CN117070454A - 一种car-t细胞的制备方法 - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种T细胞制备方法,包括:用含有NAD+或其前体的培养基处理T细胞的步骤,培养基还可以含有PQQ和DHQ,其中,所述T细胞还含有嵌合抗原受体的编码序列和/或能够表达嵌合抗原受体。培养基中加入上述小分子,可以提高T细胞增殖、CAR阳性率和抑制T细胞衰老。
Description
技术领域
本发明涉及免疫细胞治疗技术领域,更具体地涉及一种CAR-T细胞的制备方法。
背景技术
肿瘤是严重威胁人类健康和生命的主要疾病之一,每年全球罹患癌症的人数超过1400万,而死于癌症的人数超过800万。高发病率和高死亡率在一定程度上反映了临床上缺少有效防治手段的现状,这对肿瘤的预防和治疗提出了挑战。随着对肿瘤认识的不断深入,肿瘤治疗技术取得了很大的进步,显著改善了整体肿瘤治疗效果,但多或少存在副作用大、易产生耐药性等问题。目前,肿瘤生物免疫治疗已成为一种新的治疗手段。
免疫细胞治疗包括CAR-T免疫疗法、NK免疫疗法、DC免疫疗法、TIL免疫疗法等。免疫细胞治疗需要进行细胞增殖,细胞增殖一般包括细胞复苏、细胞活化、基因编辑、传代培养等。然而肿瘤患者免疫细胞状态较差,而且一般会经过冻存,进一步降低细胞活性,影响免疫细胞的扩增倍数。
NAD+(烟酰胺腺嘌呤二核苷酸,简称为辅酶Ⅰ)是体内很多脱氢酶的辅酶,连接三羧酸循环和呼吸链,其功能是将代谢过程中脱下来的氢传递给黄素蛋白。NAD+能够报证细胞能量供给,促进细胞再生。CN112472710A公开了补充NAD+前体物质能增强多种肿瘤对免疫检查点抑制剂的敏感性。
NAD+对免疫细胞培养的影响,特别是肿瘤患者免疫细胞培养的影响,目前还未有文献报道。
发明内容
本发明的目的在于使用含有NAD+或其前体的培养基处理免疫细胞,促进免疫细胞的扩增与提高细胞活性。
本发明提供了一种免疫细胞制备方法,其特征在于,包括:用含有NAD+或其前体的培养基处理免疫细胞的步骤。
在一些实施方案中,所述免疫细胞是免疫效应细胞(杀伤细胞)或抗原呈递细胞,例如选自T细胞、肿瘤浸润淋巴(TIL)细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、DC细胞、巨噬细胞和B细胞。
在一些实施方案中,所述免疫细胞为免疫效应细胞和抗原呈递细胞。
在一些实施方案中,所述免疫效应细胞还含有嵌合抗原受体的编码序列和/或能够表达嵌合抗原受体。
在一些实施方案中,所述抗原呈递细胞还负载有肿瘤抗原或含有肿瘤抗原的编码序列。
在一些实施方案中,所述免疫细胞还含有抗体的编码序列和/或能够表达抗体。在一些实施方案中,所述抗体是单域抗体。在一些实施方案中,所述单域抗体是多特异性单域抗体。
在一些实施方案中,所述方法包括如下步骤:
(1)从对象获得免疫细胞;
(2)使用含有激活剂的第一培养基处理免疫细胞,获得激活的免疫细胞,
(3)将含有嵌合抗原受体编码序列的核酸引入该激活的免疫细胞中,获得基因修饰的免疫细胞;
(4)使用第二培养基培养该基因修饰的免疫细胞;
其中,所述第一培养基和/或所述第二培养基为含有NAD+或其前体的培养基。
在一些实施方案中,所述方法包括如下步骤:
(1)使抗原呈递细胞负载肿瘤抗原或引入肿瘤抗原的编码序列;
(2)从对象获得免疫效应细胞;
(3)在含有NAD+或其前体的培养基中,将免疫效应细胞与所述抗原呈递细胞共培养足以激活所述免疫效应细胞的时间,得到激活的免疫效应细胞。
在一些实施方案中,所述免疫效应细胞选自T细胞或NK细胞,优选地为T细胞。
在一些实施方案中,所述抗原呈递细胞在负载肿瘤抗原或引入肿瘤抗原的编码序列之前和/或之后使用第三培养基培养。第三培养基也可以是含有NAD+或其前体的培养基。
在一些实施方案中,所述NAD+前体选自色氨酸、喹啉酸、烟酸(NA)、烟酰胺(NAM)、烟酰胺单核苷酸(NMN)、烟酰胺核糖核苷(NR)、或其食品学或药学上可接受的盐、衍生物或前药中的一种或多种;优选为烟酰胺单核苷酸(NMN)。
在一些实施方案中,所述培养基中NAD+或其前体的浓度为1-300μM,优选为50-200μM。
在一些实施方案中,所述培养基还含有类黄酮和/或辅酶;
在一些实施方案中,所述类黄酮选自槲皮素、二氢槲皮素(DHQ)、非瑟酮、儿茶素、没食子素、白藜芦醇、橙皮素中的一种或多种,优选为槲皮素和/或二氢槲皮素;
在一些实施方案中,所述辅酶选自谷胱甘肽或其衍生物、三磷酸腺苷及其衍生物、吡咯喹啉醌或其衍生物、腺苷蛋氨酸或其衍生物、辅酶A或其衍生物、辅酶Q类或其衍生物中的至少一者,优选为吡咯喹啉醌和/或辅酶Q10。
在一些实施方案中,所述培养基中类黄酮的浓度为0-10nM,优选为0.1-10nM,更优选为1-5nM。
在一些实施方案中,所述培养基中辅酶的浓度为0-10nM,优选为0.1-10nM,更优选为0.1-1nM。
在一些实施方案中,所述对象为健康人或肿瘤患者,优选为肿瘤患者。
在一些实施方案中,所述方法具备如下任一或多个特征:
A、步骤(2)中,激活剂选自以下的一种或多种:CD3抗体、CD28抗体、4-1BB抗体、4-1BBL抗原,
