A TNFR2-Specific TNF Fusion Protein With Improved In Vivo Activity

Abstract

Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.MethodsSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.ResultsSTAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.ConclusionsNewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.

A TNF receptor 2 agonist ameliorates neuropathology and improves cognition in an Alzheimer’s disease mouse model

“…Since IgG1(N297A) still interacts with the neonatal Fc receptor, NewStar2 [irrIgG1(N297A)-HC:sc(muTNF80)], however, has enhanced serum retention and, thus, much better in vivo activity than Star2. NewStar2 is produced by transient transfection of Hek293 cells with expression plasmids encoding the light chain and the sc(mu)TNF80 fusion protein of the heavy chain of the irrelevant IgG1(N297A) ( 59 ). NewStar2 was purified by affinity purification on anti-Flag agarose by help of internal Flag tags present in the antibody.…”

“…TNFR2 Stimulation. To selectively engage TNFR2, we used a new TNFR2 agonist, designated as NewStar2 or irrIgG1(N297A)-HC:sc(mu)TNF80 (59), which is based on Star2 [TNC-sc(mu)TNF80], a fusion protein of the trimerization domain of tenascin-C and three peptide-linker-connected (thus single-chain (sc) encoded) murine TNF (muTNF) protomers with mutations conferring specificity for TNFR2 (40). To generate NewStar2, the TNFR2-specific, sc-encoded sc(mu)TNF80 domain of Star2 was fused to the C terminus of the heavy chain of a human IgG1 with a mutation (N297A) avoiding interaction with FcγRs and irrelevant specificity in mice.…”

Basic characterization of antibodies targeting receptors of the tumor necrosis factor receptor superfamily

“…HEK293T, HT1080 (both ATCC, Rockville, USA), HT1080-CD40, HT1080-41BB, HT1080-GITR (all ref ( 39 )., HT1080-OX40 ( 40 ) and HT1080-Bcl2-TNFR2 cells ( 41 ) were cultivated in RPMI 1640 medium (Sigma-Aldrich, Steinheim, Germany) supplemented with 10% fetal calf serum (FCS; GIBCO) under standard conditions (37°C, 5% CO 2 ). To obtain expression plasmids encoding the various antibodies constructs, fusion proteins and ligands used, corresponding synthetic DNA fragments and PCR amplicons have been cloned in the expression vector pCR3 (Invitrogen, Germany).…”

Fibroblastic reticular cells mitigate acute GvHD via MHCII-dependent maintenance of regulatory T cells

“…In vivo BLI was performed using the IVIS Spectrum CCD-imaging system (PerkinElmer) as previously described ( 92 ). Briefly, mice were anesthetized with an i.p.-injected mixture of ketamine (50 μg/g body weight) and xylazine (5 μg/g body weight) in PBS in a total volume of 10 μL/g body weight.…”

“…In vivo bioluminescence imaging was performed using IVIS Spectrum CCD-imaging system (PerkinElmer, Waltham, MA, USA) as previously described (92). Briefly, mice were anesthetized with an i.p.…”