Nawaz Saima, Mullen Gregory E D, Sunassee Kavitha, Bordoloi Jayanta, Blower Philip J, Ballinger James R
Division of Imaging Sciences and Biomedical Engineering, King's College London, St Thomas' Hospital, London, UK.
Department of Nuclear Medicine, Guy's and St Thomas' Hospital, London, UK.
EJNMMI Res. 2017 Oct 25;7(1):86. doi: 10.1186/s13550-017-0336-6.
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 Ge/Ga 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. Ga-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.
使用双功能螯合剂用镓-68标记蛋白质通常存在问题，因为标记条件如低pH或高温过于苛刻，可能需要标记后纯化。为了确定三（羟基吡啶酮）（THP）双功能螯合剂是否能解决这个问题，我们评估了一种THP偶联物与一种针对前列腺特异性膜抗原（PSMA）的新型单链抗体的标记和生物分布，PSMA是前列腺癌（PCa）分期的一个有吸引力的靶点。制备了识别PSMA外部表位的单克隆抗体J591的单链可变片段（scFv），以实现与镓-68半衰期相匹配的生物动力学。对scFv J591c-scFv进行工程改造，使其C端带有半胱氨酸。
J591c-scFv在HEK293T细胞中产生，并通过尺寸排阻色谱法纯化。将马来酰亚胺THP衍生物（THP-mal）位点特异性地偶联到C端半胱氨酸残基上。THP-mal-J591c-scFv偶联物在室温及中性pH条件下，用来自锗/镓发生器的醋酸铵缓冲镓-68进行标记。在PCa细胞系DU145及其过表达PSMA的变体中对标记的偶联物进行体外评估，并将其接种到SCID小鼠体内形成异种移植瘤。J591c-scFv的产量为每升培养上清液4 - 6毫克，并能有效地与THP-mal双功能螯合剂偶联。5μg偶联物与镓-68孵育5分钟后，在室温下无需标记后纯化即可实现>95%的标记率。Ga-THP-mal-J591c-scFv在血清中稳定，对DU145-PSMA细胞系具有选择性结合，未修饰的J591c-scFv的IC50值为31.5 nM。连续PET/CT成像显示通过肾脏排泄实现快速、特异性的肿瘤摄取和清除。注射后90分钟，DU145-PSMA异种移植瘤中的蓄积为5.4±0.5%ID/g，而DU145肿瘤中的蓄积为0.5±0.2%ID/g（n = 4）。
双功能螯合剂THP-mal能够在中性pH条件下于室温进行简单、快速、定量的一步法用镓-68对蛋白质进行放射性标记，无需标记后纯化。所得的镓-68复合物在PCa异种移植模型中对PSMA具有高亲和力和良好的体内靶向特性。
