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

Novel cofactors via post-translational modifications of enzyme active sites

AbstractRecent crystallographic and biochemical studies have revealed the existence of numerous novel post-translational modifications within enzyme active sites. These modifications create structural and functional diversity. Although the function and biosynthesis of some of these modifications are well understood, others need further investigation

Metabolic Engineering of Monoclonal Antibody Carbohydrates for Antibody–Drug Conjugation

The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody–drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides

The Fucosylation Inhibitor, 2-Fluorofucose, Inhibits Vaso-Occlusion, Leukocyte-Endothelium Interactions and NF-ĸB Activation in Transgenic Sickle Mice

<div><p>2-Fluorofucose (2FF) blocks the fucosylation and the tethering of sialyl-Lewis^x tetrasaccharide and structural variants on leukocytes and red blood cells to P- and E-selectins on activated endothelial cell surfaces. Because P- and E-selectin are required for vaso-occlusion in murine sickle cell disease (SCD), we investigated whether 2FF would inhibit vaso-occlusion in SCD mice. Microvascular stasis was measured in subcutaneous venules in NY1DD and HbSS-Townes SCD mice with dorsal skin-fold chambers after infusion of hemoglobin or exposure to hypoxia/reoxygenation. 2FF in drinking water or administered by gavage inhibited stasis in sickle mice in a dose-responsive manner. Significant inhibitory effects on stasis were seen 1 day post-treatment. 2FF treatment of SCD mice also significantly reduced leukocyte rolling and adhesion along the vessel walls of SCD mice and the static adhesion of neutrophils and sickle red blood cells isolated from 2FF-treated SCD mice to resting and activated endothelial cells. Total white blood cell counts increased in response to 2FF. NF-ĸB activation and VCAM-1 and E-selectin expression were inhibited in the livers of SCD mice consistent with an overall decrease in vascular inflammation and ischemia-reperfusion physiology. Pretreatment with 2FF completely eliminated heme-induced lethality in HbSS-Townes mice, consistent with the observed anti-inflammatory and anti-adhesive properties of 2FF in SCD mice. These data suggest that 2FF may be beneficial for preventing or treating vaso-occlusive crises in SCD patients.</p></div

2FF prevents heme-induced lethality in HbSS-Townes mice.

<p>All HbSS-Townes mice (n = 6 mice/treatment) were given a bolus infusion of heme (32 μmols/kg) at time zero. Mice were given water or 100 mM 2FF in drinking water for 7 days prior to heme infusion. Time of death after heme infusion was recorded.</p

2FF inhibits microvascular stasis and leukocyte rolling and adhesion in sickle mice <i>in vivo</i>.

<p>(<b>A</b>) NY1DD transgenic sickle mice (n = 4/group) were given the indicated concentration of 2FF in their drinking water for 7 days. Microvascular stasis was measured on day 7, one hour after the infusion of stroma-free Hb (3.2 μmols heme/kg). <b>*</b>P≤.05 for all pairwise comparisons. (<b>B</b>) NY1DD sickle mice were gavaged with water or 150 mg/ml 2FF (.01 ml/g, twice per day) for 1 or 3 days. After the indicated 2FF treatment period, stroma-free Hb (3.2 μmols heme/kg body weight) was infused intravenously and microvascular stasis was measured 1 hour later. <b>*</b>P≤.01 for water vs 2FF. (<b>C</b>) NY1DD and HbSS-Townes sickle mice (n = 3/group) were given 100 mM 2FF in their drinking water for 7 days. Stasis was measured on day 7 after exposure to 1h of hypoxia and 1h of reoxygenation (H/R). <b>*</b>P<.001 for water vs 100 mM 2FF. (<b>D</b>) Leukocyte rolling flux was measured in venules in the DSFC windows of NY1DD mice before (baseline) and 1h after infusion of Hb (3.2 μmol/kg). Half of the mice (n = 4) were treated with 2FF (100 mM in drinking water X 7 days) prior to baseline measurements. Control mice (n = 4) were given drinking water without 2FF. The rolling flux was determined as the total number of leukocytes rolling through a given section of vessel per minute. Values are means + SD. ^<b>#</b>P<.001 baseline vs Hb and <b>*</b>p<.001 for water vs 2FF. (<b>E</b>) Leukocyte adhesion was measured in the same venules as described in (D). Values are mean number of adherent cells per 100 μm + SD. ^<b>#</b>P<.001 for baseline vs Hb and <b>*</b>p<.001 for water vs 2FF. (<b>F</b> and <b>G</b>) PMNs (<b>F</b>) and SS-RBCs (<b>G</b>) were isolated from HbSS-Townes mice (n = 2 mice twice) given drinking water or 100 mM 2FF in drinking water for 7 days. Fluorescently labeled PMNs or SS-RBCs were incubated with resting and activated HUVEC (4 wells/treatment) for 30 minutes. Activated HUVEC, which express P-selectin on their surface, were prepared by incubating HUVEC with 10 μM heme for 30 minutes. Values are mean % area of HUVEC cell surface covered by PMNs (<b>F</b>) or SS-RBCs (<b>G</b>) + SD after HUVEC washing. ^<b>#</b>P<.001 for resting vs activated and <b>*</b>p<.001 for water vs 2FF.</p

2FF increases the total, neutrophil (PMN) and lymphocyte white blood cell (WBC) counts in sickle mice.

<p>2FF increases the total, neutrophil (PMN) and lymphocyte white blood cell (WBC) counts in sickle mice.</p