Enhancing production and cytotoxic activity of polymeric soluble FasL-based chimeric proteins by concomitant expression of soluble FasL.
Enhancing production and cytotoxic activity of polymeric soluble FasL-based chimeric proteins by concomitant expression of soluble FasL.
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DOI：
10.1371/journal.pone.0073375
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Taupin JL
中科院分区：
文献类型：
--
作者：
Morello A;Daburon S;Castroviejo M;Moreau JF;Dechanet-Merville J;Taupin JL
膜FasL是Fas介导的细胞凋亡的天然触发器。可溶性同源三聚体对应物（sFasL）也存在，其活性非常弱，并且需要寡聚化超过其三聚体状态以诱导细胞凋亡。我们最近产生了可溶性FasL嵌合体融合的免疫球蛋白样结构域的白血病抑制因子受体gp 190的人FasL，使自发的十二聚体的同型聚合的FasL的胞外区。这种可溶性的人FasL（pFasL）在体外和体内均显示出抗肿瘤活性，而在小鼠体内无全身毒性。在目前的工作中，我们专注于pFasL的改进，有两个互补的目标。首先，我们开发了更复杂的基于pFasL的嵌合体，其中包含细胞靶向模块。其次，我们试图提高pFasL和细胞靶向嵌合体的生产和/或比活性。我们通过将HLA-A2分子或人γ-δ TCR的细胞外部分融合到pFasL上设计了两种嵌合体，并分析了共表达这些分子或pFasL与sFasL对其异位细胞产生的影响。该策略显著增强了pFasL和两种嵌合体的产生，以及两种嵌合体的细胞毒性活性，但pFasL没有。这些结果提供了用于治疗用途的基于FasL的嵌合蛋白的优化的概念证明。
Membrane FasL is the natural trigger of Fas-mediated apoptosis. A soluble homotrimeric counterpart (sFasL) also exists which is very weakly active, and needs oligomerization beyond its trimeric state to induce apoptosis. We recently generated a soluble FasL chimera by fusing the immunoglobulin-like domain of the leukemia inhibitory factor receptor gp190 to the extracellular region of human FasL, which enabled spontaneous dodecameric homotypic polymerization of FasL. This polymeric soluble human FasL (pFasL) displayed anti-tumoral activity in vitro and in vivo without systemic cytotoxicity in mouse. In the present work, we focused on the improvement of pFasL, with two complementary objectives. First, we developed more complex pFasL-based chimeras that contained a cell-targeting module. Secondly, we attempted to improve the production and/or the specific activity of pFasL and of the cell-targeting chimeras. We designed two chimeras by fusing to pFasL the extracellular portions of the HLA-A2 molecule or of a human gamma-delta TCR, and analyzed the consequences of co-expressing these molecules or pFasL together with sFasL on their heterotopic cell production. This strategy significantly enhanced the production of pFasL and of the two chimeras, as well as the cytotoxic activity of the two chimeras but not of pFasL. These results provide the proof of concept for an optimization of FasL-based chimeric proteins for a therapeutic use.
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影响因子：
56.9
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