Decoy Protein Research Articles

A tetrameric ACE2 protein broadly neutralizes SARS-CoV-2 spike variants of concern with elevated potency

The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.

A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection

Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.

Therapeutic Interleukin-6 Trans-signaling Inhibition by Olamkicept (sgp130Fc) in Patients With Active Inflammatory Bowel Disease

A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade invivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).

Institutional Experience of Using Andexanet Alfa.

Given their ease of use, safety, and efficacy, direct-acting oral anticoagulants (DOACs) are nowadays widely used in patients with atrial fibrillation or venous thromboembolism, with or without an association with malignancy. Andexanet alfa (andexanet) is a recombinant modified human factor Xa decoy protein that reverses the inhibition of factor Xa. After Food and Drug Administration (FDA) approval in May 2018, andexanet has been used for life-threatening bleeding in patients treated with apixaban or rivaroxaban.In this article, we present a single institutional retrospective review of patients receiving andexanet alfa at Guthrie Robert Packer Hospital. A total of four patients in a period of 10 months received andexanet for intracranial bleeding, 50% (2) had excellent hemostasis, 30 days mortality was 75% (3), and 25% (1) had a thromboembolic event. Anticoagulation was never started in all patients. This review tends to show the real-world utilization data of andexanet in a community hospital setting.

Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.

Optimizing the PBS1 Decoy System to Confer Resistance to Potyvirus Infection in Arabidopsis and Soybean.

The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. We have previously shown that replacing seven amino acids at the cleavage site of PBS1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell-death response too slow to contain the virus. We theorized this could result from a positional mismatch within the cell between PBS1TuMV, RPS5, and the NIa protease. To test this, we relocalized PBS1TuMV and RPS5 to cellular sites of NIa accumulation. These experiments revealed that relocation of RPS5 away from the plasma membrane compromised RPS5-dependent cell death in Nicotiana benthamiana, even though PBS1 was efficiently cleaved. As an alternative approach, we tested whether overexpression of plasma membrane-localized PBS1TuMV could enhance RPS5 activation by TuMV. Significantly, overexpressing the PBS1TuMV decoy protein conferred complete resistance to TuMV when delivered by either agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens. Lastly, we have now extended this PBS1 decoy approach to soybean by modifying a soybean PBS1 ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that we can use endogenous PBS1 proteins in crop plants to engineer economically relevant disease resistant traits.

Assay-Based Differentiation in the Neutralization Profile of Unfractionated Heparin, Enoxaparin, and Fondaparinux by Andexanet Alfa.

Andexanet alfa is a recombinant factor Xa decoy protein, designed to reverse bleeding associated with oral anti-Xa agents. Andexanet alfa is also reported to neutralize the effects of heparin-related drugs. This study focused on the neutralization profiles of unfractionated heparin (UFH), enoxaparin, and, a chemically synthetic pentasaccharide, fondaparinux by andexanet alfa. Whole blood clotting studies were carried out using thromboelastography (TEG) and activated clotting time (ACT). The anticoagulant profile of UFH, enoxaparin, and fondaparinux was studied using the activated partial thromboplastin time (aPTT), thrombin time (TT), and amidolytic anti-Xa, and anti-IIa methods. Thrombin generation inhibition was studied using the calibrated automated thrombogram system. Reversal of each of these agents was studied by supplementing andexanet alfa at 100 µg/mL. In the TEG, andexanet alfa produced almost a complete reversal of the anticoagulant effects of UFH and enoxaparin; however, it augmented the effects of fondaparinux. In the ACT, aPTT, and TT, UFH produced strong anticoagulant effects that were almost completely neutralized by andexanet alfa. Enoxaparin produced milder anticoagulant responses that were partially neutralized, whereas fondaparinux did not produce any sizeable effects. In the anti-Xa and anti-IIa assays, UFH exhibited partial neutralization whereas enoxaparin and fondaparinux did not show any neutralization. All agents produced varying degrees of the inhibition of thrombin generation, which were differentially neutralized by andexanet alfa. These results indicate that andexanet alfa is capable of differentially neutralizing anticoagulant and antiprotease effects of UFH and enoxaparin in an assay-dependent manner. However, andexanet alfa is incapable of neutralizing the anti-Xa effects of fondaparinux.

Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

579 The Efficacy and Safety of Andexanet Alfa in Patients With Acute Gastrointestinal Bleeding While Taking Factor Xa Inhibitors: An ANNEXA-4 Sub-Analysis

INTRODUCTION: Andexanet alfa is a recombinant modified human Factor Xa (FXa) decoy protein developed to reverse the anticoagulant effect of FXa inhibitors. METHODS: ANNEXA-4 was a prospective single-arm study of andexanet alfa in patients with acute major bleeding while taking a FXa inhibitor. Patients with major gastrointestinal (GI) bleeding were eligible if they had (1) signs/symptoms of hemodynamic compromise or (2) bleeding associated with a hemoglobin (Hb) drop ≥2 g/dL or baseline Hb ≤8 g/dL. Upon enrollment, patients were treated with one of two andexanet dosing regimens based on the type, timing, and dose of the most recent inhibitor intake. Efficacy was evaluated in all patients who met the bleeding criteria above and had a baseline anti-FXa activity ≥75 ng/mL (≥0.25 IU/mL for enoxaparin). Co-primary endpoints were the change in anti-FXa activity and the achievement of hemostasis, as adjudicated by an independent committee, at 12 hours after andexanet treatment. Hemostasis was defined as: excellent if the red blood cell transfusion-corrected Hb at 12 hours was ≤10% lower than baseline, good if Hb was 10-20% lower, and poor/none if Hb was >20% lower. RESULTS: Of 352 patients enrolled in the study, 90 had GI bleeding, of whom 62 were included in the efficacy analysis. Median age was 76 years (range 24-95); 51 (57%) were male. Patients were receiving rivaroxaban (n = 44), apixaban (n = 5), and edoxaban (n = 3) at study entry. Bleeding location was identified in 48 patients (upper n=27, lower n = 21) and unknown in 42 patients. 66 (73%) patients had some evidence of hemodynamic compromise, including 38 (42%) patients with hypotension. Mean Hb at the time of dosing was 4.9 ± 1.4 g/dL. In patients taking rivaroxaban, the baseline anti-FXa activity was 267 ± 168.5 ng/mL and the percent reduction with andexanet was 94% (95%CI 89-96). In apixaban patients, the baseline anti-FXa activity was 244 ± 215.0 ng/mL and the percent reduction with andexanet was 92% (95%CI 88-95). Excellent/good hemostasis was achieved in 85% of evaluable patients. 58 (94%) of 62 patients received red blood cell transfusions, 3 (5%) received platelet transfusion, and 2 (3%) received a coagulation factor product. 30-day rates of thrombosis and death were 6% and 13%, respectively. CONCLUSION: In patients with major GI bleeding treated with andexanet, a high rate of effective hemostasis was achieved. The thrombosis and mortality rates are consistent with prior studies in this patient population.

Effects of N-Glycosylation on the Structure, Function, and Stability of a Plant-Made Fc-Fusion Anthrax Decoy Protein.

Protein N-glycosylation is an important post-translational modification and has influences on a variety of biological processes at the cellular and molecular level, making glycosylation a major study aspect for glycoprotein-based therapeutics. To achieve a comprehensive understanding on how N-glycosylation impacts protein properties, an Fc-fusion anthrax decoy protein, viz rCMG2-Fc, was expressed in Nicotiana benthamiana plant with three types of N-glycosylation profiles. Three variants were produced by targeting protein to plant apoplast (APO), endoplasmic reticulum (ER) or removing the N-glycosylation site by a point mutation (Agly). Both the APO and ER variants had a complex-type N-glycan (GnGnXF) as their predominant glycans. In addition, ER variant had a higher concentration of mannose-type N-glycans (50%). The decoy protein binds to the protective antigen (PA) of anthrax through its CMG2 domain and inhibits toxin endocytosis. The protein expression, sequence, N-glycosylation profile, binding kinetics to PA, toxin neutralization efficiency, and thermostability were determined experimentally. In parallel, we performed molecular dynamics (MD) simulations of the predominant full-length rCMG2-Fc glycoform for each of the three N-glycosylation profiles to understand the effects of glycosylation at the molecular level. The MAN8 glycoform from the ER variant was additionally simulated to resolve differences between the APO and ER variants. Glycosylation showed strong stabilizing effects on rCMG2-Fc during in planta accumulation, evidenced by the over 2-fold higher expression and less protein degradation observed for glycosylated variants compared to the Agly variant. Protein function was confirmed by toxin neutralization assay (TNA), with effective concentration (EC50) rankings from low to high of 67.6 ng/ml (APO), 83.15 ng/ml (Agly), and 128.9 ng/ml (ER). The binding kinetics between rCMG2-Fc and PA were measured with bio-layer interferometry (BLI), giving sub-nanomolar affinities regardless of protein glycosylation and temperatures (25 and 37°C). The protein thermostability was examined utilizing the PA binding ELISA to provide information on EC50 differences. The fraction of functional ER variant decayed after overnight incubation at 37°C, and no significant change was observed for APO or Agly variants. In MD simulations, the MAN8 glycoform exhibits quantitatively higher distance between the CMG2 and Fc domains, as well as higher hydrophobic solvent accessible surface areas (SASA), indicating a possibly higher aggregation tendency of the ER variant. This study highlights the impacts of N-glycosylation on protein properties and provides insight into the effects of glycosylation on protein molecular dynamics.

Engineering a Decoy Substrate in Soybean to Enable Recognition of the Soybean Mosaic Virus NIa Protease.

In Arabidopsis, recognition of the AvrPphB effector protease from Pseudomonas syringae is mediated by the disease resistance (R) protein RPS5, which is activated by AvrPphB-induced cleavage of the Arabidopsis protein kinase PBS1. The recognition specificity of RPS5 can be altered by substituting the AvrPphB cleavage site within PBS1 with cleavage sequences for other proteases, including proteases from viruses. AvrPphB also activates defense responses in soybean (Glycine max), suggesting that soybean may contain an R protein analogous to RPS5. It was unknown, however, whether this response is mediated by cleavage of a soybean PBS1-like protein. Here, we show that soybean contains three PBS1 orthologs and that their products are cleaved by AvrPphB. Further, transient expression of soybean PBS1 derivatives containing a five-alanine insertion at their AvrPphB cleavage sites activated cell death in soybean protoplasts, demonstrating that soybean likely contains an AvrPphB-specific resistance protein that is activated by a conformational change in soybean PBS1 proteins. Significantly, we show that a soybean PBS1 decoy protein modified to contain a cleavage site for the soybean mosaic virus (SMV) NIa protease triggers cell death in soybean protoplasts when cleaved by this protease, indicating that the PBS1 decoy approach will work in soybean, using endogenous PBS1 genes. Lastly, we show that activation of the AvrPphB-dependent cell death response effectively inhibits systemic spread of SMV in soybean. These data also indicate that decoy engineering may be feasible in other crop plant species that recognize AvrPphB protease activity.

Differential Neutralization of Unfractionated Heparin, Enoxaparin and Fondaparinux by Andexanet Alpha

IntroductionAndexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh‐zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the inhibition of factor Xa in humans to control bleeding associated with newer oral anti‐Xa agents. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma‐carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti‐Xa agents AA is also reported to neutralize the effects of heparin and related drugs. The purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA.Materials and MethodsAPI versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombinase activated clotting time (PICT) in a concentration range of 0 – 10 ug/ml in pooled human plasma. The anti‐Xa and anti‐IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti‐protease effects of each of these agents was studied by supplementing this agent at 100 ug/ml.ResultsAll agents produce a concentration dependent effect in the anticoagulant and anti‐protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti‐IIa actions and strong anti‐Xa activity. In the anti‐Xa assay the IC‐50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). There was no change in the IC50 for F. The anticoagulant effects of UFH and E were neutralized at variable levels and were assay dependent. However there was no neutralization of the anti‐Xa effects of the F by AA. At high concentrations of greater than 2.5 ug/ml (1.47 um) the mild anticoagulant effects of F were neutralized in an assay dependent fashion.ConclusionThese results indicate that AA is capable of differentially neutralizing anticoagulant and antiprotease effects of UFH and E in an assay dependent manner. However AA is incapable of neutralizing the anti‐Xa effects of F. This may be due to the relatively strong affinity of F AT complex to factor Xa rendering it inhibited in the presence of AA.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Random Forest Refinement of the KECSA2 Knowledge-Based Scoring Function for Protein Decoy Detection.

Knowledge-based potentials generally perform better than physics-based scoring functions in detecting the native structure from a collection of decoy protein structures. Through the use of a reference state, the pure interactions between atom/residue pairs can be obtained through the removal of contributions from ideal-gas state potentials. However, it is a challenge for conventional knowledge-based potentials to assign different importance factors to different atom/residue pairs. In this work, via the use of the "comparison" concept, Random Forest (RF) models were successfully generated using unbalanced data sets that assign different importance factors to atom pair potentials to enhance their ability to identify native proteins from decoy proteins. Individual and combined data sets consisting of 12 decoy sets were used to test the performance of the RF models. We find that RF models increase the recognition of native structures without affecting their ability to identify the best decoy structures. We also created models using scrambled atom types, which create physically unrealistic probability functions in order to test the ability of the RF algorithm to create useful models based on inputted scrambled probability functions. From this test, we find that we are unable to create models that are of similar quality relative to the unscrambled probability functions. Next, we created uniform probability functions where the peak positions are the same as the original, but each interaction has the same peak height. Using these uniform potentials, we were able to recover models as good as the ones using the full potentials suggesting all that is important in these models are the experimental peak positions. The KECSA2 potential along with all codes used in this work are available at https://github.com/JunPei000/protein_folding-decoy-set .

A Systematic and Evidence-Based Review of Published and Pending Reports of Andexanet Alfa

Andexanet alfa is the newly approved factor Xa inhibitor reversal agent for the treatment of life-threatening or uncontrolled bleeding from apixaban or rivaroxaban. This decoy protein directly binds factor Xa inhibitors reversing their action. A systematic and evidence-based evaluation of the available clinical trials is lacking in the literature. This research will provide a systematic and evidence-based evaluation of published clinical trials for andexanet alfa. It will also present an evaluation of active research. Reports of research were identified through multiple databases using search terms andexanet alfa, andexanet, or PRT064445. The level of evidence and strength of recommendation were accomplished using the Strength of Recommendation Taxonomy. There are 2 published articles presenting 3 clinical trials. Two trials had a low level of evidence and the third trial, a preliminary report of results, showed a moderate level of evidence. The review of active research found 3 unique studies and a preliminary results study. The level of evidence is low for 2 of these studies, moderate for a third, and potentially high for the fourth. The strength of recommendation for all 6 studies is a C. Four of the studies present disease-oriented evidence resulting in a low level of evidence. Another study is unblinded and uncontrolled but presents patient-oriented evidence resulting in a moderate level of evidence. Only one study could score high because the outcome is patient-oriented evidence, and it could achieve follow-up of 80%. There is a need for well-controlled and blinded evaluation to improve the recommendation strength.

Andexanet alfa for the treatment of hemorrhage

ABSTRACTIntroduction: While associated life-threatening and fatal bleeding events are less frequent with the direct factor Xa inhibitors compared to vitamin K antagonists, significant concern surrounding management of major bleeds and urgent periprocedural interruption of these agents exists among clinicians. Andexanet alfa is a recombinant human factor Xa decoy protein developed in response to this clinical gap in the care of patients receiving anticoagulation with factor Xa inhibitors.Areas covered: This paper reviews results from preclinical and healthy-volunteer studies demonstrating the ability of andexanet to rapidly and reliably normalize coagulation indices in patients treated with both direct and indirect factor Xa inhibitors. An interim analysis from an ongoing phase 3/4b clinical study assessing the efficacy and safety of andexanet in patients experiencing life-threatening hemorrhage in association with factor Xa inhibitors is discussed. It also provides an overview of the major safety concerns reported in these trials which include allergic and infusion reactions, development of anti-andexanet antibodies and, importantly, thrombosis.Expert commentary: While initial reports on restoration of hemostasis and safety are promising, further study of andexanet is required to gauge its efficacy and toxicity, including a potential prothrombotic effect. Further, its use in patients requiring urgent surgery should be studied.

Abstract 11: Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor

Abstract Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm3 whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm3, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 11.

MP70-16 SEMA3C DRIVES CANCER GROWTH AND TREATMENT RESISTANCE VIA COGNATE LIGAND-INDEPENDENT ACTIVATION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2018MP70-16 SEMA3C DRIVES CANCER GROWTH AND TREATMENT RESISTANCE VIA COGNATE LIGAND-INDEPENDENT ACTIVATION Ario Takeuchi, Masaki Shiota, Junichi Inokuchi, Katsunori Tatsugami, and Masatoshi Eto Ario TakeuchiArio Takeuchi More articles by this author , Masaki ShiotaMasaki Shiota More articles by this author , Junichi InokuchiJunichi Inokuchi More articles by this author , Katsunori TatsugamiKatsunori Tatsugami More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2260AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our gene expression profiling data identified a member of the secreted class 3 semaphorins, SEMA3C, to be highly expressed in CRPC and AR pathway inhibitor-recurrent tumors. Increased expression of SEMA3C is associated with poor prognosis and tumor progression in a number of cancers. In prostate cancer, SEMA3C promotes cell migration and invasion in vitro and SEMA3C expression is a predictive marker for biochemical recurrence (BCR). Therefore, we investigated whether SEMA3C could be a key growth factor that drives CRPC progression and treatment resistance. METHODS The objective of this study was to evaluate SEMA3C as a therapeutic target for advanced prostate cancer and the physiological consequences of loss- and gain-of-function of SEMA3C. The expression of SEMA3C in a tissue microarray representing a total of 280 prostate cancer specimens was evaluated to determine association of SEMA3C expression with castration and treatment resistance. For in vivo orthotopic model, when mice bearing LNCaP tumors reached a tumor volume over 200 mm3 or serum PSA levels reached greater than 75 ng/ml, castration was performed via the scrotum under isoflurane anesthesia. Treatment commenced when PSA recovered to pre-castration levels. Mice were randomized into 4 groups for treatment with SEMA3C ASO or Scr with or without ENZ. For ASO treatment, Scr ASO or SEMA3C ASO was intra-peritoneally injected once daily for 7 days followed by three weekly treatments thereafter. For ENZ treatment, ENZ or vehicle was intragastrically administered once daily 5 days per week. RESULTS Here we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and c-MET in a cognate ligand-independent manner. High SEMA3C expression is associated with metastatic castration-resistant prostate cancer (CRPC). SEMA3C promotes cancer growth and castrate resistant progression while SEMA3C inhibition delays CRPC and Enzalutamide-resistant progression. Proteolytic processing of SEMA3C generates a dominant negative sema domain-containing decoy fragment that suppresses cell growth and RTK signaling. Intratumoral lentiviral delivery of this decoy protein inhibits CRPC progression. CONCLUSIONS SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e940 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Ario Takeuchi More articles by this author Masaki Shiota More articles by this author Junichi Inokuchi More articles by this author Katsunori Tatsugami More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Andexanet alfa to reverse the anticoagulant activity of factor Xa inhibitors: a review of design, development and potential place in therapy.

