CN117143254B - 一种嵌合抗原受体(car)以及在抗癌中的应用 - Google Patents
一种嵌合抗原受体(car)以及在抗癌中的应用 Download PDFInfo
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Abstract
本发明提供了一种嵌合抗原受体(CAR)及其在抗癌中的应用,所述嵌合抗原受体为靶向CD44和MSLN的双特异性嵌合抗原受体,所述双特异性嵌合抗原受包括信号肽、第一抗原结合区、第二抗原结合区、铰链区、跨膜区、共刺激因子和CD3ζ,所述第一抗原结合区靶向CD44,所述第二抗原结合区靶向MSLN。选用作为双靶标,可提高嵌合抗原受体的识别特异性,防止脱靶效应产生,抑制耐药细胞株的出现。本发明提供的CAR‑T细胞可与多种化学治疗药物联用,产生协同效应,能够参与调节免疫因子分泌,可有效杀死肿瘤细胞。
Description
技术领域:
本发明属于生物技术研发领域,具体提供了一种嵌合抗原受体(CAR)及其在抗癌中的应用。
背景技术:
胰腺癌(pancreatic cancer,PC)是一种常见的消化系统肿瘤,具有死亡率高、转移率高、难治愈、易复发等特点。与前几十年风险高、难度大的手术方法不同,现代手术技术已提高了胰腺癌患者的生存率,但只适合15-20%的病例。此外,胰腺癌术后复发率高,局部复发率超过50%。随机对照试验显示,接受肿瘤切除术的患者5年生存率约为28%,中位生存时间仅为18个月,上述数据表明手术治疗方法在治疗胰腺癌中有较大的局限性(参见Neoptolemos J.P.,Kleeff J.,Michl P.,Costello E.,Greenhalf W.,PalmerD.H.Therapeutic developments in pancreatic cancer:Current and future perspectives.Nat.Rev.Gastroenterol.Hepatol.2018,15:333–348)。
为弥补手术疗法的不足,逐渐发展出了辅助治疗策略来提高生存率,其中化疗是不可或缺的一部分。在众多化疗药物中,吉西他滨(Gemcitabine,GEM)通常作为胰腺癌患者的一线治疗药物,它是一种脱氧胞苷的核苷类似物,于1996年获得FDA批准,目前广泛用于治疗各种实体瘤(如乳腺癌、卵巢癌和非小细胞肺癌等)。吉西他滨的细胞毒活性基于其对DNA合成的多种影响,由于呋喃糖环2'位氟取代基的结构差异,吉西他滨在细胞药理学、代谢和作用机制等方面较其他核苷酸类似物具有明显的治疗优势(参见Mini E.,Nobili S.,Caciagli B.,Landini I.,Mazzei T.Cellular pharmacologyofgemcitabine.Ann.Oncol.2006,17((Suppl.S5)):v7–v12)。然而,吉西他滨对其无活性代谢物dFdU具有快速脱氨基作用,并且需要一系列磷酸化步骤才能获得相关活性,使得它具有一定的局限性,特别是由于其高剂量和重复剂量的毒性以及化疗耐药性的出现(参见Amrutkar M.,Gladhaug I.P.Pancreatic Cancer Chemoresistance toGemcitabine.Cancers.2017;9:157)。
