Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy

Abstract

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two‐ and three‐phase system in which an antibody to a tumor‐associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic‐antigen antibody fragment A5B7‐F(ab′)2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate‐specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor. The second approach involved galactosylation of the conjugate so that it cleared rapidly from blood via the asialoglycoprotein receptors in the liver. Localization of the radiolabeled conjugate was achieved by blocking this receptor for about 8 hours with a single injection (8 mg/mouse) of an inhibitor that binds competitively to the receptor. This allowed tumor localization of the conjugate followed by a rapid clearance of the galactosylated conjugate from blood as the inhibitor was consumed. A tumor‐to‐blood ratio of 45:1 was obtained at 24 hours, which increased to 100:1 at 72 hours after the conjugate injection. These accelerated clearance mechanisms have been applied in antitumor studies in ADEPT. Cancer 1994; 73:1114–20.

Plasma clearance of an antibody-enzyme conjugate in ADEPT by monoclonal anti-enzyme: its effect on prodrug activation in vivo

“…Regimen 1: 5 h clearance time The standard protocol for using SB43-GAL clearance has been to give this 19 h after the enzyme conjugate when sufficient SB43-GAL was administered to reduce circulatory enzyme levels to <0.1 units ml-' of blood (Sharma et al, 1994). Prodrug was then administered at 20 or 24 h after the conjugate without toxicity.…”

“…To reduce possible inactivation of enzyme at the tumour site, SB43 was covalently linked to galactose to facilitate its rapid uptake by receptors in the liver and minimise the circulatory dwell time. Given to mice bearing the LS174T colon tumour xenograft 19 h after the conjugate, SB43 linked to galactose had the predicted effect of reducing CPG2 concentrations in the blood without appreciably affecting the concentration of enzyme at the tumour site (Sharma et al, 1990(Sharma et al, , 1991(Sharma et al, , 1994. This regimen allowed therapy doses of prodrug to be injected within 24 h of giving the conjugate.…”

“…logistical advantage of this has been demonstrated in experimental therapy studies (Sharma et al, 1991(Sharma et al, , 1994 and SB43-GAL has been applied in clinical investigations in which, given as an infusion, it successfully reduced plasma enzyme concentrations and toxicity (Bagshawe et al, 1991). Anti-…”

“…Time after prodrug injection (min) Figure 7 Tumour-normal tissue ratios of generated active drug in mice (solid symbol) given A5-CPG2 followed by SB43-GAL at 19 h and prodrug at 72 h (regimen 3) and in mice (open symbol) given A5-CPG2 and prodrug only. Plasma CPG2 clearance and prodrug turnover GT Rogers et al 1362 (Sharma et al, 1994), use of anti-idiotypes or second antibodies (Pedley et al, 1989) and use of biotin-avidin constructs (Paganelli et al, 1991). Depletion of residual prodrug from the liver is somewhat faster when SB43-GAL anti-enzyme clearance is used, suggesting that SB43-enzyme conjugate complexes in this organ are capable of dissociation, thus supplementing enzyme activity already present in the liver.…”

A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours

“…Also, the targeting system, although giving high tumour to blood ratios of antibody-enzyme conjugate (10 000 : 1), was complicated, requiring additional administration of a mouse monoclonal clearing antibody directed to the active site of CPG2 (Napier et al, 2000). This antibody was galactosylated to accelerate its clearance from the circulation (Sharma et al, 1990(Sharma et al, , 1994.…”