Strategic internal covalent cross-linking of TNF produces a stable TNF trimer with improved TNFR2 signaling
© Ban et al. 2015
Received: 15 May 2015
Accepted: 17 July 2015
Published: 12 August 2015
Soluble TNF superfamily (TNFSF) ligands are less stable and less active than their transmembrane (tm) analogues. This is a problem for the therapeutic use of recombinant TNFSF ligands in diverse diseases including cancer and autoimmunity. Creating TNFSF ligand analogues with improved targeting of their respective receptors is important for research and therapeutic purposes.
Covalent internal cross-linking of TNF monomers by double mutations, S95C/G148C, results in stable trimers with improved TNFR2 function. The resulting mutein induced the selective death of autoreactive CD8 T cells in type-1 diabetic patients and demonstrates targeted proliferation and expansion of human CD4 Tregs.
Stable TNF trimers, created by internal covalent cross-linking, show improved signaling. The high structural homology within the TNF superfamily provides an opportunity to extend internal cross-linking to other TNF superfamily proteins to produce active trimers with improved stability and receptor signaling, and with potential applications for cancer, autoimmunity, infections, and transplantation.
KeywordsTNF Trimer Cell signaling TNFR2 Transmembrane TNF Tumor necrosis factor Tumor necrosis factor receptor
The tumor necrosis factor (TNF) superfamily, composed of both ligands and receptors, is one of the most successfully and frequently targeted protein families with clinical relevance to diverse diseases as cancer, autoimmunity, infections, graft rejection, inflammation and osteoporosis [1, 2]. Depending on the clinical situation, there is a need to both agonize and antagonize TNF superfamily (TNFSF) pathways. Most TNF superfamily (TNFSF) ligands are type II transmembrane proteins that act through more than one receptor, and like TNF can be cleaved to produce a soluble ligand that is less effective at signaling. All TNFSF ligands share the TNF homology domain and form non-covalent homotrimers . Ligand trimerization is essential for signaling by TNFSF members. However, the trimers tend to dissociate at low concentrations such as in serum resulting in their degradation. Creating stable trimers of TNF and other TNFSF ligands is thus an essential step in generating functionally active receptor specific muteins [4, 5]. Various methods of secondary cross-linking of TNF ligands are also desirable but have faced challenges in increasing stability, half-life and improved signaling [6, 7]. Many TNF superfamily protein design approaches have been based on the construction of fusion proteins .
TNF is a pleiotropic cytokine with diverse functions in cell proliferation, inflammation, apoptosis and morphogenesis . TNF is expressed as a tight trimeric transmembrane protein (tmTNF) that can be cleaved to produce soluble TNF (sTNF) [8, 9]. tmTNF and sTNF exert their functions via two receptors, TNFR1 and TNFR2. TNFR1 is ubiquitously expressed in the lymphoid system and in nearly all cells of the body, whereas TNFR2 has more limited cellular expression. sTNF binds with high affinity to both TNFR1 and TNFR2 but signals almost exclusively through TNFR1. Only tmTNF can effectively activate TNFR2 . The picture that emerges from the literature indicates that the membrane serves at least two functions: 1) it anchors TNF, and increases its stability and bioavailability; and 2) it provides a platform for the arrangement of multiple TNF trimers for simultaneous signaling. The therapeutic use of recombinant sTNF has been hampered by its systemic toxicity and has been limited to localized limb and liver perfusions in certain cancers [11, 12]. TNFR2 agonism may provide a more targeted immunotherapy with reduced toxicity because of the more limited cellular expression of TNFR2. TNFR2 agonists and TNF-inducers have recently emerged as potentially new treatment strategies for specific and select autoimmune diseases such as multiple sclerosis and type-1 diabetes. TNF agonism, especially through the TNFR2 receptor in select autoimmune disease eliminates autoreactive T cells and induces beneficial T-regulatory cells [13–15].
Additional file 1: Methods description.
Building on our earlier research [13, 15], we set out to create sTNF muteins with TNFR2 agonism. As a first step, we created stable trimeric sTNF ligands by introducing an intermolecular Cys-Cys covalent bond. To our surprise, the S95C/G148C (TNF07) double mutant, in addition to being a stable trimer has also shown significantly improved TNFR2 signaling even without the addition of TNFR2 selective mutations. The double mutations in TNF07 (Fig. 1) were chosen after careful consideration of several factors. Ser95 sits on a structurally conserved beta strand and the isosteric Ser to Cys mutation is one of the most chemically conservative choices. Gly148 is found at the edge of loop 145–147 that participates in receptor binding but Gly148 itself is not involved in receptor interactions. A comparison of known sTNF structures shows that the position of residue 148 changes little between the free and receptor bound forms, maintaining at all times the ideal distance with residue 95 for S-S double bond formation. In addition, residues 95 and 148 are hidden from the surface (Fig. 1a) and are thus unlikely to induce immunogenicity or cause toxicity, unlike bulky external additions or chemical cross-linkers [16, 17].
tmTNF is the preferred ligand for TNFR2 signaling. In contrast, sTNF poorly signals through TNFR2. Past work has shown murine and human Treg cells expand with TNFR2 agonism and the TNFR2 receptor is the controlling receptor for Treg expansion [15, 18, 19]. We studied the effects of the new TNF07 mutein in Treg expansion assays.
