Tumor Uptake In Mice Research Articles

GRPR-Antagonists Carrying DOTAGA-Chelator via Positively Charged Linkers: Perspectives for Prostate Cancer Theranostics.

Gastrin-releasing peptide receptor (GRPR)-antagonists have served as motifs in the development of theranostic radioligands for prostate cancer. Our efforts have been focused on the development of radiolabeled RM26 (H-DPhe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) analogs, such as [111In]In-DOTAGA-PEG2-RM26. We recently showed that its Gly11/Sar11-substituted version, [111In]In-AU-RM26-M1, resisted degradation by neprilysin (NEP) while in circulation and achieved higher tumor uptake in mice. We herein introduce the following three new AU-RM26-M1 mimics labeled with In-111, with basic residues in the linker: (i) AU-RM26-M2 (PEG2-Pip), (ii) AU-RM26-M3 (PEG2-Arg), and (iii) AU-RM26-M4 (Arg-Arg-Pip). These analogs were compared in PC-3 cells and animal models vs. AU-RM26-M1 (reference). The new analogs showed high affinity and specificity for the GRPR, exhibiting an uptake and distribution pattern in PC-3 cells typical for a radiolabeled GRPR-antagonist. They showed high stability in peripheral mice blood, except for [111In]In-AU-RM26-M3. AU-RM26-M4 achieved the highest tumor uptake and promising background clearance, followed by [111In]In-RM26-M2, showing lower background levels. These findings were confirmed for [111In]In-AU-RM26-M2 and [111In]In-AU-RM26-M4 by micro-SPECT/CT at 4 and 24 h post-injection. Hence, the type of positively charged residues in the linker of AU-RM26-M1 mimics strongly influenced biological behavior. The analogs with Pip next to DPhe6 demonstrated the best overall characteristics and warrant further investigation.

Radiolabeling Optimization and Preclinical Evaluation of the New PSMA Imaging Agent [18F]AlF-P16-093.

Prostate-specific membrane antigen (PSMA)-targeted radioligands have played an increasing role in the diagnosis of prostate cancer. [68Ga]Ga-P16-093 is a PSMA-targeting agent for positron emission tomography imaging, currently under a Phase 2 clinical trial. In the present study, P16-093 was labeled with 18F via [18F]AlF2+ complex formation, and the biological properties of [18F]AlF-P16-093 were evaluated. Optimization of radiolabeling efficiency was performed by testing a series of parameters, including the amount of free ligand; the amount of Al3+; and the influence of solvent, pH, temperature, reaction time, and reaction volume. Optimal labeling results were achieved at pH 5 by reacting at 60 °C for 15 min in a vial containing 74-370 MBq of [18F]fluoride, 46 nmol of P16-093, 40 nmol of AlCl3·6 H2O, and 50% EtOH. [18F]AlF-P16-093 was prepared with a non-decay-corrected radiochemical yield of 54.4 ± 4.4% (n = 9) within 30 min (final radiochemical purity ≥95%). In vitro, [18F]AlF-P16-093 showed PSMA-specific high uptakes in PIP-PC3 cells. The binding affinity of [18F]AlF-P16-093 to PSMA was determined as Kd of 12.4 ± 2.0 nM. The tumor uptake in mice with a xenografted PSMA-expressing PIP-PC3 tumor was high (18.8 ± 5.14% ID/g at 1 h postinjection) and retained without washout for 2 h. In addition, tumor uptake was almost completely blocked by coinjecting a PSMA inhibitor, 2-PMPA. The bone activity at 1 h post injection was higher with [18F]AlF-P16-093 (2.83 ± 0.49% ID/g) in comparison to that of [68Ga]Ga-P16-093 (0.26 ± 0.07% ID/g). In summary, an efficient and simple radiosynthesis of [18F]AlF-P16-093 was achieved. [18F]AlF-P16-093 showed desirable in vivo pharmacokinetics and excellent PSMA-targeting properties for imaging PSMA expression in prostate cancer.

Development and characterization of a theranostic multimodal anti-PSMA targeting agent for imaging, surgical guidance, and targeted photodynamic therapy of PSMA-expressing tumors.

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent 111In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light.Methods: The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with 111In. To determine the optimal dose and time point for tPDT, BALB/c nude mice with PSMA-expressing (PSMA+) s.c. LS174T-PSMA xenografts received the conjugate (24-240 µg/mouse) intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and ex vivo biodistribution at 24, 48, 72 and 168 h p.i. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 1-3 times of NIR light irradiation (50, 100, 150 J/cm2) 24 h after injection of 80 µg of DTPA-D2B-IRDye700DX was compared to control conditions.Results: Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72 and 168 h p.i. PSMA+ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Overall survival in mice treated with 80 µg of DTPA-D2B-IRDye700DX and 1x 150 J/cm2 of NIR light at 24 h p.i. was significantly improved compared to the control group receiving neither conjugate nor NIR light (73 days vs. 16 days, respectively, p=0.0453). Treatment with 3x 150 J/cm2 resulted in significantly prolonged survival compared to treatment with 3x 100 J/cm2 (p = 0.0067) and 3x 50 J/cm2 (p = 0.0338).Principal conclusions: 111In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA+ tumors with radionuclide and NIR fluorescence imaging and PSMA-targeted PDT. PSMA-tPDT using this multimodal agent resulted in significant prolongation of survival and shows great potential for treatment of (metastasized) prostate cancer.

