Protein Conjugation with Desferrioxamine

A 3-fold equivalence of DFO-Bz-SCN (Macrocylics, Inc.) in DMSO (<5% total volume) was added to a solution of Mb or Cys-Db. huJ591 was reacted with DFO-Bz-SCN with a 1:5 mAb:chelate ratio. All reactions were incubated at 37 °C for 45 min with occasional mild stirring. Subsequent purification using a PD10 size exclusion column removed any unreacted DFO-Bz-SCN and DMSO with 0.9% saline as the mobile phase. The purified product was concentrated to a volume of ~500 μL using a Vivaspin 500 (MWCO: 10 kDa) centrifugal filter. SDS gel electrophoresis was performed to analyze purified DFO conjugates of the Mb and Cys-Db (see the Supporting Information and SI Figure 1 in the Supporting Information).

Radiolabeling Experiments with ⁸⁹Zr

⁸⁹Zr-oxalate (~37 MBq) was produced as previously described (see the Supporting Information).²⁰ The pH of ⁸⁹Zr was adjusted to ~7.0–7.2 with 1 M Na₂CO₃. To each separate reaction, approximately 125–140 μg of Mb/Cys-Db-DFO (Mb-DFO, 1.56–1.75 nmol; Cys-Db-DFO, 2.5–2.8 nmol) or 250 μg (1.7 nmol) of huJ591-DFO was added and incubated at room temperature with intermittent mild shaking. After 1–1.5 h, the reaction was quenched with 10 μL of 50 mM DTPA (pH ~ 7) to remove any nonspecifically bound ⁸⁹Zr. Crude radiolabeling yields were determined to be >95% using iTLC with the ⁸⁹Zr-labeled proteins remaining close to the origin at R_f = 0.30 while free Zr-89 is found near the solvent front at R_f = 0.65. Purification of ⁸⁹Zr-proteins was performed using a PD10 size exclusion column with saline as the eluting buffer. The final radiochemical purity was >99% based on iTLC analysis. To address retained affinity for the antigen, both radiolabeled constructs ⁸⁹Zr-Mb and ⁸⁹Zr-Cys-Db were evaluated for immunoreactivity via a protocol established by Lindmo et al.²¹

Cell Lines and Small Animal Xenografts

LNCaP (PSMA(+)) and PC3 (PSMA(−)) human prostate cancer (PC) cell lines (American Type Culture Collection) were cultured in a sterile environment with 5% CO₂ at 37 °C and grown as described.²²

All animal experiments were conducted in accordance with the guidelines set by MSKCC Animal Care and Use Committee and Research Animal Resource Center. For imaging experiments, male athymic nude (nu/nu) mice (6–8 week old, Taconic) were subcutaneously inoculated with dual tumors. LNCaP cells (3 × 10⁶) in 1:1 medium:Matrigel (BD Sciences) were implanted on the left shoulder. PC3 cells (3 × 10⁶ in 1:1 medium:Matrigel) were injected on the right shoulder. Tumor growth was monitored weekly and measured using vernier calipers with the volume calculated using the formula length × width × height × 0.52. Tumor volumes were allowed to reach 150–300 mm³ prior to use.

Internalization Assay

Internalization of ⁸⁹Zr-Mb, ⁸⁹Zr-Cys-Db, and fully human ⁸⁹Zr-huJ591 was investigated on LNCaP and PC3 cells. Approximately 1 × 10⁵ cells were seeded in a 12-well plate and incubated overnight. A volume of 2 mL of radiolabeled protein (37 kBq/mL) was added to each well. The plates were incubated at 37 and 4 °C for 0.5–24 h. Following each incubation period, the medium was collected and the cells were rinsed with 1 mL of phosphate buffered saline (PBS) twice. Surface-bound activity was collected by washing the cells in 1 mL of 100 mM acetic acid + 100 mM glycine (1:1, pH 3.5) at 4 °C. The adherent cells were then lysed with 1 mL of 1 M NaOH. Each wash was collected and counted for activity. The % internalized activity was calculated as the ratio of the activity of the lysate and the total activity from the medium, PBS, acid, and base washes.

In Vitro Blocking Study

In 12-well plates, 1 × 10⁵ LNCaP cells were seeded and incubated overnight to facilitate adherence. huJ591 (100 μg, 0.67 nmol) was either coadministered with the radioactive probes or preincubated for 1 h at 37 °C. After addition of ⁸⁹Zr-Mb (1–2 μg, 12.5–25 pmol) and ⁸⁹Zr-Cys-Db (1–2 μg, 20–40 pmol) in separate wells, the cells were incubated at 37 °C for 1 h and then carefully washed with medium to remove any excess unbound activity. Cells were lysed with 1 mL of 1 M NaOH and then measured for activity. The level of bound radioligands was calculated as % bound, normalized to the amount of activity added.

