CN114788870B - 靶向免疫细胞长效补充精氨酸并中和酸环境的组合物及应用 - Google Patents
靶向免疫细胞长效补充精氨酸并中和酸环境的组合物及应用 Download PDFInfo
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- CN114788870B CN114788870B CN202210473866.9A CN202210473866A CN114788870B CN 114788870 B CN114788870 B CN 114788870B CN 202210473866 A CN202210473866 A CN 202210473866A CN 114788870 B CN114788870 B CN 114788870B
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Abstract
本发明提供靶向免疫细胞长效补充精氨酸并中和酸环境的组合物及应用。由L‑精氨酸、阳离子氨基酸转运蛋白2抑制剂及药学上可接受的缓释药物储库载体组成。本发明通过将L‑精氨酸和阳离子氨基酸转运蛋白2抑制剂共载于缓释药物储库中制备成抗肿瘤药物。本发明通过局部注射于肿瘤部位,抑制癌细胞及免疫抑制细胞的阳离子氨基酸转运蛋白2,同时在瘤内长效缓释碱性L‑精氨酸,实现靶向的肿瘤杀伤性免疫细胞L‑精氨酸补充和肿瘤细胞及免疫抑制细胞L‑精氨酸饥饿,并利用L‑精氨酸的碱性中和酸性环境,在酸性和营养两个层面完成对肿瘤微环境的促免疫改善,可以显著增强抗肿瘤免疫反应和提高肿瘤治疗效果。
Description
技术领域
本发明属于药物制剂领域,涉及一种靶向免疫细胞长效补充精氨酸并中和酸环境的组合物及应用,主要用于制备抗肿瘤药物。
背景技术
肿瘤细胞在氧气充足的情况下,依旧偏向于无氧糖酵解的糖代谢模式以合成自身所需的能量,并随之产生大量的乳酸,该现象被称为“Warburg”效应。为维持胞内稳态,肿瘤细胞会把产生的乳酸外排到细胞间质中,造成间质酸化。由此产生的弱酸环境被认为与癌细胞的高侵袭性和转移性,及对包括化疗、免疫治疗在内的多种疗法的耐受性有高度关联。更重要的是,偏低的pH值对淋巴细胞具有毒性,会抑制白介素2(IL-2)刺激的淋巴细胞增殖过程,且已经证明酸性条件下细胞毒性T淋巴细胞对各种肿瘤细胞系的杀伤作用减弱。环境pH值降低到6.0-6.5时就足以在人和小鼠肿瘤特异性CD8+T细胞中引起无反应状态T淋巴细胞,这些细胞的细胞溶解活性和细胞因子分泌能力受损,IL-2受体α(IL-2Rα)和T细胞受体(TCR)的表达降低,以及TCR激活后信号转导和转录激活因子5(STAT5)和细胞外信号调节激酶(ERK)的激活减少。酸性环境引起的免疫抑制效果也同样作用在自然杀伤细胞(NK细胞)和巨噬细胞上。因此,中和酸性肿瘤微环境可以为免疫治疗提供环境基础(CancerRes.2016 Mar15;76(6):1381-90)。
L-精氨酸(L-arg)是一种碱性氨基酸,在多种代谢产物合成过程中起着纽带的作用,其中包括NO,多胺,氨基酸和嘧啶等对细胞存活和增殖都必不可少的关键物质。而肿瘤环境的特征之一就是L-arg缺乏。一方面,因为肿瘤组织中异常的血管生成造成血管畸形,血流灌注异常和物质运输障碍,导致L-arg供给不足。而另一方面,肿瘤组织中快速增殖的癌细胞,高表达精氨酸酶1(ARG1)的髓源性抑制细胞(MDSC)和M2型巨噬细胞,又会造成L-arg的高消耗。