title: Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.
abstract: Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery.
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Martha
authors.initials: M
authors.lastname: Tan
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Vernes
authors.forename: Joyce
authors.initials: J
authors.lastname: Chan
authors.forename: Trinna L
authors.initials: TL
authors.lastname: Cuellar
authors.forename: Aarati
authors.initials: A
authors.lastname: Asundi
authors.forename: Christopher
authors.initials: C
authors.lastname: Nelson
authors.forename: Victor
authors.initials: V
authors.lastname: Yip
authors.forename: Ben
authors.initials: B
authors.lastname: Shen
authors.forename: Richard
authors.initials: R
authors.lastname: Vandlen
authors.forename: Christian
authors.initials: C
authors.lastname: Siebel
authors.forename: Y Gloria
authors.initials: YG
authors.lastname: Meng
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D034741:RNA, Small Interfering / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000906:Antibodies; D000941:Antigens; D034741:RNA, Small Interfering