title: Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein.
abstract: Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme selectively to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Previous clinical work using antibody-enzyme chemical conjugates has been limited by the moderate efficiency of tumor targeting of these 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. MFE-23::CPG2 fusion protein was overexpressed in Escherichia coli and purified using CEA affinity 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 colon adenocarcinoma xenografts. Recombinant MFE-23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to plasma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr). (125)I-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 concentrations and selective tumor retention of active enzyme delivered by MFE-23::CPG2 establish that this recombinant fusion protein has potential to give improved clinical efficiency for ADEPT.
authors.affiliation: Department of Oncology, Royal Free and University College Medical School, London, UK. jeet@rfhsm.ac.uk
authors.forename: J
authors.initials: J
authors.lastname: Bhatia
authors.forename: S K
authors.initials: SK
authors.lastname: Sharma
authors.forename: K A
authors.initials: KA
authors.lastname: Chester
authors.forename: R B
authors.initials: RB
authors.lastname: Pedley
authors.forename: R W
authors.initials: RW
authors.lastname: Boden
authors.forename: D A
authors.initials: DA
authors.lastname: Read
authors.forename: G M
authors.initials: GM
authors.lastname: Boxer
authors.forename: N P
authors.initials: NP
authors.lastname: Michael
authors.forename: R H
authors.initials: RH
authors.lastname: Begent
mesh_terms: D000818:Animals; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D008819:Mice, Nude; D011355:Prodrugs / Q000493:pharmacokinetics / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics / Q000627:therapeutic use; D014018:Tissue Distribution; D014183:Transplantation, Heterologous; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism / Q000627:therapeutic use*
chemical_list: D002272:Carcinoembryonic Antigen; D007135:Immunoglobulin Variable Region; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D011623:gamma-Glutamyl Hydrolase