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Radiotheranostics: a roadmap for future development.

Elsevier Ltd., 2020, DOI: 10.1016/S1470-2045(19)30821-6 publiziert am 04.05.2020
Radiotheranostics, injectable radiopharmaceuticals with antitumour effects, have seen rapid development over the past decade. Although some formulations are already approved for human use, more radiopharmaceuticals will enter clinical practice in the next 5 years, potentially introducing new therapeutic choices for patients. Despite these advances, several challenges remain, including logistics, supply chain, regulatory issues, and education and training. By highlighting active developments in the field, this Review aims to alert practitioners to the value of radiotheranostics and to outline a roadmap for future development. Multidisciplinary approaches in clinical trial design and therapeutic administration will become essential to the continued progress of this evolving therapeutic approach.

 

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Development of a high affinity Anticalin® directed against human CD98hc for theranostic applications

Ivyspring International Publisher, 2020, doi: 10.7150/thno.38968, publiziert am 22.01.2020
Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target.

Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation® technology - was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice.

Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8.

Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.

 

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Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin – with a tuned plasma half-life using PASylation® technology – was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice.

Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8.

Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.

 

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Acquisition strategies for spatially resolved magnetic resonance detection of hyperpolarized nuclei

Springer, 2019, doi.org/10.1007/s10334-019-00807-6 publiziert am 21.11.2019
Hyperpolarization is an emerging method in magnetic resonance imaging that allows nuclear spin polarization of gases or liquids to be temporarily enhanced by up to fve or six orders of magnitude at clinically relevant feld strengths and administered at high concentration to a subject at the time of measurement. This transient gain in signal has enabled the non-invasive detection and imaging of gas ventilation and difusion in the lungs, perfusion in blood vessels and tissues, and metabolic conversion in cells, animals, and patients. The rapid development of this method is based on advances in polarizer technology, the availability of suitable probe isotopes and molecules, improved MRI hardware and pulse sequence development. Acquisition strategies for hyperpolarized nuclei are not yet standardized and are set up individually at most sites depending on the specifc requirements of the probe, the object of interest, and the MRI hardware. This review provides a detailed introduction to spatially resolved detection of hyperpolarized nuclei and summarizes novel and previously established acquisition strategies for diferent key areas of application.

 

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Development of a high affinity Anticalin® directed against human CD98hc for theranostic applications

2019, publiziert am 19.11.2019
Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin – with a tuned plasma half-life using PASylation® technology – was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-toblood ratio of 11.8. Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.

 

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In-Vivo Imaging of Early Stages of Rheumatoid Arthritis by α5β1- Integrin Targeted Positron Emission Tomography

Institute of Pathology, TUM MRI Munich, 2019, publiziert am 23.05.2019
Background: Rheumatoid Arthritis (RA) is one of the most common rheumatic diseases. Joint inflammation and pathological growth of joint cartilage causes swollen, painful joints, which severely diminishes the patients' life quality. There is no causal treatment. Symptomatic therapies should start

as early as possible to take maximal effect. Hence, diagnostic procedures capable of detecting affected joints before the onset of clinical symptoms are highly desirable. We explored the value of PETimaging of integrin subtypes αvβ3 and α5β1 for early detection of RA foci in collagen-induced

arthritis (CIA) mouse models.

Results: Development of RA in CIA mice was monitored by paw scoring, and αvβ3- and α5β1- integrin expression was quantified by µPET using 68Ga-Avebetrin and 68Ga-Aquibeprin. For consecutive sections of selected decalcified joints (knee, ankle), arthritic degeneration and integrin

expression were assessed by MOVAT staining and β3/α5 immunohistochemistry (IHC), respectively. β3- and α5-IHC revealed elevated levels of both αvβ3- and α5β1-integrin in arthritic joints. Unlike αvβ3, α5β1 is strongly expressed in the proliferating synovial lining layer, which suggests that its presence is directly related to RA development. For mice with advanced RA (6 weeks after CIA), PET signals for α5β1-integrin were substantially stronger (>300% of baseline) than that of αvβ3-integrin (<200%). A longitudinal PET follow-up revealed that the manifestation of clinical symptoms of RA is preceded by upregulation of α5β1- but not of αvβ3-integrin.

