CN116284448A - 一种超抗原参与的三功能t细胞衔接器及其应用 - Google Patents
一种超抗原参与的三功能t细胞衔接器及其应用 Download PDFInfo
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Abstract
本发明公开了一种超抗原参与的三功能T细胞衔接器的制备及其应用,涉及生物制药技术领域。本发明超抗原参与的三功能T细胞衔接器在细胞水平上具有较好的特异性T细胞激活、增殖活性及T细胞抗肿瘤活性,而且对实体瘤具有不错的疗效。因此,本发明的三功能T细胞衔接器或其编码的基因可用于制成抗肿瘤药物。该超抗原参与的三功能T细胞衔接器的制备方法有着制备简便且通过哺乳动物细胞表达的优点。
Description
技术领域
本发明涉及生物制药技术领域,特别是涉及一种超抗原参与的三功能T细胞衔接器的制备及其应用。
背景技术
双功能T细胞衔接器(Bifunctional T-cell engager,BiTE)能同时识别肿瘤细胞和T细胞,有效地介导T细胞对肿瘤产生免疫应答,在血液肿瘤治疗中取得重要突破,其中blinatumomab对CD19+的急性B淋巴母细胞瘤有显著疗效,且已获批上市。尽管如此,BiTE在实体肿瘤中的作用相当有限。
一方面,实体肿瘤缺乏合适靶点。传统BiTE仅能靶向膜蛋白胞外区域,靶点选择范围十分有限(膜蛋白占细胞总蛋白的比例低),靶向CD19、CD20等血液肿瘤的BiTE虽能损伤正常B细胞或骨髓细胞,但是该类细胞往往可再生,而EGFR、HER2等实体肿瘤靶点表达于多种组织/器官,可能引起不可逆的组织/器官损伤,on-target off-tumor的风险较大。因此,新靶点探寻成为BiTE研发的重要任务之一。胞内蛋白可在蛋白酶体中降解成抗原肽,再由MHC I类分子递呈到细胞表面形成多肽-MHC复合物(peptide-MHC complex,pMHC),被T细胞受体(T-cell receptor,TCR)识别。由于TCR与pMHC识别的高度特异性,探寻肿瘤特异性pMHC和其相匹配的TCR已成为肿瘤免疫研究的热点,包括KRASG12D、p53R175H等热点突变在内的多种特异性TCR已被报导。此外,Immunocore公司研发基于TCR的新型BiTE(ImmTAC),已有三种进入临床研究阶段,其中Tebentafusp(靶向gp100)被证明对实体肿瘤有明显的临床疗效,已被FDA批准用于治疗HLA-A*02:01阳性不可切除性或转移性葡萄膜黑色素瘤(mUM)成人患者。T-BiTE由肿瘤靶向的单克隆TCR与抗CD3ε抗体组成,能介导T细胞免疫反应,有良好的抗肿瘤效果。理论上所有肿瘤抗原均有被MHCI分子递呈的可能,其中包括体细胞突变产生的肿瘤新生抗原、肿瘤细胞异常表达的分化抗原以及癌胚抗原等。因此,T-BiTE靶点选择范围广,是极具潜力的肿瘤免疫治疗药物。
另一方面,肿瘤微环境(Tumor microenvironment,TME)中存在大量免疫抑制细胞(如调节性T细胞、骨髓来源抑制细胞、肿瘤相关纤维母细胞等),不仅能抑制肿瘤浸润T细胞(Tumorinfiltrating lymphocyte,TIL)的活化、增殖等从而影响抗肿瘤活性,其分泌的物质可形成化学或物理屏障,阻碍血液中T细胞的浸润。因此,有效地活化TIL或增加T细胞浸润对BiTE活性至关重要。天然T细胞通过TCR-pMHC识别促发第一信号,共刺激信号进一步影响T细胞的激活、存活及分化,从而影响其活性。研究表明,嵌合抗原受体T细胞(Chimericantigen receptor T cell,CAR-T)对血液瘤的疗效优于BiTE,而这很大程度上得益于共刺激信号的参与。BiTE通过模拟第一信号促发T细胞激活以达到杀伤肿瘤的目的,Skokos D等和Wu L等证明CD28信号的参与能有效增强BiTE疗效,表明充分激活T细胞能提高BiTE的疗效,且共刺激信号的参与有利于BiTE对T细胞的活化。此外,Scheidt BV等发现,金黄色葡萄球菌肠毒素B(Staphylococcal enterotoxin B,SEB)能增加CAR-T细胞的增殖和浸润,从而增强CAR-T对实体肿瘤的杀伤。
