CN116063532B - 结合抗间皮素的抗体及其应用 - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C07K14/7051—T-cell receptor (TcR)-CD3 complex
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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Abstract
本发明提供结合抗间皮素的抗体及其应用。所述抗体的CDR3的氨基酸序列包括SEQ ID NO.9所示的序列。本发明的结合抗间皮素的抗体可特异性的结合间皮素抗原,且亲和力较好,将其作为抗原结合结构域构建嵌合抗原受体和CAR‑T细胞,对间皮素阳性的肿瘤细胞具有明显的杀伤活性。
Description
本申请是申请号为202111643701.3专利申请的分案申请(原申请的申请日为2021年12月29日,发明名称为一种抗间皮素的纳米抗体及其应用)。
技术领域
本发明属于生物医药技术领域,具体涉及结合抗间皮素的抗体及其应用。
背景技术
间皮素(mesothelin,MSLN)是一种细胞表面糖蛋白,MSLN的cDNA包含一段1884bp的开放阅读框,共有17个外显子,编码由628个氨基酸组成的相对分子质量约为69kDa的前体蛋白,经糖基化后被弗林蛋白酶切割产生相对分子质量分别约为40kDa和31kDa的2个蛋白,羧基端的疏水序列被糖基磷脂酰肌醇取代并锚定在细胞膜上,分子量约为40kDa,称为间皮素;氨基端部分释放进入血液或进一步降解,分子量约为31kDa,称为巨核细胞集落刺激因子(megakaryocyte-potentiating factor,MPF)(参见:Chang,K.&Pastan,I.Molecular cloning of mesothelin,a differentiation antigen present onmesothelium,mesotheliomas,and ovarian cancers.Proceedings of the NationalAcademy of Sciences of the United States of America 93,136-140,doi:10.1073/pnas.93.1.136(1996).)。目前研究表明MSLN参与调控细胞的存活、增殖以及迁移,并且能使细胞抵抗部分细胞毒性药物的杀伤(Tang,Z.,Qian,M.&Ho,M.The role ofmesothelinin tumor progression and targeted therapy.Anti-cancer agents in medicinalchemistry 13,276-280,doi:10.2174/1871520611313020014(2013).)。
MSLN是一种分化抗原,在胰腺癌、恶性间皮瘤、卵巢癌和非小细胞肺癌等肿瘤组织中高表达(参见:Baldo,P.&Cecco,S.Amatuximab and novel agents targetingmesothelin for solid tumors.OncoTargetsand therapy 10,5337-5353,doi:10.2147/ott.s145105(2017).)。免疫组织化学染色表明:在大多数的胰腺癌组织中(75%-100%)MSLN表达呈阳性,然而,在大多数的慢性胰腺炎组织(80%-100%)和几乎所有正常胰腺组织中MSLN表达呈阴性(参见:Baruch,A.C.et al.Immunocytochemical study oftheexpression of mesothelin in fine-needle aspirationbiopsy specimensofpancreatic adenocarcinoma.Diagnostic cytopathology 35,143-147,doi:10.1002/dc.20594(2007).;Ibrahim,D.A.&Abouhashem,N.S.Diagnostic value of IMP3 andmesothelin in differentiating pancreatic ductal adenocarcinoma from chronicpancreatitis.Pathology,research and practice 212,288-293,doi:10.1016/j.prp.2016.01.007(2016).)。上皮恶性胸膜间皮瘤(Epithelial malignant肋膜间皮瘤,MPM)普遍表达间皮素,而肉瘤样胸膜间皮瘤不表达间皮素,大多数浆液性上皮性卵巢癌和相关的原发性腹膜癌表达间皮素。
MSLN也存在于部分正常组织中,使用由Pastan小组开发的小鼠抗人间皮素抗体K1,在腹膜、胸膜和心包腔的间皮细胞中已经证实了强烈的K1反应性,但其水平低于在恶性组织中达到的水平(参见:Chang K,Pai L H,Batra J K,et al.Characterization oftheantigen(CAK1)recognized by monoclonal antibody K1 present on ovarian cancersand normal mesothelium.[J].Cancer Research,1992,52(1):181-186.);在输卵管上皮、气管基底上皮和扁桃体上皮中检测到弱K1反应;在角膜的各个层都可以发现间皮素,然而,在肝脏、肾脏、脾脏、骨髓、淋巴结、胸腺、心肌、舌头、骨骼肌、皮肤、大脑皮层、小脑、脊髓、周围神经、垂体、肾上腺、唾液腺、乳腺、甲状腺、甲状旁腺、睾丸、前列腺、附睾、宫颈上皮、肺实质、食道、小肠上皮、结肠上皮、膀胱上皮、胆囊上皮等大多数正常组织中未检测到K1反应(参见:Chang K,Pai L H,Batra J K,et al.Characterization ofthe antigen(CAK1)recognized by monoclonal antibody K1 present on ovarian cancers and normalmesothelium.[J].Cancer Research,1992,52(1):181-186.)。
