Dr Gabriela Kramer-Marek
Group Leader: Preclinical Molecular Imaging
Biography
Dr Gabriela Kramer-Marek specialises in the use of molecular imaging for early prediction of treatment response with emphasis on new targeted therapies. She received her PhD in Medical Physics from the Silesian University, Poland. Afterwards, she worked as a postdoctoral research fellow in the National Cancer Institute (NCI), National Institute of Health, USA, and was then appointed as an assistant professor at Indiana University, USA.
Her post-doctoral work at the NCI resulted in the development of a new approach for non-invasive assessment of HER2 expression in breast cancer using PET imaging and radiolabelled HER2-specific Affibody molecules. The publication arising from this initial body of work was selected to receive the 2008 Annual European Association of Nuclear Medicine Springer Prize for best basic science paper.
In addition, one of her papers showing that Affibody molecules could be used to monitor possible changes in HER2 expression in response to therapeutic intervention was the subject of an Invited Opinion (editorial) in the Journal of Nuclear Medicine, press releases by the NIH and the Society for Nuclear Medicine, and was featured in the October 2009 issue of RSNA News.
In May 2012, Molecular Imaging News featured her most recent paper showing that 18F-labeled, HER2-specific Affibody allows early detection of HER2-positive pulmonary metastases with more specificity than 18F-FDG (Kramer-Marek et al. J Nucl Med 2012). This publication has been also announced by the editors of the Journal of Nuclear Medicine to be the best basic science investigation manuscript published in their journal in 2012.
Dr Kramer-Marek has been an invited speaker at several national and international meetings and has won a variety of scientific awards over the course of her career, including in 2010 Outstanding Postdoctoral Fellow of the NCI. She has also a strong commitment to training and mentoring students at both graduate and undergraduate educational levels.
Dr Kramer-Marek is a member of the Cancer Research UK Convergence Science Centre, which brings together leading researchers in engineering, physical sciences, life sciences and medicine to develop innovative ways to address challenges in cancer.
BSc Medical Physics, University of Silesia; Department of Physics; Katowice, Poland.
MSc (Medical Physics, graduated with honors), University of Silesia; Department of Physics; Katowice, Poland.
Ph.D. (Medical Physics, graduated with honors), University of Silesia, Department of Physics, Katowice, Poland.
PhD Medical Physics, University of Silesia, Katowice, Poland.
Editors’ Choice Award for one of the best basic science investigation manuscripts published in JNM in 2012, The Journal of Nuclear Medicine, 2012.
2010 Outstanding Postdoctoral Fellow of National Cancer Institute, National Cancer Institute/ National Institute of Health, 2010.
Springer Prize for Best Basic Science Paper in 2008, European Association of Nuclear Medicine, 2008.
Editorial BoardsTomography Journal.
The Society of Nuclear Medicine, Member, SNM, 2011.
The American Association for Cancer Research, Member, AACR, 2007.
Related pages
Types of Publications
Journal articles
The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.
Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice.
<h4>Background</h4>Accurate quantification in molecular imaging is essential to improve the assessment of novel drugs and compare the radiobiological effects of therapeutic agents prior to in-human studies. The aim of this study was to investigate the challenges and feasibility of pre-clinical quantitative imaging and mouse-specific dosimetry of <sup>111</sup>In-labelled radiotracers. Attenuation, scatter and partial volume effects were studied using phantom experiments, and an activity calibration curve was obtained for varying sphere sizes. Six SK-OV-3-tumour bearing mice were injected with <sup>111</sup>In-labelled HER2-targeting monoclonal antibodies (mAbs) (range 5.58-8.52 MBq). Sequential SPECT imaging up to 197 h post-injection was performed using the Albira SPECT/PET/CT pre-clinical scanner. Mice were culled for quantitative analysis of biodistribution studies. The tumour activity, mass and percentage of injected activity per gram of tissue (%IA/g) were calculated at the final scan time point and compared to the values determined from the biodistribution data. Delivered <sup>111</sup>In-labelled mAbs tumour absorbed doses were calculated using mouse-specific convolution dosimetry, and absorbed doses for <sup>90</sup>Y-labelled mAbs were extrapolated under the assumptions of equivalent injected activities, biological half-lives and uptake distributions as for <sup>111</sup>In.<h4>Results</h4>For the sphere sizes investigated (volume 0.03-1.17 ml), the calibration factor varied by a factor of 3.7, whilst for the range of tumour masses in the mice (41-232 mg), the calibration factor changed by a factor of 2.5. Comparisons between the mice imaging and the biodistribution results showed a statistically significant correlation for the tumour activity (r = 0.999, P < 0.0001) and the tumour mass calculations (r = 0.977, P = 0.0008), whilst no correlation was found for the %IA/g (r = 0.521, P = 0.29). Median tumour-absorbed doses per injected activity of 52 cGy/MBq (range 36-69 cGy/MBq) and 649 cGy/MBq (range 441-950 cGy/MBq) were delivered by <sup>111</sup>In-labelled mAbs and extrapolated for <sup>90</sup>Y-labelled mAbs, respectively.<h4>Conclusions</h4>This study demonstrates the need for multidisciplinary efforts to standardise imaging and dosimetry protocols in pre-clinical imaging. Accurate image quantification can improve the calculation of the activity, %IA/g and absorbed dose. Diagnostic imaging could be used to estimate the injected activities required for therapeutic studies, potentially reducing the number of animals used.
The future of <sup>89</sup>Zr-based immuno-PET is reliant upon the development of new chelators with improved stability compared to the currently used deferoxamine (DFO). Herein, we report the evaluation of the octadentate molecule DFO-HOPO (3) as a suitable chelator for <sup>89</sup>Zr and a more stable alternative to DFO. The molecule showed good potential for the future development of a DFO-HOPO-based bifunctional chelator (BFC) for the radiolabelling of biomolecules with <sup>89</sup>Zr. This work broadens the selection of available chelators for <sup>89</sup>Zr in search of improved successors to DFO for clinical <sup>89</sup>Zr-immuno-PET.
<h4>Introduction</h4>Radioimmunotherapy (RIT) with monoclonal antibodies and their fragments labelled with radionuclides emitting α -particles, β-particles or Auger electrons have been used for many years in the development of anticancer strategies. While RIT has resulted in approved radiopharmaceuticals for the treatment of hematological malignancies, its use in solid tumors still remains challenging.<h4>Areas covered</h4>In this review, we discuss the exciting progress towards elucidating the potential of current and novel radioimmunoconjugates and address the challenges for translation into clinical practice.<h4>Expert opinion</h4>There are still technical and logistical challenges associated with the use of RIT in routine clinical practice, including development of novel and more specific targeting moieties, broader access α to α-emitters and better tailoring of pre-targeting approaches. Moreover, improved understanding of the heterogeneous nature of solid tumors and the critical role of tumor microenvironments will help to optimize clinical response to RIT by delivering sufficient radiation doses to even more radioresistant tumor cells.
