CN118027194A - 靶向cd123的全人源抗体及其应用 - Google Patents
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Abstract
本发明提供的CD123嵌合抗原受体中的scFv是全新的人源抗体序列,避免鼠源抗体的免疫原性;本发明提供的CD123嵌合抗原受体与肿瘤细胞上的CD123结合后,显示出了明显的抗肿瘤活性,其中的CD123scFv是对人CD123抗原具有高亲和力的全人源化抗体。
Description
技术领域
本发明属于细胞工程技术领域,具体涉及CD123抗体及其应用。
背景技术
过继性免疫细胞治疗(adoptive cell therapy,ACT)是指从肿瘤患者分离免疫活性细胞,在体外进行扩增与功能鉴定后,然后再回输给患者,从而直接杀伤肿瘤或激发机体的免疫应答杀伤肿瘤细胞。
免疫治疗(Immunotherapy)是继手术、放疗、化疗之后的第四种肿瘤治疗方法。嵌合抗原受体(chimeric antigen receptor,CAR)T细胞技术是新近迅速发展的一种细胞免疫治疗技术,CAR是一个人工合成的融合受体,在结构上包括胞外抗原结合区、跨膜区、胞内信号转导区与共刺激信号区。胞外区为识别肿瘤相关抗原的单克隆抗体序列(singlechain variable fragment,scFv),跨膜区连接胞外区与胞内区,常用的跨膜区分子选自CD3、CD4、CD8与CD28等基因的跨膜区域及其变体。胞内区具有免疫受体酪氨酸活化基序(ITAM),最常用的是T细胞受体TCR/CD3ζ链和免疫球蛋白FC受体FCεRIγ,主要负责信号转导功能;人们通过分子克隆方法将上述元件在体外进行重组,形成重组质粒,通过病毒载体、电穿孔等手段将重组质粒转导入T细胞,继之在体外培养扩增,最后回输到病人体内,起到治疗肿瘤的目的,这种经过基因修饰与改造的T细胞即称为CAR-T细胞。CAR-T细胞通过scFv段识别并结合肿瘤抗原,再通过CD3ζ链和共刺激信号区激活T细胞,这样改造的CAR-T细胞识别、杀伤肿瘤细胞就具有了两大特点:①靶向性;②克服了MHC限制性,即不再依赖MHC,因此突破了肿瘤细胞通过下调MHC的表达而发生免疫逃逸这一难题。
除了T细胞,NK细胞在肿瘤治疗方面也扮演着重要的角色。将CAR结构通过病毒载体、电穿孔等手段将重组基因转导入NK细胞后也可以形成相应的CAR-NK细胞,CAR-NK细胞不受HLA配型的限制,可以实现同种异体的免疫过继治疗。此外,NK细胞来源广泛,可来源于造血干细胞、外周血、脐带血、诱导多能干细胞等等,因此CAR-NK也可以作为免疫治疗的重要方式之一。
急性髓细胞白血病(Acute Myeloid Leukemia,AML)是一种髓系造血干/祖细胞恶性疾病,长期以来,一直采用蒽环类和阿糖胞苷化疗治疗轻度患者,使用造血干细胞转移来治疗中、高危患者,其中,老年患者(≥60岁)和临床表现不佳的患者死亡率较高,5年总生存率约为10%。由于急性髓细胞白血病的异质性,导致目前对于AML患者的治疗仍是一种挑战。近年来,研究人员发现,以CD33、CD123及CLEC12A为靶点的免疫靶向疗法来治疗AML效果显著。
CD123是异二聚体白细胞介素-3受体(IL-3R)的亚基,IL-3R是β共同受体家族成员。这种膜受体家族在调节造血细胞的生长、增殖、存活和分化中发挥重要作用,以及免疫和炎症反应。CD123在各种血液系统恶性肿瘤中广泛表达。目前,很多单抗药物也将CD123作为靶抗原,因此开发一种CD123抗体应用于过继性免疫细胞治疗中也成为一个研究热点。
发明内容
本发明提供一种CD123抗体以及靶向CD123的嵌合抗原受体。
具体的,所述CD123抗体为抗CD123的scFv,所述抗CD123的scFv包含重链可变区和轻链可变区,所述轻链可变区包含L-CDR1、L-CDR2和L-CDR3,所述重链可变区包含H-CDR1、H-CDR2和H-CDR3;所述L-CDR1、L-CDR2和L-CDR3分别为如下序列QSVSSN、GAS和QQRSNWPPALT;所述H-CDR1、H-CDR2和H-CDR3分别为如下序列:GYSFTSYW、IYPGDSDT和ARIRFDKEQLFYYYYGMDV。
进一步的,所述抗CD123的scFv包含如Sequence NO.1所示的氨基酸序列或其功能性变体。
本发明提供的嵌合抗原受体,所述嵌合抗原受体包含如权利要求1或2所述的抗CD123的scFv。
进一步的,所述嵌合抗原受体包含胞外结构域、铰链区、跨膜区和胞内信号域。
所述铰链区来源于CD28、CD3、CD40、4-1BB、OX40、CD84、CD166、CD8α、CD8β、ICOS、ICAM-1、CTLA-4、CD27、CD40、NKGD2、CD7、IgG1、IgG4或免疫球蛋白中的CH2/CH3结构域。所述CD8α的氨基酸序列如Sequence NO.3所示或其功能性变体,上述其余可作为铰链的蛋白的铰链区的序列采用可以采用能在NCBI查询到的序列。其中IgG4铰链的核苷酸序列如下:
gcaccacctcgggccagcgccctgcctgcaccacccaccggctccgccctgccagaccctcagacagcatctgccctgccagatcctccagcagcaagcgccctgccc;其中CD7铰链:
gcaccacctcgggccagcgccctgcctgcaccacccaccggctccgccctgccagaccctcagacagcatctgccctgccagatcctccagcagcaagcgccctgccc。
进一步的,所述跨膜区来源于CD2、CD3D、CD3E、CD3G、CD3ζ、CD4、CD8α、CD8β、CD16、CD27、CD28、CD28H、CD40、CD84、CD166、4-1BB、OX40、ICOS、ICAM-1、CTLA4、PD1、LAG3、2B4、BTLA、DNAM1、DAP10、DAP12、FcERIγ、IL7、IL12、IL15、KIR2DL4、KIR2DS1、KIR2DS2、NKp30、NKp44,、NKp46、NKG2C、NKG2D、CS1、或T细胞受体多肽SIRPβ1或NKp44的跨膜区,其中CD8α、SIRPβ1、NKp44氨基酸序列分别如Sequence NO.4、Sequence NO.5和Sequence NO.6所示或其功能性变体,其余可作为跨膜区的蛋白跨膜区的序列可以采用能在公开数据库,如NCBI、ENA、DDBJ上可查询到的序列。
进一步的,所述胞内信号域来源于CD28、OX40、CD27、DAP10、CD3γ、FcεRI、CD2、CD16、TCRζ、FcRβ、CD30、CD40、ICOS、LFA-1、IL-2受体、Fcγ受体、KIRDS2、SLAMF7、NKp80(KLRF1)、信号传导淋巴细胞活化分子(SLAM蛋白)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、DAP12、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、LFA-1(CD11a/CD18)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、CD137、2B4、CD3ζ、DAP10的功能域,所述CD137的功能域的氨基酸序列如SequenceNO.7所示或其功能性变体,所述2B4的功能域的氨基酸序列如SequenceNO.8所示或其功能性变体,所述CD3ζ的氨基酸序列如Sequence NO.9所示或其功能性变体,所述DAP10功能域的氨基酸序列如Sequence NO.10所示或其功能性变体,其余可作为胞内信号域的蛋白的胞内功能域可以采用能在公开数据库,如NCBI、ENA、DDBJ上可查询到的序列,其中CD28的核苷酸序列还包括如下序列:
aggagcaagcggagcagaggcggccacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggacttcgccgcctaccggagc。
进一步的,所述嵌合抗原受体还包括信号肽,所述信号肽来源于CD8α或GM-CSF或GM-CSFR的信号肽功能域,所述CD8α以及GM-CSFR的功能域的氨基酸序列分别如SequenceNO.11和Sequence NO.12所示或其功能性变体。
本发明还提供一种核酸分子,所述核酸分子编码如上所述任意一项所述的嵌合抗原受体,其核酸序列如Sequence NO.13、Sequence NO.14、Sequence NO.15、SequenceNO.16和Sequence NO.17所示。
本发明还提供一种表达载体,所述表达载体包含如上所述的核酸分子。
进一步的,所述表达载体选自慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、DNA载体,RNA载体、质粒中的任一种。
本发明还提供一种工程化细胞,所述细胞中转导有如上所述的核酸分子或如上所述的表达载体。
进一步的,所述细胞为T细胞、T细胞前体、γδT细胞或NK细胞。
本发明还提供一种细胞制品,所述细胞制品包括如上所述的工程化细胞。
如上所述任意一项所述的嵌合抗原受体或如上所述的核酸分子或如上所述的表达载体或如上所述的工程化细胞或如上所述的细胞制品在制备抗肿瘤药物中的应用。
进一步的,所述抗肿瘤药物为抗表达CD123肿瘤的药物。
进一步的,所述抗表达CD123肿瘤的药物包括抗急性淋巴样白血病药物、抗慢性淋巴细胞白血病药物、抗慢性髓性白血病药物、抗非霍奇金淋巴瘤药物和抗霍奇金淋巴瘤药物和抗急性髓系白血病的药物。
本发明中的CD123scFv适用于所有的CAR结构,不仅仅局限与如下实施例中列举的这些结构。
本发明中,术语“功能性变体”通常是指包括与其具有基本上相同的功能(例如,可以具备所述嵌合抗原受体的性质),且与其具有至少85%(例如,至少85%,至少90%,至少91%,至少92%,至少93%,至少94%,至少95%,至少96%,至少97%,至少98%,至少99%,或至少100%)序列同一性的氨基酸序列。在某些实施方式中,所述氨基酸序列的变体为与其具有基本上相同的功能。
本发明中的CD123序列可以与现有的所有公开的其他靶点序列一起构建双靶点的嵌合抗原受体,如与现有的所有公开的CD19、CLL-1、CD33等scFv一起构建双靶点的嵌合抗原受体。
本发明中的细胞制品还包括其他可增强CAR表达活性的活性剂。
在某些具体实施例中,增强CAR表达细胞活性的活性剂可以是阻断抑制性分子的活性剂。抑制性分子如PD1可以在一些实施方案中降低CAR表达细胞发动免疫效应子反应的能力。抑制性分子包括PD1、PD-L1、CTLA4、TIM3、LAG3、VISTA、BTLA、TIGIT,LAIR1、CD160、2B4、CEACAM(CEACAM-1、CEACAM-3、CEACAM-5)、LAG3、VISTA、BTLA、TIG、LAIR1、CD160、2B4、CD80、CD86、B7-H3(CD276)、B7-H4(VTCN1)、HVEM(TNFRSF14或CD270)、KIR、A2aR、MHC I类、MHC II类、GAL9、腺苷、TGFR(TGFRβ)和TGFRβ。所述抑制性分子的胞外结构域可以融合到跨膜结构域和胞内信号传导结构域,比如PD1 CAR。在某些具体实施例中,增强CAR表达细胞活性的活性剂还可以是细胞因子受体趋化因子受体的活性剂。细胞因子受体可衍生自I型细胞因子受体,例如IL-2、IL-4、IL-7、IL-9、IL-13、IL-15或IL-21。