To further demonstrate the improved TNFR2 activity of TNF07, a competitive dose response experiment was set up with escalating doses of a TNFR2 antagonistic antibody. The data clearly shows that addition of a TNFR2 antagonistic antibody in a dose response fashion blocked TNFR2 agonism and Treg expansion (Fig. 3c).
TNFR2 agonism is an improved proliferative signal for overall CD8 T cell expansion over TNFR1 or sTNF signaling. TNFR2 agonism also promotes selective autoreactive CD8 T cell directed death in type 1 diabetic cells . We set out in CD8 proliferative (WST-1) and cell death (LDH release) assays to functionally test if TNF07 preferentially target TNFR2. In the WST-1 proliferation assay both control and diabetic CD8 T cells expanded slightly with media supplemented with IL2 overnight although the control proliferation was slightly greater (24+/−2.1 versus 15 +/− 1.7; p = 0.03) (Fig. 4a). The addition of sTNF equally induced greater proliferation of control and diabetic CD8 T cells over media (46+/−5.4 versus 41+/−4.1, p = 0.56) (Fig. 4a). In marked contrast, the application of TNF07, a punitive TNFR2 specific agonist, to control and type 1 diabetic cells caused marked CD8 expansion; again the control proliferation was slightly greater than the type 1 diabetic proliferation (79 +/−3.8 versus 59+/−4.6, p = 0.03) (Fig. 4a). This proliferation data suggested functionally TNF07 was acting as a TNFR2 agonist for CD8 proliferation.
To further test whether TNF07 was acting as a functional TNFR2 agonist, we sought evidence that the less robust proliferation of CD8 T cells with TNF or TNF07 in Fig. 4a was due to TNF07 induced autoreactive T cell death in the diabetic through TNFR2 agonism [13, 15]. It was suspected that TNF or even better the TNF07 mutein was killing a minor subpopulation of autoreactive CD8 T cells in the culture of the type 1 diabetic cells. To prove this, isolated CD8 T cells were studied in the LDH cell death assay. In normal donors, CD8 T cells showed background cell death with either sTNF or TNF07 (7.3+/−1.5, 8.3+/−0.9, respectively) (Fig. 4b). In contrast, selective death of autoreactive CD8 T cells of type 1 diabetic T cells could be observed with sTNF and even more pronounced with TNF07 (Fig. 4b). This cell death assay again points to TNF07 showing preferential TNFR2 signaling.
Here we show that the covalent cross-linking of sTNF at a strategic location between TNF monomers can generate a stable trimer with improved functional agonistic consequences on human CD4 or CD8 T cells. The results of the human functional T cell assays indicate that TNF07 has improved TNFR2 agonism, more potent than sTNF. Internal cross-linking at a different location did not result in improved TNFR2 activity. TNF07 not only induced the selective death of autoreactive CD8 T cells in type-1 diabetic patients, it also demonstrated impressive proliferation of normal CD8 T cells. TNF07 was also capable of expanding CD4-Treg cells, again a function of TNFR2 agonism. Various defects in TNF signaling pathways occur in both human and mouse models of several autoimmune disorders. sTNF, TNF inducers or TNFR2 agonism have shown promise for treating these subtypes of autoimmune diseases . Due to the more limited expression of TNFR2, TNFR2 agonism provides a potentially more targeted therapy with fewer side effects. Another TNF construct referred to as TNFR2-specific sc-TNF variant has been shown to rescue human neurons from cell death highlighting the potential application of TNF analogues, especially with preferential TNFR2 agonism in neurodegenerative diseases . TNFR2 agonism is also known to facilitate diverse forms of end organ repair . TNF07 was not designed to have TNFR2 selectivity. Future efforts will focus on removing TNFR1 activity to reduce systemic toxicity for potential therapeutic applications.
Due to the high structural homology, the concept of disulfide cross-linking is broadly applicable to all TNFSF ligands. It provides an alternative to other trimer stabilizing methods as a more naturally appearing, and less immunogenic structure upon human exposure. It is an expandable system that can be combined with receptor specific mutations to selectively target various TNFSF receptors, and also with secondary cross-linking methods of either the TNFSF ligands or their receptors [6, 7, 23].
soluble Tumor necrosis factor
T regulatory cells
type 1 diabetic
The authors thank The Iacocca Foundation for research support.
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