B、步骤(2)中,处理时间为12-84小时,优选24-72小时,
C、步骤(3)中,引入方法为电转,
D、步骤(3)中,含有多肽编码序列的核酸为核酸构建体,例如表达载体,优选地,所述表达载体是非病毒载体,
E、步骤(4)中,培养时间为3-10天。
本申请另一方面提供一种T细胞培养基,其含有NAD+或其前体。
在一些实施方案中,所述NAD+前体选自色氨酸、喹啉酸、烟酸(NA)、烟酰胺(NAM)、烟酰胺单核苷酸(NMN)、烟酰胺核糖核苷(NR)、或其食品学或药学上可接受的盐、衍生物或前药中的一种或多种;优选为烟酰胺单核苷酸(NMN)。
在一些实施方案中,所述培养基中NAD+或其前体的浓度为1-300μM,优选为50-200μM。
在一些实施方案中,所述培养基还含有类黄酮和/或辅酶。
在一些实施方案中,所述类黄酮选自槲皮素、二氢槲皮素、非瑟酮、儿茶素、没食子素、白藜芦醇、橙皮素中的一种或多种,优选为槲皮素和/或二氢槲皮素。
在一些实施方案中,所述辅酶选自谷胱甘肽或其衍生物、三磷酸腺苷及其衍生物、吡咯喹啉醌或其衍生物、腺苷蛋氨酸或其衍生物、辅酶A或其衍生物、辅酶Q类或其衍生物中的至少一者,优选为吡咯喹啉醌和/或辅酶Q10。
在一些实施方案中,所述培养基中类黄酮的浓度为0-10nM,优选为0.1-10nM,更优选为1-5nM。
在一些实施方案中,所述培养基中辅酶的浓度为0-10nM,优选为0.1-10nM,更优选为0.1-1nM。
附图说明
图1为NAD+/NADH检测试剂盒的标准曲线;
图2为病人S01CAR-T细胞的增殖和CAR阳性率结果;
图3为病人CAR+细胞中衰老标志物KLRG1+的变化曲线。
具体实施方式
定义
除非另外定义,否则本文使用的所有技术和科学术语具有本发明所属领域的普通技术人员通常所理解的相同的含义。
在本发明中,术语“表达框”是指表达一个基因所需的完整元件,包括启动子和基因编码序列。
术语“编码序列”在文中定义为核酸序列中直接确定其蛋白产物(例如CAR,单链抗体,铰链区和跨膜区)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括,但不限于DNA、cDNA和重组核酸序列。
术语“共刺激分子”是指存在于抗原提呈细胞表面,能与Th细胞上的共刺激分子受体结合,产生协同刺激信号的分子。淋巴细胞的增殖不仅需要抗原的结合,还需要接受共刺激分子的信号。共刺激信号传递给T细胞主要是通过表达在抗原呈递细胞表面的共刺激分子CD80,CD86与T细胞表面的CD28分子结合。B细胞接受共刺激信号可以通过一般的病原体成分例如LPS,或者通过补体成分,或者通过激活了的抗原特异性的Th细胞表面的CD40L。
术语“特异性结合”是指抗体或者抗原结合片段与其所针对的抗原之间的反应。在某些实施方式中,特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指,抗体以小于大约10-5M,例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。“特异性识别”具有类似的含义。
术语“嵌合抗原受体”(CAR)是人工改造受体,能够将识别细胞表面抗原的特异性分子(如抗体)锚定在免疫细胞上,使免疫细胞识别细胞表面抗原(例如肿瘤抗原或病毒抗原)和杀死肿瘤细胞或病毒感染的细胞。CAR通常依次包含任选的信号肽、细胞外抗原结合结构域(简称胞外识别区,例如抗体)、铰链区、跨膜区和胞内信号区。通常,结合肿瘤细胞膜抗原的多肽能够以中等亲和力结合肿瘤细胞广泛表达的膜抗原。结合肿瘤细胞膜抗原的多肽可以是天然多肽或人工合成多肽。
本文所述“重链抗体”是源自骆驼科生物或软骨鱼科生物的抗体。相比上述4链抗体,重链抗体缺失轻链和重链恒定区1(CH1),仅包含2条由可变区(VHH)和其他恒定区组成的重链,可变区通过类似铰链区结构与恒定区相连。骆驼科重链抗体的每条重链包含1个可变区(VHH)和2个恒定区(CH2和CH3),软骨鱼科重链抗体的每条重链含有1个可变区和5个恒定区(CH1-CH5)。重链抗体的抗原结合片段包括VHH或单链重链抗体。通过与人IgG Fc的恒定区融合,重链抗体可以具有人IgG Fc的CH2和CH3。
“抗体片段”包含完整抗体的一部分,优选完整抗体的抗原结合区和/或可变区。抗体片段优选为抗体的抗原结合片段。重链抗体片段的例子包括Fv片段;双抗体;线性抗体;单域抗体(VHH);单链抗体分子;scFv-Fc片段;以及通过化学修饰或通过掺入脂质体中应能够增加半衰期的任何片段。
如本文所用,术语“纳米抗体”、“单域抗体”、“重链抗体的重链可变区结构域”、“VHH”可互换使用,指特异性识别和结合于抗原的VHH。VHH是重链抗体的可变区。通常,VHH含有三个CDR和四个FR。
术语“NAD+前体”为导致NAD+增加的任何小分子,小分子可以还原或非还原形式存在。
本发明的目的之一在于使用含有NAD+或其前体的培养基处理免疫细胞,促进免疫细胞的扩增与提高细胞活性。
本发明提供了一种免疫细胞制备方法,其特征在于,包括:用含有NAD+或其前体的培养基处理免疫细胞的步骤。
