Vascular infarction by subcutaneous application of tissue factor targeted to tumor vessels with NGR-peptides: Activity and toxicity profile

Abstract

Abstract. 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 · v ß 3 (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 (LD 10 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.

Targeting Tissue Factor to Tumor Vasculature to Induce Tumor Infarction

“…In 1998, stimulated by P. Thorpe’s work [ 28 ], our laboratory started to design a group of new bifunctional tTF fusion proteins to target procoagulatory activity selectively into the tumor vasculature and to induce tumor infarction [ 48 , 51 , 63 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ].…”

“…tTF-NGR retained the procoagulatory activity of TF in a FX/FXa assay [ 69 , 70 , 71 ] and it revealed specific binding to the respective target molecules (CD13) on stimulated endothelial cells in vitro [ 70 , 71 , 72 ] and to tumor endothelium in vivo [ 65 ]. Multiple imaging techniques showed in vivo accumulation of tTF-NGR in tumor tissues and its in vivo-induced activation of coagulation with subsequent tumor vascular occlusion and inhibition of tumor vessel blood flow upon systemic application [ 67 , 68 , 69 , 70 , 71 , 72 , 75 , 80 ]. In contrast, these central activities were not observed when using tTF without a targeting moiety.…”

The Evolution of Safe and Effective Coaguligands for Vascular Targeting and Precision Thrombosis of Solid Tumors and Vascular Malformations

“…In the same model, Huang et al later demonstrated induction of significant intravascular thrombosis and tumor necrosis, tumor growth inhibition, and increased survival using a construct with three RGD repeats fused to tTF [75]. Multiple other studies have investigated the efficacy of RGD and NGR-targeted coaguligands, primarily fused to the C-terminal region of tTF, with pre-clinical testing performed in human breast cancer (SKBR3 and MDA-MB-435), lung adenocarcinoma (A549 and MAD109), human fibrosarcoma (HT1080), glioblastoma (U87), melanoma (M21), small cell lung cancer (HTB119), and colorectal (CT26) cancer models (Table S1) [14,[76][77][78][79][80][81][82][83]. A study by Hallahan et al used embolic nanoparticles rather than tTF to target integrin α2bβ3 on radiation-primed tumor vessels [84].…”

“…Numerous molecules expressed exclusively at the surface of tumor endothelium have since been identified [12,13]. However, while the use of targeted agents increased selectivity, the issue of off-target toxicity related to the delivery of cytotoxic agents still remained [14,15].…”

Fusion protein tTF-EG3287 induces occlusion of tumor vessels and impairs tumor growth in human colon cancer

“…Kessler, et al has proven that the fusion of integrin α5β1 with tTF could induce embolization of the tumor blood vessel in adenocarcinoma, melanoma and fibrosarcoma, thereby suppressing the tumor growth [13]. The tTF-NGR designed by Christian, et al reduced the growth of fibrosarcoma transplanted tumors [14]. As an effector molecule targeting tumor vascular embolism, we need to explore tTF-related targeted drugs for colon cancer treatment.…”