Targeting HER2

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A”-DTPA yielded a chelate:protein ratio of 3.4±0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian, and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models, and the highest tumor %ID/g (51.18±13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85±273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts, was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected, but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.     

Evaluation of 99mTc-CN5DG as a broad-spectrum SPECT probe for tumor imaging

Previously, we reported a [^99mTc(ǀ)]⁺ labeled d-glucoamine derivative (^99mTc-CN5DG) and evaluated it as a tumor imaging agent in mice bearing A549 tumor xenografts. In this paper, ^99mTc-CN5DG was further studied in U87 MG (human glioma cells), HCT-116 (human colon cancer cells), PANC-1 (human pancreatic cancer cells) and TE-1 (human esophageal cancer cells) tumor xenografts models to verify its potential application for imaging of different kinds of tumors. The biodistribution data showed that ^99mTc-CN5DG had a similar biodistribution pattern in four tumor models at 2 h post-injection with high accumulation in tumors and kidneys. The tumor/muscle ratios (from 4.08 ± 0.42 to 9.63 ± 3.53) and tumor/blood ratios (from 17.18 ± 7.40 to 53.17 ± 16.16) of ^99mTc-CN5DG in four tumor models were high. All four kinds of tumors could be clearly seen on their corresponding SPECT/CT images. Pharmacokinetic study in healthy CD-1 mice demonstrated that ^99mTc-CN5DG cleared fast from blood (2 min, 12.97 ± 0.88%ID/g; 60 min, 0.33 ± 0.06%ID/g) and the blood distribution, elimination half-life was 5.81 min and 21.16 min, respectively. No abnormality was observed through the abnormal toxicity study. All of the above results demonstrated that ^99mTc-CN5DG could be a broad-spectrum SPECT probe for tumor imaging and its further clinical application is warranted.     

Development and preclinical studies of 64Cu-NOTA-pertuzumab F(ab′)2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT

We previously reported that microSPECT/CT imaging with ¹¹¹In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab′)₂ of pertuzumab modified with NOTA chelators for complexing ⁶⁴Cu. The effect of the administered mass (5–200 µg) of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to ¹¹¹In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to ¹¹¹In-labeled pertuzumab.     

Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of ^99mTc-MAG-aptamer and ¹¹¹In-DOTA-F3B. ^99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of ¹¹¹In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for ¹¹¹In-DOTA-F3B with 2.0%ID/g than for the ¹¹¹In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.     

Fractionated Therapy of HER2-Expressing Breast and Ovarian Cancer Xenografts in Mice with Targeted Alpha Emitting (227)Th-DOTA-p-benzyl-trastuzumab

Background

The aim of this study was to investigate therapeutic efficacy and normal tissue toxicity of single dosage and fractionated targeted alpha therapy (TAT) in mice with HER2-expressing breast and ovarian cancer xenografts using the low dose rate radioimmunoconjugate ²²⁷Th-DOTA-p-benzyl-trastuzumab.

Methodology/Principal Findings

Nude mice carrying HER2-overexpressing subcutaneous SKOV-3 or SKBR-3 xenografts were treated with 1000 kBq/kg ²²⁷Th-trastuzumab as single injection or four injections of 250 kBq/kg with intervals of 4–5 days, 2 weeks, or 4 weeks. Control animals were treated with normal saline or unlabeled trastuzumab. In SKOV-3 xenografts tumor growth to 10-fold size was delayed (p<0.01) and survival with tumor diameter less than 16 mm was prolonged (p<0.05) in all TAT groups compared to the control groups. No statistically significant differences were seen among the treated groups. In SKBR-3 xenografts tumor growth to 10-fold size was delayed in the single injection and 4–5 days interval groups (p<0.001) and all except the 4 weeks interval TAT group showed improved survival to the control groups (p<0.05). Toxicity was assessed by blood cell counts, clinical chemistry measurements and body weight. Transient reduction in white blood cells was seen for the single injection and 4–5 days interval groups (p<0.05). No significant changes were seen in red blood cells, platelets or clinical chemistry parameters. Survival without life threatening loss of body weight was significantly prolonged in 4 weeks interval group compared to single injection group (p<0.05) for SKOV-3 animals and in 2 weeks interval group compared with the 4–5 days interval groups (p<0.05) for SKBR-3 animals.

Conclusions/Significance

The same concentration of radioactivity split into several fractions may improve toxicity of ²²⁷Th-radioimmunotherapy while the therapeutic effect is maintained. Thus, it might be possible to increase the cumulative absorbed radiation dose to tumor with acceptable toxicity by fractionation of the dosage.     

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of ⁸⁹Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [⁸⁹Zr]Mab#58 specifically bound to HER3/RH7777 cells (K_d = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [⁸⁹Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [⁸⁹Zr]Mab#58 but not with the ⁸⁹Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with ⁸⁹Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.     

Comparative biodistribution studies of DTPA-derivative bifunctional chelates for radiometal labeled monoclonal antibodies

Biodistribution of five different backbone-substituted derivatives of SCN-Bz-DTPA (1B4M-DTPA, 1M3B-DTPA, 1B3M-DTPA, GEM-DTPA and 2B-DTPA) linked to MAb B72.3 were compared to that of the parent molecule after labeling with ¹¹¹indium. Athymic mice, bearing human colon carcinoma xenografts (LS-174T) were injected i.v. to determine the biodistribution of the MAb chelate conjugates. Three of the MAb metal chelate conjugates (1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA), labeled with ¹¹¹In showed efficient and stable tumor localization as well as a slower blood clearance rate than SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. Major differences were also seen in normal organ uptake, especially liver and spleen. Tumor-to-liver ratios rose as a function of time for 1B4M-DTPA, 1M3B-DTPA and 1B3M-DTPA MAb chelate conjugates with virtually no accumulation of the radiometal into this organ, as revealed by no increase in the liver-to-blood values. Small accretion in normal liver was noted for SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. The results reviewed here, and described previously (Roselli et al., 1991) demonstrate that the use in vivo of backbone-substituted forms of the SCN-Bz-DTPA, such as 1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA bound to MAbs, can reduce uptake of indium to normal organs while maximizing the dose to tumor.     

Immune parameters of mice bearing human head and neck cancer

A xenogeneic human head and neck squamous cell carcinoma (HNSCC) model in immunocompetent mice was evaluated for its requirement of cyclosporine for progressive tumor growth. Tumor growth and T cell functions were assessed in mice receiving cyclosporine treatment for various lengths of time. Tumor cells were injected s.c. on day 1 and cyclosporine was injected i.p. daily on days 1, 1–7, 1–14, 1–21, or for the entire 28 days of tumor growth. All mice developed tumors. These tumors were confirmed to be squamous carcinomas of human origin histologically and by positive staining for human MHC class I antigen expression. Tumors were largest in mice that received cyclosporine for days 1–21 or days 1–28. Increased tumor size was associated with increased serum levels of tumor-reactive antibodies, an increased intratumoral frequency of CD4⁺ and CD8⁺ cells, but a diminished production of interleukin-2 (IL-2) by the tumor infiltrate. Also correlating with increasing tumor size was splenomegaly, a decline in the frequency, but not the absolute levels, of splenic CD4⁺ and CD8⁺ cells, and a diminished capacity to proliferate in response to concanavalin A and to be stimulated to secrete IL-2. The HNSCC tumors contributed to the immune decline since T cell functions were more depressed in the tumor bearers than in control mice receiving only cyclosporine treatment. These results demonstrate that human HNSCC tumor xenografts can grow in mice even with limited cyclosporine treatment, and that the survival of these xenografts may, in part, be due to a tumor-induced decline in select T cell functions.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

Evaluation of 131I-Anti-Angiotensin II Type 1 Receptor Monoclonal Antibody as a Reporter for Hepatocellular Carcinoma

Background

Finding a specific agent is useful for early detection of tumor. Angiotensin II type 1 receptor (AT₁R) was reported to be elevated in a variety of tumors and participate in tumor progression. The aim of our study was to evaluate whether ¹³¹I-anti-AT₁R monoclonal antibody (mAb) is an efficient imaging reporter for the detection of hepatocellular carcinoma.

Methodology/Principal Findings

AT₁R mAb or isotype IgG was radioiodinated with ¹³¹I and the radiochemical purity and stability of the two imaging agents and the affinity of ¹³¹I-anti-AT₁R mAb against AT₁R were measured. 3.7 MBq ¹³¹I-anti-AT₁R mAb or isotype ¹³¹I-IgG was intravenously injected to mice with hepatocellular carcinoma through tail vein, and then the whole-body autoradiography and biodistribution of the two imaging agents and the pharmacokinetics of ¹³¹I-anti-AT₁R mAb were studied. ¹³¹I-anti-AT₁R mAb and ¹³¹I-IgG were successfully radioiodinated and both maintained more stable in serum than in saline. The ¹³¹I-anti-AT₁R mAb group showed much clearer whole-body images for observing hepatocellular carcinoma than the ¹³¹I-IgG group. The biodistributions of the two imaging agents suggested that hepatocellular carcinoma tissue uptook more ¹³¹I-anti-AT₁R mAb than other tissues (%ID/g = 1.82±0.40 and T/NT ratio = 7.67±0.64 at 48 h), whereas hepatocellular carcinoma tissue did not selectively uptake ¹³¹I-IgG (%ID/g = 0.42±0.06 and T/NT ratio = 1.33±0.08 at 48 h). The pharmacokinetics of ¹³¹I-anti-AT₁R mAb was in accordance with the two-compartment model, with a rapid distribution phase and a slow decline phase. These results were further verified by real-time RT-PCR, immunohistochemistry staining and Western blot.

Conclusions/Significance

¹³¹I-anti-AT₁R mAb may be a potential target for early detection of tumor.     

Inhibition of Epstein-Barr-virus-transformed human chronic lymphocytic leukaemic B cells with monoclonal-antibody-Adriamycin (doxorubicin) conjugates

The anthracyclin antineoplastic agent doxorubicin (Adriamycin) was linked by four different methods of linkage to DalB02, an IgG1 murine monoclonal antibody (mAb) against surface-associated antigens on human chronic lymphocytic leukaemia (CLL) B cells. All the four conjugates fully retained the immunoreactivity of the parent DalB02. When the inhibitory effect of these conjugates was evaluated in vitro against the target D_10–1 cells (a clone derived from an Epstein-Barr-virus-transformed human CLL B cell line that binds DalB02) it was observed that one conjugate was more potent than the free drug but the others were not. When¹³¹I-labelled unmodified DalB02 and the¹³¹I-labelled DalB02-containing conjugate that was found to be potent were injected i.v. into nude mice bearing a subcutaneous D_10–1 xenograft, the percentages of the injected dose (%ID) of both¹³¹I-DalB02 and the¹³¹I-DalB02-containing conjugate that localized in the tumour were much higher than the %ID of the respective preparations that localized in normal tissues of D_10–1-xenografted mice. The systemic toxicity of the conjugate was less than that of the free drug. At an equitoxic dose level, this conjugate was a more effective inhibitor of established D_10–1 xenografts than the free drug.This study was supported by grants from the Medical Research Council of Canada (grant MT 10964) and the Cancer Research Society Inc., Montreal, Canada     

Noninvasive Assessment of Losartan-Induced Increase in Functional Microvasculature and Drug Delivery in Pancreatic Ductal Adenocarcinoma

PURPOSE: Losartan, an angiotensin II receptor blocker, can reduce desmoplasia and enhance drug delivery and efficacy through improving interstitial transport and vascular perfusion in pancreatic ductal adenocarcinoma (PDAC) models in mice. The purpose of this study was to determine whether magnetic resonance imaging (MRI) of magnetic iron oxide nanoparticles (MNPs) and micro–positron emission tomography (PET) measurements could respectively detect improvements in tumor vascular parameters and drug uptake in orthotopic PDAC in mice treated with losartan. METHOD AND MATERIALS: All experiments were approved by the local Institutional Animal Care and Use Committee. FVB mice with orthotopic PDAC were treated daily with an i.p. injection of losartan (70 mg/kg) or saline (control vehicle) for 5 days. In order to calculate the fractional blood volume, vessel size index, and vessel density index, MRI was performed at 4.7 T following the injection of 3 mg/kg iron ferumoxytol (i.v.). Dynamic PET images were also acquired for 60 minutes using an ¹⁸F-5FU tracer dose of 200 μCi and analyzed for time activity curves normalized to muscle. Statistical analyses compared both cohorts using an unpaired two-tailed t test. RESULTS: In comparison to the control treatment, the losartan administration significantly increased the fractional blood volume (mean ± SEM) [12.1 ± 1.7 (n = 19) vs 6.7 ± 1.1 (n = 20); P < .02] and vessel size index (128.2 ± 35.6 vs 57.5 ± 18; P < .05). Losartan also induced a significant increase in the intratumoral uptake of ¹⁸F-5FU by 53% (P < .0001). CONCLUSION: MRI using FDA-approved MNPs provides a noninvasive, translatable means of assaying microvascular parameters induced by losartan in pancreatic cancer. PET measurements demonstrated that losartan significantly increased the uptake of ¹⁸F-5FU.     

