A novel Mutein of TNFα Containing the Arg-Gly-Asp Sequence Shows Reduced Toxicity in Intestine

The effects of human tumour necrosis factor-α (TNFα), or its mutein (F4168) having the cell adhesive Arg-Gly-Asp sequence at the N-terminus, on intestinal injury, were examined. Histopathological examination revealed that an intravenous injection of TNFα resulted in marked haemorrhage or oedema in the caecum of rats, whereas F4168 showed no such effects even at the same therapeutic dose. Moreover, the number of neutrophils that adhered to endothelial cells or infiltrated the mucosal tissue was much higher after TNFα injection compared with F4168 in vivo. The enhanced adhesion of neutrophils on to human umbilical vein endothelial cells also occurred when the latter were pre-stimulated with TNFα but not with F4168 in vitro. The expression of the cell adhesion molecules including endothelial leukocyte adhesion molecule-1 or intercellular adhesion molecule-1 on F4168- stimulated human umbilical vein endothelial ceils was significantly lower than that stimulated with TNFα. These results suggest that the Arg-Gly-Asp sequence introduced into the TNFα molecule abrogates the side effect of this cytokine such as tissue injury or shock, and that F4168 could be useful for systemic therapy.


Introduction
TNFot is a multi-functional cytokine which was originally identified as a serum factor that induced necrotic responses of solid tumours in vivo. Although TNFot exerts antitumour effects against various tumour cell lines, >3 it also has hypotensive 4,5 or tumour metastasis-enhancing effects. Moreover, TNFot induces shock and injury including intestinal necrosis in rats or mice when it is administered with or without LPS. 4'5'9-1 Thus, for clinical applications, it is necessary to reduce these side-effects of TNF0t while maintaining its antitumour potential. It was demonstrated previously that a TNF mutein (F4236) having the laminin derived cell-adhesive peptide (YIGSR) did not enhance experimental pulmonary metastasis in contrast to wild-type TNF. An Arg-Gly-Asp (RGD) containing mutein (F4168) also showed similar properties. Here, the effect of F4168 on inflammatory reactions, including shock and tissue injury in rats, has been examined.

Materials and Methods
Construction of expression plasmids: The human TNF0t gene prepared with synthetic oligonucleotides was used to construct the expression plasmid pKF4102 for wild-type TNF0t. The expression plasmid pKF4168 was used for the mutein (F4168) having the tripeptide Arg-Gly-Asp (RGD) near the N-terminus of human TNF. This was introduced into the wild-type TNF gene by site-directed mutagenesis. 1" The construction of pKF4168 involved the substitution of Gly-Asp for the 5Thr-6Pro of wildtype TNF0t. Both pKF4102 and pKF4168 have the tac promoter.
Production and purification of TNF and F4168: The expression plasmids were induced by adding IPTG (isopropyl--D-thio-galactopyranoside) into the Escherichia coli (K12 JM103) culture medium (M9). These proteins were expressed in E. coli, extracted by sonication and purified to homogeneity by sequential anion exchange chromatography using Sepabeads FP-DA13 (Mitsubishi Chemical Industries Co., Tokyo, Japan) and Mono Q (Pharmacia LKB Biotechnology, Uppsala, Sweden). The specific activity of both TNF and F4168 was 3 x 107 units/mg protein. The protein content was determined by a dye-binding method. 13 The effect of TNF or F4168 on tissue injury: Male Sprague-Dawley rats (220+_25 g; Crj; CD) were housed in standard cages and given food and water ad libitum in a temperature controlled room (24 _+ 1C) with a 12 h dark/light cycle until surgery. TNF and F4168 dissolved in saline were intravenously injected at a dose of 20 l.tg/500 l.d/rat. The animals were sacrificed 6 h after injection using CO,.

