CN105968188A - 一种关于cd30免疫原多肽及其用途 - Google Patents
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Abstract
本发明公开了一种关于CD30免疫原多肽，属于抗癌疫苗领域。具体涉及用作抗癌疫苗的CD30免疫原的优化多肽，增强T细胞免疫应答。该氨基酸序列为全新的序列，在制备用于治疗肿瘤药物中的应用。尤其在制备用于肿瘤免疫细胞，增强T细胞免疫应答药物中的应用，以及在制备用于CAR‑T细胞免疫治疗的应用。本发明的有益效果在于为全新的序列，可用于肿瘤免疫细胞技术中的免疫原，能（1）促进T细胞增殖，（2）抑制小鼠外周白细胞表面CD30表达，（3）能与T细胞表面的MHC有效的特异性结合，作为细胞疗法的靶标分子，应用于CAR‑T等细胞疗法。
Description
技术领域
本发明是有关抗癌疫苗领域。具体来说，本发明涉及用作抗癌疫苗的CD30免疫原的优化多肽，增强T细胞免疫应答。
背景技术
CD30(TNFRSF8)是最初发现于何杰金氏病(Hodgkin’s disease)中的里丝氏细胞(Reed Sternberg cell)上的跨膜糖蛋白。近年发现活化T细胞高表达该分子。尤其是，CD30可表达于多种恶性淋巴系统疾病，如霍奇金淋巴瘤(HL)、间变性大细胞性淋巴瘤(ALCL)、外周T细胞淋巴瘤(PTCL)、成人T细胞性淋巴瘤(ATL)等，其中以T细胞淋巴瘤占大多数，如ALCL肿瘤细胞中CD30表达可达100％。
相对于近年来B细胞淋巴瘤治疗领域中的异彩纷呈，如多种化疗方案，干细胞移植，抗CD20单抗、硼替佐米、来那度胺、Ibrutinib等新型药物不断优化着治疗格局，外周T细胞淋巴瘤的治疗则进展不大。主要方案以化疗为主要治疗手段，辅以放疗。目前，靶向治疗复发/难治性CD30+T细胞淋巴瘤成为热门话题，已有诸多单药治疗研究见诸报道。有阿仑单抗、硼替佐米、brentuximabvedotin(sALCL)、环抱素(AITL)、吉西他滨、普拉曲沙、罗米地辛。食品药品监督管理局(FDA)批准的药物有普拉曲沙、罗米地辛、brentuximabvedotin(对ALCL)，有效率分别为29％、25％、86％。
肿瘤细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法，来激发，增强机体自身免疫功能，从而达到治疗肿瘤的目的。肿瘤细胞免疫疗法是继手术、放疗和化疗之后的第四大肿瘤治疗技术。
CD30可以作为肿瘤疫苗的特异性免疫原。然而，CD30由595个氨基酸残基组成，分子量较大，较难得到纯度高的CD30。这样，不仅会给后期的特异性T细胞免疫带来困难，而且会导致患者的自身免疫。因此，本发明提供了一种特异性强，纯度较高的小分子多肽作为免疫原。
发明内容
发明目的
本发明提供一种CD30免疫原多肽，可用于肿瘤细胞免疫的免疫原，增强T细胞免疫应答，具有分子量小、特异性免疫原性强的特点。
技术方案
本发明的技术方案在于提供一种CD30免疫原多肽，序列为APPVSPATMPVRDEYPPKQCEPDYYLDEAGRCTACVA(SEQ ID NO：1)。该氨基酸序列为全新的序列，在制备用于治疗肿瘤药物中的应用。尤其在制备用于肿瘤免疫细胞，增强T细胞免疫应答药物中的应用，以及在制备用于CAR-T细胞免疫治疗的应用。
有益效果
本发明的CD30免疫原多肽，为全新的序列，可用于肿瘤免疫细胞技术中的免疫原。有益效果在于(1)促进T细胞增殖，(2)抑制小鼠外周白细胞表面CD30表达，(3)能与T细胞表面的MHC有效的特异性结合，(4)能提高胸腺淋巴瘤小鼠生存率。作为细胞疗法的靶标分子，应用于CAR-T等细胞疗法。
具体实施方式
多肽由上海生工吉尔合成。
实施例1
用小鼠胸腺淋巴瘤模型检测CD30免疫原多肽的体内活力。
6-8w龄C57BL/6小鼠，小鼠随机分成4组，雌雄各半，每组10只。(1)空白组；(2)多肽低剂量组；(3)多肽中剂量组；(4)多肽高剂量组。腹腔注射DNA烷化剂N-甲基-N-亚硝基脲(MNU)诱导小鼠胸腺淋巴瘤，建立相应动物模型，在造模后的第1、7、14、21天，分别进行免疫。方案为：空白组加入相同体积的溶剂，实验组多肽设3个剂量：5、10、20mg/Kg，在肿瘤周围多点注射。28天后，观察小鼠存活数量，计算存活率。结果显示，多肽可有效提高荷瘤小鼠的生存率，其中，剂量为20mg/Kg组的小鼠生存率达到67.90％。
实施例2
应用竞争性受体-配体亲和法测试多肽与MHC的结合能力：
将多肽与HLA各型的结合能力由竞争性受体-配体亲和法判断。将固定浓度为50μmol/L的标记125I的多肽以及不同浓度(1-50μmol/L)的多肽加入反应体系(磷酸盐缓冲液和MHC，所述的MHC试剂盒购自上海泛柯生物科技有限公司，试剂盒内有HLA-A1、A2、A3、A11和A24)，在反应体系中形成两种复合物，即待测多肽MHC复合物和125I标记多肽复合物。待测多肽与125I标记多肽竞争与MHC的结合。用超过滤(产品型号Microcon 30，Amicon公司)分离游离多肽和多肽MHC复合物，然后测定多肽MHC复合物的125I放射量，将此放射量与没有竞争性抑制的多肽MHC复合物(没有加待测多肽的样本)的放射量比较，求待测多肽在抑制50％标记多肽与MHC结合时的浓度，即IC50。由此得到，多肽对HLA-A1、A2、A3、A11和A24的IC50值分别为5.21、4.83、6.51、4.97和7.25μmol/L，均符合阳性多肽的标准(≤10μM)。因此，多肽为具有有效结合能力的免疫原多肽。