目前研究表明MSLN可作为治疗的靶点(参见:Argani,P.,et al.,Mesothelin isoverexpressed in the vast majority of ductal adenocarcinomas of the pancreas:identification of a new pancreatic cancer marker by serial analysis ofgeneexpression(SAGE).Clin Cancer Res,2001.7(12):3862-8.),Pastan和他的同事们使用一种与免疫毒素结合的抗间皮素抗体的可溶性抗体片段来治疗间皮素阳性肿瘤的癌症患者,证明了这种方法在胰腺癌治疗中具有足够的安全性和一定的临床活性(参见:Hassan R,etal.Preclinical evaluation of MORAb-009,a chimeric antibody targeting tumor-associated mesothelin.Cancer Immun.2007;7:20–29;Hassan R,et al.Phase I Studyof SS1P,a Recombinant Anti-Mesothelin Immunotoxin Given as a BolusI.V.Infusion to Patients with Mesothelin-Expressing Mesothelioma,Ovarian,andPancreatic Cancers.Clin Cancer Res.2007;13(17):5144-5149.),在卵巢癌中,这种治疗策略根据RECIST标准产生了一个轻微的反应,并在另一个患者腹水完全缓解的情况下病情稳定。
Stromnes等人构建了表达识别MSLN的TCR-T细胞,在体外实验能诱导胰腺癌细胞的大量死亡,在体内实验能显著延长荷瘤小鼠的生存时间(参见:Stromnes,I.M.et al.TCells Engineered against a Native Antigen Can Surmount Immunologic andPhysical Barriers to Treat Pancreatic Ductal Adenocarcinoma.Cancer cell 28,638-652,doi:10.1016/j.ccell.2015.09.022(2015).)。然而,此项研究只是进行了小鼠体内实验,并没有进行临床研究。2015年,宾夕法尼亚大学的研究人员完成了一例靶向MSLN的CAR-T细胞(meso-CAR-T)靶向治疗胰腺导管腺癌患者的临床试验(NCT01897415),该实验控制了患者的肿瘤生长,但是由于移植物抗宿主反应,降低了CAR-T的治疗效果,造成控制肿瘤生长的时间非常短暂(参见:DeSelm,C.J.,Tano,Z.E.,Varghese,A.M.&Adusumilli,P.S.CAR T-cell therapy for pancreatic cancer.Journal of surgical oncology116,63-74,doi:10.1002/jso.24627(2017).)。2018年宾夕法尼亚大学的研究团队又发表了一项临床实验报告,评估了6例使用了meso-CAR-T靶向治疗的难治性胰腺导管腺癌转移复发患者,结果显示2例患者疾病稳定,4例患者发生了疾病进展,6例患者均未发生细胞因子释放综合征或神经症状的不良反应(参见:Beatty,G.L.et al.Activity ofMesothelin-Specific Chimeric Antigen Receptor T Cells AgainstPancreaticCarcinoma Metastases in a Phase 1Trial.Gastroenterology 155,29-32,doi:10.1053/j.gastro.2018.03.029(2018).)。
综上所述,间皮素可作为肿瘤治疗的靶点,提供具备高特异性和亲和力的抗间皮素抗体,对于治疗肿瘤领域具有重要意义。
发明内容
针对现有技术的不足和实际需求,本发明的提供结合抗间皮素的抗体及其应用。所述抗体具有高亲和力,能够作为嵌合抗原受体分子的抗原结合结构域制备CAR-T细胞,所得CAR-T细胞在肿瘤治疗方面具有较好的应用前景。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种抗间皮素的纳米抗体,所述纳米抗体的CDR1的氨基酸序列包括SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的序列,所述纳米抗体的CDR2的氨基酸序列包括SEQ ID NO.5、SEQ ID NO.6或SEQ ID NO.7所示的序列,所述纳米抗体的CDR3的氨基酸序列包括SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示的序列。
