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.     

Cytosine residues influence kidney accumulations of 99mTc-labeled morpholino oligomers

Watson-Crick pairing between complementary oligomers is proving to be an effective means for rapidly directing radioisotopes specifically to the exterior surface of cancer cells in vivo. In such pretargeting applications, it is highly desirable that the excess of isotopically labeled oligomers, which do not bind to the cancer cells, be rapidly cleared from the body. In this context, understanding the influence of chain length and base sequence of the radiolabeled oligomers is critical. We had earlier determined that the kidneys are the principal targets of short-chain radiolabeled morpholino oligomers (MORFs). To explain these observations, MORFs consisting of uniform cytosines (Cs), uniform thymines (Ts), uniform adenines (As), and uniform AAG repeat were labeled with technetium-99m (99mTc) and studied in normal mice. In a limited investigation of the influence of oligomer backbone, a 20-mer MORF (MORF20) with a base sequence rich in Cs was compared with a phosphoromonothioate DNA (S-DNA20) of the same sequence. The in vivo behavior of the labeled MORFs was nearly identical in all organs, with the exception of kidneys. The kidney accumulations were about 25- to 80-fold higher for the uniform Cs relative to the other three uniform MORFs at 3 hours. The S-DNA20 rich in Cs showed only modest kidney accumulations compared with the equivalent MORF20, presumably because of preferential clearance of the S-DNA20 through the liver. Urine analysis showed no evidence of intact labeled S-DNA20 in contrast to fully intact labeled MORF20. We conclude that the high kidney levels observed by us previously for MORFs are most likely due largely to the C residues in the base sequence. In the case of S-DNAs, this phenomenon is partly disguised by the increased hepatic excretion and degradation. These results show that the base sequences of MORFs, and probably other oligomers as well, are an important determinant of biodistribution.     

Affinity enhancement bivalent morpholinos for pretargeting: surface plasmon resonance studies of molecular dimensions

Bivalent effectors have been reported to provide superior pretargeting by affinity enhancement. We recently reported that one bivalent MORF (phosphorodiamidate morpholino, a DNA analogue oligomer) exhibited affinity enhancement (ratio of bivalent to monovalent equilibrium constants for binding) to immobilized complementary DNA (cDNA) by surface plasmon resonance (SPR). Because bivalent effectors using oligomers are easily synthesized with molecular spacing between binding sites, an important determinant of binding, adjustable simply by selecting linkers of different dimensions and/or lengthening or shortening the oligomer chain length, they may have advantages over existing bivalent effectors. We synthesized four bivalent MORFs with different dimensions between binding sites and measured binding affinities and affinity enhancement by SPR. An 18 mer (MORF18) was made bivalent by dimerizing both with disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG) linkers. By again using DSS but adding seven nonbinding adenine bases and by eliminating six binding bases, a total of four bivalent effectors, DSS-MORF12, DSG-MORF18, DSS-MORF18, and DSS-MORF25, were prepared with two different hybridization affinities (i.e. MORF12 and MORF18/25) and three different spacings (i.e. 20, 25, and 100 angstroms) between binding sites. The biotinylated cDNA was immobilized on a sensor chip at 500 and 100 RU coating densities providing an average cDNA separation of 25 and 80 angstroms. As expected, bimolecular binding dominated monomolecular binding in all cases, especially at lower MORF effector concentrations and at higher coating densities. All bivalent MORFs showed equilibrium constants superior to their monovalent form and therefore affinity enhancement. DSS-MORF25 showed the highest equilibrium constant for bimolecular binding presumably because of its larger separation between binding sites. Nevertheless, DSS-MORF12 showed the largest affinity enhancement of almost 3 orders of magnitude presumably because the shorter chain lowered the equilibrium constant for monomolecular binding. We have shown that bivalent effectors may be easily synthesized using MORF. The results provide further evidence that the use of bivalent effectors can greatly improve MORF pretargeting and, finally, that bivalent MORFs with reduced equilibrium constants may actually exhibit higher affinity enhancement.     

