Comparative Evaluation of Novel 177Lu-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting

Simple Summary Affibody molecules are small, engineered affinity proteins based on a nonimmunoglobulin scaffold. Affibody-based radionuclide imaging probes have demonstrated excellent tumor targeting. However, the renal clearance of affibody molecules is accompanied by high reabsorption and retention of activity in the kidney, which prevents their use for radionuclide therapy. We have previously shown the feasibility of overcoming the high renal uptake using a pretargeting approach for affibody-mediated therapy based on peptide nucleic acid (PNA) hybridization. In this study, we test the hypothesis that shortening the PNA pretargeting probes would further increase the difference between the accumulation of radiometals in tumor xenografts and in kidneys. A series of novel PNA probes has been designed and evaluated in vitro and in vivo. We have found that a variant containing 9 nucleobases enables a two-fold increase of the tumor-to-kidney dose ratio compared with a variant containing 15 nucleobases. This creates preconditions for more efficient therapy of cancer. Abstract Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe ZHER2:342-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with 177Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [177Lu]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all 177Lu-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe’s size and decreased with an increased number of nucleobases. The shortest PNA probe, [177Lu]Lu-HP16, showed the highest tumor-to-kidney ratio. [177Lu]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.

. Analytical RP-HPLC chromatograms of the purified PNA probes HP16, HP17 and HP18 monitored at 220 nm (green line) and 260 nm (blue line). Peaks at 220 nm after 20 min (at 100% B: 0.1% TFA in CH3CN) were found also in blank injections.

Characterization of the affibody-PNA conjugate and the complementary PNA probes
In order to verify simultaneous binding of ZHER2:342-SR-HP15 to HP18 and the HER2 receptor, HER2-Fc (Sino Biological) was immobilized to a dextran chip Series S Sensor CM5 at 757 RU. PNA probe HP18 and ZHER2:342-SR-HP15 were mixed at a 1:1 concentration ratio to allow for hybridization prior to injection to HER2-Fc. Pre-hybridized ZHER2:342-SR-HP15:HP18 were injected at 6 concentrations; 0.78, 1.56, 3.13, 6.25, 12.5 and 25 nM ( Figure  S9). Association was allowed for 300 s and dissociation was allowed for 2400 s (40 min), followed by regeneration by injection of 20 mM HCl for 20 s. For the highest injected concentration (25 nM), a dissociation time of 7200 s (2 h) was used. All runs were performed in PBST (0.05 % Tween-20) pH 7.4 using a flow rate of 50 µl/min at 25 °C. After regeneration with HCl, running buffer was passed over the sensor surface for 2 h before injection of the next sample.
The equilibrium dissociation constant (KD) for ZHER2:342-SR-HP15:HP18 binding to HER2-Fc was calculated to 276 pM, with an association rate constant (ka) of 2.5 x 10 6 M -1 s -1 and a dissociation rate constant (kd) of 7.0 x 10 -4 s -1 . This compares well with previously reported kinetic constants for ZHER2:342-SR-HP1:HP2 binding to HER2, where KD was estimated to 212 pM, with the association rate constant ka= 1.4 x 10 6 M -1 s -1 and the dissociation rate constant kd=2.9 x 10 -4 s -1 . 1 The KD for the PNA-conjugated affibodies is approximately 10-fold higher than the KD for unmodified ZHER2:342, which has been reported to be 22 pM. 2 This supports the idea that PNA conjugation only marginally interferes with ZHER2:342 binding to HER2. CD spectroscopy (Chirascan, Applied Photophysics) was used to investigate the secondary structure of the PNA:PNA hybridization complexes. CD signal was recorded at wavelengths ranging from 195 to 300 nm. All CD spectra were recorded at a protein/PNA concentration of 0.15-0.2 mg/ml in 20 mM potassium phosphate buffer with 100 mM KCl at pH 7.4.
The hybridization between HP15 and the secondary probes H16, HP17 or HP18 gave rise to CD spectra with minima at approximately 215 nm and 260 nm ( Figure S10). The induced signals are the result of PNA:PNA double helix formation upon hybridization between complementary PNA probes carrying C-terminal L-amino acids.     Table S1. Apparent equilibrium dissociation (KD) constants for the interaction between [ 177 Lu]-Lu-PNA probes and HER2expressing SKOV3 cells determined using an Interaction Map analysis of the LigandTracer sensorgrams.

Cellular processing and retention
Cells were seeded in cell-culture dishes with a density of 10 6 cells/dish for all experiments. A set of three dishes was used for each data point.
To study processing of the primary probe, cells were incubated with [ 177 Lu]Lu-ZHER2:342-SR-HP15 (1 nM) for 1 h at 4 °C. The medium was removed, the cells were washed on ice, new medium was added and the cells were placed in a humidified incubator at 37 °C. At 1, 2, 4, 8 and 24 h, the medium was collected, cells were washed and treated with 0.2 M glycine buffer containing 4 M urea, pH 2.0, for 5 min on ice. The acidic solution was collected and cells were additionally washed with 1 mL glycine buffer. The cells were then incubated with 1 mL of 1 M NaOH at 37 °C for 10 min and collected with 1 mL of 1 M NaOH. The radioactivity in acidic fractions was considered as membrane-bound, and in the alkaline fractions as internalized.
For cellular processing and retention of [ 177 Lu]Lu-secondary probes by SKOV3 and BT474, cells were incubated with ZHER2:342-SR-HP15 (1 nM) for 1 h at 4 °C, then the medium was removed, the cells were washed on ice with cold medium, [ 177 Lu]Lu-secondary probe (10 nM in cold medium) was added, and the cells were incubated for 30 min at 4 °C. Then the medium was removed, the cells were washed on ice, new cold medium was added and the cells were placed in a humidified incubator at 37 °C. At 1, 2, 4, 8 and 24 h, a group of three dishes was removed from the incubator and treated as described above.