Bioconjugate Chem. 2001, 12, 807−816
807
Pretargeting with Amplification Using Polymeric Peptide Nucleic Acid Y. Wang, F. Chang, Y. Zhang, N. Liu, G. Liu, S. Gupta, M. Rusckowski, and D. J. Hnatowich* Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655. Received March 8, 2001; Revised Manuscript Received June 13, 2001
One goal of this investigation was to develop a polymer conjugated with multiple copies of peptide nucleic acid (PNA) and with pharmacokinetic properties suitable for applications in vivo. The second goal was to establish whether the multiple copies of PNA on the polymer could be targeted by hybridization in vitro and in vivo with 99mTc-labeled complementary PNA (cPNA). If successful, this approach could then be considered in further investigations as an alternative to existing pretargeting approaches because of the potential for signal amplification in the target. A 80 KDa poly(methyl vinyl ether-alt-maleic acid) (PA) polymer was conjugated with multiple copies of PNA and with multiple copies of poly(ethylene glycol) (PEG) by reacting the NHS derivative of PA with the amine derivatives of PNA and PEG. Using 99mTc-MAG3-cPNA, targeting of PNA-PA-PEG was studied in vitro and in vivo in inflammation and tumor mouse models, in both cases relying upon nonspecific diffusion for localization. In addition, cPNA-avidin was considered as a clearing agent with biotinylated PNA-PAPEG. About 80 PNAs could be conjugated to PA provided that about 200 PEGs were also conjugated to raise the aqueous solubility of the PNA-PA-PEG polymer lowered by the addition of the PNAs. About 70% of the PNAs on this polymer in vitro either in solution or attached to beads could be successfully targeted with 99mTc-cPNA. In both the inflammation and tumor mouse models, between 35 and 60% of these PNAs could be targeted in the lesions. The advantage of amplification was evident when less favorable results were obtained with PNA-PA-PEG conjugated with only six PNAs. We conclude that amplification can be achieved in vivo using polymers of PNA followed by radiolabeled complementary PNA and that the application of pretargeting using polymers of PNA for amplification can improve localization.
INTRODUCTION
Since pretargeting was first introduced (1), an improvement in target/nontarget radioactivity ratios over conventional imaging has been documented in many animal and patient imaging studies. With few exceptions (2, 3), pretargeting has been achieved through the use of (strept)avidin and biotin (4-7). Mathematical modeling of pretargeting has shown clearly the importance to pretargeting of high affinities between the molecular pair employed (8). (Strept)avidin and biotin admirably satisfy this criteria as a result of an affinity of approximately 10-15 M (9). Despite the obvious success of pretargeting applications with (strept)avidin and biotin, use of this molecular pair is not without its difficulties. Unless properly designed, biotin analogues carrying the radiolabel are subject to biotinadase degradation (10). Strepavidin has been found to be immunogenic when conjugated to antibodies (11). Finally, endogenous biotin (vitamin H), present in all living tissues, can saturate the biotin binding sites of (strept)avidin located in a tumor and thus prevent targeting by radiolabeled biotin (12, 13). Complementary oligonucleotides such as deoxyribonucleic acids (DNAs) may offer an alternative to (strept)avidin and biotin for pretargeting that avoids these difficulties. Depending upon chain length and base sequence, the affinities for duplex formation of comple* To whom correspondence should be addressed. Phone: (508) 856-4256, Fax: (508) 856-4572, donald.hnatowich@ umassmed.edu.
mentary DNAs in serum environments may approach or even exceed that of (strept)avidin for biotin. Furthermore, single-strand DNAs have been administered repeatedly to patients in large dosages in connection with antisense chemotherapy without evidence of immunogenicities or serious toxicities (14). Finally, there will be no interference from endogenous substances. Native DNA with its phosphodiester backbone is generally considered unsuitable for pretargeting and other in vivo applications due to rapid degradation by nucleases (15, 16). Fortunately, numerous chemically modified olignucleotides have been prepared and shown to be stable to nucleases (17). One such modification that, in our view, may be particularly attractive as an alternative to (strept)avidin and biotin for pretargeting, has been named “peptide nucleic acid” (PNA) (18, 19). This synthetic oligomer consists of (2-aminoethyl) glycines to which the nitrogenous based (nucleobases) are attached via methylenecarbonyl linkers. PNAs are reported to be stable to proteases as well as nucleases (20), to show hybridization affinities greater than that of DNA (21) and to be nontoxic (20). This laboratory has previously considered PNAs for pretargeting applications (22). In that study, pretargeting was investigated in both an inflammation and tumor mouse model in which streptavidin saturated with biotinylated PNAs was administered first and allowed sufficient time to accumulate by nonspecific diffusion in the target. At that point, the radiolabeled complementary PNA was administered. For both models, pretargeting was successfully accomplished to judge by improved
10.1021/bc0100307 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/25/2001
808 Bioconjugate Chem., Vol. 12, No. 5, 2001
lesion accumulation and improved lesion/normal tissue ratios relative to controls. One property of (strept)avidin and biotin for pretargeting provides the potential of signal amplification by a factor of 4 when the radioactivity is attached to biotin since (strept)avidin possesses four biotin binding sites. However, among the advantages of using oligonucleotides for pretargeting is the potential of amplification far in excess of four. This could be achieved if the first administration consisted of a polymer to which multiple copies of oligonucleotides were bound. There would then be the potential that multiple copies of the radiolabeled complementary oligonucleotide within a second administration could bind with the resulting amplification of the radioactivity localized in a target containing the polymer (23). Amplification, as defined above, obviously requires a multivalent oligonucleotide polymer. In studies preliminary to this report, we considered a number of polymers in a search for platforms upon which a suitable number of PNAs could be attached. These preliminary studies helped to define more clearly the properties needed in a polymer useful for pretargeting PNA applications: the polymer must be large enough to carry sufficient numbers of PNAs; the PNA polymer should be water soluble and stable both in saline and serum; the pharmacokinetic properties must be favorable, and the PNAs must be arranged on the polymer such that binding is not unduly hindered sterically. One of the polymers we considered was found to possess suitable properties and became the focus of this investigation. Poly(methyl vinyl ether-alt-maleic acid) (PA), as used in this research is a 80 KDa (number average) linear polymer in which carboxylate groups alternate with methyl ether groups. The ethers and the negatively charged carboxylates provide aqueous solubility while the carboxylates are required for conjugation to the aminederivatized PNAs. There are approximately 920 carboxyl groups per PA polymer molecule. In the course of preliminary studies, it was observed that conjugation of PA with PNA to the desired degree considerably reduced the aqueous solubility of the polymer unless the number of PNAs per polymer was reduced. Since reducing the number of PNAs could render the polymer less suitable for pretargeting with amplification, the PNA-PA was also conjugated with multiple copies of poly(ethylene glycol) (PEG) to improve water solubility. Ultimately, the polymer used in this investigation was conjugated with an average of 80 PNAs and 200 PEGs per PA molecule. The molecular weight was calculated to be 1400 KDa. Pretargeting studies by their nature are complex. Among the variable are at least two dosages as well as two time periods, that between injections and that between the last injection and sacrifice or imaging. The complexity increases further if a clearing agent is used. Additional variables unique to this investigation include the nature of the polymer, its molecular weight, the number of PNAs and PEGs attached, and the length and base sequence of both the PNA and the radiolabeled complementary PNA. This report describes our first pretargeting study using PA-PEG conjugates as platforms for PNA designed to display suitable pharmacokinetics in vivo, to localize nonspecifically (i.e., by passive diffusion) in mouse models of focal infection/inflammation and tumor and to provide there a target for 99mTc labeled complementary PNA (cPNA) within a second administration. As is described, in the course of this investigation, one clearing agent was also considered to improve upon the pharmacokinetics of the radiolabel.
