CN116693687B - 一种抗人cd25工程抗体及应用 - Google Patents
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Abstract
本发明提供了一种抗人CD25工程抗体及应用,重链可变区的CDR1的氨基酸序列如SEQ ID NO.1所示;重链可变区的CDR2的氨基酸序列如SEQ ID NO.2所示;重链可变区的CDR3的氨基酸序列如SEQ ID NO.3所示。本发明所述的抗人CD25工程抗体可用于调节性T细胞的检测,从而辅助多发性硬化症、类风湿关节炎、I型糖尿病的诊断。
Description
技术领域
本发明属于医药领域,尤其是涉及一种抗人CD25工程抗体及应用。
背景技术
CD25在Tregs中高水平表达,但在Teff细胞中不表达。消除或逆转Tregs的免疫抑制作用是肿瘤免疫治疗的关键。CD25是实现Treg耗竭的潜在靶点,它在肿瘤微环境(TME)和外周微环境中的作用受到了广泛的关注。
Treg细胞的耗竭直接促进肿瘤消退。尤其是在CD25靶向药物对CD25+肿瘤的研究中,它们与Treg细胞和Teff细胞的结合亲和力之间的平衡对于治疗效果至关重要。CD25靶向药物的给药剂量和时间也对治疗有深远影响,结果已经开发了有效地消耗肿瘤浸润Treg细胞的策略,包括使用Fc优化的抗CD25抗体,这增强了ADCC效应。此外,当将抗PD-1/PD-L1抗体与Fc优化的抗CD25抗体结合治疗小鼠肿瘤模型时,已观察到协同效应。
目前只有一个CD25靶向ADC ADCT-301进入临床试验。ADCT-301含有一种IL-2阻断抗CD25抗体,名为HuMax TAC。该ADC不仅可以靶向表达CD25的肿瘤细胞以治疗CD25+人类淋巴瘤,还可以通过靶向肿瘤内Treg细胞来治疗CD 25实体瘤。目前,还没有IL-2非阻断性抗CD25单抗用于ADC。然而,这种类型的ADC被认为具有更强的抗肿瘤活性。
发明内容
有鉴于此,本发明旨在克服现有技术中的缺陷,提出一种抗人CD25工程抗体及应用。
为达到上述目的,本发明的技术方案是这样实现的:
本发明提供一种抗人CD25工程抗体,该工程抗体包括重链可变区与轻链可变区;
所述的重链可变区的互补决定区包括CDR1、CDR2与CDR3;所述的重链可变区的CDR1的氨基酸序列如SEQ ID NO.1所示;所述的重链可变区的CDR2的氨基酸序列如SEQ IDNO.2所示;所述的重链可变区的CDR3的氨基酸序列如SEQ ID NO.3所示;
所述的轻链可变区的互补决定区包括CDR1、CDR2与CDR3;所述的轻链可变区的CDR1的氨基酸序列如SEQ ID NO.4所示;所述的轻链可变区的CDR2的氨基酸序列如SEQ IDNO.5所示;所述的轻链可变区的CDR3的氨基酸序列如SEQ ID NO.6所示。
进一步,所述的重链可变区的骨架区包括FR1、FR2、FR3和FR4;所述的重链可变区的FR1的氨基酸序列如SEQ ID NO.7所示;所述的重链可变区的FR2的氨基酸序列如SEQ IDNO.8所示;所述的重链可变区的FR3的氨基酸序列如SEQ ID NO.9所示;所述的重链可变区的FR4的氨基酸序列如SEQ ID NO.10所示;
所述的轻链可变区的骨架区包括FR1、FR2、FR3和FR4;所述的轻链可变区的FR1的氨基酸序列如SEQ ID NO.11所示;所述的轻链可变区的FR2的氨基酸序列如SEQ ID NO.12所示;所述的轻链可变区的FR3的氨基酸序列如SEQ ID NO.13所示;所述的轻链可变区的FR4的氨基酸序列如SEQ ID NO.14所示。
进一步,所述的工程抗体的重链可变区的氨基酸序列如SEQ ID NO.15所示,轻链可变区的氨基酸序列如SEQ ID NO.16所示。
本发明还提供一种核酸分子,该核酸分子编码所述的抗人CD25工程抗体。
进一步,所述的核酸分子编码所述的抗人CD25工程抗体的重链可变区的核苷酸序列如SEQ ID NO:17所示,编码轻链可变区的核苷酸序列如SEQ ID NO:18所示。
