The first recombinant endostatin that elicited strong antitumor activity was expressed in Escherichia coli and administered as a suspension. Under these conditions, the protein retained its full antiangiogenic activity. Lack of requirement for a folded structure prompted us to investigate antitumor properties of synthetic peptides corresponding to different regions of endostatin. Here, we show that the entire antitumor, antimigration, and antipermeability activities of endostatin are mimicked by a 27-amino-acid peptide corresponding to the NH 2 -terminal domain of endostatin. This peptide contains three histidines that are responsible for zinc binding. Mutations of the zinc-binding histidines abolished its antitumor and antimigration activities, but not antipermeability properties. (Cancer Res 2005; 65(9): 3656-63)

Purpose: The half-life of the antiangiogenic molecule endostatin that has been used in clinical trial is short (f2 h). In addition, f50% of the clinical grade endostatin molecules lack four amino acids at their NH 2 termini. Lack of these amino acids gives rise to a molecule that is devoid of zinc, resulting in no antitumor activity. Our goal was to develop a new version of endostatin that does not show such deficiency. Experimental Design: A recombinant human endostatin conjugated to the Fc domain of IgG was constructed and expressed in mammalian cell culture. The presence of Fc has been shown by previous investigators to play a major role in increasing the half-life of the molecule. Fc-endostatin was tested in tumor-bearing mice, and its half-life was compared with the clinical grade endostatin. Results: The antitumor dose of Fc-endostatin was found to be f100 times less than the clinical grade endostatin. The half-life of Fc-endostatin in the circulation was found to be weeks rather than hours, as observed for endostatin alone. In addition, a U-shaped curve was observed for antitumor activity of endostatin as a function of endostatin concentration delivered to the animals. Conclusion: Fc-endostatin is a superior molecule to the original clinical endostatin. Due to its long half-life, the amount of protein required is substantially reduced compared with the clinically tested endostatin. Furthermore, in view of the U-shaped curve of efficacy observed for endostatin, we estimate that the requirement for Fc-endostatin is f700-fold less than endostatin alone. The half-life of endostatin is similar to that of vascular endothelial growth factor^Trap and Avastin, two other antiangiogenic reagents. We conclude that a new clinical trial of endostatin, incorporating Fc, may benefit cancer patients.Endostatin, a proteolytic fragment from collagen 18, has been shown to be a potent antiangiogenic protein (1, 2). The antitumor activity of endostatin is well-established. At the time of this writing, there are >950 publications on endostatin. The antitumor properties of endostatin have been confirmed in the vast majority of these publications using a large variety of tumor models (2). The protein does not cause toxicity in patients.The mechanism of endostatin action is still not clear. Integrin a5h1 has been implicated by binding endostatin and inhibiting angiogenesis (3). Its generation from collagen 18 is regulated by cell pathways initiated by the master tumor suppressor p53 (4, 5). P53 up-regulates transcription of a (II) collagen prolyl-4-hydroxylase, releasing tumstatin and endostatin from collagen 4 and 18, respectively.Clinical trials of human endostatin in phase I and II used a recombinant molecule that was expressed in yeast. This formulation of endostatin carried two major handicaps. The half-life of the protein in circulation was very short. The decay of endostatin represented a biexponential model that resulted in a half-life (t 1/2 ) of 42.3 min and a h t 1/2 of 12.9 h (6). The second problem, which has...

Developing continuous systemic delivery of endostatin has been a goal of many laboratories. We have employed a method of gene therapy utilizing different viral constructs. Here, we report that a new serotype of adeno-associated viruses, which incorporates canine endostatin, provides dose-dependent transgene expression in the circulation after intramuscular injection in mice. Elevated levels of endostatin remained stable in the circulation for at least 4 months. In vitro assays determined that the protein expressed was biologically active. Antitumor activities of the above construct demonstrated a U-shape curve, where the maximum activity was observed within a certain critical concentration range. These data suggest that an optimum dose range may be required to achieve therapeutic efficacy in large animal models.