CN109593721B - 具有自杀基因开关的靶向人间皮素的工程化免疫细胞 - Google Patents
具有自杀基因开关的靶向人间皮素的工程化免疫细胞 Download PDFInfo
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Abstract
本发明提供了一种靶向间皮素(MSLN)的工程化的免疫细胞。具体地,本发明提供了一种靶向间皮素的嵌合抗原受体T细胞,所述细胞的CAR结构中包含细胞自杀元件,并且所述细胞中的PD1基因表达是被沉默的。具体地,本发明的CAR结构中同时包含CAR基本结构和细胞自杀元件,二者各自独立,其具有相应功能互不干扰。此外,本发明细胞中的PD1基因表达被沉默,与所述的CAR结构具有协同作用,肿瘤杀伤作用增强,不易复发。
Description
技术领域
本发明涉及免疫细胞治疗领域,更具体地涉及一种具有自杀基因开关的靶向人间皮素的工程化免疫细胞。
背景技术
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式,是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法,来激发、增强机体自身免疫功能,从而达到治疗肿瘤的目的。
嵌合免疫抗原受体(Chimeric antigen receptors,CARs)由胞外抗原识别区域,通常是scFv(single-chain variable fragment),跨膜区以及胞内共刺激信号区域组成。CARs的胞外段可识别一个特异的抗原,随后通过胞内结构域转导该信号,引起T细胞的活化增殖、细胞溶解毒性和分泌细胞因子,进而清除靶细胞。首先分离病人自体T细胞(或者异源供体),激活并进行基因改造产生CAR-T细胞,随后注入同一病人体内。这种方式患移植物抗宿主病概率极低,抗原被T细胞以非MHC限制方式识别。此外,一种CAR-T就可以治疗表达该抗原的所有癌症。
CAR-T细胞治疗在血液恶性肿瘤治疗中取得了非常高的临床反应率,这样的高反应率是以往任何一种治疗手段都无法达到的,在世界各引发了临床研究的热潮。但是在实体肿瘤治疗中,Moon等发现在小鼠中注射meso-cart能够约束肿瘤的生长,但是不能治疗肿瘤。他们认为是肿瘤微环境中负调控因子的上调导致cart功能的减退,如在肿瘤微环境中,T细胞表面PD-1(programmed death protein-1)表达上调限制了T细胞的功能。有研究表明,在小鼠上用抗体阻断PD1后,CAR-T细胞功能得到增强。PD-1抗体可以提高CAR-T细胞功能,但是注射抗体后全身阻断PD1,增强了自体所有反应T细胞的激活,导致毒性较大。将CAR-T中PD-1的表达沉默只特异性解除肿瘤细胞对CART细胞的抑制作用,而不会对其他T细胞发挥作用,从而降低毒性,更好的发挥CART细胞抗肿瘤的效果。
间皮素是一种肿瘤相关抗原,最早是Ira Pastan和Mark Willingham在卵巢癌中发现。他们发现间皮素高表达于卵巢癌细胞,但除了间皮细胞,在其他正常组织中不表达。目前研究发现,间皮素在间皮瘤、肺癌、胰腺癌、乳腺癌、卵巢癌及其他肿瘤组织中高表达,而在正常组织中有限表达。鉴于此,间皮素被列为一个免疫治疗的潜在靶点。目前,靶向间皮素的抗体及靶向间皮素的嵌合抗原受体T细胞(CART)等免疫治疗手段已有报道,但是发现用小鼠的抗人间皮素抗体所构建的CART在临床中出现抗鼠抗体及过敏反应等毒副作用以及相关肿瘤的治疗中疗效不理想等情况,因此需要进一步完善现有治疗技术,增强其疗效并控制好风险。
综上所述,本领域仍然需要进一步的研究,开发一种能更有效、特异性好、副作用小地治疗血液肿瘤和实体瘤的CAR-T细胞。
发明内容
本发明的目的在于提供一种具有自杀基因开关的靶向人间皮素的工程化免疫细胞。
具体地,本发明的目的在于提供一种靶向间皮素(MSLN)的嵌合抗原受体T细胞,通过基因沉默的方法将其表面分子PD-1表达沉默,增加其肿瘤杀伤作用,同时加有自杀基因开关,以阻断未知的或不可控的远期毒性,保证患者的安全。
在本发明的第一方面,提供了一种工程化的免疫细胞,所述工程化的免疫细胞为T细胞或NK细胞,并且所述的免疫细胞细胞具有以下特征:
(a)所述细胞表达靶向间皮素的CAR或外源TCR,并且所述的CAR或TCR结构中包含细胞自杀元件,所述的细胞自杀元件包含选自下组的自杀基因开关:HSV-TK、iCasp9、ΔCD20、mTMPK、ΔCD19、EGFRt、或其组合;和
(b)所述细胞中的PD1基因表达是被沉默的。
在另一优选例中,所述的工程化的免疫细胞选自下组:
(i)嵌合抗原受体T细胞(CAR-T细胞);
(ii)嵌合抗原受体NK细胞(CAR-NK细胞);或
(iii)外源T细胞受体(TCR)T细胞(TCR-T细胞)。
在另一优选例中,提供了一种嵌合抗原受体T细胞(CAR-T细胞),所述CAR-T细胞具有以下特征:
(a)所述细胞表达靶向间皮素的CAR,并且所述的CAR结构中包含细胞自杀元件,所述的细胞自杀元件包含选自下组的自杀基因开关:HSV-TK、iCasp9、ΔCD20、mTMPK、ΔCD19、EGFRt、或其组合;和
(b)所述细胞中的PD1基因表达是被沉默的。
在另一优选例中,所述的CAR的结构如下式I所示:
L-scFv-H-TM-C-CD3ζ-A-K (I)
式中,
各“-”独立地为连接肽或肽键;
L为任选的信号肽序列;
scFv为靶向间皮素的抗体单链可变区序列;
H为任选的铰链区;
TM为跨膜结构域;
C为共刺激信号分子;
CD3ζ为源于CD3ζ的胞浆信号传导序列;
A为任选的蛋白标签;
K为细胞自杀元件。
在另一优选例中,所述的蛋白标签与CD3ζ通过P2A连接。
在另一优选例中,所述的A为选自下组的一种或多种蛋白标签:
绿色荧光蛋白(GFP)、NGFRt、EGFRt、ΔCD19、ΔCD20、或其组合。
在另一优选例中,所述的细胞自杀元件K与元件A通过P2A连接。
在另一优选例中,所述的细胞自杀元件的结构如下式II所示:
B-D-F (II)
式中,
各“-”独立地为连接肽或肽键;
B为自杀基因诱导元件;
D为柔性接头;
F为自杀基因。
在另一优选例中,所述的细胞自杀元件包含iCasp9。
在另一优选例中,所述的自杀基因为半胱氨酸天冬氨酸蛋白酶-9的编码基因(Caspase9基因)。
在另一优选例中,所述的B为FKBP12-F36V结构域。
在另一优选例中,所述的FKBP12-F36V结构域包含FKBP结构域,且FKBP结构域的第36位氨基酸由苯丙氨酸突变为缬氨酸。
在另一优选例中,所述D的序列如SEQ ID NO.:8所示(Ser-Gly-Gly-Gly-Ser)。
在另一优选例中,所述的L为选自下组的蛋白的信号肽:CD8、GM-CSF、CD4、CD137、或其组合。
在另一优选例中,所述的L为CD8。
在另一优选例中,所述的scFv的氨基酸序列如SEQ ID NO.:2的第30-287位所示。
在另一优选例中,所述自杀基因元件的氨基酸序列如SEQ ID NO.:2的第792-1191位所示。
在另一优选例中,所述的H为选自下组的蛋白的铰链区:CD8、CD28、CD137、或其组合。
在另一优选例中,所述的H为CD8来源的铰链区。
在另一优选例中,所述的TM为选自下组的蛋白的跨膜区:CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中,TM包括CD8来源的跨膜区,和/或CD28来源的跨膜区。
在另一优选例中,所述的C为选自下组的蛋白的共刺激信号分子:OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
在另一优选例中,C包括4-1BB来源的共刺激信号分子,和/或CD28来源的共刺激信号分子。
在另一优选例中,编码所述CAR的核苷酸序列如SEQ ID NO.:1所示。
在另一优选例中,所述CAR的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中,所述“PD1基因表达是被沉默的”指PD1基因不表达或低表达。
在另一优选例中,所述“低表达”指所述CAR-T细胞PD1基因的表达量G1与正常T细胞PD1基因的表达量G0的比值,即G1/G0≤0.5,较佳地G1/G0≤0.3,更佳地≤0.2,更佳地≤0.1,最佳地为0。
在本发明的第二方面,提供了一种制备本发明第一方面所述的工程化免疫细胞的方法,包括以下步骤:
(A)提供一待改造的免疫细胞;和
(B)对所述的免疫细胞进行改造,从而使得所述的免疫细胞表达所述的CAR或外源TCR,所述的CAR或TCR结构中包含细胞自杀元件,并且使得所述的T细胞中PD1基因的表达沉默,从而获得本发明第一方面所述的免疫细胞。
在另一优选例中,提供了一种制备本发明第一方面所述的CAR-T细胞的方法,包括以下步骤:
(A)提供一待改造的T细胞;和
(B)对所述的T细胞进行改造,从而使得所述的T细胞表达所述的CAR,所述的CAR结构中包含细胞自杀元件,并且使得所述的T细胞中PD1基因的表达沉默,从而获得本发明第一方面所述的CAR-T细胞。