Wang et al. MATERIALS AND METHODS
The 15-base single-stranded PNAs were provided as a gift (Michael Egholm, PerSeptive Biosystems, Framingham, MA). Both PNA and cPNA were derivatized with a primary amine on the amino terminus (i.e., 5′ equivalent) end via a 17-member ethylene-ether linkage. The cPNA was also prepared occasionally with a biotin group on this end via the same linker. The linker and base sequence of PNA was NH2(CH2)2O(CH2)2O CH2CONH(CH2)2O(CH2)OCH2CO-TGT-ACG-TCA-CAA-CTA-CONH2 and that for cPNA was (biotin)- or (NH2)-(CH2)2O(CH2)2OCH2CONH(CH2)2O (CH2)OCH2CO-TAG-TTG-TGA-CGT-ACACO NH2 as previously described (19). The expected molecular masses were 4337.23, 4634.57, and 4408.27 Da, respectively, and were observed by mass spectrometry to be 4336.90, 4633.48 and 4407.65 Da. Purity was established by reverse phase HPLC (in all cases showing a single peak) and mass spectrometry (showing one predominant peak). Avidin and methoxypolyoxyethylene amine (MW 5000) were purchased (Sigma Chemical Co., St. Louis, MO). Poly(methyl vinyl ether-alt-maleic acid) (PA, number average MW ca. 80 KDa), anhydrous dimethyl formamide (DMF), N-methyl-2-pyrrolidinone (NMP), N-hydroxysuccinimide (NHS), dicyclocarbodiimide (DCC), and diisopropylethylamine (DIEA) were purchased (Aldrich Chemical Co., Milwaukee, WI). All reagents were used as received without further purification. NHS-MAG3 was synthesized according to published procedures (24). 99mTc-pertechnetate was obtained from a 99Mo-99mTc radionuclide generator (Dupont, Billerica, MA). Preparation of MAG3-Coupled cPNA. Because of different base sequences, PNA is more soluble in aqueous solution than is cPNA. As a consequence, in this investigation PA was always conjugated with PNA while the radiolabel was usually attached to cPNA. Amine-derivitized cPNA was conjugated with NHS-MAG3 as previously described (24). Briefly, a solution of cPNA was prepared at a concentration of 2-5 mg/mL in 0.25 M sodium bicarbonate-1 M sodium chloride-1 mM EDTA, pH 8.5, with 10-20% (v/v) of acetonitrile occasionally added to improve the solubility of cPNA. A solution of NHS-MAG3 in anhydrous DMF (50 mg/mL) was then added dropwise to the stirred cPNA solution until a MAG3 : cPNA molar ratio of 20:1 was reached. This solution was then incubated at room temperature for 30 min. The conjugated oligomer was purified on a 0.7 × 20 cm P4 column (BioRad, Melville, NY) eluted with 0.25 M ammonia acetate buffer, pH 5.2. Fractions off the P4 column were collected, and the absorbency of each was measured (U-2000, Hitachi Instruments, Danbury, CT) using an extinction coefficient at 260 nm determined in this laboratory of 35 µL/µg. The modified cPNA was stored at -20 °C for further use. Radiolabeling of cPNA-MAG3. A fresh 50 mg/mL sterile solution of sodium tartrate was prepared in 0.5 M sodium bicarbonate, 0.25 M ammonium acetate, 0.18 M ammonium hydroxide, pH 9.2. The high pH of the tartrate solution was necessary to adjust the final pH to approximately 7.6. In addition, a 1 mg/mL solution of SnCl2.2H2O in 10 mM HCl was prepared just prior to use. To MAG3-coupled cPNA (about 10-50 µg) contained in a sterile test tube was added the tartrate solution to a final concentration of about 7 µg/µL. To this was added sufficient 99mTc-pertechnetate solution to provide about 100 µCi/µg of PNA. The stannous ion solution was added immediately thereafter such that 3-4 µg of SnCl2‚2H2O was added for each 10 µL of 99mTc-pertechnetate solution. Higher activities of 99mTc required proportionately larger
Pretargeting,
99mTc-PNA
Polymers
Bioconjugate Chem., Vol. 12, No. 5, 2001 809
Figure 1. Schematic illustrating the preparation of NHS-PA and its conjugation with PNA and PEG.
volumes of the tin solution. After 40 min at room temperature, the labeled cPNA was purified on a 0.7 × 20 cm P4 gel filtration column using sterile pH 7.2 PBS eluant. Radioactivity and absorbency at 260 nm were used to identify and quantitate peak fractions. Preparations were routinely analyzed by size exclusion HPLC using a 1 × 30 cm Superose 12 column (Pharmacia, Piscataway, NJ). Control labelings were performed in which the native, unconjugated cPNA was subjected to the identical labeling procedure to assess the extent of nonspecific labeling. In all cases, nonspecific radiolabeling was less than 5%. PA Conjugation. Figure 1 illustrates the method of PNA and PEG conjugation to PA. As the first step, the NHS derivative of PA was prepared. With stirring, 20 mg of PA (0.230 mmol carboxyl groups) and 26 mg of NHS (0.226 mmol) were dissolved in 1 mL of anhydrous DMF, and the solution was cooled to 0 °C. To this was added 48 mg of DCC (0.233 mmol) in 1 mL of anhydrous DMF. The molar ratio of NHS and DCC to carboxylate groups on PA was therefore about 1:1. Immediately after the addition of DCC, the solution was put on ice and kept at 0 °C for 24 h. The dicyclohexylurea precipitated slowly and was filtered off at the end of the reaction. The NHSPA solution at a concentration of 10 mg/mL was stored at -20 °C and was used without further purification. Typically, about 200 µg of the amine-derivatized PNA (0.046 µmol) was dissolved in 150 µL of anhydrous DMF and 150 µL of anhydrous NMP. To this solution were added 466 µg methoxypolyoxyethylene amine (Me-PEGNH2, MW 5000, Sigma, 0.093 µmole) and 3 µL anhydrous DIEA. After thorough vortexing, 100 µg of the NHS derivatized PA in dry DMF (1.15 µmol carboxyl groups) was added, and the solution thoroughly votexed again. After 2 h at room temperature, the unreacted NHS ester was hydrolyzed by the addition of water to the reaction mixture. Early studies showed that 99mTc-labeled PAPNA polymer localized in the liver of normal mice to an unacceptable extent (>40% id/g). This unfavorable biodistribution was considered to be due to the decreased aqueous solubility of PA when conjugated with 80 PNAs per molecule. Rather than reducing the number of PNA groups, which could adversely influence amplification, the biodistribution properties of the polymer were improved by conjugating the PA with poly(ethylene glycol) (PEG) as shown in Figure 1. The conjugation of PA with PNA and PEG was accomplished simultaneously. The molar ratio of PNA to PA was usually 100:1. In most cases, the molar ratio of PEG to PA was 200:1. The average number of accessible PNA groups per PA was estimated using an aliquot of the conjugated polymer solution prior to purification. After adding tracer radiolabeled cPNA, the areas under the polymer peak and under the free PNA-cPNA duplex peak were compared in the radioactivity HPLC profile (Figure 2). The synthesized polymer was purified over a Sephadex G100
Figure 2. Size exclusion HPLC profiles of conjugated but unpurified PNA-PA-PEG after the addition of 99mTc-cPNA. Both UV and radioactivity traces are presented.