Conclusion: α5β1-integrin PET could add a new functional imaging aspect to the portfolio of RA diagnostics because it appears to be a specific biomarker for early RA development. We propose α5β1-integrin PET as a valuable tool to achieve a higher precision for early diagnosis of RA, including initial staging, monitoring of the disease course and drug treatment, and for planning of radiosynoviorthesis (RSO).

Keywords: Rheumatoid arthritis; Positron emission tomograph; Integrins; Animal models; Collagen

induced arthritis; Immunohistochemistry

 

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pH-Dependency of the Spin-Lattice Relaxation Constant (T1) of 13C-Labelled Hyperpolarized Biomolecules

2018, publiziert am 12.12.2018
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Dimer formation of GdDO3A-arylsulfonamide complexes causes loss of pH-dependency of relaxivity

Dalton Trans, 2017, DOI: 10.1039/c7dt02985c publiziert am 06.11.2017
Gadolinium(III) complexes with pH-dependent relaxivities have been proposed as responsive magnetic resonance imaging (MRI) contrast agents (CA) for mapping of pH value in living subjects. The latter is clinically relevant because hypoxia-induced reduction of interstitial pH (acidosis) is a hallmark of tumor progression and resistance against chemotherapy. In order to obtain versatile building blocks for integration of a pH-responsive MRI-CA functionality into larger molecular assemblies, such as bioconjugates, micelles or nanoparticles, we equipped the structural motif GdDO3A-ethylene(arylsulfonic acid) with additional carboxylic acid moieties in the aromatic para-position. Two novel compounds were characterized concerning their pH-dependent relaxivity as well as by 17O NMR and 1H NMRD, augmented by determination of luminescence lifetimes of the respective Eu(III) complexes and structural modelling by density functional theory (TPSSh/LCRECP/6-31G(d)). Unexpected involvement of the peripheral carboxylates into metal ion complexation effected self-assembly of the lanthanide(III) complexes, resulting in dimeric species comprising two lanthanide ions, two symmetrically bridging ligands, and one coordinated water molecule per Gd(III) (q = 1). These structures are stable even at low concentrations and in presence of competing anions like phosphate. The pH-sensitive sulfonamide moieties are not involved into Gd(III) coordination, resulting in virtually constant relaxivities of r1 = 6.7 mM−1 s−1 (298 K, 20 MHz) over the biologically relevant pH range (4 to 9). Since further functionalisation on the peripheral carboxylates would effectively inhibit dimer formation, the compounds are nonetheless suited for the initially envisaged field of application.

 

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Simultaneous characterization of tumor cellularity and the Warburg effect with PET, MRI and hyperpolarized 13C-MRSI