Design and Preclinical Evaluation of an Albumin-Binding PSMA Ligand for 64Cu-Based PET Imaging.

Recently, we developed an albumin-binding radioligand (177Lu-PSMA-ALB-56), which showed higher PSMA-specific tumor uptake in mice than the previously developed 177Lu-PSMA-617 under the same experimental conditions. Such a radioligand may be of interest also for PET imaging, possibly enabling better visualization of even small metastases at late time-points after injection. The aim of this study was, therefore, to modify PSMA-ALB-56 by exchanging the DOTA chelator with a NODAGA chelator for stable coordination of 64Cu ( T1/2 = 12.7 h; Eβ+av = 278 keV). The resulting NODAGA-functionalized PSMA-ALB-89 ligand, and the previously establish DOTA-functionalized PSMA-ALB-56 ligand were labeled with 64Cu and evaluated in vitro and in vivo. Both radioligands showed plasma protein-binding properties in vitro and PSMA-specific uptake in PC-3 PIP cells. Biodistribution studies, performed in tumor-bearing mice, revealed high accumulation of 64Cu-PSMA-ALB-89 in PSMA-positive PC-3 PIP tumor xenografts (25.9 ± 3.41% IA/g at 1 h p.i.), which was further increased at later time-points (65.1 ± 7.82% IA/g at 4 h p.i. and 97.1 ± 7.01% IA/g at 24 h p.i.). High uptake of 64Cu-PSMA-ALB-89 was also seen in the kidneys, however, 64Cu-PSMA-ALB-89 was efficiently excreted over time. Mice injected with 64Cu-PSMA-ALB-56 showed increased accumulation of radioactivity in the liver (25.3 ± 4.20% IA/g) when compared to the liver uptake of 64Cu-PSMA-ALB-89 (4.88 ± 0.21% IA/g, at 4 h p.i.). This was most probably due to in vivo instability of the 64Cu-DOTA complex, which was also the reason for lower tumor uptake (49.7 ± 16.1% IA/g at 4 h p.i. and 28.3 ± 3.59% IA/g at 24 h p.i.). PET/CT imaging studies confirmed these findings and enabled excellent visualization of the PSMA-positive tumor xenografts in vivo after injection of 64Cu-PSMA-ALB-89. These data indicate that 64Cu-PSMA-ALB-89 is favorable over 64Cu-PSMA-ALB-56 with regard to the in vivo stability and tissue distribution profile. Moreover, 64Cu-PSMA-ALB-89 outperformed previously developed 64Cu-labeled PSMA ligands. Further optimization of long-circulating PSMA-targeting PET radioligands will be necessary before translating this concept to the clinics.

Abstract 3028: PET imaging with the bispecific 89Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse models

Abstract BACKGROUND: ERY974, a modified monoclonal IgG4 bispecific antibody directed against human CD3 on T cells and glypican 3 (GPC3) on tumor cell, is currently in phase I clinical trial. The oncofetal protein GPC3 is overexpressed in several tumor types. Radiolabeling ERY974 with positron emission tomography (PET) isotope zirconium-89 (89Zr) enables non-invasive molecular imaging of tumor targeting and whole-body distribution. We aimed to evaluate 89Zr-ERY974 tumor targeting and effect of T cells on tumor uptake in mouse models, including a humanized mouse model. METHODS: ERY974 and two control molecules namely bispecific CD3xkeyhole limpet hemocyanin (KLH) and KLHxKLH antibodies were radiolabeled with 89Zr. Studies were performed in immunodeficient NOD/Shi-SCID/IL-2Rgnull (NOG) as well as human CD34+ hematopoietic stem cell engrafted NOG mice (huNOG), all subcutaneously inoculated with GPC3 overexpressing human hepatocellular carcinoma HepG2 cells. Mice received 10 µg 89Zr-ERY974, 89Zr-CD3xKLH or 89Zr-KLHxKLH intravenously, with subsequent µPET scanning at 24, 72, 120 and 168 h followed by ex vivo biodistribution. Organs of interest were quantified on µPET scans as mean standardized uptake value (SUVmean) and with ex vivo biodistribution as % injected dose/gram of tissue (%ID/g). Tumor, spleen and lymph nodes were analyzed with autoradiography and immunohistochemical CD3 staining. RESULTS: µPET imaging revealed increased tumor-to-blood ratio (TBR) of 89Zr-ERY974 in NOG over time with maximal TBR of 2.2±0.3 at 168 h post tracer injection (pi). At 168 h, tumor uptake was specific as 89Zr-CD3xKLH and 89Zr-KLHxKLH showed a TBR of only 0.6±0.2 and 0.8±0.3, respectively. In huNOG mice human CD3+ T cells were present in tumor, spleen and lymph nodes. In huNOG mice tumor uptake of 89Zr-ERY974 was higher than in NOG mice as measured on µPET scans (SUVmean at 168 h pi 6.9±2.6 vs 2.9±0.2; P<0.01) and with ex vivo biodistribution (60.9±26.2 %ID/g vs 16.7±2.3 %ID/g; P<0.001), whereas 89Zr-CD3xKLH tumor uptake in both mouse models was lower (P<0.05) but were similar in these mouse models. Autoradiography 168 h following 89Zr-ERY974 administration to huNOG mice showed 89Zr in extensive T cell infiltrate areas in the tumors of huNOG mice, whereas T cell infiltrate was lower in tumors of 89Zr-CD3xKLH and 89Zr-KLHxKLH injected huNOG mice. Spleens of huNOG mice showed CD3+ specific uptake as 89Zr-ERY974 and 89Zr-CD3xKLH uptake were higher than 89Zr-KLHxKLH uptake(P<0.05), whereas spleen uptake in NOG mice of the 3 tracers was similar. Moreover, in huNOG CD3+ mesenteric lymph nodes 89Zr-ERY974 uptake was higher than 89Zr-KLHxKLH uptake (P<0.05) CONCLUSION: 89Zr-ERY974 demonstrates specific tumor uptake in NOG and huNOG mice, while in huNOG mice tumor uptake colocalized with T cell rich infiltrate and also uptake in in spleen and lymph nodes was observed. Citation Format: Stijn J. Waaijer, Danique Giesen, Takahiro Ishiguro, Yuji Sano, Norihisa Ohishi, Athos Gianella-Borradori, Carolien P. Schröder, Elisabeth G. de Vries, Marjolijn N. Lub-de Hooge. PET imaging with the bispecific 89Zr-antibody ERY974 targeting CD3 and glypican 3 in tumor-bearing mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3028.

