Publications by authors named "Robert E Reeves"

8 Publications

  • Page 1 of 1

Specific Imaging of Bacterial Infection Using 6″-F-Fluoromaltotriose: A Second-Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria.

J Nucl Med 2017 10 10;58(10):1679-1684. Epub 2017 May 10.

Department of Radiology, Stanford University School of Medicine, Stanford, California

6″-F-fluoromaltotriose is a PET tracer that can potentially be used to image and localize most bacterial infections, much like F-FDG has been used to image and localize most cancers. However, unlike F-FDG, 6″-F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in gram-positive and gram-negative strains of bacteria. 6″-F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains in cultures and in living mice. 6″-F-fluoromaltotriose was taken up in both gram-positive and gram-negative bacterial strains. 6″-F-fluoromaltotriose was also able to detect in a clinically relevant mouse model of wound infection. The utility of 6″-F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. 6″-F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients with infectious diseases of bacterial origin.
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http://dx.doi.org/10.2967/jnumed.117.191452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632736PMC
October 2017

Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model.

Mol Imaging Biol 2017 12;19(6):903-914

Department of Radiology, School of Medicine, Stanford University, Stanford, CA, USA.

Purpose: It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade.

Procedures: We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγ (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo.

Results: [Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3-5, 3.2 ± 0.4 MBq/15-16 μg/200 μl) and [Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 μg/200 μl) were administered in mice. PET-CT scans were performed over 1-144 h ([Zr] Keytruda) and 1-48 h ([Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([Zr] Keytruda), and 6.4 ± 0.7 ([Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([Zr] Keytruda) and 48 h ([Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively.

Conclusions: Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.
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http://dx.doi.org/10.1007/s11307-017-1060-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624852PMC
December 2017

Novel Radiotracer for ImmunoPET Imaging of PD-1 Checkpoint Expression on Tumor Infiltrating Lymphocytes.

Bioconjug Chem 2015 Oct 10;26(10):2062-9. Epub 2015 Sep 10.

Radiology, School of Medicine, ‡Bioengineering and §Materials Science and Engineering, Stanford University , Stanford, California 94305, United States.

Immune checkpoint signaling through the programmed death 1 (PD-1) axis to its ligand (PD-L1) significantly dampens anti-tumor immune responses. Cancer patients treated with checkpoint inhibitors that block this suppressive signaling have exhibited objective response rates of 20-40% for advanced solid tumors, lymphomas, and malignant melanomas. This represents a tremendous advance in cancer treatment. Unfortunately, all patients do not respond to immune checkpoint blockade. Recent findings suggest that patients with tumor infiltrating lymphocytes (TILs) expressing PD-1 may be most likely to respond to αPD-1/PD-L1 checkpoint inhibitors. There is a compelling need for diagnostic and prognostic imaging tools to assess the PD-1 status of TILs in vivo. Here we have developed a novel immunoPET tracer to image PD-1 expressing TILs in a transgenic mouse model bearing melanoma. A (64)Cu labeled anti-mouse antibody (IgG) PD-1 immuno positron emission tomography (PET) tracer was developed to detect PD-1 expressing murine TILs. Quality control of the tracer showed >95% purity by HPLC and >70% immunoreactivity in an in vitro cell binding assay. ImmunoPET scans were performed over 1-48 h on Foxp3+.LuciDTR4 mice bearing B16-F10 melanoma tumors. Mice receiving anti-PD-1 tracer (200 ± 10 μCi/10-12 μg/200 μL) revealed high tracer uptake in lymphoid organs and tumors. BLI images of FoxP3(+) CD4(+) Tregs known to express PD-1 confirmed lymphocyte infiltration of tumors at the time of PET imaging. Biodistribution measurements performed at 48 h revealed a high (11×) tumor to muscle uptake ratio of the PET tracer (p < 0.05). PD-1 tumors exhibited 7.4 ± 0.7%ID/g tracer uptake and showed a 2× fold signal decrease when binding was blocked by unlabeled antibody. To the best of our knowledge this data is the first report to image PD-1 expression in living subjects with PET. This radiotracer has the potential to assess the prognostic value of PD-1 in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.
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http://dx.doi.org/10.1021/acs.bioconjchem.5b00318DOI Listing
October 2015

A Systematic Comparison of 18F-C-SNAT to Established Radiotracer Imaging Agents for the Detection of Tumor Response to Treatment.