Theranostics 2018, Vol. 8, Issue 17, 2018, doi: 10.7150/thno.25162 publiziert am 09.09.2018
Modern oncology aims at patient-specific therapy approaches, which triggered the development of biomedical imaging techniques to synergistically address tumor biology at the cellular and molecular level. PET/MR is a new hybrid modality that allows acquisition of high-resolution anatomic images and quantification of functional and metabolic information at the same time. Key steps of the Warburg effect-one of the hallmarks of tumors-can be measured non-invasively with this emerging technique. The aim of this study was to quantify and compare simultaneously imaged augmented glucose uptake and LDH activity in a subcutaneous breast cancer model in rats (MAT-B-III) and to study the effect of varying tumor cellularity on image-derived metabolic information. Methods: For this purpose, we established and validated a multimodal imaging workflow for a clinical PET/MR system including proton magnetic resonance (MR) imaging to acquire accurate morphologic information and diffusion-weighted imaging (DWI) to address tumor cellularity. Metabolic data were measured with dynamic [18F]FDG-PET and hyperpolarized (HP) 13C-pyruvate MR spectroscopic imaging (MRSI). We applied our workflow in a longitudinal study and analyzed the effect of growth dependent variations of cellular density on glycolytic parameters. Results: Tumors of similar cellularity with similar apparent diffusion coefficients (ADC) showed a significant positive correlation of FDG uptake and pyruvate-to-lactate exchange. Longitudinal DWI data indicated a decreasing tumor cellularity with tumor growth, while ADCs exhibited a significant inverse correlation with PET standard uptake values (SUV). Similar but not significant trends were observed with HP-13C-MRSI, but we found that partial volume effects and point spread function artifacts are major confounders for the quantification of 13C-data when the spatial resolution is limited and major blood vessels are close to the tumor. Nevertheless, analysis of longitudinal data

with varying tumor cellularity further detected a positive correlation between quantitative PET and 13C-data.

Conclusions: Our workflow allows the quantification of simultaneously acquired PET, MRSI and DWI data in rodents on a clinical PET/MR scanner. The correlations and findings suggest that a major portion of consumed glucose is metabolized by aerobic glycolysis in the investigated tumor

model. Furthermore, we conclude that variations in cell density affect PET and 13C-data in a similar manner and correlations of longitudinal metabolic data appear to reflect both biochemical processes and tumor cellularity.

 

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Cell Metabolism. Non-invasive measurement of brown fat metabolism based on optoacoustic imaging of hemoglobin gradients

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation, 2018, publiziert am 23.02.2018
Metabolism is a fundamental process of life. However, non-invasive measurement of local tissue metabolism is limited today by a deficiency in adequate tools for in vivo observations. We designed a multi-modular platform that explored the relation between local tissue oxygen consumption, determined by label-free optoacoustic measurements of hemoglobin, and concurrent indirect calorimetry obtained during metabolic activationc of brown adipose tissue (BAT). By studying mice and humans, we show how video-rate handheld multi-spectral optoacoustic tomography (MSOT) in the 700-970 nm spectral range enables non-invasive imaging of BAT activation, consistent with Positron Emission Tomography findings. Moreover, we observe BAT composition differences between healthy and diabetic tissues. The study consolidates hemoglobin as a principal label-free biomarker for longitudinal non-invasive imaging of BAT morphology and bioenergetics in situ. We also resolve water and fat components in volunteers, and contrast MSOT readouts to Magnetic Resonance Imaging data.

 

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Multi-Spectral Optoacoustic Tomography (MSOT) of human breast cancer.

2017, DOI: 10.1158/1078-0432.CCR-16-3200 publiziert am 06.07.2018
Purpose:

In a pilot study, we introduce fast handheld Multi-Spectral Optoacoustic Tomography (MSOT) of the breast at 28 wavelengths, aiming to identify high-resolution optoacoustic (photoacoustic) patterns of breast cancer and non-cancerous breast tissue.

Experimental Design:

We imaged 10 female patients aged 48-81 years with malignant non-specific breast cancer or invasive lobular carcinoma. Three healthy volunteers aged 31-36 years were also imaged. Fast- MSOT was based on unique single-frame-per-pulse (SFPP) image acquisition employed to improve the accuracy of spectral differentiation over using a small number of wavelengths. Breast tissue was illuminated at the 700 - 970 nm spectral range over 0.56 seconds total scan time. MSOT data were guided by ultrasonography and X-ray mammography or MRI.

