The Importance of Human FcγRI in Mediating Protection to Malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria

Cause of Death and Predictors of All-Cause Mortality in Anticoagulated Patients With Nonvalvular Atrial Fibrillation : Data From ROCKET AF

M. Kaste on työryhmän ROCKET AF Steering Comm jäsen.Background-Atrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all-cause mortality may guide interventions. Methods and Results-In the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all-cause mortality in the 14 171 participants in the intention-to-treat population. The median age was 73 years, and the mean CHADS(2) score was 3.5. Over 1.9 years of median follow-up, 1214 (8.6%) patients died. Kaplan-Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all-cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33-1.70, P= 75 years (hazard ratio 1.69, 95% CI 1.51-1.90, P Conclusions-In a large population of patients anticoagulated for nonvalvular atrial fibrillation, approximate to 7 in 10 deaths were cardiovascular, whereasPeer reviewe

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

The Fully human anti-CD47 antibody SRF231 exerts dual-mechanism antitumor activity via engagement of the activating receptor CD32a

Background CD47 is a broadly expressed cell surface glycoprotein associated with immune evasion. Interaction with the inhibitory receptor signal regulatory protein alpha (SIRPα), primarily expressed on myeloid cells, normally serves to restrict effector function (eg, phagocytosis and immune cell homeostasis). CD47/SIRPα antagonists, commonly referred to as ‘macrophage checkpoint’ inhibitors, are being developed as cancer interventions. SRF231 is an investigational fully human IgG4 anti-CD47 antibody that is currently under evaluation in a phase 1 clinical trial. The development and preclinical characterization of SRF231 are reported here.Methods SRF231 was characterized in assays designed to probe CD47/SIRPα blocking potential and effects on red blood cell (RBC) phagocytosis and agglutination. Additionally, SRF231-mediated phagocytosis and cell death were assessed in macrophage:tumor cell in vitro coculture systems. Further mechanistic studies were conducted within these coculture systems to ascertain the dependency of SRF231-mediated antitumor activity on Fc receptor engagement vs CD47/SIRPα blockade. In vivo, SRF231 was evaluated in a variety of hematologic xenograft models, and the mechanism of antitumor activity was assessed using cytokine and macrophage infiltration analyses following SRF231 treatment.Results SRF231 binds CD47 and disrupts the CD47/SIRPα interaction without causing hemagglutination or RBC phagocytosis. SRF231 exerts antitumor activity in vitro through both phagocytosis and cell death in a manner dependent on the activating Fc-gamma receptor (FcγR), CD32a. Through its Fc domain, SRF231 engagement with macrophage-derived CD32a serves dual purposes by eliciting FcγR-mediated phagocytosis of cancer cells and acting as a scaffold to drive CD47-mediated death signaling into tumor cells. Robust antitumor activity occurs across multiple hematologic xenograft models either as a single agent or in combination with rituximab. In tumor-bearing mice, SRF231 increases tumor macrophage infiltration and induction of the macrophage cytokines, mouse chemoattractant protein 1 and macrophage inflammatory protein 1 alpha. Macrophage depletion results in diminished SRF231 antitumor activity, underscoring a mechanistic role for macrophage engagement by SRF231.Conclusion SRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a

Sequencing of V Genes and SPR Analysis

<div><p>(A) Amino acid sequences of MSP1₁₉-binding scFvs. Sequences of six selected scFvs obtained by panning phage display libraries with recombinant MSP1₁₉ (C1) or P. falciparum merozoites in which secondary processing had been allowed to proceed (E9). Amino acids in bold represent residues in the E9 sequence differing to C1.</p><p>(B) SPR association and dissociation curves of Ab binding to MSP1₁₉ immobilized on a CM5 sensor chip. Abs were injected into flow at time 0 and replaced with buffer at the point indicated by vertical arrow [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030072#ppat-0030072-b024" target="_blank">24</a>].</p></div

JS1 and JS2 Are Fully Functional Human Antibodies

<div><p>(A) Human neutrophil-mediated phagocytosis of GST-<i>Pf</i>MSP1₁₉ coated fluorescent 1-μm microspheres by JS1. A control human IgG1 recognizing the homologous GST-MSP1₁₉ from P. yoelii was unable to opsonize beads and no ingestion was observed. Phagocytosed beads (red) were visualized in the cytoplasm of neutrophils (arrowed) whose nuclear DNA was counterstained in blue by DAPI.</p><p>(B) Stimulation of neutrophil NADPH oxidative bursts using JS1 and JS2 attached to GST-MSP1₁₉-coated microtiter plates. Chemiluminescence (CL; arbitary units) was induced by anti-<i>Pf</i>MSP1₁₉ human IgG1 JS1 (⋄), JS2 (○), human IgA1 (□), or no antibody (▵). All antibodies at 1 × 10⁻⁷ M. Data are presented as mean arbitary units from duplicate wells with neutrophils from a single donor.</p></div

Characterization of Purified Abs

<div><p>(A) 5 μg purified anti-MSP1₁₉ human IgG1 (JS1) was subjected to SDS-PAGE under nonreducing (lane 1) or reducing (lane 2) conditions on 4%–15% polyacrylamide gradient gels and stained with Simply Blue or immunoblotted with anti-human IgG-HRP.</p><p>(B) Under nonreducing conditions and after transfer to nitrocellulose, the human anti-MSP1₁₉ IgG1 (JS1) detects the recombinant GST-MSP1₁₉ fusion protein (lane 3) but not the GST alone control (lane 4). Localization of MSP1₁₉ by IFA.</p><p>(C) Schizont- and merozoite-stage parasites from the transgenic PbPbM19 and PbPfM19 lines were incubated with human Abs JS1 or JS2 (1:100), rabbit αPbM19 (1:1,000), or αPfM19 (1:1,000). After incubation with goat anti-rabbit Alexa-conjugated Ig (1:1,000) and FITC-conjugated anti-human IgG Fc (1:200), slides were washed and mounted in Vectrashield anti-fade. Parasites were visualized by fluorescence microscopy ×100 magnification, with the same fields photographed using filters to detect Alexa and FITC.</p><p>(D) JS1 reactive with MSP1₁₉ on methanol-acetone-fixed smears of merozoites and erythrocytes infected with P. falciparum (strain 3D7) ×40 magnification. JS2 gave similar results. No specific fluorescence was detected with an irrelevant human IgG1 (B10) recognizing MSP1₁₉ from P. yoelii [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030072#ppat-0030072-b024" target="_blank">24</a>].</p></div

Inhibition of Binding of mAbs 12.10 and 12.8 by Fully Human Anti-MSP1₁₉ IgG1s (JS1 and JS2) by Competition ELISA

<p>The binding of 12.8 is reduced by over 60% and the binding by 12.10 reduced by 30% suggesting that JS1 and JS2 compete with mAbs 12.8 and 12.10 for similar or overlapping epitopes. mAb 12.8, JS1, and JS2 were used at 0.5 μg/ml and mAb 12.10 at 0.05 μg/ml. Similar results were also observed when saturating concentrations of antibodies were used.</p