CN107988164B - 一种pd-1 car nk-92细胞及其制备方法与应用 - Google Patents
一种pd-1 car nk-92细胞及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种PD‑1 CAR NK‑92细胞及其制备方法与应用，PD‑1 CAR NK‑92细胞是在NK‑92细胞内表达PD‑1‑CD8TM‑4‑1BB‑CD3ζ融合蛋白。本发明的PD‑1 CAR NK‑92是将PD‑1 CAR分子侵染NK92细胞株并经过流式筛选获得单一克隆细胞，将性状稳定杀伤活性高的CAR NK92单克隆细胞株培养并扩增获得。该细胞可以用于大规模生产制备，可以用于不同病人且不会产生GVHR排斥，其杀伤活性特异，肿瘤治疗效果明显。
Description
技术领域
本发明涉及一种细胞及其制备方法与应用，特别涉及一种PD-1 CAR NK-92细胞及其制备方法与应用。
背景技术
随着肿瘤免疫治疗研究的逐步进展，程序性死亡生长因子-1(PD-1/CD 279)及其配体PD-L1/2(B7-H1/CD274)作为肿瘤微环境中的重要成员获得众多研究者的青睐。2014年9月4号，美国食品及药物管理局(food and drug adm-inistration，FDA)批准了Keytruda(pembrolizum ab)用于治疗对其它药物治疗无效的晚期或无法切除的黑色素瘤患者，成为阻断PD-1细胞通路的首款获得FDA批准的药物。1992年PD-1首次被发现，其主要表达于T细胞、调节性T细胞、“耗尽”的T细胞、B细胞、活化单核细胞、树突细胞、自然杀伤细胞、自然杀伤T细胞…。PD-1一般表达于活化的T细胞，其包括跨膜区、茎区、1个Ig超家族区和1个包含ITIM、ITSM的细胞内区。PD-1属于协同抑制受体，有2个配体，分别为PD-L1、PD-L2。PD-L1在不同的恶性肿瘤中异常表达，如肺、食管、头颈部的鳞状细胞癌以及其他类型的恶性肿瘤，如卵巢癌、膀胱癌、恶性黑色素瘤和胶质瘤中表达，从结构上看，PD-L2与PD-L1类似，同属于I型跨膜蛋白，其包括信号肽、IgV样区、IgC样区、茎区、跨膜区和胞质区。PD-1与配体PD-L1/2结合使ITIM和ITSM内的酪氨酸磷酸化，ITIM、IT SM与SH P-1和SH P-2结合，通过BC L·XL的下调表达和T细胞的分化传递T细胞抑制性信号，间接导致细胞死亡。PD-1 PD-L 1/2途径也被认为是介导免疫抑制的路径，PD-1作为一个负性调控点起作用。PD-1和PD-L1途径的抑制作用在体外能加强T细胞应答；在体内PD-1与特异性配体(PD-L1、PD-L2)结合起作用，下调抗原刺激的淋巴细胞增殖及细胞因子的产生，最终导致淋巴细胞“耗尽”以及诱导免疫耐受。实体肿瘤中的肿瘤细胞可上调PD-L1的表达，继而提供下调激活T细胞的抑制信号，最终关闭免疫反应并诱导免疫耐受性。PD-Ll高表达的患者生存率显著下降，与大多数报道肿瘤细胞上PD-L1的高表达与不良预后相关且具有一致性。除在恶性黑色素瘤表达外，PD-L1还可在其他不同的肿瘤中表达，包括胶质母细胞瘤、胰腺癌、卵巢癌、乳腺癌、肾细胞癌、头颈部及食管鳞状细胞癌和非小细胞肺癌，且肿瘤细胞上PD-Ll的高表达与不良预后相关。
自然杀伤(NK)细胞是用于过继癌症免疫治疗的重要效应细胞类型。类似于T细胞，NK细胞可修饰以表达嵌合抗原受体(CARs)，以增强抗肿瘤活性。CD19 CAR-T细胞在CD19阳性恶性肿瘤患者的成功应用，证明这种方法用于癌症免疫治疗(see e.g.Grupp et al,2013)的可行性。CAR-T细胞靶向多种不同的肿瘤抗原正在积极的进行临床开发(Kalos etal.,2013)。自然杀伤(NK)细胞的CAR NK细胞免疫治疗尝试较少，到目前为止，对于这种做法没有临床数据可用。NK细胞在癌症免疫监视中起重要作用，并且代表用于过继癌症免疫疗法的重要效应细胞类型。与T细胞相比，它们不需要事先活化和识别复合体MHC分子呈递的肽抗原。相反，通过编码的细胞表面受体偶联CD3ζ分子，在适当的刺激下，NK细胞可表现出杀伤活性。因此，在NK细胞中，包含CD3ζ-CARs元件容易连结到内源性的信号通路中，并触发细胞裂解活性。尽管取得了这些进展，CAR NK细胞临床开发经验仍然有限。
I期研究表明未修饰的NK-92细胞，其被应用之前辐照，已经证明临床应用的安全性。然而并未表现出明显抗肿瘤活性，可能是由未修改的NK-92细胞缺乏肿瘤特异性受体而无法识别肿瘤。
本发明人前期制备了PD-1 CAR-T细胞来治疗肿瘤，但PD-1 CAR-T细胞存在以下缺点：CAR-T细胞不同个体间存在移植物抗宿主反应(graft versus host reaction,GVHR)；需要个性定制且只能用于单一病人，造成时间长；病人T细胞状态不佳容易制备失败；不同病人来源的T细胞制备的CAR-T活性不一致导致药物效果不稳定等，上述问题亟待解决。
发明内容
本发明提供一种PD-1 CAR NK-92细胞及其制备方法与应用，解决不同病人来源的T细胞制备的CAR-T活性不一致导致药物效果不稳定的问题。
