Background:
B cells play an important role in a number of autoimmune diseases through the production of antibodies that bind to tissues and cause end organ damage. Rituximab is a highly effective treatment for antibody driven autoimmune disease and targets CD20 that is expressed on the surface of B cells. Rituximab causes death of B cells by a number of mechanisms, including complement activation and cell mediated death. Administration of rituximab results in potent B cell depletion in the blood but it does not result in the clearance of tissue resident B cells. This is likely to be the reason why it fails to cure autoimmune disease, as pathogenic B cells can ‘hide’ in tissues such as skin. The reasons for incomplete tissue clearance remain unknown and may relate to reduced complement or cell mediated B cell apoptosis in the tissue microenvironment.
We have recently developed a 3D skin model which allows the study of anti-CD20 antibodies in the skin microenvironment. We have also engineered antibodies that target proteins found on cancer cells that have modifications that enhance cell mediated cytotoxicity.
Project aims:
This project will aim to bioengineer a next generation anti-CD20 antibody with significantly greater cytotoxicity than rituximab. The key project aims are:
1. Bioengineer a range of fully human anti-CD20 antibodies with modifications to the Fc region that increase cell mediated cytotoxicity. Plasmids would be created and human embryonic kidney (HEK293/Expi293F) cells transfected to clone antibodies (Year 1)
2. Assess CD20 binding by the bioengineered antibody using flow cytometry and antibody dependent cytotoxicity and phagocytosis assays (Years 2)
3. Investigate the ex vivo activity of rituximab using a 3D skin model. This will involve populating the skin model with B cells and assessing the cytotoxicity of bio-engineered anti-CD20 antibodies versus rituximab in the presence or absence of complement and other immune cells (Years 2-4).
The outcome of this project could therefore lead to the generation of a novel anti-CD20 therapeutic antibody that could treat a wide range of autoimmune diseases.
Techniques and skills
Antibody engineering and cloning, cell culture, cytotoxicity assays, flow cytometry, ELISA.
3-month project outline?
Human embryonic kidney cells will be transfected with commercially manufactured plasmids encoding preliminary antibody sequences. A small range of cloned antibodies would then be tested for CD20 binding by flow cytometry and their cell mediated killing will be assessed initially using antibody dependent cytotoxicity and phagocytosis assays.
