Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are molecular imaging strategies that typically use radioactively labeled ligands to selectively visualize molecular targets. The nanomolar sensitivity of PET and SPECT combined with the high specificity and affinity of monoclonal antibodies have shown great potential in oncology imaging. Over the past decades a wide range of radio-isotopes have been developed into immuno-SPECT/PET imaging agents, made possible by novel conjugation strategies (e.g., site-specific labeling, click chemistry) and optimization and development of novel radiochemistry procedures. In addition, new strategies such as pretargeting and the use of antibody fragments have entered the field of immuno-PET/SPECT expanding the range of imaging applications. Non-invasive imaging techniques revealing tumor antigen biodistribution, expression and heterogeneity have the potential to contribute to disease diagnosis, therapy selection, patient stratification and therapy response prediction achieving personalized treatments for each patient and therefore assisting in clinical decision making.

RANK ligand (RANKL) is a member of the tumor necrosis factor alpha superfamily of cytokines. It is the only known ligand binding to a membrane receptor named receptor activator of nuclear factor-kappa B (RANK), thereby triggering recruitment of tumor necrosis factor (TNF) receptor associated factor (TRAF) adaptor proteins and activation of downstream pathways. RANK/RANKL signaling is controlled by a decoy receptor called osteoprotegerin (OPG), but also has additional more complex levels of regulation. The existing literature on RANK/RANKL signaling in cervical cancer was reviewed, particularly focusing on the effects on the microenvironment. RANKL and RANK are frequently co-expressed in cervical cancer cells lines and in carcinoma of the uterine cervix. RANKL and OPG expression strongly increases during cervical cancer progression. RANKL is directly secreted by cervical cancer cells, which may be a mechanism they use to create an immune suppressive environment. RANKL induces expression of multiple activating cytokines by dendritic cells. High RANK mRNA levels and high immunohistochemical OPG expression are significantly correlated with high clinical stage, tumor grade, presence of lymph node metastases, and poor overall survival. Inhibition of RANKL signaling has a direct effect on tumor cell proliferation and behavior, but also alters the microenvironment. Abundant circumstantial evidence suggests that RANKL inhibition may (partially) reverse an immunosuppressive status. The use of denosumab, a monoclonal antibody directed to RANKL, as an immunomodulatory strategy is an attractive concept which should be further explored in combination with immune therapy in patients with cervical cancer.

The objective of this study was to evaluate in vitro and in vivo drug release from in situ forming gels prepared with poloxamer 338 (P338) and/or 407 (P407) in N-methyl-2-pyrrolidone (NMP)/water mixtures for the model compound bedaquiline fumarate salt. The impact of total poloxamer concentration (20%-25% (w/w)), P338/P407 ratio (100/0%-0/100% (w/w)) and NMP/water ratio (0/100%-25/75% (v/v)) on gel point temperature (GPT) was investigated via a design of experiments (DoE), showing that GPT decreased mainly with increasing poloxamer concentration and decreasing P338/P407 ratio, while the relation with NMP/water ratio was more complex resulting in a flexion. Based on the DoE, four formulations with 10 mg/g bedaquiline fumarate salt, a fixed NMP/water ratio of 25/75% (v/v) and varying total poloxamer concentration and P338/P407 ratio were selected for evaluation of gel erosion in vitro. The fastest eroding formulation had the lowest total poloxamer concentration (20% (w/w)) and the lowest P338/P407 ratio (20.4/79.6% (w/w)), while the formulation with the highest total poloxamer concentration (23.5% (w/w)) and highest P338/P407 ratio (100/0% (w/w)) showed the lowest gel erosion rate. These fast and slow eroding formulations showed a similar trend for in vitro drug release and in vivo pharmacokinetics after intramuscular (IM) injection in rats. In vivo tmax of the IM administered poloxamer in situ forming gels was about 6 h and a short-term sustained drug release was observed in vivo in rats up to 24 h after dosing, similar to a solution of bedaquiline fumarate salt in polyethylene glycol (PEG400)/water. In conclusion, IM administration of the evaluated poloxamer in situ forming gels may be useful for drugs that require a short-term sustained release, but is not able to extend drug release rates up to weeks or months.

Background: The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a trans-cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse ¹⁸F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel ¹⁸F-labeled d-TCO radiotracers were developed, from which [¹⁸F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [¹⁸F]MICA-214 in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). Conclusions: A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel ¹⁸F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the ¹⁸F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.

Purpose: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [⁸⁹Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [⁶⁴Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. Experimental design: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with ⁶⁴Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. Results: The radiolabeling yield of [⁶⁴Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [⁶⁴Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [⁶⁴Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. Conclusions: Here, we report on a novel RANKL PET imaging agent, [⁶⁴Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

Bone metastases remain a common feature of advanced cancers and are associated with significant morbidity and mortality. Recent research has identified promising novel treatment targets to improve current treatment strategies for bone metastatic disease. This review summarizes the well-known and recently discovered molecular biology pathways in bone that govern normal physiological remodeling or drive the pathophysiological changes observed when bone metastases are present. In the rapidly changing world of targeted cancer treatments, it is important to recognize the specific treatment effects induced in bone by these agents and the potential impact on common imaging strategies. The osteoclastic targets (bisphosphonates, LGR4, RANKL, mTOR, MET-VEGFR, cathepsin K, Src, Dock 5) and the osteoblastic targets (Wnt and endothelin) are discussed, and the emerging field of osteo-immunity is introduced as potential future therapeutic target. Finally, a summary is provided of available trial data for agents that target these pathways and that have been assessed in patients. The ultimate goal of research into novel pathways and targets involved in the tumor-bone microenvironment is to tackle one of the great remaining unmet needs in oncology, that is finding a cure for bone metastatic disease.