Burley, T.A., Da Pieve, C., Martins, C.D., Ciobota, D.M., Allott, L., Oyen, W.J., Harrington, K.J., Smith, G. & Kramer-Marek, G.
(2019). Affibody-Based PET Imaging to Guide EGFR-Targeted Cancer Therapy in Head and Neck Squamous Cell Cancer Models. Journal of nuclear medicine,
Burley, T.A., Mączyńska, J., Shah, A., Szopa, W., Harrington, K.J., Boult, J.K., Mrozek-Wilczkiewicz, A., Vinci, M., Bamber, J.C., Kaspera, W., et al.
(2018). Near-infrared photoimmunotherapy targeting EGFR-Shedding new light on glioblastoma treatment. International journal of cancer,
Martins, C.D., Kramer-Marek, G. & Oyen, W.J.
(2018). Radioimmunotherapy for delivery of cytotoxic radioisotopes: current status and challenges. Expert opinion on drug delivery,
Martins, C.D., Da Pieve, C., Burley, T.A., Smith, R., Ciobota, D.M., Allott, L., Harrington, K.J., Oyen, W.J., Smith, G. & Kramer-Marek, G., et al.
(2018). HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-Based PET Imaging. Clinical cancer research,
Szopa, W., Burley, T.A., Kramer-Marek, G. & Kaspera, W.
(2017). Diagnostic and Therapeutic Biomarkers in Glioblastoma: Current Status and Future Perspectives. Biomed research international,
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..
Kramer-Marek, G. & Oyen, W.J.
(2016). Targeting the Human Epidermal Growth Factor Receptors with Immuno-PET: Imaging Biomarkers from Bench to Bedside. Journal of nuclear medicine,
Mansfield, D.C., Kyula, J.N., Rosenfelder, N., Chao-Chu, J., Kramer-Marek, G., Khan, A.A., Roulstone, V., McLaughlin, M., Melcher, A.A., Vile, R.G., et al.
(2016). Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer. Gene therapy,
Denis-Bacelar, A.M., Cronin, S.E., Da Pieve, C., Paul, R.L., Eccles, S.A., Spinks, T.J., Box, C., Hall, A., Sosabowski, J.K., Kramer-Marek, G., et al.
(2016). Pre-clinical quantitative imaging and mouse-specific dosimetry for 111In-labelled radiotracers. Ejnmmi research,
Da Pieve, C., Allott, L., Martins, C.D., Vardon, A., Ciobota, D.M., Kramer-Marek, G. & Smith, G.
(2016). Efficient [(18)F]AlF Radiolabeling of ZHER3:8698 Affibody Molecule for Imaging of HER3 Positive Tumors. Bioconjug chem,
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..
Xu, Z., Bagci, U., Mansoor, A., Kramer-Marek, G., Luna, B., Kubler, A., Dey, B., Foster, B., Papadakis, G.Z., Camp, J.V., et al.
(2015). Computer-aided pulmonary image analysis in small animal models. Medical physics,
Adesina, S.K., Holly, A., Kramer-Marek, G., Capala, J. & Akala, E.O.
(2014). Polylactide-based paclitaxel-loaded nanoparticles fabricated by dispersion polymerization: characterization, evaluation in cancer cell lines, and preliminary biodistribution studies. J pharm sci,
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..
Mohd Nafi, S.N., Generali, D., Kramer-Marek, G., Gijsen, M., Strina, C., Cappelletti, M., Andreis, D., Haider, S., Li, J.-., Bridges, E., et al.
(2014). Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer. Oncotarget,
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. .
Nafi, S., Generali, D., Kramer-Marek, G., Gijsen, M., Strina, C., Cappelletti, M., Andreis, D., Haider, S., Li, J.L., Bridges, E., et al.
(2014). Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer. Oncotarget,
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..
Feldinger, K., Generali, D., Kramer-Marek, G., Gijsen, M., Ng, T.B., Wong, J.H., Strina, C., Cappelletti, M., Andreis, D., Li, J.-., et al.
(2014). ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer. Oncotarget,
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..
Kramer-Marek, G., Gore, J. & Korc, M.
(2013). Molecular imaging in pancreatic cancer--a roadmap for therapeutic decisions. Cancer lett,
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. .
Bagci, U., Kramer-Marek, G. & Mollura, D.J.