为了达到上述目的，本发明是通过以下技术方案实现的：
本发明提供一种PD-1 CAR NK-92细胞，所述PD-1 CAR NK-92细胞是在NK-92细胞内表达PD-1-CD8TM-4-1BB-CD3ζ融合蛋白，所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述PD-1蛋白的胞外段具有：
a)如SEQ ID NO：5所示的氨基酸序列，或
b)将a)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD8TM具有如SEQ ID NO：1所示的氨基酸序列；或者，是将SEQ ID NO：1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述4-1BB的氨基酸序列如SEQ ID NO：2所示；或者，是将SEQ ID NO：2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中的所述4-1BB能替换为CD28，所述CD28的分子序列如SEQ ID NO：3所示；或者，是将SEQ ID NO：3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD3ζ的氨基酸序列如SEQ ID NO：4所示；或者，是将SEQ ID NO：4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白的氨基酸序列如SEQ ID NO：6所示；或者，是将SEQ ID NO：6所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
本发明还提供一种编码融合蛋白PD-1-CD8TM-4-1BB-CD3ζ的基因，所述的融合蛋白PD-1-CD8TM-4-1BB-CD3ζ的基因序列如SEQ ID NO：7所示；或者，在严格条件下与SEQ ID NO：7所示的序列杂交且编码具有预防和/或治疗肿瘤症功能的相关蛋白的DNA分子；或者，与SEQ ID NO：7所示的序列具有至少90％以上或95％以上或98％以上同源性且编码具有预防和/或治疗肿瘤症功能的相关蛋白的DNA分子。
所述融合蛋白PD-1-CD8TM-4-1BB-CD3ζ中PD-1的基因序列如SEQ ID NO：8所示；或者，在严格条件下与SEQ ID NO：8所示的序列杂交且编码具有相应功能的相关蛋白的DNA分子；或者，与SEQ ID NO：8所示的序列具有至少90％以上或95％以上或98％以上同源性且编码具有相应功能的相关蛋白的DNA分子。
所述融合蛋白PD-1-CD8TM-4-1BB-CD3ζ中CD 8Hinge的基因序列如SEQ ID NO：9所示；或者，在严格条件下与SEQ ID NO：9所示的序列杂交且编码具有相应功能的相关蛋白的DNA分子；或者，与SEQ ID NO：9所示的序列具有至少90％以上或95％以上或98％以上同源性且编码具有相应功能的相关蛋白的DNA分子。
所述融合蛋白PD-1-CD8TM-4-1BB-CD3ζ中CD8TM的基因序列如SEQ ID NO：10所示；或者，在严格条件下与SEQ ID NO：10所示的序列杂交且编码具有相应功能的相关蛋白的DNA分子；或者，与SEQ ID NO：10所示的序列具有至少90％以上或95％以上或98％以上同源性且编码具有相应功能的相关蛋白的DNA分子。
所述融合蛋白PD-1-CD8TM-4-1BB-CD3ζ中4-1BB的基因序列如SEQ ID NO：11所示；或者，在严格条件下与SEQ ID NO：11所示的序列杂交且编码具有相应功能的相关蛋白的DNA分子；或者，与SEQ ID NO：11所示的序列具有至少90％以上或95％以上或98％以上同源性且编码具有相应功能的相关蛋白的DNA分子。
所述融合蛋白PD-1-CD8TM-4-1BB-CD3ζ中CD3ζ的基因序列如SEQ ID NO：12所示；或者，在严格条件下与SEQ ID NO：12所示的序列杂交且编码具有相应功能的相关蛋白的DNA分子；或者，与SEQ ID NO：12所示的序列具有至少90％以上或95％以上或98％以上同源性且编码具有相应功能的相关蛋白的DNA分子。
本发明还提供了一种与融合蛋白PD-1-CD8TM-4-1BB-CD3ζ相关的生物材料，包括含有本发明所述融合蛋白PD-1-CD8TM-4-1BB-CD3ζ编码基因的重组载体、表达盒、重组细胞、重组菌、重组病毒。优选的，本发明所述的重组载体为重组表达载体或重组克隆载体。
本发明所述的融合蛋白可人工合成，也可先合成其编码基因，再进行生物表达得到。
本发明还提供一种PD-1 CAR NK-92细胞的制备方法，所述PD-1 CAR NK-92细胞的制备方法包括步骤为：
(1)合成和扩增PD-1-CD8TM-4-1BB-CD3ζ融合蛋白基因，将所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白基因克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染293T细胞，包装和制备慢病毒；
