Phase I dose-escalation study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of an inhaled recombinant human ACE2.

Background: APN01 is a soluble recombinant human angiotensin-converting enzyme 2 (rhACE2), a key player in the renin-aldosterone-angiotensin system (RAAS). In clinical studies, APN01 was administered intravenously only, so far. The aim of this study (ClinicalTrials.gov: NCT05065645) was to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of inhaled APN01. Methods: This was a phase I, double-blind, placebo-controlled, dose-escalation study. Inhalation was conducted via a nebuliser over 15 min in three single ascending dose (SAD) cohorts (n=24) and two multiple ascending dose (MAD) cohorts (n=16: every 12 h for 7 days). Doses in the SAD cohort were 1.25, 2.5 and 5 mg·mL-1; doses in the MAD cohort were 2.5 and 5 mg·mL-1. Safety (including adverse events (AEs), laboratory findings and lung function results), PK and PD data were assessed. Results: In the SAD and MAD cohorts, treatment-related AEs were slightly more frequent in the active treatment group than in the placebo group. AEs were mild to moderate, with no dose-limiting toxicities. No clinically relevant changes in lung function and laboratory results were observed. The mean maximum observed plasma concentration (Cmax) values after single and multiple doses of 5 mg·mL-1 APN01 were 1.88 and 6.61 ng·mL-1, respectively. Among the PD variables, significance was found for ACE2 and angiotensin 1-5. Conclusions: The application of aerosolised APN01 is safe and well tolerated after single and multiple doses. By achieving a high local concentration in the lungs and low systemic bioavailability, inhaled rhACE2 may present a therapeutic option in ACE2-related diseases.

Local osteo-enhancement of osteoporotic vertebra with a triphasic bone implant material increases strength-a biomechanical study.

PURPOSE: The aim of this study was to assess the biomechanical properties of intact vertebra augmented using a local osteo-enhancement procedure to inject a triphasic calcium sulfate/calcium phosphate implant material. METHODS: Twenty-one fresh frozen human cadaver vertebra (Th11-L2) were randomized into three groups: treatment, sham, and control (n = 7 each). Treatment included vertebral body access, saline lavage to displace soft tissue and marrow elements, and injection of the implant material to fill approximately 20% of the vertebral body by volume. The sham group included all treatment steps, but without injection of the implant material. The control group consisted of untreated intact osteoporotic vertebra. Load at failure and displacement at failure for each of the three groups were measured in axial compression loading. RESULTS: The mean failure load of treated vertebra (4118 N) was significantly higher than either control (2841 N) or sham (2186 N) vertebra (p < 0.05 for: treatment vs. control, treatment vs. sham). Treated vertebra (1.11 mm) showed a significantly higher mean displacement at failure than sham vertebra (0.80 mm) (p < 0.05 for: treatment vs. sham). In the control group, the mean displacement at failure was 0.99 mm. CONCLUSIONS: This biomechanical study shows that a local osteo-enhancement procedure using a triphasic implant material significantly increases the load at failure and displacement at failure in cadaveric osteoporotic vertebra.

Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines.

Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)-y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi-mature stage (smDCs) retaining the capacity to secret interleukin (IL)-12 and thus support cytolytic T-cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 +/- 4% recovery, which was approximately 5-fold higher as the most frequently used adherence protocol (8 +/- 1%), and a very good purity (92 +/- 5%) of smDCs. Immune phenotype and IL-12 secretion (adherence: 1.4 +/- 0.4; selection: 20 +/- 0.6; depletion: 1 +/-0.5; elutriation: 3.6 +/- 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL-12 (9.4 +/- 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.

Semi-mature IL-12 secreting dendritic cells present exogenous antigen to trigger cytolytic immune responses.

Dendritic cells (DC) are candidates for antigen-presenting cells that present exogenous antigen on MHC class I molecules to cytotoxic T lymphocytes (CTL), a process referred to as cross-priming. We triggered interleukin (IL)-12 release from DC, which was limited to the first day after maturation induction, by a combination of lipopolysaccharide (LPS) and interferon (IFN)-gamma. To stimulate T lymphocytes, we used soluble protein derived from lysis of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) or ovalbumin loaded onto DC. Co-culture was initiated 2-6 or 48 h after maturation corresponding to "semi-mature" actively IL-12-secreting type 1 DC (sm-DC1) or a "fully mature" DC1 that had lost the ability to release IL-12 (fm-DC1), respectively. IL-12-secreting sm-DC1 but not fm-DC1 efficiently triggered cytolytic activity in autologous T lymphocytes. The combination of IL-1beta, IL-6, TNF-alpha, and prostaglandin E2 generated type 2 DC that did not secrete IL-12 (DC2) and could not prime T-cell cytolytic activity. However, supplementation of cultures using DC2 with IL-12 resulted in CTL activity while the presence of anti-IL-12 monoclonal antibodies in cultures using IL-12 secreting sm-DC1 suppressed CTL activity. Thus, actively IL-12-secreting sm-DC1 are necessary and sufficient for the antigen-specific expansion of CTL in response to exogenously provided soluble antigen.