CN103044552A - 人源化的抗树突状细胞表面dec-205分子的单克隆抗体 - Google Patents
人源化的抗树突状细胞表面dec-205分子的单克隆抗体 Download PDFInfo
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Abstract
本发明公开了一种人源化的抗树突状细胞表面DEC-205分子的单克隆抗体，重链可变区的互补决定区CDR具有如下序列：CDR1:序列表中SEQIDNO3；CDR2:序列表中SEQIDNO4；以及CDR3:序列表中SEQIDNO5；轻链可变区的互补决定区CDR具有如下序列：CDR1:序列表中SEQIDNO6；CDR2:序列表中SEQIDNO7；以及CDR3:序列表中SEQIDNO8。本发明的有益效果：以该抗体作为载体能够特异性地并且高效地将抗原物质运输至抗原递呈细胞表面，并引起抗原递呈细胞对该抗原的内吞、加工、递呈，从而增强抗原递呈细胞的递呈效率，加强机体对该抗原的免疫反应。该抗体作为载体在免疫反应底下为特点的各种疾病中具有潜在的应用前景。
Description
技术领域
本发明属于细胞免疫学、分子生物学领域，具体涉及一种人源化的抗树突状细胞表面DEC-205分子的单克隆抗体。
背景技术
治疗性抗体药物已成为生物制药领域的热点。细胞融合筛选单克隆抗体技术自发明以来受到广泛的应用，目前已成为制备抗体的一种最基本也是最高效的方法。然而细胞融合的方法大多仅限于动物，如大鼠、兔、羊等。通过免疫动物获得较丰富的抗原特异性的浆细胞，然后进行脾细胞融合，筛选出分泌抗体的单克隆杂交瘤细胞。这种方法所产生的抗体为非人源抗体，如果作为药物应用于人体会产生较强的排斥反应，因此必须进行人源化改造。
抗体人源化改造是指将抗体中非人源的氨基酸替换成人源的氨基酸。为了不改变抗体的结合特异性，通常保留抗体的互补决定区CDR。因此，常见的方法是CDR-graft：即将抗体的CDR区移植到人源抗体的骨架区之中，这样既保留抗体的结合特异性，又最低程度的减少非人源氨基酸，避免机体的针对抗体本身的免疫反应。抗体经过CDR-graft之后亲和力通常会下降，通过对抗体构象关键的氨基酸改造可以恢复抗体的亲和力。人源化成功的抗体应该具备与鼠源亲本抗体相当的亲和力。
DEC-205分子是表达于树突状细胞表面的一种模式识别分子，它能够识别特定种类的抗原物质。机体免疫系统要消灭外源物质的入侵，必须依靠抗原呈递细胞识别外源物质，并将抗原加工呈递给免疫反应细胞，诱导针对抗原的特异的免疫反应。DEC-205分子能够通过受体介导的内吞将抗原分子吞噬到细胞内进行加工处理，然后和MHCI类分子及MHCII类分子结合呈递至细胞表面。将抗原分子与DEC-205抗体偶联，可以通过DEC-205抗体的靶向识别使得抗原分子能够主动的识别抗原递呈细胞，从而增加抗原递呈细胞的递呈效率，加强机体的免疫反应强度。
在一些慢性感染性疾病中，如HBV，HIV等，机体的免疫功能低下，细胞免疫缺陷。通过将病毒特异性抗原与DEC-205抗体偶联，能够诱导出针对病毒的特异的体液及细胞免疫反应，加强机体对病毒的清除。DEC-205抗体作为载体在慢性感染性疾病的预防与治疗中发挥作用。目前针对癌症的免疫治疗中，以提高癌症细胞特异性细胞免疫反应为主要策略。DEC-205抗体在其中也具有潜在的应用价值。
发明内容
本发明目的是提供一种人源化的抗树突状细胞表面DEC-205分子的单克隆抗体，该抗体作为病原抗原的载体，能和抗原偶联，促进抗原被机体免疫系统的识别，增强机体的免疫应答反应。
为达到上述目的，通过以下技术方案来实现：
一种人源化的抗树突状细胞表面DEC-205分子的单克隆抗体，该抗体的重链可变区的互补决定区CDR具有如下序列：CDR1:序列表中SEQ ID NO 3；CDR2:序列表中SEQ ID NO 4；以及CDR3:序列表中SEQ ID NO 5；该抗体的轻链可变区的互补决定区CDR具有如下序列：CDR1:序列表中SEQ ID NO 6；CDR2:序列表中SEQ ID NO 7；以及CDR3:序列表中SEQ ID NO 8。
该抗体的重链具有如序列表中SEQ ID NO 1所示的氨基酸序列，该抗体的轻链具有如序列表中SEQ ID NO 2所示的氨基酸序列。
本发明所述的人源化的抗树突状细胞表面DEC-205分子的单克隆抗体的制备方法，包括以下步骤：
用RT-PCR的方法从人外周血扩增出DEC-205基因，克隆到pCDNA3.1表达载体上，在YB2/0细胞中表达；
利用细胞免疫大鼠，然后分离脾细胞，与杂交瘤细胞进行融合，通过流式检测的方法筛选出分泌抗体的杂交瘤细胞克隆；