金黄色葡萄球菌肠毒素(Staphylococcal enterotoxin,SE)是一类典型的超抗原(Superantigen,SA),其可直接结合MHC II分子和TCR,有效地激活T细胞。我们在之前研究中发现,SE不仅可通过活化T细胞以增强T-BiTE的活性,其刺激T细胞分泌的物质能促进MHCI分子表达从而增加肿瘤表面T-BiTE的靶点数目,进一步提高T-BiTE对肿瘤的杀伤。因此,超抗原参与只是在理论上对BiTE在实体瘤中的疗效有积极作用。所以,一种超抗原参与的三功能T细胞衔接器有待开发。
发明内容
本发明提供了一种葡萄球菌肠毒素A超抗原(SEA)参与的以NY-ESO-1SLL/HLA-A*02:01为靶抗原的三功能T细胞衔接器的制备方法和应用。
本发明的具体的技术方案如下:
本发明提供了一种超抗原参与的三功能T细胞衔接器,包括一个包含IgG抗体恒定区的骨架,所述骨架包括两条重链和两条轻链,重链包括重链恒定区CH1、CH2、CH3,轻链包括CL,所述超抗原参与的三功能T细胞衔接器为异源三聚体,具体为:
第一亚基包括T细胞受体α链的可变区、恒定区及所述骨架中的一条轻链,第二亚基包括T细胞受体β链的可变区、恒定区及所述骨架中的一条重链,第三亚基包括葡萄球菌肠毒素A的编码序列;所述第一亚基或第三亚基上还连接有抗CD3抗体的重链可变区和轻链可变区;
第一亚基中的T细胞受体α链的可变区和恒定区与第二亚基中的T细胞受体β链的可变区和恒定区构成用于识别抗原肽-HLA复合物的T细胞受体结构域。所述IgG抗体为人源IgG抗体。所述重链包括恒定区CH1、CH2、CH3,恒定区中还有铰链区。
本发明三功能T细胞衔接器中第一亚基和第二亚基中含有用于识别抗原肽-HLA复合物的T细胞受体,抗原肽为肿瘤突变蛋白在蛋白酶体中降解而成,同时含有抗CD3抗体,以pHLA和传统肿瘤表面高表达抗原为靶点的三特异性T细胞衔接器通过葡萄球菌肠毒素A超抗原可直接结合MHC II分子和TCR,有效地激活T细胞。本发明实施例结果表示,该肿瘤双靶向的三功能T细胞衔接器在细胞水平上具有更好的特异性T细胞激活、增殖活性及T细胞抗肿瘤活性,能够有效的导致肿瘤细胞死亡。
作为示例,所述多肽-HLA复合物加载的多肽序列为SLLMWITQC;在本发明实施例中作为示例,选用所述抗CD3抗体为抗CD3ε抗体。但不限于此。
优选的,第二亚基和第三亚基中的两个CH3之间分别使用knob-into-hole技术设计成knob结构和hole结构。其中,第二亚基和第三亚基的CH2结构域引入LALA-PG突变。使抗体Fc片段沉默,失去与Fc受体结合的功能,避免Fc造成的非特异性杀伤的毒副作用。
具体的,第一亚基的氨基酸序列如SEQ ID No.9所示,第二亚基的氨基酸序列如SEQ ID No.10所示,第三亚基的氨基酸序列如SEQ ID No.11所示。
本发明还提供了编码所述三功能T细胞衔接器的基因,第一亚基的核苷酸序列如SEQ ID No.1所示,第二亚基的核苷酸序列如SEQ ID No.2所示,第三亚基的核苷酸序列如SEQ ID No.3所示。
本发明还提供了所述三特异性T细胞衔接器的制备方法,包括以下步骤:(1)构建表达第一亚基的编码基因、第二亚基的编码基因和第三亚基的编码基因的编码基因载体;(2)将步骤(1)表达第一亚基的编码基因、第二亚基的编码基因和第三亚基的编码基因载体转染到哺乳动物细胞中,培养后进行蛋白纯化,得到所述三特异性T细胞衔接器。优选的,步骤(1)所述载体为pLVX-Puro;步骤(2)所述哺乳动物细胞为293F细胞。