Saturation Binding Assay

To determine the dissociation constant (K_d) of each of the ⁸⁹Zr-mAbs, saturation binding studies were conducted using LNCaP cells. Briefly, varying concentrations of the ⁸⁹Zr-mAbs (0.03–3 μg/mL, 0.2–60 pmol/mL) were added to 5 × 10⁵ cells in PBS containing 1% bovine serum albumin. The same experiment was repeated with additions of cold mAbs (140 μg/mL for the Mb (1.75 nmol/mL) and Cys-Db (2.8 nmol/mL) and 300 μg/mL for huJ591 (2 nmol/mL)) to determine nonspecific binding (NSB). After 1 h incubation at room temperature, the cells were washed twice with 1 × PBS. Specific binding (SB, nM), derived by subtracting NSB from total bound activity, was plotted against the amount of ⁸⁹Zr-mAbs added. The dissociation constant, K_d, was calculated by nonlinear regression fitting of the resulting plot using GraphPad Prism v. 6.02. The study was conducted in duplicate.

PET Imaging Experiments

Mice (n = 3–4) bearing LNCaP xenografts were administered intravenously (iv) with 7.4–10.2 MBq of either ⁸⁹Zr-Mb (28–38 μg, 0.35–0.48 nmol) or ⁸⁹Zr-Cys-Db (25–34 μg, 0.50–0.68 nmol) in saline. The mice were anesthetized with 2% isofluorane in oxygen prior to imaging. Small-animal PET studies were conducted using microPET-R4 and Focus 120 scanners (Concorde Microsystems). PET images were acquired between 1 and 24 h after dose administration. Images were reconstructed via filter back projection. Using ASIPro VM software (Concorde Microsystems), volumes of interest (VOIs) were measured on various planar sections of the acquired image by manually drawing on the tumor site and on select organs. The mean VOI was calculated and expressed as % injected dose per gram of tumor tissue (% ID/g).

Tissue Biodistribution Studies

Single tumor bearing mice were administered intravenously with 370–555 kBq of either ⁸⁹Zr-Mb (1–2 μg, 12.5–25 pmol) or ⁸⁹Zr-Cys-Db (1–2 μg, 20–40 pmol). Competitive inhibition studies were performed with coadministration of ~200–500 μg (2.5–10 nmol) of nonradioactive Mb or Cys-Db in LNCaP tumor-bearing mice (n = 3–5). In a separate cohort of mice (n = 4) bearing the PSMA(+) tumor, the parent huJ591 (500 μg, 3.3 nmol) was administered 36 h prior to dosing with ⁸⁹Zr-Mb. Mice were euthanized by CO₂ asphyxiation after 1, 4, 12, and 24 h p.i. (n = 4–5 per group). Select tissues including the tumor were harvested and weighed with bound activity measured using a gamma counter (PerkinElmer). Activity measurements were background- and decay-corrected. The tissue uptake (% ID/g) was calculated against the total net activity injected.

Autoradiography and Histology

For autoradiographic studies, separate animals were administered with ⁸⁹Zr-Mb, ⁸⁹Zr-Cys-Db, and ⁸⁹Zr-huJ591 (7.4–10.2 MBq) as described above, 24 h before sacrifice. Five minutes prior to sacrifice, animals were injected with Hoechst 33342 (1 mg/mL PBS, iv). Immediately following sacrifice, tumors were removed and frozen in OCT mounting medium (Sakura, Europe) and a series of 10 μm tissue sections obtained. Autoradiographic distribution of ⁸⁹Zr at 25 μm pixel resolution was obtained by exposing the sections to a BAS MS2025 phosphor plate (FujiFilm), followed by reading on a GE Typhoon 7000IP plate reader. The same sections were then used to obtain high-magnification whole-mount images of Hoechst 33342 distribution and H&E staining as previously described.²³

Following autoradiography, sections were fixed in 4% paraformaldehyde and stained for PSMA expression using rabbit anti-PSMA antibody (clone EPR6253, Abcam, 1:100 dilution) overnight at 4 °C, followed by goat anti-rabbit-Alexa-568 (Invitrogen, 1:100) for 1 h at room temperature. Sections were then imaged for Alexa-568 and Hoechst 33342 signals as previously described.²⁴ Finally, sections were stained with H&E, and digital microsopic images obtained in the same manner.