进一步,多种另外的治疗剂可以与本文所述的组合物联合使用。例如,潜在有用的另外的治疗剂包括PD-1抑制剂,例如纳武单抗(nivolumab)、派姆单抗(pembrolizumab)派姆单抗、阿巴伏单抗(pidilizumab)和阿特珠单抗(Atezolizumab)。
具体地,适合于与本发明组合使用的另外的治疗剂包括但不限于伊布替尼(ibrutinib)、奥法木单抗(ofatumumab)、利妥昔单抗(rituximab)、贝伐单抗(bevacizumab)、曲妥珠单抗(trastuzumab)、伊马替尼(imatinib)、西妥昔单抗(cetuximab)、帕尼单抗(panitumumab)、卡妥索单抗(Catumaxomab)、替伊莫单抗(ibritumomab)、托西莫单抗(tositumomab)、本妥昔单抗(brentuximab)、阿仑单抗(alemtuzumab)、吉妥珠单抗(gemtuzumab)、厄洛替尼(erlotinib)、吉非替尼(gefitinib)、凡德他尼(vandetanib)、阿法替尼afatinib)、拉帕替尼(lapatinib)、来那替尼(neratinib)、阿昔替尼(axitinib)、马赛替尼(masitinib)、帕唑帕尼(pazopanib)、舒尼替尼(sunitinib)、索拉非尼(sorafenib)、toceranib、来他替尼(lestaurtinib)、阿昔替尼(axitinib)、西地尼布(cediranib)、乐伐替尼(lenvatinib)、尼达尼布(nintedanib)、帕唑帕尼(pazopanib)、瑞格非尼(regorafenib)、司马沙尼(semaxanib)、索拉非尼(sorafenib)、舒尼替尼(sunitinib)、替沃扎尼(tivozanib)、toceranib、凡德他尼(vandetanib)、恩曲替尼(entrectinib)、卡博替尼(cabozantinib)、伊马替尼(imatinib)、达沙替尼(dasatinib)、尼洛替尼(nilotinib)、帕纳替尼(ponatinib)、雷多替尼(radotinib)、博舒替尼(bosutinib)、来他替尼(lestaurtinib)、鲁索替尼(ruxolitinib)、帕克替尼(pacritinib)、考比替尼(cobimetinib)、司美替尼(selumetinib)、曲美替尼(trametinib)、比美替尼(binimetinib)、阿雷替尼(alectinib)、色瑞替尼(ceritinib)、克唑替尼(crizotinib)、阿柏西普(aflibercept)、adipotide、地尼白介素。
进一步,所述细胞制品还可以与一些治疗手段组合使用,所述治疗手段可以是手术、化疗、放射。
已经开发了许多基于病毒的系统用于将基因转移到哺乳动物细胞中。例如,逆转录病毒为基因递送系统提供了便利的平台。可以使用本领域已知的技术将选择的基因插入到载体中并且包装在逆转录病毒颗粒中。然后可以分离重组病毒并在体内或离体递送至受试者的细胞中。许多逆转录病毒系统在本领域中是已知的。在一些实施例中,使用了腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施例中,使用慢病毒载体。
用于将多核苷酸引入到宿主细胞中的物理方法包括磷酸钙沉淀、脂质转染、粒子轰击、显微注射、电穿孔等。产生包含载体和/或外源性核酸的细胞的方法在本领域中是熟知的。参见例如,Sambrook等人,2012,MOLECULAR CLONING:A LABORATORY MANUAL[分子克隆实用指南],第1-4卷,Cold Spring Harbor Press[冷泉港实验出版社],纽约)。将多核苷酸引入到宿主细胞中的优选方法是磷酸钙转染。
现有技术中,制备基因修饰免疫细胞的方案包括如下步骤中的至少一步:①取血样,②富集,③分选,④活化,⑤转染,⑥再次富集。
某些实施例中,所采用的CAR-T制备方法为快速制备方法,步骤如下:1、外周血核细胞分离:采集的全血或单采有核细胞,可进行冷冻;2、外周血核细胞活化0-72h:分离得到的或者复苏后的单个核细胞采用免疫磁珠分选得到T淋巴细胞或NK细胞,可进行冷冻;具体的,分选后的T淋巴细胞或NK细胞或者复苏后的T淋巴细胞或NK细胞,与复合物的抗体(可含磁珠)或小分子和/或刺激细胞表面上的共刺激分子的抗体(可含磁珠)接触;3、转染0-72h:T淋巴细胞或NK细胞与慢病毒载体接触;4、富集14-72h:收集最后获得的CAR-T/CAR-NK细胞。
在上述快速制备的任何一个环节中都可以使用密度梯度离心,采用的溶液可以是连续或不连续的密度差的溶液,具体可以为比重为1.077±0.001的聚蔗糖-泛影葡胺,并且离心步骤中的重悬液和洗涤液都可以为维持细胞渗透压的缓冲液,比如含Na离子(如生理盐水或含有0.5wt%~2wt%HSA的生理盐水)或K离子(如KCL溶液或者含有0.5wt%~2wt%HSA的KCL水溶液)或含蛋白成分(如不同浓度的人血白蛋白、不同浓度的人自体血浆)。
本发明有益效果在于:
1)本发明提供的CD123嵌合抗原受体中的scFv是全新的人源抗体序列,避免鼠源抗体的免疫原性。
2)本发明提供的CD123嵌合抗原受体与肿瘤细胞上的CD123结合后,显示出了明显的抗肿瘤活性,其中的CD123-scFv是对人CD123抗原具有高亲和力的全人源化抗体。
附图说明
图1为CAR-1—CAR-3的体外杀伤效率;
图2为CAR-1、CAR-2、CAR-4的体外杀伤效率;
图3为CAR-1、CAR-2、CAR-4因子分泌;
图4为CAR-1、CAR-2、CAR-5、CAR-6体外杀伤效率;