本发明还提供了所述三功能T细胞衔接器在制备抗肿瘤药物中的应用。
本发明还提供了所述的基因在制备抗肿瘤药物中的应用。
本发明还提供了一种抗肿瘤药物,活性成分为所述三功能T细胞衔接器或所述的基因。肿瘤细胞类型为HLA-A*02:01分型的NY-ESO-1阳性细胞。
本发明的有益效果:
本发明超抗原参与的三功能T细胞衔接器在细胞水平上具有较好的特异性T细胞激活、增殖活性及T细胞抗肿瘤活性,而且对实体瘤具有不错的疗效。因此,本发明的三功能T细胞衔接器或其编码的基因可用于制成抗肿瘤药物。该超抗原参与的三功能T细胞衔接器的制备方法有着制备简便且通过哺乳动物细胞表达的优点。
附图说明
图1为超抗原参与的三功能T细胞衔接器及其对照药物的分子形式图。
图2为超抗原参与的三功能T细胞衔接器及其对照药物的去糖基化分析图;其中,Lane 1:PNGase处理的tNY-aCD3/SEA;Lane 2:tNY-aCD3/SEA;Lane 3:PNGase处理的tCrtl-aCD3/SEA;Lane 4:tCrtl-aCD3/SEA;Lane 5:PNGase处理tNY/SEA;Lane 6:tNY/SEA;Lane7:PNGase处理的tNY-aCD3/aCtrl;Lane 8:tNY-aCD3/aCtrl。
图3为超抗原参与的三功能T细胞衔接器及其对照药物对NY-ESO-1 SLL/HLA-A*02:01的特异性亲和力分析图。(a)tNY-aCD3/SEA及其对照药物对NY-ESO-1 SLL/HLA-A*02:01的亲和力分析图。(b)tNY-aCD3/SEA及其对照药物对NY-ESO-1 SLL/HLA-A*02:01的特异性亲和力分析图。
图4为超抗原参与的三功能T细胞衔接器及其对照药物对Jurkat细胞(CD3+)的亲和力分析图。
图5为超抗原参与的三功能T细胞衔接器介导PBMC杀伤肿瘤细胞图。(a)用PBMC和2nM tNY-aCD3/SEA、tNY-aCD3/aCtrl、tCtrl-aCD3/SEA或tNY/SEA处理2天后A375-SLL细胞的形态变化。(b)tNY-aCD3/SEA和tCtrl-aCD3/SEA重定向T细胞在体外杀死A375细胞。(c-d)tNY-aCD3/SEA、tNY-aCD3/aCtrl、tCtrl-aCD3/SEA和tNY/SEA重定向T细胞以在体外杀死A375-SLL细胞(c)或K562-A2-SLL细胞(d)。
图6为tNY-aCD3/SEA刺激T细胞特异性激活图。(a)T细胞早期激活比例图。(b)T细胞晚期激活比例图。
图7为tNY-aCD3/aEGFR刺激T细胞扩增图。
图8为tNY-aCD3/SEA刺激T细胞释放γ干扰素图。
图9为tNY-aCD3/SEA的体内特异性抗肿瘤活性图。(a)tNY-aCD3/SEA及其对照药物对异源移植小鼠的抗肿瘤活性图。(b)tNY-aCD3/SEA及其对照药物对异源移植小鼠的生存曲线图。
具体实施方式
本申请相关动物实验经浙江大学实验动物福利伦理审查委员会审批通过,申请编号12435。本申请中涉及人的相关实验经浙江大学药学院医学伦理委员会审批通过,审批件编号为:(2018)伦研批第003号。
实施例1
超抗原参与的三功能T细胞衔接器的制备。
TCR选择NY-TCR(靶向SLLMWITQC多肽与HLA-A*0201复合物的TCR,简称tNY),来自专利:US20110014169A1。抗CD3抗体选择HXR32,来自专利:WO2012162067A2。超抗原参与的三功能T细胞衔接器示意图如图1所示,构建人肾上皮细胞(293F)表达质粒9个。
tNY-aCD3/SEA:tNYECDα-GS-CL-(G4S)3-aCD3VH-aCD3VL(基因序列如SEQ IDNo.17和SEQ ID No.1所示),tNYECDβ-GS-CH1-CH2-CH3(knob)-His6(基因序列如SEQ IDNo.17、SEQ ID No.2和SEQ ID No.20所示),SEA-CH2-CH3(hole)-Flag(基因序列如SEQ IDNo.17、SEQ ID No.3和SEQ IDNo.22所示)。