Affinity enhancement bivalent morpholino for pretargeting: initial evidence by surface plasmon resonance

Pretargeting with bivalent effectors capable of bridging antitumor antibodies has been reported to provide superior results by affinity enhancement. 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, likely to be a determinant of binding, may be adjusted simply by lengthening the chain. The goal of this investigation was to synthesize a bivalent MORF and to determine by surface plasmon resonance (SPR) whether the bivalent MORF exhibited bimolecular binding and whether the MORFs showed improved in vitro hybridization affinity in its bivalent form compared to its monovalent form. An 18 mer amino-derivitized MORF was made bivalent by dimerizing with disuccinimidyl suberate (DSS) in 1-methyl-2-pyrrolidinone (NMP) with N,N-diisopropylethylamine (DIEA) followed by purification by ion exchange chromatography. The in vitro hybridization affinity of bivalent compared to monovalent MORF was then measured by SPR. For these measurements, the complementary biotinylated cDNA was immobilized at coating densities that provided an average spacing of 20-100 angstroms and used to investigate the influence of this spacing on binding of the bivalent MORF with its binding regions separated by 25 A. The yield of bivalent MORF was as high as 45%, and the structure was confirmed by MALDI-TOF mass spectroscopy. When the sensograms obtained by SPR were analyzed using different binding models, the evidence was consistent with bimolecular binding of the bivalent MORF. The dissociation rate constant of the bivalent compared to monovalent MORF was more than 10-fold lower at 2.14 compared to 0.27 x 10(-5) (1/s) (p < 0.05), and since the association rate constants were similar at 8.53 and 5.64 x 10(5) (1/M.s) (p = 0.08), the equilibrium constant for hybridization to the immobilized cDNA of the bivalent compared to the monovalent MORF was almost 20-fold higher at 3.99 compared to 0.21 x 10(10) (1/M) (p < 0.05). In addition, qualitative evidence for bivalent binding of the bivalent MORF was apparent in the lower concentrations necessary to saturate the cDNA. Finally, the stoichiometry interpretation of the binding data provided estimates of the fraction of bivalent MORF binding bimolecularly. Under one set of conditions, this value was 20%. In conclusion, a bivalent MORF was easily synthesized by dimerization of a monovalent MORF. A lower dissociation rate constant and higher equilibrium constant was measured by SPR for the bivalent compared to monovalent MORF in their binding to an immobilized cDNA. These results show that bimolecular binding was occurring in the case of the bivalent MORF and suggest that bivalency may be superior to monovalency in MORF pretargeting applications.     

A comparison of in vitro and in vivo stability in mice of two morpholino duplexes differing in chain length

