Establishing Micro Physiological Systems by means of a radiolabeled anti-EGFR antibody for the evaluation of new radioligands


Establishing Micro Physiological Systems by means of a radiolabeled anti-EGFR antibody for the evaluation of new radioligands

Sihver, W.; Nitt-Weber, A.-K.; Behrens, S.; Schmieder, F.; Ullrich, M.; Bachmann, M.; Kopka, K.; Pietzsch, H.-J.; Sonntag, F.

Introduction: Potential radiopharmaceuticals are usually evaluated in small animals. Aligned with the 3R principle, animal numbers could be reduced by using “Micro-Physiological Systems (MPS)” which is an organ-on-chip technology. In MPS modules, cultured cells and human organoids can be analysed in a circulatory system under defined conditions [1,2].
In this study, preliminary tests with radiolabeled anti-EGFR antibody cetuximab (C225) were performed using 2D or 3D (spheroid) cultures of A431 cells. The aim was to determine selected pharmacological parameters such as binding affinity of radiolabeled C225 using the MPS.
Methods: 104 A431 cells were placed in the 6-well chamber of the MPS module, either as monolayer or as spheroids (0.8 ± 0.3 mm) and cultured for 24 hours. The conjugate NOTA-C225 was radiolabeled with 68Ga or 64Cu (molar activity: 81.1 ± 14.7 MBq/nmol). An integrated micropump, driven by a controller, generates a pulsatile fluid flow through the fluidic channels upon the cells (Fig.1).
Medium (total binding) and medium with an EGFR blocking C225 concentration of 2 µM (nonspecific binding) (1mL) was pumped through the MPS for 5 min (80 bpm at a volume flow of 6.4 µL/s). Under saturation conditions, 1.2 to 15 nM of radiolabeled C225 were applied to cells and spheroids. Using the micropump, a total volume of 1 mL of the compound solutions were distributed through the MPS over 15 min. After washing with PBS for 10 min, the MPS modules were exposed to an imaging plate, and bound radiolabeled C225 was determined by phosphor imaging. Evaluation was realised with AIDA /GraphPadPrism.
Results/Discussion: We demonstrate that a MPS environment can be employed to determine radioligand binding parameters. The first saturation assays show high binding affinity of the radiolabeled C225 with Kd values in the low nanomolar range (1.7 to 25 nM), being in the range of previous reports [3,4]. Kd of A431 monolayers was similar to that of A431 spheroids. Nonspecific binding on the integrated channels and on empty wells was < 15% of the specifically bound tracer molecule.
Our data provides a rationale to pursue further studies with multi-organ chips using MPS in order to reduce animal numbers used in preclinical radiotracer evaluation. Further steps include trials with kidney- and liver organoids, and an investigation of how radiolabeled C225 is bound, trapped and metabolized using this preclinical model.
Conclusion: Herein, we show that binding parameters of the radiolabeled C225 can be determined in a MPS environment using 2D and 3D cell culture systems. Further studies are required to confirm these data for other EGFR positive and negative cells and for other antibody receptor pairs.
Acknowledgement: W.S. and F.S. acknowledge the financial support by the Federal Ministry of Education and Research of Germany (BMBF) in the project MPS-RP (project number AKZ161L0275A/B).
References: [1] Busek et al., J Sens Sens Syst 2016, 5, 228. [2] Schmieder et al., Proc SPIE 2020, 11268, 1126804_1. [3] Eiblmaier et al., J Nucl Med 2008, 9, 1472. [4] Bellaye et al., Clin Transl Oncol 2018, 12, 1557.

  • Contribution to proceedings
    17th European Molecular Imaging Meeting, EMIM 2022, 15.-18.03.2022, Thessaloniki, Greece
  • Open Access Logo Poster
    17th European Molecular Imaging Meeting, EMIM 2022, 15.-18.03.2022, Thessaloniki, Greece
    DOI: 10.24406/publica-13

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