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

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, 24 h 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.     

New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2′-deoxycytidine in human urine

Abstract

Etheno–DNA adducts are generated from exogenous carcinogens such as vinyl chloride and urethane and also from endogenous lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE). The present authors and others have established that 1,N⁶-ethenodeoxyadenosine (εdA) and 3,N⁴-ethenodeoxycytidine (εdC) are present in human urine and could be explored as biomarkers for monitoring whole-body oxidative stress. The present study reports on a new ultrasensitive ³²P-postlabelling/thin-layer chromatography (TLC) method for the analysis of εdC as deoxynucleoside in human urine. The urine samples were purified and enriched on a solid-phase silica C-18 column followed by a semi-preparative reverse-phase high-performance liquid chromatography. The purified sample was labelled with a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) in the presence of 5′-bromo-2′-deoxyuridine (BrdU) as internal standard. The absolute sensitivity of the method was 0.1 fmol εdC detectable in 500 µl of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean εdC level of 2.49±1.76 (SD) fmol µmol^?1 creatinine (range 0.66–6.42). By this non-invasive method, εdC in human urine could be explored as a biomarker for oxidative stress-related human diseases.     

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell''s stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.     

Chicken egg yolk antibodies for cytokinin analysis

The production, isolation, and purification of specific chicken immunoglobulins (Igs) against three main groups of naturally occurring cytokinins are reported. The specific Igs directed against, respectively, zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine are extracted from the egg yolk and used in radioimmunoassays that allow the quantification in parallel of pmol of the cytokinins in plant extracts. As little as 50 fmol of zeatin riboside, 20 fmol of isopentenyladenosine, and 40 fmol of dihydrozeatin riboside can be detected. The levels of cytokinins measured in the radio-immunoassay correlate well with physicochemical analysis methods such as high performance liquid chromatography (HPLC) with UV spectrum detection and HPLC-coupled mass spectrometric detection. Cross-reactivity studies indicate that the assay is not affected by most of the structurally related compounds. The respective antibody preparations recognized zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine and the corresponding free bases. The results obtained when analyzing crude plant extracts are expressed as zeatin riboside equivalents, dihydrozeatin riboside equivalents, and isopentenyladenosine equivalents.Abbreviations B binding activity - B ₀ maximal binding - B ₁ unspecific binding - GC gas chromatography - HPLC high performance liquid chromatography - LC-MS HPLC-coupled mass spectrometry - MOPS 4-morpholinepropanesulfonic acid - RIA radioimmunoassay - TBS Tris-buffered saline - (diH)Z dihydrozeatin - (diH) [9R]Z dihydrozeatin riboside - iP isopentenyladenine - [9R]iP isopentenyladenosine - Z zeatin - [9R]Z zeatin riboside - [9G]iP isopentenyladenine-9-glucoside - [9R-5P]iP isopentenyladenosine-5-monophosphate