WO2024037477A1 - 双特异性嵌合抗原受体和免疫细胞、制备方法、应用及肿瘤治疗药品 - Google Patents
双特异性嵌合抗原受体和免疫细胞、制备方法、应用及肿瘤治疗药品 Download PDFInfo
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Abstract
涉及双特异性嵌合抗原受体和免疫细胞、制备方法、应用及肿瘤治疗药品。提供了双特异性嵌合抗原受体,该嵌合抗原受体含有B细胞表面抗原结合分子结构域和肿瘤抗原结合分子结构域;表达该嵌合抗原受体的受体细胞能对肿瘤细胞进行杀伤,有良好的治疗效果。
Description
本公开请求2022年08月15日向中国国家知识产权局提交的专利申请号为202210977266.6,发明名称为“双特异性嵌合抗原受体和免疫细胞、制备方法、应用及肿瘤治疗药品”的中国专利申请的优先权和权益,并且通过参照将其全文并入本公开。
本公开涉及生物技术领域,尤其是涉及双特异性嵌合抗原受体和免疫细胞、制备方法、应用及肿瘤治疗药品。
嵌合抗原受体T细胞免疫疗法,简称CART技术,是通过体外改造病人的T细胞,使病人的T细胞具备识别肿瘤细胞的能力,体外扩大培养后回输到病人体内进行治疗的一种方法。在CAR-T疗法的领域中,对于血液肿瘤效果最为显著,CAR-T疗法在治疗难治复发性B细胞急性淋巴细胞白血病时可以达到约90%完全缓解率,而在治疗B细胞淋巴瘤时可以达到约70%完全缓解率。除此之外,部分临床显示,大约有20%的患者接受CAR-T治疗后可以获得长期生存,CAR-T疗法对于血液肿瘤展现出令人惊叹疗效,但在实体瘤治疗效果上仍需改进。
鉴于此,特提出本公开。
发明内容
本公开的目的在于,提供一种双特异性嵌合抗原受体和免疫细胞、制备方法、应用及肿瘤治疗药品,其具有良好的实体瘤治疗效果。
为了解决上述技术问题,实现上述目的,本公开提供以下技术方案:
第一方面,本公开提供了双特异性嵌合抗原受体,所述嵌合抗原受体含有B细胞表面抗原结合分子结构域和肿瘤抗原结合分子结构域;所述肿瘤为实体瘤;
所述B细胞表面抗原结合分子结构域与肿瘤抗原结合分子结构域经连接肽串联。
本公开的发明人发现,血液肿瘤和实体瘤最主要的区别就是实体瘤有固定的生长地方,血液中没有或者非常少的肿瘤细胞;而血液瘤的肿瘤细胞是大多数都是在血液中循环的。当CAR-T细胞静脉回输给患者后,对于血液瘤来说,CAR-T细胞就能够立刻接触到肿瘤细胞,通过CAR分子上靶向的单链抗体识别肿瘤细胞表面的抗原分子,从而快速激活CAR-T细胞,让绝大部分CAR-T细胞都能够受到刺激并开始增殖,直到杀灭全部肿瘤细胞。对于实体瘤来说就不一样了,当CAR-T细胞静脉回输给患者后,由于血液中没有或者非常少的肿瘤细胞,CAR-T细胞回输后,基本上接触不到肿瘤细胞,无法通过CAR分子上靶向结构域识别肿瘤细胞表面的抗原分子,导致CAR-T细胞不能被激活,在接触肿瘤细胞前,过早凋亡,逐渐凋亡殆尽,最终无法正常发挥疗效。
本文中“CAR”、“嵌合抗原受体”,是人工改造受体,例如其包括能够将识别肿瘤细胞表面抗原或病毒抗原的特异性分子(如抗体或其抗原结合片段)锚定在免疫效应细胞(如T细胞)上,使免疫细胞识别肿瘤抗原或病毒抗原并杀死肿瘤细胞或病毒的受体。表达CAR的T细胞称为CAR-T(或CART)。通常,嵌合抗原受体依次包含胞外结构域、跨膜区域和胞内信号结构域。可采用本领域已知的用于构建CAR的跨膜区域和胞内信号结构域来构建本公开的嵌合抗原受体。肿瘤细胞表面的抗原(受体)与嵌合抗原受体的抗体(配体)结合时,可以通过铰链区和跨膜区将信号传递至胞内,胞内信号域再将信号转化为活化信号,激活效应细胞,效应细胞通过分泌穿孔素或者产生细胞因子杀伤肿瘤细胞,同时效应细胞本身也发生扩增,进一步扩大免疫杀伤作用。胞外结构域可包含抗原结合结构域(特异性结合抗原的抗体部分),抗原结合结构域可以是特异性结合抗原的单链可变区片段(也即single-chain variable fragment,scFv或单链抗体)。单链可变区片段可作为CAR的胞外结构域,该结构域决定CAR-免疫细胞的特异性。铰链区(Hinge)通常由一段起连接作用的肽构成,例如铰链区连接CAR的单链抗体和跨膜结构域,它通常维持效应细胞中稳健的CAR表达和活性所需的稳定性,大多数CAR的铰链区由IgG的铰链或CD8α/CD28胞外区衍变而来。跨膜结构域将CAR的细胞外结构域与细胞内信号转导结构域连接。常用的跨膜结构域例如来源于CD4,CD8α,CD28和CD3ζ。跨膜结构域的选择可影响CAR结构在细胞功能上的活化程度。胞内结构域由共刺激结构域和信号转导结构域构成。“共刺激分子”或“共刺激信号传导结构域”是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。T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。可采用本领域已知的用于构建CAR的共刺激分子,包括但不限于CD27、CD28、4-1BB、OX40、ICOS、B7-H3、CD16、NKp44、NKp46、DNAM-1、NKG2D和/或它们的任何组合。
本公开提供的双特异性嵌合抗原受体能够靶向B细胞和实体瘤细胞。而将该嵌合抗原受体转化进免疫细胞制备得到的CAR细胞在施用进入机体内后,能够被体内B细胞刺激激活,使CAR细胞在体内大量增殖,避免在接触肿瘤细胞前,过早凋亡。而后在接触肿瘤细胞后,对肿瘤细胞进行杀伤,实现良好的治疗效果。从而解决CAR细胞在治疗肿瘤患者时由于体内CAR细胞如CAR-T细胞数量不足而导致的疗效不佳的问题。
将B细胞表面抗原结合分子结构域与肿瘤抗原结合分子结构域经连接肽串联构建在同一个CAR上,该设计结构相较于将不同抗原结合结构域分别设计在不同CAR上的设计结构,可避免或减少CAR细胞在表达CAR时抗原结合结构域表达不完全的情况,提高双阳性率,提高实体瘤治疗效果。
