Therefore, two mouse-specific scDuokines (trans-acting msc4-1BBL-mscCD40L and cis-acting msc4-1BBL-mscCD27L) were generated

Therefore, two mouse-specific scDuokines (trans-acting msc4-1BBL-mscCD40L and cis-acting msc4-1BBL-mscCD27L) were generated. generated that act on the same or adjacent T cells, while combining CD40L with 4-1BBL, OX40L and CD27L resulted in trans-acting Duokines acting simultaneously on APCs and T cells. stability of the novel Duokine and scDuokine protein formats was assessed by incubation in human serum at 37C. Most of the Duokines retained 30% or more of their binding activity after 7?days, with the exception of three Duokines, all comprising CD27L, with remaining 10% activity after 7?days (Fig. S3a). In contrast, the plasma stability of scDuokines was more consistent with on average 24C58% intact protein remaining after 7?days (Fig. S3b). This finding indicated a stabilizing effect for some of the TNFSF members after conversion into a single-chain derivative. Bioactivity of Duokines and single-chain Duokines (using the orientation with favorable integrity, stability and receptor binding, thereby reducing the total number of tested proteins to 6 Duokines and 6 scDuokines) was investigated using HT1080 cells stably transfected with CD40, CD27, 4-1BB or OX40 as reporter cell lines. Upon ligand binding, activated TNFRSF receptors induced NF-kB signaling, which resulted in measurable IL-8 release into the supernatant (Fig. S4).21 In their soluble homotrimeric form, neither CD27L, 4-1BBL nor OX40L induced IL-8 release, but both CD40L and scCD40L as well as the other single-chain variants scCD27L, sc4-1BBL and scOX40L resulted in receptor activation. While the single-chain ligands predominantly required higher protein concentrations, the conversion of the ligands in both the Duokine and scDuokine format clearly enhanced receptor activation properties (Fig. S4). IL-8 release and therefore receptor activation was stronger for the single-chain Duokines (Fig. S4b), an effect especially prominent in case of targeting CD27 and 4-1BB, which were only weakly activated by Duokines. Bioactivity, as detected by induction of IL-8 release, was confirmed for all tested Duokines and scDuokines; sc4-1BBL-scCD40L induced strongest activation of both, CD40 and 4-1BB. Immuno-stimulatory activity of scduokines Because the single-chain Duokines appeared to be more stable and more bioactive, the immuno-stimulatory activity was analyzed for three trans-acting (scCD40L-scCD27L, sc4-1BBL-scCD40L, scOX40L-scCD40L) and two cis-acting (sc4-1BBL-scCD27L, scOX40L-scCD27L) scDuokines using freshly isolated PBMC. Comp First, expression of the receptors CD40, CD27, 4-1BB and OX40 was measured on the various target cell types present in PBMC and the binding of scDuokines to these cell populations was identified. Regardless of pre-stimulation, CD40 and CD27 were constitutively expressed on all B cells and all T cells (CD4+ and CD8+), respectively. Moreover, about 30% B cells constitutively expressed CD27, too. In contrast, 4-1BB and OX40 were upregulated on both CD4+ and CD8+ T cells only upon CD3-mediated stimulation. Here, 4-1BB was predominantly upregulated on CD8+ T cells, whereas OX40 was stronger induced on CD4+ T cells (Figure 2A). In accordance with the observed receptor expression patterns, the three trans-acting scDuokines (scCD40L-scCD27L, sc4-1BBL-scCD40L and scOX40L-scCD40L) bound almost exclusively to B cells (Figure 2B). The trans-acting scCD40L-scCD27L targeting constitutively expressed receptors also bound to a minor fraction of T cells. In contrast, the cis-acting scDuokines Pungiolide A were detected solely on T Pungiolide A cells, with an increase in binding of scOX40L-scCD27L upon T cell activation, in accordance with the observed upregulation of OX40 under these conditions (Figure 2B). Generally, trans-acting scDuokines targeted B cells, while cis-acting scDuokines targeted activated CD8+ and CD4+ T cells. Open in a separate window Figure 2. Selected scDuokines bind to human immune cells. (a) Subset populations of human bulk PBMCs were analyzed for expression of TNFRSF receptors with or without antigen-unspecific stimulation via an anti-human Pungiolide A CD3 antibody (UCHT-1). (b) Binding of five different trans- and cis-acting scDuokines (10?nM) to the immune cell populations was analyzed by flow cytometry. Mean SD, n?= 3 different PBMC donors. All trans-acting scDuokines were able to activate B cells as determined by upregulation of the activation marker CD69 and proliferation induction. ScCD40L-scCD27L, sc4-1BBL-scCD40L and scOX40L-scCD40L increased the proliferation rate of CD20+ B cells about 5-fold above the level of mock-treated cells, while no effects were observed for cis-acting scDuokines (Figure 3C). Strongest proliferation with 84% proliferating B cells was observed for sc4-1BBL-scCD40L. Likewise, upregulation of CD69 was promoted only by trans-acting but not cis-acting scDuokines (Figure 3C), in this case, Pungiolide A independent of the presence of an immobilized anti-IgM antibody, which is a known polyclonal activator of B cells (Figure 3C). Open in a separate window Figure 3. Selected scDuokines activate unstimulated.