CN119097725B - 一种cd8抗体修饰的抗tim-3的脂质纳米颗粒、制备方法及其应用 - Google Patents
一种cd8抗体修饰的抗tim-3的脂质纳米颗粒、制备方法及其应用 Download PDFInfo
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Abstract
本发明提供一种CD8抗体修饰的抗TIM‑3的脂质纳米颗粒、制备方法及其应用，属于医用配制品技术领域，所述脂质纳米颗粒中包含嵌合抗原受体；所述嵌合抗原受体包括单链抗体TIM‑3；所述单链抗体TIM‑3的核酸人工序列如SEQ ID NO.5所示。本申请对TIM‑3靶点进行序列优化，制备连接双刺激因子和自杀基因的CAR结构，并且构建由CD8抗体修饰的抗TIM‑3的纳米脂质颗粒，递送至细胞内，能够在机体内有效的捕获CD8+T细胞，产生CAR‑T细胞，省去体外制备的复杂过程，在高效识别抗原的同时提高T细胞的激活程度，发挥高效抗肿瘤作用，明显延长乳腺癌小鼠的存活期，抑制肿瘤的生长。
Description
技术领域
本发明涉及一种CD8抗体修饰的抗TIM-3的脂质纳米颗粒及其应用，属于医用配制品技术领域。
背景技术
T细胞免疫球蛋白及粘蛋白结构域分子3（简称为TIM-3），也被称为甲型肝炎病毒细胞受体2，属于Tim基因家族。TIM-3靶点在肿瘤免疫治疗中具有重要潜力，因为它可以与多种配体结合，如半乳糖凝集素-9(Gal-9)、癌胚抗原细胞粘附分子-1(CEACAM-1)、高迁移率族蛋白B1(HMGB1)和磷脂酰丝氨酸(PS)，这些配体与TIM-3的相互作用在调节T细胞免疫中起到关键作用。TIM-3的抑制可以增强抗肿瘤免疫反应，因此，多个制药企业正在开发以TIM-3为靶点的免疫治疗药物。
脂质纳米颗粒（LNP）是一种高效的药物递送系统，广泛应用于核酸药物、小分子化合物等的封装与递送。其主要成分包括可电离脂质、胆固醇、辅助脂质和聚乙二醇（PEG）脂质，这些成分共同作用，保护核酸药物免受降解、提高药物的溶解性和疗效，并实现靶向药物递送，在核酸药物递送、癌症治疗以及疫苗开发等领域展现出广泛的应用前景。
目前，以TIM-3为靶点的临床研究主要集中在急性髓系白血病（AML）和骨髓增生异常综合症（MDS）、非小细胞肺癌等实体瘤的治疗上，大多数TIM-3药物处在临床阶段。以TIM-3为靶点的CAR细胞疗法正在临床试验中探索其治疗潜力。一项临床试验（NCT06125652）正在评估抗TIM-3/CD123 CAR-T细胞治疗复发难治性急性髓系白血病的安全性和有效性，但TIM-3抗体在临床前及临床阶段显示出单药效果一般，即使和PD-1抗体联用，效果也低于预期。
现有技术中尚未报道CD8抗体修饰的抗TIM-3的脂质纳米颗粒。
发明内容
针对现有技术存在的不足，本发明提供一种CD8抗体修饰的抗TIM-3的脂质纳米颗粒、制备方法及其应用，实现以下发明目的：CD8抗体修饰的抗TIM-3的脂质纳米颗粒体外转染CD8+T细胞后，提高对靶细胞MCF-7的杀伤率，CD8抗体修饰的抗TIM-3的脂质纳米颗粒体内能够捕获CD8+T细胞，产生CAR-T细胞，提高抗肿瘤效果。
为解决上述技术问题，本发明采取以下技术方案：
一种CD8抗体修饰的抗TIM-3的脂质纳米颗粒，所述脂质纳米颗粒中包含嵌合抗原受体；所述嵌合抗原受体包括单链抗体TIM-3；所述单链抗体TIM-3的核酸人工序列如SEQID NO.5所示。
所述嵌合抗原受体由以下模块依次连接得到：导引子、单链抗体TIM-3、CD8 Hinge区、CD28跨膜区、CD28-4-1BB共刺激区、CD3ζ胞内区、自剪切区T2A、自杀基因。
所述嵌合抗原受体的核酸人工序列如SEQ ID NO.2所示。
所述CD8抗体的重链氨基酸序列如SEQ ID NO.12所示，轻链氨基酸序列如SEQ IDNO.13所示。
所述CD8抗体修饰的抗TIM-3的脂质纳米颗粒的制备方法为，将嵌合抗原受体构建得到重组质粒，采用LNP包载后，再偶联CD8抗体，制得CD8抗体修饰的抗TIM-3的脂质纳米颗粒。
所述的脂质纳米颗粒在制备抗乳腺癌药品中的应用。
与现有技术相比，本发明取得以下有益效果：
本申请对TIM-3靶点进行序列优化，制备连接双刺激因子和自杀基因的CAR结构，并且构建由CD8抗体修饰的抗TIM-3的纳米脂质颗粒，递送至细胞内，能够在机体内有效的捕获CD8+T细胞，产生CAR-T细胞，省去体外制备的复杂过程，在高效识别抗原的同时提高T细胞的激活程度，发挥高效抗肿瘤作用，明显延长乳腺癌小鼠的存活期，抑制肿瘤的生长。
本发明制备的CD8抗体修饰的抗TIM-3的纳米脂质颗粒，体外转染CD8+T细胞得到的CAR-T细胞，对靶细胞MCF-7的杀伤率达到67.9%。
附图说明
图1为AntiCD8-LNP-TIM-3-1对活化CD8+T细胞的感染率的流式图；
图2为AntiCD8-LNP-TIM-3-2对活化CD8+T细胞的感染率的流式图；
图3为CAR-T-1细胞、CAR-T-2细胞、活化CD8+T细胞对MCF-7细胞杀伤率的柱状图；
图4为实施例6中小鼠存活期的测定图；
图5为实施例6中小鼠肿瘤生长曲线测定图。
具体实施方式
实施例1目的基因的设计及重组质粒构建
CAR结构各模块及其核酸人工序列如下所示：
（1）导引子，其核酸人工序列如序列表中SEQ ID NO.3所示；
（2）单链抗体TIM-3，其优化前的核酸人工序列如序列表中SEQ ID NO.4所示；优化后的核酸人工序列如序列表中SEQ ID NO.5所示；
（3）CD8 Hinge区，其核酸人工序列如序列表中SEQ ID NO.6所示；
（4）CD28跨膜区，其核酸人工序列如序列表中SEQ ID NO.7所示；
（5）CD28-4-1BB共刺激区，其核酸人工序列如序列表中SEQ ID NO.8所示；
（6）CD3ζ胞内区，其核酸人工序列如序列表中SEQ ID NO.9所示；
（7）自剪切区T2A，其核酸人工序列如序列表中SEQ ID NO.10所示；
（8）自杀基因，其核酸人工序列如序列表中SEQ ID NO.11所示。
依次将SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.6、SEQ IDNO.7、SEQ ID NO.8、SEQID NO.9、SEQ ID NO.10、SEQ IDNO.11的核酸人工序列连接，连接后的序列如序列表中SEQID NO.1所示。委托南京金斯瑞生物科技有限公司合成其整个表达框并插入pUC57载体上，命名为pUC-CAR-1。将pUC-CAR载体和PVAX1载体进行Fast Digest BamH I(购自ThermoFisher公司)和Fast Digest Not I(购自ThermoFisher公司)双酶切，切胶回收获得线性化的DNA片段，16℃进行过夜连接形成PVAX1-CAR-TIM-3-1表达载体。将上述PVAX1-CAR-TIM-3-1表达载体转化到E.coli(DH5α)，挑取阳性克隆，进行PCR鉴定，然后提取质粒并送南京金斯瑞生物科技有限公司进行测序，测序鉴定正确，即PVAX1-CAR-TIM-3-1构建成功，从阳性克隆中提取PVAX1-CAR-TIM-3-1重组质粒，稀释至2μg/μL，保存于-80℃待用。