The stability of hybridized duplexes is an important criterion for any radiopharmaceutical application of DNAs or their analogues such as phosphorodiamidate morpholinos (MORFs). OBJECTIVE: The stabilities in vitro and in mice of the duplex between MORF and its complement (cMORF) were investigated for two different chain lengths, a 15-mer MORF compared to the identical MORF but elongated to a 25-mer. METHODS: The hybridization characteristics of the 15-mer MORF with its complementary 15-mer and that of the 25-mer with its complementary 25-mer MORF were measured using surface plasmon resonance (SPR) analysis. For radiolabeling with (99m)Tc, the 15- and 25-mer MORF, both with a primary amine via a 10-member linker on the 3' equivalent end, were conjugated with NHS-MAG(3). The 15- and 25-mer cMORFs were conjugated via their amines to carbodiimidazole treated poly(methyl vinyl ether-alt-maleic acid) (PA) such that about 50 cMORFs were attached to each polymer molecule in both cases (estimated MWs about 300 and 450 kDa, respectively). After hybridization in vitro, both the PA-cMORF15-(99m)Tc-MORF15 and PA-cMORF25-(99m)Tc-MORF25 homoduplexes were evaluated by size exclusion HPLC in saline, after incubation in 37 degrees C serum and in urine obtained 30 min post IV administration to normal mice. Biodistributions were obtained up to 18 h post administration. RESULTS: By SPR, the affinity constants for the homoduplexes were both about 10(9) M(-)(1) with the 25/25 only about 25% higher than the 15/15. However, the affinity constants for the 15/25 and 25/15 heteroduplexes showed a surprisingly 13-fold difference. By HPLC analysis, all duplexes were stable in saline; however, analysis of serum incubates and urine containing PA-cMORF15-(99m)Tc-MORF15 showed an immediate and pronounced low molecular weight peak that was identified by a shift assay to be (99m)Tc-MORF15. The comparable peak in both fluids was much less pronounced in the case of PA-cMORF25-(99m)Tc-MORF25. Whole body radioactivity levels also fell much more rapidly in mice receiving the 15-mer conjugate (65 vs 30% eliminated at 18 h) and biodistribution results showed higher kidney levels for the 15-mer conjugate. Results with the PA-cMORF25-(99m)Tc-MORF15 heteroduplex were more similar to that obtained with the 15-mer homoduplex than the 25-mer homoduplex. CONCLUSION: Despite what is reported to be high hybridization affinities, both the homoduplex and heteroduplexes prepared with (99m)Tc-MORF15 were found to be unstable in serum and in vivo toward dissociation to free (99m)Tc-MORF15. By contrast, homoduplex prepared with (99m)Tc-MORF25 showed higher stability. These differences in hybridization stability may be important considerations in radiopharmaceutical design.     

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.     

99mTc- and Re-labeled 6-dialkylamino-2-naphthylethylidene derivatives as imaging probes for β-amyloid plaques

Based on the conjugate strategy, two neutral ^99mTc labeled 2-(1-(6-(dialkylamino)naphthalen-2-yl)ethylidene)malononitrile (DDNP) and 1-(6-(dialkylamino)naphthalen-2-yl)ethanone (ENE) derivatives, and their corresponding rhenium complexes were synthesized. In vitro fluorescent staining indicated that the corresponding rhenium derivatives selectively stained the β-amyloid (Aβ) plaques in the brain sections of AD model mice with low background. Compared with FDDNP and FENE, the affinities of the corresponding rhenium derivatives to Aβ aggregates decreased about 10-14-fold. In vivo biodistribution experiments in normal mice showed that ^99mTc-MAMA-ENE displayed medium initial brain uptake (0.65 %ID/g at 2 min) with a reasonable washout from the brain (0.19 %ID/g at 2 h) while ^99mTc-MAMA-DDNP showed a low brain uptake (0.28 %ID/g at 2 min). Further optimize these ^99mTc-labeled tracers in order to improve their binding affinities to Aβ plaques and diffusion through the blood brain barrier may generate useful imaging agents for SPECT.     

Role of biotin-binding affinity in streptavidin-based pretargeted radioimmunotherapy of lymphoma

