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 CEAproducing 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.Int.

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

“…M12599, the nucleotide substitutions resulting in alanine replacement equate to substitution of CGC (nt 678 -680) with GCG (R162A), and substitution of the G at nt 1429 with C (G412A). The pPM331 and pDP161 plasmids were expressed in E. coli TG1 cells and MFECP and MFEdmCP protein were purified on activated CH Sepharose 4B crosslinked to CEA as described previously (Bhatia et al, 2000). The eluted samples were pooled and dialysed overnight against four changes of PBS and 20 Â concentrated at 41C in a stirred cell ultrafiltration system (Amicon, UK) using a YM10 membrane (Millipore, UK).…”

“…Samples were concentrated to approximately 0.15 mg ml À1 and stored at À801C. Purity and identity were confirmed by sodium dodecylsulphate -polyacrylamide gel electrophoresis (SDS -PAGE) and Western blot, CEA binding was confirmed by enzyme-linked immunosorbent assay (ELISA) (Bhatia et al, 2000).…”

“…Endotoxins were removed on a 10 ml polymyxin column (Pierce, UK) according to the manufacturer's instructions. SDS -PAGE, Western blot and ELISA were performed to confirm expression of the correct protein and binding to CEA (Bhatia et al, 2000).…”

Professor Tom Connors and the development of novel cancer therapies by the Phase I/II Clinical Trials Committee of Cancer Research UK

“…However, the system was complex, the antibodyenzyme conjugate difficult to make reproducibly, and the antibodies and enzymes were immunogenic, preventing more than two or three administrations even with immunosuppressive therapy. These problems have been addressed by making a genetic fusion protein of a single chain Fv (sFv) (Begent et al, 1996) antibody with CPG2 (Bhatia et al, 2000). The design features of this molecule include phage library-derived sFv antibody of appropriate affinity linked to CPG2 enzyme, low immunogenicity, dimeric structure, stability in vitro and in vivo, therapeutic levels of enzyme localising to tumour, clearance from normal tissues in both mice and man due to glycosylation (by fermentation in Pichia pastoris), low toxicity, and prodrug activation with therapeutic activity.…”

A33scFv–cytosine deaminase: a recombinant protein construct for antibody-directed enzyme-prodrug therapy

“…Several recombinant constructs based on F(ab) and F(ab 0 ) 2 fragments have been described. Constructs based on single-chain variable fragments (scFv) may have favourable diffusion characteristics in solid tumours, but few descriptions of this approach have been published (Bhatia et al, 2000). Here, we report on a new ADEPT concept based on the A33 antigen and recombinant scFv -CD constructs.…”