在另一优选例中,在步骤(B)中,包括(B1)将表达所述CAR的第一表达盒导入所述T细胞;和(B2)将表达用于沉默PD1基因的第二表达盒导入所述T细胞,
其中,所述的步骤(B1)和(B2)的次序无任何限定。
在另一优选例中,所述的“次序无任何限定”指对于两个步骤而言,可以依次、同时、或以相反次序进行。
在另一优选例中,所述的步骤(B1)在步骤(B2)之前进行。
在另一优选例中,所述的第一表达盒和第二表达盒位于相同或不同的载体上。
在另一优选例中,所述的第一表达盒和第二表达盒位于同一载体。
在另一优选例中,所述的载体为病毒载体。
在另一优选例中,所述的载体选自下组:DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。
在另一优选例中,所述的第二表达盒包含CRISPR/Cas9(sgRNA和Cas9)、反义RNA、或其组合。
在另一优选例中,所述的sgRNA靶向PD1,且sgRNA的序列如SEQ ID NO.:3、4、5、6、或7所示。
在另一优选例中,所述的反义RNA包括miRNA、siRNA、shRNA、抑制性mRNA、或dsRNA。
在本发明的第三方面,提供了一种制剂,所述制剂含有本发明第一方面所述的工程化免疫细胞,以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中,提供了一种制剂,所述制剂含有本发明第一方面所述的CAR-T细胞,以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中,所述制剂为液态制剂。
在另一优选例中,所述制剂的剂型为注射剂。
在另一优选例中,所述制剂中所述CAR-T细胞的浓度为1×103-1×108个细胞/ml,较佳地1×104-1×107个细胞/ml。
在本发明的第四方面,提供了本发明第一方面所述的工程化免疫细胞的用途,用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中,提供了本发明第一方面所述的CAR-T细胞的用途,用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中,所述肿瘤选自下组:血液肿瘤、实体瘤、或其组合。
在另一优选例中,所述血液肿瘤选自下组:急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中,所述实体瘤选自下组:胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在另一优选例中,所述实体瘤选自下组:卵巢癌、间皮瘤、肺癌、胰腺癌、乳腺癌、子宫内膜癌、或其组合。
在本发明的第五方面,提供了一种用于制备本发明第一方面所述工程化免疫细胞的试剂盒,所述试剂盒含有容器,以及位于容器内的:
(1)第一核酸序列,所述第一核酸序列含有用于表达所述CAR或TCR的第一表达盒;
(2)第二核酸序列,所述第二核酸序列含有用于沉默PD1的第二表达盒或sgRNA。
在另一优选例中,提供了一种用于制备本发明第一方面所述的CAR-T细胞的试剂盒,所述试剂盒含有容器,以及位于容器内的:
(1)第一核酸序列,所述第一核酸序列含有用于表达所述CAR的第一表达盒;
(2)第二核酸序列,所述第二核酸序列含有用于沉默PD1的第二表达盒或sgRNA。
在另一优选例中,所述的第一和第二核酸序列为独立的或相连的。
在另一优选例中,所述的第一和第二核酸序列位于相同或不同的容器内。
在另一优选例中,所述的第一和第二核酸序列位于相同或不同表达载体。
在另一优选例中,所述的试剂盒还含有:(4)第三核酸序列,所述第三核酸序列含有用于表达Cas9蛋白的表达盒;或Cas9蛋白。
在本发明的第六方面,提供了本发明第一方面所述工程化免疫细胞的用途,用于预防和/或治疗癌症或肿瘤。
在另一优选例中,提供了本发明第一方面所述的CAR-T细胞的用途,用于预防和/或治疗癌症或肿瘤。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了meso-CAR的结构示意图,根据CAR共刺激信号的有无及不同分别命名为P4-BBz CAR,P4-28z CAR,P4-28BBz CAR,P4-z CAR,对应的共刺激信号来源于:4-1BB、CD28、4-1BB+CD28、无共刺激信号。
图2显示了不同CART细胞的表达情况。其中,BBZ,28Z,28BBZ,Z分别表示利用图1中相应P4-BBz CAR,P4-28z CAR,P4-28BBz CAR,P4-z CAR制备的CAR-T细胞。CD19表示靶向CD19的嵌合抗原受体T细胞,T表示未经改造的T细胞。
图3显示了不同培养天数细胞增殖倍率及CAR阳性率细胞的比例。
图4显示了不同car细胞中免疫检查点的表达情况。
图5显示了四种不同的CAR-T细胞对靶细胞裂解程度有差异,且PDL-1过表达时CART细胞的杀伤作用减弱。
图6显示了P4-28Z CAR-T细胞与靶细胞作用后细胞因子的分泌(IL-2、IFN-γ)情况。其中,各横坐标分别表示为:P4代表P4-28Z结构的CAR-T细胞,19代表CD19CAR-T细胞,T代表未经改造过的T淋巴细胞,K代表K562细胞,KM代表表达MSLN的K562细胞,K19代表表达CD19的K562细胞,293M代表表达MSLN的293细胞。“-”代表共培养。以P4-K为例,指的是带P4的CAR-T细胞与K562细胞共培养。
图7显示了靶向PD-1的sgRNA序列。
图8显示了Surveyor assay的结果。
图9显示了PD-1敲除后CART细胞的杀伤作用。
图10显示了CART细胞小鼠体内实验结果。
具体实施方式
本发明人经过广泛而深入地研究,首次意外地发现一种靶向间皮素(MSLN)的嵌合抗原受体T细胞,所述细胞的CAR结构中包含细胞自杀元件,并且所述细胞中的PD1基因表达是被沉默的。具体地,本发明的CAR结构中同时包含CAR基本结构和细胞自杀元件,二者各自独立,其具有相应功能互不干扰。此外,本发明细胞中的PD1基因表达被沉默,与所述的CAR结构具有协同作用,肿瘤杀伤作用增强。再次在此基础上完成了本发明。
本发明以CAR-T细胞为例,代表性地对本发明的工程化的免疫细胞进行详细说明。本发明的工程化的免疫细胞不限于上下文所述的CAR-T细胞,本发明的工程化的免疫细胞具有与上下文所述的CAR-T细胞相同或类似的技术特征和有益效果。具体地,当免疫细胞表达嵌合抗原受体CAR时,NK细胞等同于T细胞(或T细胞可替换NK细胞);当免疫细胞为T细胞时,TCR等同于CAR(或CAR可替换为TCR)。
术语
为了可以更容易地理解本公开,首先定义某些术语。如本申请中所使用的,除非本文另有明确规定,否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成,其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者,包括静脉内,肌内,皮下,腹膜内,脊髓或其它肠胃外给药途径,例如通过注射或输注。
术语“抗体”(Ab)应包括但不限于免疫球蛋白,其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链,或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区,其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR,从氨基末端到羧基末端按照以下顺序排列:FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
间皮素(Mesothelin,MSLN)
间皮素基因编码一种69KDa的前体蛋白,经加工形成一个40kDa的膜结合蛋白(即间皮素,与GPI连接锚定在膜表面)和一个31kDa称之为巨核细胞促进因子(MPF)的脱落片断并释放出细胞外。
间皮素是一种肿瘤相关抗原,最早是Ira Pastan和Mark Willingham在卵巢癌中发现。他们发现间皮素高表达于卵巢癌细胞,但除了间皮细胞,在其他正常组织中不表达。目前研究发现,间皮素在间皮瘤、肺癌、胰腺癌、乳腺癌、卵巢癌及其他肿瘤组织中高表达,而在正常组织中有限表达。鉴于此,间皮素被列为一个免疫治疗的潜在靶点。
自杀基因开关
为进一步控制CAR-T细胞非肿瘤靶向和细胞因子释放综合征等不良,本发明中的CART细胞皆带有自杀基因开关,在外源性药物的作用下,可以有效清除体内的CAR-T细胞,阻断未知的或不可控的远期毒性,以保证患者的安全。
本发明中所用自杀基因开关可以为单纯疱疹病毒胸苷激酶(the herpes symplexvirus thymidine kinase,HSV-TK)、可诱导的半胱氨酸天冬氨酸蛋白酶9(induciblecaspase 9,iCasp9)、CD20、突变型人胸苷酸激酶(mutated human thymidylate kinase,mTMPK)等。比较而言,HSV-TK、iCasp9和CD20对T细胞的清除能力等同,但是iCasp9和CD20的清除较迅速,HSV-TK清除速度较慢。
