Imaging of tumor angiogenesis using 99mTc-labeled human recombinant anti-ED-B fibronectin antibody fragments.
The aim of this study was to target the angiogenesis-associated extracellular matrix protein ED-B fibronectin for molecular imaging of solid tumors. Recombinant and chemically modified derivatives of the single-chain antibody fragment (scFv) L19, capable of being labeled with 99mTc, were synthesized and radiolabeled. The resulting compounds 99mTc-AP39, 99mTc-L19-His, and 99mTc-L19-Hi20 were assessed for their imaging properties in vivo.


METHODS
L19 was genetically modified by inserting either the (Gly)3-Cys-Ala (AP39) or a (His)6 tag (L19-His) sequence at the C-terminal end. Chemical modifications were performed by conjugating the bifunctional chelator Hi20 (L19-Hi20) at epsilon-Lys-NH2 residues of the molecule to allow for a direct chelator-based labeling with 99mTc. Tumor-targeting, pharmacokinetic, and scintigraphic imaging properties of the radiolabeled scFvs were evaluated in nude mice bearing murine F9 teratocarcinoma.


RESULTS
99mTc labeling of the L19 derivatives yielded radiochemically pure proteins maintaining high immunoreactivity to ED-B fibronectin, as measured by affinity chromatography. Size-exclusion high-performance liquid chromatographic analysis of labeled L19 derivatives demonstrated either dimeric species (L19-His) or a mixture of predominantly associative dimeric and monomeric species (AP39, L19-Hi20). 99mTc-AP39 showed the most favorable biodistribution and imaging properties with high and fast tumor uptake (8.3 percentage injected dose per gram at 3 h after injection), rapid blood clearance and renal excretion, leading to high signal-to-noise ratios (tumor-to-blood ratio of 6.4 at 3 h after injection), and excellent planar scintigraphy in vivo.


CONCLUSIONS
ED-B fibronectin can be efficiently targeted by 99mTc-AP39 and scintigraphically visualized in tumor-bearing mice, providing a potentially useful clinical tool for imaging of angiogenesis-associated ED-B fibronectin-expressing human tumors.
Berndorff
Dietmar
D
Research Laboratories, Schering AG, Berlin, Germany. dietmar.berndorff@schering.de
Borkowski
Sandra
S
Moosmayer
Dieter
D
Viti
Francesca
F
Müller-Tiemann
Beate
B
Sieger
Stephanie
S
Friebe
Matthias
M
Hilger
Christoph S
CS
Zardi
Luciano
L
Neri
Dario
D
Dinkelborg
Ludger M
LM
D000818:Animals; D045744:Cell Line, Tumor; D005353:Fibronectins / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000098:blood supply*; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D013667:Technetium / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
C067625:FN1 protein, human; D005353:Fibronectins; D007135:Immunoglobulin Variable Region; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins; D013667:Technetium