tCrtl-aCD3/SEA:tCtrlECDα-GS-CL-(G4S)3-aCD3VH-aCD3VL(基因序列如SEQ IDNo.18和SEQ ID No.4所示),tCtrlECDβ-GS-CH1-CH2-CH3(knob)-His6(基因序列如SEQ IDNo.19、SEQ ID No.5和SEQ ID No.20所示),SEA-CH2-CH3(hole)-Flag(基因序列如SEQ IDNo.17、SEQ ID No.3和SEQ ID No.22所示)。
tNY-aCD3/aCtrl:tNYECDα-GS-CL-(G4S)3-aCD3VH-aCD3VL(基因序列如SEQ IDNo.17和SEQ ID No.1所示),tNYECDβ-GS-CH1-CH2-CH3(knob)-His6(基因序列如SEQ IDNo.17、SEQ ID No.2和SEQ ID No.20所示),aCtrlVH-CH1-CH2-CH3(hole)-Flag(基因序列如SEQ ID No.17、SEQ ID No.6和SEQ ID No.22所示),aCtrlVL-CL(基因序列如SEQ IDNo.17和SEQ ID No.7所示)。
tNY/SEA:tNYECDα-GS-CL(基因序列如SEQ ID No.17和SEQ ID No.8所示),tNYECDβ-GS-CH1-CH2-CH3(knob)-His6(基因序列如SEQ ID No.17、SEQ ID No.2和SEQ ID No.20所示),SEA-CH2-CH3(hole)-Flag(基因序列如SEQ ID No.17、SEQ ID No.3和SEQ ID No.22所示)。
其中,tNYECDα和tNYECDβ分为tNY的α链和β链的胞外结构域,tCtrlECDα和tCtrlECDβ分别为tCtrl的α链和β链的胞外结构域,(G4S)3为三个GGGGS长度的柔性接头,aCD3VH和aCD3VL分别为抗CD3ε抗体的重链可变区和轻链可变区,aCtrlVH和aCtrlVL分别为对照抗体的重链可变区和轻链可变区,SEA为葡萄球菌肠毒素A,His6是用于蛋白纯化的6个组氨酸组成的标签,Flag标签是用于蛋白纯化的8个氨基酸DYKDDDDK组成的标签,CL、CH1、CH2、CH3均为IgG1的结构,两个CH3之间分别使用knob-into-hole技术设计成knob和hole结构,同时在CH2引入LALAPG突变使Fc沉默。
tNY和tCtrl的α链和β链胞外区域,aCD3和aCtrl的重链可变区和轻链可变区,SEA的序列,IgG1型抗体重链恒定区以及κ轻链恒定区由上海生工生物技术有限公司合成。通过PCR以及重叠PCR,将各段基因按重组质粒要求组合,并在重组基因的上游引入Xho I酶切位点(CTCGAG)、kozaka序列(ACCACC),在下游引入终止密码子(TGA)及EcoR I酶切位点(GAATTC),由T4连接酶连接到pLVX-Puro质粒(质粒也经Xho I和EcoR I双酶切)。之后,将重组质粒瞬转至293F细胞中,并用HisTrap HP亲和柱(购自GE公司,货号:17-5248-02)和抗-M1亲和凝胶(购自Sigma公司,货号:A4596)进行柱层析纯化,获得相应蛋白。用PNGase(购自Sigma公司,货号:PP0201)对纯化产物进行去糖基化处理,再用SDS-PAGE分析(以未经PNGase处理的纯化产物为对照)。
如图2所示,经PNGase处理的T细胞衔接器的各亚基均出现在理论大小的位置(T细胞衔接器各亚基的理论分子量大小见表1),其中TCR的β链和α链在PNGase处理前后位置发生变化,表明有明显的糖基化修饰,α链变动更大,糖基化修饰更为复杂。