iCasp9自杀基因开关包含FKBP12-F36V结构域,可通过柔性Ser-Gly-Gly-Gly-Ser(SEQ ID NO.:8)连接半胱氨酸天冬氨酸蛋白酶9,后者不含募集结构域。FKBP12-F36V包含一个FKBP结构域,在第36个氨基酸残基位点上苯丙氨酸替代了缬氨酸。它具有高选择性和亚纳摩尔亲和力,能够结合二聚合成配基,如其他惰性小分子AP1903。当加入小分子后,能够促使其二聚话,从而诱导细胞的凋亡,而对未携带自杀基因的正常细胞无效用。
嵌合抗原受体T(CAR)
CARs的设计经历了以下过程:第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子,由于胞内只有一个活化结构域,因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌,而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应,所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子,如CD28、4-1BB、OX40、ICOS,与一代CARs相比功能有很大提高,进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134,发展成为三代和四代CARs。
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子,而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间,或在CAR的胞浆结构域和跨膜结构域之间,可并入接头。如本文所用的,术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸,优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中,本发明提供的CAR的胞外结构域包括靶向间皮素的抗原结合结构域。本发明的CAR当在T细胞中表达时,能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时,影响肿瘤细胞,导致肿瘤细胞不生长、被促使死亡或以其他方式被影响,并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地,抗原结合结构域与CD28/4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
对于绞链区和跨膜区(跨膜结构域),CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中,使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中,可选择跨膜结构域,或通过氨基酸置换进行修饰,以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域,从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR中的胞内结构域包括CD28/4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
嵌合抗原受体T细胞(CAR-T细胞)
如本文所用,术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”均指本发明第一方面所述的CAR-T细胞。本发明的CAR-T细胞表达靶向间皮素的CAR,并且所述的CAR结构中包含细胞自杀元件,且所述细胞中的PD1基因表达是被沉默的。
外源T细胞抗原受体
如本文所用,外源T细胞抗原受体(T cell receptor,TCR)为通过基因转移技术从肿瘤反应性T细胞中克隆出TCR的α链和β链,通过基因工程的手段,以慢病毒或逆转录病毒为载体,外源性转入到T细胞内的TCR。
外源TCR修饰的T细胞能够特异性识别和杀伤肿瘤细胞,并通过优化TCR与肿瘤性特异性抗原的亲和力,可以提高T细胞与肿瘤的亲和力,提高抗肿瘤效果。
嵌合抗原受体NK细胞(CAR-NK细胞)
如本文所用,术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明第一方面所述的CAR-NK细胞。本发明CAR-NK细胞可用于治疗CD47高表达的肿瘤,如B细胞淋巴瘤、非霍奇金淋巴瘤、卵巢癌等。
自然杀伤(NK)细胞是一类主要的免疫效应细胞,通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能,包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与自体CAR-T细胞相比,CAR-NK细胞还具有一下优点,例如:(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞,而对机体正常的细胞没有杀伤作用;(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险;(3)体外极易扩增及发展为“现成的”产品。除此之外,与CAR-T细胞治疗类似。
免疫检查点
免疫检查点是指免疫系统中存在的一些抑制性信号通路,通过调节外周组织中免疫反应的持续性和强度避免组织损伤,并参与维持对于自身抗原的耐受。利用免疫检查点的抑制性信号通路抑制T细胞活性是肿瘤逃避免疫杀伤的重要机制。因此,通过不同的策略增强T细胞的激活对肿瘤免疫治疗具有重要意义。本发明通过阻断PD1信号来增强T细胞激活。
如本文使用,术语“PD-1”包括人PD-1(hPD-1),hPD-1的变体(突变的hPD-1)、同种型和物种同源物,以及与hPD-1具有至少80%、85%、90%、95%、96%、97%、98%、99%相同性的类似物。完整的hPD-1序列可以在GenBank登录号U64863下找到。
如本文使用,术语“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种是PD-L2),其在结合PD-1时下调T细胞活化和细胞因子分泌。本文所用的术语“PD-L1”包括人PD-L1(hPD-L1),hPD-L1的变体、同种型和物种同源物,以及与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列可以在GenBank登录号Q9NZQ7下找到。
PD1基因表达下调或沉默
如本文所用,“PD1基因表达是被沉默的”指PD1基因不表达或低表达。“低表达”指所述CAR-T细胞PD1基因的表达量G1与正常T细胞PD1基因的表达量G0的比值,即G1/G0≤0.5,较佳地G1/G0≤0.3,更佳地≤0.2,更佳地≤0.1,最佳地为0。
本发明中PD1基因表达下调或沉默方法有CRISPR/Cas9、RNA干扰技术、转录激活因子样效应物核酸酶TALENs(transcription activator-like(TAL)effector nucleases)和锌指核酸酶Zinc finger nucleases(ZFNs)。优选地,本发明通过CRISPR/Cas9、RNA干扰技术使PD1基因下调或沉默。在本发明的一个实施例中,采用CRISPR/Cas9使PD1基因下调或沉默。
基因沉默方法
目前常用的基因沉默方法有CRISPR/Cas9、RNA干扰技术、TALENs(transcriptionactivator-like(TAL)effector nucleases)和Zinc finger nucleases(ZFNs),其中CRISPR/Cas9目前应用前景和效果最好。
CRISPR(clustered regularly interspersed short palindromic repeats)/Cas(CRISPR-associated)系统是原核生物特有的一种天然免疫系统,用于抵抗病毒或外源性质粒的侵害。Ⅱ型CRISPR/Cas系统作为RNA直接介导的基因组编辑工具已经在许多真核生物和原核生物体内成功应用。CRISPR/Cas9系统的发展彻底改变了人们编辑DNA序列和调控目标基因表达水平的能力,从而为生物体的精确基因组编辑提供了有力的工具。简化后的CRISPR/Cas9系统由两部分组成:Cas9蛋白和sgRNA。其作用原理为sgRNA通过自身的Cas9把手与Cas9蛋白形成Cas9-sgRNA复合体,Cas9-sgRNA复合体中sgRNA的碱基互补配对区序列与目标基因的靶序列通过碱基互补配对原则进行配对结合,Cas9利用自身的核酸内切酶活性对目标DNA序列进行切割。与传统的基因组编辑技术相比,CRISPR/Cas9系统具有几大明显的优势:易用性、简便性、低成本、可编程性以及可同时编辑多个基因。
