New insight into strategies used to develop long-acting G-CSF biologics for neutropenia therapy

Abstract

Over the last 20 years, granulocyte colony-stimulating factors (G-CSFs) have become the major therapeutic option for the treatment of patients with neutropenia. Most of the current G-CSFs require daily injections, which are inconvenient and expensive for patients. Increased understanding of G-CSFs' structure, expression, and mechanism of clearance has been very instrumental in the development of new generations of long-acting G-CSFs with improved efficacy. Several approaches to reducing G-CSF clearance via conjugation techniques have been investigated. PEGylation, glycosylation, polysialylation, or conjugation with immunoglobulins or albumins have successfully increased G-CSFs' half-lives. Pegfilgrastim (Neulasta) has been successfully approved and marketed for the treatment of patients with neutropenia. The rapidly expanding market for G-CSFs has increased demand for G-CSF biosimilars. Therefore, the importance of this review is to highlight the principle, elimination's route, half-life, clearance, safety, benefits, and limitations of different strategies and techniques used to increase the half-life of biotherapeutic G-CSFs. Understanding these strategies will allow for a new treatment with more competitive manufacturing and lower unit costs compared with that of Neulasta.

Keywords: G-CSF; long-acting; neutropenia; strategy; therapy.

Figure 1

Plan structures of pegfilgrastim. Covalently bound of a single 20-kDa PEG molecule attached to the recombinant N-terminal methionine (r-met) residue of rG-CSF.

Figure 2

Biosynthesis and structure of O- and N-linked glycans. (A) Initial biosynthetic pathway of O-glycans with an O-GalNAc moiety starts in the Golgi apparatus, where a GalNAc residue is attached by different polypeptide polypeptide GalNAc transferases (GlcNAcT). Later, a few glycosyltransferases can convert the resulting glycoprotein into different core structures. (B) The biosynthetic pathway of the early input glycan (9-mannose glycan) begins in the endothelial reticulum (Top). Then, glycoprotein migrates to the Golgi, where the mannose group is removed, and other monosaccharides are added in a hybrid type process (mid). The biosynthetic process is then finished as a completely sialylated glycan complex in the Golgi (Bottom). Man, mannose; Gal, galactose; GlcNAc, N-acetylglucosamine; Fuc, fucose; NeuAc, sialic acid; GalNAc, N-acetylgalactosamine.

Figure 3

Analysis of Western blot expressing and comparing G-CSF tandems and their controls. First lane, GCSF2QAT_control (2 × QAT). Second lane, GCSF2NAT (2 × NAT). Third lane, GCSF4QAT_control (4 × QAT). Fourth lane, GCSF4NAT (4 × NAT). Fifth lane, GCSF8QAT_control (8 × QAT). Sixth lane, GCSF8NAT (8 × NAT). Analysis of Western blot displays an obvious increase in molecular weight for glycosylated GCSF tandems (GCSF2NAT, GCSF5NAT, and GCSF8NAT) when compared with non-glycosylated controls (GCSF2QAT, GCSF4QAT, and GCSF8QAT).

Figure 4

Plan structures of lipegfilgrastim. Conjugation of a single 20-kDa PEG–sialic acid (Sia) to a natural O-linked glycan moiety bound at the Thr-134 residue site of rG-CSF.

Figure 5

Proposed model of albumin to FcRn in a pH-dependent manner at the cell membrane. Green (domain I), blue (domain II), and red (domain III) indicate the three domains of albumin. Domain III albumin binds to the FcRn receptor at an acidic pH of 6.0, protecting it from lysosomal destruction, and then recycles to the blood circulation, where it is released when exposed to physiological pH 7.4.

Figure 6

Fusion of G-CSF to Fc and CH domains. (A) The carboxy-terminus of human GCSF is fused to the amino termini of the IgG-Fc and IgG-CH domains by a 7–amino acid fixable linker (L). (B) The CH1, CH2, and CH3 sections of the IgG domains, as well as the hinge (H), are also shown. The presence of disulfide bonds (SS) that occur between cysteine residues causes fusion proteins in the IgG hinge region to be dimeric (15, 35).

Figure 7

The mechanism of action of eflapegrastim. The human IgG4 Fc fragment of Eflapegrastim interacts with FcRn, which is expressed and internalized on different cells such as bone marrow and assumed to protect eflapegrastim from lysosome leads to elongate its retention in bone marrow.