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

Ligands of the tumor necrosis factor superfamily (TNFSF) interact with members of the TNF receptor superfamily (TNFRSF). TNFSF ligand-TNFRSF receptor interactions have been intensively evaluated by many groups. The affinities of TNFSF ligand-TNFRSF receptor interactions are highly dependent on the oligomerization state of the receptor, and cellular factors (e.g. actin cytoskeleton and lipid rafts) influence the assembly of ligand-receptor complexes, too. Binding studies on TNFSF ligand-TNFRSF receptor interactions were typically performed using cell-free assays with recombinant fusion proteins that contain varying numbers of TNFRSF ectodomains. It is therefore not surprising that affinities determined for an individual TNFSF ligand-TNFRSF interaction differ sometimes by several orders of magnitude and often do not reflect the ligand activity observed in cellular assays. To overcome the intrinsic limitations of cell-free binding studies and usage of recombinant receptor domains, we performed comprehensive binding studies with Gaussia princeps luciferase TNFSF ligand fusion proteins for cell-bound TNFRSF members on intact cells at 37 °C. The affinities of the TNFSF ligand G. princeps luciferase-fusion proteins ranged between 0.01 and 19 nm and offer the currently most comprehensive and best suited panel of affinities for in silico studies of ligand-receptor systems of the TNF family.

Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand.

For many ligands of the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. However, for the immunostimulatory ligands CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand (GITRL) detailed information regarding these requirements is lacking. Here, we comprehensively evaluated the effect of trimer stability and oligomerization on receptor activation by these ligands. Treatment with soluble Flag-tagged CD27L, 41BBL, and GITRL minimally activated receptor signaling, while Flag-CD40L was highly active. Oligomerization with anti-Flag Abs further enhanced the specific activity of Flag-CD40L 10-fold and of Flag-41BBL more than 200-fold, but it failed to activate Flag-CD27L and Flag-GITRL. We next investigated the relevance of trimer stability by introducing the tenascin-C (TNC) trimerization domain, yielding stabilized Flag-TNC-ligand trimers. Oligomerization with anti-Flag Ab potently activated signaling by Flag-TNC-CD27L and Flag-TNC-GITRL and, albeit to a lesser extent, Flag-TNC-CD40L and Flag-TNC-41BBL. Forced hexamerization, by introducing an Ig Fc domain, revealed that hexameric derivatives of Flag-TNC-41BBL, Flag-CD40L, and Flag-TNC-GITRL all activate receptor signaling with high efficiency, whereas hexameric Flag-CD27L variant left inactive. Finally, we attempted to selectively activate receptor signaling on targeted cells, by using Ab fragment (single-chain fragment variable region, scFv)-ligand fusion proteins, an approach previously applied to other TNF ligands. Target cell surface Ag-selective activation was achieved for scFv-41BBL, scFv-CD40L, and scFv-GITRL, although the latter two displayed already significant activity toward Ag-negative cells. In conclusion, our data establish that trimeric CD40L is active, 41BBL requires hexamerization, GITRL requires trimer stabilization, and CD27L requires trimer stabilization and oligomerization. Furthermore, surface immobilization might be exploited to gain locally enhanced ligand activity.

Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization.

OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.

Production, purification, and characterization of scFv TNF ligand fusion proteins.

Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins.