Circular permutation of the granulocyte colony-stimulating factor receptoragonist domain of myelopoietin

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) a nd granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety att ached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using th e original sequence numbering) were residue 39, which is at the beginning o f the first loop connecting helices 1 and 2; residue 97, which is in the tu rn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helic es 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were const rained, either by direct linkage of the termini (L0) or by replacement of t he:amino-terminal 10-residue segment with a seven-residue linker composed o f SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of bo th IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the fi rst (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a pro tein comprised of two linked four-helix bundles. The MPOs retained the abil ity to bind to the IL-3R with affinities similar to that of the parental MP O. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17 ). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemi stry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-me diated proliferative activities and conformational stabilities than those w hich had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the CpG-CSF domain. Import ant exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (lin kage at residue 39) the G-CSFR-mediated proliferative activity. MPO in whic h the IL-3R agonist domain is attached to the cpG-CSE(L1)[133/132] domain w as shown to be more potent than the coaddition of the IL-3R agonist and G-C SF in stimulating the production of CFU-GM Colonies in a human bone marrow- derived CD34+ colony-forming unit assay. Several MPOs also had decreased pr oinflammatory activity in a leukotriene C-4 release assay using N-formyl-Me t-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, d emonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.