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Purpose: Despite recent approvals for checkpoint inhibitors and antibody–drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain–containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies. Experimental Design: CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376). Results: CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively. Conclusions: These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach, that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-D-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganismsin vivobased on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with β-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the α-1,4 and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (β-1,3), and cellobiose (β-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSKin vitro,showing accumulation by several clinically relevant pathogens includingStaphylococcus aureusandAcinetobacter baumannii,and demonstrated their specific uptakein vivo.The lead sakebiose-derived tracer [18F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [18F]FSK toS. aureusincluding methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Purpose:Despite recent approvals for checkpoint inhibitors and antibody–drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain–containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies.Experimental Design:CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376).Results:CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively.Conclusions:These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

CDCP1 mRNA is not correlated NECTIN4 or TROP2 mRNA, related to Figure 2C. Spearman correlation analyses were performed between CDCP1 and NECTIN4 or TROP2 for the Seiler, Sjodahl 2017, and Sjodahl 2012 datasets, with p values and correlation coefficients shown. Points are colored by consensus subtype as in Figure 1.

Supplemental Figure 1: Biodistribution data reflecting the accumulation of 68Ga-citrate over time in the tissues of male nu/nu mice bearing PC3 xenografts. Peak radiotracer uptake was observed at 4 hours post injection. Supplemental Figure 2: Biodistribution data reflecting the accumulation of 68Ga-citrate at 4 hours post injection in the tissues of male nu/nu mice bearing 22Rv1 xenografts. Supplemental Figure 3: Biodistribution data collected 4 hours post injection of 68Ga-citrate in male nu/nu mice bearing subcutaneous PC3 xenografts, and 48 hours post injection of 89Zr-Tf in male nu/nu mice with subcutaneous PC3 xenografts. Supplemental Figure 4. BET inhibitors suppress MYC and transferrin uptake in MYC positive prostate cancer cell lines. Supplemental Figure 5: Biodistribution data collected 4 hours post injection of 68Ga-Tf in male nu/nu mice bearing subcutaneous PC3 or 22Rv1 xenografts. Mice were treated with vehicle, (+)-JQ1 (25 mg/kg, BID), or iBET (50 mg/kg, daily oral gavage) for 5 days prior to radiotracer injection. Supplemental Figure 6: Representative PET data collected 4 hours post injection of 68Ga-citrate or 48 hours post injection of 89Zr-Tf in male nu/nu mice bearing subcutaneous 22Rv1 xenografts. Supplemental Figure 7: Paired PET imaging plus CT-guided biopsy of tracer-avid liver metastasis in patient-003 with mCRPC. Supplemental Figure 8: H&E staining of the PDX tissue isolated from the liver metastasis of patient-003 shows morphological features consistent with the liver biopsy. Supplemental Figure 9. Biodistribution data acquired 4 hours post injection of 68Ga-citrate shows the accumulation of the radiotracer in the PDX tissue and selected normal mouse tissues. Supplemental Table 1. A summary of the tumor:blood and tumor: muscle ratios for 68Ga-citrate Supplemental Table 2. A summary of the patient characteristics in this study. Supplemental Table 3. A summary of the test/re-test data for 68Ga-citrate in normal tissues. Data are presented as SUVmax. Supplementary Table 4. A summary of the histological subclassifications for the prostate cancer biopsies acquired during this study.

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mp3 file (37.5 MB). "Personalized Medicine: Late-breaking Science from AACR Journals," Saturday, March 31, 2012. Hosted by Lewis C. Cantley, Co-Editor-in-Chief of Cancer Discovery, this press conference held during the AACR Annual Meeting covers the following topics:Lack of Oxygen Influenced Tumor Behavior and Patient Outcome in Intermediate-risk Prostate CancerNovel Technology Allows for Noninvasive Imaging of Prostate Cancer (Ulmert et al., April issue of Cancer Discovery) EGFR Mutation Unique to Glioblastoma May Explain Lack of Response to Traditional EGFR Inhibitors