Immuno-PET in Pontine Glioma: More Than Meets the Eye?

610 Objectives: The success of PET imaging with 68Ga-labeled somatostatin and PSMA ligands has created a markedly increased demand for 68Ga that is challenging to meet with existing 68Ge/68Ga generators. In addition, preparation of patient doses is relatively expensive, because only small batches of the ligands can be prepared that each need to undergo QC procedures. The objective of this study was therefore to investigate the cyclotron production of 68Ga from a solid 68Zn target and the feasibility of using cyclotron produced 68Ga for preparing 68Ga-PSMA-11. We also evaluated how a decrease of specific activity - as would occur during the shipment of larger batches of 68Ga-PSMA-11 - affects tumor and normal organ uptake of [asterisk]Ga-PSMA-11 in mouse models of prostate cancer. Methods: Enriched 68Zn foils were irradiated were irradiated with 11.5 MeV protons for 1-3 h at a beam current of 30-75 µA in a GET PETTRACE cyclotron. The irradiated foils were dissolved in 10 N HCl and the solution passed through a cation exchanging resin to trap 68Ga and 68Zn. 68Zn was then eluted from the column using HBr in 80% acetone. Finally, 68Ga was eluted with 3 N HCl and used to label 68Ga-PSMA-11. In order to study the impact of injected mass on tumor and normal organ of [asterisk]Ga-PSMA-11 we injected mice bearing PSMA expressing PC3-PIP or LNCaP tumors with 3.3 - 10000 pmol of total ligand (67Ga-PSMA-11 and PSMA-11) labeled with 100-200 µCi of (67Ga) and performed a biodistribution study 1 h post injection. We also injected tumor bearing with cyclotron produced and generator produced 68Ga-PSMA-11 and performed PET imaging studies 1h post injection. Results: Up to 4.2 Ci of 68Ga were produced from the 68Zn foil before purification and, 1.8 Ci after purification. The 67Ga content was less than 0.1%. Specific activity of the produced 68Ga-PSMA-11 was 2.2-7.0 mCi/µg and radiochemical purity > 99%. The 68Ga-PSMA-11 met all the usual criteria for PET radiopharmaceuticals (bubble, LAL, and sterility test). There was no significant change in tumor uptake of 67Ga-PSMA-11 up to an injected mass of 1000 pmol for PC3-PIP and LNCaP tumors (333-fold range). PET images of tumor bearing mice injected with 800 pmol of 68Ga-PSMA11 demonstrated identical biodistribution in normal organs and tumors. Conclusions: 68Ga can be produced in Ci amounts using a medical cyclotron. The cyclotron produced 68Ga product appears suitable for labeling PSMA11 for future clinical trials.The injected mass of PSMA-11 (on a per weight basis) had no significant impact on tumor uptake in mice up to a more than 300-fold higher mass than used for human PET/CT studies with 68Ga-PSMA11. This suggests that batches of cyclotron produced 68Ga-PSMA11 may be shipped for several hours after production.

Synthesis and evaluation of 99mTc/Re-tricarbonyl complexes of the triphenylphosphonium cation for mitochondrial targeting

