Effective treatment with the selective cytokine inhibitor BNZ-1 reveals the cytokine dependency of T-LGL leukemia.

T-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes that can result in severe neutropenia, anemia, and bone marrow failure. Strong evidence from patients and mouse models demonstrate the critical role of interleukin-15 (IL-15) in T-LGLL pathogenesis. BNZ-1 is a pegylated peptide that selectively inhibits the binding of IL-15 and other γc cytokines to their cellular receptor complex, which has demonstrated efficacy in ex vivo T-LGLL cells and transgenic mice in preclinical studies. We conducted a phase 1/2 trial of BNZ-1 in patients with T-LGLL who had hematocytopenias (anemia or neutropenia) and required therapy. Clinical responses were assessed using hematologic parameters (improvement in hematocytopenias) based on response criteria from the Eastern Cooperative Oncology Group 5998 T-LGLL trial. BNZ-1 demonstrated clinical partial responses in 20% of patients with T-LGLL with minimal toxicity and the maximum tolerated dose was not reached. Furthermore, T-LGL leukemic cells showed significantly increased apoptosis in response to BNZ-1 treatment as early as day 2, including in clinical nonresponders, with changes that remained statistically different from baseline throughout treatment (P < .005). We report first-in-human proof that T-LGL leukemic cells are dependent on IL-15 and that intervention with IL-15 inhibition with BNZ-1 in patients with T-LGLL shows therapeutic effects, which carries important implications for the understanding of the pathogenesis of this disease. This trial was registered at www.clinicaltrials.gov as #NCT03239392.

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.

Identification of a γc Receptor Antagonist That Prevents Reprogramming of Human Tissue-resident Cytotoxic T Cells by IL15 and IL21.

BACKGROUND & AIMS: Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. METHODS: We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8+ T-cell lines, or CD8+ T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. RESULTS: Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. CONCLUSIONS: We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.

IL-2 and IL-15 blockade by BNZ-1, an inhibitor of selective γ-chain cytokines, decreases leukemic T-cell viability.

Interleukin-15 (IL-15) and IL-2 drive T-cell malignancies including T-cell large granular lymphocyte leukemia (T-LGLL) and HTLV-1 driven adult T-cell leukemia (ATL). Both cytokines share common γ-chain receptors and downstream signaling pathways. T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with abnormal JAK/STAT signaling. ATL is an aggressive CD4+ T-cell neoplasm associated with HTLV-1. T-LGLL and ATL share dependence on IL-2 and IL-15 for survival and both diseases lack effective therapies. BNZ-1 is a pegylated peptide designed to specifically bind the γc receptor to selectively block IL-2, IL-15, and IL-9 signaling. We hypothesized that treatment with BNZ-1 would reduce cytokine-mediated proliferation and viability. Our results demonstrated that in vitro treatment of a T-LGLL cell line and ex vivo treatment of T-LGLL patient cells with BNZ-1 inhibited cytokine-mediated viability. Furthermore, BNZ-1 blocked downstream signaling and increased apoptosis. These results were mirrored in an ATL cell line and in ex vivo ATL patient cells. Lastly, BNZ-1 drastically reduced leukemic burden in an IL-15-driven human ATL mouse xenograft model. Thus, BNZ-1 shows great promise as a novel therapy for T-LGLL, ATL, and other IL-2 or IL-15 driven hematopoietic malignancies.

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.

Immunostimulation by induced expression of NKG2D and its MIC ligands in HTLV-1-associated neurologic disease.