Noninvasive biomarkers that detect the activity of important oncogenic drivers could significantly improve cancer diagnosis and management of treatment. The goal of this study was to determine whether 68Ga-citrate (which avidly binds to circulating transferrin) can detect MYC-positive prostate cancer tumors, as the transferrin receptor is a direct MYC target gene. PET imaging paired with 68Ga-citrate and molecular analysis of preclinical models, human cell-free DNA (cfDNA), and clinical biopsies were conducted to determine whether 68Ga-citrate can detect MYC-positive prostate cancer. Importantly, 68Ga-citrate detected human prostate cancer models in a MYC-dependent fashion. In patients with castration-resistant prostate cancer, analysis of cfDNA revealed that all patients with 68Ga-citrate avid tumors had a gain of at least one MYC copy number. Moreover, biopsy of two PET avid metastases showed molecular or histologic features characteristic of MYC hyperactivity. These data demonstrate that 68Ga-citrate targets prostate cancer tumors with MYC hyperactivity. A larger prospective study is ongoing to demonstrate the specificity of 68Ga-citrate for tumors with hyperactive MYC.Implications: Noninvasive measurement of MYC activity with quantitative imaging modalities could substantially increase our understanding of the role of MYC signaling in clinical settings for which invasive techniques are challenging to implement or do not characterize the biology of all tumors in a patient. Moreover, measuring MYC activity noninvasively opens the opportunity to study changes in MYC signaling in patients under targeted therapeutic conditions thought to indirectly inhibit MYC. Mol Cancer Res; 15(9); 1221–9. ©2017 AACR.

CDCP1 mRNA is overexpressed in basal/squamous subtypes of bladder cancer, related to Figure 1A-D. Pairwise comparisons of CDCP1 expression between bladder cancer consensus subtypes using the Wilcoxon rank-sum test across four different datasets are shown.

Supplementary Methods Additional methodology of experimental methods.

Supplementary Table from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Supplementary Figure 2. A) In vitro comparison of LNCaP and LNCaP-AR and DNA damage post-radiotherapy. B) In vivo correlation of baseline AR-output using bioluminescence to tumor progression.

