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

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 NF-ĸB (nuclear factor kappa-light-chain-enhancer of activated B-cells) 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 pre-dimerization 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 into trimers but also into higher-order oligomers. A functional mutation in the pre-ligand assembly domain (PLAD) 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 wildtype TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-ĸB signaling

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.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

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

Membrane lymphotoxin-α2_2β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist

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

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

Junctional Adhesion Molecule-C expression specifies a {CD}138low/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 (JAM)-C, a member of the Ig-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 marrow and co-localizes with blood vessels within the bone marrow of mice and humans. Additionally, JAM-C upregulation 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