Redox cycling of iron powers various enzyme functions crucial for life, making the study of iron acquisition, storage, and disposition in the whole organism a worthy topic of inquiry. However, despite its important role in biology and disease, imaging iron in animals with oxidation-state specificity remains an outstanding problem in biology and medicine. Here we report a first-generation reactivity-based probe of labile ferrous iron suitable for positron emission tomography studies in live animals. The responses of this reagent to systemic changes in labile iron disposition were revealed using iron supplementation and sequestration treatments in mice, while the potential of this approach for in vivo imaging of cancer was demonstrated using genetically and pathologically diverse mouse models, including spontaneous tumors arising in a genetically engineered model of prostate cancer driven by loss of PTEN.

Although cancer has been known for decades to harbor an insatiable appetite for iron, only recently has the chemistry emerged to exploit this altered state therapeutically, by targeting the expanded cytosolic labile iron pool (LIP) of the cancer cell. The state of the art includes therapies that react with the LIP to produce cytotoxic radical species (in some cases also releasing drug payloads) and molecules that exacerbate LIP-induced oxidative stress to trigger ferroptosis. Effectively implementing LIP-targeted therapies in patients will require biomarkers to identify those tumors with the most elevated LIP and thus most likely to succumb to LIP-targeted interventions. Toward this goal, we tested whether tumor uptake of the novel LIP-sensing radiotracer ¹⁸F-TRX aligns with tumor sensitivity to LIP-targeted therapies. Methods: ¹⁸F-TRX uptake was assessed in vivo among 10 subcutaneous and orthotopic human xenograft models. Glioma and renal cell carcinoma were prioritized because these tumors have the highest relative expression levels of STEAP3, the oxidoreductase that reduces ferric iron to the ferrous oxidation state, in the Broad Institute Cancer Cell Line Encyclopedia. The antitumor effects of the LIP-activated prodrug TRX-CBI, which releases the DNA alkylator CBI, were compared in mice bearing U251 or PC3 xenografts, tumors with high and intermediate levels of ¹⁸F-TRX uptake, respectively. Results: ¹⁸F-TRX showed a wide range of tumor accumulation. An antitumor assessment study showed that the growth of U251 xenografts, the model with the highest ¹⁸F-TRX uptake, was potently inhibited by TRX-CBI. Moreover, the antitumor effects against U251 were significantly greater than those observed for PC3 tumors, consistent with the relative ¹⁸F-TRX-determined LIP levels in tumors before therapy. Lastly, a dosimetry study showed that the estimated effective human doses for adult male and female mice were comparable to those of other ¹⁸F-based imaging probes. Conclusion: We report the first evidence-to our knowledge-that tumor sensitivity to an LIP-targeted therapy can be predicted with a molecular imaging tool. More generally, these data bring a new dimension to the nuclear theranostic model by showing a requirement for imaging to quantify, in situ, the concentration of a metastable bioanalyte toward predicting tumor drug sensitivity.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of ⁶⁴Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that ⁶⁴Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of ⁶⁴Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that ⁶⁴Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying ⁶⁴Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

The complexity of glucocorticoid receptor (GR) signaling cannot be measured with direct tissue analysis in living subjects, which has stifled our understanding of GR's role in human physiology or disease and impeded the development of selective GR modulators. Herein, we report ¹⁸F-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]quinoline (¹⁸F-YJH08), a radioligand that enables noninvasive measurements of tissue autonomous GR expression levels in vivo with positron emission tomography (PET). YJH08 potently binds GR (K_i ∼ 0.4 nM) with ∼100-fold selectivity compared to nuclear hormone receptors in the same subfamily. ¹⁸F-YJH08 was prepared via Cu(OTf)₂(py)₄-mediated radiofluorination of an arylboronic acid pinacol ester with ∼12% decay corrected radiochemical yield from the starting ¹⁸F-fluoride ion. We applied treatment with the tissue-wide GR agonist dexamethasone and adrenalectomy and generated an adipocyte specific GR knockout mouse to show that ¹⁸F-YJH08 specifically binds GR in normal mouse tissues, including those for which aberrant GR expression is thought to drive severe diseases (e.g., brain, adipose tissue, kidneys). Remarkably, ¹⁸F-YJH08 PET also revealed that JG231, a potent and bioavailable HSP70 inhibitor, selectively degrades GR only in the adipose tissue of mice, a finding that foreshadows how GR targeted PET might be integrated into drug discovery to screen for selective GR modulation at the tissue level, beyond the historical screening that was performed at the transcriptional level. In summary, ¹⁸F-YJH08 enables a quantitative assessment of GR expression levels in real time among multiple tissues simultaneously, and this technology is a first step toward unraveling the daunting complexity of GR signaling and rationally engineering tissue specific therapeutic modulators in vivo.

