGF-β过表达与肿瘤进行的相关性的数据而开发。TGF-β过表达与肿瘤进行的相关性已在多数的体外(in-vitro)及体内(in-vivo)非临床模型中得到评价(Schlingensiepen KH et al,Cancer Sci.2011;102:1193-200)。
作为细胞因子(cytokine)之一的IL-2(白细胞介素-2)刺激免疫系统,广泛增加免疫细胞的规模。
发明的效果
本发明的抗癌剂配合组合物在减小给药时副作的同时,协同增进抗癌效果。
附图说明
图1图示了对多样癌细胞株的TGF-β1mRNA及TGF-β2mRNA的相对定性分析结果。
图2图示了对多样癌细胞株的TGF-β1mRNA的定量分析结果。
图3图示了对多样癌细胞株的TGF-β2mRNA的定量分析结果。
图4至图8分别图示了利用EZCYTOX的曲贝德生单独处理在乳腺癌、胰腺癌、黑色素瘤、肺癌、直肠癌(colorectal cancer)细胞株中的生长抑制分析结果。
图9至图13图示了以L-2激活的人PBMC与曲贝德生联合使用的细胞凋亡效果。
图14及图15图示了以人PBMC激活(humanize:人源化)的免疫缺陷小鼠(NSGmouse)中IL-2及曲贝德生联合使用的体内抗癌效果。
具体实施方式
本发明涉及一种包含曲贝德生及IL-2的抗癌用配合组合物。本发明的组合物尤其对实体癌(例如乳腺癌、胰腺癌、黑色素瘤、肺癌或直肠癌等)的治疗有用。
本发明的组合物在给药时,曲贝德生及IL-2可以以依次或同时给药或个别给药的方法联合给药。因此,本发明的组合物在给药时,各成分在生物体内以治疗学上有效剂量同时存在,表现出彼此协同效应。
在本发明另一方面,本发明涉及一种含有曲贝德生、用于与IL-2联合给药的抗癌用组合物。这种本发明的组合物尤其对实体癌(例如乳腺癌、胰腺癌、黑色素瘤、肺癌或直肠癌等)的治疗有用。
在本发明又一方面,本发明涉及一种含有IL-2、用于与曲贝德生联合给药的抗癌用组合物。本发明的组合物尤其对实体癌(例如乳腺癌、胰腺癌、黑色素瘤、肺癌或直肠癌等)的治疗有用。
下面根据实施例,详细说明本发明。不过,以下实施例只用于对本发明举例,并非本发明的思想或范围限定于此。
为了确认本发明的组合物的抗癌效果,在小鼠实体癌模型中实施了作为TGF-β2抑制剂的曲贝德生与IL-2联合使用的体外(in-vitro)抗癌效果实验。例如,在多样的实体癌细胞株中确认TGF-βmRNA表达量,在他们中确认了曲贝德生单独处理及与以IL-2激活免疫细胞(activation)的PBMC(外周血单核细胞:peripheral blood mononuclear cell)联合处理下的癌细胞凋亡效果。另外,在加入人PBMC而实现人源化的免疫缺陷小鼠中,确认了曲贝德生与IL-2联合使用的人癌细胞(xenograft model)的生长抑制效果。
实验例1:以多样癌为对象的TGF-β2mRNA level定量及相对定性分析
1)TGF-β2mRNA表达分析
使用如下表1所示的细胞株进行实验。
[表1]
使用RNA prep kit(德国凯杰公司)从上表1的各细胞株1x107个中提取(preparation)RNA,然后,以提取的RNA 1μg为模版,在37℃下,在1小时期间合成cDNA。
利用TGF-β2特异性引物(specific primer)F(ATCGATGGCACCTCCACATATG)及R(GCGAAGGCAGCAATTATGCTG),执行对cDNA 10μl的PCR(变性95℃1分钟、退火57℃1分钟、延伸72℃1分钟、35周期及最终延伸72℃5分钟)。然后,与对GAPDH(Glyceraldehyde 3-phosphate dehydrogenase:3-磷酸甘油醛脱氢酶)的PCR结果对比,确认了利用TGF-β1及TGF-β2谱带强度的TGF-β1及TGF-β2的mRNA表达。
图1至图3图示了TGF-β1或TGF-β2mRNA的相对定性分析及定量分析结果。
实验结果可知,在皮肤癌细胞、肺癌细胞、乳房癌细胞中,TGF-βmRNA表达相对较多。
实验例2:多样实体癌细胞株中曲贝德生单独处理的细胞凋亡效果
在多样实体癌细胞株中,确认了曲贝德生单独处理的细胞凋亡效果。
1)细胞培养
如上表1所示,根据细胞类型,使用了RPMI1640(10%FBS,抗菌-抗真菌剂,HEPES)、DMEM(10%FBS,抗菌-抗真菌剂)等培养基,细胞在37℃、CO2 5%环境下培养。
