In vitro response of ¹⁷⁷Lu-PSMA-617 with two different specific activities

Introduction: PSMA-617 radiolabeled with lutetium-177 has shown good results in compassionate studies around the world. Being a receptor-specific radiopharmaceutical, the specific activity (SA) of the preparation may represent an important factor for therapeutic efficacy. Lutetium-177 can be produced by two different routes: with ytterbium-176 (Non-carrier-added or NCA) and with lutetium-176 (Carrier-added or CA). The SA (MBq/ug) of the labeled PSMA varies accordingly to each lutetium. For NCA lutetium, the radiolabeling procedure is based on the SA of 74 MBq/ug. When the radiolabeling is performed with CA lutetium, SA is determined by the molar ratio of 2.1:1 (PSMA moles/lutetium moles declared in the certificate), resulting in lower SA than NCA. This work evaluated the influence of specific activity of 177Lu-PSMA-617 on in vitro specific binding assays (saturation, competition and internalization). Materials and Methods: Radiolabeling of PSMA-617 (ABX, Germany) with lutetium-177 was performed in heating block at 90°C for 30 minutes with sodium ascorbate (0.5 M pH 4.7) as buffer. For NCA lutetium (JSC, Russia) the radiopharmaceutical specific activity was 74 MBq/ug. For CA lutetium (IDB, Netherlands), the specific activity was 41 MBq/ug. The radiochemical purity was analyzed with HPLC. For all experiments, 6-well plates were used for adherence cells with 200,000 LNCaP per well. Molar concentration of saturation curves experiments were 0.01; 0.05; 0.6; 1.5; 3.0 and 3.5 for CA lutetium and 0.1; 0.6; 1.5; 2.0; 2.5 and 3.0 for NCA lutetium. After 1 hour of incubation at 8 ºC, supernatant was removed, then washed with PBS (phosphate buffer saline) and finally cells were burst with NaOH 1 M, and activity was measured in gama counter; the experiments were performed in octuplicate. Competition experiments were performed adding in all wells 5 nM of radiolabeled PSMA-617 and in the competition well (non-specific binding) were added an excess of 15 times (76 ug) of non radiolabeled PSMA-617. After 1 hour of incubation at 8 ºC, supernatant was removed, then washed with PBS and finally cells were burst with NaOH 1 M, and activity was measured in gama counter, these experiments were performed in triplicate. The specific binding was obtained by the difference between total binding and non-specific binding. Internalization experiments were performed at Kd of NCA and CA lutetium. After 1 hour of incubation at 37 ºC, supernatant was removed, washed with PBS, then washed again with 0.05 M glycine solution pH 2.8 and finally cells were burst with NaOH 1 M. Activity was measured in gama counter, these experiment were performed in sextuplicate. Results and discussion: The radiochemical purity were 98% and 99% for labeling with NCA and CA lutetium, respectively. Saturation curve assay with NCA lutetium shown a Kd of 0.7 ± 0.15 nM and a Bmax of 857 ± 55.79 pMol/ng, and with CA lutetium resulted in a Kd of 1.71 ± 0.45 nM and a Bmax of 1156 ± 113.8 pMol/ng. The variation between both Kd curves were statistically different (P value = 0.0058). Competition assay demonstrated an effective blocking for both types of lutetium, for NCA unpaired T test shown a P value of 0.0011. For CA lutetium, the unpaired test disclosed a P value of 0.0258. The comparison between both results revealed a P value of 0.01 at the specific binding. Internalization assay shown for both types of lutetium similar results, 27.1 ± 2.45% and 30.6 ± 4.97%, for CA and NCA lutetium, respectively, and was not statistically significant (P value = 0.17)

Conclusions: These experiments demonstrated that the type of lutetium (CA or NCA) directly affects in vitro binding of ¹⁷⁷Lu-PSMA-617 to receptors expressed in LNCaP cells. It was statistically demonstrated that the higher specific activity of ¹⁷⁷Lu-PSMA-617, more radiolabeled peptide can bind to cells at saturation and competition assays. Acknowledgments: Grant 2018/12965-4, São Paulo Research Foundation (FAPESP) and National Commission of Nuclear Energy.