4EASO transfection suppresses mesothelioma proliferation.

<p>Normal mesothelial cells (LP9) and mesothelioma cell lines H2373, H2461 and H2596 were treated with the indicated concentration of mmASO (open columns) and 4EASO (filled columns), and viable cells were counted. Normal LP9 cells were most resistant to 4EASO treatment, while growth of mesothelioma cell lines was reduced extensively. <i>Columns</i>, the mean of three independent determinations of cell number normalized to untreated cells, <i>bars</i>, s.d.</p

eIF4E expression is reduced by 4EASO treatment in mesothelioma.

<p>Cultured normal mesothelial and mesothelioma cells were transfected with mmASO or 4EASO and eIF4E and β-actin protein expression was evaluated by immunoblot analysis from lysates harvested after 72 hours. The mmASO does not alter eIF4E levels in mesothelial cells or mesothelioma cells. The band intensity levels for eIF4E following 4EASO treatment was normalized to untreated cells for each cell line and was determined using ImageJ. β-actin level does not change upon treatment and serves as a loading control.</p

4EASO transfection induces apoptosis in mesothelioma.

<p>Mesothelioma cell lines H2373 and H2461 along with human mesothelial cells (LP9) were treated with the indicated concentration of 4EASO for 48 hours. Lysates were immunoblotted with anti-PARP antibody. In mesothelioma cell lines PARP cleavage was substantially increased in 4EASO treated compared to untreated cells.</p

Enhanced susceptibility of mesothelioma cells treated with 4EASO to cytotoxic drugs.

<p>Mesothelioma cell lines transfected with mmASO or 4EASO were treated with the indicated concentration of gemcitabine (GEM) or pemetrexed (PEM) and viable cells were counted. Elevated cell death was found for cells treated in combination with 4EASO and pemetrexed or gemcitabine compared to each treatment alone. Concurrent treatment of mesothelioma cells with 4EASO combined with pemetrexed or gemcitabine suppresses proliferation compared to treatment with mmASO combined with pemetrexed or gemcitabine. <i>Columns</i>, the mean of three independent determinations of cell number normalized to untreated cells, <i>bars</i>, s.d. Averages of combination treatment was compared to either agent alone by Student’s <i>t</i>-test. * denotes a p value <0.05. NS = not statistically significant.</p

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation

<div><p>Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.</p></div

Imprime binding requires ABA.

<p>(A) Imprime binding (10 μg/mL) was measured in WB from 143 healthy donors. The serum from the same blood donation was used to measure relative ABA concentrations by ELISA with RAU/mL as the reportable value. The Pearson’s correlation (r) and <i>P</i>-value of Imprime binding to neutrophils and monocytes binding and ABA IgG and IgM concentrations are shown. (B) The IgG and IgM ABA level of each of the 143 healthy subjects identified as HB (>5% binding) and LB (≤ 5% binding) are shown in the scatter plot with the mean IgG or IgM ABA concentration of each group indicated. Imprime binding in a LB was evaluated by (C) adding increasing amount of HB serum and increasing concentration of ABA. (D) Imprime binding in a HB was evaluated in ABA-depleted serum (FT; flow through from the Imprime-conjugated bead column) and subsequently rescued binding by exogenous supplementation of ABA. MFI and percentages of BfD IV-positive cells are indicated on the contour plots. Data presented in (C) and (D) are representative of at least 3 independent experiments performed with different donors.</p

Imprime binding to neutrophils and monocytes in whole blood is complement-dependent.

<p>(A) Binding of increasing concentration of Imprime (0, 0.1, 1, 10, and 25 μg/mL) to human neutrophils and monocytes was measured by flow cytometry after incubation of WB with Imprime or vehicle at 37°C for 30 mins. (B) Imprime binding in the absence of serum (serum removed), heat-inactivated serum (HI serum), presence of anti-C3 peptide, compstatin (100 μM) or α-CR1 mAb (10 μg/mL) (right column in each respective row) are compared to the binding to vehicle or Imprime of untreated WB (left and middle column in each respective row). For compstatin and α-CR1 mAb treatment, WB was pre-treated with the blocking agents at 4°C for 30 mins prior to the incubation with 10 μg/mL Imprime at 37°C for 30 mins. (C) The role of classical pathway in Imprime binding to neutrophils and monocytes was evaluated by blocking with the anti-C1q mAb (50 μg/mL) at 4°C for 30 mins prior to the incubation with Imprime or vehicle. (D) Imprime binding to enriched human neutrophils and monocytes in 20% serum, 20% C1q-depleted serum (C1q-dep serum), or 20% C1q-depleted serum replenished with 100 μg/mL purified human C1q protein (C1q-dep serum + C1q protein) was determined by flow cytometry. The MFI and percentage of BfD IV positive cells are indicated on the contour plots. Data shown for each part are representative of 3–5 independent experiments performed with different donors.</p

Imprime interacts with endogenous IgG and IgM ABA in human serum.

