CN116925225A - 特异性结合cd7的抗体及其在制备嵌合抗原受体中的应用 - Google Patents
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Abstract
本发明公开了特异性结合CD7的抗体。本发明提供的抗CD7抗体及抗原结合部分均与CD7蛋白具有高亲和性,且结合特异性强。本发明构建的表达靶向CD7的通用型嵌合抗原受体的T细胞。通过实验验证了该嵌合抗原受体修饰的T细胞对表达CD7的急性淋巴细胞白血病(T‑ALL)细胞系及表达CD7的患者原代细胞有较强杀伤作用,对不表达CD7的细胞几乎无杀伤作用,有效防止了脱靶效应。本发明嵌合抗原受体CD7scFv‑CD8α‑4‑1BB‑CD3ζ可用于CD7抗原阳性血液肿瘤的治疗,以及与抗CD5CAR‑T细胞等进行联合治疗。
Description
交叉引用说明
本申请要求于2022年3月29日提交中国专利局、申请号为202210317175.X,发明名称为“特异性结合CD7的抗体及其在制备嵌合抗原受体中的应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域,特别涉及特异性结合CD7的抗体及其在制备嵌合抗原受体中的应用。
背景技术
人CD7分子为免疫球蛋白超家族一员,表达于胸腺细胞、NK细胞、90%以上的T细胞表面。目前已有CD7偶联免疫毒素、CD7纳米抗体以靶向白血病/淋巴瘤肿瘤细胞或人体T细胞,以达到肿瘤清除或预防/治疗移植物抗宿主病的目的。此外,亦有研究通过偶联CD7分子与相应蛋白向T细胞递送特定siRNA,以期实现人类免疫缺陷病(HIV)的控制与治疗。虽然CD7分子并非细胞生存必须,但CD7分子在T/NK细胞中执行的具体功能仍未知,仅有少量早期研究提示CD7分子或与TCR激活通路存在相互作用,影响T细胞活化。阐释CD7分子在免疫细胞/肿瘤细胞中发挥的生理/病理功能仍需大量的研究探索。
近年来,急性T淋巴细胞白血病(T-ALL)患者发病率渐增,患者整体5年生存率仅约50%,儿童患者治愈率略优于成年患者,而成年复发难治患者达长期生存者不足10%。对于复发难治患者,新药的应用效果有限,造血干细胞移植往往成为唯一的治疗选择,但其应用受限于患者疾病状态、骨髓配型结果、移植相关毒性及副作用等诸多因素。因此,此类患者亟需行之有效的新型治疗药物以减轻其疾病负荷,达到疾病缓解或者桥接骨髓移植的治疗目的。
T-ALL患者其肿瘤细胞多高表达CD2、CD5、CD7等抗原,其中CD7抗原普遍高表达,为抗体治疗及嵌合抗原受体T细胞(CAR-T)免疫疗法提供了治疗靶点。但是,由于患者肿瘤细胞即为异常的T细胞,且患者体内高肿瘤负荷亦抑制正常T细胞增殖,临床治疗中采集患者自体T细胞以制备CAR-T困难重重,潜在肿瘤细胞混杂的问题亦为治疗的安全隐患之一。因此,构建异体来源通用型CAR-T产品以应用于T-ALL的治疗尤为关键。另一方面,由于正常T细胞亦表达CD7抗原(表达阳性率在90%以上),为了实现CAR-T细胞有效增殖,需要避免细胞间“自相残杀”。现有技术仍有待探索与完善以缺乏解决上述问题,并且效果更优的抗体及嵌合抗原受体仍需进一步研发。因此,寻找该抗体及嵌合抗原受体成为当务之急。
发明内容
本发明的一个方面,是针对现有技术中特异性结合CD7的抗体亲和力差的问题,提供了一种特异性结合CD7的抗体及其在制备嵌合抗原受体中的应用。
本发明提供的技术方案为:
特异性结合CD7的抗体,所述抗体包含如SEQIDNo.1-3所示的轻链可变区的CDR1、CDR2、CDR3序列,和如SEQIDNo.4-6所示的重链可变区的CDR1、CDR2、CDR3序列,或与所述序列具有80%以上同源性的序列。
在本发明的某些实施方式中,可以对上述互补决定区(CDRs)进行点突变,其目的可以为,例如获得更好的亲和力和/或解离性质,获得更好的表达水平,等,只要其能够与抗原决定簇形成互补结合的结构即可,该突变后的氨基酸序列也视为包含在本发明的保护范围之内。
本发明的另一个方面,是提供了一种特异性结合CD7的抗原结合部分,所述抗原结合部分包含如SEQIDNo.1-3所示的轻链可变区的CDR1、CDR2、CDR3序列,和如SEQIDNo.4-6所示的重链可变区的CDR1、CDR2、CDR3序列,或与上述序列具有80%以上同源性的序列。
在本发明中,上述与其90%以上同源性的,是指与本发明中所述核苷酸、氨基酸序列具有90%以上同源性,其可以为具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的同源性,可以以合适的方式对本发明中所述核苷酸、氨基酸序列进行随机或者工程化的点突变,其目的可以为,例如,获得更好的表达水平、亲和力和/或解离性质,而这些突变后的核苷酸或氨基酸序列均包含在本发明的保护范围之内。
作为优选,在本发明的某些实施方式中,上述抗原结合部分可以为Fab、Fab′、F(ab′)2、Fd、Fv、scFv或单结构域抗体(dAB),其能够与靶蛋白形成抗原-抗体复合结构。
更优选地,在本发明的一个实施方式中,上述抗原结合部分为scFv。
在本发明的某些实施方式中,上述抗CD7 scFv包含重复个数的VL和VH序列,上述VL和VH序列包含上述CDRs。上述VL和VH之间可以通过合适的连接体/连接肽(Linker)连接,其通常可以由甘氨酸(Gly)和丝氨酸(Ser)构成,例如,GGGGS。
在本发明的某些实施方式中,上述抗CD7 scFv为一价抗体。在本发明的另一些实施方式中,上述轻链可变区(VL)和重链可变区(VH)的重复序列数可以为2或3。当上述重复序列数为2时,上述抗CD7scFv形成二价Diabody的形式;当上述重复序列数为3时,上述抗CD7scFv形成三价Triabody的形式。上述轻链可变区(VL)和重链可变区(VH)可以以任意合适的顺序连接,例如,N-VL-VH-C、N-VH-VL-C。
作为优选,在本发明的某些实施方式中,上述单价的scFv的轻链可变区的氨基酸序列如SEQIDNo.7所示,上述述单价的scFv的重链可变区的氨基酸序列如SEQIDNo.8所示,或与所述序列具有80%以上同源性的序列。
更优选地,在本发明的一个实施方式中,上述单价的scFv的氨基酸序列如SEQIDNo.9所示,或与其具有80%以上同源性的序列。
上述特异性结合CD7的抗原结合部分可使用本领域技术人员公知的方法获得,如利用化学试剂处理的方法,或利用蛋白酶消化的方法,如木瓜蛋白酶、胃蛋白酶等,或构建载体在表达系统中表达,或通过合成的方法获得。
在本发明中,可以采用Kohler等在Nature 256:495(1975)中报道的杂交瘤制备方法来制备所述抗体。首先用免疫原(必要时候添加佐剂)免疫注射小鼠或其它合适的宿主动物。
免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。佐剂可以利用弗氏佐剂(弗氏完全性佐剂或者弗氏不完全性佐剂)或MPL-TDM等。动物在接受免疫后,体内会产生分泌特异性结合免疫原的抗体的淋巴细胞。收集目的淋巴细胞,并用合适的融合剂(如PEG4000)将其与骨髓瘤细胞融合,从而获得杂交瘤细胞(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,AcademicPress,1996)。
将上述制备的杂交瘤细胞接种到合适的培养基中进行生长,所述培养基中含有一种或多种能够抑制未融合的、母体骨髓瘤细胞生长的物质。例如,对于缺乏次黄嘌呤鸟嘌呤磷酸转移酶(HGPRT或HPRT)的母体骨髓瘤细胞,在培养基中添加次黄嘌呤、氨基喋呤和胸腺嘧啶(HAT培养基)等物质将可以抑制HGPRT-缺陷细胞的生长。
优选的骨髓瘤细胞应该具有融合率高,抗体分泌能力稳定,对HAT培养基敏感等能力。其中,骨髓瘤细胞首选鼠源骨髓瘤,如MOP-21和MC-11小鼠肿瘤衍生株(THE SalkInstitute Cell Distribution Center,San Diego,Calif.USA),以及SP-2/0或X63-Ag8-653细胞株(American Type Cμlture Collection,Rockville,Md.USA)。另外,还可以利用人骨髓瘤和人鼠异源骨髓瘤细胞株制备人单抗(Kozbor,J.Immunol.,133:3001(1984);Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63,Marcel Dekker,Inc.,New York,1987)。
