Ligandtracer

Dr. Carlsson F. is affiliated researcher at the Department of Immunology, Genetics and Pathology and an employee of Corline Biomedical AB, Uppsala that manufacturers Corline Heparin Conjugate. Dr. Buijs J. is affiliated researcher at the Department of Immunology, Genetics and Pathology and an employee and shareholder of Ridgeview Instruments AB, Uppsala that manufactures and sells LigandTracer.

The binding kinetics of radiolabeled DARPins to living SKOV-3 cells was measured using LigandTracer (Ridgeview Instruments, Vänge, Sweden) as described previously [29 (link)]. Kinetics of binding to and dissociation from living cells was recorded at room temperature in real time. The TraceDrawer Software (Ridgeview Instruments, Vänge, Sweden) was used to calculate the affinity based on the association and dissociation rates. Increasing concentrations of each radioconjugate (1, 4, and 8 nM) were added to the cells followed by the change of media and measurements of retention in the dissociation phase.

The real time kinetics of [^99mTc]Tc-(HE)₃-G3 interaction with living SKOV3 cells was measured using a LigandTracer (Ridgeview Instruments, Vänge, Sweden) using the protocol described earlier [37 (link)]. For the measurements, 1.5 × 10⁶ of SKOV-3 cells were seeded on a segment of a cell culture dish one day before measurement. Kinetics of binding to the cells were measured at [^99mTc]Tc-(HE)₃-G3 concentrations 0.5 and 2 nM followed by measurements of dissociation kinetics. The measurements were performed at the room temperature. All measurements were performed in triplicates in the absence of trastuzumab. In another series of experiments (triplicate), trastuzumab (100 nM) was added simultaneously with the addition of [^99mTc]Tc-(HE)₃-G3, and further measurements were performed in the presence of trastuzumab using the same protocol. InteractionMap software (Ridgeview Diagnostics AB, Vänge, Sweden) was used to analyze the data in more detail. An unpaired t-test was used to determine if there is a statistically significant difference between equilibrium dissociation constants of [^99mTc]Tc-(HE)₃-G3 interaction with SKOV-3 cells in the presence or absence of trastuzumab. Analysis of binding of [^99mTc]Tc-ADAPT6 was not performed because the saturation experiments (see above) showed clearly that its interaction with SKOV-3 cells is suppressed by adding trastuzumab.

Binding affinity of [¹⁷⁷Lu]Lu-ABY-027 to HER2-expressing SKOV-3 cells was evaluated in the absence and presence of 100 nM HSA, using LigandTracer (Ridgeview Instruments AB, Uppsala, Sweden). Briefly, SKOV-3 cells were seeded on a local area of a cell culture dish (Nunclon^TM, Size 100 mmNUNC A/S, Roskilde, Denmark). [¹⁷⁷Lu]Lu-ABY-027 with concentrations of 0.25, 0.75, and 2.25 nM was added to the cells to estimate the association rate. The kinetics of association and dissociation were measured in real time, as described in [23 (link)]. To evaluate the effect of trastuzumab on binding of [¹⁷⁷Lu]Lu-ABY-027 to HER2-expressing SKOV-3 cells, 20 µg/mL (135 nM) of trastuzumab was added to the media to mimic the concentration of trastuzumab in the extracellular fluid of mice after injection of clinically used loading dose of trastuzumab (4 mg/kg). The experiment was performed as described above, both in the absence and presence of HSA. The data were analyzed by the InteractionMap software (Ridgeview Diagnostics AB, Uppsala, Sweden) to calculate equilibrium dissociation constant (KD).

Binding to mTfR1 was also investigated after iodine radiolabelling with LigandTracer (Ridgeview Instruments AB, Uppsala, Sweden). A small circular area in a high binding petri dish was coated with 200 nM of mTfR1 (BioArctic AB, Stockholm, Sweden) and incubated overnight at 4 °C. The plate was blocked with 5% BSA for 1 h at room temperature. The blocking buffer was replaced with 3 ml of 0.1% BSA solution and the plate was placed in a LigandTracer Grey instrument (Ridgeview Instruments AB) for a 10 min baseline measurement at room temperature. Two association phases were then measured: first with 2 nM of ¹²⁵I-labelled antibody for 3 h and the second with 6 nM of ¹²⁵I-labelled antibody for 4 h. The association phases were then followed by a dissociation phase overnight.
Binding kinetics were analysed with TraceDrawer software version 1.8.1 (Ridgeview Instruments AB). [¹²⁵I]mAb3D6-scFv8D3 binding curves were analysed with a 1:1 binding model while [¹²⁵I]di-scFv3D6-8D3 binding curves were analysed with a 1:2 binding model.

The binding kinetics of radiolabeled affibody molecules to living BxPC-3 cells was measured using LigandTracer and evaluated using the TraceDrawer Software (all from Ridgeview Instruments, Vänge, Sweden) as described earlier [51 (link)]. Increasing concentrations of radiolabeled affibody molecules (1 and 5 nM) were added to cells, followed by the change of media and measurements of retention in the dissociation phase. Kinetics was recorded at room temperature and dissociation constants were calculated based on association and dissociation rates.