图5为CAR-1、CAR-2、CAR-5、CAR-6因子分泌;
图6为CAR-2、CAR-6、CAR-7、CAR-8体外杀伤效率;
图7为CAR-2、CAR-6、CAR-7、CAR-8因子分泌;
图8为CAR-1、CAR-2修饰的T细胞体外杀伤效率;
图9为CAR-1、CAR-2因子分泌;
图10为CAR-1、CAR-2修饰的NK细胞体内药效评价;
图11为CAR-1、CAR-8和CAR-9体外杀伤效率;
图12为CAR-1、CAR-8和CAR-9的因子分泌。
具体实施方式
实施例1 CD123 scFv的制备
一、噬菌体展示
采用Ficoll分离液进行PBMC分离,将Ficoll分离液缓慢加入正常人血液使得Ficoll分离液与正常人血液保持清晰的分离界面。将装有所述血液和分离液的50mL离心管于15℃左右离心20min。离心之后,整个液面分为四层,上层为血浆混合物,下层为红细胞和粒细胞,中层为Ficoll液体,其中在上、中层交界处有以PBMC为主的白色云雾层狭窄带,即PBMC细胞层。用无菌巴氏吸管小心地吸去上层的血浆混合物,然后再用新的无菌巴氏吸管吸取PBMC,获得分离的PBMC。
通过常规方法提取总RNA并反转录成cDNA。再根据Sblattero,D.,Bradbury,A.(1998)A definitive set of oligonucleotide primers for amplifying human Vregions.Immunotechnology 3,271-278文章中的方法进行引物设计,其中VL在前或后,VH在后或前,中间由柔性Linker进行连接,PCR得到抗体的重链可变区基因片段和轻链可变区基因片段,通过常规的重叠PCR的方法扩增得到scFv核酸片段(PCR方法参考《分子克隆实验指南(Molecular Cloning:A Laboratory Manual)》(第三版),美国,Joe Sambrook,DavidRussell.科学出版社),将scFv核酸片段与噬菌粒载体pComb3XSS进行连接,通过电转仪将产物转化至TGI菌株中,得到全人源单链抗体库。
将文库菌液加入到新鲜的LB液体培养基中,进行复苏,培养至OD600≈0.5再以VCSM13:菌=50:1的感染复数添加VSCM13辅助噬菌体,充分混匀,静置后在摇床中继续培养。将培养物离心弃上清,沉淀则以氨苄青霉素、卡那霉素双抗性SOB培养基重悬,培养过夜。菌液于离心20min,收集上清并加入PEG 80002.5mol/L NaCl溶液,冰上孵育1小时,再离心30min,将沉淀的噬菌体用PBS重悬并用0.22μm滤膜过滤。
将带有His-Fc标签的CD123蛋白与proteinG磁珠共孵育,制备为CD123-proteinG偶联磁珠,将偶联磁珠抽入到制备得到的全人源单链抗体文库噬菌体淘选中。经历3-4轮的共孵育、清洗和洗脱的淘选过程,即可富集得到针对抗原的特异性单克隆抗体。
本发明中抗原淘选的具体实施过程如下:
a.封闭:5%脱脂奶粉以PBS溶解,0.45μm滤膜过滤后作为封闭液,以适量封闭液分别重悬噬菌体与CD123-proteinG偶联磁珠,室温160rpm滚转混匀1h;
b.共孵育:将CD123-proteinG偶联磁珠置于磁力架,弃上清后以噬菌体将磁珠重悬,室温160rpm滚转共孵育1h;
c.清洗:将磁珠噬菌体混合液置于磁力架,弃上清后加入适量体积0.1%Tween-80PBST清洗液对磁珠进行清洗,根据不同的淘选轮数进行清洗次数的确定,一轮淘选由PBST清洗5次,PBS清洗5次,二轮淘选由PBST清洗6次,PBS清洗6次,三轮淘选由5%FBS清洗3次,由PBS清洗1次;
d.洗脱:将清洗后的磁珠置于磁力架,吸干上清,加入pH2.2 Gly-HCl 400μL,混匀后室温孵育7min后加入约20μL PH9.0 Tris-HCl调节pH接近中性,最终将混合物置于磁力架,将噬菌体上清转移至新的1.5mL EP管完成一轮淘选;
e.富集:将噬菌体接入到OD600约为0.5的TGI菌液中进行感染,37℃静止30min后离心,保留200μL培养基重悬沉淀,涂布于2YTAG板,倒置过夜培养;
f.洗板:将过夜培养的平板由培养基洗下菌斑,作为下一轮文库包装的种子菌液,具体过程与单链抗体文库制备时的过程一致。
淘选结束后,挑取最终出库的单克隆菌斑进行ELISA检测筛选,得到与CD123抗原有结合的噬菌体克隆应用于下游的研究,淘选得到以下1株scFv,编号:FK-22-C08;具备人CD123抗原的特异性结合能力。
将100ng阳性噬菌体克隆的pComb3xss质粒与100μL感受态Rosetta gami(DE3)细菌混匀并冰浴,热激90s,冰浴后涂布至含有氨苄抗性的LB平板上,置于37℃恒温培养箱中过夜培养;挑取形成的单克隆菌斑置于LB培养基中,摇床培养后将菌液转接200mL LB培养基,37℃250rpm培养,待菌液OD达到0.5-1.0时加入IPTG并调整终浓度为1mM,诱导表达。离心收集菌体并加入PBS重悬沉淀,超声破碎2min,裂解液离心,弃去沉淀,收集上清进行蛋白纯化。
菌液裂解上清0.22μm过滤,以等体积PBS稀释后以GE Ni Sepharose excel纯化柱富集scFv,5倍柱体积PBS流洗后以5倍柱体积的含咪唑PBS溶液流洗去除杂质,含咪唑的PBS溶液洗脱蛋白,收集洗胶液以3KDa的超滤管浓缩,GE分子排阻色谱柱上样,PBS流洗并收集紫外吸收峰,SDS-PAGE鉴定scFv纯化效果后进行流式染色特异性检测。
淘选结束后,挑取最终出库的单克隆菌斑进行化学发光检测筛选,再用ELISA检测复检,得到与CD123抗原有结合的噬菌体克隆应用于下游的研究,筛选到抗体,编号分别为h13和h20。
二、鼠源人源化改造