表达盒
如本文所用,“表达盒”或“本发明表达盒”包括第一表达盒和第二表达盒。所述第一表达盒包含编码所述CAR的核酸序列。所述第二表达盒包含用于沉默PD1的核酸序列。在另一优选例中,本发明还包括用于表达Cas9蛋白的第三表达盒。
在一个实施方式中,所述第一表达盒、第二表达盒和第三表达盒分别还包括启动子。在一个实施方式中,所述第一表达盒、第二表达盒和第三表达盒分别还包括终止子。
在一个实施方式中,所述的第一表达盒、第二表达盒和第三表达盒位于相同或不同的载体上。优选地,所述的第一表达盒、第二表达盒和第三表达盒位于同一载体。优选地,所述的载体选自下组:DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。优选地,所述的载体为病毒载体。
在一个实施方式中,所述的第二表达盒包含CRISPR/Cas9(sgRNA和Cas9)、反义RNA、或其组合。优选地,所述的sgRNA靶向PD1,且sgRNA的序列如SEQ ID NO.:3、4、5、6、7所示。优选地,所述的反义RNA包括miRNA、siRNA、shRNA、抑制性mRNA、或dsRNA。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得,诸如例如通过从表达基因的细胞中筛选文库,通过从已知包括该基因的载体中得到该基因,或通过利用标准的技术,从包含该基因的细胞和组织中直接分离。可选地,感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具,因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点,因为它们可转导非增殖的细胞,诸如肝细胞。它们也具有低免疫原性的优点。
简单概括,通常可操作地连接本发明的表达盒或核酸序列至启动子,并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案,用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466,在此通过引用全文并入。在另一个实施方式中,本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如,该核酸可被克隆入如此载体,其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地,表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,ColdSpring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常,合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如,WO01/96584;WO01/29058;和美国专利号6,326,193)。
已经开发许多基于病毒的系统,用于将基因转移入哺乳动物细胞。例如,逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中,使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中,使用慢病毒载体。
额外的启动子元件,例如增强子,可以调节转录开始的频率。通常地,这些位于起始位点上游的30-110bp区域中,尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的,以便当元件相对于另一个被倒置或移动时,保持启动子功能。在胸苷激酶(tk)启动子中,启动子元件之间的间隔可被增加隔开50bp,活性才开始下降。取决于启动子,表现出单个元件可合作或独立地起作用,以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而,也可使用其他组成型启动子序列,包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子,诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地,本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关,其能够当这样的表达是期望的时,打开可操作地连接诱导型启动子的多核苷酸序列的表达,或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达,被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者,以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面,可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列,以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因,诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地,报道基因为以下基因:其不存在于受体有机体或组织或由受体有机体或组织进行表达,并且其编码多肽,该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后,报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因(例如,Ui-Tei等,2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常,显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中,载体可通过在本领域中的任何方法容易地引入宿主细胞,例如,哺乳动物、细菌、酵母或昆虫细胞。例如,表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring HarborLaboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体,特别是逆转录病毒载体,已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统,诸如大分子复合物、纳米胶囊、微球、珠;和基于脂质的系统,包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如,人造膜囊)。
在使用非病毒传递系统的情况下,示例性传递工具为脂质体。考虑使用脂质制剂,以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面,该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中,散布在脂质体的脂双层内,经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体,陷入脂质体,与脂质体复合,分散在包含脂质的溶液中,与脂质混合,与脂质联合,作为悬浮液包含在脂质中,包含在胶束中或与胶束复合,或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如,它们可存在于双分子层结构中,作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中,可能形成大小或形状不均一的聚集体。脂质为脂肪物质,其可为天然发生或合成的脂质。例如,脂质包括脂肪小滴,其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中,所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第一方面所述的CAR-T细胞,以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中,所述制剂为液态制剂。优选地,所述制剂为注射剂。优选地,所述制剂中所述CAR-T细胞的浓度为1×103-1×108个细胞/ml,更优地1×104-1×107个细胞/ml。
在一个实施方式中,所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等;碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇;蛋白质;多肽或氨基酸诸如甘氨酸;抗氧化剂;螯合剂诸如EDTA或谷胱甘肽;佐剂(例如,氢氧化铝);和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明核酸构建物的慢病毒载体(LV)转导的细胞(例如,T细胞)进行的治疗性应用。转导的T细胞可引起CAR-介导的T-细胞应答,并且转导的T细胞的ACTC1基因被抑制,从而显著提高其对肿瘤细胞的杀伤效率。