IntroductionLipophilic delocalized cations accumulate in tumor cell mitochondria due to their higher transmembrane potential. In this work, this strategy was adopted for the development of 99mTc tumor-targeted imaging agents. MethodsTwo tridentate ligands containing the triphenylphosphonium cation, L1 (S-cysteinyl) and L2 (N-iminodiacetate) as well as the respective 99mTc/ReL1 and 99mTc/ReL2 tricarbonyl complexes were synthesized. The effect of the ligands and the Re complexes on cell growth in U-87 MG glioblastoma cells was assessed. In vitro stability studies and measurement of logP of the 99mTc tracers was performed. The cellular and mitochondrial uptake of the 99mTc tracers in U-87 MG cells was evaluated. Biodistribution of 99mTcL1 and 99mTcL2 were performed on SCID mice bearing U-87 MG tumors. ResultsThe ligands L1, L2 and the Re1 and ReL2 complexes were characterized spectroscopically. Single products 99mTcL1 and 99mTcL2, >90% stable in rat serum, were obtained. LogP was 0.40±0.14 for 99mTcL1 and −0.02±0.07 for 99mTcL2. L1, ReL1 and ReL2 caused no notable cytotoxicity and L2 was found to infer 40% inhibition of cellular growth at 10−5M as well as 80% cell death in culture at 10−4M. The cell uptake of 99mTcL1 and 99mTcL2 over 4h was 1.26±0.08% and 0.06±0.01% respectively, of which 13.41±3.63% and 18.61±6.19% was distributed in the mitochondria respectively. The initial tumor uptake in mice was found to be >1% ID/g for both 99mTc tracers. ConclusionsIn vitro mitochondrial and in vivo tumor targeting was observed, better in 99mTcL1, however these properties should be optimized in future studies.Advances in Knowledge and Implications for Patient Care: Continuous efforts in this direction may lead to a suitable mitochondrial-targeted 99mTc imaging agent for tumor detection.

Human Epidermal Growth Factor Receptor 3-Specific Tumor Uptake and Biodistribution of 89Zr-MSB0010853 Visualized by Real-Time and Noninvasive PET Imaging.

The human epidermal growth factor receptor 3 (HER3) is an interesting target for antitumor therapy. For optimal HER3 signaling inhibition, a biparatopic Nanobody construct (MSB0010853) was developed that binds 2 different HER3 epitopes. In addition, MSB0010853 contains a third HER3 epitope that binds albumin to extend its circulation time. MSB0010853 is cross-reactive with HER3 and albumin of mouse origin. We aimed to gain insight into MSB0010853 biodistribution and tumor uptake by radiolabeling the Nanobody construct with 89Zr. Methods: MSB0010853 was radiolabeled with 89Zr. Dose- and time-dependent tumor uptake was studied in nude BALB/c mice bearing a subcutaneous HER3 overexpressing H441 non-small cell lung cancer xenograft. Dose-dependent biodistribution of 89Zr-MSB0010853 was assessed ex vivo at 24 h after intravenous injection. Protein doses of 5, 10, 25, 100, and 1,000 μg were used. Time-dependent biodistribution of MSB0010853 was analyzed ex vivo at 3, 6, 24, and 96 h after intravenous administration of 25 μg of 89Zr-MSB0010853. PET imaging and biodistribution were performed 24 h after administration of 25 μg of 89Zr-MSB0010853 to mice bearing human H441, FaDu (high HER3 expression), or Calu-1 (no HER3 expression) tumor xenografts. Results: Radiolabeling of MSB0010853 with 89Zr was performed with a radiochemical purity of greater than 95%. Ex vivo biodistribution showed protein dose- and time-dependent distribution of 89Zr-MSB0010853 in all organs. Uptake of 89Zr-MSB0010853 in H441 tumors was only time-dependent. Tumor could be visualized up to at least 96 h after injection. The highest mean SUV of 0.6 ± 0.2 was observed at 24 h after injection of 25 μg of 89Zr-MSB0010853. 89Zr-MSB0010853 tumor uptake correlated with HER3 expression and was highest in H441 (6.2 ± 1.1 percentage injected dose per gram [%ID/g]) and lowest in Calu-1 (2.3 ± 0.3 %ID/g) xenografts. Conclusion:89Zr-MSB0010853 organ distribution and tumor uptake in mice are time-dependent, and tumor uptake correlates with HER3 expression. In contrast to tumor uptake except for kidney uptake, organ distribution of 89Zr-MSB0010853 is protein dose-dependent for the tested doses. 89Zr-MSB0010853 PET imaging gives insight into the in vivo behavior of MSB0010853.

Dual-Modality Imaging of Prostate Cancer with a Fluorescent and Radiogallium-Labeled Gastrin-Releasing Peptide Receptor Antagonist.