(3)将NK-92细胞进行PD1抗体染色，分选PD1-CAR NK-92阳性细胞并扩大培养，得到PD-1 CAR NK-92细胞。
所述步骤(3)中将NK-92细胞密度调整至2-3×105/ml。
本发明还提供一种药物组合物，包括由PD-1-CD8TM-4-1BB-CD3ζ融合蛋白制备的PD-1 CAR NK-92细胞，以及药学上可接受的辅料。
本发明所述的药物组合物可以为片剂(包括糖衣片剂，膜包衣片剂，舌下片剂，口腔崩解片，口腔片剂等等)，丸剂，粉剂，颗粒剂，胶囊剂(包括软胶囊，微胶囊)，锭剂，糖浆剂，液体，乳剂，混悬剂，控制释放制剂(例如，瞬时释放制剂，缓释制剂，缓释微囊)，气雾剂，膜剂(例如，口服崩解膜剂，口腔粘膜-粘附膜剂)，注射剂(例如，皮下注射，静脉注射，肌内注射，腹膜内注射)，静脉滴注剂，透皮吸收制剂，软膏剂，洗剂，粘附制剂，栓剂(例如，直肠栓剂，阴道栓剂)，小药丸，鼻制剂，肺制剂(吸入剂)，眼睛滴剂等等，口服或胃肠外制剂(例如，静脉内，肌内，皮下，器官内，鼻内，皮内，滴注，脑内，直肠内等给药形式，给药至肿瘤的附近和直接给药至病变处)。优选的，所述的药物组合物为注射剂。
本发明所述的药学上可接受的辅料优选为药学上可接受的注射剂辅料，例如等渗的无菌盐溶液(磷酸二氢钠、磷酸氢二钠、氯化钠、氯化钾、氯化钙、氯化镁等，或上述盐的混合物)，或干燥的例如是冷冻干燥的组合物，其适当地通过加入无菌水或生理盐水形成可注射溶质。
本发明还提供一种由PD-1-CD8TM-4-1BB-CD3ζ融合蛋白制备的PD-1 CAR NK-92细胞在制备预防和/或治疗肿瘤药物中的应用。优选地，所述PD-1 CAR NK-92细胞在制备预防和/或治疗治疗高表达PDL-1分子的肿瘤药物中的应用。
本发明中术语“预防”或“治疗”包括治疗性或预防性处理或措施，目标是预防或减慢靶向的病理性状况或病症。如果根据本发明的方法接收治疗量的本发明所述融合蛋白后，对象表现出可观察和/或可测量的特定疾病一个或多个体征和症状的减少或消失，则该对象被成功“预防”或“治疗”。
本发明的优点和有益效果在于：本发明提供一种PD-1 CAR NK-92细胞，PD-1 CARNK-92细胞与CAR-T细胞相比，无需分离病人外周血单核细胞(PBMC)，无需特异活化T细胞和制备CAR-T细胞(该过程需要病人等待10天以上时间)，不需要个性定制且能用于多个病人，缩短了时间，PD-1 CAR-NK92细胞可以大批量制备培养，病人即来即用；另一方面，常规制备的CAR-T细胞，由于是分离病人的T细胞经病毒感染而制备的，T细胞不是同一个单克隆来源，而分选出的CAR-NK92细胞来源于同一个单克隆，性状和活性均一且稳定，便于大规模生产和质控；同时与NK92细胞相比，由于进行了PD-1 CAR载体导入，其杀伤活性特异，肿瘤治疗效果明显。
本发明的PD-1 CAR NK-92是将PD-1 CAR分子侵染NK92细胞株并经过流式筛选获得单一克隆细胞，将性状稳定杀伤活性高的CAR NK92单克隆细胞株培养并扩增获得。该细胞可以用于大规模生产制备，可以用于不同病人且不会产生GVHR排斥。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1是慢病毒质粒载体PRRLSIN-PD-1图。
图2是PD-1 CAR NK-92流式检测图谱。
图3是CCK-8方法检测PD-1 CAR-T和PD-1 CAR NK-92细胞对H1299肺癌细胞的杀伤效果图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
实施例一：
慢病毒表达载体制备
基因合成PD-1-CD8TM-4-1BB-CD3ζ融合基因序列，通过酶切转化连接到PRRSLIN载体中，基因上游为EP-1α启动子。载体转化Stbl3大肠杆菌菌株，氨苄青霉素筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，获得pRRSLIN-PD-1慢病毒转染载体，载体构建图见图1。
实施例二：
慢病毒制备
(1)转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。
(2)准备溶液A和溶液B
溶液A：6.25ml 2×HEPES buffer缓冲液(5个大皿一起包装的量，效果最好)。
溶液B：分以加入以下质粒的混合物：112.5ug pRRSLIN-PD-1(target plasmid)；39.5ug pMD2.G(VSV-G envelop)；73ug pCMVR8.74(gag,pol,tat,rev)；625μl 2M钙离子溶液。溶液B总体积：6.25ml。