吉西他滨耐药性的机制尚不清楚,可能与p53、STAT3、ABC转运蛋白、Bcl-xl等多种通路有关,但CD44却是一种主要途径。CD44是一种重要的细胞标记,是透明质酸(HA)、骨桥蛋白、软骨素、胶原蛋白、纤连蛋白和serglycin/硫酸化蛋白聚糖的膜受体,其参与细胞粘附、血管生成、细胞因子释放和肌肉修复等过程,有证据表明该蛋白可能在PC癌的发生发展中发挥重要作用(参见Hong SP,Wen J,Bang S et al,CD44-positive cells areresponsible for gemcitabine resistance in pancreatic cancer cells.Int JCancer.2009,125:2323–2331.)。研究人员表示,高CD44 PC在治疗12周后对吉西他滨产生耐药性,而低CD44 PC病例在治疗22周后对吉西他滨敏感,这一事实表明CD44+PC细胞可能对吉西他滨具有耐药性。此外,CD44过表达的水平有可能预测化疗耐药的时间,PC的复发能力可以通过以下观察结果来解释,即CD44+PC细胞的特征更多在于其致瘤潜力,而不是具有更高吉西他滨敏感性的细胞。抗CD44疗法对复发性PC有效,PC细胞中CD44的敲除会导致侵袭性降低和对吉西他滨的敏感性增加(参见Zhao S,Chen C,Chang K et al,CD44expression level and isoform contributes to pancreatic cancer cellplasticity,invasiveness,and response to therapy.Clin Cancer Res.2016,22:5592–5604)。
T细胞输注作为实体瘤的治疗已被证明是有效的,基因工程的进步和对T细胞生物学的深入了解使我们现在能够将复杂的细胞毒性T细胞反应转化为可引入T细胞的合成分子,嵌合抗原受体(CAR)靶向肿瘤抗原是一种最为有效的T细胞修饰方式。CAR直接与癌细胞表面蛋白、碳水化合物或糖脂结合,第一代CAR于1989年开发,由附着在单链可变抗体片段上的TCR刺激结构域组成;随后添加的共刺激结构域(第二代CAR)通过提高T细胞在遇到表达抗原的靶细胞时的存活和增殖能力,大大提高了其成功率;多个研究人员利用两个共刺激结构域CD28和4-1BB连接到CD3z结构域,CD3z结构域是一种细胞内信号传导结构域,可赋予T细胞溶细胞能力,然而将两个共刺激结构域(CD28和4-1BB)添加到CD3z结构域(被指定为第三代CAR)并不总是比第二代CAR表现更好(参见Zhong XS,Matsushita M,Plotkin J,et al.Chimeric antigen receptors combining 4-1BB and CD28 signaling domainsaugment PI3kinase/AKT/Bcl-XL activation and CD8+T cell-mediated tumoreradication.Molecular therapy:the journal of the American Society of GeneTherapy.2010,18:413–420)。CAR-T技术已被用于治疗胰腺癌,如在宾夕法尼亚大学的一项间皮素(mesothelin,MSLN)靶向CAR-T细胞临床试验中,一名胰腺癌患者在完成3周的静脉CAR-T细胞治疗后病情稳定。FDG PET/CT成像显示,治疗后所有疾病部位的最大标准化摄取值(SUVmax)短暂下降,治疗开始后第3天和第15天的腹水分析显示,共表达MSLN和c-Met的肿瘤细胞浓度下降了40%(参见Beatty GL,Haas AR,Maus MV,et al.Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumoractivity in solid malignancies.Cancer immunology research.2014,2:112–120)。