We designed and synthesized an IRDye 650 and DOTA-conjugated GRPr antagonist, HZ220 (DOTA-Lys(IRDye 650)-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2), by reacting DOTA-Lys-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2 (HZ219) with IRDye 650 N-hydroxysuccinimide (NHS) ester. Receptor-specific binding of gallium-labeled HZ220 was characterized in PC-3 prostate cancer cells (PC-3), and tumor uptake in mice was imaged with PET/CT and fluorescence imaging. Receptor binding affinity, in vivo tumor uptake, and biodistribution were compared with the GRPr antagonists HZ219, DOTA-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2 (DOTA-AR), and DOTA-(4-amino-1-carboxymethyl-piperidine)-[D-Phe6, Sta13]-BN(6-14)NH2 (DOTA-RM2). After hydrophilic-lipophilic balance cartridge purification, 68Ga-HZ220 was obtained with a radiochemical yield of 56% ± 8% (non-decay-corrected), and the radiochemical purity was greater than 95%. Ga-HZ220 had a lower affinity for GRPr (inhibitory concentration of 50% [IC50], 21.4 ± 7.4 nM) than Ga-DOTA-AR (IC50, 0.48 ± 0.18 nM) or Ga-HZ219 (IC50, 0.69 ± 0.18 nM). Nevertheless, 68Ga-HZ220 had an in vivo tumor accumulation similar to 68Ga-DOTA-AR (4.63 ± 0.31 vs. 4.07 ± 0.29 percentage injected activity per mL [%IA/mL] at 1 h after injection) but lower than that of 68Ga-DOTA-RM2 (10.4 ± 0.4 %IA/mL). The tumor uptake of 68Ga-HZ220 was blocked significantly with an excessive amount of GRP antagonists. IVIS spectrum imaging also visualized PC-3 xenografts in vivo and ex vivo with a high-contrast ratio. Autoradiography and fluorescent-based microscopic imaging with 68Ga-HZ220 consistently colocated the expression of GRPr. 68Ga-HZ220 displayed a higher kidney uptake than both 68Ga-DOTA-AR and 68Ga-DOTA-RM2 (16.9 ± 6.5 vs. 4.48 ± 1.63 vs. 5.01 ± 2.29 %IA/mL). 68Ga-HZ220 is a promising bimodal ligand for noninvasive PET imaging and intraoperative optical imaging of GRPr-expressing malignancies. Bimodal nuclear/fluorescence imaging may not only improve cancer detection and guide surgical resections, but also improve our understanding of the uptake of GRPr ligands on the cellular level.

99mTc-labeled gastrins of varying peptide chain length: Distinct impact of NEP/ACE-inhibition on stability and tumor uptake in mice

IntroductionIn situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three 99mTc-labeled gastrins of varying peptide chain length: [99mTc]SG6 ([99mTc-N4-Gln1]gastrin(1–17)), [99mTc]DG2 ([99mTc-N4-Gly4,DGlu5]gastrin(4–17)) and [99mTc]DG4 ([99mTc-N4-DGlu10]gastrin(10–17)). MethodsMouse blood samples were collected 5min after injection of each of [99mTc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/−) xenografts at 4h postinjection (pi). [99mTc]SG6 or [99mTc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [99mTc]DG2 control and PA animal groups were only included. ResultsTreatment of mice with PA induced significant stabilization of 99mTc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 [99mTc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only [99mTc]DG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [99mTc]DG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios. ConclusionsIn situ NEP/ACE-inhibition diversely affected the in vivo profile of 99mTc-radioligands based on different-length gastrins. Truncated [99mTc]DG4 exhibited overall the most attractive profile during combined NEP/ACE-inhibition in mouse models, providing new opportunities for CCK2R-expressing tumor imaging in man with SPECT.

In vitro and in vivo evaluation of a 64Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer

Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with 64Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate 64Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with 64Cu and to evaluate the resulting peptide. p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and 125I-Tyr4-BN to determine the IC50 value. The peptide was radiolabeled with 64Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies. The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC50 of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, the peptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported 64Cu-labeled BN analogues. These studies demonstrate that 64Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer.

PET and MRI of Metastatic Peritoneal and Pulmonary Colorectal Cancer in Mice with Human Epidermal Growth Factor Receptor 1–Targeted 89Zr-Labeled Panitumumab

Human epidermal growth factor receptor 1 (HER1) plays an important role in the pathogenesis of colorectal cancer. Panitumumab is an anti-HER1 monoclonal antibody approved for use in colorectal cancer. However, few data exist regarding HER1 status in the corresponding distant metastases, and little corresponding information is available regarding the localization of panitumumab at primary and metastatic lesions. The utility of PET and MRI using (89)Zr-panitumumab to assess the status of HER1 in distant metastases with different metastasis models is presented in this study. In vivo biodistribution and PET studies were performed in HER1-expressing LS-174T and HER1-negative A375 tumor xenografts. Additionally, studies were performed in different models of intraperitoneal and pulmonary metastases. MRI studies were performed for metastatic models to characterize the targeting potential of (89)Zr-panitumumab at different lesion sites. HER1-mediated targeting was achieved in all HER1-expressing models. The LS-174T tumor area under the curve (AUC) was 3.7-fold greater than the AUC for A375. The LS-174T tumor AUC of 204.13 ± 9.67 was significantly greater (P < 0.001) than the LS-174T tumor AUC of 36.45 ± 1.39 obtained from mice coinjected with 0.1 mg of panitumumab for blocking the target. Differences were observed in 2 intraperitoneal models; tumor uptake in mice with a 3-d tumor burden was more than 2-fold greater than the mice with a 7-d tumor burden. PET and MRI studies revealed HER1-mediated tumor targeting in all metastatic models. However, significant differences were observed between different LS-174T tumor models. Peak tumor uptake of approximately 40 percentage injected dose per gram (%ID/g) was observed at 3-4 d after injection for the subcutaneous tumor model, in contrast to approximately 75 %ID/g at 2 d after injection for the thoracic tumors and approximately 95 %ID/g at 1-2 d after injection for the intraperitoneal tumors. The potential utility of (89)Zr-panitumumab in assessing HER1 status in distant metastases and understanding the variations in antibody uptake at different lesion sites is demonstrated in this study. (89)Zr-panitumumab can play a vital role in patient stratification and immunotherapy and therefore warrants further investigation for clinical translation.