采用同样的方法，依次将SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.6、SEQ IDNO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ IDNO.11的核酸人工序列连接，连接后的序列如序列表中SEQ ID NO.2所示，按照上述方法成功构建PVAX1-CAR-TIM-3-2表达载体，从阳性克隆中提取PVAX1-CAR-TIM-3-2重组质粒，稀释至2μg/μL，保存于-80℃待用。
实施例2制备包载重组质粒的LNP
（1）取10.9μL实施例1中制备的PVAX1-CAR-TIM-3-1重组质粒(浓度2.0μg/μL)，使用pH＝4、浓度为10mM的柠檬酸缓冲液稀释至300μL，得到重组质粒混合液；
（2）将2.18mg可电离脂质DLin-MC3-DMA、1.00mg胆固醇、0.27mg的DMG-PEG2000和0.54mg的DSPC、0.1mg的DSPE-PEG-马来酰亚胺溶于1mL乙醇中，得到混合脂质溶液；
（3）向微流控设备中加入重组质粒混合液和混合脂质溶液，重组质粒混合液中的DNA和混合脂质溶液中的可电离脂质DLin-MC3-DMA的质量比为1:10，通过微流控法制备得到包载重组质粒的LNP纳米颗粒。
将获得的LNP纳米颗粒在PBS溶液( pH7.4)中透析24h，透析完使用Amicon超离心过滤器浓缩，过0.22μm滤膜2次，得到包载重组质粒的LNP，命名为LNP-TIM-3-1。
以上述同样的方法制备包载重组质粒PVAX1-CAR-TIM-3-2的LNP，命名为LNP-TIM-3-2。
使用动态光散射粒径电位测定仪测定LNP-TIM-3-1和LNP-TIM-3-2的粒径大小，使用Quant-iT 1X dsDNA 试剂盒(Invitrogen)测定包封率。
结果显示， LNP-TIM-3-1的包封率为85.3±4.36％，包封率较高，粒径为105.6±5.0nm，粒径均一； LNP-TIM-3-2的包封率为87.2±4.17%，包封率较高，粒径为108.2±5.8nm，粒径均一。
实施例3偶联CD8抗体
所述CD8抗体的重链氨基酸序列如序列表中SEQ ID NO.12所示，轻链氨基酸序列如序列表中SEQ ID NO.13所示，按照常规方法制备得到CD8抗体。
将CD8抗体在含有1mM DTT和5mM EDTA的PBS中室温放置1h使其还原，随后使用7KZeba脱盐离心柱（购自ThermoFisher Scientific）通过缓冲交换到含有5mM EDTA的PBS中，将DTT去除。缓冲液交换后，立即将还原后的CD8抗体加入LNP-TIM-3-1中（以每毫升LNP-TIM-3-1中加入0.67mg的CD8抗体），将混合物在室温下轻摇孵育2小时，然后在4℃下孵育过夜。第二天，以PBS为流动相，用琼脂糖凝胶CL-4B过滤柱上从游离抗体中分离CD8抗体偶联的LNP-TIM-3-1，使用100KMillipore超滤离心管（购自索莱宝）浓缩至LNP-TIM-3-1加入的初始体积，得到偶联CD8抗体的LNP-TIM-3-1，命名为AntiCD8-LNP-TIM-3-1。
LNP-TIM-3-2以同样的方法偶联CD8抗体，得到AntiCD8-LNP-TIM-3-2。
利用 Triton X-100分别裂解得到的AntiCD8-LNP-TIM-3-1和AntiCD8-LNP-TIM-3-2，使用Qubit™dsDNA BR Assay Kit试剂盒测量DNA的量，结果显示AntiCD8-LNP-TIM-3-1中DNA的浓度为52ng/μL， AntiCD8-LNP-TIM-3-2中DNA的浓度为54ng/μL，AntiCD8-LNP-TIM-3-1和AntiCD8-LNP-TIM-3-2中DNA的浓度调整为50ng/μL，保存备用。
