CN114369581B - 一种具有抗肿瘤免疫功能重组腺病毒、制备方法及应用 - Google Patents
一种具有抗肿瘤免疫功能重组腺病毒、制备方法及应用 Download PDFInfo
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Abstract
本发明提供了一种具有抗肿瘤免疫功能重组腺病毒、制备方法及应用，本发明将一种表达融合蛋白CD137L‑PP4基因插入腺病毒载体，构建获得具有抗肿瘤免疫功能重组腺病毒，所述融合蛋白CD137L‑PP4包含CD137L蛋白和具有T细胞激活功能的PP4多肽。该重组腺病毒能在体内表达，可以诱导产生特异性的抗肿瘤细胞免疫，实现肿瘤的免疫治疗。因而可以用于制备用于诱导机体产生针对CD137L的特异性细胞免疫反应的药物和/或诱导机体产生抑制肿瘤细胞增殖的药物。
Description
技术领域
本发明属于医学生物制品领域，特别适用于一种具有抗肿瘤免疫功能重组腺病毒的制备方法及应用。
背景技术
CD137L(CD137配体)，又名4-1BBL，属于肿瘤坏死因子超家族成员，是一种Ⅱ型跨膜糖蛋白。4-1BBL，以同源三聚体存在，以延伸的三叶样螺旋结构为特征，发挥其生物学功能。它作为CD137的高亲和力配体,表达于活化的抗原递呈细胞(APC)表面,包括巨噬细胞、B细胞和树突状细胞及多种肿瘤细胞的表面等。作为一对重要的共刺激分子，CD137L与CD137通过在免疫细胞间传递活化、增殖或者凋亡信号来调节T细胞介导的免疫反应。
CD137L与其受体结合，可启动双向信号转导：正向信号可刺激T细胞活化和增殖；反向信号可刺激B细胞增殖。CD137L及其受体在正常组织及炎症中表达不显著，而在肿瘤组织中呈现高水平表达，这种表达异常与肿瘤发生、恶化存在一定的相关性。肿瘤细胞介导的CD137L/CD137异常表达已在很多肿瘤中被发现，包括直肠癌、黑色素瘤、卵巢癌等恶性肿瘤。因此，以CD137L与其受体结合作为靶点开展相关研究已成为当前抗肿瘤免疫研究领域的一大热点。因而，以CD137L/CD137为靶抗原开展药物研究，通过诱导机体产生特异性细胞毒性T淋巴细胞(CTL)来实现抗肿瘤免疫治疗有着很强的临床应用潜力。目前，已有针对CD137L/CD137的抗肿瘤治疗主要以抗体治疗为主，如辉瑞公司研发的全人源化CD137单抗Utomilumab(PF-05082566)，被批准用于晚期实体瘤和恶性B细胞白血病的Ⅰ期临床(NCT01307267)试验，该药物临床耐受性良好，且展现出一定的抗肿瘤治疗效果。但是，由于肿瘤细胞的免疫逃逸，使得单抗类药物的治疗存在较大几率的耐药性。因此，通过激活机体自身的细胞免疫来实现抗肿瘤治疗成为了一种研发思路。重组腺病毒载体作为一种新型的治疗手段，在肿瘤治疗领域已有一些应用，借助重组腺病毒载体诱导机体产生针对CD137L/CD137的特异性细胞免疫是肿瘤生物治疗的一种新思路。
发明内容
本发明的目的是提供一种具有抗肿瘤免疫功能的重组腺病毒，将CD137L-PP4的核苷酸序列通过同源重组插入腺病毒载体构建得到一个能够表达CD137L-PP4蛋白的重组腺病毒(rAd-CD137L-PP4)，该重组腺病毒诱导机体产生针对CD137L的特异性细胞免疫反应，并且具有诱导机体产生针对CD137L的特异性细胞免疫和抑制肿瘤细胞增殖的功能。
本发明采用的技术方案如下：
一种具有抗肿瘤免疫功能重组腺病毒，是表达融合蛋白CD137L-PP4的重组腺病毒，所述融合蛋白CD137L-PP4是将人CD137L序列的蛋白与一个通过筛选获得的具有T细胞激活功能的多肽PP4通过连接序列进行连接获得。
