RESUMEN

We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker. The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10%. Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule. The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin. However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin. Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.

Asunto(s)

ADP Ribosa Transferasas/farmacología , Toxinas Bacterianas/farmacología , Disulfuros/química , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Mesotelioma/inmunología , Neoplasias Ováricas/inmunología , Factores de Virulencia/farmacología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/farmacocinética , Animales , Anticuerpos Monoclonales , Antígenos de Neoplasias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Exotoxinas/farmacocinética , Femenino , Proteínas Ligadas a GPI , Expresión Génica , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Inmunotoxinas/genética , Inmunotoxinas/farmacocinética , Glicoproteínas de Membrana/metabolismo , Mesotelina , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Plásmidos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/farmacocinética , Exotoxina A de Pseudomonas aeruginosa