EMIM 2018 ControlCenter

Online Program Overview Session: PW-28

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Oncology | Solutions to unmet clinical need

Session chair: Ferdia Gallagher - Cambridge, UK; Camiel Rosman - Nijmegen, The Netherlands
 
Shortcut: PW-28
Date: Friday, 23 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 243

18F-FDG PET as Diagnosis and Therapy Monitoring Tool for Indolent Non Hodgkin Lymphoma Mice Model (#342)

C. Penalba1, G. Garaulet1, A. Ortega-Gil1, G. Visdomine1, S. Leal1, T. Álvarez1, A. Efeyan1, F. Mulero1

1 Centro Nacional de Investigaciones Oncológicas, Molecular Imaging Unit, Madrid, Spain

Introduction

Despite recent therapeutic advances, B cell-Non-Hodgkin lymphomas remains mostly incurable. For that reason, is important search to and improve new tools for detection and therapy monitoring in order to test new available therapies.

18F-FDG PET has become a useful tool for diagnosis and staging of Non-Hodgkin lymphoma in the clinical and preclinical field it serves also to asses response to therapy.

The aim of this study is to elucidate the usefulness of PET for tumor detection and therapy monitoring in a mouse model for indolent Non-Hodgkin lymphoma.

Methods

A genetically modified mouse model for Indolent Non Hodgkin Lymphoma was used. The mice were scanned by PET-CT to diagnose lymphoma and the positives ones were treated during 15 days with a compound/drug and a new PET-CT was performed 15 days after the treatment to evaluate the possible response to the therapy.

18F-FDG was injected in the tail vein at a 14Mbq dosage. After an uptake period of 45 minutes, a PET-CT study was performed. Acquisition values were: CT- 150mA intensity, 45kV voltage, 8 shots in 2 beds, with a 360º ring loop and 200µM resolution; PET acquisition time 15 min/bed. Animals were anesthetized with Sevofluorane. PET Images were reconstructed with 3D OSEM and CT with Filtered-back Projection Algorithm and analyzed with Amide software.

Results/Discussion

Our study showed that is possible to detect Indolent Non-Hodgkin lymphoma in a mouse model being able to detect lymph nodes in different locations, axillary, inguinal, mesenteric etc (Figure 1) with 18F-FDG uptake. The quantification before and after treatment demonstrates a decrease of FDG uptake in lymph nodes (mesenteric ones) in the responders mice (Figure 2) and also non decrease or even an increase in the group of non responder mice (ongoing results). The presence of lymphoma were confirmed by histological analysis after PET-CT in the control mice and in the non-responder ones

Conclusions

We can conclude that de 18F-FDG PET combined with CT scans is a feasible tool to detect Indolent Non-Hodgkin lymphoma and to monitor therapy in preclinical assays.

Figure 1

Figure 1: 18F-FDG PET-CT scans showing coronal projection of a LNH mice. Arrow pointing lymph nodes with high 18F-FDG uptake, asterisk pointed high liver and spleen uptake

Figure 2

Figure 2: 18F-FDG PET-CT scans. A: Pretreatment images arrow pointed a mass of mesenteric lymph nodes. B: 15 days post treatment we can observe a dramatic decrease of mesenteric uptake 

Keywords: PET, FDG, Oncology, Lymphoma, Therapy monitoring
# 244

Boron-rich oil-in-water emulsions as drug nanocarriers for Boron Neutron Capture Therapy (#550)

M. Navascuez1, 2, D. Dupin1, I. Loinaz1, H. Jürgen-Grande1, U. Cossio2, J. Llop2

1 CIDETEC, Nanomedicine, San Sebastian, Spain
2 CIC biomaGUNE, Radiochemistry and Nuclear Imaging, San Sebastian, Spain

Introduction

Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy which relies in the accumulation of boron-rich drugs in the tumor, followed by irradiation with thermal neutrons to trigger a nuclear reaction that creates selective damage in the tumor tissue. In this work, we propose the use of cobalt bis(dicarbollide)-stabilized, o-carborane-loaded oil-in-water (o/w) nanoemulsions as potential boron-rich BNCT drug candidates.

Methods

To prepare the nanoemulsions, a dissolution containing o-carborane in a triglyceride oil based on docosahexanoic acid was prepared and poured into a second solution containing cobalt bis(dicarbollide) in pure water, and the mixture was sonicated under vigorous stirring in an ice bath. Hydrodynamic diameter and colloidal stability were investigated using dynamic light scattering. For imaging experiments, the solution containing o-carborane was spiked with 1-[18F]fluorocarborane, prepared using a previously reported method. The biodistribution of the labelled nanoemulsions after intravenous administration in rats was followed up to 6 hours post-administration using positron-emission tomography. Blood samples were withdrawn at different time points to assess circulation time.

Results/Discussion

Stable nanoemulsions with average hydrodynamic diameter around 160 nm could be efficiently prepared. The nanoemulsionproved to be stable up to 3 months after preparation. Distribution studies performed in rats showed significant accumulation in the liver at short times after administration, and the activity progressively translocated to the gastrointestinal track at later time points. Blood samples confirmed that around 0.1% of the radioactivity remained in blood at 3 hours after administration, suggesting that circulation time should be sufficient to enable accumulation in the tumour in future experiments. The lack of radioactivity in bones suggests negligible defluorination of the labelled carborane.    

Conclusions

Boron-rich, stable nanoemulsions could be prepared using a simple and robust methodology. In vivo biodistribution studies in rats suggest potential application of the novel nanosystems as BNCT drugs. Future studies in xenograft mouse tumour models will be carried out in the near future. 

References

Gona KB, Gómez-Vallejo V, Padro D, Llop J. [18F]Fluorination of o-carborane via nucleophilic substitution: towards a versatile platform for the preparation of 18F-labelled BNCT drug candidates. Chem. Commun. 2013;49:11491.

Figure 1
left: schematic representation of the boron-rich nanoemulsions; right: representative PET-CT images (coronal projection) at 3 min at 3 hours after intravenous administration of the labelled nanoemulsion. 
Keywords: boron, therapy, PET, nanoemulsion
# 245

Ferumoxytol as an MR imaging surrogate marker for liposomal drug deposition and longitudinal efficacy in a preclinical model of breast cancer. (#26)

M. Ventura1, N. Bernards1, W. Foltz1, H. Lee2, B. S. Hendriks2, J. Fitzgerald2, J. Zheng1, 3

1 TECHNA Institute for the Advancement of Technology for Health, University Health Network., Toronto, Ontario, Canada
2 Merrimack Pharmaceuticals, Inc., Cambridge, Massachusetts, United States of America
3 Institute of Biomaterials and Biomedical Engineering., University of Toronto, Toronto, Ontario, Canada

Introduction

The variability of individual patient’s response to nanomedicines remains a topic of interest, and accurate identification of patients who are most likely to benefit from such therapies would be advantageous. In this study, we investigate the use of FMX, an FDA-approved iron replacement therapy, used off-label as an MRI contrast agent, as a macromolecular agent to predict the efficacy of liposomal irinotecan (nal-IRI, ONIVYDE®), in a mouse xenograft model of breast cancer, motivated by the comparable PK and tumor deposition profiles of FMX and nal-IRI.

