(99m)Tc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents.

PURPOSE: Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents. METHODS AND RESULTS: Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of (99m)Tc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8pmol, respectively, per 10(10) cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90min for both (99m)Tc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of (99m)Tc-study MORF was significantly higher (p=0.009) than that of the control in the thigh with sterile inflammation. CONCLUSION: A (99m)Tc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.

Detection of Aspergillus fumigatus pulmonary fungal infections in mice with (99m)Tc-labeled MORF oligomers targeting ribosomal RNA.

PURPOSE: Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised patients. The fungus Aspergillus fumigatus (A. fumigatus) is the primary causative agent of invasive aspergillosis. However, A. fumigatus infections remain difficult to diagnose particularly in the early stages due to the lack of a rapid, sensitive and specific diagnostic approach. In this study, we investigated (99m)Tc labeled MORF oligomers targeting fungal ribosomal RNA (rRNA) for the imaging detection of fungal infections. PROCEDURES: Three phosphorodiamidate morpholino (MORF) oligomer (a DNA analogue) probes were designed: AGEN, complementary to a sequence of the fungal 28S ribosomal RNA (rRNA) of Aspergillus, as a genus-specific probe; AFUM, complementary to the 28S rRNA sequence of A. fumigatus, as a fungus species-specific probe; and cMORF, irrelevant to all fungal species, as a control probe. The probes were conjugated with Alexa Fluor 633 carboxylic acid succinimidyl ester (AF633) for fluorescence imaging or with NHS-mercaptoacetyl triglycine (NHS-MAG3) for nuclear imaging with (99m)Tc and then evaluated in vitro and in vivo. RESULTS: The specific binding of AGEN and AFUM to fungal total RNA was confirmed by dot blot hybridization while specific binding of AGEN and AFUM in fixed and live A. fumigatus was demonstrated by both fluorescent in situ hybridization (FISH) analysis and accumulation in live cells. SPECT imaging of BALB/c mice with pulmonary A. fumigatus infections and administered (99m)Tc labeled AGEN and AFUM showed immediate and obvious accumulation in the infected lungs, while no significant accumulation of the control (99m)Tc-cMORF in the infected lung was observed. Compared to non-infected mice, with sacrifice at 1h, the accumulation of (99m)Tc-AGEN and (99m)Tc-AFUM in the lungs of mice infected with A. fumigatus was 2 and 2.7 fold higher respectively. CONCLUSIONS: In vivo targeting fungal ribosomal RNA with (99m)Tc labeled MORF probes AGEN and AFUM may be useful for A. fumigatus infection imaging and may provide a new strategy for the noninvasive diagnosis of invasive aspergillosis and other fungal infections.

Her2/neu small interfering RNA delivered in culture by a streptavidin nanoparticle.

A three-component nanoparticle consisting of biotinylated Trastuzumab antiHer2 antibody, tat transferring peptide and radiolabeled antisense oligomer, linked together through streptavidin, have shown promise in the delivery to Her2+ tumor in mice following intravenous administration and with evidence of radiotherapeutic efficacy. These results have encouraged us to consider the nanoparticle as a delivery vehicle for RNA interference therapy in which the radiolabeled antisense oligomer is replaced with an unlabeled siRNA duplex. The siRNA stability within the nanoparticle was first confirmed by incubation with RNase A. The interferon responses, that indicate off-target cytotoxicity, were evaluated by quantitative real-time RT-PCR in BT-474 (Her2+) human breast cancer cells by measuring the mRNA expression of 2', 5'-oligoadenylate synthetase (OAS1) and Stat-1, two key interferon-responsive genes. Thereafter the cytotoxicity induced by the siRNA nanoparticle was evaluated by a clonogenic survival assay in BT-474 cells while the Her2 expression of these target cells was evaluated for evidence of specific gene silencing. The siRNA within the three-component anti- Her2/neu siRNA nanoparticle was largely protected from RNase-dependent degradation and did not activate an interferon response. The nanoparticle effectively and significantly inhibited colony formation of the target cells and silenced the Her2 gene expression at 5 nM compared with the identical nanoparticle with a scrambled siRNA. Our delivery nanoparticle, with tumor targeting provided by the antibody and its accumulation without entrapment, possibly due to the transfecting peptide, delivered an siRNA duplex to the proper subcellular localization for specific and effective gene silencing in culture by what appears to be an siRNA mechanism.