column (1 × 30 cm) using sterile water as eluant. The conjugated PA was stored in sterile water solution at -20 °C. The average number of PEGs per PA molecule was estimated using an aliquot of the conjugated polymer solution prior to purification. Using TNBS (trinitrobenzene sulfonic acid) (25), the number of free amine groups present due to unreacted PEG was quantitated and corrected for the contribution of free amine groups by PNA (the nitrogenous bases of PNA do not contribute in this assay). An aliquot of the reaction mixture containing a total of about 200 µg of PEG was removed to a glass tube and the solvent completely evaporated away with dry nitrogen. The residue was dissolved in 1 mL of 0.1 M sodium bicarbonate. To this solution was added 0.5 mL of 0.01% TNBS in 0.1 M sodium bicarbonate. After vortexing, the solution was incubated at 37 °C for 2 h. After adding 0.5 mL of 10% SDS and 0.25 mL of 1 M HCl, the absorbance was measured at 335 nm. Free PEG was used to construct a standard curve. Under these conditions, the conjugation of PA with PEG was virtually quantitative. Surface Plasmon Resonance Measurements. Recent improvements in instrumentation for biomolecular interaction analysis (BIA) has made it easier to measure rates of association and dissociation directly. Thus, BIA may be used to measure the rate of binding of an analyte on a surface to which its partner has been immobilized (26). The detection principle relies on surface plasmon resonance, an optical phenomenon that arises when light illuminates thin conducting films under specific conditions. Surface plasmon resonance forms the basis of BIA and permits the generation of sensorgrams in which the
810 Bioconjugate Chem., Vol. 12, No. 5, 2001
refractive index changes due to this binding are measured in resonance units (RUs) (27). In this investigation, surface plasmon resonance was used to estimate the association rate constants for duplex formation between immobilized cPNA and PNA both free and attached to PA polymer. In addition, the association rate constant between immobilized cPNA and PNA-PAPEG with an average of 6 and 80 PNAs per PA was measured. The analysis was performed on a BIAcore1000 (BIAcore, Piscataway, NJ) instrument operating at room temperature. Biotinylated-cPNA was added to a streptavidin-dextran-coated sensor chip only until a response of about 200 RUs was reached. The absence of mass transfer effects was confirmed by running separately one concentration of free PNA and one concentration of each PA polymer at three different flow rates (10, 30, and 75 µL/min) and demonstrating identical RU responses in all cases. Solutions of free PNA and both PA polymers were prepared at six concentrations (0.0 to 5.0 µM with respect to PNA) in the same running buffer (10 mM HEPES, 150 mM sodium chloride, 3.4 mM Na2EDTA, 0.005% P20, pH 7.4). Each analysis consisted of two measurements. A sample was first applied to a control surface consisting of an identical streptavidindextran-sensor chip but to which biotinylated PNA rather than cPNA had been added. Subtraction of the sensorgram obtained with the control surface from that obtained with the test surface at each concentration corrected for bulk refractive index changes. Following subtraction, the resulting sensograms were analyzed using instrument software (BIAevaluation 3.0, BIAcore) by assuming 1:1 Langmuir interaction. Radiolabeling PNA-PA-PEG. To determine the biodistribution of PNA-PA-PEG, the polymer was radiolabeled with 99mTc by adding a trace amount (about 10 µCi) of 99mTc-cPNA to 16 µg of the polymer in saline. The solution was repeatedly vortexted and incubated at room temperature for 20 min before administration. In Vitro Stability of PNA-PA-PEG. Size exclusion HPLC was used to assess the stability of PNA-PA-PEG in serum. The polymer was incubated in fresh human serum at 37 °C at a concentration of 40 µg/mL, and aliquots were removed at different intervals over 24 h. To each aliquot, 99mTc-labeled cPNA was added in the identical fashion to radiolabel the polymer by hybridization. Clearing Agents. Avidin conjugated with biotinylated cPNA was added as a clearing agent to reduce levels of the polymer in blood and in most normal tissues just prior to the administration of the radiolabeled cPNA. The cPNA-avidin was prepared by adding 200 µg of avidin to 40 µg of biotin-cPNA in 400 µL saline. The solution was gently vortexed, incubated at room temperature for 1 h and used without purification. In Vitro Studies. The concept of pretageting with amplification was tested in vitro using streptavidincoated magnetic beads 0.96 µm in diameter (Bangs Laboratories, Inc., Fishers, IN). The in vitro study consisted of three parts: involving beads without PNA (nonspecific), beads with PNA but without the addition of PNA-PA-PEG (specific without amplification), and beads with cPNA but with the addition of PNA-PA-PEG (specific with amplification). In Vivo Biodistribution Studies. Normal CD-1 male mice (mean weight 26 g, Charles River, Wilmington, MA) were injected via a tail vein with 0.15 mL of 50 mM PBS containing 16 µg (10-12 µCi) of labeled PNA-PA-PEG. At 3 h, the animals were anesthetized with metofane (Schering-Plough, Omaha, NE) and sacrificed by cervical
Wang et al.
dislocation. Following sacrifice, animals were dissected to provide samples of tissue for counting in a NaI (Tl) well counter along with a standard of the injectate. Results were expressed as percent of injected dose per gram of tissue (% id/g). To prepare the inflammation mouse model, a clinical isolate of Escherichia coli (American Type Culture Collection, Rockville, MD) was grown in LB medium (Sigma) to a density of 108 organisms/mL. Male CD-1 mice (Charles River) weighing approximately 30 g were anesthetized by inhalation of metofane and inoculated subcutaneously in the posterior right thigh with 0.1 mL of the bacterial suspension. Swelling was apparent after 14 h. At this time 16 µg of PNA-PA-PEG was administered through a tail vein. After 3 h, each animal received 2 µg (about 100 µCi) of radiolabeled cPNA by the same route. Two control groups of animals were used. One group (control 1) received in identical fashion the radiolabeled cPNA without having previously received the PNA-PAPEG polymer. Another group (control 2) received in identical fashion PNA-PA-PEG but received 99mTc-labeled PNA instead of labeled cPNA. The anesthetized animals were imaged at 3 h post administration of the radioactivity and were then killed by cervical dislocation. Along with other tissues, the entire infected right leg and, for comparison, the whole left leg, was removed for counting. To prepare the mouse tumor model, ACHN cells (American Type Culture Collection, Manassas, VA) were grown in minimal essential medium (Gibco, Grand Island, NY). The cells were removed from the culture flask by trypsinization and then washed in the culture media. Swiss male nu/nu mice (Taconic Labs, Germantown, NY) were each injected subcutaneously in the posterior left thigh with 3 × 106 cells each in 0.1 mL of medium. Two weeks later, when the tumors were approximately 0.6 cm in diameter, each animal received 16 µg of PNA-PA-PEG and, 3 h later, received 2 µg of 99m Tc-cPNA. The animals were imaged 3 and 18 h later. Control animals received the radiolabeled cPNA without the prior administration of PNA-PA-PEG. After 18 h, all mice were sacrificed and tissues obtained for counting. When under investigation, the clearance agent was administered in different dosages but always 2 h after the administration of PNA-PA-PEG polymer and 1 h before the administration of 99mTc-cPNA. Imaging Studies. Scintigrams were acquired using a portable large field of view scintillation camera (Elscint APEXD 409M, Hackensack, NJ) equipped with a parallelhole, medium-energy collimator and under computer control (Elscint APEX F1). Animals were anesthetized with nembutal (Abbott Laboratories, North Chicago, IL) and were then positioned directly on the collimator. Typically, several animals were imaged simultaneously at 3 h post administration of the radiolabel. RESULTS
Conjugation Efficiencies. The HPLC profiles of PNA-PA-PEG, after conjugation but before purification are presented in Figure 2 for both radioactivity and UV detection at 260 nm. In both profiles, the peaks at 3234 min are due to free PNA while that at 14 min is due to PNA-PA-PEG. A value of 80 for the average number of PNAs per PA was calculated on the basis of the ratio of UV peak areas. In this research, the PA-PEG polymer was also conjugated at a 10:1 PNA to PA molar ratio. By applying the same analysis, a value of 6 PNAs/PA was determined for this polymer. At 260 nm, the absorbance of PA and PEG are negligible.