充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液B，静置5-15分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。(不要旋转培养皿)放置于培养箱中培养16-18小时。更换新鲜培养基，继续培养，分别在48小时和72小时后收集含病毒的上清液。500g，25℃离心10分钟。PES膜(0.45μm)过滤。以70％乙醇消毒贝克曼库尔特Ultra-clear SW28centrifuge tubes，并置于紫外灯下消毒30分钟。将已过滤的含慢病毒的上清液转移至离心管中。在离心管底部小心铺上一层20％蔗糖(每8ml上清液加1ml蔗糖)。以PBS平衡离心管，25000rpm(82,700g)，4℃离心2小时。小心取出离心管，倒掉上清液，倒置离心管去掉残余液体。加入100μl PBS，密封离心管，在4℃放置2小时，每20分钟轻轻涡旋一次，500g离心1分钟(25℃)，收集病毒上清。冰上冷却后，置于-80℃保存。
实施例三：
PD-1 CAR-NK-92细胞制备
将NK-92细胞密度调整至2-3×105/ml，按体积比(V/V)virus vector：cellMedium＝1:5-10比例添加virus vector，同时添加polybrene 8ug/ml。4h之后，补加等量的新鲜的完全培养基(完全培养基配方参见ATCC说明)将细胞密度调整至1×105/ml继续培养。次日，将所有的细胞离心，加入新鲜的培养基，继续培养。每隔1-2天进行补液，使维持细胞密度在2-3×105/ml。72h后进行PD1抗体染色，同时流式分选PD1-CAR NK-92阳性细胞并扩大培养。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。
如图2A-F:流式检测结果；A图流式检测进样的样品为普通NK92细胞，区域圈出的为活细胞，该群细胞用于B图和C图的分析。D图流式检测进样的样品为PD-1 CAR NK-92细胞，区域圈出的为活细胞，该群细胞用于E图和F图的分析。B/E图:该图分析的细胞为用anti-PD1抗体染色，该抗体偶联PE荧光分子，图中横坐标PE-H数值越大，表明细胞仪检测到被抗体染色获得的阳性细胞数越多，即：表达PD-1分子的细胞数越多。由于B图分析的是未经转染的NK92细胞，所以以此做对照画门，方框内表示PD-1分子染色阳性，方框左侧区域表示PD-1分子检测阴性。E图为转染筛选后的PD-1 CAR NK92细胞检测，检测发现其细胞分布在方框内，表示细胞表面表达PD-1分子，证明PD-1 NK-92细胞制备成功；C图为未转染NK92，不用抗体染色，用于对照。F图为转染制备的PD-1 CAR NK-92细胞用anti-CD3抗体(鉴定是否为T细胞)和anti CD56抗体(用于鉴定是否为NK细胞)检测，上述抗体分别偶联APC和FITC荧光分子。横坐标和纵坐标数值越大表明CD56和CD3分子表达越高，C图为对照，未用抗体染色，则将其细胞分布的位置用于设置对照，即左下区域认为CD3和CD56分子表达阴性。F图检测显示，PD-1 CAR NK92细胞与对照细胞相比，CD3分子无表达，染色后为阴性。而CD56抗体染色后，FTIC数值升高，CD56分子有表达为阳性，证明筛选获得NK-92细胞未发生变化，依然是NK细胞。
实施例四：PD-1 CAR-NK细胞体外活性检测
CCK-8方法检测，即检测PD-1 CAR-T和PD-1 CAR NK-92细胞对H1299肺癌细胞的杀伤效应。
(1)在24孔板中配制1ml H1299细胞悬液(2X10^4个/孔)。将培养板在培养箱预培养12h。
(2)弃去24孔板的培养上清，每孔加入1ml效应细胞，效应细胞与靶细胞数目的比例为1:1。培养基对照孔只加1ml培养基，每个实验置三个复孔。效应细胞与靶细胞共孵育4小时。
(3)每孔加入100ul CCK-8溶液，将培养板在培养箱内孵育2h。
(4)用酶标仪测定在450nm处的吸光度。
(5)杀伤率＝(As-Ab)/(Ac-Ab)X100％
As：试验孔(含有H1299细胞的培养基、CCK-8、CAR-T或CAR-NK)
Ac：对照孔(含有H1299细胞的培养基、CCK-8)
Ab：空白对照(不含细胞和CAR-T或CAR-NK的培养基、CCK-8)
如图3所示实验结果显示，制备的PD-1 CAR NK-92细胞能够显著杀伤高表达PDL1的H1299靶细胞株，并且其杀伤效果好于PD-1 CAR-T细胞。
本发明的PD-1 CAR NK-92是将PD-1 CAR分子侵染NK92细胞株并经过流式筛选获得单一克隆细胞，将性状稳定杀伤活性高的CAR NK92单克隆细胞株培养并扩增获得。该细胞可以用于大规模生产制备，可以用于不同病人且不会产生GVHR排斥。PD-1 CAR NK-92细胞与CAR-T细胞相比，无需分离病人外周血单核细胞(PBMC)，无需特异活化T细胞和制备CAR-T细胞(该过程需要病人等待10天以上时间)，不需要个性定制且能用于多个病人，缩短了时间，PD-1 CAR-NK92细胞可以大批量制备培养，病人即来即用；另一方面，常规制备的CAR-T细胞，由于是分离病人的T细胞经病毒感染而制备的，T细胞不是同一个单克隆来源，而分选出的CAR-NK92细胞来源于同一个单克隆，性状和活性均一且稳定，便于大规模生产和质控；同时与NK92细胞相比，由于进行了PD-1 CAR载体导入，其杀伤活性特异，肿瘤治疗效果明显。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360
tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420
gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480
aggccagccg gccagttcca aaccctggtg accacgacgc cagcgccgcg accaccaaca 540
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 600
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 660
cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaac 720
cacaggaaca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 780
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 840
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 900
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 960
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1020
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1080
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1140
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaa 1194
<210> 8
<211> 510
<212> DNA
<213> 人工序列
<400> 8
atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60
ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120
ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180
gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240
gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300
cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360
tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420
gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480
aggccagccg gccagttcca aaccctggtg 510
<210> 9
<211> 135
<212> DNA
<213> 人工序列
<400> 9
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 10
<211> 84
<212> DNA
<213> 人工序列
<400> 10
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gcaaccacag gaac 84
<210> 11
<211> 126
<212> DNA
<213> 人工序列
<400> 11
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 12
<211> 339
<212> DNA
<213> 人工序列
<400> 12
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339
Claims (14)
1.一种PD-1 CAR NK-92细胞，其特征在于，所述PD-1 CAR NK-92细胞是在NK-92细胞内表达PD-1-CD8TM-4-1BB-CD3ζ融合蛋白，所述NK-92细胞来源于
CRL2407TM；所述PD-1蛋白的胞外段的氨基酸序列如SEQ ID NO：5所示。
2.根据权利要求1所述的PD-1 CAR NK-92细胞，其特征在于：所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD8TM的氨基酸序列如SEQ ID NO：1所示。
3.根据权利要求1所述的PD-1 CAR NK-92细胞，其特征在于：所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述4-1BB的氨基酸序列如SEQ ID NO：2所示。
