Publications by authors named "Katharina Frey"

23 Publications

  • Page 1 of 1

Nintedanib and a bi-specific anti-VEGF/Ang2 nanobody selectively prevent brain metastases of lung adenocarcinoma cells.

Clin Exp Metastasis 2020 Dec 12;37(6):637-648. Epub 2020 Sep 12.

Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120, Heidelberg, Germany.

Brain metastases (BM) are an ever-increasing challenge in oncology, threatening quality of life and survival of many cancer patients. The majority of BM originate from lung adenocarcinoma, and stage III patients have a risk of 40-50% to develop BM in the first years of disease onset. As therapeutic options are limited, prevention of their occurrence is an attractive concept. Here we investigated whether Nintedanib (BIBF 1120), a tyrosine kinase inhibitor (TKI) targeting the VEGF pathway approved for lung adenocarcinoma, and the dual anti-VEGF-A/Ang2 nanobody BI836880 have the potential to prevent BM formation. A mouse model of brain metastasis from lung adenocarcinoma was used in which tumor cells were injected intracardially. Metastases formation occurred inside and outside of the brain and was followed by MRI, IVIS, and immunohistochemistry. BM were reduced in volume and number by both Nintedanib and the dual anti-VEGF-A/Ang2 nanobody, which translated into improved survival. Both compounds were able to normalize cerebral blood vessels at the site of brain metastatic lesions. Extracranial metastases, however, were not reduced, and meningeal metastases only partially. Interestingly, unspecific control IgG also lead to brain vessel normalization and reduction of brain and meningeal metastases. This data indicates a brain-specific group effect of antiangiogenic compounds with respect to metastasis prevention, most likely by preventing an early angiogenic switch. Thus, Nintedanib and BI836880 are promising candidates for future BM preventive study concepts in lung adenocarcinoma patients.
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http://dx.doi.org/10.1007/s10585-020-10055-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666285PMC
December 2020

Amphibious vision - Optical design model of the hooded merganser eye.

Vision Res 2020 10 28;175:75-84. Epub 2020 Jul 28.

Ernst-Abbe-Hochschule, University of Applied Sciences Jena, Germany; Fraunhofer Institute of Applied Optics and Precision Engineering (IOF), Jena, Germany. Electronic address:

A comprehensive schematic eye model of the hooded merganser is introduced for the first time to advance the understanding of amphibious vision. It is comprised of two different configurations, the first one modeling its visual system in air (unaccommodated state) and the second one representing the case where the eye is immersed in water (accommodated state). The model was designed using available data of former studies, image analysis and the implementation of feasible assumptions that serve as starting values. An optimization process incorporating an optical design program is used to vary the starting values with the aim of finding the setup offering the best acuity. The image quality was measured using the root-mean-square radius of the focal spot formed on the retina. The resulting schematic eye model comprises all relevant optical specifications, including aspherical geometrical parameters for cornea and lens, distances between the surfaces, the gradient index distribution of the lens, the retinal specifications and the object distance in both media. It achieves a spot radius of 4.20 μm for the unaccommodated state, which meets the expectations derived by the mean ganglion cell density and comparison with other animals. In contrast, under water the determined spot radius of 11.48 µm indicates an acuity loss. As well as enhancing our understanding of the vision of the hooded merganser, the schematic eye model may also serve as a simulation basis for examing similar animal eyes, such as the cormorant or other fish hunting birds.
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http://dx.doi.org/10.1016/j.visres.2020.05.011DOI Listing
October 2020

Risk incidence of fractures and injuries: a multicenter video-EEG study of 626 generalized convulsive seizures.

J Neurol 2020 Dec 10;267(12):3632-3642. Epub 2020 Jul 10.

Center of Neurology and Neurosurgery, Epilepsy Center Frankfurt Rhine-Main, Goethe-University Frankfurt, Schleusenweg 2-16 (Haus 95), 60528, Frankfurt am Main, Germany.