本发明中,以间皮素重组蛋白免疫一只未经免疫的羊驼,构建噬菌体展示纳米抗体库,根据该噬菌体展示纳米抗体库对抗间皮素抗体进行筛选,得到可特异性的结合间皮素抗原的单克隆抗体。
SEQ ID NO.1:GFTFRNYG。
SEQ ID NO.2:GFTFSRYG。
SEQ ID NO.3:GRTFSTYS。
SEQ ID NO.4:GFTFRRYG。
SEQ ID NO.5:IFSDGST。
SEQ ID NO.6:ISWIGGST。
SEQ ID NO.7:IYSDGST。
SEQ ID NO.8:ATDPGSGTTTLHRPFGS。
SEQ ID NO.9:AATKSEWRDPDY。
SEQ ID NO.10:ATDPGSGTTTLHRSFGS。
优选地,所述纳米抗体的CDR1的氨基酸序列包括SEQ ID NO.1所示的序列,CDR2的氨基酸序列包括SEQ ID NO.3所示的序列,CDR3的氨基酸序列包括SEQ ID NO.8所示的序列。
优选地,所述纳米抗体的CDR1的氨基酸序列包括SEQ ID NO.2所示的序列,CDR2的氨基酸序列包括SEQ ID NO.3所示的序列,CDR3的氨基酸序列包括SEQ ID NO.8所示的序列。
优选地,所述纳米抗体的CDR1的氨基酸序列包括SEQ ID NO.3所示的序列,CDR2的氨基酸序列包括SEQ ID NO.6所示的序列,CDR3的氨基酸序列包括SEQ ID NO.9所示的序列。
优选地,所述纳米抗体的CDR1的氨基酸序列包括SEQ ID NO.2所示的序列,CDR2的氨基酸序列包括SEQ ID NO.7所示的序列,CDR3的氨基酸序列包括SEQ ID NO.10所示的序列。
优选地,所述纳米抗体的CDR1的氨基酸序列包括SEQ ID NO.4所示的序列,CDR2的氨基酸序列包括SEQ ID NO.7所示的序列,CDR3的氨基酸序列包括SEQ ID NO.8所示的序列。
优选地,所述纳米抗体的重链可变区还包括:SEQ ID NO.11或SEQ ID NO.12所示的框架区1(FR1)、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16或SEQ IDNO.17所示的框架区2(FR2)、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20或SEQ ID NO.21所示的框架区3(FR3)和SEQ ID NO.22所示的框架区4(FR4)。
SEQ ID NO.11:QVQLVESGGGLVQPGGSLRLSCAAS。
SEQ ID NO.12:QVQLVESGGGLVPAGGSLRLSCAAS。
SEQ ID NO.13:MGWARQVPGQGLEWVSG。
SEQ ID NO.14:MGWARQVPGKELEWVSG。
SEQ ID NO.15:MGWFRQAPGKEREPVAV。
SEQ ID NO.16:MGWARQVPGKGLEWVSG。
SEQ ID NO.17:MGWARQVSGKGLEWVSG。
SEQ ID NO.18:
YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC。
SEQ ID NO.19:
YYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYC。
SEQ ID NO.20:
YYADSVKGRFTISRDYAKNTLYLQMNSLKPADTAVYYC。
SEQ ID NO.21:
YYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYC。
SEQ ID NO.22:WGQGTQVTVSS。
优选地,所述纳米抗体的重链可变区包括SEQ ID NO.23、SEQ ID NO.24、SEQ IDNO.26或SEQ ID NO.27所示的氨基酸序列。
SEQ ID NO.23:
QVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMGWARQVPGQGLEWVSGIFSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDPGSGTTTLHRPFGSWGQGTQVTVSS。
SEQ ID NO.24:
QVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMGWARQVPGKELEWVSGIFSDGSTYYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCATDPGSGTTTLHRPFGSWGQGTQVTVSS。
SEQ ID NO.25:
QVQLVESGGGLVPAGGSLRLSCAASGRTFSTYSMGWFRQAPGKEREPVAVISWIGGSTYYADSVKGRFTISRDYAKNTLYLQMNSLKPADTAVYYCAATKSEWRDPDYWGQGTQVTVSS。
SEQ ID NO.26:
QVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMGWARQVPGKGLEWVSGIYSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDPGSGTTTLHRSFGSWGQGTQVTVSS。
SEQ ID NO.27:
QVQLVESGGGLVQPGGSLRLSCAASGFTFRRYGMGWARQVSGKGLEWVSGIYSDGSTYYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYCATDPGSGTTTLHRPFGSWGQGTQVTVSS。
