We propose to clone the genes for the heavy and light chains of mouse hybridoma T84.66, which produces a monoclonal antibody with the best available affinity and specificity to CEA. The variable regions of these genes will be characterized and then used to make transfectomas (lymphoid cells expressing cloned immunoglobulin genes). We will use exon shuffling techniques to construct transfectomas producing chimeric antibodies with the T84.66 variable regions and human constant regions. Yield improvement experiments will be conducted using a colony screening technique. We will make additional plasmid constructions for the production of hypervariable loop grafted antibodies. The starting point will be synthetic genes for the variable regions of the heavy and light chains of another CEA antibody, CEA.66. These experiments include molecular modeling and site-directed mutagenesis to test the ability to convert an antibody of one epitope specificity to that of another (T84.66) by changing only the hypervariable regions. The synthetic antibody has built-in restriction sites facilitating the replacement of the complement determining regions (CDRs). Refinement of the model for the antigen-combining site will be performed by incorporating results from these experiments with those on the antigen structure performed in Project 1. This work should lead to the production of antibodies with improved specificity and affinity for CEA.
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