Radiolabeled Zn-DPA as a potential infection imaging agent.

INTRODUCTION: A zinc-dipicolylamine analog (Zn-DPA) conjugated with a fluorophore (PSVue®794) has been shown to image bacterial infections in mice. However, radiolabeled Zn-DPA has not previously been considered for nuclear imaging of infection. METHODS: Both 111In-labeled DOTA-biotin and Zn-DPA-biotin were combined using streptavidin (SA) as a noncovalent linker. Mice injected intramuscularly with Streptococcus pyogenes (infection model) or with lipopolysaccharide (LPS) (inflammation model) were coinjected intravenously with 6 µg of DPA as PSVue794 and as 111In-DOTA-biotin/SA/biotin-Zn-DPA. Periodic fluorescent and SPECT (single photon emission computed tomography)/CT (computed tomography) images were acquired, and biodistributions were obtained at 22 h. RESULTS: Histological examination confirmed the validity of both the infection and inflammation animal models. Both the whole-body optical and nuclear images showed obvious accumulations in the target thigh in both models at all time points. At 22 h, the average target thigh accumulation of 111In was 1.66%ID/g (S.D. 0.15) in the infection mice compared to 0.58%ID/g (S.D. 0.07) in the inflammation mice (P<.01), and the 111In target/normal thigh ratio was 2.8 fold higher in the infection animals compared to the inflammation animals. CONCLUSIONS: These preliminary results show that Zn-DPA within streptavidin targets S. pyogenes-infected mice similarly to its free fluorescent analogue. The significantly higher accumulation in the live bacterial infection thigh compared to that of the LPS-induced inflammation thigh suggests that Zn-DPA may be a promising imaging agent to distinguish between bacterial infections and sterile inflammations.

Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation.

INTRODUCTION: Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus. METHODS: As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared. The three-component noncovalent nanoparticle consisted of streptavidin linking three biotinylated components: a Cy3-labeled anti-RIα antisense phosphorodiamidate morpholino (MORF) oligomer, a tat transfecting peptide and the anti-Her2 herceptin antibody. The covalent constructs included an anti-RIα antisense DNA conjugated to a radiolabeled herceptin and a fluorescent DNA conjugated to native herceptin. Fluorescence microscopy in SK-BR-3 (Her2+) cells was used to evaluate the fate of the fluorescent Cy5.5-DNA and Cy3-MORF, while the subcellular accumulation of the (111)In-labeled herceptin and herceptin-DNA in both SK-BR-3 and MDA-MB-231 (Her2) cells was determined by isolating and counting the nuclear fractions. RESULTS: Previously, we demonstrated that when incubated as the three-component nanoparticle consisting of herceptin and streptavidin and (99m)Tc-labeled antisense MORF, only the MORF accumulated in the nucleus of Her2+ cells. In this investigation, clear evidence was observed of nuclear accumulation of the antisense oligomer within the noncovalent nanoparticle as before, but when incubated as the covalent construct, by both fluorescence microscopy and nuclear counting, no evidence of nuclear accumulation was observed. CONCLUSION: The weaker noncovalent biotin-streptavidin bond may be essential for adequate delivery of the radiolabeled antisense oligomer to the nucleus of tumor cells.

A brief evaluation of tumor imaging in mice with 99mTc-glucarate including a comparison with 18F-FDG.