杂交瘤细胞生长的培养基用于检测针对特异抗原的单抗的产生。可以使用下列方法来测定杂交瘤细胞产生的单抗的结合特异性:免疫沉淀或体外结合试验,如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如,利用Munson等在Anal.Biochem.107:220(1980)中描述的Scatchard分析法可测定单抗的亲和力。
在确定杂交瘤产生的抗体的特异性、亲和力和反应性之后,目的细胞株可以通过Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,AcademicPress,1996描述的有限稀释法进行亚克隆化。合适的培养基可以是DMEM或RPMI-1640等。另外,杂交瘤细胞还可以腹水瘤的形式在动物体内生长。
利用传统的免疫球蛋白纯化方法,如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等,可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来,进而得到单克隆抗体。
本发明的另一个方面,是提供了一种多核苷酸,所述多核苷酸编码上述抗体,或上述抗原结合部分。
进一步地,本发明的多核苷酸序列可以以合适的方法插入到任意合适的表达载体中,例如,细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。同样地,在合适的具有遗传功能的宿主细胞中也可包含上述多核苷酸或上述表达载体,例如,表达嵌合抗原受体的T细胞或NK细胞。
本发明的另一个方面,是提供了一种载体,所述载体包含上述多核苷酸的序列。
本发明的另一个方面,是提供了一种分离的细胞,所述细胞包含上述抗体、上述抗原结合部分、上述多核苷酸或上述载体;
所述细胞为选自SP2/0、YB2/0、IR983F、PERC6、CHO细胞系或昆虫细胞系。
本发明的另一个方面,是提供了上述抗体或上述抗原结合部分在制备检测或治疗肿瘤的产品中的用途。
作为优选,在本发明的某些实施方式中,所述肿瘤为血液肿瘤。
更优选地,在本发明的某些实施方式中,所述血液肿瘤为急性T淋巴细胞白血病、T淋巴母细胞白血病/淋巴瘤、外周T细胞淋巴瘤、血管免疫母细胞性T细胞淋巴瘤、间变性大细胞淋巴瘤、CD7表达阳性急性髓系白血病。
上述检测肿瘤的产品可以为检测样本中CD7含量的试剂、试剂盒或细胞培养板。
本发明的另一个方面,是提供了一种药物组合物,所述药物组合物包含上述抗体,或上述抗原结合部分,或上述分离的细胞,或上述多核苷酸,以及药学上可接受的载体。
上述药物组合物可以为口服剂或注射剂。
本发明的另一个方面,是提供了一种免疫偶联物,所述免疫偶联物包括:
a)上述抗体,或上述抗原结合部分;
b)选自药物、酶、可检测标记物、毒素、细胞因子或放射性核素的偶联部分;
c)部分a和部分b的连接体。
本发明中的抗体或抗原结合部分可以以任意合适的方式与偶联部分进行偶联,形成偶联物,例如,抗体药物偶联物(antibody-drug conjugate,ADC),用来预防或治疗肿瘤。
本发明的另一个方面,是提供了一种嵌合抗原受体,包含胞外区、跨膜区和胞内区,所述胞内区包含胞内信号转导区,所述嵌合抗原受体的胞外区包含CD7结合结构域,所述CD7结合结构域包含上述的抗原结合部分。
作为优选,在本发明的一个实施方式中,上述抗原结合部分的氨基酸序列如SEQIDNo.9所示,或与其具有80%以上同源性的序列。
在本发明的某些实施方式中,上述胞外区还包含信号肽。信号肽可引导抗原识别区及铰链区转移到胞外。任意合适的信号肽或信号肽的组合均可实现本发明的目的。
作为优选,在本发明的某些实施方式中,上述胞外区还包含构建在所述的嵌合抗原受体氨基末端的信号肽或与所述信号肽具有90%以上同源性的氨基酸序列;
更优选地,在本发明的一个实施方式中,所述信号肽为CD8α中的信号肽序列或GM-CSF。
进一步优选地,在本发明的一个实施方式中,所述信号肽为如SEQ ID NO.10所示的信号肽。
在本发明的某些实施方式中,本发明所述嵌合抗原受体包含的所述CD7结合结构域通过铰链区与其编码的所述跨膜区连接。任意合适的铰链区序列均可实现本发明的目的。作为优选,在本发明的一个实施方式中,所述铰链区为CD8α中的铰链区序列。
在本发明的某些实施方式中,上述嵌合抗原受体还包括跨膜结构域。任意合适的跨膜结构域均能实现本发明的目的。作为优选,在本发明的某些实施方式中,所述跨膜区可以为选自以下蛋白质的跨膜结构域或与所述蛋白质具有90%以上同源性的氨基酸序列:T细胞受体的α、β或ζ链、CD2、CD3ε、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、CD18、CD19、CD27、CD28、CD29、CD30、CD40、CD48、CD49a、CD49d、CD49f、CD66a、CD66b、CD66c、CD66d、CD66e、CD69、CD79A、CD79B、CD84、CD96、CD100、CD103、CD134、CD137、CD150、CD158A、CD158B1、CD158B2、CD158C、CD158D、CD158F1、CD158F2、CD158K、CD160、CD162、CD226、CD229、CD244、CD247、CD258、CD268、CD270、CD272、CD276、CD279、CD314、CD319、CD335、CD336、CD337、CD352、CD353、CD355、CD357、LFA-1、NKG2C、DAP-10、ICAM-1、NKp80、IL-2R beta、IL-2Rgamma、IL-7R alpha、LFA-1、SLAMF9、LAT、GADS、SLP-76、PAG1/CBP、CD83配体、Fc gamma受体、整联蛋白、激活性NK细胞受体或Toll配体受体,或其组合。
更优选地,在本发明的一个实施方式中,所述跨膜区为CD8α中的跨膜区序列。
在本发明的某些实施方式中,上述嵌合抗原受体包含的所述胞内区还包含共刺激因子;
作为优选,在本发明的某些实施方式中,所述共刺激因子可以为通过选自以下蛋白质或与所述蛋白质具有90%以上同源性的氨基酸序列获得的功能性信号结构域的一种或几种:整联蛋白、BTLA、Toll配体受体、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1、4-1BB、B7-H3、CD278、GITR、BAFFR、LIGHT、HVEM、KIRDS2、SLAMF7、NKp80、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49α、IA4、CD49D、ITGA6、VLA6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11α、ITGAM、CD11b、ITGAX、CD11c、CD29、ITGB1、ITGB2、CD18、ITGB7、NKG2D、NKG2C、TNFR2、CD226、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、CD162、LTBR、LAT、GADS或SLP-76;
更优选地,在本发明的一个实施方式中,所述共刺激因子为CD28或4-1BB,或与其具有90%以上同源性的氨基酸序列。
在本发明的某些实施方式中,上述嵌合抗原受体还包含胞内信号转导区。作为优选,在本发明的某些实施方式中,所述胞内信号转导区可以为选自以下蛋白质或与所述蛋白质具有90%以上同源性的氨基酸序列:4-1BB、B7-H3、BAFFR、BLAME、BTLA、CD100、CD103、CD160、CD18、CD19、CD19a、CD2、CD247、CD27、CD276、CD28、CD29、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8alpha、CD8beta、CD96、CDS、CEACAM1、CRTAM、DAP-10、DNAM1、Fc gamma受体、GADS、GITR、HVEM、IA4、ICAM-1、ICAM-1、Ig alpha、IL2R beta、IL2R gamma、IL7R alpha、整联蛋白、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、KIRDS2、LAT、LFA-1、LFA-1、LIGHT、LIGHT、LTBR、Ly9、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80、OX-40、PAG/Cbp、PD-1、PSGL1、SELPLG、SLAMF4、SLAMF6、SLAMF7、SLP-76、TNFR2、Toll配体受体、TRANCE/RANKL、VLA1或VLA-6,或其组合。
