Bremer Edwin, de Bruyn Marco, Samplonius Douwe F, Bijma Theo, ten Cate Bram, de Leij Lou F M H, Helfrich Wijnand
Laboratory for Tumor Immunology, Section Medical Biology, Department of Pathology and Laboratory Medicine, University Medical Center Groningen, Hanzeplein 1, Groningen, The Netherlands.
J Mol Med (Berl). 2008 Aug;86(8):909-24. doi: 10.1007/s00109-008-0348-9. Epub 2008 May 27.
Previously, we have shown that epidermal growth factor receptor (EGFR)-selective delivery of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), by genetic fusion to antibody fragment scFv425, enhances the tumor-selective pro-apoptotic activity of sTRAIL. Insight into the respective contribution of the agonistic receptors TRAIL-R1 and TRAIL-R2 to TRAIL-induced apoptosis may provide a rational approach to further optimize TRAIL-based therapy. Recently, this issue has been investigated using sTRAIL mutants designed to selectively bind to either receptor. However, the relative contribution of the respective TRAIL receptors, in particular TRAIL-R1, in TRAIL signaling is still unresolved. Here, we fused scFv425 to designed sTRAIL mutant sTRAILmR1-5, reported to selectively activate TRAIL-R1, and investigated the therapeutic apoptotic activity of this novel fusion protein. EGFR-specific binding of scFv425:sTRAILmR1-5 potently induced apoptosis, which was superior to the apoptotic activity of scFv425:sTRAIL-wt and a nontargeted MOCK-scFv:sTRAILmR1-5. During cotreatment with cisplatin or the histone deacetylase inhibitor valproic acid, scFv425:sTRAILmR1-5 retained its superior pro-apoptotic activity compared to scFv425:sTRAIL-wt. However, in catching-type Enzyme-Linked ImmunoSorbent Assays with TRAIL-R1:Fc and TRAIL-R2:Fc, scFv425:sTRAILmR1-5 was found to not only bind to TRAIL-R1 but also to TRAIL-R2. Binding to TRAIL-R2 also had functional consequences because the apoptotic activity of scFv425:sTRAILmR1-5 was strongly inhibited by a TRAIL-R2 blocking monoclonal antibody. Moreover, scFv425:sTRAILmR1-5 retained apoptotic activity upon selective knockdown of TRAIL-R1 using small inhibitory RNA. Collectively, these data indicate that both agonistic TRAIL receptors are functionally involved in TRAIL signaling by scFv425:sTRAILmR1-5 in solid tumor cells. Moreover, the superior target cell-restricted apoptotic activity of scFv425:sTRAILmR1-5 indicates its therapeutic potential for EGFR-positive solid tumors.
此前，我们已经表明，通过与抗体片段scFv425进行基因融合，可实现可溶性肿瘤坏死因子相关凋亡诱导配体（sTRAIL）对表皮生长因子受体（EGFR）的选择性递送，从而增强sTRAIL的肿瘤选择性促凋亡活性。深入了解激动性受体TRAIL-R1和TRAIL-R2对TRAIL诱导凋亡的各自贡献，可能为进一步优化基于TRAIL的治疗提供合理方法。最近，已使用设计用于选择性结合任一受体的sTRAIL突变体对此问题进行了研究。然而，在TRAIL信号传导中，各个TRAIL受体，特别是TRAIL-R1的相对贡献仍未解决。在此，我们将scFv425与设计的sTRAIL突变体sTRAILmR1-5融合，据报道该突变体可选择性激活TRAIL-R1，并研究了这种新型融合蛋白的治疗性凋亡活性。scFv425:sTRAILmR1-5的EGFR特异性结合有效诱导了凋亡，其凋亡活性优于scFv425:sTRAIL-wt和非靶向的MOCK-scFv:sTRAILmR1-5。在与顺铂或组蛋白去乙酰化酶抑制剂丙戊酸联合治疗期间，与scFv425:sTRAIL-wt相比，scFv425:sTRAILmR1-5保留了其优越的促凋亡活性。然而，在使用TRAIL-R1:Fc和TRAIL-R2:Fc进行捕获型酶联免疫吸附测定中，发现scFv425:sTRAILmR1-5不仅与TRAIL-R1结合，还与TRAIL-R2结合。与TRAIL-R2的结合也产生了功能后果，因为scFv425:sTRAILmR1-5的凋亡活性被TRAIL-R2阻断单克隆抗体强烈抑制。此外，在使用小干扰RNA选择性敲低TRAIL-R1后，scFv425:sTRAILmR1-5仍保留凋亡活性。总体而言，这些数据表明，在实体瘤细胞中，两种激动性TRAIL受体在scFv425:sTRAILmR1-5的TRAIL信号传导中均发挥功能作用。此外，scFv425:sTRAILmR1-5优越的靶细胞限制性凋亡活性表明其对EGFR阳性实体瘤具有治疗潜力。
