Activation of TNFR2 with a novel agonist expands T reg cells in vivo and protects allo-HCT recipients from acute GvHD while sparing antilymphoma and antiinfectious properties of transplanted donor T cells.

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 (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.

Multiple myeloma remains a largely incurable disease of clonally expanding malignant plasma cells. The bone marrow microenvironment harbors treatment-resistant myeloma cells, which eventually lead to disease relapse in patients. In the bone marrow, CD4+FoxP3+ regulatory T cells (Tregs) are highly abundant amongst CD4+ T cells providing an immune protective niche for different long-living cell populations, e.g., hematopoietic stem cells. Here, we addressed the functional role of Tregs in multiple myeloma dissemination to bone marrow compartments and disease progression. To investigate the immune regulation of multiple myeloma, we utilized syngeneic immunocompetent murine multiple myeloma models in two different genetic backgrounds. Analyzing the spatial immune architecture of multiple myeloma revealed that the bone marrow Tregs accumulated in the vicinity of malignant plasma cells and displayed an activated phenotype. In vivo Treg depletion prevented multiple myeloma dissemination in both models. Importantly, short-term in vivo depletion of Tregs in mice with established multiple myeloma evoked a potent CD8 T cell- and NK cell-mediated immune response resulting in complete and stable remission. Conclusively, this preclinical in-vivo study suggests that Tregs are an attractive target for the treatment of multiple myeloma.

Acute graft versus host disease (aGVHD) remains a major complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), the only curative treatment for many malignant hematologic diseases. After initial priming in secondary lymphoid organs, alloreactive donor T cells efficiently migrate to the intestinal tract, liver and skin. We observed that alloreactive effector T cells infiltrating and attacking the lamina propria of the small and large intestines closely interact with intestinal myeloid cells of host origin. Here we asked whether these intimate interactions regulate alloreactive effector T cell responses and how they impact intestinal aGvHD. To address these questions, we employed non-invasive bioluminescence imaging, fluorescence and confocal microscopy, clinical and histopathologic scoring, flow cytometry and single cell RNA sequencing in murine models of myeloablative, MHC-mismatched allo-HSCT. In the intestinal lamina propria, we observed that allogeneic T cells closely interacted with CD11b+CD11c+CD103- radio-resistant host type hematopoietic myeloid antigen presenting cells. Selective depletion of intestinal CD11chi or CX3CR1+CD11chi host cells 3 or 8 days after allo-HSCT accelerated alloreactive T cell infiltration, increased T cell mediated inflammatory cytokine production and exacerbated tissue damage resulting in hyperacute lethal aGvHD. These results suggested a strong immunoregulatory effect of these intestinal host-type myeloid cells. Single cell RNA-Seq analysis and flow cytometry (e.g. MHC II, CD11c, F4/80, CD26, CD64, CCR2, CX3CR1), lineage reporter- and defined knockout mice determined these cells as non-classical monocyte derived macrophages as the development and differentiation of these cells did not depend on Flt3, Zbtb46, and CCR2 but rather on CSF-1R and CX3CR1. Adoptive transfer, bone marrow chimeras and parabiosis experiments revealed that these non-classical monocyte derived macrophages differentiated from non-circulating non-classical monocytic precursors. Finally, PD-L1 expression on these intestinal host non-classical monocyte derived macrophages but not on stroma or other hematopoietic cells regulated alloreactive T cell responses in the intestinal tract. Based on these findings we postulate that a specialized and persistent subpopulation of host non-classical monocyte derived macrophages can potently suppress alloreactive T cells in the lamina propria of the intestinal tract. Fostering the differentiation and function of these tissue resident cells may represent an attractive therapeutic strategy to prevent intestinal aGvHD. Disclosures No relevant conflicts of interest to declare.

No abstract