Supplemental Methods, Supplementary Figure 1. A. Real time PCR data showing the suppression of PTEN mRNA levels by an anti PTEN shRNA in GBM and PCa cell lines. The suppression of PTEN mRNA was statistically significant (P < 0.05). B. Immunoblot data showing suppression of PTEN protein in the cell line cohort. Supplementary Figure 2. A. Real time PCR data showing the upregulation of PIK3CA wild type and mutant mRNA in HEK293 cells. Lysates were collected for analysis 48 hours after transfection. The upregulation was statistically significant (P < 0.05). SSupplementary Figure 3. A. Real time PCR data showing the upregulation of mTOR wild type and mutant mRNA in HEK293 cells. Lysates were collected for analysis 48 hours after transfection. The upregulation was statistically significant compared to mock (P < 0.05). Supplemental Figure 4. Right: In vitro uptake data showing that transient knockdown of TSC1 or TSC2 results in higher intracellular uptake of 125I-transferrin in NIH-3T3 cells. The cells were transfected for 48 hours prior to running the uptake assay for 1 hour. Left: In vitro uptake data showing that 125I-transferrin is internalized into TSC1-/- and TSC2-/- MEFs to a greater extent than the respective wild type subline. The uptake assay was conducted for 1 hour. The uptake was statistically significant compare to the respective parental line (P < 0.05). Supplemental Figure 5. Real time PCR data showing the knockdown of TSC1 or 2 mRNA by siRNA. Dharmacon smartPOOL siRNAs were transfected into NIH-3T3 cells for 48 hours prior to harvest. The gene knockdown was statistically significant (P < 0.01). Supplemental Figure 6. A photograph and PET images of wild type and PTEN null prostate tissues show higher uptake of the radiotracer in the PTEN null tissues. The contralateral lobes of the prostate are arranged with bilateral symmetry along a vertical axis cutting through the middle of the petri dish. Additional normal tissues from the urogenital tract are shown for comparison. Abbreviations: AP = anterior prostate, DP = dorsal prostate, VP = ventral prostate, LP = lateral prostate, B = bladder, SV = seminal vesicle, Ur = urethra. Supplemental Figure 7. A summary of the biodistribution data for all tissues in the Pb-cre PTENlox/lox mice. The statistics for the prostate lobes were described in the text. Supplementary Figure 8. Immunoblot data showing changes in phosphoprotein abundance in the PI3K/Akt/mTOR signaling axis, indicative of bioactivity for the respective kinase inhibitor in PTEN null GBM and PCa cell lines. Note no change in phosphoprotein levels for doxorubicin, as expected. Lysates were harvested 6 hours after the initiation of therapy. Supplementary Figure 9. A summary of the 125I-transferrin uptake data for all doses and treatment durations of BEZ235 in the PTEN null cell lines. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05). Supplementary Figure 10. A summary of the 125I-transferrin uptake data for all doses and treatment durations of INK128 in the PTEN null cell lines. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05). Supplementary Figure 11. A summary of the 125I-transferrin uptake data for all doses and treatment durations of RAD001 in the PTEN null cell lines. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05). Supplementary Figure 12. A summary of the 125I-transferrin uptake data for all doses and treatment durations of doxorubicin in the PTEN null cell lines. Supplementary Figure 13. Immunoblot data showing changes in phosphor-S6 (Ser 240/44), indicative of bioactivity for the respective kinase inhibitor in T47D or HCT115 cells. Note no change in phosphoprotein levels for doxorubicin, as expected. Lysates were harvested 24 hours after the initiation of therapy. Supplementary Figure 14. A summary of the 125I-transferrin uptake data for all doses and treatment durations of BEZ235, INK128, RAD001 and doxorubicin in the T47D cells. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05 for all treatments except doxorubicin). Supplementary Figure 15. A summary of the 125I-transferrin uptake data for all doses and treatment durations of BEZ235, INK128, RAD001 and doxorubicin in the HCT115 cells. The suppression of Tf uptake was statistically significant compared to vehicle treated cells (P < 0.05 for all treatments except doxorubicin). Supplementary Figure 16. Real time PCR data showing the efficacy of siRNA knockdown using the Dharmacon smartPOOL reagents. These data were collected 48 hours post transfection. The knockdown was statistically significant compared to cells treated with non-targeting (NT) siRNA (P < 0.01). Supplementary Figure 17. Pharmacological inhibition of mTORC1 activity suppresses TFRC transcription in vitro. (Left). Real time PCR data collected in vitro after 48 hours of treatment shows a substantial reduction in TFRC mRNA by treatment with targeted therapies, and no effect of doxorubicin, as expected, in U87MG, LNCaP-AR and PC3 cells. The doses of drugs were BEZ235 (100 nM), INK128 (100 nM), RAD001 (10 nM) and Doxorubicin (500 nM). (Right). An in vitro binding assay with 125I-DF1513 (a monoclonal antibody recognizing an extracellular epitope on human TFRC) shows that the abundance of cell surface TFRC is reduced by BEZ235 (100 nM), INK128 (100 nM), and RAD001 (10 nM) compared to vehicle. No effect was observed with Doxorubicin (500 nM). Cells were treated for 48 hours with drug, and incubated with 125I-DF1513 for 30 min at 4o C before isolating and counting the cell associated activity. In all cases, treatment with kinase inhibitors resulted in statistically significant decreases compared to vehicle and doxorubicin (P < 0.01). Supplementary Figure 18. Genetic inhibition of mTORC1 activity suppresses TFRC transcription in vitro. ( Left).Real time PCR data collected in vitro after 48 hours of transfection shows a substantial reduction in TFRC mRNA induced by siRNA to mTOR and RAPTOR, but not the mTORC2 component PRR5. Knockdown was confirmed with rtPCR. (Right) An in vitro binding assay with 125I-DF1513 shows that the abundance of cell surface TFRC is reduced by siRNAs (100 nmol) targeting mTOR and RPTOR compared to a nontargeting (NT) siRNA. No effect was observed with siRNA targeting PRR5, as expected. Cells were transfected for 48 hours, and incubated with 125I-DF1513 for 30 min at 4o C before isolating and counting the cell associated activity. In all cases, siRNA to mTOR or RPTOR resulted in statistically significant decrease in TFRC mRNA or protein compare to NT and siRNA against PRR5 (P < 0.01). Supplemental Figure 19. A summary of the biodistribution data for mice bearing subcutaneous U87 tumors, and treated with BEZ235, INK128, RAD001, or doxorubicin. The statistics for the tumor associated values were described in the text. Supplementary Figure 20. ROI analysis of the U87MG tumors in mice treated with vehicle or the indicated inhibitor. This data was derived from the same cohort used to generate the biodistribution data. The changes associated with the kinase inhibitors were statistically significant compare to vehicle and doxorubicin (P < 0.01). Supplementary Figure 21. A summary of the biodistribution data for mice bearing subcutaneous LNCaP tumors, and treated with BEZ235, INK128, RAD001, or doxorubicin. The statistics associated with the tumor values were described in the text. Supplementary Figure 22. A summary of the biodistribution data for mice bearing subcutaneous PC3 tumors, and treated with BEZ235, INK128, RAD001, or doxorubicin. The statistics associated with the tumor values were described in the text. Supplemental Figure 23. In vitro uptake data showing the impact of treatment dosing and changing exposure time in LNCaP-AR cells. The values on the X-axis refer to the amount of time LNCaP-AR was exposed to drug prior to 1 hour incubation with 125I-Tf. The cell surface and internalized activity was quantified, and normalized to vehicle treated cells. All treatments with the exception of ARN (1 �M) and ARN + RAD were statistically significant compared to vehicle (P < 0.01). Supplemental Figure 24. Biodistribution data from mouse tissues and tumors treated with vehicle, RAD001, ARN-509, or the combination for two days prior to injection with 89Zr-Tf. The biodistribution was collected 48 hours post injection of radiotracer. The statistics associated with the tumor values were described in the text.

Supplementary Figure legends for Supplementary Figure 1 and 2

Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival. Cancer Res; 75(22); 4688–96. ©2015 AACR.

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.

Supplementary Figure from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Purpose:With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets.Experimental Design:CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post–enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice.Results:CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4–2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4–2B xenografts.Conclusions:The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

Supplementary Data file with additional experimental and figures. SUPPLEMENTAL FIGURE 1: Radiolabeling of [89Zr]Zr-transferrin as shown by instant thin layer chromatography. Radiochemical yield ranged from 60-70% and all radiochemical purity was >99%. SUPPLEMENTAL FIGURE 2: Protein analysis of drug treatment with BRD4 and ERK inhibitors in human PDAC cells via western blot. SUPPLEMENTAL FIGURE 3: Ex vivo immunohistochemistry of resected murine iKras*p53* tumors. SUPPLEMENTAL FIGURE 4: Dosing regimen for BRD4 and ERK inhibitors (indicated as "drug" in the scheme) over the course of 7 days, with a total of 12 doses, 12 hours apart. The timeline of [89Zr]Zr-transferrin radiotracer administration is indicated on day 5, with PET imaging and biodistribution on day 7, 48 h post-injection with the radiotracer. SUPPLEMENTAL FIGURE 5: Ex vivo immunohistochemistry of resected human PDAC tumors pre and post-treatment with JQ1. Differences in MYC and TfR protein expression can be observed in tumors that responded to JQ1 treatment with regards to [89Zr]Zr-transferrin uptake in vivo. SUPPLEMENTAL FIGURE 6: PET imaging of [89Zr]Zr-Transferrin uptake in vehicle vs. SCH772984-treated animals bearing BxPC-3 (KRAS-WT) xenografts.

Supplemental methods, figures, and tables

Supplemental

Purpose: mTOR regulates many normal physiological processes and when hyperactive can drive numerous cancers and human diseases. However, it is very challenging to detect and quantify mTOR signaling noninvasively in clinically relevant animal models of disease or man. We hypothesized that a nuclear imaging tool measuring intracellular mTOR activity could address this unmet need.Experimental Design: Although the biochemical activity of mTOR is not directly amenable to nuclear imaging probe development, we show that the transferrin receptor can be used to indirectly measure intracellular changes in mTOR activity.Results: After verifying that the uptake of radiolabeled transferrin (the soluble ligand of the transferrin receptor) is stimulated by active mTORC1 in vitro, we showed that 89Zr-labeled transferrin (Tf) can measure mTORC1 signaling dynamics in normal and cancerous mouse tissues with PET. Finally, we show that 89Zr-Tf can detect the upregulation of mTORC1 by tumor cells to escape the antitumor effects of a standard-of-care antiandrogen, which is to our knowledge the first example of applying PET to interrogate the biology of treatment resistant cancer.Conclusions: In summary, we have developed the first quantitative assay to provide a comprehensive measurement of mTOR signaling dynamics in vivo, in specific normal tissues, and during tumor development in genetically engineered animal models using a nuclear imaging tool that is readily translatable to man. Clin Cancer Res; 23(12); 3045–52. ©2016 AACR.