在缺乏L-arg的情况下,T细胞会出现CD3ζ链表达减少,产生细胞因子的能力受损的情况,还会经由GCN2/eIF2α途径造成激活后进入***期的T细胞发生细胞周期阻滞引起增殖抑制。而与之相反,充足的L-arg水平可以诱导T细胞整体代谢模式发生变化,表现为激活的T细胞产能方式从糖酵解转变为氧化磷酸化,进而促进具有较高生存能力的中央记忆细胞的生成,在小鼠模型中能够表现出更强的抗肿瘤活性。此外,L-arg还是M1型巨噬细胞中诱导型一氧化氮合酶(iNOS)的底物,通过iNOS将L-arg催化为一氧化氮(NO)是M1细胞发挥免疫功能的重要方式,L-arg不足将限制M1生成NO的能力。
所以,向肿瘤组织中补充碱性L-arg可以在为免疫细胞提供需要营养支持的同时,利用其碱性改造原抑制性酸性环境,从而进一步促进免疫反应。已有研究证明,口服补充L-arg可以提升免疫治疗效果(Cancer Biol Ther.2017 Feb;18(2):94-100),有利于手术预后(Clin Nutr.2014 Dec;33(6):951-7)。但是口服L-arg所需剂量极大,病人不易耐受,且无法利用其碱性,而局部的溶液注射因为快速扩散无法实现持续的L-arg水平提高(Nature.2021 Oct;598(7882):662-666)。故能够局部且持续在肿瘤释放碱性L-精氨酸的制剂手段更适合L-arg补充疗法。
此外,L-arg同时也是肿瘤细胞增殖过程必需的一种营养物质,早先设计的人为消耗全身L-arg的饥饿策略虽对部分肿瘤有生长抑制作用,但是其治疗效果对肿瘤基因表型有严重的依赖性(JAMA Oncol.2017 Jan 1;3(1):58-66)且对免疫***具有显著抑制效果(Cancer Res.2015 Jan 15;75(2):275-83),不利于治疗。综上所述,为使该策略的效果最大化,就必须阻止癌细胞从改善的环境中获益,即要在补充L-arg的环境下实现肿瘤细胞的营养饥饿。
细胞对环境L-arg的可用性和转运体的表达情况有关,哺乳动物细胞的L-arg转运主要由SLC7家族中的阳离子氨基酸转运蛋白(CAT)中的CAT-1(SLC7a1)和CAT-2(SLC7a2)主导。T细胞在活化阶段,会上调CAT-1的表达满足自身对L-arg的需求,且在L-arg不足的情况下增强,但不会上调CAT-2或其他转运体(Eur J Immunol.2016 Jan;46(1):92-103)。然而,对BRAF抑制剂产生耐药的黑色素瘤细胞,表现出从葡萄糖到精氨酸依赖的代谢转变并伴随CAT-2表达水平提高,沉默CAT-2表达显减弱其增殖能力(Mol Oncol.2017 Dec;11(12):1806-1825)。局部炎症和肿瘤部位的MDSCs表现出了CAT-2,ARG1和NOS2的协同上调,并通过消耗环境中的L-arg抑制T细胞免疫反应,在没有CAT-2的情况下,MDSCs在***炎症和癌症模型中控制T细胞免疫的能力减弱(J Immunol.2015 Dec 1;195(11):5237-50.)。同样的,CAT-2缺陷小鼠的肺炎模型中可以观察到树突状细胞活化增加和记忆T细胞数量的增加(Proc Natl Acad Sci U S A.2006 Oct 3;103(40):14895-900)。这种CAT-1和CAT-2的差异表达情况,还没有在肿瘤环境调控中被利用,通过抑制肿瘤环境中的CAT-2,有望在为肿瘤杀伤性免疫细胞补充L-arg的同时实现肿瘤细胞特异性的L-arg饥饿。
发明内容
本发明的目的是提供一种靶向免疫细胞长效补充精氨酸并中和酸环境的组合物,所述组合物由L-精氨酸、阳离子氨基酸转运蛋白2抑制剂以及药学上可接受的缓释药物储库载体组成。
