RESUMEN
Aminopeptidase N (CD13) is expressed on tumor vasculature and tumor cells. It represents a candidate for targeted therapy, e.g., by truncated tissue factor (tTF)-NGR, binding to CD13, and causing tumor vascular thrombosis. We analyzed CD13 expression by immunohistochemistry in 97 patients with STS who were treated by wide resection and uniform chemo-radio-chemotherapy. Using a semiquantitative score with four intensity levels, CD13 was expressed by tumor vasculature, or tumor cells, or both (composite value, intensity scores 1-3) in 93.9% of the STS. In 49.5% tumor cells, in 48.5% vascular/perivascular cells, and in 58.8%, composite value showed strong intensity score 3 staining. Leiomyosarcoma and synovial sarcoma showed low expression; fibrosarcoma and undifferentiated pleomorphic sarcoma showed high expression. We found a significant prognostic impact of CD13, as high expression in tumor cells or vascular/perivascular cells correlated with better relapse-free survival and overall survival. CD13 retained prognostic significance in multivariable analyses. Systemic tTF-NGR resulted in significant growth reduction of CD13-positive human HT1080 sarcoma cell line xenografts. Our results recommend further investigation of tTF-NGR in STS patients. CD13 might be a suitable predictive biomarker for patient selection.
RESUMEN
tTF-NGR consists of the extracellular domain of the (truncated) tissue factor (tTF), a central molecule for coagulation in vivo, and the peptide GNGRAHA (NGR), a ligand of the surface protein aminopeptidase N (CD13). After deamidation of the NGR-peptide moiety, the fusion protein is also a ligand for integrin αvß3 (CD51/CD61). Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR showed binding to specific binding sites on endothelial cells in vitro as shown by flow cytometry. Subcutaneous injection of tTF-NGR into athymic mice bearing human HT1080 fibrosarcoma tumors induced tumor growth retardation and delay. Contrast enhanced ultrasound detected a decrease in tumor blood flow in vivo after application of tTF-NGR. Histological analysis of the tumors revealed vascular disruption due to blood pooling and thrombotic occlusion of tumor vessels. Furthermore, a lack of resistance was shown by re-exposure of tumor-bearing mice to tTF-NGR after regrowth following a first cycle of treatment. However, after subcutaneous (s.c.) push injection with therapeutic doses (1-5 mg/kg bw) side effects have been observed, such as skin bleeding and reduced performance. Since lethality started within the therapeutic dose range (LD10 approximately 2 mg/kg bw) no safe therapeutic window could be found. Limiting toxicity was represented by thrombo-embolic events in major organ systems as demonstrated by histology. Thus, subcutaneous injection of tTF-NGR represents an active, but toxic application procedure and compares unfavourably to intravenous infusion.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Infarto/inducido químicamente , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Tromboplastina/administración & dosificación , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/efectos adversos , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Línea Celular Tumoral , Células Cultivadas , Humanos , Inyecciones Subcutáneas , Ratones , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Oligopéptidos/efectos adversos , Oligopéptidos/química , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/química , Tromboplastina/efectos adversos , Tromboplastina/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A variety of fusion proteins consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) fused to the peptides GRGDSP (abbr. RGD), GNGRAHA (abbr. NGR) or derivates of these peptides, have been synthesized. These binding motif peptides target av-integrins or aminopeptidase N (CD13), respectively, on tumor endothelial cells. After expression and deposition as inclusion bodies in Escherichia coli BL21 (DE3), the tTF-fusion proteins were refolded and purified in a multi-step chromatography process. The upscaling process of fusion protein synthesis in order to produce amounts needed for clinical studies is presented. The proteins retained their specific proteolytic ability to activate FX by FVIIa and were able to bind to endothelial cells in vitro. Western blot analysis, analytic chromatography, FX coagulation assay and in vivo experiments have been performed to test for the in vitro stability of the tTF-NGR protein after long-term incubation at 5 degrees C or 25 degrees C, respectively. In vivo xenograft studies in nude mice bearing different malignant human tumors (mammary carcinoma SKBR3, adenocarcinoma of the lung A549) revealed that intravenous or subcutaneous administration of tTF-NGR or -RGD fusion proteins, but not the tTF protein without binding motif, induced thrombosis of tumor vessels which led to significant tumor growth retardation or regression. The anti-vascular mechanism of the tTF fusion proteins was verified by the molecular imaging methods such as magnetic resonance imaging (MRI) and fluorescence reflectance imaging (FRI); MRI showed a reduction of the relative tumor blood volume (BV) and FRI the formation of fibrin in the tTF-fusion protein treated tumors.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Tromboplastina/uso terapéutico , Animales , Antineoplásicos/farmacología , Vasos Sanguíneos/efectos de los fármacos , Escherichia coli/genética , Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias/patología , Oligopéptidos/genética , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Tromboplastina/genética , Tromboplastina/farmacologíaRESUMEN
We induced thrombosis of blood vessels in solid tumors in mice by a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA, targeting aminopeptidase N (CD13) and the integrin alpha(v)beta(3) (CD51/CD61) on tumor vascular endothelium. The designed fusion protein tTF-NGR retained its thrombogenic activity as demonstrated by coagulation assays. In vivo studies in mice bearing established human adenocarcinoma (A549), melanoma (M21), and fibrosarcoma (HT1080) revealed that systemic administration of tTF-NGR induced partial or complete thrombotic occlusion of tumor vessels as shown by histologic analysis. tTF-NGR, but not untargeted tTF, induced significant tumor growth retardation or regression in all 3 types of solid tumors. Thrombosis induction in tumor vessels by tTF-NGR was also shown by contrast enhanced magnetic resonance imaging (MRI). In the human fibrosarcoma xenograft model, MRI revealed a significant reduction of tumor perfusion by administration of tTF-NGR. Clinical first-in-man application of low dosages of this targeted coagulation factor revealed good tolerability and decreased tumor perfusion as measured by MRI. Targeted thrombosis in the tumor vasculature induced by tTF-NGR may be a promising strategy for the treatment of cancer.