Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
A dual click 18F-labeling strategy for pretargeted PET imaging of the tumor-targeting monoclonal antibody CC49 (#290)
E. Johanna L. Stéen1, 2, Jesper T. Jørgensen2, 3, Kamilla Nørregaard2, 3, Raffaella Rossin5, Christoph Denk4, Martin Wilkovitsch4, Dennis Svatunek4, Klas Bratteby1, 2, Patricia E. Edem1, 2, 3, Claudia Kuntner6, Thomas Wanek6, Marc Robillard5, Jesper L. Kristensen1, Andreas Kjær2, 3, Hannes Mikula4, Matthias M. Herth1, 2
1 University of Copenhagen, Department of Drug Design and Pharmacology, Copenhagen, Denmark
Pretargeted PET imaging using the ligation between a radiolabeled tetrazine (Tz) and a monoclonal antibody (mAb) modified with a trans-cyclooctene (TCO) allows for the use of short-lived radionuclides, such as fluorine-18.1,2 The objective of the present study was to develop a library of 18F-labeled tetrazines via indirect radiolabeling for subsequent evaluation in pretargeted PET imaging of the TAG-72 targeting mAb CC49 in murine colon carcinoma.3
A library of Tz-derivatives was obtained via Cu-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) between alkyne-modified Tz-derivatives and 18F/19F-fluorinated azides (Fig. 1). To assess their potential as PET tracers for pretargeting, a blocking assay was carried out in BALB/c mice bearing colon carcinoma LS174T xenografts. Here, the ability of each Tz to block a previously described 111In-labeled Tz was studied.1 The mice were pretreated with TCO-modified CC49 (100 µg, 6.7 nmol, ~7 TCO/mAb) 72 h prior to the administration of the non-radioactive Tz. After 2 h, the 111In-labeled Tz was injected and any potential blocking of TCO-moieties on the mAb in the tumor could be determined. Pretargeted PET/CT imaging was performed using the same tumor model and mAb as for the blocking assay.
Radiolabeling via the CuAAC provided 18F-labeled tetrazines in decay corrected radiochemical yields of up to 68% and high radiochemical purity (˃94%). The performances of the non-radioactive tetrazines in the blocking assay were correlated to parameters such as reaction kinetics and lipophilicity. In general, high rate constants (>200 M-1s-1) with a calculated distribution coefficient (clogD7.4) below 0.5 were favorable for in vivo pretargeting. Pretargeted PET imaging was performed with six 18F-labeled tetrazines from the library to assess the predictive ability of the assay. The best tetrazine in the blocking assay (99% blocking) also showed the highest tumor uptake (2.5 ± 1.0 % ID/g 1 h after tracer administration) in pretargeting studies using the 18F-labeled analog.
In the present work we have developed a library of 18F-labeled tetrazines. Evaluation of the library revealed a tetrazine lead compound, which bound to the mAb at the tumor-site using high molar activity conditions. Current efforts are directed toward improving tumor-to-background ratios by using a clearing agent that can remove remaining mAb in the blood pool prior to injection of the 18F-labeled tetrazine.
The authors greatly acknowledge the H2020 project Click-it, under grant agreement no. 668532 for financial support.
 Rossin, R.; Verkerk, P. R.; van den Bosch, S. M.; Vulders, R. C. M.; Verel, I.; Lub, J.; Robillard, M. S. 2010, In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice. Angew. Chem. Int. Edit., 49 (19), 3375-3378.
 Denk, C.; Svatunek, D.; Filip, T.; Wanek, T.; Lumpi, D.; Frohlich, J.; Kuntner, C.; Mikula, H. 2014, Development of a F-18-Labeled Tetrazine with Favorable Pharmacokinetics for Bioorthogonal PET Imaging. Angew. Chem. Int. Edit., 53 (36), 9655-9659.
 Rossin, R.; van den Bosch, S. M.; Ten Hoeve, W.; Carvelli, M.; Versteegen, R. M.; Lub, J.; Robillard, M. S. 2013, Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems. Bioconjug. Chem., 24 (7), 1210-7.
Figure 1. Illustration of the dual click strategy for the 18F-labeling of CC49-TCO
A dual click strategy for the 18F-labeling of CC49-TCO in murine colon carcinoma. The CuAAC was performed between each tetrazine building block and a 18F-labeled azide synthon (click 1), respectively. The 18F-labeled click product was injected into tumor bearing mice, where it reacted with CC49-TCO that has accumulated at the tumor-site (click 2).