Methods

Female SCID mice were inoculated with triple negative MDA-MB-231 breast cancer cells. When tumors reached a size of ≥250 mm3, mice were injected with 50 mg/kg FMX and imaged on a 7T  MRI, pre-contrast injection and at 4 and 24h thereafter. After imaging, 6 weekly doses of nal-IRI (20 mg/kg) were administered. Response to treatment was monitored by MRI and caliper-based tumor volume measurements. The variability of FMX tumor deposition assessed by ICP-AES and MRI was further explored in the SUM190 inflammatory breast, HT29 colorectal, and the H2170 and A549 lung cancer models. Agarose phantoms containing FMX-labeled J774A.1 macrophage cells, or corresponding concentrations of extracellular FMX, were also used to explore the effect of cell bound vs. free FMX on T2 and T2* MR signal.

Results/Discussion

A positive correlation between intratumoral concentration of FMX and free irinotecan was observed at 24h post-injection. In vivo FMX quantification from MR T2 maps was not as sensitive in detecting small concentration changes and more variable compared to ex vivo ICP measurements. The limited range of FMX tumor uptake, and its heterogeneous spatial distribution, were potential causes of the higher variability of MR quantification. Our phantom study confirmed that spatial clustering of FMX (i.e. macrophage internalization) affects R2* more than R2, independent of the MR magnetic field strength. Therefore, if low propensity for macrophage engulfment is expected for the nanotherapeutic, R2 measurements may be preferable to avoid overestimation of FMX concentrations. R2 MRI provided effective short-term (1 week) and long-term (5 weeks) prediction of treatment outcome (p < 0.005) between FMX tumor uptake measured prior to treatment initiation and treatment response.

Conclusions

FMX-MR imaging showed potential as an effective tool for predicting the treatment outcome of nanomedicines.

# 246

MET as Potential Target for Molecular Fluorescence Guided Surgery in Papillary Thyroid Carcinoma (#71)

P. K. C. Jonker2, 1, M. Sywak2, D. Leeuw1, G. M. van Dam1, 3, A. F. Gill4, P. J. van Diest5, 7, S. F. Oosting6, R. S. N. Fehrmann6, S. Kruijff1

1 University of Groningen, University Medical Center Groningen, Surgical Oncology, Groningen, Netherlands
2 University of Sydney, Royal North Shore Hospital, Endocrine Surgery and Surgical Oncology, Sydney, Australia
3 University of Groningen, University Medical Center Groningen, Nuclear and Molecular Imaging, Intensive Care, Groningen, Netherlands
4 University of Sydney, Royal North Shore Hospital, Anatomical Pathology, Sydney, Australia
5 University of Utrecht, University Medical Center Utrecht, Pathology, Utrecht, Netherlands
6 University of Groningen, Medical Oncology, Groningen, Netherlands
7 Johns Hopkins, Oncology Center, Baltimore, United States of America

Introduction

Central compartment lymph node metastases in papillary thyroid cancer (PTC) are difficult to demonstrate preoperatively. A reliable diagnostic tool to identify clinically significant nodal metastasis may help to individualise the surgical approach and minimise operative complications. Molecular fluorescence guided surgery (MFGS) might improve patient selection by visualizing central lymph node metastases. This study aims to identify potential target antigens in PTC for clinically available, near infrared fluorescent (NIRF) tracers currently used for MFGS.

 

Methods

Functional Genomic mRNA (FGmRNA) profiling was applied to microarray expression data from the public domain of PTC and normal thyroid tissue (NTT).1 Class comparison followed by multivariate permutation testing (MVP) between PTC and NTT FGmRNA-expression profiles was performed to identify significantly upregulated genes. Twelve clinical available NIRF tracers where identified from literature.2 Protein expression of the most significantly expressed gene targeted by these tracers was validated by immunohistochemistry (CST8198S antibody) using a tissue microarray (671 PTC/108 NTT cases). An H-score was calculated per patient from available cores (H-score ≥150 = positive staining) and compared between PTC and NTT. The relation between H-scores and PTC recurrence rates was assessed.

Results/Discussion

Class comparison between FGmRNA expression profiles of 97 PTC and 80 NTT samples identified 1,702 significantly upregulated genes in PTC after MVP (false discovery rate 5%, confidence interval 80%). Within these significantly upregulated genes we identified 5 genes that are targeted by one of the twelve clinical available NIRF tracers: MET (ranked 11; target of EMI-137), PSMA 7 & PSMA 1 (ranked 685 & 1070; targets of MDX1201-A488), CTSK and CTSH (ranked 668 & 1672; targets of LUM015). IHC validation for MET showed that the H-score was significantly higher in PTC compared to NTT (p<0.0001). An overview of staining intensities is provided in figure 1. 76% of PTC samples and 3% NTT samples stained positive for MET. Higher H-scores were associated with a higher risk of PTC recurrence rates (p=0.001) as illustrated in figure 2.

Conclusions

Based on the FGmRNA expression level, protein overexpression, association of increased H-scores with PTC recurrence and the clinical availability of a NIRF tracer, MET is an interesting target for MFGS in PTC.

References

  1. Fehrmann RSN, Karjalainen JM, Krajewska M, et al. Gene expression analysis identifies global gene dosage sensitivity in cancer. Nat Genet. 2015;47(2):115-125. doi:10.1038/ng.3173.
  2. Zhang RR, Schroeder AB, Grudzinski JJ, et al. Beyond the margins: real-time detection of cancer using targeted fluorophores. Nat Rev Clin Oncol. 2017;13:653–18. doi:10.1038/nrclinonc.2016.21
Cell Signaling 8198S staining intensities in PTC tissue

Figure 1

Overview of Cell Signaling 8198S (targeting MET) staining intensities in PTC tissue. Staining intensities are provided at 10x and 40x magnifications (L/N = low/negative).

Association between H-score and Disease Free Survival

Disease free survival per staining intensity and corresponding H-scores. The number of patients with follow up data are provided per time interval. Patients lost in follow up were censored.

Keywords: Papillary Thyroid Cancer, Molecular Imaging, Molecular Fluorescence Guided Surgery, bioinformatics
# 247

[18F]-FET and [18F]-FAZA PET guided irradiation of glioblastoma in rats (#129)

J. Verhoeven1, J. Bolcaen2, B. Descamps3, T. Baguet1, G. Hallaert4, K. Kersemans2, T. Boterberg5, K. Deblaere6, C. Vanhove3, F. De Vos1, I. Goethals2

1 Ghent University, Lab for radiopharmacy, Ghent, Belgium
2 Ghent University Hospital, Department of Nuclear Medicine, Ghent, Belgium
3 iMinds-IBiTech-MEDISIP Ghent University, Department of Electronics and Information Systems, Ghent, Belgium
4 Ghent University Hospital, Department of Neurosurgery, Ghent, Belgium
5 Ghent University Hospital, Department of Radiation Oncology, Ghent, Belgium
6 Ghent University Hospital, Department of Radioloy and Medical Imaging, Ghent, Belgium

Introduction

Glioblastoma (GB) is the most common primary malignant brain tumor of the central nervous system1. The standard therapy consists of maximal possible surgical resection with concomitant radiation (RT) and chemotherapy. An accurate definition of the tumor volume is of utmost importance for guiding radiation therapy. Currently the target volume delineation is based on CT and MRI2,3. In this project we investigated the feasibility of incorporating [18F]FET and [18F]-FAZA for guiding RT and the impact on treatment outcome by applying subvolume boosting to a PET-defined tumor part.