在可选的实施方式中,前述实施方式所述B细胞表面抗原结合分子包括抗B细胞表面抗原的scFv分子,所述肿瘤抗原结合分子包括抗肿瘤抗原的scFv分子。
术语“scFv”、“单链抗体”是包含轻链可变区和重链可变区的融合蛋白,其中轻链可变区(VL)和重链可变区(VH)通过连接肽连接,能够表达为单链多肽,并且scFv保持其所源自的完整抗体的抗原结合活性。除非特别指出,否则在本文中scFv可以以任何一种顺序具有VL和VH可变区,例如scFv多肽的N端至C端,可以包含:a)VL-接头-VH或b)VH-接头-VL。
术语“连接子”、“连接肽”、“连接多肽”、“Linker”或“接头”指连接两个多肽片段的连接单元,通常具有一定的柔性,接头的使用不会使蛋白质结构域原有的功能丧失。连接子可以是肽连接子,其包含一个或多个氨基酸,典型的约1-30个、2-24个或3-15个氨基酸;在一些实施方案中,所述连接子为选自(GxS)y连接子,其中,x选自1-5的整数,y选自1-6的整数。
在可选的实施方式中,前述实施方式所述串联方式为(a)~(d)中任一种:
(a)所述抗B细胞表面抗原的scFv分子的重链可变区与抗肿瘤抗原的scFv分子的重链可变区经连接肽串联;
(b)所述抗B细胞表面抗原的scFv分子的重链可变区与抗肿瘤抗原的scFv分子的轻链可变区经连接肽连接;
(c)所述抗肿瘤抗原的scFv分子的轻链可变区与抗肿瘤抗原的scFv分子的重链可变区经连接肽连接;
(d)所述抗肿瘤抗原的scFv分子的轻链可变区与抗肿瘤抗原的scFv分子的轻链可变区经连接肽连接。
可选地,前述实施方式所述连接肽的氨基酸组成为(G4S)n,所述n为1、2、3、4或5。
在可选的实施方式中,前述实施方式所述B细胞表面抗原选自CD19、BCMA、CD20或CD22。
需要说明的是,所述B细胞表面抗原结合分子结构域目的在于能够识别B细胞表面抗原,从而使得当表达上述嵌合抗原受体的免疫细胞进入人体后能够被血液中的B细胞激活从而避免过早凋亡,因此,凡是能够靶向B细胞表面抗原,能够被B细胞激活的表面抗原结合分子结构域均可用于本公开。
在可选的实施方式中,所述实体瘤包括但不限于间皮瘤、胃肠道腺瘤、肝细胞癌、胶质瘤、膀胱癌、结直肠癌、神经母细胞瘤、胶质母细胞瘤、胃癌、大肠癌、食道癌、胰腺癌、宫颈癌、甲状腺肿瘤、尿路上皮癌、肾癌、子宫内膜癌、前列腺癌、卵巢癌、转移性癌症、胆管癌、淋巴瘤、黑色素瘤、甲状腺癌、肉瘤、肺癌、胰腺肿瘤、卵巢透明细胞癌、非小细胞肺癌、星形细胞瘤、肝癌、结肠癌、卵黄囊癌、视网膜母细胞瘤、肺鳞状细胞癌、胸腺癌、或乳腺癌。
在可选的实施方式中,所述肿瘤抗原包括但不限于GUCY2C、MSLN、Claudin18.2、GPC3、EGFR、HER2、CEA、GD2、EGFRvⅢ、MUC1、PRLR、CLCA1、MUC12、GPR35、CR1L、MUC17、TMPRSS11B、MUC21、TMPRSS1IE、CD207、SLC30A8、CFC1、SLC12A3、SSTR1、GPR27、FZD10、TSHR、SIGLEC15、SLC6A3、KISSIR、QRFPR、GPR119、CLDN6、UPK2、ADAM12、SLC45A3、ACPP、MUC21、MUC16、MS4A12、ALPP、EphA2、FAP、IL13-Ra2、PSMA、ROR1、VEGFR-II、FR-a、EpCAM、EGFRvII、tMUC1、PSCA、FCER2、GPR18、FCRLA、CXCR5、FCRL3、FCRL2、HTR3A、CLEC17A、TRPMI、SLC45A2、SLC24A5、DPEP3、KCNK16、LIM2,KCNV2、SLC26A4、CD171、Glypican-3、IL-13、CD79a/b或MAGEA4。
而所述肿瘤抗原结合分子结构域则可根据治疗目的,选择具体肿瘤细胞靶点,例如针对实体瘤细胞,则可选择在实体瘤中特异表达的表面抗原,同时为了应对肿瘤细胞表面抗原的突变,还可选择保守的实体瘤细胞表面抗原作为靶点。
胃肠道肿瘤(胃癌、结肠癌等)是国内的高发癌症(第二、三大高发),是治疗需求高度未被满足的适应症,目前大多数结直肠癌是微卫星稳定的(约占晚期CRC的95%),并伴有KRAS或BRAF突变,预后非常差。
GUCY2C在各种胃肠道恶性肿瘤中均有表达,包括95%以上的大肠癌和50%以上的胃或胃食管交界癌,GUCY2C在包括肝转移在内的所有阶段的大多数结肠癌中都具有高表达,中等分化和高分化的胃肠道肿瘤往往与GUCY2C的更高的膜表达相关,但在其他非胃肠道肿瘤中基本不表达,转移性结直肠癌细胞中GUCY2C的表达为正常肠上皮细胞的2~10倍。
GUCY2C由于很大程度上限制性表达于正常组织肠上皮紧密连接的顶端,而由于肿瘤破坏了正常的紧密连接结构,将GUCY2C更彻底地暴露出来,因此与正常组织相比,只结合GUCY2C抗体,从而不会对正常组织的影响。
正常情况下,MSLN(间皮素)在间皮细胞当中表达,其功能与细胞粘附有一定的关联。MSLN在多种实体瘤中过表达,例如包括间皮瘤、胰腺癌、卵巢癌、肺癌、胆管癌、胃癌、结肠癌、胸腺癌、食管癌、乳腺癌以及子宫内膜癌等。
Claudin18.2(CLDN18.2)是Claudin蛋白质家族的一员,位于细胞膜表面,正常情况下仅低水平表达于胃粘膜分化上皮细胞,但在病理状态下,Claudin18.2在多种肿瘤中有的表达显著上调,包括80%的胃肠道腺瘤、60%的胰腺肿瘤。此外,CLDN 18.2活化还可见于食管癌、卵巢癌和肺腺癌中。
磷脂酰肌醇蛋白聚糖3(glypican 3,GPC3)在调控细胞生长和分化方面起重要作用,与肝癌的发生、发展密切相关。GPC3在肝细胞癌、卵巢透明细胞癌、卵黄囊癌等生殖系统肿瘤中表达,其在肝细胞癌中的表达率达74.8%,而在正常肝组织内几乎无表达。
