The main objective of this research project is to study structure- function relationships of recombinant immunoglobulin molecules and to develop novel immunological reagents for the diagnosis and therapy of human carcinomas. Several murine monoclonal antibodies (MAbs) with selective reactivity to carcinoma associated antigens, including tumor associated glycoprotein (TAG)-72, have been developed in this laboratory. Among those MAbs which react with the TAG-72 antigen, MAb CC49 has shown excellent tumor localization in recent clinical trials and is thus serving as a prototype recombinant immunoglobulin. The thrust of our effort is to obviate the problem of the immunogenicity of the murine CC49 in patients, and to optimize Ig phamacokinetics and metabolism. To this end, we cDNA cloned the genes encoding the heavy and light chains of the MAb CC49, and subsequently developed a mouse- human chimeric (c) CC49. We have now developed, purified and characterized a """"""""humanized"""""""" CC49 (HuCC49), which is expected to have even a greater reduced immunogenicity in humans. Previous clinical trials with murine CC49 have also shown antivariable region responses. Therefore, one of the current efforts is aimed at identifying those MAb CC49 CDRs, which, in part or in their entirety, may be dispensable for binding to the TAG-72 antigen. To maximize the human content of the HuCC49, we wish to replace the dispensable CDRs, or parts of them, with sequences derived from human antibody CDRs. To optimize the pharmacokinetics of MAb CC49, we have developed a CH2 domain-deleted cCC49 and evaluated its plasma clearance and tumor targeting properties. More recently, we have also developed a CH2 domain-deleted HuCC49. Attempts are also underway to use the baculovirus system to express and purify these potential clinical reagents, in a cost effective way. To facilitate ex vivo transfection and in vivo expression of the recombinant CC49, we have developed single-gene constructs encoding single-chain immunoglobulin (SCIg) molecules. These SCIgs have been expressed, purified and characterized from both transfected SP2/0 mouse myeloma and insect cells. One such molecule, a single-chain immunoglobulin-IL-2 fusion protein, SCIg-IL-2, has been expressed in mice, using both a transcutaneous gene gun and intramuscular injection. The fusion protein was readily detected at the site of the gene delivery as well as in the animal sera. In addition, cellular and humoral immune responses to the in vivo expressed fusion protein have been detected. This technique could thus also provide a unique approach for the activation of cytotoxic lymphocytes for targeting antigen bearing cancer cells.