NAD+是一种在新陈代谢中发挥核心作用的辅助因子,且NAD+水平随着年龄的增长而下降,NAD+可以帮助修复DNA、调节免疫细胞传导、提供细胞能量降低衰老等。免疫细胞体外制备过程中,添加NAD+处理,可以为免疫细胞扩增提供充足的能量,提高免疫细胞的扩增和降低细胞衰老指标。NAD+前体是通过化学转化变成 NAD+的化合物。
在一些实施方案中,所述免疫细胞是免疫效应细胞(杀伤细胞)或抗原呈递细胞,例如选自T细胞、肿瘤浸润淋巴(TIL)细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、DC细胞、巨噬细胞和B细胞。
在一些实施方案中,所述免疫细胞为免疫效应细胞(杀伤细胞),所述杀伤细胞为T细胞、TIL细胞或NK细胞。
在一些实施方案中,所述杀伤细胞还含有嵌合抗原受体的编码序列和/或能够表达嵌合抗原受体。该CAR含有任选的信号肽序列、胞外靶标识别区(抗原结合域)、铰链区、跨膜区、胞内共刺激域和胞内信号域。所述胞外识别区包含靶向抗原(例如肿瘤抗原)的抗体,例如全长抗体、抗原结合片段、单域抗体(纳米抗体)、单链可变片段(scFv)。
所述嵌合抗原受体的靶向抗原没有限制,包括但不限于:CD19、CD22、CD23、骨髓增生性白血病蛋白(MPL)、CD30、CD32、CD20、CD70、CD79b、CD99、CD123、CD138、CD179b、CD200R、CD276、CD324、Fc受体样5(FcRH5)、CD171、CS-1(信号传导淋巴细胞激活分子家族7,SLAMF7)、C型凝集素样分子-1(CLL-1)、CD33、钙粘蛋白1、钙粘蛋白6、钙粘蛋白16、钙粘蛋白17、钙粘蛋白19、表皮生长因子受体变体III(EGFRviii)、神经节苷脂GD2、神经节苷脂GD3、人白细胞抗原A2(HLA-A2)、B细胞成熟抗原(BCMA)、Tn抗原、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、FMS样酪氨酸激酶3(FLT3)、成纤维细胞激活蛋白(FAP)、肿瘤相关糖蛋白(TAG)-72、CD38、CD44v6、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、B7-H3(CD276)、KIT、白介素13受体亚基α-2(IL-13Ra2)、白介素11受体亚基α(IL11Ra)、间皮素(MSLN)、前列腺干细胞抗原(PSCA)、血管内皮生长因子受体2(VEGFR2)、Lewis Y、CD24,血小板衍生生长因子受体β(PDGFR-β)、蛋白酶丝氨酸21(PRSS21)、唾液酸糖脂阶段特异性胚胎抗原4(SSEA-4)、CD20、免疫球蛋白Fc区、组织因子、叶酸受体α、表皮生长因子受体2(ERBB2)、粘蛋白1(MUC1)、表皮生长因子受体(EGFR)、神经小粘附分子(NCAM)、蛋白酶、前列腺酸性磷酸酶(PAP)、延伸因子2突变型(ELF2M)、Ephrin B2、胰岛素样生长因子I受体(IGF-1受体)、碳酸酐酶IX(CAIX)、潜伏膜蛋白2(LMP2)、黑素细胞蛋白gpl00、bcr-abl、酪氨酸酶、促红细胞生成素产生肝细胞癌A2(EphA2)、岩藻糖基化单唾液酸神经节苷脂(岩藻糖基GM1)、唾液酸化Lewis a(sLea)、神经节苷脂GM3、转谷氨酰胺酶5(TGS5)、高分子量黑色素瘤相关抗原(HMWMAA)、邻乙酰基GD2神经节苷脂、叶酸受体β、TEM1/CD248、肿瘤内皮标记相关蛋白7(TEM7R)、claudin 6(CLDN6)、甲状腺刺激激素受体(TSHR)、T细胞受体(TCR)-β1恒定链、TCRβ2恒定链、TCRγ-δ、G蛋白偶联受体C类5组成员D(GPRC5D)、CXORF61蛋白、CD97、CD179a、间变性淋巴瘤激酶(ALK)、聚唾液酸、胎盘特异性1(PLAC1)、糖类抗原GloboH、乳腺分化抗原NY-BR-1、uroplakin-2(UPK2)、甲型肝炎病毒细胞受体1(HAVCR1)、肾上腺素受体β3(ADRB3)、泛连接蛋白3(PANX3)、G蛋白偶联受体20(GPR20)、淋巴细胞抗原6家族成员K(LY6K)、嗅觉受体家族51亚家族E成员2(OR51E2)、T细胞受体γ链可变阅读框蛋白(TARP)、Wilms肿瘤抗原1蛋白(WT1)、肿瘤-睾丸抗原NY-ESO-1、肿瘤-睾丸抗原LAGE-1a、legumain、人乳头瘤病毒(HPV)E6、HPV E7、人T淋巴营养病毒(HTLV1)-Tax、卡波西氏肉瘤相关疱疹病毒糖蛋白(KSHV)K8.1蛋白、爱泼斯坦-巴尔病毒(EBV)编码的糖蛋白350(EBB gp350)、HIV1包膜糖蛋白gp120、多元自动化基因组工程(MAGE)-A1、易位-Ets-白血病病毒(ETV)蛋白6-AML、精子蛋白17、X抗原家族成员(XAGE)1、跨膜酪氨酸蛋白激酶受体Tie 2、黑色素瘤肿瘤-睾丸抗原MAD-CT-1、黑色素瘤肿瘤-睾丸抗原MAD-CT-2、Fos相关抗原1、p53、p53突变体、prostein、生存速和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)/半乳糖凝集素8、MelanA/MART1、Ras突变体、人端粒酶逆转录酶(hTERT)、δ-样3(DLL3)、滋养层细胞表面抗原2(TROP2)、蛋白酪氨酸激酶7(PTK7)、鸟苷酸环化酶C(GCC)、甲胎蛋白(AFP)、肉瘤易位断裂点、黑色素瘤凋亡抑制剂(ML-IAP)、ERG(TMPRSS2 