皮生长因子受体(EGFR)是一种酪氨酸激酶受体,约在50%的成人原发性胶质母细胞瘤中存在,此外,也可作为非小细胞肺癌、肝癌、胃癌、结直肠癌等癌种的靶点。
PSMA是一种II型跨膜蛋白,通常在肾小管和十二指肠中表达,并特异性地表达于前列腺癌上皮细胞。在前列腺癌组织中,PSMA的表达明显上调。PSMA称为前列腺癌诊断及治疗的重要靶点。
表皮生长因子受体-2(HER2)是一种大分子的I型跨膜蛋白,又称ErbB2,是ErbB受体家族的一员。HER2最早出现在乳腺癌的研究中,乳腺癌红基因突变容易导致HER2阳性,并且HER2高表达欲肿瘤低预后、低无病生存期有关,HER2也高表达在部分膀胱癌、卵巢癌、子宫内膜癌、胰腺癌和非小细胞肺癌中。
胚抗原(CEA)最初发现于结肠癌和胎儿肠组织中,后被证实广泛存在于内胚叶起源的消化系统肿瘤,比如胃癌、肝癌、胰腺癌、结直肠癌等。
神经节苷脂2(GD2)是一种表面糖脂抗原,在神经母细胞瘤、星形细胞瘤、视网膜母细胞瘤、肉瘤、黑色素瘤等多种肿瘤细胞中高表达,而在正常组织中表达受限。
表皮生长因子受体变体Ⅲ(EGFRvⅢ)是一种胶质母细胞瘤相关特异性抗原,是胶质母细胞瘤治疗的理想靶点。
MUC1是一种跨膜黏蛋白,广泛过表达在胃癌、肝癌、胰腺癌。
实体瘤抗原以及对应的相关肿瘤见下表:
需要说明的是,本领域技术人员可以根据治疗需要选择合适的实体瘤抗原与B细胞抗原搭配组合,以实现更好的治疗效果,其无论以何种靶点搭配,均属于本公开的保护范围。
在可选的实施方式中,前述实施方式所述抗B细胞表面抗原的scFv分子为靶向CD19的scFv分子;所述抗肿瘤抗原的scFv分子为靶向GUCY2C的scFv分子或靶向MSLN的scFv分子。
在可选的实施方式中,前面任一实施方式所述的嵌合抗原受体,其中所述靶向CD19的scFv分子包括SEQ ID NO:2中的scFv分子的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;可选地,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义,或由Kabat编号系统定义,或由Chothia编号系统定义,或由Contact编号系统定义,或由AbM编号系统定义;
术语“互补性决定区”、“CDR”是指免疫球蛋白的重链和轻链的高度可变区,其是抗体可变结构域内主要促成与抗原特异性结合的区域。重链互补决定区用HCDR表示,重链可变区中含有的3个CDR区:HCDR1、HCDR2和HCDR3;轻链互补决定区用LCDR表示,轻链可变区中含有的3个CDR区:LCDR1、LCDR2和LCDR3。可以通过各种公知方案来确定CDR的氨基酸序列边界,例如:“Kabat”编号规则(参见Kabat等(1991),“Sequences of Proteins of Immunological Interest”,第5版,Public Health Service,National Institutes of Health,Bethesda,MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc,M.P.等,Dev.Comp.Immunol.,27,55-77(2003);Front Immunol.2018 Oct 16;9:2278)等。除非特别说明,否则本公开的一些实施方式中,所述CDR根据Kabat编码规则确定。
在可选的实施方式中,其中所述靶向CD19scFv分子,其中,LCDR1序列为SEQ ID NO:2的第45-55位氨基酸残基,LCDR2序列为SEQ ID NO:2的第71-77位氨基酸残基,LCDR3序列为SEQ ID NO:2的第110-118位氨基酸残基,HCDR1序列为SEQ ID NO:2的第174-178位氨基酸残基,HCDR2序列为SEQ ID NO:2的第193-208位氨基酸残基,HCDR3序列为SEQ ID NO:2的第241-252位氨基酸残基;
在可选的实施方式中,其中所述靶向CD19scFv分子,其中,轻链可变区序列为SEQ ID NO:2的第22-128位氨基酸残基,重链可变区序列为SEQ ID NO:2的第144-263位氨基酸残基;
在可选的实施方式中,所述靶向CD19的scFv分子的氨基酸序列如SEQ ID NO:2中第22~263位所示。
在可选的实施方式中,前面任一实施方式所述的嵌合抗原受体,其中所述靶向GUCY2C的scFv分子包括SEQ ID NO:4中的scFv分子的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;可选地,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义,或由Kabat编号系统定义,或由Chothia编号系统定义,或由Contact编号系统定义,或由AbM编号系统定义;
在可选的实施方式中,所述靶向GUCY2C的scFv分子,其中LCDR1序列为SEQ ID NO:4的第183-197位氨基酸残基,LCDR2序列为SEQ ID NO:4的第213-219位氨基酸残基,LCDR3序列为SEQ ID NO:4的第252-260位氨基酸残基,HCDR1序列为SEQ ID NO:4的第52-56位氨基酸残基,HCDR2
序列为SEQ ID NO:4的第71-87位氨基酸残基,HCDR3序列为SEQ ID NO:4的第120-133位氨基酸残基;
在可选的实施方式中,所述靶向GUCY2C的scFv分子,其中轻链可变区序列为SEQ ID NO:4的第160-271位氨基酸残基,重链可变区序列为SEQ ID NO:4的第22-144位氨基酸残基;
在可选的实施方式中,所述靶向GUCY2C的scFv分子的氨基酸序列如SEQ ID NO:4中第22~271位所示。