本发明中,利用噬菌体展示技术对间皮素免疫骆驼VHH文库进行筛选,得到的纳米抗体具备高亲和性高,在构建靶向间皮素的嵌合抗原受体方面具有重要的应用前景。
第二方面,本发明提供一种核酸分子,所述核酸分子包括第一方面所述的抗间皮素的纳米抗体的编码基因。
第三方面,本发明提供一种嵌合抗原受体,所述嵌合抗原受体包括信号肽、抗原结合结构域、铰链区、跨膜区和信号转导结构域,所述抗原结合结构域包括第一方面所述的抗间皮素的纳米抗体。
优选地,所述信号肽包括CD8α信号肽。
优选地,所述铰链区包括CD8α铰链区。
优选地,所述跨膜区包括CD8α跨膜区、CD28跨膜区或DAP10跨膜区中的任意一种或至少两种的组合。
优选地,所述信号转导结构域包括免疫受体酪氨酸活化基序(CD3ζ)。
优选地,所述信号转导结构域还包括共刺激分子,所述共刺激分子包括4-1BB、CD28胞内区、OX40、ICOS或DAP10胞内区中的任意一种或至少两种的组合。
优选地,所述嵌合抗原受体包括CD8α信号肽、第一方面所述的抗间皮素的纳米抗体、CD8α铰链区、CD8α跨膜区和免疫受体酪氨酸活化基序。
第四方面,本发明提供一种表达载体,所述表达载体包括第三方面所述的嵌合抗原受体的编码基因。
优选地,所述表达载体为含有第三方面所述的嵌合抗原受体的编码基因的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种,优选为慢病毒载体。
第五方面,本发明提供一种重组慢病毒,所述重组慢病毒含有第四方面所述的表达载体。
优选地,所述重组慢病毒由转染第四方面所述的表达载体和辅助质粒的哺乳细胞制备得到。
第六方面,本发明提供一种嵌合抗原受体免疫细胞,所述嵌合抗原受体免疫细胞表达第三方面所述的嵌合抗原受体。
优选地,所述嵌合抗原受体免疫细胞包括第四方面所述的表达载体和/或底物方面所述的重组慢病毒。
优选地,所述嵌合抗原受体免疫细胞包括T细胞、B细胞、NK细胞、肥大细胞或巨噬细胞中的任意一种或至少两种的组合。
第七方面,本发明提供一种药物组合物,所述药物组合物包括第六方面所述的嵌合抗原受体免疫细胞。
优选地,所述药物组合物还包括药学上可接受的辅料。
优选地,所述辅料包括载体、稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
第八方面,本发明提供第一方面所述的抗间皮素的纳米抗体、第二方面所述的核酸分子、第三方面所述的嵌合抗原受体、第四方面所述的表达载体、第五方面所述的重组慢病毒、第六方面所述的嵌合抗原受体免疫细胞或第七方面所述的药物组合物在制备肿瘤治疗药物中的应用。
优选地,所述肿瘤包括表达间皮素的肿瘤。
与现有技术相比,本发明至少具有以下有益效果:
(1)本发明以间皮素重组蛋白免疫未经免疫的羊驼,构建噬菌体展示纳米抗体库,根据该噬菌体展示纳米抗体库对抗间皮素抗体进行筛选,所得纳米抗体可特异性的结合间皮素抗原,且亲和力较好,通过抗体亲和力的测定可知,其KD(M)分别为2.83×10-8、1.01×10-8、1.41×10-8、6.34×10-8和5.82×10-8;
(2)本发明提供的抗间皮素的纳米抗体具有较好的亲和力,将其作为抗原结合结构域构建嵌合抗原受体,并利用该嵌合抗原受体制备T细胞,所述CAR-T细胞对间皮素阳性的肿瘤细胞具有杀伤活性,且与间皮素阳性细胞共培养后,高效分泌细胞因子IFN-γ和TNF-α,说所述纳米抗体能够有效应用于免疫治疗,对于开发肿瘤治疗药物具有重要意义。
附图说明
图1A为实施例2中采用Biacore检测抗间皮素纳米抗体VHH-1的亲和力曲线图;
图1B为实施例2中采用Biacore检测抗间皮素纳米抗体VHH-3的亲和力曲线图;
图1C为实施例2中采用Biacore检测抗间皮素纳米抗体VHH-9的亲和力曲线图;
图1D为实施例2中采用Biacore检测抗间皮素纳米抗体VHH-28的亲和力曲线图;
图1E为实施例2中采用Biacore检测抗间皮素纳米抗体VHH-29的亲和力曲线图;
图2为实施例3中抗间皮素纳米抗体识别间皮素抗原的FACS检测结果图;
图3为实施例4中靶向间皮素的嵌合抗原受体慢病毒载体质粒图谱;
图4为实施例4中表达间皮素的嵌合抗原受体结构示意图;
图5为实施例6中T淋巴细胞的嵌合抗原受体表达率的流式检测结果图;
图6A为实施例6中FACS检测T细胞和CAR-T细胞表型(CD3+CD4+)所得到的结果图;
图6B为实施例6中FACS检测T细胞和CAR-T细胞表型(CD3+CD8+)所得到的结果图;
图7为实施例7中CAR-T细胞对293T细胞的杀伤效果曲线图;
图8为实施例7中CAR-T细胞对胰腺癌细胞AsPC1-Mesothelin的杀伤效果曲线图;
图9为实施例7中CAR-T细胞对胰腺癌细胞PANC1-Mesothelin杀伤效果曲线图;
图10为实施例8中CAR-T细胞分泌TNFα的柱状图;
图11为实施例8中CAR-T细胞分泌IFNγ的柱状图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案,但下述的实例仅仅是本发明的简易例子,并不代表或限制本发明的权利保护范围,本发明的保护范围以权利要求书为准。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例构建噬菌体纳米抗体库及淘选,并使用ELISA进行初步筛选,具体步骤如下:
(1)噬菌体纳米抗体库的构建
