Generic design principles for antibody-based tumour necrosis factor (TNF) receptor 2 (TNFR2) agonists with FcγR-independent agonism.

Background: Selective TNFR2 activation can be used to treat immune pathologies by activating and expanding regulatory T-cells (Tregs) but may also restore anti-tumour immunity by co-stimulating CD8+ T-cells. Oligomerized TNFR2-specific TNF mutants or anti-TNFR2 antibodies can activate TNFR2 but suffer either from poor production and pharmacokinetics or in the case of anti-TNFR2 antibodies typically from the need of FcγR binding to elicit maximal agonistic activity. Methods: To identify the major factor(s) determining FcγR-independent agonism of anti-TNFR2 antibodies, we systematically investigated a comprehensive panel of anti-TNFR2 antibodies and antibody-based constructs differing in the characteristics of their TNFR2 binding domains but also in the number and positioning of the latter. Results: We identified the domain architecture of the constructs as the pivotal factor enabling FcγR-independent, thus intrinsic TNFR2-agonism. Anti-TNFR2 antibody formats with either TNFR2 binding sites on opposing sites of the antibody scaffold or six or more TNFR2 binding sites in similar orientation regularly showed strong FcγR-independent agonism. The affinity of the TNFR2 binding domain and the epitope recognized in TNFR2, however, were found to be of only secondary importance for agonistic activity. Conclusion: Generic design principles enable the generation of highly active bona fide TNFR2 agonists from nearly any TNFR2-specific antibody.

Corrigendum: A TNFR2-specific TNF fusion protein with improved in vivo activity.

[This corrects the article DOI: 10.3389/fimmu.2022.888274.].

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

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.

Tumor Necrosis Factor Receptor 2 (TNFR2): An Emerging Target in Cancer Therapy.

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.

CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism.

Background: A strategy to broaden the applicability of checkpoint inhibitors is the combined use with antibodies targeting the immune stimulatory receptors CD40 and 41BB. However, the use of anti-CD40 and anti-41BB antibodies as agonists is problematic in two ways. First, anti-CD40 and anti-41BB antibodies need plasma membrane-associated presentation by FcγR binding to exert robust agonism but this obviously limits their immune stimulatory efficacy by triggering ADCC, CDC or anti-inflammatory FcγRIIb activities. Second, off tumor activation of CD40 and 41BB may cause dose limiting systemic inflammation. Methods: To overcome the FcγR-dependency of anti-41BB and anti-CD40 antibodies, we genetically fused such antibodies with a PDL1-specific blocking scFv as anchoring domain to enable FcγR-independent plasma membrane-associated presentation of anti-CD40- and anti-41BB antibodies. By help of GpL-tagged variants of the resulting bispecific antibodies, binding to their molecular targets was evaluated by help of cellular binding studies. Membrane PDL1-restricted engagement of CD40 and 41BB but also inhibition of PDL1-induced PD1 activation were evaluated in coculture assays with PDL1-expressing tumor cell lines and 41BB, CD40 and PD1 responsible cell lines or T-cells. Results: The binding properties of the bispecific antibody fusion proteins remained largely unchanged compared to their parental molecules. Upon anchoring to membrane PDL1, the bispecific antibody fusion proteins activated CD40/41BB signaling as efficient as the parental anti-CD40/anti-41BB antibodies when bound to FcγRs or cells expressing membrane-bound CD40L/41BBL. PD1 inhibition remained intact and the anti-41BB fusion protein thus showed PDL1-restricted costimulation of T-cells activated in vitro with anti-CD3 or a BiTe. Conclusions: Targeting of anti-CD40 and anti-41BB fusion proteins to membrane PDL1 with a blocking PDL1 scFv links PD1-PDL1 checkpoint blockade intrinsically with engagement of CD40 or 41BB.

Junctional adhesion molecule C expression specifies a CD138low/neg multiple myeloma cell population in mice and humans.