更优选地,在本发明的一个实施方式中,所述胞内信号转导区为CD3ζ。
作为优选,在本发明的某些实施方式中,上述嵌合抗原受体是以CD7 scFv抗原识别区、CD8α铰链区和跨膜区、4-1BB共刺激分子及CD3ζ胞内信号结构域串联而成的结构为信号传导结构域,其串联序列如SEQ ID NO.12所示核酸序列、SEQ ID NO.11所示氨基酸序列。
另外,在上述抗原识别区、铰链区、跨膜区以及胞内信号区之间合适的位置可插入任意肽链作为间隔区,所述肽链可以为寡肽或多肽。
本发明的另一个方面,是提供了一种多核苷酸,所述多核苷酸编码上述的嵌合抗原受体;
作为优选,在本发明的一个实施方式中,上述多核苷酸的序列如SEQIDNo.12所示。
对于上述核酸分子的制备方法,可基于上述抗原识别区、铰链区、跨膜区以及胞内信号区等结构域的碱基序列,通过化学合成或PCR扩增等已知技术制备。通常,可以对编码上述结构域的氨基酸的密码子进行优化,以优化其在宿主细胞中的表达。上述碱基序列的信息可通过检索已知文献或NCBI(https://www.ncbi.nlm.nih.gov/)等数据库来获得。
在本发明的一个实施方式中,发明人采用PCR扩增的方法获得CD7结合结构域CD7scFv抗原识别区的碱基序列。具体为,提取小鼠抗人CD7单克隆抗体杂交瘤细胞株的总RNA,逆转录合成cDNA第一链,使用“小鼠抗体scFv基因扩增试剂盒”PCR扩增合成鼠抗人CD7scFv。
本发明的另一个方面,是提供了一种载体,所述载体包含上述核酸分子。
在本发明中,所述载体可以为直链载体,也可以为环状载体。可以为质粒等非病毒载体,也可以为病毒载体,还可以为利用转座子的载体。所述载体中可含有启动子、终止子等调控序列,以及耐药基因、报告基因等标记序列。另外,上述载体也可包含编码自杀基因的序列,可根据治疗过程,通过给予激活自杀基因的物质,从而控制体内CAR-T细胞的数目。
作为上述病毒载体,可以为逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体等。在本发明的一个实施方式中,使用的是慢病毒表达载体。
本发明的另一个方面,是提供了一种分离的细胞,所述细胞中包含上述的抗原结合部分、上述的嵌合抗原受体,或上述的多核苷酸的序列;
所述细胞为T细胞或NK细胞;
作为优选,在本发明的某些实施方式中,上述T细胞为自体来源的T细胞或异体来源的T细胞。
在本发明的某些实施方式中,上述细胞为人的T细胞。所述T细胞可以来自血液、骨髓等体液,也可以来自脾脏、胸腺、淋巴等组织,或者原发肿瘤、转移性肿瘤、癌性腹水等癌症组织,经分离、纯化后得到。作为优选,所述T细胞为自体T细胞。同时,所述T细胞可以为CD4+T细胞、CD8+T细胞、αβT细胞或γδT细胞,也可以为上述多种T细胞的混合物。所述T细胞可以以合适的方式替换为NK细胞,其也视为包含在本发明的保护范围之内。
为了实现本发明的一个目的,在本发明的某些实施方式中,上述细胞为异体来源的T细胞,所述T细胞为通用型嵌合抗原受体T细胞(CAR-T);
作为优选,在本发明的一个实施方式中,所述T细胞缺失CD7基因序列;
更优选地,在本发明的一个实施方式中,所述T细胞还缺失TRAC基因序列。
本发明的另一个方面,是提供了一种通用型CAR-T细胞的构建方法,所述方法为以电穿孔方式向T细胞中导入靶向TRAC基因的核蛋白复合物,以实现TRAC基因的敲除和TCRα/β抗原表达的缺失。
本发明的另一个方面,是提供了一种CD7-/-CAR-T的构建方法,所述方法为以电穿孔方式向T细胞中导入靶向CD7基因的核蛋白复合物,以实现CD7基因的敲除和CD7膜抗原表达的缺失。
作为优选,在本发明的一个实施方式中,利用CRISPR/Cas9系统对上述基因进行敲除,其中,所使用的gRNA(guideRNA)的序列为:
CD7 gRNA:GGGGTCAATGTCTACGGCTC
TRACgRNA:AGAGTCTCTCAGCTGGTACA
本发明的另一个方面,是提供了上述的抗体、上述的抗原结合部分、上述的多核苷酸、上述的载体、上述的分离的细胞,或上述的嵌合抗原受体在制备治疗肿瘤的药物中的用途;
作为优选,在本发明的某些实施方式中,所述肿瘤为血液肿瘤;
更优选地,在本发明的某些实施方式中,所述肿瘤为T细胞血液肿瘤;
进一步优选地,在本发明的某些实施方式中,所述肿瘤为CD7抗原阳性的急性T淋巴细胞性白血病或淋巴瘤;
进一步优选地,在本发明的一个实施方式中,所述肿瘤为难治或复发的CD7抗原阳性的急性T淋巴细胞性白血病或淋巴瘤。
本发明的另一个方面,是提供了一种药物组合物,所述药物组合物包含上述的抗体、上述的抗原结合部分、上述的多核苷酸、上述的嵌合抗原受体、上述的载体或上述的细胞,以及药学上接受的载体。
本发明药物组合物除包含上述成分以外,还可包含任意药学上允许的添加剂,例如,生理盐水、细胞培养基、葡萄糖、注射用水、甘油、乙醇以及它们的组合物、稳定剂、表面活性剂、防腐剂、等渗剂等。
同样,本发明药物组合物也可以和其他合适的抗癌剂联合应用。例如,长春新碱、柔红霉素、门冬酰胺酶、环磷酰胺、泼尼松等。
作为优选,在本发明的一个实施方式中,上述药物组合物还包含抗CD5的CAR-T细胞。
本发明的另一个方面,是提供了一种治疗血液肿瘤的方法,向患者施用上述抗体、上述抗原结合部分、上述细胞或上述药物组合物。
本发明的另一个方面,是提供了上述药物组合物在制备治疗肿瘤的药物中的用途;
作为优选,在本发明的某些实施方式中,所述肿瘤为血液肿瘤;
更优选地,在本发明的某些实施方式中,所述肿瘤为T细胞血液肿瘤;
进一步优选地,在本发明的某些实施方式中,所述肿瘤为CD7抗原阳性的急性T淋巴细胞性白血病或淋巴瘤;
进一步优选地,在本发明的一个实施方式中,所述肿瘤为难治或复发的CD7抗原阳性的急性T淋巴细胞性白血病或淋巴瘤。
本发明的有益效果为:
本发明提供的抗CD7抗体及抗原结合部分均与CD7蛋白具有高亲和性,且结合特异性强。本发明构建的表达靶向CD7的通用型嵌合抗原受体的T细胞。通过实验验证了该嵌合抗原受体修饰的T细胞对表达CD7的急性淋巴细胞白血病(T-ALL)细胞系及表达CD7的患者原代细胞有较强杀伤作用,对不表达CD7的细胞几乎无杀伤作用,有效防止了脱靶效应。本发明嵌合抗原受体CD7scFv-CD8α-4-1BB-CD3ζ可用于CD7抗原阳性血液肿瘤的治疗,以及与抗CD5CAR-T细胞等进行联合治疗。
附图说明
图1为本发明实施例中鼠抗人CD7单克隆抗体轻链可变区(VL)和重链可变区(VH)的PCR扩增电泳图,其中,1为2Kbp核酸分子量标准泳道,2、3为VL泳道,4、5为VH泳道;
图2为VL和VH序列进行胶回收纯化,对两者进行overlap PCR扩增鼠源CD7 VL-VH(mLH)片段序列的电泳图,其中,1为2Kbp marker分子量标记泳道,2-5为CD7 VL-VH片段泳道;
图3为CD7靶向嵌合抗原受体质粒结构示意图;
图4为未经敲除与慢病毒感染的原代T细胞(记作primary T)、未敲除但慢病毒感染的CAR-T细胞(记作WT-7CAR)、敲除但未经慢病毒感染的T细胞(记作T-knockout)、敲除并进行慢病毒感染的CAR-T细胞(记作KO-7CAR),细胞扩增倍数统计图;
图5为TRAC与CD7敲除后T细胞CD7CAR感染效率检测结果图;
图6为实验组T细胞CD7、TCRα/β表达结果检测图,其中,A为单次实验敲除CD7、TCRα/β效率示意结果,B为多次实验敲除效率统计结果;
图7为primary T、WT-7CAR、T-knockout、KO-7CAR细胞抑制性受体PD-1、LAG-3、TIM-3表达结果统计图;
图8为primary T、WT-7CAR、T-knockout、KO-7CAR细胞活化相关受体CD25、CD69表达结果统计图;
图9为primary T、WT-7CAR、T-knockout、KO-7CAR细胞活化诱导后细胞凋亡(AICD)检测结果图;
图10为primary T、WT-7CAR、T-knockout、KO-7CAR细胞CD8表达及细胞分群统计结果图;
图11为靶细胞系及患者原代细胞CD7抗原表达检测结果图;
