89Zr-Cobalamin PET Tracer: Synthesis, Cellular Uptake, and Use for Tumor Imaging

Vitamin B12, or cobalamin (Cbl), is an essential nutrient. Acquisition, transport, and cellular internalization of Cbl are dependent on specific binding proteins and associated receptors. The circulating transport protein transcobalamin (TC) promotes cellular uptake via binding to specific receptors such as CD320, a receptor upregulated in several cancer cell lines. In this study, we report the successful synthesis of 89Zirconium-labeled Cbl that was derivatized with desferrioxamine (89Zr-Cbl). We document the purity of the tracer and its binding to TC compared with that of unmodified cyano-Cbl (CN-Cbl). In vitro studies employing the CD320 receptor-positive breast cancer cell line MDA-MB-453 showed a 6- to 10-fold greater uptake of 89Zr-Cbl when compared with the uptake in the presence of 200-fold excess of CN-Cbl at 37 °C. We used nude mice with MDA-MB-453 tumors to study the feasibility of employing the tracer to visualize CD320 positive tumors. In vivo positron emission tomography images displayed a clear visualization of the tumor with 1.42 ± 0.48 %ID/g uptake (n = 3) at 4 h after injection (p.i.) with the tracer retained at 48 h p.i. Ex vivo biodistribution studies using 89Zr-Cbl exhibited the highest uptake in kidney and liver at 48 h p.i. Results document the feasibility of synthesizing a Cbl-based tracer suitable for both in vivo and ex vivo studies of Cbl trafficking and with the potential to visualize tumors expressing TC receptors, such as CD320.


■ INTRODUCTION
Vitamin B 12 (cobalamin, Cbl) is a critical nutrient that is physiologically required to maintain cell growth and differentiation. 1−4 Cbl is involved in the biosynthesis of nucleic acids, lipids, and proteins, and its deficiency leads to a reduction in functional methionine synthase and metabolism of methylmalonic acid in humans, leading to megaloblastic anemia and/ or various neurological disorders. 1−4 Cbl gains entry into cells upon binding to transport proteins and subsequent receptor mediated transport. Cbl in blood is bound to the transport protein transcobalamin (TC) (holo-TC), which, in turn, is recognized by specific receptors such as CD320. 1−4 Upregulation of CD320 receptors has been reported in several malignancies including breast, prostate, thyroid, cervical, colorectal, and stomach cancers. 5 The important role of Cbl in cellular proliferation and the upregulation of CD320 in tumor cells has made Cbl uptake an attractive candidate for tumor imaging, mainly using singlephoton emission computed tomography with 99m Technetiumor III Indium-labeled Cbl. 6−13 One Cbl-positron emission tomography (PET) imaging agent, labeled with 64 Cu (t 1/2 ∼ 12.7 h), has also been reported. 14 Herein, the utility of Cbl as a vector was explored for delivering the PET radionuclide 89 Zr (t 1/2 ∼ 3.27 days). We hypothesized that 89 Zr would (1) retain the sensitivity of PET imaging and (2) provide a longer visualization window by providing a greater signal-to-noise ratio compared with prior tracers reported, allowing for an improved tumor targeting and imaging. Following radiosynthesis, we evaluated the in vitro uptake and in vivo pharmacokinetics of the CD320-positive MDA-MB-453 breast cancer in athymic nude mice.

■ RESULTS
Synthesis of Cbl-Desferrioxamine (Cbl-DFO). Cbldesferrioxamine (Cbl-DFO) was synthesized by forming a carbamate linkage between the 5′-hydroxyl of deoxyribose moiety in Cbl and the amine group of DFO (Scheme 1, Figure  S1). 15 Cbl was activated using 1,1′-carbonyl-di-(1,2,4-triazole) (CDT), followed by the addition of DFO, which links through the primary amine. Purification and characterization confirmed that the conjugate was of ≥97% purity ( Figures S2−S4 Radiolabeling of Cbl with 89 Zr. Cbl-DFO was labeled with 89 Zr ( 89 Zr-Cbl) using a previously established protocol (Scheme 1). 16 A radiolabeling efficiency of ∼97% was determined by instant thin-layer chromatography (iTLC, Figure S5a). The specific activity of the tracer was determined by titrating 89 Zr 4+ and Cbl at different mole ratios with an
In Vitro Stability of 89 Zr-Cbl. Stability of the tracer was analyzed by incubating 89 Zr-Cbl in saline and in human serum at 37°C. Bound versus unbound radio metal was analyzed at 0, 4, 24, and 48 h after incubation using iTLC (Figure 1a,b). The intact tracer was located closer to the origin (40−80 mm), whereas unbound tracer was found at 100−140 mm. After 48 h of incubation, free 89 Zr was <1% in both saline and serum.
TC Binding Studies. Mouse TC binding of 91 Zr-Cbl was studied by radiometric chase assay using 57 Co-labeled Cbl employing a previously described design. 17 91 Zr-Cbl was synthesized similar to 89 Zr-Cbl, but with 91 ZrCl 4 . Mouse TC binding of 91 Zr-Cbl displaced 57 Co-labelled Cbl in a manner comparable to that of CN-Cbl (Figure 1c), indicating that the modification of Zr-Cbl did not compromise binding to TC. The same results were obtained for binding to human intrinsic factor (data not shown).

