Production of Multimeric Prostate-Specific Membrane Antigen Small-Molecule Radiotracers Using a Solid-Phase ^99mTc Preloading Strategy

Reagents

MGI Pharma compound 11245-36 (GPI) was synthesized as described previously (8). Ultradry dimethyl sulfoxide (DMSO) was purchased from Acros Organics. HPLC-grade triethylammnonium acetate, pH 7 (TEAA), was from Glen Research. HPLC-grade water was from American Bioanalytic. Triserine was from Bachem. N-succinimidyl-S-acetylthioacetate (SATA) was from Pierce. N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) was from Advanced Chemtech. All other chemicals were purchased from Fisher Scientific and were American Chemical Society (ACS)- or HPLC-grade.

HPLC/Mass Spectrometry Platform

The HPLC/mass spectrometry platform used for purification of both nonradioactive and radioactive tumor-targeting small molecules and peptides has been described in detail previously (7). Briefly, the system is composed of a Waters model 1525 binary pump, a model 2487 ultraviolet detector (Waters), a Sedex model 75 (Richards Scientific) evaporative light scatter detector (ELSD) with the nebulizer modified to reduce band broadening at low flow rates, a model FC-3200 high-sensitivity photomultiplier tube γ-detector (Bioscan), and a Waters fraction collector, all housed within a Capintec hot cell equipped with a model CRC-15R (Capintec) dose calibrator. For nonradioactive reactions, column eluate was split into a Waters LCT electrospray time-of-flight (ES-TOF) mass spectrometer.

Preparative Synthesis of S-Acetylmercaptoacetyltriserine (MAS₃)

Ten milligrams (36 μmol) of triserine were dissolved in 350 μL of water. One equivalent (5 μL, 36 μmol) of the base triethylamine (Et₃N) was added, followed by 2 equivalents (16.6 mg, 72 μmol dissolved in 160 μL N,N-dimethylformamide [DMF]) of SATA. The reaction mixture was vortexed at room temperature for 3 h. An additional equivalent (8.3 mg, 36 μmol dissolved in 80 μL DMF) of SATA was then added, and vortexing was continued for an additional 2 h.

To confirm completion of the reaction, a 10-μL sample was analyzed by reverse-phase HPLC (RP-HPLC) using a 4.6 × 150 mm Symmetry (Waters) C₁₈ column and a linear gradient from 0% to 15% B over 35 min, starting 2 min after injection, at 1 mL/min, where A = H₂O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid. MAS₃ eluted at a retention time (R_t) = 11.6 min as detected by the ELSD, with its mass confirmed by ES-TOF mass spectrometry. Preparative purification was performed on an HPLC system described in detail previously (7) and equipped with a 5-mL sample loop, after dilution into a final volume of 5 mL of H₂O + 0.1% trifluoroacetic acid (TFA), and using a 19 × 150 mm Symmetry C₁₈ column. The gradient consisted of 0% B for 3.5 min, then 0%–15% B over 35 min at 7 mL/min, where A = H₂O + 0.1% TFA and B = acetonitrile + 0.1% TFA. MAS₃ eluted at R_t = 21.8 min using ELSD detection. Fractions containing product were pooled and lyophilized. MAS₃ was obtained as a white powder in 57% isolated yield (8.0 mg, 20.5 μmol), with the expected mass confirmed by ES-TOF mass spectrometry, and a purity of ≥98%.

Solid-Phase Labeling of MAS₃ with ^99mTc-Pertechnetate

One-hundred fifty microliters of a 50% slurry of Chelex 100 resin (Bio-Rad) in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.0, were added to an empty micro Bio-Spin (Bio-Rad) chromatography column, washed once with MES buffer, and centrifuged at 3,000g for 10 s; 1.2 mg (3 μmol) of MAS₃ were dissolved in 1 mL of water. One milligram (4 μmol) of stannous (II) chloride dihydrate was dissolved in 1 mL of 10 mM HCl. Then 100 μL of MAS₃ and 35 μL of stannous chloride were mixed well and added to the Chelex resin. ^99mTc-pertechnetate (185–370 MBq [5–10 mCi]) in 100 μL of saline, eluted directly from a ⁹⁹Mo generator with saline, were added to the tube. The tube was capped, wrapped in parafilm, and boiled for 10 min in a water bath. ^99mTc loading of MAS₃ was monitored by RP-HPLC using a 4.6 × 75 mm Symmetry C₁₈ column with a linear gradient from 0% to 60% B over 30 min, starting 2 min after injection, at 1 mL/min, where A = 10 mM TEAA and B = absolute MeOH. ^99mTc-MAS₃ eluted at R_t = 14.1 min. In separate experiments, the limits of this reaction were determined to be 370 MBq (10 mCi) ^99mTc-pertechnetate and 1.3 μmol of MAS₃ using 150 μL of 50% Chelex 100 slurry.

A 10-mg Oasis HLB cartridge (catalog no. 186000383) was activated using methanol, then H₂O, and equilibrated with H₂O, pH 4.0 (pH adjusted with 10 mM HCl). The ^99mTc-MAS₃ solution was diluted with 1 mL of H₂O, pH 4.0, and bound to the cartridge. The cartridge was washed with 10 mL of H₂O, pH 4.0, to remove ^99mTc-pertechnetate, tin, and free MAS_3; then it was purged completely with nitrogen. Finally, ^99mTc-MAS₃ was eluted with 400 μL dry DMSO or DMF.

Synthesis of [^99mTc-MAS₃]-NHS

After 200 μg (0.7 μmol) of TSTU powder were placed in a 1.5-mL Eppendorf tube, 185–370 MBq (5–10 mCi) of purified ^99mTc-MAS₃ in 400 μL DMSO or DMF and 0.5 μL (2.9 μmol) of neat (5.74 M) N,N-diisopropylethylamine (DIEA) were added. The top was sealed and the solution was incubated at 60°C for 10 min. The reaction was diluted to 1 mL final volume by the addition of 600 μL of dichloromethane:hexane (6:4). The reactants and by-products were removed by passage through Waters Oasis MCX (catalog no. 186000252) and MAX (catalog no. 186000366) cartridges attached in tandem. The flow-through, containing pure [^99mTc-MAS₃]-NHS ester, was concentrated by loading on an Oasis HLB cartridge, washing with of 1 mL of dichloromethane:hexane (6:4), and eluting using 500 μL of either DMF or DMSO. The purity of the compound was assessed by RP-HPLC using a 4.6 × 75 mm Symmetry C₁₈ column with a linear gradient from 0% to 60% B over 30 min, starting 2 min after injection, at 1 mL/min, where A = 10 mM TEAA and B = absolute MeOH. [^99mTc-MAS₃]-NHS eluted at R_t = 23.5 min. The specific activity of [^99mTc-MAS₃]-NHS was estimated by conjugation to tryptophan and measurement of 280-nm absorbance of the product using the same RP-HPLC conditions as those used to assess purity.

Synthesis and Purification of Radiolabeled PSMA Ligands

Covalent conjugation of GPI derivatives with [^99mTc-MAS₃]-NHS was performed by the addition of 100 μL (10 μmol) of 100 mM triethylamine in dry DMSO to 10 μL (0.1 μmol) of a 10 mM solution of GPI derivative in a total of 400 μL dry DMF/DMSO, followed by the addition of 200 μL of [^99mTc-MAS₃]-NHS (185–259 MBq [5–7 mCi]) in dry DMSO. Constant stirring at room temperature was maintained for 40–90 min, until the reaction was completed. The radiolabeled ligands were analyzed by RP-HPLC on a 4.6 × 75 mm Symmetry C₁₈ column using a linear gradient from 0% to 60% B over 25 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = 10 mM TEAA, pH 7.0, and B = absolute MeOH.

Synthesis of Re-MAS₃

A procedure reported previously (9) was used with slight modification. MAS₃ (4.8 mg, 16 μmol) was dissolved in 1.5 mL of water. Stannous chloride dihydrate (5.4 mg, 24 μmol) in 1.5 mL of 0.1 M citrate buffer, pH 5.0, and NaReO₄ (6.6 mg, 24 μmol) in 1.5 mL of H₂O were added to the MAS₃ solution. The reaction mixture was stirred at 90°C for 1 h. After cooling to room temperature, Re-MAS₃ was purified on an Oasis HLB cartridge and eluted with DMSO. The purity was assessed by liquid chromatography/mass spectrometry (LC/MS) on a 4.6 × 150 mm Symmetry C₁₈ column using a linear gradient from 0% to 50% B over 15 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = H₂O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid. Retention time was 6.4 min.

