Anti-JL1 immunoliposomes encapsulating doxorubicin for leukemia-directed therapy

Abstract

[한글]

[영문]Doxorubicin-loaded long-circulating (stealth) liposomes (Doxil®, Alza Pharmaceuticals), PEGylated liposomes encapsulating doxorubicin, are commercially available to treat a variety of cancers. It has been well documented that the liposomal formulation of doxorubicin exhibits reduced side-effects, resulting in enhanced therapeutic efficacy. However, the liposomal doxorubicin still has substantial adverse side-effects due to nonselective toxicity and nonspecific distribution of the drug to normal cells. To address these problems, anti-JL1 immunoliposomes encapsulating doxorubicin were prepared for targeted delivery of doxorubicin to leukemia cells over-expressing JL1 antigens. It was previously reported that JL1 antigens were uniquely expressed on the cell surface of most T leukemias. In this study, stealth immunoliposomes (SILs) were prepared by two different methods, (1) direct coupling of anti-JL1 antibody to 1,2-distearoyl-sn-glycero-3-phosphoethanol- amine-N-[maleimide(polyethylene glycol)2000] (DSPE-PEG2000-MAL) in preformed liposomes encapsulating doxorubicin or (2) post-insertion of DSPE-PEG-anti-JL1 antibody conjugates to preformed liposomes encapsulating doxorubicin. The SILs prepared by the two different methods were compared to each other in terms of in vitro binding affinity to T-leukemic cells, cellular internalization, and cytotoxicity depending on the presence of JL1 antigens and concentration of anti-JL1 antibody on the liposomal surface. According to FACS analysis and fluorescence microscopy, anti-JL1 SILs were able to specifically bind to JL1-positive CEM and HL60 cells, not to control H9 cells (JL1-negative). With a confocal laser scanning microscope, effective internalization of anti-JL1 SILs into the cytoplasm of CEM and HL60 cells was verified, which was presumably due to JL1 antigen-mediated recognition and endocytosis of the liposomes. As previously shown, doxorubicin encapsulated in liposomes was less cytotoxic than free doxorubicin, regardless presence of JL1 antigen on the cell surface. However, the reduced cytotoxicity was significantly recovered by coupling of anti-JL1 antibodies to the liposomal surface, presumably resulting from JL1-mediated internalization of doxorubicin to tumor cells. Meanwhile, the SIL-DOX formulations prepared by the two different methods did not exhibited significant differences in terms of cell binding, internalization, and cytotoxicity. The experimental results in this thesis provide valuable information that the anti-JL1 SIL-DOX would be applicable as a noble clinical modality for treatment of leukemia expressing JL1 antigens after preclinical and clinical evaluation.