Recombinant proteins are gaining wide use as pharmaceuticals to treat patients with cancer and its sequelae. One problem with the use of these agents is the frequency with which injections must be made in order to maintain a therapeutic level in the circulation. It has been shown that erythropoietin dimers produced by chemical crosslinking or in the form of a fusion protein have enhanced potency and efficacy in vitro and in vivo. It is proposed that the molecular, cellular and pharmacological mechanisms underlying these observations be studied in detail in vivo studies in rodents and monkeys. The interaction of erythropoietin with soluble erythropoietin receptor and the demonstration that a stoichiometry of two receptors per erythropoietin molecule exists will be documented. The principal investigator will also show that an inactive mutant erythropoietin binds only one receptor. The principal investigator will study the interaction of erythropoietin with the membrane receptor and provide direct proof of membrane receptor dimerization by erythropoietin. They will study the affinity and binding stoichiometry of crosslinked erythropoietin dimmers and the fusion protein for the soluble erythropoietin receptor and will compare them with these values for both Site 1 and Site 2 mutants. The interaction of crosslinked dimers and fusion protein with the membrane bound receptor including affinity, internalization and degradation, and demonstration of receptor tetramer formation will be studied. Enhanced signaling from these tetramers will be studied using reporter constructs induced by the Ras/MAPK and JAK/STAT pathways, respectively. The pharmacokinetics and pharmacodynamic characteristics of dimmer and fusion protein will be compared with those of the monomer in rats and monkeys. The potential immunogenicity, structure and stability of these compounds will be studied. The results of these experiments will lay the foundation for the design of novel recombinant proteins for cancer therapy.