Methods

F98 GB cells inoculated in the rat brain were imaged using T2- and contrast-enhanced T1-weighted (CE-T1w) MRI. After tumor growth, a 30 min [18F]-FET (30min p.i.) or [18F]-FAZA (2h p.i.) PET was acquired. Subsequently, a treatment planning CT was obtained on the small animal radiation research platform (SARRP). A dose of 20 Gy (3x3 mm) was delivered to the target volume delineated based on CE-T1w-MRI (group 1-3). In group 2 and 3 an additional radiation boost of 5 Gy (1x1 mm) was delivered to the region with maximal PET tracer uptake. Temozolomide (TMZ, 5 mg i.p.) was administered in groups 1-3 on five consecutive days. The 4th group received sham injections with saline. Tumor volumes on follow-up MRI were determined by drawing volumes of interest around the tumor on CE-T1w-MRI (PMOD).

Results/Discussion

CE-T1w-MRI showed a heterogeneous tumor, enabling to select the MRI target volume. Both [18F]-FET and [18F]-FAZA showed an increased tumor uptake. After co-registration of the planning CT with the CE-T1w-MRI and PET images, PET-guided RT was performed. Using three non-coplanar arcs, the dose delivered to the normal surrounding brain tissue was minimized. The average, minimum and maximum dose, as well as the D90-, D50- and D2- values were calculated for nine rats with both RT plans. The dose volume histograms (DVH) are represented in figure 1. A slight shift to the right for the graph could be noted of the DVH based on the RT with PET based subvolume boosting. The evolution of the normalized tumor volumes is shown in figure 2. Significant differences were found between the therapy and control groups. No significant differences were observed between the different therapy groups.

Conclusions

MRI guided irradiation with PET subvolume boosting is feasible, but very labor-intensive. Tools for faster and more accurate image co-registration would be helpful. Based on tumor growth, a significant difference was found between therapy and no therapy, but no significant difference could be observed between the three treatment groups. Additional information from molecular imaging techniques enables the visualization of metabolically highly active regions. As GB are highly heterogeneous solid tumors, the concept of biological target volume and multidimensional conformal RT seems promising.

References

1. Louis, D.N. et al., 2016. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathologica, 131(6), pp.803–820.

2. Stupp, R. et al., 2005. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. New England Journal of Medicine, 352(10), pp.987–996.

3. Wen, P.Y. & Kesari, S., 2008. Malignant gliomas in adults. The New England journal of medicine, 359(5), pp.492–507.

Acknowledgement

Financial support by the Belgian Association for Neuro Oncology (BANO) with the Belgian Brain Tumor Support (BBTS) award 2017.

Evolution of normalized tumor volumes

Figure 2: Graphical representation of the median change of the tumor volume by day.

Dose volume histograms

Figure 1: Indication of the overlapping volume of the three rotating bundles (green, A). Dose volume histogram with isocenter based on the center of the gadolinium containing contrast uptake on the T1-weighted MRI (B). Dose volume histogram with the second isocenter based on the hot spot showed by the PET scan (C).

Keywords: Irradition, glioblastoma, PET, FET, FAZA
# 248

Photoimmunotherapy for HER2+ve tumors - The red-light for cancer. (#23)

J. Mączyńska1, 2, C. Da Pieve2, T. A. Burley2, A. Shah2, D. Ciobota2, J. Bamber2, J. Saczko1, G. Kramer-Marek2

1 Wroclaw Medical University, Department of Medical Biochemistry, Wrocław, Poland
2 The Institute of Cancer Research, Division of Radiotherapy and Imaging, London, United Kingdom

Introduction

Development of HER2-targeted drugs like trastuzumab, lapatinib, trastuzumab-emtansine (T-DM1) has significantly improved the overall survival of HER2 positive cancer patients. Nevertheless, regardless of the initial promising response to treatment, many of these patients relapse due to small cohorts of resistant tumour cells surviving the therapy. Therefore, we investigated the efficacy of photoimmunotherapy (PIT) using a HER2 specific affibody-based conjugate to selectively eliminate residual HER2 positive cancer cells.

Methods

HER2-specific affibody molecules (ZHER2:2891) were conjugated to IR700-maleimide. The specificity of conjugate binding was confirmed by flow cytometry and confocal microscopy in a panel of cancer cell lines with different HER2 expression levels. The therapeutic efficacy of ZHER2:2891-IR700DX (PIT, 16 J/cm2) was tested in adherent cells and 3D spheroids by assessing cellular viability with the CellTiter-Glo assay. Oxidative stress was determined via DCFDA/H2DCFDA assay. Immuno-cell death markers (e.g. calreticulin, HMGB1) were measured by flow cytometry and Western blot. Proof-of-concept in vivo studies were performed using subcutaneous HER2+ve xenografts (PIT, 100 J/cm2). Post-treatment, tumours were dissected for ex vivo analysis.

Results/Discussion

The binding of the ZHER2:2891-IR700DX, as seen by flow cytometry and confocal microscopy, correlated with HER2 expression level. Blocking with an excess of non-labelled affibody effectively reduced the median fluorescence in all tested cell lines, further confirming probe specificity.  Phototoxicity studies in vitro showed a significant decrease in cell viability in a dose-dependent manner. Irradiation of spheroids resulted in swelling and release of cellular content shortly after, which indicated that necrotic cell death was occurring. Induced ROS generation post PIT, triggered immunogenic cell death leading to HMGB1 release, and translocation of calreticulin to the cellular membrane (Fig.1). In vivo studies confirmed that the conjugate specifically targets HER2+ve tumours and significantly inhibits tumour growth post-irradiation.

Conclusions

The ZHER2:2891-IR700DX showed therapeutic efficacy in HER2+ve cell lines and 3D spheroids. The proof of concept in vivo studies demonstrated that the conjugate specifically targets HER2+ve tumours and significantly inhibits tumour growth post-irradiation.

Acknowledgement

The authors gratefully thank AffibodyAB (Stockholm, Sweden) for supplying the affibody molecules.

This work was supported by the funding from National Center of Science Poland (UMO-2015/19/N/NZ7/01336), the Institute of Cancer Research and Cancer Research UK-Cancer Imaging Centre (C1060/A16464).