其中L-精氨酸浓度为10-160mg/mL,所述阳离子氨基酸转运蛋白2抑制剂可以为抑制性多肽和抗体,或者为基于阳离子氨基酸转运蛋白2基因序列的shRNA或siRNA。所述shRNA为基于阳离子氨基酸转运蛋白2基因序列的短发夹RNA,其序列可以为:
(1)gctttatgccctatggctttattcaagagataaagccatagggcataaagctttttt;
(2)cgtccttacttgtctgctttattcaagagataaagcagacaagtaaggacgtttttt;
(3)cggcctttgctatgctgaatttcaagagaattcagcatagcaaaggccgtttttt。
所述siRNA为基于阳离子氨基酸转运蛋白2基因序列的小干扰RNA,其序列可以为:
(1)cgaaauacgggauaccgaaau;
(2)gcaggaaugaacagacgaaau;
(3)gccggaaacgauacgacuua。
当阳离子氨基酸转运蛋白2抑制剂为抑制性多肽和抗体时,其浓度为1-20mg/mL;当阳离子氨基酸转运蛋白2抑制剂为shRNA时,其浓度为0.1-5mg/mL;当阳离子氨基酸转运蛋白2抑制剂为siRNA时,其浓度为10-200nmol/mL。当阳离子氨基酸转运蛋白2抑制剂为shRNA或siRNA时,需复合使用基因递送载体,包括但不限于阳离子脂质体、阳离子纳米乳、固体脂质纳米粒。所述缓释药物储库载体的类型包括但不限于微球、凝胶和多囊脂质体。
本发明的另一个目的是提供所述组合物在制备抗肿瘤药物中的应用,所述肿瘤为黑色素瘤。
本发明所述抗肿瘤是由所述组合物药物单独施用,或组合物与免疫治疗、手术治疗、化疗及放疗联合施用实现。
本发明的抗肿瘤作用是通过阳离子氨基酸转运蛋白2抑制剂,抑制肿瘤组织中阳离子氨基酸转运蛋白2的功能,限制肿瘤细胞和免疫抑制性细胞对肿瘤环境中L-精氨酸的利用能力,避免其从改善的环境中获益,甚至迫使其处于L-精氨酸饥饿状态。利用L-精氨酸的碱性和缓释药物储库的释放特性,实现持续性的对肿瘤杀伤性免疫细胞的L-精氨酸靶向补充和弱酸环境中和,从pH环境和营养两个层面同时改善肿瘤微环境,实现对抗肿瘤免疫反应的增强。
本发明设计了一种靶向免疫细胞长效补充精氨酸并中和酸环境的组合物。该组合物通过抑制肿瘤组织中的CAT-2,限制肿瘤细胞及免疫抑制细胞对环境中的L-arg的摄取利用,联合局部碱性L-精氨酸缓释补充,可长时间改善肿瘤微环境中的营养条件和pH环境,同时实现强化抗肿瘤免疫、饥饿肿瘤和减弱免疫抑制的效果,并且可用于辅助免疫疗法、手术治疗、化疗及放疗,具备良好的临床应用潜力。
本发明通过将L-精氨酸和阳离子氨基酸转运蛋白2抑制剂共载于缓释药物储库中制备成抗肿瘤药物。本发明通过局部注射于肿瘤部位,抑制癌细胞及免疫抑制细胞的阳离子氨基酸转运蛋白2,同时在瘤内长效缓释碱性L-精氨酸,实现靶向的肿瘤杀伤性免疫细胞L-精氨酸补充和肿瘤细胞及免疫抑制细胞L-精氨酸饥饿,并利用L-精氨酸的碱性中和酸性环境,在酸性和营养两个层面完成对肿瘤微环境的促免疫改善,可以显著增强抗肿瘤免疫反应和提高肿瘤治疗效果。
本发明的创新性在于利用缓释制剂技术,完成了对高浓度碱性L-精氨酸和阳离子氨基酸转运蛋白2抑制剂的包封,实现了对弱酸肿瘤微环境的中和,对抗肿瘤免疫细胞的靶向L-精氨酸补充,对肿瘤细胞和免疫抑制细胞的特异性L-精氨酸饥饿。本发明的组合物,突破了肿瘤组织中环境和营养介导的双重免疫抑制作用,可显著提高机体的抗肿瘤免疫的响应性,除单独使用外,也可用于辅助化疗、放疗、免疫治疗和手术治疗,具备良好的临床应用前景,涉及的L-精氨酸药物储库制备技术,工艺简单成熟,物料成本低,稳定可靠,可用于商业化生产。