图12为primary T、T-knockout、KO-7CAR细胞与靶细胞系按照效靶比5:1、1:1共培养24h、48h残余靶细胞比例3次独立重复实验的检测结果汇总图;
图13为T/CAR-T细胞与3例患者原代细胞共培养检测结果,其中,A为primary T、WT-7CAR、T-knockout、KO-7CAR细胞与3例患者原代细胞以效靶比1:1、1:2、1:4共培养24h、48h后残余靶细胞比例,B为T/CAR-T细胞与患者原代细胞共培养48h后,患者原代细胞CD7抗原表达检测结果;
图14为T/CAR-T细胞脱颗粒检测结果图,其中,A为primary T、T-knockout、KO-7CAR细胞与靶细胞系共培养脱颗粒检测结果,B为primary T、WT-7CAR、T-knockout、KO-7CAR细胞与患者原代细胞共培养脱颗粒检测结果;
图15为通用型CAR-T体内实验小动物活体成像检测结果图;
图16为本发明抗体或抗原结合部分(APC荧光通道)、CD7-6B7商品化抗体(PE/Cy7荧光通道)与CD7抗原阴性的K562及Raji细胞的结合情况结果图;
图17为本发明抗体或抗原结合部分(APC荧光通道)、CD7-6B7商业化抗体(PE/Cy7荧光通道)与CD7抗原阳性的Jurkat、Molt4、CEM及Hut78细胞表面CD7抗原的结合情况结果图;
图18为FACS法检测本发明抗体或抗原结合部分与CD7-6B7同CD7抗原竞争结合的情况结果图。
序列说明
SEQIDNo.1-3为本发明抗体或抗原结合部分的轻链可变区的CDR1-3的氨基酸序列;
SEQIDNo.4-6为本发明抗体或抗原结合部分的重链可变区的CDR1-3的氨基酸序列;
SEQIDNo.7为本发明实施例中单链抗体scFv的轻链可变区的氨基酸序列;
SEQIDNo.8为本发明实施例中单链抗体scFv的重链可变区的氨基酸序列;
SEQIDNo.9为本发明实施例中单链抗体scFv的氨基酸序列;
SEQIDNo.10为本发明实施例中信号肽的氨基酸序列;
SEQIDNo.11为本发明实施例中嵌合抗原受体CD8α信号肽-CD7结合域-CD8α铰链区-CD8α跨膜区-41BB共刺激区-CD3ζ信号转导区的氨基酸序列;
SEQIDNo.12为本发明实施例中嵌合抗原受体CD8α信号肽-CD7结合域-CD8α铰链区-CD8α跨膜区-41BB共刺激区-CD3ζ信号转导区的核苷酸序列。
具体实施方式
本发明公开了一种特异性结合CD7的抗体及其在制备嵌合抗原受体中的应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。需要特别指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明,并且相关人员明显能在不脱离本发明内容、精神和范围的基础上对本文所述内容进行改动或适当变更与组合,来实现和应用本发明技术。
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。除非另有其它明确表示,否则在整个说明书和权利要求书中,术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分,而并未排除其它元件或其它组成部分。术语“如”、“例如”等旨在指示例性实施方案,而不意图限制本公开的范围。
下面就本发明中出现的部分术语作以解释。
术语“抗体”,是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε,并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内,可变区和恒定区通过大约12或更多个氨基酸的“J”区连接,重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子,包括免疫系统的各种细胞(例如,效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR)),其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。。术语“抗体”不受任何特定的产生抗体的方法限制。例如,其包括,特别地,重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体,例如,IgG(例如,IgG1,IgG2,IgG3或IgG4亚型),IgA1,IgA2,IgD,IgE或IgM抗体。
术语“抗原结合部分”,是指包含全长抗体的片段的多肽,其保持特异性结合全长抗体所结合的相同抗原的能力,和/或与全长抗体竞争对抗原的特异性结合,其也被称为“抗原结合片段”。通常参见,Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版,RavenPress,N.Y.(1989),其以其全文通过引用合并入本文,用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下,抗原结合片段包括Fab、Fab′、F(ab′)2、Fd、Fv等。
其中,术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段;术语“F(ab′)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fd片段”意指由VH和CH1结构域组成的抗体片段;术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段。
在本文中,除非上下文明确指出,否则当提及术语“抗体”时,其不仅包括完整抗体,而且包括抗体的抗原结合片段。
术语“特异性结合”,是指两分子间的非随机的结合反应,如抗体和其所针对的抗原之间的反应。
术语抗体的“可变区”,是指抗体轻链的可变区或抗体重链的可变区,单独或组合。如本领域已知的,重链和轻链的可变区各自由通过也称作高变区的3个互补决定区(CDR)连接的4个框架区(FR)组成。每条链中的CDR通过FR紧紧保持在一起,以及与来自其他链的保持在一起,有助于形成抗体的抗原结合位点。有至少两种技术用于确定CDR:(1)基于跨物种序列变异性的方法(即,Kabat et al.Sequences of Proteins of ImmunologicalInterest,(5th ed.,1991,National Institutes of Health,Bethesda MD));以及(2)基于抗原-抗体复合物的晶体学研究的方法(Al-lazikani et al.,1997,J.Molec.Biol.273:927-948)。如本文所用,CDR可以指通过任一种方法或通过两种方法的组合定义的CDR。
术语“共刺激”,是指为免疫细胞激活免疫应答的次级信号事件;免疫细胞在抗原呈递细胞存在下依靠共刺激来激活免疫应答,对于T细胞,需要两个刺激才能完全激活其免疫应答,就是说在淋巴细胞活化过程中,共刺激通常对有效免疫应答的发展至关重要,除了来自其抗原受体的抗原特异性信号之外,还需要共刺激。第二个信号使活化的T细胞免于无反应性,使T细胞产生额外T细胞生长所必需的淋巴因子。
为了使本领域的技术人员更好地理解本发明的技术方案,下面结合具体实施例对本发明作进一步的详细说明。
实施例1:CD7-HIT7单克隆抗体的制备
1.小鼠杂交瘤单克隆抗体筛选
