CN116751310B - 靶向cd19和gprc5d配体的嵌合抗原受体及其应用 - Google Patents
靶向cd19和gprc5d配体的嵌合抗原受体及其应用 Download PDFInfo
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Abstract
本发明实施例公开了一种靶向CD19和GPRC5D配体的嵌合抗原受体及其应用。本发明提供的GPRC5D‑CD19‑CAR‑T双特异性CAR‑T能同时识别GPRC5D和CD19抗原，相比单靶点CAR‑T，双特异性CAR‑T的杀伤能力更强，能够有效清除CD19和GPRC5D配体单/双阳性肿瘤细胞，减少抗原逃逸，在多发性骨髓瘤的治疗中具有极大的临床转化价值。
Description
技术领域
本发明实施例涉及肿瘤药物技术领域，具体涉及一种靶向CD19和GPRC5D配体的嵌合抗原受体及其应用。
背景技术
多发性骨髓瘤(MM)是一种克隆性浆细胞异常增殖的恶性疾病，发病率正逐年增高。几十年来，多发性骨髓瘤的治疗出现了大量新进展，包括蛋白酶体抑制剂、免疫调节药物、单克隆抗体等，显著延长了患者生存，但目前尚无法根治，几乎所有的患者最终会进入疾病的复发难治阶段。针对这部分患者，目前仍存在大量未被满足的治疗需求。
嵌合抗原受体T(CAR-T)细胞是近年来新兴的细胞免疫治疗手段。采用患者本身的T淋巴细胞，在体外通过病毒转染的方法，将针对肿瘤靶点的CAR分子整合到T细胞中从而制备CAR-T细胞。靶向B细胞成熟抗原(BCMA)的CAR-T细胞在难治复发多发性骨髓瘤中取得了重要突破，为这部分患者带来了生存的新希望，目前国外已有两款BCMACAR-T细胞产品获批上市。黄河教授团队于2021年发表于Clinicalcancerresearch的论文总结了BCMA CAR-T细胞治疗难治复发多发性骨髓瘤的临床数据，总体反应率98.3％，完全缓解率70.3％。但仍有约60％的患者面临疾病的复发，复发后预后极差，缺乏有效的治疗手段。因此亟需寻找有效的免疫治疗新靶点，这也是目前全球该领域的研究热点。
发明内容
本发明是基于发明人对以下事实和问题的发现和认识做出的：
GPRC5D(Gprotein-coupledreceptorclassCgroup5memberD)是G蛋白偶联受体家族C组5成员D，其是一种G蛋白偶联孤儿受体，并且是7次跨膜的膜蛋白。在CCLE数据库中分析多发性骨髓瘤细胞中高表达GPRC5D(见图1)。研究表明，GPRC5D主要是在浆细胞中表达，包括大部分来自MM患者的恶性浆细胞。在MM患者中，GPRC5DmRNA的表达量与浆细胞负荷和基因畸变相关，而在正常组织中，其仅在表达硬角蛋白的细胞如毛囊中有表达。GPRC5D的过表达与多发性骨髓瘤患者的不良预后和肿瘤负荷有关，这种关联使其成为治疗多发性骨髓瘤的潜在候选靶点。此外，GPRC5D与BCMA独立表达，不具有相关性；联合靶向BCMA和GPRC5D已被证明可以防止BCMA逃逸的复发，可以发挥互补效应或者开发双抗、双靶点CAR-T等。
一小部分MM细胞克隆可能表达CD19，这被认为是低分化的MM细胞或骨髓瘤样干细胞。针对该亚组的疗法可能与传统的骨髓瘤疗法具有协同作用。抗CD19CART细胞联合自体干细胞移植(ASCT)在R/RMM患者中也显示出潜在活性。此外，通过应用超分辨率显微镜，CD19在大多数患者的一小部分骨髓瘤细胞上以超低密度表达，每个骨髓瘤细胞的超低CD19分子可以触发抗CD19CART细胞的消除。
