31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

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

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

Role of CR3 and FcgRIIA in binding of Imprime.

<p>(A) Imprime binding was measured in the presence of a combination of α-CR3 blocking mAbs (LM2/1, a mAb against the I-domain of CR3 at 10 μg/mL; VIM12, a mAb against the lectin-domain of CR3 at 10 μg/mL; IB4, a mAb against the β2 chain of CR3 at 5 μg/mL). WB was incubated with the antibodies at 4°C for 30 mins prior to the incubation with 10 μg/mL Imprime at 37°C for 30 mins. Representative results from 3 independent experiments are shown. (B) Total serum IgG ABA level from 143 healthy subjects was measured by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165909#pone.0165909.g004" target="_blank">Fig 4</a>. Subclass ELISA was performed similarly with the exception of using IgG1 and IgG2 specific secondary antibodies. The Pearson’s correlation (r) and <i>P</i>-value of total IgG ABA and IgG1 or IgG2 ABA are shown. (C) Plasmid DNA for individual FcgR, including FcgRIIIB (CD16B), FcgRIIA (CD32A), and FcgRI (CD64) as well as vector DNA (data not shown) was transfected into HEK293 cells, and Imprime binding was then assessed in these FcgR—expressing HEK293 transfectants by incubating with 10 μg/mL Imprime and enriched ABA (100 RAU/mL) in the presence or absence of serum at 37°C for 30 mins. Representative results from 3 independent experiments are shown. (D) Imprime binding in WB was assessed in the presence of respective FcR blocking mAbs, 20 μg/mL of anti-CD16 (FcgRIII), anti-CD32 (FcgRII), or anti-CD64 (FcgRI) by flow cytometry. Data are shown as mean ± SEM of multiple donors (N = 12). (E) Imprime binding in the presence of enriched ABA (400 RAU/mL) was measured in 3 LB identified to have H131 (HH) or R131 (RR) or the heterozygous HR allotype of FcgRIIA by PCR. Representative results are shown here from 3 independent experiments.</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

List of antibodies used in the study.

<p>List of antibodies used in the study.</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

Structure of Imprime.

<p>Imprime is composed of a β-(1–3)-linked glucopyranose backbone with periodic β-(1–3)-linked glucopyranose side chains linked to the backbone via β-(1–6) glycosidic linkages.</p

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