参照本公司申请的专利201811125919.8以及专利201811125906.0中的方法,最后制备出编号为1h7的scFv。
实施例2靶向CD123的CAR质粒构建
通过PCR扩增获得scFv序列,再通过限制性内酶酶切的方式,将序列连接于含不同铰链,不同跨膜,不同共刺激信号的慢病毒载体中,即获得不同结构的靶向CD123的CAR质粒,结构如下(下面结构中的8h表示铰链区为源自人CD8α的铰链区,8TM表示跨膜区源自人CD8α的跨膜去,BB表示源自人CD137的胞内信号功能域,z表示CD3ζ链,134表示CD134的胞内信号功能域,2B4 leader表示源自2B4的信号肽功能域,2B4表示2B4分子的胞内信号功能域,2B4h表示铰链区为源自2B4的胞外铰链区,2B4TM表示跨膜区为源自2B4的跨膜区,NKp44TM表示跨膜区为源自NKp44的跨膜区,SIRPβ1TM表示跨膜区为源自SIRPβ1TM的跨膜区,GMR表示GM-CSFR的信号肽,2A为来自P2A自剪切多肽):
CAR-1:CD8α-CD123 scFv(1h7)-8h-8TM-BBz
CAR-2:CD8α-CD123 scFv(h13)-8h-8TM-BBz
CAR-3:CD8α-CD123 scFv(h20)-8h-8TM-BBz
CAR-4:CD8α-CD123 scFv(1h7)-8h-8TM-2B4z
CAR-5:GMR-CD123 scFv(1h7)-8h-8TM-2B4z
CAR-6:GMR-CD123 scFv(h13)-8h-8TM-2B4z
CAR-7:GMR-CD123 scFv(h13)-8h-SIRPβ1TM-2B4z
CAR-8:GMR-CD123 scFv(h13)-8h-NKp44TM-2B4z
CAR-9:GMR-CD123 scFv(h13)-8h-NKp44TM-2B4-DAP10
表1:核苷酸序列
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表2氨基酸序列表
实施例3制备慢病毒及感染T/NK淋巴细胞
本实施例中,CAR-1至CAR-9转染NK细胞,CAR-1与CAR-2转染T细胞。
本实施例包装慢病毒采用磷酸钙法,具体为:用含10% FBS(w/v)的DMEM培养基培养293T细胞至较佳状态,包装质粒(RRE:REV:2G)和表达质粒按一定比例加入到1.5mL的离心管中,加入CaCl2和2×HBS,混匀后室温静置后加入到处理好的293T细胞培养液中,3-5h后再次换液至10mL含10%FBS的DMEM培养基,48h或72h后收集细胞上清,纯化病毒,进行滴度测定。
对于NK细胞的包装细胞系制备如下:将CAR-1至CAR-9目的质粒分别与三个慢病毒包装质粒pMDLg/pRRE、pRSV-Rev、pMD2.G按照3:1:1:1的比例混个,通过钙转法转染慢病毒包装细胞系,转染6小时,更换新鲜的5%FBS-DMEM培养基,转染后48小时收获病毒粗提液。
制备的慢病毒滴度测定时用CHO细胞检测,用1e5/孔的CHO感染待测病毒48h后用Protein-L检测总的CAR表达,并计算阳性率。阳性率计算公式为:滴度(Tu/ml)=1e5×阳性率×稀释倍数×1000÷病毒体积(uL)。上述CAR结构病毒滴度表3所示。
表3 CD123-CAR慢病毒滴度列表
质粒编号 | 病毒滴度(Tu/ml) |
CAR-1 | 5.83E+08 |
CAR-2 | 2.70E+08 |
CAR-3 | 2.41E+08 |
CAR-4 | 5.87E+08 |
CAR-5 | 6.81E+08 |
CAR-6 | 6.85E+08 |
CAR-7 | 2.38E+08 |
CAR-8 | 1.98E+08 |
CAR-9 | 1.25E+08 |
利用梯度离心法进行淋巴细胞分离;离心后,取第二层白色淋巴细胞层,生理盐水洗涤,加入含有10%FBS的RPMI 1640完全培养基培养,获得人PBMC细胞。获得的PBMC细胞经抗CD3、CD28单克隆抗体活化24h后,按一定的感染复数(MOI)感染已活化的PBMC,在病毒感染的第12天检测CAR-T的阳性率,检测方法为流式检测,抗体为:Protein-L-PE,Protein-L可识别抗体轻链,CAR抗原识别区的scFv序列的轻链可被Protein-L识别,因此利用Protein-L可检测CAR阳性率和CAR表达强度。
对于NK细胞而言,获得PBMC后需要使用CD56抗体标记的Microbeads进行磁力分选以获得CD56阳性的NK细胞。NK细胞活化24h后可以进行慢病毒转导,转导后3天,CAR-NK的阳性率需使用CD123-6×His(ACRO-Biosystems)或靶向相应CD123-scFv的单克隆抗体进行检测。NK细胞用CD3-PE/Cy7和CD56-BV510(BioLegend)标记CAR-T/NK阳性率检测结果如表4所示,如下表所示,构建的所有结构均能较好的表达出来。
表4 CD123-CAR-NK/T阳性率检测结果
实施例4 CAR-1—CAR-3修饰的NK细胞体外杀伤实验以及细胞因子分泌实验
以CD123+KG1a-Luc-GFP、MV-4-11-Luc-GFP细胞作为阳性靶细胞。将CAR-NK细胞按1:1的比例铺于靶细胞中,24小时后通过luciferase检测杀伤。Luciferase原理:检测时将靶细胞用裂解液裂解后,其中的luciferase会分解底物发出荧光,利用荧光值分析结果公式为:CAR-NK细胞杀伤率=1-(实验组荧光值÷空白对照组荧光值)。
结果如图以及下表5以及图1所示,由CD123(h13)-scFv组成的CAR结构CAR-2修饰的CAR-NK细胞在对于KG1a-Luc-GFP的杀伤中体现出了明显优势。
表5 CAR-1、CAR-2和CAR-3体外杀伤数据