采用间皮素(MSLN)胞外段重组蛋白免疫羊驼,ELISA检测血清效价后,抽取外周血;分离淋巴细胞,提取总RNA,再反转录为cDNA,然后利用巢式PCR扩增VHH基因;将VHH基因插入pShort噬菌粒,电转化感受态细胞,扩增后,用PEG8000/NaCI沉淀法分离纯化噬菌休,获得抗体库;调整浓度,分装冻存于-80℃冰箱备用;
(2)噬菌体纳米抗体库的筛选
首先取293T细胞与抗体库共孵育进行负筛,然后取上清,分别与293T-MSLN细胞(间皮素阳性)、293T细胞进行孵育;采用4℃预冷的PT缓冲液,清洗4次;感染NEB alpha 5F’细胞,加入辅助噬菌体后,过夜培养;Drop法涂板,第二天统计富集程度;PEG8000/NaCI沉淀法分离纯化噬菌休,进入下一轮筛选;出现富集后,以所得的噬菌体为模板,扩增VHH区域,进行二代测序,获得5种抗间皮素的纳米抗体,氨基酸序列分别如SE1 ID NO.22、SE1 IDNO.23、SE1 ID NO.24、SE1 ID NO.25和SE1 ID NO.26所示,分别命名为VHH-1、VHH-3、VHH-9、VHH-28和VHH-29。
实施例2
本实施例对实施例1筛选的抗间皮素的纳米抗体(VHH-mIgG2a Fc纳米抗体)进行表达和纯化,并进行抗体亲和力的测定。为了进一步对筛选得到的抗体进行鉴定,需要通过哺乳动物细胞表达抗体,因此,首先构建了带有小鼠Fc标签表达VHH的质粒载体,记为C-4pCP.Stuffer-mCg2a-FC,具体步骤如下:
(1)使用PCR扩增VHH片段,其反应体系如表1所示,扩增程序如下表2所示;
表1
表2
(2)酶切体系如表3所示,酶切温度为37℃,时间为6h,酶切后的载体用PCR纯化试剂盒进行纯化,回收得到的DNA溶解于45μL水中,检测DNA的浓度;
表3
试剂 | 使用量 |
C-4pCP.Stuffer-mCg2a-FC | 5μg |
10×酶切缓冲液(10×Reaction Buffer) | 5μL |
FspA I | 2μL |
PfI 23II | 2μL |
ddH2O | 补足至50μL |
(3)将PCR扩增产物采用同源重组的方式连接入酶切的线性化载体中,体系如表4所示,条件为37℃水浴30min;
表4
试剂 | 体积(μL) |
Exnase II | 1 |
2×Exnase II buffer | 2 |
线性化载体(linearized vector) | 4 |
插入片段(Insert fragment) | 3 |
(4)取全部同源重组反应体系加入DH5α感受态细胞中,转化DH5α感受态细胞,转化条件如表5所示;
表5
程序 | 温度 | 时间 |
冰浴 | 0℃ | 5min |
热击 | 42℃ | 1min |
冰浴 | 0℃ | 3min |
加入500μLLB培养基,220rpm振荡培养 | 37℃ | 1.5h |
吸取200μL,涂布于LB/Amp平板 | 37℃ | 过夜 |
(5)转化平板挑选单克隆PCR预鉴定,PCR鉴定体系条件如表6所示;条件为95℃预变性3min;95℃变性30s、55℃退火30s、72℃延伸30s,35个循环;72℃延伸5min,4℃保存,送测序公司测序鉴定,测序结果符合预期,说明书本实施例中成功构建了带有小鼠Fc标签表达VHH的质粒载体。
表6
在质粒转染前约24h,传代293E细胞,使细胞密度约为0.6×106cells/mL;当细胞密度为1.2×106cells/mL、活率>95%时,以150μg DNA/100mL 293E的比例,用PEI法转染293E细胞,其中,质粒DNA和PEI的比例为1:2;
37℃、130rpm、8%CO2摇床培养6天,3000rpm、30min收集细胞培养物上清,把收集到的包含目的抗体的上清经0.45μm滤器过滤后,加样品到纯化柱MabSelectTM SuReTM,加5倍的PBS洗涤柱体,0.1M Gly-HCL(pH 3.0)洗脱蛋白,并用1/10体积且pH为8.5的Tris-HCL中和,随后4℃透析过夜后,用NanoDrop 2000测定A280的方法定量,SEC-HPLC测定抗体纯度。
另外,本实施例中还将纯化后的VHH抗体通过Biacore进行亲和力的测定。
Biacore是基于表面等离子共振(surface Plasmon resonance,SPR)开发的生物分析传感技术,可检测跟踪溶液中的分子与固定在芯片表面的分子结合、解离的整个变化过程,以传感图的形式进行记录,并提供动力学和亲和力数据。
测定过程中,将抗体固化到芯片表面,流动相为含有抗原(间皮素)的溶液,测定结果如表7和图1A-图1E所示,5条抗体的亲和力均达到亚纳摩尔级,分别为2.83E-8、1.01E-8、1.41E-8、6.34E-8和5.82E-8。
表7
纳米抗体 | Ka(1/Ms) | Kd(1/s) | KD(M) |
VHH-1(SEQ ID NO.23) | 5.53E+04 | 1.56E-03 | 2.83E-8 |
VHH-3(SEQ ID NO.24) | 6.54E+04 | 6.6E-04 | 1.01E-8 |
VHH-9(SEQ ID NO.25) | 4.95E+04 | 6.98E-03 | 1.41E-8 |
VHH-28(SEQ ID NO.26) | 6.83E+04 | 4.33E-03 | 6.34E-8 |
VHH-29(SEQ ID NO.27) | 6.25E+04 | 3.64E-03 | 5.82E-8 |
实施例3
本实施例对实施例抗间皮素的纳米抗体进行流式测定。
将293T(Mesothelin-)、293T-Mesothelin细胞与纯化的抗间皮素的纳米抗体混合,在冰浴下孵育30min,然后用APC标记的羊抗鼠IgG抗体孵育30min,采用流式细胞仪检测,结果如图2所示,表明本发明的抗间皮素的纳米抗体可识别细胞表面的间皮素抗原。
