IRX-2, a Novel Immunotherapeutic, Enhances Functions of Human Dendritic Cells

Background: In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC). Methodology/Principal Findings: Monocyte-derived DC obtained from 18 HNSCC patients and 12 healthy donors were matured using IRX-2 or a mix of TNF-α, IL-1β and IL-6 ("conv. mix"). Multicolor flow cytometry was used to study the DC phenotype and antigen processing machinery (APM) component expression. ELISPOT and cytotoxicity assays were used to evaluate tumor-reactive cytotoxic T lymphocytes (CTL). IL-12p70 and IL-10 production by DC was measured by Luminex® and DC migration toward CCL21 was tested in transwell migration assays. IRX-2-matured DC functions were compared with those of conv. mix-matured DC. IRX-2-matured DC expressed higher levels (p<0.05) of CD11c, CD40, CCR7 as well as LMP2, TAP1, TAP2 and tapasin than conv. mix-matured DC. IRX-2-matured DC migrated significantly better towards CCL21, produced more IL-12p70 and had a higher IL12p70/IL-10 ratio than conv. mix-matured DC (p<0.05 for all). IRX-2-matured DC carried a higher density of tumor antigen-derived peptides, and CTL primed with these DC mediated higher cytotoxicity against tumor targets (p<0.05) compared to the conv. mix-matured DC. Conclusion: Excellent ability of IRX-2 to induce ex vivo DC maturation in HNSCC patients explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DC generated for therapy. © 2013 Schilling et al

Pathologic and gene expression features of metastatic melanomas to the brain: Biology of Metastatic Melanomas to the Brain

The prognosis of MBM is variable with prolonged survival in a subset. It is unclear whether MBMs differ from extracranial metastases (EcM) and primary melanomas (PrM)

IRX-2, a novel biologic, favors the expansion of T effector over T regulatory cells in a human tumor microenvironment model

IRX-2, a natural cytokine biological with multiple components, has been used in preclinical and clinical studies to promote antitumor activity of T lymphocytes. To define cellular mechanisms responsible for antitumor effects of IRX-2, its ability to induce effector T cells (Teff) was examined in a model simulating the tumor microenvironment. An in vitro model containing conventional CD4+CD25− cells co-cultured with autologous immature dendritic cells, irradiated tumor cells, and cytokines was used to study differentiation and expansion of regulatory T cells (Treg) and Teff in the presence and absence of IRX-2. Phenotype, suppressor function, signaling, and cytokine production were serially measured using flow cytometry, Western blots, CFSE-based suppressor assays, and Luminex-based analyses. The presence of IRX-2 in the co-cultures promoted the induction and expansion of IFN-γ+Tbet+ Teff and significantly (p < 0.01) decreased the induction of inducible IL-10+TGF-β+ Treg. The responsible mechanism involved IFN-γ-driven T cell polarization towards Teff and suppression of Treg differentiation. In an in vitro model simulating the human tumor microenvironment, IRX-2 promoted Teff expansion and antitumor activity without inducing Treg. Thus, IRX-2 could be considered as a promising component of future antitumor therapies

IRX-2, a Novel Immunotherapeutic, Enhances Functions of Human Dendritic Cells

Abstract Background: In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC)

Arginase-1 in Plasma-Derived Exosomes as Marker of Metastasis in Patients with Head and Neck Squamous Cell Carcinoma

Immunoregulatory Arginase-1 (Arg-1) is present in the tumor microenvironment of solid tumors. Its association to clinicopathology and its prognostic impact are inconsistent among different tumor types and biological fluids. This study evaluated Arg-1 protein levels in tumors and the circulation of patients with head and neck squamous cell carcinoma (HNSCC) in relation to clinical stage and prognosis. Tumor Arg-1 expression was monitored via immunohistochemistry while plasma Arg-1 levels via ELISA in 37 HNSCC patients. Arg-1 presence in plasma-derived exosomes was assessed using Western blots in 20 HNSCC patients. High tumor Arg-1 expression correlated with favorable clinicopathology and longer recurrence-free survival (RFS), while high plasma Arg-1 levels were associated with unfavorable clinicopathology. All patients with low tumor and high plasma Arg-1 had nodal metastases and developed recurrence. This discrepancy was attributed to the presence of Arg-1-carrying exosomes. Arg-1 was found in plasma-derived exosomes from all HNSCC patients. High exosomal Arg-1 levels were associated with positive lymph nodes and short RFS. Circulating Arg-1+ exosomes represent a mechanism of active Arg-1 export from the tumor to the periphery. Exosomes reflected biologically relevant Arg-1 levels in metastatic HNSCC and emerged as potentially more accurate biomarkers of metastatic disease and RFS than tissue or plasma Arg-1 levels

Clinicopathologic characteristics of patients with HNSCC who donated blood for this study.

<p>Clinicopathologic characteristics of patients with HNSCC who donated blood for this study.</p

Cytotoxicity of CTL generated in IVS cultures.

<p>CTL were induced and expanded using iDC and DC matured either with IRX-2 or the conventional cocktail from HLA-A2⁺ HNSCC patients. CTL primed by conventionally-matured or IRX-2-matured DC were more effective than CTL primed with iDC. CTL generated using IRX-2-matured DC showed higher cytotoxicity than those primed with conventional DC. Blocking MHC-class-I recognition with the mAb w6.32 abrogated cytotoxicity. The data are mean percentages ± SEM of specific killing at different E:T ratios obtained from 4 independent experiments.</p

Phenotype and migration of DC matured in IRX-2 or conventional cytokines.

<p>(<b><u>A</u></b>) DC obtained from HNSCC patients were matured for 48 h either with IRX-2 or the conventional maturation cocktail. While conventionally matured DC (white bars) expressed higher levels of CD80, CD83 and CD86 (*, p<0.05), IRX-2 matured DC (black bars) showed higher expression of CD11c, CD40 and CCR7 (*, p<0.05). The data are mean x-fold of MFI ± SEM for cells obtained from 12 different HNSCC patients. (<b><u>B</u></b>) Representative histograms showing expression of DC markers after maturation with IRX-2 or the conventional cytokine cocktail in DC generated from monocytes of one HNSCC patient. The shaded peaks represent isotype controls. (<b><u>C</u></b>) Migration of mDC <i>in vitro</i>: Migration assays were performed as described in Materials & Methods using DC generated from peripheral blood monocytes of HNSCC patients. While iDC showed very little migration in response to CCL21, both conventional- and IRX-2-matured DC migrated significantly better. Results are shown as the mean absolute numbers of migrated cells ± SEM obtained from 5 different HNSCC patients.</p

APM expression in mDC.

<p>moDC from HNSCC patients were matured for 48 h either with IRX-2 or the conventional maturation cocktail. (A) IRX-2-matured DC (black bars) expressed significantly higher levels of TAP1, TAP2, LMP2 and Tapasin than conventional DC (white bars, *, p<0.05). APM expression was determined by flow cytometry. The data are mean x-fold of MFI ± SEM for cells obtained from 12 different HNSCC patients.</p