实施例5 CAR-1、CAR-2、CAR-4修饰的NK细胞体外杀伤实验以及细胞因子分泌实验
以CD123+KG1a-Luc-GFP、MV-4-11-Luc-GFP细胞作为阳性靶细胞,将CAR-NK细胞按1:1的比例铺于靶细胞中,24小时后通过luciferase检测杀伤。luciferase原理:检测时将靶细胞用裂解液裂解后,其中的luciferase会分解底物发出荧光,利用荧光值分析结果公式为:CAR-NK细胞杀伤率=1-(实验组荧光值÷空白对照组荧光值)。
结果如图2和表6所示:加入CAR-NK细胞24小时后,由CD123(h13)-scFv组成的CAR结构CAR-2修饰的CAR-NK细胞仍体现出了明显的功能优势,其次在CD123(1h7)-scFv构成的CAR结构修饰的CAR-NK中,CAR-4现出了较好的体外功能。
表6 CAR-1、CAR-2、CAR-4体外杀伤数据
MV-4-11-Luc-GFP | KG-1α-Luc-GFP | |
CAR-1 | 72.93% | 21.06% |
CAR-2 | 94.91% | 72.14% |
CAR-4 | 90.51% | 44.93% |
C-NK | 71.37% | 7.398% |
在杀伤24小时后收集细胞上清,进行CAR-NK细胞受到靶细胞刺激后IFN-γ分泌能力的检测。收集的上清,利用ELISA(酶联免疫)方法检测IFN-γ的分泌情况。结果如图3和表7所示,经抗原刺激后CAR-2具有更强的IFN-γ分泌能力,CD123(1h7)-scFv构成的CAR结构修饰的CAR-NK,CAR-2有较高的因子分泌水平,因此,优选采用CD123(h13)-scFv构建的CAR-2。
表7 CAR-1、CAR-2、CAR-4的细胞因子分泌数据
MV-4-11-Luc-GFP | KG-1α-Luc-GFP | |
CAR-1 | 6504.33 | 3991.33 |
CAR-2 | 8385 | 6227.67 |
CAR-4 | 3854.33 | 1622.33 |
C-NK | 2072 | 950.07 |
实施例6 CAR-1、CAR-2、CAR-5、CAR-6饰的NK细胞体外杀伤实验以及细胞因子分泌实验
以CD123+KG1a-Luc-GFP、MV-4-11-Luc-GFP细胞作为阳性靶细胞,将CAR-NK细胞按1:1的比例铺于靶细胞中,24小时后通过luciferase检测杀伤。luciferase原理:检测时将靶细胞用裂解液裂解后,其中的luciferase会分解底物发出荧光,利用荧光值分析结果公式为:CAR-NK细胞杀伤率=1-(实验组荧光值÷空白对照组荧光值)。
结果如图4和表8所示:加入CAR-NK细胞24小时后,由CD123(h13)-scFv组成的CAR结构CAR-2、CAR-6饰的CAR-NK细胞仍体现出了明显的功能优势,其次在CD123(1h7)-scFv构成的CAR结构修饰的CAR-NK中,CAR-5体现了较好的体外功能。
表8 CAR-1、CAR-2、CAR-5、CAR-6的细胞因子分泌数据
MV-4-11-Luc-GFP | KG-1a-Luc-GFP | |
CAR-1 | 57.038% | 26.59% |
CAR-2 | 74.60% | 60.97% |
CAR-5 | 70.76% | 47.38% |
CAR-6 | 93.11% | 86.88% |
C-NK | 29.40% | 12.07% |
在杀伤24小时后收集细胞上清,进行CAR-NK细胞受到靶细胞刺激后IFN-γ分泌能力的检测。收集的上清,利用ELISA(酶联免疫)方法检测IFN-γ的分泌情况。结果如图5和表9所示,经抗原刺激后CAR-2与CAR-6具有更强的IFN-γ分泌能力,充分说明了CD123-scFv(h13)在CAR分子构成方面的优势。
表9 CAR-1、CAR-2、CAR-5、CAR-6因子分泌(单位pg/ml)
MV-4-11-Luc-GFP | KG-1a-Luc-GFP | |
CAR-1 | 2814.982 | 1496.2 |
CAR-2 | 5752.046 | 2288.47 |
CAR-5 | 3122.022 | 1364.496 |
CAR-6 | 4232.311 | 2256.261 |
C-NK | 735.649 | 347.269 |
实施例7 CAR-2、CAR-6、CAR-7、CAR-8修饰的NK细胞体外杀伤实验以及细胞因子分泌实验
以CD123+KG1a-Luc-GFP、MV-4-11-Luc-GFP、MOLM-13-Luc-GFP细胞作为阳性靶细胞,KG-1a-CD123(-)-Luc-GFP(KG1a-Luc-GFP敲除CD123)作为阴性靶细胞。将CAR-NK细胞按1:1的比例铺于靶细胞中,24小时后通过luciferase检测杀伤。Luciferase原理:检测时将靶细胞用裂解液裂解后,其中的luciferase会分解底物发出荧光,利用荧光值分析结果公式为:CAR-NK细胞杀伤率=1-(实验组荧光值÷空白对照组荧光值)。
结果如图6和表10所示:加入CAR-NK细胞24小时后,由CD123(h13)-scFv组成的CAR结构CAR-2、6、7、8修饰的CAR-NK细胞仍体现出很好的体外功能,其中CAR-2、6、7、8均有较好的体外功能。
表10 CAR-2、CAR-6、CAR-7、CAR-8体外杀伤效率
MOLM-13-Luc-GFP | MV-4-11-Luc-GFP | KG-1a-Luc-GFP | KG-1a-CD123(-)-Luc-GFP | |
CAR-2 | 77.75% | 71.29% | 65.89% | 5.20% |
CAR-6 | 88.41% | 87.55% | 79.44% | 11.34% |