Objective: To evaluate the incidence and risk factors of generalized convulsive seizure (GCS)-related fractures and injuries during video-EEG monitoring.

Methods: We analyzed all GCSs in patients undergoing video-EEG-monitoring between 2007 and 2019 at epilepsy centers in Frankfurt and Marburg in relation to injuries, falls and accidents associated with GCSs. Data were gathered using video material, EEG material, and a standardized reporting form.

Results: A total of 626 GCSs from 411 patients (mean age: 33.6 years; range 3-74 years; 45.0% female) were analyzed. Severe adverse events (SAEs) such as fractures, joint luxation, corneal erosion, and teeth loosening were observed in 13 patients resulting in a risk of 2.1% per GCS (95% CI 1.2-3.4%) and 3.2% per patient (95% CI 1.8-5.2%). Except for a nasal fracture due to a fall onto the face, no SAEs were caused by falls, and all occurred in patients lying in bed without evidence of external trauma. In seven patients, vertebral body compression fractures were confirmed by imaging. This resulted in a risk of 1.1% per GCS (95% CI 0.5-2.2%) and 1.7% per patient (95% CI 0.8-3.3%). These fractures occurred within the tonic phase of a GCS and were accompanied by a characteristic cracking noise. All affected patients reported back pain spontaneously, and an increase in pain on percussion of the affected spine section.

Conclusions: GCSs are associated with a substantial risk of fractures and shoulder dislocations that are not associated with falls. GCSs accompanied by audible cracking, and resulting in back pain, should prompt clinical and imaging evaluations.
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http://dx.doi.org/10.1007/s00415-020-10065-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674387PMC
December 2020

Animal, Fungi, and Plant Genome Sequences Harbor Different Non-Canonical Splice Sites.

Cells 2020 02 18;9(2). Epub 2020 Feb 18.

Genetics and Genomics of Plants, Center for Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany.

Most protein-encoding genes in eukaryotes contain introns, which are interwoven with exons. Introns need to be removed from initial transcripts in order to generate the final messenger RNA (mRNA), which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides, which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5' end and AG at the 3' end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations, which have been known for years. Recently, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here, we expand systematic investigations of non-canonical splice site combinations in plants across eukaryotes by analyzing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences, such as an apparently increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts are a likely explanation for this observation, thus indicating annotation errors. In addition, high numbers of GA-AG splice site combinations were observed in and . A variant in one U1 small nuclear RNA (snRNA) isoform might allow the recognition of GA as a 5' splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3' splice site compared to the 5' splice site across animals, fungi, and plants.
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http://dx.doi.org/10.3390/cells9020458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072748PMC
February 2020

Homologous Proteins of the Manganese Transporter PAM71 Are Localized in the Golgi Apparatus and Endoplasmic Reticulum.

Plants (Basel) 2020 Feb 13;9(2). Epub 2020 Feb 13.

Molekularbiologie der Pflanze (Botanik), Department Biologie I, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany.

Chloroplast manganese transporter 1 (CMT1) and photosynthesis-affected mutant 71 (PAM71) are two membrane proteins that function sequentially to mediate the passage of manganese across the chloroplast envelope and the thylakoid membrane. CMT1 and PAM71 belong to a small five-member protein family in . The other three, photosynthesis-affected mutant 71 like 3 (PML3), PML4 and PML5 are not predicted to reside in chloroplast membranes. In this study, the subcellular localization of PML3:GFP, PML4:GFP and PML5:GFP was determined using transient and stable expression assays. PML3:GFP localizes to the Golgi apparatus, whereas PML4:GFP and PML5:GFP are found in the endoplasmic reticulum. We also examined patterns of , and promoter activity. Although the precise expression pattern of each promoter was unique, all three genes were expressed in the leaf vasculature and in roots. Greenhouse grown single mutants , , and the double mutant did not exhibit growth defects, however an inspection of the root growth revealed a difference between and the other genotypes, including wild-type, in 500 µM manganese growth conditions. Strikingly, overexpression of PML3 resulted in a stunted growth phenotype. Putative functions of PML3, PML4 and PML5 are discussed in light of what is known about PAM71 and CMT1.
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http://dx.doi.org/10.3390/plants9020239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076475PMC
February 2020

A chromosome-level sequence assembly reveals the structure of the Arabidopsis thaliana Nd-1 genome and its gene set.