ETS融合基因)、N-乙酰氨基葡萄糖基转移酶V(NA17)、配对盒蛋白Pax-3(PAX3)、雄激素受体、细胞周期蛋白B1、v-myc禽骨髓细胞瘤病毒癌基因神经母细胞瘤衍生同源物(MYCN)、Ras同源家族成员C(RhoC)、酪氨酸酶相关蛋白2(TRP-2)、细胞色素P4501B1(CYP1B1)、CCCTC结合因子(锌指蛋白)样(BORIS或印记位点的调节物的兄弟(Brother of the Regulator of Imprinted Sites))、T细胞识别的鳞状细胞癌抗原3(SART3)、PAX5、前顶体蛋白结合蛋白sp32(OY-TES1)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、A激酶锚蛋白4(AKAP-4)、滑膜肉瘤、X断裂点2(SSX2)、晚期糖基化终产物受体(RAGE-1)、肾ubiquitous 1(RU1)、RU2、肠道羧基酯酶、热休克蛋白70-2突变型(mut hsp70-2)、CD79a、CD79b、CD72、白细胞相关免疫球蛋白样受体1(LAIR1)、IgA受体Fc片段(FCAR)、白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)、CD300分子样家族成员f(CD300LF)、C型凝集素结构域家族12成员A(CLEC12A)、骨髓基质细胞抗原2(BST2)、含EGF样模块粘蛋白样激素受体样2(EGF-like module-containing mucin-like hormone receptor-like 2,EMR2)、淋巴细胞抗原75(LY75)、磷脂酰肌醇蛋白聚糖-3(GPC3)、Fc受体样5(FCRL5)、免疫球蛋白λ样多肽1(IGLL1)、FITC、促黄体生成激素受体(LHR)、卵泡刺激素受体(FSHR)、绒毛膜促性腺激素受体(CGHR)、CC趋化因子受体4(CCR4)、神经节苷脂GD3、信号传导淋巴细胞激活分子(SLAM)家族成员6(SLAMF6)、SLAMF4、促黄体生成激素受体(LHR)、卵泡刺激素受体(FSHR)、绒毛膜促性腺激素受体(CGHR)或它们的任意组合。
在一些实施方案中,所述胞外识别区为抗间皮素单域抗体,例如WO2022143550A1中所述。在一些实施方案中,所述胞外识别区为抗MUC1单域抗体。在一些实施方案中,所述嵌合抗原受体如PCT/CN2022/143407中所述。上述文献上述专利申请的全文以其中各实施方案具体和单独地被引用而纳入本文。
CAR上任选的信号肽可以根据需要选择。例如CD8信号肽、CD28信号肽、CD4信号肽或轻链信号肽,其序列在本领域技术人员的知识范围内。适用于本发明的CD8信号肽可以是本领域常用于CAR的各种人CD8信号肽序列。
CAR的铰链区选自CD8α铰链区、IgD铰链区、IgG1 Fc CH2CH3铰链区或IgG4 FcCH2CH3铰链区,其序列在本领域技术人员的知识范围内。适用于本发明的CD8铰链区可以是本领域常用于CAR的各种人CD8铰链区序列。
CAR的跨膜区选自CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种;优选为CD28跨膜区或CD8跨膜区,其序列在本领域技术人员的知识范围内。适用于本发明的人CD28跨膜区或CD8跨膜区可以是本领域常用于CAR的各种人CD28跨膜区或CD8跨膜区序列。
可以根据需要选择适合的胞内共刺激域,包括具有共刺激信号分子的胞内结构域,例如CD28、CD134/OX40、CD137/4-1BB、淋巴细胞特异性蛋白酪氨酸激酶、诱导性T细胞共刺激因子(ICOS)和DNAX激活蛋白10的胞内结构域,优选为CD28共刺激域或4-1BB共刺激域。适用于本发明的人CD28共刺激域或4-1BB共刺激域可以是本领域常用于CAR的各种人CD28共刺激域序列或4-1BB共刺激域序列。
类似地,CAR的胞内信号域可以根据需要选择,包括但不限于CD3ζ胞内信号域或FcεRIγ胞内信号域。适用于本发明的CD3ζ胞内信号域可以是本领域已知的各种用于CAR的CD3ζ胞内信号域。
形成本发明的嵌合抗原受体的上述各部分,如CD8信号肽、抗原结合域、CD8铰链区、CD28跨膜区、CD28共刺激域、CD3ζ胞内信号域等,相互之间可直接连接,或者可通过接头序列连接。接头序列可以是本领域周知的适用于抗体的接头序列,例如含G和S的接头序列。通常,接头含有一个或多个前后重复的基序。例如,该基序可以是GGGS、GGGGS、SSSSG、GSGSA和GGSGG。优选地,该基序在接头序列中是相邻的,在重复之间没有插入氨基酸残基。接头序列可以包含1、2、3、4或5个重复基序组成。接头的长度可以是3~25个氨基酸残基,例如3~15、5~15、10~20个氨基酸残基。在某些实施方案中,接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制,通常为2~20个,例如2~15、2~10、2~8个。除甘氨酸和丝氨酸来,接头中还可含有其它已知的氨基酸残基,例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。在某些实施方案中,接头序列为(GGGGS)n连接,其中n为1~5的整数。