Pretargeting,
99mTc-PNA
Polymers
Bioconjugate Chem., Vol. 12, No. 5, 2001 811
Figure 3. Typical room-temperature sensograms after subtraction for the hybridization of PNA-PA-PEG with cPNA immobilized on the sensor chip. Traces are presented at one concentration for PA conjugated with 80 and with 6 PNAs per polymer.
The HPLC profiles presented in Figure 2 were also used to determine the number of PNAs conjugated to PA that are accessible in solution. The 99mTc-cPNA added to unpurified PNA-PA-PEG distributes between free PNAs and PNAs accessible on the polymer according to their relative concentrations. Since the free PNA is unquestionably accessible, the radioactivity under the 32-34 min peaks was used to calculate the number of counts per accessible PNA. This value was then applied to the radioactivity of the 14 min peak. In this way the number of PNAs on PNA-PA-PEG accessible in solution was estimated to be about 60. The average number of PEGs per PA molecule was determined as described above to be about 200. Surface Plasmon Resonance. Figure 3 presents typical sensograms for the hybridization of PNA-PA-PEG to immobilized cPNA obtained at room temperature and after subtraction showing association during injection followed by dissociation. Curves are presented at one concentration for both PNA-PA-PEG conjugated with 80 and 6 PNAs/PA. The higher response observed for PA conjugated with 80 PNAs compared to 6 PNAs was expected and confirms that a higher number of PNAs are available for hybridization in the case of this polymer. As was observed previously for DNAs (28), the slow dissociation rate of these oligomers is below the detection limits of the method. Accordingly, it was not possible to measure an equilibrium constant although, as before, it was possible to measure the association rate constants. For the 80 and 6 PNAs/PA, respectively, the association rate constant was measured as 1.5 and 1.1 × 105 M-1 s-1 (N ) 3). Because each polymer presumably attaches to several immobilized cPNAs on the sensor chip, the absolute value of the association rate constant based on 1:1 Langmuir binding may be unreliable. Nevertheless, that these values are of the same order of magnitude suggests that the kinetics of binding probably is the same for both polymers. Furthermore, since the association rate constant for free PNA was measured as 1.2 × 104 M-1 s-1, the hybridization of the polymer may therefore have been about 10 fold faster than that of free PNA. A faster association may be expected for polymer with multiple PNAs. Finally, these values suggest that the method used to conjugate PNA to PA probably have not chemically altered the nitrogenous bases of the oligomer. Stability of PNA-PA-PEG in Serum. The results presented in Figure 4 demonstrate that PNA-PA-PEG is stable in 37 °C serum environments. The addition of 99mTc-cPNA to aliquots of serum containing PNA-PA-PEG
Figure 4. Size exclusion HPLC radiochromatograms of 99mTc-cPNA added at different times to a 37 °C serum incubate of PNA-PA-PEG.
shows essentially no change in the distribution of the radiolabel between hybridized polymer and free 99mTccPNA even after 24 h of incubation. In Vitro Amplification Studies. The concept of pretageting with amplification was tested in vitro using streptavidin-coated magnetic beads. Radiolabeled cPNA was added identically to beads prepared in three different manners: beads without PNA (nonspecific binding), beads with PNA but without the addition of PNA-PAPEG (specific binding and including nonspecific binding), and beads with cPNA and the prior addition of PNA-PAPEG (specific binding with amplification). Table 1 summarizes the protocol and lists the results. About 50 µL of packed magnetic beads with a total biotin capacity of greater than 100 pmol of biotinylated PNA, according to the manufacturer, was washed and resuspended with the aid of a magnetic holder. Over a period of 20 min at room temperature with periodic vortexing, the beads were bound with 1.0 × 10-3 µg (0.22 pmol) of either biotin-derivatized PNA or biotin-derivatized cPNA. Since this addition would occupy only 0.3% of the biotin binding sites, complete PNA or cPNA binding was assumed. After additional washes, the beads were resuspended. To the cPNA beads only was added 0.4 µg of PNA-PA-PEG (i.e. 0.29 pmol PA, 23 pmol of PNA) followed by another 20 min incubation at room temperature. The 0.29 pmol of polymer may be expected to saturate the 0.22 pmol of cPNA on the beads leaving about (0.22 × 80) or 18 pmol of PNA on the beads. These beads were then washed as before and resuspended. To each of the three samples of beads was added 0.12 µg (24.2 µCi, 25 pmol) of 99mTc-cPNA. After incubation for 30 min at room temperature with periodic voretexing, the beads were thoroughly washed for counting in a dosage calibrator and, after sufficient decay, in a NaI(Tl) well counter. These results show that nonspecific binding is an important contributor to what is referred to above as “specific”. Thus, true “specific” binding (minus nonspecific) amounted to only a little more than 0.1 µCi (0.350.23). This result is reasonable since about 0.12 µg of labeled cPNA was added to beads containing about 10-3 µg of PNA for a theoretic maximum binding of only (10-3/
812 Bioconjugate Chem., Vol. 12, No. 5, 2001
Wang et al.