Keywords: Pretargeting, immuno-PET, fluorine-18, click chemistry
Tissue penetration and retention of gallium-68 labeled dendrimer-based nanoparticles in pancreatic tumor-bearing mice (#455)
Béatrice Louis1, 2, Philippe Garrigue1, 2, Anaïs Moyon1, 2, Ahlem Bouhlel1, 2, Laure Balasse2, Samantha Fernandez2, Stéphanie Simoncini1, Ling Ding3, Eric Mas4, Françoise Dignat-George1, Ling Peng3, Benjamin Guillet1, 2
1 Aix-Marseille Université, C2VN, UMR INSERM 1263 INRA 1260, Marseille, France
The incidence of pancreatic cancer keeps increasing with a poor survival. For an earlier diagnosis, new medical imaging tools are needed. Nanometric size promotes penetration of chemicals into solid tumors. In this context, our group has developed a new nanosized imaging agent based on gallium-68 labeled dendrimers: [68Ga]Ga-dend. The aim of the present study was to characterize the [68Ga]Ga-dend blood and tissue distribution in orthotopic pancreatic tumor-bearing mice.
Athymic nude mice, either healthy or bearing orthotopic SOJ-6 human pancreatic tumor cells were injected intravenously with [68Ga]Ga-dend. Anesthetized mice were then imaged using Positron Emission Tomography (PET). PET quantifications were realized at 5, 10, 15, 20, 30, 60, 90 and 120 min post-injection in the organs of interest: brain, heart, liver, kidneys, bladder, spleen, lungs, gut and tumor. Tumor blood flow was assessed by echo-Doppler.
The dendrimer formulation of [68Ga]Ga allowed a longer residence time in the organism, which can be ascribed to a slower elimination. The uptake of [68Ga]Ga-dend in tumors was steadily increased, while that in the liver was significantly decreased within the 2-h imaging period. This finding is also in line with the characteristic size and charge features of the [68Ga]Ga-dend nanoparticles. Indeed, their nanosize allowed them to be readily trapped and enriched in the tumor via the EPR effect, and in the liver through the reticuloendothelial system, whereas their elimination was balanced between the kidneys and the intestines. We observed a significant negative correlation between the tumor uptake of [68Ga]Ga-dend and tumor blood flow as measured by 3D- Doppler. This suggests that [68Ga]Ga-dend is more likely to accumulate in low-turbulent blood flow tumors, where the EPR effect contribution takes effect.
Collectively, the combination of high tumor signal stemming from the EPR effect, short biological half-life, and the absence of accumulation in most of the organs except the liver, all contribute to the favorable pharmacokinetic properties of the [68Ga]Ga-dend nanoparticles for PET imaging and quantification of the EPR effect.
AcknowledgmentThe authors are thankful to Michel Skandalovski, Samy Vigier and Sandrine Pons for technical support.
 Garrigue P, Tang J, Ding L, Bouhlel A, Tintaru A, Laurini E, Huang Y, Lyu Z, Zhang M, Fernandez S, Balasse L, Lan W, Mas E, Marson D, Weng Y, Liu X, Giorgio S, Iovanna J, Pricl S, Guillet B, Peng L (2018) Self-assembling supramolecular dendrimer nanosystem for PET imaging of tumors. PNAS 201812938 . https://doi.org/10.1073/pnas.1812938115
 Wei T, et al. (2015) Anticancer drug nanomicelles formed by self-assembling amphi- philic dendrimer to combat cancer drug resistance. Proc Natl Acad Sci USA 112:
 Liu X, et al. (2015) Promoting siRNA delivery via enhanced cellular uptake using an arginine-decorated amphiphilic dendrimer. Nanoscale 7:3867–3875.
 Filippi M, et al. (2014) Dendrimersomes: A new vesicular nano-platform for MR- molecular imaging applications. Chem Commun (Camb) 50:3453–3456.
 Filippi M, et al. (2015) Novel stable dendrimersome formulation for safe bioimaging applications. Nanoscale 7:12943–12954.