Glioblastomas (GBMs) are high-grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12-15 months following standard therapy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR-specific affibody (Z<sub>EGFR:03115</sub> ) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near-infrared light produces a cytotoxic response. Z<sub>EGFR:03115</sub> -IR700DX EGFR-specific binding was confirmed by flow cytometry and confocal microscopy. The conjugate showed effective targeting of EGFR positive GBM cells in the brain. The therapeutic potential of the conjugate was assessed both in vitro, in GBM cell lines and spheroids by the CellTiter-Glo® assay, and in vivo using subcutaneous U87-MGvIII xenografts. In addition, mice were imaged pre- and post-PIT using the IVIS/Spectrum/CT to monitor treatment response. Binding of the conjugate correlated to the level of EGFR expression in GBM cell lines. The cell proliferation assay revealed a receptor-dependent response between the tested cell lines. Inhibition of EGFRvIII+ve tumor growth was observed following administration of the immunoconjugate and irradiation. Importantly, this response was not seen in control tumors. In conclusion, the Z<sub>EGFR:03115</sub> -IR700DX showed specific uptake in vitro and enabled imaging of EGFR expression in the orthotopic brain tumor model. Moreover, the proof-of-concept in vivo PIT study demonstrated therapeutic efficacy of the conjugate in subcutaneous glioma xenografts.
<b>Purpose:</b> Recent studies have highlighted a role of HER3 in HER2-driven cancers (e.g., breast cancer), implicating the upregulation of the receptor in resistance to HER-targeted therapies and Hsp90 inhibitors (e.g., AUY922). Therefore, we have developed an affibody-based PET radioconjugate that quantitatively assesses HER3 changes induced by Hsp90 inhibition <i>in vivo</i><b>Experimental Design:</b> Z<sub>HER3:8698</sub> affibody molecules were conjugated via the C-terminus cysteine to DFO-maleimide for <sup>89</sup>Zr radiolabeling. The probe was characterized <i>in vitro</i> and <i>in vivo</i> in a panel of human breast cell lines and xenograft models with varying HER3 receptor levels. In addition, the radioconjugate was investigated as a tool to monitor the outcome of AUY922, an Hsp90 inhibitor, in an MCF-7 xenograft model.<b>Results:</b> We demonstrated that <sup>89</sup>Zr-DFO-Z<sub>HER3:8698</sub> can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our <i>in vitro</i> findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to treatment with AUY922 through HER3/IGF-1Rβ-mediated signaling. Of note, the lack of response <i>in vitro</i> due to HER3 recovery was confirmed <i>in vivo</i> using <sup>89</sup>Zr-DFO-Z<sub>HER3:8698</sub>-based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 upregulation concomitant with an increase in IGF-1Rβ expression.<b>Conclusions:</b> These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not responding to targeted therapies due to HER3 recovery. <i>Clin Cancer Res; 24(8); 1853-65. ©2018 AACR</i>.
Trifluoromethyl groups are widespread in medicinal chemistry, yet there are limited 18F-radiochemistry techniques available for the production of the complementary PET agents. Herein, we report the first radiosynthesis of the anticancer nucleoside analogue trifluridine, using a fully automated, clinically-applicable 18F-trifluoromethylation procedure. [18F]Trifluridine was obtained after two synthetic steps in <2 hours. The isolated radiochemical yield was 3% ± 0.44 (n = 5), with a radiochemical purity >99%, and a molar activity of 0.4 GBq μmol-1 ± 0.05. Biodistribution and PET-imaging data using HCT116 tumour-bearing mice showed a 2.5 %ID g-1 tumour uptake of [18F]trifluridine at 60 minutes post-injection, with bone uptake becoming a prominent feature thereafter. In vivo metabolite analysis of selected tissues revealed the presence of the original radiolabelled nucleoside analogue, together with deglycosylated and phosphorylated [18F]trifluridine as the main metabolites. Our findings suggest a potential role for [18F]trifluridine as a PET radiotracer for elucidation of drug mechanism of action.
<b>Background:</b> Overexpression of EGFR is a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). Patients with HNSCC who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Strategies to identify HNSCC patients likely to benefit from EGFR-targeted therapies, together with biomarkers of treatment response, would have clinical value. <b>Methods:</b> Functional MRI and <sup>18</sup>F-FDG PET were used to visualize and quantify imaging biomarkers associated with drug response within size-matched EGFR TKI-resistant CAL 27 (CAL<sup>R</sup>) and sensitive (CAL<sup>S</sup>) HNSCC xenografts <i>in vivo</i>, and pathological correlates sought. <b>Results:</b> Intrinsic susceptibility, oxygen-enhanced and dynamic contrast-enhanced MRI revealed significantly slower baseline R2∗ , lower hyperoxia-induced ΔR2∗ and volume transfer constant K<sup>trans</sup> in the CAL<sup>R</sup> tumors which were associated with significantly lower Hoechst 33342 uptake and greater pimonidazole-adduct formation. There was no difference in oxygen-induced ΔR<sub>1</sub> or water diffusivity between the CAL<sup>R</sup> and CAL<sup>S</sup> xenografts. PET revealed significantly higher relative uptake of <sup>18</sup>F-FDG in the CAL<sup>R</sup> cohort, which was associated with significantly greater Glut-1 expression. <b>Conclusions:</b> CAL<sup>R</sup> xenografts established from HNSCC cells resistant to EGFR TKIs are more hypoxic, poorly perfused and glycolytic than sensitive CAL<sup>S</sup> tumors. MRI combined with PET can be used to non-invasively assess HNSCC response/resistance to EGFR inhibition.
In head and neck squamous cell cancer, the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR monoclonal antibody used for the treatment of head and neck squamous cell cancer, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate noninvasive measurement of EGFR expression across the entire tumor burden and allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. <b>Methods:</b> EGFR-specific Affibody molecule (Z<sub>EGFR:03115</sub>) was radiolabeled with <sup>89</sup>Zr and <sup>18</sup>F. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting <sup>89</sup>Zr-deferoxamine-Z<sub>EGFR:03115</sub> (2.4-3.6 MBq, 2 μg) either together with or 30 min after increasing amounts of unlabeled Z<sub>EGFR:03115</sub> (1, 5, 10, 15, and 20 μg). PET images were acquired at 3, 24, and 48 h after injection, and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot, and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed, and <sup>18</sup>F-aluminium fluoride-NOTA-Z<sub>EGFR:03115</sub> (12 μg, 1.5-2 MBq/mouse) was used to monitor receptor changes. <b>Results:</b> In vivo studies demonstrated that coinjecting 10 μg of nonlabeled molecules with <sup>89</sup>Zr-deferoxamine-Z<sub>EGFR:03115</sub> allows for clear tumor visualization 3 h after injection. The radioconjugate tumor accumulation was EGFR-specific, and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration, and receptor expression in tumor tissues. Additionally, <sup>18</sup>F-aluminium fluoride-NOTA-Z<sub>EGFR:03115</sub> could measure receptor downregulation in response to EGFR inhibition. <b>Conclusion:</b> Z<sub>EGFR:03115</sub>-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.
Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH<sub>2</sub>) analogue infraluciferin (iLH<sub>2</sub>), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5'-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH<sub>2</sub> reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH<sub>2</sub> is superior to LH<sub>2</sub> for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.
Site-selective bioconjugation of cysteine-containing peptides and proteins is currently achieved via a maleimide-thiol reaction (Michael addition). When maleimide-functionalized chelators are used and the resulting bioconjugates are subsequently radiolabeled, instability has been observed both during radiosynthesis and post-injection in vivo, reducing radiochemical yield and negatively impacting performance. Recently, a phenyloxadiazolyl methylsulfone derivative (PODS) was proposed as an alternative to maleimide for the site-selective conjugation and radiolabeling of proteins, demonstrating improved in vitro stability and in vivo performance. Therefore, we have synthesized two novel PODS-bearing bifunctional chelators (NOTA-PODS and NODAGA-PODS) and attached them to the EGFR-targeting affibody molecule Z<sub>EGFR:03115</sub>. After radiolabeling with the aluminum fluoride complex ([<sup>18</sup>F]AlF), both conjugates showed good stability in murine serum. When injected in high EGFR-expressing tumor-bearing mice, [<sup>18</sup>F]AlF-NOTA-PODS-Z<sub>EGFR:03115</sub> and [<sup>18</sup>F]AlF-NODAGA-PODS-Z<sub>EGFR:03115</sub> showed similar pharmacokinetics and a specific tumor uptake of 14.1 ± 5.3% and 16.7 ± 4.5% ID/g at 1 h post-injection, respectively. The current results are encouraging for using PODS as an alternative to maleimide-based thiol-selective bioconjugation reactions.
The presence of human epidermal growth factor type 2 (HER2) on 20-30% of human breast cancer is a prognostic indicator of more rapid disease progression and a therapeutic indicator for anti-HER2 monoclonal antibodies. Because the literature has demonstrated some discordance between primary and metastatic tumors in the same patient for expression of the HER2 marker, we set out to develop an imaging agent that could be used to assess the marker concentration in vivo in an individual patient. The pharmaceutical company Affibody AB has optimized the specificity of Affibody molecules for HER2. Two Affibody molecules, a 7 kD and an 8 kD protein, were designed with a single carboxy terminal cysteine in order to provide a specific location for the purposes of labeling for various types of imaging. We have prepared N-[2-(4-[(18)F]fluorobenzamido)ethyl]maleimide utilizing a coupling reaction between [(18)F]fluorobenzoic acid and aminoethylmaleimide. We then optimized the conjugation of this radiolabeled maleimide to the free sulfhydryl of cysteine by incubating at pH 7.4 in phosphate buffered saline containing 0.1% sodium ascorbate. An overall uncorrected yield of radiolabeled Affibody molecule of approximately 10% from [(18)F]fluoride was achieved in a 2 h synthesis. These conjugated Affibody molecules were obtained with a specific activity of 2.51 +/- 0.92 MBq/microg. Characterization of the product by HPLC-MS supported the conjugation of [(18)F]FBEM with the Affibody molecule. The radiolabeled Affibody molecule retained its binding specificity as demonstrated by successful imaging of xenografts expressing HER2.
PURPOSE: The expression of human epidermal growth factor receptor-2 (HER2) receptors in cancers is correlated with a poor prognosis. If assessed in vivo, it could be used for selection of appropriate therapy for individual patients and for monitoring of the tumor response to targeted therapies. We have radiolabeled a HER2-binding Affibody molecule with fluorine-18 for in vivo monitoring of the HER2 expression by positron emission tomography (PET). MATERIALS AND METHODS: The HER2-binding Z(HER2:342)-Cys Affibody molecule was conjugated with N-2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). The in vitro binding of the resulting radioconjugate was characterized by receptor saturation and competition assays. For in vivo studies, the radioconjugate was injected into the tail vein of mice bearing subcutaneous HER2-positive or HER2-negative tumors. Some of the mice were pre-treated with non-labeled Z(HER2:342)-Cys. The animals were sacrificed at different times post-injection, and the radioactivity in selected tissues was measured. PET images were obtained using an animal PET scanner. RESULTS: In vitro experiments indicated specific, high-affinity binding to HER2. PET imaging revealed a high accumulation of the radioactivity in the tumor as early as 20 min after injection, with a plateau being reached after 60 min. These results were confirmed by biodistribution studies demonstrating that, as early as 1 h post-injection, the tumor to blood concentration ratio was 7.5 and increased to 27 at 4 h. Pre-saturation of the receptors with unlabeled Z(HER2:342)-Cys lowered the accumulation of radioactivity in HER2-positive tumors to the levels observed in HER2-negative ones. CONCLUSION: Our results suggest that the [18F]FBEM-Z(HER2:342) radioconjugate can be used to assess HER2 expression in vivo.
PURPOSE: HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, that is, whose binding does not interfere with the binding of the therapeutic agents and whose effect on the target cells is minimal. EXPERIMENTAL DESIGN: We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semiquantitative in vivo NIR optical imaging studies were carried out using mice with s.c. xenografts of HER2-positive tumors. RESULTS: The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. CONCLUSION: Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific NIR probe for the noninvasive semiquantitative imaging of HER2 expression in vivo.
Glioblastoma (GBM) is a primary neuroepithelial tumor of the central nervous system, characterized by an extremely aggressive clinical phenotype. Patients with GBM have a poor prognosis and only 3-5% of them survive for more than 5 years. The current GBM treatment standards include maximal resection followed by radiotherapy with concomitant and adjuvant therapies. Despite these aggressive therapeutic regimens, the majority of patients suffer recurrence due to molecular heterogeneity of GBM. Consequently, a number of potential diagnostic, prognostic, and predictive biomarkers have been investigated. Some of them, such as IDH mutations, 1p19q deletion, MGMT promoter methylation, and EGFRvIII amplification are frequently tested in routine clinical practice. With the development of sequencing technology, detailed characterization of GBM molecular signatures has facilitated a more personalized therapeutic approach and contributed to the development of a new generation of anti-GBM therapies such as molecular inhibitors targeting growth factor receptors, vaccines, antibody-based drug conjugates, and more recently inhibitors blocking the immune checkpoints. In this article, we review the exciting progress towards elucidating the potential of current and novel GBM biomarkers and discuss their implications for clinical practice.