Table 1. Experimental Protocol and Results for in Vitro Pretargeting with Amplification Using Three Different Conditions. The Radioactivity on the Beads Follows the Identical Addition of Labeled CPNA, N ) 3 beads first addition second addition mean (range) radioactivity (µCi)
nonspecific
specific
specific with amplification
native beads nothing 99mTc-cPNA 0.23 (0.22-0.23)
PNA-beads nothing 99mTc-cPNA 0.35 (0.33-0.37)
cPNA-beads PNA-PA-PEG 99mTc-cPNA 15.1 (15.0-15.3)
Table 2. The Biodistribution of PNA-PA-PEG with 0, 100, and 200 PEGs per Polymer Molecule In Normal Mice at 3 h. %id/g (s.d.), N ) 5 tissue
0 PEGs/ polymer
100 PEGs/ polymer
200 PEGs/ polymer
liver heart kidney lung spleen stomach sm. int. lg. int. blood muscle
44 (0.7) 1.2 (0.2) 5.1 (0.3) 1.4 (0.2) 15 (0.8) 0.8 (0.3) 0.7 (0.1) 0.8 (0.3) 1.1 (0.2) 0.3 (0.02)
35 (0.4) 1.1 (0.1) 3.5 (0.3) 1.0 (0.04) 11 (2.3) 0.7 (0.1) 0.8 (0.2) 1.1 (0.1) 0.6 (0.1) 0.3 (0.1)
13 (1.7) 3.5 (0.5) 5.5 (0.8) 4.7 (0.7) 7.5 (1.5) 1.1 (0.3) 1.7 (0.3) 1.1 (0.1) 25 (1.5) 0.8 (0.2)
0.12 × 24 µCi) or about 0.20 µCi. In the case referred to above as “specific with amplification”, the 25 pmol of labeled cPNA was added to beads which should contain 18 pmol of PNA if amplified by the presence of 80 PNAs per PA. If 18 pmol of 99mTc-cPNA was now to bind (i.e., saturation of PNA on PA), there will be about ([24 µCi/ 25 pmol] x 18 pmol) or 17 µCi on the beads. This value is in agreement with the 15 µCi observed and suggests that the radiolabeled cPNA has access to PNA on PA-PEG and the accessible PNAs on each immobilized PNA-PA-PEG molecule is about ([15/17] × 80) or 70. Since a value of 60 accessible PNAs per polymer molecule was determined from the UV and radioactivity HPLC profiles, the value of 70 PNAs per polymer molecule measured in the bead study suggests that the PNAs on immobilized polymer are equally accessible to the polymer in solution. Normal Mouse Biodistributions. The biodistributions of PNA-PA-PEG with 80 PNAs but with 0, 100 and 200 PEG per polymer are shown in Table 2. The molecular weights of the polymers were calculated to be 427, 927 and 1427 KDa, respectively. The results of Table 2 show that when PA is conjugated with 80 PNAs, the polymer must also be conjugated with more than 100 PEGs to achieve what may be a satisfactory biodistribution. Otherwise, liver levels are excessively high at about 40% id/g, blood clearance is rapid with blood levels showing only about 1% id/g at 3 h. However, when the polymer is conjugated with 200 PEGs, the liver level drops to 13% and the blood level rises to 25%. Influences on Inflammation Imaging of PNAs per Polymer. To investigate the influence of the number of PNAs per polymer, PA-PEG was conjugated with either 80 or 6 PNAs. The molecular weights were calculated as 1427 and 1106 KDa, respectively. Figure 5 presents representative images of inflammation mice at 3 h post administration of 99mTc-cPNA in animals receiving the polymer conjugated with 6 PNAs (left) vs 80 PNAs (right). Table 3 lists the biodistribution results. In the above table, differences in tissue values between the two polymers are significant in all cases (P < 0.005). These results show clearly the advantage of increasing the number of PNAs per PA. While the accumulation of radioactivity increased in all tissues sampled (for example, the target in the infected thigh increased about
Figure 5. Whole body images of two infected mice at 3 h post administration of 99mTc-cPNA and 6 h post administration of PNA-PA-PEG with 6 (left image) and 80 (right image) PNAs per polymer molecule. The location of the inflammation in the thigh is indicated by the arrows. Table 3. The Biodistribution of 99mTc in the Infected Mouse Model 3 h Post Administration of Radiolabeled CPNA and 6 h Post Administration of PNA-PA-PEG. Results Are Presented for Polymer Conjugated with 6 and 80 PNAs. %id/g (s.d), N ) 5 tissue
6 PNA/PA
target/ nontarget
80 PNA/PA
target/ nontarget
liver heart kidneys lung spleen stomach sm. int. lg. int. blood normal thigh infected thigh
1.5 (0.2) 0.45 (0.05) 2.1 (0.2) 1.0 (0.2) 2.1 (0.5) 1.0 (0.2) 0.38 (0.05) 0.75 (0.11) 9.4 (1.0) 0.3 (0.1) 0.8 (0.2)
0.5 1.8 0.4 0.8 0.4 0.8 2.1 1.1 0.1 2.7 -
7.8 (1.1) 2.5 (0.6) 4.2 (1.2) 6.4 (1.5) 9.8 (1.6) 1.6 (0.3) 1.5 (0.2) 1.3 (0.1) 11.1 (0.9) 1.2 (0.2) 5.6 (0.5)
0.7 2.2 1.3 0.9 0.6 3.5 3.7 4.3 0.5 4.7 -
Table 4. Biodistribution of 99mTc in Infected Mice 3 h Post Administration of Radiolabeled cPNA and 6 h Post Administration of PNA-PA-PEG. Results Presented for Study Animals as well as Two Controls (see text). %id/g (s.d.), N ) 5-6 tissue
study mice
control mice 1
control mice 2
liver heart kidney lung spleen stomach sm. int. lg. int. blood normal thigh infected thigh
5.8 (0.5) 1.8 (0.2) 2.9 (0.3) 5.4 (0.5) 5.5 (0.7) 0.9 (0.2) 1.2 (0.2) 0.6 (0.2) 15.6 (1.0) 0.8 (0.1) 3.2 (0.4)
0.6 (0.1) 0.08 (0.02) 2.0 (0.3) 0.2 (0.1) 0.6 (0.1) 0.5 (0.1) 0.2 (0.1) 0.4 (0.1) 0.22 (0.05) 0.09 (0.02) 0.08 (0.01)
0.4 (0.3) 0.1 (0.1) 2.2 (0.9) 0.2 (0.3) 0.3 (0.2) 0.9 (0.1) 0.3 (0.4) 0.7 (0.2) 0.3 (0.3) 0.09 (0.01) 0.13 (0.03)
7-fold), the target/nontarget ratio are more favorable in all cases. The 22% difference in molecular weight between the two polymers is unlikely in itself to alter the biodistribution to the extent shown. Infection Studies without Clearance Agents. Table 4 presents biodistribution results in mice infected in one thigh with E. coli and obtained 3 h. post iv administration of labeled cPNA and 6 h post iv administration of PNAPA-PEG. Results are presented for the study animals and for two sets of control animals. One set of animals (control 1) did not receive the first administration of PNA-PAPEG while the second set (control 2) received the PNAPA-PEG in the identical manner but received radiola-
Pretargeting,
99mTc-PNA
Polymers
Bioconjugate Chem., Vol. 12, No. 5, 2001 813 Table 5. the Biodistribution Results in Tumored Nude Mice 18 h Post Administration of Radiolabeled CPNA and 21 h Post Administration of PNA-PA-PEG. ID%/gm (s.d.), N ) 5
Figure 6. Two pairs of images of infected mice receiving PNAPA-PEG followed by radiolabeled cPNA 3 h later. Whole body images obtained at 3 h post administration of 99mTc-cPNA. In each pair, the control animals is on the left. The location of the inflammation in the thigh is indicated by the arrows.
