A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models.
A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models.
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DOI：
10.18632/oncotarget.6925
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发表时间：
2016-03-29
期刊：
影响因子：
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通讯作者：
Accardi L
中科院分区：
文献类型：
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作者：
Amici C;Visintin M;Verachi F;Paolini F;Percario Z;Di Bonito P;Mandarino A;Affabris E;Venuti A;Accardi L
表达为“细胞内抗体”（胞内抗体）的单链可变片段（scFv）可以靶向细胞内抗原以有效且特异性地阻碍其功能。在这里，我们使用靶向人乳头瘤病毒16（HPV 16）的E6癌蛋白的胞内抗体来解决HPV癌症患者的非侵入性治疗问题。利用胞内抗体捕获技术筛选出抗HPV 16 E6的单链抗体，并在HPV 16阳性的SiHa、HPV阴性的C33 A和293 T细胞核中表达为I7 nuc。在不同的细胞室中观察到I7 nuc和重组E6的共定位，获得归因于I7 nuc的E6离域的证据。在SiHa细胞中，pLNCX逆转录病毒载体表达的I7 nuc能够部分抑制主要E6靶点p53的降解，并诱导p53在细胞核中积聚。当分析对细胞增殖和存活的体外活性时，I7 nuc能够降低生长，诱导SiHa细胞的晚期凋亡和坏死。最后，在HPV肿瘤的两个临床前模型中证明了I7 nuc的抗肿瘤活性。C57 BL/6小鼠皮下注射HPV 16阳性TC-1或C3肿瘤细胞，用表达或不表达I7 nuc的pLNCX逆转录病毒载体感染。所有注射了表达I7 nucc的细胞的小鼠都表现出明显的肿瘤发病延迟;分别有60%和40%的接受TC-1和C3细胞的小鼠在17周的随访中保持无肿瘤，而100%的对照组在接种后20天都有肿瘤。我们的数据支持E6靶向I7 nuc对HPV肿瘤的治疗潜力。
Single-chain variable fragments (scFvs) expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients. A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells. Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors.