免疫促凋亡分子用于治疗HBsAg阳性肝细胞癌的研究

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors occurring worldwide. High recurrence rates of surgical treatment and non-specific side-effects of radiotherapy and chemotherapy on normal tissues lead to limited clinical benefits of HCC therapy. Targeted therapy is needed for patients with HCC. We have established a novel antitumor strategy by generating a series of immunoproapoptotic molecules (IPMs) and confirmed its antitumor activity against HER2-positive tumors in vitro and in vivo. The hepatitis B virus surface antigen (HBsAg) is overexpressed in HBV-related HCC. We previously reported that scFv15, a human single-chain antibody, specifically recognized and entered HBsAg-positive HCC cells. Here, we switched our well-established immunoproapoptotic strategy from HER2-targeting to HBsAg-targeting and explored its killing capacity after introducing an endosomolytic HA2 motif. scFv15 was conjugated to a truncated form of the proapoptotic protein Bid (tBid) via the furin-cleavable motif from diphtheria toxin (Fdt) and the HA2 peptide of influenza hemagglutinin to generate the HBsAg-directed immunoproapoptotic molecule. The purified immunoproapoptotic molecules were tested for HBsAg-binding activity, endosome escape activity, and tumor cell killing efficacy using cultured HCC cells. In vivo imaging of antitumor activity was performed in athymic nude mice bearing orthotopic human HCC. This study will extend the targeted immunoproapoptotic strategy to the treatment of HBsAg-positive HCC and show the significant potential of HA2 to accelerate endosome escape and enhance the antitumor effects of immunoproapoptotic proteins.

肝细胞癌（HCC）是最常见的恶性肿瘤之一，手术治疗复发率高，传统放、化疗对正常组织毒害大。本课题组曾将抗HER2单链抗体与促凋亡分子融合表达，构建的免疫促凋亡分子在HER2阳性肿瘤模型中证实具有明确的抗肿瘤作用。通过筛选改造，本课题组获得一株对HBsAg具有亲和活性并可经由内吞体进入HBsAg阳性肿瘤细胞的单链抗体（scFv15），为构建靶向HBsAg的免疫促凋亡分子奠定了基础。本课题拟将scFv15作为靶向识别结构域、促凋亡分子tBid作为杀伤效应结构域，构建靶向HBsAg的免疫促凋亡分子，并加入HA2序列作为转位结构域，以提高免疫促凋亡分子释放效率，增强对靶细胞的杀伤效果。通过细胞水平和动物水平实验，检测免疫促凋亡分子对HBsAg的亲和活性、内吞体破坏活性，及对HBsAg阳性HCC肿瘤的靶向性和杀伤活性。本课题具有扩展免疫促凋亡策略的理论意义，并对HBV相关HCC的治疗具有实际意义。

肝细胞癌是最常见的恶性肿瘤之一，手术治疗复发率高，传统放、化疗对正常组织毒害大。本课题组将单链抗体、转位结构域（Fdt）和促凋亡分子（tBid）融合构建的免疫促凋亡分子，已在HER2肿瘤模型中证实具有明确的抗肿瘤作用，该分子靶向性高、副作用小，有希望推广应用于肝细胞癌治疗。.本课题组筛选获得一株对HBsAg具有亲和活性并可内吞进入HBsAg阳性肿瘤细胞的人源单链抗体scFv15，构成了靶向HBsAg免疫促凋亡分子的基础。免疫促凋亡分子发挥作用的关键步骤是从内吞体转位释放到胞浆。前期研究发现，流感病毒血凝素中HA2序列暴露于多肽链N末端时具有极强的内吞体膜破坏能力。.本研究将上述结构域融合表达，构建靶向HBsAg的免疫促凋亡分子scFv15-Fdt-HA2-tBid。该分子可在内吞体中被Furin蛋白酶切割，将HA2暴露于多肽链N末端，进而破坏内吞体膜，释放tBid进入胞浆发挥杀伤效应。细胞水平实验表明，免疫促凋亡分子可以特异性结合HBsAg阳性肿瘤细胞；融合有HA2序列的免疫促凋亡分子能更快速的突破内吞体限制，转位进入胞浆。免疫促凋亡分子对HBsAg阴性细胞无明显毒性，但能通过诱导凋亡显著降低HBsAg阳性肿瘤细胞存活率。免疫促凋亡分子的促凋亡效应与线粒体凋亡通路的激活有关，伴随着线粒体膜电位降低，caspase-9和caspase-3的活化。动物水平实验表明，免疫促凋亡分子经尾静脉注入肝原位荷瘤裸鼠后，能够向肿瘤部位富集，在其他正常组织脏器累积分布较少，表明该分子具有良好的在体靶向性。免疫促凋亡分子治疗后，瘤体增长速度显著减缓；scFv15-Fdt-HA2-tBid具有更强的肿瘤增殖抑制作用，能够更高效地诱导肿瘤细胞发生凋亡。以治疗剂量的10倍对Balb/c小鼠注射免疫促凋亡分子，未表现出对脏器的严重损伤，未引起明显的炎症反应，提示该分子具有良好的应用安全性。免疫原性检测结果显示，转位结构域HA2的引入，并未显著增加免疫促凋亡分子的免疫原性。.综上所述，scFv15-Fdt-HA2-tBid在体内外均可以靶向结合HBsAg阳性肿瘤细胞，具有较强的特异性和良好的渗透性；HA2序列可以促进凋亡效应分子向胞浆释放，显著提高诱导肿瘤细胞凋亡的效率；该分子安全性较好，并且对人体的潜在免疫原性低，扩展了免疫促凋亡策略的理论意义，并对乙肝病毒相关的HCC治疗具有实际意义。