Clin Cancer Res 2015 Sep 13;21(17):3896-905. Epub 2015 May 13.

Department of Radiology, Stanford University, Stanford, California.

Purpose: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of (18)F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3-triggered nanoaggregation.

Experimental Design: Here, we compared the preclinical utility of (18)F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo.

Results: In drug-treated lymphoma cells, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R(2) > 0.8; P < 0.001), with no correlation measured for (18)F-ML-10 (R(2) = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of (99m)Tc-Annexin V and (18)F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although (99m)Tc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either (18)F-FDG or (18)F-ML-10.

Conclusions: We have demonstrated here that (18)F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with (18)F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-3176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558304PMC
September 2015

Thimerosal as discrimination: vaccine disparity in the UN Minamata Convention on mercury.

Indian J Med Ethics 2014 Oct-Dec;11(4):206-18. Epub 2014 Apr 11.

CoMeD, Inc, Silver Spring, MD; Institute of Chronic Illnesses, Inc, Silver Spring, MD United States.

When addressing toxins, one unmistakable parallel exists between biology and politics: developing children and developing nations are those most vulnerable to toxic exposures. This disturbing parallel is the subject of this critical review, which examines the use and distribution of the mercury (Hg)-based compound, thimerosal, in vaccines. Developed in 1927, thimerosal is 49.55% Hg by weight and breaks down in the body into ethyl-Hg chloride, ethyl-Hg hydroxide and sodium thiosalicylate. Since the early 1930s, there has been evidence indicating that thimerosal poses a hazard to the health of human beings and is ineffective as an antimicrobial agent. While children in the developed and predominantly western nations receive doses of mostly no-thimerosal and reduced-thimerosal vaccines, children in the developing nations receive many doses of several unreduced thimerosal-containing vaccines (TCVs). Thus, thimerosal has continued to be a part of the global vaccine supply and its acceptability as a component of vaccine formulations remained unchallenged until 2010, when the United Nations (UN), through the UN Environment Programme, began negotiations to write the global, legally binding Minamata Convention on Hg. During the negotiations, TCVs were dropped from the list of Hg-containing products to be regulated. Consequently, a double standard in vaccine safety, which previously existed due to ignorance and economic reasons, has now been institutionalised as global policy. Ultimately, the Minamata Convention on Hg has sanctioned the inequitable distribution of thimerosal by specifically exempting TCVs from regulation, condoning a two-tier standard of vaccine safety: a predominantly no-thimerosal and reduced-thimerosal standard for developed nations and a predominantly thimerosal-containing one for developing nations. This disparity must now be evaluated urgently as a potential form of institutionalised discrimination.
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http://dx.doi.org/10.20529/IJME.2014.054DOI Listing
June 2016

Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects.

Proc Natl Acad Sci U S A 2012 Sep 15;109(37):E2476-85. Epub 2012 Aug 15.

Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA 94305, USA.

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/β)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90β/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/β)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.
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http://dx.doi.org/10.1073/pnas.1205459109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443147PMC
September 2012

Single-nucleotide-specific siRNA targeting in a dominant-negative skin model.

J Invest Dermatol 2008 Mar 11;128(3):594-605. Epub 2007 Oct 11.

TransDerm Inc., Santa Cruz, California, USA.

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.
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http://dx.doi.org/10.1038/sj.jid.5701060DOI Listing
March 2008

Development of therapeutic siRNAs for pachyonychia congenita.

J Invest Dermatol 2008 Jan 30;128(1):50-8. Epub 2007 Aug 30.

Epithelial Genetics Group, Human Genetics Unit, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.
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http://dx.doi.org/10.1038/sj.jid.5701040DOI Listing
January 2008