表1超抗原参与的三功能T细胞衔接器及其对照药物各亚基理论分子量大小
实施例2
(1)亲和力验证
对NY-ESO-1 SLL/HLA-A*02:01的特异性亲和力验证(ELISA)。
首先通过复性法制备生物素化的NY-ESO-1 SLL/HLA-A*02:01复合物(SLLMWITQC与HLA-A*02:01的复合物,称为SLL/A02-Biotin),WT1 RMF/HLA-A*02:01复合物(RMFPNAPYL与HLA-A*02:01的复合物,称为RMF/A02-Biotin)和Tax LLF/HLA-A*02:01复合物(LLFGYPRYV与HLA-A*0201的复合物,称为LLF/A02-Biotin)。
在96孔EIA/RIA板(购自Corning公司,货号:03619013)中4℃过夜包被100μL,1μg/mL的链霉亲和素(购自上海生工生物技术有限公司,货号:A100497)。PBST(含0.05%吐温20的PBS溶液)清洗4次后,37℃封闭1h(封闭液:5%脱脂奶粉的PBST溶液)。PBST清洗4次后,37℃孵育100μL,1μg/mL的SLL/A02-Biotin、RMF/A02-Biotin或LLF/A02-Biotin 1h。PBST清洗4次后,37℃孵育100μL浓度梯度的超抗原参与的三功能T细胞衔接器及对照药物1h。PBST清洗4次后,37℃孵育辣根过氧化物酶标记山羊抗人IgG(H+L)(购自碧云天生物技术有限公司,货号:A0201)1h。PBST清洗5次后,加入100μL TMB液体底物(购自Sigma公司,货号:T4444)显色约10min,加入100μL,2M的硫酸终止反应,并测定OD450值。
结果表明,tNY-aCD3/SEA、tNY-aCD3/aCtrl及tNY/SEA因含有tNYECD亚基从而对NY-ESO-1 SLL/HLA-A*02:01有较高的亲和力,且呈浓度依赖性;而tCrtl-aCD3/SEA对NY-ESO-1 SLL/HLA-A*02:01没有亲和,可以在后续药物评价中作为对照药物(图3a)。tNY-aCD3/SEA、tNY-aCD3/aCtrl及tNY/SEA对WT1 RMF/HLA-A*02:01和Tax LLF/HLA-A*02:01没有亲和,表明其对NY-ESO-1 SLL/HLA-A*02:01的亲和特异性良好(图3b)。
(1)对CD3抗原的亲和力分析
500×g,5min离心收集处于对数生长期的Jurkat细胞(CD3+细胞),4℃分别孵育200μL,不同浓度的超抗原参与的三功能T细胞衔接器及其对照药物30min(以孵育PBS为对照)。PBS清洗2遍后,4℃孵育200μL FITC标记山羊抗人IgG(H+L)(购自碧云天生物技术有限公司,货号:A0556)。PBS清洗2遍后,用400μL PBS重悬细胞,再用ACEA NovoCyteTM流式细胞仪检测。
结果如图4所示,tNY-aCD3/SEA和tNY-aCD3/aCtrl的aCD3对Jurkat(CD3+)有相近的亲和力,且都弱于tCtrl-aCD3/SEA的aCD3对Jurkat的亲和力。
实施例3:体外特异性抗肿瘤活性
稳定转染EGFP以及递呈多肽SLLMWITQC的A375-SLL细胞、与PBMC(购自上海赛笠生物科技有限公司,货号:2001030)按1∶4铺于24孔细胞培养板,并加入系列梯度浓度的tNY-aCD3/SEA、tNY-aCD3/aCtrl、tCtrl-aCD3/SEA或tNY/SEA(n=3),混匀后置于37℃,5%CO2培养箱孵育。通过观察A375-SLL细胞形态变化,初步探索tNY-aCD3/SEA、tNY-aCD3/aCtrl、tCtrl-aCD3/SEA及tNY/SEA的体外抗肿瘤活性。