Antibodies have become the most important and rapidly expanding class of biotherapeutics. There are 12 FDA approved antibodies for human use and 25% of the drugs currently under development are based on antibodies. While most of the antibodies in use and in clinical trials are for systemic administration, the greatest potential for antibodies lies in their topical administration for both prevention and treatment of infectious diseases at the mucosal surfaces. This is because a) most of the infections are initiated at the mucosal surfaces, b) from a public heath perspective prevention is very important, c) direct application of antibody to the site of infection will block the pathogen entry, d) local administration of antibodies will provide effective concentrations of the drug immediately, and e) topical application is an easy and attractive mode of administration. ? Secretory IgA (S-lgA) is the effector antibody at the mucosal surfaces. Production of S-lgA requires the expression of four polypeptides; heavy, light, J chain and secretory component. As a first step towards the commercial production of S-lgA, we have obtained the proof of concept that heavy, light, J chain and secretory component can be expressed in a single cell system for the production of S-lgA. However, using this cell line, the cost of large-scale production of S-lgA is estimated to be about $100,000 per gram of antibody. We now hypothesize that by using vectors containing glutamine synthetase (GS) for selection and expressing SC and J chain and H and L chains as a dicistronic mRNAs in non-lymphoid cells, it is possible to produce at least 1000 fold more S-lgA than the current cell line can produce. To test this hypothesis, we propose the following specific aims. First, to construct optimal expression vectors to express H, L, J chains and SC in mammalian cells to produce large quantities of S-lgA. We will generate two vectors containing the GS gene amplification system to co-express SC and J chain and H and L chains connected by the internal ribosomal entry site sequence (IRES). Second, to produce stable transfectants of Chinese hamster ovary and the human kidney 293T cells to produce gram quantities of S-lgA. Finally, to characterize recombinant S-lgA with respect to carbohydrates present on S-lgA, its binding to antigen and its stability in presence of enzymes usually present in mucosal secretions. ? ?