Photoimmunotherapy (PIT) with ZHER2:2891-IR700DX selectively causes necrosis of HER2+ve cells:

A. Confocal microscopy images demonstrating target-specific binding of ZHER2:2891-IR700DX

B. Specificity of ZHER2:2891-IR700DX binding to HER2 confirmed by flow cytometry.

C. HER2 expression in the selected cell lines.

D. SKOV3 cells viability 24h after PIT.

E. Changes in morphology of SKOV3 spheroids 96h after PIT.

F. Translocation of calreticulin to the cell membrane after PIT by flow cytometry.

Keywords: Photoimmunotherapy, HER2, Affibody, Optical Imaging
# 249

Liposomal Irinotecan Injection (nal-IRI) Achieves Significant Survival and Tumor Burden Control in a Triple Negative Breast Cancer Model of Spontaneous Metastasis (#17)

N. Bernards1, M. Ventura1, I. B. Fricke1, B. S. Hendriks2, J. Fitzgerald2, H. Lee2, J. Zheng1, 3

1 UHN, TECHNA, Toronto, Ontario, Cameroon
2 Merrimack Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America
3 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Toronto, Ontario, Canada

Introduction

Triple negative breast cancer (TNBC) represents a significant treatment challenge due to its highly aggressive & metastatic nature. Non-liposomal irinotecan (IRI) has been used in combination with other chemotherapeutics to treat metastatic breast cancer & TNBC, but showed toxicity & limited activity in patients(1). Liposomal chemotherapeutics address these shortcomings allowing for decreased toxicity & increased drug exposure at the target site. We investigate the therapeutic benefit of nal-IRI for the treatment of advanced TNBC in a clinically relevant mouse model of spontaneous metastasis.

Methods

Female SCID mice were inoculated with TNBC LM2-4-luc cells in the lower right mammary fat pad. The primary tumors were resected at 2-3 weeks post inoculation at a mean tumor volume of 230 ± 60 mm3, and metastasis formation was followed using bioluminescence imaging (BLI). Mice were randomized into the following 3 groups: (1) no treatment control (n=13), (2) IRI (50 mg/kg i.v.) once per week (n=13), (3) nal-IRI (10 mg/kg, hydrochloride salt, i.v.) once per week (n=16). Randomization occurred between day 5 and 9 post primary tumor resection, when animals presented with at least one distant metastasis detected via BLI. Treatment with either IRI or nal-IRI was administered i.v. every 7 days until study endpoint, and animals were monitored 2-3 times per week using BLI.

Results/Discussion

No significant body weight changes were observed, showing no adverse effects from the treatment(Fig.1). Nal-IRI provided survival benefit with 50% of the animals surviving until d66 post treatment initiation, while 50% of the control&IRI-treated animals survived until d14(Fig.2). The survival benefit of nal-IRI(4.7x longer than control&IRI treated groups)was supported by a significant delay in primary tumor regrowth for the treated animals(Fig.3,4). BLI measured mean whole body photon flux at 50% survival time for the nal-IRI treated animals(d68)was an order of magnitude lower (2.2x108±3.4x108p/s) than for the IRI treated group (2.6x109±2.7x109p/s)(p =0.0286,d14). Lower signal (nal-IRI,d14)(2.6x109±1.01x109p/s) than IRI (p = 0.0286,d14)(Fig. 5), indicated effective control of metastasis.

Conclusions

In a highly aggressive TNBC model of spontaneous metastasis, nal-IRI demonstrated superior anti-tumor activity and provided significant survival benefit compared to IRI-treated and untreated groups, while being well-tolerated. BLI imaging was successfully used as a non-invasive and high-throughput technique to monitor whole body disease progression, metastatic spread and response to therapy in mice longitudinally. These results indicate that nal-IRI has potential for the treatment of patients with TNBC and warrants clinical evaluation.

References

1.        Kümler I, Brünner N, Stenvang J, Balslev E, Nielsen DL. A systematic review on topoisomerase 1 inhibition in the treatment of metastatic breast cancer. Breast Cancer Res. Treat. 2013. page 347–58.

Keywords: TNBC, Liposome, Spontaneous Metastasis, Irinotecan, Preclinical Imaging
# 250

Magnetic Particle Imaging Guided Heating In Vivo : Gradient Fields Localize Heating To Tumor while Sparing Non-specific Nanoparticle Accumulations (#192)

Z. W. Tay1, P. Chandrasekharan1, D. Hensley1, X. Y. Zhou1, B. Zheng1, R. Dhavalikar2, A. Chiu-Lam2, C. Rinaldi2, S. M. Conolly1

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 University of Florida, Gainesville, Florida, United States of America

Introduction

Magnetic Fluid Hyperthermia (MFH) is promising for cancer therapy but one challenge is focusing AC fields for selective heating of tumors with magnetic nanoparticles (MNP) while sparing healthy tissues with non-specific accumulations such as the clearance organs (liver) [1]. Magnetic Particle Imaging (MPI) is a new tracer imaging modality [2] whose signal generation is similar to how MFH generates heat [3-6]. MPI gradients localize signal & heating by locking MNP rotation. We show the first MPI image-guided localized heating of mice xenograft tumors while sparing MNPs cleared to the liver.

Methods

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) from University of Florida was used. The in vitro study used a 3 x 3 grid of 0.1 ml vials separated edge-to-edge by 7 mm. The in vivo study used 7 – 9 week old athymic nude mice with MDA-MB-231-luc xenografts. 1.5 mg Fe of SPION was injected intratumorally and/or injected in the tail-vein and allowed to clear to the liver. MPI imaging used a 6.3 T/m field-free-line scanner at 20 kHz 20 mT. Based on the MPI image-guidance, targeted heating was performed on a co-registered 2.35 T/m field-free-line MPI setup optimized for 354 kHz 14 mT excitation [4]. In-vivo temperatures was measured by NeoptixTM fiber optic temperature probes. IVIS Lumina VivovisionTM  was used for bioluminescence and KubtecTM Cabinet X-ray for the anatomic references.

Results/Discussion

Fig 1 shows a MPI theranostics workflow where we achieved in vivo heating of tumor but spared the liver. Step 1) MPI image scan visualizing the SPION biodistribution in the tumor(s) (and clearance organs).  2) User selects a region to heat, and MPI gradients shift the field-free-line to target  3) Thermal dose planning based on MPI image 4) Localized heating. In vitro results (Fig 2a) show heating of the central vial with negligible temperature rise in the surrounding vials spaced 7 mm away, demonstrating highly localized heating to < 7 mm. With a 6.3 T/m gradient, this can be improved to 3 mm localization. In vivo results (Fig 2b) show good correlation of SAR deposition with MPI image intensity, enabling thermal dose planning from the MPI image alone. Fig 2c: To evaluate localization of therapy, we used dual tumor mice (both with SPIONs) but heating was targeted at only one tumor. Bioluminescence images show a dramatic decrease in activity only at the targeted tumor.

Conclusions

MPI enables (1) high contrast imaging of SPIONs  (2) thermal dose planning due to the quantitative nature of MPI images  (3) spatial localized heating of SPIONs through user-directed location of the field-free-line. In addition, real-time temperature monitoring could be achieved through MPI thermometry [7], enabling closed-loop feedback during treatment. Furthermore, localized heating / actuation could be leveraged with thermally or mechanically sensitive liposomes for user-directed localization of drug release in vivo. All these features make MPI a promising cancer theranostics platform. 

References

1. Thiesen B, Jordan A. Clinical applications of magnetic nanoparticles for hyperthermia. Int J Hyperthermia. 2008 Sep;24(6):467–474.