Supplementary Figure 1. In vitro experiments demonstrating increase AR protein level post-radiotherapy; A) Nuclear AR staining, B) 18F-FDHT uptake. C) Isodose distribution of multibeam animal EBRT.

New understanding of turbulent jet flames of natural gas, and blends of natural gas with ethylene (C2H4), issuing into a range of preheated coflowing oxidisers with reduced oxygen (O2) concentrations are reported. Comparisons are made for coflow O2 concentrations of 3 %, 4 %, 5 %, 6 %, 9 % and 11 % and coflow temperatures of 1250 K, 1315 K and 1385 K. Instantaneous and simultaneously planar imaging measurements of temperature, hydroxyl radicals (OH) and formaldehyde (CH2O) were taken at eight locations ranging from 9 mm to 75 mm downstream of the jet exit plane. The experimental data is supplemented with chemical reaction modelling. It is shown that increasing the proportion of C2H4 in the fuel supply resulted in an increase in the flame front temperature. At 3 % O2, CH2O signal did not very between the three fuels containing C2H4, whilst at 9 %, the signal increased with increasing C2H4. OH number density was highest for a blend of 66 % NG and 33 % C2H4. The fuel composition made little effect on the jet spread rate. Reaction modelling found evidence of changes in the reaction process, both in the suppression of reactions with different fuels, and in the suppression of reactions, and the locations they occur, with reducing O2 concentrations. Reaction modelling also found a significant decrease in ignition delay when C2H4 was present in the fuel mixture, and an increase in ignition delay as all four fuels transition to MILD combustion. Refereed/Peer-reviewed

The elevated temperature of hydrogen combustion increases the formation of thermal NOx. Moderate or intense low oxygen dilution (MILD) combustion is known to reduce NOx emissions and increase thermal efficiency. Pressure is often also used for increasing thermal efficiency. The impact that pressure has on fluid dynamics and chemical kinetics is especially relevant in MILD combustion conditions. Hydrogen jet flames issuing into a hot and vitiated coflow were imaged using OH∗ chemiluminescence at different pressures (1–7 bar) and oxygen levels (3–9% by vol.). Laminar flame simulations complemented the experiments. The observed mean radial OH∗ width increased with increased pressure, but only at O2 content less than 9%, suggesting that pressure has greater influence on kinetics when oxygen is reduced. The integrated OH∗ signal strength remained constant at 3% coflow O2, despite an apparent increase in flame width, suggesting a spatial broadening of the flame with pressure. Numerical results indicate that at 3–6% O2, conditions for MILD combustion of H2 are met across a wide range of strains and pressures, supporting the experimental observations for 3% O2. Refereed/Peer-reviewed

ObjectiveUsing the American College of Rheumatology Rheumatology Informatics System for Effectiveness (RISE) registry, our objective was to examine performance on rheumatoid arthritis (RA) quality measures and to assess the association between practice characteristics and changes in performance over time among participating practices.MethodsWe analyzed data from practices enrolled in RISE between January 1, 2015 and December 31, 2017. Eight quality measures in the areas of RA disease management, cardiovascular risk reduction, and patient safety were examined. Variability in performance was evaluated at the practice level. Multivariate linear models were used to predict change in measure performance by year and to determine the effect of practice characteristics on change in performance over time.ResultsData from 59,986 patients from 54 practices were examined. The mean ± SD age was 62 ± 14 years, 77% were female, 69% were Caucasian, and most patients were seen in a single-specialty group practice (46%). The average performance on measures related to RA treatments was consistently high (>90%) across the study period. Measures related to RA functional status and disease activity assessment had the greatest improvements over time (8.4% and 13.0% increase per year, respectively; P

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., A protease-activated chemosensor for in vivo molecular imaging enables the detection of activated immune cells by specifically harnessing granzyme B catalysis.