PLoS One 2019 21;14(5):e0216233. Epub 2019 May 21.

Bielefeld University, Faculty of Biology & Center for Biotechnology, Bielefeld, Germany.

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species. Here we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences. Detailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216233PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529160PMC
January 2020

Upright MRI after decompression of spinal stenosis and concurrent spondylolisthesis.

Neurosurg Focus 2019 05;46(5):E14

1Department of Neurosurgery, University of Frankfurt; and.

OBJECTIVEThe treatment of patients with spinal stenosis and concurrent degenerative spondylolisthesis is controversial. Two large randomized controlled clinical trials reported contradictory results. The authors hypothesized that a substantial number of patients will show evidence of micro-instability after a sole decompression procedure.METHODSThis study was a retrospective analysis of all cases of lumbar spinal stenosis treated at the Frankfurt University Clinic (Universitätsklinik Frankfurt) from 2010 through 2013. Patients who had associated spondylolisthesis underwent upright MRI studies in flexion and extension for identification of subtle signs of micro-instability. Clinical outcome was assessed by means of SF-36 bodily pain (BP) and physical functioning (PF) scales.RESULTSA total of 21 patients were recruited to undergo upright MRI studies. The mean duration of follow-up was 65 months (SD 16 months). Of these 21 patients, 10 (47%) showed signs of micro-instability as defined by movement of > 4 mm on flexion/extension MRI. Comparison of mean SF-36 BP and PF scores in the group of patients who showed micro-instability versus those who did not showed no statistically significant difference on either scale.CONCLUSIONSThere seems to be a substantial subset of patients who develop morphological micro-instability after sole decompression procedures but do not experience any clinically significant effect of the instability.
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http://dx.doi.org/10.3171/2019.3.FOCUS18730DOI Listing
May 2019

Comprehensive optical design model of the goldfish eye and quantitative simulation of the consequences on the accommodation mechanism.

Vision Res 2019 01 29;154:115-121. Epub 2018 Nov 29.

Ernst-Abbe-Hochschule, University of Applied Sciences Jena, Germany; Fraunhofer Institute of Applied Optics and Precision Engineering (IOF), Jena, Germany. Electronic address:

To further extent our understanding of aquatic vision, we introduce a complete optical model of a goldfish eye, which comprises all important optical parameters for the first time. Especially a spherical gradient index structure for the crystalline lens was included, thus allowing a detailed analysis of image quality, regarding spot size, and wavelength dependent aberration. The simulation results show, that our realistic eye model generates a sufficient image quality, with a spot radius of 4.9 μm which is below the inter cone distance of 5.5 μm. Furthermore, we optically simulate potential mechanical processes of accommodation and compare the results with contradictory findings of previous experimental studies. The quantitative simulation of the accommodation capacity shows that the depth of field is strongly dependent on the resting position and becomes significantly smaller when shorter resting positions are assumed. That means, to enable an extended depth perception with high acuity for the goldfish an adaptive, lens shifting mechanism would be required. In addition, our model allows a clear prediction of the expected axial lens-shift, which is necessary to ensure a sufficient resolution over a large object range.
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http://dx.doi.org/10.1016/j.visres.2018.11.005DOI Listing
January 2019

Automated detection of the choroid boundary within OCT image data using quadratic measure filters.

J Biomed Opt 2017 02;22(2):25004

Leipzig University Hospital, Department of Ophthalmology, Leipzig, Germany.

