(621b) Treatment Tests of Breast Cancer in Mice Using a Vascular-Targeted Enzyme Prodrug Therapy

Metastatic breast cancer is usually not curable.  The median survival of women with metastatic breast cancer is approximately two years after documentation of metastasis.  Treatment for most patients with metastatic breast cancer is considered palliative in nature, although durable complete remissions have been achieved in a small number of patients (< 5%).  Some disease response has been demonstrated with various chemotherapeutic agents; however, these agents are almost always associated with undesirable side effects such as nausea, vomiting, hair loss, cognitive dysfunction, and fatigue.  Recent research has centered on therapies that are directed only towards malignant cells. 

This work is on a novel enzyme prodrug therapy using a fusion protein containing annexin V to target only tumor vascular endothelial cells and cancer cells, reducing unwanted toxicity to healthy tissue.  The L-methioninase enzyme will convert the inert prodrug selenomethionine to toxic methylselenol and also deplete the cancer cells of methionine necessary for protein synthesis and continued growth.  The L-methioninase enzyme is linked to human annexin V protein in a fusion protein.  This therapy takes advantage of the fact that phosphatidylserine (PS), an anionic phospholipid, is exposed almost exclusively on the outer membrane of cancer and endothelial cells of the tumor vasculature and that annexin V binds specifically to PS. 

Recombinant technology was used to express and purify the methioninase-annexin V fusion protein.  During purification, a (His)₆ tag was cleaved from the fusion protein, and endotoxin was removed.  Pharmacokinetic testing in mice revealed complete clearance of the fusion protein from the bloodstream to occur within 12 hours following intraperitoneal (i.p.) injection.  The enzyme prodrug system was tested in SCID (severe combined immune deficient) female mice with implanted with MDA-MB-231 human breast tumors and was found to significantly inhibit tumor growth compared to three control groups of mice (untreated, fusion protein only, and selenomethionine only).  The fusion protein and the prodrug were delivered by i.p. injection.  The MDA-MB-231 cancer cells had been transfected with green fluorescent protein (GFP) to follow the growth of the tumors using a small animal fluorescence imaging system.  Results will also be reported of a separate study of this enzyme prodrug therapy using mice fed a diet deficient in methionine but supplemented with homocysteine.