本发明为开发胰腺癌的新型治疗药物,尤其是克服胰腺癌的耐药性缺陷,提供了一种新型嵌合抗原受体,其能够靶向CD44和间皮素,有效对抗耐药性胰腺癌细胞,改善抗肿瘤效果。
发明内容
本发明中的第一方面提供了一种双特异性嵌合抗原受体CAR,所述双特异性嵌合抗原受包括信号肽、第一抗原结合区、第二抗原结合区、铰链区、跨膜区、共刺激因子和CD3ζ,所述第一抗原结合区靶向CD44,包括氨基酸序列如SEQ ID NO.1所示的重链可变区和氨基酸序列如SEQ ID NO.2所示的轻链可变区;所述第二抗原结合区靶向MSLN,包括氨基酸序列如SEQ ID NO.3所示的重链可变区和氨基酸序列如SEQ ID NO.4所示的轻链可变区。
嵌合抗原受体可有效识别目标肿瘤细胞,进而介导抗肿瘤治疗,目前已经在白血病、淋巴瘤、骨髓瘤等液体肿瘤中获得了巨大成功,但在实体肿瘤方面却疗效欠佳,主要原因在于难以获得理想的抗肿瘤靶点。本发明基于耐药性胰腺癌的特点,选用CD44和MSLN作为双靶标,其中CD44在吉西他滨耐药细胞中高表达,而间皮素是一种细胞表面抗原,在间皮瘤、卵巢癌和胰腺癌中高度表达,在正常组织中仅在胸膜、心包和腹膜中低水平表达,在癌细胞中,MSLN参与细胞增殖、粘附、细胞信号传导和转移。间皮素在80%的胰腺癌中表达,在阳性肿瘤中25-100%的细胞表达该抗原(参见Hassan R,Laszik ZG,Lerner M,etal.Mesothelin is overexpressed in pancreaticobiliary adenocarcinomas but notin normal pancreas and chronic pancreatitis.Am J Clin Pathol.2005,124:838–845)。因此,选用CD44和MSLN作为双靶标,可提高嵌合抗原受体的识别特异性,防止脱靶效应产生,抑制耐药细胞株的出现。而且,利用重组抗原筛选并获得了具有高度亲和力的新型抗原结合物结构,能够满足临床需求。
进一步的,所述共刺激因子为4-1BB。
进一步的,所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.5所示。
进一步的,所述双特异性嵌合抗原受体的核苷酸序列如SEQ ID NO.6所示。
本发明中的第二方面提供了一种重组载体,其特征在于包含所述嵌合抗原受体的核苷酸序列。
进一步的,所述重组载体为慢病毒、腺病毒、腺联病毒或逆转录病毒中的一种或几种。
本发明中的第三方面提供了一种嵌合抗原受体T细胞,所述细胞膜表面表达有所述的嵌合抗原受体。
本发明中的第四方面提供了一种药物组合物,包括所述的嵌合抗原受体T细胞和化学抗癌药,所述化学抗癌药选自吉西他滨、紫杉醇、5-FU、奥沙利铂、伊立替康中的一种或几种。
由于单一药物治疗在胰腺癌方面的局限性,研究人员已经尝试将免疫检查点抑制剂(如PD-1抗体,CTLA4抗体)、溶瘤病毒、放射疗法、化学疗法(如顺铂、奥沙利铂、吉西他滨、替吉奥、紫杉醇)联合使用,能够有效控制给药剂量,防止肿瘤复发,提高患者生活质量。因此,本发明也试图通过联合给药的方式,进一步抑制肿瘤生长,降低毒副作用。
本发明中的第五方面提供了所述的双特异性嵌合抗原受体或嵌合抗原受体T细胞在制备抗肿瘤药物中的应用。
进一步的,所述的肿瘤为高表达CD44的耐药性胰腺癌。
有益效果
本发明提供了一种嵌合抗原受体(CAR)及其在抗癌中的应用,具有以下优势:
(1)选用CD44和MSLN作为双靶标,可提高嵌合抗原受体的识别特异性,防止脱靶效应产生,抑制耐药细胞株的出现;
(2)利用重组抗原筛选并获得了新型抗原结合物结构,对目标抗原具有高度亲和力;