Abstract 5303: Serial HER2-PET imaging with 89Zr-trastuzumab-F(ab’)2 of lapatinib and 17AAG treated mice bearing SKBR3 xenografts

Abstract Background: Understanding molecular responses involved in human epidermal growth factor receptor-2 (HER2) targeted drugs may improve treatment of HER2 positive breast cancer. In vivo assessment of effects on HER2 expression is of value in this setting. The EGFR/HER2 tyrosine kinase inhibitor lapatinib inhibits HER2 signaling and stabilizes HER2 at the cell membrane, while the heat shock protein-90 (HSP90) inhibitor 17AAG degrades HER2/EGFR. We therefore evaluated the effect on HER2 dynamics in vivo with 89Zr-trastuzumab-F(ab’)2 HER2-PET imaging in human tumor bearing mice treated with lapatinib and 17AAG. Materials and methods: Effects of lapatinib and 17AAG on EGFR and HER2 membrane expression were evaluated in vitro in the human breast cancer cell line SKBR3 using flow cytometry. In vivo effects on HER2 expression were evaluated with 89Zr-trastuzumab-F(ab’)2 μPET imaging in SKBR3 xenograft bearing mice. 89Zr-trastuzumab-F(ab’)2 (5 MBq; 20 μg) was injected two times with a 7 day interval and μPET imaging was performed 24 hours after tracer injection. Lapatinib was dosed 100 mg/kg/day orally for 6 days, starting 1 day after the first μPET scan. 17AAG was dosed 3×50 mg intraperitoneal in the 24 hours prior to the second tracer injection. A non-treated group of mice served as control and for validation of 89Zr-trastuzumab-F(ab’)2 μPET imaging. Imaging data were compared with ex vivo biodistribution analysis, immunohistochemistry, and with an in vitro HER2 internalization experiment. Results: On flow cytometry, lapatinib induced upregulation of EGFR (+70%; P = 0.0011), but not HER2 (+19%; P = 0.1275). 17AAG treatment lowered both EGFR (−41%; P = 0.016) and HER2 (−76%; P = 0.022) expression. This EGFR/HER2 reduction by 17AAG was inhibited by lapatinib. In vivo, 89Zr-trastuzumab-F(ab’)2 tumor uptake on the second μPET scan was lowered by lapatinib (−32%; P = 0.00004), 17AAG (−34%; P = 0.0022) and the lapatinib plus 17AAG combination (−53%; P = 0.011). 89Zr-trastuzumab-F(ab’)2 tumor uptake in mice treated with lapatinib plus 17AAG was lower than with either lapatinib or 17AAG alone (P = 0.0043 and P = 0.0022, respectively). In vitro, 89Zr-trastuzumab-F(ab’)2 internalization was reduced by lapatinib (−25%; P = 0.0022), explaining the lower 89Zr-trastuzumab-F(ab’)2 tumor uptake after lapatinib. Conclusion: 89Zr-trastuzumab-F(ab’)2 μPET visualized treatment induced impact on HER2 expression and -dynamics. Lapatinib inhibited HER2 internalization, and thereby decreased 89Zr-trastuzumab-F(ab’)2 tumor uptake. HSP90 inhibition lowered HER2 membrane expression and thereby 89Zr-trastuzumab-F(ab’)2 tumor uptake. This knowledge on HER2 dynamics ameliorates understanding of efficacy of several combinations of HER2 targeted agents. Supported by grant 2007-3739 of the Dutch Cancer Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2011-5303

In Vitro and In Vivo Evaluation of 64Cu-Labeled SarAr-Bombesin Analogs in Gastrin-Releasing Peptide Receptor–Expressing Prostate Cancer

Bombesin is a 14-amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7-14) for radiolabeling with (64)Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin(7-14), radiolabel the conjugate with (64)Cu, and evaluate in vitro and in vivo. SarAr was synthesized as previously published and conjugated to bombesin(7-14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7-14) to yield the resulting SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and (125)I-Tyr(4)-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with (64)Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with (64)Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported (64)Cu-labeled bombesin analogs. The (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) had more rapid blood clearance and lower tumor and normal-tissue uptake than (64)Cu-SarAr-SA-Aoc-bombesin(7-14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h). These studies demonstrate that (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.

PET imaging of tumor angiogenesis in mice with VEGF‐A–targeted 86Y‐CHX‐A″‐DTPA‐bevacizumab