OBJECTIVE: Recently 99mTc-glucarate, a radiolabeled glucose analogue, has been considered as a SPECT alternative to 18F-FDG and PET for non-invasive detection of certain tumors. Thus far there have been few studies on (99m) Tcglucarate for tumor imaging and fewer, if any, studies comparing (99m)Tc-glucarate with 18F-FDG. As a preliminary indication of the properties of (99m)Tc-glucarate as a possible substitute for 18F-FDG in animal studies, we have imaged mice bearing xenografts of four tumor types with (99m)Tc-glucarate and have compared in two mice with one of these tumor types the 99mTc and 18F biodistributions. METHODS: Two mice bearing SUM190 breast cancer xenografts received 1 mCi of (99m)Tc-glucarate and were imaged on a NanoSPECT/CT small animal camera. One day later, the same animals received 1 mCi of 18F-FDG and were imaged on a MosaicHP PET small animal camera. In addition, 0.5-1 mCi of (99m)Tc-glucarate only was administered to mice bearing xenografts induced by BxPC3 pancreatic cancer cells, HEK-293 renal cell carcinomas cells or HCT-116 colorectal tumor cells. NanoSPECT/CT acquisitions were performed in these mice to evaluate tumor accumulations. RESULTS: In the SUM190 xenografted mice, the average tumor accumulation was 1.4 % (ID%/cm3) for (99m)Tc-glucarate and 2.1 % (ID%/cm3) for 18F-FDG. While slightly higher than (99m)Tc-glucarate, the tumor accumulation of 18F-FDG was accompanied by higher bone marrow and muscle accumulations at levels that could interfere with the tumor image depending upon location. The whole body clearance of (99m)Tc-glucarate was faster than that of 18F-FDG. Tumor accumulation of (99m)Tc-glucarate varied among tumor types but the tumors were readily visible in all images. CONCLUSION: In a direct comparison in the same two SUM190 tumored animals, SPECT images obtained with (99m)Tcglucarate compared favorably with PET images obtained with 18F-FDG. Tumor images with 99mTc-glucarate were also positive in three additional tumor mouse models. While further comparison studies are necessary, we conclude that (99m)Tcglucarate may be a more convenient and less expensive alternative to 18F-FDG for tumored mouse studies.

90Y labeled phosphorodiamidate morpholino oligomer for pretargeting radiotherapy.

While (188)Re has been used successfully in mice for tumor radiotherapy by MORF/cMORF pretargeting, previous radiolabeling of the amine-derivatized cMORF with (90)Y, a longer physical half-life nuclide, was not very successful. After developing a method involving a prepurification heating step during conjugation that increases labeling efficiency and label stability, the biodistribution of (90)Y-DOTA-Bn-SCN-cMORF ((90)Y-DOTA-cMORF) was measured in normal mice and in MORF-CC49 pretargeted mice that bear LS174T tumors. Absorbed radiation doses were then estimated and compared to those estimated for (188)Re. The pharmacokinetics of the (90)Y-DOTA-cMORF in normal mice and in the pretargeted nude mice was similar to that observed previously with (99m)Tc- and (188)Re-MAG(3)-cMORFs. While the (90)Y-DOTA-cMORF cleared rapidly from normal tissues, tumor clearance was very slow and tumor radioactivity accumulation was constant for at least 7 days such that the tumor/blood (T/B) ratio increased linearly from 6 to 25 over this period. Therefore, by extrapolation, normal tissue toxicities following administration of therapeutic doses of (90)Y may be comparable to that observed for (188)Re in which the T/B increased from 5 to 20. In conclusion, radiolabeling of DOTA-cMORF with (90)Y was improved by introducing a prepurification heating step during conjugation. The (90)Y-DOTA-cMORF provided a similar T/B ratio and biodistribution to that of (188)Re-MAG(3)-cMORF and was retained well in the tumor pretargeted with MORF-CC49. Because of the longer physical half-life, the T/NT absorbed radiation dose ratios were improved in most organs and especially in blood.

Improving the quantitation accuracy in noninvasive small animal single photon emission computed tomography imaging.