在可选的实施方式中,前面任一实施方式所述的嵌合抗原受体,其中所述靶向MSLN的scFv分子包括SEQ ID NO:8中的scFv分子的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;可选地,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义,或由Kabat编号系统定义,或由Chothia编号系统定义,或由Contact编号系统定义,或由AbM编号系统定义;
在可选的实施方式中,所述靶向MSLN的scFv分子,其中LCDR1序列为SEQ ID NO:8的第187-200位氨基酸残基,LCDR2序列为SEQ ID NO:8的第216-226位氨基酸残基,LCDR3序列为SEQ ID NO:8的第261-269位氨基酸残基,HCDR1序列为SEQ ID NO:8的第52-58位氨基酸残基,HCDR2序列为SEQ ID NO:8的第73-90位氨基酸残基,HCDR3序列为SEQ ID NO:8的第123-133位氨基酸残基;
在可选的实施方式中,所述靶向MSLN的scFv分子,其轻链可变区序列为SEQ ID NO:8的第165-280位氨基酸残基,其重链可变区序列为SEQ ID NO:8的第22-144位氨基酸残基;
在可选的实施方式中,所述靶向MSLN的scFv分子的氨基酸序列如SEQ ID NO:8中第22~280位所示。
在可选的实施方式中,前述实施方式所述嵌合抗原受体还包括信号肽结构域和跨膜结构域。
在可选的实施方式中,前面任一项所述跨膜结构域为选自以下蛋白质的跨膜结构域:T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154;
在可选的实施方式中,所述信号肽结构域选自CD8SP结构域;
在可选的实施方式中,所述人CD8信号肽其序列为SEQ ID NO:2的第1-21位氨基酸残基;
在可选的实施方式中,所述跨膜结构域为CD8-TM;
在可选的实施方式中,所述CD8-TM的序列为SEQ ID NO:2的第264-332位氨基酸残基;
在可选的实施方式中,所述嵌合抗原受体包括初级信号传导结构域和共刺激信号传导结构域。
在可选的实施方式中,所述信号传导结构域为选自以下的蛋白质的至少一个功能性信号结构域:MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CDS、ICAM-1、LFA-1、4-1BB、B7-H3、CDS、ICAM-1、ICOS、GITR、BAFFR、LIGHT、HVEM、KIRDS2、SLAMF7、NKp80、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、RANKL、CD226、SLAMF4、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a和CD83配体;
在可选的实施方式中,所述初级信号传导结构域选自CD3-ζ刺激性结构域。
在可选的实施方式中,所述初级信号传导结构域序列为SEQ ID NO:2的第335-486位氨基酸残基;
在可选的实施方式中,所述共刺激信号传导结构域选自4-1-BB胞内结构域、CD28结构域、CD16结构域、NKp44结构域、NKp46结构域、DNAM-1结构域或NKG2D结构域中的至少一种。
在可选的实施方式中,所述4-1-BB胞内结构域的序列为SEQ ID NO:2的第333-374位氨基酸残基。
在可选的实施方式中,所述嵌合抗原受体氨基酸序列如SEQ ID NO:6所示或如SEQ ID NO:10所示。
第二方面,本公开提供了生物材料,所述生物材料包括以下(ⅰ)~(Ⅳ)任一项:
(ⅰ)核酸分子,包括编码前述实施方式任一项所述嵌合抗原受体的DNA分子或RNA分子;
(ⅱ)重组载体,包括慢病毒载体和(ⅰ)核酸分子;
(ⅲ)转化体,包括(ⅱ)重组载体转染宿主细胞后得到的慢病毒;
(Ⅳ)嵌合抗原受体细胞,包括细胞膜表达前述实施方式任一项所述嵌合抗原受体的嵌合抗原受体细胞,或者,将(ⅲ)转化体转化进免疫细胞得到的嵌合抗原受体免疫细胞,所述免疫细胞选自NK细胞或T细胞。
在可选的实施方式中,前述嵌合抗原受体细胞为NK细胞或T细胞。
第三方面,本公开提供前述实施方式所述生物材料(ⅰ)~(ⅲ)任一项在制备嵌合抗原受体细胞中的应用。
第四方面,本公开提供双特异性嵌合抗原受体细胞的制备方法,将编码前述实施方式任一项所述嵌合抗原受体的DNA分子插入慢病毒载体得到重组载体,而后转染宿主细胞包装成慢病毒,再使用慢病毒转化免疫细胞得到双特异性嵌合抗原受体细胞。
在可选的实施方式中,所述DNA分子包含编码双特异性结合分子的DNA片段,所述双特异性结合分子由靶向CD19的scFv分子、和靶向GUCY2C的scFv分子或靶向MSLN的scFv分子串联组成。
在可选的实施方式中,所述编码双特异性结合分子的DNA片段的核苷酸序列如SEQ ID NO:5所示或如SEQ ID NO:9所示。
在可选的实施方式中,所述宿主细胞选自293细胞。
在可选的实施方式中,所述免疫细胞选自NK细胞或T细胞。
第五方面,本公开提供前述实施方式任一项所述嵌合抗原受体、前述实施方式所述生物材料或采用前述实施方式所述制备方法得到的双特异性嵌合抗原受体细胞在制备肿瘤治疗药品中的应用。
第六方面,本公开提供肿瘤治疗药品,所述药品含有前述实施方式所述(Ⅳ)嵌合抗原受体细胞或采用前述实施方式所述制备方法得到的双特异性嵌合抗原受体细胞。
第七方面,本公开提供一种治疗肿瘤的方法,包括向需要治疗的主体施用前述实施方式所述的肿瘤治疗药品。
在可选的实施方式中,前面任一项所述药品用于治疗肿瘤,可选地,所述肿瘤为表达CD19的肿瘤,表达GUCY2C的肿瘤,或表达MSLN的肿瘤。