鉴于CD19和GPRC5D配体参与到多发性骨髓瘤的发生及进展过程，都可作为治疗多种癌症的良好靶点。为此，本发明构建了可同时靶向CD19和GPRC5D配体的双特异性CAR结构，经改造后的抗CD19和GPRC5D配体双靶点CAR-T细胞可专一性、高效杀灭肿瘤细胞，可以对肿瘤更有效和全面的覆盖，以消除绝大部分多发性骨髓瘤细胞，降低肿瘤复发的风险。
根据本发明实施例第一方面，本发明提供一种靶向CD19和GPRC5D配体的嵌合抗原受体，所述嵌合抗原受体包括：抗原结合结构域、铰链区、跨膜结构域和胞内结构域，其中，所述抗原结合结构域包括抗CD19scFv和抗GPRC5DscFv，所述抗CD19scFv氨基酸序列如SEQID NO:1所示，所述抗GPRC5DscFv氨基酸序列如SEQ ID NO:2所示。
进一步地，所述铰链区为CD8铰链区；所述跨膜结构域为CD8跨膜结构域；所述胞内结构域包括4-1BB胞内区和CD3ζ胞内区。
进一步地，所述嵌合抗原受体的氨基酸序列如SEQ ID NO:3所示。
根据本发明实施例第二方面，本发明提供用于编码如上所述的嵌合抗原受体的核酸分子。
进一步地，所述核酸分子包括：编码抗CD19scFv的如SEQ ID NO:4所示的核苷酸序列，和；编码抗GPRC5DscFv的如SEQ ID NO:5所示的核苷酸序列。
根据本发明实施例第三方面，本发明提供一种重组表达载体，含有如上所述的核酸分子。
进一步地，其核苷酸序列如SEQ ID NO:6所示。
根据本发明实施例第四方面，本发明提供一种靶向CD19和GPRC5D配体的嵌合抗原受体T细胞，其表达有如上所述的嵌合抗原受体。
根据本发明实施例第五方面，本发明提供如上所述的嵌合抗原受体、如上所述的核酸分子、如上所述的重组表达载体，或如上所述的嵌合抗原受体T细胞在制备治疗多发性骨髓瘤药物中的应用。
本发明实施例具有以下优点：
本发明提供的GPRC5D-CD19-CAR-T双特异性CAR-T能同时识别GPRC5D和CD19抗原，相比单靶点CAR-T，双特异性CAR-T的杀伤能力更强，能够有效清除CD19和GPRC5D配体单/双阳性肿瘤细胞，减少抗原逃逸，在多发性骨髓瘤的治疗中具有极大的临床转化价值。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案，下面将对实施方式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示例性的，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图引伸获得其它的实施附图。
图1为本发明提供的在CCLE数据库中分析多发性骨髓瘤细胞中GPRC5D配体的表达情况；
图2为本发明提供的CD19-GPRC5D-CAR的结构示意图；
图3为本发明提供的CD19-CAR的结构示意图；
图4为本发明提供的GPRC5D-CAR的结构示意图；
图5为本发明提供的慢病毒CD19-GPRC5D-CAR载体的酶切片段的电泳鉴定图；
图6为本发明提供的慢病毒CD19-CAR载体的酶切片段的电泳鉴定图；
图7为本发明提供的慢病毒GPRC5D-CAR载体的酶切片段的电泳鉴定图；
图8为本发明提供的CD19-GPRC5D-CAR的表达情况；
图9为本发明提供的CAR-T表达阳性率检测结果；
图10为本发明提供的CAR-T对Raji-luc的杀伤率检测结果；
图11为本发明提供的CAR-T对MM1.S的杀伤率检测结果。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1基因合成和慢病毒载体的构建