附图说明
图1是组合物(微球剂型)的体外释放曲线。
图2是组合物(多囊脂质体机型)的体外释放曲线。
图3是组合物(凝胶剂型)的体外释放曲线。
图4是CAT-2 shRNA质粒/Lipo8000复合物抑制B16细胞CAT-2蛋白。
图5是CAT-2蛋白抑制对B16细胞增殖的影响。
图6是CAT-2蛋白抑制对CD8+ T细胞增殖的影响。
图7是CAT-2蛋白抑制对CD8+ T细胞分化的影响。
图8是组合物中和肿瘤酸性微环境的效果。
图9是组合物对肿瘤生长的抑制作用。
图10是组合物对小鼠体重的影响。
图11是组合物对小鼠生存期的影响。
图12是组合物对肿瘤中浸润CD8+ T细胞数量的影响。
图13是组合物对肿瘤中浸润CD8+ T细胞分化的影响。
图14是组合物对肿瘤相关巨噬细胞极化的影响。
图15是组合物对肿瘤组织中细胞因子的影响。
图16是组合物联合抗PD-1治疗对肿瘤生长的抑制作用。
图17是组合物联合抗PD-1治疗对小鼠体重的影响。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图和实施例对本发明进行具体描述,有必要指出的是,以下实施例仅用于对本发明进行解释和说明,并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整,仍属于本发明的保护范围。
实施例1一种组合物(微球剂型)的制备方法
组合物(微球剂型)的组成:
L-精氨酸 5mg
CAT-2 siRNA/Lipo8000复合物 7nmol(以siRNA含量计)
Poly(lactic-co-glycolic)acid,1.5w,50:50 200mg。
将处方量的L-精氨酸和CAT-2 siRNA/Lipo8000复合物溶于200μL的纯水中,作为内水相;处方量的Poly(lactic-co-glycolic)acid溶于4mL二氯甲烷中,作为油相。将内水相加入油相中,涡旋30秒后再经100W探头超声乳化2分钟。所得W/O乳剂加入到40mL含2%PVA的水溶液中,高速搅拌后,将其加入到100mL含0.2%PVA的水溶液中,低速搅拌过夜,除去二氯甲烷并完成微球固化。所得悬液经高速离心后,收集底部沉淀,即得组合物(微球剂型)。超纯水洗涤3遍后,于4℃储存。
实施例2组合物(微球剂型)的体外释放
本实施例在实施例1的基础上,取组合物(微球剂型),重悬于pH为7.4的PBS溶液中,置于37℃恒温振荡箱中,于设定时间取样,高速离心后收集上清,经HPLC测定其中L-精氨酸含量,考察组合物(微球剂型)的体外释放行为。释放曲线表明,37℃条件下,组合物(微球剂型)在pH为7.4的PBS溶液中表现出了长达21天的缓释能力(图1)。
所用HPLC条件为:
流动相:甲醇:0.01M戊烷磺酸钠=(10:90),1mL/min;检测波长:206nm;色谱柱:Agilent ZORBAX C18 column(4.6×250mm,5μm)。
实施例3一种组合物(多囊脂质体剂型)的制备方法
组合物(多囊脂质体剂型)的组成:
将处方量的L-arg和CAT-2 shRNA质粒/Lipo8000复合物溶于1mL纯水中,作为第一水相;处方量的蛋黄卵磷脂E80、胆固醇和α-生育酚溶于2mL二氯甲烷中,作为油相。将第一水相加入油相中,200W探头超声乳化4分钟,制得W/O乳剂。将含2%十二烷基硫酸钠的5mg/mL氯化钠水溶液作第二水相,将制得的W/O乳剂加入到10mL第二水相中,低速搅拌制得W/O/W型复乳,向其中加入5mg/mL的氯化钠溶液20mL,25℃水浴搅拌5小时,挥发去除有机溶剂,制得乳白色均匀状组合物(多囊脂质体剂型)悬液。所得悬液经低速离心富集组合物(多囊脂质体剂型),弃去上清并加入20mL的5mg/mL氯化钠溶液洗涤,重复3次。洗涤完毕后,收集组合物(多囊脂质体剂型),于4℃储存。