Variable In Vitro Internalization Rates of ⁸⁹Zr-Labeled huJ591, Mb, and Cys-Db

With reports showing PSMA possessing a putative endocytic functionality,^25,26 we conducted in vitro internalization studies using LNCaP cells to compare the rates of internalization of ⁸⁹Zr-Mb, ⁸⁹Zr-Cys-Db, and ⁸⁹Zr-huJ591. The internalization patterns of all three radiotracers appear to be rapid, however, initial slow uptake was displayed by ⁸⁹Zr-Mb at 4 h at 37 °C with lower fractions internalized (3.7 ± 0.5%, P = 0.002) (Figure ​(Figure1A,1A, left panel). This was then followed by rapid internalization, peaking at 12 h with 15.1 ± 1.0% (P = 0.027), which reached a plateau at 24 h (16.6 ± 1.0%). In Figure ​Figure1A1A (middle panel), ⁸⁹Zr-Cys-Db showed consistent linear internalization, spanning all time points (1.0 ± 0.1% at 0.5 h, 3.5 ± 0.2% at 1 h, 4.5 ± 0.5% at 4 h, 8.9 ± 0.7% at 12 h, and 11.9 ± 0.1% at 24 h) at 37 °C. At 37 °C, ⁸⁹Zr-huJ591 demonstrated internalization with 1.4 ± 0.1% at 0.5 h, 1.8 ± 0.8% at 1 h, 7.3 ± 0.6% at 4 h, 12.1 ± 1.3% at 12 h, and 10.1 ± 1.3% at 24 h (Figure ​(Figure1A,1A, right panel). Minimal internalization was demonstrated by all three imaging probes at 4 °C, evidence of an endocytic uptake process.

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Figure 1

In vitro assays in LNCaP prostate cancer cells. Both antibody fragment radiotracers were added to LNCaP prostate cancer cells and incubated at 37 and 4 °C at 0.5, 1, 4, 12, and 24. (A) The internalization rates of ⁸⁹Zr-Mb (left) and ⁸⁹Zr-huJ591 (right) follow a nonlinear trend with slow uptake rates after 4 h of incubation; a subsequent increase in intracellular radioactivity with higher internalized fractions was observed for ⁸⁹Zr-Mb after 12 h. ⁸⁹Zr-Cys-Db (middle) displays a linear rate of internalization. All three radiotracers display minimal intracellular accumulation at all time periods at 4 °C. (B) In LNCaP prostate cancer cells, 1 h preincubation or coaddition of huJ591 (100 μg) with the radiotracer diminished binding of ⁸⁹Zr-Mb (1–2 μg, left) and ⁸⁹Zr-Cys-Db (1–2 μg, right).

In Vitro Blocking Study of ⁸⁹Zr-Mb and ⁸⁹Zr-Cys-Db by huJ591

The specificities of both ⁸⁹Zr-Mb and ⁸⁹Zr-Cys-Db to PSMA were evaluated by competitive inhibition with nonradioactive huJ591. In Figure ​Figure1B1B (left panel), ⁸⁹Zr-Mb demonstrated as much as 4.96 ± 0.73% binding to LNCaP cells with no added huJ591. This level of bound Mb was satisfactorily blocked as much as 10-fold with both preincubated (0.55 ± 0.19%, P < 0.0001) and coadded (0.56 ± 0.3%, P < 0.0001) huJ591. Similar outcomes were observed with ⁸⁹Zr-Cys-Db (Figure ​(Figure1B,1B, right panel) where, in the absence of huJ591, the radiotracer displayed 4.77 ± 0.21% PSMA-binding activity; this uptake was significantly diminished by preincubation (0.16 ± 0.08%, P < 0.0001) and coaddition (0.15 ± 0.07%, P < 0.0001) of huJ591.

Saturation Binding Assays

⁸⁹Zr-huJ591, ⁸⁹Zr-Mb, and ⁸⁹Zr-Cys-Db demonstrated specific binding and high affinities for PSMA localized on the surface of LNCaP cells. The saturation binding studies showed apparent K_d values that are similar for all three radiotracers: ⁸⁹Zr-huJ591 (K_d = 2.31 ± 0.27 nM), ⁸⁹Zr-Mb (K_d = 2.18 ± 0.50 nM), and ⁸⁹Zr-Cys-Db (K_d = 2.59 ± 0.41 nM).

