摘要
B7-H3是在多种肿瘤表面过表达的免疫检查点分子,是肿瘤免疫治疗的理想靶点。利用本实验室前期设计的硝基化T细胞表位为基础,构建了可靶向免疫检查点B7-H3的表位疫苗。该疫苗能在CT26结肠癌模型中显著抑制肿瘤生长,且与PD-L1蛋白疫苗有明显的协同作用。B7-H3疫苗可以增加脾脏T淋巴细胞中CD
B7-H3(也称CD276)是一种免疫检查点分子,属于B7超家族,在正常组织中广泛低表达,同时在多种恶性肿瘤中过表达,如黑色素瘤、白血病、乳腺癌、前列腺癌、卵巢癌、结直肠癌
目前针对B7-H3分子设计的药物如enoblituzumab(MGA271)为单克隆抗体,在临床Ⅰ期阶段表现出了一定的抗肿瘤活
本研究以实验室前期设计的硝基化T细胞表位为基
3,3',5,5'-四甲基联苯胺(TMB)、辣根过氧化物酶(HRP)标记的羊抗小鼠IgG(北京索莱宝科技有限公司);小鼠脾脏淋巴细胞分离液、小鼠肿瘤淋巴细胞分离液(天津灏洋生物制品科技有限责任公司);RPMI 1640培养基、胎牛血清FBS(美国Gibco公司);PE标记的抗小鼠CD3抗体、FITC标记的抗小鼠CD4抗体、APC标记的抗小鼠CD8抗体、PerCP/Cyanine5.5标记的抗小鼠IFN-γ抗体、PE标记的抗小鼠IL-4抗体、APC标记的抗小鼠CD25抗体、PE标记的抗小鼠Foxp3抗体(美国Biolegend公司);固定破膜液、洗涤缓冲液(美国BD公司)。
多肽和佐剂均为南京金斯瑞生物科技有限公司合成。
通过Uniprot数据库(http://www.uniprot.org)查询得到免疫检查点B7-H3分子胞外区氨基酸序列为:B7-H3(29 ~ 248):VEVQVSEDPVVALVDT DATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRLQRVRVTDEGSYTCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCSSYQGYPEAEVFWKDGQGVPLTGNVTTSQMANERGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPLTFPPEA通过IEDB(http://www.iedb.org)表位在线预测网站对免疫检查点分子B7-H3的B表位进行预测,并综合评价ParkerHydrophilicity、Chou & FasmanBeta-Turn、EminiSurfaceAccessibility、Karplus &SchulzFlexibility、BepipredLinearEpitope多种B表位预测方法结果,综合考虑选择表位的氨基酸长度。用柔性肽将硝基化T表位与B7-H3分子胞外区B表位连接,构建靶向B7-H3分子的表位疫苗。所有多肽送至南京金斯瑞生物科技有限公司合成。
BALB/c小鼠随机分为3组:PBS组、2种不同氨基酸长度的表位疫苗组,每组6只,确保免疫前组间小鼠体质量不产生显著性差异。免疫方式:腹股沟皮下免疫,疫苗每只50 μg,CpG佐剂每只10 μg,每周1次,共3次。免疫后每周对小鼠进行眼底静脉丛取血,6 000 r/min离心20 min,分离上清液即为所需血清,对血清适当稀释后检测第1次免疫后第21和28天抗体滴度。
每只BALB/c小鼠于右前肢腋下处皮下注射5 × 1
处死小鼠,在75%乙醇中浸泡5~10 min,无菌条件下取出脾脏,并用PBS对脾脏进行清洗,使用2 mL注射器内芯研磨,研磨后组织通过70 μm筛网;用PBS对细胞和筛网进行冲洗,转移至离心管中,430 r/min离心10 min。弃上清液,加入PBS 1 mL重悬;缓慢滴加细胞悬液至3 mL淋巴细胞分离液上层,保持淋巴细胞分离液与细胞悬液液面清晰分层;1 032 r/min离心20 min,小心吸取淋巴细胞层;加入PBS 8 mL,430 r/min离心5 min,弃上清液,重复1次;用PBS重悬细胞至浓度为每毫升5×1
流式抗体标记:CD
剪取适量大小的肿瘤样本(约1 g),用2 mL注射器内芯研磨,边研磨边加入PBS冲洗,使得肿瘤组织经过70 μm筛网润洗至6孔板中。将细胞转移到15 mL离心管,按照体积比1∶1加入等量的淋巴细胞分离液。2 000 r/min离心15 min。将乳白色淋巴细胞层转移到新的15 mL 离心管中,向离心管中加入10 mL PBS,2 000 r/min,15 min。弃上清液,用PBS重悬细胞至浓度为每毫升5 × 1
流式抗体标记:CD
B7-H3分子蛋白质胞外区预测得到的不同氨基酸长度的B表位序列如下:
B7-H3(B14):FTEGRDQGSAYSNR;B7-H3(B20):AAPYSKPSMTLEPNKDLRPG
通过柔性连接肽GPSL将预测的B7-H3蛋白分子胞外区B表位序列与通用硝基化T细胞表位(AKFVAAWTLKpNO2PheAA)序列连接,构建B7-H3表位肽疫苗候选分子。所有多肽送至南京金斯瑞生物科技有限公司合成。合成的多肽表位疫苗氨基酸序列分别为:B7-H3(B14) + T(pNO2Phe
为了筛选出免疫原性较高的多肽表位疫苗,用两种多肽表位疫苗分别免疫BALB/c小鼠,每周1次,共3次。取第1次免疫后第21、28 天的小鼠血清,使用ELISA法检测抗体滴度。结果如