Keywords: dendrimer, nanoparticle, PET, EPR effect, pancreatic cancer
Towards imaging cisplatin resistance with 64Cu PET (#244)
Fahad Al-salemee1, Joanna Bartnicka1, Timothy H. Witney1, Philip Blower1
1 King's College London, Imaging Chemistry and Biology, London, United Kingdom
Cisplatin is the backbone of several treatment regimens for various cancer types, including ovarian cancer; however, its efficacy is often abolished by the early development of resistance, especially in ovarian cancer patients. Consequently, many patients suffer the debilitating side effects of cisplatin in vain. A correlation between cellular accumulation of cisplatin and copper transporters (CTR1, ATP7A, ATP7B) has been repeatedly reported in the literature.1–4 We investigated the novel possibility of using unchelated 64Cu(II) PET imaging to predict cisplatin accumulation in tumours in vivo.
Two variants of the human ovarian carcinoma cell line A2780, wild type (WT) and cisplatin-resistant (CisR),5 were used. Cisplatin resistance was compared between the two cell lines using a Sulforhodamine B assay allowing the determination of the half-maximal inhibitory concentration (IC50). Cellular accumulation and retention of [64Cu]CuCl2 and non-radioactive cisplatin (10 μM) were compared between the two cell lines at 10, 30, 60 and 120 minutes via gamma counting and ICP-MS, respectively. PET/CT imaging of xenograft-bearing (WT or CisR) female Balb/c nu/nu mice intravenously injected with acetate-buffered [64Cu]CuCl2 (1.5-4 MBq) was used to compare 64Cu accumulation between the two types of xenograft in vivo, dynamically for 2 hours post-injection (p.i.) and again at 24 hours p.i.
The IC50 value for WT cells was 2.2(±0.55) μM, compared to 10.3(±1.44) μM for the CisR cell line (n=3, p<0.001). 64Cu accumulation was significantly lower in CisR cells in vitro compared to WT (1.2±0.4 and 4.1±0.3 Bq/10^3 cells at 120 minutes, respectively)(n=3, p<0.001). The same was true for cisplatin accumulation (0.105±0.022 vs. 0.299±0.067 ng Pt/10^3 cells at 120 minutes)(n=3, p=0.009). 64Cu retention and that of platinum were comparable between the 2 cell lines at the time points tested. PET/CT images showed that the difference in 64Cu accumulation between CisR and WT xenografts was not statistically significant over the first 2 hours p.i. (3.3±1.3 and 1.6±0.5 %ID/g respectively at 120 minutes p.i.)(n=3, p=0.107). At 24 hours p.i., 64Cu accumulation was similar between WT and CisR xenografts (2.9±1.6 and 2.9±0.5 %ID/g, respectively). Ex vivo biodistribution at 24 hours confirmed imaging results (3±1.2 vs. 2.9±0.9 %ID/g for WT and CisR, respectively).
64Cu accumulation correlates well with cisplatin accumulation and sensitivity/resistance in vitro in A2780 cells. However, A2780 WT and CisR xenografts in mice did not differ in their 64Cu uptake. This highlights the limitations of in vitro experimentation as a predictor of in vivo behaviour of molecules, due to an inherent inability to take into account key factors influencing such behaviour, particularly pharmacokinetics and metabolism.
 Ishida S, Lee J, Thiele DJ, Herskowitz I. Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc Natl Acad Sci. 2002. doi:10.1073/pnas.162491399
 Holzer AK. The Copper Influx Transporter Human Copper Transport Protein 1 Regulates the Uptake of Cisplatin in Human Ovarian Carcinoma Cells. Mol Pharmacol. 2004. doi:10.1124/mol.104.001198
 Larson CA, Blair BG, Safaei R, Howell SB. The Role of the Mammalian Copper Transporter 1 in the Cellular Accumulation of Platinum-Based Drugs. Mol Pharmacol. 2008. doi:10.1124/mol.108.052381
 Kim ES, Tang XM, Peterson DR, et al. Copper transporter CTR1 expression and tissue platinum concentration in non-small cell lung cancer. Lung Cancer. 2014. doi:10.1016/j.lungcan.2014.04.005
 Ozols RF, Masuda H, Grotzinger KR, et al. Characterization of a cis-Diamminedichloroplatinum(II)-resistant Human Ovarian Cancer Cell Line and its use in Evaluation of Platinum Analogues. Cancer Res. 1987.
In vitro Results
A) Normalised survival % of WT and CisR cells as a function of log(cisplatin concentration) used. These curves were used to calculate IC50 values for the 2 cell lines B) Cu64 accumulation in WT and CisR cells over 120 minutes, represented as Bq/cell (n=3) C) Platinum accumulation in WT and CisR cells over 120 minutes, shown as ng platinum/10^3 cells (n=3) D) Cu64 retention % in WT and CisR cells over 120 minutes E) Platinum retention % in WT and CisR cells over 120 minutes.
