CN114891038A - 磷酸抗原化合物及其制备方法和用于Vγ9Vδ2T细胞的培养方法 - Google Patents
磷酸抗原化合物及其制备方法和用于Vγ9Vδ2T细胞的培养方法 Download PDFInfo
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Abstract
本发明公开了磷酸抗原化合物及其制备方法和用于Vγ9Vδ2T细胞的培养方法。PTA都通三步法合成,然后将其用于Vγ9Vδ2T细胞的培养,具体包括(1)激活;(2)扩增;和(3)收获步骤。本发明合成的PTA是一种疏水性的药物前体,可以穿透细胞膜,进入细胞质内,在脂酶的作用下分解成为药物活性成分TA、戊醛酸、甲醛,激活γδT细胞。基于PTA进行的γδT细胞的培养,可以显著的提高了γδT细胞纯度和数量,同时降低了γδT细胞凋亡,为γδT细胞治疗肿瘤的临床应用奠定基础。
Description
技术领域
本发明属于免疫细胞体外培养技术领域,具体涉及一种磷酸抗原化合物PTA及其制备方法和用于Vγ9Vδ2T细胞的培养方法。
背景技术
肿瘤过继性免疫疗法是将致敏淋巴细胞(具有特异免疫力)或其产物输给细胞免疫功能低下者(如肿瘤病人),使其获得抗肿瘤免疫力,它是用来治疗肿瘤的一种免疫疗法。1985年美国国家癌症研究委员会将癌症的免疫疗法确立为继外科手术、放射疗法和化学药物疗法之后的第四种疗法。
根据T细胞表面TCR的类型,可将T细胞分为αβT细胞和γδT细胞两类。其中αβT细胞即通常所称的T细胞,其表面表达TCRαβ,占T细胞总数的95%以上;而γδT细胞表面表达TCRγδ。γδT细胞是1986年被发现的,在疾病初始阶段具有快速、直接反应的能力,在天然免疫反应中扮演重要角色。γδT细胞是一类不同于αβT细胞的主要T细胞亚群,是人体内联系固有免疫和适应性免疫之间的桥梁。γδT细胞的Vγ9Vδ2亚群主要存在于外周血中,对肿瘤细胞具有直接细胞毒性,能通过直接杀伤作用和释放细胞因子来共同控制肿瘤细胞的生长和转移,其杀伤作用不依赖于MHC分子。因此,近年来γδT细胞技术开始涌现入临床,用于肿瘤细胞的治疗。
然而外周血中的γδT细胞只占整个淋巴亚群的1%-5%,数量稀少,需要经过体外刺激扩增后,方可取得足够的细胞,供临床使用。目前常用含氮的膦酸抗原如唑来膦酸(zol)来激活γδT细胞。唑来膦酸,化学名为1-羟基-2-(咪唑-1-基)-亚乙基-1,1-二磷酸,是一种二膦酸盐药物,用于治疗肿瘤骨转移及预防骨骼并发症。Zol含P-C-P结构,带负电荷,因此需要液相胞吞作用,从而被巨噬细胞和递呈细胞所吞噬进入细胞质。在细胞质中与脂代谢过程中法尼基二磷酸合酶(FPPS)结合,抑制法尼基二磷酸(FPP)产生,导致代谢上游的异戊烯焦磷酸(IPP)累积,IPP通过嗜乳脂蛋白家族BTN3A1与TCRγδ结合,激活γδT细胞;然而Zol所带负电荷由于需要液相胞饮才能进入细胞,限制了zol在细胞质的浓度,从而需要较高浓度的zol才能有效激活γδT细胞。然而高浓度的zol会导致γδT细胞凋亡,从而限制了γδT细胞的扩增数量。
发明内容
本发明利用化学合成的方法,合成了PTA,PTA是一种疏水性的药物前体,可以穿透细胞膜,进入细胞质内,在脂酶的作用下分解成为药物活性成分TA、戊醛酸、甲醛,TA与FPPS结合,抑制FPP产生,导致代谢上游的IPP累积,IPP通过BTN3A1与TCRγδ结合,激活γδT细胞。本发明公开了PTA的合成方法,以及用PTA进行γδT细胞的合成,可以显著的提高了γδT细胞纯度和数量,同时降低了γδT细胞凋亡,为γδT细胞治疗肿瘤的临床应用,奠定基础。