Noninvasive methods to study glucocorticoid receptor (GR) signaling are urgently needed to elaborate the complexity of GR signaling in normal physiology and human disorders, as well as to identify selective GR modulators to treat diseases. Here, we report evidence supporting translational studies with (±)-¹¹C-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]-quinoline ((±)-¹¹C-YJH08), a radioligand for PET that engages the ligand binding domain on GR. Methods: (±)-¹¹C-YJH08 was synthesized by reacting the phenol precursor with ¹¹C-methyl iodide. The biodistribution was studied in vivo. Specific binding was tested in vivo with adrenalectomy and ligand competition. A library of analogs was synthesized and studied in vitro and in vivo to understand the (±)-¹¹C-YJH08 structure-activity relationship. Rodent dosimetry studies were performed to estimate the human-equivalent doses of (±)-¹¹C-YJH08. Results: (±)-¹¹C-YJH08 was synthesized by reaction of the phenolic precursor with ¹¹C-methyl iodide, giving a radiochemical yield of 51.7% ± 4.7% (decay-corrected to starting ¹¹C-methyl iodide). Specific binding was observed in many tissues, including the brain. An analysis of the (±)-YJH08 structure-activity relationship showed that (R)- and (S)-enantiomers are equally avid for GR by occupying discrete binding modes. A focused chemical screen revealed that the aryl fluoride motif on YJH08 is essential for high-affinity GR binding in vitro, high tissue uptake in vivo, and efficient passage across the blood-brain barrier. Lastly, we performed dosimetry studies on rodents, from which we estimated the human-equivalent doses of (±)-¹¹C-YJH08 to be commensurate with the widely used ¹¹C and ¹⁸F tracers. Conclusion: These studies reveal the molecular determinants of a high-affinity and high-selectivity ligand-receptor interaction and support the use of (±)-¹¹C-YJH08 PET to make the first measurements of GR expression in human subjects.

Purpose

The recent emergence of radioligand therapies for cancer treatment has increased enthusiasm for developing new theranostic strategies coupling both imaging and cytotoxicity in the same entity. In this study, we evaluated whether CUB domain containing protein 1 (CDCP1), a single-pass transmembrane protein highly overexpressed in diverse human cancers, might be a target for cancer theranostics.

Experimental design

The ectodomain of CDCP1 was targeted using radiolabeled forms of 4A06, a potent and specific recombinant human antibody that we developed. Imaging and antitumor assessment studies were performed in animal models of pancreatic cancer, including two patient-derived xenograft models we developed for this study. For antitumor assessment studies, the endpoints were death due to tumor volume >3,000 mm³ or ≥20% loss in body weight. Specific tracer binding or antitumor effects were assessed with an unpaired, two-tailed Student t test and survival advantages were assessed with a log rank (Mantel-Cox) test. Differences at the 95% confidence level were interpreted to be significant.

Results

⁸⁹Zr-4A06 detected a broad dynamic range of full length or cleaved CDCP1 expression on seven human pancreatic cancer tumors (n = 4/tumor). Treating mice with single or fractionated doses of ¹⁷⁷Lu-4A06 significantly reduced pancreatic cancer tumor volume compared with mice receiving vehicle or unlabeled 4A06 (n = 8; P < 0.01). A single dose of ²²⁵Ac-4A06 also inhibited tumor growth, although the effect was less profound compared with ¹⁷⁷Lu-4A06 (n = 8; P < 0.01). A significant survival advantage was imparted by ²²⁵Ac-4A06 (HR = 2.56; P < 0.05).

Conclusions

These data establish that CDCP1 can be exploited for theranostics, a finding with widespread implications given its breadth of overexpression in cancer.

Purpose

We recently identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted PET radiopharmaceutical, [⁸⁹Zr]DFO-YS5, and evaluated its performance for immunoPET imaging in murine prostate cancer models.

Experimental design

[⁸⁹Zr]DFO-YS5 was prepared and its in vitro binding affinity for CD46 was measured. ImmunoPET imaging was conducted in male athymic nu/nu mice bearing DU145 [AR^-, CD46⁺, prostate-specific membrane antigen-negative (PSMA^-)] or 22Rv1 (AR⁺, CD46⁺, PSMA⁺) tumors, and in NOD/SCID gamma mice bearing patient-derived adenocarcinoma xenograft, LTL-331, and neuroendocrine prostate cancers, LTL-331R and LTL-545.

Results

[⁸⁹Zr]DFO-YS5 binds specifically to the CD46-positive human prostate cancer DU145 and 22Rv1 xenografts. In biodistribution studies, the tumor uptake of [⁸⁹Zr]DFO-YS5 was 13.3 ± 3.9 and 11.2 ± 2.5 %ID/g, respectively, in DU145 and 22Rv1 xenografts, 4 days postinjection. Notably, [⁸⁹Zr]DFO-YS5 demonstrated specific uptake in the PSMA- and AR-negative DU145 model. [⁸⁹Zr]DFO-YS5 also showed uptake in the patient-derived LTL-331 and -331R models, with particularly high uptake in the LTL-545 neuroendocrine prostate cancer tumors (18.8 ± 5.3, 12.5 ± 1.8, and 32 ± 5.3 %ID/g in LTL-331, LTL-331R, and LTL-545, respectively, at 4 days postinjection).

Conclusions

[⁸⁹Zr]DFO-YS5 is an excellent PET imaging agent across a panel of prostate cancer models, including in both adenocarcinoma and neuroendocrine prostate cancer, both cell line- and patient-derived xenografts, and both PSMA-positive and -negative tumors. It demonstrates potential for clinical translation as an imaging agent, theranostic platform, and companion biomarker in prostate cancer.