title: Humanized Lewis-Y specific antibody based delivery of STAT3 siRNA.
abstract: The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Le(y)), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Le(y)-expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Le(y)-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a noncovalent construct based on (d-arginine)(9) (9r) modified hu3S193. Le(y)-specific binding and internalization of both the covalent and noncovalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the noncovalent system led to efficient STAT3 silencing in Le(y)-positive cancer cells (A431) but not in Le(y)-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified noncovalent construct induced ∼70% STAT3 knockdown at submicromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ∼50% inhibition of cell proliferation of Le(y)-positive cells. Noncovalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, California 91010, United States.
authors.forename: Yuelong
authors.initials: Y
authors.lastname: Ma
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Kowolik
authors.forename: Piotr M
authors.initials: PM
authors.lastname: Swiderski
authors.forename: Marcin
authors.initials: M
authors.lastname: Kortylewski
authors.forename: Hua
authors.initials: H
authors.lastname: Yu
authors.forename: David A
authors.initials: DA
authors.lastname: Horne
authors.forename: Richard
authors.initials: R
authors.lastname: Jove
authors.forename: Otavia L
authors.initials: OL
authors.lastname: Caballero
authors.forename: Andrew J G
authors.initials: AJ
authors.lastname: Simpson
authors.forename: Fook-Thean
authors.initials: FT
authors.lastname: Lee
authors.forename: Vinochani
authors.initials: V
authors.lastname: Pillay
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scott
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D020868:Gene Silencing; D006801:Humans; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D008954:Models, Biological; D015394:Molecular Structure; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000172:deficiency / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human