RESUMEN
We have shown previously that high-affinity receptors for interleukin-13 (IL-13Ralpha2) are overexpressed on a variety of solid cancer cells, diseased fibroblasts, and other cells, and a chimeric fusion protein composed of human IL-13 and mutated Pseudomonas exotoxin (IL-13-PE38) is highly and specifically cytotoxic to these cells in vitro and in vivo. To improve the specificity for the target, we isolated specific antibodies against IL-13Ralpha2 from human single-chain Fv (scFv) antibody phage library and developed immunotoxin by selecting two high-affinity clones of scFv and fused to PE. The fusion chimeric gene was expressed in Escherichia coli, and highly purified IL-13R-specific immunotoxin, termed anti-IL-13Ralpha2(scFv)-PE38, was tested for its cytotoxicity. This molecule was highly cytotoxic to U251 glioma and PM-RCC renal cell carcinoma cell lines in vitro. The cytotoxic activity was neutralized by purified extracellular domain of IL-13Ralpha2 but not by IL-13, indicating that cytotoxic activity is specific. Anti-IL-13Ralpha2(scFv)-PE38 showed significant antitumor activity in immunodeficient mice with s.c. glioma tumors. Both i.p. and i.t. routes of administration showed antitumor activity in a dose-dependent manner. The maximum tolerated dose of anti-IL-13Ralpha2(scFv)-PE38 was 200 microg/kg i.p. twice daily for 5 days. These results indicate that anti-IL-13Ralpha2(scFv)-PE38 is a highly selective therapeutic agent for cancer therapy and should be further tested in animal models of human cancer.
Asunto(s)
Anticuerpos/farmacología , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Mutación/genética , Neoplasias/metabolismo , Pseudomonas/metabolismo , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Células Clonales , Electroforesis en Gel de Poliacrilamida , Glioblastoma/metabolismo , Humanos , Región Variable de Inmunoglobulina , Inmunotoxinas/aislamiento & purificación , Subunidad alfa2 del Receptor de Interleucina-13/química , Ratones , Ratones Desnudos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A high-affinity receptor for interleukin (IL)-13 (interleukin-13R alpha 2) is over-expressed in disease-related fibroblasts and neoplastic cells and is involved in cancer, allergic, and inflammatory diseases. The extracellular domain of IL-13R alpha2 (ECD alpha2) could be cleaved, which serves as a decoy receptor. We have expressed and purified ECD alpha2 in both Escherichia coli (E. coli) and mammalian systems as a soluble fragment and studied its biological activities. Although both products of ECD alpha2 showed IL-13 inhibitory activities, mammalian cell-derived ECD alpha2 appeared to be superior compared with purified protein from E. coli. When expressed in E. coli, ECD alpha2 appeared to be a monomer of 42 but a 60 kDa protein when purified from mammalian cells due to heavy glycosylation. The purified glycosylated ECD alpha2 efficiently inhibited IL-13-induced STAT6 phosphorylation in immune and Hodgkin's lymphoma cell lines, IL-13 binding, and cytotoxicity of IL-13 cytotoxin in various cancer cell lines. The improved potency of mammalian cell-derived ECD alpha2 was shown over ECD alpha2/Fc fusion protein. The N-linked glycosylation of ECD alpha2 was found to be essential for optimal IL-13 inhibitory activity as deglycosylation by PNGase F showed lower activity. ECD alpha2 did not inhibit IL-4-induced STAT6 phosphorylation, indicating that inhibitory effects of ECD alpha2 are receptor specific. These results indicate that glycosylated ECD alpha2 can serve as a potent inhibitor of IL-13 in a variety of conditions in which IL-13 is a key mediator, e.g., pulmonary, allergic, fibrotic, and neoplastic diseases.