本发明还提供一种表达载体,该表达载体包含所述的核酸分子。所述的表达载体为pcDNA3.4或Simple-T。
本发明还提供一种抗人CD25工程抗体的应用,所述的抗人CD25工程抗体在制备检测调节性T细胞的试剂中的应用。
调节性T细胞是维持机体免疫耐受的重要因素之一,它由胸腺产生后输出至外周,并通过主动调节的方式抑制存在于正常机体内潜在的自身反应性T细胞的活化与增殖,从而调节机体的免疫力,如防止自身免疫性疾病的发生。调节性T细胞的数量减少或功能异常均有可能导致自身免疫病的发生,一些自身免疫性疾病如多发性硬化症、活动性类风湿关节炎、I型糖尿病等调节性T细胞的数量和功能均会发生变化。在许多恶性疾病如肺、胰腺和乳腺癌等已经发现调节性T细胞明显增高。调节性T细胞也阻断抗肿瘤免疫反应从而导致死亡率上升,也可用于骨髓移植术前术后的跟踪。
进一步,所述的抗人CD25工程抗体在制备诊断多发性硬化症、类风湿关节炎或I型糖尿病的试剂中的应用;所述的抗人CD25工程抗体在制备治疗肿瘤的药物中的应用或在制备检测骨髓移植指标的试剂中的应用。
本发明还提供一种表达载体的应用,所述的表达载体在制备检测调节性T细胞的试剂中的应用;所述的表达载体在制备诊断多发性硬化症、类风湿关节炎或I型糖尿病的试剂中的应用;所述的表达载体在制备治疗肿瘤的药物中的应用或在制备检测骨髓移植指标的试剂中的应用。
本发明还提供一种免疫检测试剂盒,该试剂盒含有所述的抗人CD25工程抗体和/或所述的表达载体。
相对于现有技术,本发明具有以下优势:
本发明所述的抗人CD25工程抗体可用于调节性T细胞的检测,从而辅助多发性硬化症、类风湿关节炎、I型糖尿病的诊断。
本发明所述的抗人CD25单克隆抗体的轻链可变区基因和重链可变区基因及其表达产物,两者重组后表达产生的抗CD25抗体,能够特异性结合人CD25分子。
附图说明
图1为本发明实施例3所述的抗人CD25工程抗体的纯化电泳图;
图2为本发明实施例4所述的抗人CD25工程抗体的活性检测图;
图3为本发明实施例6所述的活性检测的阴性对照图:其中A图为阴性对照CD45-PerCP,B图为阴性对照淋巴细胞中CD4-PE,C图为阴性对照CD127-APC;
图4为本发明实施例6所述的鼠源抗体的活性检测图:其中A图为鼠源抗体的CD45-PerCP,B图为鼠源抗体的CD4-PE,C图为CD127-APC与鼠源抗体CD25-FITC;
图5为本发明实施例6所述的工程抗体的活性检测图:其中A图为工程抗体的CD45-PerCP,B图为工程抗体的CD4-PE,C图为CD127-APC与工程抗体CD25-FITC。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例来详细说明本发明。
实施例1 小鼠抗人CD25抗体的轻重链可变区基因克隆
1、RNA提取:采用Trizol一步法,(1)取杂交瘤细胞约1×106个,加入1ml Trizol,吹打混匀,室温静置 10 分钟;(2)加入 0.2ml 氯仿,剧烈振荡 15 秒,室温静置 2-3 分钟;(3)12000rpm,4℃,离心 15 分钟(4)取上清,加入 0.5ml 异丙醇室温静置 15 分钟;(5)12000rpm,4℃,离心15 分钟;(6)弃上清,加入 1ml 75%的乙醇洗,7500rpm,4℃,离心5 分钟;(7)弃上清,沉淀晾干,加入 30μLDEPC 水溶解;
2、逆转录为 cDNA(40μl) :使用TransScript All-in-one First-Strand cDNASynthesis SuperMix for qPCR试剂盒进行逆转录反应,取总RNA 1.0 μg,TransScriptAll-in-one SuperMix for qPCR 4μl,gDNA remover 4μl,DEPC水补充至20μl,反应42℃,15min后,85℃孵育5min,于20℃保存;
