Two areas of research both related to killing of human tumor cells by monoclonal antibody and complement are proposed in this grant application. One area concentrates on molecular mechanisms of complement-mediated killing initiated by either a complement-activating monoclonal antibody or antibody-cobra venom factor (CVF) conjugates. This project includes the analysis of cellular defense mechanisms against complement attack and their modulation by metabolic inhibitors. In particular, we want to investigate the role of membrane components in the slow degradation of C3b on complement-susceptible cells and of the rapid degradation on complement-resistant cells. Additional studies will elucidate the role of antigen expression and internalization of surface-bound complement components in determining complement susceptibility. The kinetics of binding of complement components C4, C5 and C9 will be studied. We will investigate the effect of adriamycin to enhance complement susceptibility. These studies are directed to identify the membrane targets of the adriamycin action and the mechanism by which adriamycin achieves the enhanced complement susceptibility. Based on the observation that immobilized adriamycin can enhance the complement susceptibility in vitro we wish to generate antibody conjugates with adriamycin, and to test them for their in vitro activity, their in vivo pharmacokinetic properties, and their therapeutic activity in tumor-bearing animals. We also wish to identify other complement-enhancing drugs and their mechanisms of action. The second area of research will focus on covalent conjugates of monoclonal antibodies with CVF as cytotoxic agents. Initially, we wish to find a structural basis for the recently discovered activity differences of CVF isolated from different Naja naja subspecies. The investigations on the effect of heterobifunctional cross-linking reagents on activity and pharmacokinetic properties of antibody-CVF conjugates will be continued with new cross-linking reagents. Another project is to analyze the localization of antibody-CVF conjugates into tumors. Subsequently, the immunotherapeutic effect of antibody-CVF treatment in tumor-bearing animals will be studied. Finally, monoclonal antibodies to CVF will be used as a tool to identify immunogenic epitopes and functional sites on CVF, cobra C3, and human C3 as an approach to study the structure/function relationships of these homologous proteins.
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