Comparison of several linear fluorophore- and quencher-conjugated oligomer duplexes for stability, fluorescence quenching, and kinetics in vitro and in vivo in mice.

A useful property of optical imaging is the potential to modulate the detectable signal to improve target/nontarget ratios. When administered as a dimer of a fluorophore- and a quencher-conjugated duplex arranged to inhibit fluorescence but designed to dissociate only in the presence of its target, the fluorescence signal should in principle appear only in the target. This laboratory has demonstrated the feasibility of this approach by using a duplex consisting of a linear oligomer conjugated with Cy5.5 (emitter) hybridized to another linear oligomer conjugated with Iowa Black (quencher) in a pretargeting optical study. Now eight duplexes consisting of combinations of 18 mer linear phosphodiester (PO) and phosphorothioate (PS) DNAs and phosphorodiamidate morpholinos (MORFs) conjugated with Cy5.5 (emitter) and Iowa Black (quencher) were variously screened for in vitro duplex stability. The MORF/PO duplex was selected for further study based on evidence of stability in 37 degrees C serum. Simultaneously, the kinetics of quenching were investigated in vitro and in vivo in mice. Thereafter, mice were implanted in one thigh with MORF/PO Cy 5.5 microspheres and the complementary PS Iowa Black administered iv to measure the extent and kinetics of duplex formation in the target. While all duplexes were stable in buffer, only the MORF/PO duplexes and possibly all PS containing duplexes were stable in 37 degrees C serum for at least 4 h. The kinetics of quenching were found to be rapid in vitro, with a 80-90% decrease in Cy5.5 fluorescence immediately following formation of a PS/PS homoduplex, and in vivo, with a 27 to 38% decrease in target thigh/nontarget ratio within 1 h following administration of the complementary PS Iowa Black complementary DNA but not the random control DNA to mice implanted with MORF/PO Cy5.5 microspheres. This investigation has provided additional evidence that Cy5.5 may be efficiently and rapidly quenched by Iowa Black when both are conjugated to complementary oligomers and that the resulting inhibition of fluorescence is sufficiently persistent for imaging.

Evaluation of 99mTc-MAG3-annexin V: influence of the chelate on in vitro and in vivo properties in mice.

We conjugated mercaptoacetyltriglycine (MAG(3)) to rh-annexin V to permit radiolabeling with (99m)Tc in an effort to decrease the high kidney and liver accumulation observed for (99m)Tc-labeled Hynic-annexin V. The 36-kDa protein was conjugated at a 5:1 molar ratio with NHS-MAG(3) in HEPES buffer pH 7.8 at room temperature, then quenched with glycine and purified by dialysis. The biopotency of the resulting MAG(3)-annexin was similar to that of Hynic-annexin as determined by a sensitive red blood cell membrane affinity binding assay and a surface plasmon resonance (SPR) assay. The (99m)Tc radiolabeling of MAG(3)-annexin resulted in radiochemical yields of 90% under mildly basic pH conditions. Biodistribution data in normal mice clearly showed a significant decrease in kidney and liver uptake at 1 h postinjection for the (99m)Tc MAG(3)-annexin compared to the (99m)Tc Hynic-annexin (from 24% ID to 4% ID for the liver, and 45% ID to 15% ID for the kidneys, respectively). Autoradiography of the kidneys showed retention of radioactivity in the collecting tubules following administration of both labeled annexins. The (99m)Tc MAG(3)-annexin biodistribution was also characterized by a lower retention of radioactivity in the whole body, but with small intestine accumulation over fivefold higher than observed with (99m)Tc Hynic-annexin. These findings show a definite improvement in renal and hepatic clearance of the MAG(3) radioligand. However, due to the increased radioactivity uptake in the small intestines, the early in vivo detection of ongoing apoptosis in the lower abdomen might be more difficult with (99m)Tc MAG(3)-annexin. Nevertheless, (99m)Tc MAG(3)-annexin may be an attractive alternative to (99m)Tc Hynic-annexin for the in vivo imaging of phosphatidylserine receptors.

Amplification targeting: a modified pretargeting approach with potential for signal amplification-proof of a concept.

UNLABELLED: Conventional nuclear medicine imaging with large radiolabeled molecules such as antitumor antibodies suffers from slow localization and clearance. Pretargeting is under active investigation as an alternative using either (strept)avidin/biotin, bispecific antibodies, or oligomers. However, only the use of oligomers such as phosphorodiamidate morpholinos (MORFs) in pretargeting offers the potential of signal amplification at the target. Amplification targeting is a multistep procedure with the potential to greatly improve target localization of radioactivity (and eventually drugs) through the intermediate use of polymers conjugated with multiple copies of oligomers. OBJECTIVE: This study was conducted to prove the concept in vivo in tumored mice of amplfication targeting. METHODS: Nude mice bearing LS174T tumors received in order: the anti-CEA antibody MN14 conjugated with MORF, a polymer conjugated with multiple copies of complementary MORFs (cMORFs), and, finally, (99m)Tc-MORF. RESULTS: In tumored animals, dual radiolabels ((99m)Tc and (111)In) were used to demonstrate that, after 18 h, about 25% of antibody MORFs in tumor were targeted with polymeric cMORFs and, after 3 h, about 12% of the polymeric cMORFs in tumor were targeted with (99m)Tc-MORF. Therefore, hybridization in tumor in both cases (i.e., polymeric cMORF to antibody MORF and radiolabeled MORF to polymeric cMORF) was surprisingly efficient given the barriers to targeting in vivo and the competition between targeting and clearance. Moles of radiolabeled MORF accumulating in tumor were more than tripled for study animals receiving all 3 injections compared with control animals not receiving the antibody or the polymer. Furthermore, MORF expression (on antibody) and cMORF expression (on polymer) were rapidly lost in normal organs such as liver, spleen, and kidneys but not in tumor, thus improving the target-to-nontarget ratios. CONCLUSION: Although signal amplification has not yet been convincingly demonstrated and amplification targeting will require further studies for optimization, the concept has now been shown to be feasible.

