Aim/Introduction: Broadly neutralizing antibodies (bNAbs) targeting several HIV-1 envelope (Env) epitopes are currently being investigated as a new curative approach against HIV-1 infection. Radiolabeled bNAbs are prospective novel tracers for translational investigation of HIV-1 infection in vivo imaging. In this study, the bNAb VRC07 was conjugated with the versatile macrocyclic ligand p-SCN-Bn-PCTA. The construct was radiolabeled with indium-111 and evaluated in vitro and in vivo in a tumour bearing mice HIV-1 model. Materials and Methods: VRC07 was conjugated with ten equivalents of p-SCN-Bn-PCTA at pH 9.5. The immunoaffinity of PCTA-VRC07 was tested by flow cytometry using HeLa243 cells that stably express the HIV-1 NL4.3 Env and Tat proteins. Chelation of PCTA-VRC07 was obtained with 113 MBq indium-111 for one hour at 37 °C and pH 4.5 in NH4OAc. Immunoreactive fraction assay with 111In-PCTA-VRC07 was done using serial dilution of HIV-1 Env glycoprotein gp120 heptamer coated on microplate. Biodistribution of 111In-PCTA-VRC07 was studied in female BALB/c nude mice bearing xenografts of HeLa243 and control HeLa tumours. Three animals per time point were sacrificed at 24, 48 and 120 hours after injection of 0.5 Mbq 111In-PCTA-VRC07. SPECT/CT scans were acquired at the same time points after injection of 15 Mbq 111In-PCTA-VRC07 in three additional mice. The expression of gp120 was confirmed by immunoblotting ex vivo in the same tumours. Results: Flow cytometry histograms of immunolabeled HeLa243 with PCTA-VRC07 and VRC07 were overlapping showing retained affinity for HIV-1 Env proteins after PCTA conjugation. As shown in SEC-HPLC and iTLC, the radioimmunoconjugate 111In-PCTA-VRC07 was obtained in 99% radiolabeling yield and thus used without purification. An immunoreactive fraction of 111In-PCTA-VRC07 for gp120 greater than 70% was calculated. At 48 hours, 111In-PCTA-VRC07 uptake was significantly higher in the HeLa243 tumours than in HeLa tumours (control), respectively 16.8 ± 1.0 versus 11.7 ± 2.0 % IA/g (t test, P = 0.017, n = 3). At 120 hours, 9.8 ± 2.0 % IA/g of 111In-PCTA-VRC07 was found in the HeLa243 tumour, 10.0 ± 0.9 % IA/g in the liver, 5.1 ± 2.0 % IA/g in the control tumour, 4.3 ± 0.3 % IA/g in the spleen and 3.5 ± 0.4 % IA/g in the blood. SPECT images confirmed the preferential uptake of 111In-PCTA-VRC07 in HeLa243 tumours compared to HeLa control tumours. Conclusion: Using the innovative radioimmunoconjugate 111In-PCTA-VRC07 directed against gp120, in vivo SPECT imaging of HIV-1 Env proteins was possible in a HeLa243 tumour bearing mice model.
In-111-PCTA-VRC07 a Radiolabeled Broadly Neutralizing Antibody For HIV Imaging in Mice
F Cicone;
2019-01-01
Abstract
Aim/Introduction: Broadly neutralizing antibodies (bNAbs) targeting several HIV-1 envelope (Env) epitopes are currently being investigated as a new curative approach against HIV-1 infection. Radiolabeled bNAbs are prospective novel tracers for translational investigation of HIV-1 infection in vivo imaging. In this study, the bNAb VRC07 was conjugated with the versatile macrocyclic ligand p-SCN-Bn-PCTA. The construct was radiolabeled with indium-111 and evaluated in vitro and in vivo in a tumour bearing mice HIV-1 model. Materials and Methods: VRC07 was conjugated with ten equivalents of p-SCN-Bn-PCTA at pH 9.5. The immunoaffinity of PCTA-VRC07 was tested by flow cytometry using HeLa243 cells that stably express the HIV-1 NL4.3 Env and Tat proteins. Chelation of PCTA-VRC07 was obtained with 113 MBq indium-111 for one hour at 37 °C and pH 4.5 in NH4OAc. Immunoreactive fraction assay with 111In-PCTA-VRC07 was done using serial dilution of HIV-1 Env glycoprotein gp120 heptamer coated on microplate. Biodistribution of 111In-PCTA-VRC07 was studied in female BALB/c nude mice bearing xenografts of HeLa243 and control HeLa tumours. Three animals per time point were sacrificed at 24, 48 and 120 hours after injection of 0.5 Mbq 111In-PCTA-VRC07. SPECT/CT scans were acquired at the same time points after injection of 15 Mbq 111In-PCTA-VRC07 in three additional mice. The expression of gp120 was confirmed by immunoblotting ex vivo in the same tumours. Results: Flow cytometry histograms of immunolabeled HeLa243 with PCTA-VRC07 and VRC07 were overlapping showing retained affinity for HIV-1 Env proteins after PCTA conjugation. As shown in SEC-HPLC and iTLC, the radioimmunoconjugate 111In-PCTA-VRC07 was obtained in 99% radiolabeling yield and thus used without purification. An immunoreactive fraction of 111In-PCTA-VRC07 for gp120 greater than 70% was calculated. At 48 hours, 111In-PCTA-VRC07 uptake was significantly higher in the HeLa243 tumours than in HeLa tumours (control), respectively 16.8 ± 1.0 versus 11.7 ± 2.0 % IA/g (t test, P = 0.017, n = 3). At 120 hours, 9.8 ± 2.0 % IA/g of 111In-PCTA-VRC07 was found in the HeLa243 tumour, 10.0 ± 0.9 % IA/g in the liver, 5.1 ± 2.0 % IA/g in the control tumour, 4.3 ± 0.3 % IA/g in the spleen and 3.5 ± 0.4 % IA/g in the blood. SPECT images confirmed the preferential uptake of 111In-PCTA-VRC07 in HeLa243 tumours compared to HeLa control tumours. Conclusion: Using the innovative radioimmunoconjugate 111In-PCTA-VRC07 directed against gp120, in vivo SPECT imaging of HIV-1 Env proteins was possible in a HeLa243 tumour bearing mice model.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
