研究目的：食管癌是世界上最常见的恶性肿瘤之一，其中食管鳞状细胞癌 (ESCC)在中国的发病率相对较高，因此，迫切需要探索新的有效治疗药物和有前途的治疗策略。表皮生长因子受体(EGFR)是癌症治疗的理想靶点之一，以EGFR为治疗靶点，制备靶向EGFR的重组融合蛋白以及它的抗体偶联药物治疗食管癌或许可以带来良好的临床效益。在前期的研究中，我们构建了一种新型的重组融合蛋白（Fv-LDP-D3）以及它的抗体偶联药物（Fv-LDP-D3-AE），其中Fv是抗EGFR单链可变区片段，D3是人血清白蛋白（HSA）的结构域III，LDP是Lidamycin (LDM)的辅基蛋白，AE是LDM的活性烯二炔发色团。前期研究结果表明其对K-Ras突变型胰腺癌显示出了较显著的抗肿瘤效果。然而，Fv-LDP-D3和Fv-LDP-D3-AE是否对食管癌具有抗癌活性以及潜在的分子机制尚未得到研究。在本研究中，我们主要在体外和体内评估Fv-LDP-D3和Fv-LDP-D3-AE对食管癌的抗肿瘤活性，以及探索潜在的分子机制。

研究方法：通过基因工程和发酵技术制备重组融合蛋白Fv-LDP-D3，在体外将力达霉素的活性烯二炔发色团AE和辅基蛋白LDP拆分，再通过分子组装将AE组装到Fv-LDP-D3上，从而制备抗体偶联药物（Fv-LDP-D3-AE）。通过细胞ELISA实验和细胞免疫荧光法分析Fv-LDP-D3与食管癌细胞的结合活性。通过荧光显微镜检测食管癌细胞对重组融合蛋白Fv-LDP-D3的巨胞饮摄取作用。应用小动物活体成像仪观察重组融合蛋白的体内靶向肿瘤部位能力。采用CCK-8法，流式细胞术、western blot等实验评价重组融合蛋白和它的抗体偶联药物的体外活性和作用机制。最后，在体内评价药物对KYSE150，KYSE520裸鼠移植瘤模型的抗肿瘤效果。

研究结果：细胞ELISA实验和细胞免疫荧光实验均揭示出重组融合蛋白在体外与食管癌细胞具有结合活性。经荧光显微镜检测，人食管癌细胞通过巨胞饮介导的途径摄取重组融合蛋白Fv-LDP-D3。重组融合蛋白Fv-LDP-D3通过抑制p-EGFR和IMPDH2信号的传导，从而抑制食管癌细胞增殖。此外，Fv-LDP-D3可诱导细胞凋亡。小动物活体成像表明，Fv-LDP-D3在KYSE520裸鼠移植瘤模型体内显示出肿瘤部位特异性的生物分布，靶向富集在肿瘤部位持续时间达到624 h。组装活性烯二炔发色团AE后制备的抗体偶联药物Fv-LDP-D3-AE，在所有食管癌测试细胞系中，均显示出了强大的抗细胞增殖作用，Fv-LDP-D3-AE可在体外抑制食管癌细胞迁移和侵袭作用，诱导自噬，破坏线粒体结构，诱导食管癌细胞凋亡，引起DNA损伤，阻滞细胞G2/M期。体内药效学实验结果显示，在KYSE150裸鼠移植瘤模型中，Fv-LDP-D3-AE（0.2 mg/kg）抑瘤率为64.3%。在KYSE520裸鼠移植瘤模型中，Fv-LDP-D3（20 mg/kg）和Fv-LDP-D3（40 mg/kg）抑瘤率分别为72.9%，81.2%；Fv-LDP-D3-AE（0.25 mg/kg）和Fv-LDP-D3-AE（0.5 mg/kg）抑瘤率分别为69.4%，88.5%；联合给药组抑瘤率为81.0%。组织切片苏木精-伊红（H&E）染色的检查结果表明，重组融合蛋白和其抗体偶联药物均未造成裸鼠主要脏器的明显损伤，表明在剂量耐受情况下，重组融合蛋白及其抗体偶联药物均显示出显著的体内抗肿瘤疗效。

研究结论：本研究表明，由EGFR单链抗体片段，力达霉素的辅基蛋白和白蛋白第三结构域，制备而成的的重组融合蛋白（Fv-LDP-D3），以及重组融合蛋白和力达霉素的烯二炔发色团制备的抗体偶联药物（Fv-LDP-D3-AE），不仅在体外对食管癌癌细胞具有强大的抗肿瘤活性，而且在体内能高效抑制裸鼠异种移植瘤的生长，揭示了重组融合蛋白Fv-LDP-D3及其抗体偶联药物Fv-LDP-D3-AE可能是治疗ESCC的潜在候选药物。