In vivo results
A) Represantative PET/CT images showing 64Cu accumulation in WT and CisR xenografts at 10, 30, 60, 90 and 120 minutes. B) Imaging data represented as %ID/g of 64Cu in WT and CisR xenografts over 2 hours and at 24 hours p.i. (n=3). C) Ex vivo biodistribution at 24 hours showing the %ID/g in the two types of xenograft (n=5).
Keywords: Cu-64, cisplatin, PET, resistance, ovarian cancer
Development of an orthotopic mouse model of Pancreatic Ductal Adenocarcinoma (PDAC) by ultrasound(US)-guided injection (USGI) of MiaPaCa-1 cells (#481)
Tiziano Lottini1, Matteo Stefanini1, Claudia Duranti1, Rayhana Bouazzi1, Annarosa Arcangeli1
1 University of Florence, Department of Experimental and Clinical medicine, Florence, Italy
PDAC is one of the cancers with worst prognosis, with a five-year survival rate less than 6%1,2. This occurs not only because PDAC is often diagnosed at advanced stages, but also because of an intrinsic resistance of PDAC to the most common chemotherapeutic drugs. Moreover, to date for this type of tumor no molecular-target drugs have been identified. In preclinical research, the use of animal models, that mimic as much as possible the development of the disease, is mandatory. To this purpose we have developed an orthotopic mouse model of PDAC by using the not invasive USGI method.
The orthotopic PDAC mouse model was developed by USGI of 1 X 106 MiaPaCa-1 cells on the pancreas of athymic nude mice. A 3D acquisition of the mouse abdominal region was performed prior to xenografting to establish a baseline for comparative analysis. A 50 µL bolus of cells suspended in PBS was injected directly into the pancreas using 1 mL syringe with a 27 g needle previously placed in the mechanical syringe holder and lined up parallel to the transducer and perpendicular to the body. VevoLAZR-X system was used for USGI and for monitoring the tumor development.
The development of the PDAC has been characterized and monitored by US imaging. After one weeks, in all the mice we observed a pancreatic lesion of 6-7 mm3. After eleven days we detected a defined pancreatic tumor with a mean volume of 12 mm3. Further experiment for evaluating the therapeutic effects of a bifunctional antibody (scDb) that target the oncogenic hERG1 channel/β1 integrin complex are ongoing.
The USGI is a feasible and minimally invasive method for the establishment of orthotopic PDAC mouse model. In a preliminary set of experiment was observed a decrease of pAKT activation in mice treated with scDb suggesting a deregulation of proliferation and survival pathways.
AcknowledgmentWe would like to thank Visualsonics group for the training
 Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. 2013. IARC CancerBase No. 11. Available from http://globocan.iarc.fr accessed on 26 February 2014.
 -Partensky C. Toward a better understanding of pancreatic ductal adenocarcinoma: glimmers of hope. Pancreas. 2013;42:729–739.
Keywords: Ultrasound-guided injection, PDAC cancer, VevoLAZR-X
Multimodal imaging of melanoma spheroids (#81)
Bartosz Leszczynski1, Hanieh Karimi1, Tomasz Kolodziej1, Andrzej Wrobel1, Ewa Stepien1
1 Jagiellonian University, M. Smoluchowski Institute of Physics, Krakow, Poland
In many aspects, 2D cell cultures are not sufficient for tumor studies. Introduction of 3D spheroids allowed to mimic physical structure of the tumor, with a better representation of physiological characteristics and limitation of use of laboratory animals. A typical spheroid consists of three distinct cell layers of different proliferation rates, caused by diverse access to oxygen and nutrients: necrotic core, quiescent cells middle layer and proliferating cells external layer. Microtomography (micro-CT) adds third dimension to classic microscopic studies of spheroids.
Primary melanoma cell line WM115 and malignant melanoma cell line WM266 with initial cell densities of 500, 1000 and 1500 cells/15 µl of RPMI 1640 medium were used to form spheroids by hanging drop method. Spheroids were investigated after 24 to 72 h depending on cell line and initial density. Shape analysis and growth rate were calculated based on optical microscopy images. Fluorescein diacetate (FDA), calcein and propidium iodide (PI) were used to stain the spheroids for viability analysis by fluorescence microscopy. For micro-CT scanning spheroids were stained in Lugol solution (I3K) for 1.5 h. Samples were scanned with Bruker SkyScan 1172 with X-ray of 40 keV without filtering and pixel size of 1-2 µm. Image processing and analysis were performed using ImageJ, CTAn and CTVox software.