A novel method for the automated detection of the outer choroid boundary within spectral-domain optical coherence tomography image data, based on an image model within the space of functions of bounded variation and the application of quadratic measure filters, is presented. The same method is used for the segmentation of retinal layer boundaries and proves to be suitable even for data generated without special imaging modes and moderate line averaging. Based on the segmentations, an automated determination of the central fovea region and choroidal thickness measurements for this and two adjacent 1-mm regions are provided. The quality of the method is assessed by comparison with manual delineations performed by five trained graders. The study is based on data from 50 children of the ages 8 to 13 that were obtained in the framework of the LIFE Child study at Leipzig University.
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http://dx.doi.org/10.1117/1.JBO.22.2.025004DOI Listing
February 2017

Antibody-based delivery of interleukin-2 to neovasculature has potent activity against acute myeloid leukemia.

Sci Transl Med 2013 Sep;5(201):201ra118

Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.

Acute myeloid leukemia (AML) is a rapidly progressing disease that is accompanied by a strong increase in microvessel density in the bone marrow. This observation prompted us to stain biopsies of AML and acute lymphoid leukemia (ALL) patients with the clinical-stage human monoclonal antibodies F8, L19, and F16 directed against markers of tumor angiogenesis. The analysis revealed that the F8 and F16 antibodies strongly stained 70% of AML and 75% of ALL bone marrow specimens, whereas chloroma biopsies were stained with all three antibodies. Therapy experiments performed in immunocompromised mice bearing human NB4 leukemia with the immunocytokine F8-IL2 [consisting of the F8 antibody fused to human interleukin-2 (IL-2)] mediated a strong inhibition of AML progression. This effect was potentiated by the addition of cytarabine, promoting complete responses in 40% of treated animals. Experiments performed in immunocompetent mice bearing C1498 murine leukemia revealed long-lasting complete tumor eradication in all treated mice. The therapeutic effect of F8-IL2 was mediated by both natural killer cells and CD8(+) T cells, whereas CD4(+) T cells appeared to be dispensable, as determined in immunodepletion experiments. The treatment of an AML patient with disseminated extramedullary AML manifestations with F16-IL2 (consisting of the F16 antibody fused to human IL-2, currently being tested in phase 2 clinical trials in patients with solid tumors) and low-dose cytarabine showed significant reduction of AML lesions and underlines the translational potential of vascular tumor-targeting antibody-cytokine fusions for the treatment of patients with leukemia.
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http://dx.doi.org/10.1126/scitranslmed.3006221DOI Listing
September 2013

Combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy.

J Immunother 2012 Jun;35(5):418-29

Institut für Zellbiologie und Immunologie, Universität Stuttgart, Stuttgart, Germany.

Initiation of a tumor-directed immune response and appropriate modulation of its progress are key issues in cancer immunotherapy. Combinatorial strategies addressing both aspects might therefore be especially suitable. Here, we report a targeted approach combining a bispecific antibody with 2 costimulatory antibody-ligand fusion proteins. According to the concept, the bispecific antibody (scDbFAP×CD3) retargets T cells in a MHC-independent manner to tumor cells, providing an artificial first signal that allows the costimulatory antibody-ligand fusion proteins (B7.2-Db and scFv-4-1BBL) likewise targeted to the tumor cells to modulate the T-cell response. In our model system, the target cells coexpress the fibroblast activation protein (FAP) and endoglin as antigens. ScDbFAPCD3 and B7.2-Db are targeted to FAP although by different antibody moieties, whereas scFv-4-1BBL is directed against endoglin. ScDbFAPCD3-induced T-cell stimulation could be enhanced by the addition of either B7.2-Db or scFv-4-1BBL and even further by the combination of both as shown in terms of cytokine release (interleukin-2/interferon γ), proliferation and activation marker expression (CD25). By combined costimulation, overall T-cell population strongly increased in activation-experienced memory phenotype accompanied by a decrease in naive phenotype. ScFv-4-1BBL-mediated costimulation of naive CD8+ T cells promoted the expansion and development of cytotoxic T cells with strong effector potential. Thus, combining a bispecific antibody with antibody-ligand fusion protein-mediated CD28 and 4-1BB costimulation in a targeted approach shows great potential to generate and shape an immune response at the tumor site. Therefore, the adaptation of this approach to other immune modulatory ligands and tumor-relevant targets seems to be promising.
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http://dx.doi.org/10.1097/CJI.0b013e3182594387DOI Listing
June 2012

Analysis of optical side-effects of fs-laser therapy in human presbyopic lens simulated with modified contact lenses.