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案,下面将对具体实施方式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,下面描述中的附图是本公开的一些实施方式,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为本公开实施例1中提供的五种嵌合抗原受体元件组成示意图;
图2为本公开实施例3得到的第一组三种效应细胞的阳性率检测结果;
图3为本公开实施例3得到的第二组三种效应细胞的阳性率检测结果;
图4为本公开实施例3得到的第一组三种效应细胞的体外细胞因子分泌结果;
图5为本公开实施例3得到的第一组三种效应细胞的杀瘤流式检测结果;
图6为本公开实施例3得到的第一组三种效应细胞的体外杀瘤效果对比图;
图7为本公开实施例3得到的第二组三种效应细胞的体外杀瘤效果对比图;
图8为本公开实施例3得到的第二组三种效应细胞的体外细胞因子分泌结果;
图9为本公开实施例3得到的第二组三种效应细胞的体外杀瘤效果对比图;
图10为本公开实施例1中插入了CAR-CD19 DNA片段的慢病毒载体PCDHF质粒图谱。
为使本公开实施例的目的、技术方案和优点更加清楚,下面将结合本公开实施方式中的附图,对本公开实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式是本公开一部分实施方式,而不是全部的实施方式。实施方式中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
因此,以下对在附图中提供的本公开的实施例的详细描述并非旨在限制要求保护的本公开的范围,而是仅仅表示本公开的选定实施方式。基于本公开中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本公开保护的范围。
下面结合附图,对本公开的一些实施方式作详细说明。在不冲突的情况下,下述的实施例及实施例中的特征可以相互组合。
实施例1双CAR分子的慢病毒包装载体的构建
采用基因合成的方法,分别合成DNA片段CAR-CD19(结合CD19的CAR)、CAR-GUCY2C(结合GUCY2C的CAR)、CAR-MSLN(结合MSLN的CAR)、CAR-CD19&GUCY2C(结合CD19和GUCY2C的CAR)和CAR-CD19&MSLN(结合CD19和MSLN的CAR),五种嵌合抗原受体元件组成示意图见图1,具体核苷酸序列和对应的氨基酸序列如下:
CAR-CD19核苷酸序列为SEQ ID NO:1,氨基酸序列为SEQ ID NO:2;
CAR-GUCY2C核苷酸序列为SEQ ID NO:3,氨基酸序列为SEQ ID NO:4;
CAR-CD19&GUCY2C核苷酸序列为SEQ ID NO:5,氨基酸序列为SEQ ID NO:6。
CAR-MSLN核苷酸序列为SEQ ID NO:7,氨基酸序列为SEQ ID NO:8;
CAR-CD19&MSLN核苷酸序列为SEQ ID NO:9,氨基酸序列为SEQ ID NO:10。
将上述DNA片段CAR-CD19、CAR-GUCY2C和CAR-CD19&GUCY2C作为分组一,DNA片段CAR-CD19、CAR-MSLN和CAR-CD19&MSLN作为分组二,各片段分别构建至慢病毒载体上(参考图10),设计引物,通过测序结果验证载体构建的正确性,构建完成的慢病毒包装载体培养3代保存待用。
实施例2包装并浓缩慢病毒
将293t按照5×106cells/10cm培养皿的密度接种,次日观察细胞的状态,用PEI转染的方法将实施例1得到的慢病毒包装载体与辅助质粒PCDH D(PMD2.G)、PCDH M(pMDLg-pRRE)和PCDH N(pRSV-Rev)共转至293t,转染完6小时后换液,按照15mL/150mm2培养皿添加含有10%胎牛血清的DMEM培养基,转染完48小时与72小时收集病毒上清,2000rpm 4℃10min离心,去除细胞碎片,之后0.45微米的滤器过滤杂质,过滤后的病毒悬液用25000rpm 4℃2小时浓缩慢病毒,浓缩后的病毒加入适量的培养基重悬,置于-80℃保存。
实施例3生产CAR-T及对照细胞
抽志愿者的外周血血20mL,Ficoll梯度离心分离PBMC,用Stemcell公司T细胞阴选试剂盒(货号:19051)分离T细胞,分离后的T细胞用添加5%人AB血清及300单位/mL IL-2X-VIVO 15培养基重悬T细胞至1×106cells/mL,用含1%FBS X-VIVO 15清洗beads,按照数量比,磁珠:T细胞=2:1比例加入预先清洗过的磁珠(Cat#40203D,10mL,Life technology),2~3days后用新鲜的培养基重悬T细胞至(3~5)×106cells/mL,按照MOI=10值加入慢病毒(参见实施例2),同时加入8ug/mL的Polybrene,4~6小时后,补加培养基稀释细胞至1×106cells/mL,次日更换新鲜培养基,使细胞浓度维持在(0.2~0.3)×106PBMC/mL,之后每隔2~3天更换一次培养基,病毒侵染完72小时后流式分析细胞阳性率。
第一组三种效应细胞的阳性率检测结果如图2所示,结果显示,CART-CD19(表达结合CD19的CAR的T细胞)效应细胞的阳性率为62.69%,CART-GUCY2C(表达结合GUCY2C的CAR的T细胞)效应细胞的阳性率为73.48%,CART-CD19&GUCY2C(表达结合CD19和GUCY2C的CAR的T细胞)效应细胞的阳性率为51.11%,结果见图2。
第二组三种效应细胞的阳性率检测结果如图3所示,结果显示,CART-CD19(表达结合CD19的CAR的T细胞)效应细胞的阳性率为41.82%,CART-MSLN(表达结合MSLN的CAR的T细胞)效应细胞的阳性率为71.94%,CART-CD19&MSLN(表达结合CD19和MSLN的CAR的T细胞)效应细胞的阳性率为25.22%,结果见图3。