以人CD7蛋白为免疫原,采用腹腔注射的方式免疫Balb/c小鼠,分别在初次免疫后的第3周和第5周进行加强免疫,在加强免疫后的第8天取小鼠尾血,室温静置1小时,4℃,12000rpm离心10分钟,收集血清,以PBS稀释成不同浓度:1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800。收集Jurkat细胞,PBS洗1次,细胞计数,每个样品用1×106个细胞,将100μl不同稀释度的血清加入到细胞中,阴性对照组以未免疫小鼠血清代替抗血清,4℃孵育1小时,PBS洗两次;加入PE标记大鼠抗小鼠IgG抗体2μl,4℃避光孵育40分钟;细胞重悬于500μl PBS缓冲液中,流式细胞仪检测血清中抗体与细胞的结合百分率及荧光强度;以平均荧光强度为阴性对照两倍以上为有效效价,效价高于6400时,方可进行融合。融合前3天,对免疫小鼠通过尾静脉注射免疫原的方式进行冲击免疫。取成功免疫的小鼠脾脏细胞与骨髓瘤SP2/0细胞进行细胞融合(以10:1比例),融合时在37℃水浴的环境下,将50%的PEG在1min之内加入混匀且弃去上清的脾脏细胞与骨髓瘤细胞细胞团之中,37℃水浴震荡1min,再在2min之内加入10ml无血清1640培养基。800rpm,6min离心,弃去上清,用含有HAT的1640培养基重悬细胞,并移液入96孔板(2.5×107细胞/板)。在37℃、5%CO2条件下培养细胞。当融合板中克隆足够大时,每孔取100μl上清液与2×105个Jurkat细胞共孵育,进行检测,方法与检测效价相同。平均免疫荧光强度为阴性孔两倍以上作为阳性孔,进行下一步克隆化培养。将筛选阳性的杂交瘤克隆从96孔板扩至24孔板培养3-5天,再次进行培养上清筛选检测,检测阳性的克隆再进行下一步的亚克隆培养,剩余细胞冻存。收集24孔板中杂交瘤细胞,细胞计数,将细胞密度调整为10个/mL;将细胞铺到96孔板中,每孔100μl,37℃、5%CO2孵箱培养;培养10天左右,可见克隆形成,选取只有单个克隆的孔,吸取培养上清,检测方法同前,选取阳性克隆,扩至24孔板培养,再次上清检测后,选择阳性克隆进行第二轮的亚克隆培养,一般进行多轮亚克隆培养后,直至所有检测孔均为阳性为止,即获得稳定的杂交瘤细胞株。选取阳性杂交瘤培养上清,采用抗体亚型检测试纸检测抗体的亚型,本发明中的单克隆抗体编号为HIT7,为鼠源IgG1亚型,轻链为κ链。
2.腹水制备及纯化
无菌PBS溶液洗涤杂交瘤细胞,以5×106/0.5ml/只的细胞量腹腔注射到液体石蜡预致敏的Balb/c小鼠体内。7至10天后收集腹水,室温3000rpm,10min,收集上清。以终浓度为33%的饱和硫酸铵对抗体进行粗纯,方法为取1份腹水加1份PBS,滴加1份饱和硫酸铵,边加边搅拌,4℃过夜,10000rpm离心10min除去上清,用少量PBS溶解沉淀,在4℃环境下用PBS透析除盐24h,期间换液3次。粗纯后的抗体利用AKTA蛋白纯化系统,经1ml Protein G纯化预装柱进行进一步的纯化。所得抗体纯品用于后续的抗体检测及功能实验。
3.通过蛋白质测序得到上述单克隆抗体HIT7的轻链可变区的CDR1、CDR2、CDR3序列如SEQIDNo.1-3所示,重链可变区的CDR1、CDR2、CDR3序列如SEQIDNo.4-6所示。
实施例2:嵌合抗原受体中抗原识别区CD7scFv的克隆
1)提取实施例1中制备的鼠抗人CD7单克隆抗体杂交瘤细胞株的总RNA:在5×106细胞中加入RNA iso Plus(Takara)1ml,吹打混匀。加入200μl三氯甲烷,上下颠倒、涡旋振荡混匀。4℃,12000rpm,离心5分钟。吸取上清至1.5ml EP管,加入同体积异丙醇,轻轻上下颠倒混匀。4℃,12000rpm,离心15分钟。4℃预冷75%乙醇沉淀RNA,50μl DEPC水溶解总RNA。
2)逆转录合成cDNA第一链:配制PCR反应体系(20μl)如下:Oligo d(T)15Primers:2μl;M-MLV(200u/μl):1μl;dNTP(each 2.5mM):1μl;DTT(0.1M):2μl;First strand buffer(5×):4μl;BCMA-RNA:2μg;DEPC水:补足至20μl。反应条件:37℃,60分钟,70℃10分钟。
3)使用“小鼠抗体scFv基因扩增试剂盒”(Public Protein/Plasmid Library)PCR扩增鼠抗人CD7单克隆抗体轻链(VL)和重链(VH)的基因片段:
扩增VL链:配制PCR反应体系(50μl)如下:MVL mix:45μl;DNA polymerase:0.3μl;cDNA:400ng;ddH2O:补足至50μl。反应条件:94℃预变性3分钟;重复如下循环30次:94℃30秒,56℃30秒,72℃45秒;72℃延伸10分钟;
扩增VH链:配制PCR反应体系(50μl)如下:MVH mix:45μl;DNA polymerase:0.3μl;cDNA:400ng;ddH2O:补足至50μl。反应条件:94℃预变性3分钟;重复如下循环30次:94℃30秒,56℃30秒,72℃45秒;72℃延伸10分钟;
琼脂糖凝胶电泳分离并回收VL、VH片段。结果如图1所示。
4)将VL、VH连接至pMD19-simple T载体并测序,根据测序结果确定BCMA单克隆抗体VL、VH的核酸序列。而后用计算机软件进行人源化模拟,得到人源化的VL、VH核酸序列并将其合成。
5)overlapPCR方法构建VL-linker-VH方向CD7 scFv片段:
P1:CTAGCTAGCGACATTGTGCTGACACAGTCTCC
P2:GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGGTGCAGCTGCAGGAGTC
P3:AGAACCACCACCACCGGAGCCGCCGCCGCCAGAACCACCACCACCAGCCCGTTTTATTTCCAGCTTGG
P4:CCGGAATTCCTGAAGAGACGGTGACCGTG
配制第一轮PCR反应体系,获得NheⅠ-VL-linker片段(50μl):
2×Pfu PCR Master Mix(TianGen公司):25μl;
10μMP1+P2:2μl;
pMD19-VL质粒:100ng;
ddH2O:补足至50μl。
反应条件:94℃预变性5分钟;重复如下循环32次:94℃30秒,60℃30秒,72℃45秒;最后,72℃延伸10分钟;
配制第一轮PCR反应体系,获得linker-VH-EcoRⅠ片段(50μl):
2×Pfu PCR Master Mix(TianGen公司):25μl;
10μMP3+P4:2μl;
pMD19-VH质粒:100ng;
ddH2O:补足至50μl。
反应条件:94℃预变性5分钟;重复如下循环32次:94℃30秒,60℃30秒,72℃45秒;72℃延伸10分钟;
琼脂糖凝胶电泳分离并回收NheⅠ-VL-linker、linker-VH-EcoRⅠ片段;
配制第二轮PCR反应体系,获得CD7 scFv(VL-linker-VH方向)片段(50μl):
2×Pfu PCR Master Mix(TianGen公司):25μl;
10μMP1+P4:2μl;
NheⅠ-VL-linker片段:100ng;
linker-VH-EcoRⅠ片段:100ng;
ddH2O:补足至50μl。
反应条件:
94℃预变性5分钟;重复如下循环32次:94℃30秒,58℃90秒;72℃延伸10分钟。
琼脂糖凝胶电泳分离并回收VL-linker-VH方向CD7 scFv片段,结果如图2所示。
也可以通过商业化合成本实施例中所述的序列。
实施例3:CD7靶向嵌合抗原受体的构建
1)采用Nhe I、EcoR I核酸内切酶酶切含有CD8α-4-1BB-CD3ζ片段的载体质粒(质粒系实验室前期构建),获得含CD8α-4-1BB-CD3ζ的载体片段。所述含有CD8α-4-1BB-CD3ζ片段的质粒可通过现有技术中任意合适的方法制得。
2)将实施例1中得到的CD7 scFv(mLH)和CD7scFv(mHL)片段与目的载体进行连接,将构建的CD7-VL-VH-CD8α-4-1BB-CD3ζCAR目的载体用NheⅠ和NotⅠ进行酶切鉴定,并送菌液测序鉴定,序列比对正确即为质粒构建成功。质粒示意图如图3所示。
实施例4:慢病毒制备与转染
1)质粒提取:
取以上测序正确的菌液以1:100的比例加入新鲜的含有氨苄青霉素的LB液体培养基(20-30ml),在37℃恒温摇床中以200rpm/min的速度震荡培养12-16小时,利用TIANGEN公司的质粒提取试剂盒提取质粒;
1.柱平衡步骤:向吸附柱CP4中(吸附柱放入收集管中)加入500μl的平衡液BL,12,000rpm(~13,400g)离心1min,倒掉收集管中的废液,将吸附柱重新放回收集管中。
2.取菌液加入离心管中,12,000rpm(~13,400g)离心1min,尽量吸除上清。
3.向留有菌体沉淀的离心管中加入500μl溶液P1(已加入RNaseA),使用移液器或涡旋振荡器彻底悬浮细菌细胞沉淀。
4.向离心管中加入500μl溶液P2,温和地上下翻转6-8次使菌体充分裂解。
5.向离心管中加入500μl溶液P4,立即温和地上下翻转6-8次,充分混匀,可见出现白色絮状沉淀,后室温放置10min左右,12,000rpm(~13,400g)离心10min。
6.将上一步收集的上清液分次加入过滤柱CS(过滤柱放入收集管中),12,000rpm(~13,400g)离心2min,滤液收集在干净的2ml离心管中。
7.向滤液中加入0.3倍滤液体积的异丙醇,上下颠倒混匀后转移到吸附柱CP4中(吸附柱放入收集管中)。吸附柱CP4的最大容积为700μl,根据需要分次过柱。