Results:

The extended spectral range allowed the computation of oxygenated hemoglobin (HBO2), deoxygenated hemoglobin (HB), total blood volume (TBV), lipid and water contributions, allowing first insights into in-vivo high-resolution breast tissue MSOT cancer patterns. TBV and Hb/HBO2 images resolved marked differences between cancer and control tissue, manifested as a vessel rich tumor periphery with highly heterogeneous spatial appearance compared to healthy tissue. We observe significant TBV variations between different tumors and between tumors over healthy tissues. Water and fat lipid layers appear disrupted in cancer vs. healthy tissue; however offer weaker contrast compared to TBV images.

Conclusion:

In contrast to optical methods, MSOT resolves physiological cancer features with high resolution and revealed patterns not offered by other radiological modalities. The new features relate to personalized and precision medicine potential.

 

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Imaging of Extracellular pH Using Hyperpolarized Molecules

Isr. J. Chem., 2017, DOI: 10.1002/ijch.201700017 publiziert am 20.07.2017

Many diseases can overrule natural pH regulatory mechanisms and alter the extracellular pH (pHe). A noninvasive method that resolves pHe in vivo with high spatial and temporal resolution could therefore improve diagnosis and monitoring of diseases, contributing to the concept of precision medicine. During the last decades, several techniques have been proposed to image pHe non-invasively. The majority of these methods rely on magnetic resonance because of its good spatial resolution, high penetration depth, non-ionizing radiation and excellent complimentary soft tissue contrast. Dissolution dynamic nuclear polarization (DNP) is an emerging concept to enhance nuclear magnetic resonance (NMR) signals by more than four orders of magnitude, making it possible to observe in vivo metabolic processes in real-time. Here, we summarize and review recent developments in pHe imaging techniques based on hyperpolarization methods and give an overview of recently discovered hyperpolarized pH sensor molecules that have been applied in vitro and in vivo.

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Anticalins directed against vascular endothelial growth factor receptor 3 (VEGFR-3) with picomolar affinities show potential for medical therapy and in vivo imaging

Biological Chemistry, 2016, DOI: 10.1515/hsz-2016-0195 publiziert am 16.08.2016
Members of the vascular endothelial growth factor receptor (VEGFR) family play a central role in angiogenesis as well as lymphangiogenesis and are crucial for tumor growth and metastasis. In particular, VEGFR-3 expression is induced in endothelial cells during tumor angiogenesis. We report the design of Anticalins that specifically recognize the ligandbinding domains 1 and 2 of VEGFR-3. To this end, a library of the lipocalin 2 scaffold with 20 randomized positions distributed across its binding site was subjected to phage display selection and ELISA screening using the VEGF-C binding fragment (D1-2) or the entire extracellular region (D1-7) of VEGFR-3 as target proteins. Promising Anticalin candidates were produced in E. coli and biochemically characterized. Three variants with different receptor binding modes were identified, and two of them were optimized with regard to target affinity as well as folding efficiency. The resulting Anticalins show dissociation constants down to the single-digit picomolar range. Specific recognition of VEGFR-3 on cells was demonstrated by immunofluorescence microscopy. Competitive binding versus VEGF-C was demonstrated for two of the Anticalins with Ki values in the low nanomolar range. Based on these data, VEGFR-3 specific Anticalins provide promising reagents for the diagnosis and/or therapeutic intervention of tumor-associated vessel growth.

 

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Tumor uptake of anti‐CD20 Fabs depends on tumor perfusion