Synthesis of [Re-MAS₃]-NHS

Fifty microliters (3 μmol) of a 60 mM TSTU solution in DMSO and 200 μL (2 μmol) of a 10 mM Re-MAS₃ solution in DMSO were mixed together; then 25 μL (5 μmol) of 200 mM N,N-diisopropylethylamine and 25 μL of DMSO were added. The solution was vortexed at room temperature for 40 min. After dilution with dichloromethane, the NHS ester was purified using Oasis MCX, MAX, and HLB cartridges connected in series as described earlier. The purity was assessed by LC/MS on a 4.6 × 150 mm Symmetry C₁₈ column using a linear gradient from 0% to 50% B over 15 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = H₂O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid. Retention time was 10.4 min.

Synthesis and Purification of [Re-MAS₃]-Conjugates

Covalent conjugation of GPI derivatives with [Re-MAS₃]-NHS was performed by the addition of 0.1 mL (10 μmol) of 100 mM triethylamine in dry DMSO to 0.1 mL (1 μmol) of a 10 mM solution of GPI derivatives in dry DMSO or DMF, followed by addition of 0.2 mL (2 μmol) of a 10 mM [Re-MAS₃]-NHS solution in dry DMSO. Constant stirring at room temperature was maintained for 2 h. The conjugates were purified by preparative HPLC (Symmetry C₁₈ column, 19 × 150 mm, 7 μm) using a linear gradient from 0% to 50% B over 27 min, starting 5 min after injection, with a flow rate of 10 mL/min. The collected fractions were lyophilized, and purity was assessed by LC/MS on a 4.6 × 150 mm Symmetry C₁₈ column using a linear gradient from 0% to 50% B over 15 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = H₂O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid.

Quantification of Serum Stability

The stabilities of [^99mTc-MAS₃]-GPI compounds were tested by incubation in the absence (phosphate-buffered saline [PBS] only) or presence of 100% calf serum for 4 h at 37°C. Stability and transmetallation were quantified using high-resolution chromatography. For PBS experiments, a 4.6 × 75 Symmetry C₁₈ column was used as described earlier for radiolabeled PSMA ligands. For serum experiments, an 8 × 300 mm, 200-Å Diol (YMC, catalog no. DL20S053008WT) gel-filtration column was used with PBS as the mobile phase. Gel-filtration molecular weight (M.W.) markers (Bio-Rad) were: M₁ = thyroglobulin (670 kDa), M₂ = γ-globulin (158 kDa), M₃ = ovalbumin (44 kDa), M₄ = myoglobin (17 kDa), and M₅ = vitamin B₁₂ (1.3 kDa).

High-Throughput, Radioactive Live Cell Binding and Affinity Assay

Human prostate cancer cell lines, PSMA-positive LNCaP and PSMA-negative PC-3, were obtained from the American Type Culture Collection. The PSMA-negative human bladder cancer cell line TsuPR1 was a generous gift of Dr. John T. Isaacs (Johns Hopkins University, Baltimore, MD). Cell lines were cultured at 37°C under humidified 5% CO₂ in RPMI 1640 medium (Mediatech Cellgro) supplemented with 10% fetal bovine serum (Gemini Bio-Products) and 5% penicillin/streptomycin (Cambrex Bioscience). Cells were split onto 96-well filter plates (model MSHAS4510; Millipore) and grown to 50% confluence (approximately 35,000 cells per well) over 48 h.

To assign absolute affinity to each compound, a homologous competition assay was used with the ^99mTc-labeled version as the tracer and the relabeled version as the test compound. To avoid internalization of the radioligand due to constitutive endocytosis (2), live cell binding was performed at 4°C. Cells were washed 2 times with ice-cold Tris-buffered saline (TBS), pH 7.4, and incubated for 20 min at 4°C with 0.02 MBq (0.5 μCi) of radiotracer in the presence or absence of the test compound. Cells were then washed 3 times with TBS using a Millipore vacuum manifold (catalog no. MSVMHTS00), and the well contents was transferred directly to 12 × 75 mm plastic tubes placed in γ-counter racks. Transfer was accomplished using a modified (Microvideo Instruments) 96-well puncher (MAMP09608; Millipore) and disposable punch tips (MADP19650; Millipore). Well contents were counted on a model 1470 Wallac Wizard (Perkin Elmer) 10-detector γ-counter, and curves were fit using Prism version 4.0a (GraphPad) software.

