Characterization of the receptor binding kinetics of PSMA-specific peptides by Surface Plasmon Resonance Spectroscopy

1128 Objectives: bjectives Targeting the prostate-specific membrane antigen (PSMA) is a highly promising diagnostic and therapeutic approach. Pharmacokinetic data of the targeting process are highly relevant for the development and the optimization of new ligands as well as for the improvement of individualized molecular radiotherapy by adaption of Physiologically-based pharmacokinetic (PBPK) models. Binding characteristics of PSMA-specific peptides are often obtained by cell assays at non-physiological conditions unsuitable to determine kinetics parameters, e.g. association rate (kon) and dissociation rate (koff). With surface plasmon resonance (SPR) spectroscopy a reliable cell-free tool is available to determine these kinetic rates and the resulting equilibrium dissociation constant (KD). Determination and comparison at physiological conditions of these essential data for peptide development and optimization as well as improvement of individualized treatment was aim of the presented studies. Materials and Methods Using a Biacore X100 (GE Healthcare, Freiburg, Germany) the PSMA protein (R&D Systems) was immobilized on the surface of various CM5 sensor chips (GE Healthcare). Concentration ranges of 0.1-fold to 10-fold of the expected KD of the peptides of interests, PSMA-11, (Ga)PSMA-11, PSMA-617 and (Ga)PSMA-617 (ABX, Radeberg, Germany) was applied in ascending order performing the device-specific single-cycle kinetics program. All measurements were performed at least in triplicate and the sensor chips were regenerated and reused multiple times. Using the Biacore evaluation software by assumption of an 1:1 interaction model the obtained data were analyzed and statistically evaluated using the Kruskal-Wallis H test and Mann-Whitney U test (p<0.05 was assumed significant). Results Kinetic parameters were in the range of kon: (2.0 x 106 to 5.5 x 106) (Ms)-1 and koff: (5.4 x 10-5 to 4.2 x 10-4) s-1. KD calculation for PSMA-11 and GaPSMA-11 resulted in (0.07±0.02) nM and (0.04±0.02) nM at 25°C, and (0.07±0.04) nM and (0.10±0.08) nM at 37°C. For PSMA-617 (0.01±0.01) nM at 25°C and (0.1±0.06) nM at 37°C were determined. No significant differences were observed for PSMA-11 and GaPSMA at 37°C and 25°C, while at these temperatures the KD of PSMA-617 differed significantly. Conclusions: The highly reproducible characterization of binding kinetic parameters at physiological conditions by SPR spectroscopy is of eminent value for the development and optimization of peptides. Knowledge of precise kinetic rates will improve individualized treatment planning of molecular radiotherapy by implementation in a physiologically-based pharmacokinetic (PBPK) model.