Shape analysis was carried out using circularity and roundness parameters. Both showed clear differences between cell lines. WM266 spheroids are more condensed, circularity and roundness increases with time. WM115 cell line features are not that predictable and have unstable growth rate between 36 to 49 µm/day. WM266 growth rate is more stable, and ranges from 33 to 38 µm/day. Fluorescence analysis of spheroid viability clearly shows the increase of the necrotic region in the spheroid core over growth time. In malignant WM266 spheroids the necrotic process is more visible. Micro-CT analysis shows that WM266 spheroids have more round well defined shapes and reach higher diameters than WM115. WM115 cell line rather forms clusters of decentralized low-diameter spheroids.
The 3D cell cultures such as spheroids provide a great model of the tumor structure. They mimic various features of the real tumor. There are clear differences in the geometry, viability and growth of the spheroids formed from different cell lines. Micro-CT visualization and analysis of spheroids in 3D is a powerful add to classical microscopic investigation methods.
Keywords: melanoma, micro-CT, spheroids
Site-selective antibody modification for in vivo imaging (#516)
Vera F. Ferreira1, Bruno L. Oliveira2, Alice D'Onofrio1, António Paulo1, 4, Gonçalo Bernardes2, 3, Filipa Mendes1, 4
1 Instituto Superior Técnico, Center for Nuclear Sciences and Technologies (C2TN), Lisboa, Portugal
Site-selective protein modification methods have been crucial for the development of new biologically active protein conjugates for in vivo applications.
A carbonylacrylic derivative bearing a TCO moiety was synthesized and conjugated to THIOMAB, an antibody engineered to contain a free cysteine at the light chain. To confirm the bioorthogonal reaction, the conjugate reacted with 6-methyl-tetrazine. Reactions were assessed by liquid chromatography-mass spectrometry (LC-MS). The conjugate reacted with the fluorescent click partner 6-methyl-tetrazine-sulfo-cy3 and labelling in the light chain was confirmed by LC-MS and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Click reaction between the TCO-modified antibody and a fluorescent or 111In-labelled tetrazine were performed for visualization/detection of HER2 in high and low HER2-expressing cells (SK-BR-3 and MDA-MB-231, respectively).
The conjugation of the cysteine-selective reagent bearing a TCO moiety to THIOMAB led to a single modification in the light chain of the antibody, while the heavy chain remained unmodified. Reaction of the conjugate with 6-methyl-tetrazine confirmed that the reaction bioorthogonality was preserved.
In summary, here we report a new methodology for antibody labelling using a simple carbonylacrylic derivative and taking advantage of the IEDDA reaction. We believe that the conjugate homogeneity and labelling under biologically friendly conditions obtained with this method allows for the preparation of well-defined imaging conjugates for in vivo applications.
AcknowledgmentThis work is funded by Fundação para a Ciência e Tecnologia - FCT (PTDC/BTM-TEC/29256/2017 (co-funded by Lisboa2020 - EU FEDER) and UID/Multi/04349/2019). Vera Ferreira also acknowledges FCT for the PhD fellowship (SFRH/BD/108623/2015) and Federation of European Biochemical Societies for a Summer Fellowship.
 Bernardim, B, Cal, PM, Matos, MJ, Oliveira, BL, Martinez-Saez, N, Albuquerque, IS, Perkins, E, Corzana, F, Burtoloso, AC, Jimenez-Oses, G, Bernardes, GJ 2016, "Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents", Nat Commun. 7, 13128.
Keywords: Click chemistry, Antibody modification, PET/SPECT
Disorder measurements from backscattering spectra through an optical fiber (#75)
Roberto Fernández1, 2, Asier Marcos1, Manuel Desco Menéndez1, 3, 5, Jorge Ripoll1
1 Universidad Carlos III of Madrid, Department of Bioengineering and Aerospace Engineering, Leganés, Madrid, Spain
Partial wave spectroscopy (PWS) is a technique which allows the detection of nanoscale structural changes related to different diseases based on the calculation of a statistical parameter, the disorder strength (Ld). This technique is usually applied acquiring the backscattering spectra for each wavelength of a range using a camera. Here, we explore the possibility of applying this method to the spectrum of the averaged spatial information measured by a spectrometer and an optical fiber, enabling this technique to be used in systems with restricted measurement conditions, such as endoscopes.