One pretargeting approach to cancer radioimmunotherapy utilizes an antibody-streptavidin conjugate that is first localized to the tumor. A "clearing agent" is then administered to remove the excess bioconjugate from blood, followed by injection of the radiolabeled biotin therapeutic. In this study, the role of streptavidin-biotin affinity in this pretargeting system was investigated for the first time in vivo, with a reduced affinity, site-directed streptavidin mutant and with radiolabeled bis-biotin reagents. The S45A streptavidin mutant (SA-S45A), which displays a faster off-rate for biotin, was utilized with a bivalent biotin carrier that retains high avidity for the streptavidin mutant. Mice were fed either a normal or biotin-deficient diet, yielding serum endogenous biotin concentrations of 31 nM and 5 nM, respectively. Lymphoma-bearing nude mice pretargeted with 1F5 Antibody-SA-Wild Type (WT) bioconjugates produced (125)I-bis-biotin tumor concentrations of 2.2%ID/g and 7.0%ID/g in mice fed normal diets vs biotin-deficient diets. (125)I-bis-biotin tumor concentrations of mice pretargeted with 1F5-SA-S45A were 12%ID/g and 10%ID/g for mice fed normal and biotin-deficient diets, respectively. However, poor clearance of the 1F5-SA-S45A with the biotinylated clearing agent led to high normal organ concentrations of (125)I-bis-biotin. A galactosylated human serum albumin (HSA) modified with bis-biotin was then tested, and normal organ (125)I-bis-biotin concentrations were significantly reduced. Tumor-to-organ ratios achieved for 1F5-SA-S45A with the HSA-bis-biotin clearing agent in mice with high serum biotin were similar to those achieved with 1F5-SA-WT in mice with low serum biotin. These results demonstrate that exchange of bound endogenous biotin with lower affinity streptavidin mutants is possible, and that corresponding use of bis-biotin carriers can nearly eliminate the differences in therapeutic radioactivity at the tumor site in animals on normal vs biotin-deficient diets. The results also interestingly demonstrate, however, that improved clearance agents capable of removing the lower affinity streptavidin-antibody conjugate are needed to achieve comparable specificity in tumor to blood or normal organ ratios.     

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.     

Synthesis and biological evaluation of novel oxindole derivatives for imaging neurofibrillary tangles in Alzheimer's disease

This letter describes the synthesis and biological evaluation of a novel series of radioiodinated oxindole (OI) derivatives for detecting neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer's disease (AD). In binding experiments in vitro, 2-oxindole (2-OI) and 3-oxindole (3-OI) derivatives showed affinity for tau aggregates. The 3-OI derivative 14 showed the highest affinity of these derivatives. In biodistribution experiments using normal mice, the OI derivatives displayed good uptake (2.4-2.5%ID/g at 2min) and clearance from the brain with time (0.6-1.4%ID/g at 30min). In fluorescence staining experiments using AD brain sections, 14 clearly stained NFTs. 3-OI may serve as a new molecular scaffold for developing novel NFT imaging agents.     

Biological distribution of 99mTc-labeled YIGSR and IKVAV laminin peptides in rodents: 99mTc-IKVAV peptide localizes to the lung

Two laminin-derived peptides containing either YIGSR or IKVAV (single amino acid code) sequences were radiolabeled with ^99mTc and their biological distribution evaluated in rodents. Both ^99mTC-peptides cleared rapidly from the circulation though the kidney, and to a lesser extent, through the liver. ^99mTC-YIGSR peptide did not accumulate in any organ examined in normal, tumored, and emphysemic mice. The ^99mTc-IKVAV peptide localized within 10 min to the lung of normal animals, resulting in lung-to-blood ratios of approximately 23:1. The ^99mTc-IKVAV peptide localized to lung after submicron filtration and after intraperitoneal injection, suggesting that particulates do not play a major role in localization. Pre-incubation of ^99mTc-IKVAV peptide in whole blood decreased lung localization, suggesting that margination of radiolabeled blood cells does not play a major role in the lung localization. When ^99mTc-IKVAV was injected into mice with tumored lungs (melanoma), the lung uptake was markedly increased (up to 20% injected dose higher than control lungs) at all time points examined (10, 30, and 120 min). When ^99mTc-IKVAV was injected into mice with genetic emphysema, the lung uptake was markedly decreased at all time points. The localization of the ^99mTc-IKVAV-containing peptide to the lung is consistent with a receptor-based mechanism.     

PEGylation and biodistribution of an anti-MUC1 aptamer in MCF-7 tumor-bearing mice

Aptamers are characterized by a rapid renal clearance leading to a short in vivo circulating half-life. In order to use aptamers as anticancer therapeutic agents, their exposure time to the tumor has to be enhanced via increasing residency in the bloodstream. A way to achieve this goal is by conjugating the aptamer to poly(ethylene glycol) (PEG). Herein, we present the conjugation of a bifunctionalized anti-MUC1 aptamer (NH(2)-AptA-SR) with the (99m)Tc coordinating moiety MAG2 and either a conventional branched PEG or the comb-shaped PolyPEG via a two-step synthesis. The isolated products were radiolabeled with (99m)Tc and their biodistribution and tumor-targeting properties in MCF-7 tumor bearing mice were analyzed and compared.     