Asunto(s)
Subunidad alfa1 del Receptor de Interleucina-13/fisiología , Interleucina-13/fisiología , Animales , Secuencia de Bases , Células COS , División Celular , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Glicosilación , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/genética , Ratones , Plásmidos , Proteínas Recombinantes/farmacología , RetroviridaeRESUMEN
Ionizing radiation (IR) and concomitant angiostatin (AS) produce greater than additive local antitumor effects. We examined whether prolonged AS treatment added to IR reduces proliferation of lung metastases from LLC primary tumors. Flank tumors were treated with 40 Gy with or without AS (25 mg/kg/day). IR plus a 14-day course of AS improved local tumor control and blocked the increase in lung weights observed in the group receiving IR plus a 2-day course of AS group. Animals treated with prolonged AS exhibited no increase in lung weight and no macrometastases. These findings suggest that long-term treatment with antiangiogenic compounds may be effective in preventing metastases from IR-treated tumors as well as increasing the local antitumor effects of radiotherapy.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/radioterapia , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Fragmentos de Péptidos/farmacología , Plasminógeno/farmacología , Angiostatinas , Animales , Carcinoma Pulmonar de Lewis/secundario , Terapia Combinada , Femenino , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , Ratones , Ratones Endogámicos C57BLRESUMEN
PURPOSE: We examined the interaction between cyclophosphamide (CPA) and angiostatin (AS) on the growth of primary Lewis lung carcinoma (LLC) tumors and on the development of LLC pulmonary metastases. We studied the effects of AS and CPA on the stages of angiogenesis employing in vitro assays. METHODS: Primary tumor growth and pulmonary metastases were measured to evaluate the effects of treatment with AS alone, CPA alone or the combination of CPA and AS. We examined the effects of CPA plus AS on endothelial cell (HUVEC) survival, migration and tube formation. RESULTS: Combined treatment with CPA and AS did not significantly affect primary tumor growth when compared with CPA treatment alone. However, a significant decrease in the number of pulmonary metastases was observed following CPA plus AS treatment when compared with CPA treatment alone ( P<0.001). AS did not enhance CPA-mediated HUVEC cytotoxicity, and CPA failed to enhance AS-mediated inhibition of migration. However, tube formation was inhibited following combined treatment with CPA and AS when compared with either treatment alone. CONCLUSIONS: AS enhanced the antimetastatic effects of CPA without significantly influencing the effects of CPA on primary tumor growth. CPA plus AS inhibited tube formation, suggesting that interrupting specific steps in the angiogenesis process might be an effective approach to the treatment of subclinical distant metastases.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacología , Metástasis de la Neoplasia/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Plasminógeno/farmacología , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Angiostatinas , Animales , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/secundario , Movimiento Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Ciclofosfamida/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Modelos Lineales , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos C57BL , Morfogénesis/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Plasminógeno/administración & dosificación , Inhibidores de la Síntesis de la Proteína/administración & dosificación , Inhibidores de la Síntesis de la Proteína/farmacología , Método Simple Ciego , Células Tumorales Cultivadas/trasplanteRESUMEN
BACKGROUND: The ability to infect tumor cells limits the antitumor effects of gene therapy. The addition of radiotherapy to treatment with Ad.Egr.TNF.11D, a replication-deficient adenovirus containing a radiation-inducible promoter, early growth response-1, and the tumor necrosis factor-alpha (TNFalpha) complementary DNA may enhance the therapeutic ratio. METHODS: Seg-1 human esophageal adenocarcinoma cells were treated with Ad.Egr.TNF.11D with or without radiation. TNFalpha levels were quantified with enzyme-linked immunosorbent assay. Athymic nude mice bearing Seg-1 tumors were randomized to buffer, ionizing radiation, Ad.Egr.TNF.11D, and combination therapy. Tumor growth delay was used to compare treatment regimens. TNFalpha levels were measured in tumor homogenates and plasma. RESULTS: Seg-1 cells treated with Ad.Egr.TNF.11D and ionizing radiation demonstrated increased TNFalpha levels at 72 hours compared with cells exposed to vector alone (124 +/- 0 pg/mL vs. 31.11 +/- 22 pg/mL; P =.008). In vivo, Ad.Egr.TNF.11D-treated tumors expressed low TNFalpha levels (151.5 +/- 107.11 pg/mg protein) compared with tumors receiving combined treatment (793.92 +/- 489.13 pg/mg protein; P =.067). Increased TNFalpha levels were associated with increased tumor growth delay after combined treatment (P <.05). CONCLUSIONS: Radiotherapy enables focal stimulation of TNFalpha expression in Ad.Egr.TNF.11D-infected cells and thus improves local tumor control.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/radioterapia , Proteínas de Unión al ADN/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Proteínas Inmediatas-Precoces , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Adenoviridae , Animales , Terapia Combinada , Proteínas de Unión al ADN/biosíntesis , Progresión de la Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz , Vectores Genéticos , Humanos , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas , Radioterapia/métodos , Factores de Transcripción/biosíntesis , Trasplante Heterólogo , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
Infection of tumors with an adenoviral vector expressing a chimeric gene composed of the CArG elements of the Egr-1 promoter and a cDNA encoding TNF-alpha (Ad.Egr-TNF) has previously been shown to result in the production of high intratumoral levels of TNF-alpha and thereby tumor regression. The antitumor effects of TNF-alpha were ascribed to vascular thrombosis. We and others, have reported that inhibition of tumor vessel thrombosis using anticoagulation therapy does not abrogate the antitumor effects after TNF-alpha treatment. To investigate the potential antiangiogenic effects of TNF-alpha, we studied the generation of angiostatin after intratumoral injection of Ad.Egr-TNF. We report an increase in plasma angiostatin levels both during and after treatment with Ad.Egr-TNF that parallel tumor regression. We also report that TNF-alpha enhances angiostatin production by inducing the activity of plasminogen activator and the release of MMP-9 by tumor cells. These studies support a model in which the antiangiogenic effects of TNF-alpha on the tumor microvasculature are mediated by generation of angiostatin.