PBMC和2nM的药物共同作用2天后,tNY-aCD3/SEA组的A375-SLL细胞明显皱缩、成团,且细胞数量显著低于对照组,表明tNY-aCD3/SEA介导PBMC发挥抗肿瘤活性;tNY-aCD3/aCtrl及tNY/SEA的A375-SLL细胞出现少量皱缩,大部分肿瘤细胞状态及细胞密度与对照组无显著差别,表明tNY-aCD3/aCtrl及tNY/SEA在2nM浓度下抗肿瘤效果不显著;而tCtrl-aCD3/SEA组的A375-SLL细胞状态、密度与对照组无肉眼可见差别,表明tCtrl-aCD3/SEA在2nM浓度下无可见的抗肿瘤效果(图5a),以上结果初步表明,tNY-aCD3/SEA具备一定特异性的抗肿瘤活性。
接着,将A375细胞、A375-SLL细胞、稳定转染HLA-A*02:01和EGFP以及递呈多肽SLLMWITQC的K562-A2-SLL细胞(均NY-ESO-1 SLL/HLA-A*02:01+)分别与PBMC按1∶4铺于24孔细胞培养板,并加入系列梯度浓度的tNY-aCD3/SEA、tNY-aCD3/aCtrl、tCtrl-aCD3/SEA或tNY/SEA(n=3),混匀后置于37℃,5%CO2培养箱孵育。48h后收集细胞,用AnnexinV,633凋亡检测试剂盒(购自东仁化学科技(上海)有限公司,货号:AD11)染色,再用ACEA NovoCyteTM流式细胞仪检测肿瘤细胞的凋亡情况。发现tNY-aCD3/SEA能介导PBMC杀伤A375细胞,但是其活性并不理想(>2nM才有显著杀伤,图6b);虽然tCtrl-aCD3/SEA介导PBMC杀伤A375细胞的能力弱于tNY-aCD3/SEA,但是差异并不大(图5b),猜测aCD3/SEA对T细胞的非特异性激活可能主导该活性。为了进一步验证tNY-aCD3/SEA的活性及特异性,选择SLL/A02表达相对较高的A375-SLL细胞及K562-A2-SLL细胞进行评价。发现提高SLL/A02表达量之后,tNY-aCD3/SEA的活性得到显著提升的同时,tCtrl-aCD3/SEA的活性无显著变化,即当给药浓度在8-200pM之间,tNY-aCD3/SEA已展示显著的抗肿瘤活性,而tCtrl-aCD3/SEA未体现出抗肿瘤活性(图5c-d);此外,tNY-aCD3/aCtrl及tNY/SEA抗肿瘤活性与tCtrl-aCD3/SEA相当,且均显著弱于tNY-aCD3/SEA(图5c-d),也表明tNY-aCD3/SEA高活性是其三个组分共同作用的结果。以上结果表明,tNY-aCD3/SEA能介导PBMC杀伤SLL/A02阳性肿瘤细胞,其活性由三个组分共同决定,而过低的SLL/A02可能导致特异性识别肿瘤的TCR组分作用强度不够,影响tNY-aCD3/SEA活性发挥。
实施例4:T细胞特异性激活检测
A375-SLL细胞分别与PBMC按照1∶4铺于48孔细胞培养板,并加入系列梯度浓度的tNY-aCD3/SEA及其对照药物(n=3),混匀后置于37℃,5%CO2培养箱孵育。培养72h后,600×g,5min离心收集细胞,用1×PBS清洗1次后重悬于1×PBS,加入5μL APC标记的小鼠抗人CD3抗体(购自ThermoFisher Scientific公司,货号17-0037-42),5μL Super Bright 436标记的小鼠抗人CD69抗体(购自ThermoFisher Scientific公司,货号62-0699-42)和1μLPE标记的小鼠抗人CD25抗体(购自ThermoFisher Scientific公司,货号12-0259-80),4℃避光孵育30min。1×PBS清洗2次后,重悬于200μL的1×PBS中,用Cytomic FC 500MCL流式细胞仪检测CD69和CD25阳性T细胞(CD3阳性)的比例。