CO2 mineralization via aqueous Mg/Ca/Na-carbonate (MgCO3/CaCO3/Na2CO3) formation represents a huge opportunity for the utilization of captured CO2. However, large-scale mineralization is hindered by slow kinetics due to the highly hydrated character of the cations in aqueous solutions (Mg2+ in particular). Reaction conditions can be optimized to accelerate carbonation kinetics, for example by the inclusion of selected additives that promote competitive dehydration of Mg and subsequent agglomeration, nucleation and crystallization. Towards tracking mineralisation and these steps of the reaction, neutron scattering presents unprecedented advantages over traditional techniques for time-resolved in situ measurements. However, a setup providing continuous solution circulation to ensure reactant system homogeneity for industrially relevant CO2-mineralizsation was currently available for use on neutron beamlines. We therefore undertook the design, construction, testing and implementation of such a self-contained reactor rig for use on selected neutron beamlines at the ISIS Neutron and Muon Source (Chilton, UK). The design ensured robust attachment via suspension from the covering flange to stabilize the reactor assembly and all fittings, as well as facilitating precise alignment of the entire reactor and sample (test) cell with respect to beam dimension and direction. The assembly successfully accomplished the principal tasks of providing a continuous flow of the reaction mixture for homogeneity, quantitative control of CO2 flux into the mixture as well as temperature and pressure regulation throughout the reaction and measurements. The design is discussed, with emphasis placed on the reactor including its geometry, components and all technical specifications. Descriptions of the offbeamline bench tests, safety and functionality, as well as the installation on beamlines and trial experimental procedure are provided, together with representative raw neutron scattering results.

Background Behçet’s disease (BD), a chronic systemic vasculitis, has distinct geographical and ethnic variation. Data regarding the epidemiology of patients with BD in the U.S. are limited; therefore, we sought to describe BD patient characteristics and medication use in the U.S., and compared them with data from patients from endemic regions. Methods We conducted a cross-sectional study using data from the RISE registry (2014–2018). Patients aged ≥ 18 years with BD were included. Sociodemographic and treatment information was extracted. We compared patients from the RISE registry to data from other published studies of patients with BD from endemic areas. Results One thousand three hundred twenty-three subjects with BD from the RISE registry were included. Mean age was 48.7 ± 16.3 years, female to male ratio was 3.8:1, and 66.7% were White. The most frequently used medications included glucocorticoids (67.6%) and colchicine (55.0%). Infliximab and adalimumab were the most used biologics (14.5% and 14.1%, respectively); 3.2% of patients used apremilast. The RISE registry had more women (79.3%), and patients were older compared to previously published BD studies from endemic areas. Methotrexate and TNFi were more commonly reported in RISE (21.8% and 29.4%) compared to studies from Egypt and Turkey. Colchicine, cyclosporine, and cyclophosphamide were more commonly used in cohorts from Egypt, Turkey, and Iran. Conclusions Findings from the largest BD dataset in the U.S. suggest that BD patients are predominantly female. Further research is needed to explore the reasons for the higher prevalence of BD among women in the U.S. and its possible impact on disease severity and management.

Molecular imaging with PET offers an alternative method to quantify programmed-death-ligand 1 (PD-L1) to accurately select patients for immunotherapies. More and more clinical and preclinical trials involve radiolabeling of antibody fragments for their desirably fast clearance and high tumor penetration. As the radiolabeling strategy can significantly impact pharmacokinetics and biodistribution, we explored in this work a site-specific radiofluorination strategy on an anti-PD-L1 fragment antigen-binding (Fab) and compared the pharmacokinetic and biodistribution properties with the same Fab labeled using stochastic radiolabeling chemistry. We applied an enzymatic bioconjugation mediated by a variant of the lipoic acid ligase (LplA) that promotes the formation of an amide bond between a short peptide cloned onto the C terminus of the Fab. A synthetic analogue of the enzyme natural substrate, lipoic acid, was radiolabeled with fluorine-18 for site-specific conjugation by LplA. We compared the biodistribution of the site-specifically labeled Fab with a stochastically labeled Fab on lysine side chains in tumor-bearing mice. The two methods of fluorination demonstrate a comparable whole-body biodistribution. The