The human epidermal growth factor receptor 3 (HER3) is overexpressed in several cancers, being linked to a more resistant phenotype and hence leading to poor patient prognosis. Imaging HER3 is challenging owing to the modest receptor number (<50000 receptors/cell) in overexpressing cancer cells. Therefore, to image HER3 in vivo, high target affinity PET probes need to be developed. This work describes two different [(18)F]AlF radiolabeling strategies of the ZHER3:8698 affibody molecule specifically targeting HER3. The one-pot radiolabeling of ZHER3:8698 performed at 100 °C and using 1,4,7-triazanonane-1,4,7-triacetate (NOTA) as chelator resulted in radiolabeled products with variable purity attributed to radioconjugate thermolysis. An alternative approach based on the inverse electron demand Diels-Alder (IEDDA) reaction between a novel tetrazine functionalized 1,4,7-triazacyclononane-1,4-diacetate (NODA) chelator and the trans-cyclooctene (TCO) functionalized affibody molecule was also investigated. This method enabled the radiolabeling of the protein at room temperature. The [(18)F]AlF-NOTA-ZHER3:8698 and [(18)F]AlF-NODA-ZHER3:8698 conjugates showed a specific uptake at 1 h after injection in high HER3-expressing MCF-7 tumors of 4.36 ± 0.92% ID/g and 4.96 ± 0.65% ID/g, respectively. The current results are encouraging for further investigation of [(18)F]AlF-NOTA-ZHER3:8698 as a HER3 imaging agent.
Pancreatic ductaladeno carcinoma (PDAC) is a deadly cancer characterized by multiple molecular alterations, the presence of an intense stroma, poor perfusion, and resistance to therapy. In addition to standard imaging techniques, experimental imaging strategies, such as those utilizing molecular probes, nanoparticle-based agents, and tagged antibodies are actively being explored experimentally. It is hoped that advances in these technologies will allow for detecting PDAC at an early stage, and could serve to validate experimental therapies, rapidly identify non-responders, and assist in the design of novel therapeutic strategies tailored to the patient's molecular profile.
BACKGROUND: Care providers use complementary information from multiple imaging modalities to identify and characterize metastatic tumors in early stages and perform surveillance for cancer recurrence. These tasks require volume quantification of tumor measurements using computed tomography (CT) or magnetic resonance imaging (MRI) and functional characterization through positron emission tomography (PET) imaging. In vivo volume quantification is conducted through image segmentation, which may require both anatomical and functional images available for precise tumor boundary delineation. Although integrating multiple image modalities into the segmentation process may improve the delineation accuracy and efficiency, due to variable visibility on image modalities, complex shape of metastatic lesions, and diverse visual features in functional and anatomical images, a precise and efficient segmentation of metastatic breast cancer remains a challenging goal even for advanced image segmentation methods. In response to these challenges, we present here a computer-assisted volume quantification method for PET/MRI dual modality images using PET-guided MRI co-segmentation. Our aims in this study were (1) to determine anatomical tumor volumes automatically from MRI accurately and efficiently, (2) to evaluate and compare the accuracy of the proposed method with different radiotracers (18F-Z HER2-Affibody and 18F-flourodeoxyglucose (18F-FDG)), and (3) to confirm the proposed method's determinations from PET/MRI scans in comparison with PET/CT scans. METHODS: After the Institutional Administrative Panel on Laboratory Animal Care approval was obtained, 30 female nude mice were used to construct a small-animal breast cancer model. All mice were injected with human breast cancer cells and HER2-overexpressing MDA-MB-231HER2-Luc cells intravenously. Eight of them were selected for imaging studies, and selected mice were imaged with MRI, CT, and 18F-FDG-PET at weeks 9 and 10 and then imaged with 18F-Z HER2-Affibody-PET 2 days after the scheduled structural imaging (MRI and CT). After CT and MR images were co-registered with corresponding PET images, all images were quantitatively analyzed by the proposed segmentation technique.Automatically determined anatomical tumor volumes were compared to radiologist-derived reference truths. Observer agreements were presented through Bland-Altman and linear regression analyses. Segmentation evaluations were conducted using true-positive (TP) and false-positive (FP) volume fractions of delineated tissue samples, as complied with the state-of-the-art evaluation techniques for image segmentation. Moreover, the PET images, obtained using different radiotracers, were examined and compared using the complex wavelet-based structural similarity index (CWSSI). (continued on the next page) (continued from the previous page) RESULTS: PET/MR dual modality imaging using the 18F-Z HER2-Affibody imaging agent provided diagnostic image quality in all mice with excellent tumor delineations by the proposed method. The 18F-FDG radiotracer did not show accurate identification of the tumor regions. Structural similarity index (CWSSI) between PET images using 18F-FDG and 18F-Z HER2-Affibody agents was found to be 0.7838. MR showed higher diagnostic image quality when compared to CT because of its better soft tissue contrast. Significant correlations regarding the anatomical tumor volumes were obtained between both PET-guided MRI co-segmentation and reference truth (R2=0.92, p<0.001 for PET/MR, and R2=0.84, p<0.001, for PET/CT). TP and FP volume fractions using the automated co-segmentation method in PET/MR and PET/CT were found to be (TP 97.3%, FP 9.8%) and (TP 92.3%, FP 17.2%), respectively. CONCLUSIONS: The proposed PET-guided MR image co-segmentation algorithm provided an automated and efficient way of assessing anatomical tumor volumes and their spatial extent. We showed that although the 18F-Z HER2-Affibody radiotracer in PET imaging is often used for characterization of tumors rather than detection, sensitivity and specificity of the localized radiotracer in the tumor region were informative enough; therefore, roughly determined tumor regions from PET images guided the delineation process well in the anatomical image domain for extracting accurate tumor volume information. Furthermore, the use of 18F-FDG radiotracer was not as successful as the 18F-Z HER2-Affibody in guiding the delineation process due to false-positive uptake regions in the neighborhood of tumor regions; hence, the accuracy of the fully automated segmentation method changed dramatically. Last, we qualitatively showed that MRI yields superior identification of tumor boundaries when compared to conventional CT imaging.
<h4>Purpose</h4>To develop an automated pulmonary image analysis framework for infectious lung diseases in small animal models.<h4>Methods</h4>The authors describe a novel pathological lung and airway segmentation method for small animals. The proposed framework includes identification of abnormal imaging patterns pertaining to infectious lung diseases. First, the authors' system estimates an expected lung volume by utilizing a regression function between total lung capacity and approximated rib cage volume. A significant difference between the expected lung volume and the initial lung segmentation indicates the presence of severe pathology, and invokes a machine learning based abnormal imaging pattern detection system next. The final stage of the proposed framework is the automatic extraction of airway tree for which new affinity relationships within the fuzzy connectedness image segmentation framework are proposed by combining Hessian and gray-scale morphological reconstruction filters.<h4>Results</h4>133 CT scans were collected from four different studies encompassing a wide spectrum of pulmonary abnormalities pertaining to two commonly used small animal models (ferret and rabbit). Sensitivity and specificity were greater than 90% for pathological lung segmentation (average dice similarity coefficient > 0.9). While qualitative visual assessments of airway tree extraction were performed by the participating expert radiologists, for quantitative evaluation the authors validated the proposed airway extraction method by using publicly available EXACT'09 data set.<h4>Conclusions</h4>The authors developed a comprehensive computer-aided pulmonary image analysis framework for preclinical research applications. The proposed framework consists of automatic pathological lung segmentation and accurate airway tree extraction. The framework has high sensitivity and specificity; therefore, it can contribute advances in preclinical research in pulmonary diseases.
