CN117659195A - 一种识别cd38的单克隆抗体、重组基因表达载体、嵌合抗原受体nk细胞及其应用 - Google Patents
一种识别cd38的单克隆抗体、重组基因表达载体、嵌合抗原受体nk细胞及其应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,具体公开了一种识别CD38的单克隆抗体、重组基因表达载体、嵌合抗原受体NK细胞及其应用,所述单克隆抗体的轻链可变区基因序列如SEQ ID NO.1所示,其重链可变区基因序列如SEQ ID NO.2所示。本发明提供嵌合抗原受体NK细胞用于CD38阳性肿瘤(多发性骨髓瘤)的靶向杀伤,且杀伤率显著高于普通NK细胞(NK92细胞)。
Description
技术领域
本发明涉及肿瘤免疫细胞治疗技术领域,具体涉及一种识别CD38的单克隆抗体、重组基因表达载体、嵌合抗原受体NK细胞及其应用。
背景技术
多发性骨髓瘤(MM)是一种难以治愈的恶性浆细胞肿瘤。它的特征是骨髓中单克隆浆细胞不受控制的生长,导致非功能性完整免疫球蛋白或免疫球蛋白链的过度产生。这些免疫球蛋白的积累以及异常单克隆浆细胞与骨髓中其他细胞的相互作用会导致一系列问题,包括贫血、骨病变、感染、高钙血症、肾衰竭、疲劳和疼痛。目前多发性骨髓瘤(MM)虽有治疗和缓解的途径,但是复发率高。对于,多发性骨髓瘤的免疫治疗十分迫切。
人CD38抗原是一种46kDa的II型跨膜糖蛋白,具有短的N末端细胞质尾和长细胞外结构域。CD38可以内化和脱落,以39kDa可溶形式存在于体液中。CD38在多发性骨髓瘤(MM)细胞上高度均匀地表达。CD38在正常淋巴细胞和骨髓细胞以及一些非造血来源的组织中也以相对较低的水平表达。因此,该靶点已被作为MM免疫治疗的重要靶点,并且已经有多个抗CD38抗体的靶向药物用于临床的治疗。
近年来,一种新的肿瘤免疫治疗方法——嵌合抗原受体(chimeric antigenreceptor,CAR)修饰的T细胞(CAR-T)及NK细胞(CAR-NK)治疗受到了广泛的关注。CAR的结构主要包括肿瘤抗原结合区、跨膜区、胞内信号活化区三大基本构造,是将细胞外肿瘤抗原结合域与一个或多个细胞内免疫细胞活化信号域结合的基因工程融合蛋白。目前针对CD38蛋白的抗体较少,构建新的针对CD38蛋白的抗体对多发性骨髓瘤的治疗意义重大。
发明内容
为获得新的针对CD38蛋白的抗体,本发明提供了一种识别CD38的单克隆抗体、重组基因表达载体、嵌合抗原受体NK细胞及其应用。CD38-CAR基因表达载体经慢病毒包装后导入NK细胞。依此方法制备的NK细胞称之为CD38-CAR-NK细胞。与普通NK细胞相比,CD38-CAR-NK细胞对CD38蛋白阳性肿瘤细胞(如多发性骨髓瘤细胞)具有更强的杀伤活性。
本发明提供了一种识别CD38蛋白的单克隆抗体,其轻链可变区基因序列如SEQ IDNO.1所示,其重链可变区基因序列如SEQ ID NO.2所示。
进一步地,所述抗体的轻链可变区的氨基酸序列如SEQ ID NO.5所示,所述抗体的重链可变区的氨基酸序列如SEQ ID NO.6所示。
本发明还提供了一种识别CD38蛋白的嵌合抗原受体,所述嵌合抗原受体为CD38-CAR,其含有所述的识别CD38蛋白抗体的轻链可变区和重链可变区。
本发明还提供了一种特定的核酸分子,其编码识别CD38蛋白的抗体可变区,或者编码识别CD38蛋白的嵌合抗原受体。
本发明还提供了一种重组基因表达载体,所述重组基因表达载体为CD38-CAR基因表达载体,所述CD38-CAR基因表达载体含有上述核酸分子。
本发明还提供了一种免疫细胞,其表达所述CD38-CAR基因表达载体。
本发明还提供了一种识别CD38蛋白的嵌合抗原受体NK细胞,所述嵌合抗原受体NK细胞为CD38-CAR-NK细胞,其表达所述的嵌合抗原受体。
进一步地,所述CD38-CAR-NK细胞对多发性骨髓瘤细胞的杀伤率为59.1%±2.7%。
进一步地,所述多发性骨髓瘤细胞为RPMI 8226细胞。
本发明还提供了所述的单克隆抗体、所述的嵌合抗原受体、所述的核酸分子、所述的免疫细胞或所述的嵌合抗原受体NK细胞在制备治疗多发性骨髓瘤药物中的应用。