2)曲贝德生转染(transfection)
将表1记载的各细胞株如下表2所示进行接种(seeding)后,在37C、5%CO2条件下培养24小时,转染了曲贝德生。
具体的转染方法如下:
将曲贝德生在无血清培养液(Serum free media)(培养液A:RPMI 1640或DMEM)中稀释,使得在最终制备的培养液(即,包含以下说明的培养液A、培养液B及细胞培养液的培养液)中,分别达到20nM、40nM、80nM及160nM的浓度。
将脂质体(Lipofectamine)2000既定量添加于无血清培养液(培养液B:RPMI 1640或DMEM),使得成为下表2所示实验条件。
使培养液A及培养液B分别在常温下反应5分钟后,按12孔板基准,将培养液A 50ul与培养液B 50ul(1:1体积比)混合,再在常温下反应20分钟。
将所述“1)细胞培养”步骤中准备的细胞的培养液更换为新的10%FBS培养液后,将结束反应的培养液A及B的混合物加入于细胞培养液。
[表2]
3)细胞毒性分析(Cytotoxicity Assay)
为了细胞毒性分析,进行曲贝德生转染后第二天,利用EZCYTOX(DAEILLAB服务公司),以O.D 450nm值测量了细胞的活性度,通过Annexin V及PI染色,利用FACS(Fluorescence activated cell sorter:荧光激活细胞分类器),进行了细胞凋亡(Apoptosis)/坏死(Necrosis)分析。
图4至图8是利用EZCYTOX分析曲贝德生单独处理在癌细胞株中的生长抑制的结果。可知,借助于曲贝德生给药,癌细胞株的活性被抑制。不过,在Capan-2及SK-MEL-28细胞株中,观察到细胞凋亡不大。
实验例3:乳房癌细胞株中以IL-2激活的人PBMC与曲贝德生联合使用的细胞凋亡效果试验
1)IL-2激活
利用ficoll-plaque plus(购自通用电气医疗集团)分离外周血液的PBMC(外周血单核细胞:peripheral blood mononuclear cell)后,在添加了10%FBS、IL-2(20ng/ml)的培养液中按4x106/ml浓度培养,第五天利用FACS在CD4+/CD8+T cell中确认了CD69(T cellactivation marker:T细胞激活标记)的表达。
2)IL-2及曲贝德生联合给药分析
如上所述,利用ficoll-plaque plus(通用电气医疗集团)分离外周血液的PBMC后,在添加10%FBS、IL-2(20ng/ml)的培养液中,按4x106/ml浓度培养。在培养第三天,将曲贝德生转染(transfection)于癌细胞后,24小时后,将目标癌细胞的培养液更换为10%FBS培养液,在IL-2培养第五天,将PBMC按癌细胞株(seeding):效应器(免疫效应细胞)=1:10比率(细胞数比率)置于10%FBS RPMI media 2ml后,添加于孔板进行培养。
非激活PBMC另行利用聚蔗糖分离,用作对照组。另外,不加入任何PBMC的细胞株也用作对照组。曲贝德生在转染时变化培养液中的浓度(即,分别针对0nM、10nM、40nM浓度)进行了实验。
在24小时后,通过PI染色分析了细胞毒性(cytotoxicity)结果。
实验例4:胰脏癌细胞株中以IL-2激活的人PBMC与曲贝德生联合使用的细胞凋亡效果试验
以与实验例3类似的方法,对胰脏癌细胞株(AsPC-1)实施了实验。
实验例5:黑色素瘤细胞株中以IL-2激活的人PBMC与曲贝德生联合使用的细胞凋亡效果实验
以与实验例3类似的方法,针对黑色素瘤细胞株(Hs 294T)实施了实验。
实验例6:肺癌细胞株中以IL-2激活的人PBMC与曲贝德生联合使用的细胞凋亡效果实验
以与实验例3类似的方法,对肺癌细胞株(A549)实施了实验。
实验例7:直肠癌细胞株中以IL-2激活的人PBMC与曲贝德生联合使用的细胞凋亡效果实验
以与实验例3类似的方法,对直肠癌细胞株(HT29、DLD-1、LS174T)实施了实验。
将实验例3至7的实验结果图示于图9至图13。如图所示,可知曲贝德生与IL-2联合给药组(IL-2activated PBMC)相比曲贝德生单独给药组(非激活PBMC及DMEM或RPMI)或IL-2单独给药组(IL-2activated PBMC、曲贝德生0nM),细胞凋亡效果极大增加。