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Article versus 83 ± 15 cpm/10 5 cells at 24 h (p < 0.0001). Competitive assays using excess Cbl at 37°C displayed lower binding at all time points (p < 0.01). All of the data are reported as mean ± standard deviation of four independent measurements.

■ DISCUSSION
In this proof-of-concept study, we report the successful production of a Cbl-derived 89 Zr tracer suitable for use in PET studies. Studies on nude mice bearing a human breast cancer cell tumor allow us to demonstrate the use of the tracer for PET visualization of the tumor.
In agreement with previous data, 17,18 we found that 91 Zr-Cbl bound to mouse TC in a manner comparable to that of CN-Cbl ( Figure 1c). Next, an in vitro assay was performed in the breast cancer cell line MDA-MB-453 to demonstrate a specific uptake of 89 Zr-Cbl. 19 We demonstrated a greater than 4-fold uptake of 89 Zr-Cbl at 37°C versus 4°C; blocking with 200-fold excess unlabeled Cbl had a similar reduced uptake (Figure 1d). These results support the idea that the targeting properties of 89 Zr-Cbl in MDA-MB-453 cells rely on a Cbl-dependent internalization mechanism, likely through the CD320 receptor.
In vivo imaging with 89 Zr-Cbl showed an uptake in MDA-MB-453 tumors with 1.42 ± 0.48 of %ID/g, whereas ex vivo tissue distribution studies showed a tumor uptake of 5.11 ± 1.33 %ID/g at 4 h p.i. (Figures 2 and 3). Notable uptake was also observed in the liver and kidneys with 4.27 ± 0.51 and 8.92 ± 1.45 %ID/g at 4 h p.i., respectively (Figure 2c). An in vivo block using 200-fold excess of unradiolabeled Cbl showed a significantly reduced uptake (p < 0.001) of the tracer, indicating that the in vivo tumor initialization is Cbl dependent, supported in the in vitro internalization assay. Muscle-to-tumor ratio showed that the maximum tumor-to-background ratio was achieved at 4 h p.i.
To compare the uptake values with those described in the literature, the biodistribution data will be used for a more accurate comparative analysis for the rest of the discussion. Tumor uptake persisted throughout the 4−48 h imaging period without a significant change (5.11 ± 1.33 at 4 h vs 3.78 ± 0.77 at 48 h, p > 0.1), whereas blood clearance was evident between 4 and 24 h with approximately ∼80% decrease (5.28 ± 0.62 at 4 h vs 0.92 ± 0.25 %ID/g at 24 h) in the circulating 89 Zr-Cbl; the final blood activity was observed to be 0.39 ± 0.06 %ID/g at 48 h p.i.
The tumor uptake achieved in our model is comparable to that of the other Cbl-based tracers reported thus far. 6−14 Ikotun et al. investigated the tumor uptake of 64 Cu-labeled Cbl in pancreatic, ovarian, murine melanoma, and colorectal tumor models, with the highest %ID/g being 4.84 ± 0.32 at 6 h p.i. in the colorectal tumor models. 14 In the melanoma model, the tumor uptake was highest (3.43 ± 0.87 %ID/g) at 1 h, which decreased to 2.64 ± 0.10 %ID/g after 24 h, whereas 89 Zr-Cbl had a higher accumulation and did not show a significant decrease in tumor accumulation over 4−48 h, p > 0.1.
One of the limitations of our 89 Zr-Cbl tracer is the observed kidney uptake (94.42 ± 4.27 %ID/g at 4 h), a problem across all of the Cbl tracers to date. Renal processing is the most prominent route for Cbl accumulation/storage and/or reabsorption and is driven by megalin, a known TC-Cbl receptor expressed in kidney proximal tubuli. 20,21 In addition, this is the first positively charged PET tracer reported, the effect of which is unknown. An overall positive charge on the Cbl (as 89 Zr-Cbl conjugate), or indeed simply the result of modification of the β-axial position, may affect the Cbl cellular processing across tissues, as has been shown previously for two forms with varying α-ligands (OH-Cbl and CN-Cbl) Cbl species, and warrants further investigation. 12,22−24 89 Zr-Cbl shows feasibility as a PET tracer to identify MDA-MB-453 tumors in vivo. The longer window for PET imaging allowed for reduced uptake in the kidneys, a problem to date in Cbl radiotracers, while still maintaining a moderate tumor uptake over 48 h p.i. This tracer is promising since, to our knowledge, tumor uptake is the highest reported to date for a B12 based PET probe.