In vivo assessment of 111in-labeled hematoporphyrin derivative in breast tumor-bearing animals

The biological behavior of ¹¹¹In-labeled HPD has been investigated in tumor-bearing animals. Mice mammary adenocarcinomas and 7,12-dimethylbenz(a)anthracine induced breast tumors in Sprague-Dawley female rats were clearly visualized by ¹¹¹In-HPD nuclear scintigraphy. Optimal scans were obtained after a 48 h delay. In normal and tumor-bearing animals, the highest uptake of ¹¹¹In-HPD 72 h post-injection was found in the liver, the spleen and the kidneys. Depending on the size and the extent of necrosis, the uptake of ¹¹¹In-HPD by malignant breast tumors varied from 2.5% injected dose (ID) (range 0.14–5.3% ID) in mice to 1% ID (range 0.22–8.1% ID) in rats. Benign breast tumor uptake of ¹¹¹In-HPD was less that 1%ID. No significant amount of the radiopharmaceutical was found in pulmonary abscesses and abdominal cysts (< 0.1 % ID). Scintigrams of these infectious or inflammatory lesions were normal. Malignant tumor to blood, heart and lung ratios averaged 50:1, 10:1 and 3:1 respectively. Tumor to brain ratio ranged from 72 to 444:1.     

(R)-NODAGA-PSMA: A Versatile Precursor for Radiometal Labeling and Nuclear Imaging of PSMA-Positive Tumors

Purpose

The present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with ¹¹¹In for SPECT and intraoperative applications as well as ⁶⁸Ga and ⁶⁴Cu for PET imaging.

Methods

The PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)₃), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with ¹¹¹In, ⁶⁸Ga and ⁶⁴Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with ⁶⁸Ga-HBED-CC-PSMA (HBED-CC: N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid) and ¹¹¹In-PSMA-617 for comparison.

Results

⁶⁸Ga-CC34, ⁶⁴Cu-CC34, and ¹¹¹In-CC34 were prepared in radiochemical purity >95%. ^68/natGa-CC34, ^64/natCu-CC34, ^111/natIn-CC34, ^68/natGa-HBED-CC-PSMA, and ^111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with K_d values of 19.3±2.5 nM, 27.5±2.7 nM, 5.5±0.9 nM, 2.9±0.6 nM and 5.4±0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. ⁶⁸Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of ⁶⁸Ga-CC34 (14.5±2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to ⁶⁸Ga-HBED-CC-PSMA (15.8±1.4% IA/g) (P = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of ⁶⁸Ga-CC34 were also comparable to ⁶⁸Ga-HBED-CC-PSMA (P>0.05). Tumor uptake of ¹¹¹In-CC34 (28.5±2.6% IA/g) at 1 h p.i. was lower than ¹¹¹In-PSMA-617 (52.1±6.5% IA/g) (P = 0.02). The acquisition of PET-images with ⁶⁴Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time.

Conclusions

⁶⁸Ga-CC34 is comparable to ⁶⁸Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. ⁶⁴Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. ⁶⁴Cu-CC34 is currently being prepared for clinical translation.     

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

Background

The positron-emitting radionuclide ⁸⁹Zr (t _1/2 = 3.17 days) was used to prepare ⁸⁹Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.

Methodology/Principal Findings

Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [⁸⁹Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in ⁸⁹Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. ⁸⁹Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of ⁸⁹Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.

Conclusions/Significance

The results indicate that ⁸⁹Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.     

Imaging of Orthotopic Glioblastoma Xenografts in Mice Using a Clinical CT Scanner: Comparison with Micro-CT and Histology

PurposeThere is an increasing need for small animal in vivo imaging in murine orthotopic glioma models. Because dedicated small animal scanners are not available ubiquitously, the applicability of a clinical CT scanner for visualization and measurement of intracerebrally growing glioma xenografts in living mice was validated.ResultsTumor volumes (mean±SD mm³) were similar between both CT-modalities (micro-CT: 19.8±19.0, clinical CT: 19.8±18.8; Wilcoxon signed-rank test p = 0.813). Moreover, between reader analyses for each modality showed excellent agreement as demonstrated by correlation analysis (Spearman-Rho >0.9; p<0.01 for all correlations). Histologically measured tumor volumes (11.0±11.2) were significantly smaller due to shrinkage artifacts (p<0.05). CNR and SNR were 2.1±1.0 and 1.1±0.04 for micro-CT and 23.1±24.0 and 1.9±0.7 for the clinical CTscanner, respectively.ConclusionClinical CT scanners may reliably be used for in vivo imaging and volumetric analysis of brain tumor growth in mice.     