Figure 1 Immunogenicity of B7-H3 epitope vaccine ()
A:Antibody titers produced by different B7H3 vaccines after immunizing mice; B:Liver and kidney HE staining of mice immunized with PBS and B7-H3-NitraTh (200 × )
对PBS组和经过3次疫苗免疫后的B7-H3-NitraTh疫苗组小鼠肝肾进行HE染色,结果显示,B7-H3-NitraTh表位疫苗组小鼠肝肾细胞形态无明显异常(
在BALB/c小鼠皮下构建CT26实体瘤模型,设置PBS组、B7-H3-NitraTh疫苗组,同时将B7-H3-NitraTh与实验室前期筛选得到的PD-L1-NitraTh蛋白疫苗联用,以期获得更好的抑瘤效果。结果如

Figure 2 Anti-tumor activity of B7-H3-NitraTh vaccine in mice with colon cancer
A:Picture of the tumor removed from the sacrificed mice after the treatment (n = 3); B:Mouse tumor volume growth curve measured after immunization with B7-H3-NitraTh vaccine (); C:Curve of the weight change of mice measured after immunization with B7-H3-NitraTh vaccine ()
为了探究肿瘤疫苗的作用机制,本实验首先检测脾脏中CD

Figure 3 Differentiation of CD
A:Frequency of CD
研究疫苗治疗后对结肠癌小鼠的脾脏和肿瘤中CD

Figure 4 Frequency of CD
A:Frequency of CD
为了进一步探究B7-H3-NitraTh疫苗治疗后对免疫抑制性肿瘤微环境的影响,检测了肿瘤中Treg细胞的浸润情况。与PBS组相比,B7-H3-NitraTh组可以明显减少抑制性Treg细胞的比例(P < 0.01),而相对于单给药组,B7-H3-NitraTh+PD-L1-NitraTh联合给药又能进一步显著降低肿瘤中Treg细胞浸润,Treg细胞的比例达到(1.04 ± 0.16)%(P < 0.001)。结果表明B7-H3-NitraTh疫苗可以减少肿瘤浸润的抑制性Treg细胞的比例,从而改善肿瘤免疫抑制微环境。

Figure 5 Frequency of Treg cells in the tumor microenvironment of mice with colon cancer after immunization with B7H3 vaccine ()
**P < 0.01,
免疫检查点是治疗性肿瘤疫苗设计的良好靶
此外,考虑到免疫检查点是自身蛋白,存在免疫耐受,较低的免疫原性限制了疫苗的开发。而本课题组前期筛选得到一种硝基化通用Th表位,该表位可以和多种小鼠MHC Ⅱ类分子以及人的HLA Ⅱ类分子结合,打破MHC限制
临床前研究表明,免疫检查点的同时阻断可产生协同抗肿瘤效
通过检测脾脏中淋巴细胞变化情况探究肿瘤疫苗的作用机制,发现两种给药方式都在一定程度上刺激了T细胞活化并提高CD
实体肿瘤中浸润的效应T细胞,一定条件下可控制肿瘤的发展,临床研究表明,肿瘤组织高CD
综上所述,本研究筛选得到的B7-H3表位疫苗可以产生高滴度特异性抗体,有效抑制肿瘤生长,并能增加肿瘤浸润CD
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