RESUMEN
A recombinant immunotoxin was constructed from the hybridoma antibody TH-69 directed against human CD7, a surface antigen of leukemic T cells. The antibody was subcloned as a single chain Fv (scFv) fragment and genetically linked to a truncated Pseudomonas exotoxin A fragment containing the catalytic domains II and III but lacking the receptor binding domain I. Domain I was replaced by the scFv, thus conferring restricted specificity for CD7-positive cells. The bacterially expressed and purified toxin retained binding specificity for CD7-positive cells. It promoted apoptosis in two CD7-positive cell lines derived from T-lineage acute lymphoblastic leukemias, CEM and Jurkat, but not in the CD7-negative B-lymphoid lines REH, Nalm-6, and SEM. Maximum killing in excess of 95% was reached after 96 h in CEM and Jurkat cells with a single dose of 100 ng/ml. Cells treated with a similarly constructed scFv-exotoxin A immunotoxin against melanoma-associated chondroitin sulfate proteoglycan, an antigen absent from leukemic T cells, remained unaffected. Lysis of target cells occurred via apoptosis as evidenced by staining with Annexin V and specific cleavage of poly(ADP-ribose) polymerase. Approximately 20% of leukemic cells from a patient with CD7-positive acute T-cell leukemia kept in long-term primary culture for 30 cell generations were killed within 96 h after treatment with the toxin. These findings justify further evaluation of the agent in view of potential therapeutic applications.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Antígenos CD7/inmunología , Apoptosis/efectos de los fármacos , Toxinas Bacterianas , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Leucemia de Células T/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasas , Factores de Virulencia , Animales , Especificidad de Anticuerpos , Apoptosis/inmunología , Células COS , Niño , Epítopos/inmunología , Femenino , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/farmacología , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Lactante , Células Jurkat/citología , Células Jurkat/efectos de los fármacos , Leucemia de Células T/inmunología , Leucemia de Células T/patología , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMEN
A family of interleukin-10 (IL-10)-related cytokines has emerged, comprising a series of herpesviral and poxviral members and several cellular sequence paralogs, including IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)] and IL-26 (AK155). Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. This leads, through the activation of signal transducer and activator of transcription (STAT) factors, to diverse biological effects. For example, whereas IL-10 is a well-studied pleiotropic immunosuppressive and immunostimulatory cytokine, IL-22/IL-TIF mediates acute-phase response signals in hepatocytes and IL-20 induces the hyperproliferation of keratinocytes, which has been proposed as a pathogenic mechanism of psoriasis.