3、PCR 扩增CD25抗体的轻重链可变区基因
轻链可变区基因PCR扩增反应体系(50μl) :设计通用简并引物:上游引物5’-GACATT GTG CTC ACC CAG WCT SMH-3’(SEQ ID NO:19),下游引物5’-CCG TTAGAT CTC CARBTT KGT SCS-3’ (SEQ ID NO:20);以cDNA 为模板,高保真 pfu DNA 聚合酶扩增;PCR 循环程序为 94℃ 5 分钟 ;94℃ 30 秒,55℃ 30秒,72℃ 30 秒,共 30 个循环 ;最后 72℃延伸 10 分钟;
重链可变区基因 PCR 扩增反应体系 (50μl) :上游引物 5’-CAG GTS MARCTGCAGSAG TCW GG-3’(SEQ ID NO:21);下游引物 5’-TGA GGA GAC KGT GAC HGT GGT SCC-3’(SEQ ID NO:22);以 cDNA 为模板,高保真 pfu DNA 聚合酶扩增;PCR 循环程序为 94℃ 5分钟 ;94℃,30 秒,55℃,30 秒,72℃,30 秒,共 35 个循环 ;最后 72℃延伸 10 分钟;
4、测序载体的构建 :pClone007 Blunt Simple Vector Kit购自擎科生物;将轻重链可变区基因PCR 产物回收,与 pClone007 Blunt Simple Vector载体连接后,按常规方法氯化钙转化,以 100μg/ml 氨苄浓度筛选阳性克隆,送测序,该基因完全符合蛋白数据库中抗体所具有的若干保守的框架氨基酸的特点,该序列为抗体基因序列。分别命名为pClone007-VH及pClone007-VL。
实施例2 工程抗体表达载体pcDNA3.4-H和pcDNA3.4-L的构建
根据轻、重链可变区序列及构建载体pcDNA3.4的序列,设计并合成了用于扩增重链可变区的引物(PDH-F和PDH-R)、用于扩增轻链可变区的引物(PDL-F和PDL-R)、用于扩增重链载体的引物(ZTH-F和ZTH-R)和用于扩增轻链载体的引物(ZTL-F和ZTL-R)。
PDH-F:
5’-TTCCAGGTTCCACTGGTGACGAGGTGAAGCTTCACCAGTCTGG-3’(SEQ ID NO:23)
PDH-R:
5’-GATGGGGGTGTCGTTTTGGCTGAAGAGACGGTGACTGAGGTTC-3’(SEQ ID NO:24)
PDL-F:
5’-TTCCAGGTTCCACTGGTGACGATATCCAGATGACACAGACTACTTCCT-3’(SEQ ID NO:25)
PDL-R:
5’-ACAGTTGGTGCAGCATCAGCCCGTTTTATTTCCAGCTTGGTC-3’(SEQ ID NO:26)
ZTH-F:
5’-GCCAAAACGACACCCCCA-3’(SEQ ID NO:27)
ZTH-R:
5’-GTCACCAGTGGAACCTGGAACC-3’(SEQ ID NO:28)
ZTL-F:
5’-GCTGATGCTGCACCAACTGTAT-3’(SEQ ID NO:29)
ZTL-R:
5’-GTCACCAGTGGAACCTGGAACC-3’(SEQ ID NO:30)
轻链可变区基因PCR扩增反应体系(50μl) :上游引物PDL-F;下游引物PDL-R;以pClone007-VL为模板,高保真 pfu DNA 聚合酶扩增;PCR 循环程序为 94℃ 5 分钟 ;94℃30 秒,58℃ 60秒,72℃ 30 秒,共 30 个循环 ;最后 72℃延伸 10 分钟。
重链可变区基因PCR扩增反应体系(50μl) :上游引物PDH-F;下游引物PDH-R;以pClone007-VH为模板,高保真 pfu DNA 聚合酶扩增;PCR 循环程序为 94℃ 5 分钟 ;94℃30 秒,58℃ 60秒,72℃ 30 秒,共 30 个循环 ;最后 72℃延伸 10 分钟。
