Shuping Dou

Successful radiotherapy of tumor in pretargeted mice by 188Re-radiolabeled phosphorodiamidate morpholino oligomer, a synthetic DNA analogue.

Purpose: Pretargeting has been attracting increasing attention as a drug delivery approach. We recently proposed Watson-Crick pairing of phosphorodiamidate morpholino oligomers (MORF) for the recognition system in tumor pretargeting. MORF pretargeting involves the initial i.v. injection of a MORF-conjugated antitumor antibody and the subsequent i.v. injection of the radiolabeled complement. Our laboratory has reported on MORF pretargeting for diagnosis using 99mTc as radiolabel. We now report on the use of MORF pretargeting for radiotherapy in a mouse tumor model using 188Re as the therapeutic radiolabel. Experimental Design: An initial tracer study was done to estimate radiation dose, and was followed by the radiotherapy study at 400 μCi per mouse with three control groups (untreated, MORF antibody alone, and 188Re complementary MORF alone). Results: Tracer study indicated rapid tumor localization of 188Re and rapid clearance from normal tissues with a tumor area under the curve (AUC) about four times that of kidney and blood (the normal organs with highest radioactivity). Tumor growth in the study group ceased 1 day after radioactivity injection, whereas tumors continued to grow at the same rate among the three control groups. At sacrifice on day 5, the average net tumor weight in the study group was significantly lower at 0.68 ± 0.29 g compared with the three control groups (1.24 ± 0.31 g, 1.25 ± 0.39 g, and 1.35 ± 0.41 g; Ps < 0.05), confirming the therapeutic benefit observed by tumor size measurement. Conclusions: MORF pretargeting has now been shown to be a promising approach for tumor radiotherapy as well as diagnosis.

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.

Investigation of four 99mTc-labeled bacteriophages for infection-specific imaging

INTRODUCTION This study investigated radiolabeled bacteriophages for specific detection of infection through gamma imaging. Previously, a (99m)Tc-labeled M13 phage demonstrated specific binding for its host Escherichia coli in vitro and in mice through imaging. METHODS This study was extended to phages P22, E79, VD-13 and phage 60. Each was radiolabeled with (99m)Tc using the chelator MAG(3), and were evaluated for binding to host and non-host bacteria in vitro and in a mouse infection model. RESULTS In vitro, each (99m)Tc-phage bound to its host at least 4-fold higher than to non-host bacteria. For example, (99m)Tc-E79 showed 10- to 20-fold greater binding to host Pseudomonas aeruginosa compared to non-host Escherichia coli and Salmonella enterica, and (99m)Tc-phage 60 showed 20-fold greater binding to host Klebsiella pneumoniae over non-hosts. Mice received host or non-host bacteria in one thigh, and 3 h later, the (99m)Tc-phages were administered intravenously. After a further 3 h, the tissues were counted. Liver accumulation was highest for (99m)Tc-E79, averaging 39% compared to an average of 13% for the other (99m)Tc-phages. Animals infected with host bacteria showed infected thigh/normal thigh ratios of 14.2 for (99m)Tc-E79, 2.9 for (99m)Tc-P22, 3.5 for (99m)Tc-VD-13 and 2.1 for (99m)Tc-phage 60. CONCLUSIONS Although specific host binding was observed in vitro for each of these four (99m)Tc-phages, only (99m)Tc-E79 showed specificity for its host in an in vivo model.

A new TAG-72 cancer marker peptide identified by phage display

Radiolabeled peptides as markers of cancer targets have demonstrated their value in diagnostic imaging and radiotherapy. The 16 mer f88-4/Cys6 phage display library was applied to affinity purified TAG-72 and three consensus peptides were identified: VHHSCTKLTHCCQNWH (A2-13), GGVSCMQTSPVCENNL (A2-6) and TKRDCSAQNYGCQKAI (A2-11). The A2-13 and A2-6 phages showed the highest percent binding to LS-174T cells by flow cytometry and were 3-fold higher than a control phage, while fluorescence microscopy showed that both A2-6 and A2-13 phages bound to the LS-174T cell membrane. However, only the A2-6 phage demonstrated specificity by low binding to the TAG-72 negative cell HT-29. Furthermore, the synthesized free A2-6 peptide demonstrated specific binding to LS-174T cells by flow cytometry and by immunohistochemical staining of xenograft tumor compared to normal colon. These data indicate that the A2-6 peptide is specific for the TAG-72 cancer target.

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.

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.