实施例4 CART-CD19&GUCY2C体外杀伤功能评估
将实施例3得到的T细胞、CART-CD19、CART-GUCY2C和CART-CD19&GUCY2C效应细胞和靶细胞共培养72h后,根据检测靶细胞残留情况来观察这三种CAR-T细胞的体外杀瘤功能情况。具体步骤为:取K562-CD19(细胞膜上表达CD19的K562细胞)和K562-GUCY2C细胞(细胞膜上表达GUCY2C的K562细胞),计数离心后用X-VIVO 15培养基重悬,向24孔板中的15个孔中分别加入1×105个K562-CD19和1×105个K562-GUCY2C细胞,取T-cell、CART-19效应细胞、CART-GUCY2C效应细胞和CART-CD19&GUCY2C效应细胞,计数离心,用X-VIVO 15培养基重悬,分别往孔中加入1×106个细胞,共培养72h后,收集细胞悬液并离心,上清用于检测细胞因子IFN-γ含量,检测试剂盒为Human IFN Gamma Elisa RSG(Invitrogen,88-7316-88),按照检测试剂盒说明书进行检测。
三种效应细胞的体外细胞因子分泌结果如图4所示,结果显示,CAR-T与靶细胞共培养72h后,上清中含有大量的IFN-γ,说明CAR-T细胞在杀伤靶细胞的同时分泌了大量的IFN-γ,也间接证明了CAR-T细胞对靶细胞具有杀伤功能。
细胞沉淀采用anti-CD19-FITC和anti-GUCY2C-APC流式抗体染色后上机检测。
三种效应细胞的杀瘤流式检测结果如图5所示,三种效应细胞的体外杀瘤效果对比图如图6所示,图5和图6结果显示,CART-CD19效应细胞只对K562-CD19细胞具有杀伤功能,对K562-GUCY2C细胞没有杀伤功能,CART-GUCY2C效应细胞只对K562-GUCY2C细胞具有杀伤功能,对K562-CD19细胞没有杀伤功能,而CART-CD19&GUCY2C效应细胞对K562-CD19细胞和K562-GUCY2C细胞同时具有杀伤功能。
实施例5 CART-CD19&MSLN体外杀伤功能评估
将实施例3得到的T细胞、CART-CD19、CART-MSLN和CART-CD19&MSLN效应细胞在体外杀伤Nalm6(人急性B淋巴白血病细胞)和HGC27靶细胞(人胃癌细胞),杀伤Nalm6靶细胞实验方法为:先利用cytocalceinTM violet 550对靶细胞进行染色,其次调整效应细胞密度为5×106个/mL,靶细胞密度为5×105个/mL,将上述效应细胞与靶细胞按照0:1、0.5:1、1:1、2:1、4:1及8:1加入96孔板中混匀共培养6h,在上述时间段显微镜下观察凋亡情况,并将混合细胞离心,沉淀部分用100μL binding buffer重悬,300g离心5min,添加1.2μL APC-Annexin V和1.2μL PI染料,避光孵育15min,添加100μL binding buffer流式检测凋亡效率,结果显示CART-CD19和CART-CD19&MSLN对表达CD19的Nalm6靶细胞具有显著的杀瘤效果,体外杀瘤效果见图7。
杀伤HGC-27靶细胞实验方法为:xCELLigence实时、细胞介导的细胞毒性系统(Acea Biosciences Inc.)用于评估CAR-T细胞介导的细胞毒性,2E4个HGC-27或A375细胞在E-Plate 16(Acea Biosciences)的每个孔中的150μL生长培养基中,并在37℃培养箱中培养过夜,使用RTCA DP Analyzer系统每15分钟量化一次电阻抗和RTCA软件版本2.0(Acea Biosciences Inc.)。24小时后,加入50μL CAR-T细胞(E:T比例为2.5:1)或50μL培养基作为阴性,在接下来的24小时内量化细胞介导的杀伤,每15分钟读取一次电阻抗,48h后收集细胞上清检测IFN-r细胞因子分泌情况,结果见图8。结果显示CART-MSLN和CART-CD19&MSLN对表达MSLN的HGC-27靶细胞具有显著的杀伤效果,结果见图9。
实施例6 CART-CD19&GUCY2C体内杀瘤功能评估
NSG小鼠,雌性,6-8周,在第0天皮下接种LS1034肿瘤细胞,5.0E+06/只,第9天根据量瘤结果分成4组,分别为PBS组,T-cell组,CART-GUCY2C组,CART-CD19&GUCY2C组,第9天给CART-CD19&GUCY2C组尾静脉注射2.0E+6/只的Nalm6肿瘤细胞(ATCC,CRL-3273),第14天,PBS组尾静脉注射PBS 200μL,T-cell组尾静脉注射2.0E+6/只的T-cell(实施例3获得),CART-GUCY2C组尾静脉注射2.0E+6/只的CART-GUCY2C(实施例3获得),CART-CD19&GUCY2C组尾静脉注射2.0E+6/只的CART-CD19&GUCY2C(实施例3获得),随后每周测量两次肿瘤体积,用游标卡尺测量肿瘤长径和短径。以公式:(1/2)X长径X(短径)2计算肿瘤体积。实验结果见表1,实验结果表明,CART-CD19&GUCY2C组比CART-GUCY2C组具有显著的抑瘤效果。
表1.CART-CD19&GUCY2C在LS1034小鼠肿瘤模型的抗肿瘤活性
上述实施例涉及的序列如下:
SEQ ID NO:1
SEQ ID NO:2