beled PNA instead of radiolabeled cPNA in the second injection. The PA was conjugated with 200 PEGs and 80 PNAs. With the exception of stomach and large intestines, tissue radioactivity levels are significantly higher in the study animals compared to the control animals. This increase is related to the presence in infected and normal tissues of PNA-PA-PEG in concentrations sufficient to bind the radiolabeled cPNA in the second injection. These values may be used to calculate the percentage of PNAs on PA in the lesion which are targeted with radiolabeled cPNA. From the biodistribution of radiolabeled PNA-PA-PEG in infected mice (data not presented) about 4% id/g of the polymer accumulates in the target at 3 h and, as such, about 35% of the PNAs on PA were targeted in each gram of lesion. This value represents a decrease from the 60-70% of PNAs targeted on the polymer when in solution or when bound to beads in vitro but is still considerable for in vivo targeting. The mean inflamed to normal thigh ratios from the results presented in Table 4 are 4.0, 0.89, and 1.4 for the study and two groups of control animals, respectively. The differences between study and each control animals are significant. Thus, this ratio has been more than doubled by the prior administration of the polymer. Moreover, the mean uptake in the infected thigh of animals receiving the polymer has been increased a factor of 40 over the infected thighs of control animals not receiving the polymer. Finally the inflamed thigh to tissue radioactivity ratios are higher for the study animals in all tissues compared to the control animals, except for blood. The two pairs of whole body images in Figure 6 are representative of the infected animals. Both control animals show radioactivity in bladder with faint accumulation in the target thigh. In each pair, the image on the left is of the study animals showing similar activity in bladder, increased activity in liver and, in particular, increased uptake in the infected thigh. Tumor Studies without Clearing Agents. Table 5 presents biodistribution results in nude mice implanted in one thigh with the ACHN tumor. The results were obtained at 18 h post administration of labeled cPNA and 21 h post iv administration of PNA-PA-PEG. Results are presented for both the study and control animals (animals not receiving PNA-PA-PEG). Tissue radioactivity levels are again significantly higher in most tissues for the study animals compared to the control animals. This increase is again related to the presence of PNA-PA-PEG in concentrations sufficient to bind the radiolabeled cPNA in the second injection. The mean tumored to normal thigh ratios from the results presented in Table 5 are 14.6 and 2.0 for the study and control animals, respectively. As in the study of the
tissue
study mice
control mice
liver heart kidneys lung spleen stomach sm. int. lg. int. blood muscle tumor
4.0 (0.6) 0.4 (0.1) 0.6 (0.1) 0.4 (0.1) 3.9 (0.5) 0.3 (0.1) 0.5 (0.1) 3.7 (0.8) 1.1 (0.2) 0.1 (0.02) 1.9 (0.4)
0.2 (0.04) 0.01 (0.01) 0.31 (0.03) 0.02 (0.01) 0.06 (0.02) 0.3 (0.2) 0.08 (0.03) 1.8 (0.5) 0.03 (0.02) 0.01 (0.003) 0.02 (0.01)
Figure 7. Two pairs of images of tumored mice receiving PNAPA-PEG and radiolabeled cPNA 3 h later. Whole body images obtained at 3 h (left images) and 18 h (right images) post administration of 99mTc cPNA. In each pair, the control animal not receiving the polymer is on the left. The location of the tumor in the thigh is indicated by the arrows.
infected mice, prior administration of the PNA-PA-PEG has significantly improved the uptake ratio. Moreover, the mean uptake in the tumored thighs has been increased a factor of 90 over that in the tumored thighs of control animals not receiving the PNA-PA-PEG polymer. Finally, the tumored thigh to tissue radioactivity ratios are higher for the study animals compared to the control animals in all tissues. These values may be used to calculate the percentage of PNAs on PA in the tumor that are targeted with radiolabeled cPNA. From the biodistribution of radiolabeled PNA-PA-PEG in tumored mice (data not presented), at 3 h post administration, about 2.5% id/g of the polymer accumulates in the target and, as such, about 58% of the PNAs on PA were targeted by radiolabeled cPNA. This is compared to the 35% targeting in the inflammation model and is equal to the 60-70% of PNAs targeted on the polymer in vitro. The whole body images of Figure 7 were obtained at 3 and 18 h post administration of radiolabeled cPNA and are representative of the tumored animals. On the left of each pair is the image of a control animal not receiving the polymer. Inflammation Studies with Clearing Agents. Avidin-cPNA was prepared by mixing avidin with biotincPNA as described above. Mice were administrated IV either 0, 1, 3, 6, or 15 µg of avidin-cPNA 2 h after the iv injection of PNA-PA-PEG and 1 h before the administration of radiolabeled cPNA. The results are presented in Table 6 while Figure 8 presents representative images of animals receiving 0, 3, and 6 µg of this clearing agent. Under these circumstances, a 6 µg dosage appears to be optimum in terms of improving the target/nontarget ratios without excessively decreasing accumulation in the target. Tumored Studies with Clearing Agent. Mice were administrated IV either 0 and 6 µg of avidin-cPNA 2 h after the first injection of PNA-PA-PEG and 1 h before the administration of radiolabeled cPNA. Table 7 presents the biodistribution results.
814 Bioconjugate Chem., Vol. 12, No. 5, 2001
Wang et al.
Table 6. Biodistribution at 3 h Post Administration of Radiolabeled CPNA and 6 h Post Administration of PNA-PA-PEG to Infected Mice Receiving Varying Amount of Avidin-cPNA as Clearance Agent. %id/g (s.d.), N ) 5 dosage of Avidin-cPNA tissue
0 µg
1 µg
3 µg
6 µg
15 µg
liver heart kidney lung spleen stomach sm. int. lg. int. blood normal thigh inflamed thigh
10.7 (0.9) 1.7 (0.1) 3.7 (0.2) 4.1 (0.4) 9.8 (1.0) 1.9 (0.3) 1.7 (0.2) 0.8 (0.2) 10.4 (0.9) 1.1 (0.1) 3.3 (0.5)
7.7 (0.3) 1.7 (0.3) 4.1 (0.3) 4.8 (0.3) 14.3 (0.6) 1.6 (0.3) 1.4 (0.1) 0.8 (0.1) 12.2 (0.2) 1.03 (0.03) 3.7 (0.2)
7.7 (1.2) 1.14 (0.02) 3.6 (0.3) 3.0 (0.5) 10.9 (2.0) 1.6 (0.4) 1.4 (0.3) 0.8 (0.2) 7.2 (0.9) 0.8 (0.1) 2.7 (0.4)
3.2 (0.3) 0.5 (0.1) 3.8 (1.4) 1.3 (0.3) 4.2 (0.3) 1.5 (0.2) 0.8 (0.2) 0.6 (0.3) 1.3 (0.3) 0.3 (0.2) 2.3 (0.3)
1.6 (0.2) 0.13 (0.01) 1.6 (0.2) 0.64 (0.05) 2.6 (0.4) 0.9 (0.2) 0.17 (0.02) 0.52 (0.04) 0.42 (0.02) 0.11 (0.02) 0.45 (0.05)
Figure 8. Whole body images of infected mice 3 h post administration of 99mTc cPNA and 6 h post administration of PNA-PA-PEG. Each mouse received the indicated dosage of avidin-cPNA as clearance agent 2 h earlier. The location of the inflammation in the thigh is indicated by the arrows. Table 7. Biodistribution at 3 h Post Administration of Radiolabeled CPNA and 6 h Post Administration of PNA-PA-PEG to Tumored Mice Receiving Varying Amount of Avidin-cPNA as Clearance Agent. %id/g (s.d.), N ) 5 dosage of avidin-cPNA tissue
0 µg
6 µg
liver heart kidney lung spleen stomach sm. int. lg. int. blood muscle Tumor
8.1 (0.6) 1.5 (0.1) 3.1 (0.2) 2.8 (0.1) 7.4 (2.1) 1.9 (0.2) 1.5 (0.2) 0.9 (0.2) 11.8 (0.6) 0.4 (0.2) 2.9 (0.6)