实施例4
本实施例制备表达靶向MSLN的嵌合抗原受体(MSLN CAR)的慢病毒载体。
首先,构建携带MSLN CAR嵌合抗原受体的慢病毒载体pSIN03 MSLN CAR,载体图谱如图3所示,嵌合抗原受体的示意图如图4所示,包括CD8α信号肽、抗间皮素的纳米抗体(anti-MSLN VHH)、CD8α铰链区、跨膜区和免疫受体酪氨酸活化基序(CD3ζ)。
其中,信号肽的氨基酸序列(SEQ ID NO.28)为:
MALPVTALLLPLALLLHAARP。
anti-MSLN VHH的氨基酸序列如SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQID NO.26或SEQ ID NO.27所示。
CD8α铰链区和跨膜区的氨基酸序列(SEQ ID NO.29)为:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC。
4-1BB胞内区氨基酸序列(SEQ ID NO.30)为:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。
CD3ζ氨基酸序列(SEQ ID NO.31)为:
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
具体的制备方法如下:
(1)按照表8(表中试剂来源于TOYOBO Inc.)配制PCR反应体系,扩增各抗间皮素的纳米抗体片段,按照表9所示的PCR程序进行反应,引物序列为:
MS1-F(SEQ ID NO.32):
tgccgctggccttgctgctccacgccgccaggccgcaggtgcagctggtggag;
MS1-R(SEQ ID NO.33):cgctggcgtcgtggtgctagacactgtcacctg;
MS9-R(SEQ ID NO.34):cgctggcgtcgtggtagagctcactgtcacctg。
表8
试剂 | 体积(μL) |
10×buffer | 5 |
2mM dNTP | 5 |
25mM MgSO4 | 3 |
10μM MF1-F | 1 |
10μM MS1-R或MS9-R | 1 |
模板DNA(cDNA clone) | 1 |
无菌去离子水(PCR grade water) | 33 |
KOD-Plus-Neo高保真PCR酶 | 1 |
表9
(2)按照表10配制PCR反应体系,在所得扩增产物前加CD8α信号肽,按照表9所示的PCR程序进行反应,使用引物为:
BamH-CD8αsig-F(SEQ ID NO.35):
GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC;
表10
反应结束后,PCR产物行1%琼脂糖凝胶电泳,回收500bp左右的片段,紫外吸收法定量;
(3)按照表11配制PCR反应体系,配制结束后按照表9所示的PCR程序进行PCR反应,扩增CD8αhinge-TM-41BB-CD3Z片段,使用引物如下:
CD8αH-F(SEQ ID NO.36):ACCACGACGCCAGCGCCGCGAC;
Vector-R(SEQ ID NO.37):TCGATAAGCTTGATATCG;
表11
试剂 | 体积(μL) |
10×buffer | 5 |
2mM dNTP | 5 |
25mM MgSO4 | 3 |
10μM上游引物CD8αH-F | 1 |
10μM下游引物Vector-R | 1 |
模板DNA(HD CD19 CAR) | 1 |
无菌去离子水(PCR grade water) | 33 |
KOD-Plus-Neo高保真PCR酶 | 1 |
PCR结束后行1%琼脂糖凝胶电泳,回收780bp左右的片段,紫外吸收法定量;
(4)将5μg实验室构建的HD SIN03 CD19 CAR质粒进行BamHI和EcoRI双酶切,37℃水浴反应2h后,回收载体;
将上述回收的3个片段和载体骨架用重组酶连接,重组反应体系如表12所示,配制结束后37℃水浴反应0.5h,按常规方法转化至大肠杆菌stbl3感受态细胞。
表12
试剂 | 使用量 |
HD CD19 CAR | 184.54ng |
CD8αsingal MSLN VHH | 31.32ng |
CD8αhinge-TM-41BB-CD3Z | 29.72ng |
5×CE buffer | 2μL |
ExnaseTMII | 1μL |
无菌去离子水(PCRgradewater) | 补足至10μL |
从固体培养基上挑选单克隆,过夜培养,进行PCR鉴定,PCR反应体系配制如表13所示,PCR程序如表14所示,PCR结束后挑选阳性克隆进一步测序鉴定,测序结果符合预期。
表13
试剂 | 体积(μL) |
Taq PCR Master Mix | 10 |
10μM F Seq-trEF1a-F | 1 |
10μM R Vector-R | 1 |
Template DNA菌液 | 1 |
无菌去离子水(PCR grade water) | 7 |
表14
实施例5
本实施例中对实施例4中制备得到的慢病毒载体HD SIN03-MSLN CAR进行慢病毒包装、浓缩和滴度检测,包括以下步骤:
(1)慢病毒包装
以1.6×107细胞数接种293T细胞于15cm培养皿中,37℃,5%CO2培养过夜准备包装病毒,培养基为含10%胎牛血清的DMEM;将慢病毒载体14.5μg HD SIN03-MSLN CAR、16.7μg辅助质粒pMDLg-RRE、16.7μg辅助质粒pRSV-REV与6.5μg包膜质粒VSVg溶入2mL无血清DMEM培养液,混匀;