Graefes Arch Clin Exp Ophthalmol 2012 Dec 29;250(12):1813-25. Epub 2012 Feb 29.

Department of Ophthalmology, HELIOS Klinikum Erfurt, Nordhäuserstrasse 74, 99089, Erfurt, Germany.

Background: In a single-blinded study, optical side-effects of a potential femtosecond (fs)-laser therapy in presbyopic human lenses were tested. Simulation of this therapy was carried out by applying fs-laser patterns into standard contact lenses (CL).

Methods: In the first part of the study, the influence of the numerical aperture on optical side-effects was investigated by comparing a typical fs-LASIK configuration to a fs-presbyopia treatment (n = 11). The second part focused on a possible improvement of visual performance by comparing a regular grid pattern to a randomly chosen spacing of the laser spots (n = 16). Visual acuity was measured with ETDRS charts, contrast sensitivity with F.A.C.T. charts and mesopic vision with Mesotest II. Forward scattered light was measured with the C-Quant (both instruments: Oculus Optikgeräte GmbH, Germany). A questionnaire detected subjective quality of vision. Differences between laser-treated and untreated CL and among the modifications were analyzed.

Results: The laser-treated and standard CL indicated no significant difference in visual acuity, contrast sensitivity and mesopic vision without glare. While wearing modified lenses with a regular grid, quality of vision decreased significantly by means of mesopic vision with glare and subjective straylight. These modifications also caused an impairment of subjective quality of vision. In contrast, there was no significant difference between the random pattern and standard CL.

Conclusion: The increase of optical side-effects was reproducibly dependent on the geometry of the laser-structure. A randomized grid induced the least limitation. The study results are useful for planning possible laser-patterns in fs-laser therapy of the presbyopic lens.
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http://dx.doi.org/10.1007/s00417-012-1965-0DOI Listing
December 2012

The targeted delivery of IL17 to the mouse tumor neo-vasculature enhances angiogenesis but does not reduce tumor growth rate.

Angiogenesis 2012 Mar 4;15(1):165-9. Epub 2011 Nov 4.

Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.

There has been a long controversy as to whether interleukin-17 (IL17) has an impact on tumor growth. In order to assess whether IL17 may affect tumor growth, it would be convenient to achieve high levels of this pro-inflammatory cytokine at the tumor neo-vasculature, since IL17 is known to promote angiogenesis. Here, we have generated and tested in vivo a fusion protein, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis) and of murine IL17 (mIL17). The resulting immunocytokine (termed F8-mIL17) was shown to selectively localize at the tumor neo-vasculature and to vigorously promote tumor angiogenesis, without however reducing or enhancing tumor growth rate both in immunocompetent and in immunodeficient mice.
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http://dx.doi.org/10.1007/s10456-011-9239-8DOI Listing
March 2012

Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

PLoS One 2011 9;6(6):e20820. Epub 2011 Jun 9.

Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riss, Germany.

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/principal Findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111438PMC
November 2011

Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions.

Exp Dermatol 2011 Aug 7;20(8):685-8. Epub 2011 Jun 7.

