Recombinant
methioninase (rMETase) is an
enzyme active in preclinical mouse models of human
cancer. The efficacy of rMETase is due to depletion of plasma
methionine, an
amino acid for which
tumors generally have an abnormally high
methionine requirement. Furthermore, transient
methionine depletion results in a markedly increased sensitivity of the
tumors to several chemotherapeutic agents. This study characterized methods to prolong the half-life of rMETase to extend the in vivo period of depletion of plasma and
tumor methionine. In the present study, rMETase was coupled to
methoxypolyethylene glycol succinimidyl glutarate-5000 in order to prolong the half-life of rMETase and thus extend the in vivo period of depletion of plasma and
tumor methionine. Matrix-assisted
laser desorption ionization mass spectrometry indicated that one sub-unit of rMETase was modified by approximately 4, 6 and 8 PEG molecules when rMETase was PEGylated at molar ratios of PEG/rMETase of 30/1, 60/1, and 120/1, respectively. PEG-rMETase (120/1) had a serum half-life increase of 20-fold, and
methionine depletion time increased 12-fold compared to unmodified rMETase. The increase in in vivo half-life depended on the extent of PEGylation of rMETase. In addition, a remarkable prolongation of in vivo activity and effective
methionine depletion by the PEG-rMETase was achieved by the simultaneous administration of
pyridoxal 5'-phosphate. PEGylation also reduced the immunogenicity of rMETase. The extent of reduction in immunogenicity depended on the number of residues PEGylated. PEG-rMETase 30/1 had a 10-fold decrease in
IgG titer while PEG-rMETase 120/1 had a 10(4)-fold decreased titer compared to naked rMETase. Thus, the molecular modification of PEGylation confers critical new properties to rMETase for development as a
cancer therapeutic.