CAR-7 | 89.37% | 91.06% | 84.02% | 14.87% |
CAR-8 | 91.88% | 93.37% | 74.69% | 17.24% |
CNK | 38.13% | 44.37% | 5.90% | 1.45% |
在杀伤24小时后收集细胞上清,进行CAR-NK细胞受到靶细胞刺激后IFN-γ分泌能力的检测。收集的上清,利用ELISA(酶联免疫)方法检测IFN-γ的分泌情况。结果如图7和表11所示,CAR-2、6、7、8因子分泌水平相当。
表11 CAR-2、CAR-6、CAR-7、CAR-8因子分泌
MOLM-13-Luc-GFP | MV-4-11-Luc-GFP | KG-1a-Luc-GFP | KG-1a-CD123(-)-Luc-GFP | |
CAR-2 | 4001.491 | 8287.013 | 4294.896 | 1971.616 |
CAR-6 | 2163.523 | 5111.036 | 2146.177 | 385.418 |
CAR-7 | 2922.99 | 6397.685 | 2954.751 | 1258.33 |
CAR-8 | 3053.724 | 6775.104 | 2837.154 | 998.721 |
CNK | 542.765 | 1217.419 | 389.023 | 347.816 |
实施例8 CAR-1、CAR-2修饰的T细胞体外杀伤实验以及细胞因子分泌实验
以CD123+细胞KG1a-Luc-GFP、MOLM-13-Luc-GFP作为阳性靶细胞,用Raji-Luc-GFP阴性靶细胞。将CAR-T细胞按1:1的比例铺于靶细胞中,24小时后通过luciferase检测杀伤。Luciferase原理:检测时将靶细胞用裂解液裂解后,其中的luciferase会分解底物发出荧光,利用荧光值分析结果公式为:CAR-T细胞杀伤率=1-(实验组荧光值÷空白对照组荧光值)。
结果如图以及下表12以及图8所示,CAR-1、CAR-2修饰的CAR-T细胞在对于KG1a-Luc-GFP、MOLM-13-Luc-GFP有明显且有效的杀伤,但对于阴性细胞Raji-Luc-GFP杀伤水平极低。
表12 CAR-1、CAR-2修饰的T细胞体外杀伤效率
MV-4-11-Luc-GFP | MOLM-13-Luc-GFP | Raji-Luc-GFP | |
CT | 41.01% | 9.86% | 18.86% |
CAR-1 | 97.65% | 99.61% | 29.2% |
CAR-2 | 99.42% | 99.97% | 13.24% |
在杀伤24小时后收集细胞上清,进行CAR-T细胞受到靶细胞刺激后IFN-γ分泌能力的检测。收集的上清,利用ELISA(酶联免疫)方法检测IFN-γ的分泌情况。结果如图9和表13所示,经抗原刺激后相比于CAR-1,CAR-2修饰的T细胞具有更强的IFN-γ分泌能力。
表13 CAR-1、CAR-2修饰的T细胞因子分泌
MV-4-11-Luc-GFP | MOLM-13-Luc-GFP | Raji-Luc-GFP | |
CT | 685.869 | 326.63 | 2545.829 |
CAR-1 | 21704.481 | 10675.533 | 5017.71 |
CAR-2 | 55577.524 | 22214.741 | 3314.78 |
实施例9 CAR-1、CAR-2修饰的NK细胞体内药效评价
以CD123+细胞MV-4-11-Luc-GFP作为阳性靶细胞,按1e6/只小鼠的剂量通过尾静脉注射NCG小鼠进行预成瘤,预成瘤时间为8天。CAR-1、CAR-2修饰的NK体外培养13天后按6e6/只小鼠的剂量通过尾静脉注射已经预成瘤的NCG小鼠,未进行CAR修饰的NK细胞以相同剂量及方式进行注射作为对照组。此后每7天腹腔注射一次luciferase的荧光底物通过小动物活体成像仪进行活体成像,检测瘤荷情况。
结果如图10所示,CAR-2修饰的NK细胞相对于CAR-1修饰的NK细胞及未修饰的NK具有相对最好的体内药效优势。
实施例10 CAR-1、CAR-8、CAR-9修饰的NK细胞体外杀伤实验以及细胞因子分泌实验
以CD123+KG1a-Luc-GFP、MV-4-11-Luc-GFP细胞作为阳性靶细胞,将CAR-NK细胞按1:1的比例铺于靶细胞中,24小时后通过luciferase检测杀伤。Luciferase原理:检测时将靶细胞用裂解液裂解后,其中的luciferase会分解底物发出荧光,利用荧光值分析结果公式为:CAR-NK细胞杀伤率=1-(实验组荧光值÷空白对照组荧光值)。
结果如图11和表14所示:加入CAR-NK细胞24小时后,由CD123(h13)-scFv组成的CAR结构CAR-8修饰的CAR-NK细胞仍体现出很好的体外功能,CAR-8与CAR-9功能相当。
表14 CAR-1、CAR-2修饰的NK细胞体外杀伤效率
MV-4-11-Luc-GFP | KG-1a-Luc-GFP | |
CAR-1 | 58.70 | 24.065 |
CAR-8 | 78.9 | 49.14 |
CAR-9 | 57.25 | 22.69 |
Control NK | 41.19 | -3.40 |
在杀伤24小时后收集细胞上清,进行CAR-NK细胞受到靶细胞刺激后IFN-γ分泌能力的检测。收集的上清,利用ELISA(酶联免疫)方法检测IFN-γ的分泌情况。结果如图12和表15所示,经抗原刺激后CAR-8与CAR-9修饰的NK细胞均具有较强的IFN-γ分泌能力。
表15 CAR-1、CAR-8、CAR-9修饰的NK细胞因子分泌
MV-4-11-Luc-GFP | KG-1a-Luc-GFP | |
CAR-1 | 3354.552 | 1884.587 |