实施例4组合物(多囊脂质体剂型)的体外释放
本实施例在实施例3的基础上,取组合物(多囊脂质体剂型),稀释于pH为7.4的PBS溶液中,置于37℃恒温振荡箱中,于设定时间取样,低速离心后弃去上清将收集的组合物用甲醇溶解后,以实施例2中的HPLC条件测定L-精氨酸含量,检测组合物(多囊脂质体剂型)体外释放行为。释放曲线表明,37℃条件下,组合物(多囊脂质体剂型)在pH为7.4的PBS溶液中表现出了长达216小时的缓释能力(图2)。
实施例5一种组合物(凝胶剂型)的制备方法
组合物(凝胶剂型)的组成:
将处方量的Poly(lactic-co-glycolic)acid和N-甲基吡咯烷酮混合后,静置2小时。待其完全溶胀后,加入处方量的L-精氨酸和anti-CAT-2抗体,混匀后得到组合物(凝胶剂型)前体溶液,于4℃储存。
实施例6组合物(凝胶剂型)的体外释放
本实施例在实施例5的基础上,取组合物(凝胶剂型)前体溶液,注射于透析袋中,将含有凝胶前体溶液的透析袋悬于pH为7.4的PBS溶液中,置于37℃恒温振荡箱中,于设定时间从外部释放介质中取样,以实施例2中的HPLC条件测定L-精氨酸含量,检测组合物(凝胶剂型)体外释放行为。释放曲线表明,37℃条件下,组合物(凝胶剂型)在pH为7.4的PBS溶液中表现出了长达30天的缓释能力(图3)。
实施例7对肿瘤细胞阳离子氨基酸转运体2(CAT-2)的抑制
实施例描述的体外细胞实验中,CAT-2 shRNA质粒工作均为1μg/mL,使用的阳离子载体剂量根据说明书中质粒与载体比例计算。
将B16细胞接种于6孔板中,过夜贴壁后,分成3组分别进行如下处理:①加入生理盐水(Blank)共孵育24小时,②加入CAT-2 shRNA质粒/Lipo8000复合物(shRNA)共孵育24小时,③加入CAT-2 shRNA质粒/Lipo8000复合物(shRNA)共孵育24小时,再经嘌呤霉素筛选(shRNA-p)24小时。处理完成后收集细胞提取蛋白,经westernblot检测CAT-2表达的变化。实验结果显示,shRNA(CAT-2 shRNA质粒-Lipo8000复合物)可以下调B16的CAT-2表达水平约50%,经嘌呤霉素筛选再培养后,下调率可提高到80%,并且全过程中未见CAT-1表达补偿性增加(图4)。
将B16细胞接种于96孔板中,过夜贴壁后,分成4组分别进行如下处理:①加入生理盐水(Blank),②加入Lipo8000(Lipo),③scramble shRNA质粒/Lipo8000复合物(Scramble),④CAT-2 shRNA质粒/Lipo8000复合物(shRNA),共孵育24小时后MTT法检测细胞活性。B16的CAT-2表达下调后,其在L-arg充足时的增殖活性变得与L-arg不足时的相似(图5),该结果证明CAT-2蛋白是B16细胞较为依赖的L-arg转运体,抑制该蛋白,可实现肿瘤在富精氨酸环境下的饥饿化。
实施例8抑制肿瘤细胞阳离子氨基酸转运体2对免疫细胞的影响