在示例性实施方案中,CAR从N端到C端依次含有CD8信号肽、抗MSLN单域抗体或抗MUC1单域抗体、CD8铰链区、CD28跨膜区或CD8跨膜区、CD28共刺激域或4-1BB共刺激域、CD3ζ胞内信号域。
在一些实施方案中,所述免疫细胞为抗原呈递细胞。
在一些实施方案中,所述免疫细胞为免疫效应细胞和抗原呈递细胞。抗原呈递细胞摄取、加工处理抗原,并将抗原呈递给杀伤细胞,从而激活杀伤细胞。将杀伤细胞和抗原呈递细胞共同培养可以用于激活杀伤细胞。
在一些实施方案中,所述抗原呈递细胞还负载有肿瘤抗原或含有肿瘤抗原的编码序列。
在一些实施方案中,所述免疫细胞还含有抗体的编码序列和/或能够表达抗体。在一些实施方案中,所述抗体是单域抗体。在一些实施方案中,所述单域抗体是多特异性单域抗体。
所述肿瘤抗原选自肿瘤相关抗原、肿瘤新抗原;优选地,所述抗原呈递细胞表达所述肿瘤抗原;更优选地,所述肿瘤抗原选自以下的一个或多个:hTERT、p53、Her2、Survivin、CEA、MAGE-A1、MAGE-A2、MAGE-A3、MAGE-C1、MAGE-C2、MUC1、肾母细胞瘤1(WT1)、Her2-neu、P53、NY-ESO-1、hTERT、Mammaglobin-A、Folate Receptor α (FR-α)、HPV16/18-E6、HPV16/18-E7、甲胎蛋白 (AFP)、Glypican3 (GPC3)、前列腺特异性抗原 (PSA)、前列腺酸性磷酸酶(PAP)、前列腺特异性膜抗原 (PSMA)、前列腺干细胞抗原 (PSCA)、前列腺6跨膜上皮抗原1(STEAP1)、B细胞成熟抗原 (BCMA)、CMV pp65、gp100、PRAME;进一步优选地,所述肿瘤抗原包括CEA、survivin和p53中的任意两种或三种。
在一些实施方案中,所述肿瘤抗原的编码序列为RNA,优选为mRNA。
在一些实施方案中,所述多特异性单域抗体含有分别靶向多个靶点的多个功能区,且所述多个功能区分别是单域抗体。
在一些实施方案中,所述多个靶点选自:免疫检查点蛋白、免疫细胞相关抗原、肿瘤相关抗原、免疫共刺激分子或其受体。免疫检查点蛋白包括但不限于:PD-1、CTLA4、PDL1、PDL2、PDL3、TIM3、LAG3、CD47、BTLA、TIGIT、CD160、LAIR1、B7-H1、B7-1、VSIR、CD244。免疫细胞相关抗原包括但不限于:CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、CD80、CD86。肿瘤相关抗原包括但不限于:EIIIB纤连蛋白、Siglecl5、VEGF(R)、HER2、PSMA、AXL、MUC1、MUC16。
在一个或多个实施方案中,所述多个功能区中的至少一个功能区是免疫检查点抑制性单域抗体。
在一个或多个实施方案中,所述多个功能区中的至少一个功能区是免疫共刺激分子或其受体的激活型抗体。
在一个或多个实施方案中,所述多个功能区分别为不同的免疫检查点抑制性单域抗体。
在一个或多个实施方案中,所述多特异性单域抗体含有分别靶向2个或3个靶点的2个或3个功能区。
在一个或多个优选实施方案中,所述多特异性单域抗体含有:靶向PD-1的第一功能区,和靶向CTLA4的第二功能区;所述第一功能区是抗PD-1单域抗体,所述第二功能区是抗CTLA-4单域抗体。示例性的PD-1单域抗体如WO2022143552A1中所述,示例性的CTLA-4单域抗体如WO2023051618A1中所述。上述文献上述专利申请的全文以其中各实施方案具体和单独地被引用而纳入本文。
在一些实施方案中,所述多特异性单域抗体的编码序列为DNA或RNA,优选为mRNA或saRNA(自复制RNA)。
在一个或多个实施方案中,多特异性纳米抗体还含有Fc区;优选地,所述Fc区为IgG1、IgG2、IgG3或IgG4的Fc区;所述IgG1、IgG2、IgG3或IgG4为人源。
由于Fc区会引起显著的ADCC、CDC效应,可能会导致免疫细胞损伤,具有药理学的负面效应。因而通常会对Fc进行改造(本文还称为变体Fc),通过位点突变,多特异性纳米抗体与FcγRIIIa和/或C1q的亲和力常数相比突变前降低,提高抗体药物药效。目前的已经公开了的突变位点,按照EU编号系统,包括:IgG1中Fc区突变位点包括L234A、L235A、L235E、L235G、G236A、G237A、N297A、G318A、L320A、L322A;IgG3中Fc区突变位点包括V234A、G237A、P238S、H28A、V309L、A330S、P331S;IgG3中Fc区突变位点包括Leu281、Leu282、Gly283、Gly284、Asn344、Pro378;IgG4中Fc区突变位点包括S228P、E233P、F234V、L235A、F243L、D254A、R292P、Y300L、L309V、R409K。本文的变体Fc包括但不限于具有上述突变位点的各IgG的Fc。