The NKG2D receptor costimulates effector/memory CD8 T cells and is normally absent on CD4 T cells but can be induced by T cell antigen receptor complex stimulation and interleukin-15 (IL-15). Among its ligands are the human major histocompatibility complex class I-related MICA and MICB, which have a restricted tissue distribution but are frequently associated with malignancies and some microbial infections. Moreover, aberrant expression of MIC may promote autoimmune disease progression. Human T cell lymphotropic virus type I (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease of the central nervous system that resembles multiple sclerosis. Disease progression involves production of IL-15 and its receptor through transactivation by the viral Tax regulator protein, an activated immune response state, and local cytokine production and T cell fratricide by Tax-specific cytotoxic T lymphocytes (CTL). This study shows that as with CD8 T cells, substantial proportions of HAM/TSP patient CD4 T cells are positive for NKG2D and that large numbers of T cells from both subsets express MIC, which can be transactivated by Tax independent of nuclear factor kappaB. Engagement of MIC by NKG2D promotes spontaneous HAM/TSP T cell proliferation and, apparently, CTL activities against HTLV-1-infected T cells. These results reveal a viral strategy that may exploit immune stimulatory mechanisms to negotiate a balance between promotion and limitation of infected host T cell expansions.

Repression of IRF-4 target genes in human T cell leukemia virus-1 infection.

The human T cell leukemia/lymphotropic virus-1 (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes. Interferon regulatory factor-4 (IRF-4) was shown previously to be constitutively expressed in T cells infected with HTLV-1. In this study, we investigated the role of IRF-4 gene regulation in the context of HTLV-1 infection using gene array technology and IRF-4 expressing T cells. Many potential IRF-4 regulated genes were identified, the vast majority of which were repressed by IRF-4 expression. Cyclin B1, a G2-M checkpoint protein identified as an IRF-4 repressed gene in the array, was further characterized in the context of HTLV-1 infection. All HTLV-1 infected cell lines and ATL patient lymphocytes demonstrated a dramatic decrease in cyclin B1 levels; subsequent analysis of the cyclin B1 promoter identified two sites important in IRF-4 binding and repression of cyclin B1 expression. Furthermore, IRF-4-mediated repression of cyclin B1 led to a significant decrease in CDC2 kinase activity in HTLV-1 infected T cells. IRF-4 expression in HTLV-1 infected T cells also downregulated other genes implicated in the mitotic checkpoint as well as genes involved in actin cytoskeletal rearrangement, DNA repair, apoptosis, metastasis and immune recognition. Several of the identified genes are dysregulated in ATL and may provide important mechanistic information concerning pathways critical to the emergence of ATL.

Regulation of IFN regulatory factor 4 expression in human T cell leukemia virus-I-transformed T cells.

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-kappaB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the kappaB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the -617 to -209 region of the IRF-4 promoter. Furthermore, mutation of either the kappaB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4(+) T lymphocytes.

IFN regulatory factor 4 participates in the human T cell lymphotropic virus type I-mediated activation of the IL-15 receptor alpha promoter.

IL-15Ralpha mRNA and protein levels are increased in human T cell lymphotropic virus type-I (HTLV-I)-associated adult T cell leukemia. Previously, we demonstrated that IL-15Ralpha expression was activated by HTLV-I Tax, in part, through the action of NF-kappaB. However, there appeared to be additional motifs within the IL-15Ralpha promoter that were responsive to HTLV-I Tax. In this study, we demonstrated that IL-15Ralpha mRNA expression was activated in human monocytes by IFN treatment, suggesting a role for IFN regulatory factors (IRFs) in IL-15Ralpha transcription. In addition, an IRF element within the Tax-responsive element of the IL-15Ralpha promoter was necessary for maximal Tax-induced activation of this promoter. Furthermore, we demonstrated that IRF-4, a transcription factor known to be elevated in HTLV-I-infected cells, activated the IL-15Ralpha promoter. Inhibition of IRF-4 action lead to reduced Tax-induced activation of the IL-15Ralpha promoter, while inhibition of both IRF-4 and NF-kappaB severely inhibited the Tax-induced activation of this promoter. These findings suggest a role for both NF-kappaB and IRF-4 in the transcriptional regulation of IL-15Ralpha by HTLV-I Tax. It is possible that the HTLV-I Tax-mediated induction of IL-15Ralpha and IL-15 may lead to an autocrine cytokine-mediated stimulatory loop leading to the proliferation of HTLV-I infected cells. This loop of proliferation may facilitate viral propagation and play a role in HTLV-I-mediated disease progression.