轻链线性化载体的扩增:上游引物ZTL-F;下游引物ZTL-R;以pcDNA-CD15-L(合成序列,含鼠IgG1恒定区核苷酸序列)为模板,高保真 pfu DNA 聚合酶扩增;PCR 循环程序为94℃ 5 分钟 ;94℃ 30 秒,58℃ 60秒,72℃ 4 min,共 30 个循环 ;最后 72℃延伸 10分钟;
将PCR产物分别回收,将回收轻链可变区PCR产物和重链可变区PCR产物分别与回收线性化载体产物使用重组酶进行重组,重组后转化DH5α克隆菌株,并挑取单菌落扩大培养,送样测序,测序正确的克隆用于转染CHO细胞。
重链线性化载体的扩增:上游引物ZTH-F;下游引物ZTH-R;以pcDNA-CD15-H(合成序列,含鼠IgG1恒定区核苷酸序列)为模板,高保真 pfu DNA 聚合酶扩增;PCR 循环程序为94℃ 5 分钟 ;94℃ 30 秒,58℃ 60秒,72℃ 4 min,共 30 个循环 ;最后 72℃延伸 10分钟;
将PCR产物分别回收,将回收轻链可变区PCR产物和重链可变区PCR产物分别与回收线性化载体产物使用重组酶进行重组,重组后转化DH5α克隆菌株,并挑取单菌落扩大培养,送样测序,测序正确的克隆用于转染CHO细胞。
实施例3 抗人CD25工程抗体的表达、纯化
1、工程抗体 CD25抗体的表达:将准备好的CHO细胞从培养箱中取出,准备两支15ml 无菌离心管,在其中一支中加入 5ml SPM培养基 和 100μg 无菌质粒 DNA(含轻链表达载体和重链表达载体),轻轻吹打混匀;取另一支离心管,加入 5ml SPM 和 320μl转染试剂,轻轻吹打混匀;将含有转染试剂的离心管中所有液体转移至含质粒的离心管中,轻轻吹打混匀;室温下静置5分钟制备出质粒-载体复合物;从恒温摇床中取出细胞,边摇边加入制备好的质粒-载体复合物,放回 CO2恒温摇床中震荡培养;第1天和第5天补加辅料,第12天收获细胞上清;
2、抗人CD25工程抗体的纯化:将上步收集的细胞上清使用Protein A亲和柱进行纯化,PBS平衡柱子后,上样,使用pH值为3.0的甘氨酸进行洗脱,洗脱纯抗使用G25柱置换溶液,将溶液置换为PBS,然后分装后于-20℃冻存。结果如图1所示(从左侧至右侧,第三个泳道是非还原电泳,第6个泳道是maker,第9个泳道是还原电泳)。
实施例 4 抗人CD25工程抗体的活性测定
PHA细胞系检测:每管加入PHA细胞悬液 100μl,分别加入不同量的工程抗体CD25和腹水来源的杂交瘤抗体CD25,室温避光孵育 30 分钟; 1000 转/分钟离心 5 分钟,弃上清;加入 2ml 冷 PBS 缓冲液,重悬,1000 转/分钟离心 5 分钟,弃上清;再加入 0.5μgAPC 标记的鼠二抗,室温避光孵育 30 分钟;加入 2ml 冷 PBS 缓冲液,重悬,1000 转/分钟离心 5 分钟,弃上清;加入 250μl PBS 缓冲液,流式细胞仪检测。结果如图2所示。
实施例 5 抗人CD25工程抗体的FITC标记
制备溶于pH值为 9.5的碳酸盐缓冲液的待标记抗体溶液,浓度≥2mg/ml;用无水DMSO溶解FITC配制成10mg/ml的溶液;按1mg抗体加入60μg FITC溶液的比例加入所需FITC,混匀,25℃生化培养箱内避光反应90min;反应结束,通过G25层析柱对标记抗体进行纯化,去除游离FITC,收集标记好的CD25-FITC溶液,用于流式检测。
实施例 6 抗人CD25-FITC工程抗体的活性测定
正常人外周血检测:每管加入正常人抗凝外周血 100μl,分别加入不同量的鼠源抗体CD25-FITC(0.5μg、0.025、0.125μg)和不同量的工程抗体CD25-FITC(0.5μg、0.025、0.125μg),再加入抗体CD45-PerCP,CD4-PE,CD127-APC,室温避光孵育 30 分钟;加入 2ml溶血素室温避光反应 10 分钟后 1000 转/分钟离心 5 分钟,弃上清;加入 2ml 冷 PBS缓冲液,1000 转/分钟离心 5 分钟,弃上清;加入 300μl PBS 缓冲液,流式细胞仪检测。
