Catalytic activity of an in vivo tumor targeted anti-CEA scFv :: carboxypeptidase G2 fusion protein

Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme selectiv ely to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Previous clinical work using antibody-enzyme chemical conj ugates has been limited by the moderate efficiency of tumor targeting of th ese molecules. To address this a recombinant fusion protein composed of MFE -23, an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody , fused to the amino-terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA-producing tumors. MPE-23::CPG2 fus ion protein was overexpressed in Escherichia coil and purified using CEA af finity chromatography, Efficacy of MFE-23::CPG2 delivery to tumors in vivo was assessed by measuring catalytic activity after intravenous injection of purified MFE-23::CPG2 into nude mice bearing CEA-positive LS174T human col on adenocarcinoma xenografts. Recombinant MFE-23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to pl asma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr), I -125-MFE-23::CPG2 was retained in kidney, liver and spleen but MFE-23::CPG2 catalytic activity was not, resulting in excellent tumor to normal tissue enzyme ratios 48 hr after injection. These were 371:1 (tumor to liver), 450 :1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1 ,618:1 (tumor to spleen). Favorable tumor:normal tissue ratios occurred at early time points when there was still 21% (24 hr) and 9.5% (48 hr) of the injected activity present per gram of tumor tissue. The high tumor concentr ations and selective tumor retention of active enzyme delivered by MFE-23:: CPG2 establish that this recombinant fusion protein has potential to give i mproved clinical efficiency for ADEPT. Int. J. Cancer 85:571-577, 2000. (C) 2000 Wiley-Liss, Inc.