与现有技术相比,本发明的有益效果在于:
1、本发明通过淋巴细胞杂交瘤技术得到了具有特异识别CD38蛋白的单克隆抗体重、轻链可变区基因序列,具有其唯一性和独特性。在此基础上构建与制备的CD38-CAR-NK细胞在识别CD38蛋白阳性肿瘤细胞(如多发性骨髓瘤细胞)上也有其唯一性和独特性。
2、本发明提供的CD38-CAR-NK细胞对多发性骨髓瘤细胞‘RPMI 8226’的杀伤率为59.1%±2.7%,普通NK细胞(NK92细胞)对多发性骨髓瘤细胞‘RPMI 8226’的杀伤率为32.7%±2.5%;可见,本发明制备得到的CD38-CAR-NK细胞对多发性骨髓瘤细胞‘RPMI8226’的杀伤活性显著高于普通NK细胞(NK92细胞)对多发性骨髓瘤细胞‘RPMI 8226’的杀伤活性(P<0.01)。有制备治疗多发性骨髓瘤药物的潜力。
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的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例3中的慢病毒表达载体103的图谱。
具体实施方式
下面对本发明的具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。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的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。本发明各实施例中所述实验方法,如无特殊说明,均为常规方法,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明提供了一种识别CD38蛋白的单克隆抗体,所述单克隆抗体轻链可变区基因序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.5所示;所述单克隆抗体的重链可变区基因序列如SEQ ID NO.2所示,其编码的氨基酸序列如SEQ ID NO.6所示。
本发明还提供了一种识别CD38蛋白的嵌合抗原受体(CD38-CAR),其含有所述的识别CD38蛋白的轻链可变区和重链可变区。
本发明还提供了一种特异序列的核酸分子,其编码所述的抗体可变区,或者其编码所述的嵌合抗原受体。
本发明还提供了一种重组基因表达载体,所述重组基因表达载体为CD38-CAR基因表达载体,其含有上述的核酸分子。
本发明还提供了一种免疫细胞,所述免疫细胞表达CD38-CAR基因表达载体。
本发明还提供了一种识别CD38蛋白的嵌合抗原受体NK细胞(CD38-CAR-NK细胞),其表达所述的CD38-CAR嵌合抗原受体。
本发明还提供了所述的单克隆抗体、嵌合抗原受体、核酸分子、免疫细胞或嵌合抗原受体NK细胞在制备治疗多发性骨髓瘤药物中的应用。
实施例1:一种CD38单克隆抗体杂交瘤细胞筛选制备。
抗CD38单克隆抗体杂交瘤细胞筛选制备过程如下:
1、抗原免疫
8周龄BALB/c小鼠3只,体重25-29g;CD38重组蛋白与福氏完全佐剂混合至油包水状态。每只小鼠分4点皮下注射,每点注射50μL(含50μg人CD38蛋白)。每间隔1周进行第二、第三和第四次加强免疫,每次每只BALB/c小鼠腹腔注射100μg CD38蛋白。第四次免疫后第四天,取小鼠脾脏细胞,用于后续与骨髓瘤细胞的融合。
2、骨髓瘤细胞制备
SP2/0骨髓瘤细胞在与脾细胞融合前2周复苏,用含有20%胎牛血清的1640培养基培养,生长良好,待用于融合。
3、饲养细胞制备
正常BALB/c小鼠,体重25g,脱颈处死,无菌操作取出脾脏。200目不锈钢目筛研磨脾脏组织,无血清1640培养基冲洗,收集脾细胞并计数。3块96孔板,每孔加入2×105个脾细胞,37℃二氧化碳培养箱培养至次日。
4、B淋巴细胞与骨髓瘤细胞融合
经CD38抗原免疫的BALB/c小鼠脾脏细胞制备:在细胞融合当天,将CD38抗原免疫的BALB/c小鼠脱颈处死,无菌操作取出脾脏。200目不锈钢目筛研磨脾脏组织,1640培养基冲洗收集脾细胞。取含1×107个脾细胞与3×107个SP2/0骨髓瘤细胞用50%的PEG1500融合剂融合。融合后细胞用HAT培养液重悬浮,加入已含有饲养细胞的96孔细胞培养板中,每孔0.1mL,继续培养。每日观察杂交瘤生长状况,第三日用HT培养液进行半换液。
