Presentation_1_Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies.PDF

<p>Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.</p

DataSheet_1_Basic characterization of antibodies targeting receptors of the tumor necrosis factor receptor superfamily.pdf

Many new immunotherapeutic approaches aim on the stimulatory targeting of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) using antibodies with intrinsic or conditional agonism. There is an initial need to characterize corresponding TNFRSF receptor (TNFR)-targeting antibodies with respect to affinity, ligand binding, receptor activation and the epitope recognized. Here, we report a collection of simple and matched protocols enabling the detailed investigation of these aspects by help of Gaussia princeps luciferase (GpL) fusion proteins and analysis of interleukin-8 (IL8) production as an easily measurable readout of TNFR activation. In a first step, the antibodies and antibody variants of interest are transiently expressed in human embryonal kidney 293 cells, either in non-modified form or as fusion proteins with GpL as a reporter domain. The supernatants containing the antibody-GpL fusion proteins can then be used without further purification in cell-free and/or cellular binding studies to determine affinity. Similarly, binding studies with mutated TNFR variants enable the characterization of the antibody binding site within the TNFR ectodomain. Furthermore, in cellular binding studies with GpL fusion proteins of soluble TNFL molecules, the ability of the non-modified antibody variants to interfere with TNFL-TNFR interaction can be analyzed. Last but not least, we describe a protocol to determine the intrinsic and the Fc gamma receptor (FcγR)-dependent agonism of anti-TNFR antibodies which exploits i) the capability of TNFRs to trigger IL8 production in tumor cell lines lacking expression of FcγRs and ii) vector- and FcγR-transfected cells, which produce no or only very low amounts of human IL8. The presented protocols only require standard molecular biological equipment, eukaryotic cell culture and plate readers for the quantification of luminescent and colorimetric signals.</p

Genetically engineered IgG1 and nanobody oligomers acquire strong intrinsic CD40 agonism

Most anti-CD40 antibodies show robust agonism only upon binding to FcγR+ cells, such as B cells, macrophages, or DCs, but a few anti-CD40 antibodies display also strong intrinsic agonism dependent on the recognized epitope and/or isotype. It is worth mentioning, however, that also the anti-CD40 antibodies with intrinsic agonism can show a further increase in agonistic activity when bound by FcγR-expressing cells. Thus, conventional antibodies appear not to be sufficient to trigger the maximum possible CD40 activation independent from FcγR-binding. We proved here the hypothesis that oligomeric and oligovalent anti-CD40 antibody variants generated by genetic engineering display high intrinsic, thus FcγR-independent, agonistic activity. We generated tetra-, hexa- and dodecavalent variants of six anti-CD40 antibodies and a CD40-specific nanobody. All these oligovalent variants, even when derived of bivalent antagonistic anti-CD40 antibodies, showed strongly enhanced CD40 agonism compared to their conventional counterparts. In most cases, the CD40 agonism reached the maximum response induced by FcγR-bound anti-CD40 antibodies or membrane CD40L, the natural engager of CD40. In sum, our data show that increasing the valency of anti-CD40 antibody constructs by genetic engineering regularly results in molecules with high intrinsic agonism and level out the specific limitations of the parental antibodies.</p

The induction of cardiac ruptures by HSA-Flag-TWEAK depends on neutrophils.

<p>Mice were treated with an anti-Ly6G antibody to deplete neutrophils (A) in the myocardium as measured by immunohistological staining and (B) in the peripheral blood as measured by FACS analysis. (C) Neutrophil depletion did not affect survival after MI in PBS and HSA-Flag-TWEAK treated mice. (D) The occurrence of cardiac ruptures was significantly reduced after anti-Ly6G antibody treatment in the HSA-Flag-TWEAK treated group in comparison to the neutrophil intact control.</p

HSA-Flag-TWEAK treatment modulates the expression of cytokines and chemokines via NFκB and JAK/STAT-signalling.

<p>The protein-protein interaction network maps cytokine and chemokine data from the protein microarrays onto the human interactome. Circles indicate proteins; kinases are depicted in triangular shape, each specified by gene names. They are connected either by gray lines indicating protein-protein interactions or red arrows denoting phosphorylation reactions. The functional entities are highlighted (interleukins, chemokines, JAK/STAT pathway). Green-colored nodes show up-regulation in the TWEAK stimulation (orange) condition.</p

HSA-Flag-TWEAK increases immune cell infiltration into the infarcted heart.

<p>(A) Exogenous administration of HSA-Flag-TWEAK amplified the infiltration of leukocytes into the infarcted myocardium 3 days after MI as determined by FACS analysis. (B) Neutrophils highly infiltrated the border zone of HSA-Flag-TWEAK treated mice.</p

HSA-Flag-TWEAK modulates the production of cytokines in the infarcted heart after MI.

<p>u.d. = under detection limit.</p><p>IFN-γ: <11 pg/ml; IL-12: <5 pg/ml; MCP-1: <5 pg/ml; MCP-5: <1 pg/ml; RANTES: <3 pg/ml.</p><p>A cytokine array for the detection of the protein expression of different cytokines was performed 3 days after MI. HSA-Flag-TWEAK treated mice showed statistically significant up-regulation of interleukin 12 (IL-12), interleukin 5 (IL-5), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and regulated on activation, normal T cell expressed and secreted (RANTES) (u.d. = under limit of detection).</p

HSA-Flag-TWEAK does not induce cardiomyocyte apoptosis.

<p>The percentage of apoptotic cells 3(A) TUNEL-positive cells and (B) cleaved PARP was similar between HSA-Flag-TWEAK and PBS treated mice.</p

HSA-Flag-TWEAK does not affect echocardiographic measurements after MI.

<p>Animals underwent echocardiography on day 1, day 3, day 21 (data not shown) and day 56 (data not shown) after MI. All measurements were recorded at the midpapillary level which shows changes in the dimensions of the surviving non-infarcted myocardium, as well as on the apical level depicting changes in scar formation.</p><p>Data are means ± sem; <i>n</i> indicates number of animals studied. EDA, end-diastolic area; ESA, end-systolic area; FS, fractional shortening; 2D, 2-dimensional.</p

HSA-Flag-TWEAK fails to modulate extracellular matrix remodeling after MI.

<p>mRNA-expression of (A) collagen1α1, collagen1α2, (B) MMP-2, MMP-3, MMP-8, and MMP-9 were unaffected in the scar region of HSA-Flag-TWEAK challenged mice as were the (C) zymographic activities of MMP-2 and MMP-9 (measured as gel band intensity) and (D) TIMP-2, TIMP-3, and VEGF mRNA expression.</p