将163.2μg PEI(1μg/μL)溶解于2mL的无血清DMEM培养液中,1000rpm涡旋5秒钟,25℃孵育5min;将PEI混合液加入DNA混合液中,加入后立即涡旋混合或轻轻混匀,25℃下孵育20min,形成转染复合物;再将转染复合物4mL滴加入含25mL含293T细胞的DMEM培养基中,4h小时后,更换新鲜培养基;48h后,收集病毒液上清;
(2)慢病毒浓缩
将病毒上清用0.45μm滤膜过滤后收集到50mL离心管中,加入1/4的PEG-NaCl病毒浓缩液,上下颠倒混匀,4℃放置过夜;4℃,3500rpm,离心30min;去上清,加入适量的RPMI1640培养基(含10%FBS),溶解重悬病毒沉淀;浓缩后的慢病毒悬液分装成50μL每份,保存在成品管中,储存在-80℃中;
(3)慢病毒滴度检测
将500μL K562细胞(1×105个细胞)接种细胞于24孔培养板,再将浓缩后的慢病毒分别以1μL、0.2μL和0.04μL的体积添加到细胞悬液中,并添加polybrene至终浓度5μg/mL,37℃,5%CO2培养过夜后,更换新鲜培养基;
感染72h后,400×g离心5min,弃上清收集细胞,加100μL PBS+2%FBS重悬细胞,加入0.5μg的MSLN-his蛋白,冰上孵育30min;流式缓冲液(含2%FBS的PBS)缓冲液清洗1次后,加入100μL缓冲液重悬细胞,加入APC anti-His抗体,冰上孵育30min;流式缓冲液清洗2次后,加入300μL流式缓冲液重悬细胞,采用流式细胞仪检测感染效率;计算滴度,滴度计算公式如下:滴度(TU/mL)=细胞数量(105)×阳性率/病毒体积(mL)。
实施例6
本实施例使用实施例5中制备得到的慢病毒转导T淋巴细胞,包括以下步骤:
(1)用PBS将抗人CD3抗体和抗人CD28抗体稀释,终浓度分别为1μg/mL和0.5μg/mL,包被孔板,4℃冰箱静置过夜;弃去孔板中的抗体包被液,1mL PBS洗板;
(2)用T细胞培养基(X-VIVO+10%FBS+300U/mL IL-2)将人PBMC调整至密度为1×106/mL,接种到CD3和CD28抗体包被的孔板中活化24h;收集活化的T细胞,调整细胞密度为1×106/mL,按照感染复数(multiplicity of infection,MOI)为10加入慢病毒,添加polybrene至终浓度为5μg/mL;于37℃、5%CO2环境中培养过夜后更换新鲜培养基,每3天进行传代;
(3)T细胞感染5天后,取3×105的T细胞,4℃、400×g离心5min,弃上清,流式缓冲液清洗一次;加100μL缓冲液重悬细胞,加入0.5μg的MSLN-his蛋白,冰上孵育30min;加缓冲液清洗1次后,加入100μL缓冲液重悬细胞,加入APC anti-His抗体,冰上孵育30min;加缓冲液清洗2次后,加入300μL缓冲液重悬细胞;
采用流式细胞仪检测T淋巴细胞的嵌合抗原受体表达率,结果如图5所示,各组CAR-T细胞的感染效率分别为:41.1%、41.8%、39.2%、47.1%和36.4%,表明成功构建CAR-T细胞。
此外,本实施例中还使用流式细胞仪检测淋巴细胞表型,包括以下步骤:
(1)T细胞感染5天后,取3×105的T细胞,4℃、400×g离心5min,弃上清,PBS(含质量分数2%的FBS)缓冲液清洗一次;
(2)加50μL缓冲液重悬细胞,加入1μL FITC标记的Anti-CD3 Ab、Percp-Cy5.5标记的Anti-CD4Ab和PE-Cy7标记的Anti-CD8 Ab,冰上孵育30min;缓冲液清洗两次后,加入300μL缓冲液重悬细胞,采用流式细胞仪检测细胞表型,结果如图6A和图6B所示,CD3+CD4+细胞群占比40%左右,CD3+CD8+细胞群占比60%左右。
实施例7
本实施例中进行CAR-T细胞体外毒性实验,包括以下步骤:
(1)靶细胞接种
将293-GPF-luci(MSLN阴性,MSLN-)、AsPC1-MSLN-luci、PANC1-MSLN-luci作为靶细胞,调整靶细胞浓度为2×105/mL,取50μL接种到白色96孔板;
(2)效应细胞接种
MSLN CAR-T以及对照T细胞为效应细胞,按效靶比0.3:1和1:1向96孔板中加入CAR-T细胞及对照T细胞;
(3)各组均设3个复孔,取3个复孔的平均值,其中各实验组和各对照组如下:
实验组:各靶细胞+CAR-T;
对照组:只接种靶细胞;
(4)检测方法:
效应细胞与靶细胞共培养18h后,向各孔中加入100μL试剂(Promega,Cat#E2520),反应5min后,采用多功能酶标仪检测生物发光信号;
(5)CAR-T杀伤效率计算公式为:
杀伤效率%=(对照组-实验组)/对照组×100%
结果如图7-图9所示,本发明构建的CAR-T细胞对MSLN阴性的293T细胞无杀伤作用,对MSLN阳性的肿瘤细胞具有杀伤活性,表明本发明构建的CAR-T细胞不仅具备高效肿瘤杀伤活性,还具备高特异性。
实施例8
本实施例中检测CAR-T细胞因子TNF-α和IFN-γ的分泌情况,所用试剂盒分别为Human TNF-αELISAKit(联科生物,货号:EK182-96)和Human IFN-γELISAKit(联科生物,货号:EK180-96)。
1、细胞培养上清
将效靶比1:1的细胞培养物400×g离心10min去除沉淀物,取上清置于-80℃贮存待检。
2、试剂准备
检测前将所有的试剂、样本恢复至25℃,如果浓缩的试剂出现结晶,37℃温浴,直至结晶全部溶解,按照使用说明配制1×洗液,1×检测缓冲液。
3、标准品及样品配制
标准品:使用5%1640培养基2倍稀释标准品原液,一共8个稀释梯度,包括零浓度。
样品:使用5%1640培养基按比稀释样品。
