(490d) Targeting L-Methioninase to the Vasculature of Tumors

It is well known that tumor cells of all types have an elevated growth requirement for methionine compared to normal cells. Many cancer cell lines are unable to survive and grow when methionine is replaced in the medium with homocystine. However, normal adult cell lines survive and grow well with this substitution. A means of eliminating methionine without harming normal cells is by addition of the enzyme L-methioninase, which converts methionine to alpha-ketobutyrate.

Recently, it has been discovered that phosphatidylserine (PS) is exposed on the surface of the vascular endothelium of blood vessels in tumors but not on normal endothelium. Human annexin V, a natural ligand that binds to anionic phospholipids, specifically localizes to vascular endothelium of tumor tissue, but none is detected on endothelium of normal tissue.

The basic idea of this project is that a fusion protein (FP) consisting of L-methioninase linked to annexin V that is injected into the bloodstream will bind to the vascular endothelium in tumors only and will prevent methionine in the bloodstream from reaching the tumor, thereby killing the tumor and/or inhibiting its growth. The use of this FP has the great advantage that it does not have to be delivered directly to the tumor cells but only to the bloodstream, thus overcoming a major disadvantage of protein-based therapeutics for cancer treatment.

A process was developed to produce the FP in Escherichia coli and then purify it. The gene for L-methioninase from Pseudomonas putida was joined to the gene for human annexin V, and the fusion gene was ligated to the pET 30 Ek/LIC vector and transformed into E. coli strain BL21(DE3). This vector adds an amino-terminal 6xHis tag to the overexpressed protein. In the cloning, the gene for the HRV 3C protease was added just ahead of the ATF gene. The FP was overexpressed and then was purified to homogeneity by immobilized metal affinity chromatography. The 6xHis tag was cleaved off using HRV 3C protease. The L-methioninase activity of the FP was measured to be 8.6 U/mg protein.

The purified FP was tested in two binding assays to demonstrate specific binding to PS. In the first assay, binding was measured over a wide range of FP concentrations using PS immobilized on plastic microtiter plates. An L-methioninase primary antibody and a secondary antibody coupled to horseradish peroxidase were used for detection. Absorbance at 450 nm of the chromogenic substrate O-phenylenediamine increased as the concentration of the FP increased; negligible absorbance was obtained when L-methioninase was used instead of FP at the highest concentration. In the second assay, MCF-7 breast cancer cells were grown to confluence in microtiter plates, and hydrogen peroxide at a concentration of 1 mM was added to induce the exposure of PS on the external surface of the cells. The cells were then fixed with glutaraldehyde, and binding of FP was measured over a wide range of FP concentrations. The same procedure with an L-methionionase primary rabbit antibody and secondary rabbit antibody coupled to horseradish perixiodase was used for detection of bound FP. Again, absorbance at 450 nm increased with FP concentration, and there was negligible absorbance when L-methioninase was used instead of FP at the highest concentration.