Novel imaging agents for β-amyloid plaque based on the N-benzoylindole core

We report the synthesis and evaluation of a series of N-benzoylindole derivatives as novel potential imaging agents for β-amyloid plaques. In vitro binding studies using Aβ(1-40) aggregates versus [(125)I]TZDM showed that all these derivatives demonstrated high binding affinities (K(i) values ranged from 8.4 to 121.6 nM). Moreover, two radioiodinated compounds [(125)I]7 and [(125)I]14 were prepared. Autoradiography for [(125)I]14 displayed intense and specific labeling of Aβ plaques in the brain sections of AD model mice (C57, APP/PS1) with low background. In vivo biodistribution in normal mice exhibited sufficient initial brain uptake for imaging (2.19% and 2.00%ID/g at 2 min postinjection for [(125)I]7 and [(125)I]14, respectively). Although additional modifications are necessary to improve brain uptake and clearance from the brain, the N-benzoylindole may be served as a backbone structure to develop novel β-amyloid imaging probes.     

Site-specifically traced drug release and biodistribution of a paclitaxel-antibody conjugate toward improvement of the linker structure

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.     

Reduction of kidney uptake in radiometal labeled peptide linkers conjugated to recombinant antibody fragments. Site-specific conjugation of DOTA-peptides to a Cys-diabody

Arano and co-workers (Arano et al. (1999) Cancer Res. 59, 128-134) have synthesized peptides with an N-terminal radioiodinated hippuric acid and a C-terminal lysine linked to antibody fragments via the epsilon-amino group of lysine that show reduced kidney uptake compared to antibody fragments directly radioiodinated. This approach takes advantage of the lysine specific carboxypeptidase activity of the kidney brush border enzymes that cleave off the radiolabeled peptide linker from the antibody fragment prior to uptake by proximal tubule cells. On the basis of their approach, we have synthesized a tetrapeptide with an N-terminal DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and a C-terminal (N(epsilon)-maleoyl)lysine that was site-specifically conjugated to an anti-CEA diabody (Yazaki et al. (2001) Bioconjugate Chem. 12, 220-228) that was engineered to contain a C-terminal cysteine (Cys-diabody). Biodistributions of the In-111-radiolabeled conjugate in nude mice show significantly reduced kidney uptake (a maximum of 82%ID/g at 6 h) compared to In-111 radiolabeled DOTA-diabody (184%ID/g at 6 h) in which DOTA was conjugated to endogenous lysine residues using DOTA-active ester chemistry. To further reduce kidney uptake, a homologous compound with a C-terminal (N(epsilon)-amino-1,6-hexane-bis-vinyl sulfone)lysine was synthesized and site-specifically conjugated to the Cys-diabody. Biodistributions of this In-111-labeled conjugate reduced kidney uptake to 54%ID/g at 6 h. To explore the effect of the relative positions of the chelate vs the cys-diabody on kidney uptake, we also synthesized a tetrapeptide with an N-terminal bromoacetate for conjugation to Cys-diabody and a C-terminal (N(epsilon)-amidino-propyl-3-thio-vinylsulfonyl-DO3A)lysine. This peptide essentially reverses the positions of the chelate and Cys-diabody attachment points on the peptide, while retaining the linker length on the epsilon-amino group of the lysine. In this case, biodistributions of the In-111-radiolabeled conjugate in nude mice showed high kidney uptake (189%ID/g at 6 h), comparable to that obtained with the In-111-radiolabeled active ester conjugated DOTA-diabody (184%ID/g at 6 h). We conclude that the peptide linker strategy of Arano and co-workers to reduce kidney uptake can be successfully applied to chelate/radiometal complexes and requires that the chelate/radiometal be located at the N-terminus of the peptide and the antibody fragment attachment site on the epsilon-amino group of the lysine. Furthermore, we demonstrated a role for the attachment chemistry to the epsilon-amino group of the lysine on the magnitude of kidney uptake.     