首先基因合成抗CD19和GPRC5D双靶点嵌合抗原受体的编码基因，依次包括：CD8L,CD19VH-VL,(G4S)3,GPRC5DVH-VL,CD8HT,4-1BB,CD3ζ(结构见图2)。在编码基因的C端和N端分别添加限制性内切酶Tth111i酶切位点及其保护碱基和限制性内切酶Msci酶切位点及其保护碱基，利用限制性内切酶Tth111i和Msci对编码基因进行双酶切，琼脂凝胶电泳回收获得含有粘性末端的酶切产物，连接入同样经Tth111i和Msci双酶切的线性化pUC质粒(含黏性末端)中，连接反应在T4DNA聚合酶(Invitrogent公司)的参与下进行，将CAR编码基因连接到MND启动子的下游，得到含有靶向GPRC5D和CD19双靶点CAR编码基因的慢病毒CD19-GPRC5D-CAR载体，酶切片段电泳鉴定结果见图5，泳道6为CD19-GPRC5D-CAR载体，所构建的CD19-GPRC5D-CAR载体全长核苷酸序列如SEQ ID NO:6所示。
同时构建抗原结合结构域分别为抗CD19scFv的CAR和抗GPRC5DscFv的对照CAR：CD19-CAR(结构见图3)和GPRC5D-CAR(结构见图4)，并构建相应的慢病毒CD19-CAR载体和GPRC5D-CAR载体，酶切电泳鉴定结果分别见图6-7，图6中泳道5为CD19-CAR载体，图7中泳道94为GPRC5D-CAR载体。
实施例2CAR-T细胞的制备
(1)分离PBMC
采集50mL外周血；在超净工作台中向2支50mL灭菌离心管中分别加入15mL淋巴细胞分离液，将25～30mL外周血缓慢注入含有淋巴细胞分离液的离心管中，按照淋巴细胞分离液的使用说明，将离心管在700g下室温离心20min；离心后，血液分为4层，由血浆(上层)、血浆和分离液之间的单个核细胞(第2层)、分离液(第3层)和红细胞(底层)构成，用吸管将第2层单个核细胞收集至新的离心管中，加入20mLPBS稀释细胞悬液，500g离心10min；移除上清液，加入20mLPBS稀释细胞悬液，混匀，取20μL细胞悬液计数，余下悬液500g离心10min备用。
(2)T细胞增殖
加入10mLT激培养基重悬PBMC，T激活培养基包含GT-T551H3培养基、2mL灭活自体血浆、适宜浓度IL-2等；取20μL细胞悬液计数；将上述细胞悬液转入细胞培养瓶中，用T激活培养基调整细胞密度至(2～3)×106个/mL，37℃、5％CO2培养箱中培养3～4天。
(3)慢病毒包装
分别对实施例1中的慢病毒载体进行慢病毒包装，采用四质粒系统，具体步骤如下：四质粒系统分别表达慢病毒载体包装所需的gag/pol、Rev、包膜质粒及实施例1构建的CD19-GPRC5D-CAR载体、CD19-CAR载体和GPRC5D-CAR载体：将质粒进行瞬时转染293T细胞，DNA含量为2μg/mL；将上述质粒与PEI转染试剂混合，加入至一定体积的无血清的DMEM中，混匀后放置15分钟，将上述混合液加入至铺有293T细胞的T75培养瓶中，轻轻混匀，于37℃、5％CO2细胞培养箱培养6h；6h后更换新鲜培养基，继续进行培养，并且加入10mM的丁酸钠溶液，72小时后收集慢病毒的培养上清进行纯化并检测。
(4)慢病毒感染和扩大培养
T细胞激活3～4天后，将包含CAR基因的病毒过夜感染；感染后更换T细胞扩增培养基(含10％血清，500IU/mLIL-2等)，调整细胞密度为(1～2)×106个/mL；每2～3天观察细胞生长状态；扩大培养7天后，将培养瓶中的细胞悬液移入培养袋，添加T细胞扩增培养基在37℃、5％CO2培养箱中继续培养；培养至第10天，收集细胞进行检测。
实施例3流式检测抗CD19、GPRC5D双靶点CAR-T表达