4.根据权利要求1所述的PD-1 CAR NK-92细胞，其特征在于：所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中的所述4-1BB能替换为CD28，所述CD28的分子序列如SEQ ID NO：3所示。
5.根据权利要求1所述的PD-1 CAR NK-92细胞，其特征在于：所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白中所述CD3ζ的氨基酸序列如SEQ ID NO：4所示。
6.根据权利要求1所述的PD-1 CAR NK-92细胞，其特征在于：所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白的氨基酸序列如SEQ ID NO：6所示。
7.一种编码融合蛋白PD-1-CD8TM-4-1BB-CD3ζ的基因，其特征在于，所述的融合蛋白PD-1-CD8TM-4-1BB-CD3ζ的基因序列如SEQ ID NO：7所示。
8.包含权利要求7所述的编码融合蛋白PD-1-CD8TM-4-1BB-CD3ζ的基因的重组载体、表达盒、重组细胞、重组菌或重组病毒。
9.一种权利要求1所述的PD-1 CAR NK-92细胞的制备方法，其特征在于，所述PD-1 CARNK-92细胞的制备方法包括步骤为：
(1)合成和扩增PD-1-CD8TM-4-1BB-CD3ζ融合蛋白基因，将所述PD-1-CD8TM-4-1BB-CD3ζ融合蛋白基因克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染293T细胞，包装和制备慢病毒；
(3)将NK-92细胞进行PD1抗体染色，分选PD1-CAR NK-92阳性细胞并扩大培养，得到PD-1 CAR NK-92细胞。
10.根据权利要求9所述的PD-1 CAR NK-92细胞的制备方法，其特征在于，所述步骤(3)中将NK-92细胞密度调整至2-3×105/ml。
11.一种药物组合物，其特征在于，包括表达PD-1-CD8TM-4-1BB-CD3ζ融合蛋白的权利要求1所述的PD-1 CAR NK-92细胞，以及药学上可接受的辅料。
12.根据权利要求11所述的一种药物组合物，其特征在于，所述药物组合物为注射剂。
13.一种表达PD-1-CD8TM-4-1BB-CD3ζ融合蛋白的权利要求1所述的PD-1 CAR NK-92细胞在制备预防和/或治疗肿瘤药物中的应用。
14.根据权利要求13所述的表达PD-1-CD8TM-4-1BB-CD3ζ融合蛋白的权利要求1所述的PD-1 CAR NK-92细胞在制备预防和/或治疗肿瘤药物中的应用，其特征在于，所述PD-1 CARNK-92细胞在制备预防和/或治疗治疗高表达PDL-1分子的肿瘤药物中的应用。
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| CN201610944672.7A CN107988164B (zh) | 2016-10-26 | 2016-10-26 | 一种pd-1 car nk-92细胞及其制备方法与应用 |
| PCT/CN2016/104493 WO2018076391A1 (zh) | 2016-10-26 | 2016-11-04 | 一种pd-1 car nk-92细胞及其制备方法与应用 |
| US16/389,954 US11331345B2 (en) | 2016-10-26 | 2019-04-21 | PD-1 CAR NK-92 cell and preparation method and use thereof |
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| IL321548A (en) | 2018-05-22 | 2025-08-01 | Immunitybio Inc | Recombinant NK cells expressing a chimeric antigen receptor (CAR) for epsilon-FC and uses thereof |
| CN109939127A (zh) * | 2018-06-13 | 2019-06-28 | 阿思科力(苏州)生物科技有限公司 | Nk细胞的应用及包括该nk细胞的药物组合物及其应用 |
| IL314189A (en) | 2018-08-01 | 2024-09-01 | Immunitybio Inc | A QUADRICISTRONIC SYSTEM COMPRISING A HOMING RECEPTOR OR A CYTOKINE, AND CHIMERIC ANTIGEN RECEPTOR FOR GENETIC MODIFICATION OF IMMUNOTHERAPIES- Expedited Examination |