具体采用以下的技术方案:
一种磷酸抗原化合物PTA,其结构如式I所示:
本发明还提供了上述磷酸抗原化合物PTA的制备方法,包括以下步骤:通过二乙胺、甲醛、亚甲基二磷酸四甲酯反应合成亚乙烯基-1,1-二膦酸四甲酯,将其溶于乙腈溶液中后,加入碘化钠和新戊酸氯甲酯,合成得到四新戊酰氧基甲基亚乙烯基-1,1-二膦酸酯;然后将其溶于三氯甲烷溶液中后再与2-氨基噻唑反应,得到PTA。
其中,二乙胺:甲醛:亚甲基二磷酸四甲酯=60mmol:300mmol:60mmol;碘化钠:新戊酸氯甲酯:亚乙烯基-1,1-二膦酸四甲酯=68mmol:85mmol:17mmol;新戊酰氧基甲基亚乙烯基-1,1-二膦酸酯:2-氨基噻唑=0.1mmol:0.2mmol。
本发明的另一个目的在于提供一种扩增Vγ9Vδ2T细胞的培养方法,包括以下步骤:
(1)激活:从外周血中分离PBMC,重悬于含10%FBS的RPMI-1640培养基中,使外周血单个核细胞的细胞浓度为1×106个/mL,同时添加上述的PTA、IL-2、IL-15、静置培养3天;
(2)扩增:添加培养基稀释细胞浓度至5×105个/mL,同时添加IL-2、IL-15,静置培养6至11天;
(3)收获:将培养的Vγ9Vδ2T细胞培养基收集,离心,所得沉淀即是成熟的Vγ9Vδ2T细胞。
优选地,步骤(1)中,添加组分后的培养基PTA浓度为1-100μM,IL-2浓度为100-1000IU/mL,IL-15浓度为100-1000IU/mL。
优选地,步骤(2)中,添加组分后的培养基IL-2浓度为100-1000IU/mL,IL-15浓度为100-1000IU/mL。
优选地,所述步骤(1)和(2)中的静置培养条件为:温度为37℃、CO2体积百分比含量为5%、相对饱和湿度为100%。
优选地,所述步骤(3)还包括将制得的Vγ9Vδ2T细胞经离心后,重悬于保存液中,所述保存液含有如下组分:体积百分比为20%的人血清白蛋白5mL,0.9wt%氯化钠溶液定容至100mL。
本发明的有益效果为:本发明合成的PTA是一种疏水性的药物前体,可以穿透细胞膜,进入细胞质内,在脂酶的作用下分解成为药物活性成分TA、戊醛酸、甲醛,激活γδT细胞。基于PTA进行的γδT细胞的培养,可以显著的提高了γδT细胞纯度和数量,同时降低了γδT细胞凋亡,为γδT细胞治疗肿瘤的临床应用奠定基础。
附图说明
图1所示为PTA的HPLC检测结果;
图2所示为PTA的MS检测结果;
图3所示为PTA的HNMR检测结果;
图4所示为培养至14天的Vγ9Vδ2T细胞表型;
图5所示为培养至14天的Vγ9Vδ2T细胞绝对数量;
图6所示为培养至14天的Vγ9Vδ2T细胞凋亡结果。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述,以充分地理解本发明的目的、方案和效果。相关术语说明如下:
PBMC:外周血单个核细胞;IL-2:白细胞介素2;IL-15:白细胞介素15;ZOL:唑来膦酸;FPPS:法尼基二磷酸合酶;FPP:法尼基二磷酸;IPP:异戊烯焦磷酸;BTN3A1:嗜乳脂蛋白家族蛋白。
实施例1:PTA的合成
一种磷酸抗原化合物PTA的制备方法,包括以下步骤:
PTA的结构如式I所示:
(1)合成亚乙烯基-1,1-二膦酸四甲酯
