Polyethylene Glycol Conjugation of Recombinant Methioninase for Cancer Therapy

• Published in: Protein expression and purification 1998-02, Vol.12 (1), p.45-52
• Main Authors: Tan, Yuying, Sun, Xinghua, Xu, Mingxu, An, Zili, Tan, Xuezhong, Tan, Xiuying, Han, Qinghong, Miljkovic, Dusan A., Yang, Meng, Hoffman, Robert M.
• Format: Article
• Language: English
• Subjects: Animals; Carbon-Sulfur Lyases - genetics; Carbon-Sulfur Lyases - isolation & purification; Carbon-Sulfur Lyases - therapeutic use; Cell Division - drug effects; Half-Life; Humans; In Vitro Techniques; Male; Methionine - blood; Mice; Neoplasms - drug therapy; Neoplasms - metabolism; Polyethylene Glycols; Rats; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - therapeutic use; Tissue Distribution; Tumor Cells, Cultured
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1997.0805

Recombinant methioninase (rMETase) is a homotetrameric pyridoxal 5′-phosphate enzyme of 172-kda molecular mass derived fromPseudomonas putidaand cloned inEscherichia coli.rMETase has been found previously to be an effective, anti-tumor agentin vitroandin vivo.The enzyme targets the elevated minimal methionine requirement seen in all tumor types. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, theN-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. Molar ratios of M-SPA-PEG-5000 (PEG) to rMETase from 10 to 40 were used for PEGylation of rMETase. PEGylation reactions were run at 20°C for 30 to 60 min in reaction buffer (20 mM sodium phosphate buffer, pH 8.3). The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG /rMETase of 30/1 had enzyme activities of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum methionine levels to less than 0.1 μM for approximately 8 h compared to 2 h for rMETase in rats. Efficacy studies of PEG-rMETase on human lung cancer and kidney cancer cellsin vitrodemonstrated a 50% inhibitory concentration (IC50) of 0.04 and 0.06 units/ml, respectively. These IC50values were almost identical to unmodified rMETase, thus indicating maintenance of antitumor efficacy in the PEGylated enzyme. PEG-rMETase had an IC50for normal lung and kidney cells of 0.8 and 1.5 units/ml, respectively, similar to rMETase. The efficacy data indicated that PEG-rMETase maintained the high level tumor selectivity of rMETase. PEG-rMETase injected intravenously in mice demonstrated a tumor/blood retention ratio of approximately 1/6 compared to 1/10 of unmodified enzyme, indicating that PEG-rMETase distri