采用流式细胞仪检测制备的抗CD19-GPRC5D-CAR分子的表达，利用APC-anti-CD3抗体标记T细胞群，然后用FITC-ProteinL(AcroBiosystems公司)检测CAR表达阳性率。结果显示：慢病毒感染继续培养第3天和第4天的抗CD19-GPRC5D-CAR-T表达率分别为63.2％和80.2％(图8)。
实施例4CAR的表达及其功能评估
采用流式细胞仪检测CAR-T细胞表面CAR分子的表达及其与对应抗原蛋白的结合能力，利用APC-anti-CD3抗体标记T细胞群，然后用CD19蛋白和GPRC5D蛋白(ACROBiosystems公司)检测CAR表达阳性率。结果显示：CD19-GPRC5D双特异性CAR-T(s-CAR-T)识别CD19和GPRC5D抗原蛋白的能力，其阳性率分别为14.73％和39.73％，表明双特异性CAR-T能有效识别两种抗原(图9)。
用过表达荧光素酶基因的人CD19阳性Raji作为靶细胞，进行杀伤试验。Raji铺板后，按1:1的效靶比共孵育6小时后，加入荧光素酶底物，在多功能酶标仪上检测细胞中的荧光素酶与底物反应的发光信号。结果显示，GPRC5D-CD19-CAR-T细胞和CD19-CAR-T细胞都能杀伤Raji，在E:T＝4:1时，杀伤率分别为86和79％，说明GPRC5D-CD19-CAR-T更优的杀肿瘤效果。
用过表达荧光素酶基因的人多发性骨髓瘤细胞MM1.S作为靶细胞，进行杀伤试验。MM1.S铺板后，按1:1的效靶比共孵育6小时后，加入荧光素酶底物，在多功能酶标仪上检测细胞中的荧光素酶与底物反应的发光信号。结果显示，GPRC5D-CAR-T细胞和GPRC5D-CD19-CAR-T细胞都能杀伤MM1.S，在E:T＝4：1时候的杀伤率分别为87.333％和96％。表明双特异性CAR-T比单靶点CAR-T对MM1.S的杀伤能力更强(图11)。
综上所述，GPRC5D-CD19-CAR-T双特异性CAR-T能同时识别GPRC5D和CD19抗原，相比单靶点CAR-T，双特异性CAR-T的杀伤能力更强。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
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Claims (7)
1.一种靶向CD19和GPRC5D配体的嵌合抗原受体，其特征在于，所述嵌合抗原受体包括：抗原结合结构域、铰链区、跨膜结构域和胞内结构域，其中，所述抗原结合结构域包括抗CD19 scFv和抗GPRC5D scFv，所述抗CD19scFv氨基酸序列如SEQ ID NO:1所示，所述抗GPRC5D scFv氨基酸序列如SEQ ID NO:2所示；所述嵌合抗原受体的氨基酸序列如SEQ IDNO:3所示。
2.用于编码如权利要求1所述的嵌合抗原受体的核酸分子。
3.根据权利要求2所述的核酸分子，其特征在于，所述核酸分子包括：
编码抗CD19 scFv的如SEQ ID NO:4所示的核苷酸序列，和；
编码抗GPRC5D scFv的如SEQ ID NO:5所示的核苷酸序列。
4.一种重组表达载体，其特征在于，含有权利要求2或3所述的核酸分子。
5.根据权利要求4所述的重组表达载体，其特征在于，其核苷酸序列如SEQ ID NO:6所示。
6.一种靶向CD19和GPRC5D配体的嵌合抗原受体T细胞，其特征在于，其表达有如权利要求1所述的嵌合抗原受体。
7.权利要求1所述的嵌合抗原受体、权利要求2所述的核酸分子、权利要求4所述的重组表达载体，或权利要求6所述的嵌合抗原受体T细胞在制备治疗多发性骨髓瘤药物中的应用。
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