在室温下将二乙胺(6.3mL,60mmol)加入到甲醛(9.0g,300mmol)的甲醇(230mL)溶液中,并在65℃下搅拌30分钟;加入在甲醇(10mL)中的亚甲基二磷酸四甲酯(14g,60mmol)的溶液,并将反应混合物保持回流1.5小时;将所得混合物真空浓缩,得到粗产物,将其用于下一步反应,无需进一步纯化;将粗产物溶解在甲苯(200mL)中,并用对甲苯磺酸一水合物(114mg,0.6mmol)处理;然后用CHCl3稀释;用盐水洗涤所得混合物,浓缩,得到纯化的粗产物通过硅胶柱层析,得到8.9g(60%)目标化合物,为无色油状物;
(2)四新戊酰氧基甲基亚乙烯基-1,1-二膦酸酯((新戊酰氧基)甲基亚乙烯基-1,1-二膦酸酯)的合成
将NaI(10.2g,68mmol)和新戊酸氯甲酯(POMCl,12.4mL,85mmol)加入到四甲基亚乙烯基-1,1-二膦酸酯(4.2g,17mmol)的MeCN(85mL)溶液中,然后保持回流4小时;加入H2O后,将所得混合物用EtOAc萃取,并将有机层用盐水洗涤,经Na2SO4干燥,过滤并真空浓缩,得到粗产物,将其通过硅胶柱纯化得到3.8g(35%)目标化合物,为淡黄色油状物;
(3)PTA的合成
将2-氨基噻唑(20mg,0.2mmol)加入到四新戊酰氧基甲基亚乙烯基-1,1-二膦酸酯(64mg,0.1mmol)的CHCl3(0.4mL)溶液中,并在室温下搅拌1小时;真空浓缩所得混合物,得到粗产物,用硅胶纯化柱层析,得到69mg(92%)目标化合物为无色固体。
对制得的PTA分别进行HPLC、MS、HNMR检测,其结果如图1-3所示。本实施例最终合成出的PTA,纯度达到98.8%。由于PTA是疏水性的化合物,因此,将PTA溶解于DMSO中备用。
实施例2:
1、一种提取外周血单个核细胞的方法,包括如下步骤:
(1)吸取5mL Ficoll淋巴细胞分离液于15mL离心管中,将肝素钠采集的外周血混匀均匀缓慢地加至Ficoll上层,形成完整的界面;
(2)800g,离心20min,可见明显分层;
(3)收集单个核细胞,用生理盐水洗涤2次;
(4)用生理盐水重悬细胞并计数,备用。
2、Vγ9Vδ2T细胞的免疫表型检测,包括步骤如下:
(1)分别取Zol组与PTA组培养第14天的细胞,转移入流式检测管,加1mL PBS充分混匀,离心洗涤细胞(离心条件:1000rpm,5min)。倾去离心上清,根据细胞样本浓度和体积,用适量PBS悬浮细胞,调整细胞浓度为1*105个/mL;
(2)荧光标记细胞:将细胞按照空白对照管、同型对照、实验组分别移入相应试管中,每管约5*105个细胞。对照管和检测管按照抗体说明书分别配制荧光抗体染色液:抗人CD3-FITC、抗人TCR-Vδ2-PE或抗人TCR-Vδ2-PC5.5。用含1%BSA的PBS 100μL重悬细胞,加入相应的荧光抗体,每种抗体量为5μL;
(3)漩涡振荡器上轻轻混匀,室温避光孵育15min;
(4)加含1%BSA的1mL PBS洗涤,离心,1000rpm,5min,丢弃上清,加入500uL PBS悬浮沉淀,充分混匀;
(5)上机检测,结果见图4。
由上述图4的结果可知,PTA组的Vγ9Vδ2T细胞表型要显著优于Zol组;表面PTA对于Vγ9Vδ2T细胞的刺激要优于Zol。
3、Vγ9Vδ2T细胞扩增倍数检测
在第0天的时候,分成Zol组与PTA组分别接种1*106个PBMC于12孔板中,分别取培养的第3、6、9、12、14天的Vγ9Vδ2T细胞,用细胞计数板进行细胞计数,并与当天流式细胞仪所测CD3+Vδ2+细胞相乘,计算出绝对的Vγ9Vδ2T细胞数量,结果间见图5。由图5的结果可以看出,在培养至12天后,PTA组的Vγ9Vδ2T细胞数量要显著高于Zol组的Vγ9Vδ2T细胞数量,说明PTA相对于Zol,对Vγ9Vδ2T细胞的刺激作用更强。
