Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Lang, Isabell; Füllsack, Simone; Wyzgol, Agnes; Fick, Andrea; Trebing, Johannes; Arana, José Antonio Carmona; Schäfer, Viktoria; Weisenberger, Daniela; Wajant, Harald

doi:10.1074/jbc.m115.683946

Cited by 52 publications

(53 citation statements)

References 69 publications

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“…In soluble form, 4-1BBL and OX40L have been described to be basically inactive and to require molecular cross-linking in order to regain their activity. 15,16,30 We observed that our recombinant 4-1BBL and OX40L in both formats were indeed not capable of or very inefficient in activating the corresponding receptor-transfected HT1080 cell lines (Fig. 2C).…”

Section: Discussionmentioning

confidence: 87%

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

et al. 2016

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Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e., three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumordirected scFv antibody fragments directed against EpCAM or FAP to co-stimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT), a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-TNFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced co-stimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.Abbreviations: EpCAM, epithelial cell adhesion molecule; ED-A, fibronectin extradomain A; FAP, fibroblast activation protein; GITRL, glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand; LIGHT, homologous to lymphotoxins, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes; mAb, monoclonal antibody; scFv, single-chain fragment variable; TNF, tumor necrosis factor; TNFSF, tumor necrosis factor superfamily; TRAIL, TNF-related apoptosis inducing ligand

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Section: Discussionmentioning

confidence: 87%

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

et al. 2016

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“…Thus, the competition between these cytokines’ receptors on the cell surface and the antibody for DBT-generated IL-1β and TNFα might greatly favor the receptors. Additionally, it has been shown that the receptors for TNF generally have significantly higher affinities for the cytokine than do many antibodies (Lang et al ., 2016; Song et al ., 2008). …”

Section: Discussionmentioning

confidence: 99%

“…Additionally, it has been shown that the receptors for TNF generally have significantly higher affinities for the cytokine than do many antibodies (Lang et al, 2016;Song et al, 2008).…”

Section: Ability Of Tumor Necrosis Factor-α and Interleukin-1β To Smentioning

confidence: 99%

Butyltin compounds alter secretion of interleukin 6 from human immune cells

Brown

Wilburn

Martin

et al. 2017

J of Applied Toxicology

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Butyltins (BTs), tributyltin (TBT) and dibutyltin (DBT) are organotin compounds that have been used in a variety of industrial applications; as a result, these compounds have been found in human blood. Interleukin (IL)-6 is a proinflammatory mediator that is produced by T lymphocytes and monocytes. It is responsible for immune response regulation as well as tissue repair and cellular growth. Both BTs decrease the ability of human natural killer cells to destroy tumor cells and alter the secretion of proinflammatory cytokines tumor necrosis factor alpha, interferon gamma and IL-1 beta (β) from human lymphocytes ex vivo. Here, we show that BTs alter the secretion of IL-6 from increasingly reconstituted preparations of human immune cells. IL-6 secretion was examined after 24 hour, 48 hour or 6 day exposures to TBT and DBT in highly enriched human natural killer cells, monocyte-depleted peripheral blood mononuclear cells (PBMCs), PBMCs, granulocytes and a preparation combining both PBMCs and granulocytes (PBMCs + granulocytes). The results indicated that both BTs altered IL-6 secretion from all cell preparations. Significant decreases of IL-6 secretion were seen at the highest concentration of TBT (200 nm) and DBT (5-2.5 μm) while the lower concentrations of DBT (0.05 and 0.1 μm) caused elevation of IL-6 secretion. The data indicate that BT-induced alterations of IL-6 secretion from immune cells may be a significant consequence of BT exposures that may potentially affect immune competence.

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“…[9][10][11][12][13][14][15] Accordingly, various scFvGpL and GpL-TNFSF ligand fusion proteins proved to be highly suitable for cellular binding studies and tracer applications. We therefore evaluated whether GpL-tagging is also applicable and useful for conventional antibodies.…”

Section: Resultsmentioning

confidence: 99%

Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins

Kums

Nelke

Rüth

et al. 2017

mAbs

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Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometrybased analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcgRs). In accordance with this, the GpL (LC-CT) -18D1-IgG1 antibody fusion protein showed basically the same FcgR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTbR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.Abbreviations: GpL, Gaussia princeps luciferase; FcgR, Fc-gamma receptor; Fn14, fibroblast growth factor-inducible 14; TWEAK, tumor necrosis factor (TNF)-related weak inducer of apoptosis

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Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Cited by 52 publications

References 69 publications

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

Butyltin compounds alter secretion of interleukin 6 from human immune cells

Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins

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