实验例8:(体内试验)三阴性乳房癌细胞株中以人PBMC人源化的免疫缺陷小鼠(NSG mouse)中IL-2与曲贝德生联合使用的癌生长抑制效果试验
在对NSG(NOD/SCID/Gamma)免疫缺陷小鼠注入人PBMC而实现人源化的小鼠中,实施了用于确认人三阴性乳腺癌(TNBC:triple negative breast cancer)细胞株MDA-MB-231的生长在曲贝德生单独给药时、IL-2单独给药时及曲贝德生与IL-2联合给药时分别受到何种影响的实验
实验方法
准备了冻结干燥的粉末形态的曲贝德生(TBD)及液剂形态的基因重组人IL-2(rhIL-2,韩国JW CreaGene;Cat.No.JW-H031-0025)。曲贝德生的保管条件为2~8℃,基因重组人IL-2的保管条件为-80℃。
将曲贝德生溶解于灭菌生理盐水使用。基因重组人IL-2将浓度25μg/250μl(2x108IU/mg)溶液在临给药前稀释使用。
将NSG小鼠分成10组,每组6只,按下表3的用量,进行腹腔内给药(IP)。
[表3]
异种移植模型制作
使MDA-MB-231细胞在37℃5%CO2培养环境下增殖。使用了包含10%FBS的RPMI1640(包括左旋谷酰胺、25mM HEPES)培养基,当细胞占25T烧瓶表面的70~80%时,按1:5比率进行转种培养。
在肿瘤移植日,将所述培养的肿瘤细胞按1×107细胞/100μl悬浮于PBS,按1×107细胞/小鼠,在小鼠右肋(flank)部位皮下移植100μl。
人-PBMC(Hu-PBMC)NSG小鼠模型制作
将人PBMC在37℃水槽(water bath)中解冻(thawing),利用包含20%FBS的RPMI1640(含DNAse I),进行2次清洗(washing),在37℃、5%CO2培养环境下稳定30分钟。
培养30分钟后,将PBMC在PBS中稀释为5×107至10×107细胞/ml的浓度后,1只小鼠按1×107细胞/只的用量给药(癌细胞给药后第三天进行PBMC给药)。
给药方法
针对如上所述进行PBMC给药的小鼠,在PBMC给药后第二天(癌细胞移植后第五天),按表3的用量,按1次/2日间隔,将曲贝德生进行腹腔内给药共8次,在PBMC给药后第四天(癌细胞移植后第七天),按表3的用量,按1次/1日间隔,将IL-2给药4天后停药3天,以此为一个周期,共腹腔内给药两个周期。
接种、PBMC给药、曲贝德生给药及IL-2给药日程如下:
肿瘤体积(Tumor Volume)测量
从曲贝德生及/或IL-2给药后起,按1次/2日测量了肿瘤体积。即,肿瘤体积自肿瘤接种(0天)后第5天起,按1次/2日测量。肿瘤体积利用卡尺(Caliper:CD-15CPX,数显卡尺,日本三丰公司),测量了在注入部分产生的癌细胞块的大小,按下述数学式计算了体积:
[数学式]
肿瘤体积(TV:mm3)=(W2×L)/2
在上式中,W为短轴长度,L为长轴长度。
所述实验结果可知,与只单独给药曲贝德生或只单独给药IL-2相比,曲贝德生及IL-2联合给药时,协同发挥癌细胞的生长抑制效果(参照图14及图15)。
工业实用性
本发明的抗癌配合组合物可以用于抗癌给药。
Claims (10)
1.一种包含曲贝德生及IL-2的抗癌用配合组合物。
2.根据权利要求1所述的抗癌用配合组合物,其特征在于,所述曲贝德生及IL-2同时给药,或依次给药,或个别给药。
3.根据权利要求1所述的抗癌用配合组合物,其特征在于,用于治疗实体癌。
4.根据权利要求3所述的抗癌用配合组合物,其特征在于,所述实体癌为乳腺癌、胰腺癌、黑色素瘤、肺癌或直肠癌。
5.一种含有曲贝德生、用于与IL-2联合给药的抗癌用组合物。
6.根据权利要求5所述的用于治疗实体癌的抗癌用组合物。
7.根据权利要求6所述的抗癌用组合物,其特征在于,所述实体癌为乳腺癌、胰腺癌、黑色素瘤、肺癌或直肠癌。
8.一种含有IL-2、用于与曲贝德生联合给药的抗癌用组合物。
9.根据权利要求8所述的用于治疗实体癌的抗癌用组合物。
10.根据权利要求9所述的抗癌用组合物,其特征在于,所述实体癌为乳腺癌、胰腺癌、黑色素瘤、肺癌或直肠癌。
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