Although most patients with cancer have some form of immune dysfunction, these patients often have immune effector cells that can readily be activated with cytokines including IL-2 and GM-CSF. These activated cells, however, do not have the capability of selectively recognizing or destroying neoplastic tissue. Certain tumor-reactive monoclonal antibodies and their engineered derivatives can recognize determinants preferentially expressed by certain cancer cells. Some of these tumor bound monoclonal antibodies can activate Fc receptor-bearing effector cells. Enhanced stimulation of these, as well as FcR-, effector cells at the site of tumor cell surfaces can be induced by fusion proteins that link tumor-reactive monoclonal antibodies to cytokines. In this project a new antibody-IL2 fusion protein will be tested in pre-clinical in vitro and in vivo model systems. Of primary importance will be the testing of novel mechanisms for in vivo delivery of the fusion protein (FP) based on the use of particle mediated gene transfer (PMGT). The antibody moiety is an engineered construct fusing the variable region from the CC49 monoclonal antibody, recognizing the tumor associated antigen TAG-72, with human constant heavy domains. By splicing this single chain antibody gene to the IL-2 gene, a fusion protein has been created that should deliver IL-2 to the surface of TAG-72+ tumors, thereby activating IL-2 inducible effector functions utilizing human immune cells at the site of these tumors. The main goal of this project is to test the immunologic activity of this CC49-IL2 fusion protein in vitro and in vivo and to determine the feasibility of using PMGT as a means of delivering this protein to local and disseminated sites of TAG-72+ tumors. It is specifically proposed: (1) to analyze the expression for distinct vector systems expressing the CC49-IL2 gene, in order to identify regulatory elements that increase transgene expression, and to delineate a useful vector for subsequent experiments; (2) to test distinct in vivo PMGT delivery regimens to identify additional variables that may influence local and systemic expression levels; (3) to utilize the most promising vector delivery system in tumor-bearing mice to assess the ability of PMGT delivered protein to bind and penetrate TAG-72+ tumor. Tumor sites located adjacent to as well as distant from the PMGT sites will be studied and (4) to establish an immunotherapy using TAG-72+ xenografts in combination with adoptively transferred human effector cells to test whether PMGT can result in protein levels sufficient for immunotherapeutic effects.