4、检测步骤
(1)浸泡酶标板:加入300μL 1×洗液静置浸泡30s,弃掉洗液,在吸水纸上将微孔板拍干;
(2)加标准品:标准品孔加入100μL 2倍倍比稀释的标准品,空白孔加入100μL标准品稀释液(血清/血浆样本);
(3)加样本:样本孔加入100μL细胞培养上清;
(4)加检测抗体:每孔加入50μL稀释的检测抗体(1:100稀释);
(5)孵育:使用封板膜封板,300rpm振荡,25℃孵育2h;
(6)洗涤:弃掉液体,每孔加入300μL洗液洗板,洗涤6次;
(7)加酶孵育:每孔加入100μL稀释的辣根过氧化物酶标记的链霉亲和素(1:100稀释);
(8)孵育:使用新的封板膜封板,300rpm振荡,25℃孵育45min,洗涤;
(9)加底物显色:每孔加入100μL显色底物TMB,避光,25℃孵育20min;
(10)加终止液:每孔加入100μL终止液;
(11)检测读数:在30min之内,使用酶标仪进行双波长检测,测定450nm最大吸收波长和参考波长下的OD值;校准后的OD值为450nm的测定值减去参考波长下的测定值。
TNF-α、IFN-γ因子分泌结果分别如图10和图11所示,其中,自发为单独的CAR-T细胞组,未检测到细胞因子;CAR-T细胞与293T细胞共培养组,也没有检测到细胞因子;与过表达MSLN的靶细胞共培养后,CAR-T细胞分泌的TNF-α超过500pg/mL,IFN-γ超过3000pg/mL。本发明构建的CAR-T细胞对MSLN阳性的肿瘤细胞释放细胞因子,而对MSLN阴性细胞无明显细胞因子分泌。
综上所述,本发明筛选并制备具备高亲和力的抗间皮素的纳米抗体,能够高效与间皮素特异性结合,将其作为抗原结合结构域构建嵌合抗原受体和CAR-T细胞,所得CAR-T细胞对间皮素阳性的肿瘤细胞具有明显的杀伤活性和特异性,且能够分泌杀伤肿瘤的细胞因子,表明本发明的纳米抗体能够有效应用于免疫治疗,对于开发肿瘤治疗药物具有重要意义。
申请人声明,以上所述仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,所属技术领域的技术人员应该明了,任何属于本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,均落在本发明的保护范围和公开范围之内。
Claims (17)
1. 一种结合抗间皮素的纳米抗体,其特征在于,所述纳米抗体的CDR1的氨基酸序列如SEQ ID NO.3所示;
所述纳米抗体的CDR2的氨基酸序列如SEQ ID NO.6所示;
所述纳米抗体的CDR3的氨基酸序列如SEQ ID NO.9所示。
2.一种核酸分子,其特征在于,所述核酸分子编码权利要求1所述的结合抗间皮素的纳米抗体。
3.一种嵌合抗原受体,其特征在于,所述嵌合抗原受体包括信号肽、抗原结合结构域、铰链区、跨膜区和信号转导结构域;
所述抗原结合结构域包括权利要求1所述的结合抗间皮素的纳米抗体。
4.根据权利要求3所述的嵌合抗原受体,其特征在于,所述信号肽包括CD8α信号肽。
5.根据权利要求3所述的嵌合抗原受体,其特征在于,所述铰链区包括CD8α铰链区。
6.根据权利要求3所述的嵌合抗原受体,其特征在于,所述跨膜区包括CD8α跨膜区、CD28跨膜区或DAP10跨膜区中的任意一种或至少两种的组合。
7.根据权利要求3所述的嵌合抗原受体,其特征在于,所述信号转导结构域包括免疫受体酪氨酸活化基序。
8.根据权利要求7所述的嵌合抗原受体,其特征在于,所述信号转导结构域还包括共刺激分子,所述共刺激分子包括4-1BB、CD28胞内区、OX40、ICOS或DAP10胞内区中的任意一种或至少两种的组合。
9.根据权利要求3所述的嵌合抗原受体,其特征在于,所述嵌合抗原受体包括CD8α信号肽、权利要求1所述的结合抗间皮素的纳米抗体、CD8α铰链区、CD8α跨膜区和免疫受体酪氨酸活化基序。
10.一种表达载体,其特征在于,所述表达载体包括权利要求3所述的嵌合抗原受体的编码基因。
11.根据权利要求10所述的表达载体,其特征在于,所述表达载体为含有权利要求3-9任一项所述的嵌合抗原受体的编码基因的慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种。
12.根据权利要求11所述的表达载体,其特征在于,所述表达载体为含有权利要求3-9任一项所述的嵌合抗原受体的编码基因的慢病毒载体。
13.一种重组慢病毒,其特征在于,所述重组慢病毒含有权利要求10-12任一项所述的表达载体。
14.一种嵌合抗原受体免疫细胞,其特征在于,所述嵌合抗原受体免疫细胞表达权利要求3-9任一项所述的嵌合抗原受体;
所述嵌合抗原受体免疫细胞为T细胞或NK细胞。
15.根据权利要求14所述的嵌合抗原受体免疫细胞,其特征在于,所述嵌合抗原受体免疫细胞包括权利要求10-12任一项所述的表达载体和/或权利要求13所述的重组慢病毒。
16.一种药物组合物,其特征在于,所述药物组合物包括权利要求14或15所述的嵌合抗原受体免疫细胞;
所述药物组合物还包括药学上可接受的辅料;
所述辅料包括载体、稀释剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
17.如权利要求1所述的结合抗间皮素的纳米抗体、权利要求2所述的核酸分子、权利要求3-9任一项所述的嵌合抗原受体、权利要求10-12任一项所述的表达载体、权利要求13所述的重组慢病毒、权利要求14或15所述的嵌合抗原受体免疫细胞或权利要求16所述的药物组合物在制备肿瘤治疗药物中的应用;
所述肿瘤为表达间皮素的肿瘤。
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