Objective: Esophageal cancer is one of the most common malignant tumors in the world, and the incidence of esophageal squamous cell carcinoma (ESCC) is relatively high in China. Therefore, there is an urgent need to explore new effective therapeutic drugs and promising therapeutic strategies. Epidermal growth factor receptor (EGFR) is one of the ideal targets for cancer therapy. Taking EGFR as the therapeutic target, the preparation of EGFR targeted recombinant fusion protein and its antibody-drug conjugate for the treatment of esophageal cancer may bring good clinical benefits. In previous studies, we constructed a novel recombinant fusion protein (Fv-LDP-D3) and its antibody-drug conjugate (Fv-LDP-D3-AE), in which Fv is an anti-EGFR single-chain variable fragment, D3 is the domain III of human serum albumin (HSA), LDP is the apoprotein of Lidamycin (LDM), and AE is the active enediyne chromophore of LDM. The results of previous studies showed that it had a significant antitumor effect on K-Ras mutant pancreatic cancer. However, the anticancer efficacy of Fv-LDP-D3 and Fv-LDP-D3-AE against esophageal cancer and the mechanism of action have not yet investigated. In this study, we evaluated the antitumor activity of Fv-LDP-D3 and Fv-LDP-D3-AE against esophageal cancer in vitro and in vivo, and explored the underlying molecular mechanisms.

Research methods: The recombinant fusion protein Fv-LDP-D3 was prepared by genetic engineering and fermentation technology. The active enediyne chromophore AE and the apoprotein LDP of lidamycin were separated in vitro, and then AE was assembled into Fv-LDP-D3 through molecular assembly, so as to prepare the antibody-drug conjugate (Fv-LDP-D3-AE). The binding activity of Fv-LDP-D3 to esophageal cancer cells was analyzed by cell ELISA and cellular immunofluorescence. The macropinocytosis mediated uptake of the recombinant fusion protein Fv-LDP-D3 by esophageal cancer cells was detected by fluorescence microscope. Small animal in vivo imager was used to observe the capability of the recombinant fusion protein to accumulate in tumor sites. The in vitro activity and mechanism of action of the recombinant fusion protein and its antibody-drug conjugate were determined by CCK-8 assay, flow cytometry, western blot and other experiments. Finally, the in vivo anti-tumor effect of the drug was evaluated with esophageal squamous cell carcinoma KYSE150 and KYSE520 xenografts in athymic mice.

Research results: ELISA and immunofluorescence analysis indicated that recombinant fusion protein could bind to esophageal cancer cells in vitro. Fluorescence microscopy showed that the recombinant fusion protein Fv-LDP-D3 entered into human esophageal cancer cells through a macropinocytosis-mediated pathway. The recombinant fusion protein Fv-LDP-D3 suppressed the proliferation of esophageal cancer cells by inhibiting the signaling of p-EGFR and IMPDH2. In addition, Fv-LDP-D3 induced apoptosis. In vivo imaging showed that Fv-LDP-D3 exhibited tumor site-specific biodistribution in the KYSE520 nude mouse xenograft model, with targeted enrichment at the tumor site for 624 h. Fv-LDP-D3-AE, a relevant antibody drug conjugate prepared after assembling the active enediyne chromophore AE, displayed highly potent anti-proliferative effect against all tested esophageal cancer cell lines. Fv-LDP-D3-AE inhibited migration and invasion of esophageal cancer cell, induced autophagy, damaged mitochondrial structure, induced apoptosis, incurred DNA damage, and caused G2/M arrest. The results of in vivo experiment showed that the tumor inhibition rate of Fv-LDP-D3-AE (0.2 mg/kg) was 64.3% in KYSE150 xenograft model. Moreover, in the KYSE520 xenograft model, the tumor inhibition rates of Fv-LDP-D3 at 20 mg/kg and 40 mg/kg were 72.9% and 81.2%; and those of Fv-LDP-D3-AE at 0.25 mg/kg and 0.5 mg/kg were 69.4% and 88.5%, respectively. The tumor inhibition rate of the combined administration group was 81.0%. Histo-pathological observation showed that the recombinant fusion protein and its antibody drug conjugate did not cause obvious damage to major organs of the treated mice, indicating that the recombinant fusion protein and its antibody drug conjugate exerted strong anti-tumor effect in vivo at a tolerated dosage level.

Conclusions: The present study demonstrated that the recombinant fusion protein Fv-LDP-D3 which consists of the Fv fragment of an anti-EGFR antibody, the apoprotein of lidamycin (LDP), and the third domain of human serum albumin (D3) and its pertinent drug conjugate (Fv-LDP-D3-AE) which is prepared by integrating the active enediyne chromophore (AE) into the fusion protein molecule both exhibited strong anti-tumor activity against esophageal cancer cells in vitro and effectively inhibited the growth of cancer xenografts in vivo. The current findings revealed that the recombinant fusion protein Fv-LDP-D3 and its antibody-drug conjugates Fv-LDP-D3-AE may be potential candidates for the treatment of esophageal squamous cell carcinoma.