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

We have investigated the staining patterns of primary and metastatic melanoma lesions using F8, L19 and F16. These three clinical-stage antibodies are currently being studied in clinical trials for the pharmacodelivery of cytokines or therapeutic radionuclides to neoplastic sites in patients with cancer. Frozen sections of 24 primary and 29 metastatic melanoma lesions were stained, using immunofluorescence procedures, with biotinylated preparations of the F8, L19 and F16 antibodies, which are specific to the alternatively spliced extra domain A and extra domain B domains of fibronectin and A1 domain of tenascin-C, respectively. Blood vessels were costained using von Willebrand factor-specific antibodies. In primary cutaneous melanoma lesions, F16 and F8 (but not L19) strongly stained the basal lamina at the interface between epidermis and dermis, with a strikingly complementary pattern. By contrast, metastatic melanoma lesions displayed a strong and diffuse pattern of immunoreactivity with all three antibodies. It was found that the extracellular matrix in melanoma undergoes extensive remodelling during the transition from primary to metastatic lesions. The intense staining of metastatic melanoma lesions by the F8, L19 and F16 antibodies provides a strong rationale for the use of these antibodies and their derivatives for the treatment of melanoma patients and possibly for the personalized choice of the best performing antibody in individual patients.
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http://dx.doi.org/10.1111/j.1600-0625.2011.01314.xDOI Listing
August 2011

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7.

J Biotechnol 2011 Jun 16;154(1):84-92. Epub 2011 Apr 16.

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse, Zurich, Switzerland.

We generated and characterized novel antibody-cytokine fusion proteins ("immunocytokines") based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed "F8-mIL7") of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed "F8-mIL7-F8"), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio=16:1, 24h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.
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http://dx.doi.org/10.1016/j.jbiotec.2011.04.003DOI Listing
June 2011

Antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation.

Integr Biol (Camb) 2011 Apr 11;3(4):468-78. Epub 2011 Jan 11.

Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerland.

The antibody-mediated targeted delivery of cytokines, growth factors and immunomodulators offers great potential for the therapy of cancer and other serious conditions. Interferon-alpha has long been used in the clinic for the treatment of patients with certain malignancies or with viral disease. Promising anticancer activity has recently been reported for two fusion proteins consisting of immunoglobulins bearing the interferon-alpha polypeptide at the C-terminal end of the molecule. Here we describe the design, production and characterization of a novel immunocytokine, in which murine interferon-alpha2 was sequentially fused with the tumor-targeting antibody fragment scFv(F8), specific to the alternatively-spliced EDA domain of fibronectin. The resulting fusion protein (F8-IFNa) could be produced to homogeneity and was shown to retain both antigen binding activity and interferon-alpha activity. Biodistribution studies in tumor-bearing mice with radioiodinated protein preparations confirmed the ability of F8-IFNa to selectively localize at the tumor site. However, using two different murine models of cancer (F9 teratocarcinomas and Cloudman S91 melanomas in immunocompetent mice), we could not detect a striking superiority for the therapeutic performance of F8-IFNa as compared to KSF-IFNa, a fusion protein of irrelevant specificity in the mouse which was used as negative control. In the paper, we present hypotheses why the antibody-based pharmacodelivery of interferon-alpha fails to eradicate tumors, in contrast to the situation observed by our group for other immunocytokines, which benefit from a selective localization at the tumor site.
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http://dx.doi.org/10.1039/c0ib00099jDOI Listing
April 2011

Selection of Carbonic Anhydrase IX Inhibitors from One Million DNA-Encoded Compounds.

ACS Chem Biol 2011 Apr 12;6(4):336-44. Epub 2011 Jan 12.

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland.

DNA-encoded chemical libraries, i.e., collections of compounds individually coupled to distinctive DNA fragments serving as amplifiable identification barcodes, represent a new tool for the de novo discovery of small molecule ligands to target proteins of pharmaceutical interest. Here, we describe the design and synthesis of a novel DNA-encoded chemical library containing one million small molecules. The library was synthesized by combinatorial assembly of three sets of chemical building blocks using Diels-Alder cycloadditions and by the stepwise build-up of the DNA barcodes. Model selections were performed to test library performance and to develop a statistical method for the analysis of high-throughput sequencing data. A library selection against carbonic anhydrase IX revealed a new class of submicromolar bis(sulfonamide) inhibitors. One of these inhibitors was synthesized in the absence of the DNA-tag and showed accumulation in hypoxic tumor tissue sections in vitro and tumor targeting in vivo.
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http://dx.doi.org/10.1021/cb1003477DOI Listing
April 2011

Comparative in vivo analysis of the atherosclerotic plaque targeting properties of eight human monoclonal antibodies.