INTRODUCTION: Noninvasive imaging of small animals to measure biodistributions and pharmacokinetics of radiolabeled agents is increasingly seen as an effective alternative to external counting of tissues obtained by sacrifice and dissection. However, we have observed important disagreements in measuring the accumulation of (111)In-labeled antibodies in organs such as liver and kidneys when comparing imaging to ex vivo counting in the same animals. This study was conducted to establish whether this discrepancy could be minimized by selecting the region of interest (ROI) in images at the appropriate color threshold and by correcting for the estimated radioactivity within the blood pool of these organs during imaging. METHODS: Vials with known concentrations of (111)In as phantoms were imaged on a Bioscan NanoSPECT/CT. Thereafter, an (111)In-DTPA-IgG antibody as the test agent was administered intravenously to normal rats, and whole body acquisitions were obtained at 2, 24 or 48 h. Immediately following imaging, the animals were sacrificed, the tissues were removed for ex vivo counting and the radioactivity accumulations were then compared. RESULTS: The phantom measurements showed that accuracy depended upon setting the correct ROI and that, in turn, depended upon setting the appropriate threshold of the color scale. Under the most unfavorable conditions, this error did not exceed 60%. Compared to the results of ex vivo counting, quantitation by imaging provided high values in liver and kidneys at all three time points by as much as 140%. However, by using the blood radioactivity at the time of sacrifice and the known blood volume in these organs, the disagreement was reduced in all cases to below 25%. CONCLUSION: In this study, the discrepancy in quantitating organ radioactivity accumulations between noninvasive imaging and necropsy was primarily due to blood pool radioactivity contributing to the in vivo images. The discrepancy may be minimized by subtracting an estimate of this contribution.

Comparing two TAG-72 binding peptides previously identified by phage display as potential imaging agents.

AIM: To evaluate the targeting property in vitro and in vivo of two tumor-associated glycoprotein 72 (TAG-72) binding peptides, previously identified in this laboratory by phage selection using different elution conditions. MATERIALS AND METHODS: The peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were radiolabeled with technetium-99m ((99m)Tc) using N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine (NHS-MAG(3)) as a chelator or were biotinylated. The specificity of the two peptides for the TAG-72 positive LS-174T cancer cells was demonstrated in vitro both by flow cytometry analysis using the biotinylated peptides and by competitive binding using the (99m)Tc-labeled peptides. The in-vivo biodistributions of the peptides were evaluated in TAG-72 positive LS-174T tumor-bearing mice by small-animal single photon emission computed tomography/computed tomography imaging. RESULTS: As evidence of specific binding, both peptides showed a significant increase in percentage binding with increasing peptide concentration by flow cytometry analysis to LS-174T cells, but not to TAG-72 negative HT-29 cells. The (99m)Tc-labeled A2-6 peptide bound LS-174T cells with an inhibition constant at 50% of 46.5 nmol/l compared with 420 nmol/l for the A3-10 peptide. In mice, accumulation of both peptides was highest in kidneys and gallbladder. Tumors were clearly visible by single photon emission computed tomography imaging for both (99m)Tc-labeled peptides through 60 min, although the tumor accumulation was higher for the A3-10 peptide. CONCLUSION: The A3-10 peptide, with lower, yet reasonable binding affinity compared with the A2-6 peptide, showed sufficiently favorable specific binding and tumor accumulation to be considered further as a potential imaging agent for TAG-72 positive cancers.

A nanoparticle for tumor targeted delivery of oligomers.

The tissue-specific delivery nanoparticle consists of an antisense oligomer, a cell-penetrating peptide, and an antitumor antibody, each biotinylated and each linked via streptavidin. Within the nanoparticle, the antibody provides specific targeted delivery and binding to the target cells, the peptide improves cell membrane transport, and the antisense oligomer, through its mRNA-binding ability, provides specific retention of the radioactivity in the target cell nucleus. The use of streptavidin as linker eliminates the need for covalent conjugation without appearing to interfere with the in vitro and in vivo properties of each component. The delivery nanoparticle is under development to improve tumor targeting with unlabeled siRNAs as well as radiolabeled antisense oligomers in a variety of tumor types. The anti-HER2 Trastuzumab (Herceptin) antibody, the tat peptide, and a radiolabeled antisense oligomer against the RIα mRNA have been used in this report as an example.

Human islet cell MORF/cMORF pretargeting in a xenogeneic murine transplant model.