| KR102533540B1 (ko) * | 2018-10-31 | 2023-05-17 | 난트퀘스트, 인크. | Pd-l1 키메라 항원 수용체-발현 nk 세포에 의한 pd-l1-양성 악성종양의 제거(elimination of pd-l1-positive malignancies by pd-l1 chimeric antigen receptor-expressing nk cells) |
| US12109238B2 (en) | 2018-11-06 | 2024-10-08 | Immunitybio, Inc. | Chimeric antigen receptor-modified NK-92 cells |
| AU2019375375B2 (en) | 2018-11-06 | 2022-12-01 | Immunitybio, Inc. | Chimeric antigen receptor-modified NK-92 cells |
| AU2019459423B2 (en) | 2019-08-01 | 2023-06-01 | Immunitybio, Inc. | Anti-B7-H4 chimeric antigen receptor-modified NK-92 cells |
| US11230699B2 (en) | 2020-01-28 | 2022-01-25 | Immunitybio, Inc. | Chimeric antigen receptor-modified NK-92 cells targeting EGFR super-family receptors |
| CN112301059B (zh) * | 2020-09-23 | 2023-04-25 | 杭州美中疾病基因研究院有限公司 | 一种基于复制缺陷性重组慢病毒的car-nk转基因载体及其构建方法和应用 |
| CN114807042B (zh) * | 2021-01-22 | 2024-09-03 | 南京助天中科科技发展有限公司 | 一种嵌合抗原受体改造的nk细胞及其制备方法与应用 |
| CN118359727A (zh) * | 2023-01-17 | 2024-07-19 | 上海以慈生物科技有限公司 | 突变的pd1胞外域片段及含有该片段的car和nk细胞 |
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| WO2015193411A1 (en) * | 2014-06-18 | 2015-12-23 | Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus | Car-expressing nk-92 cells as cell therapeutic agents |
| CN105246504A (zh) * | 2013-03-15 | 2016-01-13 | 纪念斯隆-凯特琳癌症中心 | 用于免疫疗法的组合物和方法 |
| CN105907719A (zh) * | 2016-04-18 | 2016-08-31 | 李华顺 | Anti ROBO1 CAR-T细胞及其制备和应用 |
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| WO2015090229A1 (en) * | 2013-12-20 | 2015-06-25 | Novartis Ag | Regulatable chimeric antigen receptor |
| CN106163547A (zh) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | 使用嵌合抗原受体治疗癌症 |
| CN105132445B (zh) * | 2015-10-23 | 2018-10-02 | 马健颖 | 一种特异识别肿瘤细胞的受体蛋白、t淋巴细胞及nk细胞 |
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| CN105246504A (zh) * | 2013-03-15 | 2016-01-13 | 纪念斯隆-凯特琳癌症中心 | 用于免疫疗法的组合物和方法 |
| WO2015193411A1 (en) * | 2014-06-18 | 2015-12-23 | Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus | Car-expressing nk-92 cells as cell therapeutic agents |
| CN105907719A (zh) * | 2016-04-18 | 2016-08-31 | 李华顺 | Anti ROBO1 CAR-T细胞及其制备和应用 |
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| US20190240258A1 (en) | 2019-08-08 |
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