The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.
Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.
The macromonomer method was used to prepare cross-linked, paclitaxel-loaded polylactide (PLA)-polyethylene glycol (stealth) nanoparticles using free-radical dispersion polymerization. The method can facilitate the attachment of other molecules to the nanoparticle surface to make it multifunctional. Proton nuclear magnetic resonance and Fourier transform infrared spectra confirm the synthesis of PLA macromonomer and cross-linking agent. The formation of stealth nanoparticles was confirmed by scanning and transmission electron microscopy. The drug release isotherm of paclitaxel-loaded nanoparticles shows that the encapsulated drug is released over 7 days. In vitro cytotoxicity assay in selected breast and ovarian cancer cell lines reveal that the blank nanoparticle is biocompatible compared with medium-only treated controls. In addition, the paclitaxel-loaded nanoparticles exhibit similar cytotoxicity compared with paclitaxel in solution. Confocal microscopy reveals that the nanoparticles are internalized by MCF-7 breast cancer cells within 1 h. Preliminary biodistribution studies also show nanoparticle accumulation in tumor xenograft model. The nanoparticles are suitable for the controlled delivery of bioactive agents.
BACKGROUND: Near infrared (NIR) photoimmunotherapy (PIT) is a new type of cancer treatment based on a monoclonal antibody (mAb)-NIR phthalocyanine dye, (IR700) conjugate. In vitro cancer-specific cell death occurs during NIR light exposure in cells previously incubated with mAb-IR700 conjugates. However, documenting rapid cell death in vivo is more difficult. METHODS: A luciferase-transfected breast cancer cell (epidermal growth factor receptor+, MDA-MB-468luc cells) was produced and used for both in vitro and in vivo experiments for monitoring the cell killing effect of PIT. After validation of cytotoxicity with NIR exposure up to 8 J/cm2in vitro, we employed an orthotopic breast cancer model of bilateral MDA-MB-468luc tumors in female athymic mice, which subsequently received a panitumumab-IR700 conjugate in vivo. One side was used as a control, while the other was treated with NIR light of dose ranging from 50 to 150 J/cm2. Bioluminescence imaging (BLI) was performed before and after PIT. RESULTS: Dose-dependent cell killing and regrowth was successfully monitored by the BLI signal in vitro. Although tumor sizes were unchanged, BLI signals decreased by >95% immediately after PIT in vivo when light intensity was high (>100 J/cm2), however, in mice receiving lower intensity NIR (50 J/cm2), tumors recurred with gradually increasing BLI signal. CONCLUSION: PIT induced massive cell death of targeted tumor cells immediately after exposure of NIR light that was demonstrated with BLI in vivo.
UNLABELLED: The overexpression and overactivation of hepatocyte growth factor receptor (Met) in various cancers has been linked to increased proliferation, progression to metastatic disease, and drug resistance. Developing a PET agent to assess Met expression would aid in the diagnosis and monitoring of responses to Met-targeted therapies. In these studies, onartuzumab, the experimental therapeutic 1-armed monoclonal antibody, was radiolabeled with (76)Br or (89)Zr and evaluated as an imaging agent in Met-expressing cell lines and mouse xenografts. METHODS: (89)Zr-desferrioxamine (df)-onartuzumab was synthesized using a df-conjugate; (76)Br-onartuzumab was labeled directly. Met-binding studies were performed using the human tumor-derived cell lines MKN-45, SNU-16, and U87-MG, which have relatively high, moderate, and low levels of Met, respectively. Biodistribution and small-animal PET studies were performed in MKN-45 and U87-MG xenografts. RESULTS: (76)Br-onartuzumab and (89)Zr-df-onartuzumab exhibited specific, high-affinity Met binding (in the nanomolar range) that was concordant with established Met expression levels. In MKN-45 (gastric carcinoma) xenografts, both tracers cleared slowly from nontarget tissues, with the highest uptake in tumor, blood, kidneys, and lungs. (76)Br-onartuzumab MKN-45 tumor uptake remained relatively constant from 18 h (5 percentage injected dose per gram of tissue [%ID/g]) to 48 h (3 %ID/g) and exhibited tumor-to-muscle ratios ranging from 4:1 to 6:1. In contrast, (89)Zr-df-onartuzumab MKN-45 tumor uptake continued to accumulate from 18 h (10 %ID/g) to 120 h (23 %ID/g), attaining tumor-to-muscle ratios ranging from 20:1 to 27:1. MKN-45 tumors were easily visualized in imaging studies with both tracers at 18 h, but after 48 h (89)Zr-df-onartuzumab image quality improved, with at least 2-fold-greater tumor uptake than nontarget tissues. MKN-45 tumor uptake for both tracers correlated significantly with tumor mass and Met expression and was not affected by the presence of plasma shed Met. CONCLUSION: (89)Zr-df-onartuzumab and (76)Br-onartuzumab specifically targeted Met in vitro and in vivo; (89)Zr-df-onartuzumab achieved higher tumor uptake and tumor-to-muscle ratios than (76)Br-onartuzumab at later times, suggesting that (89)Zr-df-onartuzumab would be better suited to image Met for diagnostic and prognostic purposes.
UNLABELLED: Targeted therapies often depend on the expression of the target present in the tumor. This expression can be difficult to ascertain in widespread metastases. (18)F-FDG PET/CT, although sensitive, is nonspecific for particular tumor markers. Here, we compare the use of a human epidermal growth factor receptor 2 (HER2)-specific (18)F-Z(HER2)(:342)-Affibody and (18)F-FDG in HER2-expressing pulmonary metastases in a murine model of breast cancer. METHODS: The lung metastasis model was established by intravenous injection of MDA-MB-231(HER2)-Luc human breast cancer cells into the tail vein. Bioluminescence imaging was used to evaluate metastasis progression. Uptake of (18)F-Z(HER2)(:342)-Affibody and (18)F-FDG was confirmed by coregistration of the PET images with MR and CT images. At the end of the study, the presence of neoplastic cells and HER2 expression in lung tissues, and distribution of the tracer, were assessed ex vivo by immunohistochemistry and autoradiography. RESULTS: (18)F-Z(HER2)(:342)-Affibody successfully targeted HER2-positive lesions in the lung and allowed detection of metastases as early as 9 wk after injection of cells. In contrast, (18)F-FDG uptake was often masked by surrounding inflammatory changes and was nonspecific for HER2 expression. HER2 expression at a cellular level correlated well with tracer uptake on autoradiography. CONCLUSION: (18)F-Z(HER2)(:342)-Affibody is a promising tracer for evaluation of HER2 status of breast cancer metastases and is more specific for detecting HER2-positive lesions than (18)F-FDG.