8.室温12,000rpm(~13,400g)离心1min,倒掉收集管中的废液,将吸附柱重新放回收集管中。
9.向吸附柱CP4中加入500μl去蛋白液PD,12,000rpm(~13,400g)离心1min,倒掉收集管中的废液,将吸附柱CP4放入收集管中。
10.向吸附柱CP4中加入600μl漂洗液PW(已加入无水乙醇),室温静置2-5min,12,000rpm(~13,400g)离心1min,倒掉收集管中的废液,将吸附柱CP4放入收集管中。
11.向吸附柱CP4中加入600μl漂洗液PW,12,000rpm(~13,400g)离心1min,倒掉收集管中的废液。
12.将吸附柱CP4重新放回收集管中,12,000rpm(~13,400g)离心2min,将吸附柱CP4开盖,置于室温放置数分钟,以彻底晾干吸附材料中残余的漂洗液。
13.将吸附柱CP4置于一个干净的离心管中,向吸附膜的中间部位悬空滴加100-300μl洗脱缓冲液TB或无菌双蒸水,室温放置2min,12,000rpm(~13,400g)离心1min将质粒溶液收集到离心管中。
14.提取的质粒溶液利用NanoDrop2000测定浓度、A260/A280、A260/A230。
15.质粒溶液于-20℃储存,以备后续转染使用。
2)慢病毒制备与浓缩:
1.胰酶消化处于对数生长期的293T细胞,将5×106个HEK293T细胞接种于10cm细胞培养皿(培养基为DMEM+15%FBS),总体系为10ml。晃匀细胞后将细胞培养皿置于37℃、5%CO2的孵箱中培养24-36h。在细胞汇合度达到80-90%时,进行转染操作。
2.在15ml离心管中,用无血清DMEM培养基稀释DNA,转染体系如下:
上述混合体系配置后静置15min。吸取10cm细胞培养皿中的旧培养基,加入7ml新鲜的含15%FBS的DMEM培养基,将上述转染体系缓慢逐滴加入培养皿中,培养皿置于37℃、5%CO2孵箱培养,12h后更换完全新鲜的含15%FBS的DMEM培养基。
3.收集换液后48h的病毒原液,于4℃离心,3000rpm,15min,收集上清。
4.利用0.45μm滤器过滤病毒上清,收集于超速离心管中,精密天平严格配平后,超速离心机离心,4℃,50000g,2h30min。
5.离心结束后,弃上清,加入100μl无血清培养基(KBM581)重悬病毒,分装于1.5mlEP管,-80℃储存备用。
实施例5:CRISPR技术进行T细胞CD7、TRAC基因敲除
1)T细胞的制备:
取新鲜健康人外周血10ml,采用RosetteSep T cell enrichment Cocktail(Stemcell公司)和Ficoll-Paque PLUS(GE Healthcare公司)提取T细胞(具体步骤按照RosetteSep T cell enrichment Cocktail说明书)。按细胞:磁珠=1:1比例加入抗CD3/CD28磁珠(Gibco公司),培养48小时即为基因敲除前的T细胞。
2)T细胞CD7、TRAC基因敲除:
1.取抗CD3/CD28磁珠刺激48h后的T细胞,离心后去除磁珠,计数用3×106T细胞,加入过量DPBS,1500rpm、10min离心以清洗细胞;
2.取2个200μl离心管,分别加入3μgCD7 gRNA+3μgCas9 protein、3μgTRACgRNA+3μgCas9 protein(gRNA序列系实验室前期筛选高敲除效率序列,通过TAKARAGuide-itTMsgRNA In Vitro Transcription Kit制备,Cas9蛋白为TakaraGuide-itTM RecombinantCas9)。PCR仪孵育:37℃,5min,随后4℃维持。
CD7 gRNA:GGGGTCAATGTCTACGGCTC
TRACgRNA:AGAGTCTCTCAGCTGGTACA
3.采用Lonza 2b电转仪,电转试剂盒系Human T Cell NucleofectorTM Kit。按照操作说明书配置100μl电转液,现配现用。
4.用100μl电转液重悬细胞,并与孵育后的gRNA与Cas9蛋白复合物混合,加入电转杯中,移至电转仪进行电转操作。
5.将电转后的细胞液小心吸取加入37℃预热的T细胞培养基中,放入37℃、5%CO2孵箱继续培养。
实施例6:T细胞慢病毒感染与扩增
1)慢病毒感染T细胞及感染后T细胞的培养:电转结束4-6h后,从-80℃中取出病毒上清,冰上融化,按每(0.5-1)×106T细胞加入100μl病毒上清,加入Polybrene至终浓度为8μg/ml。离心:32℃,1800rpm,1.5h,结束后将培养孔板放入5%CO2、37℃孵箱培养。
2)留取未经敲除及未经慢病毒感染的原代T细胞(记作primary T)、未敲除但慢病毒感染的CAR-T细胞(记作WT-7CAR)、敲除但未经慢病毒感染的T细胞(记作T-knockout)、敲除并进行慢病毒感染的CAR-T细胞(记作KO-7CAR),分别在第2、6、8、11、14、18、21天计数T细胞数,以反映细胞扩增情况。图4为以上细胞倍增情况统计图,如图所示,未经敲除的抗CD7CART细胞无法实现细胞扩增,而CD7敲除后的抗CD7 CAR-T细胞可持续扩增至第21天,其第21天扩增倍数达千倍。TRAC与CD7敲除与未敲除T细胞扩增无差别,说明基因敲除对细胞扩增无显著影响,CAR-T制备方案可行。
实施例7:通用型CAR-T细胞的表型检测
在T细胞培养第7天(基因敲除及慢病毒感染后第5天),分别取~1×105细胞/管通过流式细胞术进行以下表型检测:
1)T/CAR-T细胞CAR表达检测及CD7、TCRα/β敲除效率检测:
收集细胞,标记山羊抗鼠IgG F(ab')2抗体(APC检测通道),室温孵育15min,过量PBS洗涤两次,后标记anti-human PE/Cy7 CD7、anti-human PE TCRα/β流式抗体,室温孵育15min,过量PBS洗涤细胞,重悬上机进行流式分析。图5为T细胞感染效率结果显示,KO-7CAR感染效率波动于15%-40%。图6为CD7、TCRα/β表达结果显示,图6的A为单次实验敲除效率示意结果,图6的B为多次实验敲除效率统计结果,结果显示敲除后CD7-TCRα/β-细胞比例均在90%以上。
2)T/CAR-T细胞抑制性受体表达检测:
收集细胞,标记anti-human APC/Cy7 PD-1、anti-human PETIM3、anti-human PE/Cy7 LAG3流式抗体,室温孵育15min,过量PBS洗涤细胞,重悬上机进行流式分析。图7为primary T、WT-7CAR、T-knockout、KO-7CAR抑制性受体表达结果统计图。如图所示,未经CD7敲除的WT-7CAR的抑制性受体表达显著升高,主要体现于PD-1、TIM-3的表达升高,LAG-3的表达略微升高,但尚未达到显著统计学差异。
3)T/CAR-T细胞活化受体表达检测:
收集细胞,标记anti-human CD25(T细胞晚期活化标记抗体)、anti-human CD69(T细胞早期活化标记抗体)流式抗体,室温孵育15min,过量PBS洗涤细胞,重悬上机进行流式分析。图8为primary T、WT-7CAR、T-knockout、KO-7CAR活化相关受体表达结果统计图。如图所示,抗CD7 CAR-T细胞CD25表达明显高于T细胞(primary T、T-knockout),WT-7CAR与KO-7CAR在活化相关受体的表达方面无明显差异。
4)T/CAR-T细胞活化诱导的细胞凋亡(AICD)检测:
收集细胞,加入1mlAnnexinVbinding buffer洗涤细胞,洗涤结束后弃去上清,用100μlAnnexinVbinding buffer重悬细胞,标记AnnexinV抗体,室温孵育15min,加入1μl500μg/ml PI,混匀后低速上机,进行流式检测。图9为AICD检测结果,如图所示,KO-7CAR早期凋亡及晚期凋亡均低于WT-7CAR。
5)T/CAR-T细胞CD8表达及CD45RA、CCR7细胞分群检测:
收集细胞,标记anti-human PerCPCD8、anti-human APC/Cy7 CD45RA、anti-humanPECCR7流式抗体,室温孵育15min,过量PBS洗涤细胞,重悬上机进行流式分析。图10为T/CAR-T细胞CD8表达及细胞分群统计结果,如图所示,WT-7CAR、KO-7CARCD8表达均低于原代T细胞;整体而言,细胞分群在各组间无明显差异。