■ CONCLUSIONS
We have successfully developed and evaluated the first 89 Zrlabeled Cbl tracer as a viable tool for visualizing TC-mediated Cbl uptake into a CD320 positive tumor. 89 Zr-Cbl displayed retained tumor uptake up to 48 h p.i., allowing for a longer imaging window. Our data paves the road for future studies to understand the kinetics of Cbl transport and to study the use as a tool for visualizing tumors capable of accumulating Cbl. 89 Zr-Radiochemistry. Optimum conditions for radiolabeling of Cbl-DFO were tested by titrating with 89 Zr and analyzing the incubated solution using iTLC. Optimum labeling activity was found to be 250 ± 20 mCi/μmol (9250 ± 740 MBq/ micromole). Approximately 1 mCi (37 MBq) of 89 Zr(C 2 O 4 ) 2 (3D Imaging, LLC) was diluted with 0.9% saline and the pH was adjusted to 7−7.5 by adding 1 M Na 2 CO 3 . A solution of Cbl-DFO (0.004 μmol, 10.8 μg) was added to the pH-adjusted 89 Zr solution and incubated for 20 min at ambient temperature (Scheme 1). Radiolabeling efficiency of >97% was determined by iTLC using silica iTLC strips and EDTA mobile phase ( Figure S5a). The identity of the tracer was characterized via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis using Cbl-DFO labeled with cold Zr 4+ as standard. Expected: 2030.  Figure S5b).
Mouse TC Binding to 91 Zr-Cbl. Nonradioactive 91 Zr-Cbl was synthesized for TC binding studies by reacting Cbl-DFO with 91 ZrCl 4 as described above. The conjugate was characterized by ESI-MS (data not shown). Mouse TC binding of 91 Zr-Cbl was confirmed by radiometric chase assay using 57 Co-labeled Cbl and compared with free Cbl (cyanocobalamin) employing a previously described protocol. 17 Mouse TC was derived as previously described. 25 In Vitro Uptake. Modified internalization assay was performed. MDA-MB-453 cells were cultured in Cbl-free media (DMEM with 10% charcoal stripped FBS) and plated in six-well plates. Each well contained 200 000 cells plated and incubated overnight. To each well, 89 Zr-Cbl (0.1 μCi, 3.7 KBq, 0.4 pmol of Cbl per well) was added. For the blocking experiment, unmodified Cbl was added (40 pmol per well). Plates were incubated for 1, 4, and 24 h intervals at either 37 or 4°C. At the end of each time point, wells were serially washed with phosphate-buffered saline (1×), acid (1 mM acetic acid and 1 mM glycine), and base (1 M NaOH, 1 mL, 5 min). Each wash was collected and measured for bound activity using a γ counter (Perking Elmer 2480 WIZARD). Control wells were trypsinized and counted using a cell counter (Contessa II). Internalized activity was normalized to 10 5 cells.
Cell Lines and Small Animal Xenografts. All of the animal handling and manipulations were conducted in accordance with the guidelines set by WSU Animal Care and Use Committee (IACUC). For imaging and in vivo uptake experiments, female nude mice (Envigo) were kept under Cbldeficient diet (Teklad Cbl-free custom diet, Envigo) for 3 weeks. Cells were subcutaneously implanted on the shoulder with MDA-MB-453 cancer cells (5 × 10 6 cells/mouse) after 2 weeks of Cbl-free diet. Cells were injected in 1:1 media/ matrigel (Corning LLC) at a volume of 200 μL. The tumor volume until was calculated using the formula length × width 2 × 0.52. Mice with tumors of 100−200 mm 3 dimensions were used for imaging experiments.
Ex Vivo Distribution and Competitive Saturation. The tissue distribution of 89 Zr-Cbl was studied by administering 10−25 μCi (0.37−0.93 MBq, 0.04−0.1 nmol) of the tracer on the lateral tail vain of the rodent. For the competitive saturation assay, ∼20 nmol/mouse of cold CN-Cbl was co-injected with 89 Zr-Cbl. Euthanasia was performed via CO 2 asphyxiation at 4, 24, and 48 h p.i.