Dataset Information

Manipulating the In Vivo Behaviour of 68Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

ABSTRACT: Pretargeting is widely explored in immunoPET as a strategy to reduce radiation exposure of non-target organs and allow the use of short-lived radionuclides that would not otherwise be compatible with the slow pharmacokinetic profiles of antibodies. Here we investigate a pretargeting strategy based on gallium-68 and the chelator THPMe as a high-affinity pair capable of combining in vivo. After confirming the ability of THPMe to bind 68Ga in vivo at low concentrations, the bifunctional THPMe-NCS was conjugated to a humanised huA33 antibody targeting the A33 glycoprotein. Imaging experiments performed in nude mice bearing A33-positive SW1222 colorectal cancer xenografts compared pretargeting (100 ?g of THPMe-NCS-huA33, followed after 24 h by 8-10 MBq of 68Ga3+) with both a directly labelled radioimmunoconjugate (89Zr-DFO-NCS-huA33, 88 ?g, 7 MBq) and a 68Ga-only negative control (8-10 MBq of 68Ga3+). Imaging was performed 25 h after antibody administration (1 h after 68Ga3+ administration for negative control). No difference between pretargeting and the negative control was observed, suggesting that pretargeting via metal chelation is not feasible using this model. However, significant accumulation of "unchelated" 68Ga3+ in the tumour was found (12.9 %ID/g) even without prior administration of THPMe-NCS-huA33, though tumour-to-background contrast was impaired by residual activity in the blood. Therefore, the 68Ga-only experiment was repeated using THPMe (20 ?g, 1 h after 68Ga3+ administration) to clear circulating 68Ga3+, producing a three-fold improvement of the tumour-to-blood activity concentration ratio. Although preliminary, these results highlight the potential of THPMe as a 68Ga clearing agent in imaging applications with gallium citrate.

SUBMITTER: Imberti C

PROVIDER: S-EPMC7073083 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Manipulating the In Vivo Behaviour of 68Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

Imberti Cinzia C Adumeau Pierre P Blower Julia E JE Al Salemee Fahad F Baguña Torres Julia J Lewis Jason S JS Zeglis Brian M BM Terry Samantha Y A SYA Blower Philip J PJ

International journal of molecular sciences 20200222 4

Pretargeting is widely explored in immunoPET as a strategy to reduce radiation exposure of non-target organs and allow the use of short-lived radionuclides that would not otherwise be compatible with the slow pharmacokinetic profiles of antibodies. Here we investigate a pretargeting strategy based on gallium-68 and the chelator THPMe as a high-affinity pair capable of combining in vivo. After confirming the ability of THPMe to bind 68Ga in vivo at low concentrati ...[more]

PMID: 32098299

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Project description: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.

Project description:BACKGROUND:68Ga-labelled peptides targeting somatostatin receptor 2 (SSTR2) have demonstrated encouraging results in managing patients with neuroendocrine tumours (NETs). In addition to metal chelation, bifunctional chelators have also been found to impact imaging outcomes due to their differences in stability, charge, hydrophilicity, etc. In the present work, a comparative pharmacokinetic evaluation and imaging characteristics were performed between 68Ga-labelled somatostatin analogues (TATE) using NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as bifunctional chelating agents (BFCAs). RESULTS:Both 68Ga-NOTA-TATE and 68Ga-DOTA-TATE were obtained with high radiochemical purity. 68Ga-NOTA-TATE demonstrated higher in vitro stability (≥ 99%) than 68Ga-DOTA-TATE (≥ 95%) after 3 h of incubation. The water solubilities (partition coefficients, - 1.76 ± 0.06 vs. - 2.72 ± 0.16) and plasma protein binding rates (12.12% vs. 30.6%) were lower for 68Ga-NOTA-TATE than for 68Ga-DOTA-TATE. Differential pharmacokinetics and comparable tumour affinities (within 1 h) were observed in AR42J tumour-bearing mice. Healthy volunteer imaging studies showed comparable distribution patterns of these two imaging agents. However, the maximum standardized uptake values (SUVmax) of the two tracers varied in each organ. The two PET agents demonstrated almost identical SUVmax values in the kidneys. 68Ga-NOTA-TATE did have a lower SUVmax in most other organs compared with 68Ga-DOTA-TATE, including the liver (4.2 vs. 10.1), potentially due to the lower protein binding rate. CONCLUSION:68Ga-NOTA-TATE and 68Ga-DOTA-TATE demonstrated comparable tumour uptake in an AR42J mouse model. An initial clinical study revealed that 68Ga-NOTA-TATE may have reduced background uptake in the major organs such as the liver. Although the subject numbers were limited, further investigation of 68Ga-NOTA-TATE is warranted for detecting SSTR2-positive neuroendocrine tumours.

Project description:Due to its long half-life (78 h) and decay properties (77% electron capture, 23% ?(+), Emax = 897 keV, Eav = 397 keV, E? = 909 keV, I? = 100%) (89)Zr is an appealing radionuclide for immunoPET imaging with whole IgG antibodies. Derivatives of the siderophore desferrioxamine-B (H3DFO) are the most widely used bifunctional chelators for coordination of (89)Zr(4+) because the radiolabeling of the resulting immunoconjugates is rapid under mild conditions. (89)Zr-DFO complexes are reportedly stable in vitro but there is evidence that (89)Zr(4+) is released in vivo, and subsequently taken up by the skeleton. We have evaluated a novel tripodal tris(hydroxypyridinone) chelator, H3CP256 and its bifunctional maleimide derivative, H3YM103, for coordination of Zr(4+) and compared the NMR spectra, and the (89)Zr(4+) radiolabeling, antibody conjugation, serum stability and in vivo distribution of radiolabelled immunoconjugates with those of H3DFO and its analogues. H3CP256 coordinates (89)Zr(4+) at carrier-free concentrations forming [(89)Zr(CP256)](+). Both H3DFO and H3CP256 were efficiently radiolabelled using [(89)Zr(C2O4)4](4-) at ambient temperature in quantitative yield at pH 6-7 at millimolar concentrations of chelator. Competition experiments demonstrate that (89)Zr(4+) dissociates from [(89)Zr(DFO)](+) in the presence of one equivalent of H3CP256 (relative to H3DFO) at pH 6-7, resulting largely in [(89)Zr(CP256)](+). To assess the stability of H3DFO and H3YM103 immunoconjugates radiolabelled with (89)Zr, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Both immunoconjugates were labelled with (89)Zr(4+) in >98% yield at high specific activities and the labeled immunoconjugates were stable in serum with respect to dissociation of the radiometal. In vivo studies in mice indicate that (89)Zr(4+) dissociates from YM103-trastuzumab with significant amounts of activity becoming associated with bones and joints (25.88 ± 0.58% ID g(-1) 7 days post-injection). In contrast, <8% ID g(-1) of (89)Zr activity becomes associated with bone in animals administered (89)Zr-DFO-trastuzumab over the course of 7 days. The tris(hydroxypyridinone) chelator, H3CP256, coordinates (89)Zr(4+) rapidly under mild conditions, but the (89)Zr-labelled immunoconjugate, (89)Zr-YM103-trastuzumab was observed to release appreciable amounts of (89)Zr(4+)in vivo, demonstrating inferior stability when compared with (89)Zr-DFO-trastuzumab. The significantly lower in vivo stability is likely to be a result of lower kinetic stability of the Zr(4+) tris(hydroxypyridinone complex) relative to that of DFO and its derivatives.

Dataset Information

Manipulating the In Vivo Behaviour of 68Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

Publications

Manipulating the In Vivo Behaviour of <sup>68</sup>Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

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