The main objective of this research project is to genetically engineer immunoglobulin genes to generate useful reagents for in vivo localization and therapy of human tumors. Several hybridoma cell lines have been developed in this laboratory that produce monoclonal antibodies (MAbs) that selectively react with tumor-associated antigens. These include the carcinoembryonic antigen and a tumor-associated glycoprotein, TAG-72, which is present in a variety of carcinomas. The MAbs are currently being used in a number of diagnostic and therapeutic trials on breast, colon and ovarian cancers. In clinical tests, two such MAbs, B72.3 and CC49, which were raised against the tumor associated antigen TAG-72, have shown promise for being developed into diagnostic and therapeutic agents. The usefulness of mouse MAbs for in vivo therapy and diagnosis of human tumors, however, is limited because of their immunogenicity in patients. This problem can be reduced by replacing the constant region of the mouse antibody with the constant region of the human antibody, using recombinant DNA techniques. To that end, we have cloned the rearranged heavy and light chain variable regions as well as the cDNA copies of the heavy and light chain gene messages of the MAb, B72.3. Chimeric heavy and light chain immunoglobulin genes have been constructed, inserted separately into appropriate expression vectors, and stable chimeric antibody-producing cell lines were developed by introducing these constructs into both fibroblast and myeloma cell lines. Chimeric antibody produced either from cDNA clones or clones of the rearranged genes retained specificity and binding properties of the parental antibody. A novel type of recombinant molecule, a single chain antigen binding protein, has also been constructed and characterized. The molecule, also termed a single chain Fv, was constructed and expressed in E. coli, and is composed of a variable light chain amino acid sequence tethered to a variable heavy chain sequence by a designed peptide.