Near-Infrared (NIR) Fluorescence and γ-Radioscintigraphic Imaging

To assess viability and to verify confluence, living cells were loaded with the NIR fluorophore IR-786 by adding it to the cell culture medium at 1 μM for 30 min at 37°C before the start of the experiment (10). Cells were incubated as described earlier with 3.7 MBq (100 μCi) of ^99mTc-labeled compound per well, in the buffer being tested, for 20 min at 4°C, before extensive washing of the filter plate. Simultaneous white light and NIR fluorescence imaging of plates was performed as described in detail previously (11,12). γ-Radioscintigraphy was performed with an Isocam Technologies Research Digital Camera equipped with a ½-in. NaI crystal, 86 photomultiplier tubes, and a high-resolution, low-energy lead collimator.

High-Affinity, Multimeric Small Molecules for Targeting PSMA

Our group has previously developed a high-affinity (9 nM), monomeric small molecule (311 Da) termed GPI, which is engineered to bind the active site of PSMA (2). Unfortunately, inorganic anions in serum—such as phosphate—compete with monomeric GPI, rendering it marginal for use as an in vivo diagnostic (2). The work of Whitesides and colleagues (13) suggests that by decreasing the off-rate, multivalency can permit a small molecule to “out-compete” endogenous competitors. To test this hypothesis, while maintaining overall small size, we have developed a tri-NHS ester derivative of adamantane (14), which permits conjugation of up to 3 targeting ligands (such as GPI) in addition to a contrast agent or radiotracer. Recently, we have added a 6-carbon linker to improve radiotracer conjugation and have purified a series of GPI derivatives with increasing valency (Fig. 1). As shown in Table 1, conversion of monomeric GPI (I) to an adamantane trimer (IV) results in an affinity improvement of over 1.5 logarithms and retention of affinity in 100% serum.

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FIGURE 1. 

Highly anionic PSMA-targeting small-molecule ligands: chemical structures and M.W. for PSMA-targeting ligands used in this study.

Solid-Phase Prelabeling for Preparation of ^99mTc-MAS₃ Conjugates

Unfortunately, highly anionic small molecules such as IV are difficult to use as ^99mTc diagnostic radiotracers because the 12 net negative charges after conjugation to a chelator can potentially act as coligands for ^99mTc. Furthermore, traditional pre- and postlabeling approaches for ^99mTc (Fig. 2A) involve the use of excess quantities of an exchange ligand such as Na⁺/K⁺ tartrate. This creates 2 major problems. First, separation of ^99mTc-tartrate away from a desired radiolabeled chelator is extremely difficult and requires high-resolution HPLC (Fig. 2B). For this reason, the radiolabeled tartrate peak is typically just accepted as “contaminant” and the entire labeling reaction is used (even in clinical settings). Second, nonradioactive exchange ligand is used in excess, hence, all subsequent chemical steps, such as solution-phase prelabeling (Fig. 2A) are competed by the exchange ligand's carboxylic acids, unless great care, and time-consuming separation, is used. Finally, if not purified to completion, nonradioactive tartrate can form reactive NHS esters and conjugate covalently to the targeting ligand, again requiring careful purification, and risking competition with radiotracer.

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FIGURE 2. 

^99mTc labeling strategies, solid-phase prelabeling, and purification of ^99mTc-/Re-labeled MAS₃ and MAS₃-NHS. (A) General approaches for creating ^99mTc-radiolabeled derivatives of small molecules and peptides include postlabeling (route 1) in presence of excess exchange ligand such as tartrate, solution-phase prelabeling using excess of exchange ligand (route 2), or solid-phase prelabeling (route 3) using Chelex 100 resin as described. (B) C₁₈ HPLC radiochromatograms of conventional solution-phase prelabeling of ^99mTc-MAS₃ with excess tartrate (top left) and solid-phase prelabeling with Chelex 100 (bottom left). Solid-phase formation and purification of [^99mTc-MAS₃]-NHS (top right) and hydrolysis of NHS ester at pH 10 (bottom right). ^99mTc-MAS₃ and [^99mTc-MAS₃]-NHS elute at 14.1 and 23.5 min, respectively. ^99mTc-Tartrate elutes at 13.5 min. (C) Simple, cartridge-based labeling and purification protocol to produce [^99mTc-MAS₃]-NHS is shown. Total elapsed time (brackets) and yield for each step are also shown. (D) C₁₈ HPLC ELSD tracings (top) and mass spectra of major peak (bottom) for Re-MAS₃ (left) and its NHS ester (right). Expected isotopic patterns for these Re derivatives are shown as insets. AU = absorbance units.