实施例8:通用型CAR-T细胞的功能检测
1)靶细胞CD7抗原表达检测:
选择Jurkat、Molt4、Hut78及CEM细胞系作为杀伤靶细胞(靶细胞过表达截短型CD19抗原以便检测),CD7表达阴性K562细胞系(K562细胞系过表达红色荧光蛋白RFP以便检测)作为对照组细胞,取细胞标记CD7抗体及其同型对照,室温孵育15min,过量PBS洗涤后,重悬细胞进行流式检测。
复苏T-ALL患者原代细胞,标记CD7抗体及其同型对照,室温孵育15min,过量PBS洗涤后,重悬细胞进行流式检测。
靶细胞系及患者原代细胞CD7抗原表达结果如图11所示。
2)CAR-T细胞杀伤功能实验:
1.T/CAR-T细胞与靶细胞系共培养
将上述细胞Jurkat、Molt4、CEM、Hut78、K562细胞系按5×104细胞/孔接种48孔培养板(总培养体系500μl),分别加入2.5×105(E:T=5:1)、5×104(E:T=1:1)的CAR修饰的T细胞,以primaryT、T-knockout为对照组,于孵箱中共培养,分别于0h、24h、48h通过流式细胞术,将共培养后的细胞用anti-human PE CD19单抗(Biolegend公司)标记,检测残余靶细胞比例。3次独立的重复实验结果汇总如图12所示。杀伤结果显示,KO-7CAR在E:T=5:1、1:1的效靶比下均可介导CD7表达阳性细胞系的特异性杀伤。
2.T/CAR-T细胞与T-ALL患者原代肿瘤细胞共培养
在共培养前,使用DiD膜染料对患者原代细胞(n=3,记作P1、P2、P3)进行染色,以区分T/CAR-T细胞与靶细胞。将患者原代细胞按2×105细胞/孔接种24孔培养板(总培养体系1ml),分别加入2×105(E:T=1:1)、1×105(E:T=1:2)、5×104(E:T=1:4)的CAR修饰的T细胞,以primaryT、T-knockout为对照组,于孵箱中共培养,分别于0h、24h、48h通过流式细胞术检测残余靶细胞比例,培养后48h检测时,标记PEanti-human CD7抗体,以检测残余靶细胞CD7表达情况。3例患者原代细胞杀伤结果如图13所示,图13的A结果显示,相较于primary T与T-knockout,WT-7CAR与KO-7CAR均能介导针对患者原代细胞的杀伤,KO-7CAR杀伤效果由于WT-7CAR;图13的B结果显示,于共培养48h后标记患者原代细胞CD7抗原表达,与KO-7CAR共培养组无法检测到CD7抗原,认为KO-7CAR可介导针对肿瘤细胞的特异性杀伤,而其余对照组(primary T、T-knockout、WT-7CAR)均有CD7抗原表达阳性细胞残留。
3)T/CAR-T细胞脱颗粒检测:
1.T/CAR-T细胞与靶细胞系共培养检测
将primaryT、T-knockout、KO-7CAR细胞分别与Jurkat、Molt4、CEM、Hut78、K562细胞系按照效靶比1:1于96孔板进行共培养,靶细胞数为1×105,培养体系为200μl,在共培养体系中加入1μl PE/Cy7 anti-human CD107a抗体、0.5μl50U/ml IL-2,共培养1h后,避光,向96孔板体系中加入莫能霉素(Monensin,终浓度为2μM);4h后应用流式细胞仪检测T细胞表面CD107a的表达水平。每组设置3个复孔,3次重复实验结果如图14的A所示,相较于primaryT和T-knockout组,KO-7CAR细胞与CD7阳性靶细胞系共培养时,CD107a表达组显著升高,三组T细胞与K562细胞共培养CD107a表达无显著差异。
2.T/CAR-T细胞与患者原代细胞共培养检测
将primaryT、T-knockout、WT-7CAR、KO-7CAR细胞分别与2例患者原代细胞(患者原代细胞预先进行DiD膜染色)按照效靶比1:1于96孔板进行共培养,靶细胞数为1×105,培养体系为200μl,在共培养体系中加入1μl PE/Cy7anti-human CD107a抗体、0.5μl 50U/mlIL-2,共培养1h后,避光,向96孔板体系中加入莫能霉素(Monensin,终浓度为2μM);4h后应用流式细胞仪检测DiD阴性细胞(即T细胞)表面CD107a的表达水平。每组设置3个复孔,细胞脱颗粒结果如图14的B所示,相较于primaryT、T-knockout,CD7 CAR-T组CD107a表达显著升高,且WT-7CAR脱颗粒水平略高于KO-7CAR。
实施例9:通用型CAR-T细胞体内功能检测
取8-10周龄NSG小鼠,尾静脉接种5×105Jurkat-luc(表达荧光素)细胞(记作day0),分别于day4、day8尾静脉接种2.5×106T-knockout细胞(对照组)或KO-7CAR细胞(实验组),通过小动物活体成像技术,每周监测对照组及实验组小鼠体内肿瘤负荷情况,以反映通用型CAR-T细胞在体内的肿瘤清除功能。小动物活体成像结果如图15所示。
实施例10:抗体或抗原结合部分与人CD7阴性细胞的交叉反应
将实施例1中的单克隆抗体HIT7以0.15μg/100μl的浓度分别与2×105Raji细胞(Burkitt淋巴瘤细胞系,CD7阴性)及2×105K562细胞(慢性髓系白血病细胞系,CD7阴性)孵育,以CD7商品化抗体(克隆号:CD7-6B7)为对照。4℃避光孵育30分钟,PBS洗两次,100μl重悬细胞,上机进行FACS检测。结果如图16所示,结果显示,本发明单克隆抗体或scFv及对照的商品化抗体CD7-6B7与Raji细胞、K562细胞均不能结合,无交叉反应。
实施例11:抗体或抗原结合部分与人CD7阳性细胞(Jurkat、Molt4、CCRF-CEM、Hut78细胞)特异性结合
FACS检测实施例1中的单克隆抗体HIT7、商品化CD7-6B7抗体(Biolegend,PE/Cyanine7抗人CD7抗体,克隆号CD7-6B7)与高表达人CD7的Jurkat、Molt4、CCRF-CEM、Hut78细胞表面CD7的结合:抗体以0.15μg/100μl的浓度分别与2×105Jurkat、Molt4、CEM及Hut78细胞进行孵育,4℃避光孵育30分钟,PBS洗两次;100μl重悬细胞,上机进行FACS检测。结果如图17所示,结果表明本发明单克隆抗体或scFv能有效结合Jurkat、Molt4、CEM、Hut78细胞,亲和性强,结合能力与商品化抗体CD7-6B7无差异。
实施例12:FACS法检测抗体或抗原结合部分与商品化抗体CD7-6B7的竞争实验
将实施例1中的单克隆抗体HIT7用PBS稀释至100μM、50μM、25μM、12.5μM、6.25μM,同时每管加入2.5μl商品化抗体,混匀,与2×105CEM细胞4℃避光孵育30分钟,PBS洗两次,100μl重悬细胞,上机进行FACS检测。结果如图18所示,结果表明,本发明单克隆抗体或scFv不能与商品化抗体CD7-6B7(Biolegend,PE/Cyanine7抗人CD7抗体,克隆号CD7-6B7)竞争与CD7抗原的结合,提示这两种抗体的抗原结合表位不同。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国医学科学院血液病医院( 中国医学科学院血液学研究所)
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gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 1200
taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1260
gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1320
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1380
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1440
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1500
Claims (18)
1.特异性结合CD7的抗体,其特征在于,所述抗体包含如SEQ ID No.1-3所示的轻链可变区的CDR1、CDR2、CDR3序列,和如SEQ ID No.4-6所示的重链可变区的CDR1、CDR2、CDR3序列,或与所述序列具有80%以上同源性的序列。
