Convalently crosslinked hybrid toxins containing a toxin active chain and a receptor-binding protein may be potential pharmacological agents for site specific cancer chemotherapy. A number of hybrid toxins have been synthesized and shown to achieve cell type specificity but low toxicity. The overall objective of this proposal is to seek an understanding of the mechanism of cell intoxication by hybrid toxins in vitro so as to enable us to redesign a new generation of hybrid toxins with both cell type specificity and high toxicity. Initially the mechanism of intoxication of isolated rat hepatocytes by the disulfide and thioether conjugates of diptheria toxin fragment A (DTA) and asialoorosomucoid (ASOR) will be studied as a model system. The intracellular compartment(s) from which the hybrid DTA is released into its cell cytosol site of action will be identified kinetically by incubating the cells with the hybrid toxin in the absence and presence of inhibitors of ligand transport and degradation which are capable of enhancing the toxicity of the hybrid and measuring the rates of cell intoxication. The site of DTA release can be deduced from the known site of action of the inhibitor producing maximum enhancement of toxicity of the hybrids. This will be confirmed by measuring the rates of DTA release into cell cytosol under the same experimental condition using a sensitive DTA enzymatic assay and radioimmunoassay for the conjugate subunits. The rate of appearance of the hybrid toxin in various subcellular fractions also will be determined by Percoll density gradient centrifugation using the same assays. The kinetics of uptake and degradation of radioactively labelled conjugates will be studied and the results will be compared with those obtained from the above experiments. Conjugates of DTA with glycopeptide fragments of ASOR will be prepared and similarly studied to see if alteration in receptor-ligand interaction will change the toxicity of the conjugate. The conjugates of intact diphtheria toxin (DT) and DTA to ASOR as well as glycoconjugates of DT and DTA also will be prepared and studied for their toxicity so as to see if inclusion of the toxin binding chain will enhance toxicity of the conjugates. The toxicity of conjugates of DT and DTA will be studied in the toxin sensitive guinea pig hepatocytes to determine if there is a species difference in the entry mechanism of these hybrid toxins. The information obtained from these studies will be useful for future design of more effective hybrid toxin for studying site-specific cancer chemotherapy.