如图3-图5所示,CD25-FITC在Treg细胞上反应正常,特异性好,标记后的CD25抗体活性没有受到影响。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (15)
1.一种抗人CD25工程抗体,其特征在于:该工程抗体包括重链可变区与轻链可变区;
所述的重链可变区的互补决定区包括CDR1、CDR2与CDR3;所述的重链可变区的CDR1的氨基酸序列如SEQ ID NO.1所示;所述的重链可变区的CDR2的氨基酸序列如SEQ ID NO.2所示;所述的重链可变区的CDR3的氨基酸序列如SEQ ID NO.3所示;
所述的轻链可变区的互补决定区包括CDR1、CDR2与CDR3;所述的轻链可变区的CDR1的氨基酸序列如SEQ ID NO.4所示;所述的轻链可变区的CDR2的氨基酸序列如SEQ ID NO.5所示;所述的轻链可变区的CDR3的氨基酸序列如SEQ ID NO.6所示。
2.根据权利要求1所述的抗人CD25工程抗体,其特征在于:所述的重链可变区的骨架区包括FR1、FR2、FR3和FR4;所述的重链可变区的FR1的氨基酸序列如SEQ ID NO.7所示;所述的重链可变区的FR2的氨基酸序列如SEQ ID NO.8所示;所述的重链可变区的FR3的氨基酸序列如SEQ ID NO.9所示;所述的重链可变区的FR4的氨基酸序列如SEQ ID NO.10所示;
所述的轻链可变区的骨架区包括FR1、FR2、FR3和FR4;所述的轻链可变区的FR1的氨基酸序列如SEQ ID NO.11所示;所述的轻链可变区的FR2的氨基酸序列如SEQ ID NO.12所示;所述的轻链可变区的FR3的氨基酸序列如SEQ ID NO.13所示;所述的轻链可变区的FR4的氨基酸序列如SEQ ID NO.14所示。
3.根据权利要求1所述的抗人CD25工程抗体,其特征在于:所述的工程抗体的重链可变区的氨基酸序列如SEQ ID NO.15所示,轻链可变区的氨基酸序列如SEQ ID NO.16所示。
4.一种核酸分子,其特征在于:该核酸分子编码权利要求1-3中任一项所述的抗人CD25工程抗体。
5.根据权利要求4所述的核酸分子,其特征在于:所述的核酸分子编码所述的抗人CD25工程抗体的重链可变区的核苷酸序列如SEQ ID NO:17所示,编码轻链可变区的核苷酸序列如SEQ ID NO:18所示。
6.一种表达载体,其特征在于:该表达载体包含权利要求4或5所述的核酸分子。
7.权利要求1-3中任一项所述的抗人CD25工程抗体的应用,其特征在于:所述的抗人CD25工程抗体在制备检测调节性T细胞的试剂中的应用。
8.权利要求1-3中任一项所述的抗人CD25工程抗体的应用,其特征在于:所述的抗人CD25工程抗体在制备诊断多发性硬化症、类风湿关节炎或I型糖尿病的试剂中的应用。
9.权利要求1-3中任一项所述的抗人CD25工程抗体的应用,其特征在于:所述的抗人CD25工程抗体在制备检测骨髓移植指标的试剂中的应用。
10.权利要求1-3中任一项所述的抗人CD25工程抗体的应用,其特征在于:所述的抗人CD25工程抗体在制备治疗肿瘤的药物中的应用。
11.权利要求6所述的表达载体的应用,其特征在于:所述的表达载体在制备检测调节性T细胞的试剂中的应用。
12.权利要求6所述的表达载体的应用,其特征在于:所述的表达载体在制备诊断多发性硬化症、类风湿关节炎或I型糖尿病的试剂中的应用。
13.权利要求6所述的表达载体的应用,其特征在于:所述的表达载体在制备治疗肿瘤的药物中的应用。
14.权利要求6所述的表达载体的应用,其特征在于:所述的表达载体在制备检测骨髓移植指标的试剂中的应用。
15.一种免疫检测试剂盒,其特征在于:该试剂盒含有权利要求1-3中任一项所述的抗人CD25工程抗体和/或权利要求6所述的表达载体。
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