Noninvasive measurement of human islet cell mass in pancreas or following islet transplantation by nuclear imaging has yet to be achieved. It has been shown using mouse tumor models that pretargeting imaging strategies are sensitive and can greatly increase target to nontarget signal ratios. The objective now is to demonstrate the specific pretargeting of human islet cells in mice. Our pretargeting strategy uses an anti-human islet cell antibody HPi1, conjugated to a phosphorodiamidate morpholino oligomer (MORF) that binds specifically to a (99m)Tc labeled complementary MORF (cMORF). Sensitivity and specificity of the pretargeting were first validated in culture using a human beta cell line (betalox5) and a negative control human cell line (HEK293). Pretargeting was then used to target and visualize these two cell lines and human islets transplanted subcutaneously in NOD-scid IL2rγ(null) mice. In culture, (99m)Tc accumulation on the betalox5 cells pretargeted by MORF-HPi1 was 100-fold higher than on untreated betalox5 cells or following treatment with native HPi1 and much higher than on the MORF-HPi1 pretargeted control HEK293 cells. Small animal imaging readily localized the transplanted betalox5 cells and human islets, but not the HEK293 cells. Ex vivo counting demonstrated 3-fold higher (99m)Tc accumulation in the transplanted betalox5 cells and human islets than in the control HEK293 cells. The target accumulation was also shown to increase linearly with increased numbers of the implanted betalox5 cells. These results demonstrate specific binding of radioactivity and successful imaging of human betalox5 cells and human islets transplanted in mice. Thus MORF/cMORF pretargeting may be useful to measure noninvasively human islet cell mass within the pancreas or following islet transplantation.

Unexpected side products in the conjugation of an amine-derivatized morpholino oligomer with p-isothiocyanate benzyl DTPA and their removal.

In connection with pretargeting, an amine-derivatized morpholino phosphorodiamidate oligomer (NH(2)-cMORF) was conjugated conventionally with p-isothiocyanate benzyl-DTPA (p-SCN-Bn-DTPA). However, after (111)In radiolabeling, unexpected label instability was observed. To understand this instability, the NH(2)-cMORF and, as control, the native cMORF without the amine were conjugated in the conventional manner. Surprisingly, the (111)In labeling of the native cMORF conjugate was equally effective as that of the NH(2)-cMORF conjugate (>95%) despite the absence of the amine group. Furthermore, heating the radiolabeled NH(2)-cMORF and native cMORF conjugates resulted in a 35% loss and a complete loss of the label, respectively. Since the (111)In labeled DTPA is known to be stable, the instability in both cases must be due to some unstable association of DTPA to the cMORF, presumably unstable association to some endogenous sites in cMORF. Based on this assumption, a postconjugation-prepurification heating step was introduced, and labeling efficiency and stability were again investigated. By introducing the heating step, the side products were dissociated, and after purification and labeling, the NH(2)-cMORF conjugate provided a stable label and high labeling efficiency with no need for postlabeling purification. The biodistribution of this radiolabeled conjugate in normal mice showed significantly lower backgrounds compared with the labeled unstable native cMORF conjugate. In conclusion, the conventional conjugation procedure to attach the p-SCN-Bn-DTPA to NH(2)-cMORF resulted in side product(s) that were responsible for the (111)In label instability. Adding a postconjugation-prepurification heating step dissociated the side products, improved the label stability and lowered tissue backgrounds in mice.

Identification of a high affinity TAG-72 binding peptide by phage display selection.

PURPOSE: Phage display was used to select novel peptides that specifically bind the TAG-72 antigen and with properties suitable for imaging TAG-72 positive cancers. RESULTS: After three rounds of selection against TAG-72 and using two different elution conditions including a long elution, the consensus sequences FRERCDKHPQKCTKFL and DPRHCQKRVLPCPAWL were expressed on phages G3-15 and T3-15 respectively. ELISA, fluorescence-activated cell sorting analysis and fluorescence microscopy provided evidence that both phages specifically bound TAG-72 in vitro. Both peptides are stable in 37oC serum. By a cell binding competition assay, the IC50 for T3-15 was measured as 0.29 nM and therefore 36-fold higher affinity than G3-15 at 10.32 nM. The biodistribution in mice carrying LS-174T tumors in one thigh were similar for both 99mTc-peptides at 30 min, but at 90 min the 99mTc-T3-15 peptide accumulated almost three times higher in the tumor. The SPECT/CT images were consistent with the biodistribution results. PROCEDURES: The f88-4/Cys6 phage library and two different elution conditions were used to identify two new higher affinity binding peptides for the TAG-72 antigen. One, was a single brief elution with pH 2.2 glycine buffer, and the second began with the glycine elution but was followed with a longer elution with Tris buffered saline (TBS) at pH 7.4. The phages that bound TAG-72 were evaluated by fluorescence-activated cell sorting analysis using TAG-72 positive LS-174T cells and confirmed by immunofluorescence imaging. The consensus peptides displayed on the selected phages were synthesized and conjugated with NHS-MAG3 for radiolabeling with 99mTc. The IC50 for TAG-72 binding was evaluated by cell binding competition in vitro while binding affinity was evaluated in vivo by necropsy and SPECT/CT imaging in a tumor mouse model. CONCLUSION: We have identified a peptide with a sub nanomolar inhibition constant for the TAG-72 antigen that may have application in cancer imaging.

