A rapid bead-based radioligand binding assay for the determination of target-binding fraction and quality control of radiopharmaceuticals

Abstract

Introduction: Determination of the target-binding fraction (TBF) of radiopharmaceuticals using cell-based assays is prone to inconsistencies arising from several intrinsic and extrinsic factors. Here, we report a cell-free quantitative method of analysis to determine the TBF of radioligands.

Methods: Magnetic beads functionalized with Ni-NTA or streptavidin were incubated with 1 μg of histidine-tagged or biotinylated antigen of choice for 15 min, followed by incubating 1 ng of the radioligand for 30 min. The beads, supernatant and wash fractions were measured for radioactivity on a gamma counter. The TBF was determined by quantifying the percentage of activity associated with the magnetic beads.

Results: The described method works robustly with a variety of radioisotopes and class of molecules used as radioligands. The entire assay can be completed within 2 h.

Conclusion: The described method yields results in a rapid and reliable manner whilst improving and extending the scope of previously described bead-based radioimmunoassays.

Advances in knowledge: Using a bead-based radioligand binding assay overcomes the limitations of traditional cell-based assays. The described method is applicable to antibody as well as non-antibody based radioligands and is independent of the effect of target antigen density on cells, the choice of radioisotope used for synthesis of the radioligand and the temperature at which the assay is performed.

Implications for patient care: The bead-based radioligand binding assay is significantly easier to perform and is ideally suited for adoption by the radiopharmacy as a quality control method of analysis to fulfill the criteria for release of radiopharmaceuticals in the clinic. The use of this assay is likely to ensure a more reliable validation of radiopharmaceutical quality and result in fewer failed doses, which could ultimately translate to an efficient release of radiopharmaceuticals for administration to patients in the clinic.

Keywords: Immunoreactivity; Lindmo assay; Magnetic beads; Target-binding fraction.

Figure 1.. Overview of the bead-based radioligand binding assay.

The first step of the assay involves preparation of the antigen-coated magnetic beads. The second step allows the radioligand to bind with the target antigen on the beads. The final step isolates the beads from the supernatant and washes to quantify the target-binding fraction of the radioligand.

Figure 2.. Applicability of bead-based radioligand binding assay for targets having low versus high cell-surface abundance.

A) Inconsistent results obtained from cell-based assays to determine the target-binding fraction (TBF) of [⁸⁹Zr]Zr-DFO-SC16.56 (molar activity = 14 MBq/nmol) at end of synthesis (EOS) and 24 h after storage of the sample at −80 °C in 4 consecutive experimental trials done over a period of 4 weeks; B) Demonstration of high TBF for [⁸⁹Zr]Zr-DFO-SC16.56 using Ni-NTA beads coated with His-tagged DLL3 and minimum non-specific binding (NSB) to naked Ni-NTA beads at (EOS) and 24 h later; C) High TBF demonstrated by [¹⁷⁷Lu]Lu-CHX-A”-DTPA-SC16.56 (molar activity = 22.4 MBq/nmol) – a radioimmunotherapeutic variant of the DLL3-targeting antibody. Specificity of binding to antigen-coated beads for both the DLL3-targeting radioligands was validated in the presence of a 3500-fold excess of unlabeled SC16.56 immunoconjugates; D) Evaluation of the TBF of [⁸⁹Zr]Zr-DFO-Trastuzumab (molar activity = 14 MBq/nmol) using Ni-NTA beads coated with His-tagged Her2 protein showed ~25% NSB of the radioligand to naked Ni-NTA beads; E) Using streptavidin beads coated with biotinylated Her2 to evaluate the TBF of [⁸⁹Zr]Zr-DFO-Trastuzumab eliminated the NSB and demonstrated high specific binding, which could be blocked by excess unlabeled DFO-Trastuzumab, but not DFO-Pertuzumab; F) Using ¹³¹I-labeled biotinylated Her2 to determine the degree of loading of the streptavidin beads demonstrated linearity with increasing concentration of the antigen over at least 4 orders of magnitude. The error bars on each data point represent 95% CI. The plot validates that the bead-based assay is being operated in the pre-saturation concentration range when 1 μg (10 pmoles) of biotinylated Her2 was used with 20 μL (200 μg) of streptavidin beads.

Figure 3.. Comparative analysis of the performance of cell-based radioligand binding assay and the bead-based assay.

A) Poor and progressively decreasing immunoreactive fractions obtained from Lindmo assay of [¹³¹I]I-Omburtamab (molar activity = 392 MBq/nmol) with B7-H3-expressing LAN-1 cells at EOS, 48 h, 72 h and 76 h after radiosynthesis (radioligand samples/aliquots stored at −80 °C; B) High TBFs obtained for the same preparation of [¹³¹I]I-Omburtamab with His-tagged B7-H3 in a bead-based assay performed at EOS and 76 h after storage of the sample at −80 °C; C-D) Bar graphs showing a comparative analysis of the means from multiple experiments to evaluate the performance of cell-based radioligand binding assay (RBA) versus bead-based assay (BBA); E) Summarized statistics for the two methods of analyses showing superior intermediate precision obtained from use of BBA (%CV <5; F-test of equality of variances p-value <0.005).

Figure 4.. Applicability of the bead-based assay to evaluate the TBF of radioimmunoconjugates that bind shed antigens and small molecule radioligands.

A) High TBF of [⁸⁹Zr]Zr-DFO-5B1 (molar activity =13 MBq/nmol) with polyvalent biotinylated CA19.9 antigen; C) Demonstration of the utility of the bead-based assay with small molecule radioligand – [¹⁷⁷Lu]Lu-PSMA-617 (molar activity = 2.6 MBq/nmol) showing high (~80%) TBF and minimum NSB at 24 h post radiosynthesis.