结果如图6所示,CD69和CD25分别是T细胞(CD3+)早期和晚期激活的主要标志,在20-2000pM药物作用下,T细胞中CD25+或CD69+的细胞比例随药物浓度增加而上调,表明tNY-aCD3/SEA、tNY-aCD3/aCtrl、tCtrl-aCD3/SEA或tNY/SEA均对T细胞有激活作用。tNY-aCD3/SEA上调CD25+或CD69+的T细胞比例显著高于三种对照药物,表明tNY-aCD3/SEA的T细胞激活能力最强,其三个组分对T细胞的激活均有贡献。三个对照药物中,tNY-aCD3/aCtrl上调CD25+或CD69+的T细胞比例显著高于tCtrl-aCD3/SEA或tNY/SEA,表明特异性识别肿瘤对tNY-aCD3/SEA活性发挥至关重要,在SLL/A02表达相对较高情况下,tNY-aCD3/SEA的特异性较好。
实施例5:T细胞增殖检测
1000rpm,10min离心收集PBMC,用室温预热的1×PBS清洗2次后,重悬于1×PBS中,加入CFSE(购自ThermoFisher Scientific公司,货号:65-0850-84)至终浓度为2.5μM,混匀,37℃避光孵育15min。1000rpm,10min离心收集A375-SLL和CFSE处理的PBMC,PBS清洗1次后,A375-SLL分别与CFSE处理的PBMC按照1∶4铺于48孔细胞培养板,并加入系列梯度浓度的tNY-aCD3/SEA及其对照药物(n=3),混匀后置于37℃,5%CO2培养箱孵育。
培养96h后,600×g,5min离心收集细胞,用1×PBS清洗2次后重悬于100μL 1×PBS中,加入5μL PI母液(1mg/mL),室温避光孵育15min后,600×g,5min离心,加400μL 1×PBS重悬,用ACEA NovoCyteTM流式细胞仪根据细胞形态选取PBMC检测其增殖情况。
T细胞激活后,可分裂增殖。CFSE可非特异性的结合细胞内的蛋白,产生绿色荧光,并随着细胞分裂平均分配到两个子代细胞中,绿色荧光随着分裂次数增加逐级递减。结果如图7所示,在20-2000pM药物作用4天后,tNY-aCD3/SEA能有效诱导T细胞增殖,且tNY-aCD3/SEA诱导T细胞增殖的能力显著强于同等浓度的三个对照药物,表明tNY-aCD3/SEA的三个组分均参与诱导T细胞增殖。三个对照药中,tNY-aCD3/aCtrl诱导T细胞增殖能力强于tCtrl-aCD3/SEA或tNY/SEA,表明特异性识别肿瘤对tNY-aCD3/SEA活性发挥至关重要。
实施例6:γ干扰素检测
A375-SLL细胞分别与PBMC按照1∶4铺于48孔细胞培养板,并加入系列梯度浓度的tNY-aCD3/SEA及其对照药物(n=3),混匀后置于37℃,5%CO2培养箱孵育。
培养72h后,600×g,5min离心收集上清,按一定浓度稀释后,通过γ干扰素检测试剂盒(购自ThermoFisher Scientific公司,货号:88-7316-88)检测T细胞γ干扰素的释放情况。
结果如图8所示,γ干扰素是T细胞激活后所释放的一种细胞因子,具有较强的抑制肿瘤作用,tNY-aCD3/SEA诱导T细胞分泌IFN-γ的能力显著优于三种对照药物,且tNY-aCD3/aCtrl诱导T细胞分泌IFN-γ的能力又优于tCtrl-aCD3/SEA或tNY/SEA,表明tNY-aCD3/SEA的T细胞激活能力最强,且具备较高的肿瘤特异性。
实施例7:tNY-aCD3/SEA的体内特异性抗肿瘤活性
A375-SLL细胞与PBMC按照2×106:2×106接种于8周龄雌性SCID-Beige小鼠(购自江苏集萃药康生物科技股份有限公司,SPF级)右侧腋下皮下。接种1h后随机分5组(生理盐水组、tCrtl-aCD3/SEA组(100μg/kg)、tNY-aCD3/aCtrl组(100μg/kg)、tNY-aCD3/SEA组(100μg/kg)和tNY-aCD3/SEA低剂量组(25μg/kg)),每组5只小鼠,并通过尾静脉给药,每隔3天给药一次,总共给药3次。测量小鼠肿瘤大小并记录(肿瘤体积=肿瘤长×肿瘤宽×肿瘤宽/2)。