进一步地，所述CD137L-PP4的氨基酸序列如SEQ ID NO.1所示，其中第1-246位为CD137L蛋白，第247-250位为两个蛋白之间的连接序列，第251-265位为PP4多肽，所述多肽PP4的氨基酸序列如SEQ ID NO.3所示，该序列来自白喉毒素无毒突变体中的第113-127位序列，该序列具有促进淋巴细胞增殖的作用。
进一步地，所述编码CD137L-PP4蛋白的核苷酸序列如SEQ ID NO.2所示。
进一步地，所述腺病毒载体是5型腺病毒载体、26型腺病毒载体或28型腺病毒载体。
本发明所述rAd-CD137L-PP4重组腺病毒载体的构建方法，具体为：
将人工合成编码融合蛋白CD137L-PP4的核苷酸序列(SEQ ID NO.2)插入通过分子克隆技术插入腺病毒载体。所得重组腺病毒再经过细胞培养、增殖和层析柱纯化得到所需的重组腺病毒样品。
进一步地，具体包括以下步骤：
1)人工合成SEQ ID NO.2序列，通过同源重组的方法插入腺病毒载体，构建得到重组腺病毒rAd-CD137L-PP4。
2)用HEK293细胞培养步骤1)所述重组腺病毒获得病毒培养液，经三次反复冻融后收集上清，用超滤方法对所得上清进行浓缩，获得病毒浓缩液。
3)将步骤2)所述病毒浓缩液，先用阴离子交换层析进行纯化，再用分子筛层析进行纯化得到病毒纯化液。
进一步地，步骤2)所述超滤方法，可以是切向流超滤方法。
进一步地，步骤3)所述阴离子交换层析所用的填料可以是SOURCE30Q或者DEAE。
进一步地，步骤3)所述分子筛层析所用的填料可以是Superdex200pg。
本发明的有益效果是：本发明的一种表达CD137L-PP4蛋白基因插入腺病毒载体，使得该重组腺病毒能在体内表达，诱导产生特异性的抗肿瘤细胞免疫，实现肿瘤的免疫治疗。
附图说明
图1为白喉毒素无毒突变体蛋白的T细胞活性多肽对T细胞增殖作用的结果；
图2为pDC316-CD137L-PP4重组穿梭质粒图；
图3为免疫荧光检测表达结果图(A:空白细胞，B:表达CD137L-PP4重组腺病毒感染后细胞，C：空白细胞免疫荧光照片，D:表达CD137L-PP4重组腺病毒感染后细胞免疫荧光照片)
图4为小鼠特异性细胞免疫检测结果图(A：表达CD137L-PP4重组腺病毒组，B：表达CD137L重组腺病毒组、C：空腺病毒组，D：空白组)；
图5为TC-1小鼠肿瘤模型肿瘤细胞增殖抑制实验结果图(A：表达CD137L-PP4重组腺病毒组，B：空腺病毒组、C：空白组)
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，并给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
所述HEK293细胞、DEMM培养基、pDC316质粒、腺病毒骨架质粒pBHGloxΔE1,3Cre、脂质体Lipofectamine 2000、300kD膜胞、SOURCE30Q阴离子交换填料、Superdex200pg分子筛纯化填料、CD137L单克隆抗体、兔抗羊-FITC、预包被IFN-γ抗体的酶联细胞免疫检测板、TC-1肿瘤细胞均为市售。
实施例1多肽PP4的筛选
1)分析白喉毒素无毒突变体蛋白序列，通过NetMHCⅡpan4.0Server软件预测CRM197的CD4+T细胞表位，筛选出中、强亲和力的多肽，初步筛选得到9条多肽，如表1所示：
表1白喉毒素无毒突变体蛋白的T细胞活性多肽初步筛选结果
2)人工合成步骤1)所述的9条多肽，将所得9条多肽混匀后与等体积弗氏完全佐剂乳化后免疫8只，在免疫后第10天处死小鼠，取脾脏制备单核淋巴细胞。