备注:SEQ ID NO:2中,第22~263位氨基酸残基为靶向CD19的scFv分子的氨基酸序列,靶向CD19的scFv分子的轻链可变区序列为SEQ ID NO:2的第22-128位氨基酸残基,重链可变区序列为SEQ ID NO:2的第144-263位氨基酸残基;LCDR1序列为SEQ ID NO:2的第45-55位氨基酸残基,LCDR2序列为SEQ ID NO:2的第71-77位氨基酸残基,LCDR3序列为SEQ ID NO:2的第110-118位氨基酸残基,HCDR1序列为SEQ ID NO:2的第174-178位氨基酸残基,HCDR2序列为SEQ ID NO:2的第193-208位氨基酸残基,HCDR3序列为SEQ ID NO:2的第241-252位氨基酸残基;人CD8信号肽的序列为SEQ ID NO:2的第1-21位氨基酸残基,人CD8铰链-跨膜区的序列为SEQ ID NO:2的第264-332位氨基酸残基;4-1BB分子胞内区的序列为SEQ ID NO:2的第333-374位氨基酸残基,人CD3Zeta分子胞内区序列为SEQ ID NO:2的第335-486位氨基酸残基;所述CDR根据Kabat编号系统定义。
SEQ ID NO:3
SEQ ID NO:4
备注:SEQ ID NO:4中,第22~271位氨基酸残基为靶向GUCY2C的scFv分子的氨基酸序列,靶向GUCY2C的scFv分子的轻链可变区序列为SEQ ID NO:4的第160-271位氨基酸残基,重链可变区序列为SEQ ID NO:4第22-144位氨基酸残基;LCDR1序列为SEQ ID NO:4的第183-197位氨基酸残基,LCDR2序列为SEQ ID NO:4第213-219位氨基酸残基,LCDR3序列为SEQ ID NO:4第252-260位氨基酸残基,HCDR1序列为SEQ ID NO:4第52-56位氨基酸残基,HCDR2序列为SEQ ID NO:4第71-87位氨基酸残基,HCDR3序列为SEQ ID NO:4第120-133位氨基酸残基;所述CDR根据Kabat编号系统定义。
SEQ ID NO:5
SEQ ID NO: 6
SEQ ID NO: 7
SEQ ID NO: 8
备注:SEQ ID NO:8中,第22~280位氨基酸残基为靶向MSLN的scFv分子的氨基酸序列,靶向MSLN的scFv分子的轻链可变区序列为SEQ ID NO:8的第165-280位氨基酸残基,重链可变区序列为SEQ ID NO:8的第22-144位氨基酸残基;LCDR1序列为SEQ ID NO:8的第187-200位氨基酸残基,LCDR2序列为SEQ ID NO:8的第216-226位氨基酸残基,LCDR3序列为SEQ ID NO:8的第261-269位氨基酸残基,HCDR1序列为SEQ ID NO:8的第52-58位氨基酸残基,HCDR2序列为SEQ ID NO:8的第73-90位氨基酸残基,HCDR3序列为SEQ ID NO:8的第123-133位氨基酸残基;所述CDR根据Kabat编号系统定义。
SEQ ID NO:9
SEQ ID NO:10
以上所述仅为本公开的优选实施例而已,并不用于限制本公开,对于本领域的技术人员来说,本公开可以有各种更改和变化。凡在本公开的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本公开的保护范围之内。
Claims (13)
- 双特异性嵌合抗原受体,所述嵌合抗原受体含有B细胞表面抗原结合分子结构域和肿瘤抗原结合分子结构域;所述肿瘤为实体瘤;所述B细胞表面抗原结合分子结构域与肿瘤抗原结合分子结构域经连接肽串联。
- 根据权利要求1所述的嵌合抗原受体,其中所述B细胞表面抗原结合分子包括抗B细胞表面抗原的scFv分子,所述肿瘤抗原结合分子包括抗肿瘤抗原的scFv分子。
- 根据权利要求1或2所述的嵌合抗原受体,所述串联方式为(a)~(d)中任一种:(a)所述抗B细胞表面抗原的scFv分子的重链可变区与抗肿瘤抗原的scFv分子的重链可变区经连接肽串联;(b)所述抗B细胞表面抗原的scFv分子的重链可变区与抗肿瘤抗原的scFv分子的轻链可变区经连接肽连接;(c)所述抗肿瘤抗原的scFv分子的轻链可变区与抗肿瘤抗原的scFv分子的重链可变区经连接肽连接;(d)所述抗肿瘤抗原的scFv分子的轻链可变区与抗肿瘤抗原的scFv分子的轻链可变区经连接肽连接;可选地,所述连接肽的氨基酸组成为(G4S)n,所述n为1、2、3、4或5。
- 根据权利要求1~3任一项所述的嵌合抗原受体,所述B细胞表面抗原选自CD19、BCMA、CD20或CD22。
- 根据权利要求1~4任一项所述的嵌合抗原受体,所述实体瘤选自间皮瘤、胃肠道腺瘤、肝细胞癌、胶质瘤、膀胱癌、结直肠癌、神经母细胞瘤、胶质母细胞瘤、胃癌、大肠癌、食道癌、胰腺癌、宫颈癌、甲状腺肿瘤、尿路上皮癌、肾癌、子宫内膜癌、前列腺癌、卵巢癌、转移性癌症、胆管癌、淋巴瘤、黑色素瘤、甲状腺癌、肉瘤、肺癌、胰腺肿瘤、卵巢透明细胞癌、非小细胞肺癌、星形细胞瘤、肝癌、结肠癌、卵黄囊癌、视网膜母细胞瘤、肺鳞状细胞癌、胸腺癌、或乳腺癌;可选地,所述肿瘤抗原选GUCY2C、MSLN、Claudin18.2、GPC3、EGFR、HER2、CEA、GD2、EGFRvⅢ、MUC1、PRLR、CLCA1、MUC12、GPR35、CR1L、MUC17、TMPRSS11B、MUC21、TMPRSS1IE、CD207、SLC30A8、CFC1、SLC12A3、SSTR1、GPR27、FZD10、TSHR、SIGLEC15、SLC6A3、KISSIR、QRFPR、GPR119、CLDN6、UPK2、ADAM12、SLC45A3、ACPP、MUC21、MUC16、MS4A12、ALPP、EphA2、FAP、IL13-Ra2、PSMA、ROR1、VEGFR-II、FR-a、EpCAM、EGFRvII、tMUC1、PSCA、FCER2、GPR18、FCRLA、CXCR5、FCRL3、FCRL2、HTR3A、CLEC17A、TRPMI、SLC45A2、SLC24A5、DPEP3、KCNK16、LIM2,KCNV2、SLC26A4、CD171、Glypican-3、IL-13、CD79a/b或MAGEA4。