由于肿瘤体积大于500mm3后,部分小鼠出现消瘦等现象,以500mm3为标准绘制小鼠生存曲线。与对照组相比,100μg/kg给药剂量的tNY-aCD3/SEA、tCtrl-aCD3/SEA、tNY-aCD3/aCtrl均能抑制移植瘤生长,其中tNY-aCD3/SEA组仅一只小鼠观察到肿瘤生长,tCtrl-aCD3/SEA和tNY-aCD3/aCtrl组的5只小鼠均观察到肿瘤生长(图9a),表明tNY-aCD3/SEA的抗肿瘤效果显著优于tCtrl-aCD3/SEA和tNY-aCD3/aCtrl,tNY-aCD3/SEA的三个组分均参与体内抗肿瘤活性发挥,该结果与上述体外活性及激活一致。tCtrl-aCD3/SEA组小鼠肿瘤的生长速率显著慢于tNY-aCD3/aCtrl组,且tCtrl-aCD3/SEA组小鼠生存时间显著长于tNY-aCD3/aCtrl组(图9a-b),表明tCtrl-aCD3/SEA的体内抗肿瘤活性可能优于tNY-aCD3/aCtrl。tCtrl-aCD3/SEA虽然缺乏SLL/A02识别功能,但是其可通过移植瘤中的PBMC靶向肿瘤,从而激活T细胞杀伤肿瘤,这一方面体现tNY-aCD3/SEA可能存在由aCD3/SEA引起的部分非特异性杀伤,同时也表明SEA的参与可能协助免疫细胞克服实体肿瘤的抑制。此外,比较100μg/kg和25μg/kg的tNY-aCD3/SEA体内抗肿瘤活性差异,发现剂量较高的tNY-aCD3/SEA活性优于低剂量组(图9a-b),表明tNY-aCD3/SEA的体内抗肿瘤活性在25-100μg/kg呈剂量依赖。
Claims (10)
1.一种超抗原参与的三功能T细胞衔接器,包括一个包含IgG抗体恒定区的骨架,所述骨架包括两条重链和两条轻链,重链包括重链恒定区CH1、CH2、CH3,轻链包括CL,其特征在于,所述超抗原参与的三功能T细胞衔接器为异源三聚体,具体为:
第一亚基包括T细胞受体α链的可变区、恒定区及所述骨架中的一条轻链,第二亚基包括T细胞受体β链的可变区、恒定区及所述骨架中的一条重链,第三亚基包括葡萄球菌肠毒素A的编码序列;所述第一亚基或第三亚基上还连接有抗CD3抗体的重链可变区和轻链可变区;
第一亚基中的T细胞受体α链的可变区和恒定区与第二亚基中的T细胞受体β链的可变区和恒定区构成用于识别抗原肽-HLA复合物的T细胞受体结构域。
2.如权利要求1所述的三功能T细胞衔接器,其特征在于,所述IgG抗体为人源IgG抗体。
3.如权利要求1所述的三功能T细胞衔接器,其特征在于,所述抗CD3抗体为抗CD3ε抗体。
4.如权利要求1所述的三功能T细胞衔接器,其特征在于,第二亚基和第三亚基中的两个CH3之间分别使用knob-into-hole技术设计成knob结构和hole结构。
5.如权利要求4所述的三功能T细胞衔接器,其特征在于,第二亚基和第三亚基的CH2结构域引入LALA-PG突变。
6.如权利要求5所述的三功能T细胞衔接器,其特征在于,第一亚基的氨基酸序列如SEQID No.9所示,第二亚基的氨基酸序列如SEQ ID No.10所示,第三亚基的氨基酸序列如SEQID No.11所示。
7.编码权利要求1~6任一所述三功能T细胞衔接器的基因,第一亚基的核苷酸序列如SEQ ID No.1所示,第二亚基的核苷酸序列如SEQ ID No.2所示,第三亚基的核苷酸序列如SEQ ID No.3所示。
8.权利要求1~6任一所述三功能T细胞衔接器在制备抗肿瘤药物中的应用。
9.如权利要求7所述的基因在制备抗肿瘤药物中的应用。
10.一种抗肿瘤药物,其特征在于,活性成分为权利要求1~6任一所述三功能T细胞衔接器或权利要求7所述的基因。
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