2. Gleich B, Weizenecker J. Tomographic imaging using the nonlinear response of magnetic particles. Nature. 2005 Jun 30;435(7046):1214–1217.

3. Dhavalikar R, Rinaldi C. Theoretical predictions for spatially-focused heating of magnetic nanoparticles guided by magnetic particle imaging field gradients. J Magn Magn Mater. 2016 Dec 1;419:267–273.

4. Hensley DW, Tay ZW, Dhavalikar R, Zheng B, Goodwill P, Rinaldi C, Conolly S. Combining magnetic particle imaging and magnetic fluid hyperthermia in a theranostic platform. Phys Med Biol. 2016 Dec 29;

5. Murase K, Aoki M, Banura N, Nishimoto K, Mimura A, Kuboyabu T, Yabata I. Usefulness of Magnetic Particle Imaging for Predicting the Therapeutic Effect of Magnetic Hyperthermia. Open Journal of Medical Imaging. Scientific Research Publishing; 2015;5(02):85.

6. Tasci TO, Vargel I, Arat A, Guzel E, Korkusuz P, Atalar E. Focused RF hyperthermia using magnetic fluids. Med Phys. 2009 May;36(5):1906–1912.

7. Perreard IM, Reeves DB, Zhang X, Kuehlert E, Forauer ER, Weaver JB. Temperature of the magnetic nanoparticle microenvironment: estimation from relaxation times. Phys Med Biol.

Acknowledgement

We would like to acknowledge NIH funding and the A*STAR NSS-PhD and Siebel Scholars fellowship (ZW Tay).

Experimental Data from a Theranostics Workflow for MPI Image-Guided, Localized Heating
First, an MPI image at low frequency (20 kHz) is taken of the mouse. Negligible heating occurs. Thermal dose planning based on MPI image intensity is performed. Next, the field-free-line gradient (FFL) is aligned to the target tumor. This prevents SPION rotation & heating everywhere but the target during the 354 kHz heat scan, localizing heat to the tumor while sparing the liver.
Spatial Localization of Tumor Therapy in a Dual Tumor Mouse
a.  3 x 3 phantom shows MPI gradients (2.3 T/m) can localize heating to < 7 mm.    b.  MPI image intensity correlates well with measured in vivo SAR, enabling thermal dose planning from the MPI image.   c. MPI localized heating of one out of two tumors. Tumor bioluminescence images show marked decrease in bioluminescence in the targeted tumor only, verifying spatial localization of therapy.
Keywords: Magnetic Particle Imaging, Localized Heating, Magnetic Fluid Hyperthermia, Magnetic Nanoparticles, Image-Guided Therapy, Theranostics
# 251

Evaluation of self-assembled nanogels containing boron for tumor imaging and therapy (#234)

R. Poirot1, B. Busser2, 3, F. Garcia1, R. Auzély-Velty1, J. - L. Coll2, L. Sancey2

1 Grenoble Alpes University, Centre de Recherches sur les Macromolécules Végétales, Grenoble cedex 9, France
2 Institute for Advanced Biosciences, Grenoble Alpes University/INSERM U1209/ CNRS UMR5309, La Tronche, France
3 Grenoble Alpes University Hospital, Grenoble, France

Introduction

The development of well-tolerated nanoparticles based on biopolymers, and particularly hyaluronic acid (HA), might be interesting for tumor targeting and further delivery of anti-cancer drugs. In the context of theranostic applications, such nano-structures might be used for optimization of accelerator based-neutron capture therapy (AB-NCT), an emerging radiotherapeutic modality, due to their efficient tumor-targeting. Here, we investigated the properties of innovative nanogels based on hyaluronic acid and maltose for efficient tumor targeting.

Methods

The biological behavior and tumor-targeting properties of the native nanogels were determined using optical imaging in vitro and in vivo, for cell expressing low (i.e. TS/A-pc cells) and high-levels of CD44 (i.e. HeLa cells), the natural receptor of the hyaluronic acid. The tumor-to-liver ratios were quantified and compared to the one of the native hyaluronic acid, labeled with Cyanine5.5, until 48 hours post injection.

Similar investigations were then performed after addition of phenylboronic acid (PBA) onto the hyaluronic acid moieties. Cell internalization was evaluated and AB-NCT was performed at the Institute Lauë-Langevin facility. In vivo, the precise distribution of boron was determined using laser-induced breakdown spectrometry (LIBS).

Results/Discussion

In vitro, we demonstrated the efficient internalization of the nanogels, and observed that this phenomenon was independent of the CD44 status. Then, the in vivo properties of the nanogels were investigated on tumor–bearing mice. Two tumors models were used: the CD44-positive HeLa cells and the CD44-negative TS/A-pc cells. Similarly to the in vitro results, the tumor targeting was not driven by the CD44 status, but rather relies on the passive enhanced permeability and retention (EPR) effect. This result indicated the efficient cross-linking of the 2 biopolymers that constituted the nanogel. This later was mainly eliminated by the hepatobiliary route. The tumor-to-liver ratios were determined in vivo and ex-vivo for both tumor types. For TS/A-pc, this ratio reached 1 from 24 to 48 hours post-injection. After addition of PBA into the nanogel, the cell internalization was still highly efficient, leading to efficient cell death after neutron capture therapy.

Conclusions

The biocompatible nanogels, based on hyaluronic acid, were efficiently and rapidly internalized by various cell types in vitro. After systemic administration in tumor-bearing mice, the nanogels circulated in the bloodstream, were eliminated through the liver, and strongly accumulated into the tumor tissues. The addition of PBA into the nanogel allowed the vectorization boron-atoms into the tumor sites. LIBS allowed to localize the boron into different organs, including the tumor. Promising in vitro results of AB-NCT were obtained and are encouraging for the next experiments of therapy in mice.

Acknowledgement

This work was partly supported by the framework of the Glyco@Alps project of the "Investissements d’avenir” program (ANR-15-IDEX-02). 

Near Infrared tumor imaging
In vivo near-infrared fluorescence (NIRF) images of the time dependent biodistribution of Cy5.5-labeled crosslinked nanogels in sub-cut breast TS/A-pc  tumor-bearing mice. The fluorescence was measured before injection and at the following time elapse after administration: 30 min, 1h, 2 h 30, 5 h, 24 h, and 48 h. The tumor locations are indicated by the arrows.
Keywords: vectorization, AB-NCT, Biopolymers
# 252

α-camera imaging of Astatine-211 labeled anti-HER2 nanobodies (#352)

Y. Dekempeneer1, M. D'Huyvetter1, E. Aneheim2, C. Xavier1, T. Lahoutte1, 3, T. Bäck2, H. Jensen4, V. Caveliers1, 3, S. Lindegren2

1 Vrije Universiteit Brussel, Brussels, Belgium, Laboratory of In Vivo Cellular and Molecular Imaging, Jette, Brussels, Belgium
2 University of Gothenburg, Targeted Alpha Therapy group, Gothenburg, Gothenburg, Sweden
3 UZ Brussel, Nuclear Medicine Department, Jette, Brussels, Belgium
4 Copenhagen University Hospital, Cyclotron and PET Unit, Copenhagen, Copenhagen, Denmark

Introduction

This study investigates a novel targeted therapy which combines the α-emitter Astatine-211 and HER2-targeting nanobodies to selectively kill HER2+ metastases. α-particle radiation is characterized by a very short path length. This means that high amounts of cytotoxic radiation are released in a small and focused area. α-camera imaging might give us more insight in the distribution of radiolabelled nanobodies at cellular level. Two different radiochemical methodologies were validated in vitro and in vivo using three different prosthetic groups.