Predicting the biodistribution of radiolabeled cMORF effector in MORF-pretargeted mice

PurposePretargeting with phosphorodiamidate morpholino oligomers (MORFs) involves administration of a MORF-conjugated anti-tumor antibody such as MN14 as a pretargeting agent before that of the radiolabeled complementary MORF (cMORF) as the effector. The dosages of the pretargeting agent and effector, the pretargeting interval, and the detection time are the four pretargeting variables. The goal of this study was to develop a semiempirical description capable of predicting the biodistribution of the radiolabeled effector in pretargeted mice and then to compare predictions with experimental results from pretargeting studies in tumored animals in which the pretargeting interval and the detection time were both fixed but the dosages of both the effector and the pretargeting agent were separately varied.MethodsPretargeting studies in LS174T tumored mice were performed using the anti-CEA antibody MN14 conjugated with MORF and the cMORF radiolabeled with 99mTc. A description was developed based on our previous observations in the same mouse model of the blood and tumor levels of MORF-MN14, accessibility of MORF-MN14 to labeled cMORF, the tumor accumulation of labeled cMORF relative to MORF-MN14 levels therein, and the kidney accumulation of labeled cMORF. The predicted values were then compared with the experimental values.ResultsThe predicted biodistribution of the radiolabeled effector and the experimental data were in gratifying agreement in normal organs, suggesting that the description of the pretargeting process was reliable. The tumor accumulations occasionally fell outside two standard deviations of that predicted, but after tumor size correction, good agreement between predicted and experimental values was observed here as well.ConclusionA semiempirical description of the biodistribution of labeled cMORF was capable of predicting the biodistribution of the radiolabeled effector in the pretargeted tumored mouse model, demonstrating that the underlying pretargeting concepts are correct. We believe that the approach described herein may be applied to any of the alternative pretargeting approaches and animal tumor models currently under investigation. Furthermore, appreciation of the concepts may provide a rationale for selecting dosages and timings in human pretargeting studies as an alternative to pure empirical means.

Synthesis and in vitro characterization of a dendrimer-MORF conjugate for amplification pretargeting

Amplification pretargeting can play an important role in molecular imaging by significantly increasing the accumulation of signal in target tissues. Multiple-step amplification pretargeting offers the potential to greatly improve target localization of effector molecules through the intermediate use of polymers conjugated with multiple copies of complementary oligomers. In this study, PAMAM dendrimer generation 3 (G3) was conjugated with multiple copies of a phosphorodiamidate morpholino (MORF) oligomer. Characterization of the conjugate by native-PAGE and SE-HPLC demonstrated that the conjugation was successful. The average numbers of MORF groups in the G3-MORF conjugate, both attached and accessible to the (99m)Tc labeled complementary MORF (cMORF), were determined. The antitumor antibody CC49 was conjugated with both MORF and cMORF (collectively (c)MORF) at an average of about one group per molecule. Nine of the 32 carboxyl groups of the dendrimer were modified with MORF, of which 90% were accessible in solution to (99m)Tc-cMORF. After purification, the G3-MORF was radiolabeled with tracer (99m)Tc-labeled cMORF (i.e., G3-MORF/(99m)Tc-cMORF) and added to the antibody CC49 previously conjugated with cMORF (i.e., CC49-cMORF/G3-MORF/(99m)Tc-cMORF), the complex demonstrated a single peak on SE-HPLC as evidence of complete hybridization between G3-MORF/(99m)Tc-cMORF and CC49-cMORF. The CC49-(c)MORF were bound to both Protein G and Protein L coated plates, and G3-MORF was added to hybridize with CC49-cMORF before the (99m)Tc-cMORF was added to test amplification pretargeting. In comparison to conventional pretargeting without the G3-MORF, the signal was amplified about 6 and 14 times, respectively, showing that the G3-MORF participated in amplifying the signal. Further amplification studies using the CC49-(c)MORF for LS174T tumor cells in tissue culture also demonstrated clear evidence of signal amplification.

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.

An experimental and theoretical evaluation of the influence of pretargeting antibody on the tumor accumulation of effector

In treating tumors by pretargeting, the antitumor antibody and the cytotoxic effector (e.g., toxins and radioactivity) are separately administered. Therefore, pretargeting is more complicated with many variables. We are conducting studies to understand the influence of each variable using a novel recognition pair of mutually complementary phosphorodiamidate morpholino oligomers (MORF/cMORF). Earlier we developed a semi-empirical model capable of accurately predicting the behavior of a radiolabeled cMORF effector with variations in dosages and timing. We have now extended the model to predict the effector behavior, in particular, its maximum percent tumor accumulation (MPTA) in mice pretargeted with three different MORF-conjugated antibodies (MN14, B72.3, and CC49). The MN14 and the CC49 target different antigens in the same tumor, whereas the CC49 and the B72.3 target the same antigen but with very different tumor accumulation. By comparing the pretargeting results of these three antibodies with our prediction, we confirmed that the MPTA of the radiolabeled cMORF effector in the LS174T tumor is independent of the antibodies. In conclusion, the MPTA cannot be improved through the use of different pretargeting antibodies, although different antibodies may improve the maximum absolute tumor accumulation, the heterogeneity, and/or the tumor-to-normal tissue ratios of the effector. This conclusion will apply equally well to effectors carrying a fluorescent probe, an anticancer agent, or a radioactive imaging agent. [Mol Cancer Ther 2008;7(5):1025–32]