Positron emission tomography (PET) has become an indispensable tool in the study of cancer biology, as well as in the clinical management of patients with cancer. Quantitative measurements of tracer accumulation enable researchers to detect tumor changes much earlier than by conventional imaging modalities. ¹⁸F-FDG has been widely utilized for staging and restaging of cancer, evaluation of response to the treatment, and assessment of prognosis; however, recently target-specific PET tracers have raised even more attention. This overview discusses the current status of PET imaging in optimization of cancer therapies in preclinical and clinical studies.
UNLABELLED: Currently, an alteration in the gross volume of a tumor is used to assess its response to trastuzumab; however, this approach provides only a late indication of response. Tissue-sample ex vivo assays are potentially valuable, but their procurement through biopsies is invasive and might be biased by tumor heterogeneity. We studied the feasibility of using PET to quantify changes in ErbB2 (HER2/neu) expression and to predict the response to trastuzumab in BT474 breast cancer xenografts with N-[2-(4-(18)F-fluorobenzamido)ethyl]maleimide ((18)F-FBEM)-HER(2:342) Affibody. METHODS: Mice bearing BT474 tumors were given trastuzumab (50 mg/kg loading dose, 25 mg/kg maintenance dose, administered intraperitoneally twice a week) or saline (control) for a total of 5 doses. Tumor size was monitored twice a week. Animals were scanned before the treatment, at 48 h, and 2 wk after the beginning of therapy. After the final scan, PET results were correlated with tumor response and immunohistochemical assessment of ErbB2 level, as well as with vasculature in the treated tumors. RESULTS: Analysis of PET images indicated that tracer uptake was significantly reduced after 1 dose of trastuzumab, compared with baseline, suggesting applicability as an early indicator of changes in ErbB2 expression. After 5 doses of trastuzumab, the overall decrease in (18)F-FBEM-HER(2:342) Affibody uptake also correlated with tumor response and downregulation of ErbB2 expression by immunohistochemical assessment. However, individual animals had different responses. There was a correlation between bigger PET changes and a higher vessel count in the tumors, suggesting that an increased number of vessels could lead to better trastuzumab delivery. We confirmed that the difference in average vessel count in the tumors was not related to the size of the tumors and therefore was not due to the selection of more vascular tumors. This finding is consistent with previous findings demonstrating that the number of vessels in a tumor could be a useful prognostic marker for treatment response. CONCLUSION: Our data suggest that Affibody-based PET can noninvasively provide specific information on changes in receptor expression and could be a valuable strategy for predicting tumor response to trastuzumab.
The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell-surface receptors/tyrosine kinases. EGFR- and HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683-696). We have created photostable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/ receptors.
PURPOSE: Overexpression of HER2/neu in breast cancer is correlated with a poor prognosis. It may vary between primary tumors and metastatic lesions and change during the treatment. Therefore, there is a need for a new means to assess HER2/neu expression in vivo. In this work, we used (68)Ga-labeled DOTA-Z(HER2:2891)-Affibody to monitor HER2/neu expression in a panel of breast cancer xenografts. METHODS: DOTA-Z(HER2:2891)-Affibody molecules were labeled with (68)Ga. In vitro binding was characterized by a receptor saturation assay. Biodistribution and PET imaging studies were conducted in athymic nude mice bearing subcutaneous human breast cancer tumors with three different levels of HER2/neu expression. Nonspecific uptake was analyzed using non-HER2-specific Affibody molecules. Signal detected by PET was compared with ex vivo assessment of the tracer uptake and HER2/neu expression. RESULTS: The (68)Ga-DOTA-Z(HER2:2891)-Affibody probe showed high binding affinity to MDA-MB-361 cells (K (D) = 1.4 ± 0.19 nM). In vivo biodistribution and PET imaging studies demonstrated high radioactivity uptake in HER2/neu-positive tumors. Tracer was eliminated quickly from the blood and normal tissues, resulting in high tumor-to-blood ratios. The highest concentration of radioactivity in normal tissue was seen in the kidneys (227 ± 14%ID/g). High-contrast PET images of HER2/neu-overexpressing tumors were recorded as soon as 1 h after tracer injection. A good correlation was observed between PET imaging, biodistribution estimates of tumor tracer concentration, and the receptor expression. CONCLUSION: These results suggest that PET imaging using (68)Ga-DOTA-Z(HER2:2891)-Affibody is sensitive enough to detect different levels of HER2/neu expression in vivo.
UNLABELLED: In vivo imaging of human epidermal growth factor receptor type 2 (HER2) expression may allow direct assessment of HER2 status in tumor tissue and provide a means to quantify changes in receptor expression after HER2-targeted therapies. This work describes the in vivo characterization of the HER2-specific N-2-(4-(18)F-fluorobenzamido)ethyl]maleimide ((18)F-FBEM)-Z(HER2:342) Affibody molecule and its application to study the effect of 17 (dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on HER2 expression by PET. METHODS: To assess the correlation of signal observed by PET with receptor expression, we administered the tracer to athymic nude mice bearing subcutaneous human breast cancer xenografts with different levels of HER2 expression. To study the downregulation of HER2, we treated the mice with 4 doses (40 mg/kg) of 17-DMAG, an inhibitor of heat-shock protein 90, known to decrease HER2 expression. The animals were scanned before and after treatment. After the last scan, the mice were euthanized and tumors were frozen for receptor analysis. RESULTS: The tracer was eliminated quickly from the blood and normal tissues, providing high tumor-to-blood and tumor-to-muscle ratios as early as 20 min after injection. The high-contrast images between normal and tumor tissue were recorded for BT474 and MCF7/clone18 tumors. Low but still detectable uptake was observed for MCF7 tumors, and none for MDA-MB-468. The signal correlated with the receptor expression as assessed by immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay. The levels of HER2 expression estimated by post-treatment PET decreased 71% (P < 4 x 10(-6)) and 33% (P < 0.002), respectively, for mice bearing BT474 and MCF7/clone18 tumors. These changes were confirmed by the biodistribution studies, enzyme-linked immunosorbent assay, and Western blot. CONCLUSION: Our results suggest that the described (18)F-FBEM-Z(HER2:342) Affibody molecule can be used to assess HER2 expression in vivo by PET and monitor possible changes of receptor expression in response to therapeutic interventions.
Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25% to 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin-a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A, as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using surface plasmon resonance technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by Pseudomonas exotoxin A activity domain-mediated ADP-ribosylation of translation elongation factor 2 and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65+/-2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56+/-0.1 pM), and almost 3 orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7+/-5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.
