The objective of this proposal is threefold: A) To investigate the varieties of functional antibodies that plant cells are capable of assembling: B) to initiate a critical evaluation of the potential of plant produced antibodies for possible therapeutic applications and C) to train students in advanced molecular biology techniques. The specific scientific aim of the proposal is to determine whether isotypes of the MAb 14.18 (lgG3), which recognizes ganglioside GD2 on the surface of tumors of neuroectodermal origin, can be independently expressed in plants, the gene for the k chain and the gene for four g chain isotype variants will be cloned as full length cDNAs and used to transform tobacco and alfalfa cells. We will then evaluate the efficacy of plant-produced MAb 14.18 for expressing Fc-mediated effector functions. Each isotype will be tested in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) assays. These results will be compared with those of identical assays performed with the parental murine antibody of each isotype. To analyze the in vivo behavior of these antibodies, they will be tested for serum clearance and for biodistribution in athymic (nu/nu) mice bearing M21 tumor xenografts expressing the GD2 antigen. Students working on this project will learn the most advanced techniques in molecular biology and biotechnology. This project provides a unique spectrum that spans both plant and animal molecular biology. This proposal is significant since it provides the opportunity to explore for the first time some of the unique capabilities of plant cells for assembly of various immunoglobulin structures that could ultimately be of therapeutic value. These structures will include light chains assembled with: 1) heavy chains derived from inhibitor treated plant cell cultures in which glycosylation will be absent or modified; 2) isotypes of a murine monoclonal antibody (14.18) which recognize GD2 and will afford a direct in vivo comparison of the potential therapeutic efficacy of the plant and hybridoma antibodies. We have focused on these objectives in order to address the critical questions of 1) the scope of the plant cell's ability to process and assemble immunoglobulins and 2) the fidelity of function preserved in plant produced antibodies. The answers to these questions will help determine the therapeutic utility of plant produced antibodies. It has been shown that plant cells provide an efficient system for the synthesis and assembly of antibodies which possess the antigen-binding functions of the corresponding hybridoma-produced antibodies. this establishes plants as a novel heterologous system for an in-depth study of the structural factors affecting antibody processing and assembly as well as determinants of antibody function. All techniques and methods necessary to carry out the proposed research have been developed and are currently available.
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