(2013). Automated computer quantification of breast cancer in small-animal models using PET-guided MR image co-segmentation. Ejnmmi res,
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..
Kramer-Marek, G., Gijsen, M., Kiesewetter, D.O., Bennett, R., Roxanis, I., Zielinski, R., Kong, A. & Capala, J.
(2012). Potential of PET to predict the response to trastuzumab treatment in an ErbB2-positive human xenograft tumor model. J nucl med,
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..
Capala, J., Kong, A., Kramer-Marek, G. & Gijsen, M.
(2012). Reply. J nucl med,
Kramer-Marek, G., Bernardo, M., Kiesewetter, D.O., Bagci, U., Kuban, M., Aras, O., Zielinski, R., Seidel, J., Choyke, P. & Capala, J., et al.
(2012). PET of HER2-positive pulmonary metastases with 18F-ZHER2:342 affibody in a murine model of breast cancer: comparison with 18F-FDG. J nucl med,
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..
Kramer-Marek, G. & Capala, J.
(2012). The role of nuclear medicine in modern therapy of cancer. Tumor biology,
Mitsunaga, M., Nakajima, T., Sano, K., Kramer-Marek, G., Choyke, P.L. & Kobayashi, H.
(2012). Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy. Bmc cancer,
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..
Jagoda, E.M., Lang, L., Bhadrasetty, V., Histed, S., Williams, M., Kramer-Marek, G., Mena, E., Rosenblum, L., Marik, J., Tinianow, J.N., et al.
(2012). Immuno-PET of the hepatocyte growth factor receptor Met using the 1-armed antibody onartuzumab. J nucl med,
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..
Kramer-Marek, G., Longmire, M.R., Choyke, P.L. & Kobayashi, H.
(2012). Recent Advances in Optical Cancer Imaging of EGF Receptors. Current medicinal chemistry,
Kramer-Marek, G. & Capala, J.
(2012). Can PET imaging facilitate optimization of cancer therapies?. Curr pharm des,
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..
Dahl, M., Bouchelouche, P., Kramer-Marek, G., Capala, J., Nordling, J. & Bouchelouche, K.
(2011). Sarcosine induces increase in HER2/neu expression in androgen-dependent prostate cancer cells. Molecular biology reports,
Kramer-Marek, G., Shenoy, N., Seidel, J., Griffiths, G.L., Choyke, P. & Capala, J.
(2011). 68Ga-DOTA-affibody molecule for in vivo assessment of HER2/neu expression with PET. Eur j nucl med mol imaging,
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..
Gijsen, M., Kramer-Marek, G., Bennett, R., Harris, A., Capala, J. & Kong, A.
(2011). Abstract 1530: Trastuzumab treatment induces ADAM17 and HER ligands in vivo. Tumor biology,
Kramer-Marek, G., Shenoy, N., Seidel, J., Griffiths, G., Choyke, P. & Capala, J.
(2011). Abstract 5284:68Ga-radiolabeled DOTA-affibody molecule for in vivo assessment of HER2 expression with PET. Tumor biology,
Jagoda, E., Bhadrasetty, V., Lixin, L., Williams, M., Histed, S., Gabriela, K.-., Marik, J., Tinianow, J., Merchant, M., Szajek, L., et al.
(2011). Comparison of [Zr-89] and [Br-76] labeled MetMAb, a one-armed monoclonal antibody targeting the Met receptor tyrosine kinase (Met), in human gastric carcinoma (MKN-45) and glioblastoma (U87 MG) cells and xenografts. Journal of labelled compounds & radiopharmaceuticals,
Lyakhov, I., Zielinski, R., Kuban, M., Kramer-Marek, G., Fisher, R., Chertov, O., Bindu, L. & Capala, J.
(2010). HER2- and EGFR-specific affiprobes: novel recombinant optical probes for cell imaging. Chembiochem,
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..
Lyakhov, I., Kuban, M., Zielinski, R., Kramer-Marek, G. & Capala, J.
(2010). Abstract 1151: HER2- and EGFR-specific Affiprobes - Novel recombinant optical probes for cell imaging. Cellular and molecular biology,
Watson, R.P., Kramer-Marek, G. & Capala, J.
(2010). Instrumental neutron activation analysis of gold in mouse tissues. Abstracts of papers of the american chemical society,
Morgan, N.Y., Kramer-Marek, G., Smith, P.D., Camphausen, K. & Capala, J.