1.8 (0.2) 0.2 (0.02) 2.0 (0.3) 0.5 (0.1) 0.8 (0.2) 1.4 (0.2) 0.6 (0.4) 0.3 (0.03) 1.0 (0.05) 0.07 (0.01) 0.8 (0.3)
The use of a 6 µg dosage of avidin-cPNA as clearing agent reduced the radioactivity levels in all tissues including the tumor, such that tumor/normal tissue ratios were improved only in liver, heart, lung, spleen, blood, and muscle. In contrast to the use of the same dosage in the infected mice where the lesion lost only about 30% of its radioactivity, in the tumored mice, the lesion lost more than 70%. Clearly, the optimal dosage of clearing agent must be determined for each model system. DISCUSSION
Since the goal of this investigation was to determine whether polymers of PNA could be targeted with radiolabeled cPNA to improve the localization of radioactivity using a pretargeting strategy, it was often necessary in the course of this study to select among a number of possibilities. Apart from the selection of the polymer scaffold itself, the selection of PNA as oligomer was significant. By surface plasmon resonance measure-
ments, the association rate for hybridization of this 15 base PNA pair was found to be similar to that for a pair of 18 base uniformly phosphorothioate DNAs (29). Accordingly, both DNAs and PNAs were considered to display the hybridization kinetics required for in vivo targeting. However, PNA was selected because of its stability in vivo and its high binding affinity toward its complement. Furthermore, the high charge of DNA was considered a disadvantage compared to the electrically neutral PNAs if charge neutrality permits closer packing on polymers. In fact, much greater difficulties were experienced in conjugating uniform phosphorothioate DNAs to PA and other polymers, and this was attributed to charge repulsion. It may be calculated that the average spacing of PNA groups on the PNA-PA-PEG polymer used in this investigation was 3.5 nm. This is closer than that used in DNA microarrays where about 10 nm separates each DNA (30). Even at this larger spacing, charge repulsion effects and steric hindrance are occasionally encountered (FR Ortigao, Hybain, private communciation, 2000). Thus the short 3.5 nm spacing might be expected to severely restrict targeting. However, PA is a linear molecular with a backbone permitting free rotation. This fact may help explain that over 70% of the PNAs on the polymer were targeted in solution and even when the polymer was immobilized on beads. That targeting was 35-58% in vivo is only slightly less impressive considering the barriers which exist in vivo compared to the in vitro situation. The selection of PNA introduced one difficulty as the poor aqueous solubility of PNA and, in particular, of cPNA, limited the number of PNAs that could be conjugated to PA. Biodistributions of radiolabeled PA, even with limited numbers of PNA, showed rapid blood clearance and excessive accumulation in liver (data not presented), properties which were only exasperated by increasing the number of PNAs per polymer to the required levels. Fortunately, the large number of carboxylate groups on PA (about 920) over that needed for PNA conjugation permitted the addition to this polymer of sufficient numbers of PEGs to increase circulation time and decrease liver accumulation. As shown in Table 2, liver accumulation at 3 h in normal mice was 44 id%/g with no PEG on PA molecule, while this value fell to 13 id%/g at 200 PEGs/PA. An addition consideration was the number of PNAs per polymer molecule. Fortunately, the addition of PEGs permitted up to 80 PNAs per PA to be attached without adversely influencing the biodistribution of PA. That the number of PNAs is an important determinant to the success of pretargeting with this polymer is evident from the biodistribution results of PAs conjugated with 6 vs 80 PNAs (Table 3 and Figure 5). The absolute uptake
Pretargeting,
99mTc-PNA
Polymers
was 7 fold higher and the target/nontarget ratios were more favorable in all tissues when the polymer conjugated with the higher number of PNAs was administered. Most pretargeting studies employ an intermediate administration of a clearing agent to remove as much of the first injectate remaining in circulation as possible before the radioactivity is administered. This process can significantly improve the target/nontarget radioactivity ratios (7). Therefore a number of clearing agents were considered in this investigation. With biotin conjugated to PNA-PA-PEG, it was possible to use either avidin or streptavidin as a clearing agent since both proteins are tetravalent for biotin and are therefore capable of crosslinking biotinylated PA. Accumulation in the infected thigh was unchanged by either clearing agent as was expected since neither protein should interfere with the target. However, liver, kidney, and spleen levels decreased only in the case of streptavidin, but by much less than desired (data not presented). Avidin-cPNA was also investigated since cPNA on this potential clearing agent will hybridize to PNA-PA-PEG in circulation while the avidin should encourage clearance of the complex into the liver. However, avidin-cPNA can also reduce the number of available PNAs in the target. Any clearing agent containing cPNA may hybridize to PNA-PA-PEG in the target, thereby reducing the number of PNAs available to hybridize 99mTc-cPNA. Table 6 presents biodistribution results showing the effect of dosage of avidin-cPNA. The infected thigh target shows minimal decrease with increasing dosage of this clearing agent until 15 µg are reached. However all normal tissues except kidneys show dosage-related decreases within this range. The images of Figure 8 also show this improvement. Thus, the results of this research show that pretargeting may be accomplished with PNA polymers. Using the hyperperfusion of denatured capillaries at sites of inflammation and tumor in mice, we have observed that the PNA-PA-PEG polymer of this investigation accumulates in both lesion types. Thereafter, the polymer may be targeted with a subsequent administration of radiolabeled cPNA. This investigation is similar to those conducted in this laboratory previously using PNA but with streptavidin-PNA as the first injectate followed by 99mTc-cPNA without a clearing agent (22). Previously, in both the inflammation and tumored mouse models, it was necessary to inject a large dosage of both the streptavidinPNA and radiolabeled cPNA (150 and 50 µg, respectively) to achieve positive pretargeting. By contrast, in this research, only 16 µg of the PNA-PA-PEG and 2 µg of the radiolabeled cPNA were required to achieve superior ratios even in the absence of a clearing agent. With the use of 6 µg of avidin-cPNA in the inflammation model, almost all target/nontarget ratios were further improved (Table 6). In conclusion, the results of this investigation demonstrate that PA may be conjugated with a large number of PNAs and, when conjugated with sufficient PEGs as well, the polymer displays pharmacokinetic properties suitable for in vivo applications. In particular the conjugated PA was shown to accumulate by nonspecific diffusion in lesion of both inflammation and tumored mouse models. We were also able to demonstrate that the PNAs on PA may be successfully targeted in vitro and, in particular, in vivo by 99mTc-labeled cPNA. Finally, the advantage of including amplification in pretargeting applications was also shown. Although a number of polymer scaffolds were considered preliminary to this work, it is inconceivable that the ideal polymer was found