Bevacizumab is a humanized monoclonal antibody that binds to tumor-secreted vascular endothelial growth factor (VEGF)-A and inhibits tumor angiogenesis. In 2004, the antibody was approved by the US Food and Drug Administration (FDA) for the treatment of metastatic colorectal carcinoma in combination with chemotherapy. This report describes the preclinical evaluation of a radioimmunoconjugate, (86)Y-CHX-A″-DTPA-bevacizumab, for potential use in Positron Emission Tomography (PET) imaging of VEGF-A tumor angiogenesis and as a surrogate marker for (90)Y-based radioimmunotherapy. Bevacizumab was conjugated to CHX-A″-DTPA and radiolabeled with (86)Y. In vivo biodistribution and PET imaging studies were performed on mice bearing VEGF-A-secreting human colorectal (LS-174T), human ovarian (SKOV-3) and VEGF-A-negative human mesothelioma (MSTO-211H) xenografts. Biodistribution and PET imaging studies demonstrated highly specific tumor uptake of the radioimmunoconjugate. In mice bearing VEGF-A-secreting LS-174T, SKOV-3 and VEGF-A-negative MSTO-211H tumors, the tumor uptake at 3 days postinjection was 13.6 ± 1.5, 17.4 ± 1.7 and 6.8 ± 0.7 % ID/g, respectively. The corresponding tumor uptake in mice coinjected with 0.05 mg cold bevacizumab were 5.8 ± 1.3, 8.9 ± 1.9 and 7.4 ± 1.0 % ID/g, respectively at the same time point, demonstrating specific blockage of the target in VEGF-A-secreting tumors. The LS-174T and SKOV3 tumors were clearly visualized by PET imaging after injecting 1.8-2.0 MBq (86)Y-CHX-A″-DTPA-bevacizumab. Organ uptake quantified by PET closely correlated (r(2) = 0.87, p = 0.64, n = 18) to values determined by biodistribution studies. This preclinical study demonstrates the potential of the radioimmunoconjugate, (86)Y-CHX-A″-DTPA-bevacizumab, for noninvasive assessment of the VEGF-A tumor angiogenesis status and as a surrogate marker for (90)Y-CHX-A″-DTPA-bevacizumab radioimmunotherapy.

Preparation, Biological Evaluation, and Pharmacokinetics of the Human Anti-HER1 Monoclonal Antibody Panitumumab Labeled with 86Y for Quantitative PET of Carcinoma

Panitumumab, a human monoclonal antibody that binds to the epidermal growth factor receptor (HER1), was approved by the Food and Drug Administration in 2006 for the treatment of patients with HER1-expressing carcinoma. In this article, we describe the preclinical development of (86)Y-CHX-A''-diethylenetriaminepentaacetic acid (DTPA)-panitumumab for quantitative PET of HER1-expressing carcinoma. Panitumumab was conjugated to CHX-A''-DTPA and radiolabeled with (86)Y. In vivo biodistribution, PET, blood clearance, area under the curve, area under the moment curve, and mean residence time were determined for mice bearing HER1-expressing human colorectal (LS-174T), prostate (PC-3), and epidermoid (A431) tumor xenografts. Receptor specificity was demonstrated by coinjection of 0.1 mg of panitumumab with the radioimmunoconjugate. (86)Y-CHX-A''-DTPA-panitumumab was routinely prepared with a specific activity exceeding 2 GBq/mg. Biodistribution and PET studies demonstrated a high HER1-specific tumor uptake of the radioimmunoconjugate. In mice bearing LS-174T, PC-3, or A431 tumors, the tumor uptake at 3 d was 34.6 +/- 5.9, 22.1 +/- 1.9, and 22.7 +/- 1.7 percentage injected dose per gram (%ID/g), respectively. The corresponding tumor uptake in mice coinjected with 0.1 mg of panitumumab was 9.3 +/- 1.5, 8.8 +/- 0.9, and 10.0 +/- 1.3 %ID/g, respectively, at the same time point, demonstrating specific blockage of the receptor. Normal organ and tumor uptake quantified by PET was closely related (r(2) = 0.95) to values determined by biodistribution studies. The LS-174T tumor had the highest area under the curve (96.8 +/- 5.6 %ID d g(-1)) and area under the moment curve (262.5 +/- 14.9 %ID d(2) g(-1)); however, the tumor mean residence times were identical for all 3 tumors (2.7-2.8 d). This study demonstrates the potential of (86)Y-CHX-A''-DTPA-panitumumab for quantitative noninvasive PET of HER1-expressing tumors and represents the first step toward clinical translation.

Radiochemical Synthesis, Rodent Biodistribution and Tumor Uptake, and Dosimetry Calculations of [11C] Methylated LY2181308

The aim of the study was to develop a rapid and reproducible method to label LY2181308, an antisense oligonucleotide to Survivin, with carbon-11 in order to study its in vivo biodistribution and tumor uptake in rodents and its human dosimetry based on baboon data. Randomly [¹¹C] methylated LY2181308 was produced with [¹¹C] methyl iodide. The biodistribution was performed in female Sprague-Dawley (SD) rats and EMT-6 tumor-bearing mice in the presence of nonradioactive LY2181308. Human dosimetry calculations were based on baboon PET studies. In SD rats, the kidney and liver were the organs with the most accumulation of radioactivity. Tumor uptake in mice was also relatively high after 5 min and remained constant for up to 1 h. Baboon dosimetry suggested that up to 42 mCi of radioactivity could be administered to human with a dose-limiting organ being the kidneys with a radiation dose of 32 µGy/MBq (0.118 rad/mCi). [¹¹C] methylated LY2181308 to rodents and baboons showed its biodistribution, tumor uptake, and human dosimetry evaluation. These results should facilitate the understanding of the pharmacokinetics of LY2181308 prior to use as a potential new therapeutic agent in oncology as well as to warrant more in vivo validations as a potentially useful tumor-imaging agent.