Direct oral anticoagulants are associated with rates of major bleeding which are not negligible, albeit lower than those associated with vitamin K antagonists. No specific reversal agent for factor Xa (FXa) direct inhibitors is currently available for clinical use. A modified activated human FXa decoy protein, andexanet alfa, is being developed that binds FXa direct inhibitors in their active site, thus reversing their anticoagulant effect. The purpose of this article is to review the design, development and clinical trials of andexanet alfa. Andexanet alfa was shown to reverse FXa inhibitors anticoagulant activity both in thrombosis animal models, healthy volunteers and patients with acute major bleeding. Andexanet alfa has been studied in double-blind, placebo-controlled phase II and III studies. A preliminary report of the phase III study showed that an effective hemostasis was obtained after andexanet alfa infusion in the majority of the patients with acute major bleeding associated with FXa inhibitors. Additional studies are ongoing and andexanet alfa is expected to be launched in the market in the near future.

Abstract 3675: Novel immune oncology strategy for targeted cytotoxic lymphocyte activation

Abstract High-dose interleukin 2 (IL2) therapy was the first FDA-approved immunotherapy, and still has an exceptionally high true long-term cure rate for metastatic cancer. However, IL2 therapy has fallen out of favor due to systemic toxicity and off-target side effects. We have recently described a novel fusion cytokine comprised of a mutant form of IL2 with inhibited α-receptor binding and an NKG2D-binding viral decoy protein called Orthopox Major Histocompatibility Complex Class I-like Protein (OMCP). Unlike wild-type IL2, our fusion cytokine (OMCP-mutIL2) binds to and activates only NKG2D expressing cytotoxic lymphocytes. This targeted IL2 activity results in superior efficacy and decreased toxicity over wild-type IL2 therapy. Here, we present data which further establishes the superior efficacy of OMCP-mutIL2 therapy as measured by biomarkers such as systemic cytokine production and tumor lymphocyte infiltration. Additionally, we present data which evaluates OMCP-mutIL2 therapy as a potentiator of PD1 checkpoint inhibitor therapy. To evaluate tumor lymphocyte infiltration mice with established Lewis Lung Carcinoma (LLC) flank tumors were treated for three days with either saline, wild type IL2, or OMCP-mutIL2 and tumor infiltrating lymphocytes evaluated flow cytometrically. A trend for increased tumor infiltrating NK cells was evident for both IL2 and OMCP-mutIL2 treated groups over saline controls, and OMCP-mutIL2 tended to increase tumor infiltrating CD8+ T cells over either IL2 or saline controls was evident as well. Additionally, tumor infiltrating CD8+ T cells in IL2-treated mice had a higher level of exhaustion over OMCP-mutIL2 treated mice as measured by PD-1 expression. Tumor infiltrating CD4+ T cells also showed a trend toward a Foxp3 regulatory phenotype in IL2 treated mice over OMCP-mutIL2. To study cytokine serum profile mice were treated with high dose OMCP-mutIL2 with or without NK depletion and serum cytokine levels measured by cytometric bead array. Serum cytokine analysis of mice treated with supra-therapeutic doses of OMCP-mutIL2 did not reveal an increase in pro-inflammatory cytokines TNF-α, IFN-γ, and IL-6. A significant increase in serum levels of G-CSF was evident in NK cell sufficient mice treated with OMCP-mutIL2. Combination immunotherapy was initiated by the addition of PD-1 blockade (clone RMPI-14) to OMCP-mutIL2 therapy in a lung LLC tumor model. Combination therapy led to near complete control of LLC growth in the lung compared to OMCP-mutIL2 or PD-1 blockade alone. The data presented here suggest that in addition to our previously published data suggesting increased safety of OMCP-mutIL2 over high-dose IL2 therapy, OMCP-mutIL2 therapy may be functionally superior to classic high-dose IL2 therapy. Continued development of OMCP-mutIL2 is ongoing. Citation Format: Eric Lazier, Sarah Hein, John Westwick, Dan Watkins, Alexander S. Krupnick. Novel immune oncology strategy for targeted cytotoxic lymphocyte activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3675. doi:10.1158/1538-7445.AM2017-3675