本实施例在实施例7的基础上,使用Transwell***,将B16细胞接种于下室,激活的CD8+ T细胞接种于上室,两种细胞各20万。调整培养体系中L-精氨酸的浓度为50μM或100μM,并向下室中加入生理盐水或CAT-2 shRNA质粒/Lipo8000复合物,共培养48小时。结果表明,在L-arg不充足(50μM)的情况下共培养,T细胞的增殖受到严重抑制,向环境中补充L-arg可以缓解这种抑制效果,而对肿瘤细胞CAT-2的下调也起到了促进T细胞增殖的效果,且二者效果可以叠加,实现更好的免疫促进作用(图6)。并且二者的联合使用使得效应T细胞(CD44+CD62L-)和记忆T细胞(CD44+CD62L+)的数量都出现了显著增加,提示具有协同的促免疫效果(图7)。
实施例9组合物中和肿瘤酸性微环境效果考察
本实施例在实施例3的基础上,将1×106的B16细胞接种于成年雌性C57b/c小鼠皮下建立肿瘤模型,接种7天后,随机将小鼠分为2组(n=5),分别接受以下治疗:①生理盐水组(Saline),瘤内注射生理盐水50μL;②组合物组(Com),瘤内注射组合物(多囊脂质体剂型)50μL(L-arg剂量:20mg/kg;CAT-2 shRNA质粒剂量:0.7mg/kg)。治疗一次后,利用针式pH计测量肿瘤组织中的pH变化。对小鼠肿瘤组织中pH值的监测结果说明,组合物在释放期间,可以持续中和肿瘤组织内部的酸性环境,将环境pH提升至中性水平(图8)。
实施例10组合物对肿瘤生长的抑制作用考察
本实施例在实施例3的基础上,将1×106的B16细胞接种于成年雌性C57b/c小鼠皮下建立肿瘤模型,接种7天后,随机将小鼠分为4组(n=5),分别接受以下治疗:①生理盐水组(Saline),瘤内注射生理盐水50μL,一周一次;②纯L-精氨酸组合物组(L-arg),瘤内注射不含阳离子氨基酸转运蛋白2抑制剂的纯L-精氨酸组合物(多囊脂质体剂型)50μL(L-arg剂量:20mg/kg),一周一次;③纯阳离子氨基酸转运蛋白2抑制剂组合物组(iCAT-2),瘤内注射不含L-精氨酸的纯阳离子氨基酸转运蛋白2抑制剂组合物(多囊脂质体剂型)50μL(CAT-2shRNA质粒剂量:0.7mg/kg),一周一次;④组合物组(Com),瘤内注射组合物(多囊脂质体剂型)50μL(L-arg剂量:20mg/kg;CAT-2 shRNA质粒剂量:0.7mg/kg),一周一次,共治疗两个周期。治疗期间记录体重和肿瘤体积变化,当体重下降率超过10%或者肿瘤体积大于1500mm3时,判定小鼠死亡。肿瘤体积曲线表明单独的L-arg或iCAT-2的治疗,都无法引起有效的肿瘤排斥,因为前者具备支持肿瘤生长的作用,而后者无法对免疫抑制环境进行改善,但是组合物有效抑制了黑色素瘤的生长(图9)。治疗过程中,组合物组的小鼠体重没有发生明显下降(图10),生存期大幅延长(图11)。上述结果表明,本发明的组合物从环境改善,营养补充和癌细胞竞争力限制方面实现了对肿瘤生长的抑制效果。
实施例11组合物抗肿瘤机制的研究
本实施例在实施例9的基础上,取各组小鼠肿瘤组织,切片后进行免疫荧光染色,观察组织中T细胞的浸润和分化情况。并取组织样品,处理成单细胞悬液后,通过流式分析M1型巨噬细胞和M2型巨噬细胞的比例变化。将组织样品匀浆后,离心收集上清液,通过ELISA试剂盒,检测组织中细胞因子变化情况。切片结果表明,组合物显著提高了肿瘤组织中CD8+ T细胞的数量,并且多为效应细胞(CD44+CD62L-,红)和记忆细胞(CD44+CD62L+,黄),而不是在其他三组中观察到的T细胞(CD44-CD62L+,绿)(图12,13)。流式结果说明,组合物改变了肿瘤组织中巨噬细胞的极化模式,M1型巨噬细胞比例显著提高,具有免疫抑制和促瘤发展的M2型巨噬细胞比例降低(图14)。细胞因子水平结果与上述实验结果相符,干扰素γ(IFN-γ)及NO水平在组合物组中明显提高,提示组合物产生了显著的抗肿瘤免疫反应,且白介素4(IL-4)水平没有明显变化,说明没有显著的免疫抑制反应发生(图15)。
