ASK-lAb

Therapeutic monoclonal antibodies have had a tremendous impact on treating difficult diseases and conditions with precise targeted therapies. However, in some individuals the occurrence of anti-drug antibodies following administration of the therapeutic monoclonal antibody occurs. This is a growing problem that is attracting attention from frontline clinicians and drug developers. We developed a platform for the detection of IgG anti-drug antibodies that may provide an initial screen for all therapeutic monoclonal antibodies.

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In the ASK-Lab, The variable heavy and light domain encoding region of known antibody drugs are combined with nanoluciferase reporter to generate recombinant GloBodies, which retain the drug antibody paratopes and Nanoluciferase activities. In the presence of anti-drug antibody molecules, the GloBody is bound by specific IgG in the sample. These complexes are captured on immobilised protein G and the luciferase activity determined. The amount of light generated being indicative of the anti-drug antibody levels in serum. It should be possible to assemble GloBody reagents for all therapeutic monoclonal antibodies and adapt the capture phase to include additional specific isotypes. The assay has been developed for use with plasma, serum and IgG eluted from dried blood spots.

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Our areas of interest for ADA are biologic drugs used in the treatment of multiple sclerosis, haemophilia A and asthma.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signalling pathways involved in the progression and metastasis of cancer where it is found to be upregulated. Expression of Dsg3 impacts the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB light, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 seemed to be Dsg3 specific since neither E-cadherin nor desmoplakin knockdown rendered similar effect. Analysis of Dsg3-/- mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 compared to Dsg3+/- controls.

We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies that demonstrated the treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied with disruption of cell-cell adhesion. Collectively, our findings implicate a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and suggest this pathway is disrupted in PV.

Chronic periodontal disease and related systemic pathologies are initiated and perpetuated by specialist pathogens such as Porphyromonas gingivalis . Small exosome-like structures released in the form of outer membrane vesicles (OMVs) are a reservoir for key virulence factors of this perioodontopathogen. OMVs have invasive properties permitting them to cause local tissue destruction and to travel to other parts of the
body to contribute to the development of chronic life-threatening systemic disorders such as Alzheimer's
Neurodegeneration.

In the ASK-Lab, we harnessed the natural diversity of the human antibody response and assembled a scFv antibody library (VH/VL orientation) using ARepCatch™ technology. Using A2RT™ selection against OMVs, we selected a panel of monoclonal antibodies against the OMVs of P. gingivalis. Briefly, OMV immune serum IgG was captured on immobilised Protein G. After washing OMVs were captured by the immobilised antibodies. The captured OMVs were used to select the invitro transcribed and translated antibody mRNA ribosome complexes. The bound complexes were recovered and cDNA encoding the binding scFvs prepared.

We aim to expand this scFv antibody library, test their efficacy in mammalian cells and to identify actual OMV inhibitors that will serve as candidates for pre-clinical evaluation. The ultimate goal is to develop a therapeutic monoclonal antibody that will provide utility in preventing or reversing periodontal disease orchestrated by Porphyromonas gingivalis.

Behçet’s syndrome is an inflammatory condition that almost affects any of the parts of the body. The mouth, skin and genital areas are the most commonly affected. Often the skin and the lining mucosa of the mouth and genital organs become inflamed causing ulcers and rashes. Inflammation of the eyes and blood vessels is common to occur. Pain in the joints (arthritis), headache and stomach pain may also happen. In very rare conditions, the brain, spinal cord or nerves become involved. These clinical manifestations of Behçet’s syndrome are believed to be due to vasculitis. The inflammation occurs most commonly in veins of the lower extremities resulting in deep venous thrombosis while blood clots are less common to occur in arteries.

Behçet’s syndrome is more common along the old silk trading routes extending from the Far East to the Mediterranean Sea. There is no cure for Behçet’s syndrome, but the aim of any treatment is to resolve signs and symptoms of the disease and prevent the occurrence of any complications. Therefore, developing a better understanding of the syndrome at the molecular and cellular level is key for the development of interventions to reduce the burden of disease and to enhance the quality of life