Two photosensitizing systems: (1) tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) encapsulated in sterically stabilized liposomes (SSL) and (2) p-THPP functionalized by covalent attachment of poly(ethylene glycol) (p-THPP-PEG(2000)) were studied in vitro. The dark and photo cytotoxicity of these systems were evaluated on two cell lines: HCT 116, a human colorectal carcinoma cell line, and DU 145, a prostate cancer cell line and compared with these determined for free p-THPP. It was demonstrated that both encapsulation in liposomes as well as attachment of PEG chain result in pronounced reduction of the dark cytotoxicity of the parent porphyrin. The liposomal formulation showed higher than p-THPP-PEG(2000) photocytotoxicity towards both cell lines used in the studies.
The recent demonstration of nanoscale scintillators has led to interest in the combination of radiation and photodynamic therapy. In this model, scintillating nanoparticles conjugated to photosensitizers and molecular targeting agents would enhance the targeting and improve the efficacy of radiotherapy and extend the application of photodynamic therapy to deeply seated tumors. In this study, we calculated the physical parameters required for these nanoparticle conjugates to deliver cytotoxic levels of singlet oxygen at therapeutic radiation doses, drawing on the published literature from several disparate fields. Although uncertainties remain, it appears that the light yield of the nanoscintillators, the efficiency of energy transfer to the photosensitizers, and the cellular uptake of the nanoparticles all need to be fairly well optimized to observe a cytotoxic effect. Even so, the efficacy of the combination therapy will likely be restricted to X-ray energies below 300 keV, which limits the application to brachytherapy.
There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (Z<sub>HER2:2395</sub>-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of Z<sub>HER2:2395</sub>-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated Z<sub>HER2:2395</sub>-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated Z<sub>HER2:2395</sub>-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the Z<sub>HER2:2395</sub>-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that Z<sub>HER2:2395</sub>-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.
Targeted radiotherapy with <sup>131</sup>I-mIBG, a substrate of the human norepinephrine transporter (NET-1), shows promising responses in heavily pre-treated neuroblastoma (NB) patients. Combinatorial approaches that enhance <sup>131</sup>I-mIBG tumour uptake are of substantial clinical interest but biomarkers of response are needed. Here, we investigate the potential of <sup>18</sup>F-mFBG, a positron emission tomography (PET) analogue of the <sup>123</sup>I-mIBG radiotracer, to quantify NET-1 expression levels in mouse models of NB following treatment with AZD2014, a dual mTOR inhibitor. The response to AZD2014 treatment was evaluated in MYCN amplified NB cell lines (Kelly and SK-N-BE(2)C) by Western blot (WB) and immunohistochemistry. PET quantification of <sup>18</sup>F-mFBG uptake post-treatment in vivo was performed, and data correlated with NET-1 protein levels measured ex vivo. Following 72 h AZD2014 treatment, in vitro WB analysis indicated decreased mTOR signalling and enhanced NET-1 expression in both cell lines, and <sup>18</sup>F-mFBG revealed a concentration-dependent increase in NET-1 function. AZD2014 treatment failed however to inhibit mTOR signalling in vivo and did not significantly modulate intratumoural NET-1 activity. Image analysis of <sup>18</sup>F-mFBG PET data showed correlation to tumour NET-1 protein expression, while further studies are needed to elucidate whether NET-1 upregulation induced by blocking mTOR might be a useful adjunct to <sup>131</sup>I-mIBG therapy.
Near-InfraRed PhotoImmunoTherapy (NIR-PIT) is a novel cancer-targeted treatment effected by a chemical conjugation between a photosensitiser (e.g. the NIR phthalocyanine dye IRDye700DX) and a cancer-targeting moiety (e.g. a monoclonal antibody, moAb). Delivery of a conjugate in vivo leads to accumulation at the tumour cell surface by binding to cell surface receptors or antigens. Upon deployment of focal NIR-light, irradiation of the conjugate results in a rapid, targeted cell death. However, the mechanisms of action to produce the cytotoxic effects have yet to be fully understood. Herein, we bring together the current knowledge of NIR-PIT from preclinical and clinical studies in a variety of cancers highlighting the key unanswered research questions. Furthermore, we discuss how to enhance the local control of solid cancers using this novel treatment regimen.
Neuroblastoma (NB) is the most common extracranial solid tumour in childhood, accounting for approximately 15% of all cancer-related deaths in the paediatric population1. It is characterised by heterogeneous clinical behaviour in neonates and often adverse outcomes in toddlers. The overall survival of children with high-risk disease is around 40-50% despite the aggressive treatment protocols consisting of intensive chemotherapy, surgery, radiation therapy and hematopoietic stem cell transplantation2,3. There is an ongoing research effort to increase NB's cellular and molecular biology knowledge to translate essential findings into novel treatment strategies. This review aims to address new therapeutic modalities emerging from preclinical studies offering a unique translational opportunity for NB treatment.
Recently, the demand for hybrid PET/MRI imaging techniques has increased significantly, which has sparked the investigation into new ways to simultaneously track multiple molecular targets and improve the localization and expression of biochemical markers. Multimodal imaging probes have recently emerged as powerful tools for improving the detection sensitivity and accuracy-both important factors in disease diagnosis and treatment; however, only a limited number of bimodal probes have been investigated in preclinical models. Herein, we briefly describe the strengths and limitations of PET and MRI modalities and highlight the need for the development of multimodal molecularly-targeted agents. We have tried to thoroughly summarize data on bimodal probes available on PubMed. Emphasis was placed on their design, safety profiles, pharmacokinetics, and clearance properties. The challenges in PET/MR probe development using a number of illustrative examples are also discussed, along with future research directions for these novel conjugates.
Junctional adhesion molecules (JAMs) play a critical role in cell permeability, polarity and migration. JAM-A, a key protein of the JAM family, is altered in a number of conditions including cancer; however, consequences of JAM-A dysregulation on carcinogenesis appear to be tissue dependent and organ dependent with significant implications for the use of JAM-A as a biomarker or therapeutic target. Here, we test the expression and prognostic role of JAM-A downregulation in primary and metastatic colorectal cancer (CRC) (n = 947). We show that JAM-A downregulation is observed in ~60% of CRC and correlates with poor outcome in four cohorts of stages II and III CRC (n = 1098). Using JAM-A knockdown, re-expression and rescue experiments in cell line monolayers, 3D spheroids, patient-derived organoids and xenotransplants, we demonstrate that JAM-A silencing promotes proliferation and migration in 2D and 3D cell models and increases tumour volume and metastases in vivo. Using gene-expression and proteomic analyses, we show that JAM-A downregulation results in the activation of ERK, AKT and ROCK pathways and leads to decreased bone morphogenetic protein 7 expression. We identify MIR21 upregulation as the cause of JAM-A downregulation and show that JAM-A rescue mitigates the effects of MIR21 overexpression on cancer phenotype. Our results identify a novel molecular loop involving MIR21 dysregulation, JAM-A silencing and activation of multiple oncogenic pathways in promoting invasiveness and metastasis in CRC.