Deregulation such as overexpression of adhesion molecules influences cancer progression and survival. Metastasis of malignant cells from their primary tumor site to distant organs is the most common reason for cancer-related deaths. Junctional adhesion molecule-C (JAM-C), a member of the immunoglobulin-like JAM family, can homodimerize and aid cancer cell migration and metastasis. Here we show that this molecule is dynamically expressed on multiple myeloma (MM) cells in the bone marrow and co-localizes with blood vessels within the bone marrow of patients and mice. In addition, upregulation of JAM-C inversely correlates with the downregulation of the canonical plasma cell marker CD138 (syndecan-1), whose surface expression has recently been found to dynamically regulate a switch between MM growth in situ and MM dissemination. Moreover, targeting JAM-C in a syngeneic in vivo MM model ameliorates MM progression and improves outcome. Overall, our data demonstrate that JAM-C might serve not only as an additional novel diagnostic biomarker but also as a therapeutic target in MM disease.

Membrane lymphotoxin-α2ß is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist.

In the early 1990s, it has been described that LTα and LTß form LTα2ß and LTαß2 heterotrimers, which bind to TNFR1 and LTßR, respectively. Afterwards, the LTαß2-LTßR system has been intensively studied while the LTα2ß-TNFR1 interaction has been ignored to date, presumably due to the fact that at the time of identification of the LTα2ß-TNFR1 interaction one knew already two ligands for TNFR1, namely TNF and LTα. Here, we show that LTα2ß interacts not only with TNFR1 but also with TNFR2. We furthermore demonstrate that membrane-bound LTα2ß (memLTα2ß), despite its asymmetric structure, stimulates TNFR1 and TNFR2 signaling. Not surprising in view of its ability to interact with TNFR2, LTα2ß is inhibited by Etanercept, which is approved for the treatment of rheumatoid arthritis and also inhibits TNF and LTα.

Quantitative single-molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα-induced NF-ĸB signaling.

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.

Analysis of FcγR-Dependent Agonism of Antibodies Specific for Receptors of the Tumor Necrosis Factor (TNF) Receptor Superfamily (TNFRSF).

In vivo research of the last decade revealed that the anchoring of antitumor necrosis factor (TNF) receptor superfamily (TNFRSF) receptor antibodies to cell-expressed Fcγ receptors (FcγR) can be of decisive relevance for their receptor-stimulatory activity. Indeed, FcγR anchoring may even result in the conversion of antagonistic to agonistic anti-TNFR antibody activity. The knowledge on this issue is obviously not only relevant to understand the in vivo effects of anti-TNFR antibodies but also of overwhelming importance for the rational clinical development of antibodies and antibody derivatives. Based on the fact that with exception of the decoy TNFRSF receptors (TNFRs) all TNFRs are able to trigger proinflammatory NFκB signaling, resulting in the production of chemokines and cytokines, we established an easy and broadly applicable coculture assay for the evaluation of the FcγR-dependency of the agonism of anti-TNFR antibodies. In this assay, TNFR responder cells, which produce high amounts of IL8 in response to TNFR stimulation, were pairwise incubated with empty vector- and FcγR-transfected HEK293 cells, which produce only very low amounts of IL8. This cocultures were then comparatively analyzed with respect to anti-TNFR antibody-induced IL8 production as a readout for TNFR activation to uncover proagonistic effects of FcγR binding.

Analysis of Ligand-Receptor Interactions Using Bioluminescent TNF Superfamily (TNFSF) Ligand Fusion Proteins.

