Circular Permutation of the Granulocyte Colony-Stimulating Factor Receptor Agonist Domain of Myelopoietin

Abstract

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and 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 attached 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 the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both 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 first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. 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) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which 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. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-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, demonstrating that it is a useful tool in engineering chimeric protein...

The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist

“…33 In BaF3/G-CSFR cells, leridistim and rhG-CSF exhibit similar affinity for the receptor, however, stabilization of the G-CSF moiety of the chimeric molecule may account for the moderate increase in potency exhibited with leridistim. 32 In TF-1 cells, leridistim is less potent than rhIL-3 whereas SC-65303, and the combination of rhG-CSF plus SC-65303 were more potent than rhIL-3. This may be due to a decrease in the association rate with a corresponding decrease in the affinity of leridistim for IL-3R.…”

“…26,27,32 Recombinant G-CSF (Neupogen) and rhEPO (Procrit) were obtained from Amgen (Thousand Oaks, CA, USA).…”

“…32,39 IL-3R-dependent proliferative activity was measured using TF-1 cells while AML-193.1.3 cells were utilized to measure dual IL-3R and G-CSFR proliferative activity, as previously described. 27 …”

“…Leridistim is a specific MPO consisting of the SC-65303 sequence at the N terminus linked to a G-CSF receptor agonist (G-CSF, Ser 17 ) with a 35 amino acid sequence based on the IgG 2b hinge region. 32 In this paper, we report in vitro proliferation activity of leridistim in BaF3/G-CSFR (G-CSF receptor positive), TF-1 (IL-3 receptor positive) and AML-193.1.3 (IL-3 and G-CSF receptor positive) cell lines. The biological activity of leridistim in bone marrow CD34 + CFU assays demonstrated that leridistim was more potent than either rhG-CSF or SC-65303 alone or in equimolar combination.…”

“…31 These observations provided the rationale for the development of a family of engineered proteins termed myelopoietins (MPOs), which result from the combination of an early-acting multilineage factor (rhIL-3) with a lineage-specific factor (rhG-CSF) within a single molecule thereby engendering it with unique biological properties. 32 A prototype MPO (SC-68420) was created containing the IL-3 receptor agonist moiety present in daniplestim linked to rhG-CSF that was shown to enhance the proliferation and suppress apoptosis of responsive cell lines. [33][34][35][36] Treatment with this prototype MPO in a non-human primate model of radiation-induced myelosuppression resulted in a dramatic, dose-related improvement in the neutrophil and platelet nadir, a reduction in the duration of neutropenia and thrombocytopenia and a reduced time to recovery of neutrophil and platelet values.…”