Using PWS technique, developed and validated by Backman et al [1-4], we measured the spectral fluctuations in the backscattering spectra to obtain the disorder strength (Ld) statistical parameter of a sample. This method is usually applied using a combination of a liquid crystal tunable filter (LCTF) and a CCD camera to acquire a backscattered image in a range between 400–700 nm and calculate the Ld for each pixel on the image. By means of a dual acquisition setup (Figure 1a), we compared this acquisition method to one based on a spectrometer to measure the full spectrum of backscattered light gathered by an optical fiber. Thus, Ld parameter was calculated from a single mean intensity for each wavelength instead of doing it for each pixel of the images corresponding to each wavelength .
Different areas of microscope slides (corner, corner-center and center) containing histological section samples (Figure 1b) were measured using both methods simultaneously. Then, Ld parameter was calculated for the data obtained through each method. The results obtained were compared to validate the fiber-based measurements. In Figure 2a, the normalized results obtained for the sample shown in Figure 1b are depicted. These values shown the ability of the spectrometer to separate different disorder areas and are similar to the ones obtained through Backman’s method. In Figure 2b, raw and normalized results obtained for each method are detailed. The lower values obtained by the spectrometer can be related to the rounding done by this device or to optical fiber loses. However, the results allowed us to identify and separate the three measured areas. The relationship between the Ld of these areas is similar in both methods.
The results obtained through proposed method were in accordance with the ones provided by original PWS method for different areas of the same sample. Thus, we were able to separate them based on the Ld value obtained, greater for areas with more tissue presence. The flexibility given by the fiber on this method enables the identification of distinct disease stages in tissue inflammation and cancer in areas only accessible by endoscopic devices.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801347 SENSITIVE and Spanish Ministry of Economy and Competitiveness (MINECO) Grant FIS2016-77892-R. R.F acknowledges funding from Generalitat Valenciana and European Social Fund through postdoctoral grant APOSTD/2018/A/084. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).
Keywords: Partial wave spectroscopy, disorder strength, backscattering
Exploring the limits of antibody-based intranuclear PET/SPECT imaging (#550)
Mathew Veal1, Gemma Dias1, Deborah Sneddon1, Bart Cornelissen1
1 University of Oxford, UKRI Medical Research Council Oxford Institute for Radiation Oncology, Oxford, United Kingdom
Antibody-based intranuclear imaging using cell penetrating peptides (CPPs), such as Tat, has great potential for the investigation of a range of cellular processes, otherwise not possible with conventional antibody-based imaging techniques1. Previous research from our group demonstrated in vivo imaging of the nucleus bound phosphorylated histone γH2AX, using 111In-anti-γH2AX-Tat2. Here, we establish a lower limit of potential intranuclear targets, we used a H2B-EGFP model.
H1299 lung adenocarcinoma cells were stably transfected with varying levels of histone-2B fused to enhanced green fluorescent protein (H2B-EGFP). The number of H2B-GFP copies was determined by ELISA, and H2B-EGFP transfected cells were tested for antibiotic free stability. We assessed the selectivity and sensitivity of an antibody GPF-G1 by flow cytometry and immunofluorescence microscopy. Conjugation of GFP-G1 IgG with the CPP Tat and a chelating agent, DTPA, was optimised, along with subsequent radiolabelling with 111In. Radiolabelled 111In-DTPA-GFP-G1-Tat uptake into stably transfected H2B-EGFP H1299 cells was evaluated.
H1299 cells were transfected with H2B-EGFP and underwent selection to produce four stably transfected H2B-EGFP cell lines, as confirmed by fluorescence microscopy and flow cytometry (Fig 1). The level of H2B-EGFP was measured in each cell line and ranged from 1.8 x 105 to approximately 1.0 x 106 copies per cell. The monoclonal antibody GFP-G1 was produced and purified in house. A dissociation constant (KD) of GFP-G1 was determined by flow cytometry at 10 ± 6 nM. Uptake assays demonstrated uptake of 111In-DTPA-GFP-G1-Tat correlated with increasing levels of H2B-GFP (R2 = 0.94, one phase association model), suggesting a possible lower detection limit in vitro of 40,000 copies per cell (Fig 2). Furthermore, percentage radioactive compound uptake of 111In-DTPA-GFP-G1-Tat was significantly higher than 111In-GFP-G1 without the Tat-peptide (1.022 ± 0.102% vs. 0.430 ± 0.021 %; P < 0.001).