Methods

Using the “random labelling method”, the primary amines of lysine’s on the Nb are used as conjugation sites. Via the “site-specific labelling approach” the carboxyl-terminal cysteine of anti-HER2 His6-Linker-Cys is used as a conjugation site. The anti-HER2-Nb was thus randomly labelled with 211At via the prosthetic groups m-eATE and SGMAB, while the anti-HER2-His6-Linker-Cys Nb was site-specifically labelled using MSB. The immunoreactive fraction (IRF) was evaluated on HER2+ cells using the Lindmo assay. In vivo biodistribution of the three different 211At-Nbs was assessed in HER2+-SKOV-3 xenografts. Furthermore, ex vivo α-camera imaging was done on cryosections of tumor, kidneys, stomach and spleen.

Results/Discussion

Astatination of anti-HER2 Nbs using m-eATE and a specific activity of 118 MBq/mg resulted in a 75% RCY and 99% RP. The use of SGMAB with a specific activity of 69 MBq/mg resulted in a 31% RCY and a 98% RP. 211At-MSB-Nb using a specific activity of 271 MBq/mg resulted in a 73% RCY and 96% RP. The tumor uptake of 211At-SGMAB-Nb was 9; 5; 5; 0,9 %ID/g at 1, 3, 6 and 24h. Except for the kidneys, rapid clearance of 211At-SGMAB-Nb from normal tissues was observed. Although higher activity levels were measured in stomach, lungs of m-eATE and MSB coupled Nbs. α-camera images of tumors targeted by 211At-SGMAB-Nb revealed high-intensity areas in the periphery and the core of the tumor 1hpi injection. The activity distribution becomes more uniform after 3h p.i., showing a homogeneous tumor distribution. At 1hpi, the images of the kidneys revealed a pronounced uptake of 211At-SGMAB-Nb in the renal cortex. At 3hpi, 211At was mainly located in the medulla, showing an efficient wash out from kidneys.

Conclusions

211At-SGMAB-Nb is the preferred radioimmunoconjugate for targeting HER2+ malignancies, due to its superior in vivo biodistribution. α-camera imaging allowed us to quantitatively analyze the time-dependent activity distributions for several tissues. Intra-renal measurements of 211At-SGMAB-Nb revealed concentrated activity in the renal cortex and more specifically in the glomeruli. Activity then quickly migrated to the kidney medulla and later to urine. Overall, the results are highly encouraging and suggest that 211At-labeled anti-HER2-Nbs require further investigation as probes for TAT.

alpha-camera images tumors 1h p.i. and 3h p.i of Astatinated nanobodies
α-camera images of tumors targeted by 211At-SGMAB-Nb, 211At-meATE-Nb and 211At-MSB-Nb revealed high-intensity areas both in the periphery and the core of the tumor 1h p.i. injection. The radioactivity distribution becomes more uniform after 3h p.i., showing a homogeneous tumor distribution.
Alpha-camera images of 211At-SGMAB-Nb 1h p.i and 3h p.i

At 1h p.i the images of the kidneys revealed a pronounced uptake of 211At-SGMAB-Nb in the renal cortex. After 3h p.i. the 211At activity was mainly located in the medulla, showing an efficient wash out of 211At-SGMAB-Nb from the kidneys.

# 253

Detection threshold and reproducibility of 68Ga-PSMA11 PET/CT in a mouse model of prostate cance (#42)

K. Lückerath1, 2, A. D. Stuparu1, L. Wei1, W. Kim1, C. G. Radu1, C. Mona1, J. Calais1, J. Czernin1, R. Slavik1, K. Herrmann1, 3, M. Eiber4, W. P. Fendler1

1 University of California Los Angeles, Molecular & Medical Pharmacology, Los Angeles, California, United States of America
2 University Hospital Wuerzburg, Nuclear Medicine, Wuerzburg, Germany
3 University Hospital Essen, Nuclear Medicine, Essen, Germany
4 Technical University Munich, Nuclear Medicine, Muenchen, Germany

Introduction

PET/CT with 68Ga-prostate specific membrane antigen (PSMA) ligands (re)stages prostate cancer with high accuracy. However, the characteristics of 68Ga-PSMA PET/CT, such as the association of absolute cell surface PSMA and image signal, have not been defined  yet. This study aimed at (i) exploring the relationship between quantitative cell surface PSMA and image signal and ii) determining reproducibility of 68Ga-PSMA11 PET/CT measurements in a murine prostate cancer model.

Methods

Murine prostate cancer cells (RM1) expressing different levels of human PSMA (RM1-YFP, -LOW, -MEDIUM, and -HIGH/PGLS) were injected into the shoulder of C57Bl/6 mice. PSMA expression was determined by 68Ga-PSMA11 PET/CT and quantitative flow cytometry of tumor fine needle aspiration biopsies on days 7 and 8 after tumor inoculation. Absolute number of cell surface PSMA was correlated with PET signal. The threshold for PET positivity was determined based on clinical PROMISE criteria. Inter reader (reader 1 vs. 2) as well as inter day (day 7 vs. 8) reproducibility was determined by intraclass correlation (ICC).

Results/Discussion

Tumor size was comparable across groups (mean±SEM 76±8 mm3). In our model, absolute cell surface PSMA expression was correlated with 68Ga-PSMA11 PET/CT signal. Approximately 20,000 PSMA receptors per tumor cell surface were identified as threshold for positive PET readings. This suggests that the threshold for PET positivity is rather low; on the other hand, correlation of the PET signal flattened at high PSMA levels.
Inter observer agreement critically determines feasibility of a method for clinical and research applications. When determined by two independent readers/on two separate days, maximum and average %IA/g 68Ga-PSMA11 were almost perfectly correlated (ICC 1.00/0.89 and 0.95/0.88, respectively). Visual PSMA expression score (0.69/0.57) and CT tumor volume (0.71/0.76) were substantially reproducible; the lower repeatability here might have been caused by overlap of kidney uptake with the reference organ liver/guts, which occurs to a greater degree in mice than in patients.