Journal of Nuclear Medicine, 2016, doi:10.2967/jnumed.116.176784 publiziert am 14.07.2016
Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends on both the presence and accessibility of the tumor antigen by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non‐Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL‐ 4 and Granta, using multimodal imaging techniques. Methods: To investigate in vivo biodistribution, two differently modified CD20 antigen‐binding fragments (Fab), prepared (i) by PASylation and (ii) by fusion with an albumin‐binding domain, were radiolabeled with 125I and intravenously injected into immunocompromised mice bearing corresponding xenografts. Results: Validation with 18F‐FDG revealed similar distribution of vital tumor tissue 1 h p.i. However, large differences in tumor uptake were observed when applying the CD20‐specific radiotracers 125I‐Fab‐ABD and 125I‐Fab‐PAS200 with 12.3 and 2.4 % ID/g, respectively, for Granta in comparison with 3.5 and 0.75 % ID/g, respectively, for SUDHL‐4 xenografts 24 h p.i. 3D light‐sheet fluorescence microscopy with Cy5‐Fab‐PAS200 confirmed better tracer extravasation in the Granta tumors. Moreover, dynamic contrast enhanced MRI imaging revealed significantly reduced tumor perfusion in the SUHDL‐4 xenografts. Conclusion: Tracer uptake was highly dependent on local tumor perfusion as well as Fab permeation in the SUDHL‐4 and Granta tumors. Thus, the SUDHL‐4 xenograft offers an excellent model system to investigate the influence of therapies affecting tumor angiogenesis.

 

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Probing lactate secretion in tumours with hyperpolarised NMR

NMR Biomed, 2016, doi: 10.1002/nbm.3574. publiziert am 27.06.2016
Most tumours exhibit a high rate of glycolysis and predominantly produce energy by lactic acid fermentation. To maintain energy production and prevent toxicity, the lactate generated needs to be rapidly transported out of the cell. This is achieved by monocarboxylate transporters (MCTs), which therefore play an essential role in cancer metabolism and development. In vivo experiments were performed on eight male Fisher F344 rats bearing a subcutaneous mammary carcinoma after injection of hyperpolarised [1-(13) C]pyruvate. A Gd(III)DO3A complex that binds to pyruvate and its metabolites was used to efficiently destroy the extracellular magnetisation after hyperpolarised lactate had been formed. Moreover, a pulse sequence including a frequency-selective saturation pulse was designed so that the pyruvate magnetisation could be destroyed to exclude effects arising from further conversion. Given this preparation, metabolite transport out of the cell manifested as additional decay and apparent cell membrane transporter rates could thus be obtained using a reference measurement without a relaxation agent. In addition to slice-selective spectra, spatially resolved maps of apparent membrane transporter activity were acquired using a single-shot spiral gradient readout. A considerable increase in decay rate was detected for lactate, indicating rapid transport out of the cell. The alanine signal was unaltered, which corresponds to a slower efflux rate. This technique could allow for better understanding of tumour metabolism and progression, and enable treatment response measurements for MCT-targeted cancer therapies. Moreover, it provides vital insights into the signal kinetics of hyperpolarised [1-(13) C]pyruvate examinations.

 

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Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA)

Oxford University Press, 2016, vol. 29 no. 3, pp. 105–115, doi: 10.1093/protein/gzv065 publiziert am 21.01.2016
Although prostate carcinoma (PCa) is by far the most commonly diagnosed neoplasia in men, corresponding diagnostic and therapeutic modalities have limited efficacy at present. Anticalins comprise a novel class of binding proteins based on a non-immunoglobulin scaffold that can be engineered to specifically address molecular targets of interest. Here we report the selection and characterization of Anticalins that recognize human prostate-specific membrane antigen (PSMA), a membrane-tethered metallopeptidase constituting a disease-related target for imaging and therapy of PCa as well as solid malignancies in general.We used a randomized lipocalin library based on the human lipocalin 2 (Lcn2) scaffold together with phage display and ELISA screening to select PSMAspecific variants. Five Anticalin candidates from the original panning were expressed in Escherichia coli as soluble monomeric proteins, revealing affinities toward PSMA down to the low nanomolar range. Binding characteristics of the most promising candidate were further improved via affinity maturation by applying error-prone PCR followed by selection via phage display as well as bacterial surface display under more stringent conditions. In BIAcore measurements, the dissociation constant of the best Anticalin was determined as ∼500 pM, with a substantially improved dissociation rate compared with the first-generation candidate. Finally, immunofluorescence microscopy revealed specific staining of PSMA-positive tumor cell lines while flowcytometric analysis confirmed the ability of the selected Anticalins to detect PSMA on live cells. Taken together, Anticalins resulting from this study offer a viable alternative to antibody-based PSMA binders for biomedical applications, including in vivo imaging of PCa or neovasculature of solid tumors.

