P-487. [18F]-labeled J3 Nanobody is a Specific PET Tracer for Molecular Imaging of HIV-1 gp120

Abstract Background Targeted molecular imaging of HIV-1 can potentially identify viral reservoirs and provide a better understanding of disease pathogenesis and management. Nanobody-based radiolabeled probes offer several advantages over full antibodies, including smaller size, deep tissue penetration, and high stability. In this study, three fluorine-18 labeled nanobodies targeting the well-conserved CD4-binding site on HIV-1 envelope protein (gp120) were evaluated in gp120-expressing flank tumor models. Methods Binding affinities to gp120 protein were determined by bio-layer interferometry. For in vivo PET imaging, an animal model with wild type (WT) and/or gp120-expressing HEK293T cells (gp120+) flank tumors was developed in nude mice. Radiolabeled nanobodies were prepared by an indirect labeling method using 6-fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) prosthetic group. Dynamic PET/CT images up to 60 minutes and static images at 120 minutes were obtained. Tumors were evaluated ex vivo using immunofluorescent staining. Unpaired t test was used for statistical analysis. Results J3 nanobody showed higher binding affinity to gp120 compared to 3E3 and B9, with KD values of 1.1nM, 3.4nM, and 23.7nM, respectively. PET/CT imaging revealed selective binding (measured as SUVpeak) to gp120+ compared to WT tumors with J3 (0.64 ± 0.14 vs. 0.29 ± 0.08) and 3E3 (0.61 ± 0.27 vs. 0.22 ± 0.12) but not with B9 (0.21 ± 0.14 vs 0.25 ± 0.12). After 2 hours, J3 and 3E3 had minimal background signal, whereas B9 showed poor ligand clearance and off target binding (Fig. 1). [18F]J3 time activity curves (TACs) from dynamic and static scans showed selective binding to gp120+ tumors compared to WT tumors (Fig. 2). To validate our PET findings, ex vivo gp120 tumor expression was confirmed by immunofluorescence staining. J3 nanobody showed specific immunoreactivity that colocalized with VRC01, an anti-gp120 full antibody (Fig. 3). Conclusion We evaluated the binding characteristics of three 18F-labeled nanobodies to gp120+ cells. Overall, J3 nanobody had higher specificity and binding to its target compared to 3E3 and B9, demonstrating a higher potential for non-invasive identification of HIV-infected cells in infected models. Disclosures All Authors: No reported disclosures