Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells.
We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His. This 3D8 mAb interacted with an epitope localized on the 156-197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.
Guillaume-Rousselet
Nathalie
N
Immunochimie des Régulations Cellulaires et des Interactions Virales, INSERM U.354, Centre INSERM, Hôpital Saint-Antoine, 75012 Paris, France.
Jean
Didier
D
Frade
Raymond
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008207:Lymphatic Metastasis; D008545:Melanoma / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C063905:procathepsin L; D056668:Cathepsin L