在一个或多个实施方案中,所述Fc区为IgG1的Fc区域,且按照EU编号系统,所述Fc区具有如下一种或多种突变:L234A、L235A、G237A。在一个或多个实施方案中,所述Fc区为IgG4的Fc区域,且按照EU编号系统,所述Fc区具有如下一种或多种突变:S228P、E233P、F234V、L235A、D254A、L309V、R409K。
在一些实施方案中,所述方法包括如下步骤:
(1)从对象获得免疫细胞;
(2)使用含有激活剂的第一培养基处理免疫细胞,获得激活的免疫细胞,
(3)将含有嵌合抗原受体编码序列的核酸引入该激活的免疫细胞中,获得基因修饰的免疫细胞;
(4)使用第二培养基培养该基因修饰的免疫细胞;
其中,所述第一培养基和/或所述第二培养基为含有NAD+或其前体的培养基。
在一些实施方案中,所述方法包括如下步骤:
(1)使抗原呈递细胞负载肿瘤抗原或引入肿瘤抗原的编码序列;
(2)从对象获得免疫效应细胞;
(3)在含有NAD+或其前体的培养基中,将免疫效应细胞与所述抗原呈递细胞共培养足以激活所述免疫效应细胞的时间,得到激活的免疫效应细胞。
在一些实施方案中,所述免疫效应细胞选自T细胞或NK细胞,优选地为T细胞。
在一些实施方案中,所述抗原呈递细胞为DC细胞,所述免疫效应细胞选自T细胞或NK细胞,优选地为T细胞。
在一些实施方案中,所述抗原呈递细胞在负载肿瘤抗原或引入肿瘤抗原的编码序列之前和/或之后使用第三培养基培养。第三培养基也可以是含有NAD+或其前体的培养基。
本文中,在将多肽编码序列导入免疫细胞之前使用“激活剂”将免疫细胞活化,有助于缓解细胞制备中的细胞损伤,提高细胞活力和存活比例。理论上,可使用本领域已知的任何可用于免疫细胞活化的试剂。在免疫细胞是T细胞(特别是CD3+T细胞)的示例性实施方案中,所述激活剂包括选自以下的一种或多种:CD3抗体、CD28抗体、4-1BB抗体、4-1BBL抗原。
激活的过程包括在激活剂和细胞接触的条件下孵育免疫细胞。在孵育混合物中,激活剂终浓度、免疫细胞的浓度以及二者的比例不受限制。例如,激活剂可以包被在固相载体上,包被液的终浓度可为1-20μg/ml,例如1、5、10、15或20μg/ml。激活剂与免疫细胞的浓度比可为1-20μg/ml:2.45-2.8×108个免疫细胞。所述激活的温度为适合免疫细胞生长的任何温度,优选27-45℃,更优选37℃。通常,所述激活在含有CO2的环境中进行,例如5%的CO2。
激活孵育所需的培养基可以是任何适合免疫细胞(例如T细胞)生长的商用或自制培养基。在一个或多个实施方案中,使用含有或不含5%血清或其替代物的AIM-V培养基进行激活孵育。优选地,所述培养基中还含有细胞因子,例如IL-2、IL-15、IL-21、IL-7、IL-6、LSD1抑制剂、MALT1抑制剂。当免疫细胞为T细胞时,细胞因子为IL-7和/或IL-15,优选地,IL-7的终浓度为1-50ng/mL,IL-15的终浓度为1-50ng/mL。当免疫细胞为NK细胞时,细胞因子为IL-2和/或IL-15。
所述激活剂可以溶质的形式存在于孵育混合物中,或者其可固定在固相载体上。可用于固定激活剂(例如抗体)的固相载体本领域周知,例如磁珠或容器壁。在一些实施方案中,所述激活剂为固定在磁珠上的CD3抗体和CD28抗体;优选地,所述激活剂为MiltenyiMACS GMP TransAct CD3/28磁珠和/或CTS Dynabeads CD3/28。在一些实施方案中,所述激活剂为固定在容器壁上的CD3抗体,CD3抗体和CD28抗体,CD3抗体和4-1BB抗体,或CD3抗体和4-1BBL抗原;优选地,所述容器为T75瓶。
在一些实施方案中,使用容器包被的CD3抗体激活分选的T细胞24-72小时后电转,优选激活48小时;使用容器包被的CD3抗体和CD28抗体激活分选的T细胞24-72小时后电转,优选激活48小时;使用容器包被的CD3抗体和4-1BBL抗原激活分选的T细胞24-72小时后电转,优选激活48小时;使用容器包被的CD3抗体和4-1BB抗体激活分选的T细胞24-72小时后电转;使用包被CD3抗体和CD28抗体的Miltenyi MACS GMP TransAct CD3/28磁珠激活分选的T细胞24-72小时后电转,优选激活72小时;使用包被CD3抗体和CD28抗体的DynabeadsCD3/28磁珠激活分选的T细胞24-72小时后电转,优选激活48-72小时。在一个或多个实施方案中,Miltenyi MACS GMP TransAct CD3/28磁珠的终浓度为4mL/1×108个T细胞,6mL/1×108个T细胞,8mL/1×108个T细胞。在一个或多个实施方案中,CTS Dynabeads CD3/28磁珠与T细胞的比例为1:1。
激活前,免疫细胞经过分选,例如T细胞、NK细胞。本领域知晓通常用于分选免疫细胞特别是T细胞的方法,例如抗体分选或流式细胞术。在一个或多个实施方案中,所述T细胞是CD3+T细胞。