PSMA-Specific Tumor Uptake and Tissue Distribution of ⁸⁹Zr-Mb and ⁸⁹Zr-Cys-Db

Tissue distributions of ⁸⁹Zr-Mb were analyzed in mice with single implants of PSMA(+) LNCaP (Figure ​(Figure2A;2A; SI Table 1 in the Supporting Information) or PSMA(−) PC3 tumors (SI Table 2 in the Supporting Information). Tumor uptake of ⁸⁹Zr-Mb at 1 h (2.3 ± 0.5% ID/g) was observed to steadily increase at 4 h p.i. (4.7 ± 1.0% ID/g). Accumulation of the probe in the tumor increased at 12 h p.i. with 6.2 ± 2.5% ID/g and at 24 h p.i. (12.1 ± 3.6% ID/g). To establish the specificity of ⁸⁹Zr-Mb for PSMA in vivo, we performed blocking assays with various doses of nonradioactive Mb (SI Table 3 in the Supporting Information). At 12 h p.i., the uptake of ⁸⁹Zr-Mb was moderately decreased by 2-fold at 200 μg of coinjected cold Mb with 3.73 ± 1.27% ID/g (P = 0.21). Increasing the nonradioactive dose to ~500 μg did not significantly block the uptake either with 3.82 ± 0.70% ID/g (P = 0.19). We then employed huJ591 to block PSMA epitopes required for binding. A 500 μg huJ591 dose was administered iv 36 h prior to injection of ⁸⁹Zr-Mb with the mice (n = 4) euthanized 24 h p.i. of the radiotracer, which mitigated tumor uptake (5.0 ± 2.4% ID/g, P = 0.025). The in vivo specificity of this probe was further strengthened with differences between ⁸⁹Zr-Mb accumulation in PSMA(−) PC3 tumors against LNCaP tumors in (Figure ​(Figure2A,2A, inset). The PC3 tumor sustained a constant accretion of 2.3 ± 0.4% ID/g at 1 h (P = 0.28), 2.1 ± 0.3% ID/g at 4 h (P = 0.03), 1.7 ± 0.5% ID/g at 12 h (P = 0.04), and 2.4 ± 0.3% ID/g at 24 h. This nonspecific tumor uptake is attributed to the tumor’s leaky vasculature.

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Figure 2

Ex vivo tissue distribution studies of ⁸⁹Zr-Mb and ⁸⁹Zr-Cys-Db administered intravenously in LNCaP (PSMA(+)) and PC3 (PSMA(−)) prostate tumor-bearing mice. (A) Biodistribution of ⁸⁹Zr-Mb at different time points (1–24 h) in select tissues including the tumor; a blocking dose of ~500 μg of huJ591 36 h prior to intravenous injection of ⁸⁹Zr-Mb effectively mitigated tumor uptake at 24 h p.i. (A, inset) Direct comparison between LNCaP and PC3 tumors displays cumulative ⁸⁹Zr-Mb uptake in the PSMA(+) xenograft, which was significantly higher as early as 4 h p.i., whereas nonspecific binding in the negative tumor is observed. (B) ⁸⁹Zr-Cys-Db exhibits selective tumor tissue targeting, which peaks at 12 h p.i. A blocking dose of 200 μg of nonradioactive Cys-Db decreases the radiotracer tumor uptake at 12 h p.i. (B, inset) Higher uptake of ⁸⁹Zr-Cys-Db in LNCaP tumors compared to PC3 implants at 12 and 24 h p.i. confirms the specificity of this imaging probe for PSMA.

Assessment of ⁸⁹Zr-Cys-Db localization in the PSMA(+) tumor demonstrated uptake with 4.1 ± 1.0% ID/g at 1 h, 7.0 ± 3.0% ID/g at 4 h, and 12.3 ± 2.5% ID/g at 12 h (Figure ​(Figure2B;2B; SI Table 4 in the Supporting Information). A decrease in tumor accretion at 24 h (6.5 ± 1.0% ID/g) was observed. Blocking with 200 μg of cold Cys-Db at 12 h p.i. effectively lowered the tumor accumulation to 5.64 ± 1.75% ID/g (P = 0.044). PC3 tumor uptake (Figure ​(Figure2B2B inset; SI Table 5 in the Supporting Information) displayed lower accretion across all time points with 2.36 ± 0.48% ID/g (1 h, P = 0.068), 3.65 ± 1.54% ID/g (4 h, P = 0.12), 2.75 ± 0.51% ID/g (12 h, P = 0.007), and 3.44 ± 0.85% ID/g (24 h, P = 0.0011).