CAR-8 | 2520.282 | 1147.98 |
CAR-9 | 1609.368 | 1060.005 |
Control NK | 840.51 | 487.456 |
Claims (16)
1.抗CD123的scFv,其特征在于:所述抗CD123的scFv包含重链可变区和轻链可变区,所述轻链可变区包含L-CDR1、L-CDR2和L-CDR3,所述重链可变区包含H-CDR1、H-CDR2和H-CDR3;所述L-CDR1、L-CDR2和L-CDR3分别为如下序列:QSVSSN、GAS和QQRSNWPPALT;所述H-CDR1、H-CDR2和H-CDR3分别为如下序列:GYSFTSYW、IYPGDSDT和ARIRFDKEQLFYYYYGMDV。
2.如权利要求1所述的抗CD123的scFv,其特征在于:所述轻链可变区的氨基酸序列如Sequence NO.1所示或其功能性变体;所述重链可变区的氨基酸序列如Sequence NO.2所示或其功能性变体。
3.一种嵌合抗原受体,其特征在于:所述嵌合抗原受体包含如权利要求1或2所述的抗CD123的scFv。
4.如权利要求3所述的嵌合抗原受体,其特征在于:所述嵌合抗原受体包含胞外结构域、铰链区、跨膜区和胞内信号域。
5.如权利要求4所述的嵌合抗原受体,其特征在于:所述铰链区来源于CD28、CD3ζ、CD40、4-1BB、OX40、CD84、CD166、CD8α、CD8β、ICOS、ICAM-1、CTLA-4、CD27、CD40、NKGD2、IgG1或免疫球蛋白中的CH2/CH3结构域,所述CD8α的氨基酸序列如Sequence NO.3所示或其功能性变体。
6.如权利要求5所述的嵌合抗原受体,其特征在于:所述跨膜区来源于CD2、CD3D、CD3E、CD3G、CD3ζ、CD4、CD8α、CD8β、CD16、CD27、CD28、CD28H、CD40、CD84、CD166、4-1BB、OX40、ICOS、ICAM-1、CTLA4、PD1、LAG3、2B4、BTLA、DNAM1、DAP10、DAP12、FcERIγ、IL7、IL12、IL15、KIR2DL4、KIR2DS1、KIR2DS2、NKp30、NKp44,、NKp46、NKG2C、NKG2D、CS1、或T细胞受体多肽SIRPβ1或NKp44的跨膜区,所述CD8α、SIRPβ1、NKp44氨基酸序列分别如Sequence NO.4、Sequence NO.5和Sequence NO.6所示或其功能性变体。
7.如权利要求6所述的嵌合抗原受体,其特征在于:所述胞内信号域来源于CD28、OX40、CD27、DAP10、CD3γ、FcεRI、CD2、CD16、TCRζ、FcRβ、CD30、CD40、ICOS、LFA-1、IL-2受体、Fcγ受体、KIRDS2、SLAMF7、NKp80(KLRF1)、信号传导淋巴细胞活化分子(SLAM蛋白)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、DAP12、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、LFA-1(CD11a/CD18)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、CD137、2B4、CD3ζ、DAP10的信号功能域,所述CD137功能域的氨基酸序列如Sequence NO.7所示或其功能性变体,所述2B4功能域的氨基酸序列如Sequence NO.8所示或其功能性变体,所述CD3ζ功能域的氨基酸序列如Sequence NO.9所示或其功能性变体,所述DAP10功能域的氨基酸序列如Sequence NO.10所示或其功能性变体。
8.如权利要求3-7任意一项所述的嵌合抗原受体,其特征在于:所述嵌合抗原受体还包括信号肽,所述信号肽为来源于CD8α或GM-CSF或GM-CSFR的信号肽功能域,所述CD8α以及GM-CSFR的功能域的氨基酸序列分别如SequenceNO.11和Sequence NO.12所示或其功能性变体。
9.一种核酸分子,其特征在于:编码权利要求3-8任意一项所述的嵌合抗原受体,其核酸序列如Sequence NO.13、Sequence NO.14、Sequence NO.15、Sequence NO.16和SequenceNO.17所示。
10.一种表达载体,其特征在于,所述表达载体包含如权利要求9所述的核酸分子。
11.如权利要求10所述的表达载体,其特征在于:所述表达载体选自慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、DNA载体,RNA载体、质粒中的任一种。
12.一种工程化细胞,其特征在于:所述细胞中转导有权利要求9所述的核酸分子或权利要求10或11所述的表达载体,优选地,所述细胞为T细胞、T细胞前体、γδT细胞或NK细胞。
13.一种细胞制品,其特征在于;所述细胞制品包括如权利要求12所述的工程化细胞。
14.权利要求3-8任意一项所述的嵌合抗原受体或权利要求9所述的核酸分子或权利要求10或11所述的表达载体或权利要求13所述的工程化细胞或权利要求13所述的细胞制品在制备抗肿瘤药物中的应用。
15.如权利要求14所述的应用,其特征在于:所述抗肿瘤药物为抗表达CD123肿瘤的药物。
16.如权利要求15所述的应用,其特征在于:所述抗表达CD123肿瘤的药物包括抗急性淋巴样白血病药物、抗慢性淋巴细胞白血病药物、抗慢性髓性白血病药物、抗非霍奇金淋巴瘤药物、抗霍奇金淋巴瘤药物和抗急性髓系白血病药物。
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