因此,本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法,其包括以下步骤:施用给哺乳动物表达本发明核酸构建物的T细胞。
在一个实施方式中,本发明包括一类细胞疗法,其中T细胞被基因修饰以表达本发明的核酸构建物,得到ACTC1基因被抑制的CAR-T细胞,且CAR-T细胞被注入需要其的接受者中。注入的细胞能够杀死接受者的肿瘤细胞。不像抗体疗法,CAR-T细胞能够体内复制,产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中,本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外,CAR介导的免疫应答可为过继免疫疗法步骤的一部分,其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如,靶向间皮素CAR-T细胞引起抗表达间皮素的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括靶向间皮素的scFv、铰链和跨膜区、和4-1BB/CD28和CD3ζ信号传导结构域的慢病毒载体,但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤,以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤,例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤,和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤,例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病,包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌、。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地,哺乳动物为人。
对于离体免疫,以下中的至少一项在将细胞施用进入哺乳动物前在体外发生:i)扩增细胞,ii)将编码CAR的核酸引入细胞,和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的,并在以下更完全地进行讨论。简单地说,细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即,体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者,以提供治疗益处。哺乳动物接受者可为人,和CAR-修饰的细胞可相对于接受者为自体的。可选地,细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外,本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法,其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说,本发明的药物组合物可包括如本文所述的靶细胞群,与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等;碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇;蛋白质;多肽或氨基酸诸如甘氨酸;抗氧化剂;螯合剂诸如EDTA或谷胱甘肽;佐剂(例如,氢氧化铝);和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定,如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时,待施用的本发明组合物的精确量可由医师确定,其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出:包括本文描述的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量,优选105至106个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等,NewEng.J.of Med.319:1676,1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行,包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中,本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中,本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤,淋巴结或感染位置。
在本发明的某些实施方式中,利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞,与任何数量的有关治疗形式结合(例如,之前、同时或之后)施用给患者,所述治疗形式包括但不限于用以下试剂进行治疗:所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中,本发明的T细胞可与以下结合使用:化疗、辐射、免疫抑制剂,诸如,环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506,抗体或其他免疫治疗剂。在进一步的实施方式中,本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如,之前、同时或之后)而施用给患者。例如,在一个实施方式中,对象可经历高剂量化疗的标准治疗,之后进行外周血干细胞移植。在一些实施方式中,在移植后,对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中,扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常,每次治疗或每个疗程,可将1×106个至1×1010个本发明经修饰的T细胞(如,CAR-T20细胞),通过例如静脉回输的方式,施用于患者。
本发明的主要优点包括:
(1)本发明的CAR-T细胞带有自杀基因开关,在发生毒副作用时,能够在极短的时间内有效清除CAR-T细胞,为安全提供保障。
(2)本发明所设计的如表1所示的sgRNA序列,可以有效的实现沉默PD1基因。
(3)相比使用抗体阻断PD1免疫抑制作用,本发明CAR-T细胞中PD1基因沉默,克服肿瘤免疫抑制的效果更好,在体内存活时间较长,并且可以避免自体反应T细胞的激活,不干扰体内正常T细胞,更安全、毒副作用更小。
(4)本发明的CAR-T细胞特异性的靶向间皮素阳性的肿瘤,且CAR-T细胞PD-1表达沉默能够阻断肿瘤细胞对CAR-T细胞的功能抑制作用,使得肿瘤杀伤效果更强,体内存活时间更久,疗效更佳。
(5)本发明CAR-T细胞的CAR结构中同时包含CAR基本结构和细胞自杀元件,并且该CAR-T细胞的PD-1基因沉默,上述改造各自独立地发挥功能,互不干扰。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
实施例1
从供体血液中分离PBMC和扩增T细胞
从脐带血中分离单核细胞,使用Histopaque-1077(Sigma-Aldrich)进行密度梯度离心,并富集T细胞(EasySep human T cell enrichment kit,Stemcell Technologies),使用偶联anti-CD3/anti-CD28的磁珠激活培养和扩增T细胞;培养基使用X-vivo15(含5%FBS,2mM L-glutamine,1mM丙酮酸钠,300IU/ml rhIL2);所有细胞均置于37℃,5%CO2恒温培养箱中培养。
实施例2
细胞培养及构建
表达MSLN的细胞系:OVCAR3细胞(人卵巢癌细胞系,HTB-161TM)、HCT116(人结肠癌细胞系,CCL-247TM)、CRL5826(人肺癌细胞,CRL-5826,H226);表达MSLN/CD19的K562细胞(人红白血病细胞系,-CCL-243),以上细胞均使用RPMI 1640培养基培养;293T(人肾上皮细胞系细胞,CRL-3216)使用DMEM培养基培养。所有培养基均添加10%(v/v)胎牛血清和100U/ml的青霉素和链霉素,2mM L-谷氨酰胺,1mM丙酮酸钠
其中,表达MSLN以及CD19的K562细胞及高表达PDL-1的CRL5826、HCT116和OVCAR3是通过慢病毒载体将MSLN、CD19及PDL-1抗原转入,再经过单克隆筛选后获得的稳转细胞系,能够特异性的表达MSLN、CD19及PDL-1等蛋白分子。