A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, <sup>68</sup>Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of <sup>68</sup>Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded <sup>68</sup>Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of <sup>68</sup>Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.
Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system tumour in adults. It has extremely poor prognosis since the current standard of care, comprising of gross total resection and temozolomide (TMZ) chemoradiotherapy, prolongs survival, but does not provide a durable response. To a certain extent, this is due to GBM's heterogeneous, hostile and cold tumour microenvironment (TME) and the unique ability of GBM to overcome the host's immune responses. Therefore, there is an urgent need to develop more effective therapeutic approaches. This review provides critical insights from completed and ongoing clinical studies investigating novel immunotherapy strategies for GBM patients, ranging from the use of immune checkpoint inhibitors in different settings of GBM treatment to novel combinatorial therapies. In particular, we discuss how treatment regimens based on single antigen peptide vaccines evolved into fully personalised, polyvalent cell-based vaccines, CAR-T cell, and viral or gene therapies. Furthermore, the results of the most influential clinical trials and a selection of innovative preclinical studies aimed at activating the immunologically cold GBM microenvironment are reviewed.
Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.
Fluorescence-guided surgery is set to play a pivotal role in the intraoperative management of pediatric tumors. Shortwave infrared imaging (SWIR) has advantages over conventional near-infrared I (NIR-I) imaging with reduced tissue scattering and autofluorescence. Here, two NIR-I dyes (IRDye800CW and IR12), with long tails emitting in the SWIR range, were conjugated with a clinical-grade anti-GD2 monoclonal antibody (dinutuximab-beta) to compare NIR-I and SWIR imaging for neuroblastoma surgery. A first-of-its-kind multispectral NIR-I/SWIR fluorescence imaging device was constructed to allow an objective comparison between the two imaging windows. Conjugates were first characterized in vitro. Tissue-mimicking phantoms, imaging specimens of known geometric and material composition, were used to assess the sensitivity and depth penetration of the NIR-I/SWIR device, showing a minimum detectable volume of ∼0.9 mm3 and depth penetration up to 3 mm. In vivo, fluorescence imaging using the NIR-I/SWIR device showed a high tumor-to-background ratio (TBR) for both dyes, with anti-GD2-IR800 being significantly brighter than anti-GD2-IR12. Crucially, the system enabled higher TBR at SWIR wavelengths than at NIR-I wavelengths, verifying SWIR imaging enables high-contrast delineation of tumor margins. This work demonstrates that by combining the high specificity of anti-GD2 antibodies with the availability and translatability of existing NIR-I dyes, along with the advantages of SWIR in terms of depth and tumor signal-to-background ratio, GD2-targeted NIR-I/SWIR-guided surgery could improve the treatment of patients with neuroblastoma, warranting investigation in future clinical trials.<h4>Significance</h4>Multispectral near-infrared I/shortwave infrared fluorescence imaging is a versatile system enabling high tumor-to-background signal for safer and more complete resection of pediatric tumors during surgery.
There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (Z<sub>PD-L1</sub>) radiolabelled with F-18 (Al<sup>18</sup>F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. Z<sub>PD-L1</sub> was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that <sup>18</sup>F-AlF-NOTA-Z<sub>PD-L1</sub> can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292<sub>PD-L1Ko</sub>). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. <sup>68</sup>Ga-NOTA-Z<sub>PD-L1</sub> showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients.
<h4>Background</h4>Surgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients' outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response.<h4>Methods</h4>EGFR-specific affibody molecule (Z<sub>EGFR:03115</sub>) was conjugated to IR700. The response to Z<sub>EGFR:03115</sub>-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used.<h4>Results</h4>In vitro findings confirmed the ability of Z<sub>EGFR:03115</sub>-IR700 to produce reactive oxygen species upon light irradiation. Z<sub>EGFR:03115</sub>-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs) (calreticulin, ATP, HSP70/90, and HMGB1) into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T<sub>2</sub>*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours.<h4>Conclusions</h4>Our data underline the potential of Z<sub>EGFR:03115</sub>-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.
Patents
The invention provides a radiolabeled af?body molecule comprising a fragment of an IgG-binding domain of proteinA from Staphylococcus aureus, a bifunctional linker, and a radiolabel comprising 18F or 76Br, Wherein the bifunctional linker links the fragment and the radiolabel. The af?body molecule binds With high ai?nity to select receptors, Which are over-expressed in certain cancers. Since the radionuclides emit a positron, the in vitro and in vivo binding characteristics of the radiolabeled af?body can be assessed using diagnostic imaging.
Conferences
<h4>Background</h4>Immune checkpoint inhibitors (ICPIs) have proven to restore adaptive anti-tumor immunity in many cancers; however, no noteworthy therapeutic schedule has been established for patients with glioblastoma (GBM). High programmed death-ligand 1 (PD-L1) expression is associated with immunosuppressive and aggressive phenotypes in GBM. Presently, there is no standardized protocol for assessing PD-L1 expression levels to select patients and monitor their response to ICPI therapy. The aim of this study was to investigate the use of 89Zr-DFO-Atezolizumab to image the spatio-temporal distribution of PD-L1 in preclinical mouse models and in patients with newly diagnosed GBM treated with/without neoadjuvant Pembrolizumab.<h4>Methods</h4>The immunoreactivity, binding affinity, and specificity of 89Zr-DFO-Atezolizumab were confirmed in vitro. Mice-bearing orthotopic GBM tumors or patients with newly diagnosed GBM treated with/without Pembrolizumab were intravenously injected with 89Zr-DFO-Atezolizumab, and PET/CT images were acquired 24, 48, and 72 hours in mice and at 48 and 72 post-injection in patients. Radioconjugate uptake was quantified in the tumor and healthy tissues. Ex vivo immunohistochemistry (IHC) and immunophenotyping were performed on mouse tumor samples or resected human tumors.<h4>Results</h4>89Zr-DFO-Atezolizumab was prepared with high radiochemical purity (RCP > 99%). In vitro cell-associated radioactivity of 89Zr-DFO-Atezolizumab corroborated cell line PD-L1 expression. PD-L1 in mouse GBM tumors was detected with high specificity using 89Zr-DFO-Atezolizumab and radioconjugate uptake correlated with IHC. Patients experienced no 89Zr-DFO-Atezolizumab-related side effects. High 89Zr-DFO-Atezolizumab uptake was observed in patient tumors at 48 hours post-injection, however, the uptake varied between patients treated with/without Pembrolizumab.<h4>Conclusions</h4>89Zr-DFO-Atezolizumab can visualize distinct PD-L1 expression levels with high specificity in preclinical mouse models and in patients with GBM, whilst complementing ex vivo analysis.