Interaction of Differently Designed Immunoliposomes with Colon Cancer Cells and Kupffer Cells. An in Vitro Comparison Gerben A. Koning 1, 5 , Henriëtte W. M. Morselt 1 , Arko Gorter 2 , Theresa M. Allen 3 , Samuel Zalipsky 4 , Gerrit L. Scherphof 1 and Jan A. A. M. Kamps 1,6 Received March 5, 2003; accepted April 30, 2003 Purpose. Evaluate the effectiveness of distal-end coupling of a tumor- specific antibody to liposomal polyethylene glycol (PEG) chains to improve target binding and reduce interference by macrophage up- take. Methods. Monoclonal antibody CC52, specific for CC531 rat colon carcinoma, was coupled to the bilayer of PEG-liposomes (type I) or to the distal end of bilayer-anchored PEG-chains (type II). Uptake of both (radiolabeled)liposome types by CC531 cells and rat liver mac- rophages was determined. Results. With increasing antibody density, both immunoliposome types showed increased binding to target cells, but type II liposomes displayed better target recognition than type I. Uptake by macro- phages increased with antibody density for both liposome types. Low- est uptake by macrophages was found for type II liposomes at low antibody densities. Unexpectedly, not only for type I but also for type II liposomes, in which the antibody is coupled via its Fc moiety, uptake by macrophages was inhibited by aggregated IgG, indicating involvement of Fc receptors. Also polyinosinic acid, an inhibitor of scavenger receptors, reduced uptake of type II liposomes. Conclusion. Although distal end coupling of antibodies to bilayer- anchored PEG chains in liposomes through the Fc moiety enhances target cell binding, it does not prevent the recognition by Fc receptors on macrophages. KEY WORDS: Immunoliposomes; Poly(ethylene glycol); Colon cancer; Kupffer cells; Tumor specific antibody; Drug-targeting. INTRODUCTION Delivery of drugs to target cells by means of liposomes is hindered by the uptake of these particles by macrophages in liver and spleen. Attachment of antibodies to liposomes for targeting purposes may result in increased macrophage up- take and thus in an even lower availability of the drug for the target cells. Characterization of the effects of antibody cou- pling to liposomes on the target binding potential vs. uptake by macrophages is therefore a major issue. PEG coupled to the liposomal surface increases the cir- culation half-life of liposomes (1). The method of coupling and the orientation of the antibody, combined with liposome size and the absence or presence of PEG, mainly determine the fate of systemically applied immunoliposomes. Efficient localization of immunoliposomes at sites not readily acces- sible, like tumor cells in solid tumors, is determined by blood circulation time and liposome size (2–5). Increased circulation time results in enhanced extravasation in the tumor, allowing increased anti-tumor activity. Not only liver and spleen macrophages interfere with tumor-cell specific delivery. Following extravasation at the tumor site, tumor-associated macrophages may also compete with the target cells for uptake of the immunoliposomes (6). Several reports describe decreased circulation half-lives following the coupling of antibodies to PEG-liposomes, espe- cially when the antibody is attached to the distal end of the PEG-chains either via a hydrazone-bond (3,5,7), by means of PDP-PEG-DSPE (8) or by using a PEG-terminal carboxyl- group (9). Increased clearance rates paralleled enhanced up- take by (macrophages of) liver and spleen (7,8). Liposomes with antibodies attached to the distal end of the PEG-chains were cleared from circulation much faster than liposomes with the antibody coupled directly to the bilayer of PEG- DSPE containing liposomes (5,9). Circulation time of the lat- ter was similar to that of control PEG-liposomes without an- tibody suggesting that the antibody was masked by the PEG- chains (5,9,10). Most antibody coupling methods result in a random orientation of the antibody on the liposomes. The resulting exposure of the Fc portion of the antibody facilitates recognition by Fc receptors on macrophages. Coupling of an- tibodies to the distal end of the PEG-chain by means of Hz- PEG-DSPE specifically involves the Fc portion of the anti- body: in this way diminished Fc-exposition can be achieved (11). In this paper we compare liposomes with varying amounts of the antibody CC52, specific to rat CC531 colon cancer cells with regard to their target-binding capacity and their uptake by Kupffer cells. The antibody was attached ei- ther directly to the bilayer of liposomes with or without mPEG-DSPE (type I) or to the distal end of bilayer-anchored PEG-chain (type II). MATERIALS AND METHODS Materials N-succinimidyl-S-acetylthioacetate (SATA), sodium per- iodate, N-acetylmethionine, cholesterol (Chol), polyinosinic acid (poly I) and human IgG were obtained from Sigma (St. Louis, MO, USA). Egg yolk phosphatidylcholine (PC), ma- 1 Department of Cell Biology, Section Liposome Research, Gronin- gen University Institute for Drug Exploration (GUIDE), Faculty of Medical Sciences, University of Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands. 2 Department of Pathology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. 3 Department of Pharmacology, University of Alberta, Edmonton, Canada T6G 2H7. 4 ALZA Corporation, Palo Alto, California 94303-0802, USA. 5 Present address: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O Box 80 082, 3508 TB Utrecht, The Netherlands. 6 To whom correspondence should be addressed. J.A.A.M. Kamps, Department of Cell Biology, Faculty of Medical Sciences, Univer- sity of Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands. Tel: 31 50 363 2655; Fax: 31 50 363 8171. (e-mail: j.a.a.m.kamps@med.rug.nl) ABBREVIATIONS: CC52, Colon Carcinoma CC531 specific anti- body; PC, Phosphatidylcholine; Chol, Cholesterol; PEG-DSPE, me- thoxypoly(ethylene glycol) 2000 -distearoylphosphatidylethanolamine; MPB-PE, maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine; Hz-PEG, hydrazide-PEG; BSA, bovine serum albumin; FCS, fetal calf serum; poly I, polyinosinic acid; TL, total lipid (phospholipids and cholesterol). Pharmaceutical Research, Vol. 20, No. 8, August 2003 (© 2003) Research Paper 1249 0724-8741/03/0800-1249/0 © 2003 Plenum Publishing Corporation