title: Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.
abstract: Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in vitro cell study, and in vivo validation.
authors.affiliation: †Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, United States.
authors.forename: Shuping
authors.initials: S
authors.lastname: Dou
authors.affiliation: ‡Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States.
authors.forename: John
authors.initials: J
authors.lastname: Virostko
authors.affiliation: §Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, United States.
authors.forename: Dale L
authors.initials: DL
authors.lastname: Greiner
authors.affiliation: ∥Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States.|⊥Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States.|@Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37232, United States.
authors.forename: Alvin C
authors.initials: AC
authors.lastname: Powers
authors.affiliation: †Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, United States.
authors.forename: Guozheng
authors.initials: G
authors.lastname: Liu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism*; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D001710:Biotin; D004247:DNA; D019809:Streptavidin
keywords: antibody conjugate; clearance; in vitro assay; in vivo properties; morpholino oligomers; quantitative correlation; trifunctional antibody