实施例4两种AntiCD8-LNP-TIM-3转染CD8+T细胞
（1）活化CD8+T细胞的制备
取75mL患者自体外周血，用Ficoll-Paque淋巴细胞分离液分离外周血单个核细胞，分离后的细胞用BD公司提供的CD8+分选试剂分选出CD8+T细胞，进行细胞计数，按1×106个细胞/mL进行接种，加入含有终浓度为1500IU/mL IL-2的KBM551细胞培养基(购自Corning，货号：88-551-CM)，然后加入与细胞相同数量的CD3CD28磁珠进行活化T细胞，活化24h后取出磁珠，得到活化CD8+T细胞。
（2）转染CD8+T细胞
调整活化CD8+T细胞的数量至1×107个/mL，得到细胞悬液，取0.1mL细胞悬液接种于6孔板中，置于37℃、5％CO2、饱和湿度培养箱中过夜培养，然后加入100ng实施例3制备的AntiCD8-LNP-TIM-3-1，37℃下孵育48h，离心收集AntiCD8-LNP-TIM-3-1-CAR-T细胞，简称CAR-T-1。
同样的方法得到AntiCD8-LNP-TIM-3-2-CAR-T细胞，简称CAR-T-2。
通过流式细胞术检测AntiCD8-LNP-TIM-3对活化CD8+T细胞的感染率，如图1和图2所示，AntiCD8-LNP-TIM-3-1对活化CD8+T细胞的感染率为58.7%，AntiCD8-LNP-TIM-3-2对活化CD8+T细胞的感染率为65.6%。
实施例5体外抗肿瘤实验
（1）靶细胞
以人乳腺癌细胞株MCF-7细胞(购自中国科学院细胞库)为目标细胞，复苏-80℃冷冻保存的MCF-7细胞，接种于含有10％vol%FBS、100U/mL青霉素、100U/mL链霉素的RPMI1640培养基中，37℃、5％CO2、饱和湿度培养箱中进行培养，待细胞融合度达80％以上时，进行传代培养；共传5代，以便充分活化细胞。
（2）细胞共培养
用含有10vol%FBS的DMEM培养基调整效应细胞（1×105/孔）与靶细胞（1×105/孔）的浓度，并接种于96孔板，效应细胞分别选择实施例4中制备的CAR-T-1细胞、CAR-T-2细胞，对照组选择实施例4制备的活化CD8+T细胞。
分为三组，具体分组如下：
实验组A：CAR-T-1细胞和MCF-7细胞共培养；
实验组B：CAR-T-2细胞和MCF-7细胞共培养；
对照组：活化CD8+T细胞和MCF-7细胞共培养；
每组三个重复，置于5%CO2、37℃培养箱培养，24h后每孔加入20mL的CCK-8，继续孵育2h后，酶标仪检测450nm波长，读取OD值。计算细胞杀伤率：杀伤率/%＝[1-(空白组OD值-效应细胞组OD值)/空白组OD值]×100%。
杀伤率的结果如图3所示：实验组A、实验组B的杀伤率分别是42.5%、67.9%，对照组的杀伤率为14.8%。实验组B的杀伤率高于实验组A，说明ScFv(TIM-3)序列的优化对于最终制备的CAR-T细胞的肿瘤杀伤效果有显著的影响。
实施例6体内抗肿瘤实验
（1）动物模型制备
将周龄6-8周的Balb/c裸鼠适应饲养一周后，离心收集生长状态良好的对数生长期的MCF-7细胞，使用无菌生理盐水重悬细胞，调节细胞浓度1×107个/mL，使用无菌注射器将0.5mL细胞悬液注射到裸鼠右侧肩胛骨下；然后每天观测肿瘤大小，记录肿瘤的长径(L)与短径(W)，肿瘤体积(V)计算公式如下：V肿瘤＝(L×W2)/2，当肿瘤体积至200-300mm3时，造模成功用于后续实验。
（2）动物存活期观测
将造模成功的裸鼠随机分为4组，每组12只，分别为：
对照组：注射200μL生理盐水；
实验A组：注射1×106个活化CD8+T细胞；
实验B组：注射200μL AntiCD8-LNP-TIM-3-1；