Atherosclerosis 2011 Feb 27;214(2):325-30. Epub 2010 Nov 27.

Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.

Objective: The selective in vivo localization of antibody derivatives in atherosclerotic plaques may open novel diagnostic and therapeutic applications. Here, we present a comparative in vivo localization analysis of eight radioiodinated human monoclonal antibodies in apolipoprotein E-deficient (ApoE(-/-)) mice.

Methods: Animals were fed with a cholesterol-rich diet, followed by harvesting of the aorta 24h after intravenous antibody injection and investigated by autoradiographic analysis. Localization of F8 antibody on atherosclerotic plaque structures was further studied in three-color fluorescence microscopy.

Results: The study revealed that the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin, exhibited the highest plaque-targeting potential among the antibodies analyzed in this study, with an ability to preferentially localize to all plaques within the aorta. Targeting results were confirmed by injection of fluorescein-labeled F8 antibody, followed by three-color fluorescence microscopy analysis.

Conclusion: These findings open novel biomolecular avenues for the in vivo imaging of atherosclerotic plaques and for pharmacodelivery applications, since F8 had previously been reported by our group to strongly stain atherosclerotic plaques in human carotid arteries.
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http://dx.doi.org/10.1016/j.atherosclerosis.2010.11.027DOI Listing
February 2011

The immunocytokine F8-IL2 improves the therapeutic performance of sunitinib in a mouse model of renal cell carcinoma.

J Urol 2010 Dec 28;184(6):2540-8. Epub 2010 Oct 28.

Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland.

Purpose: We investigated the therapeutic action of F8-IL2, a fusion protein consisting of the F8 antibody specific to the alternatively spliced extradomain-A of fibronectin, in diabody format and of human interleukin-2 in the Caki-1 (ATCC®) model of human renal cell carcinoma grafted subcutaneously in nude mice.

Materials And Methods: F8-IL2 was cloned, expressed in CHO cells and purified to homogeneity. This immunocytokine was administered alone or combined with 3 standard drugs commonly used as therapy for kidney cancer, including sunitinib, sorafenib and interferon-α, in 2 sets of doses and treatment schedules.

Results: Neither F8-IL2 nor any other therapeutic agent cured tumor bearing mice when used as a single agent. The best therapeutic results were observed for the combination of sunitinib with F8-IL2 in a continuous administration schedule, which yielded a 28% cure rate and substantial tumor growth retardation.

Conclusions: Considering that recombinant interleukin-2 based immunocytokines are now being investigated in several clinical trials in patients with cancer alone or combined with chemotherapy our preclinical results provide a motivation to study F8-IL2 combined with sunitinib in clinical trials in patients with kidney cancer.
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http://dx.doi.org/10.1016/j.juro.2010.07.030DOI Listing
December 2010

Tumour-targeting properties of antibodies specific to MMP-1A, MMP-2 and MMP-3.

Eur J Nucl Med Mol Imaging 2010 Aug 20;37(8):1559-65. Epub 2010 Apr 20.

Department of Chemistry and Applied Biosciences, ETH Zürich, Institute of Pharmaceutical Sciences, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

Purpose: Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are upregulated in many diseases, but several attempts to use radiolabelled MMP inhibitors for imaging tumours have proved unsuccessful in mouse models, possibly due to the limited specificity of these agents or their unfavourable pharmacokinetic profiles. In principle, radiolabelled monoclonal antibodies could be considered for the selective targeting and imaging of individual MMPs.

Methods: We cloned, produced and characterized high-affinity monoclonal antibodies specific to murine MMP-1A, MMP-2 and MMP-3 in SIP (small immunoprotein) miniantibody format using biochemical and immunochemical methods. We also performed comparative biodistribution analysis of their tumour-targeting properties at three time points (3 h, 24 h, 48 h) in mice bearing subcutaneous F9 tumours using radioiodinated protein preparations. The clinical stage L19 antibody, specific to the alternatively spliced EDB domain of fibronectin, was used as reference tumour-targeting agent for in vivo studies.