Conclusions

In summary, we characterize 68Ga-PSMA11 PET/CT in a murine model of prostate cancer by demonstrating a) for the first time a relationship between image signal and PSMA expression level with a low expression threshold for PET positivity; b) the reproducibility of image findings across readers and time points. Understanding these characteristics is crucial for image interpretation

Keywords: 68Ga-PSMA, PSMA, PET/CT, mouse model, radio ligand therapy, detection threshold, prostate cancer
# 254

In vivo imaging of polymersome uptake and distribution in cells and tumor bearing mice (#5)

S. J. Roobol1, 2, T. A. Hartjes3, R. M. de Kruijff6, J. D. M. Molkenboer-Kuenen7, S. Heskamp7, G. Torrelo-Villa6, R. Kanaar4, 1, A. B. Houtsmuller3, D. C. van Gent1, A. G. Denkova6, M. E. van Royen3, J. Essers1, 4, 5

1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands
2 Erasmus MC, Radiology & Nuclear Medicine, Rotterdam, Netherlands
3 Erasmus MC, Optical Imaging Centre, Rotterdam, Netherlands
4 Erasmus MC, Radiation Oncology, Rotterdam, Netherlands
5 Erasmus MC, Vascular Surgury, Rotterdam, Netherlands
6 Delft University of Technology, Radiation Science and Technology, Delft, Netherlands
7 Radboud University Medical Center, Radiology & Nuclear Medicine, Nijmegen, Netherlands

Introduction

Polymersomes, composed of amphiphilic block copolymers PB-b-PEO (polybutadiene - d - polyethylene oxide), have emerged as  promising robust customizable nano-carriers for high-LET radionuclides in radionuclide therapy. In this study, we analyzed on the uptake mechanism of polymersomes in cells in vitro using live cell microscopy and distribution in vivo using optical and SPECT/CT imaging of these novel nano-carriers.

Methods

Polymersomes were formed using a solvent displacement methodology. Characterization was done by Cryo-TEM and Dynamic Light Scattering. Polymersomes were  loaded with 111In or fluorescent PKH dyes for subsequent in vivo optical and SPECT imaging in MDA-MB-231 tumor bearing and control Balb/c nude mice. Confocal microscopy and fluorescence flow cytometry were used to quantify cellular uptake and analyze kinetics of polymersomes. We compared fibroblast, cancer and macrophage cell lines. Co-localization experiments were performed using PNT2C2 cells transfected and incubated with Rab4A, Rab7 or Lysotracker, respectively.

Results/Discussion

Polymersomes of 80nm were cleared from circulation within 1 hour. Interestingly, control mice showed longer circulating polymersomes compared to tumor bearing mice (139 v.s. 7 minute half-life, respectively). Biodistribution analysis showed high uptake in liver, spleen and bone-marrow compared to limited uptake in the tumor.  Live cell imaging demonstrated gradual intracellular uptake over time, cell cycle-dependent uptake kinetics and in addition we analyzed microtubule-mediated processing of polymersomes.  Co-localization showed polymersomes entering the endocytosis pathway through early endosomes (Rab4) and were transferred to late-endosomes (Rab7) and lysosomes (Lysotracker) after which they reside in a peri-nuclear localization. High-throughput analysis showed cell line specific uptake kinetics, evidenced in competition assays using macrophage cell lines.

Conclusions

Whole animal imaging showed very fast clearance of polymersomes by both the liver and spleen. With a combination of confocal, spinning disk and high-throughput microscopy we determined the timeframe, cell cycle-specificity and localization of fluorescent polymersome uptake in various cell types by endocytosis. To avoid healthy-organ damage using polymersomes this a specific accumulation might be countered by increasing the PEG-chain length. Future experiments using active targeting of the vesicles will give more insight in how polymersomes could be used in the clinic.

References

Wang G, de Kruijff RM, Rol A, Thijssen L, Mendes E, Morgenstern A, et al. Retention studies of recoiling daughter nuclides of 225Ac in polymer vesicles. Appl Radiat Isot. 2014 Feb;85:45-53.

 

 

In vitro analysis of polymersome uptake

(A,B) PNT2C2 cells stably expressing CAAX-GFP as membrane marker were incubated with polymersomes and followed over time. A, uptake over a time course of three hours. B, stills of dividing cells showing dramatic increase of uptake after division. (C) Competition assay using U2OS and J774 (white arrows) indicating heavy competition between cell lines. Polymersomes were labelled with PKH26 (red).

In vivo analysis of polymersome distribution and blood circulation.
(A) Balb/c nude mice bearing MDA-MB-231 tumors were injected with polymersomes (80nm, 111In-labaled, 15-20 MBq) and imaged at 4  and 24 hours post injection (h.p.i.) using SPECT/CT. (B) Both healthy control and tumor bearing mice were injected as before. At indicated time-points blood was sampled. Plot shows measured activity in blood samples. 
Keywords: nanoparticles, SPECT, CT, optical, polymersomes, confocal, fluorescence, therapy, tumor, imaging
# 255

Quantitative optoacoustic imaging detects known changes in vascularity in relationship to oestrogen receptor status: implications for clinical practice (#279)

O. Abeyakoon1, T. Torheim4, R. Manavaki1, I. Mendichovszky2, N. Dalhaus3, S. Morscher3, N. Burton3, M. Wallis2, P. Moyle2, J. Joseph4, I. Quiros Gonzales4, S. Bohndiek4, F. Gilbert1

1 University of Cambridge, Department of Radiology, Cambridge, Cambridge, United Kingdom
2 Cambridge University Hospital NHS Foundation Trust, Radiology, Cambridge, Cambridge, United Kingdom
3 iThera Medical GmbH, Munich, Germany
4 Li Ka Shing Centre University of Cambridge, CRUK, Cambridge, Cambridge, United Kingdom

Introduction

Oestrogen receptor (ER) positive breast cancer maybe treated with targeted hormonal therapy (e.g. tamoxifen/letrozole). An ER+ receptor status is known to be associated with increased vascularity (1). Receptor status is established by histopathology (core biopsy/post surgery), but this requires an invasive procedure with a real risk of undersampling or sampling error which fails to appreciate the heterogeneity of receptor expression. Our aim was to determine if optoacoustic imaging could detect vascularity changes in patients with ER+ breast cancer.

Methods

Following IRB approval 23 patients with breast cancer undergoing primary surgery were enrolled. All patients underwent an optoacoustic scan using OPUS Acuity iThera Medical GmbH at 800nm (isosbestic point of oxy- and deoxyhaemoglobin absorption spectra). The 800nm signal intensity was used as a surrogate measure of vascularity. ROIs were drawn in MATLAB using the US image to outline the lesion and an automated 3mm ROI was then propagated to the optoacoustic images to obtain a measure of vascularity in the peri-lesional boundary zone. The goal standard for determining ER receptor status was surgical histopathology. Statistical analysis was performed using Mann Whitney U test in GraphPad Prism.

Results/Discussion

19 patients with ER+ disease and 4 patients with ER- disease were included in the study. Results are presented as mean and standard error of the mean. Whole lesions measurements of 800nm were statistically significantly higher (p=0.012) in ER positive disease (50.86 AU +/- 6.06) in comparison to ER negative disease ( 23.18 AU +/- 5.65). A similar statistically significant pattern (p=0.02) was observed in the peri-lesional boundary zone vascularity at 800nm. The measurements were (96.69 AU +/- 15.59) in ER positive disease and (42.52 AU +/- 14.23) in ER negative disease.

Conclusions

Preliminary results are promising suggesting the possibility of non-invasive characterization of vascularity in relation to ER status.

