Asunto(s)
Membrana Celular/metabolismo , Neoplasias/inmunología , Transducción de Señal , Linfocitos T/inmunología , Anticuerpos Biespecíficos/inmunología , Antígenos de Neoplasias/inmunología , Humanos , Sinapsis Inmunológicas/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Neoplasias/patología , Células THP-1RESUMEN
T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.
Asunto(s)
Anticuerpos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/metabolismo , Inmunoterapia/métodos , Linfocitos T/inmunología , Animales , Anticuerpos/genética , Antineoplásicos Inmunológicos/inmunología , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Efecto Espectador , Línea Celular Tumoral , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Medicina de Precisión/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF(SKP2) (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G(1) arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2(V617F), and TEL-PDGFRbeta, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2(-/-) marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2(+/+) marrow. SKP2(-/-) leukemic cells demonstrated higher levels of nuclear p27 than SKP2(+/+) counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF(SKP2) may be therapeutically useful.
Asunto(s)
Genes abl , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Animales , Secuencia de Bases , Trasplante de Médula Ósea , Ciclo Celular , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Cartilla de ADN/genética , Proteínas de Fusión bcr-abl , Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/etiología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos Mieloproliferativos/etiología , Trastornos Mieloproliferativos/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.