(2009). Nanoscintillator conjugates as photodynamic therapy-based radiosensitizers: calculation of required physical parameters. Radiat res,
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..
Nawalany, K., Rusin, A., Kepczyński, M., Mikhailov, A., Kramer-Marek, G., Snietura, M., Połtowicz, J., Krawczyk, Z. & Nowakowska, M.
(2009). Comparison of photodynamic efficacy of tetraarylporphyrin pegylated or encapsulated in liposomes: in vitro studies. J photochem photobiol b,
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..
Kramer-Marek, G., Kiesewetter, D.O. & Capala, J.
(2009). Changes in HER2 expression in breast cancer xenografts after therapy can be quantified using PET and (18)F-labeled affibody molecules. J nucl med,
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..
Zielinski, R., Lyakhov, I., Jacobs, A., Chertov, O., Kramer-Marek, G., Francella, N., Stephen, A., Fisher, R., Blumenthal, R. & Capala, J., et al.
(2009). Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors. J immunother,
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..
Capala, J., Kramer-Marek, G., Lee, S.B., Hassan, M., Kiesewetter, D.O., Puri, A., Chernomordik, V., Gandjbakhche, A., Griffiths, G. & Blumenthal, R., et al.
(2009). Molecular targeting of HER2 for diagnosis and therapy of breast cancer. Cancer research,
Watson, R., Popelka-Filcoff, R., Kramer-Marek, G., Mackey, E.A., Spatz, R.O. & Capala, J.
(2009). ANYL 96-Quantitation of gold in biological tissues using neutron activation analysis. Abstracts of papers of the american chemical society,
Kramer-Marek, G., Kiesewetter, D.O., Martiniova, L., Jagoda, E., Lee, S.B. & Capala, J.
(2008). [18F]FBEM-Z(HER2:342)-Affibody molecule-a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography. Eur j nucl med mol imaging,
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..
Kiesewetter, D.O., Krämer-Marek, G., Ma, Y. & Capala, J.
(2008). Radiolabeling of HER2 specific Affibody(R) molecule with F-18. J fluor chem,
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..
Lee, S.B., Hassan, M., Fisher, R., Chertov, O., Chernomordik, V., Kramer-Marek, G., Gandjbakhche, A. & Capala, J.
(2008). Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging. Clin cancer res,
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..
Puri, A., Kramer-Marek, G., Campbell-Massa, R., Yavlovich, A., Tele, S.C., Lee, S.-., Clogston, J.D., Patri, A.K., Blumenthal, R. & Capala, J., et al.
(2008). HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents. Journal of liposome research,
Kramer-Marek, G., Kiesewetter, D.O., Martiniova, L., Rodriguez, Y. & Capala, J.
(2007). F-18-Z(HER2)-Affibody: A new tracer for molecular imaging of HER2-expressing tumors by PET. Molecular cancer therapeutics,
Evaluation of DFO-HOPO as an octadentate chelator for zirconium-89. Chemical communications,
Oyen, W. & kramer-marek, G.
Radioimmunotherapy for delivery of cytotoxic radioisotopes - current status and challenges. Expert opinion on drug delivery,
Smith, G., King, A., Doepner, A., Turton, D., Ciobota, D., Da Pieve, C., Wong Te Fong, A.-., Kramer-Marek, G. & Chung, Y.
Radiosynthesis of the anticancer nucleoside analogue Trifluridine using an automated 18F-trifluoromethylation procedure. Organic and biomolecular chemistry,
Baker, L.C., Sikka, A., Price, J.M., Boult, J.K., Lepicard, E.Y., Box, G., Jamin, Y., Spinks, T.J., Kramer-Marek, G., Leach, M.O., et al.
Evaluating Imaging Biomarkers of Acquired Resistance to Targeted EGFR Therapy in Xenograft Models of Human Head and Neck Squamous Cell Carcinoma. Frontiers in oncology,
Stowe, C.L., Burley, T.A., Allan, H., Vinci, M., Kramer-Marek, G., Ciobota, D.M., Parkinson, G.N., Southworth, T.L., Agliardi, G., Hotblack, A., et al.
Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin. Elife,
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 (LH2) analogue infraluciferin (iLH2), 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-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 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..