To solve the problems associated with traditional radiolabeling, we have developed a simple and rapid, cartridge-based method for converting readily available and inexpensive ^99mTc-pertechnetate (Na^99mTcO₄) into a chemically pure, reactive NHS ester in organic solvent (Fig. 2C). The first key to this strategy is the elimination of soluble exchange ligand in favor of a solid-phase source of carboxylic acids (Chelex 100 resin). As described in detail earlier, only 8 min is required to fully load (99% radiochemical yield) a ^99mTc chelator, such as MAS₃ (9), with ^99mTc (Fig. 2C). MAS₃ was chosen over MAG₃ and other ^99mTc chelates given its low-background binding in primates after conjugation to targeting molecules (15).

The second key to this strategy is the transfer of ^99mTc-MAS₃ from aqueous buffer to nonaqueous buffer, as the final NHS ester is highly susceptible to attack by the hydroxyl ion of H₂O. To accomplish this, and to remove unwanted molecules, the supernatant from the Chelex 100 resin is bound to a C₁₈ mixed-bed cartridge, washed, and then eluted with desired organic solvent (DMSO or DMF). The NHS ester of ^99mTc-MAS₃ is then formed rapidly using TSTU and DIEA as described. Excess reactants are removed using tandem mixed-mode anion-exchange and cation-exchange cartridges, and the final product is concentrated on a C₁₈ mixed-bed cartridge (Fig. 2C). Using this chemical strategy, [^99mTc-MAS₃]-NHS is prepared in ultrapure form (>99%; Fig. 2B) in DMSO or DMF, in only 25 min, with an overall radiochemical yield of 70%–75% relative to ^99mTc-pertechnetate starting material. Reaction of the final product with basic water (pH 10) results in the rapid reformation of ^99mTc-MAS₃, thus proving the presence of an NHS ester (Fig. 2B). The typical specific activity of [^99mTc-MAS₃]-NHS produced using this strategy was 4.1 × 10⁸ MBq/mmol (1.1 × 10⁴ Ci/mmol).

To confirm, absolutely, the chemical structures produced, as well as to prepare nonradioactive derivatives of diagnostic agents for affinity measurements, the chemistry was repeated using Re. Although the exact procedure shown in Figure 2C worked well for Re-MAS₃ chelates, the yield was low (≈20%), likely reflecting the difficulty others have had in preparing Re chelates (8). Hence, for preparative purification of Re-MAS₃, solution-phase labeling was used, followed by formation and purification of [Re-MAS₃]-NHS using our solid-phase approach. A high-resolution LC/MS HPLC system (7) was also used to verify that all cartridges performed as designed and to verify the purity of final compounds (Fig. 2D).

Solid-Phase Prelabeling of Highly Anionic PSMA Small Molecules

Using this solid-phase preloading strategy, GPI-containing molecules of increasing ligand valency were labeled with either ^99mTc or Re in 1 chemical step as shown in Figure 3A. For ^99mTc-labeled molecules, no further purification was necessary, as the NHS ester reaction with an amine in dry DMSO or DMF goes to completion without side reactions (Fig. 3B). For Re-labeled molecules, which were to be used for affinity measurements, LC/MS analysis with ELSD (7) was used to ensure the highest possible purity and to confirm the expected isotopic pattern (Fig. 3B).

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FIGURE 3. 

