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01.12.2017 | Original research | Ausgabe 1/2017 Open Access

EJNMMI Research 1/2017

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

EJNMMI Research > Ausgabe 1/2017
Saima Nawaz, Gregory E. D. Mullen, Kavitha Sunassee, Jayanta Bordoloi, Philip J. Blower, James R. Ballinger
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The online version of this article (https://​doi.​org/​10.​1186/​s13550-017-0336-6) contains supplementary material, which is available to authorized users.



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.


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).


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.
Additional file 1: Table S1. Deconvoluted electrospray mass spectra of J591c-scFv pre- and post-reduction and conjugation with THP-mal. Table S2. Ex vivo biodistribution data (%ID/g) for 68Ga-THP-mal-J591-scFv in mice bearing DU145 (PSMA-) and Du145-PSMA (PSMA+) (mean and standard deviation are shown; n = 4 in each group). Figure S1. Electrospray mass spectra of J591c-scFv pre-TCEP treatment (top, 27,923.2 corresponds to disulfide formed from J591c linked to cysteine via a disulfide bond), J591c-scFv post-TCEP treatment (middle, 27,803.9 corresponds to J591-scFv), and TCEP-treated J591-scFv after incubation with THP-mal (bottom, 28,723.7 corresponds to THP-mal-J591-scFv adduct). Figure S2. The effect of different molar excesses of TCEP on dimerisation of J591-scFv. Lanes: 1: molecular weight markers; 2: non-reduced protein; 3: reduced protein (NuPAGE reducing agent); 4: 0.4:1 M ratio TCEP to protein; 5: 1:1 M ratio; 6: 2:1 M ratio; 7: 5:1 M ratio; 8: 10:1 M ratio; 9: 15:1 M ratio; 10: 20:1 M ratio; 11: 30:1 M ratio. Figure S3. FPLC purification of THP-mal-J591-scFv conjugate on Superdex 75 HR 10/30 size-exclusion column eluted with phosphate-buffered saline at 0.5 ml/min. Conjugate elutes before free THP-mal. Free THP-mal can be seen eluting at 15–18 min, monomeric THP-conjugated protein at 10 min and dimeric protein at 8 min. Figure S4. Exemplar data showing radiolabelling efficiency (% protein bound, determined by ITLC) at different protein conjugate concentrations and times. At concentrations of 0.25 mg/ml or higher, labelling efficiency was 100% at all time points from 10 s onward (the first data point in each series represents a sample taken after 10 s incubation). Figure S5. SDS-PAGE analysis of serum stability of 68Ga-THP-mal-J591-scFv. A: radioactive gel analysed with cyclone phosphor imager. B: gel stained with Coomassie blue. Lanes: 1: molecular weight markers; 2: human serum incubated with ammonium acetate-buffered 68Ga eluate; 3: radiolabelled 68Ga-THP-mal-J591-scFv conjugate control (without serum incubation); 4: conjugate incubated with human serum for 1 min; 5: 30 min; 6: 60 min; 7: 120 min; 8: 180 min; 9: 240 min; 10: 360 min. Figure S6. Time course of 68Ga-THP-mal-J591c-scFv activity (SUVmax) in DU145-PSMA xenografts derived from serial images in a single mouse determined by PET imaging. A region of interest near the centre of each organ was drawn from which SUVmax was determined in each 15 min bin. Figure S7. Ex vivo biodistribution data for 68Ga-THP-mal-J591-scFv in mice bearing DU145 (PSMA-, blue) and DU145-PSMA (PSMA+, red), 90 min post-injection. Error bars represent standard deviation (n = 4 per group). Data are the same as those shown in the main manuscript (Fig. 5) but expanded to include kidneys. (PDF 1307 kb)
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