Radiolabeling RGD peptide and preliminary biodistribution evaluation in mice bearing S180 tumors

To prepare the rhenium-188 (188Re)-arginine-glycine-aspartic acid (RGD) peptide in a convenient manner and to evaluate its potential as an agent for alphavbeta3 integrin receptor-positive tumors. Radiolabeled RGD was obtained by conjugating the His group at the end of peptide with fac-[188Re(H2O)3(CO)3]+. Chelating efficiency of fac-[188Re(H2O)3(CO)3]+ and radiolabeling efficiency of radiolabeled peptide were measured by thin-layer chromatography and high-performance liquid chromatography. In-vitro stability of the radio-complex was determined in phosphate-buffered saline (0.05 mol/l, pH 7.4), new-born calf serum, His or Cys solution at 37 degrees C or room temperature and analyzed by thin-layer chromatography. A biodistribution study was carried out in mice bearing S180 tumors. 188Re-RGD was obtained with a more than 95% of radiolabeling efficiency, and showed high stability in phosphate-buffered saline, new-born calf serum, His and Cys solution. Furthermore, this radio-complex was cleared rapidly from the blood and showed specific tumor uptake in mice bearing S180 tumors. 188Re-RGD was prepared by a simple method. Preliminary biodistribution results showed its potential as an agent for cancer therapy and encouraged further investigation.

Targeting Beta-3 Integrin Using a Linear Hexapeptide Labeled with a Near-Infrared Fluorescent Molecular Probe

Biomolecules containing the RGD peptide sequence are known to bind integrins with high affinity. Studies of hexa-and hepta-peptides labeled with a near-infrared fluorescent probe (cypate) showed that rearranging the glycine in a linear RGD peptide sequence to form the GRD analogue favored the uptake of the GRD compound by alphavbeta3 integrin receptor (ABIR)-positive A549 tumor cells and tissue. The internalization of the GRD compound in A549 cells and tumor uptake in mice were inhibited by ABIR-avid peptides, suggesting its recognition by this receptor. Further studies with functional blocking antibodies and beta3 knockout cells revealed that beta3 integrin mediates the internalization of the cypate-GRD peptide. Molecular modeling studies supported preferential interaction of the probe with the beta3 subunit of integrins relative to the alphav subunit. The results demonstrate that the cypate-GRD peptide targets beta3 integrin, thereby providing a strategy to monitor drug delivery and efficacy, and physiopathologic processes mediated by this protein.

131I-Lym-1 in mice implanted with human Burkitt's lymphoma (Raji) tumors: loss of tumor specificity due to radiolysis.

Preliminary evaluations of 125I-labeled Lym-1, an anti-lymphoma mouse IgG2a monoclonal antibody, demonstrated favorable tumor uptake in mice bearing human Burkitt's lymphoma (Raji) tumors. In this study, the pharmacokinetics of 125I- and 131I-Lym-1, and the dosimetry, efficacy, and toxicity of 131I-Lym-1 in Raji-tumored mice were evaluated. Lym-1 was radioiodinated by the chloramine-T method and analyzed for monomeric fraction and immunoreactivity (antigen cell binding, relative to unmodified Lym-1). Nude mice bearing Raji tumors (20-500 mm3) received 1.5 MBq (40 microCi) 125I-Lym-1, or 1.5, 7.4, 14.8, or 18.5 MBq (40, 200, 400, or 500 microCi) 131I-Lym-1. Pharmacokinetic data (total body and blood clearance and biodistribution) were used to estimate radiation dosimetry. Mini-thermoluminescent dosimetry (TLD) was also used to measure radiation dosimetry directly for 7 days after injection of 131I-Lym-1. Tumor size, survival, body weight, and blood counts were monitored for 60 days to evaluate therapeutic efficacy and toxicity of 131I-Lym-1. At the time of injection, the mean quality assurance (QA) values for 125I-Lym-1 were 100% monomer and 100% relative immunoreactivity; the corresponding values for 131I-Lym-1 were 73% and 66%, indicating that radiolysis had occurred during the interval between radiolabeling and injection. 125I-Lym-1 exhibited high and sustained concentration in tumors relative to normal organs, whereas 131I-Lym-1 did not. Assuming identical pharmacokinetic behavior to 125I-Lym-1, 131I-Lym-1 would deliver radiation doses of 3.45, 0.83, 1.03, 0.34, and 0.56 Gy per MBq injected (12.8, 3.1, 3.8, 1.3, and 2.1 rad/microCi), to tumor, liver, lungs, total body, and marrow, respectively. When the actual pharmacokinetic data for 131I-Lym-1 (1.5 MBq) were used to estimate dosimetry, corresponding values of 0.51, 0.72, 0.49, 0.31, and 0.41 Gy/MBq (1.9, 2.7, 1.8, 1.1, and 1.5 rad/microCi) were obtained. Similar values were obtained for mice receiving 7.4 or 14.8 MBq of 131I-Lym-1. Similarly, TLD data indicated little preferential radiation dosimetry to tumor. Response rates (cure + CR + PR) for mice receiving 0, 7.4, 14.8, and 18.5 MBq of 131I-Lym-1 were 8%, 7%, 21%, and 45%, respectively. The LD50/30 dose of 131I-Lym-1 was 12.7 MBq (343 microCi). 125I-Lym-1 exhibited high and sustained concentration in Raji tumors in mice, indicating excellent therapeutic potential for 131I-Lym-1. However, in vitro QA results for 131I-Lym-1 indicated that radiolysis had occurred, and 131I-Lym-1 demonstrated little accumulation in tumor, or preferential radiation dosimetry to tumor in the same model.