Biodistribution and Targeting of Anti-5T4 Antibody–Drug Conjugate Using Fluorescence Molecular Tomography

Abstract

Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.

Assessment of near-infrared fluorophores to study the biodistribution and tumor targeting of an IL13 receptor α2 antibody by fluorescence molecular tomography

“…Our lab is assessing the utility of fluorescence molecular tomography (FMT) in evaluating the biodistribution of biotherapeutics. For example, we recently determined the in vivo biodistribution and tumor uptake of a 5T4 antibody-drug conjugate (ADC) and the 5T4 antibody itself using FMT [ 5 ]. FMT is an advanced optical imaging technology that uses the near-infrared spectrum (NIR) (600–900 nm) for non-invasive in vivo imaging and 3D quantification of the fluorescent probes [ 5 – 7 ].…”

Evaluation of pharmacokinetic and pharmacodynamic profiles of liposomes for the cell type-specific delivery of small molecule drugs

“…The lipid solution was extruded 41 times through a syringe extruder containing a 0.2 µm Nuclepore filter (Thermo Fisher Scientific Inc., Waltham, MA). 16 The resulting liposomes were characterized by Nanosight NS300 (Malvern Instruments Ltd., Worcestershire, UK) to determine particle size and concentration. The absorbance of FAM on the peptides enabled the determination of the number of peptides that were incorporated in each liposomal formulation.…”

“…Quantitative whole body biodistribution of fluorescent particles can be assessed non-invasively using FMT. 14–16 In vivo imaging was followed by immunohistochemistry using antibodies recognizing various cell types of interest in the infarct / border zone to determine the cellular specificity. The presence of FAM on the peptides facilitated the identification of the cell types recognized by the peptide-targeted liposomes by co-localization with antibodies against CD31 (for endothelial cells; clone MEC 13.3, BD Biosciences, San Jose, CA), Hrnr (rabbit polyclonal antibody, Sigma-Aldrich, St. Louis, MO), α-smooth muscle actin (for myofibroblasts and smooth muscle cells; clone 1A4, Sigma-Aldrich, St. Louis, MO), caveolin-3 (for cardiomyocytes; sc-7665, Santa Cruz Biotechnologies, Dallas, TX), and c-Kit (progenitor cells; sc-5535, Santa Cruz Biotechnologies, Dallas, TX).…”

Molecular imaging reveals biodistribution of P-cadherin LP-DART bispecific and trafficking of adoptively transferred T cells in mouse xenograft model

“…P-cadherin LP-DART has demonstrated potent cytotoxic T-cell mediated tumor cell killing in in vitro and in vivo models [16]. To further expand our understanding of the pharmacology of P-cadherin LP-DART (i.e., the exposure at the target site and the recruitment of T cells in tumors), we have utilized fluorescence molecular tomography (FMT) imaging [17,18]. Longitudinal in vivo biodistribution, tissue exposure and tumor targeting of a P-cadherin LP-DART was assessed using FMT after labeling with near-infra red (NIR) fluorophore VivoTag ® 680XL (here after referred as VT680).…”

“…wt. of ~100 kDa) were labeled with VT680 using a VivoTag ® 680XL protein labeling kit as per the manufacturerʹs instructions (Perkin Elmer Inc) and has been previously described [17]. Briefly, the VT680 has a succinimidyl ester group, which reacts with the primary amine group on the bispecific antibodies to form a stable amide linkage.…”