实施例3
CAR结构设计与转导
meso-CAR结构,即靶向间皮素(Mesothelin)的CAR结构:
本发明方法构建了一代、二代及三代CAR,所有CAR结构中都带有可调控的开关icasp9(FKBP12-F36V-Caspase9),通过P2A连接,如图1所示。
CAR的核心结构包括CD8胞外先导序列,来自P4的scFv(特异性靶向mesothelin),来自CD8的铰链和CD8/CD28跨膜区。根据胞内段共刺激信号的有或无及不同,共构建了四种meso-CAR,分别命名为CD3ζ、4-1BB-CD3ζ、CD28-CD3ζ、CD28-4-1BB-CD3。根据胞内共刺激区域的不同分别命名,具体如表1所示:
表1 CAR-T细胞命名
命名 | 共刺激信号 | 胞内激活区 |
P4-z-CAR(简称P4-z) | N/A | CD3ζ |
P4-BBz-CAR(简称P4-BBz) | 4-1BB | CD3ζ |
P4-28z-CAR(简称P4-28z) | CD28 | CD3ζ |
P4-28BBz-CAR(简称P4-28BBz) | CD28+4-1BB | CD3ζ |
将4种meso-CAR基因克隆至FUW慢病毒载体骨架中,置于EF1α(EF-1α)的启动子下,形成Fuw-EF1α-meso-CAR,将Fuw-EF1α-mesoCAR、慢病毒包膜质粒pMD2.G(Addgene,Plasmid#12259)和慢病毒包装质粒psPAX2(Addgene,Plasmid#12260)三个质粒使用Lipofectamine3000转入293T中制备慢病毒完整表达载体;在48h和72h收集病毒上清,超离进行浓缩(Merck Millipore);浓缩后的病毒即可用于感染T细胞。
结果显示,利用四种meso-CAR基因,成功构建了四种慢病毒载体。
一种典型的包含自杀基因开关的CAR结构为P4-28z-GFP-icasp9(简称P4-28z),其DNA序列如下所示:(SEQ ID NO.:1)
其氨基酸序列如下所示:(SEQ ID NO.:2)
MDYKDDDDKALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRIDATNFSLLKQAGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKATNFSLLKQAGDVEENPGPGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSGSG
实施例4
CAR-T细胞制备
实验方法如下:
4.1慢病毒感染
分离纯化的原代T细胞在激活2天后,利用实施例3构建的4种慢病毒,按MOI(1-10)进行慢病毒载体感染,转移至细胞培养瓶,置于37℃,5%CO2恒温培养箱中培养。
4.2电转
在病毒感染后第二天,去病毒后,收集电转所需的细胞置于离心管内,300g,5min离心后,DPBS洗涤2次,然后用opti-mem重悬至细胞密度为1-3X108/ml,根据细胞的密度计算所需的Cas9/sgRNA的量,各30ug/ml。取所需的Cas9与sgRNA,并按一比一混合,室温孵育10min后用加入到电转缓冲液中与细胞混合后加入到电极杯中,用4D-NucleofectorSystem N(Lonza)电转系统,选择Program EO-115程序进行电转。电转结束后,用已经预热的培养基重悬细胞密度至1-2X106/ml,并转移至相应的培养皿中,并置于37℃,5%CO2培养箱中继续培养。
4.3细胞增殖及CAR阳性率检测
感染后第3-9天每天取样检测细胞数量及GFP阳性细胞得占比,即检测T细胞的CAR阳性率,每隔2-3天更换一半培养基。
利用四种慢病毒载体,成功构建了4种CAR-T细胞,分别命名为P4-BBz CAR-T、P4-28z CAR-T、P4-28BBz CAR-T和P4-z CAR-T。
结果如图2和图3所示,4种CAR-T细胞的CAR表达间无显著差异,且增殖速率无显著差异。
流式结果如图4所示,4种CAR-T细胞的PD-1表达强度都较高,可能会限制CAR-T细胞的功能。
实施例5
细胞体外杀伤
对实施例4获得的4种CAR-T细胞进行体外杀伤实验。通过将荧光素酶基因转入靶细胞,克隆筛选后获得稳转细胞株。进行实验时,加入荧光素底物,荧光素酶与荧光素反应即可产生荧光,通过检测荧光的强度可以测定荧光素酶的活性,检测细胞的存活比率,即可得到CART细胞的杀伤效应。
结果如图5所示,4种CAR-T细胞(P4-BBz CAR-T,P4-28z CAR-T,P4-28BBz CAR-T,P4-z CAR-T)与靶细胞(CRL5826)2:1共培养时,培养5天后,大部分靶细胞已经被杀死裂解;且P4-28z CAR-T细胞裂解作用最强,为此,后续实验中优选P4-28z CAR-T细胞进行进一步的体内外功能研究。
高表达PDL-1时,4种CAR-T细胞(P4-BBz CAR-T,P4-28z CAR-T,P4-28BBz CAR-T,P4-z CAR-T)杀伤作用都减弱。
实施例6
细胞因子释放检测
将P4-28z CAR-T细胞(实施例4获得)与肿瘤细胞(K562、MSLN+K562、CRL5826、MSLN+293T)以0.1:1混合,置于RPMI培养基中,各细胞密度配制为1X106/ml,CAR-T细胞与肿瘤细胞各100ul,置于96孔板中,共培养过夜,收集上清,离心后取上清检测细胞因子IFN-γ与IL2的释放水平,采用Elisa试剂盒(Biolegend)进行检测。
结果如图5所示,被MSLN阳性的靶细胞刺激后(P4-KM组、P4-CRL5826组、P4-293M组),P4-28z CAR-T细胞(图中简称P4)能大量分泌IFN-γ与IL-2,而与不表达MSLN抗原的K562细胞共培养后(P4-K组),只少量分泌IFN-γ与IL-2。表明P4-28z CAR-T能被肿瘤表面MSLN抗原有效且特异性的激活。
实施例7
PD-1基因敲除
利用CRISPR/Cas9系统对实施例4获得的P4-28z CAR-T细胞中的PD-1基因进行敲除,获得PD-1表达沉默的CAR-T细胞,即PD-1-/-P4-28z CAR-T细胞。
首先设计靶向PD-1的寡链sgRNA(序列见表2,此处优选SEQ ID NO.:5,如图7所示)及其配对的核酸序列,设计引物,以pX330为模板PCR扩增体外转录的模板,将PCR产物分离纯化后连接到质粒载体中,将质粒转入细菌后进行阳性克隆筛选,测序验证正确后进行扩增并抽离提纯备用。
表2靶向PD-1的sgRNA序列
Sequence ID | sgRNA序列 |
SEQ ID NO.:3 | TGTAGCACCGCCCAGACGACTGG |
SEQ ID NO.:4 | TCAGGCGGAGGTGAGCGGAAGGG |
SEQ ID NO.:5 | GTCTGGGCGGTGCTACAACTGGG |
SEQ ID NO.:6 | CGTCTGGGCGGTGCTACAACTGG |
SEQ ID NO.:7 | GGCGCCCTGGCCAGTCGTCTGGG |
将表达sgRNA的质粒及Cas9蛋白电转入细胞后48小时,收集部分细胞进行流式检测PD-1蛋白水平的敲除效率。同时使用lysis buffer裂解细胞,1000个细胞/ul裂解液,首先置于50℃孵育1hr,再90℃孵育30min,裂解后加入PCR引物进行扩增PD-1,结束后进行凝胶电泳验证,验证后取3.5ulPCR产物,加入6ul 1*accuprime assay buffer,运行以下程序:
结束后加入0.5ul Nuclease S,42℃反应20min后进行凝胶电泳。
结果如图8所示,PD-1基因敲除效率约为30%左右。
实施例8
体内药效研究
选取6-12周大的NOD-Prkdcscid IL2rgnull(NPG)小鼠,腹腔注射2×105H226-ffluc-PDL-1+细胞,50μL DPBS和50μL matrigel matrix(Corning)。两天后检测肿瘤移植物的负荷,分成肿瘤负荷相当的5组,分组后一天分别注射200uL DPBS/鼠,5×106P4-28z-CAR-T细胞/鼠,5×106T细胞/鼠,5×106PD-1-/-P4-28z-CAR-T细胞/鼠,另外,静脉注射5×106P4-28z-CAR-T cells/mouse,CAR-T处理后第26、32、37、41、48、56、62、69、76、83及90天评估小鼠肿瘤负荷,每只小鼠腹腔注射3mg d-luciferin(Perkin Elmer Life Sciences),四分钟后使用Xenogen IVIS Imaging System(Perkin Elmer Life Sciences)拍照,曝光30s。生物发光的信号按照发出的光子量计算,光子量使用曝光时间、表面积归一化,最后得出光子量/s/cm2/球面角度(p/s/cm2/sr)。