Results
The acute toxicity caused by the intravenous injection of TNFcz or F4168 in rats has been examined.
Since the maximal hypotensive effect in rats and serious tissue injury in mice were observed 6 to 8 h after TNFz injection, the rats were killed for necropsy and histopathological examinations 6 h after TNFz or F4168 injection.
At necropsy, several gross abnormalities of the caecum including marked thickening of the walls or focal haemorrhage were apparent in rats injected with TNF0t. Histopathological sections through the caecum 6 h after TNF0t injection (20 lag) revealed an inflammatory response with haemorrhage in the lamina propria mucosae or infiltration of leukocytes (Fig. 1A). The venule of the tunica submucosa was dilated, and invasion of leukocytes was also observed. A detailed histopathological examination of the venule revealed the adhesion of neutrophils on the vessel wall or infiltration into the surrounding mucosal tissues (Fig. 1B). In contrast, rats injected with F4168 had a reduced inflammatory response compared with the TNF0t administered group. Haemorrhage was rarely observed (Fig. 1D), and the number of lymphocytes or neutrophils adhering or infiltrating in mucosa was relatively small in the F4168 administered group (Fig. 1E). Table 1 shows the comparison of the effects of TNFcz and F4168 on several histopathological findings. Similar results were obtained for the duodenum of mice injected with TNFc or F4168 (data not shown).
The effect of TNF0t or F4168 on the neutrophil adhesion to HUVEC in vitro (Fig. 2) also was examined. Human neutrophils were overlaid on HUVEC prestimulated with 0.1 ng/ml of TNF0t or F4168, and the adhesion during 30 min incubation at 37C was Fig. 1. The histopathological appearance of the caecum in Sprague-Dawley rats given TNF or F4168. TNF(20 lg/rat) (Panels A, B and C) or F4168 (20 lg/rat) (Panels D and E) was administered intravenously. Six hours later, the rats were sacrificed for histopathological examination. A and D, x 100; B and E, x 400; C, x 800. There is a large number of neutrophils adhering to the vascular endothelium. evaluated. The number of adherent neutrophils onto the F4168-stimulated HUVEC was significantly lower than that in TNF{x (154 _+ 30 vs. 236 + 49 cells/mm2, p< 0.01). Similar results were also obtained when HUVEC were prestimulated with 1 ng/ml of each factor (data not shown).
The TNF0t or F4168 induced ELAM-1 on HUVEC were next examined. Although the initial expression rate of ELAM-1 by TNF or F4168 (0.1 ng/ml, each) was almost the same, the maximal expression (4 or 6 h after stimulation) by F4168 was significantly lower than that by TNF0t (Fig. 3). Moreover, the ELAM-1 induction by TNF0t was dose dependent at concentrations of 0.01 to 10 ng/ml (Fig. 4). F4168, however, induced a relatively low level of ELAM-1 expression compared with that by TNF{x at any  concentration tested except 10 ng/ml (Fig. 4). Similar results were also obtained with respect to ICAM-1 (Fig. 5B), suggesting that the RGD sequence in F4168 reduces the enhanced ELAM-1 or ICAM-1 inducibility of TNF0t.
Although the GRGDS peptide did not affect the ondary' inflammation. The 'primary inflammation' occurs immediately after TNF0t injection and includes haemorrhage, thrombus formation, congestion of capillary vessels and complete loss of blood circulation followed by the necrosis of tumour vasculature. 15 At the same time, adhesion or infiltration of neutrophils or macrophages on vessel walls is promoted, which might result in the destruction of tumour vasculature. These sequential reactions at the early stage are thought to be mainly concerned with the expression of the antitumour activity of TNF.
This study indicates that F4168 is less toxic to the intestine than is TNF. Based on the histopathological findings ( Fig. 1 and Table 1), it is speculated that this difference between F4168 and TNF is accompanied by a varied extent of secondary inflammation, especially the accumulation of activated neutrophils at the inflammatory site. It has been reported that the binding of leukocytes to endothelial cells is mediated by cell adhesion molecules belonging to the selectin or immunoglobulin family. [19][20][21] The present results show that ELAM-1 or ICAM-1 expression on the endothelial cell surface is up-regulated by TNF stimulation (Figs 3 and 4), which could be correlated with the enhanced neutrophil binding to endothelial cells (Fig. 2). Similar results have been reported by Pober et a/. [21][22][23] In contrast, F4168 induced a much lower level of ELAM-1 or ICAM-1 expression (Fig. 5) and neutrophil binding (Fig. 2) on endothelial cells compared with that of TNF0t. Therefore, F4168 should induce neither excessive rolling and infiltration of neutrophils nor excessive secondary inflammation in vivo. This may explain why F4168 does not induce serious intestinal injury.
It is of particular note that a synthetic GRGDS pentapeptide has the potential to reduce TNF0 mediated ICAM-1 expression (Fig. 5B), because this fact could provide a better understanding of the mechanisms by which F4168 shows much lower inducibility of cell adhesion molecules. Since F4168 (but not TNF00 mediates the adhesion and spreading of endothelial cells onto an inert substrate (data not shown), it is possible that RGD-directed receptor is involved in the binding of F4168 with endothelial cells. As described above, stimulation by F4168 through the TNF receptor up-regulates the expression of adhesion molecules. In contrast, stimulation through RGD receptors (integrins) may reduce the excessive TNF mediated expression of adhesion molecules. Moreover, it is suspected that the RGD domain in F4168 functions more effectively than an RGD oligopeptide, because TNF functions as an effective carrier protein for it. Thus, binding between the RGD and integrin could effectively occur. Another possibility is that truncated F4168, whose TNF activity is abolished as a result of digestion by proteases secreted from activated neutrophils, [24][25][26] may also express RGD-derived functions more effectively in vivo. In addition to the low toxic effect of F4168 on intestinal function, this novel mutein has the same antitumour potential in mice transplanted with Meth A fibrosarcoma (Fig. 6). Therefore, this mutein should be a useful antitumour drug without the side effects of wild-type TNF0t.