Simplified production of a recombinant human angiostatin derivative that suppresses intracerebral glial tumor growth

Angiostatin is an endogenous inhibitor of tumor neovascularization that inh ibits the proliferation of endothelial cells. Production of sufficient quan tities of biologically active angiostatin by the enzymatic cleavage of plas minogen has proven difficult in that it has delayed clinical testing. We ha ve cloned, expressed, and purified a recombinant human angiostatin derivati ve (K1-3) using a mammalian expression system. Through the addition of a se cretory signal and polyhistidine sequence tag, K1-3 can be purified from po st-culture medium by simple column chromatography. Purified K1-3 protein is apparently folded in an active conformation, as evidenced by its ability t o bind to lysine-Sepharose, In vitro, recombinant K1-3 significantly suppre ssed endothelial cell proliferation in a dose-dependent manner with an IC50 of 50 nM. Using an animal model of intracranial brain tumors in immune-com petent rats, systemic administration of purified recombinant K1-3 resulted in up to 85% suppression of tumor growth (P = 0.011), Growth suppression wa s accompanied by a 32% decrease (P = 0.01) in tumor neovascularization. This study demonstrates a simple method to produce a biologically active re combinant angiostatin derivative. The ability to suppress intracerebral tum or growth after systemic administration suggests that K1-3 is likely to hav e therapeutic value in the treatment of malignant glial tumors.