用5’RACE的方法从杂交瘤细胞中扩增出抗体基因，经过生物信息学分析，找出抗体的互补决定区CDR序列，经过人源化改造，产生人源化的DEC-205分子的单克隆抗体。
本发明还包括对上述抗体的氨基酸序列通过对氨基酸残基的添加、删除、修改形成的包含有本发明所述抗体的CDR区序列并具有相同功能或改造及优化的一切抗体，包括人源与非人源抗体、改造的单链（scFv）抗体；以及含有本发明所述的单个重链CDR区的其他抗体片段或含有本发明所述的单个轻链CDR区的其他抗体片段。
本发明的人源化DEC-205抗体能够识别人外周血来源的树突状细胞，并引起树突状细胞对该抗体的内吞。
本发明的人源化DEC-205抗体能够作为抗原的载体，用于病毒感染性疾病和癌症的预防性疫苗及免疫治疗性抗体药物。
本发明的有益效果：本发明所述的人源化DEC-205抗体能够主动识别表达于抗原递呈细胞表面的模式识别受体分子DEC-205，并引起抗原递呈细胞的内吞效应。以该抗体作为载体能够特异性地并且高效地将抗原物质运输至抗原递呈细胞表面，并引起抗原递呈细胞对该抗原的内吞、加工、递呈，从而增强抗原递呈细胞的递呈效率，加强机体对该抗原的免疫反应。该抗体作为载体在免疫反应底下为特点的各种疾病中具有潜在的应用前景。
附图说明
下面根据附图对本发明作进一步详细说明。
图1是流式细胞仪检测DEC1细胞系表达DEC-205分子，染色抗体MG38（PE，BD公司）。；
图2为大鼠血清中抗体检测，采集免疫后大鼠血清，与DEC1细胞孵育，加FITC标记抗大鼠二抗检测；
图3为单克隆抗体172检测，取172克隆号的细胞培养上清与DEC1细胞孵育，加FITC标记抗大鼠二抗检测；
图4为抗体表达；
图5为抗体与人DC细胞结合；
图6为DEC-205被人DC细胞吞噬，DEC-205抗体用FITC标记，人DC细胞先标记上APC-CD11c抗体，在加入DEC-205抗体，分别在37℃和4℃孵育，37℃条件下，绿色的抗体被DC细胞吞噬到胞内。
具体实施方式
本发明的实施例所述的人源化的抗树突状细胞表面DEC-205分子的单克隆抗体的制备过程包括以下步骤：
1．免疫抗原的制备
设计引物将DEC-205分子的N端SP、Fn、CR区及C端的跨膜区、胞内区分别扩增出来，用overlap PCR将两个片段连接起来，插入到表达载体pCDNA3.1中。插入位点：XbalI、EcoRI。扩增引物分别为：
DEC11：TGCTCTAGAGCCACCATGAGGACAGGCTGG，
DEC12：TATCCTCCCTCGGAGGGAGGCTTTAAGCAGATGCC，
DEC21：CTCCCTCCGAGGGAGGATACACAGCAATAGCTATC，
DEC22：CTCCCTCCGAGGGAGGATACACAGCAATAGCTATC。
将表达载体转染YB2/0细胞系，G418筛选出稳定表达的细胞系DEC1，用流式细胞仪检测细胞表达结果。如图1所示。
2．收集DEC1细胞免疫大鼠
收集生长状态良好的DEC1细胞，用生理盐水洗3遍，与免疫佐剂混合，充分乳化，用于免疫6-8周的lewis品系大鼠。大鼠免疫采取腹腔注射。免疫两次，之间间隔三周，每次免疫用10^8细胞，用1ml生理盐水混合加1ml完全免疫佐剂CFA乳化。在取大鼠做实验前一周加强免疫一次，用5×10^7细胞与不完全免疫佐剂IFA乳化。实验之前，采血检测血清中抗体含量，如图2所示。
3．细胞融合及筛选单克隆
将大鼠处死并分离脾脏细胞。采用标准的细胞融合方法，将脾脏细胞与小鼠的骨髓瘤细胞SP2/0进行细胞融合。用HAT选择培养基悬浮起细胞，接种到96孔细胞培养板之中培养。培养8-10天，显微镜下观察到有单个细胞长出的细胞团时，去细胞培养上清用流式检测分泌抗体的克隆。流式检测用细胞培养上清孵育DEC1细胞，加FITC标记的抗大鼠二抗检测。筛选出分泌抗体的细胞克隆做进一步的扩大培养。筛选出克隆号172的大鼠抗人DEC-205单克隆抗体，如图3所示。
4. 5’RACE方法扩增抗体基因
收集10^7细胞提取RNA，用oligo-T引物反转录成cDNA，将cDNA纯化后100℃加热1分钟，然后用末端核苷酸转移酶（TdT transferase，promega），加10uM dGTPs，37℃反应1小时，然后加热至70℃，10分钟灭活末端核苷酸转移酶。以此为模板扩增抗体基因。扩增抗体重链基因引物为Anchor和H，扩增抗体轻链基因引物为Anchor和L。
Anchor：CGTCGATGAGCTCTAGAATTCGCATGTGCAAGTCCGATGGTCCCCCCCCCCCCCC
L：AGGATGATGTCTTATGAACAA
H：TCACATTGAGCTTGCTGTA
