RESUMO
The emergence of targeted cancer therapy has been limited by the paucity of determinants which are tumor-specific and generally associated with disease, and have cell dynamics which effectively deploy cytotoxic payloads. Guanylyl cyclase C (GUCY2C) may be ideal for targeting because it is normally expressed only in insulated barrier compartments, including intestine and brain, but over-expressed by systemic metastatic colorectal tumors. Here, we reveal that GUCY2C rapidly internalizes from the cell surface to lysosomes in intestinal and colorectal cancer cells. Endocytosis is independent of ligand binding and receptor activation, and is mediated by clathrin. This mechanism suggests a design for immunotoxins comprising a GUCY2C-directed monoclonal antibody conjugated through a reducible disulfide linkage to ricin A chain, which is activated to a potent cytotoxin in lysosomes. Indeed, this immunotoxin specifically killed GUCY2C-expressing colorectal cancer cells in a lysosomal- and clathrin-dependent fashion. Moreover, this immunotoxin reduced pulmonary tumors>80% (p<0.001), and improved survival 25% (p<0.001), in mice with established colorectal cancer metastases. Further, therapeutic efficacy was achieved without histologic evidence of toxicity in normal tissues. These observations support GUCY2C-targeted immunotoxins as novel therapeutics for metastatic tumors originating in the GI tract, including colorectum, stomach, esophagus, and pancreas.
Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias Gastrointestinais/tratamento farmacológico , Imunotoxinas/farmacologia , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Ricina/farmacologia , Animais , Anticorpos Monoclonais/administração & dosagem , Caveolinas/metabolismo , Linhagem Celular Tumoral , Clatrina/metabolismo , Portadores de Fármacos , Endocitose , Neoplasias Gastrointestinais/patologia , Imunoterapia , Imunotoxinas/administração & dosagem , Lisossomos , Camundongos , Terapia de Alvo Molecular , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Receptores de Enterotoxina , Ricina/administração & dosagem , Ricina/genéticaRESUMO
Virulent tissue culture-derived Trypanosoma cruzi trypomastigotes were readily killed by immune IgG-ricin A chain conjugates (ITR) in vitro. Forty micrograms of ITR immobilized 10(6) trypomastigotes after 48 hours of incubation at 37 degrees C. ITR showed antibody specificity and 125I-labeled anti-T. cruzi IgG bound to parasitized host cells 9-fold more than to nonparasitized host cells. The degree of specificity was evaluated further in experiments in which 10 micrograms of ITR showed 78% inhibition of [3H]thymidine incorporation by T. cruzi. In contrast, nonimmune IgG-ricin A chain conjugate neither immobilized nor inhibited [3H]thymidine incorporation by the parasite. Furthermore, 20 micrograms of ITR significantly inhibited T. cruzi trypomastigote release from infected host cells and thus prevented reinfection of other cells in vitro.