 

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Anticalins directed against the fibronectin extra domain B as diagnostic tracers for glioblastomas

International Journal of Cancer, 2015, DOI: 10.1002/ijc.29874 publiziert am 30.09.2015
The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the c-emitter 123I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.

 

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Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin

Oxford University Press, 2015, vol. 28 no. 12, pp. 553–565, doi: 10.1093/protein/gzv048 publiziert am 23.09.2015
The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions, e.g. with aminooxy-functionalised fluorescent probes, radiometal chelates, toxins or polymers. However, the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection, i.e. the modified aminoacyl-tRNA synthetase (aaRS), the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter, we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end, both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye, thus allowing isualisation of this tumour target under the microscope.

 

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89Zr-Labeled Versus 124I-Labeled aHER2 Fab with Optimized Plasma Half-Life for High-Contrast Tumor Imaging In Vivo

THE JOURNAL OF NUCLEAR MEDICINE, 2015, 56:1112–1118, DOI: 10.2967/jnumed.114.149690 publiziert am 21.05.2015
Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)—a biologic alternative to chemical conjugation with polyethylene glycol, PEG—offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide 89Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide 124I.

 

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PET Imaging of Oncolytic VSV Expressing the Mutant HSV-1 Thymidine Kinase Transgene in a Preclinical HCC Rat Model

The American Society of Gene & Cell Therapy, 2015, doi:10.1038/mt.2015.12 publiziert am 17.02.2015
Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancerrelated death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk–treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications. Received 8 August 2014; accepted 12 January 2015; advance onlinepublication 17 February 2015. doi:10.1038/mt.2015.

 

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High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

Taylor & Francis Group, LLC, 2015, DOI: 10.4161/19420862.2014.985522 publiziert am 20.01.2015
Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized aHER2 and aCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified aHER2 and aCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for aHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for aHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding aCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/ g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

 

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Early recognition of lung cancer by integrin targeted imaging in K-ras mouse model

Int. J. Cancer: 137, 1107–1118 (2015) C 2014 UICC, 2014, DOI: 10.1002/ijc.29372 publiziert am 02.12.2014
Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex vivo and fluorescence molecular tomography-X-ray computed tomography (FMT-XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrintargeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions (PL) at all ages studied The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features weredetected by avb3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be noninvasively detected in vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated avb3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.

 

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Limited-projection-angle hybrid fluorescence molecular tomography of multiple molecules

Journal of Biomedical Optics, April 2014, 19 (4), 046016-1 publiziert am 01.04.2014
An advantage of fluorescence methods over other imaging modalities is the ability to concurrently resolve multiple moieties using fluorochromes emitting at different spectral regions. Simultaneous imaging of spectrally separated agents is helpful in interrogating multiple functions or establishing internal controls for accurate measurements. Herein, we investigated multimoiety imaging in the context of a limited-projection-angle hybrid fluorescence molecular tomography (FMT), and x-ray computed tomography implementation and the further registration with positron emission tomography (PET) data. Multichannel FMT systems may image fluorescent probes of varying distribution patterns. Therefore, it is possible that different channels may require different use of priors and regularization parameters. We examined the performance of automatically estimating regularization factors implementing priors, using data-driven regularization specific for limited-projectionangle schemes. We were particularly interested in identifying the implementation variations between hybrid FMT channels due to probe distribution variation. For this reason, initial validation of the data-driven algorithm on a phantom was followed by imaging different agent distributions in animals, assuming superficial and deep seated activity. We further demonstrate the benefits of combining hybrid FMT with PET to gain multiple readings on the molecular composition of disease.