本文中,核酸转化优选电转。通常,将电转混合物在电转装置中电击。示例性地,所述电转混合物包含:核酸、如本文所述方法激活的免疫细胞和电转液。在具体实施例中,电转装置采用为Lonza Nucleofactor 4D或Maxcyte电转仪(LONZA设备的电转液为V4XP-3024;Maxcyte设备的电转液为Gibco Opti-MEM™ I Reduced Serum Medium),电转程序为FI-115或Resting T/ Expand T4。本领域技术人员可以选择不同的电转仪并根据待电转的核酸和细胞选择电转程序和参数。
在一些实施方案中,所述NAD+前体选自色氨酸、喹啉酸、烟酸(NA)、烟酰胺(NAM)、烟酰胺单核苷酸(NMN)、烟酰胺核糖核苷(NR)、或其食品学或药学上可接受的盐、衍生物或前药中的一种或多种;优选为烟酰胺单核苷酸(NMN)。NMN作为NAD+的直接前体,可以通过NMNAT酶容易直接转化为NAD+,且容易被细胞吸收。
在一些实施方案中,所述培养基中NAD+或其前体的浓度为1-300μM,优选为50-200μM。NAD+或其前体的浓度过大,会导致细胞能量代谢过快,不利于细胞的长期增殖。
在一些实施方案中,所述培养基还含有类黄酮和/或辅酶。
类黄酮具有抗自由基和抗氧化作用,且能够延缓细胞凋亡。在一些实施方案中,所述类黄酮选自槲皮素、二氢槲皮素、非瑟酮、儿茶素、没食子素、白藜芦醇、橙皮素中的一种或多种,优选为槲皮素和/或二氢槲皮素。其中,槲皮素是一种来自多酚的类黄酮组的植物黄酮醇,其存在于许多水果、蔬菜、叶子、种子和谷物中,是一种天然存在的极性生长素转运抑制剂。二氢槲皮素是一种二氢黄酮醇类化合物,属于维生素P族,是一种应用广泛的生物活性剂,在人体中具有多种生物活性,包括抗氧化、清除自由基等作用。
辅酶是一大类有机辅助因子的总称,是酶催化氧化还原反应、基团转移和异构反应的必须因子。在一些实施方案中,所述辅酶选自谷胱甘肽或其衍生物、三磷酸腺苷及其衍生物、吡咯喹啉醌或其衍生物、腺苷蛋氨酸或其衍生物、辅酶A或其衍生物、辅酶Q类或其衍生物中的至少一者,优选为吡咯喹啉醌和/或辅酶Q10。其中,吡咯喹啉醌(PQQ)是一种氧化还原酶辅基,化学式为C14H6N2O8,参与多种氧化还原反应,通过作为辅酶的形式,介导一系列细胞和分子生化反应,从而影响人体的生理和代谢过程。
在一些实施方案中,所述培养基中类黄酮的浓度为0-10nM,优选为0.1-10nM,更优选为1-5nM。
在一些实施方案中,所述培养基中辅酶的浓度为0-10nM,优选为0.1-10nM,更优选为0.1-1nM。
在一些实施方案中,所述对象为健康人或肿瘤患者,优选为肿瘤患者。获得的免疫细胞为新鲜采集或冻存复苏后的细胞。
在一些实施方案中, 步骤(2)中,激活剂选自以下的一种或多种:CD3抗体、CD28抗体、4-1BB抗体、4-1BBL抗原。
在一些实施方案中,步骤(2)中,处理时间为12-84小时,优选24-72小时。
在一些实施方案中,步骤(3)中,引入方法为电转。
在一些实施方案中,步骤(3)中,含有多肽编码序列的核酸为核酸构建体,例如表达载体,优选地,所述表达载体是非病毒载体。
在一些实施方案中,步骤(4)中,培养时间为3-10天。
在免疫细胞培养基中加入NAD+或其前体、类黄酮和/或辅酶,可以提高细胞内NAD+含量、增加线粒体含量、提供充足能量以增强细胞功能,改善细胞代谢和活性,从而促进细胞增殖、抑制细胞衰老、使细胞的端粒和长寿蛋白增加、核酸转导阳性率、增加细胞因子分泌和靶细胞杀伤能力等。
本发明还提供一种药物组合物,包含本发明所述制备方法制备得到的免疫细胞,以及药学上可以接受的辅料。
在一些实施方案中,所述药物组合物为CAR-T制剂。
在一些实施方案中,所述药物组合物为NK细胞制剂。
在一些实施方案中,所述药物组合物为疫苗组合物,优选DC疫苗。
下文将以具体实施例的方式阐述本发明。应理解,这些实施例仅仅是阐述性的,并非意图限制本发明的范围。实施例中所用到的方法和材料,除非另有说明,否则均为本领域常规的材料和方法。
实施例
实施例1,PBMC复苏和NAD+检测
复苏2个donor(肿瘤病人)的PBMC,使用AIM-V培养基(含2% FBS)复苏PBMCs,按照5E6/mL接种进培养瓶中,放置于培养箱(37℃;5% CO2)。复苏当天进行NAD+检测,采用NAD+/NADH检测试剂盒(WST-8法,碧云天)。检测试剂盒的标准曲线如图1所示,NAD+检测结果如表1所示。根据检测结果,从病人S01(0.13uM)和病人S02(0.19uM)中选择NAD+水平较低的S01进行CAR-T细胞制备。
表1 S01和S02 PBMC的NAD+检测结果
实施例2,CAR-T制备
采用不同的CAR-T培养基,培养基的分组如下表2所示:
表2 CAR-T培养基及分组
