Assessing the binding affinity of a 64Cu-labeled PSMA ligand in a micro physiological system

Introduction:

Radiolabeled prostate-specific membrane antigen (PSMA) ligands have been successfully developed in recent years for diagnosis and therapy of prostate cancer [1]. A potential new PSMA radioligand would, after in vitro characterization, normally be further evaluated in small animals. However, the use of micro-physiological systems (MPS), an organ-on-chip technology in which human cell spheroids and organoids are treated in a circuit under defined conditions [2,3], should lead to a reduction of animal experiments.
In the present study, experiments with the novel 64Cu-labeled PSMA antagonist FR52 were performed using 2D or 3D cultures of LNCaP cells in MPS chips. Our aim was to determine binding parameters (Kd, Bmax) in the MPS chip and to compare them with the results of assays in well plates (WP).
Methods:
Derived from the compound PSMA-617 [4], instead of DOTA a cyclohexanediamine-triazole (CHDT) chelator was conjugated, resulting in the conjugate FR52 (in-house synthesis). FR52 was radiolabeled with 64Cu (molar activity: 14.2 ± 2.2 MBq/nmol).
For the assays, 5 x 104 LNCaP cells were placed in 3 wells of an MPS module (à 0.2 cm2), either as monolayer or as spheroids and cultured for 2 to 7 days. The MPS chips include an integrated micropump, driven by a controller, that generates a pulsatile flow of media that runs through the capillaries and keeps the cells alive [5] (Fig. 1).
After blocking of adjacent cell samples (PSMA pharmacophore 0.8 mM; 30 min) [64Cu]Cu-FR52 (6 to 200 nM) was added to all samples and pumped through the MPS for 10 min (volume: 0.1 mL/well; 80 bpm; volume flow 6.4 µL/s). After PBS washing and exposing the MPS chips to an imaging plate, the cells were lysed and in vials measured in a gamma counter. Evaluation was processed with the analysis program GraphPad Prism.
Results:
Saturation assays with [64Cu]Cu-FR52 in MPS revealed similar high binding affinities (Kd) for LNCaP monolayer and spheroids (MPS-monolayer Kd: 33 ± 6 nM; MPS-spheroids Kd: 36 ± 8 nM). Also the binding capacities (Bmax) were in the same range (MPS-monolayer Bmax: 0.57 ± 0.04 pmol/mg; MPS-spheroids Bmax: 0.35 ± 0.03 pmol/mg). The binding affinity to LNCaP spheroids in WP was similar to that in MPS, but the binding capacity was slightly lower than in the chip experiments (WP-spheroids Kd: 59 ± 6 nM; WP-spheroids Bmax: 0.22 ± 0.01 pmol/mg). LNCaP monolayer treatment in WP was performed in previous experiments with 18F, 68Ga or 111In labeled FR52. Those Kd values were slightly lower (WP-monolayer Kd: 18 ± 2 nM) and the Bmax values were slightly higher (WP-monolayer Bmax: 0.7 to 3.5 pmol/mg).
Conclusions:
The successful evaluation of the binding parameters of the 64Cu-labeled PSMA antagonist FR52 using MPS is a promising step to foster the reduction of animal experiments in preclinical evaluation. Moreover [64Cu]Cu-FR52 seems to be a promising candidate. Nevertheless, further experiments with kidney and liver organoids in MPS should investigate first evidence for uptake, metabolism and excretion of [64Cu]Cu-FR52.
Acknowledgements:
The authors like to express great appreciation for the financial support from the German Federal Ministry of Education and Research (BMBF) (Funding number 161L0275A/B).
References:
[1] Neels et al., Cancers 2021, 13, 6255 [2] Busek et al., J Sens Sens Syst 2016, 5, 228. [3] Schmieder et al., Proc SPIE 2020, 11268, 1126804_1. [4] Benešová et al., J Nucl Med 2015, 56, 914. [5] Sihver et al., Curr Dir Biomed Eng 2022, 8, 532.