2.特异性结合CD7的抗原结合部分,其特征在于,所述抗原结合部分包含如SEQ IDNo.1-3所示的轻链可变区的CDR1、CDR2、CDR3序列,和如SEQ ID No.4-6所示的重链可变区的CDR1、CDR2、CDR3序列,或与上述序列具有80%以上同源性的序列。
3.根据权利要求2所述的抗原结合部分,其特征在于,所述抗原结合部分为Fab、Fab′、F(ab′)2、Fd、Fv、scFv或单结构域抗体(dAB)。
4.根据权利要求3所述的抗原结合部分,其特征在于,所述抗原结合部分为scFv,所述scFv为单价、二价或三价的形式;
其中,所述单价的scFv的轻链可变区的氨基酸序列如SEQ ID No.7所示,所述单价的scFv的重链可变区的氨基酸序列如SEQ ID No.8所示,或与所述序列具有80%以上同源性的序列;
优选地,所述单价的scFv的氨基酸序列如SEQ ID No.9所示,或与其具有80%以上同源性的序列。
5.一种多核苷酸,其特征在于,所述多核苷酸编码如权利要求1所述的抗体,或如权利要求2~4任意一项中所述的抗原结合部分。
6.一种载体,其特征在于,所述载体包含如权利要求5所述的多核苷酸的序列。
7.一种分离的细胞,其特征在于,所述细胞包含如权利要求1所述的抗体、如权利要求2~4任意一项中所述的抗原结合部分、如权利要求5所述的多核苷酸或如权利要求6所述的载体;
所述细胞为选自SP2/0、YB2/0、IR983F、PERC6、CHO细胞系或昆虫细胞系。
8.一种嵌合抗原受体,包含胞外区、跨膜区和胞内区,所述胞内区包含胞内信号转导区,其特征在于,所述嵌合抗原受体的胞外区包含CD7结合结构域,所述CD7结合结构域包含如权利要求2~4任意一项中所述的抗原结合部分。
9.根据权利要求8所述的嵌合抗原受体,其特征在于,所述胞外区还包含构建在所述嵌合抗原受体的氨基末端的信号肽或与所述信号肽具有90%以上同源性的氨基酸序列;
优选地,所述信号肽为CD8α中的信号肽序列或GM-CSF;
更优选地,所述信号肽为如SEQ ID NO.10所示的信号肽。
10.根据权利要求8所述的嵌合抗原受体,其特征在于,所述CD7结合结构域通过铰链区与所述跨膜区连接;
所述铰链区优选为CD8α中的铰链区序列;
所述跨膜区为选自以下蛋白质的跨膜结构域或与所述蛋白质具有90%以上同源性的氨基酸序列:T细胞受体的α、β或ζ链、CD2、CD3ε、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、CD18、CD19、CD27、CD28、CD29、CD30、CD40、CD48、CD49a、CD49d、CD49f、CD66a、CD66b、CD66c、CD66d、CD66e、CD69、CD79A、CD79B、CD84、CD96、CD100、CD103、CD134、CD137、CD150、CD158A、CD158B1、CD158B2、CD158C、CD158D、CD158F1、CD158F2、CD158K、CD160、CD162、CD226、CD229、CD244、CD247、CD258、CD268、CD270、CD272、CD276、CD279、CD314、CD319、CD335、CD336、CD337、CD352、CD353、CD355、CD357、LFA-1、NKG2C、DAP-10、ICAM-1、NKp80、IL-2R beta、IL-2Rgamma、IL-7R alpha、LFA-1、SLAMF9、LAT、GADS、SLP-76、PAG1/CBP、CD83配体、Fc gamma受体、整联蛋白、激活性NK细胞受体或Toll配体受体,或其组合;
优选地,所述跨膜区为CD8α中的跨膜区序列。
11.根据权利要求8所述的嵌合抗原受体,其特征在于,所述胞内区还包含共刺激因子;
优选地,所述共刺激因子为通过选自以下蛋白质或与所述蛋白质具有90%以上同源性的氨基酸序列获得的功能性信号结构域的一种或几种:整联蛋白、BTLA、Toll配体受体、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1、4-1BB、B7-H3、CD278、GITR、BAFFR、LIGHT、HVEM、KIRDS2、SLAMF7、NKp80、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49α、IA4、CD49D、ITGA6、VLA6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11α、ITGAM、CD11b、ITGAX、CD11c、CD29、ITGB1、ITGB2、CD18、ITGB7、NKG2D、NKG2C、TNFR2、CD226、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、CD162、LTBR、LAT、GADS或SLP-76;
更优选地,所述共刺激因子为CD28或4-1BB,或与其具有90%以上同源性的氨基酸序列。
12.根据权利要求8所述的嵌合抗原受体,其特征在于,所述胞内信号转导区为选自以下蛋白质或与所述蛋白质具有90%以上同源性的氨基酸序列:4-1BB、B7-H3、BAFFR、BLAME、BTLA、CD100、CD103、CD160、CD18、CD19、CD19a、CD2、CD247、CD27、CD276、CD28、CD29、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8alpha、CD8beta、CD96、CDS、CEACAM1、CRTAM、DAP-10、DNAM1、Fc gamma受体、GADS、GITR、HVEM、IA4、ICAM-1、ICAM-1、Igalpha、IL2R beta、IL2R gamma、IL7R alpha、整联蛋白、ITGA4、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB2、ITGB7、KIRDS2、LAT、LFA-1、LFA-1、LIGHT、LIGHT、LTBR、Ly9、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80、OX-40、PAG/Cbp、PD-1、PSGL1、SELPLG、SLAMF4、SLAMF6、SLAMF7、SLP-76、TNFR2、Toll配体受体、TRANCE/RANKL、VLA1或VLA-6,或其组合;
优选地,所述胞内信号转导区为CD3ζ。
13.根据权利要求8所述的嵌合抗原受体,其特征在于,所述嵌合抗原受体的氨基酸序列如SEQ ID No.11所示,或与其具有90%以上同源性的氨基酸序列。
14.一种多核苷酸,其特征在于,所述多核苷酸编码如权利要求8~13任意一项中所述的嵌合抗原受体;
优选地,所述多核苷酸的序列如SEQ ID No.12所示。
15.一种分离的细胞,其特征在于,所述细胞中包含如权利要求2~4任意一项中所述的抗原结合部分、如权利要求8~13任意一项中所述的嵌合抗原受体,或如权利要求5或14中所述的多核苷酸的序列;
所述细胞为T细胞或NK细胞;
优选地,所述T细胞为自体来源的T细胞或异体来源的T细胞。
16.根据权利要求15所述的细胞,其特征在于,所述细胞为异体来源的T细胞,所述T细胞为通用型嵌合抗原受体T细胞(CAR-T);
优选地,所述T细胞缺失CD7基因序列;
更优选地,所述T细胞还缺失TRAC基因序列。
17.如权利要求1所述的抗体、如权利要求2~4任意一项中所述的抗原结合部分、如权利要求5或14中所述的多核苷酸、如权利要求6所述的载体、如权利要求7、15或16所述的分离的细胞,或如权利要求8~13任意一项中所述的嵌合抗原受体在制备治疗肿瘤的药物中的用途;
优选地,所述肿瘤为血液肿瘤;
更优选地,所述肿瘤为T细胞血液肿瘤;
进一步优选地,所述肿瘤为CD7抗原阳性的急性T淋巴细胞性白血病或淋巴瘤;
进一步优选地,所述肿瘤为难治或复发的CD7抗原阳性的急性T淋巴细胞性白血病或淋巴瘤。
18.一种药物组合物,其特征在于,所述药物组合物包含如权利要求1所述的抗体、如权利要求2~4任意一项中所述的抗原结合部分、如权利要求5或14中所述的多核苷酸、如权利要求6所述的载体、如权利要求7、15或16所述的分离的细胞,或如权利要求8~13任意一项中所述的嵌合抗原受体,以及药学上可接受的载体;
优选地,所述药物组合物还包含抗CD5的CAR-T细胞。
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