Abstract
BackgroundLabelling 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.ResultsJ591c-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).ConclusionsThe 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.
Highlights
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
Gel electrophoresis analysis was consistent with the presence of both monomeric J591c-scFv and its disulfide-linked dimer, the deconvoluted electrospray mass spectrum (Additional file 1: Table S1 and Figure S1) before TCEP treatment showed no evidence of dimeric protein and that the monomeric protein (27923) was 119 Da heavier than predicted from its amino acid sequence (27804), corresponding to a conjugate formed by a disulfide bond between the terminal Cys residue and a non-peptide bound cysteine; only minor peaks were detected corresponding to Radiolabelling THP-mal-J591c-scFv could be labelled with 98Ga acetate quantitatively within 5 min at room temperature and neutral pH at concentrations ≥ 0.25 μg/μl
We have shown that the THP bifunctional chelator is potentially a solution to the problem that labelling of proteins with gallium-68 typically entails harsh conditions such as high temperature and acidic pH, as well as post-labelling purification, opening the door to kitbased protein labelling with gallium-68
Summary
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. Prostate-specific membrane antigen (PSMA) is a well-established marker for PCa, with elevated expression in virtually all PCa but in high-grade disease [4]. The radiopharmaceutical 111In capromab pendetide (ProstaScint, EUSA Pharma, licenced in the USA since 1997) is a monoclonal antibody (mAb) that targets an internal epitope of PSMA, which limits its utility because of poor accessibility to circulating mAb [5]. The mAb J591 targets an external epitope and has shown more promise as an imaging agent, labelled with 111In for SPECT imaging, 89Zr for PET imaging and 177Lu for targeted radionuclide therapy; no commercial product has emerged yet [6–8]
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