Quantitative analysis of the binding of tumor necrosis factor (TNF) superfamily ligands (TNFLs) to TNF receptor superfamily receptors (TNFRs) is of crucial relevance for the understanding of the mechanisms of TNFR activation. Ligand binding studies are also a basic method required for the development and characterization of agonists and antagonists of TNFRs. TNFL-induced formation of fully active TNFR signaling complexes is a complex process. It involves not only reorganization of monomeric and inactive pre-assembled TNFR complexes into trimeric liganded TNFR complexes but also the secondary interaction of the latter. Moreover, various factors, e.g., TNFR modification, special membrane domains, or accessory proteins, may affect TNFL-TNFR interactions in a TNFR type-specific manner. Widely used cell-free methods for the analysis of protein-protein interactions are thus of limited value for the analysis of TNFL-TNFR interactions and makes therefore in this case cellular binding studies to the method of choice. We and others observed that the genetic fusion of monomeric protein domains to the N-terminus of soluble TNFLs has typically no effect on activity and TNFR binding. We exploited this to generate bioluminescent TNFL fusion proteins which allow simple, sensitive, and highly reproducible cellular binding studies for the investigation of TNFL-TNFR interactions. Here, we report detailed protocols for the production of TNFL fusion proteins with the luciferase of Gaussia princeps and the use of these fusion proteins in various types of cellular binding studies.

CD40- and CD95-specific antibody single chain-Baff fusion proteins display BaffR-, TACI- and BCMA-restricted agonism.

Antibodies that target a clinically relevant group of receptors within the tumor necrosis factor receptor superfamily (TNFRSF), including CD40 and CD95 (Fas/Apo-1), also require binding to Fc gamma receptors (FcγRs) to elicit a strong agonistic activity. This FcγR dependency largely relies on the mere cellular anchoring through the antibody's Fc domain and does not involve the engagement of FcγR signaling. The aim of this study was to elicit agonistic activity from αCD40 and αCD95 antibodies in a myeloma cell anchoring-controlled FcγR-independent manner. For this purpose, various antibody variants (IgG1, IgG1N297A, Fab2) against the TNFRSF members CD40 and CD95 were genetically fused to a single-chain-encoded B-cell activating factor (scBaff) trimer as a C-terminal myeloma-specific anchoring domain substituting for Fc domain-mediated FcγR binding. The antibody-scBaff fusion proteins were evaluated in binding studies and functional assays using tumor cell lines expressing one or more of the three receptors of Baff: BaffR, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA). Cellular binding studies showed that the binding properties of the different domains within the fusion proteins remained fully intact in the antibody-scBaff fusion proteins. In co-culture assays of CD40- and CD95-responsive cells with BaffR, BCMA or TACI expressing anchoring cells, the antibody fusion proteins displayed strong agonism while only minor receptor stimulation was observed in co-cultures with cells without expression of Baff-interacting receptors. Thus, our CD40 and CD95 antibody fusion proteins display myeloma cell-dependent activity and promise reduced systemic side effects compared to conventional CD40 and CD95 agonists.

Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells.

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity.

Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target.

INTRODUCTION: Tumor necrosis factor (TNF) receptor 2 (TNFR2) is one of two receptors of the cytokines, TNF and lymphotoxin-α. TNFR1 is a strong inducer of proinflammatory activities. TNFR2 has proinflammatory effects too, but it also elicits strong anti-inflammatory activities and has protective effects on oligodendrocytes, cardiomyocytes, and keratinocytes. The protective and anti-inflammatory effects of TNFR2 may explain why TNF inhibitors failed to be effective in diseases such as heart failure or multiple sclerosis, where TNF has been strongly implicated as a driving force. Stimulatory and inhibitory TNFR2 targeting hence attracts considerable interest for the treatment of autoimmune diseases and cancer. Areas covered: Based on a brief description of the pathophysiological importance of the TNF-TNFR1/2 system, we discuss the potential applications of TNFR2 targeting therapies. We also debate TNFR2 activation as a way forward in the search for TNFR2-specific agents. Expert opinion: The use of TNFR2 to target regulatory T-cells is attractive, but this approach is just one amongst many suitable targets. With respect to its preference for Treg stimulation and protection of non-immune cells, TNFR2 is more unique and thus offers opportunities for translational success.