PSMA(+) Tumor Delineation

We performed in vivo PET imaging in mice bearing PSMA(+) LNCaP tumors from 1 to 24 h on all three radiotracers (SI Figure 2 in the Supporting Information). In Figure ​Figure3A,3A, planar sections of acquired images revealed accumulation of ⁸⁹Zr-Mb at 12 and 24 h p.i. Maximal intensity projection (MIP) at 12 h showed tumor delineation with nonspecific binding observed in the liver and kidneys. Similar results were achieved with ⁸⁹Zr-Cys-Db where localized delivery of the radiotracer to the tumor provided high contrasts at earlier time points (Figure ​(Figure3B).3B). In contrast to ⁸⁹Zr-huJ591 (Figure ​(Figure3C),3C), very minimal to no activity is present in the lung and heart in both ⁸⁹Zr-antibody fragments, proof that the circulating probes were eliminated faster than the intact mAb. Quantification of tumor uptake is presented in Figure ​Figure4A4A (SI Table 6 in the Supporting Information), which displays an increasing trend in tumor uptake for all three radiotracers. Tumor VOIs at 1 and 4 h p.i. were comparable. At 12 h p.i, the accumulation of both ⁸⁹Zr-Mb (6.85 ± 0.87% ID/g) and ⁸⁹Zr-Cys-Db (9.84 ± 2.54% ID/g) plateaued, whereas ⁸⁹Zr-huJ591 (15.84 ± 1.79% ID/g) steadily increased at 12 h p.i. After 24 h p.i., the tumor uptake of the intact mAb was 3-fold higher compared to the scFv proteins. This lower tumor delivery of the engineered mAb fragments most likely stemmed from low availability of both fragments in the blood pool. As shown in the VOIs drawn on the heart (Figure ​(Figure4B;4B; SI Table 7 in the Supporting Information), a sharp decreasing trend in radiotracer activity was displayed by ⁸⁹Zr-Cys-Db followed by ⁸⁹Zr-Mb across all time points such that, at 12 h, only 2.73 ± 0.61% ID/g ⁸⁹Zr-Cys-Db and 5.06 ± 2.84% ID/g ⁸⁹Zr-Mb remained. At 24 h, residual cardiac activity from both tracers was determined (i.e., 2.00 ± 0.18% ID/g for ⁸⁹Zr-Cys-Db and 3.26 ± 1.20% ID/g for ⁸⁹Zr-Mb). ⁸⁹Zr-huJ591 heart activity remained consistently higher at all time points (i.e., 16.36 ± 1.57% ID/g at 12 h and 12.13 ± 1.26% ID/g at 24 h).

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Figure 3

PET imaging. Representative maximum intensity projections (MIPs) and planar PET images of (A) ⁸⁹Zr-Mb, (B) ⁸⁹Zr-Cys-Db, and (C) ⁸⁹Zr-huJ591 acquired in male athymic nude mice bearing LNCaP (left shoulder, T) prostate tumors xenografts at 12 and 24 h postinjection. Kidney (K) and liver (L) accumulations were observed for both ⁸⁹Zr-Mb and ⁸⁹Zr-Cys-Db and rationalized as routes of excretion. All three probes localize in the tumor, however, only ⁸⁹Zr-huJ591 remains in circulation between 12 and 24 h.

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Figure 4

Time–activity curves of ⁸⁹Zr-Mb, ⁸⁹Zr-Cys-Db, and ⁸⁹Zr-huJ591 demonstrating (A) tumor volumes of interest (VOIs), (B) blood clearance through heart VOIs, (C) tumor-to-muscle ratios, and (D) tumor-to-heart ratios from 1 to 24 h.

Scrutiny of tumor-to-muscle (T/M) ratios from the PET scans disclosed no significant differences among all three radiotracers at all time points (P > 0.05) (Figure ​(Figure4C;4C; SI Table 8 in the Supporting Information). Tumor-to-heart (T/H) ratios (Figure ​(Figure4D; SI4D; SI Table 9 in the Supporting Information) showed no distinctions between ⁸⁹Zr-Mb and ⁸⁹Zr-huJ591; however, analysis of T/H ratios for ⁸⁹Zr-Cys-Db provided notably higher values at all time points against the intact mAb, for example, 3.58 ± 0.19 vs 0.98 ± 0.19 (P = 0.0002) at 12 h and 4.91 ± 0.26 vs 2.22 ± 0.46 (P = 0.014) at 24 h.

Acknowledgments are extended to the Radiochemistry and Molecular Imaging Service Core and the grant-funding support provided by the NIH MSKCC Center Grant (P30-CA08748). This project was also funded in part with federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN261201100123C.