datasheet1_Antibody-Based Targeting of Interferon-Beta-1a Mutein in HER2-Positive Cancer Enhances Antitumor Effects Through Immune Responses and Direct Cell Killing.docx

Type I interferon (IFN) has been approved as an anticancer agent to treat some malignancies. However, IFNs have a short in vivo half-life, systemic toxicity, and poor biophysical properties, which prevent it from being widely used for cancer therapy. This study aimed to construct recombinant IFN-β-1a mutein immunocytokines that comprise a human epidermal growth factor receptor 2 (HER2)-targeting antibody and IFN-β muteins with an additional glycosylation, which can overcome the limitation of the cytokine itself. Hence, the molecular design aims to 1) enhance productivity and biophysical properties by adding secondary glycosylation in IFN-β, 2) increase the therapeutic index of IFN-β therapy by preferential retention at the tumor by possessing high affinity for HER2-expressing cancer cells, and 3) improve the pharmacokinetics and, thus, the convenience of IFN-β administration. The yield of trastuzumab-IFN-β mutein was higher than that of trastuzumab-wild-type IFN-β in the mammalian cell culture system. Trastuzumab-IFN-β mutein showed similar IFN activity and HER2-targeting ability equivalent to that of IFN-β mutein and trastuzumab, respectively. Trastuzumab-IFN-β mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-β mutein alone. Trastuzumab-IFN-β mutein and IFN-β mutein displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-β mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Tumor-targeting effect of trastuzumab-IFN-β mutein was analyzed using in vivo fluorescence imaging. The accumulation of trastuzumab-IFN-β mutein was observed in HER2-positive tumors rather than other tissues except the liver. To evaluate the both direct tumor growth inhibition effect and indirect immune cell-mediated antitumor effect, we tested the effect of trastuzumab-IFN-β mutein in HER2-positive cancer xenograft models using nude mice or humanized mice. Trastuzumab-IFN-β mutein could significantly enhance tumor regression when compared with trastuzumab or IFN-β mutein. In addition, an increase in tumor-infiltrating lymphocytes was observed in the trastuzumab-IFN-β mutein-treated group, implying that the tumor-targeting IFN-β may have an enhanced antitumor effect through increased immune response. Therefore, targeting IFN-β with an anti-HER2 monoclonal antibody makes the immunocytokine more potent than either agent alone. These novel findings suggest that trastuzumab-IFN-β mutein merits clinical evaluation as a new candidate of anticancer therapeutics.

I型干扰素（Type I interferon, IFN）已被批准作为抗癌药物，用于治疗部分恶性肿瘤。然而，干扰素体内半衰期短、存在系统毒性且生物物理特性欠佳，这极大限制了其在癌症治疗中的广泛应用。
本研究旨在构建重组IFN-β-1a突变体免疫细胞因子（immunocytokines），该分子由靶向人表皮生长因子受体2（human epidermal growth factor receptor 2, HER2）的抗体与带有额外糖基化修饰的IFN-β突变体融合而成，可克服细胞因子本身的固有局限性。因此，该分子的设计目标包括三点：1）通过在IFN-β中引入次级糖基化修饰，提升其表达产量与生物物理特性；2）凭借对HER2阳性癌细胞的高亲和力，使药物优先滞留于肿瘤部位，从而改善IFN-β治疗的治疗指数；3）优化药代动力学特性，进而简化IFN-β的给药流程。
在哺乳动物细胞培养体系中，曲妥珠单抗（trastuzumab）-IFN-β突变体的表达产量高于曲妥珠单抗-野生型IFN-β融合蛋白。曲妥珠单抗-IFN-β突变体的IFN活性与IFN-β突变体相当，其HER2靶向能力亦与曲妥珠单抗相仿。
曲妥珠单抗-IFN-β突变体可直接抑制HER2阳性胃癌细胞系的增殖，且抗肿瘤效果优于单独使用曲妥珠单抗或单独使用IFN-β突变体。曲妥珠单抗-IFN-β突变体与IFN-β突变体均能增强免疫细胞介导的细胞毒性效应。
综上，曲妥珠单抗-IFN-β突变体兼具间接免疫细胞介导的抗肿瘤活性与直接细胞增殖抑制活性。研究人员采用体内荧光成像技术分析了曲妥珠单抗-IFN-β突变体的肿瘤靶向效果，观察到该融合蛋白可在HER2阳性肿瘤部位特异性富集，而在除肝脏外的其他正常组织中富集程度极低。
为同时评估其直接肿瘤生长抑制效应与间接免疫细胞介导的抗肿瘤效应，本研究在裸鼠及人源化小鼠的HER2阳性癌症异种移植模型中测试了曲妥珠单抗-IFN-β突变体的治疗效果。
结果显示，相较于单独使用曲妥珠单抗或单独使用IFN-β突变体，曲妥珠单抗-IFN-β突变体可显著促进肿瘤消退。此外，曲妥珠单抗-IFN-β突变体处理组的肿瘤浸润淋巴细胞数量显著升高，这提示肿瘤靶向性IFN-β可通过增强机体免疫应答进一步提升抗肿瘤效果。
因此，借助抗HER2单克隆抗体靶向递送IFN-β，可使该免疫细胞因子的疗效优于任一单一药物。上述全新研究结果表明，曲妥珠单抗-IFN-β突变体作为新型抗癌治疗候选药物，值得开展临床评价。