Preparation of immunoliposomes bearing poly(ethylene glycol)-coupled monoclonal antibody linked via a cleavable disulfide bond for ex vivo applications

Several methods for the preparation of sterically stabilized immunoliposome s (SIL) have recently been described. This report examines an established m ethod for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (P EG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phospha tidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient at tachment of pyridyldithio-derivatized mAb took place (equivalent to couplin g ca. 70% of total input protein) at 2 mol percent of the functionalized PE G-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG- la and hematopoietic progenitor cells) and the extent of binding was a func tion of liposomal lipid concentration, the mAb density in the liposome surf ace and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurk at), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4 %) similar to those reported in clinical samples (such as cord blood, mobil ized peripheral blood and bone marrow) using a direct immunostaining with M y10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by tr eatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degreesC). SIL binding and subsequent dith iothreitol treatment did not influence the cell viability. Our approach sho uld contribute to the development of targetable liposomal vehicles to CD34 cells for use in ex vivo conditions as sorting of hematopoietic stem cells . (C) 2000 Elsevier Science B.V. All rights reserved.