5、阳性克隆筛选
融合后第十日起,选取有克隆生长的孔,吸取100μL上清液,采用ELISA方法检测上清液中是否含有抗CD38抗体。ELISA方法如下:ELISA板条每孔包被CD38蛋白2μg,待测孔每孔加入待测上清100μl,阴性对照孔加入100μLSP2/0细胞上清液,37℃孵育50min后洗板3次,随后加入100μL工作浓度的酶标二抗(羊抗鼠IgG-HRP),37℃孵育40min后洗板5次,每孔加入100μLTMBS显色液,37℃显色10min,450nm测吸光值(OD值)。待测孔OD值/阴性对照孔OD值≧2.1(P/N比值≧2.1)即判定为阳性。阳性克隆随后连续进行2次的克隆化,最终确定稳定的杂交瘤。本次融合筛选到了一株强阳性的杂交瘤克隆CL1。其培养上清OD值为1.15,阴性对照OD值为0.1,(P/N比值11.5)。
实施例2:CL1杂交瘤抗CD38单克隆抗体重链和轻链可变区基因测序。
1、CL1杂交瘤细胞RNA提取:
根据Trizol试剂说明书提供的方案,使用Trizol裂解实施例1中获得的杂交瘤细胞CL1。将裂解后样本全部转移至1.5mL离心管中,每1mL Trizol试剂添加0.2mL氯仿,抽提,室温下静置10min;4℃,12000rpm,离心15min。小心吸取含RNA的上层水相于无RNA酶的小管中,加入等量异丙醇混匀,4℃条件下,12000rpm离心15min,弃上清。75%乙醇洗沉淀2次,待乙醇挥发彻底后,每管加入50μL无菌、无RNA酶水溶解沉淀。取10μL RNA用于随后的RT-PCR。
2、逆转录聚合酶链式反应(Reverse transcription Polymerase chainreaction,RT-PCR)扩增重、轻链可变区基因:
以上述提取的杂交瘤细胞CL1的总RNA为模板,通过RT-PCR合成cDNA。RT-PCR引物是采用一组通用的小鼠IgG抗体重链、轻链可变区扩增引物。其中VL-F1:SAAAWTGTKCTCACCCAGTCTCC和VL-R1:TTTBAKYTCCAGCTTGGTSCC扩增出了轻链可变区基因;VH-F:AGGTCCARCTGCAGCAGYCT和VH-R:TGMRGAGACDGTGASTGARG扩增出了重链可变区基因。PCR扩增产物通过琼脂糖凝胶电泳分析条带大小,随后回收该条带,送样测序。测序结果确定了CL1杂交瘤细胞的单克隆抗体轻链可变区基因序列如SEQ ID NO.1所示,其氨基酸序列如SEQ ID NO.5所示,CL1杂交瘤细胞的单克隆抗体重链可变区基因序列如SEQ ID NO.2所示,其对应的氨基酸序列如SEQ ID NO.6所示。
SEQ ID NO.1:
ATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAG
GGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTATATCTGGCAAAA
GTTATGTATACTGGTACCTACAGAAACCAGGACAGCCACCCAAACTCCTC
ATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGG
CAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAG
GAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCGTTCAC
GTTCGGTGCTGGGACCAAGCTG
SEQ ID NO.5:IVLTQSPASLAVSLGQRATISCRASKSVSISGKSYVYWYLQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGAGTKL
SEQ ID NO.2:
GTCCAGCCTCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATTTATACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGTTTGGGGGGATTGATCCTAGCAATGGTGACACTAACTTCAATGAGAGGTTCAAGAACAAGGCCACACTGACTGTGGACAGATCCTCCAGCACAGTCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATCGGGGTTACTACCCTCTTACTTTGCTATGGACTTCTGGGGTCAAGGAACCTCACTCACCGTCTCT
SEQ ID NO.6:
VQPQQSGAELVKPGASVKLSCKASGYTFTSYYLYWVKQRPGQGLEWFGGIDPSNGDTNFNERFKNKATLTVDRSSSTVYMQLSSLTSEDSAVYYCTRSGLLPSYFAMDFWGQGTSLTVS
实施例3:103-CAR载体构建
1、按照下述的基因拼接顺序,合成CAR框架基因:
XbaI-Igk sig-EcoRI-NheI-CD8 hinge-CD28 TM-CD28 ITAM-41BB ITAM-CD3ζITAM-BamHI。
CAR载体框架基因序列如SEQ ID NO.3所示;
SEQ ID NO.3:
TCTAGA(XbaI)ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGAC(Igk-sig)GAATTC(EcoRI)TTCGAAGCTAGC(NheI)ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(CD8hinge)TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCAGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(CD28-TM+ITAM)AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG(4-1BB-ITAM)AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(CD3ζITAM)GGATCC(BamHI)
在CAR框架基因的5’端和3’分别引入XbaI和BamHI限制性酶切位点,以便于将CAR框架基因克隆至慢病毒载体‘FT103-pCDH-CMV-MCS-SV40-Neo’(简称103载体,如图1所示)。此外,在CAR框架基因Igk sig和CD8 hinge之间引入EcoRI-NheI,为单链抗体(轻链可变区+Linker+重链可变区)基因的克隆预留酶切位点。将上述的CAR框架基因用XbaI和BamHI双酶切后克隆至同样双酶切的慢病毒载体‘FT103-pCDH-CMV-MCS-SV40-Neo’,筛选阳性克隆,进一步进行基因测序予以验证。把克隆有CAR框架基因的‘FT103-pCDH-CMV-MCS-SV40-Neo’载体命名为103-CAR。
2、单克隆抗体重轻链可变区基因对应的单链抗体基因的合成
合成‘轻链可变区+Linker+重链可变区’的单链抗体基因。单链抗体基因的5’端和3’端分别引入EcoRI和NheI限制性内切酶位点,以便于克隆至103-CAR载体。
根据筛选获得的杂交瘤抗体的可变区基因序列,采用基因合成的方法获得单克隆抗体的单链抗体基因,其基因序列如SEQ ID NO.4所示。采用PCR方法扩增后克隆至T载体,筛选阳性克隆,DNA测序验证。
SEQ ID NO.4:
GAATTCATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGG
CAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTATATCTGG
CAAAAGTTATGTATACTGGTACCTACAGAAACCAGGACAGCCACCCAAA
CTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTC
AGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGG
AGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCC
GTTCACGTTCGGTGCTGGGACCAAGCTGGGCGGTGGCGGTTCTGGTGGC
GGTGGCTCCGGCGGTGGCGGTTCTGTCCAGCCTCAGCAGTCTGGGGCTG
AACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGG
CTACACCTTCACCAGCTACTATTTATACTGGGTGAAGCAGAGGCCTGGAC
AAGGCCTTGAGTGGTTTGGGGGGATTGATCCTAGCAATGGTGACACTAAC
TTCAATGAGAGGTTCAAGAACAAGGCCACACTGACTGTGGACAGATCCT
CCAGCACAGTCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGC
GGTCTATTACTGTACAAGATCGGGGTTACTACCCTCTTACTTTGCTATGGA
CTTCTGGGGTCAAGGAACCTCACTCACCGTCTCTGCTAGC
3、CD38-CAR重组表达载体的构建
采用EcoRI和NheI双酶切,从T载体上酶切并回收SEQ ID NO.4所示的单链抗体基因片段,克隆至同样双酶切的103-CAR载体,筛选阳性克隆,DNA测序验证,获得CD38-CAR基因表达载体。操作步骤如下:分别将103-CAR载体(1μg)以及含有SEQ ID NO.4的T载体(1μg)于水浴37℃下,用EcoRI和NheI双酶切2个小时,酶切反应体积20μl。酶切产物采用琼脂糖凝胶电泳,胶回收试剂盒回收相应大小的EcoRI、NheI双酶切103-CAR载体和SEQ ID NO.4。分别取2uL酶切回收的103-CAR载体和取2μL酶切回收的SEQ ID NO.4,加入同一管中,16℃过夜连接。将10μL连接产物轻轻的加入融化后的感受态细胞,放置冰上静止30min,并将冰盒放置于4℃冰箱中;随后转入42℃的水浴锅中,热击45s后立即放置冰上静止2分钟;吸取500μL预热的LB培养基加入上述管中,放置37℃恒温水浴摇床,200rpm 1h;吸取300μl菌悬液加入含有0.1mg/mL的氨苄固体平板上,并用涂布棒涂匀;待菌液完全吸收后,37℃培养12-15h。在平板上随机挑选10个单菌落,分别加入含有0.1mg/mL氨苄青霉素的LB培养液,放置37℃恒温水浴摇床,200rpm,过夜;次日吸取10μl菌液于无菌去离子水中,97℃煮10min,然后10000rpm离心1min,取上清用于PCR鉴定。PCR反应体积为25μL,其中上游引物F4-GAATTCATTGTACTCACCCAGTCC1μL,下游引物R4-GCTAGCTGACACTGTCAAAGAGG 1μL,25μM dNTP4μL,taq DNA聚合酶1μL,裂解菌液上清2μL,10×Buffer 2.5μL,去离子水13.5μL,94℃30s-55℃30s-72℃30s进行30个循环PCR;PCR阳性菌落,扩大培养,提取质粒,送交测序公司进行DNA序列测定,确定CD38-CAR重组表达载体序列完全正确。
4、将CD38-CAR基因表达载体(质粒)与逆转录病毒的VSV-G外膜蛋白质粒共转染GP2-293T细胞,培养上清中即包含携带CD38-CAR基因的假病毒颗粒。收获的假病毒颗粒采用数字微滴PCR(ddPCR)的绝对定量方法进行病毒颗粒含量测定,随后将假病毒颗粒导入NK细胞获得CD38-CAR-NK细胞。
实施例4:CD38-CAR-NK细胞体外杀伤多发性骨髓瘤细胞活性测定。
1、由于多发性骨髓瘤细胞系RPMI 8226高表达CD38分子,所以本实验选择该细胞作为靶细胞用于CD38-CAR-NK细胞的体外杀伤实验;
