A pan-influenza monoclonal antibody neutralizes H5 strains and prophylactically protects through intranasal administration

Avian A(H5N1) influenza virus poses an elevated zoonotic threat to humans, and no pharmacological products are currently registered for fast-acting pre-exposure protection in case of spillover leading to a pandemic. Here, we show that an epitope on the stem domain of H5 hemagglutinin is highly conserved and that the human monoclonal antibody CR9114, targeting that epitope, potently neutralizes all pseudotyped H5 viruses tested, even in the rare case of substitutions in its epitope. Further, intranasal administration of CR9114 fully protects mice against A(H5N1) infection at low dosages, irrespective of pre-existing immunity conferred by the quadrivalent seasonal influenza vaccine. These data provide a proof-of-concept for broad, pre-exposure protection against a potential future pandemic using the intranasal administration route. Studies in humans should assess if autonomous administration of a broadly-neutralizing monoclonal antibody is safe and effective and can thus contribute to pandemic preparedness

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

Prolonged circulation of tissue-type plasminogen activator in vivo by blocking mannose receptor- and lrp-mediated liver uptake

A major complication of the use of of tissue-type plasminogen activator (t-PA) as fibrinolytic agent is its rapid clearance from the bloodstream. We have studied whether t-PA clearance can be reduced by preventing receptor-mediated endocytosis of t-PA in vivo via the mannose receptor and the low-density lipoprotein receptor related protein (LRP) in the liver. A series of synthetic cluster mannosides has been devised and validated by in vitro binding studies. The most promising mannoside, MeL5, displaying a nanomolar affinity for the mannose receptor (K,= 0.6 nM), was found to significantly inhibit 125l-tPA clearance (1 mg/kg) in rats (apparent serum half-life t,/2= 3.1±0.2 vs 1.1±0.1 min for the control; P<0.005). Although liver endothelial cells did not contribute any longer to the hepatic uptake of 1J5l-t-PA after preinjection of MjL; (2 mg/kg), an increased uptake by hepatocytes partly compensated for the reduced endothelial cell uptake of t-PA. As this parenchymal cell uptake may be mediated by LRP, it was studied whether an additional reduction in t-PA clearance could be achieved by simultaneous blocking of LRP via preinjection of the 39kDa protein (RAP; 40 mg/kg). RAP preinjection reduced the clearance of t-PA (t1/8= 5.9±0.5 min; P<0.001) and reduced liver uptake from 82.8±1.3% to 60.4 ±1.2% of the injected dose (P<0.01) The combined pretreatment with M and RAP almost fully abolished liver uptake (10.6 ±6% of the injected dose; P<0.0001 ) and t-PA clearance (tt/2=16 ± 2.5 min; P<0.0001). In conclusion, therapeutic levels of serum t-PA can be maintained for a prolonged time-span by circumventing both LRP and the mannose receptor-mediated liver uptake of t-PA in vivo

A spontaneously in vitro transformed mesothelial cell line has a similar pattern of PDGF chain and PDGF receptor expression to malignant mesothelioma cell lines

The cell line TM-2 was obtained by spontaneous in vitro transformation of the normal mesothelial cell line NM-7. In contrast to NM-7, TM-2 was found to have an abnormal karyotype and an indefinite lifespan, and to proliferate independently of the addition of growth factors to the culture medium. TM-2 showed platelet-derived growth factor (PDGF) A-chain, B-chain and β-receptor messenger ribonucleic acid (mRNA) expression, whereas NM-7 expressed PDGF A-chain, α-receptor and β-receptor mRNA. PDGF α- and β-receptor protein expression in TM-2 and NM-7 confirmed the results obtained by Northern blot analysis. The expression pattern of PDGF chains and receptors in TM-2 strongly resembled the pattern found in malignant mesothelioma cell lines. These results support the hypothesis that PDGF-BB is an autocrine growth factor in malignant mesothelioma cell lines and plays a role in the transformation of normal mesothelial cells.</p

Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells.

Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6 +/- 2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4 +/- 0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 degrees C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH

Sub-chronic (13-week) oral toxicity study in rats with recombinant human lactoferrin produced in the milk of transgenic cows

The oral toxicity of recombinant human lactoferrin (rhLF) produced in the milk of transgenic cows was investigated in Wistar rats by daily administration via oral gavage for 13 consecutive weeks, 7 days per week. The study used four groups of 20 rats/sex/dose. The control group received physiological saline and the three test groups received daily doses of 200, 600 and 2000 mg of rhLF per kg body weight. Clinical observations, growth, food consumption, food conversion efficiency, water consumption, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy and microscopic examination of various organs and tissues were used as criteria for detecting the effects of treatment. Overall, no treatment-related, toxicologically significant changes were observed. The few findings that may be related to the treatment (lower cholesterol in high-dose females, lower urinary pH in high-dose males and females and very slightly higher kidney weight in high-dose females) were considered of no toxicological significance. Based on the absence of treatment-related, toxicologically relevant changes, the no-observed-adverse-effect level (NOAEL) was considered to be at least 2000 mg/kg body weight/day. © 2005 Elsevier Ltd. All rights reserved