Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

Myelopoietins comprise a class of chimeric cytokine receptor agonists consi sting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (hum an granulocyte colony-stimulating factor) receptor agonist linked head-to-t ail at their respective carboxy and amino termini. The combination of an ea rly acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficien t hematopoeitic reconstruction following myeloablative insult, and drives d ifferentiation of non-myelocytic lineages (ie thrombocytic lineages) that a re inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filament ous bacteriophage: both component agonists of myelopoietin were presented i n biologically functional conformations as each recognized its correspondin g receptor. Five amino acid positions in a short region of the hG-CSF recep tor agonist module of myelopoietin that had been identified as important fo r proliferative activity were mutagenized. Display was used because it allo ws very 'deep' mutagenesis at selected residues: > 10(5) substitution varia nts were affinity-screened using the hG-CSF receptor and 130 new, active va riants of myelopoietin were identified and characterized. None of the selec ted variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may alr eady be optimal at these positions for the assays used, and potentially acc ounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previou s alanine scanning, and other genetic and biochemical data for hG-CSF varia nts.