2、CD38-CAR-NK细胞以NK92细胞系为原始细胞,制备方法如下述步骤3;NK92细胞系同时也作为普通对照的NK细胞。
3、NK92细胞以每孔7×105接种于6孔板中(6孔板体系为5mL培养液)。次日弃去NK92细胞中2.5mL细胞培养液,加入10μL携带CD38-CAR基因的假病毒颗粒(ddPCR定量的假病毒颗粒数为106),继续培养48小时即为本实验的CD38-CAR-NK细胞;
4、NK细胞杀伤活性测定以多发性骨髓瘤细胞RPMI 8226作为靶细胞,效应细胞有2种:CD38-CAR-NK细胞以及作为普通对照的NK92细胞系。操作过程如下:96孔培养板,杀伤实验孔设复孔,每孔加入CD38-CAR-NK细胞1×105个、RPMI 8226细胞2×104个;只含CD38-CAR-NK孔,设复孔,每孔加入CD38-CAR-NK细胞1×105个;只含RPMI 8226细胞孔,复孔,每孔加入RPMI 8226细胞2×104个;每孔补足培养液至200μl。同时设NK92的对照,即以NK92替代CD38-CAR-NK。
5、上述培养板放置CO2培养箱37℃培养6h后,每孔加入10μL CCK-8,震荡混匀;
6、培养箱继续孵育1h,取出96孔板,多功能酶标仪测450mm波长下的OD值。
7、计算CD38-CAR-NK细胞杀伤RPMI 8226细胞的杀伤率;杀伤活性以杀伤率来表示。杀伤率公式为:杀伤率(%)=1-(A-B)/C×100%,其中A是CD38-CAR-NK细胞与RPMI8226细胞共培养孔的吸光度值;B是单独CD38-CAR-NK细胞孔的吸光度值,C是RPMI 8226细胞的吸光度值。
8、CD38-CAR-NK细胞、NK92细胞对RPMI 8226细胞的杀伤率比较
表1CD38-CAR-NK细胞及NK92细胞杀伤多发性骨髓瘤细胞活性比较
CD38-CAR-NK细胞对多发性骨髓瘤细胞‘RPMI 8226’的平均杀伤率为59.1%±2.7%;不含CD38-CAR的普通NK细胞(NK92细胞)对多发性骨髓瘤细胞‘RPMI 8226’的平均杀伤率为32.7%±2.5%。
CD38-CAR-NK细胞对多发性骨髓瘤细胞‘RPMI 8226’的杀伤率显著高于NK92细胞对RPMI 8226的杀伤率(P=0.0003,P<0.01)(表1)。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.一种识别CD38蛋白的单克隆抗体,其特征在于,其轻链可变区基因序列如SEQ IDNO.1所示,其重链可变区基因序列如SEQ ID NO.2所示。
2.根据权利要求1所述的识别CD38蛋白的单克隆抗体,其特征在于,所述抗体的轻链可变区的氨基酸序列如SEQ ID NO.5所示,所述抗体的重链可变区的氨基酸序列如SEQ IDNO.6所示。
3.一种识别CD38蛋白的嵌合抗原受体,其特征在于,所述嵌合抗原受体为CD38-CAR,其含有权利要求1-2任一项所述的识别CD38蛋白的轻链可变区和重链可变区。
4.一种特异序列的核酸分子,其特征在于,其编码权利要求1所述的抗体可变区,或者其编码权利要求3所述的嵌合抗原受体。
5.一种重组基因表达载体,其特征在于,所述重组基因表达载体为CD38-CAR基因表达载体,其含有权利要求4所述的核酸分子。
6.一种免疫细胞,其特征在于,其表达权利要求5所述的基因表达载体。
7.一种识别CD38蛋白的嵌合抗原受体NK细胞,其特征在于,所述嵌合抗原受体NK细胞为CD38-CAR-NK细胞,其表达权利要求3所述的嵌合抗原受体。
8.根据权利要求7所述的识别CD38蛋白的嵌合抗原受体NK细胞,其特征在于,所述CD38-CAR-NK细胞对多发性骨髓瘤细胞的杀伤率为59.1%±2.7%。
9.根据权利要求8所述的识别CD38蛋白的嵌合抗原受体NK细胞,其特征在于,所述多发性骨髓瘤细胞为RPMI 8226细胞。
10.一种权利要求1所述的单克隆抗体、权利要求3所述的嵌合抗原受体、权利要求4所述的核酸分子、权利要求6所述的免疫细胞或权利要求7所述的嵌合抗原受体NK细胞在制备治疗多发性骨髓瘤药物中的应用。
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