Very Late Antigen-4 (α4β1 Integrin) Targeted PET Imaging of Multiple Myeloma

Biomedical imaging techniques such as skeletal survey and ¹⁸F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30–50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α₄β₁ integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, ⁶⁴Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of ⁶⁴Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with ⁶⁴Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, ⁶⁴Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using ⁶⁴Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

Defective Chylomicron Synthesis as a Cause of Delayed Particle Clearance in Diabetes?

Chylomicron metabolism is abnormal in diabetes and the chylomicron particle may play a very important role in atherosclerosis. The aim of this study was to examine the effect of diabetes on the metabolism of chylomicrons in cholesterol-fed alloxan diabetic and nondiabetic rabbits. Five diabetic rabbits and 5 control rabbits were given [¹⁴C]linoleic acid and [³H]cholesterol by gavage. Lymph was collected following cannulation of the lymph duct and radiolabelled chylomicrons were isolated by ultracentrifugation. The chylomicrons from each animal were injected into paired control and diabetic recipients. Lymph apolipoprotein (apo) B48, apo B100, and apo E were measured using sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis. Mean blood sugar of the diabetic donors and diabetic recipients were 19.7 ± 2.3 and 17.2 ± 3.2 mmol/L. Diabetic rabbits had significantly raised plasma triglyceride (10.8 ± 13.9 versus 0.8 ± 0.5 mmol/L, P < 0.02). There was a large increase in apo B48 in lymph chylomicrons in the diabetic donor animals (0.19 ± 0.10 versus 0.04 ± 0.02 mg/h, P < 0.01) and apo B100 (0.22 ± 0.15 versus 0.07 ± 0.07 mg/h, P < 0.05) and a reduction in apo E on the lymph chylomicron particle (0.27 ± 0.01 versus 0.62 ± 0.07 mg/mg apo B, P < 0.001). Diabetic recipients cleared both control and diabetic chylomicron triglyceride significantly more slowly than control recipients (P < 0.05). Clearance of control chylomicron cholesterol was delayed when injected into diabetic recipients compared to when these chylomicrons were injected into control recipients (P < 0.005). Clearance of diabetic chylomicron cholesterol was significantly slower when injected into control animals compared to control chylomicron injected into control animals (P < 0.02). In this animal model of atherosclerosis, we have demonstrated that diabetes leads to the production of an increased number of lipid and apo E–deficient chylomicron particles. Chylomicron particles from the control animals were cleared more slowly by the diabetic recipient (both triglyceride and cholesterol). The chylomicron particles obtained from the diabetic animals were cleared even more slowly when injected into the diabetic recipient. Although there was an initial delay in clearance of chylomicron triglyceride from the diabetic particle when injected into the control animals, the clearance over the first 15 minutes was not significantly different when compared to the control chylomicron injected into the control animal. On the other hand, the cholesterol clearance was significantly delayed. Thus, diabetes resulted in the production of an increased number of lipid- and apo E–deficient chylomicron particles. These alterations account, in part, for the delay in clearance of these particles.     

Imaging of human ovarian tumour xenografts in nude mice using a novel 111In-labelled monoclonal antibody (10B)

Monoclonal antibody (mAb) 10B, directed against the human ovarian adenocarcinoma cell line, HEY, was conjugated with cyclic DTPA anhydride and labelled with ¹¹¹In. The biodistribution of ¹¹¹In-DTPA-10B was determined in non-tumour bearing mice and mice bearing subcutaneous (s.c.) and intraperitoneal (i.p.) HEY tumours. The radiolabel was preferentially targeted to s.c. and i.p. tumours in comparison with a control mAb, ¹¹¹In-DTPA-2G3, which does not bind to HEY cells. Among normal organs, the predominant uptake of radiolabel was into liver and kidney. Subcutaneous tumours were successfully imaged using external gamma scintigraphy following i.v. injection of ¹¹¹In-DTPA-10B. The results suggest that ¹¹¹In-DTPA-10B may be a useful agent for the diagnostic imaging of tumour masses in patients with ovarian cancer.