3)取步骤2)所述单核淋巴细胞，按5×105/孔加至96孔细胞培养板中，同时设立空白对照。37℃条件下培养48h后，加显色液CCK8显色，用酶标仪在波长450nm下读数。
4)结果显示(如图1所示)：PP4多肽对小鼠淋巴细胞的增殖作用最明显。
实施例2表达CD137L-PP4蛋白的重组腺病毒的构建
1)根据SEQ ID NO.2所示编码PP4多肽的核苷酸序列进行人工合成，所得合成的序列通过MluⅠ和HindⅢ两个酶切位点插入pDC316质粒，获得pDC316-CD137L-PP4重组穿梭质粒(图2)。
2)用DEMM培养基培养HEK293细胞长至单层。
3)将步骤1)所述pDC316-CD137L-PP4重组穿梭质粒和腺病毒骨架质粒pBHGloxΔE1,3Cre用脂质体Lipofectamine 2000共同转染步骤2)所述HEK293细胞中，转染后显微镜下观察细胞病变情况，待细胞病变发生完全脱落时，收获原代病毒培养液。
4)将步骤1)所述原代病毒培养液，经三次反复冻融后收集后，经过三次病毒挑斑纯化获得所需的表达CD137L-PP4蛋白的重组腺病毒。
实施例3表达CD137L-PP4蛋白的重组腺病毒的制备
1)用DEMM培养基培养HEK293细胞长至单层。
2)将表达CD137L-PP4蛋白的重组腺病毒感染步骤1)所述的HEK293细胞，待细胞病变发生完全脱落时，收获病毒培养液。
3)将步骤2)所述病毒培养液，经三次反复冻融后收集上清，用300kD膜胞进行切向超滤浓缩10倍，获得病毒浓缩液。
4)将步骤3)所述病毒浓缩液，先用SOURCE30Q阴离子交换层析进行纯化，再用Superdex200pg分子筛层析进行纯化得到病毒纯化液。
实施例4表达CD137L-PP4蛋白的重组腺病毒的表达鉴定
1)用DEMM培养基培养HEK293细胞长至单层。
2)取表达CD137L-PP4蛋白的重组腺病毒接种至步骤1)所述HEK293单层细胞中，同时以空细胞为对照。在细胞培养箱37℃、5％CO2条件下培养48小时后，用胰酶消化细胞，300g/min离心5分钟弃上清，将细胞滴在载玻片上，用封闭液作用10分钟后晾干；加CD137L单克隆抗体(1:100)4℃孵育过夜；洗涤三遍后加兔抗羊-FITC(1:200)37℃孵育1小时，洗涤三遍后加95％甘油封片，在荧光显微镜下观察结果并拍照。结果如图3所示，重组腺病毒感染后细胞与特异性抗体结合后通过荧光标记二抗的捕获，在紫外发射光下即可观察到荧光细胞，结果表明重组腺病毒表达了CD137L-PP4蛋白。
实施例5表达CD137L-PP4蛋白的重组腺病毒诱导小鼠特异性细胞免疫
1)取6-8周龄C57/BL小鼠，分成四组，每组3只小鼠，分别为表达CD137L-PP4蛋白的重组腺病毒组、表达CD137L蛋白的重组腺病毒组，空腺病毒组、空白组。其中表达CD137L-PP4蛋白的重组腺病毒组、表达CD137L蛋白的重组腺病毒组和空腺病毒组小鼠按1×108IU/只剂量接种腺病毒，空白组小鼠接种等体积PBS。
2)步骤1)所述小鼠在接种后第14天处死取脾脏，制备脾脏淋巴细胞悬液。
3)将步骤2)所述脾脏淋巴细胞悬液按每孔5×105个细胞量，接种至预包被IFN-γ抗体的酶联细胞免疫检测板中，每个样本都作复孔。用CD137L的重叠多肽库作为刺激物，按照ELISPOT试剂盒说明书操作检测。结果取两复孔的平均值作为免疫小鼠的效应细胞数。结果如图4所示，表达CD137L-PP4蛋白的重组腺病毒组(A)能够产生高水平的诱导特异性IFN-γ的效应T细胞且所诱导的细胞免疫水平高于单独表达CD137L的重组腺病毒(B)，而空腺病毒组(C)和空白组(D)小鼠几乎不产生反应诱导特异性IFN-γ的效应T细胞。
