实施例6IFN-β增加了体内的抗原特异性细胞溶解活性
虽然增殖是产生有效的免疫应答的良好指示,其并不总是转化为强有力的效应子功能。因此,发明人研究了ad-IFN-β疗法是否引起增加的体内特异性裂解。用ad-null或ad-IFN-β处理带有肿瘤的小鼠,然后向其中转移加载了经CFSE标记的肽的靶细胞。作为阴性对照,还将加载了经CFSE标记的肽的靶细胞转移到幼稚小鼠中,而如预期的,这些小鼠显示出正常的特异性裂解。通过比较发现,与经ad-null处理的小鼠相比,经ad-IFN-β处理的小鼠显示出显著更高的特异性裂解(图5e)。这些数据表明,在经ad-IFNβ处理的小鼠中观察到的抗原特异性细胞的扩增引起了强有力的效应子CTL活性。
为了确定用IFNβ靶向肿瘤是否可引起肿瘤消退,接种了TUBO-EGFR细胞并且在第14、17和20天肿瘤内注射表达IFN的腺病毒。ad-null对于肿瘤生长有温和的效果,而ad-IFNβ引起肿瘤的消退(图6)。这表明,在TUBO上局部递送ad-IFNβ也可诱导强的应答,从而引起肿瘤消退。
为了测试由CHO细胞制备的实际融合蛋白是否能够诱导快速的肿瘤消退,用编码抗体-IFN(对于抗EGFR-IFNβ,将Cetuximab的3’端与IFNβ相连,然后将其引入到载体pcDNA中,亚克隆均采用标准的分子生物学技术进行,简要地,1)将HC-Fc-IFN克隆到载体Abvec-hIgG1中;2)将抗体的轻链克隆到载体Abvec-lamda中;3)从1)中得到的产物中剪切HC-Fc-IFN,并通过平末端连接将其克隆到lonza的载体pEE6.4中;4)从步骤2)中获得的产物中剪切出轻链并通过平末端连接将其克隆到lonza的载体pEE12.4中;5)从步骤4)的产物中切出表达盒并将其克隆到步骤3)的产物中,从而得到最终的质粒pEE12.4-抗-EGFR-IFNβ)的质粒转染CHO细胞并且选择大量表达所述融合蛋白的细胞。收集含有这种质粒的CHO的上清液并且通过蛋白A柱子纯化融合蛋白,所述蛋白A柱子以高亲合力结合融合蛋白的Fc部分。用低剂量的抗体或者所述抗体-IFN处理带有TUBO-EGFR的小鼠。仅仅是融合蛋白引起了肿瘤的快速消退(图7)。从带有B16-EGFR肿瘤的小鼠也获得了类似的结果(图8)。因此,与单独的抗体相比,这种融合蛋白也可被用于更好地清除肿瘤表达EGFR的肿瘤。
为了确定这种显著的效果是否是由T细胞免疫引起的,用低剂量的所述融合蛋白和T细胞耗竭抗体处理带有EGFR-TUBO肿瘤的小鼠。虽然大幅延迟了EGFR-TUBO的生长,CD8+T细胞的耗竭引起了快速复发,而CD4+T细胞的耗竭则不会引起肿瘤复发(图9)。
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