Reducing the background fluorescence in mice receiving fluorophore/inhibitor DNA duplexes.

In principle, a DNA duplex consisting of an antisense fluorophore-conjugated major strand hybridized to a shorter complementary inhibitor-conjugated minor strand should provide fluorescence only in the tumor after intravenous administration if designed to remain intact except in the presence in tumor of its mRNA target. While we have obtained impressive tumor images in mice using this approach, there remains some background fluorescence. In this study, tissue homogenates of selected mouse organs were incubated with a test duplex and the kinetics of duplex dissociation in normal tissues were measured. In this manner we were able to identify the liver as the likely major source responsible for the duplex dissociation providing this fluorescence background. Thereafter liver homogenates were used to screen a series of duplex candidates with variable-length minor strands, and dissociation was measured by gel electrophoresis. The selected fluorophore/inhibitor duplex with improved stability displayed an insignificant (P > 0.05) background fluorescence after administration to SKH-1 normal mice and apparently without affecting target mRNA binding in vitro in cell culture or in vivo in tumor bearing mice.

A preclinical 188Re tumor therapeutic investigation using MORF/cMORF pretargeting and an antiTAG-72 antibody CC49.

The utility of MORF/cMORF pretargeting for the radiotherapy of cancer requires further validation in tumored mice before clinical trials. We now report on a therapeutic study in mice pretargeted with MORF-CC49 (an anti-TAG-72 antibody CC49 conjugated with MORF, a phosphorodiamidate morpholino oligomer) and then targeted by 188Re-cMORF (a 188Re labeled complementary MORF). Before the dose-escalating therapeutic study, a pretargeting study in LS174T tumored mice was performed at tracer levels. By both necropsy and imaging, the tracer study showed that the whole body radioactivity was largely restricted to tumor in the mice pretargeted 48 h earlier with MORF-CC49 and the tumor radioactivity was retained over 90 h. After decay correction, a best-fit to the biodistribution provided the areas under the radioactivity curves (AUCs) used for the radiation dose estimates. The tumor to normal organ AUC ratios in all cases were greater than unity and ranged from 3 (kidneys) to 48 (muscle). Tumor growth was inhibited in the therapy study. At the highest 188Re dose of 1.40 mCi, a complete but temporary tumor remission was evident in 3 out of the 5 animals. Histological examination of tissues from these animals showed no evidence of cytotoxicity to normal tissues but obvious radiation damage to tumor. In conclusion, effective radiotherapy was achieved in a mouse model by MORF/cMORF pretargeting using 188Re as the therapeutic radionuclide and CC49 as the pretargeting antibody.

Auger-mediated cytotoxicity of cancer cells in culture by an 125I-antisense oligomer delivered as a three-component streptavidin nanoparticle.