Intranuclear immuno-PET/SPECT has potential as a highly specific, sensitive and versatile imaging modality. This project determined that Tat conjugated anti-GFP radioimmunoconjugate could successfully distinguish between five cell lines with increasing amounts of nuclear expressed H2B-EGFP in-vitro from 0 to 1.0 x 106 copies per cell. The work provides significant insight into the technical possibilities of intranuclear immuno-PET/SPECT imaging.
AcknowledgmentThe authors would like to thank UKRI-MRC for funding this work.
 Cornelissen, B, 2014, 'Imaging the inside of a tumour: A review of radionuclide imaging and theranostics targeting intracellular epitopes', Journal of Labelled Compounds and Radiopharmaceuticals, 57, 310–316, https://doi.org/10.1002/jlcr.3152, Wiley Online Library.
 Cornelissen, B., Kersemans, V., Darbar, S., Thompson, J., Shah, K., Sleeth, K., Hill, M.A. and Vallis, K.A., 2011, 'Imaging DNA damage in vivo using γH2AX-targeted immunoconjugates', Cancer Research, 71(13), 4539-4549, https://doi.org/10.1158/0008-5472.CAN-10-4587, American Association for Cancer Research.
Keywords: Antibody, Imaging, Intranuclear, PET/SPECT
Development and in vivo preclinical validation of highly hydrophilic 18F-labelled 2-nitroimidazole derivatives for high performance hypoxia PET imaging (#105)
Clémence Maingueneau1, Anne-Elodie Lafargue1, Fabien Fillesoye1, Stéphane Guillouet1, Cécile Perrio1
1 Cyceron, Normandie Univ, UNICAEN, CEA, CNRS, UMR 6030-ISTCT, LDM-TEP, Caen, France
Hypoxia detection by PET imaging has been demonstrated to be useful for diagnosis, therapeutic orientation and follow-up of malignant radio/chemoresistant tumors. Several 18F-nitroimidazole derivatives ([18F]FMISO, [18F]FAZA, [18F]HX4, [18F]DIFA) have reached the clinical stage but limitations were encountered due to slow clearance and low specificity. We hypothesised that hydrophilicity of the radiotracers was a key parameter for hypoxia imaging and we designed a new class of 18F-nitroimidazole analogues bearing a sulfo group known to highly increase hydrophilic properties.
The sulfo-18F-nitroimidazole analogues were readily obtained in one step from sultone precursors by ring opening reaction with [18F]fluoride in acetonitrile for 5 to 15 min at 20-110 °C. The logP and logD values were measured using a standard shake flask method. Stability, biodistribution and pharmacokinetics were studied in healthy rats and in vivo specificity for hypoxia was evaluated in glioblastoma rat models (C6, 9L and U251 cells) using small-animal PET imaging and in comparison to [18F]FMISO. Additional histological experiments including ex vivo autoradiography and pimonidazole staining were also performed.
The radiosyntheses were carried out in 35-70% radiochemical yields and 97-99% radiochemical purity within 40-60 min total radiosynthesis time. The logP and logD values of the radiofluorinated products were in the –(1.5-3.6) range. No radiodefluorination and no radiometabolization were observed at 2.5 h post-injection. According to the structures, hepatobiliary or renal elimination were observed. [18F]FLUSONIM that displayed the highest hydrophilicity, was selected for evaluation in tumor models. Accumulation in hypoxic tumor was rapid (30-60 min versus 2.5 h for [18F]FMISO), and muscle to tumor ratios were 3 to 10 fold higher than those obtained with [18F]FMISO. Ex vivo autoradiography and immunohistostaining demonstrated a correlation between the uptake of the radiotracer and hypoxic regions.
The radiofluorination of sultone-containing 2-nitroimidazoles afforded highly hydrophilic radiotracers. Among them, [18F]FLUSONIM selectively and quickly accumulated in tumor hypoxic regions and provided a better contrast image of tumor hypoxia with shorter acquisition time compared to [18F]FMISO. The overall findings led us to consider clinical transfer as promising.
AcknowledgmentThis work was supported by Région Normandie, CNRS, CEA, Unicaen, Labex IRON (ANR-11-LABX-0018–01) and FR3038 INC3M.