实验C组：注射200μL AntiCD8-LNP-TIM-3-2；
每天观测并记录实验动物存活状态。结果如图4所示，实验A组小鼠的存活期要远远低于实验B组和实验C组，实验C组小鼠存活期大于实验B组，且生存率更高，说明ScFv(TIM-3)序列优化后制备的抗TIM-3的纳米脂质颗粒的抗肿瘤效果明显提高。
本申请制备的AntiCD8-LNP-TIM-3-1和AntiCD8-LNP-TIM-3-2，可以在体内诱导产生CAR-T细胞，可明显延长乳腺癌动物的存活期。
（3）肿瘤生长曲线测定
6-8周的雄性C57BL6小鼠(购自南京君科生物工程有限公司)于动物房饲养(室温23±2℃，湿度50%±10%)，收集对数期的MCF-7细胞，磷酸盐缓冲液(PBS)稀释至1×106个/mL。无菌条件下，小鼠左侧腋下接种0.2mL MCF-7细胞悬浮液，观察两周，待腋下出现米粒大小较硬的结节作为建模成功的标准。
C57BL6乳腺癌模型小鼠(游标卡尺量取皮下肿瘤组织块的大小为90-100mm3)随机分成5组，每组10只，开始注射治疗实验：
a.对照组，尾部静脉注射同等体积的生理盐水；
b.治疗一组，尾部静脉注射1×106个细胞/只活化后的CD8+T细胞；
c.治疗二组，尾部静脉注射200μL AntiCD8-LNP-TIM-3-1/只；
d.治疗三组，尾部静脉注射200μL AntiCD8-LNP-TIM-3-2/只；
e.治疗四组，尾部静脉注射200μL AntiCD8-LNP-TIM-3-2/只，24h后注射一次利妥昔单抗（第二次免疫后同样24h后注射一次利妥昔单抗）。
每周免疫上述各组小鼠一次，连续免疫两周，以首次免疫当天为0天记录，每三天通过游标卡尺测量各个治疗组小鼠皮下肿瘤组织块大小，用肿瘤体积均值绘制肿瘤生长曲线图，5周后解剖小鼠，将肿瘤组织利用石蜡包埋，切片。
肿瘤体积增长结果图5所示，首次免疫第30天时，对照组小鼠的肿瘤体积均值为3980mm3，治疗一组小鼠的肿瘤体积均值为1817mm3，治疗二组小鼠的肿瘤体积均值为723mm3，治疗三组小鼠的肿瘤体积均值为452mm3，治疗四组小鼠的肿瘤体积均值为3639mm3。
结果表明本申请设计的 AntiCD8-LNP-TIM-3-2在小鼠体内诱导产生的CAR-T细胞对C57BL6小鼠肿瘤生长的抑制作用最好，说明序列优化后的ScFv(TIM-3)抗体对肿瘤的杀伤效果更显著，注射利妥昔单抗后CAR-T细胞对肿瘤基本不起作用，说明利妥昔单抗可以启动机体内CAR-T细胞的自杀系统RQR8，细胞活性受自杀基因系统的控制。
Claims (2)
1.一种CD8抗体修饰的抗TIM-3的脂质纳米颗粒，其特征在于：所述脂质纳米颗粒中包含嵌合抗原受体；所述嵌合抗原受体由以下模块依次连接得到：导引子、单链抗体TIM-3、CD8 Hinge区、CD28跨膜区、CD28-4-1BB共刺激区、CD3ζ胞内区、自剪切区T2A、自杀基因；所述嵌合抗原受体的核酸人工序列如SEQ ID NO.2所示；
所述CD8抗体的重链氨基酸序列如SEQ ID NO.12所示，轻链氨基酸序列如SEQ IDNO.13所示；
所述脂质纳米颗粒的制备方法为将嵌合抗原受体构建得到重组质粒，采用LNP包载后，再偶联CD8抗体，制得CD8抗体修饰的抗TIM-3的脂质纳米颗粒；
所述LNP的组成为：可电离脂质DLin-MC3-DMA、胆固醇、DMG-PEG2000、DSPC、DSPE-PEG-马来酰亚胺，质量比为2.18：1.00：0.27：0.54：0.1。
2.权利要求1所述的脂质纳米颗粒在制备抗乳腺癌药品中的应用。
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