4、Vγ9Vδ2T细胞凋亡检测
(1)分别取Zol组与PTA组培养第14天的细胞,转移入流式检测管,加3mL PBS充分混匀,离心洗涤细胞(离心条件:1000rpm,5min);倾去离心上清,根据细胞样本浓度和体积,用适量PBS悬浮细胞,调整细胞浓度为5*106个/mL;
(2)荧光标记细胞:将细胞按照空白对照管、实验组分别移入相应试管中,每管约5*105个细胞。对照管和检测管按照试剂盒说明书分别加入荧光抗体染色液:抗人AnnexinV-FIT C、抗人PI,常温避光染色20分钟;
(3)加入0.4mL的结合缓冲液充分混匀;
(4)上机检测,结果见图6。
由图6中可以看出,培养至14天的PTA组的细胞凋亡(Annexin-V+PI+)百分比,显著低于Zol组的细胞凋亡百分比,由此可以得出PTA相对于Zol,减少了扩增的γδT细胞凋亡。
以上所述,只是本发明的较佳实施例而已,本发明并不局限于上述实施方式,只要其以相同的手段达到本发明的技术效果,都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。
Claims (8)
2.一种权利要求1所述的磷酸抗原化合物PTA的制备方法,其特征在于,包括以下步骤:通过二乙胺、甲醛、亚甲基二磷酸四甲酯反应合成亚乙烯基-1,1-二膦酸四甲酯,将其溶于乙腈溶液中后,加入碘化钠和新戊酸氯甲酯,合成得到四新戊酰氧基甲基亚乙烯基-1,1-二膦酸酯;然后将其溶于三氯甲烷溶液中后再与2-氨基噻唑反应,得到PTA。
3.根据权利要求2所述的制备方法,其特征在于,二乙胺:甲醛:亚甲基二磷酸四甲酯=60mmol:300mmol:60mmol;碘化钠:新戊酸氯甲酯:亚乙烯基-1,1-二膦酸四甲酯=68mmol:85mmol:17mmol;新戊酰氧基甲基亚乙烯基-1,1-二膦酸酯:2-氨基噻唑=0.1mmol:0.2mmol。
4.一种扩增Vγ9Vδ2T细胞的培养方法,其特征在于,包括以下步骤:
(1)激活:从外周血中分离PBMC,重悬于含10%FBS的RPMI-1640培养基中,使外周血单个核细胞的细胞浓度为1×106个/mL,同时添加权利要求1所述的PTA、IL-2、IL-15、静置培养3天;
(2)扩增:添加培养基稀释细胞浓度至5×105个/mL,同时添加IL-2、IL-15,静置培养6至11天;
(3)收获:将培养的Vγ9Vδ2T细胞培养基收集,离心,所得沉淀即是成熟的Vγ9Vδ2T细胞。
5.根据权利要求4所述的培养方法,其特征在于,步骤(1)中,添加组分后的培养基PTA浓度为1-100μM,IL-2浓度为100-1000IU/mL,IL-15浓度为100-1000IU/mL。
6.根据权利要求4所述的培养方法,其特征在于,步骤(2)中,添加组分后的培养基IL-2浓度为100-1000IU/mL,IL-15浓度为100-1000IU/mL。
7.根据权利要求4所述的培养方法,其特征在于,所述步骤(1)和(2)中的静置培养条件为:温度为37℃、CO2体积百分比含量为5%、相对饱和湿度为100%。
8.根据权利要求4所述的培养方法,其特征在于,所述步骤(3)还包括将制得的Vγ9Vδ2T细胞经离心后,重悬于保存液中,所述保存液含有如下组分:体积百分比为20%的人血清白蛋白5mL,0.9wt%氯化钠溶液定容至100mL。
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