Background: Cobitolimod (DIMS0150, Kappaproct) is an oligonucleotide that acts as a Toll Like Receptor-9 (TLR-9) agonist and is able to induce clinical remission in patients with active ulcerative colitis after topical administration. To gain further insights into the TLR-9 binding properties we studied the kinetics of the interaction between cobitolimod (GMP/non-GMP grade) and the human TLR-9 receptor using the Attana A200 system.
Methods: The Attana A200® is a dual channel, continuous-flow system for automated analysis based on the Quartz Crystal Microbalance (QCM) technology. To monitor binding, one of the interacting molecules is immobilized on the sensor surface and the sample containing the other molecule is injected over the sensor surface. The signal output is given in frequency (Hz) and is directly related to changes in mass on the sensor surface. TLR-9 was produced using a wheat-germ cell free expression system in the presence of liposomes. Proteoliposomes with TLR-9 or protein mock control were immobilized by adsorption on polystyrene surfaces and cobitolimod was injected in duplicates at different concentrations.
Results: Both GMP and non-GMP grades of cobitolimod bound specifically to the TLR-9 receptor and not to liposomes with control mock protein. Upon subtraction of the reference sensogram, cobitolimod GMP/non-GMP grade presented a 1:1 binding to TLR-9/liposomes. However, stronger binding (lower KD) was detected for cobitolimod GMP grade (KD: 11.8±3.9 μM), compared to cobitolimod non-GMP grade (KD: 24.3±9.9 μM). The calculated kinetics parameters for cobitolimod with GMP grade show medium-fast association rates (9.14±2.78×103 (1/M·s)) and fast dissociation rates (1.08±0.01×10–1 (1/s)).
Conclusions: Using the Attana A200 system the in vitro binding of cobitolimod to the human TLR-9 receptor and its affinity has been demonstrated for the first time. These data provide further support and insights in the TLR-9 mediated activity of cobitolimod, a promising compound in late stage of clinical development for the treatment of active ulcerative colitis.