Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor.
Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively.


METHODS
HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice.


RESULTS
There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively).


CONCLUSIONS
(99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.
Blankenberg
Francis G
FG
Department of Pediatrics, Stanford University, Stanford, California 94304, USA. blankenb@stanford.edu
Mandl
Stefanie
S
Cao
Yu-An
YA
O'Connell-Rodwell
Caitlin
C
Contag
Christopher
C
Mari
Carina
C
Gaynutdinov
Timur I
TI
Vanderheyden
Jean-Luc
JL
Backer
Marina V
MV
Backer
Joseph M
JM
D000230:Adenocarcinoma / Q000000981:diagnostic imaging / Q000378:metabolism; D000818:Animals; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D008325:Mammary Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D008807:Mice, Inbred BALB C; D009928:Organ Specificity; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000097:blood / Q000138:chemical synthesis / Q000493:pharmacokinetics; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics*; D012680:Sensitivity and Specificity; D013667:Technetium / Q000097:blood / Q000493:pharmacokinetics; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics*
D019275:Radiopharmaceuticals; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D013667:Technetium; D012259:Ribonuclease, Pancreatic