Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen

Background Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. Results J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68Ge/68Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4–6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 μg conjugate with gallium-68 for 5 min without post-labelling purification. 68Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145 tumours (n = 4). Conclusions The bifunctional chelator THP-mal enabled simple, rapid, quantitative, one-step room temperature radiolabelling of a protein with gallium-68 at neutral pH without a need for post-labelling purification. The resultant gallium-68 complex shows high affinity for PSMA and favourable in vivo targeting properties in a xenograft model of PCa. Electronic supplementary material The online version of this article (10.1186/s13550-017-0336-6) contains supplementary material, which is available to authorized users.


Protein purification and analysis
Size exclusion chromatography: Purification of J591c-scFv and its THP-mal conjugate was performed using FPLC size exclusion column chromatography using a Superdex 75 10/300 GL column (GE), eluting with phosphate buffered saline (pH 7) at a flow rate of 0.5 ml/min and maximum pressure of 1.8 MPa.
Polyacrylamide gel electrophoresis and Western blotting were used to evaluate purity of the size-exclusion-purified J591c-scFv, using pre-cast polyacrylamide gels (NuPAGE 12% Novex Bis-Tris mini gels with MES buffer, Life Technologies) and Western blotting. For the preparation of samples, 10 µl aliquots of J591-scFv were placed in microcentrifuge tubes and 5 µl of NuPAGE LDS loading buffer (4x) was added. For reduced samples, the reducing agent (NuPAGE reducing agent of strength 10x, 2 µl) was added along with NuPAGE LDS buffer of 4x concentration (5 µl). Samples were placed on a heating block for 5 min at 95 o C and then cooled on ice for 1 min. The NuPAGE chambers were filled with the MES buffer (50 ml of 20x MES buffer + 950 ml of deionised water). Novex sharp pre-stained protein standard was loaded in one of the wells. The gel was run for 40 min at a nominal voltage of 200 V with constant current of 125 mA, developed with SimplyBlue SafeStain (Thermo Fisher Scientific) for 2 h and destained with water overnight. In the case of radiolabelled proteins, before staining, activity markers were placed on the gels which were then imaged with a phosphorimager.
For Western blotting, the 12% gel was prepared as above; instead of staining, the gel was loaded on a nitrocellulose transfer membrane and developed in non-reducing transfer buffer (NuPAGE transfer buffer (20x) 50 ml + methanol (100 ml) + deionised water (850 ml)) for 1 h at constant 30 V and current of 170-130 mA. After 1 h the transfer membrane was removed and placed in a blocking agent (4% milk in 50 ml of PBS with 0.05% Tween 20) for 90 min, washed briefly with PBS, then placed in a solution containing antibody against Histag (6.3 µl of antibody diluted (1:4000 dilution) by adding 25 ml of PBS with 0.5% BSA, which is 0.125 g/25 ml of BSA) overnight at 4 o C with shaking. The transfer membrane was washed four times with 10 ml of PBST (1 l of phosphate buffered saline with 0.05% Tween 20) before incubating with secondary antibody (goat anti-mouse horseradish peroxidase) for 60 min

Instant thin layer radiochromatography (ITLC)
Protein labelling with gallium-68 was assessed by instant thin layer chromatography (ITLC) using ITLC-SA (Agilent) developed with 0.1 M sodium citrate buffer (pH 6) as the mobile phase. Under these conditions unbound gallium-68 moved with the solvent front (Rf = 1) and radiolabelled conjugated protein remained at the origin (Rf = 0).

High performance radiochromatography
The radiolabelled protein conjugates were analysed using a size-exclusion column (SEC-2000), sample volume 20 µl, eluting with phosphate buffered saline (pH 7), flow rate 1 ml/min, UV detection at 280 nm and gamma detection with a sodium iodide detector.

Serum stability
THP-J591c-scFv (200 µl, 0.4 µg/µl) was radiolabelled by incubation with 300 µl of 68 Ga generator eluate (60 MBq) for 20 min at room temperature and labelling efficiency found to be >99% by ITLC. Without further purification or modification, the radiolabelled conjugated J591c (100 µl) was mixed with serum (200 µl) and physiological saline (100 µl). The mixture was incubated at 37 o C and samples (5 µl each) were collected at 30, 60, 120, 180, 240 and 300 min and stored at -80 o C until all samples had been collected. The behaviour of unchelated Ga-68 in serum was determined by incubating 10 µl of ammonium acetate buffered 68 Ga generator eluate with serum as described above, incubating for up to 60 min at 37 o C, taking 5 µl samples and storing them at -80 o C until all the time points were collected for gel electrophoresis analysis. 68 Ga-THP-mal-J591c-scFv incubated in saline and sampled similarly was used as a reference. For analysis by SDS-PAGE, the samples (5 µl) were mixed with 10 µl of LDS buffer (lithium dodecyl sulfate, pH 8.4) and applied on the gel (10 µl). The gel was developed in MES buffer (2-ethanesulfonic acid) for 40 min at 200 V and 120-130 A. Afterwards the gel was removed, activity markers were placed and it was analysed by phosphorimager and then stained with Coomassie blue.

PET imaging and quantification
Dynamic imaging (n = 1 each group) was performed over the time period of 3 h from the time of injection, 25 days after tumour inoculation, using a BioScan nanoPET-CT PLUS (Mediso) scanner using their proprietary acquisition software (Nucline, version 2.00). CT was performed with an x-ray tube voltage of 45 kVp, 600 ms of exposure time, and 360 projections. This scan took 10 min to obtain. Dynamic PET scans were acquired within a 94.7-mm field of view from 0 to 190 min after tail vein injection of the tracer. Acquisition took place in 1-5 coincidence mode with a coincidence window of 5 ns and a 400-to 600-keV energy window. The dynamic PET data were reconstructed using Nucline software (version 2.00). Images were constructed based on 0.4 mm 3 voxels for PET and 0.21 mm 3 for CT. Image processing and analysis were performed using Vivoquant software (version 1.23).
Before analysis, both CT and PET images were realigned and processed to a voxel size of 5 0.21 mm 3 and the PET output calibrated to display MBq per voxel. Regions of interest (ROIs) for each data file were produced using freehand segmentation. SUV max values were obtained from regions of interest near the centre of each selected organ/tumour, using the total activity within the image (excluding activity within the tail) as the total injected dose.
Time-activity curves were produced from 15-min bins.