TY  - JOUR
A1  - Weinelt, Nadine
A1  - Karathanasis, Christos
A1  - Smith, Sonja
A1  - Medler, Juliane
A1  - Malkusch, Sebastian
A1  - Fulda, Simone
A1  - Wajant, Harald
A1  - Heilemann, Mike
A1  - van Wijk, Sjoerd J. L.
T1  - Quantitative single‐molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα‐induced NF‐ĸB signaling
JF  - Journal of Leukocyte Biology
N2  - TNFR1 is a crucial regulator of NF‐ĸB‐mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα‐ and TNFR1‐controlled NF‐ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2‐mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single‐molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1‐mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand‐independent preligand assembly domain (PLAD)‐mediated TNFR1 dimerization as well as TNFα‐induced TNFR1 oligomerization. In addition, zafirlukast‐mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF‐ĸB signaling in reconstituted TNFR1‐mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD‐mediated, ligand‐independent TNFR1 dimerization for NF‐ĸB activation, highlight the PLAD as central regulator of TNFα‐induced TNFR1 oligomerization, and demonstrate that TNFR1‐mEos2 MEFs can be used to investigate TNFR1‐antagonizing compounds employing single‐molecule quantification and functional NF‐ĸB assays at physiologic conditions.
KW  - Single‐Molecule Localization Microscopy (SMLM)
KW  - Pre‐Ligand Assembly Domain (PLAD)
KW  - Cysteine‐Rich Domain (CRD)
KW  - CysLTR1
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215960
VL  - 109
IS  - 2
SP  - 363
EP  - 371
ER  - 
TY  - JOUR
A1  - Vargas, Juan Gamboa
A1  - Wagner, Jennifer
A1  - Shaikh, Haroon
A1  - Lang, Isabell
A1  - Medler, Juliane
A1  - Anany, Mohamed
A1  - Steinfatt, Tim
A1  - Mosca, Josefina Peña
A1  - Haack, Stephanie
A1  - Dahlhoff, Julia
A1  - Büttner-Herold, Maike
A1  - Graf, Carolin
A1  - Viera, Estibaliz Arellano
A1  - Einsele, Hermann
A1  - Wajant, Harald
A1  - Beilhack, Andreas
T1  - A TNFR2-Specific TNF fusion protein with improved in vivo activity
JF  - Frontiers in Immunology
N2  - 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.

Methods
Single-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.


Results
STAR2 (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.


Conclusions
NewSTAR2 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.
KW  - agonist
KW  - GvHD
KW  - regulatory T cells
KW  - serum retention
KW  - TNF
KW  - TNFR2
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-277436
SN  - 1664-3224
VL  - 13
ER  - 
TY  - JOUR
A1  - Medler, Juliane
A1  - Kucka, Kirstin
A1  - Wajant, Harald
T1  - Tumor necrosis factor receptor 2 (TNFR2): an emerging target in cancer therapy
JF  - Cancers
N2  - Despite the great success of TNF blockers in the treatment of autoimmune diseases and the identification of TNF as a factor that influences the development of tumors in many ways, the role of TNFR2 in tumor biology and its potential suitability as a therapeutic target in cancer therapy have long been underestimated. This has been fundamentally changed with the identification of TNFR2 as a regulatory T-cell (Treg)-stimulating factor and the general clinical breakthrough of immunotherapeutic approaches. However, considering TNFR2 as a sole immunosuppressive factor in the tumor microenvironment does not go far enough. TNFR2 can also co-stimulate CD8\(^+\) T-cells, sensitize some immune and tumor cells to the cytotoxic effects of TNFR1 and/or acts as an oncogene. In view of the wide range of cancer-associated TNFR2 activities, it is not surprising that both antagonists and agonists of TNFR2 are considered for tumor therapy and have indeed shown overwhelming anti-tumor activity in preclinical studies. Based on a brief summary of TNFR2 signaling and the immunoregulatory functions of TNFR2, we discuss here the main preclinical findings and insights gained with TNFR2 agonists and antagonists. In particular, we address the question of which TNFR2-associated molecular and cellular mechanisms underlie the observed anti-tumoral activities of TNFR2 agonists and antagonists.
KW  - NFkappaB
KW  - regulatory T-cell (Treg)
KW  - tumor necrosis factor (TNF)
KW  - TNF receptor 2 (TNFR2)
KW  - TNF receptor associated factor 1 and 2 (TRAF1, TRAF2)
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275143
SN  - 2072-6694
VL  - 14
IS  - 11
ER  - 
TY  - JOUR
A1  - Kucka, Kirstin
A1  - Lang, Isabell
A1  - Zhang, Tengyu
A1  - Siegmund, Daniela
A1  - Medler, Juliane
A1  - Wajant, Harald
T1  - Membrane lymphotoxin-α\(_2\)β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist
JF  - Cell Death & Disease
N2  - In the early 1990s, it has been described that LTα and LTβ form LTα\(_2\)β and LTαβ\(_2\) heterotrimers, which bind to TNFR1 and LTβR, respectively. Afterwards, the LTαβ\(_2\)–LTβR system has been intensively studied while the LTα\(_2\)β–TNFR1 interaction has been ignored to date, presumably due to the fact that at the time of identification of the LTα\(_2\)β–TNFR1 interaction one knew already two ligands for TNFR1, namely TNF and LTα. Here, we show that LTα\(_2\)β interacts not only with TNFR1 but also with TNFR2. We furthermore demonstrate that membrane-bound LTα\(_2\)β (memLTα\(_2\)β), despite its asymmetric structure, stimulates TNFR1 and TNFR2 signaling. Not surprising in view of its ability to interact with TNFR2, LTα\(_2\)β is inhibited by Etanercept, which is approved for the treatment of rheumatoid arthritis and also inhibits TNF and LTα.
KW  - cytokines
KW  - signal transduction
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260077
VL  - 12
IS  - 4
ER  -