Abstract
AimThe aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients.Materials and methodsPhage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I.ResultsTwo affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor.ConclusionThe single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging.
Highlights
The development of molecular imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) continues to progress, owing to the ability of these techniques to allow non-invasive in vivo visualization of biological processes at the molecular and cellular levels
The small animal PET-CT study using 124I-labeled CD44v6-single chain fragment variable (scFv)-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor
The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 bind to CD44v6, and the radiolabeled counterparts provide high tumor-toblood ratios and fast clearance from organs and blood
Summary
The development of molecular imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) continues to progress, owing to the ability of these techniques to allow non-invasive in vivo visualization of biological processes at the molecular and cellular levels. Antibody-based radionuclide imaging is an attractive tool for diagnostics of HNSCC Such imaging agents combine the imaging ability of PET and SPECT with tumor specificity. Intact monoclonal antibodies (mAbs) are large (150 kDa) molecules, with generally slow pharmacokinetics, slow blood clearance, and sub optimal tumor penetration and accumulation [5,6,7,8] Antibody fragments such as single chain fragment variable (scFv) have shown great promise in imaging applications [9] including cancer diagnostics, where the scFv format has been shown to provide a good balance of system clearance, tumor penetration and tissue accumulation [7, 10,11,12]. Recombinant antibody formats provide immediate availability of recovered antibody genes, which facilitates downstream optimization [12, 16]
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