Results From a First-in-Human Study of BNZ-1, a Selective Multicytokine Inhibitor Targeting Members of the Common Gamma (γc) Family of Cytokines.

Pathologic roles of interleukin (IL)-2, IL-9, and IL-15, have been implicated in multiple T-cell malignancies and autoimmune diseases. BNZ-1 is a selective and simultaneous inhibitor of IL-2, IL-9, and IL-15, which targets the common gamma chain signaling receptor subunit. In this first-in-human study, 18 healthy adults (n = 3/cohort) received an intravenous dose of 0.2, 0.4, 0.8, 1.6, 3.2, or 6.4 mg/kg infused over ≤5 minutes on day 1 and were followed for 30 days for safety and pharmacokinetic/pharmacodynamic sample collection. No dose-limiting toxicities, infusion reactions, or serious or severe treatment-emergent adverse events were observed. Headache was the only treatment-emergent adverse event in >1 subject (n = 3). Peak and total BNZ-1 exposure was generally dose proportional, with a terminal elimination half-life of ∼5 days. Pharmacodynamic effects of BNZ-1 on regulatory T cells (Tregs, IL-2), natural killer (NK) cells (IL-15) and CD8 central memory T cells (Tcm, IL-15) were measured by flow cytometry and used to demonstrate target engagement. For Tregs, 0.2 mg/kg was an inactive dose, while a maximum ∼50% to 60% decrease from baseline was observed on day 4 after doses of 0.4 to 1.6 mg/kg, and higher doses produced an 80% to 93% decrease from baseline on day 15. Similar pharmacodynamic trends were observed for natural killer cells and CD8 Tcm, although decreases in CD8 Tcm were more prolonged. These subpopulations returned to/toward baseline by day 31. T cells (total, CD4, and CD8), B cells, and monocytes were unchanged throughout. These preliminary results suggest that BNZ-1 safely and selectively inhibits IL-2 and IL-15, which results in robust, reversible immunomodulation.

Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical spastic paraparesis.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive inflammatory myelopathy occurring in a subset of HTLV-1-infected individuals. Despite advances in understanding its immunopathogenesis, an effective treatment remains to be found. IL-2 and IL-15, members of the gamma chain (γc) family of cytokines, are prominently deregulated in HAM/TSP and underlie many of the characteristic immune abnormalities, such as spontaneous lymphocyte proliferation (SP), increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific cytotoxic CD8(+) T lymphocytes (CTLs). In this study, we describe a novel immunomodulatory strategy consisting of selective blockade of certain γc family cytokines, including IL-2 and IL-15, with a γc antagonistic peptide. In vitro, a PEGylated form of the peptide, named BNZ132-1-40, reduced multiple immune activation markers such as SP, STAT5 phosphorylation, spontaneous degranulation of CD8(+) T cells, and the frequency of transactivator protein (Tax)-specific CD8(+) CTLs, thought to be major players in the immunopathogenesis of the disease. This strategy is thus a promising therapeutic approach to HAM/TSP with the potential of being more effective than single monoclonal antibodies targeting either IL-2 or IL-15 receptors and safer than inhibitors of downstream signaling molecules such as JAK1 inhibitors. Finally, selective cytokine blockade with antagonistic peptides might be applicable to multiple other conditions in which cytokines are pathogenic.

Targeting the binding interface on a shared receptor subunit of a cytokine family enables the inhibition of multiple member cytokines with selectable target spectrum.

The common γ molecule (γc) is a shared signaling receptor subunit used by six γc-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple γc-cytokines are pathogenically involved in a single disease, thus making the shared γc-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the γc and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the γc and cytokines, we successfully designed peptides that not only inhibit multiple γc-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three γc-cytokines without affecting the other three or non-γc-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of γc-cytokines the γc-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of γc-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases.

ATP-dependent chromatin remodeling by Cockayne syndrome protein B and NAP1-like histone chaperones is required for efficient transcription-coupled DNA repair.

The Cockayne syndrome complementation group B (CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome--a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP-dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP-dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB-binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP-dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome.

Reciprocally regulated chromatin association of Cockayne syndrome protein B and p53 protein.

The Cockayne syndrome complementation group B (CSB) protein is an ATP-dependent chromatin remodeler with an essential function in transcription-coupled DNA repair, and mutations in the CSB gene are associated with Cockayne syndrome. The p53 tumor suppressor has been known to interact with CSB, and both proteins have been implicated in overlapping biological processes, such as DNA repair and aging. The significance of the interaction between CSB and p53 has remained unclear, however. Here, we show that the chromatin association of CSB and p53 is inversely related. Using in vitro binding and chromatin immunoprecipitation approaches, we demonstrate that CSB facilitates the sequence-independent association of p53 with chromatin when p53 concentrations are low and that this is achieved by the interaction of CSB with the C-terminal region of p53. Remarkably, p53 prevents CSB from binding to nucleosomes when p53 concentrations are elevated. Examining the enzymatic properties of CSB revealed that p53 excludes CSB from nucleosomes by occluding a nucleosome interaction surface on CSB. Together, our results suggest that the reciprocal regulation of chromatin access by CSB and p53 could be part of a mechanism by which these two proteins coordinate their activities to regulate DNA repair, cell survival, and aging.