POLYETHYLENE-GLYCOL CONJUGATION OF RECOMBINANT METHIONINASE FOR CANCER-THERAPY

Recombinant methioninase (rMETase) is a homotetrameric pyridoxal 8'-ph osphate enzyme of 172-kda molecular mass derived from Pseudomonas puti da and cloned in Escherichia coli. rMETase has been found previously t o be an effective, anti-tumor agent in vitro and in vivo. The enzyme t argets the elevated minimal methionine requirement seen in all tumor t ypes. In order to prevent immunological reactions which might be produ ced by multiple dosing of rMETase and to prolong the serum half-life o f rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glyc ol 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 degrees C f or 30 to 60 min in reaction buffer (20 mM sodium phosphate buffer, pH 8.3). The PEGylated molecules (PEG-rMETase) were purified from unreact ed PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DE AE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETa se subunit, from a PEG/rMETase ratio of 30/1 in the synthetic reaction , had a molecular mass of approximately 53 kda determined by matrix-as sisted laser desorption/ionization mass spectrometry, indicating the c onjugation of two PEG molecules per subunit of rMETase and eight per t etramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rM ETase of 30/1 had enzyme activities of 70% of unmodified rMETase. PEGy lation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PE G-rMETase could deplete serum methionine levels to less than 0.1 mu M for approximately 8 h compared to 2 h for rMETase in rats. Efficacy st udies of PEG-rMETase on human lung cancer and kidney cancer cells in v itro demonstrated a 50% inhibitory concentration (IC50) of 0.04 and 0. 06 units/ml, respectively. These IC50 values were almost identical to unmodified rMETase, thus indicating maintenance of antitumor efficacy in the PEGylated enzyme. PEG-rMETase had an IC50 for normal lung and k idney cells of 0.8 and 1.5 units/ml, respectively, similar to rMETase. The efficacy data indicated that PEG-rMETase maintained the high leve l tumor selectivity of rMETase. PEG-rMETase injected intravenously in mice demonstrated a tumor/blood retention ratio of approximately 1/6 c ompared to 1/10 of unmodified enzyme, indicating that PEG-rMETase dist ributes to the tumor at least as effectively as rMETase. (C) 1998 Acad emic Press.