Results: All anti-MMP antibodies and SIP(L19) strongly stained sections of F9 tumours when assessed by immunofluorescence methods. In biodistribution experiments, SIP(SP3), specific to MMP-3, selectively accumulated at the tumour site 24 and 48 h after intravenous injection, but was rapidly cleared from other organs. By contrast, SIP(SP1) and SIP(SP2), specific to MMP-1A and MMP-2, showed no preferential accumulation at the tumour site.

Conclusion: Antibodies specific to MMP-3 may serve as vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to sites of disease.
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http://dx.doi.org/10.1007/s00259-010-1446-9DOI Listing
August 2010

New strategy for the extension of the serum half-life of antibody fragments.

Bioconjug Chem 2009 Dec;20(12):2286-92

Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, and Philochem AG, ETH Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerland.

Antibody fragments can recognize their cognate antigen with high affinity and can be produced at high yields, but generally display rapid blood clearance profiles. For pharmaceutical applications, the serum half-life of antibody fragments is often extended by chemical modification with polymers or by genetic fusion to albumin or albumin-binding polypeptides. Here, we report that the site-specific chemical modification of a C-terminal cysteine residue in scFv antibody fragments with a small organic molecule capable of high-affinity binding to serum albumin substantially extends serum half-life in rodents. The strategy was implemented using the antibody fragment F8, specific to the alternatively spliced EDA domain of fibronectin, a tumor-associated antigen. The unmodified and chemically modified scFv-F8 antibody fragments were studied by biodistribution analysis in tumor-bearing mice, exhibiting a dramatic increase in tumor uptake for the albumin-binding antibody derivative. The data presented in this paper indicate that the chemical modification of the antibody fragment with the 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate albumin-binding moiety may represent a general strategy for the extension of the serum half-life of antibody fragments and for the improvement of their in vivo targeting performance.
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http://dx.doi.org/10.1021/bc9002772DOI Listing
December 2009

A novel antibody-4-1BBL fusion protein for targeted costimulation in cancer immunotherapy.

J Immunother 2008 Oct;31(8):714-22

Institut für Zellbiologie und Immunologie, Universität Stuttgart, Stuttgart, Germany.

Costimulation is an essential step in T-cell activation and hence, represents an important aspect in cancer immunotherapy. 4-1BB, a member of the tumor necrosis factor receptor family, has gained particular interest as a costimulatory molecule. Here, we investigated the potential of a targeted activation of 4-1BB-mediated costimulation at the tumor site by generating a recombinant antibody-cytokine fusion protein composed of a single-chain antibody fragment (scFv36) specific for the tumor stromal antigen fibroblast activation protein (FAP) and the extracellular domain of the 4-1BB ligand (4-1BBL). The scFv36-4-1BBL fusion protein is a homotrimeric molecule that binds specifically to FAP and the receptor 4-1BB. T-cell costimulation was demonstrated by interferon-gamma release of peripheral blood mononuclear cells cocultured with FAP-expressing HT1080 cells upon T-cell receptor triggering by monoclonal anti-CD3 antibody. Costimulatory activity of the scFv36-4-1BBL fusion protein was concentration dependent, ligand-specific, and substantially constrained to FAP-expressing target cell binding. Furthermore, scFv36-4-1BBL enhanced T-cell activation when the bispecific antibody scDb33CD3 (specific for FAP and CD3) was used as primary stimulus. Thus, target cell-dependent costimulation with scFv36-4-1BBL constitutes a new option to enhance T-cell activation by bispecific antibodies or antigen-dependent T-cell receptor triggering and should be useful to improve T cell-mediated antitumor responses.
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http://dx.doi.org/10.1097/CJI.0b013e31818353e9DOI Listing
October 2008