Preparation, purification, and analysis of PSMA-specific radiotracers. (A) Single nucleophiles (primary amines) of small molecules from Fig. 1 were conjugated in 1 step to [^99mTc-MAS₃]-NHS using solid-phase preloading strategy described in text to create PSMA-specific radiotracers. (B) RP-HPLC analysis of compounds I–IV after solid-phase prelabeling using either ^99mTc (left) or Re (right). R_t values for compounds Ia–IVa are shown, as are ES-TOF(−) mass spectra of peak for Re compounds (insets). AU = absorbance units. For comparison, R_t values for [^99mTc-MAS₃]-NHS and [Re-MAS₃]-NHS on their optimized gradients are 23.5 and 10.4 min, respectively (Figs. 2B and 2D). (C) Serum protein binding and serum stability of solid-phase prelabeled ^99mTc PSMA-specific small molecules. Compound ^99mTc-IVa was incubated for 0 or 4 h at 37°C in PBS (top) or 100% serum (bottom) and then subjected to HPLC analysis. Samples in PBS were resolved on a Symmetry C₁₈ column. Samples in serum were resolved on a 120-Å high-resolution gel-filtration column and also include absorbance (280 nm) tracings. R_t values for gel-filtration markers M₁−M₅ are shown as arrows. Marker R_t values were M₁ = 6.6 min, M₂ = 8.2 min, M₃ = 9.2 min, M₄ = 11.1 min, and M₅ = 13.4 min. (D) Live cell-binding assay. PSMA-positive LNCaP cells grown on 96-well filter plates as described in text were incubated for 20 min at 4°C with monomeric ^99mTc-Ia (top row) or trimeric ^99mTc-IVa (bottom row) in presence of increasing concentrations of homologous nonradioactive test compound. Shown are results for monomeric Re-Ia (top row) and trimeric Re-IVa (bottom row) in TBS (left), PBS (middle), and 100% serum (right). Also shown are mean affinities ± 95% confidence intervals for 3 independent assays. N.D. = none detected.

The stability of adamantine-trimerized GPI molecule (IVa) was tested by incubation at 37°C for 4 h in either PBS or 100% serum. HPLC analysis (Fig. 3C) revealed 100% stability in PBS and 99% stability in serum, with 1% appearing as an early-eluting peak.

Bioactivity and Radioscintigraphic Imaging of PSMA-Specific Radiotracers

Using the nonradioactive Re derivatives as test compounds, ^99mTc derivatives as radiotracers, and a high-throughput 96-well filter plate assay (7), the absolute affinity of each molecule for the surface of living prostate cancer cells was measured using homologous competition (Table 1). Actual raw data from the assay using monomeric [^99mTc-MAS₃]-GPI (Ia) as radiotracer (Fig. 3D, top) and trimeric [^99mTc-MAS₃]-AdamGPI (IVa) as radiotracer (Fig. 3D, bottom) confirm that both phosphate and serum compete effectively with monomeric radiotracers for the active site of PSMA but are unable to compete with trimeric radiotracers, resulting in nearly identical affinities of IVa under all physiologic conditions. The measured maximum number of binding sites (B_max) values for compounds Ia/IIa, IIIa, and IVa were 1.6 × 10⁵, 2.1 × 10⁵, and 2.6 × 10⁵ PSMA receptors per cell, respectively, which is consistent with previously published reports of 1.8 × 10⁵ to 8.0 × 10⁵ for PSMA-specific monoclonal antibodies (16,17).

To confirm that the synthesized radiotracers permit sensitive and specific radioscintigraphic imaging of prostate cancer cells, approximately 35,000 PSMA-positive (LNCaP) or PSMA-negative (PC-3 and TsuPR1) cancer cells were grown in each well of a 96-well filter plate and incubated at 4°C for 20 min with the indicated tracer. As shown in Figure 4, and confirming the results in Table 1 and Figure 3D, ^99mTc-labeled trimeric AdamGPI (IVa) bound with high affinity to living prostate cancer cells expressing PSMA in all buffers, including 100% serum, and no binding was detectable on PSMA-negative cells.

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FIGURE 4. 

Radioscintigraphic imaging of ^99mTc-labeled PSMA-specific radiotracers: Live cell binding of ^99mTc-Ia (GPI monomer; M) and ^99mTc-IVa (AdamGPI trimer, T) was performed in TBS (top row), PBS (middle row), and 100% serum (bottom row) for 20 min at 4°C using PSMA-positive LNCaP cells (left), PSMA-negative PC-3 cells (middle), and PSMA-negative TsuPR1 cells (right) grown on 96-well filter plates, followed by extensive washing. Cells were independently loaded with NIR fluorophore IR-786 to assess viability and confluence. Shown are white light (top), NIR fluorescence (middle), and γ-detector (bottom) images of cells grown on 96-well filter plates.