Cobra venom factor (CVF) is a nontoxic, complement-activating glycoprotein from cobra venom. In mammalian serum, CVF can effect exhaustive complement activation. This unique property of CVF has been used to impart a complement-activation effector function to tumor cell- specific antibodies for selective killing of target cells. However, CVF contains oligosaccharides terminating with Gal alpha 1-Gal Beta 1 epitopes, which are highly immunogenic in humans and react strongly with naturally occurring human anti-Gal alpha 1-3Gal Beta 1 antibodies. Therefore it is essential to eliminate this alpha-Gal-dependent immunoreactivity in order to study CVF-antibody conjugates as cancer therapeutic agents. Since oligosaccharides of CVF are not required for its activity, this problem can be addressed. The long term objective is to study CVF as a cancer therapeutic agent. The objectives of the current proposal are to: a) address the problem of alpha-Gal-dependent immunoreactivity, b) obtain therapeutically effective immunoconjugates by preserving the activities of both CVF and the antibody, and c) evaluate the efficacy of immunoconjugates in selective killing of tumor cells. To accomplish these goals, immunoconjugates will be prepared either by using deglycosylated CVF or by employing oligosaccharide chains of CVF as attachment sites to couple to the antibody. It is anticipated that more potent immunoconjugates will be obtained by attaching CVF to the antibody via carbohydrate chains. The efficacy of the prepared immunoconjugates will be assessed in model systems.
Three specific aims are proposed: 1. The goals of Specific Aim 1 are: a) to prepare deglycosylated CVF; b) to modify carbohydrate moieties of CVF to generate aldehyde groups which can be used as specific sites for attachment of the antibody while eliminating anti-alpha-Gal immunoreactivity; c) to study the effect of these modifications on the functional activity of CVF and its immunoreactivity to human anti-alpha Gal antibodies; d) to determine the actual glycosylation sites and structures of the oligosaccharides in CVF. 2. The CVF-antibody conjugates will be prepared by the following approaches: a) coupling deglycosylated CVF to the target-specific antibody using heterobifunctional crosslinking reagents; b) using the terminal sugar residues of oligosaccharide chains in CVF as specific sites of linkage via aldehyde groups to achieve controlled and uniform conjugation while eliminating the immunoreactivity of the carbohydrate epitope in CVF; c) coupling the antibody and CVF through the carbohydrate moieties of both glycoproteins. The activities of bound CVF and the antibody will be assessed. 3. The efficacy of the CVF-antibody conjugates will be evaluated using in vitro and in vivo models. In vitro experiments will be performed to study the stability, selective binding to tumor cells and cytotoxic potential of CVF-antibody conjugates. Nude mice bearing the target tumor cells will be used as a model to study the selective targeting of immunoconjugates to tumor cells and their ability to prevent tumor growth.
