Optical pretargeting of tumor with fluorescent MORF oligomers.
OBJECTIVE
Pretargeting with radioactivity has significantly improved tumor to normal tissue radioactivity ratios over conventional antibody imaging in both animal studies and clinical trials. This laboratory is investigating DNA analogues such as phosphorodiamidate morpholinos (MORFs) for pretargeting using technetium-99m ((99m)Tc) for detection. However, the unique properties of fluorescence activation and quenching combined with oligomers with their unique properties of hybridization may be particularly useful when used together for pretargeting with optical detection. The use of linear fluorophore-conjugated oligomer duplexes have been little used in animals, and to our knowledge, have not previously been considered for pretargeting applications.


METHODS
A MORF/cDNA pair was selected such that when hybridized, the fluorescence of the Cy5.5-conjugated 25 mer MORF (Cy5.5-MORF25) is inhibited with a BHQ3-conjugated 18 mer complementary DNA (BHQ3-cDNA18). The short BHQ3-cDNA18 was selected to dissociate in the presence of a long cMORF25 in the pretargeted tumor, thus releasing the inhibitor from the Cy5.5 emitter. In this manner, the Cy5.5 fluorescence will be inhibited everywhere but in the target. The dissociation was first examined in vitro by adding the duplex to the cMORF25 both in solution and immobilized on polystyrene microspheres and by surface plasmon resonance (SPR). Thereafter, biotinylated cMORF25 immobilized on streptavidin polystyrene microspheres were administered intramuscularly in one thigh of hairless SKH-1 mice as target while an identical weight of the identical microspheres but without the cMORF25 was administered in the contralateral thigh as control. The animals then received IV the Cy5.5-MORF25/BHQ3-cDNA18 duplex or equal molar dosage of single-chain Cy5.5-MORF25 and were imaged.


RESULTS
The SPR studies showed that the immobilized cDNA18 rapidly captured the flowing MORF25 to provide a duplex with a slow dissociation rate constant. Furthermore, when cMORF25 was next allowed to flow over the now immobilized duplex, the cDNA18 was unable to prevent dissociation of the heteroduplex and the formation and release of the cMORF25-MORF25 homoduplex. Images of animals obtained soon after receiving the Cy5.5-MORF25 singlet showed intense whole body fluorescence obscuring the target thigh. However, only 5 minutes after receiving the Cy5.5-MORF25/BHQ3-cDNA18 duplex, the target thigh was clearly visible along with only the kidneys.


CONCLUSIONS
This first study of optical pretargeting provides a proof of concept that oligomer pretargeting found to be useful with radioactivity detection is applicable with fluorescent detection as well. In addition, our results demonstrate that by using linear oligomers for optical pretargeting, chain lengths (and base sequences) may be manipulated to provide duplexes with stabilities and fluorescence inhibition optimized for pretargeting and other in vivo applications of optical imaging.
He
Jiang
J
Department of Radiology, University of California San Francisco, San Francisco, CA 94143, USA,
Rusckowski
Mary
M
Wang
Yi
Y
Dou
Shuping
S
Liu
Xinrong
X
Zhang
Surong
S
Liu
Guozheng
G
Hnatowich
Donald J
DJ
D000818:Animals; D002232:Carbocyanines; D005456:Fluorescent Dyes; D051379:Mice; D008863:Microspheres; D009025:Morpholines / Q000737:chemistry; D060172:Morpholinos; D009369:Neoplasms / Q000175:diagnosis*; D019275:Radiopharmaceuticals / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D013667:Technetium / Q000737:chemistry
C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009025:Morpholines; D060172:Morpholinos; D019275:Radiopharmaceuticals; D013667:Technetium