Characterization Of Binding Kinetics Research Articles

Abstract 160: High target binding affinity with long lasting cellular target engagement and high dose intermittent schedule of PI3K inhibitor copanlisib contribute to the potent anti-tumor activity and good safety profile

Abstract Introduction: Several generations of PI3K inhibitors have been tested in clinic. However, thus far, clinical activity has been moderate. Different from other oral PI3K inhibitors dosed continuously, copanlisib (BAY 80-6946) is an intravenous PI3K inhibitor given intermittently to patients. Copanlisib dosed once weekly demonstrated clinical benefit with an improved safety profile, and therefore challenges the concept of default continuous dosing of PI3K inhibitors. However, it is still unclear if this concept can be generalized and whether ‘micropharmacokinetic parameters’ also contributed to the potent anti-tumor profile of copanlisib. Here, we report the characterization of binding kinetics for copanlisib, as well as the functional consequence in vivo. Methods: A set of PI3K inhibitors were characterized in 1) a kinetic probe competition assay (kPCA); 2) a cellular nanoBRET target engagement assay; 3) a cellular washout study with the assessment on pathway engagement; and 4) in vivo pharmacokinetics analysis. Results: Copanlisib showed nearly diffusion-controlled on- and relatively slow off-rates with kon = 3.45E+7 [M-1*s-1] and koff = 1.67E-3 [s-1] to PI3Kα. Consequently, it exhibited very high affinity to PI3Kα ( Ki ePCA = 9.31E-11[M] and KD kPCA = 4.77E-11 [M]). In a cellular nanoBRET target engagement assay, the apparent half-life (t1/2) of ca. 2 hours greatly surpassed the 6.9 min measured using kPCA. The high affinity to PI3Kα also translated into potent cellular pathway engagement demonstrated by inhibition of p-AKT and p-PRAS40 in the PIK3CAmut KPL4 cell line. In a cellular washout study, p-AKT and p-PRAS40 were assessed till 168 h after incubation with copanlisib for 1 h followed by a washout step. A dose- and time-dependent pathway engagement was observed even at 72 h post washout. This result indicated that in cells, copanlisib engages PI3Kα for an extremely long time, likely due to rebinding effects facilitated by the fast equilibration kinetics of the compound and its micropharmacokinetic properties. Interestingly, in vivo, BAY 80-6946 levels were approximately 100-fold higher in the tumor than in plasma at 48 hours and drug clearance from the tumor occurred more slowly than from plasma. This high and prolonged tumor exposure might be explained, at least in part, by the high expression of PI3Kα and long lasting target occupancy of copanlisib in tumors. Conclusion: Copanlisib demonstrated high affinity to PI3Kα with protracted target engagement at cellular and in vivo levels. This ‘micropharmacokinetic feature’ not only supports intermittent dosing but likely also explains the high exposure in tumors vs plasma, potent anti-tumor activity and good safety profiles. Citation Format: Amaury E. Fernández-Montalván, Victoria Georgi, James Vasta, Sarah Glaeske, Vera Puetter, Matthew B. Robers, Ursula Moenning, Andrea Sturz, Julien Lefranc, Karl Ziegelbauer, Michael Brands, Christian Stegmann, William J. Scott, Ningshu Liu. High target binding affinity with long lasting cellular target engagement and high dose intermittent schedule of PI3K inhibitor copanlisib contribute to the potent anti-tumor activity and good safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 160. doi:10.1158/1538-7445.AM2017-160

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50ms to 4h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100ms with the solid-phase surface.