2种石房蛤毒素完全抗原交联方法的比较及多克隆抗体的制备

[目的]探究石房蛤毒素(STX)完全抗原制备方法和STX多克隆抗体免疫方案。[方法]通过碳二亚胺法(EDC)和高碘酸盐法(periodate reaction)2种交联方法,将小分子石房蛤毒素与牛血清白蛋白(BSA)、鸡卵清蛋白(OVA)和孔血蓝蛋白(KLH)分别进行交联,制备了6种形式STX完全抗原,并对交联物进行琼脂糖凝胶电泳鉴定和紫外吸收峰迁移变化鉴定。分别将EDC法和高碘酸盐法交联的STX-BSA、STX-KLH 4种完全抗原作为免疫原,对Balb/c小鼠进行免疫,获得STX多克隆抗体。通过间接ELISA法,对不同方法制备的多克隆抗体进行分析比较。[结果]在石房蛤毒素完全抗原的制备中,在交联方法的选择上,EDC法较高碘酸盐法更具优势;而在免疫原的选择上,STX-BSA完全抗原效果最好。[结论]本研究探究了2种制备STX完全抗原的方法,为今后多克隆抗体生产以及特异性单克隆抗体筛选提供数据支撑。     

99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.     

Evaluation of 99mTc-labeled cyclic RGD dimers: impact of cyclic RGD peptides and 99mTc chelates on biological properties

The main objective of this study is to explore the impact of cyclic RGD peptides and (99m)Tc chelates on biological properties of (99m)Tc radiotracers. Cyclic RGD peptide conjugates, HYNIC-K(NIC)-RGD(2) (HYNIC = 6-hydrazinonicotinyl; RGD(2) = E[c(RGDfK)](2) and NIC = nicotinyl), HYNIC-K(NIC)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), and HYNIC-K(NIC)-3P-RGD(2) (3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2)), were prepared. Macrocyclic (99m)Tc complexes [(99m)Tc(HYNIC-K(NIC)-RGD(2))(tricine)] (1), [(99m)Tc(HYNIC-K(NIC)-3G-RGD(2))(tricine)] (2), and [(99m)Tc(HYNIC-K(NIC)-3P-RGD(2))(tricine)] (3) were evaluated for their biodistribution and tumor-targeting capability in athymic nude mice bearing MDA-MB-435 human breast tumor xenografts. It was found that 1, 2, and 3 could be prepared with high specific activity (～111 GBq/μmol). All three (99m)Tc radiotracers have two major isomers, which show almost identical uptake in tumors and normal organs. Replacing the bulky and highly charged [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) with a smaller [(99m)Tc(HYNIC-K(NIC))(tricine)] resulted in less uptake in the kidneys and lungs for 3. Surprisingly, all three (99m)Tc radiotracers shared a similar tumor uptake (1, 5.73 ± 0.40%ID/g; 2, 5.24 ± 1.09%ID/g; and 3, 4.94 ± 1.71%ID/g) at 60 min p.i. The metabolic stability of (99m)Tc radiotracers depends on cyclic RGD peptides (3P-RGD(2) > 3G-RGD(2) ～ RGD(2)) and (99m)Tc chelates ([(99m)Tc(HYNIC)(tricine)(TPPTS)] > [(99m)Tc(HYNIC-K(NIC))(tricine)]). Immunohistochemical studies revealed a linear relationship between the α(v)β(3) expression levels and tumor uptake or tumor/muscle ratios of 3, suggesting that 3 is useful for monitoring the tumor α(v)β(3) expression. Complex 3 is a very attractive radiotracer for detection of integrin α(v)β(3)-positive tumors.