Targeting Tissue Factor to Tumor Vasculature to Induce Tumor Infarction

Abstract

Besides its central functional role in coagulation, TF has been described as being operational in the development of malignancies and is currently being studied as a possible therapeutic tool against cancer. One of the avenues being explored is retargeting TF or its truncated extracellular part (tTF) to the tumor vasculature to induce tumor vessel occlusion and tumor infarction. To this end, multiple structures on tumor vascular wall cells have been studied at which tTF has been aimed via antibodies, derivatives, or as bifunctional fusion protein through targeting peptides. Among these targets were vascular adhesion molecules, oncofetal variants of fibronectin, prostate-specific membrane antigens, vascular endothelial growth factor receptors and co-receptors, integrins, fibroblast activation proteins, NG2 proteoglycan, microthrombus-associated fibrin-fibronectin, and aminopeptidase N. Targeting was also attempted toward cellular membranes within an acidic milieu or toward necrotic tumor areas. tTF-NGR, targeting tTF primarily at aminopeptidase N on angiogenic endothelial cells, was the first drug candidate from this emerging class of coaguligands translated to clinical studies in cancer patients. Upon completion of a phase I study, tTF-NGR entered randomized studies in oncology to test the therapeutic impact of this novel therapeutic modality.

Keywords: CD13; aminopeptidase N; solid tumors; tissue factor (TF); truncated and retargeted tissue factor tTF-NGR; tumor infarction; tumor vascular occlusion; vascular targeting.

Figure 1

Schematic structure of the tTF-NGR protein. The truncated tissue factor (tTF) in red, the C-terminal NGR-peptide binding motif in green, and the N-terminal Histidin tag (His tag) in orange. The molecular weight is 30,381.98 dalton, the NGR motif is located on the C-terminus of tTF. aa, amino acids.

Figure 2

Antimetastatic effect of tTF-fusion proteins tTF-NGR (NGR) and tTF-RGD (RGD). Antimetastatic effect of two tTF-fusion proteins systemically given at 3 mg/kg body weight every 2–3 days x5 in a syngeneic B-16 melanoma model growing in C57BL6 mice. Freedom from treatment failure: spontaneous metastasis after surgical removal of the subcutaneous primary tumors was clinically observed (e.g., inactivity, dyspnea, weight loss, fur changes), subsequently the mice were sacrificed (survival time) and the lungs of the animals were examined for lung metastasis. When compared with saline controls tTF-NGR revealed significant superiority (Mann-Whitney test, p = 0.009). PBS, saline control. Unpublished data.

Figure 3

Intratumoral doxorubicin concentration with or without subsequent vascular occlusion by systemic tTF-NGR. Fluorescence-based semi-quantification of doxorubicin tumor accumulation and wash-out kinetics without tTF-NGR (black line) versus with tTF-NGR (red line) applied 6 h after doxorubicin for the first time. The combination protocol significantly retarded wash-out times of doxorubicin from the tumor with prolonged high intratumoral drug levels in the tumor tissue after 65 and 120 h upon doxorubicin-tTF-NGR sequences as compared to control sequences of doxorubicin-saline (p = 0.0043 at 65 h, p = 0.0095 at 120 h, Mann–Whitney test). AU, arbitrary units; AUC, area under the curve; kg, kilogram body weight; t_1/2, tumor elimination half-life. For methodological details see Ref. [79].

Figure 4

Contrast-enhanced ultrasound (CEUS) imaging series of a lymphnode metastasis of a medullary thyroid carcinoma. (A). Shortly before treatment. (B). After 5 days of daily treatment with 4 mg/m² bsa tTF-NGR. Upper panels for (A,B) (u): Heat maps of dynamic CEUS. Color scales at the right lower corner of each photograph semiquantify contrast perfusion (red = high, blue/black = low). The green circles represent “regions of interest” (ROI) for the “total” lesion, the yellow circles represent the “lower half” of the lesion, and the red circles represent a “high perfusion” area of the lesion. Middle panels for (A,B) (m): CEUS arbitrary units (a.u.) for the wash-in perfusion index (WIAUC/RT; for the methodological details see [88]) representing the contrast flow through the vasculature of the lesion. For color representation of the 3 different ROI measured, see upper panel. Lower panels for (A,B) (l): Graphic follow-up of the wash-in perfusion index representing the contrast flow through the vasculature of the lesion for 70 sec starting with the injection of contrast via peak-enhancement until wash-out. For color representation of the 3 different “regions of interest” (ROI) see upper and middle panels.