Dataset Information

64Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor.

ABSTRACT: This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR signaling was examined in cell culture. DOTA-Gp2 molecules were labeled with 64Cu and intravenously injected (0.6-2.3 MBq) into mice bearing EGFRhigh (n = 7) and EGFRlow (n = 4) xenografted tumors. PET/computed tomography (CT) images were acquired at 45 min, 2 h, and 24 h. Dynamic PET (25 min) was also acquired. Tomography results were verified with gamma counting of resected tissues. Two-tailed t tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (KD = 7 ± 5 nM) and murine (KD = 29 ± 6 nM) EGFR, and nontargeted Gp2 had no detectable binding. Gp2-EGFR did not agonize EGFR nor antagonize EGF-EGFR. 64Cu-Gp2-EGFR tracer effectively localized to EGFRhigh tumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFRlow tumors (0.9 ± 0.3%ID/g, p < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, p < 0.001). Nontargeted Gp2 tracer had low uptake in EGFRhigh tumors (0.5 ± 0.3%ID/g, p < 0.001). Similar data was observed at 2 h, and tumor signal was retained at 24 h (2.9 ± 0.3%ID/g). An engineered Gp2 PET imaging probe exhibited low background and target-specific EGFRhigh tumor uptake at 45 min, with tumor signal retained at 24 h postinjection, and compared favorably with published EGFR PET probes for alternative protein scaffolds. These beneficial in vivo characteristics, combined with thermal stability, efficient evolution, and small size of the Gp2 domain validate its use as a future class of molecular imaging agents.

SUBMITTER: Kruziki MA

PROVIDER: S-EPMC5519506 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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<sup>64</sup>Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor.

Kruziki Max A MA Case Brett A BA Chan Jie Y JY Zudock Elizabeth J EJ Woldring Daniel R DR Yee Douglas D Hackel Benjamin J BJ

Molecular pharmaceutics 20161010 11

This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR ...[more]

PMID: 27696863

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Project description:Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2-positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2-targeted therapies. Here, we report the synthesis and characterization of an antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo.Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results.Flow cytometry examination revealed the specific binding capacity of fluorescein isothiocyanate-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with (64)Cu, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-RamAb in VEGFR-2-positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2-negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of (64)Cu-NOTA-RamAb.This study provides initial evidence that (64)Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find potential applications in monitoring the treatment response of VEGFR-2-targeted cancer therapy.

Project description:A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

Project description:To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

Project description:UnlabelledWomen with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer.MethodsEight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax).ResultsLiver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from (64)Cu-DOTA-trastuzumab was similar to (18)F-FDG.Conclusion(64)Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a (64)Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. (64)Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.

Dataset Information

64Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor.

Publications

<sup>64</sup>Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor.

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