We reported recently that a three-component nanoparticle, consisting of a targeting antibody, a transfecting peptide and an 111In-antiRIalpha MORF antisense oligomer, provided Auger electron-mediated, antisense-mediated, cytotoxicity of cells in culture. We have now measured the cytotoxicity of the nanoparticle in culture with the 111In replaced by 125I, another attractive Auger electron emitter. The nanoparticle consisted of streptavidin linking the 125I labeled antiRIalpha mRNA antisense MORF oligomer, the tat transfecting peptide and the anti-Her2 Trastuzumab antibody. Cytotoxicity was evaluated by a clonogenic survival assay in BT-474 (Her2+) human breast cancer cells. In a dose escalation study, as measured by the surviving fraction, the cytotoxicity of tumor cells to the 125I-labeled antisense nanoparticle was significantly higher than that for the identical sense control. When compared with our previous study with 111In as label, a similar level of cytotoxicity was achieved but the observed minimal therapeutic dose for the 125I-labeled nanoparticle in BT-474 cells was lower than that for 111In-labeled nanoparticle in SK-BR-3 cells. Thus, a radiolabeled antisense MORF oligomer delivered into cells by a three-component nanoparticle is an effective vehicle for Auger radiotherapy when radiolabeled with 111In or 125I.

Comparison of 18F PET and 99mTc SPECT imaging in phantoms and in tumored mice.

Our objective was to compare the performance of a micro-single photon emission computed tomography (micro-SPECT) with that of a micro-positron emission tomography (microPET) in a Her2+ tumored mice using an anti-Her2 nanoparticle radiolabeled with (99m)Tc and (18)F. Camera performance was first compared using phantoms; then a tumored mouse administered the (99m)Tc-nanoparticle was imaged on a Bioscan NanoSPECT/CT, while another tumored mouse received the identical nanoparticle, labeled now with (18)F, and was imaged on a Philips Mosaic HP PET camera. The nanoparticle was radiolabeled with (99m)Tc via MAG(3) chelation and with (18)F via SFB as an intermediate. Phantom imaging showed that the resolution of the SPECT camera was clearly superior, but even with 4 heads and multipinhole collimators, detection sensitivity was 15-fold lower. Radiolabeling of the nanoparticle by chelation with (99m)Tc was considerably easier and safer than manual covalent attachment of (18)F. Both cameras provided accurate quantitation of radioactivity over a broad range. In conclusion, when deciding between (99m)Tc vs (18)F, an advantage rests with the chelation of (99m)Tc over covalent attachment of (18)F, achieved manually or otherwise, but with these small animal cameras, this choice also results in trading lower sensitivity for higher resolution.

Multimodality nuclear and fluorescence tumor imaging in mice using a streptavidin nanoparticle.

Combining two or more different imaging modalities in the same agent can be of considerable value in molecular imaging. We describe the use of streptavidin nanoparticle-based complexes as multimodality imaging agents to achieve tumor detection in a mouse model by both fluorescence and nuclear imaging. Up to four biotinylated functionalities can be readily attached to these streptavidin nanoparticles without apparent influence on their properties and with reasonable pharmacokinetics and therefore may be ideally suited for multimodality imaging. By binding a biotinylated anti-Her2 Herceptin antibody to provide tumor targeting, a biotinylated DOTA chelator labeled with (111)ln and a biotinylated Cy5.5 fluorophore to a streptavidin nanoparticle, we demonstrated multimodality imaging in SUM190 (Her2+) tumor bearing mice on both an IVIS fluorescence camera and a NanoSPECT/CT small animal nuclear camera. The imaging results show high tumor accumulation and strong tumor-to-normal tissue contrast by both fluorescence and nuclear imaging. The subsequent biodistribution study confirmed the specific tumor accumulation in that tumor accumulation of radioactivity at 40 h was 21 ID%/g and therefore much higher than all other tissues including liver, heart, kidney, spleen, and muscle that accumulated 8.7, 2.5, 6.9, 7.2, and 1.9 ID%/g, respectively. In conclusion, the streptavidin nanoparticle under development in this laboratory was used effectively for multimodality imaging of tumor in mice by fluorescence and nuclear detection. Presumably, other imaging modalities could also be considered.

Affinity enhancement pretargeting: synthesis and testing of a 99mTc-labeled bivalent MORF.