使用表2所示的不同组别的培养基,以及使用5ug/mL anti-CD3以及anti-CD28在4℃条件过夜包被6孔板或者培养瓶。将复苏的PBMCs接种到经过包被的6孔板或者培养瓶中,刺激48h进行活化。收集细胞并采用Lonza 4D电转仪进行电转,电转过程根据电转仪说明书操作步骤进行优化。用100uL电转液重悬5×106 到7×106PBMC细胞,将6ug pC23S-1444加入到细胞悬液中,同时电转体系中加入20ug PB mRNA(PB酶的氨基酸序列如CN105154473A中所述)和200IU Rnase inhibitor,用EO-115程序进行电转。电转后室温静置10min。之后,将PBMCs转移到相应分组的培养基的6孔板中(需要提前预热至37℃),放置到培养箱(37℃;5%CO2)中培养24h-48h,再将培养基补充至4mL。电转后的第3天进行第一次传代,第6天进行第二次传代,第10天CAR-T细胞制备完成。传代过程中的PBMCs培养于表2所示相应组别的培养基中,传代细胞密度为5×105/ml。CAR-T细胞制备过程及制备完成后,利用流式细胞仪检测细胞表面CAR阳性率及表面标志物。
上述pC23S-1444质粒为含有1444CAR(从N端到C端依次含有CD8信号肽、1444号抗间皮素纳米抗体、CD8 铰链区、CD8跨膜区、4-1BB共刺激域、CD3ζ胞内信号域)的编码序列的质粒。pC23S-1444质粒的核苷酸序列如SEQ ID NO:1所示。
通过检测CAR-T细胞的增殖和CAR阳性率变化,评价小分子NMN、PQQ和DHQ对CAR-T细胞体外制备的影响。病人S01 CAR-T细胞的增殖和CAR阳性率结果如图2所示。由图中可知,在CAR-T培养基中添加小分子NMN、PQQ和DHQ,可以促进CAR-T制备中的总细胞增殖、CAR+(即为CAR阳性)细胞的增殖和CAR+细胞的百分比。
通过检测CAR+细胞中衰老标志物KLRG1+的变化,评价小分子NMN、PQQ和DHQ对CAR-T细胞衰老的影响。病人CAR+细胞中衰老标志物KLRG1+的变化曲线如图3所示。由图中可知,加入小分子NMN、PQQ和DHQ,特别是第四组培养基,可以降低具有衰老标志物KLRG1+的细胞百分比,因而具有维持细胞的年轻状态的效果,从而提高CAR-T治疗效果。
Claims (17)
1.一种T细胞培养基,其特征在于,含有NAD+或其前体。
2.如权利要求1所述的培养基,其特征在于,所述NAD+前体选自色氨酸、喹啉酸、烟酸、烟酰胺、烟酰胺单核苷酸、烟酰胺核糖核苷、或其食品学或药学上可接受的盐、衍生物或前药中的一种或多种。
3.如权利要求2所述的培养基,其特征在于,所述NAD+前体为烟酰胺单核苷酸。
4.如权利要求1所述的培养基,其特征在于,所述培养基中NAD+或其前体的浓度为1-300μM。
5.如权利要求4所述的培养基,其特征在于,所述培养基还含有类黄酮和/或辅酶。
6.如权利要求5所述的培养基,其特征在于,所述类黄酮选自槲皮素、二氢槲皮素、非瑟酮、儿茶素、没食子素、白藜芦醇、橙皮素中的一种或多种。
7.如权利要求6所述的培养基,其特征在于,所述类黄酮为槲皮素和/或二氢槲皮素。
8.如权利要求5所述的培养基,其特征在于,所述辅酶选自谷胱甘肽或其衍生物、三磷酸腺苷及其衍生物、吡咯喹啉醌或其衍生物、腺苷蛋氨酸或其衍生物、辅酶A或其衍生物、辅酶Q类或其衍生物中的至少一种。
9.如权利要求8所述的培养基,其特征在于,所述辅酶为吡咯喹啉醌和/或辅酶Q10。
10.如权利要求5所述的培养基,其特征在于,所述培养基中类黄酮的浓度为0-10nM。
11.如权利要求5所述的培养基,其特征在于,所述培养基中辅酶的浓度为0-10nM。
12.一种T细胞制备方法,其特征在于,包括:使用权利要求1-11任一项所述的培养基处理T细胞的步骤。
13.如权利要求12所述的方法,其特征在于,所述T细胞还含有嵌合抗原受体的编码序列和/或能够表达嵌合抗原受体。
14.如权利要求13所述的方法,其特征在于,所述方法包括如下步骤:
(1)从对象获得T细胞;
(2)使用含有激活剂的第一培养基处理T细胞,获得激活的T细胞,
(3)将含有嵌合抗原受体编码序列的核酸引入该激活的T细胞中,获得基因修饰的T细胞;
(4)使用第二培养基培养该基因修饰的T细胞;
其中,所述第一培养基和/或所述第二培养基为权利要求1-11任一项所述的培养基。
15.如权利要求14所述的方法,其特征在于,所述对象为健康人或肿瘤患者。
16.如权利要求15所述的方法,其特征在于,所述对象为肿瘤患者。
17.如权利要求14所述的方法,其特征在于,具备如下A-E中的任一或多个特征:
A、步骤(2)中,激活剂选自以下的一种或多种:CD3抗体、CD28抗体、4-1BB抗体、4-1BBL抗原,
B、步骤(2)中,处理时间为12-84小时,
C、步骤(3)中,引入方法为电转,
D、步骤(3)中,含有多肽编码序列的核酸为核酸构建体,
E、步骤(4)中,培养时间为3-10天。
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FEREIDOON SHAHIDI 主编: "《贝雷油脂化学与工艺学 第6版》", 中国协和医科大学出版社, pages: 439 - 442 * |
范登霞: ""βNMN和3MOBA作为增强剂促进T细胞的增殖、分化和功能"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, vol. 2022, no. 2, pages 257 - 258 * |
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