实施例6表达CD137L-PP4蛋白的重组腺病毒抑制肿瘤细胞增殖
1)培养TC-1肿瘤细胞，按2×104个的细胞量将细胞接种于每只C57/BL小鼠左腿皮下，获得小鼠TC-1肿瘤模型。将肿瘤模型小鼠分成表达CD137L-PP4蛋白的重组腺病毒组、空腺病毒组和肿瘤模型对照组三个组别，每组10只小鼠。在肿瘤模型建立的24小时后，表达CD137L-PP4蛋白的重组腺病毒组和空腺病毒组小鼠按1×108IU/只剂量接种腺病毒于小鼠大腿右侧肌肉，肿瘤模型对照组不接种。
2)对步骤1)所述小鼠进行观察，间隔一周记录小鼠的肿瘤生长情况进行评分，连续观察7周，并检测肿瘤大小，根据肿瘤大小的半径(r)和长度(L)计算瘤块体积结果显示(如图5所示)：空腺病毒组(B)和肿瘤模型对照组(C)小鼠肿瘤生长水平差异不显著，表达CD137L-PP4蛋白的重组腺病毒组(A)的小鼠肿瘤生长受到显著的抑制。
序列表
<110> 杭州医学院
<120> 一种具有抗肿瘤免疫功能重组腺病毒、制备方法及应用
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Claims (8)
1.一种具有抗肿瘤免疫功能重组腺病毒，其特征在于，所述具有抗肿瘤免疫功能重组腺病毒表达融合蛋白CD137L-PP4，所述融合蛋白CD137L-PP4为CD137L蛋白和PP4多肽，氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的具有抗肿瘤免疫功能重组腺病毒，其特征在，编码所述融合蛋白CD137L-PP4的核苷酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的具有抗肿瘤免疫功能重组腺病毒，其特征在于，所述腺病毒载体是5型腺病毒载体、26型腺病毒载体或28型腺病毒载体。
4.一种权利要求1-3任一项所述的具有抗肿瘤免疫功能重组腺病毒的构建方法，其特征在于，具体为：
人工合成编码融合蛋白CD137L-PP4的核苷酸序列，融合蛋白CD137L-PP4的氨基酸序列如SEQ ID NO.1所示；通过同源重组的方法插入腺病毒载体，构建得到重组腺病毒rAd-CD137L-PP4。
5.根据权利要求4所述的构建方法，其特征在于，还包括纯化步骤，具体为：
用HEK293细胞培养所述构建得到的所述重组腺病毒rAd-CD137L-PP4得到病毒培养液，经多次反复冻融后收集上清，用超滤方法对所得上清进行浓缩，获得病毒浓缩液；将所述病毒浓缩液，先用阴离子交换层析进行纯化，再用分子筛层析进行纯化得到病毒纯化液。
6.根据权利要求5所述的构建方法，其特征在于，所述超滤方法为切向流超滤方法；所述阴离子交换层析所用的填料是SOURCE30Q或者DEAE；所述分子筛层析所用的填料是Superdex200pg。
7.一种权利要求1-3任一项所述的具有抗肿瘤免疫功能重组腺病毒在制备用于诱导机体产生针对CD137L的特异性细胞免疫反应的药物和/或诱导机体产生抑制肿瘤细胞增殖的药物中的应用。
8.根据权利要求7所述的具有抗肿瘤免疫功能重组腺病毒在制备用于诱导机体产生针对CD137L的特异性细胞免疫反应的药物和/或诱导机体产生抑制肿瘤细胞增殖的药物中的应用，其特征在于，所述诱导机体产生针对CD137L的特异性细胞免疫反应的药物具体为诱导特异性IFN-γ的效应T细胞的药物。
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