- 根据权利要求2~5任一项所述的嵌合抗原受体,所述抗B细胞表面抗原的scFv分子为靶向CD19的scFv分子;所述抗肿瘤抗原的scFv分子为靶向GUCY2C的scFv分子或靶向MSLN的scFv分子;可选地,所述靶向CD19的scFv分子包括SEQ ID NO:2中的scFv分子的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;可选地,所述靶向GUCY2C的scFv分子包括SEQ ID NO:4中的scFv分子的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;可选地,所述靶向MSLN的scFv分子包括SEQ ID NO:8中的scFv分子的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;可选地,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义,或由Kabat编号系统定义,或由Chothia编号系统定义,或由Contact编号系统定义,或由AbM编号系统定义;可选地,所述靶向CD19scFv分子,其中所述LCDR1、LCDR2和LCDR3的氨基酸序列分别为SEQ ID NO:2的第45-55位氨基酸残基、第71-77位氨基酸残基、第110-118位氨基酸残基;所述HCDR1、HCDR2和HCDR3的氨基酸序列分别为SEQ ID NO:2的第174-178位氨基酸残基、第193-208位氨基酸残基、第241-252位氨基酸残基;可选地,所述靶向GUCY2C的scFv分子,其中所述LCDR1、LCDR2和LCDR3的氨基酸序列分别为SEQ ID NO:4的第183-197位氨基酸残基、第213-219位氨基酸残基、第252-260位氨基酸残基; 所述HCDR1、HCDR2和HCDR3的氨基酸序列分别为SEQ ID NO:4的第52-56位氨基酸残基、第71-87位氨基酸残基、第120-133位氨基酸残基;可选地,所述靶向MSLN的scFv分子,其中所述LCDR1、LCDR2和LCDR3的氨基酸序列分别为SEQ ID NO:8的第187-200位氨基酸残基、第216-226位氨基酸残基、第261-269位氨基酸残基;所述HCDR1、HCDR2和HCDR3的氨基酸序列分别为SEQ ID NO:8的第52-58位氨基酸残基、第73-90位氨基酸残基、第123-133位氨基酸残基;可选地,所述靶向CD19scFv分子,其中轻链可变区序列为SEQ ID NO:2的第22-128位氨基酸残基,重链可变区序列为第144-263位氨基酸残基;可选地,所述靶向GUCY2C的scFv分子,其中轻链可变区序列为SEQ ID NO:4的第160-271位氨基酸残基,重链可变区序列为第22-144位氨基酸残基;可选地,所述靶向MSLN的scFv分子,其中轻链可变区序列为SEQ ID NO:8的第165-280位氨基酸残基,重链可变区序列为第22-144位氨基酸残基;可选地,所述靶向CD19的scFv分子的氨基酸序列如SEQ ID NO:2中第22~263位所示;可选地,所述靶向GUCY2C的scFv分子的氨基酸序列如SEQ ID NO:4中第22~271位所示;可选地,所述靶向MSLN的scFv分子的氨基酸序列如SEQ ID NO:8中第22~280位所示。
- 根据权利要求1~6任一项所述的嵌合抗原受体,所述嵌合抗原受体还包括信号肽结构域和跨膜结构域;可选地,所述信号肽结构域选自CD8 SP结构域;可选地,所述跨膜结构域包括初级信号传导结构域和共刺激信号传导结构域;可选地,所述初级信号传导结构域选自CD3-ζ刺激性结构域;可选地,所述共刺激信号传导结构域选自4-1-BB胞内结构域、CD28结构域、CD16结构域、NKp44结构域、NKp46结构域、DNAM-1结构域或NKG2D结构域中的至少一种;可选地,所述嵌合抗原受体氨基酸序列如SEQ ID NO:6所示或如为SEQ ID NO:10。
- 生物材料,其包括以下(ⅰ)~(Ⅳ)任一项:(ⅰ)核酸分子,包括编码权利要求1~7任一项所述嵌合抗原受体的DNA分子或RNA分子;(ⅱ)重组载体,包括慢病毒载体和(ⅰ)核酸分子;(ⅲ)转化体,包括(ⅱ)重组载体转染宿主细胞后得到的慢病毒;(Ⅳ)嵌合抗原受体细胞,包括细胞膜表达权利要求1~7任一项所述嵌合抗原受体的嵌合抗原受体细胞,或者,将(ⅲ)转化体转化进免疫细胞得到的嵌合抗原受体免疫细胞,所述免疫细胞选自NK细胞或T细胞;可选地,所述嵌合抗原受体细胞为NK细胞或T细胞。
- 权利要求8所述生物材料(ⅰ)~(ⅲ)任一项在制备嵌合抗原受体细胞中的应用。
- 双特异性嵌合抗原受体细胞的制备方法,其包括将编码权利要求1~7任一项所述嵌合抗原受体的DNA分子插入慢病毒载体得到重组载体,而后转染宿主细胞包装成慢病毒,再使用慢病毒转化免疫细胞得到双特异性嵌合抗原受体细胞;可选地,所述DNA分子包含编码双特异性结合分子的DNA片段,所述双特异性结合分子由靶向CD19的scFv分子与靶向GUCY2C的scFv分子串联组成,或由靶向CD19的scFv分子与靶向MSLN的scFv分子串联组成;可选地,所述编码双特异性结合分子的DNA片段的核苷酸序列如SEQ ID NO:5所示或如SEQ ID NO:9所示;可选地,所述宿主细胞选自293细胞;可选地,所述免疫细胞选自NK细胞或T细胞。
- 权利要求1~7任一项所述嵌合抗原受体、权利要求8所述生物材料或采用权利要求10所述制备方法得到的双特异性嵌合抗原受体细胞在制备肿瘤治疗药品中的应用。
- 肿瘤治疗药品,其含有权利要求8所述(Ⅳ)嵌合抗原受体细胞或采用权利要求10所述制备方法得到的双特异性嵌合抗原受体细胞。
- 一种治疗肿瘤的方法,包括向需要治疗的主体施用如权利要求12所述的肿瘤治疗药品。
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