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Keywords: hypoxia, fluorine-18, PET imaging, 2-nitroimidazole, sultone
Effects of penetratin and nona-arginine on the binding and internalisation of a nanobody-based theranostic platform (#228)
Estel Collado Camps1, 2, Sanne A. M. van Lith1, Jordi Lankhof2, Maarten Brom1, 3, Martin Gotthardt1, Roland Brock2
1 Radboud University Nijmegen, Radiology and Nuclear Medicine / Nuclear Medicine / Radboudumc, Nijmegen, Netherlands
Nanobodies are promising probes for molecular imaging and theranostics. We aim at improving the targeting abilities of an anti-Epidermal Growth Factor Receptor (EGFR) nanobody (7D12) using cell-penetrating peptides (CPPs). Our group previously showed that conjugation of 7D12 to the CPP hLF leads to increased internalisation1. Interesting alternative CPPs are nona-arginine and penetratin. Here we present new insights on the effects of conjugate concentration and receptor density on binding, internalization, subcellular localization and 3D penetration of the nanobody-CPP conjugates.
7D12 and a GFP-targeting control nanobody (αGFP) were conjugated to D-nona-arginine (r9) and D-penetratin (pen) via sortase-mediated coupling. A maleimide-fluorophore or maleimide-DTPA were site-specifically attached to an unpaired C-terminal cysteine. The DTPA-conjugates were labelled with 111In, and binding and internalization of low nanomolar concentrations of the various conjugates was determined in vitro using EGFR overexpressing A431 cells and EGFR negative HEK293 cells. To further investigate binding and subcellular localisation in 2D and 3D, we incubated monolayer cultures and spheroids of cells with high and moderate EGFR expression (A431 and SKOV3, respectively) with micromolar concentrations of the fluorophore-conjugates and imaged with laser scanning confocal microscopy.
In radioactivity-based assays (Fig1), binding and uptake of both conjugates were higher than those of unconjugated 7D12. Binding and uptake of 7D12-r9 were high on EGFR-negative HEK cells and were not completely blocked (27% and 55% reduction respectively) with unlabelled 7D12 on the EGFR-positive A431 cells. This indicates mostly CPP-mediated interactions. Binding and uptake of 7D12-pen appeared more EGFR-mediated, being respectively 87% and 69% lower in A431 cells when blocked. In microscopy assays, internalization of both 7D12-pen and 7D12-r9 was increased in SKOV3 cells when compared to 7D12. For A431 cells there were no differences. αGFP-D-r9 showed some internalisation, but no membrane staining, indicating receptor independent uptake. In line with the 2D experiments, we observed better retention in spheroids of 7D12-pen, 7D12-r9 and aGFP-R9 into SKOV3 spheroids when compared to 7D12 or aGFP, respectively (Fig2). In A431 spheroids, penetration of the conjugates was incomplete.
In conclusion, nona-arginine and penetratin enhance nanobody uptake. The relative importance of CPP-driven and nanobody-driven interactions depends on various factors such as the type of CPP, the conjugate concentration and receptor density on the target cells. Upcoming biodistribution experiments will help to understand how these factors affect the in vivo behaviour of the various nanobody-CPP conjugates.
We thank professor Heinrich Leonhardt (Ludwig Maximilians Univeristy Munich) for kindly providing the sequence of the αGFP nanobody. The macro for the analysis of the spheroid images was written by Christiaan Stuut.
 van Lith et al, 2017, A Conjugate of an Anti-Epidermal Growth Factor Receptor (EGFR) VHH and a Cell-Penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation. Chembiochem, 18(24):2390-2394
 Verdurmen et al, 2011, Preferential uptake of L- versus D-peptide amino acid cell-penetrating peptides in a cell type-dependent manner. Chemistry and Biology, 18:1000-1010
Figure 1.Results of a binding and internalisation assay on A431 (EGFR high) and HEK (EGFR negative)
Cell monolayers were incubated for 1h with nanobody and conjugates labelled with 111In (aprox. 0.2nM). The percentage of total activity added was calculated by measuring the activity of the different fractions and of standards in a gamma-counter.
Figure 2. Mean intensity profiles and representative confocal microscopy sections of spheroids
Each dotted line represents the mean intensity measured every three pixels along 36 radii drawn at different angles, for each spheroid. Microscopy images are pseudo coloured according to the fluorescence intensity. A: spheroids incubated with 7D12 (parent nanobody), B: incubated with 7D12-r9, C: incubated with7D12-r9, D: incubated with αGFP, E: incubated with αGFP-r9. Concentration was 500nM for all conditions. Note: αGFP-pen nanobody was not included due to a consistent failure of the sortase conjugation for this pair.
Keywords: nanobodies, cell-penetrating peptides, spheroids, theranostics