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P06.09 In vitro assessment of human IL-12Fc function on porcine and canine peripheral blood mononuclear cells

ABSTRACT: Abstract Introduction: Despite state-of-the-art surgical methods combined with radio- and chemotherapy the clinical prognosis of brain cancer patients remains poor. The brain is an immune privileged site and brain cancers are known to form a highly immunosuppressive microenvironment, suggesting that patients could benefit from immunomodulatory drugs. Multiple therapies targeting the immune system are currently under development, including application of a potent activator of immune responses, interleukin (IL)-12. This cytokine stimulates the cytotoxic function of lymphocytes and can trigger rejection of gliomas in preclinical studies. Even though IL-12 has a relatively short half-life, systemic IL-12 can quickly lead to severe adverse reactions. Local application or targeting is therefore mandatory. Local IL-12 therapy is currently tested in early stage clinical trials of intratumoral genetherapy using viral vectors (ClinicalTrials.gov identifiers NCT02026271 and NCT02062827). An alternative approach is the use of convection-enhanced delivery (CED). This way IL-12 could be dosed precisely to the tumour focus with minimal systemic leakage. Furthermore, to increase the local half-life of IL-12, we have fused it with the Fc part of human IgG4 (IL-12Fc). Using CED, we are aiming to validate efficacy and safety of local IL-12Fc monotherapy in a preclinical setting with canine brain tumours. Materials and Methods: Canine peripheral blood mononuclear cells (PBMCs) were stimulated with increasing concentrations of human IL-12Fc or with recombinant human or canine IL-12 in presence or absence of concanavalin A. Interferon-? (IFN-?) production was measured by ELISA. Phosphorylation of STAT4 was measured by western blot. Porcine PBMCs were stimulated in the presence of recombinant porcine IL-2 and IL-18. IFN-? production was measured by ELISA. Results: Human IL-12Fc is able to stimulate canine and porcine PBMCs to secrete IFN-? in a dose-dependent manner. Furthermore, stimulation with hIL-12Fc leads to phosphorylation of STAT4. Conclusions: Human IL-12 immunotherapeutics are able to stimulate canine PBMCs and thus therapy of spontaneous brain tumours in dogs can be used for preclinical testing. To estimate leakage and brain perfusion in the dog and ultimately in humans, we will next test CED of hIL-12Fc in large white landrace pigs. We are aiming to initiate a trial to assess the efficacy of local delivery of human IL-12Fc as therapy for canine brain tumours.

SUBMITTER: Schellhammer L

PROVIDER: S-EPMC5464105 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlzheimer's disease (AD) is the most common form of dementia, and it affects more women than men. Mitochondrial dysfunction (MD) plays a key role in AD, and it is detectable at an early stage of the degenerative process in peripheral tissues, such as peripheral mononuclear blood cells (PBMCs). However, whether these changes are also reflected in cerebral energy metabolism and whether sex-specific differences in mitochondrial function occur are not clear. Therefore, we estimated the correlation between mitochondrial function in PBMCs and brain energy metabolites and examined sex-specific differences in healthy participants to elucidate these issues.MethodsThe current pilot study included 9 male and 15 female healthy adults (mean age 30.8?±?7.1 years). Respiration and activity of mitochondrial respiratory complexes were measured using a Clarke-electrode (Oxygraph-2k system), and adenosine triphosphate (ATP) levels were determined using a bioluminescence-based assay in isolated PBMCs. Citrate synthase activity as a mitochondrial marker was measured using a photometric assay. Concentrations of brain energy metabolites were quantified in the same individuals using 1H-magnetic resonance spectroscopy (MRS).ResultsWe detected sex-associated differences in mitochondrial function. Mitochondrial complexes I, I+II, and IV and uncoupled respiration and electron transport system (ETS) capacity in PBMCs isolated from blood samples of females were significantly (p?<?0.05; p?<?0.01) higher compared to males. ATP levels in the PBMCs of female participants were approximately 10% higher compared to males. Citrate synthase (CS) activity, a marker of mitochondrial content, was significantly (p?<?0.05) higher in females compared to males. Sex-associated differences were also found for brain metabolites. The N-acetylaspartate (NAA) concentration was significantly higher in female participants compared to males in targeted regions. This difference was observed in white matter (WM) and an area with a high percentage (>?50%) of gray matter (GM) (p?<?0.05; p?<?0.01). The effect sizes indicated a strong influence of sex on these parameters. Sex-associated differences were found in PBMCs and brain, but the determined parameters were not significantly correlated.ConclusionsOur study revealed sex-associated differences in mitochondrial function in healthy participants. The underlying mechanisms must be elucidated in more detail, but our study suggests that mitochondrial function in PBMCs is a feasible surrogate marker to detect differences in mitochondrial function and energy metabolism in humans and it underscores the necessity of sex-specific approaches in therapies that target mitochondrial dysfunction.

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P06.09 In vitro assessment of human IL-12Fc function on porcine and canine peripheral blood mononuclear cells

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