Bioconjugate Chem., Vol. 12, No. 5, 2001 815
in PA. Furthermore, although PNAs have very attractive properties, other oligomers may eventually be shown to be superior for this application. Clearing agents other than those studied herein could be considered. Thus, this report may be viewed as describing only the first step to the use of oligomer polymers for pretargeting. Nevertheless, the goal of demonstrating that pretargeting using polymers of PNA for amplification can improve localization was realized. ACKNOWLEDGMENT
We are grateful to Mike Egholm and Ivar Jensen of PerSeptive Biosystems for providing the PNA used in this investigation and for helpful discussions. We also appreciate the help of Ken Miller of BIAcore Corp with our surface plasmon resonance measurements. Financial support for this investigation was provided in part by the National Institutes of Health (CA79507). LITERATURE CITED (1) Goodwin, D. A., Meares, C. F., McTigure, M., McCall, M. J., and Davis, G. S. (1986) Rapid localization of hapten in sites containing previously administered antibody for immunoscintigraphy with short half-life tracers. J. Nucl. Med. 27, 959 (Abstract). (2) Chatal, J. F., Faivre-Chauvet, A., and Bardies, M. (1995) Bifunctional antibodies for radioimmunotherapy. Hybridoma 14, 125-128. (3) Barbet, J., Kraeber-Bodere F., Vuillez J-P., Gautherot E., Chatal J-F.(1999) Pretargeting with the affinity enhancement system for radioimmunotherapy. Cancer Biother. Radiopharma 14, 153-166. (4) Hnatowich D. J., Virzi F., and Rusckowski M. (1987) Investigations of avidin and biotin for imaging applications. J. Nucl. Med. 28, 1294-1302. (5) Paganelli G., Malcovati M., and Fazio F. (1991) Monoclonal antibody pretargetting techniques for tumour localization: the avidin-biotin system. Nucl. Med. Commu. 12, 211-234. (6) Hnatowich, D. J. (1994) The in vivo uses of streptavidin and biotin - a short progress report. Nucl. Med. Commun. 15, 575-577 (Editorial). (7) Knox, S. J., Goris, M. L., Tempero, M., Weiden, P. L., Gentner, L., Breitz, H., Adams, G. P., Axworthy, D., Gaffigan, S., Bryan, K., Fishewr, D. R., Colcher, D., Horak, I. D., and Weiner, L. M. (2000) Phase II trial of yttrium-90-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer. Clin. Cancer Res. 6, 406-414. (8) Sung, C., van Osdol, W. W., Saga, T., Neumann, R. D., Derick, R. L., and Weinstein, J. N. (1994) Streptavidin distribution in metastatic tumors pretargeted with biotinylated monoclonal antibody: theoretical and experimental pharmacokinetics. Cancer Res. 54, 2166-2175. (9) Green, N. M. (1975) Avidin. Adv. Prot. Chem. 29, 85-133. (10) Wilbur, D. S., Hamlin, D. K., Pathare, P. M., and Weerawarma S. A. (1997) Biotin reagents for antibody pretargeting. Synthesis, radioiodination, and in vitro evaluation of water souble, biotindase resistant biotin derivatives. Bioconjugate Chem. 8, 572-584. (11) Chinol, M., Casalini, P., Maggiolo, M., Canevari, S., Omedeo, E. S., Caliceti, P., Veronese, F. M., Cremonesi, M., Chiolerio, F., Nardone, E., Siccardi, A. G., and Paganelli, G. (1998) Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce imunogenicity. Br. J. Cancer. 78, 189-197. (12) Rusckowski, M., Fogarasi, M., Fritz, B., and Hnatowich, D. J. (1995) Influence of endogenous biotin on the biodistribution of two In-111 labeled biotin derivatives in mice. Nucl. Med. Commun. 16, 38-46. (13) Rusckowski, M., Fogarasi, M., Fritz, B., and Hnatowich, D. J. (1997) Effect of endogenous biotin on the applications of streptavidin and biotin in mice. Nucl. Med. Biol. 24, 263268.
816 Bioconjugate Chem., Vol. 12, No. 5, 2001 (14) Crooke, S. T. (1997) Advances in understanding the pharmacological properties of antisense oligonucleotides. Adv Pharm. 40, 1-47. (15) Agrawal, S., Temsamani, J., Galbraith, W., and Tang, J. (1995) Pharmacokinetics of antisense oligonucleotides. Clin. Pharmacokinet. 28, 7-16. (16) Hnatowich, D. J., Mardirossian, G., Fogarasi, M., Sano, T., Smith, C. L., Cantor, C. R., Rusckowski, M., and Winnard, P., Jr. (1996) Comparative Properties of a technetium-99mlabeled single-stranded natural DNA and a phosphorothioate derivative in vitro and in vivo. J. Pharm. Exp. Ther. 276, 326-334. (17) Goodchild, J. (1990) Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjugate Chem. 1, 165-187. (18) Egholm, M., Buchardt, O., Nielsen, P. E., and Berg, R. H. (1992) Peptide nucleic acids (PNA): oligonucleotide analogues with an achiral peptide backbone. J. Am. Chem. Soc. 114, 1895-1897. (19) Mardirossian, G., Lei, K., Rusckowski, M., Chang, F., Qu, T., Egholm, M., and Hnatowich, D. J. (1997) In vivo hybridization of technetium-99m-labeled peptide nucleic acid (PNA). J. Nucl. Med. 38, 907-913. (20) Knudsen, H., and Nielsen, P. E. (1997) Application of peptide nucleic acid in cancer therapy. Anticancer Drugs 8, 113-118. (21) Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden, B., and Nielsen, P. E. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogenbonding rules. Nature 365, 566-568. (22) Rusckowski, M., Qu, T., Chang, F., and Hnatowich, D. J. (1997) Pretargeting using peptide nucleic acid (PNA). Cancer 80, 2699-2705. (23) Hnatowich, D. J., Winnard, P. Jr., Virzi, F., Niemeyer, C., Dalan, A. B., Sano, T., Smith, C. L., Cantor, C. R., and
Wang et al. Rusckowski, M. (1994). Amplification of radioactivity using complementary DNA. Proceedings, Fifth Conference on Radioimmunodetection and Radioimmunotherapy of Cancer, Princeton, NJ. (Abstract). (24) Winnard, P., Jr., Chang, F., Rusckowski, M., Mardirossian, G., and Hnatowich, D. J. (1997) Preparation and use of NHSMAG3 for technetium-99m labeling of DNA. Nucl. Med. Biol. 24, 425-432. (25) Sashidhar, R. B., Capoor, A. K., and Ramana, D. (1994) Quantitation of -amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. J. Immunol. Methods 167, 121-127. (26) Jonsson, U., Fagerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., Lofas, S., Persson, B., Roos, H., Ronnberg, I., Sjolander, S., Stenberg, E., Stahlberg, R., Urbaniczky, C., Ostlin, H., and Malmqvist, M. (1991) Realtime biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 11, 620-627. (27) Malmqvist, M. (1993) Biospecific interaction analysis using biosensor technology. Nature 36, 186-187. (28) Zhang, Y. M., Liu, N., Zhu, Z. H., Rusckowski, M., and Hnatowich, D. J. (2000) Influences of three chelators (HYNIC, MAG3 and DTPA) on the in vitro and in vivo behaviors of 99mTc attached to antisense DNA. Eur. J. Nucl. Med. 27, 1700-1707. (29) Hnatowich, D. J. Antisense and Nuclear Medicine. (2000) Where are we now? Cancer Biother. Radiopharam. 15, 447457. (30) Strother, T., Hamers, R. J., and Smith, L. M. (2000) Covalent attachment of oligodeoxyribonucleotides to amine modified Si(001) surfaces. Nucleic Acids Res. 28, 3535-3541.
BC0100307