结果如图10所示,相比对照组,注射P4-28z-CAR-T及PD-1-/-P4-28zZ-CAR-T细胞的小鼠肿瘤负荷显著减小,而注射PD-1-/-P4-28z-CAR-T细胞的小鼠存活更长,在90天后依然存活,且基本无肿瘤负荷或负荷很少,表明PD-1基因的表达沉默能够维持CART细胞的存活,并增强其抗肿瘤效果。
实施例9
iCasp9基因介导的CART细胞的清除
为进一步验证iCasp9基因介导的CART细胞的清除,利用实施例3、实施例4和实施例7中的类似方法,构建不包含icasp9基因的PD-1-/-P4-28z-CAR-T/T细胞,作为对照,进行实验。
具体地,药物诱导的CAR-T细胞体外增殖实验:体外检测加入诱导药物后PD-1-/-P4-28z-icasp9-CAR-T细胞的增殖活性。分别向CFSE标记的PD-1-/-P4-28z-icasp9-CAR-T/PD-1-/-P4-28z-CAR-T/T细胞培养基中加入10nM AP1903,测定不同时间死细胞和活细胞的数目,计算药物诱导细胞死亡比例。
体内功能实验:验证药物诱导后CAR-T细胞的体内增殖活性。使用FFLuc标记CAR-T细胞,用于标记体内T细胞的活性。向小鼠分别注射5*106T细胞、FFLuc-PD-1-/-P4-28z-icasp9-CAR-T和FFLuc-PD-1-/-P4-28z-CAR-T细胞,第7天腹腔注射AP1903(50mg/鼠),分别在Day0/2天腹腔注射3mg d-luciferin,使用Xenogen IVIS Imaging System拍照,计算得出光子量/s/cm2/球面角度值(p/s/cm2/sr)。
结果显示,在注射AP1903之后,FFLuc-PD-1-/-P4-28z-CAR-T细胞及T细胞组在加药后保持高强度荧光,而FFLuc-PD-1-/-P4-28z-icasp9-CAR-T组,Day2小鼠体内基本检测不到荧光,FFLuc-PD-1-/-P4-28z-icasp9-CAR-T细胞基本被清除,表明AP1903可快速诱导带自杀开关的移植CAR-T细胞。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 亘喜生物科技(上海)有限公司
<120> 具有自杀基因开关的靶向人间皮素的工程化免疫细胞
<130> P2017-1709
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 3576
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggattaca aagacgatga cgataaggcc ttaccagtga ccgccttgct cctgccgctg 60
gccttgctgc tccacgccgc caggccgcaa gtgcaactcc aacaatctgg ccccggtctg 120
gtcactcctt cccagactct tagtcttacc tgtgccatat caggtgattc agtgagtagt 180
aactccgcga catggaattg gatcaggcag tcccccagtc gagggttgga atggctcggt 240
agaacatact accgcagtaa atggtataat gattatgctg taagtgtgaa aagcaggatg 300
tctataaatc cagatacttc caaaaaccag ttctctcttc agcttaatag tgtaactccc 360
gaagacacag ctgtttacta ttgtgcgcgg ggtatgatga cttactacta tggtatggac 420
gtttggggac aaggtacaac cgtcaccgtg tccagtggga tcctgggctc aggcggtgga 480
ggttccggtg ggggcgggtc cggagggggg ggtagccaac ctgttctcac gcaatcatcc 540
tccctcagtg caagccccgg tgcctccgct tctttgacgt gtacactgag gtcaggaatc 600
aatgtagggc cttacaggat ctactggtat cagcaaaaac caggttctcc tccccaatac 660
cttctcaatt acaagagcga ctcagataag cagcagggat ctggagttcc atcaaggttt 720
agtggatcta aagacgcaag cgccaatgcg ggtgtgctgc tcatctcagg tctcaggtcc 780
gaggacgagg ccgattatta ctgtatgatt tggcatagtt cagccgctgt tttcggtgga 840
ggcacgcaac tgacagtgct gaccacgacg ccagcgccgc gaccaccaac accggcgccc 900
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 960
gcagtgcaca cgagggggct ggacttcgcc tgtgattttt gggtgctggt ggtggttggg 1020
ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1080
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1140
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1200
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1260
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1320
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1380
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1440
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1500
tacgacgccc ttcacatgca ggccctgccc cctcgcatcg atgccactaa cttctccctg 1560
ttgaaacaag caggggatgt cgaagagaat cccgggccaa tggtgagcaa gggcgaggag 1620
ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 1680
ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 1740
atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 1800
ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 1860
gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 1920
aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 1980
ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 2040
agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 2100
atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 2160
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 2220
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 2280
gccgggatca ctctcggcat ggacgagctg tacaaggcca ctaacttctc cctgttgaaa 2340
caagcagggg atgtcgaaga gaatcccggg ccaggagtgc aggtggaaac catctcccca 2400
ggagacgggc gcaccttccc caagcgcggc cagacctgcg tggtgcacta caccgggatg 2460
cttgaagatg gaaagaaagt tgattcctcc cgggacagaa acaagccctt taagtttatg 2520
ctaggcaagc aggaggtgat ccgaggctgg gaagaagggg ttgcccagat gagtgtgggt 2580