References

1. Lloyd et al.Vascular measurements correlate with estrogen receptor status BMC Cancer 2014, 14:279

Acknowledgement

O Abeyakoon is funded by CRUK

We would also like to thank University of Cambridge and Biomedical Research campus for funding. 

# 256

In vitro and in vivo evaluation of 177Lu-radiolabelled nanogel-bombesin conjugates. (#533)

M. Orzelowska1, M. Maurin1, P. Garnuszek1, R. Mikolajczak1, C. Dispenza2, 3, M. A. Sabatino2, L. A. Ditta2

1 National Centre for Nuclear Research Radioisotope Centre POLATOM, Otwock, Poland
2 Dipartimento dell’Innovazione Industriale e Digitale, Palermo, Italy
3 Istituto di Biofisica (IBF), Consiglio Nazionale delle Ricerche, Palermo, Italy

Introduction

Poly-N-vinyl pyrrolidone-co-acrylic acid nanogels have been developed as nanocarrier system. Their small size (80nm), minimal toxicity, stability in serum and easiness of their surface modification (by attachment of targeting molecules, fluorescent probes or chelators for radiolabelling) make them interesting candidates for innovative drug delivery system. The aim of presented work was the evaluation of biological behavior of 177Lu radiolabeled nanogel which was prior modified by conjugation of DOTA- 5-amino pentanoic acid-BBN(7-14) (DOTA-bombesin).

Methods

DOTA-bombesin-nanogels and DOTA-bombesin as reference were labelled with 177LuCl3 (LutaPol)(specific activity >500 MBq/mg). The labelling yield was assessed by TLC method. The biological activity was evaluated by carrying out in vitro binding studies in AR42J cells (rat’s pancreatic cancer) known to express bombesin receptors. AR42J cells were grown in RPMI1640 medium supplemented with 10% FBS and antibiotics (penicillin 100 U/mL, and streptomycin 100 μg/mL). The cells were seeded on 12-well plates in concentration of 0.6 mln per well and after 1 hour incubation with radiolabelled peptide were treated with glicine buffer and NaOH for determining binding and internalization, respectively.  Biodistribution was assessed  1h after intravenous injection of the radiolabelled particles  (10MBq/ 0.1mL) into tail vain of healthy Wistar rats.

Results/Discussion

[177Lu]-DOTA--bombesin-nanogels showed high affinity to the AR42J cells at the level of 8.4% with 80% internalisation, comparable to [177Lu]-DOTA-bombesin. In vivo biodistribution revealed that liver is a critical organ with over 80 % accumulation of injected dose (%ID), while renal excretion was only at the level of 4 %.

Conclusions

Comparable affinity to the AR42J cells of the [177Lu]-DOTA--bombesin-nanogels and [177Lu]-DOTA-bombesin suggests their potential for cancer diagnosis and therapy. However, the preliminary biodistribution data showed that particles accumulate in the liver. That high hepatic accumulation could be overcome by modification of administration route or by modification of nanoparticles size or/and surface.

Acknowledgement

The study is a part of the project coordinated by International Atomic Agency nr.  F22064 „Nanosized Delivery Systems for Radiopharmaceuticals”

Keywords: bombesin, nanogels, DOTA-nanogel-bombesin, radiopharmaceuticals
# 257

Development and validation of a pretargeting strategy for radio-immunotherapy with novel 188Re-labelled vector molecules. (#553)

T. Van Royen1, C. Vanhove2, F. De Vos1

1 Ghent University, Laboratorium of Radiopharmacy, Ghent, Belgium
2 Ghent University, Department of Electronics and information systems, Ghent, Belgium

Introduction

Conventional radioimmunotherapy (RIT) is based on a radiolabelled monoclonal antibody (mAb) that targets an antigen expressed on a tumor cell. Prolonged circulation in the blood increases the radiation burden to healthy tissue, limiting the maximum activity that can be administered.
Pretargeting RIT can overcome these drawbacks. First, a mAb targeting the antigen expressed on a tumor cell is injected. Second, a small radiolabelled vectormolecule with high affinity for the mAb is administered. This small molecule is cleared rapidly out of the blood stream, limiting the radiation burden1,2.

Methods

In order to achieve a rapid interaction between the mAb and the vector molecule, the inverse electron demand Diels-Alder reaction is used. This principle consists of a transcyclo-octene modified mAb (TCO-mAb) and a radiolabelled tetrazine vector3. In a first step, the radiolabelled vector molecule will be synthesized. The scheme below describes the synthetic route that will be applied. The radiolabelling will be performed with rhenium-188, since its half-life is suitable for pretargeting strategies. Secondly, the mAb will be modified with a TCO functional group. Commercially available TCO-NHS will be coupled to trastuzumab using an amine-reaction to lysine residues. In a last phase, optimization of the coupling between the TCO-modified mAb and the 188Re-vector will be performed.

Results/Discussion

Synthesis of the tetrazine tag (II) was carried out successfully with reaction yields of 33.8% pure product. In literature4, for similar reactions, yields were obtained up to 70%. This difference could be due to the use of hydrazine monohydrate instead of anhydrous hydrazine, as described in literature. Step 2 of the synthesis route was carried out starting from this tetrazine tag. Reaction yields were again lower than expected. Therefore, no further reactions were carried out. Step 1 will be performed again when the anhydrous hydrazine is commercially available. This is more closely related to data described in literature. The applied synthesis scheme is based upon the use of S-mercaptoacetyltriglycine (MAG3) as chelator. When this synthesis route does not give proper yields, other chelators can be implemented. When the in vivo behavior or the in vitro stability of the vector are not optimal for pretargeting RIT, the polyethyleneglycol moiety of the vector can be removed or extended.

Conclusions

The first steps of the synthesis of the vector were carried out successfully, but yields were lower than expected when compared to data found in literature. Future synthesis will be carried out by use of anhydrous hydrazine instead of the hydrazine monohydrate. Further optimizations and modifications will be implemented in the synthesis route for both the vector molecule as well as for the TCO-modified mAb.

References

(1) F. Kraeber-Bodéré, C. Rousseau, C. Bodet-Milin, E. Frampas, A. Faivre-Chauvet, A. Rauscher, R.M. Sharkey, D.M. Goldenberg, J.F. Chatal and J. Barbet. A pretargeting system for tumor PET imaging and radioimmunotherapy Front. Pharmacol., 2015, 6, 54.


(2) D.M. Goldenberg, C.H. Hang, E.A. Rossi, J.W. McBride and R.M. Sharkey. Pretargeted molecular imaging and radioimmunotherapy. Theranostics, 2012, 2, 523-540

(3) T. Reiner, B. Zeglis. The inverse electron demand Diels–Alder click reaction in radiochemistry. J Labelled Comp Radiopharm., 2014, 57(4), 285–290

(4) J. Yang, M.R. Karver, W. Li, S. Sahu, N.K. Devaraj. Metal-Catalyzed One-Pot Synthesis of Tetrazines Directly from Aliphatic Nitriles and Hydrazine. Angew. Chem. 2012, 51(21), 5222–5225

Synthesis route for tetrazine vector molecule: step 3
Synthesis route for tetrazine vector molecule: step 1 and 2
Keywords: Pretargeting, diels alder reaction, spect, chelators