实施例12组合物联合抗PD-1抗体的治疗效果研究
本实施例在实施例9的基础上,将1×106的B16细胞接种于成年雌性C57b/c小鼠皮下建立肿瘤模型,接种7天后,随机将小鼠分为3组(n=5),分别接受以下治疗:①抗PD-1抗体组(anti-PD-1),腹腔注射抗PD-1抗体(12.5mg/kg),一周一次;②组合物组(Com),瘤内注射组合物(多囊脂质体剂型)50μL(L-arg剂量:20mg/kg;CAT-2 shRNA质粒剂量:0.7mg/kg),一周一次;③联合组(anti-PD-1&Com),瘤内注射注射组合物(多囊脂质体剂型)50μL(L-arg剂量:20mg/kg;CAT-2 shRNA质粒剂量:0.7mg/kg),并腹腔注射抗PD-1抗体(12.5mg/kg),一周一次。共治疗两个周期,治疗期间记录体重和肿瘤体积变化,当体重下降率超过10%或者肿瘤体积大于1500mm3时,判定小鼠死亡。肿瘤体积曲线表明(图16),单独施用抗PD-1抗体并不能对B16肿瘤产生有效的抑制作用,而本发明的组合物能够逆转肿瘤对免疫疗法的无响应性,产生显著的抑制肿瘤生长作用,提高免疫治疗的效果,且没有造成明显的体重下降情况,具有良好的安全性(图17)。
序列表
<110> 浙江大学
<120> 靶向免疫细胞长效补充精氨酸并中和酸环境的组合物及应用
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Claims (3)
1.一种靶向免疫细胞长效补充精氨酸并中和酸环境的组合物,其特征在于,所述组合物由L-精氨酸、阳离子氨基酸转运蛋白2抑制剂及药学上可接受的缓释药物储库载体组成,所述阳离子氨基酸转运蛋白2抑制剂为基于阳离子氨基酸转运蛋白2基因序列的shRNA或siRNA;
其中shRNA为基于阳离子氨基酸转运蛋白2基因序列的短发夹RNA,其序列为:
(1)gctttatgccctatggctttattcaagagataaagccatagggcataaagctttttt;
(2)cgtccttacttgtctgctttattcaagagataaagcagacaagtaaggacgtttttt;
(3)cggcctttgctatgctgaatttcaagagaattcagcatagcaaaggccgtttttt;
siRNA为基于阳离子氨基酸转运蛋白2基因序列的小干扰RNA,其序列为:
(1)cgaaauacgggauaccgaaau;
(2)gcaggaaugaacagacgaaau;
(3)gccggaaacgauacgacuua;
其中L-精氨酸浓度为10-160 mg/mL,当阳离子氨基酸转运蛋白2抑制剂为shRNA时,其浓度为0.1-5 mg/mL,当阳离子氨基酸转运蛋白2抑制剂为siRNA时,其浓度为10-200 nmol/mL;
当阳离子氨基酸转运蛋白2抑制剂为shRNA或siRNA时,需复合使用基因递送载体,所述基因递送载体包括阳离子脂质体、阳离子纳米乳或固体脂质纳米粒;
所述缓释药物储库载体的类型包括微球、凝胶或多囊脂质体。
2.权利要求1所述组合物在制备抗肿瘤药物中的应用,其特征在于,所述肿瘤为黑色素瘤。
3.根据权利要求2所述的应用,其特征在于,所述抗肿瘤是通过组合物单独施用,或同时与免疫治疗、手术治疗、化疗及放疗联合施用。
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