 

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FMT-PCCT: Hybrid Fluorescence Molecular Tomography—X-Ray Phase-Contrast CT Imaging of Mouse Models

IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO. 7, JULY 2014, 2014, DOI 10.1109/TMI.2014.2313405 publiziert am 24.03.2014
The implementation of hybrid fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high accuracy. FMT affords highly sensitive 3-D imaging of fluorescence bio-distribution, but in standalone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from CT. Conversely, CT generally suffers from low soft tissue contrast. Therefore utilization of CT data as prior information in FMT inversion is challenging when different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast CT (PCCT). PCCT relies on phase shift differences in tissue to achieve soft tissue contrast superior to conventional CT. We demonstrate for the first time FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively.

The results show that FMT-PCCT expands the potential of FMT in imaging lesions with otherwise low or no CT contrast, while retaining the cost benefits of CT and simplicity of hybrid device realizations. The results point to the most accurate FMT performance to date.

 

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High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO. 7, JULY 2014, 2014, DOI 10.1109/TMI.2014.2313405 publiziert am 24.03.2014

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics,their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, 2conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal fi ltration. Humanized a HER2 and a CD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli . Cyto fl uorimetric titration analysis on tumor cell lines con fi rmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodi fi ed a HER2 and a CD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS 400 . Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodi fi ed Fabs were measured in the biodistribution analysis (48 h p.i.) for a HER2 Fab-PAS 100 and Fab-PAS 200 , respectively. These fi ndings were con fi rmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for a HER2 Fab-PAS 100 and Fab-PAS 200 . Even stronger tumor signals were obtained with the corresponding a CD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS 100 vs . 0.24% ID/ g for the unmodi fi ed Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to signi fi cantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

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Levels of p27 sensitize to dual PI3K/mTOR inhibition

Mol Cancer Ther., 2011, 10(8), 1450-9 publiziert am 01.08.2011
 
Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling cascade occurs in a variety of human malignancies, where it sustains tumor cell proliferation and survival. Pharmacologic blockade of this pathway exerts antineoplastic activity by triggering apoptosis and/or cell-cycle arrest. Pituitary adenomas show activation of the PI3K/AKT/mTOR pathway, but only a fraction of them respond in vitro to the antiproliferative action of rapamycin and RAD001 (mTOR inhibitors), possibly because of the described negative feedback loop on AKT which reactivates the signaling cascade. Rats affected by the multiple endocrine neoplasia-like syndrome (MENX) develop pituitary adenomas showing increased activated AKT. In this study, we comparatively investigated the antitumor potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235 and the single mTOR inhibitor RAD001 on rat pituitary adenoma cells in primary culture. NVP-BEZ235 inhibits the PI3K pathway both upstream and downstream of AKT, thereby preventing the negative feedback loop. NVP-BEZ235 was more effective than RAD001 in reducing cell viability of pituitary adenomas. Consistently, NVP-BEZ235 treatment decreased Akt and S6 phosphorylation and triggered apoptosis. Because MENX is caused by a germline loss-of-function mutation in the cell-cycle inhibitor p27Kip1, we investigated the relationship between this defect and response to NVP-BEZ235 treatment. The levels of p27Kip1 positively correlate with the response to NVP-BEZ235 treatment. Combined treatment with NVP-BEZ235 and the proteasome inhibitor bortezomib, which increases p27Kip1 amount, shows synergistic antiproliferative effects on pituitary adenoma cells. Our data suggest that NVP-BEZ235 may represent an effective therapeutic modality for pituitary adenomas and that p27Kip1 levels represent a potential predictor of response to dual PI3K/mTOR inhibition. ©2011 AACRv

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Int J Mol Imaging., 2011, 2011:175352, publiziert am 08.06.2011
 
Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or (11)C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of (11)C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.

 



Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma

Proc Natl Acad Sci U S A., 2010, 107(43), 18493-8 publiziert am 26.10.2010
 
Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use.




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