<p>(A) The surface IgG and IgM ABA on vehicle- or Imprime-treated neutrophils and monocytes were detected by flow cytometry using rabbit anti-human IgG- and IgM-specific Abs, respectively, after Imprime was incubated with WB at 37°C for 30 mins. The MFI and percentage of IgG- or IgM-positive cells are indicated on the contour plots. Data shown are representative of 3 independent experiments. (B) The ability of an Imprime-conjugated bead column to selectively bind and retain IgG (filled bar) and IgM (empty bar) ABA from human serum is shown. The percentage of ABA in the flow-through (FT) and the eluate was measured based on the serum ABA concentration pre-loaded on the column. The ABA eluted off the Imprime-conjugated bead column along with purified IgG and IgM controls were resolved by SDS-PAGE under reducing conditions. Protein bands corresponding to the heavy and light chain of IgG and IgM were observed by Coomassie staining (left panel), or detected by immunoblotting with an anti-IgG Ab (middle panel) or anti-IgM heavy chain Ab (right panel).</p

ABA are critical for Imprime function.

<p>(A) The generation of C5a in WB plasma (14 LB and 18 HB) after incubation with Imprime for 30 mins, and IL-8 (21 LB and 11 HB) in WB after incubation with Imprime for 24 hrs were measured by ELISA. The amounts of C5a or IL-8 produced by Imprime—treated WB are presented as fold increase for individual HB and LB relative to the vehicle control. CD11b expression on neutrophils (11 LB and 12 HB) and monocytes (8 LB and 10 HB) after incubation with Imprime for 30 mins were assessed by flow cytometry. The % increase of CD11b was calculated using the MFI of Imprime-treated cells compared with vehicle-treated cells as baseline. ROS and ADCP assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165909#pone.0165909.g006" target="_blank">Fig 6</a>. The amount of ROS activity is presented as fold increase of AUC relative to that of the vehicle-treated neutrophils. The insets show the representative ROS activity of neutrophils from a LB or a HB over a 60-mins time period. The ADCP is presented as average fold increase relative to the vehicle control. Results for ADCP and ROS are from 3 HB and 3 LB. Data represent mean ± SEM of triplicates for each treatment condition. IL-8 and MCP-1 production (B) and neutrophil ROS activity (C) were assessed in LB after exogenous addition of ABA to Imprime-treated WB. Results presented are representative of at least 3 independent experiments performed with different donors.</p

Imprime activates innate immune functions.

<p>(A) Complement activation proteins C4a, C5a, SC5b-9 in the plasma of WB treated with 10 μg/mL Imprime or vehicle for 30 mins at 37°C was measured by ELISA. Data represent mean ± SEM of triplicates for each treatment condition. (B) Modulation of CD11b, CD62L, CD88 and CXCR2 expression on neutrophils and monocytes post Imprime binding in WB was determined by flow cytometry. (C) Chemokine, IL-8 and MCP-1, production in the plasma of WB treated with Imprime or vehicle for 24 h at 37°C was measured by Luminex. Data represent mean ± SEM of duplicates from 3 independent experiments. (D) ROS production in 25:1 co-cultures of neutrophils (isolated from WB treated with 25 μg/mL Imprime or vehicle for 2 hrs at 37°C) and Raji cells treated with or without 1 μg/mL rituximab was measured by luminescence-based assay. Macrophage-mediated ADCP was measured by flow cytometry after 1:1 co-incubation of macrophages (differentiated from monocytes isolated from WB treated with 25 μg/mL Imprime or vehicle for 2 hrs at 37°C) with Raji cells treated with or without 1 μg/mL rituximab. Representative results are shown here from at least 3 independent experiments performed with three different donors.</p