PROTEIN EXPRESSION AND PURIFICATION 12, 45–52 (1998) ARTICLE NO. PT970805 Polyethylene Glycol Conjugation of Recombinant Methioninase for Cancer Therapy Yuying Tan, Xinghua Sun, Mingxu Xu, Zili An, Xuezhong Tan, Xiuying Tan, Qinghong Han, Dusan A. Miljkovic, Meng Yang, and Robert M. Hoffman AntiCancer, Inc., 7917 Ostrow Street, San Diego, California 92111 Received June 23, 1997, and in revised form September 1, 1997 ase, thus indicating maintenance of antitumor efficacy in the PEGylated enzyme. PEG-rMETase had an IC 50 Recombinant methioninase (rMETase) is a homotet- for normal lung and kidney cells of 0.8 and 1.5 units/ rameric pyridoxal 5-phosphate enzyme of 172-kda mo- ml, respectively, similar to rMETase. The efficacy data lecular mass derived from Pseudomonas putida and indicated that PEG-rMETase maintained the high cloned in Escherichia coli. rMETase has been found level tumor selectivity of rMETase. PEG-rMETase in- previously to be an effective, anti-tumor agent in vitro jected intravenously in mice demonstrated a tumor/ and in vivo. The enzyme targets the elevated minimal blood retention ratio of approximately 1/6 compared methionine requirement seen in all tumor types. In to 1/10 of unmodified enzyme, indicating that PEG- order to prevent immunological reactions which rMETase distributes to the tumor at least as effectively might be produced by multiple dosing of rMETase and as rMETase.  1998 Academic Press to prolong the serum half-life of rMETase, the N-hydro- xysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. Molar ratios of M-SPA-PEG-5000 (PEG) to A tumor-selective target with high therapeutic poten- rMETase from 10 to 40 were used for PEGylation of tial is the elevated minimum methionine dependence of rMETase. PEGylation reactions were run at 20°C for 30 most and possibly all types of tumor cells relative to to 60 min in reaction buffer (20 mM sodium phosphate normal cells (1–12). The L-methionine a-deamino-g- buffer, pH 8.3). The PEGylated molecules (PEG-rMET- mercaptomethane lyase (methioninase, METase) gene ase) were purified from unreacted PEG with Amicon from Pseudomonas putida has been previously cloned in 30 K centriprep concentrators or by Sephacryl S-300 Escherichia coli (12–14). A scale-up recombinant methi- HR gel-filtration chromatography. Unreacted rMET- oninase (rMETase) production protocol has been estab- ase was removed by DEAE Sepharose FF anion-ex- lished with high yield (60%), high purity (98%), high change chromatography. The resulting PEG-rMETase stability, and low endotoxin for preclinical and clinical subunit, from a PEG/rMETase ratio of 30/1 in the syn- studies targeting the methionine dependence of human thetic reaction, had a molecular mass of approxi- cancer (12). mately 53 kda determined by matrix-assisted laser de- Studies of the antitumor efficacy of rMETase in vitro sorption/ionization mass spectrometry, indicating the and in vivo on human tumors xenografted in nude mice conjugation of two PEG molecules per subunit of demonstrated that all types of human tumors tested rMETase and eight per tetramer. PEG-rMETase mole- were sensitive to rMETase (12). In contrast, normal cules obtained from reacting ratios of PEG /rMETase cells were relatively insensitive to rMETase in vitro of 30/1 had enzyme activities of 70% of unmodified and, correspondingly, no toxicity was detected in vivo rMETase. PEGylation of rMETase increased the serum at the effective doses (12). half-life of the enzyme in rats to approximately 160 However, as with many other bacterial polypeptides min compared to 80 min for unmodified rMETase. and proteins, rMETase may be immunogenic in higher PEG-rMETase could deplete serum methionine levels animals, which may limit the utility of rMETase espe- to less than 0.1 mM for approximately 8 h compared cially with regard to multiple dosing. Anti-METase an- to 2 h for rMETase in rats. Efficacy studies of PEG- tibodies may accelerate methioninase clearance and rMETase on human lung cancer and kidney cancer consequently reduce its therapeutic effectiveness and cells in vitro demonstrated a 50% inhibitory concentra- may also reduce the enzyme potency by binding at the tion (IC 50 ) of 0.04 and 0.06 units/ml, respectively. These IC 50 values were almost identical to unmodified rMET- active site. 45 1046-5928/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.