(3)本发明提供的CAR-T细胞可与多种化学治疗药物联用,产生协同效应;
(4)本发明提供的CAR-T细胞参与调节免疫因子分泌,可有效杀死肿瘤细胞。
附图说明
图1为嵌合抗原受体结构图;
图2为CAR-T抑制AsPC-1-GEM细胞增殖图;
图3为CAR-T联合化疗药物抑制AsPC-1-GEM细胞增殖图;
图4为裸鼠血清中IL-6表达水平图;
图5为裸鼠血清中TNF-α表达水平图;
图6为裸鼠血清中VEGF表达水平图。
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂生物材料、检测试剂盒,如无特殊说明,均可从商业途径获得。
实施例1嵌合抗原受体的设计
如本发明背景技术中所述,耐药性的胰腺癌细胞表面常呈现CD44高表达,因此筛选并设计了靶向CD44抗原的第一抗原结合域;由于胰腺癌细胞中高表达MSLN,且相关抗体已用于临床试验研究,技术资料丰富,有效性和安全性均较高,因此筛选并设计了靶向MSLN抗原的第二抗原结合域。
分别利用基因工程技术,合成人源CD44和MSLN抗原,并使用所述抗原免疫6-8周龄Balb/c健康雌性小鼠,提取免疫成功的小鼠脾脏淋巴细胞,通过细胞融合技术将淋巴细胞与小鼠骨髓瘤细胞SP2/0进行融合,融合细胞进行ELISA阳性筛选及亚克隆,取腹水,用Protein A/G抗体纯化柱纯化抗体。测定所述单克隆抗体与目标抗原的亲和力,为了提高对肿瘤细胞的靶向性和杀伤能力,选择其中亲和力较高的候选抗体进行后续研究,经测定靶向CD44的第一抗原结合区与目标抗原的亲和力为3.17nM,靶向MSLN的第二抗原结合区与目标抗原的亲和力为2.53nM。通过氨基酸测序和分析,鉴定相应抗体的氨基酸序列和结构,所述第一抗原结合区包括氨基酸序列如SEQ ID NO.1所示的重链可变区和氨基酸序列如SEQID NO.2所示的轻链可变区;所述第二抗原结合区包括氨基酸序列如SEQ ID NO.3所示的重链可变区和氨基酸序列如SEQ ID NO.4所示的轻链可变区。
在嵌合抗原受体结构方面,本发明选用了最为经典的第二代CAR结构,这种结构相对简单,仅包括一个共刺激因子,不含有外源细胞因子和刹车基因等额外调控元件,使得制备容易,成本低廉;并且已上市的CAR产品和大量临床试验中均采用了这种结构,其安全性和有效性也能得到保障。在共刺激因子选择上,本发明选用了4-1BB,一方面这种因子可延长CAR-T细胞的存续时间,延长治疗有效期间,可抑制胰腺癌等恶性肿瘤的复发,另一方面这种因子的激活程度相对温和,可避免免疫因子风暴和神经毒性等严重不良反应的发生。
本发明中所述嵌合抗原受体的结构如图1所示,依次包括信号肽、第一抗原结合区、第二抗原结合区、铰链区、跨膜区、共刺激因子和CD3ζ,所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.5所示,核苷酸序列如SEQ ID NO.6所示。
实施例2嵌合抗原受体的制备
通过PCR技术扩增并将所述CAR基因克隆入pCDH慢病毒转移载体中,酶切和核酸测序鉴定序列正确,得到pCDH-CAR。将转移质粒pCDH-CAR、包装质粒(pCMV-dR8.9)、包膜质粒(pVSV-G)以磷酸钙法共转染包装细胞HEK293T,病毒过滤、浓缩后用p24 ELISA试剂盒(购自Abcam公司)检测病毒滴度,符合后续实验要求后低温保存用于转染T细胞。
收集健康成人外周血,用Ficoll密度梯度法分离、提取单个核细胞,采用自然沉降法纯化并获得T淋巴细胞。CD3/CD28mAb(5mg/L)包被平板激活T细胞,辅以终浓度500U/mL的白细胞介素2(interleukin-2,IL-2)扩增培养。细胞生长至对数生长期后,采用所述慢病毒载体转染T细胞,转染48h后,收集细胞加入荧光抗体避光孵育30min,PBS洗涤后,用流式细胞仪检测所述CAR的表达情况,结果显示所述CAR-T细胞能够有效表达双特异性CAR。