Pharmacokinetics in mice of four oligomer-conjugated polymers for amplification targeting.

UNLABELLED: For use in amplification targeting, an oligomer-conjugated polymer must display adaptable chemistry, minimal steric hindrance, low toxicity, and favorable pharmacokinetics. In particular, the polymer must remain in circulation sufficiently long to permit target localization. OBJECTIVES: To evaluate their properties for amplification targeting, the biodistribution in normal mice was determined for four polymers conjugated with multiple copies of a phosphorodiamidate morpholino (MORF) oligomer. METHODS: An amine-derivatized 25-mer MORF oligomer was radiolabeled with 99mTc. Three polymers of succinylated polylysine (PL) with initial weight average molecular weights (Mw) of 30, 100, and 200 KDa, and one poly (methyl vinyl ether-alt-maleic acid) (PA) with initial Mw of 45 KDa polymer, were each conjugated with an amine derivatized 25-mer complementary MORF (i.e., cMORF). The average number of attached cMORF groups on each polymer molecule (i.e., gpm) was estimated by a high performance liquid chromatography (HPLC) shift assay after the addition of trace 99mTc-MORF to the unpurified polymer, while the average number of accessible cMORF on each polymer was determined by adding radiolabeled MORF at increasing concentrations to the purified cMORF polymer solution until saturation. After purification, each polymer was radiolabeled by incubation with trace 99mTc-MORF. The biodistribution was then established in normal CD1 mice at a constant dosage of 2-4 micrograms of cMORF. RESULTS: The gpm varied from about 12 on 30 KDa PL to 40 on 45 KDa PA. The biodistribution results show that the pharmacokinetics of the radiolabel is a function of both the type of polymer as well as its gpm. Of the four polymers, the 30 KDa PL showed the most favorable pharmacokinetic profile, with the lowest liver accumulation and the highest blood values compared to the remaining three polymers. CONCLUSION: The biodistribution of the four polymers showed characteristic differences, with one polymer (30 KDa PL) showing the most favorable properties for amplification targeting.

Tumor pretargeting in mice using (99m)Tc-labeled morpholino, a DNA analog.

UNLABELLED: Over the past several years, investigators in this laboratory and elsewhere have been studying tumor localization by pretargeting with streptavidin and biotin or with avidin and biotin. Despite encouraging results, difficulties related to endogenous biotin and the immunogenicities of streptavidin and avidin have made a search for alternative strategies sensible. Recently, we have considered the use of DNAs and peptide nucleic acids for this purpose because oligomers can have hybridization affinities equivalent to that of biotin for streptavidin or avidin without the associated difficulties. We now report on the use of a morpholino (MORF), another commercially available synthetic oligomer, for pretargeting applications. MORFs support the nitrogenous bases by nonionic phosphorodiamidate linkages and, besides being nuclease resistant, can display good water solubility. METHODS: An 18mer MORF and its 18mer complementary MORF (cMORF) were obtained with a primary amine through a 3-member alkyl linker on the 3' equivalent end. An anti--carcinoembryonic antigen IgG antibody (MN14) was conjugated with MORF, whereas cMORF was conjugated with N-hydroxysuccinimide-mercaptoacetyltriglycine (MAG3) to permit radiolabeling with (99m)Tc. The biodistribution of labeled cMORF was first evaluated in normal CD-1 mice. Subsequently, nude mice bearing LS174T tumors received 50 microg conjugated antibody 48 h before the administration of 1.0 microg (7.4 MBq) (99m)Tc-MAG3-cMORF. Control animals received the labeled cMORF without prior administration of the antibody. A clearing step was not used. RESULTS: Biodistributions in normal mice showed that (99m)Tc-MAG3-cMORF was excreted rapidly through the kidneys, with only 7 percentage injected dose (%ID) remaining within the whole body (excluding urine) at 3 h. In tumor-bearing mice at 24 h, only 11 %ID of the radioactivity remained in the whole body of study animals, and of this amount, 2 %ID/g was in tumor tissue. The sites with the highest %ID were the kidneys, at 4 %ID/g, and the blood, at 0.5 %ID/g; all other organs had <1 %ID/g. At the same time, values for the control animals were 5 %ID (whole body), 0.05 %ID/g (tumor), and 3 %ID (kidneys). All images reflected high uptake in the tumors and low uptake in the normal tissues of the study mice. CONCLUSION: Pretargeting using MORFs was effective in a mouse tumor model.