实施例3
T淋巴细胞的增殖作用：无菌取小鼠脾脏，1640培养基清洗3次，5ml注射器芯研磨，200目筛网过滤，制成单细胞悬液，离心(1000r/min，5min)，弃上清，Tris-NH4CL破解红细胞，冰水浴静置3-5min，离心(1000r/min,5min)，弃上清，用无菌冷PBS洗涤细胞两次。最后加入10％小牛血清的RPMI 1640培养液(5ml)悬浮细胞，细胞计数，调整细胞浓度为5×106个/ml，于96孔培养板中培养。
实验设空白对照组、模型组(刀豆蛋白A，sigma公司购买)、多肽各剂量(5、10、20mg/ml)组。各组分别加入脾脏淋巴细胞悬液100μl/孔后，空白对照组加入1640培养液100μl，模型组加入ConA(终浓度为5μg/ml)，多肽各剂量组在加入不同浓度的多肽基础上加ConA(终浓度为5μg/ml)。37℃细胞培养箱静止培养48h，培养结束后每孔加入20μl MTT，继续培养4h，最后弃掉每孔所有溶液，每孔加入100μlDMSO，震荡，用酶标仪检测570nm处OD值，每孔设5个平行。
表1多肽对T淋巴细胞的增殖作用
*P<0.05，**P<0.01与模型组相比。
实验结果见表1，与模型组比较，多肽能促进小鼠脾脏淋巴细胞的增殖，在剂量为5～20mg/ml的范围呈现很好的剂量依赖关系。
实施例4
采用流式细胞技术测定免疫原多肽对免疫动物体内T细胞表面CD30表达的影响。6-8w龄C57BL/6小鼠，小鼠随机分成4组，每组10只。(1)空白组；(2)免疫原多肽低剂量组；(3)免疫原多肽中剂量组；(4)多肽高剂量组。在试验的第0、7、14天，分别进行免疫。方案为：空白组加入相同体积的溶剂，实验组多肽设3个剂量：5、10、20mg/Kg，在小鼠背部淋巴结附近多点注射。30天后，眼球取血0.8ml，肝素抗凝，将血离心后取血细胞层，用红细胞裂解液破解红细胞，洗去裂解的红细胞，再用荧光标记的CD30+-FITC(购自北京华泰昕生物医疗技术有限公司)孵育、固定后，进行流式细胞技术测定，评价免疫原多肽对CD30+T细胞的影响。
表2多肽对CD30抗体+T淋巴细胞的作用
*P<0.05，**P<0.01与空白组相比。
实验结果见表2，与空白组比较，多肽能抑制小鼠外周白细胞表面CD30表达，在剂量为5～20mg/Kg的范围呈现很好的剂量依赖关系。
SEQUENCE LISTING
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苏州普罗达生物科技有限公司
<120>
一种关于CD30免疫原多肽及其用途
<130>
<160>
1
<170>
PatentIn version 3.3
<210>
1
<211>
37
<212>
PRT
<213>
人工序列
<400>
1
Ala Pro Pro Val
Ser Pro Ala Thr Met Pro Val Arg Asp Glu Tyr Pro
1
5
10
15
Pro Lys Gln
Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Gly Arg Cys
20
25
30
Thr Ala Cys
Val Ala
35
Claims (4)
1.一种CD30免疫原多肽，其特征在于：所述的多肽序列为SEQ ID NO：1。
2.根据权利要求1所述的多肽，其特征在于：在制备用于治疗肿瘤药物中的应用。
3.根据权利要求2所述的多肽，其特征在于：在制备用于肿瘤免疫细胞，增强T细胞免疫应答药物中的应用。
4.根据权利要求2所述的多肽，其特征在于：在制备用于CAR-T细胞免疫治疗的应用。
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Citations (2)
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|---|---|---|---|---|
| CN103336131A (zh) * | 2013-06-25 | 2013-10-02 | 中国人民解放军海军医学研究所 | 可溶性cd30作为快速估算电离辐射剂量生物指标的应用 |
| CN104592392A (zh) * | 2015-01-21 | 2015-05-06 | 武汉友芝友生物制药有限公司 | 一种双特异性抗体EpCAM×CD3的构建及应用 |
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| CN103336131A (zh) * | 2013-06-25 | 2013-10-02 | 中国人民解放军海军医学研究所 | 可溶性cd30作为快速估算电离辐射剂量生物指标的应用 |
| CN104592392A (zh) * | 2015-01-21 | 2015-05-06 | 武汉友芝友生物制药有限公司 | 一种双特异性抗体EpCAM×CD3的构建及应用 |
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