实施例3吉西他滨耐药胰腺癌细胞株的制备
本发明以人胰腺癌AsPC-1细胞系为初始材料诱导制备吉西他滨耐药胰腺癌细胞。复苏冻存保藏的AsPC-1,调整细胞浓度后将其接种于6孔板中,加入GEM至终浓度为0.1μg/mL,在培养箱内培养48h后,弃含药培养液;更换普通培养液继续培养,待存活细胞长满培养板,进入对数生长期后,重复同一剂量的药物培养;较稳定后在0.4μg/mL的GEM中继续培养,依此每次使药物浓度4倍递增,如此反复递增加药,筛选存活细胞继续培养后,直至其对高浓度的GEM产生稳定耐药性,并且在无药培养液中以及反复冻存后耐药性依然保持稳定。最终,历时6个月获得对GEM药物稳定耐受的细胞株AsPC-1-GEM,其在4μg/mL浓度的吉西他滨作用下可持续增长。
实施例4CAR-T细胞体外抑制肿瘤细胞生长
4.1CAR-T有效抑制AsPC-1-GEM细胞增殖
分别培养CAR-T和AsPC-1-GEM细胞至对数生长期,然后调节细胞密度,按CAR-T和AsPC-1-GEM为1:1、5:1和10:1比例混合,接种于96孔板中,另设GEM组,接种同数量的AsPC-1-GEM细胞(1×105个),加入终浓度为50μg/mL的吉西他滨。37℃,5%CO2恒温培养箱孵育培养24h后,弃去培养基,每孔加入以无血清培养基稀释的0.5m g/ml的MTT 100μL,37℃孵育4h,弃上清,每孔加入DMSO150μL溶解,室温振荡15分钟,于490nm处应用自动酶标仪检测OD值。每个实验组设置3个复孔,以细胞不加药孔为对照组及以无细胞不加药的孔为空白对照组。计算细胞存活率:细胞存活率(%)=(加药细胞OD值—空白对照OD值)/(对照细胞OD值一空白对照OD值)*100%。
如图2所示,吉西他滨对于耐药肿瘤细胞的抑制作用极为有限,而使用本发明中所提供的CAR-T细胞却可以显著杀死肿瘤细胞,并且呈现了一定的剂量依赖性,加入的CAR-T细胞比例越高,抑制作用越强。这可能是由于吉西他滨耐药胰腺癌细胞中CD44表达升高,使得其更容易被本发明中的CAR-T细胞所识别并杀死。
4.2CAR-T联合化疗制剂抑制AsPC-1-GEM细胞增殖
在肿瘤治疗过程中,联合用药是一种提高疗效,防止耐药性发生的常见策略,本发明中选用了吉西他滨(GEM)、紫杉醇(Paclitaxel,PTX)、5-FU、奥沙利铂(oxaliplatin,OHP)、伊立替康(Irinotecan,CPT-11)等常见抗肿瘤药,联合本发明中所提供的CAR-T细胞,以便提高其疗效。
将1×105个AsPC-1-GEM细胞接种于96孔板中,每孔按1:1比例加入本发明中所提供的CAR-T细胞,然后分别加入终浓度为50μg/mL的吉西他滨、紫杉醇、5-FU、奥沙利铂、伊立替康。37℃,5%CO2恒温培养箱孵育培养24h后,采用MTT法检测细胞存活率,具体方法参见4.1节。
结果如图3所示,CAR-T细胞与吉西他滨、5-FU联用的效果并不明显,与紫杉醇联用似乎具有优势,但未显示出显著差异,与奥沙利铂、伊立替康联用则显著优于其他化疗药物,且明显高于单独使用CAR-T细胞的情形,说明这种联合治疗是有益的。
实施例5CAR-T细胞抑制动物体内肿瘤生长
5.1肿瘤动物模型建立和给药
体外传代培养AsPC-1-GEM细胞,当所述细胞处于对数生长期时,胰酶消化制成细胞悬液,调节细胞浓度,取1×106个细胞接种于裸鼠右前肢腋窝处皮下,每天观察成瘤情况并记录,肿瘤体积生长至100mm3以上时,表示造模成功,用于后续实验。