PCR扩增产物经过电泳检测，将电泳胶中的条带进行胶回收，连接到T载体中进行测序。
5．生物信息学分析抗体序列及人源化
扩增出的抗体基因测序，经过blast比对分析找出抗体的重链和轻链完整编码区。然后经过IMGT网站分析，确定抗体重链和轻链的可变区V和恒定区C，以及互补决定区CDR1，CDR2，CDR3。通过比对分析找出与该大鼠可变区V同源性最高的人源抗体序列（NCBI No.）。以此抗体为骨架，将鼠源抗体的CDR区移植构成一个完整的抗体序列。经过生物信息学分析，做如下定点突变以恢复抗体的亲和力。
6. 抗体表达
将人源化的抗体重链和轻链基因的可变区通过PCR引物扩增出来，末端加上酶切位点，分别插入到抗体表达载体MH和MK中。将构建好的表达载体进行质粒抽提，转染HEK293T细胞。重链和轻链表达载体以摩尔比1:1混合，加入转染试剂lipofectamine2000混匀，加入到培养的HEK293T细胞中。每两天收集细胞上清，换上新鲜培养液。收集的细胞培养上清经过超滤离心浓缩，proteinG柱纯化出抗体。用蛋白胶检测抗体的表达，用流式染色的方法检测表达的抗体的亲和性。如图4所示。
7. 抗体功能验证
用流式染色的方法来检测抗DEC-205抗体对人树突状细胞DC的亲和性。
用Ficoll密度梯度离心从人的外周血分离出PBMC，磁珠分选出CD14阳性单核细胞，加细胞因子诱导成DC细胞。用DEC-205抗体与DC细胞孵育，加入FITC标记二抗，通过流式细胞仪检测抗体对人DC细胞的亲和性。结果见图5所示。
用SPR技术测定抗体的亲和力
将抗人二抗偶联到CM5芯片上，RU值达到。将抗体以六个浓度梯度1000nM、500nM、250nM、125nM、62.5nM流过芯片表面，记录信号。经过软件分析得出抗体的动力学参数。如表1：
表1 DEC-205抗体的亲和常数
| Ka (M-1·S-1) | Kd (S-1) | KD (M) | |
| Ab抗体 | 8.22±0.48E+03 | 1.37±0.06E-04 | 1.67±0.07E-08 |
用ImageStream测定抗体引起的内吞效应
人DC细胞先与APC标记CD11c抗体孵育，在加入FITC标记的DEC-205抗体，分别在37℃和4℃孵育30分钟，上ImageStream机器检测。经过 软件分析得出DC细胞对DEC-205抗体在37℃和4℃条件下的内吞效果。如图6所示。
虽然以上仅描述了本发明的具体实施方式范例，但是本领域的技术人员应当理解，这些仅是举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均落入本发明的保护范围。
序列表
<110>中国医学科学院病原生物学研究所
<120>人源化的抗树突状细胞表面DEC-205分子的单克隆抗体
<130>PI122482
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Claims (4)
1.一种人源化的抗树突状细胞表面DEC-205分子的单克隆抗体，其特征在于，该抗体的重链可变区的互补决定区CDR具有如下序列：CDR1:序列表中SEQ ID NO 3，CDR2:序列表中SEQ ID NO 4，以及CDR3:序列表中SEQ ID NO 5；该抗体的轻链可变区的互补决定区CDR具有如下序列：CDR1:序列表中SEQ ID NO 6，CDR2:序列表中SEQ ID NO 7，以及CDR3:序列表中SEQ ID NO 8。
2.根据权利要求1所述的人源化的抗树突状细胞表面DEC-205分子的单克隆抗体，其特征在于：该抗体的重链具有如序列表中SEQ ID NO 1所示的氨基酸序列，该抗体的轻链具有如序列表中SEQ ID NO 2所示的氨基酸序列。
3.权利要求1或2所述的人源化的抗树突状细胞表面DEC-205分子的单克隆抗体的制备方法，其特征在于，包括以下步骤：
用RT-PCR的方法从人外周血扩增出DEC-205基因，克隆到pCDNA3.1表达载体上，在YB2/0细胞中表达；
利用细胞免疫大鼠，然后分离脾细胞，与杂交瘤细胞进行融合，通过流式检测的方法筛选出分泌抗体的杂交瘤细胞克隆；以及
用5’RACE的方法从杂交瘤细胞中扩增出抗体基因，经过生物信息学分析，找出抗体的互补决定区CDR序列，经过人源化改造，产生人源化的DEC-205分子的单克隆抗体。
4.权利要求1或2所述的人源化的抗树突状细胞表面DEC-205分子的单克隆抗体在制备用于病毒感染性疾病和癌症的预防性疫苗及免疫治疗性抗体药物中的用途。
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