SUMMARY: Background rVIII-SingleChain is a novel B-domain truncated recombinant factor VIII (rFVIII) comprised of covalently bonded FVIII heavy and light chains, demonstrating a high binding affinity to von Willebrand factor. Objectives This phase III study investigated the safety, efficacy and pharmacokinetics of rVIII-SingleChain in previously treated pediatric patients < 12 years of age with severe hemophilia A. Patients/Methods Patients could be assigned to prophylaxis or on-demand therapy by the investigator. For patients assigned to prophylaxis, the treatment regimen and dose were based on the bleeding phenotype. For patients receiving on-demand therapy, dosing was guided by World Federation of Hemophilia recommendations. The primary endpoint was treatment success, defined as a rating of 'excellent' or 'good' on the investigator's clinical assessment of hemostatic efficacy for all treated bleeding events. Results The study enrolled 84 patients (0 to < 6 years, n = 35; ≥ 6 to < 12 years, n = 49); 81 were assigned to prophylaxis and three to an on-demand regimen. Patients accumulated a total of 5239 exposure days (EDs), with 65 participants reaching > 50 EDs. In the 347 bleeds treated and evaluated by the investigator, hemostatic efficacy was rated as excellent or good in 96.3%. The median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.00, 2.20), and the median annualized bleeding rate was 3.69 (Q1, Q3: 0.00, 7.20) across all prophylaxis regimens. No participant developed an inhibitor. Conclusions rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy and a favorable safety profile in a clinical study in children < 12 years of age with severe hemophilia A.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
AIM: To investigate the safety and efficacy of rVIII-SingleChain in previously untreated patients (PUPs).
METHODS: In an open-label, phase 3, extension study, PUPs with severe haemophilia A (FVIII <1%) received rVIII-SingleChain prophylactically or on-demand. The primary endpoints were incidence of high-titre (HT) inhibitor formation to FVIII, treatment success for major bleeding episodes and annualised spontaneous bleeding rate (AsBR).
RESULTS: Twenty-four PUPs (median age 1 year [range 0-5]) were treated with rVIII-SingleChain; median time on study was 35.0 months (range 2.4-54.0). Overall, six PUPs developed a HT inhibitor (>5 BU/mL) and six developed a low-titre (LT) inhibitor (≤5 BU/mL). The median number of exposure days at inhibitor development was 10 (interquartile range [IQR] 5.0-14.0). Of 11 inhibitor-positive PUPs (five HT, six LT) who continued rVIII-SingleChain therapy, nine (81.8%; three HT, six LT) achieved inhibitor eradication (<0.6 BU/mL). Median time to eradication was 14.3 weeks (IQR 9.8-53.8). Seventeen treatment-emergent adverse events in 12 PUPs (50.0%) were related to rVIII-SingleChain, mainly inhibitor development (14/17 events). Treatment was successful (haemostatic efficacy rated excellent or good) for 290/315 bleeding events (92.1%). During prophylactic therapy, inhibitor-negative PUPs had a median (IQR) AsBR of 0.52 (0.00-4.99) and annualised bleeding rate of 1.98 (0.77-11.23).
CONCLUSION: RVIII-SingleChain demonstrated a satisfactory benefit:risk profile in PUPs, with a high treatment success rate and a low AsBR during prophylaxis, and was effective at eradicating inhibitors.
METHODS AND PRINCIPAL FINDINGS: A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing.
CONCLUSIONS/SIGNIFICANCE: We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.