Rousselet Nathalie, Mills Lisa, Jean Didier, Tellez Carmen, Bar-Eli Menashe, Frade Raymond
Immunochimie des Régulations Cellulaires et des Interactions Virales, INSERM U.354, Genopole, Evry, France.
Cancer Res. 2004 Jan 1;64(1):146-51. doi: 10.1158/0008-5472.can-03-1717.
We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anti-cathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.
我们之前证明，人类黑色素瘤细胞从非转移性表型转变为高转移性表型与组织蛋白酶L前体的分泌直接相关。这种半胱氨酸蛋白酶是根据其切割补体第三成分人类C3的特性鉴定出来的。为了控制组织蛋白酶L前体的分泌，我们最近从一种针对重组组织蛋白酶L产生的抗组织蛋白酶L单克隆抗体中制备了抗组织蛋白酶L单链可变片段（ScFv）。我们在此选择了稳定转染该抗组织蛋白酶L ScFv的克隆，并分析它们在肿瘤生长和转移方面的变化。我们发现，在稳定转染的克隆中，抗组织蛋白酶L ScFv强烈抑制组织蛋白酶L前体的分泌，而不改变组织蛋白酶L各形式的细胞内含量或加工模式。共聚焦分析表明内源性组织蛋白酶L和抗组织蛋白酶L ScFv共定位。此外，这种ScFv的表达强烈抑制了这些人类黑色素瘤克隆在裸鼠中的肿瘤生成和转移。在体内，转染抗组织蛋白酶L ScFv的细胞产生的肿瘤血管化减少（血管生成），同时肿瘤细胞凋亡增加。基质胶实验也表明黑色素瘤的侵袭性完全被消除。因此，这首次证明了抗组织蛋白酶L ScFv可用于抑制依赖组织蛋白酶L前体分泌的人类黑色素瘤的致瘤和转移表型，因此可作为治疗性细胞方法中的一种分子工具。