cagagagcca aactgactat atctccagat tatgcctatg gtgccactgg gcacccaggc 2640
atcatcccac cacatgccac tctcgtcttc gatgtggagc ttctaaaact ggaatctggc 2700
ggtggatccg gagtcgacgg atttggtgat gtcggtgctc ttgagagttt gaggggaaat 2760
gcagatttgg cttacatcct gagcatggag ccctgtggcc actgcctcat tatcaacaat 2820
gtgaacttct gccgtgagtc cgggctccgc acccgcactg gctccaacat cgactgtgag 2880
aagttgcggc gtcgcttctc ctcgctgcat ttcatggtgg aggtgaaggg cgacctgact 2940
gccaagaaaa tggtgctggc tttgctggag ctggcgcagc aggaccacgg tgctctggac 3000
tgctgcgtgg tggtcattct ctctcacggc tgtcaggcca gccacctgca gttcccaggg 3060
gctgtctacg gcacagatgg atgccctgtg tcggtcgaga agattgtgaa catcttcaat 3120
gggaccagct gccccagcct gggagggaag cccaagctct ttttcatcca ggcctgtggt 3180
ggggagcaga aagaccatgg gtttgaggtg gcctccactt cccctgaaga cgagtcccct 3240
ggcagtaacc ccgagccaga tgccaccccg ttccaggaag gtttgaggac cttcgaccag 3300
ctggacgcca tatctagttt gcccacaccc agtgacatct ttgtgtccta ctctactttc 3360
ccaggttttg tttcctggag ggaccccaag agtggctcct ggtacgttga gaccctggac 3420
gacatctttg agcagtgggc tcactctgaa gacctgcagt ccctcctgct tagggtcgct 3480
aatgctgttt cggtgaaagg gatttataaa cagatgcctg gttgctttaa tttcctccgg 3540
aaaaaacttt tctttaaaac atcaggcagt ggctaa 3576
<210> 2
<211> 1191
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Asp Tyr Lys Asp Asp Asp Asp Lys Ala Leu Pro Val Thr Ala Leu
1 5 10 15
Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gln Val Gln
20 25 30
Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln Thr Leu Ser
35 40 45
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala Thr
50 55 60
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly
65 70 75 80
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val
85 90 95
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1 5
Claims (9)
1.一种工程化的免疫细胞,所述工程化的免疫细胞为T细胞,并且所述的免疫细胞具有以下特征:
(a) 所述细胞表达靶向间皮素的CAR,并且所述的CAR结构中包含细胞自杀元件,所述的细胞自杀元件为自杀基因开关iCasp9;和
(b) 所述细胞中的PD1基因表达是被沉默的;
并且,所述CAR的氨基酸序列如SEQ ID NO: 2所示。
2.如权利要求1所述的免疫细胞,其特征在于,编码所述CAR的核苷酸序列如SEQ IDNO: 1所示。
3.一种制备权利要求1所述的免疫细胞的方法,其特征在于,包括以下步骤:
(A) 提供一待改造的免疫细胞;和
(B) 对所述的免疫细胞进行改造,从而使得所述的免疫细胞表达所述的CAR,并且使得所述的免疫细胞中PD1基因的表达沉默,从而获得权利要求1所述的免疫细胞;
其中,所述免疫细胞为T细胞。
4.如权利要求3所述的方法,其特征在于,在步骤(B)中,包括(B1) 将表达所述CAR的第一表达盒导入所述T细胞;和(B2) 将表达用于沉默PD1基因的第二表达盒导入所述T细胞,
其中,所述的步骤(B1)和(B2)的次序无任何限定。
5.如权利要求4所述的方法,其特征在于,所述的第二表达盒包含CRISPR/Cas9即sgRNA和Cas9、反义RNA 、或其组合;其中所述的sgRNA靶向PD1,且sgRNA的序列如SEQ ID NO: 3、4、5、6、或7所示。
6.如权利要求5所述的方法,其特征在于,所述sgRNA的序列如SEQ ID NO: 6所示。
7.一种制剂,其特征在于,所述制剂含有权利要求1所述的免疫细胞,以及药学上可接受的载体。
8.一种权利要求1所述的免疫细胞的用途,其特征在于,用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
9.一种用于制备权利要求1所述的免疫细胞的试剂盒,其特征在于,所述试剂盒含有容器,以及位于容器内的:
(1) 第一核酸序列,所述第一核酸序列含有用于表达所述CAR的第一表达盒;
(2) 第二核酸序列,所述第二核酸序列含有用于沉默PD1的第二表达盒或sgRNA。
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RU2731293C1 (ru) * | 2019-04-12 | 2020-09-01 | Игорь Петрович Белецкий | Способ получения генно-модифицированных линий клеток натуральных киллеров с нокаутированным геном PD-1 и повышенной экспрессией белков семейства Фактора Некроза Опухолей для иммунотерапии онкологических заболеваний |
WO2020216238A1 (en) * | 2019-04-22 | 2020-10-29 | Nanjing Legend Biotech Co., Ltd. | Engineered cells and uses thereof |
KR20220017914A (ko) * | 2019-05-07 | 2022-02-14 | 그라셀 바이오테크놀로지스 (상하이) 컴퍼니, 리미티드 | Bcma를 표적으로 하는 조작된 면역 세포 및 그의 용도 |
CN110592014A (zh) * | 2019-08-14 | 2019-12-20 | 广东美赛尔细胞生物科技有限公司 | 一种在nk细胞治疗中免辐照体外体内持续去除饲养细胞的方法 |
CN112430578A (zh) * | 2019-08-26 | 2021-03-02 | 深圳宾德生物技术有限公司 | 携带安全开关并靶向Her2的嵌合抗原受体T细胞及其制备方法和应用 |
CN110863000B (zh) * | 2019-11-22 | 2020-10-09 | 北京鼎成肽源生物技术有限公司 | 一种转染nk细胞的基因和转染载体 |
WO2021120526A1 (zh) * | 2019-12-16 | 2021-06-24 | 四川大学华西医院 | 同时靶向间皮素和fap的双靶点嵌合抗原受体及其用途 |
KR20220118532A (ko) * | 2019-12-23 | 2022-08-25 | 셀렉티스 | 고형 종양들 암 면역요법을 위한 새로운 메소텔린 특이적 키메라 항원 수용체들 (car) |
CN115197989A (zh) * | 2021-04-09 | 2022-10-18 | 中国科学院分子细胞科学卓越创新中心 | 基于细胞死活表型的药物靶点拮抗剂的新型筛选系统 |
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