Pretargeting with bivalent effectors capable of bridging antitumor antibodies (affinity enhancement pretargeting) has been reported to provide superior results by affinity enhancement. Phosphorodiamidate morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, a determinant of binding, may be adjusted simply by lengthening the chain. We have shown by surface plasmon resonance that bivalent MORFs will provide superior affinity enhancement provided that suitable spacing exists between the binding regions. The goals of this study were to synthesize a bivalent MORF with a MAG(3) group attached for technetium-99m ((99m)Tc) radiolabeling, investigate whether the bivalent MORF showed improved cell accumulation in culture compared to its corresponding monovalent MORF and compare biodistributions in normal mice and in pretargeted tumored mice. An excess of an amine derivatized 18 mer MORF with 6 nonbinding bases for spacing was reacted with Fmoc-l-beta-homoglutamic acid to form duplexes via their carboxylate groups and, after deprotection, conjugated with NHS-MAG(3) to attach the chelator. The anti-CEA antibody MN14 was conjugated with a 12 mer complementary MORF (i.e., cMORF). The binding behavior between radiolabeled monovalent and bivalent MORFs was compared in LS174T tumor cells at 4 degrees C pretargeted with MN14-cMORF. Biodistributions of radiolabeled monovalent and bivalent MORFs at 3 h postadministration were measured in normal mice and in tumor mice pretargeted with MN14-cMORF. In the pretargeted cells in culture, the accumulation of the bivalent MORF was significantly higher than the monovalent MORF (p = 0.002), thus providing strong evidence for affinity enhancement. In normal mice, whole body clearance of the bivalent and monovalent MORFs was equally rapid. In tumored mice, tumor accumulation of the radiolabeled bivalent MORF was significantly higher than that of the monovalent MORF. In conclusion, a bivalent MAG(3)-MORF was successfully synthesized and radiolabeled with (99m)Tc. While a pharmacokinetic effect for the higher tumor accumulations in pretargeted mice of the radiolabeled bivalent MORF cannot be excluded, the results may be best explained by affinity enhancement. Thus two monovalent MORFs were covalently conjugated into a bivalent MORF effector to improve tumor targeting by both pharmacokinetics and affinity enhancement influences.

Preparation of (111)In-DTPA morpholino oligomer for low abdominal accumulation.

An ability to quantitate the beta cell mass by noninvasive nuclear imaging will be very useful in the prevention, diagnosis, and treatment of diabetes. However, to be successful, radioactivity from the pancreas must not be obscured by the background radioactivity in the abdomen. Pretargeting offers the promise of achieving high target organ to normal tissue ratios. In preparation for pancreas imaging studies by pretargeting using morpholino oligomers (MORF/cMORF), it was necessary to develop a simple and efficient method to radiolabel the cMORF effector. Because we have shown that labeling the cMORF with (111)In via DTPA reduces excretion into the intestines compared to labeling with (99m)Tc via MAG(3), the conjugation of DTPA to cMORF was investigated for (111)In labeling. The amine-derivatized cMORF was conjugated with DTPA using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as an alternative to the conventional cyclic anhydride. The conjugation efficiency (represented by the number of DTPA groups attached per cMORF) was investigated by changing the EDC, DTPA, and cMORF molar ratios. Different open columns were considered for the purification of DTPA-cMORF. Before conjugation, each cMORF molecule was confirmed to have an amine by trinitrobenzene sulfonic acid (TNBS) assay using the omega-amino butyric acid as positive standard and the non-amine derivatized cMORF as negative standard. The average number of DTPA groups per cMORF was 0.15-0.20 following the conjugation over a cMORF/DTPA molar ratio of 0.5-5 and over a cMORF/EDC molar ratio of 20-60. The conjugation efficiency was lower than expected probably due to steric hindrance. A 1 x 50cm P-4 column using ammonium acetate as eluting buffer provided an adequate separation of DTPA-cMORF from free DTPA. The (111)In labeling efficiency by transchelation from acetate exceeded 95%, thus avoiding the need for postlabeling purification. Despite the lower than expected conjugation efficiency in which no more than one-fifth of the cMORF were DTPA-derivatized, a specific radioactivity of at least 300microCi/microg or 1.90Ci/micromol of cMORF was achieved. In conclusion, a protocol is described for (111)In-DTPA-cMORF that provides the high specific activity favorable to beta cell imaging because of the low mass fraction of beta cells in pancreas (1-2%) and obviates the need for postlabeling purification.