取30只造模成功小鼠,随机分为三组,分别为:CAR-T组,尾静脉注射1×106个本发明中所提供的CAR-T细胞;联合组,尾静脉注射1×106个本发明中所提供的CAR-T细胞,每2天腹腔注射10mg/kg伊立替康,共治疗30天;对照组,每2天腹腔注射等体积生理盐水,共治疗30天。
5.2CAR-T细胞抑制体内肿瘤生长
治疗30天后,称取裸鼠体重,眼眶取血后,采用颈椎脱臼法处死裸鼠,完整地剥出瘤体,去除表面脂肪组织,称取瘤质量,并测量肿瘤体积。
结果如表1所示,CAR-T治疗可显著抑制肿瘤的体积和重量,并且在联合伊立替康后,肿瘤抑制效果更加明显,这与细胞实验结果一致,说明对于吉西他滨耐药性的胰腺癌,使用本发明中所提供的CAR-T或其联合疗法较为有效。
表1各组裸鼠的肿瘤生长情况
5.3CAR-T细胞对体内免疫因子调控的影响
裸鼠眼球取血后(参见5.2节),室温静置2h,离心分离血清,采用ELISA试剂盒(购自武汉伊莱瑞特生物科技股份有限公司)检测血清IL-6、TNF-α、VEGF水平。
如图4、5所示,CAR-T可显著提高IL-6、TNF-α的表达水平,并且这种趋势在联合治疗中得到了强化,这种情况一方面说明CAR-T或其联合疗法能够有效激活体内免疫系统,诱导表达抗肿瘤免疫因子分泌,强化抗肿瘤效果;另一方面也提示了免疫因子表达升高,可能诱发“免疫因子风暴”的风险,但在本实验中所述细胞因子水平仍处于可接受范围内,后期在临床应用中应加强临床监测与管控,必要时给予免疫因子拮抗治疗,保证临床用药安全。
如图6所示,CAR-T细胞似乎对于VEGF的表达无明显调节作用,但在联合组中VEGF表达似乎受到了抑制,这可能是源自伊立替康或二者的协同效应。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (10)
1.一种双特异性嵌合抗原受体CAR,其特征在于,所述双特异性嵌合抗原受体CAR包括信号肽、第一抗原结合区、第二抗原结合区、铰链区、跨膜区、共刺激因子和CD3ζ,所述第一抗原结合区靶向CD44,包括氨基酸序列如SEQ ID NO.1所示的重链可变区和氨基酸序列如SEQ ID NO.2所示的轻链可变区;所述第二抗原结合区靶向MSLN,包括氨基酸序列如SEQ IDNO.3所示的重链可变区和氨基酸序列如SEQ ID NO.4所示的轻链可变区。
2.如权利要求1所述的双特异性嵌合抗原受体CAR,其特征在于,所述共刺激因子为4-1BB。
3.如权利要求2所述的双特异性嵌合抗原受体CAR,其特征在于,所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.5所示。
4.如权利要求3所述的双特异性嵌合抗原受体CAR,其特征在于,所述双特异性嵌合抗原受体的编码核酸序列如SEQ ID NO.6所示。
5.一种重组载体,其特征在于包含权利要求4中所述的核酸。
6.如权利要求5所述的重组载体,其特征在于,所述重组载体为慢病毒、腺病毒或腺联病毒中的一种或几种。
7.一种嵌合抗原受体T细胞,所述细胞膜表面表达有权利要求1-4任一项所述的双特异性嵌合抗原受体CAR。
8.一种药物组合物,包括权利要求7所述的嵌合抗原受体T细胞和化学抗癌药,所述化学抗癌药选自吉西他滨、紫杉醇、5-FU、奥沙利铂、伊立替康中的一种或几种。
9.如权利要求1-4任一项所述的双特异性嵌合抗原受体CAR或权利要求7所述的嵌合抗原受体T细胞在制备抗肿瘤药物中的应用。
10.根据权利要求9所述的应用,其特征在于所述的肿瘤为高表达CD44的耐药性胰腺癌。
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